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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

• the present invention in some embodiments thereof, relates to personalized anticancer therapy by methods of predicting efficacy of anti-cancer therapies on a subject, and, more particularly, but not exclusively, to methods of treating solid tumors by predicting the efficacy of an anti-VEGFA treatment on the subject and selecting a treatment regimen based on the prediction of efficacy.
• anti-cancer drugs are efficient in some patients but exhibit no therapeutic effect on other patients having apparently the same diagnosis.
• anti-cancer therapies are associated with varying degrees of side effects, their high costs are often taken into consideration when designing a treatment regimen.
• targeted therapies for cancer specific subgroups of the cancerous tumors have to be identified in terms of specific molecular aberrations of individual tumors.
• identification of defining and recurring genetic abnormalities, which distinguish susceptible tumors, is mandatory for optimization of cancer treatment.
• Predictive biomarkers which are quantifiable parameters identifying subsets of disease that are more likely to respond to a specific treatment, are usually based on specific pathogenetic mechanisms that are related to a specific drug, and are considered the most important aspect of personalized medicine.
• Prominent examples for clinically validated cancer biomarkers include ERBB2 amplification in breast and gastric cancer and K-RAS mutations in colorectal cancer. These biomarkers serve as key determinates of treatment with Trustuzumab or Cetuximab, respectively.
• Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide, and the fifth most common cancer. It is generally accepted that HCC is most commonly the outcome of chronic injury and inflammation, resulting in hepatocyte regeneration and dysregulated growth factor signaling. In recent years it has become clear that inflammatory signaling pathways can support survival, growth and progression of cancer.
• the first line treatment for HCC is the multi-kinase inhibitor Sorafenib, which although not specific, is a strong inhibitor of VEGF receptors signaling. Sorafenib blocks several receptor tyrosine kinases including: VEGFRl, 2 and 3, PDGFR, c-Kit and RET, as well as inhibiting downstream Raf kinase isoforms (Kamimura, S. & Tsukamoto, H. Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease. Hepatology 22, 1304-1309, 1995). Sorafenib was recently shown to extend median survival from 7.9 months to 10.7 months in patients with advanced HCC (Stage C), establishing a new standard of care.
• Mdr2 is an ortholog of a human gene mutated in progressive familial intrahepatic cholestatsis (PFIC3).
• PFIC3 progressive familial intrahepatic cholestatsis
• Mdr2 deficiency (Mdr2 _/ ⁇ ) results in chronic inflammation of the portal tracts, eventually leading to inflammation-induced liver tumors that share many features with human HCC, and therefore was shown to be an effective tool for studying HCC [Mauad, T.H., et al, 1994, Am. J. Pathol. 145, 1237-45; Pikarsky, E.
• VEGF-A is a master regulator of angiogenesis whose role in tumor vessel recruitment is very well established.
• VEGF-A can act synergistically with EGFR to promote proliferation of skin cancer cells which express VEGF receptor 1 (FLT1).
• FLT1 VEGF receptor 1
• VEGF-A elicits hepatocyte proliferation by elevating the expression of several mitogens in the liver sinusoidal endothelial cells [Ding, B.S., et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468, 310-315 (Published November 11, 2010); LeCouter, J., et al. Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science 299, 890-893, 2003].
• Siegel AB et al, 2008; Zhu AX, et al, 2006; and Thomas MB, et al, 2009 describe phase II clinical trials using bevacizumab (AvastinTM) alone or in combination with additional anti-cancer drugs for the treatment of hepatocellular carcinoma.
• AvastinTM bevacizumab
• Komorowski J, et al., 2006 show that in cells the anti-angiogenic action of thalidomide [a-(N-phthalimido)-glutarimide] is due to direct inhibitory action on VEGF secretion and capillary microvessel formation.
• a method of predicting an efficacy of an anti-vascular endothelial growth factor A (VEGFA) treatment on a subject diagnosed with a solid tumor comprising: determining a presence or an absence of a genomic amplification which comprises a VEGFA gene in a sample of the solid tumor, wherein the presence or the absence of the genomic amplification predicts the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor, thereby predicting the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor.
• VEGFA anti-vascular endothelial growth factor A
• a method of treating of a subject diagnosed with a solid tumor comprising: (a) predicting the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the prediction; thereby treating of the subject diagnosed with the solid tumor.
• a method of selecting a treatment regimen for treating a subject diagnosed with a solid tumor comprising: (a) predicting the efficacy of the anti- VEGFA treatment on the subject diagnosed with the solid tumor according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the prediction; thereby selecting the treatment regimen for treating the subject diagnosed with a solid tumor.
• the solid tumor is carcinoma.
• the carcinoma is hepatocellular carcinoma.
• determining the presence or the absence of the genomic amplification is effected by comparing a ratio determined in a sample of the solid tumor between a copy number of the VEGFA and a copy number of a centromeric marker of human chromosome 6, or visa versa, to a reference ratio determined in at least one sample devoid of the solid tumor between a copy number of the VEGFA and a copy number of the centromeric marker of human chromosome 6, or visa versa, respectively.
• an increase above a predetermined threshold in the ratio determined in the sample of the solid tumor relative to the reference ratio indicates the presence of the genomic amplification.
• an identical ratio or a change below a predetermined threshold in the ratio determined in the sample of the solid tumor as compared to the reference ratio indicates the absence of the genomic amplification.
• determining a presence or an absence of a genomic amplification is effected using a DNA detection method. According to some embodiments of the invention, determining a presence or an absence of a genomic amplification is effected using a chromosomal detection method.
• the method further comprising comparing an expression level of the VEGFA in the sample of the solid tumor to a reference expression data obtained from at least one sample devoid of cancer.
• an increase above a predetermined threshold in the expression level of the VEGFA in the sample of the solid tumor relative to the reference expression data predicts the efficacy of the anti- VEGFA treatment on the solid tumor.
• the sample devoid of cancer is a liver sample.
• the expression level is determined using an RNA detection method.
• the expression level is determined using a protein detection method.
• the anti-VEGFA treatment comprises Sorafenib.
• the anti-VEGFA treatment comprises bevacizumab.
• the anti-VEGFA treatment comprises a soluble form of the VEGF-receptor.
• the anti-VEGFA treatment comprises thalidomide.
• the anti-VEGFA treatment comprises a combination of at least two anti-VEGFA drugs selected from the group consisting of Sorafenib, bevacizumab, a soluble form of the VEGF-receptor and thalidomide.
• the treatment regimen comprises administering of at least one anti-VEGFA drug selected from the group consisting of Sorafenib, bevacizumab, a soluble form of the VEGF-receptor and thalidomide.
• the treatment regimen further comprises administering a drug selected from the group consisting of erlotinib, oxaliplatin, cisp latin, platinum, rIFNa-2b, doxorubicin, fluorouracil, DX-8951f, thalidomide, doxorubicin, epirubicin, and taxol.
• the genomic amplification is of a human chromosome 6p21.
• the genomic amplification comprises the nucleotide sequence set forth in SEQ ID NO:23.
• the genomic amplification comprises the nucleotide sequence set forth in SEQ ID NO:24.
• the genomic amplification comprises the nucleotide sequence set forth in SEQ ID NO:25.
• the VEGFA comprises the genomic nucleic acid sequence set forth by SEQ ID NO: 1.
• the solid tumor is hepatocellular, and wherein the hepatocellular solid tumor is associated with hepatitis C infection.
• the efficacy of the anti- VEGFA treatment is determined by tumor regression following at least 8 weeks of the anti-VEGFA treatment.
• the efficacy of the anti- VEGFA treatment is determined by progression-free survival (PFS) time of at least one year.
• PFS progression-free survival
• the efficacy of the anti- VEGFA treatment is determined by a proliferation assay.
• the DNA detection method comprises DNA quantitative PCR (qPCR).
• FIG. 1 is an image of raw data obtained from array comparative genome hybridization (aCGH) analysis showing the amplification (right shift of dots) in a specific region of chromosome 17.
• aCGH array comparative genome hybridization
• FIGs. 2A-H are images depicting chromogenic in situ hybridization (CISH) of murine HCC tumors harboring the Chrl7qB3 genomic region amplification.
• CISH was performed using probes specific for Chrl7qB3. Two different nuclei from two different tumors from each subgroup are shown.
• FIG. 21 is a schematic representation of the murine Chrl7qB3 genomic region amplification in various HCC tumors which map the critical region of the genomic amplification.
• DNA qPCR analysis was performed using primers specific for different loci on the qB3 arm of chromosome 17. Each line represents a different Amp pos tumor. Thin line represents non-amplified region, thick line represents amplified (>2 fold increase) regions.
• the list includes several of the residing genes (the full list is presented in Table 5 in the Examples section which follows).
• FIGs. 2J-0 are graphs depicting relative mR A expression of various genes residing on the amplified region in HCC tumors with or without the genomic amplification. qPCR analysis was performed on wild type liver tissues (WT liver), HCC tumors without the amplification (Amp neg tumor) and HCC tumors harboring the genomic amplification (Amp pos tumor) using primers which specifically detect Cdc5L
• FIG. 3 is a histogram depicting real time quantitative PCR (qPCR) and ELISA analysis of VEGF mRNA and protein levels, respectively, of amplified versus non- amplified murine Mdr2 _/ ⁇ tumors. Note the significant increase in VEGFA expression level (on both RNA and protein levels) in amplified HCC tumors.
• FIGs. 4A-F are representative images of murine HCC tumors with ( Figures 4B,
• FIGs. 4G-I are histograms depicting quantization of the IHC analyses (for which representative images are shown in Figures 4A-F). IHC stainings were quantified using automated image analysis.
• FIGs. 5A-H are images of IHC for BrdU ( Figures 5A-D) and Ki67 ( Figures 5E- H of HCC tumors with or without the genomic amplification of mice which were treated with sFLT or remained untreated.
• Mdr2 _/ ⁇ mice were treated with adenovectors expressing either GFP alone ( Figures 5A, 5C, 5E and 5G) or GFP with sFLT ( Figures 5B, 5D, 5F and 5H) for 10 days. Shown are representative photomicrographs of IHC for BrdU and Ki67. Tumor infiltrating cells remain proliferative. Scale bars: Figures 5A-D - 50 ⁇ , Figures 5E-H - ⁇ .
• FIGs. 5I-J are histograms depicting quantization of the IHC results (for which representative images are shown in Figures 5A-H).
• FIGs. 5K-L are images depicting IHC for the mitosis-specific marker phospho- histone 3 (pHH3) in amplified tumors treated with adeno-sFLT ( Figure 5L) or adeno- GFP ( Figure 5K). Note the decrease in pHH3 positive hepatocytes in adeno-sFLT treated tumors, indicating reduced proliferation.
• pHH3 mitosis-specific marker phospho- histone 3
• FIGs. 5M-N are histograms depicting the results of qPCR analysis of VEGF-A (Figure 5M), HGF ( Figure 5N) of Amp neg and Amp pos tumors treated with the indicated adeno vectors (as described in Figures 5 AH above).
• Cross line signifies geometric mean (*p ⁇ 0.0001).
• sFLT anti-VEGF-A treatment
• FIGs. 50-R are histograms ( Figures 50-P) and images ( Figures 5Q-R) depicting high expression level of the HIFla target genes Glutl ( Figure 50) and PGK1 (Figure 5P) in Amp pos tumors treated with the indicated adenovectors (as described in Figures 5 AH above) and the associated necrosis ( Figures 5Q-R).
• Cross line signifies geometric mean.
• Figure 5Q - a histological section stained with H&E, showing necrosis.
• Figure 5R - a macroscopic picture of a tumor showing areas of hemorrhagic necrosis. Results are representative for three out of the six sFLT treated Amp pos tumors. Note that the tumors that show increased expression of the HIFla target genes Glutl and PGK1 (markers for hypoxia) also display necrosis on histological evaluation.
• FIGs. 6A-C are a histogram and images depicting the results of qPCR analysis of HGF mRNA (Figure 6A) and IHC of HGF in non-amplified ( Figure 6B) and amplified ( Figure 6C) tumors.
• qPCR analysis of HGF mRNA was performed on WT livers, non-amplified tumors and amplified tumors.
• Immunostaining for HGF reveals staining in endothelial and inflammatory cell populations, predominantly in the amplified tumor.
• FIG. 7 is a histogram depicting VEGF-A relative gene dose in various murine HCC tumors which is used for screening for Amp pos tumors.
• qPCR was performed on DNA extracted from Mdr2 _/ ⁇ tumors (Tumors numbers 1-42) and wild-type (WT) liver samples (samples 1-3) using a set of primers targeting the 3'-UTR (untranslated region) (white bars) and the promoter (grey bars) regions of the VEGF-A gene.
• Primers' sequences are provided in Table 4 in the EXAMPLES section which follows. The results were compared to those obtained by CGH testing of the same tumors (tumors 1- 10) and validated that the VEGF-A gene dose (determined by DNA qPCR) can detect the genomic amplification.
• FIGs. 8A-D are images of vWF IHC analysis performed on amplified ( Figures 8A-B) and non-amplified ( Figures 8C-D) HCC tumors treated with adeno-sFLT ( Figures 8B and 8D) or adeno-GFP ( Figures 8A and 8C) vectors.
• FIGs. 9A-H are images depicting representative photomicrographs of BrdU ( Figures 9A-D) or vWF ( Figures 9E-H) IHC in amplified tumors ( Figures 9C, 9D, 9G and 9H) or non-amplified tumors ( Figures 9A, 9B, 9E and 9F) which were treated with Sorafenib ( Figures 9B, 9D, 9F and 9H) or vehicle ( Figures 9A, 9C, 9E and 9G). Note the decrease in stained nuclei (as determined by BrdU staining) in the treated Amp pos group alone ( Figure 9D) while no decrease in blood vessel (as determined by vWF staining; Figure 9H) is evident.
• FIG. 91 is a histogram depicting quantization of the IHC staining for vWF using automated image analysis.
• P 0.06.
• FIGs. 9J-M are histograms depicting qPCR analysis of Amp neg and Amp pos for VEGF-A (Figure 9J), HGF (Figure 9K), PGK1 ( Figure 8L) and Glutl (Figure 9M).
• Cross line signifies geometric mean (*p ⁇ 0.05). Note that Amp pos tumors are sensitive to short term treatment with Sorafenib as indicated by a reduction in HGF mRNA levels.
• FIG. 9N is a histogram depicting quantization of the IHC staining for BrdU using automated image analysis, demonstrating the effect of Sorafenib treatment on the proliferation of mice HCC tumors.
• Mdr2 _/ ⁇ mice were treated with Sorafenib or vehicle alone for 3 days. Shown are the percentages of BrdU positive nuclei in tumors bearing the amplification ("Biomarker +") or being devoid of the amplification ("Biomarker -”), following treatment with Sorafenib or vehicle. Note the decline in the proliferation rate of tumor cells in tumors bearing the amplification which were treated with Sorafenib, as compared to the proliferation rate of the tumor cells in tumors devoid of the genomic amplification.
• FIGs. 10A-K are histograms depicting qPCR analysis of the mRNA levels of several of the 63 genes in Amp pos and Amp neg , demonstrating the expression profile of several of the amplicon residing genes.
• Cross line signifies geometric mean (*p ⁇ 0.05, **p ⁇ 0.01).
• FIGs. 11A-E are histological analyses demonstrating that Amp pos HCC hold distinct histological features.
• Figures 11 A-B Representative H&E stained sections of Amp neg and Amp pos tumors showing steatosis (lipid droplets) in the Amp pos but not Amp neg group. Scale bar 100 ⁇ .
• Figures 11C-D Representative H&E stained sections of Amp neg and Amp pos tumors demonstrating the differences in the size of tumor cell and cytoplasm between the two groups. Scale bar 50 ⁇ .
• Figure HE -H&E stained sections of Amp neg and Amp pos tumors were examined by a pathologist and evaluated for the presence of large cells and steatosis. A ⁇ 2 test was used to determine statistical significance.
• FIGs. 12 A- J are images ( Figures 12A-H) and histograms ( Figures 121- J) demonstrating that 10 days inhibition of VEGF-A in Amp pos tumors does not alter the microenvironmental content.
• Amplified tumors ( Figures 12C, 12D, 12G, 12H) and non-amplified tumors Figures 12 A, 12B, 12E, 12F) were treated for 10 days with adenovirus vector expressing GFP ( Figures 12A, 12C, 12E and 12G) or sFTL-GFP ( Figures 12B, 12D, 12F and 12H).
• Amp neg GFP treated (n 12; white bars)
• Amp neg sFLT treated (n 12; light grey bars)
• Amp pos GFP treated (n 5; dark grey bars)
• Amp pos sFLT treated (n 5; black bars).
• Differences between the Amp pos with GFP or sFLT were statistically insignificant.
• the present invention in some embodiments thereof, relates to personalized anticancer therapy by methods of predicting efficacy of anti-cancer therapies on a subject, and, more particularly, but not exclusively, to methods of treating solid tumors by predicting the efficacy of an anti-VEGFA treatment on the subject and selecting a treatment regimen based on the prediction of efficacy.
• the present inventors have uncovered that the variability in response to an anti- VEGFA treatment such as Sorafenib (a multi-tyrosine kinase inhibitor, marketed as Nexavar by Bayer) or a soluble VEGF-A receptor, depends on the presence or absence of the genomic amplification on murine chromosome 17qB3 (a syntenic region of human chromosome 6p21) which comprises the VEGFA gene, such that subjects having a solid tumor which comprises the genomic amplification respond well (i.e., in an efficient manner) to the anti-VEGFA treatment, and subjects having the same solid tumor albeit devoid of the genomic amplification respond in a less efficient manner (or do not respond at all) to the anti-VEGFA treatment.
• Sorafenib a multi-tyrosine kinase inhibitor, marketed as Nexavar by Bayer
• a soluble VEGF-A receptor depends on the presence or absence of the genomic amplification on murine chromos
• genomic amplification induces a unique tumor environment with higher expression of the macrophage marker F4/80 ( Figures 4E-F and 41, Example 3) and higher levels of hepatocyte growth factor (HGF, Figures 6A-C, Example 3).
• the Amp pos - HCC tumors were significantly more susceptible to treatment with a soluble form of the VEGF-A receptor (sFLT) as compared to Amp neg -HCC tumors, as shown by an efficient inhibition of tumor cell proliferation (Figures 5A-L, Example 4), which was accompanied by a decrease in HGF mR A levels in sFLT-treated tumors ( Figure 5N, Example 4) and with an increase in tissue hypoxia ( Figures 50-P, Example 4) and necrosis ( Figures 5Q-R, Example 4).
• sFLT soluble form of the VEGF-A receptor
• Example 6 of the Examples section which follows the present inventors found that Amp pos -HCC tumors are uniquely sensitive to Sorafenib, a multi-tyrosine kinase inhibitor (which inhibits VEGF-A activity), which is currently the first line treatment of advanced HCC in human beings, as shown by decrease in tumor cell proliferation ( Figures 9A-D, Figure 9N, Example 6) and HGF levels ( Figure 9K, Example 6).
• genomic amplification which comprises the VEGF-A gene distinguishes a subgroup of HCC tumors that are sensitive to direct VEGF-A blocking (e.g., sFLT) and Sorafenib treatment, and suggest the use of the genomic amplification as a prognostic marker to predict the efficacy of an anti-VEGF-A treatment in a subject having a solid tumor.
• sFLT direct VEGF-A blocking
• Sorafenib treatment suggest the use of the genomic amplification as a prognostic marker to predict the efficacy of an anti-VEGF-A treatment in a subject having a solid tumor.
• a method of predicting an efficacy of an anti-vascular endothelial growth factor A (VEGFA) treatment on a subject diagnosed with a solid tumor comprising determining a presence or an absence of a genomic amplification which comprises a VEGFA gene in a sample of the solid tumor, wherein the presence or the absence of the genomic amplification predicts the efficacy of the anti- VEGFA treatment on the subject diagnosed with the solid tumor, thereby predicting the efficacy of the anti- VEGFA treatment on the subject diagnosed with the solid tumor.
• presence of the genomic amplification which comprises the VEGFA gene in a solid tumor sample predicts that the anti- VEGFA treatment will be efficient in treating the solid tumor in the subject.
• the phrase "predicting efficacy of an anti- VEGFA treatment” refers to determining the likelihood that an anti-VEGFA treatment will be efficient or non-efficient in treating the solid tumor, e.g., the success or failure of the anti-VEGFA treatment in treating the solid tumor in a subject in need thereof.
• the term "efficacy” as used herein refers to the extent to which the anti-VEGFA treatment produces a beneficial result, e.g., an improvement in one or more symptoms of the pathology (caused by the solid tumor) and/or clinical parameters related to the pathology as described hereinbelow.
• the efficacy of an anti-VEGFA treatment may be evaluated using standard therapeutic indices for solid tumors.
• the efficacy of treatment is a long-term efficacy.
• long-term efficacy refers to the ability of a treatment to maintain a beneficial result over a period of time, e.g., at least about 16 weeks, at least about 26 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 48 weeks, at least about 52 weeks, at least about 18 months, at least about 24 months, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, or longer.
• the efficacy of the anti- VEGFA treatment is determined by tumor regression following at least 8 weeks of the anti-VEGFA treatment.
• an anti-VEGFA treatment is considered efficient in treating a solid tumor if it exerts an improvement in at least one relevant clinical parameter related to the solid tumor in the treated subject as compared to an untreated subject diagnosed with the same solid tumor (e.g., the same type, stage, degree and/or classification of the solid tumor), or as compared to the clinical parameters related to the solid tumor of the same subject prior to the anti-VEGFA treatment.
• Non-limiting examples of the clinical parameters related to the solid tumor which can be monitored in order to determine the efficacy of the anti-VEGFA treatment include the number of tumor lesions, dimensions (e.g., size) of each of the tumor lesion, tumor stage, differentiation state of tumor, presence and/or degree of tumor metastases, effect of the tumor on physiological function of the subject affected by the solid tumor, and the like.
• Evaluation of the efficacy of an anti-VEGFA treatment can be also performed using acceptable clinical criteria, such as the criteria proposed by the "Response Evaluation Criteria in Solid Tumors (RECIST) Committee" described in Therasse P., Arbuck SG., Eisenhauer EA et al. (New guidelines to evaluate the response to treatment in solid tumors: European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J. Natl. Cancer Inst. 92:205-216, 2000), which is incorporated by reference in its entirety.
• the (RECIST) is a set of published rules that define when cancer patients improve (“respond”), stay the same (“stabilize”), or worsen ("progression") during treatments.
• the efficacy of the anti- VEGFA treatment can be determined using at least one, two or all three of the response criteria included in the RECIST which include evaluation of target lesions [target lesions are selected on the basis of their size (lesions with the longest diameter)], evaluation of non-target lesions [all other lesions (or sites of disease) identified as non-target lesions], and evaluation of best overall response.
• Evaluation of target lesions classifies the response as follows: (i) Complete Response (CR) -Disappearance of all target lesions; (ii) Partial Response (PR) - At least a 30% decrease in the sum of the longest diameter (LD) of target lesions, taking as reference the baseline sum LD; (iii) Stable Disease (SD) - Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for Progressive Disease (PD), taking as reference the smallest sum LD since the treatment started; (iv) Progressive Disease (PD) - At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions.
• Evaluation of non-target lesions classifies the response as follows: (i) Complete Response (CR) - Disappearance of all non-target lesions and normalization of tumor marker level; (ii) Incomplete Response/ Stable Disease (SD) - Persistence of one or more non-target lesion(s) or/and maintenance of tumor marker level above the normal limits; (iii) Progressive Disease (PD) - Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions.
• CR Complete Response
• SD Incomplete Response/ Stable Disease
• PD Progressive Disease
• an anti-VEGFA treatment is considered efficient in treating the solid tumor if it results in at least a partial response (PR) according to the RECIST criteria.
• an anti-VEGFA treatment is considered efficient in treating the solid tumor if it results in a complete response (CR) according to the RECIST criteria.
• VEGFA treatment is determined by progression-free survival (PFS) time of at least one year.
• PFS progression-free survival
• determining the efficacy of an anti-VEGF-A treatment is performed by monitoring the proliferation state of the tumor cells.
• the proliferation state of tumor cells can be determined using various methods known in the art, such as using the synthetic nucleoside analogue of thymidine, bromodeoxyuridine (5-bi mo-2-deoxyuridine, BrdU).
• BrdU is commonly used in the detection of proliferating cells in living tissues.
• BrdU is incorporated into the newly synthesized DNA of replicating cells, substituting for thymidine during DNA replication.
• Antibodies specific for BrdU can then be used to detect the incorporated chemical, thus indicating cells that were actively replicating their DNA.
• Non-limiting examples of solid tumors which can be treated by the anti-VEGFA treatment according to some embodiments of the invention include tumors of the gastrointestinal tract (colon solid tumor, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small and/or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, pancreatic endocrine tumors), endometrial carcinoma, dermatofibrosarcoma protuberans, gallbladder carcinoma, Biliary tract tumors, prostate cancer, prostate adenocarcinoma, renal cancer (e.g., Wilms' tumor type 2 or type 1), liver cancer (e.g., hepato
• the solid tumor which is treated by the anti-VEGFA treatment is a carcinoma.
• carcinoma refers to any malignant tumor derived from epithelial cells or tissue.
• the carcinoma is selected from the group consisting of hepatocellular carcinoma, cervical cancer, Nasopharyngeal carcinoma (NPC), bladder cancer, lung cancer (e.g., non-small cell lung cancer), esophageal squamous cell carcinoma, multiple myeloma, kidney cancer (renal cell carcinoma), metastatic renal cell carcinoma, colon cancer, colorectal cancer, pancreatic cancer and ovarian cancer.
• NPC Nasopharyngeal carcinoma
• bladder cancer e.g., non-small cell lung cancer
• esophageal squamous cell carcinoma multiple myeloma
• kidney cancer renal cell carcinoma
• metastatic renal cell carcinoma colon cancer
• colorectal cancer pancreatic cancer and ovarian cancer.
• the carcinoma is lung cancer. According to some embodiments of the invention the carcinoma is colorectal cancer.
• the carcinoma is hepatocellular carcinoma.
• the hepatocellular solid tumor is associated with hepatitis C infection.
• the carcinoma is cancer metastases.
• Table 1 provides a non- limiting list of solid tumors which include the 6p21 genomic amplification.
• Table 2 provides a non- limiting list of solid tumors which include the 6p21-p23 gain or amplification.
• VEGFA vascular endothelial growth factor A
• VPF vascular endothelial growth factor A
• VEGF vascular endothelial growth factor A
• MVCD1, MGC70609 synthetic, recombinant and/or naturally occurring polynucleotide and polypeptide sequences assigned to the gene symbol VEGFA. These include but are not limited to the genomic sequence encoding VEGFA [nucleotides 43737953-43754224 of GenBank Accession No. NC_000006.11 as set forth by SEQ ID NO: l], the mRNA transcripts encoded thereby [e.g., GenBank Accession Nos.
• NM 001025366.1 (SEQ ID NO:2); NM 003376.4 (SEQ ID NO:3); NM 001025367.1 (SEQ ID NO:4); NM 001025368.1 (SEQ ID NO:5); NM 001033756.1 (SEQ ID NO:6); NM 001025369 (SEQ ID NO:7); NM 001025370 (SEQ ID NO: 8)] and/or the polypeptide variants encoded thereby [e.g., GenBank Accession Nos.
• NP 001020537.2 (SEQ ID NO:9); NP 003367.4 (SEQ ID NO: 10); NP 001020538.2 (SEQ ID NO: 11); NP 001020539.2 (SEQ ID NO: 12); NP 001028928.1 (SEQ ID NO: 13); NP 001020540.2 (SEQ ID NO: 14); NP 001020541.2 (SEQ ID NO: 15).
• genomic amplification which comprises a VEGFA gene refers to the presence of more than one copy per chromosome homolog of at least the genomic sequence encoding VEGFA.
• chromosome homolog refers to a single chromosome of the pair of chromosomes that pair (synapse) during meiosis.
• the genomic amplification which comprises the VEGFA gene is of the human chromosome 6p21 [nucleotide coordinates chr6:30,400,001-46,200,000 (SEQ ID NO:23) according to UCSC on Human GRCh37 Assembly (human genome 19 (hgl9)].
• the 6p21 region includes the chromosomal bands 6p21.33, 6p21.32, 6p21.31, 6p21.2, 6p21.1.
• the specific band in 6p21 region which comprises the VEGFA genomic sequence is 6p21.1, which is encompassed by nucleotide coordinates chr6:40,500,001-46,200,000 (SEQ ID NO:24) according to UCSC on Human GRCh37 Assembly [human genome 19 (hgl9)].
• the genomic amplification which comprises the VEGFA gene is set forth in SEQ ID NO:24.
• the genomic amplification which comprises the VEGFA gene is set forth in SEQ ID NO: 25 (Chr6: 43684022- 44002022 in the hgl9 assembly).
• This sequence comprises the VEGFA (SEQ ID NO: l, LOC100132354 (SEQ ID NO:405) and C6orf223 (SEQ ID NO:406) genomic sequences.
• Non-limiting examples of BACs bacterial artificial chromosomes which are derived from the 6p21.1 region and which can be used to detect the genomic amplification which comprises VEGFA gene
• RP11-710L16 [chromosome 6:43,633,251-43,817,196; according to UCSC (University California Santa Cruz) on Human GRCh37 Assembly (human genome 19 (hgl9); SEQ ID NO: 18] which fully covers the VEGFA genomic sequence
• RP11-21M9 chr6:43,743,280-43,929,157 according to UCSC on Human GRCh37 Assembly (human genome 19 (hgl9); SEQ ID NO: 19] which partially covers the VEGFA genomic sequence (data not shown).
• the genomic amplification which comprises the VEGFA gene is set forth in SEQ ID NO: 18 and/or 19.
• the genomic amplification comprises at least 2, e.g., at least 3, e.g., at least 4, e.g., at least 5, e.g., at least 6, e.g., at least 7, e.g., at least 8, e.g., at least 9, e.g., at least 10, e.g., at least 15, e.g., at least 20, e.g., at least 30, e.g., at least 40, e.g., at least 50, e.g., at least 60, e.g., at least 70, e.g., at least 80, e.g., at least 90, e.g., at least 100, e.g., at least 200, e.g., at least 300, e.g., at least 400, e.g., at least 600, e.g., at least 1000 copies per chromosome homo log, or more of the genomic sequence encompasses the
• the genomic amplification comprises at least 2, e.g., at least 3, e.g., at least 4, e.g., at least 5, e.g., at least 6, e.g., at least 7, e.g., at least 8, e.g., at least 9, e.g., at least 10, e.g., at least 15, e.g., at least 20, e.g., at least 30, e.g., at least 40, e.g., at least 50, e.g., at least 60, e.g., at least 70, e.g., at least 80, e.g., at least 90, e.g., at least 100, e.g., at least 200, e.g., at least 300, e.g., at least 400, e.g., at least 600, e.g., at least 1000 copies per chromosome homolog, or more of the genomic sequence selected from the group
• determining the presence or the absence of the genomic amplification is effected by comparing a ratio determined in a sample of the solid tumor between a copy number of the VEGFA and a copy number of a centromeric marker of human chromosome 6, or visa versa, namely, comparing a ratio determined in a sample of the cancer between the copy number of a centromeric marker of human chromosome 6 and a copy number of the VEGFA to a reference ratio determined in at least one sample devoid of the solid tumor between a copy number of the VEGFA and a copy number of the centromeric marker of human chromosome 6, or visa versa, namely, a reference ratio determined in at least one sample devoid of the solid tumor between a copy number of the centromeric marker of human chromosome 6 and a copy number of the VEGFA, respectively.
• the centromeric marker of human chromosome 6 includes nucleotide coordinates 6:60500000-63300000 according to UCSC on Human GRCh37 Assembly (human genome 19 (hgl9)).
• Non-limiting examples of BAC clones which are encompassed by the human pericentromeric chromosome 6 and which can be used for detection of the copy number of human chromosome 6 include: RP1-91N13 (SEQ ID NO:20), RP5-1194012 (SEQ ID NO:21), and RP1-271N20 (SEQ ID NO:22).
• centromeric markers which can be used to detect the copy number of human chromosome 6 include the CEP 6 SPECTRUM GREEN (ABBOTT Molecular); ZYTODOT CEN 6 probe (PD2; Zyto Vision, C-3002-400); alphoid clone 308 (D6Z1; a 3-kb DNA fragment that is repeated in centromer 6; Jabs Wang E et al., Characterization of human centromeric regions of specific chromosomes by means of alphoid DNA sequences. Am. J. Hum. Genet. 41 :374-390, 1987); and SKU CEN006 (Empire Genomics, Buffalo, NY, USA).
• CEP 6 SPECTRUM GREEN ABBOTT Molecular
• ZYTODOT CEN 6 probe PD2; Zyto Vision, C-3002-400
• alphoid clone 308 D6Z1; a 3-kb DNA fragment that is repeated in centromer 6
• Jabs Wang E et al. Character
• sample refers to any biological sample which contains a cell of a subject or a cellular component.
• Non-limiting examples of biological samples include body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk as well as white blood cells, tissue biopsies (including those obtained by fine needle aspiration, a surgical tool) e.g., of malignant tissues.
• body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk as well as white blood cells, tissue biopsies (including those obtained by fine needle aspiration, a surgical tool) e.g., of malignant tissues.
• the sample comprises tumor cells and/or tissue.
• the sample is a tissue section (e.g., a paraffin-embedded section, e.g., an archive paraffin-embedded tissue section, or a frozen tissue section).
• a tissue section e.g., a paraffin-embedded section, e.g., an archive paraffin-embedded tissue section, or a frozen tissue section.
• the sample is a fine-needle aspiration sample of a solid tumor.
• an increase above a predetermined threshold in the ratio determined in the sample of the carcinoma relative to the reference ratio indicates the presence of the genomic amplification.
• an increase above a predetermined threshold refers to an increase in the ratio determined in the sample of the carcinoma relative to the reference ratio which is higher than a predetermined threshold such as a about 10 %, e.g., higher than about 20 %, e.g., higher than about 30 %, e.g., higher than about 40 %, e.g., higher than about 50 %, e.g., higher than about 60 %, higher than about 70 %, higher than about 80 %, higher than about 90 %, higher than about 2 times, higher than about three times, higher than about four time, higher than about five times, higher than about six times, higher than about seven times, higher than about eight times, higher than about nine times, higher than about 20 times, higher than about 50 times, higher than about 100 times, higher than about 200 times, higher than about 350, higher than about 500 times, higher than about 1000 times, or more relative to the reference ratio.
• a predetermined threshold such as a about 10 %, e.g., higher than about
• a predetermined threshold refers to an increase or a decrease in the level of expression in the cell of the subject relative to the reference ratio which is lower than a predetermined threshold, such as lower than about 2 times, e.g., lower than about 90%, e.g., lower than about 80%, e.g., lower than about 70%), e.g., lower than about 60%>, e.g., lower than about 50%>, e.g., lower than about 40%o, e.g., lower than about 30%>, e.g., lower than about 20%>, e.g., lower than about 10%), e.g., lower than about 9%>, e.g., lower than about 8%, e.g., lower than about 7%o, e.g., lower than about 6%>, e.g., lower than about 5%>, e.g., lower than about 4%, e.g., lower than about 3%>, e
• a predetermined threshold such as lower
• determining a presence or an absence of a genomic amplification is effected using a chromosomal detection method.
• Non-limiting examples of chromosomal detection methods include fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), primed in situ labeling (PRINS), quantitative FISH (Q-FISH) and/or multicolor-banding (MCB).
• FISH fluorescent in situ hybridization
• CISH chromogenic in situ hybridization
• PRINS primed in situ labeling
• Q-FISH quantitative FISH
• MB multicolor-banding
• probes e.g., a probe derived from the amplified region which comprises the VEGF-A gene such as SEQ ID NO: 18 (BAC RP11-710L16), 19 (BAC RP11-21M9), 23, 24 and/or 25 labeled in one color (e.g., green) and a probe derived from a chromosome 6 centromer such as SEQ ID NO:20 (BAC RP 1-9 IN 13), 21 (BAC RP5-1194012) and/or 22 (BAC RP1-271N20) labeled in another color (e.g., red)] are mixed with hybridization buffer (e.g., LSI/WCP, Abbott) and a carrier DNA (e.g., human Cot 1 DNA, available from Abbott).
• hybridization buffer e.g., LSI/WCP, Abbott
• carrier DNA e.g., human Cot 1 DNA, available from Abbott.
• the probe solution is applied on microscopic slides containing the biological sample (e.g., tissue sections from a tumor biopsy) and the slides are covered using a covers lip.
• the probe-containing slides are denatured for 4-5 minutes at 71 °C (or 3 minutes at 70 °C) and are further incubated for 24-60 hours at 37 °C using an hybridization apparatus (e.g., HYBrite, Abbott Cat. No. 2J11-04).
• an hybridization apparatus e.g., HYBrite, Abbott Cat. No. 2J11-04.
• the slides are washed for 2 minutes at 70-72 °C in a solution of 0.3 % NP-40 (Abbott) in 60 mM NaCl and 6 mM NaCitrate (0.4XSSC).
• CISH a labeled complementary DNA or RNA strand is used to localize a specific DNA or RNA sequence in a tissue specimen.
• CISH can be used to detect various chromosomal abnormalities such as gene amplification, gene deletion, chromosome translocation, and chromosome number.
• CISH utilizes conventional peroxidase or alkaline phosphatase reactions, and is applicable to formalin- fixed, paraffin-embedded tissues, blood or bone marrow smears, metaphase chromosome spreads, and fixed cells.
• PRINS analysis has been employed in the detection of gene deletion (Tharapel SA and Kadandale JS, 2002. Am. J. Med. Genet. 107: 123-126), determination of fetal sex (Orsetti, B., et al, 1998. Prenat. Diagn. 18: 1014-1022), and identification of chromosomal aneuploidy (Mennicke, K. et al., 2003. Fetal Diagn. Ther. 18: 114-121).
• Methods of performing PRINS analysis are known in the art and include for example, those described in Coullin, P. et al. (Am. J. Med. Genet. 2002, 107: 127-135); Findlay, I., et al. (J. Assist. Reprod. Genet. 1998, 15: 258-265); Musio, A., et al. (Genome 1998, 41 : 739-741); Mennicke, K., et al. (Fetal Diagn. Ther. 2003, 18: 114- 121); Orsetti, B., et al. (Prenat. Diagn. 1998, 18: 1014-1022).
• slides containing interphase chromosomes are denatured for 2 minutes at 71 °C in a solution of 70 % formamide in 2XSSC (pH 7.2), dehydrated in an ethanol series (70, 80, 90 and 100 %) and are placed on a flat plate block of a programmable temperature cycler (such as the PTC-200 thermal cycler adapted for glass slides which is available from MJ Research, Waltham, Massachusetts, USA).
• the PRINS reaction is usually performed in the presence of unlabeled primers and a mixture of dNTPs with a labeled dUTP (e.g., fluorescein- 12-dUTP or digoxigenin-11-dUTP for a direct or indirect detection, respectively).
• a labeled dUTP e.g., fluorescein- 12-dUTP or digoxigenin-11-dUTP for a direct or indirect detection, respectively.
• sequence-specific primers can be labeled at the 5' end using e.g., 1-3 fluorescein or cyanine 3 (Cy3) molecules.
• a typical PRINS reaction mixture includes sequence-specific primers (50-200 pmol in a 50 ⁇ reaction volume), unlabeled dNTPs (0.1 mM of dATP, dCTP, dGTP and 0.002 mM of dTTP), labeled dUTP (0.025 mM) and Taq DNA polymerase (2 units) with the appropriate reaction buffer. Once the slide reaches the desired annealing temperature the reaction mixture is applied on the slide and the slide is covered using a cover slip.
• Annealing of the sequence-specific primers is allowed to occur for 15 minutes, following which the primed chains are elongated at 72 °C for another 15 minutes. Following elongation, the slides are washed three times at room temperature in a solution of 4XSSC/0.5 % Tween-20 (4 minutes each), followed by a 4-minute wash at PBS. Slides are then subjected to nuclei counterstain using DAPI or propidium iodide. The fluorescently stained slides can be viewed using a fluorescent microscope and the appropriate combination of filters (e.g., DAPI, FITC, TRITC, FITC-rhodamin).
• filters e.g., DAPI, FITC, TRITC, FITC-rhodamin
• the PRINS analysis can be used as a multicolor assay for the determination of the presence, and/or location of several genes or chromosomal loci.
• the PRINS analysis can be performed on the same slide as the FISH analysis, preferably, prior to FISH analysis.
• MMB Multicolor banding
• Q-FISH Quantitative FISH
• PNA probes are synthetic DNA mimics in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by an amine bond and to which the nucleobases are fixed (Pellestor F and Paulasova P, 2004; Chromosoma 112: 375-380).
• the hydrophobic and neutral backbone enables high affinity and specific hybridization of the PNA probes to their nucleic acid counterparts (e.g., chromosomal DNA).
• Such probes have been applied on interphase nuclei to monitor telomere stability (Slijepcevic, P. 1998; Mutat. Res. 404:215-220; Henderson S., et al, 1996; J. Cell Biol.
• Fanconi aneamia Hanson H, et al., 2001, Cytogenet. Cell Genet. 93: 203-6
• numerical chromosome abnormalities such as trisomy 18 (Chen C, et al, 2000, Mamm. Genome 10: 13-18), as well as monosomy, duplication, and deletion (Taneja KL, et al, 2001, Genes Chromosomes Cancer. 30: 57-63).
• Q-FISH can be performed by co-hybridizing whole chromosome painting probes (e.g., for chromosomes 21 and 22) on interphase nuclei as described in Truong K et al, 2003, Prenat. Diagn. 23: 146-51.
• determining a presence or an absence of a genomic amplification is effected using a DNA detection method.
• Comparative Genome Hybridization is based on a quantitative two- color fluorescence in situ hybridization (FISH) on metaphase chromosomes.
• FISH fluorescence in situ hybridization
• a test DNA e.g., DNA extracted from the biological sample which includes tumor cells, e.g., obtained from a tumor tissue biopsy
• one color e.g., green
• a reference DNA e.g., DNA extracted from a control cell
• a different color e.g., red
• genomic DNA is amplified using a degenerate oligonucleotide primer [e.g., 5 '-CCGACTCGAGNNNNNNATGTGG, SEQ ID NO: 404 (Telenius, H., et al, 1992; Genomics 13:718-25)] and the amplified DNA is labeled using e.g., the Spectrum Green-dUTP (for the test DNA) or the Spectrum Red- dUTP (for the reference DNA).
• a degenerate oligonucleotide primer e.g., 5 '-CCGACTCGAGNNNNNNATGTGG, SEQ ID NO: 404 (Telenius, H., et al, 1992; Genomics 13:718-25)
• the amplified DNA is labeled using e.g., the Spectrum Green-dUTP (for the test DNA) or the Spectrum Red- dUTP (for the reference DNA).
• the mixture of labeled DNA samples is precipitated with Cotl DNA (Gibco-BRL) and resuspended in an hybridization mixture containing e.g., 50 % formamide, 2XSSC, pH 7 and 10 % dextrane sulfate.
• the labeled DNA samples i.e., the probes
• the metaphase chromosome spreads are denatured using standard protocols (e.g., dehydration in a series of ethanol, denaturation for 5 minutes at 75 °C in 70 % formamide and 2XSSC).
• Hybridization conditions include incubation at 37 °C for 25-30 hours in a humidified chamber, following by washes in 2XSSC and dehydration using an ethanol series, essentially as described elsewhere (Wells, D., et al, 2002; Fertility and Sterility, 78: 543-549).
• Hybridization signal is detected using a fluorescence microscope and the ratio of the green-to-red fluorescence can be determined using e.g., the Applied Imaging (Santa Clara, CA) computer software. If both genomes are equally represented in the metaphase chromosomes (i.e., no deletions, duplication or insertions in the DNA derived from the tumor cells) the labeling on the metaphase chromosomes is orange. However, regions which are either deleted or duplicated in the tumor cell(s) are stained with red or green, respectively.
• CGH-array DNA array-based comparative genomic hybridization
• Hu, D.G., et al, 2004, Mol. Hum. Reprod. 10: 283- 289 is a modified version of CGH and is based on the hybridization of a 1 : 1 mixture of the test and reference DNA probes on an array containing chromosome-specific DNA libraries.
• Methods of preparing chromosome-specific DNA libraries are known in the art (see for example, Bolzer A., et al, 1999; Cytogenet. Cell. Genet. 84: 233-240).
• single chromosomes are obtained using either microdissection or flow-sorting and the genomic DNA of each of the isolated chromosomes is PCR-amplified using a degenerated oligonucleotide primer.
• the amplified DNA is subjected to affinity chromatography in combination with negative subtraction hybridization (using e.g., human Cot-1 DNA or centromer-specific repetitive sequence as subtractors), essentially as described in Craig JM., et al, 1997; Hum. Genet. 100: 472-476.
• Amplified chromosome-specific DNA libraries are then attached to a solid support [(e.g., SuperAmine slides (TeleChem, USA)], dried, baked and washed according to manufacturer's recommendation.
• Labeled genomic DNA probes (a 1 : 1 mixture of the test and reference DNAs) are mixed with non-specific carrier DNA (e.g., human Cot-1 and/or salmon sperm DNA, Gibco-BRL), ethanol-precipitated and re- suspended in an hybridization buffer such as 50 % deionized formamide, 2XSSC, 0.1 % SDS, 10 % Dextran sulphate and 5 X Denhardt's solution.
• an hybridization buffer such as 50 % deionized formamide, 2XSSC, 0.1 % SDS, 10 % Dextran sulphate and 5 X Denhardt's solution.
• the DNA probes are then denatured (80 °C for 10 minutes), pre-annealed (37 °C for 80 minutes) and applied on the array for hybridization of 15-20 hours in a humid incubator. Following hybridization the arrays are washed twice for 10 minutes in 50 % formamide/2XSSC at 45 °C and once for 10 minutes in 1XSSC at room temperature, following which the arrays are rinsed three times in 18.2 ⁇ deionized water. The arrays are then scanned using any suitable fluorescence scanner such as the GenePix 4000B microarray reader (Axon Instruments, USA) and analyzed using the GenePix Pro. 4.0.1.12 software (Axon).
• any suitable fluorescence scanner such as the GenePix 4000B microarray reader (Axon Instruments, USA) and analyzed using the GenePix Pro. 4.0.1.12 software (Axon).
• the genomic amplification which comprises the VEGFA gene can be detected using a quantitative DNA-based techniques such as quantitative DNA PCR (qDNA PCR) or quantitative Southern blot analysis.
• the quantitative DNA assays qPCR or qSouthern blot
• qPCR or qSouthern blot can determine the absolute number of gene copies or relative amount of gene copies (of a DNA sequence derived from the genomic amplification) when normalized to normalizing genes, and those of ordinary skills in the art are capable of assessing the results of such assays in order to determine presence or absence of the genomic amplification (see e.g., Figure 7).
• genomic DNA is extracted using known methods from a "test" biological sample (e.g., a tumor biopsy, or a fine needle aspiration sample for which the presence or absence of the genomic amplification is unknown) and from a reference sample with a known status with regard to presence or absence of the genomic amplification.
• the reference sample can be a positive control, i.e., a sample which is known to have the genomic amplification as determined by other methods such as FISH (e.g., a tumor having the genomic amplification), or it can be a negative control, i.e., a sample which is known to be devoid of the genomic amplification (having only a single copy per chromosome homologue).
• the negative control sample can be derived from a non-tumor tissue (derived from the same species, e.g., from human) or from a tumor tissue devoid of the genomic amplification as determined based on other methods, such as FISH.
• qPCR analyses can be carried out with SYBR green (Invitrogen) in 7900HT Fast Real-Time PCR System (Applied BioSystems), and the results can be analyzed using the qBase vl .3.5 software.
• SYBR green Invitrogen
• 7900HT Fast Real-Time PCR System
• qPCR can be performed using primers pairs specific to any of the genes encompassed in the genomic amplification (e.g., SEQ ID NOs: 1, 26-403 and 405-433). Non-limiting examples of such primers pairs are provided in Table 3 below and in SEQ ID NOs: 456-1269.
• PRR3 30524486 30532473 464 465 1274
• NRM 40 30655826 30658769 484 485 1284
• CDSN 58 31082865 31088252 520 521 1302
• HLA-C 65 31236529 31239855 534 535 1309
• HCP5 75 31430957 31433586 554 555 1319
• NCR3 90 31556660 31560762 584 585 1334
• GLP1R 306 39016557 39055520 1014 1015 1548
• MOCS1 316 39872034 39902290 1034 1035 1557
• TDRG1 320 40346163 40347631 1042 1043 1561
• TSP02 324 41010237 41012076 1050 1051 1565
• TTBK1 43211222 43255997 1170 1171 1623
• Table 3 Provided are the genes encompassed within the human chromosome 6p21 genomic amplification region [shown by gene symbol, sequence identifier and the start and end nucleotide positions on human chromosome 6 according to UCSC on Human GRCh37 Assembly (human genome 19 (hgl9)], and means to quantifying the copy number of the genomic amplification in 6p21 using PCR primers for qDNA PCR (provided by sequence identifiers) and DNA probes for quantitative Southern blot (provided by sequence identifiers).
• Genomic sequence SEQ ID NO: Genomic sequence SEQ ID NO:; "Start” - The first nucleotide corresponding to the gene as indicated by position on human Chr.6; “End” - The last nucleotide corresponding to the gene as indicated by position on human Chr. 6; "For. SEQ ID NO:” - Forward primer (5' ⁇ 3') for qDNA PCR (SEQ ID NO:); “Rev. SEQ ID NO:” - Reverse primer for qDNA PCR (SEQ ID NO:); “probe SEQ ID NO:” - Probe for qSouthern blot. #N/A - not available.
• the concentration of the DNA can be determined with a spectrophotometer, following which the DNA is digested with restriction enzyme endonucleases such as Hindlll and/or with combination of a few restriction enzymes (e.g., Hindlll and Bglll).
• restriction enzyme endonucleases such as Hindlll and/or with combination of a few restriction enzymes (e.g., Hindlll and Bglll).
• restriction enzyme endonucleases such as Hindlll and/or with combination of a few restriction enzymes (e.g., Hindlll and Bglll).
• the amount in each lane is equalized based on the intensity of DNA smear stained with ethidium bromide.
• a membrane e.g., Hybond N+; Amersham
• the blotted DNA is hybridized with a labeled DNA probe.
• the probe can be labeled using methods well known in the art with a radioactive isotope such as 32 P, or with a nonradioactive labeling such as using Digoxigenin labeling.
• Suitable DNA probes for quantitative Southern blot can be prepared from the genomic region of-interest, and/or from a complementary DNA (cDNA, complementary DNA probes, which are in the antisense direction with respect to the mRNA sense sequence) of any of the coding sequences comprised in the sequence of the genomic amplification which comprises the VEGF-A gene.
• cDNA complementary DNA
• cDNA complementary DNA probes, which are in the antisense direction with respect to the mRNA sense sequence
• the sequence of the cDNA can be obtained from the NCBI web site [Hypertext Transfer Protocol ://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/] by searching "GENE” with the "Gene Symbol” listed in Table 3 above, or by performing a sequence alignment (e.g., BLASTN) using as a "Query Sequence” any of the genomic sequences provided in SEQ ID NOs: l , 26-403 and 405-433 (Table 3 above).
• the length of the probe for Southern blot analysis can vary from few tens of nucleotides to several kilobases of nucleotides.
• Suitable polynucleotide probes are those referred to as "unique probes" which specifically hybridize to the target DNA sequence but not to other DNA sequences in the sample under the same hybridization conditions.
• hybridization of short polynucleotide probes can be effected by the following hybridization protocols depending on the desired stringency;
• hybridization of short polynucleotide probes can be effected by the following hybridization protocols depending on the desired stringency; (i) an hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 ⁇ g/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 °C below the T m , final wash solution of 3 M TMAC
• hybridization conditions e.g., hybridization solution, wash solutions and temperatures
• the hybridization/washes temperatures can be about 55-80 °C (e.g., 65 °C) and the hybridization/wash solutions can include formamide (e.g., 50 %).
• hybridization with longer DNA probes can be performed using the following hybridization solution (i) 5X Denhardt's Solution [5 OX Denhardt's Solution consists of 1% BSA, 1% Polyvinylpyrrolidone, 1% Ficoll], 100 mg/ml Salmon or Herring Sperm DNA, 0.1% SDS, 5XSSPE [SSPE (20X) consists of: 3M NaCl, 0.2M Sodium Phosphate, pH 7.4, 25 mM EDTA], 50% formamide; hybridization temperature of 20°C below the calculated Tm (melting temperature); and washes 1 x 20 minutes in IX SSC, 0.1% SDS at 45° C, followed by 3 x 20 minutes in 0.2X SSC, 0.1 % SDS at 65° C.
• 5X Denhardt's Solution consists of 1% BSA, 1% Polyvinylpyrrolidone, 1% Ficoll], 100 mg/ml Salmon or Herring Sperm DNA, 0.1% SDS
• 5XSSPE [SS
• the probe has a nucleotide sequence having as a 5' nucleotide the nucleotide at position "X" in SEQ ID NO:23 and as a 3' nucleotide the nucleotide at position "Y" in SEQ ID NO:23; wherein X is a numerator selected from nucleotide position 1-15,799,979 in SEQ ID NO:23, wherein Y is a numerator selected from nucleotide position 21-15,799,999 in SEQ ID NO:23, and wherein numerator Y is larger than the numerator X by at least about 20 nucleotides, e.g., by at least about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1
• the probe has a nucleotide sequence having as a 5' nucleotide the nucleotide at position "X" in SEQ ID NO:24 and as a 3' nucleotide the nucleotide at position "Y" in SEQ ID NO:24; wherein X is a numerator selected from nucleotide position 1-5,699,979 in SEQ ID NO:24, wherein Y is a numerator selected from nucleotide position 21-5,699,999 in SEQ ID NO:24, and wherein numerator Y is larger than the numerator X by at least about 20 nucleotides, e.g., by at least about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1
• the probe has a nucleotide sequence having as a 5' nucleotide the nucleotide at position "X" in SEQ ID NO:25 and as a 3' nucleotide the nucleotide at position "Y" in SEQ ID NO:25; wherein X is a numerator selected from nucleotide position 1-317,980 in SEQ ID NO:25, wherein Y is a numerator selected from nucleotide position 21-318,000 in SEQ ID NO:25, and wherein numerator Y is larger than the numerator X by at least about 20 nucleotides, e.g., by at least about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400
• the length of the probe varies from about 20 bases to about 10 kilobase (kb), e.g., from about 20 bases to about 7 kb, e.g., 100 bases to about 5 kb, e.g., from about 200 bases to about 2 kb, e.g., from about 300 bases to about 1.5 kb, e.g., from about 500-1300 bases.
• kb kilobase
• Various software are available to design appropriate DNA probes for genomic hybridization using Southern blot analysis.
• Examples include, but are not limited to G2C:: Software [Hypertext Transfer Protocol ://World Wide Web (dot) genes2cognition (dot) org/software/southern blot/probe tiling method (dot) html].
• a probe for Southern blot analysis should be preferably devoid of repetitive DNA sequences, such that when hybridized to genomic DNA will result in a single or a few distinct bands (see e.g., determination of gene copy number by quantitative Southern blot analysis in the Dystrophin gene, Hiraishi Y., et al., 1992, J. Med. Genet. 29: 897-901, which is hereby incorporated by reference in its entirety).
• Bioinformatic tools for removing repetitive sequences from nucleotide sequences are known in the art.
• the RepeatMasker tool Institute for System Biology, available via the Hypertext Transfer Protocol://World Wide Web (dot) repeatmasker (dot) org/ web site can be used.
• Another convenient tool for excluding repetitive sequences from a DNA sequence is via the UCSC Genome Browser [Hypertext Transfer Protocol ://genome (dot) ucsc (dot) edu/cgi-bin/hgGateway] in which one can insert the genomic region of interest (e.g., human chromosome 6p21) and in the "DNA” tool select the option of "Mask Repeats".
• the outcome of the repeat masking tools can be such that all repetitive sequences are converted to "NNN" an can be further excluded from the sequence.
• Non-limiting examples of DNA probes (devoid of repetitive sequences) from the genomic region encompassed by the amplification (6p21) are provided in SEQ ID NOs: 1270-1672 as described in the "Probe” column in Table 3 above.
• Qualifying of suitable probes for quantitative Southern blot can be done by Southern blot analysis using the identified probe (according to the teachings provided herein) using known reference DNA samples derived from a positive control biological sample (which is known to include the genomic amplification based on other methods such as FISH) and/or a negative control biological sample (which is known to be devoid of the genomic amplification based on other methods such as FISH).
• a positive control biological sample which is known to include the genomic amplification based on other methods such as FISH
• a negative control biological sample which is known to be devoid of the genomic amplification based on other methods such as FISH.
• the method further comprising comparing an expression level of the VEGFA in the sample of the carcinoma to a reference expression data obtained from at least one sample devoid of cancer.
• an increase above a predetermined threshold in the expression level of the VEGFA in the sample of the carcinoma relative to the reference expression data predicts the efficacy of the anti- VEGFA treatment on the carcinoma (i.e., that the anti-VEGFA treatment is efficient in treating the solid tumor).
• the sample devoid of cancer is a liver sample.
• VEGFA The expression level of VEGFA can be detected using various RNA detection methods such as Northern Blot analysis, RT-PCR analysis, RNA in situ hybridization stain, In situ RT-PCR stain, DNA microarrays/DNA chips, Oligonucleotide microarray.
• RNA detection methods such as Northern Blot analysis, RT-PCR analysis, RNA in situ hybridization stain, In situ RT-PCR stain, DNA microarrays/DNA chips, Oligonucleotide microarray.
• the expression and/or activity level of the VEGFA protein can be determined using methods known in the arts such as Enzyme linked immunosorbent assay (ELISA), Western blot, radio-immunoassay (RIA), fluorescence activated cell sorting (FACS), immunohistochemical analysis, in situ activity assay, in vitro activity assays.
• ELISA Enzyme linked immunosorbent assay
• RIA radio-immunoassay
• FACS fluorescence activated cell sorting
• anti-VEGFA refers to an agent (e.g., drug) which reduces, inhibits, or suppresses VEGFA levels (expression level and/or activity) in cells or tissue.
• the anti-VEGF agent inhibits
• the anti-VEGF agent is a small molecule, which blocks the VEGF-A tyrosine kinase receptor.
• the anti-VEGF agent reduces, inhibits or suppresses VEGFA levels in the tumor cells of a subject in need thereof.
• anti- VEGFA treatment refers to administration of an anti-VEGF drug into a subject in need thereof. It should be noted that administration of an anti-VEGF drug may comprise a single or multiple dosages, as well as a continuous administration, depending on the pathology to be treated and the subject receiving the treatment.
• Non-limiting examples of anti-VEGF agents which can be used according to the method of the invention include an anti-VEGF antibody (e.g., the monoclonal antibody bevacizumab such as AVASTINTM; ranibizumab), an anti- VEGFA RNA silencing agents (e.g., antisense, siRNA, shRNA), an anti- VEGFA Ribozyme, an anti- VEGFA DNAzyme, a soluble form of the VEGF-receptor (e.g., GenBank Accession No.
• an anti-VEGF antibody e.g., the monoclonal antibody bevacizumab such as AVASTINTM; ranibizumab
• an anti- VEGFA RNA silencing agents e.g., antisense, siRNA, shRNA
• an anti- VEGFA Ribozyme e.g., an anti- VEGFA DNAzyme
• a soluble form of the VEGF-receptor e.g.,
• AAC50060 SEQ ID NO: 16
• thalidomide or an analogue thereof e.g., as described in Miguel Fernandez Brana et al., European Journal of Medicinal Chemistry, Volume 44, Issue 9, 2009, Pages 3533-3542; Magdy A.-H.
• anti- VEGFA aptamers e.g., Pegaptanib, a pegylated anti-VEGF aptamer which specifically binds to VEGF 165; pegaptanib sodium
• small molecules which are anti-VEGF -A tyrosine kinase inhibitors such as Sorafenib (Nexavar®, Bayer HealthCare Pharmaceuticals), ABT-869 (Linifanib; N-[4-(3 -Amino- lH-indazol-4-yl)phenyl]-N * -(2-fluoro-5- methylphenyl)urea), Axitinib (also known as AGO 13736, a small molecule tyrosine kinase inhibitor under development by Pfizer), BIBF1120 (oral
• anti-VEGFA agents which can be used according to the method of the invention, include those described in Murukesh N., et al, 2010, which is fully incorporated herein in its entirety.
• the anti- VEGFA comprises Sorafenib.
• the anti- VEGFA comprises bevacizumab.
• the anti- VEGFA comprises a soluble form of the VEGF-receptor.
• the anti- VEGFA comprises thalidomide.
• the anti- VEGFA comprises a combination of at least two-anti VEGFA drugs selected from the group consisting of Sorafenib, bevacizumab, a soluble form of the VEGF-receptor and thalidomide.
• Non-limiting examples of such combination therapy with anti- VEGFA drugs comprises Sorafenib and bevacizumab; Sorafenib and a soluble form of the VEGF- receptor; Sorafenib and thalidomide; bevacizumab and a soluble form of the VEGF- receptor; and bevacizumab and thalidomide; a soluble form of the VEGF-receptor and thalidomide.
• the anti-VEGFA antibody comprises an antigen binding region capable of specifically binding VEGFA.
• the antigen binding region specifically binds at least one epitope of VEGFA.
• epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
• Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
• antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages.
• These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of
• Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
• Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
• antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
• This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
• a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
• an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
• cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
• Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
• sFv single-chain antigen binding proteins
• the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
• the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
• Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al, Science 242:423-426 (1988); Pack et al, Bio/Technology 11 : 1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
• CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
• Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary determining region
• donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)].
• Fc immunoglobulin constant region
• a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and coworkers [Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature 332:323- 327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991)].
• the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
• human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
• the anti-VEGFA agent is an anti-VEGFA RNA silencing agent.
• RNA silencing refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene.
• RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
• RNA silencing agent refers to an RNA which is capable of inhibiting or “silencing" the expression of a target gene (e.g., VEGFA).
• the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism.
• RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
• Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
• the RNA silencing agent is capable of inducing RNA interference.
• the RNA silencing agent is capable of mediating translational repression.
• RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
• siRNAs short interfering RNAs
• the corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi.
• the process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla.
• Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA.
• dsRNAs double-stranded RNAs
• the presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer.
• Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs).
• Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes.
• RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
• RISC RNA-induced silencing complex
• the present invention contemplates use of dsRNA to downregulate protein expression from mRNA.
• the dsRNA is greater than 30 bp.
• the use of long dsRNAs i.e. dsRNA greater than 30 bp
• the use of long dsRNAs can provide numerous advantages in that the cell can select the optimal silencing sequence alleviating the need to test numerous siRNAs; long dsRNAs will allow for silencing libraries to have less complexity than would be necessary for siRNAs; and, perhaps most importantly, long dsRNA could prevent viral escape mutations when used as therapeutics.
• the present invention also contemplates introduction of long dsRNA (over 30 base transcripts) for gene silencing in cells where the interferon pathway is not activated (e.g. embryonic cells and oocytes) see for example Billy et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides, October 1, 2003, 13(5): 381-392. doi: 10.1089/154545703322617069.
• long dsRNA over 30 base transcripts
• the present invention also contemplates introduction of long dsRNA specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression.
• Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5 '-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.
• siRNAs small inhibitory RNAs
• siRNA refers to small inhibitory RNA duplexes (generally between 18-30 basepairs) that induce the RNA interference (RNAi) pathway.
• RNAi RNA interference
• siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3 '-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100- fold increase in potency compared with 21mers at the same location.
• RNA silencing agent of the present invention may also be a short hairpin RNA (shRNA).
• RNA agent refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
• the number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop.
• oligonucleotide sequences that can be used to form the loop include 5'-UUCAAGAGA-3' (Brummelkamp, T. R. et al. (2002) Science 296: 550) and 5 * -UUUGUGUAG-3 * (Castanotto, D. et al. (2002) RNA 8: 1454). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.
• the RNA silencing agent may be a miRNA.
• miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants.
• the primary transcript (termed the “pri-miRNA") is processed through various nucleolytic steps to a shorter precursor miRNA, or "pre-miRNA.”
• the pre -miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target)
• the pre -miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex.
• miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17: 1376- 1386).
• miRNAs bind to transcript sequences with only partial complementarity (Zeng et al., 2002, Molec. Cell 9: 1327-1333) and repress translation without affecting steady-state RNA levels (Lee et al, 1993, Cell 75:843-854; Wightman et al, 1993, Cell 75:855-862). Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (Hutvagner et al., 2001, Science 293:834-838; Grishok et al, 2001, Cell 106: 23-34; Ketting et al, 2001, Genes Dev.
• RNA silencing agents suitable for use with the present invention can be effected as follows. First, the VEGFA mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245].
• UTRs untranslated regions
• siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
• potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (world wide web (dot) ncbi (dot) nlm (dot) nih (dot) gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
• sequence alignment software such as the BLAST software available from the NCBI server (world wide web (dot) ncbi (dot) nlm (dot) nih (dot) gov/BLAST/).
• Qualifying target sequences are selected as template for siRNA synthesis.
• Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %.
• Several target sites are preferably selected along the length of the target gene for evaluation.
• a negative control is preferably used in conjunction.
• Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome.
• a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
• RNA silencing agent of the present invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
• the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide.
• a "cell- penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non- endocytotic) translocation properties associated with transport of the membrane- permeable complex across the plasma and/or nuclear membranes of a cell.
• the cell- penetrating peptide used in the membrane-permeable complex of the present invention preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double-stranded ribonucleic acid that has been modified for such linkage.
• Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference.
• the cell-penetrating peptides of the present invention preferably include, but are not limited to, penetratin, transportan, plsl, TAT(48-60), pVEC, MTS, and MAP.
• RNA silencing agents include, but are not limited to, those whose expression is correlated with an undesired phenotypic trait.
• Exemplary mRNAs that may be targeted are those that encode truncated proteins i.e. comprise deletions. Accordingly the RNA silencing agent of the present invention may be targeted to a bridging region on either side of the deletion. Introduction of such RNA silencing agents into a cell would cause a down-regulation of the mutated protein while leaving the non-mutated protein unaffected.
• DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the VEGFA.
• DNAzymes are single- stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 1997;943:4262)
• a general model (the " 10-23" model) for the DNAzyme has been proposed.
• DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate- recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM [Curr Opin Mol Ther 4:119-21 (2002)].
• DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 2002, Abstract 409, Ann Meeting Am Soc Gen Ther world wide web(dot) asgt (dot) org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
• Another anti- VEGFA agent is an antisense polynucleotide capable of specifically hybridizing with an m NA transcript encoding the VEGFA.
• VEGFA must be effected while considering two aspects important to the antisense approach.
• the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
• antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin Mol Ther 1 :372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin Mol Ther 1 :297-306 (1999)].
• Another anti-VEGFA agent is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding a VEGFA.
• Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al, Curr Opin Biotechnol. 9:486-96 (1998)].
• the possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
• ribozymes In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al, Clin Diagn Virol. 10: 163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials.
• ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway.
• Ribozyme Pharmaceuticals, Inc. as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models.
• HEPTAZYME a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page).
• Qualifying agents which can reduce, inhibit or suppress the expression level and/or activity of VEGF can be performed using various in vitro (e.g., using a biological sample of the subject), ex vivo (e.g., in cells of a subject which are treated by the agent and are then injected into an animal model) or in vivo (e.g., by testing the expression level and/or activity of VEGF A in cells of a subject following treatment of the subject with the anti-VEGFA agent) methods.
• Each of the anti-VEGFA agents described hereinabove or the expression vector encoding same can be administered to the individual per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carrier.
• a pharmaceutical composition which also includes a physiologically acceptable carrier.
• the purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.
• a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• active ingredient refers to the anti-VEGFA agent accountable for the biological effect.
• physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
• An adjuvant is included under these phrases.
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
• neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
• molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
• pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
• the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
• each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
• compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
• Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
• the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen- free water based solution, before use.
• a suitable vehicle e.g., sterile, pyrogen- free water based solution
• compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (anti-VEGFA agent) effective to prevent, alleviate or ameliorate symptoms of the cancer (e.g., carcinoma) or prolong the survival of the subject being treated.
• active ingredients anti-VEGFA agent
• the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
• a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
• the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
• Dosage amount and interval may be adjusted individually to provide tissue levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
• MEC minimum effective concentration
• the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
• dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
• compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may, for example, comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
• Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
• the method further comprising informing the subject on the results of the prediction of anti-VEGFA treatment efficacy test. For example, informing the subject that based on the presence of the genomic amplification (which comprises a VEGFA gene) in the solid tumor the anti- VEGFA treatment is predicted to be efficient in treating the cancer. On the other hand, if the solid tumor is devoid of the genomic amplification (which comprises a VEGFA gene), then informing the subject that, based on the absence of the genomic amplification, the anti- VEGFA treatment is likely not to be efficient for treating the cancer, and that alternative therapeutic approaches should be explored.
• the genomic amplification which comprises a VEGFA gene
• a method of treating a subject diagnosed with a solid tumor comprising: (a) predicting the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the prediction; thereby treating of the subject diagnosed with the solid tumor.
• treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition, e.g., the cancer, e.g., the carcinoma) and/or causing the reduction, remission, or regression of a pathology.
• a pathology disease, disorder or condition, e.g., the cancer, e.g., the carcinoma
• Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
• the term "subject” includes mammals, preferably human beings at any age which suffer from the pathology.
• a method of selecting or designing a treatment regimen for treating a subject diagnosed with a solid tumor comprising: (a) predicting the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the prediction; thereby selecting or designing the treatment regimen for treating the subject diagnosed with a solid tumor.
• treatment regimen refers to a treatment plan that specifies the type of treatment, dosage, schedule and/or duration of a treatment provided to a subject in need thereof (e.g., a subject diagnosed with a pathology).
• the selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the pathology) or a more moderate one which may relief symptoms of the pathology yet results in incomplete cure of the pathology. It will be appreciated that in certain cases the more aggressive treatment regimen may be associated with some discomfort to the subject or adverse side effects (e.g., a damage to healthy cells or tissue).
• the type of treatment can include the anti-VEGFA agent of the invention alone of in combination with other chemotherapeutic drugs, a surgical intervention (e.g., removal of lesion, diseased cells, tissue, or organ), a cell replacement therapy, an administration of a therapeutic drug (e.g., receptor agonists, antagonists, hormones, chemotherapy agents) in a local or a systemic mode, an exposure to radiation therapy using an external source (e.g., external beam) and/or an internal source (e.g., brachytherapy) and/or any combination thereof.
• a therapeutic drug e.g., receptor agonists, antagonists, hormones, chemotherapy agents
• an external source e.g., external beam
• an internal source e.g., brachytherapy
• the dosage, schedule and duration of treatment can vary, depending on the severity of pathology and the selected type of treatment, and those of skills in the art are capable of adjusting the type of treatment with the dosage, schedule and duration of treatment.
• Non-limiting examples of chemotherapy agents which can be administered in combination with the anti-VEGFA agent of some embodiments of the invention include Mechlorethamine, (FiN 2), Cyclophosphamide, Ifosfamide, Melphalan, Chlorambucil, Estramustine, Hexamethyl-melamine, Thiotepa, Busulfan, Carmustine, Lomustine, Semustine, Streptozocin, dacarbazine, Procarbazine, Aziridine, Methotrexate, Trimetrexate, Fluorouracil, Floxuridine, Cytarabine, Azacitidine, Mercaptopurine, Thioguanine, Pentostatin, Fludarabine, Vinblastine (VLB), Vincristine, Vindesine, Etoposide, Teniposide, Dactinomycin, Daunorubicin, Doxorubicin, 4'-, Deoxydoxorubicin, Bleomycin, Plicamycin, Mitomycin, L-As
• Non-limiting examples of approved oncology drugs which can be administered in combination with the anti-VEGFA agent of some embodiments of the invention include Aldesleukin, Alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, Asparaginase, BCG Live, bexarotene capsules, bexarotene gel, bleomycin, busulfan intravenous, busulfan oral, calusterone, capecitabine, carboplatin, carmustine, carmustine with Polifeprosan 20 Implant, celecoxib, chlorambucil, cisplatin, cladribine, cyclophosphamide, cyclophosphamide, cytarabine, cytarabine liposomal, dacarbazine, dactinomycin, actinomycin D, dactinomycin, actinomycin D, Darbepo
• the treatment regimen further comprises administering an anti-VEGFA agent in combination with a drug selected from the group consisting of erlotinib, oxaliplatin, cisplatin, platinum, rIFNa-2b, doxorubicin, fluorouracil, DX-8951f, thalidomide, doxorubicin, epirubicin, and taxol.
• a drug selected from the group consisting of erlotinib, oxaliplatin, cisplatin, platinum, rIFNa-2b, doxorubicin, fluorouracil, DX-8951f, thalidomide, doxorubicin, epirubicin, and taxol.
• the treatment regimen further comprises administering an anti-VEGFA agent in combination with a treatment selected from the group consisting of percutaneous ethanol injection, radio frequency ablation, transcatheter arterial chemoembolization.
• a treatment selected from the group consisting of percutaneous ethanol injection, radio frequency ablation, transcatheter arterial chemoembolization.
• the term "about” refers to ⁇ 10 %.
• compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
• a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• mice - Mdr2 _/ ⁇ mice on FVB background were held in specific pathogen free conditions.
• Wild-type (WT) control mice were aged matched FVB mice. Two hours prior to sacrifice, mice were injected with 100 ⁇ BrdU (Cell Proliferation labeling reagent, Amersham; Catalogue number: RPN201) per 10 gram body weight. Mice were anesthesized using Ketamine and Xylazine and were perfused via the heart with PBS- Heparin solution followed by perfusion with 4% formaldehyde. All animal experiments were performed in accordance with the guidelines of the institutional committee for the use of animals for research.
• BrdU Cell Proliferation labeling reagent
• Adenoviral vectors and Sorafenib - Adenoviral vectors encoding green fluorescence protein (GFP) or GFP and sFLT (Soluble fms-like tyrosine kinase- 1) were prepared in GH354 cells using standard procedures. A titer of 109 transducing units was injected to mice tail veins. Sorafenib (Xingcheng Chempharm Co., Ltd Taizhou, China) was administered daily (50 mg/kg) by oral gavage. Cremophor EL/ethanol/water; (12.5: 12.5:75) was used as vehicle solution.
• GFP green fluorescence protein
• sFLT Soluble fms-like tyrosine kinase- 1
• Immunohistochemistry (IHC) and ELISA - Antibodies used for IHC were vWF (DAKO Corp, Carpinteria, CA, USA, Catalogue number A0082), Cleaved Caspase 3 [Cell Signaling, USA, Catalogue number: 9661 (Rabbit polyclonal)], F4/80 (Serotec Raleigh, NC, USA; Catalogue number MCA497GA), HGF (R&D Systems Inc, Minneapolis, MN, USA; Catalogue number: AF2207), BrdU (NeoMarkers, Thermo Scientific, Fremont, USA, BrdU Ab-3, Catalogue number: MS-1058-P), Ki67 (NeoMarkers, Thermo Scientific, Fremont, USA, Catalogue number: RM-9106), pHH3 (phospho-Histone H2A.X, MILLIPORE, Catalogue number: 05-636).
• vWF DaKO Corp, Carpinteria, CA, USA, Catalogu
• IHC was performed on 5 ⁇ paraffin sections.
• Antigen retrieval was performed in a Decloaking ChamberTM (Biocare Medical, Concord, CA, USA) in Citrate buffer for all antibodies except vWF and F4/80 for which retrieval was performed with Pronase (Sigma, St Louis, MO, USA, Catalogue number: P8038).
• VEGF-A ELISA was performed using Quantikine® mouse ELISA kit (R&D Systems Inc, Minneapolis, MN, USA). Secondary antibodies for all antibodies used were Histofme® (Nichirei Biosciences, Chuo-ku, Tokyo 104-8402 Japan), except for mouse derived antibodies that were detected with EnvisionTM (Corp, Carpenteria, CA, USA).
• In-Situ hybridization - Probes for CISH analysis were prepared from the BAC clones RP24-215A3 for Chromosome 17 and RP23-174D11 for the pericentromeric region (BACPAC resources center).
• BAC clones were labeled with Digoxigenin (DIG) using Nick-Translation mix (Roche, Indianapolis, IN, USA, Catalogue No. 11745808910).
• Mouse Cot-1 DNA Gibco-Invitrogen Corporation products, Grand Island, NY, USA
• sonicated murine genomic DNA were added to the probe for background block.
• Tissues were prepared by boiling in pretreatment buffer and digestion with Pepsin (Zymed® Catalog Number - 00-3009). Hybridization was performed overnight in 37°C after 5 minutes of denaturation in 95° C.
• the Spot-Light® detection kit (Invitrogen) was used for anti DIG antibody and secondary antibody.
• aCGH - Genomic DNA was isolated using the QIAGEN DNAeasy Tissue kit. Samples were hybridized to mouse CGH 60-mer oligonucleotides microarrays (Agilent Technologies, Santa Clara, CA, United States), washed and scanned according to Agilent Technologies instructions.
• RNA qPCR - RNA was extracted from tissues by mechanical grinding in TriReagent® (Sigma, St Louis, MO, USA) with a Polytron tissue homogenizer (Kinematica, Bohemia, NY, USA). cDNA was prepared with MMLV reverse transcriptase (Invitrogen by Life Technologies). RNA qPCR analyses were carried out with SYBR® Green qPCR Detection (Invitrogen by Life Technologies) in 7900HT Fast Real-Time PCR System (Applied BioSystems). Results were analyzed using the qBase vl .3.5 software. Primer sequences are shown in Table 4 below. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) and PPIA [peptidylprolyl isomerase A (cyclophilin A)] together were used as reference genes in all analyses.
• HPRT Hypoxanthine-guanine phosphoribosyltransferase
• PPIA peptidylprolyl isomerase A
• primer sequences 5'— >3' (with sequence identifiers) which were used in the qPCR analysis. Unless indicated otherwise, the provided primers were used for qRNA-PCR. The primers from VEGFA-3'-UTR and VEGFA-promoter regions were used for DNA qPCR.
• the aCGH revealed several recurring genomic amplifications in the qB3 band of murine chromosome 17 (Chrl7qB3, data not shown) encoding among others the VEGF-A (GenelD: 7422), Mrpsl8a (human GenelD: 55168), Pare (CCL18, human GenelD: 6362) and Exportin 5 (XP05, human GenelD: 57510) ( Figure 1).

Abstract

Description

METHODS OF PREDICTING EFFICACY OF AN ANTI-VEGFA TREATMENT

FOR SOLID TUMORS

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to personalized anticancer therapy by methods of predicting efficacy of anti-cancer therapies on a subject, and, more particularly, but not exclusively, to methods of treating solid tumors by predicting the efficacy of an anti-VEGFA treatment on the subject and selecting a treatment regimen based on the prediction of efficacy.

Many of the anti-cancer drugs are efficient in some patients but exhibit no therapeutic effect on other patients having apparently the same diagnosis. In addition, while anti-cancer therapies are associated with varying degrees of side effects, their high costs are often taken into consideration when designing a treatment regimen. In order to develop targeted therapies for cancer, specific subgroups of the cancerous tumors have to be identified in terms of specific molecular aberrations of individual tumors. Thus, identification of defining and recurring genetic abnormalities, which distinguish susceptible tumors, is mandatory for optimization of cancer treatment.

Predictive biomarkers, which are quantifiable parameters identifying subsets of disease that are more likely to respond to a specific treatment, are usually based on specific pathogenetic mechanisms that are related to a specific drug, and are considered the most important aspect of personalized medicine. Prominent examples for clinically validated cancer biomarkers include ERBB2 amplification in breast and gastric cancer and K-RAS mutations in colorectal cancer. These biomarkers serve as key determinates of treatment with Trustuzumab or Cetuximab, respectively.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide, and the fifth most common cancer. It is generally accepted that HCC is most commonly the outcome of chronic injury and inflammation, resulting in hepatocyte regeneration and dysregulated growth factor signaling. In recent years it has become clear that inflammatory signaling pathways can support survival, growth and progression of cancer.

The first line treatment for HCC is the multi-kinase inhibitor Sorafenib, which although not specific, is a strong inhibitor of VEGF receptors signaling. Sorafenib blocks several receptor tyrosine kinases including: VEGFRl, 2 and 3, PDGFR, c-Kit and RET, as well as inhibiting downstream Raf kinase isoforms (Kamimura, S. & Tsukamoto, H. Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease. Hepatology 22, 1304-1309, 1995). Sorafenib was recently shown to extend median survival from 7.9 months to 10.7 months in patients with advanced HCC (Stage C), establishing a new standard of care. Yet, the overall success of this treatment is modest and Sorafenib therapy is associated with a substantial degree of side effects (Llovet, J.M., et al. 2008, Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359, 378-390). A clear picture of the molecular pathogenesis that underlies the development of HCC is lacking; furthermore, no molecular biomarker predictive of treatment outcome has been found yet.

One of the ways to study cancer initiation and progression is to develop mouse models that faithfully recapitulate specific human malignant processes. Mdr2 is an ortholog of a human gene mutated in progressive familial intrahepatic cholestatsis (PFIC3). Mdr2 deficiency (Mdr2^_/~) results in chronic inflammation of the portal tracts, eventually leading to inflammation-induced liver tumors that share many features with human HCC, and therefore was shown to be an effective tool for studying HCC [Mauad, T.H., et al, 1994, Am. J. Pathol. 145, 1237-45; Pikarsky, E. et al, 2004, Nature 431, 461-466; Katzenellenbogen, M., et al, 2006, Cancer Res 66, 4001-10; Oude Elferink, R.P. and Groen, A.K., 1995, J. Hepatol. 23, 617-25].

VEGF-A is a master regulator of angiogenesis whose role in tumor vessel recruitment is very well established. However, other roles for VEGF-A in tumorigenesis are now emerging. It was recently shown that VEGF-A can act synergistically with EGFR to promote proliferation of skin cancer cells which express VEGF receptor 1 (FLT1). Moreover, in the context of the liver, it was shown that VEGF-A elicits hepatocyte proliferation by elevating the expression of several mitogens in the liver sinusoidal endothelial cells [Ding, B.S., et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468, 310-315 (Published November 11, 2010); LeCouter, J., et al. Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science 299, 890-893, 2003].

Murukesh N., et al. 2010 (Minireview. Biomarkers of angiogenesis and their role in the development of VEGF inhibitors; British Journal of Cancer, 102:8-18, which is hereby incorporated by reference in its entirety), review several clinical studies which aim at identifying biomarkers for predicting efficacy of VEGF inhibitors and conclude that none of the clinical studies published to date have been qualified as having a predictive value on efficacy of treatment. In addition, studies aiming at using the expression levels of VEGF as a biomarker for predicting efficacy to anti-VEGFA treatment resulted in vague results (Siegel AB, et al, 2008; Thomas MB, et al, 2009), thus excluding expression levels of VEGFA from being a predictive marker for treatment efficacy.

Chiang, D.Y., et al, 2008, describe overexpression of VEGFA via gain of 6p21 in hepatocellular carcinoma. In addition, the Chr6p21 amplification was found to be correlated with advanced stage HCC (Chiang, D.Y., et al, 2008, Cancer Res. 68, 6779- 88; Chochi, Y., et al, 2009, J. Pathol. 217, 677-84; Patil, M.A., et al, 2005, Carcinogenesis 26, 2050-7).

Siegel AB, et al, 2008; Zhu AX, et al, 2006; and Thomas MB, et al, 2009 describe phase II clinical trials using bevacizumab (Avastin™) alone or in combination with additional anti-cancer drugs for the treatment of hepatocellular carcinoma.

Komorowski J, et al., 2006 show that in cells the anti-angiogenic action of thalidomide [a-(N-phthalimido)-glutarimide] is due to direct inhibitory action on VEGF secretion and capillary microvessel formation.

Additional background art includes Ouchi, K., et al. Dig Surg 17, 42-48, 2000;

Moinzadeh, P., et al, Br J Cancer 92, 935-941, 2005; Weir, B.A., et al. 2007, Nature 450, 893-898; Tsafrir, D., et al. 2006, Cancer Res. 66, 2129-2137.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of predicting an efficacy of an anti-vascular endothelial growth factor A (VEGFA) treatment on a subject diagnosed with a solid tumor, comprising: determining a presence or an absence of a genomic amplification which comprises a VEGFA gene in a sample of the solid tumor, wherein the presence or the absence of the genomic amplification predicts the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor, thereby predicting the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor. According to an aspect of some embodiments of the present invention there is provided a method of treating of a subject diagnosed with a solid tumor, the method comprising: (a) predicting the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the prediction; thereby treating of the subject diagnosed with the solid tumor.

According to an aspect of some embodiments of the present invention there is provided a method of selecting a treatment regimen for treating a subject diagnosed with a solid tumor, the method comprising: (a) predicting the efficacy of the anti- VEGFA treatment on the subject diagnosed with the solid tumor according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the prediction; thereby selecting the treatment regimen for treating the subject diagnosed with a solid tumor.

According to some embodiments of the invention, the solid tumor is carcinoma. According to some embodiments of the invention, the carcinoma is hepatocellular carcinoma.

According to some embodiments of the invention, determining the presence or the absence of the genomic amplification is effected by comparing a ratio determined in a sample of the solid tumor between a copy number of the VEGFA and a copy number of a centromeric marker of human chromosome 6, or visa versa, to a reference ratio determined in at least one sample devoid of the solid tumor between a copy number of the VEGFA and a copy number of the centromeric marker of human chromosome 6, or visa versa, respectively.

According to some embodiments of the invention, an increase above a predetermined threshold in the ratio determined in the sample of the solid tumor relative to the reference ratio indicates the presence of the genomic amplification.

According to some embodiments of the invention, an identical ratio or a change below a predetermined threshold in the ratio determined in the sample of the solid tumor as compared to the reference ratio indicates the absence of the genomic amplification.

According to some embodiments of the invention, determining a presence or an absence of a genomic amplification is effected using a DNA detection method. According to some embodiments of the invention, determining a presence or an absence of a genomic amplification is effected using a chromosomal detection method.

According to some embodiments of the invention, the method further comprising comparing an expression level of the VEGFA in the sample of the solid tumor to a reference expression data obtained from at least one sample devoid of cancer.

According to some embodiments of the invention, an increase above a predetermined threshold in the expression level of the VEGFA in the sample of the solid tumor relative to the reference expression data predicts the efficacy of the anti- VEGFA treatment on the solid tumor.

According to some embodiments of the invention, the sample devoid of cancer is a liver sample.

According to some embodiments of the invention, the expression level is determined using an RNA detection method.

According to some embodiments of the invention, the expression level is determined using a protein detection method.

According to some embodiments of the invention, the anti-VEGFA treatment comprises Sorafenib.

According to some embodiments of the invention, the anti-VEGFA treatment comprises bevacizumab.

According to some embodiments of the invention, the anti-VEGFA treatment comprises a soluble form of the VEGF-receptor.

According to some embodiments of the invention, the anti-VEGFA treatment comprises thalidomide.

According to some embodiments of the invention, the anti-VEGFA treatment comprises a combination of at least two anti-VEGFA drugs selected from the group consisting of Sorafenib, bevacizumab, a soluble form of the VEGF-receptor and thalidomide.

According to some embodiments of the invention, the treatment regimen comprises administering of at least one anti-VEGFA drug selected from the group consisting of Sorafenib, bevacizumab, a soluble form of the VEGF-receptor and thalidomide. According to some embodiments of the invention, the treatment regimen further comprises administering a drug selected from the group consisting of erlotinib, oxaliplatin, cisp latin, platinum, rIFNa-2b, doxorubicin, fluorouracil, DX-8951f, thalidomide, doxorubicin, epirubicin, and taxol.

According to some embodiments of the invention, the genomic amplification is of a human chromosome 6p21.

According to some embodiments of the invention, the genomic amplification comprises the nucleotide sequence set forth in SEQ ID NO:23.

According to some embodiments of the invention, the genomic amplification comprises the nucleotide sequence set forth in SEQ ID NO:24.

According to some embodiments of the invention, the genomic amplification comprises the nucleotide sequence set forth in SEQ ID NO:25.

According to some embodiments of the invention, the VEGFA comprises the genomic nucleic acid sequence set forth by SEQ ID NO: 1.

According to some embodiments of the invention, the solid tumor is hepatocellular, and wherein the hepatocellular solid tumor is associated with hepatitis C infection.

According to some embodiments of the invention, the efficacy of the anti- VEGFA treatment is determined by tumor regression following at least 8 weeks of the anti-VEGFA treatment.

According to some embodiments of the invention, the efficacy of the anti- VEGFA treatment is determined by progression-free survival (PFS) time of at least one year.

According to some embodiments of the invention, the efficacy of the anti- VEGFA treatment is determined by a proliferation assay.

According to some embodiments of the invention, the DNA detection method comprises DNA quantitative PCR (qPCR).

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is an image of raw data obtained from array comparative genome hybridization (aCGH) analysis showing the amplification (right shift of dots) in a specific region of chromosome 17. Samples produced from ten different Mdr2^_/~ derived HCCs were subjected to array CGH. Note the 2 Mbp long genomic region on the qB3 band of murine chromosome 17 which includes the VEGFA gene (human GenelD: 7422), Mrpsl8a (human GenelD: 55168), Pare (CCL18, human GenelD: 6362), Exportin 5 (XP05, human GenelD: 57510). CGH reveals amplification in the genomic locus of VEGFa

FIGs. 2A-H are images depicting chromogenic in situ hybridization (CISH) of murine HCC tumors harboring the Chrl7qB3 genomic region amplification. CISH was performed using probes specific for Chrl7qB3. Two different nuclei from two different tumors from each subgroup are shown. Figures 2A-B - Tumor #1-; Figures 2C-D - Tumor #2; Figures 2E-F -Tumor #3; Figure 2G-H - Tumor #4. Note that tumors #1 and #2 are negative for the genomic amplification and tumors #3 and #4 are positive for the genomic amplification. Scale bar 3 μιη. Arrows point to positive CISH signals.

FIG. 21 is a schematic representation of the murine Chrl7qB3 genomic region amplification in various HCC tumors which map the critical region of the genomic amplification. DNA qPCR analysis was performed using primers specific for different loci on the qB3 arm of chromosome 17. Each line represents a different Amp^pos tumor. Thin line represents non-amplified region, thick line represents amplified (>2 fold increase) regions. The list includes several of the residing genes (the full list is presented in Table 5 in the Examples section which follows).

FIGs. 2J-0 are graphs depicting relative mR A expression of various genes residing on the amplified region in HCC tumors with or without the genomic amplification. qPCR analysis was performed on wild type liver tissues (WT liver), HCC tumors without the amplification (Amp^neg tumor) and HCC tumors harboring the genomic amplification (Amp^pos tumor) using primers which specifically detect Cdc5L

(Figure 2 J), HSP90abl (Figure 2K), VEGF-A (Figure 2L), Tjapl (Figure 2M), Xpo5

(Figure 2N) and Pare (Figure 20). Cross line signifies geometric mean (*p<0.01, **p<0.0001). Note the significant increase in mRNA expression of Cdc5L, VEGF-A,

Tjapl, Xpo5 and Pare in tumors harboring the genomic amplification as compared to tumors devoid of the genomic amplification.

FIG. 3 is a histogram depicting real time quantitative PCR (qPCR) and ELISA analysis of VEGF mRNA and protein levels, respectively, of amplified versus non- amplified murine Mdr2^_/~ tumors. Note the significant increase in VEGFA expression level (on both RNA and protein levels) in amplified HCC tumors.

FIGs. 4A-F are representative images of murine HCC tumors with (Figures 4B,

4D, 4F) or without (Figures 4A, 4C and 4E) the genomic amplification stained by IHC with antibodies specific to vWF (Figures 4A-B), BrdU (Figures 4C-D), and F4/80 (Figures 4E-F). Scale bar 100 μιη. Note that the Amp^pos tumors are a distinct subpopulation.

FIGs. 4G-I are histograms depicting quantization of the IHC analyses (for which representative images are shown in Figures 4A-F). IHC stainings were quantified using automated image analysis. Figure 4G - vWF; Figure 4H - BrdU; Figure 41 - F4/80. White bars = WT liver; Light grey bars = AMP^neg Tumors; Dark grey bars = AMP^pos tumors. WT n = 2, Amp^neg n = 9, Amp^pos n = 7. *p < 0.01, **p < 0.05).

FIGs. 5A-H are images of IHC for BrdU (Figures 5A-D) and Ki67 (Figures 5E- H of HCC tumors with or without the genomic amplification of mice which were treated with sFLT or remained untreated. Mdr2^_/~ mice were treated with adenovectors expressing either GFP alone (Figures 5A, 5C, 5E and 5G) or GFP with sFLT (Figures 5B, 5D, 5F and 5H) for 10 days. Shown are representative photomicrographs of IHC for BrdU and Ki67. Tumor infiltrating cells remain proliferative. Scale bars: Figures 5A-D - 50 μιη, Figures 5E-H - ΙΟΟμιη.

FIGs. 5I-J are histograms depicting quantization of the IHC results (for which representative images are shown in Figures 5A-H). The percent of positive nuclei in IHC staining for BrdU (Figure 51) and Ki67 (Figure 5 J) was quantified using automated image analysis. Mice were treated as described in Figures 5A-H above. Amp^neg GFP treated (n = 11, white bars), Amp^neg sFLT treated (n = 10, light grey bars), Amp^pos GFP treated (n = 7, dark grey bars) and Amp^pos sFLT treated (n = 6, black bars). * indicates significance of p < 0.05; ** indicates significance of p<0.05. Note that in Amp^pos sFLT there is a significant decrease in the number of BrdU or Ki67 positive nuclei, thus demonstrating a significant response to the anti-VEGF-A sFLT treatment. These results demonstrate that the VEGF-A amplicon predicts a response to VEGF blockade by sFLT.

FIGs. 5K-L are images depicting IHC for the mitosis-specific marker phospho- histone 3 (pHH3) in amplified tumors treated with adeno-sFLT (Figure 5L) or adeno- GFP (Figure 5K). Note the decrease in pHH3 positive hepatocytes in adeno-sFLT treated tumors, indicating reduced proliferation.

FIGs. 5M-N are histograms depicting the results of qPCR analysis of VEGF-A (Figure 5M), HGF (Figure 5N) of Amp^neg and Amp^pos tumors treated with the indicated adeno vectors (as described in Figures 5 AH above). Cross line signifies geometric mean (*p<0.0001). Note that in tumors harboring the amplification, the anti-VEGF-A treatment (sFLT) results in a significant decrease in HGF mRNA levels as compared to tumors harboring the amplification which were transformed with a control adenovirus GFP vector (Figure 5N).

FIGs. 50-R are histograms (Figures 50-P) and images (Figures 5Q-R) depicting high expression level of the HIFla target genes Glutl (Figure 50) and PGK1 (Figure 5P) in Amp^pos tumors treated with the indicated adenovectors (as described in Figures 5 AH above) and the associated necrosis (Figures 5Q-R). Cross line signifies geometric mean. Figure 5Q - a histological section stained with H&E, showing necrosis. Scale bar 500 μιη; Figure 5R - a macroscopic picture of a tumor showing areas of hemorrhagic necrosis. Results are representative for three out of the six sFLT treated Amp^pos tumors. Note that the tumors that show increased expression of the HIFla target genes Glutl and PGK1 (markers for hypoxia) also display necrosis on histological evaluation.

FIGs. 6A-C are a histogram and images depicting the results of qPCR analysis of HGF mRNA (Figure 6A) and IHC of HGF in non-amplified (Figure 6B) and amplified (Figure 6C) tumors. qPCR analysis of HGF mRNA was performed on WT livers, non-amplified tumors and amplified tumors. Immunostaining for HGF reveals staining in endothelial and inflammatory cell populations, predominantly in the amplified tumor.

FIG. 7 is a histogram depicting VEGF-A relative gene dose in various murine HCC tumors which is used for screening for Amp^pos tumors. qPCR was performed on DNA extracted from Mdr2^_/~ tumors (Tumors numbers 1-42) and wild-type (WT) liver samples (samples 1-3) using a set of primers targeting the 3'-UTR (untranslated region) (white bars) and the promoter (grey bars) regions of the VEGF-A gene. Primers' sequences are provided in Table 4 in the EXAMPLES section which follows. The results were compared to those obtained by CGH testing of the same tumors (tumors 1- 10) and validated that the VEGF-A gene dose (determined by DNA qPCR) can detect the genomic amplification.

FIGs. 8A-D are images of vWF IHC analysis performed on amplified (Figures 8A-B) and non-amplified (Figures 8C-D) HCC tumors treated with adeno-sFLT (Figures 8B and 8D) or adeno-GFP (Figures 8A and 8C) vectors.

FIGs. 9A-H are images depicting representative photomicrographs of BrdU (Figures 9A-D) or vWF (Figures 9E-H) IHC in amplified tumors (Figures 9C, 9D, 9G and 9H) or non-amplified tumors (Figures 9A, 9B, 9E and 9F) which were treated with Sorafenib (Figures 9B, 9D, 9F and 9H) or vehicle (Figures 9A, 9C, 9E and 9G). Note the decrease in stained nuclei (as determined by BrdU staining) in the treated Amp^pos group alone (Figure 9D) while no decrease in blood vessel (as determined by vWF staining; Figure 9H) is evident.

FIG. 91 is a histogram depicting quantization of the IHC staining for vWF using automated image analysis. Amp^neg vehicle treated (n=13; Amp^neg Veh), Amp^neg Sorafenib treated (n=13, Amp^neg Sor), Amp^pos vehicle treated (n=7; Amp^pos Veh) and Amp^pos sorafenib treated (n=5; Amp^pos Sor). P = 0.06. FIGs. 9J-M are histograms depicting qPCR analysis of Amp^neg and Amp^pos for VEGF-A (Figure 9J), HGF (Figure 9K), PGK1 (Figure 8L) and Glutl (Figure 9M). Cross line signifies geometric mean (*p<0.05). Note that Amp^pos tumors are sensitive to short term treatment with Sorafenib as indicated by a reduction in HGF mRNA levels.

FIG. 9N is a histogram depicting quantization of the IHC staining for BrdU using automated image analysis, demonstrating the effect of Sorafenib treatment on the proliferation of mice HCC tumors. Mdr2^_/~ mice were treated with Sorafenib or vehicle alone for 3 days. Shown are the percentages of BrdU positive nuclei in tumors bearing the amplification ("Biomarker +") or being devoid of the amplification ("Biomarker -"), following treatment with Sorafenib or vehicle. Note the decline in the proliferation rate of tumor cells in tumors bearing the amplification which were treated with Sorafenib, as compared to the proliferation rate of the tumor cells in tumors devoid of the genomic amplification.

FIGs. 10A-K are histograms depicting qPCR analysis of the mRNA levels of several of the 63 genes in Amp^pos and Amp^neg, demonstrating the expression profile of several of the amplicon residing genes. Cross line signifies geometric mean (*p<0.05, **p<0.01). Figure 10A - Runx2; Figure 10B - SuptSh; Figure IOC - Clic5; Figure 10D - Aars2; Figure 10E - Nfkbie; Figure 10F - Tmem63b; Figure 10G - Mrpll4; Figure 10H - Capnl 1; Figure 101 - Mrpsl8a; Figure 10J- EGFL9; Figure 10K - Gnmt.

FIGs. 11A-E are histological analyses demonstrating that Amp^pos HCC hold distinct histological features. Figures 11 A-B - Representative H&E stained sections of Amp^neg and Amp^pos tumors showing steatosis (lipid droplets) in the Amp^pos but not Amp^neg group. Scale bar 100 μιη. Figures 11C-D - Representative H&E stained sections of Amp^neg and Amp^pos tumors demonstrating the differences in the size of tumor cell and cytoplasm between the two groups. Scale bar 50 μιη. Figure HE -H&E stained sections of Amp^neg and Amp^pos tumors were examined by a pathologist and evaluated for the presence of large cells and steatosis. A χ² test was used to determine statistical significance.

FIGs. 12 A- J are images (Figures 12A-H) and histograms (Figures 121- J) demonstrating that 10 days inhibition of VEGF-A in Amp^pos tumors does not alter the microenvironmental content. Figures 12A-D - IHC of vWF; Figures 12E-H - IHC of F4/80. Amplified tumors (Figures 12C, 12D, 12G, 12H) and non-amplified tumors (Figures 12 A, 12B, 12E, 12F) were treated for 10 days with adenovirus vector expressing GFP (Figures 12A, 12C, 12E and 12G) or sFTL-GFP (Figures 12B, 12D, 12F and 12H). Figures 12I-J - quantization of the IHC for vWF (Figure 121) and F4/80 (Figure 12J) using automated image analysis. Amp^neg GFP treated (n = 12; white bars), Amp^neg sFLT treated (n = 12; light grey bars), Amp^pos GFP treated (n = 5; dark grey bars) and Amp^pos sFLT treated (n = 5; black bars). Differences between the Amp^pos with GFP or sFLT were statistically insignificant.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to personalized anticancer therapy by methods of predicting efficacy of anti-cancer therapies on a subject, and, more particularly, but not exclusively, to methods of treating solid tumors by predicting the efficacy of an anti-VEGFA treatment on the subject and selecting a treatment regimen based on the prediction of efficacy.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The present inventors have uncovered that the variability in response to an anti- VEGFA treatment such as Sorafenib (a multi-tyrosine kinase inhibitor, marketed as Nexavar by Bayer) or a soluble VEGF-A receptor, depends on the presence or absence of the genomic amplification on murine chromosome 17qB3 (a syntenic region of human chromosome 6p21) which comprises the VEGFA gene, such that subjects having a solid tumor which comprises the genomic amplification respond well (i.e., in an efficient manner) to the anti-VEGFA treatment, and subjects having the same solid tumor albeit devoid of the genomic amplification respond in a less efficient manner (or do not respond at all) to the anti-VEGFA treatment.

As shown in the Examples section which follows, the present inventors have uncovered that a subset of HCCs tumors in Mdr2-deficient mice harbor a specific amplicon at murine chromosome 17 (Chrl7qB3) spanning the VEGF-A gene (Figures 1, 2A-0, 7 and 10A-K, and Table 5, Example 1); that the genomic amplification elevates VEGF-A mRNA and protein levels (Figure 3, Example 2), that tumors harboring the amplification (Amp^pos) are characterized by larger tumor cells, extensive steatosis (Figures 11 A-E), increased vessel density (Figure 4A-B and 4G, Example 2) and increased proliferation rate (Figures 4C-D and 4H, Example 2). In addition, the present inventors found that genomic amplification induces a unique tumor environment with higher expression of the macrophage marker F4/80 (Figures 4E-F and 41, Example 3) and higher levels of hepatocyte growth factor (HGF, Figures 6A-C, Example 3). In addition, as shown in Example 4 of the Examples section which follows, the Amp^pos- HCC tumors were significantly more susceptible to treatment with a soluble form of the VEGF-A receptor (sFLT) as compared to Amp^neg-HCC tumors, as shown by an efficient inhibition of tumor cell proliferation (Figures 5A-L, Example 4), which was accompanied by a decrease in HGF mR A levels in sFLT-treated tumors (Figure 5N, Example 4) and with an increase in tissue hypoxia (Figures 50-P, Example 4) and necrosis (Figures 5Q-R, Example 4). Moreover, as shown in Example 6 of the Examples section which follows, the present inventors found that Amp^pos-HCC tumors are uniquely sensitive to Sorafenib, a multi-tyrosine kinase inhibitor (which inhibits VEGF-A activity), which is currently the first line treatment of advanced HCC in human beings, as shown by decrease in tumor cell proliferation (Figures 9A-D, Figure 9N, Example 6) and HGF levels (Figure 9K, Example 6). Altogether, these data demonstrate that the genomic amplification which comprises the VEGF-A gene distinguishes a subgroup of HCC tumors that are sensitive to direct VEGF-A blocking (e.g., sFLT) and Sorafenib treatment, and suggest the use of the genomic amplification as a prognostic marker to predict the efficacy of an anti-VEGF-A treatment in a subject having a solid tumor.

Thus, according to an aspect of some embodiments of the invention, there is provided a method of predicting an efficacy of an anti-vascular endothelial growth factor A (VEGFA) treatment on a subject diagnosed with a solid tumor, comprising determining a presence or an absence of a genomic amplification which comprises a VEGFA gene in a sample of the solid tumor, wherein the presence or the absence of the genomic amplification predicts the efficacy of the anti- VEGFA treatment on the subject diagnosed with the solid tumor, thereby predicting the efficacy of the anti- VEGFA treatment on the subject diagnosed with the solid tumor. According to some embodiments of the invention, presence of the genomic amplification which comprises the VEGFA gene in a solid tumor sample, predicts that the anti- VEGFA treatment will be efficient in treating the solid tumor in the subject.

As used herein the phrase "predicting efficacy of an anti- VEGFA treatment" refers to determining the likelihood that an anti-VEGFA treatment will be efficient or non-efficient in treating the solid tumor, e.g., the success or failure of the anti-VEGFA treatment in treating the solid tumor in a subject in need thereof. The term "efficacy" as used herein refers to the extent to which the anti-VEGFA treatment produces a beneficial result, e.g., an improvement in one or more symptoms of the pathology (caused by the solid tumor) and/or clinical parameters related to the pathology as described hereinbelow. For example, the efficacy of an anti-VEGFA treatment may be evaluated using standard therapeutic indices for solid tumors.

According to some embodiments of the invention, the efficacy of treatment is a long-term efficacy.

As used herein the phrase "long-term efficacy" refers to the ability of a treatment to maintain a beneficial result over a period of time, e.g., at least about 16 weeks, at least about 26 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 48 weeks, at least about 52 weeks, at least about 18 months, at least about 24 months, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, or longer.

According to some embodiments of the invention, the efficacy of the anti- VEGFA treatment is determined by tumor regression following at least 8 weeks of the anti-VEGFA treatment.

According to some embodiments of the invention, an anti-VEGFA treatment is considered efficient in treating a solid tumor if it exerts an improvement in at least one relevant clinical parameter related to the solid tumor in the treated subject as compared to an untreated subject diagnosed with the same solid tumor (e.g., the same type, stage, degree and/or classification of the solid tumor), or as compared to the clinical parameters related to the solid tumor of the same subject prior to the anti-VEGFA treatment.

Non-limiting examples of the clinical parameters related to the solid tumor which can be monitored in order to determine the efficacy of the anti-VEGFA treatment include the number of tumor lesions, dimensions (e.g., size) of each of the tumor lesion, tumor stage, differentiation state of tumor, presence and/or degree of tumor metastases, effect of the tumor on physiological function of the subject affected by the solid tumor, and the like.

Evaluation of the efficacy of an anti-VEGFA treatment can be also performed using acceptable clinical criteria, such as the criteria proposed by the "Response Evaluation Criteria in Solid Tumors (RECIST) Committee" described in Therasse P., Arbuck SG., Eisenhauer EA et al. (New guidelines to evaluate the response to treatment in solid tumors: European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J. Natl. Cancer Inst. 92:205-216, 2000), which is incorporated by reference in its entirety. The (RECIST) is a set of published rules that define when cancer patients improve ("respond"), stay the same ("stabilize"), or worsen ("progression") during treatments.

According to some embodiments of the invention, the efficacy of the anti- VEGFA treatment can be determined using at least one, two or all three of the response criteria included in the RECIST which include evaluation of target lesions [target lesions are selected on the basis of their size (lesions with the longest diameter)], evaluation of non-target lesions [all other lesions (or sites of disease) identified as non-target lesions], and evaluation of best overall response.

Evaluation of target lesions classifies the response as follows: (i) Complete Response (CR) -Disappearance of all target lesions; (ii) Partial Response (PR) - At least a 30% decrease in the sum of the longest diameter (LD) of target lesions, taking as reference the baseline sum LD; (iii) Stable Disease (SD) - Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for Progressive Disease (PD), taking as reference the smallest sum LD since the treatment started; (iv) Progressive Disease (PD) - At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions.

Evaluation of non-target lesions classifies the response as follows: (i) Complete Response (CR) - Disappearance of all non-target lesions and normalization of tumor marker level; (ii) Incomplete Response/ Stable Disease (SD) - Persistence of one or more non-target lesion(s) or/and maintenance of tumor marker level above the normal limits; (iii) Progressive Disease (PD) - Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions.

Evaluation of the best overall response recorded from the start of the treatment until disease progression/recurrence (taking as reference for PD the smallest measurements recorded since the treatment started). In general, the patient's best response assignment will depend on the achievement of both measurement and confirmation criteria. Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having "symptomatic deterioration". In some circumstances it may be difficult to distinguish residual disease from normal tissue. When the evaluation of complete response depends on this determination, it is recommended that the residual lesion be investigated (fine needle aspirate/biopsy) to confirm the complete response status.

According to some embodiments of the invention, an anti-VEGFA treatment is considered efficient in treating the solid tumor if it results in at least a partial response (PR) according to the RECIST criteria.

According to some embodiments of the invention, an anti-VEGFA treatment is considered efficient in treating the solid tumor if it results in a complete response (CR) according to the RECIST criteria.

According to some embodiments of the invention, the efficacy of the anti-

VEGFA treatment is determined by progression-free survival (PFS) time of at least one year.

According to some embodiments of the invention, determining the efficacy of an anti-VEGF-A treatment is performed by monitoring the proliferation state of the tumor cells.

The proliferation state of tumor cells can be determined using various methods known in the art, such as using the synthetic nucleoside analogue of thymidine, bromodeoxyuridine (5-bi mo-2-deoxyuridine, BrdU). BrdU is commonly used in the detection of proliferating cells in living tissues. BrdU is incorporated into the newly synthesized DNA of replicating cells, substituting for thymidine during DNA replication. Antibodies specific for BrdU can then be used to detect the incorporated chemical, thus indicating cells that were actively replicating their DNA. Non-limiting examples of solid tumors (cancers) which can be treated by the anti-VEGFA treatment according to some embodiments of the invention include tumors of the gastrointestinal tract (colon solid tumor, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small and/or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, pancreatic endocrine tumors), endometrial carcinoma, dermatofibrosarcoma protuberans, gallbladder carcinoma, Biliary tract tumors, prostate cancer, prostate adenocarcinoma, renal cancer (e.g., Wilms' tumor type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cells tumor, immature teratoma of ovary, uterine, epithelial ovarian, sacrococcygeal tumor, choriocarcinoma, placental site trophoblastic tumor, epithelial adult tumor, ovarian carcinoma, serous ovarian cancer, ovarian sex cord tumors, cervical carcinoma, uterine cervix carcinoma, small-cell and non-small cell lung carcinoma, nasopharyngeal, breast carcinoma (e.g., ductal breast cancer, invasive intraductal breast cancer, sporadic; breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer- 1, breast cancer-3; breast-ovarian cancer), squamous cell carcinoma (e.g., in head and neck), neurogenic tumor, astrocytoma, ganglioblastoma, neuroblastoma, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, B cell, Burkitt, cutaneous T cell, histiocytic, lymphoblastic, T cell, thymic), gliomas, adenocarcinoma, adrenal tumor, hereditary adrenocortical carcinoma, brain malignancy (tumor), various other carcinomas (e.g., bronchogenic large cell, ductal, Ehrlich-Lettre ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinocellular, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependimoblastoma, epithelioma, fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., multiforme, astrocytoma), glioma hepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), hypernephroma, insulinoma, islet tumor, keratoma, leiomyoblastoma, leiomyosarcoma, leukemia (e.g., acute lymphatic, acute lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic T cell leukemia, acute - megakaryoblastic, monocytic, acute myelogenous, acute myeloid, acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, Friend, granulocytic or myelocytic, hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic-macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm, predisposition to myeloid malignancy, acute nonlymphocytic leukemia), lymphosarcoma, melanoma, mammary tumor, mastocytoma, medulloblastoma, mesothelioma, metastatic tumor, monocyte tumor, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, nervous tissue glial tumor, nervous tissue neuronal tumor, neurinoma, neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's), papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's, histiocytic cell, Jensen, osteogenic, reticulum cell), schwannoma, subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma, testicular tumor, thymoma and trichoepithelioma, gastric cancer, fibrosarcoma, glioblastoma multiforme; multiple glomus tumors, Li-Fraumeni syndrome, liposarcoma, lynch cancer family syndrome II, male germ cell tumor, mast cell leukemia, medullary thyroid, multiple meningioma, endocrine neoplasia myxosarcoma, paraganglioma, familial nonchromaffin, pilomatricoma, papillary, familial and sporadic, rhabdoid predisposition syndrome, familial, rhabdoid tumors, soft tissue sarcoma, and Turcot syndrome with glioblastoma.

According to some embodiments of the invention, the solid tumor which is treated by the anti-VEGFA treatment is a carcinoma.

The term "carcinoma" as used herein refers to any malignant tumor derived from epithelial cells or tissue.

According to some embodiments of the invention, the carcinoma is selected from the group consisting of hepatocellular carcinoma, cervical cancer, Nasopharyngeal carcinoma (NPC), bladder cancer, lung cancer (e.g., non-small cell lung cancer), esophageal squamous cell carcinoma, multiple myeloma, kidney cancer (renal cell carcinoma), metastatic renal cell carcinoma, colon cancer, colorectal cancer, pancreatic cancer and ovarian cancer.

According to some embodiments of the invention the carcinoma is lung cancer. According to some embodiments of the invention the carcinoma is colorectal cancer.

According to some embodiments of the invention the carcinoma is hepatocellular carcinoma.

According to some embodiments of the invention the hepatocellular solid tumor is associated with hepatitis C infection.

According to some embodiments of the invention the carcinoma is cancer metastases.

Table 1 provides a non- limiting list of solid tumors which include the 6p21 genomic amplification.

Table 1

Table 1.

Table 2 provides a non- limiting list of solid tumors which include the 6p21-p23 gain or amplification.

Table 2

Number of Tumors with gain Tumors with amplification

Type of tumor

tumors in 6p21-p23 (%) in 6p21-p23 (%)

Carcinomas*

Hepatocellular

409 22.61 0.20

carcinoma

Merkel cell carcinoma 48 27.10 2.10

Basal cell carcinoma 16 40.30 0.00

Ovarian serous

56 28.60 0.00

carcinoma

Transitional cell

133 9.68 0.51

carcinoma

Lymphoid tumors

Large B cell lymphoma 360 8.36 0.07 Number of Tumors with gain Tumors with amplification

Type of tumor

tumors in 6p21-p23 (%) in 6p21-p23 (%)

Plasmacytoma 21 22.76 0.00

Sarcomas

Osteosarcoma 137 33.19 3.39

Malignant peripheral

70 23.93 0.00

nerve sheath

Leiomyosarcoma 136 9.98 1.64

Melanomas 91 31.78 0.00

Retinoblastoma 133 39.36 10.50

Glioblastoma 108 4.60 0.90

Neuroblastoma 303 22.10 0.17

Carcinosarcoma 23 21.70 6.31

Table 2. Tumors with 6p21-p23 gain or amplification. Derived from J Clin Pathol

2007, 60: 1-7 (Santos GC).

As used herein the term "vascular endothelial growth factor A (VEGFA)" (also known as VPF, VEGF, MVCD1, MGC70609) refers to synthetic, recombinant and/or naturally occurring polynucleotide and polypeptide sequences assigned to the gene symbol VEGFA. These include but are not limited to the genomic sequence encoding VEGFA [nucleotides 43737953-43754224 of GenBank Accession No. NC_000006.11 as set forth by SEQ ID NO: l], the mRNA transcripts encoded thereby [e.g., GenBank Accession Nos. NM 001025366.1 (SEQ ID NO:2); NM 003376.4 (SEQ ID NO:3); NM 001025367.1 (SEQ ID NO:4); NM 001025368.1 (SEQ ID NO:5); NM 001033756.1 (SEQ ID NO:6); NM 001025369 (SEQ ID NO:7); NM 001025370 (SEQ ID NO: 8)] and/or the polypeptide variants encoded thereby [e.g., GenBank Accession Nos. NP 001020537.2 (SEQ ID NO:9); NP 003367.4 (SEQ ID NO: 10); NP 001020538.2 (SEQ ID NO: 11); NP 001020539.2 (SEQ ID NO: 12); NP 001028928.1 (SEQ ID NO: 13); NP 001020540.2 (SEQ ID NO: 14); NP 001020541.2 (SEQ ID NO: 15).

As used herein the phrase "genomic amplification which comprises a VEGFA gene" refers to the presence of more than one copy per chromosome homolog of at least the genomic sequence encoding VEGFA. As used herein the phrase "chromosome homolog" refers to a single chromosome of the pair of chromosomes that pair (synapse) during meiosis.

According to some embodiments of the invention the genomic amplification which comprises the VEGFA gene is of the human chromosome 6p21 [nucleotide coordinates chr6:30,400,001-46,200,000 (SEQ ID NO:23) according to UCSC on Human GRCh37 Assembly (human genome 19 (hgl9)]. The 6p21 region includes the chromosomal bands 6p21.33, 6p21.32, 6p21.31, 6p21.2, 6p21.1. The specific band in 6p21 region which comprises the VEGFA genomic sequence is 6p21.1, which is encompassed by nucleotide coordinates chr6:40,500,001-46,200,000 (SEQ ID NO:24) according to UCSC on Human GRCh37 Assembly [human genome 19 (hgl9)].

According to some embodiments of the invention the genomic amplification which comprises the VEGFA gene is set forth in SEQ ID NO:24.

According to some embodiments of the invention the genomic amplification which comprises the VEGFA gene is set forth in SEQ ID NO: 25 (Chr6: 43684022- 44002022 in the hgl9 assembly). This sequence comprises the VEGFA (SEQ ID NO: l, LOC100132354 (SEQ ID NO:405) and C6orf223 (SEQ ID NO:406) genomic sequences.

Non-limiting examples of BACs (bacterial artificial chromosomes) which are derived from the 6p21.1 region and which can be used to detect the genomic amplification which comprises VEGFA gene include RP11-710L16 [chromosome 6:43,633,251-43,817,196; according to UCSC (University California Santa Cruz) on Human GRCh37 Assembly (human genome 19 (hgl9); SEQ ID NO: 18] which fully covers the VEGFA genomic sequence; and RP11-21M9 [chr6:43,743,280-43,929,157 according to UCSC on Human GRCh37 Assembly (human genome 19 (hgl9); SEQ ID NO: 19] which partially covers the VEGFA genomic sequence (data not shown).

According to some embodiments of the invention the genomic amplification which comprises the VEGFA gene is set forth in SEQ ID NO: 18 and/or 19.

According to some embodiments of the invention, the genomic amplification comprises at least 2, e.g., at least 3, e.g., at least 4, e.g., at least 5, e.g., at least 6, e.g., at least 7, e.g., at least 8, e.g., at least 9, e.g., at least 10, e.g., at least 15, e.g., at least 20, e.g., at least 30, e.g., at least 40, e.g., at least 50, e.g., at least 60, e.g., at least 70, e.g., at least 80, e.g., at least 90, e.g., at least 100, e.g., at least 200, e.g., at least 300, e.g., at least 400, e.g., at least 600, e.g., at least 1000 copies per chromosome homo log, or more of the genomic sequence encompasses the coding region of VEGFA (e.g., SEQ ID NO: l).

According to some embodiments of the invention, the genomic amplification comprises at least 2, e.g., at least 3, e.g., at least 4, e.g., at least 5, e.g., at least 6, e.g., at least 7, e.g., at least 8, e.g., at least 9, e.g., at least 10, e.g., at least 15, e.g., at least 20, e.g., at least 30, e.g., at least 40, e.g., at least 50, e.g., at least 60, e.g., at least 70, e.g., at least 80, e.g., at least 90, e.g., at least 100, e.g., at least 200, e.g., at least 300, e.g., at least 400, e.g., at least 600, e.g., at least 1000 copies per chromosome homolog, or more of the genomic sequence selected from the group consisting of SEQ ID NOs: l, 18, 19, 23, 24, and 25.

According to some embodiments of the invention, determining the presence or the absence of the genomic amplification is effected by comparing a ratio determined in a sample of the solid tumor between a copy number of the VEGFA and a copy number of a centromeric marker of human chromosome 6, or visa versa, namely, comparing a ratio determined in a sample of the cancer between the copy number of a centromeric marker of human chromosome 6 and a copy number of the VEGFA to a reference ratio determined in at least one sample devoid of the solid tumor between a copy number of the VEGFA and a copy number of the centromeric marker of human chromosome 6, or visa versa, namely, a reference ratio determined in at least one sample devoid of the solid tumor between a copy number of the centromeric marker of human chromosome 6 and a copy number of the VEGFA, respectively.

According to some embodiments of the invention, the centromeric marker of human chromosome 6 includes nucleotide coordinates 6:60500000-63300000 according to UCSC on Human GRCh37 Assembly (human genome 19 (hgl9)). Non-limiting examples of BAC clones which are encompassed by the human pericentromeric chromosome 6 and which can be used for detection of the copy number of human chromosome 6 include: RP1-91N13 (SEQ ID NO:20), RP5-1194012 (SEQ ID NO:21), and RP1-271N20 (SEQ ID NO:22). Non- limiting examples of centromeric markers which can be used to detect the copy number of human chromosome 6 include the CEP 6 SPECTRUM GREEN (ABBOTT Molecular); ZYTODOT CEN 6 probe (PD2; Zyto Vision, C-3002-400); alphoid clone 308 (D6Z1; a 3-kb DNA fragment that is repeated in centromer 6; Jabs Wang E et al., Characterization of human centromeric regions of specific chromosomes by means of alphoid DNA sequences. Am. J. Hum. Genet. 41 :374-390, 1987); and SKU CEN006 (Empire Genomics, Buffalo, NY, USA).

As used herein a "sample" refers to any biological sample which contains a cell of a subject or a cellular component.

Non-limiting examples of biological samples include body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk as well as white blood cells, tissue biopsies (including those obtained by fine needle aspiration, a surgical tool) e.g., of malignant tissues.

According to some embodiments of the invention, the sample comprises tumor cells and/or tissue.

According to some embodiments of the invention, the sample is a tissue section (e.g., a paraffin-embedded section, e.g., an archive paraffin-embedded tissue section, or a frozen tissue section).

According to some embodiments of the invention, the sample is a fine-needle aspiration sample of a solid tumor.

According to some embodiments of the invention, an increase above a predetermined threshold in the ratio determined in the sample of the carcinoma relative to the reference ratio indicates the presence of the genomic amplification.

As used herein the phrase "an increase above a predetermined threshold" refers to an increase in the ratio determined in the sample of the carcinoma relative to the reference ratio which is higher than a predetermined threshold such as a about 10 %, e.g., higher than about 20 %, e.g., higher than about 30 %, e.g., higher than about 40 %, e.g., higher than about 50 %, e.g., higher than about 60 %, higher than about 70 %, higher than about 80 %, higher than about 90 %, higher than about 2 times, higher than about three times, higher than about four time, higher than about five times, higher than about six times, higher than about seven times, higher than about eight times, higher than about nine times, higher than about 20 times, higher than about 50 times, higher than about 100 times, higher than about 200 times, higher than about 350, higher than about 500 times, higher than about 1000 times, or more relative to the reference ratio. According to some embodiments of the invention, an identical ratio or a change below a predetermined threshold in the ratio determined in the sample of the carcinoma as compared to the reference ratio indicates the absence of the genomic amplification.

As used herein the phrase "changed below a predetermined threshold" refers to an increase or a decrease in the level of expression in the cell of the subject relative to the reference ratio which is lower than a predetermined threshold, such as lower than about 2 times, e.g., lower than about 90%, e.g., lower than about 80%, e.g., lower than about 70%), e.g., lower than about 60%>, e.g., lower than about 50%>, e.g., lower than about 40%o, e.g., lower than about 30%>, e.g., lower than about 20%>, e.g., lower than about 10%), e.g., lower than about 9%>, e.g., lower than about 8%, e.g., lower than about 7%o, e.g., lower than about 6%>, e.g., lower than about 5%>, e.g., lower than about 4%, e.g., lower than about 3%>, e.g., lower than about 2%, e.g., lower than about 1% relative to the reference ratio.

According to some embodiments of the invention, determining a presence or an absence of a genomic amplification is effected using a chromosomal detection method.

Non-limiting examples of chromosomal detection methods include fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), primed in situ labeling (PRINS), quantitative FISH (Q-FISH) and/or multicolor-banding (MCB).

Methods of employing FISH analysis on interphase chromosomes are known in the art. Briefly, directly-labeled probes [e.g., a probe derived from the amplified region which comprises the VEGF-A gene such as SEQ ID NO: 18 (BAC RP11-710L16), 19 (BAC RP11-21M9), 23, 24 and/or 25 labeled in one color (e.g., green) and a probe derived from a chromosome 6 centromer such as SEQ ID NO:20 (BAC RP 1-9 IN 13), 21 (BAC RP5-1194012) and/or 22 (BAC RP1-271N20) labeled in another color (e.g., red)] are mixed with hybridization buffer (e.g., LSI/WCP, Abbott) and a carrier DNA (e.g., human Cot 1 DNA, available from Abbott). The probe solution is applied on microscopic slides containing the biological sample (e.g., tissue sections from a tumor biopsy) and the slides are covered using a covers lip. The probe-containing slides are denatured for 4-5 minutes at 71 °C (or 3 minutes at 70 °C) and are further incubated for 24-60 hours at 37 °C using an hybridization apparatus (e.g., HYBrite, Abbott Cat. No. 2J11-04). To increase the specificity, following hybridization, the slides are washed for 2 minutes at 70-72 °C in a solution of 0.3 % NP-40 (Abbott) in 60 mM NaCl and 6 mM NaCitrate (0.4XSSC). Slides are then immersed for 1 minute in a solution of 0.1 % NP- 40 in 2XSSC at room temperature, following which the slides are allowed to dry in the dark. Counterstaining is performed using, for example, DAPI II counterstain (Abbott).

For CISH a labeled complementary DNA or RNA strand is used to localize a specific DNA or RNA sequence in a tissue specimen. CISH can be used to detect various chromosomal abnormalities such as gene amplification, gene deletion, chromosome translocation, and chromosome number. Usually, CISH utilizes conventional peroxidase or alkaline phosphatase reactions, and is applicable to formalin- fixed, paraffin-embedded tissues, blood or bone marrow smears, metaphase chromosome spreads, and fixed cells.

PRINS analysis has been employed in the detection of gene deletion (Tharapel SA and Kadandale JS, 2002. Am. J. Med. Genet. 107: 123-126), determination of fetal sex (Orsetti, B., et al, 1998. Prenat. Diagn. 18: 1014-1022), and identification of chromosomal aneuploidy (Mennicke, K. et al., 2003. Fetal Diagn. Ther. 18: 114-121).

Methods of performing PRINS analysis are known in the art and include for example, those described in Coullin, P. et al. (Am. J. Med. Genet. 2002, 107: 127-135); Findlay, I., et al. (J. Assist. Reprod. Genet. 1998, 15: 258-265); Musio, A., et al. (Genome 1998, 41 : 739-741); Mennicke, K., et al. (Fetal Diagn. Ther. 2003, 18: 114- 121); Orsetti, B., et al. (Prenat. Diagn. 1998, 18: 1014-1022). Briefly, slides containing interphase chromosomes are denatured for 2 minutes at 71 °C in a solution of 70 % formamide in 2XSSC (pH 7.2), dehydrated in an ethanol series (70, 80, 90 and 100 %) and are placed on a flat plate block of a programmable temperature cycler (such as the PTC-200 thermal cycler adapted for glass slides which is available from MJ Research, Waltham, Massachusetts, USA). The PRINS reaction is usually performed in the presence of unlabeled primers and a mixture of dNTPs with a labeled dUTP (e.g., fluorescein- 12-dUTP or digoxigenin-11-dUTP for a direct or indirect detection, respectively). Alternatively, or additionally, the sequence-specific primers can be labeled at the 5' end using e.g., 1-3 fluorescein or cyanine 3 (Cy3) molecules. Thus, a typical PRINS reaction mixture includes sequence-specific primers (50-200 pmol in a 50 μΐ reaction volume), unlabeled dNTPs (0.1 mM of dATP, dCTP, dGTP and 0.002 mM of dTTP), labeled dUTP (0.025 mM) and Taq DNA polymerase (2 units) with the appropriate reaction buffer. Once the slide reaches the desired annealing temperature the reaction mixture is applied on the slide and the slide is covered using a cover slip. Annealing of the sequence-specific primers is allowed to occur for 15 minutes, following which the primed chains are elongated at 72 °C for another 15 minutes. Following elongation, the slides are washed three times at room temperature in a solution of 4XSSC/0.5 % Tween-20 (4 minutes each), followed by a 4-minute wash at PBS. Slides are then subjected to nuclei counterstain using DAPI or propidium iodide. The fluorescently stained slides can be viewed using a fluorescent microscope and the appropriate combination of filters (e.g., DAPI, FITC, TRITC, FITC-rhodamin).

It will be appreciated that several primers which are specific for several targets can be used on the same PRINS run using different 5' conjugates. Thus, the PRINS analysis can be used as a multicolor assay for the determination of the presence, and/or location of several genes or chromosomal loci.

In addition, as described in Coullin et al, (2002, Supra) the PRINS analysis can be performed on the same slide as the FISH analysis, preferably, prior to FISH analysis.

High-resolution multicolor banding (MCB) on interphase chromosomes - This method, which is described in detail by Lemke et al. (Am. J. Hum. Genet. 71 : 1051- 1059, 2002), uses YAC/BAC and region-specific microdissection DNA libraries as DNA probes for interphase chromosomes. Briefly, for each region- specific DNA library 8-10 chromosome fragments are excised using microdissection and the DNA is amplified using a degenerated oligonucleotide PCR reaction. For example, for MCB staining of chromosome 5, seven overlapping microdissection DNA libraries were constructed, two within the p arm and five within the q arm (Chudoba I., et al, 1999; Cytogenet. Cell Genet. 84: 156-160). Each of the DNA libraries is labeled with a unique combination of fluorochromes and hybridization and post-hybridization washes are carried out using standard protocols (see for example, Senger et al, 1993; Cytogenet. Cell Genet. 64: 49-53). Analysis of the multicolor-banding can be performed using the isis/mFISH imaging system (MetaSystems GmbH, Altlussheim, Germany). It will be appreciated that although MCB staining on interphase chromosomes was documented for a single chromosome at a time, it is conceivable that additional probes and unique combinations of fluorochromes can be used for MCB staining of two or more chromosomes at a single MCB analysis. Quantitative FISH (Q-FISH) - In this method chromosomal abnormalities are detected by measuring variations in fluorescence intensity of specific probes. Q-FISH can be performed using Peptide Nucleic Acid (PNA) oligonucleotide probes. PNA probes are synthetic DNA mimics in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by an amine bond and to which the nucleobases are fixed (Pellestor F and Paulasova P, 2004; Chromosoma 112: 375-380). Thus, the hydrophobic and neutral backbone enables high affinity and specific hybridization of the PNA probes to their nucleic acid counterparts (e.g., chromosomal DNA). Such probes have been applied on interphase nuclei to monitor telomere stability (Slijepcevic, P. 1998; Mutat. Res. 404:215-220; Henderson S., et al, 1996; J. Cell Biol. 134: 1-12), the presence of Fanconi aneamia (Hanson H, et al., 2001, Cytogenet. Cell Genet. 93: 203-6) and numerical chromosome abnormalities such as trisomy 18 (Chen C, et al, 2000, Mamm. Genome 10: 13-18), as well as monosomy, duplication, and deletion (Taneja KL, et al, 2001, Genes Chromosomes Cancer. 30: 57-63).

Alternatively, Q-FISH can be performed by co-hybridizing whole chromosome painting probes (e.g., for chromosomes 21 and 22) on interphase nuclei as described in Truong K et al, 2003, Prenat. Diagn. 23: 146-51.

According to some embodiments of the invention, determining a presence or an absence of a genomic amplification is effected using a DNA detection method.

Comparative Genome Hybridization (CGH) - is based on a quantitative two- color fluorescence in situ hybridization (FISH) on metaphase chromosomes. In this method a test DNA (e.g., DNA extracted from the biological sample which includes tumor cells, e.g., obtained from a tumor tissue biopsy) is labeled in one color (e.g., green) and mixed in a 1 : 1 ratio with a reference DNA (e.g., DNA extracted from a control cell) which is labeled in a different color (e.g., red). Methods of amplifying and labeling whole-genome DNA are well known in the art (see for example, Wells D, et al, 1999; Nucleic Acids Res. 27: 1214-8). Briefly, genomic DNA is amplified using a degenerate oligonucleotide primer [e.g., 5 '-CCGACTCGAGNNNNNNATGTGG, SEQ ID NO: 404 (Telenius, H., et al, 1992; Genomics 13:718-25)] and the amplified DNA is labeled using e.g., the Spectrum Green-dUTP (for the test DNA) or the Spectrum Red- dUTP (for the reference DNA). The mixture of labeled DNA samples is precipitated with Cotl DNA (Gibco-BRL) and resuspended in an hybridization mixture containing e.g., 50 % formamide, 2XSSC, pH 7 and 10 % dextrane sulfate. Prior to hybridization, the labeled DNA samples (i.e., the probes) are denatured for 10 minutes at 75 °C and allowed to cool at room temperature for 2 minutes. Likewise, the metaphase chromosome spreads are denatured using standard protocols (e.g., dehydration in a series of ethanol, denaturation for 5 minutes at 75 °C in 70 % formamide and 2XSSC). Hybridization conditions include incubation at 37 °C for 25-30 hours in a humidified chamber, following by washes in 2XSSC and dehydration using an ethanol series, essentially as described elsewhere (Wells, D., et al, 2002; Fertility and Sterility, 78: 543-549). Hybridization signal is detected using a fluorescence microscope and the ratio of the green-to-red fluorescence can be determined using e.g., the Applied Imaging (Santa Clara, CA) computer software. If both genomes are equally represented in the metaphase chromosomes (i.e., no deletions, duplication or insertions in the DNA derived from the tumor cells) the labeling on the metaphase chromosomes is orange. However, regions which are either deleted or duplicated in the tumor cell(s) are stained with red or green, respectively.

DNA array-based comparative genomic hybridization (CGH-array) - This method, which is fully described in Hu, D.G., et al, 2004, Mol. Hum. Reprod. 10: 283- 289, is a modified version of CGH and is based on the hybridization of a 1 : 1 mixture of the test and reference DNA probes on an array containing chromosome-specific DNA libraries. Methods of preparing chromosome-specific DNA libraries are known in the art (see for example, Bolzer A., et al, 1999; Cytogenet. Cell. Genet. 84: 233-240). Briefly, single chromosomes are obtained using either microdissection or flow-sorting and the genomic DNA of each of the isolated chromosomes is PCR-amplified using a degenerated oligonucleotide primer. To remove repetitive DNA sequences, the amplified DNA is subjected to affinity chromatography in combination with negative subtraction hybridization (using e.g., human Cot-1 DNA or centromer-specific repetitive sequence as subtractors), essentially as described in Craig JM., et al, 1997; Hum. Genet. 100: 472-476. Amplified chromosome-specific DNA libraries are then attached to a solid support [(e.g., SuperAmine slides (TeleChem, USA)], dried, baked and washed according to manufacturer's recommendation. Labeled genomic DNA probes (a 1 : 1 mixture of the test and reference DNAs) are mixed with non-specific carrier DNA (e.g., human Cot-1 and/or salmon sperm DNA, Gibco-BRL), ethanol-precipitated and re- suspended in an hybridization buffer such as 50 % deionized formamide, 2XSSC, 0.1 % SDS, 10 % Dextran sulphate and 5 X Denhardt's solution. The DNA probes are then denatured (80 °C for 10 minutes), pre-annealed (37 °C for 80 minutes) and applied on the array for hybridization of 15-20 hours in a humid incubator. Following hybridization the arrays are washed twice for 10 minutes in 50 % formamide/2XSSC at 45 °C and once for 10 minutes in 1XSSC at room temperature, following which the arrays are rinsed three times in 18.2 ΜΩ deionized water. The arrays are then scanned using any suitable fluorescence scanner such as the GenePix 4000B microarray reader (Axon Instruments, USA) and analyzed using the GenePix Pro. 4.0.1.12 software (Axon).

The genomic amplification which comprises the VEGFA gene can be detected using a quantitative DNA-based techniques such as quantitative DNA PCR (qDNA PCR) or quantitative Southern blot analysis. The quantitative DNA assays (qPCR or qSouthern blot) can determine the absolute number of gene copies or relative amount of gene copies (of a DNA sequence derived from the genomic amplification) when normalized to normalizing genes, and those of ordinary skills in the art are capable of assessing the results of such assays in order to determine presence or absence of the genomic amplification (see e.g., Figure 7).

Briefly, genomic DNA is extracted using known methods from a "test" biological sample (e.g., a tumor biopsy, or a fine needle aspiration sample for which the presence or absence of the genomic amplification is unknown) and from a reference sample with a known status with regard to presence or absence of the genomic amplification. The reference sample can be a positive control, i.e., a sample which is known to have the genomic amplification as determined by other methods such as FISH (e.g., a tumor having the genomic amplification), or it can be a negative control, i.e., a sample which is known to be devoid of the genomic amplification (having only a single copy per chromosome homologue). The negative control sample can be derived from a non-tumor tissue (derived from the same species, e.g., from human) or from a tumor tissue devoid of the genomic amplification as determined based on other methods, such as FISH.

Methods of performing qDNA PCR analysis are known to the ordinary skilled in the art and further exemplified in the Examples section which follows and in Figure 7. For example, qPCR analyses can be carried out with SYBR green (Invitrogen) in 7900HT Fast Real-Time PCR System (Applied BioSystems), and the results can be analyzed using the qBase vl .3.5 software. For example, for detecting the genomic amplification in chromosome 6p21, which comprises the VEGF-A gene, qPCR can be performed using primers pairs specific to any of the genes encompassed in the genomic amplification (e.g., SEQ ID NOs: 1, 26-403 and 405-433). Non-limiting examples of such primers pairs are provided in Table 3 below and in SEQ ID NOs: 456-1269.

Table 3

Genes encompassed within the human 6p21 genomic amplification region and primers and probes for quantifying the copy number of the genomic amplification

Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

LOC646

26 30436659 30438025 456 457 520 1270

RANP1 27 30453662 30454724 458 459 1271

HLA-E 28 30457183 30461982 460 461 1272

GNL1 29 30509154 30525371 462 463 1273

PRR3 30 30524486 30532473 464 465 1274

ABCF1 31 30539170 30559309 466 467 1275

MIR877 32 30552109 30552194 468 469 1276

PPP1R1

33 30568182 30585020 470 471 0 1277

MRPS1

34 30585486 30594174 472 473 8B 1278

C6orfl3

35 30594613 30614598 474 475 4 1279

PTMAP

36 30601227 30603024 476 477 1 1280

C6orfl3

37 30614816 30620987 478 479 6 1281

DHX16 38 30620896 30640830 480 481 1282

KIAA19

39 30644166 30655672 482 483 49 1283

NRM 40 30655826 30658769 484 485 1284

RPL7P4 41 30664523 30665313 486 487 1285

MDC1 42 30667584 30685458 488 489 1286

TUBB 43 30688157 30693199 490 491 1287

LOCI 00

44 30691 146 30694154 492 493 287146 1288

FLOT1 45 3069551 1 30710453 494 495 1289

IER3 46 30710976 30712327 496 497 1290

DDR1 47 30851861 30867933 498 499 1291 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

GTF2H

48 30875977 30881880 500 501 4 1292

VARS2 49 30881982 30894236 502 503 1293

SFTA2 50 30899127 30899952 504 505 1294

DPCR1 51 30908777 30921998 506 507 1295

LOCI 00

52 30929178 30929755 508 509 422429 1296

LOCI 00

53 30931992 30933937 510 51 1 420530 1297

MUC21 54 30951485 30957675 512 513 1298

HCG22 55 31021984 31027653 514 515 1299

C6orfl5 56 31079000 31080332 516 517 1300

PSORS l

57 31082608 31 107869 518 519 CI 1301

CDSN 58 31082865 31088252 520 521 1302

PSORS l

59 31 10531 1 31 107127 522 523 C2 1303

CCHCR

60 31 1 10216 31 126015 524 525 1 1304

TCF19 61 31 126303 31 131992 526 527 1305

POU5F1 62 31 1321 14 31 138451 528 529 1306

PSORS l

63 31 141512 31 145676 530 531 C3 1307

HCG27 64 31 165537 31 171745 532 533 1308

HLA-C 65 31236529 31239855 534 535 1309

LOCI 00

66 31243352 31246531 536 537 287272 1310

RPL3P2 67 31248108 31249348 538 539 131 1

WASF5

68 31255162 31256941 540 541 P 1312

HLA-B 69 31321649 31324989 542 543 1313

DHFRP

70 31331244 31334742 544 545 2 1314

LOCI 00

71 31345494 31345805 546 547 462812 1315

HLA-S 72 31349346 31350264 548 549 1316

LOCI 00

73 31367561 31433586 550 551 507436 1317

HLA-X 74 31429623 31430267 552 553 1318

HCP5 75 31430957 31433586 554 555 1319

HCG26 76 31439006 31440185 556 557 1320

MICB 77 31465855 31478901 558 559 1321

PPIAP9 78 31486654 31488179 560 561 1322

RPL15P

79 31495853 31496498 562 563 4 1323

MCCD1 80 31496739 31498008 564 565 1324

BAT1 81 31497996 31510225 566 567 1325 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

SNORD

82 31504151 31504226 568 569 1 17 1326

SNORD

83 31508878 31508955 570 571 84 1327

ATP6V

84 31512239 31514627 572 573 1G2 1328

NFKBI

85 31514628 31526606 574 575 LI 1329

LTA 86 31539876 31542098 576 577 1330

TNF 87 31543350 315461 12 578 579 1331

LTB 88 31548335 31550202 580 581 1332

LST1 89 31553956 31556686 582 583 1333

NCR3 90 31556660 31560762 584 585 1334

LOCI 00

91 31578860 31579133 586 587 130756 1335

AIF1 92 31582994 31584798 588 589 1336

BAT2 93 31588450 31605554 590 591 1337

SNORA

94 31590856 31590987 592 593 38 1338

BAT3 95 31606805 31620477 594 595 1339

APOM 96 31623671 31625987 596 597 1340

C6orf47 97 31626075 31628549 598 599 1341

BAT4 98 31629862 31633163 600 601 1342

CSNK2

99 31633657 31637843 602 603 B 1343

LY6G5

100 31638728 31640227 604 605 B 1344

LY6G5

101 31644461 31648150 606 607 C 1345

BAT5 102 31654726 31671 137 608 609 1346

LY6G6

103 31674684 31678372 610 61 1 F 1347

LY6G6

104 31679753 31681842 612 613 E 1348

LY6G6

105 31683133 31685581 614 615 D 1349

LY6G6

106 31686425 31689510 616 617 C 1350

C6orf25 107 31691 161 31692851 618 619 1351

DDAH2 108 31694817 31698039 620 621 1352

CLIC1 109 31698358 31704341 622 623 1353

MSH5 1 10 31707725 31730455 624 625 1354

C6orf26 1 1 1 31730773 31732627 626 627 1355

C6orf27 1 12 31733371 31745108 628 629 1356

VARS 1 13 31745295 31763712 630 631 1357

LSM2 1 14 31765173 31774743 632 633 1358

HSPAl

1 15 31777396 31782835 634 635 L 1359 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

HSPAl

1 16 31783291 31785719 636 637 A 1360

HSPAl

1 17 31795512 31798031 638 639 B 1361

C6orf48 1 18 31802693 31807541 640 641 1362

SNORD

1 19 31803040 31803103 #N/A #N/A 48 1363

SNORD

120 31804853 31804916 #N/A #N/A 52 1364

NEU1 121 31826829 31830709 642 643 1365

SLC44A

122 31830969 31846823 644 645 4 1366

EHMT2 123 31847536 31865464 646 647 1367

ZBTB12 124 31867394 31869769 648 649 1368

C2 125 31895266 31913449 650 651 1369

CFB 126 31913721 31919861 652 653 1370

RDBP 127 31919864 31926864 654 655 1371

MIR 123

128 31924616 31924717 656 657 6 1372

SKIV2L 129 31926581 31937532 658 659 1373

DOM3Z 130 31937588 31940032 660 661 1374

STK19 131 31938952 31949223 662 663 1375

C4A 132 31949834 31970457 664 665 1376

CYP21

133 31972719 31976761 666 667 AlP 1377

TNXA 134 31976197 31980800 668 669 1378

STK19P 135 31981047 31981961 670 671 1379

C4B 136 31982572 32003195 672 673 1380

CYP21

137 32006082 32009419 674 675 A2 1381

TNXB 138 32008932 32077153 676 677 1382

ATF6B 139 32083045 32096030 678 679 1383

FKBPL 140 32096484 32098067 680 681 1384

PRRT1 141 321 16140 321 19720 682 683 1385

LOCI 00

142 32120579 321221 19 684 685 507547 1386

PPT2 143 32121301 32131452 686 687 1387

EGFL8 144 32132405 32136062 688 689 1388

AGP AT

145 32135983 32145888 690 691 1 1389

RNF5 146 32146162 32148570 692 693 1390

AGER 147 32148746 32152023 694 695 1391

PBX2 148 32152510 32157963 696 697 1392

GPSM3 149 32158543 32163300 698 699 1393

NOTCH

150 32162620 32191844 700 701 4 1394

C6orfl0 151 32260475 32339656 702 703 1395

Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

RPL32P

179 33047076 33047788 758 759 1 1423

HLA-

180 33059259 33061091 760 761 DPA2 1424

COL 1 1

181 33071570 33074821 762 763 A2P 1425

HLA-

182 33080293 33096890 764 765 DPB2 1426

HLA-

183 33098974 33099120 766 767 DPA3 1427

COL 1 1

184 33130469 33160245 768 769

A2 1428

PvXRB 185 33161365 33168432 770 771 1429

RNY4P

186 33167378 33167469 772 773 10 #N/A

SLC39A

187 33168603 33172214 774 775 7 1430

HSD17

188 33172419 33174607 776 777 B8 1431

MIR219

189 33175612 33175721 778 779 -1 1432

RING1 190 33176286 33180499 780 781 1433

LOCI 00

191 33183351 33183960 782 783 419609 1434

VPS52 192 33218049 33239662 784 785 1435

RPS18 193 33239852 33244281 786 787 1436

B3GAL

194 33244917 33246602 788 789 T4 1437

WDR46 195 33246880 33257304 790 791 1438

PFDN6 196 33257378 3325871 1 792 793 1439

RGL2 197 33259431 33267176 794 795 1440

TAPBP 198 33267471 33282164 796 797 1441

ZBTB22 199 33282182 33285719 798 799 1442

DAXX 200 33286335 33290793 800 801 1443

MYL8P 201 33306755 33307272 802 803 1444

LYPLA

202 33332501 33334139 804 805 2P1 1445

RPL35A

203 33357133 33357256 806 807 P4 1446

KIFC1 204 33359313 33377701 808 809 1447

RPL12P

205 33367792 33368421 810 81 1 1 1448

PHF1 206 33378773 33384230 812 813 1449

CUTA 207 33384319 33386065 814 815 1450

SYNGA

208 33387847 33421466 816 817 PI 1451

ZBTB9 209 33422356 33425321 818 819 1452

BAK1 210 33540323 33548070 820 821 1453 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

GGNBP

21 1 33551476 33556803 822 823 1 1454

C6orf22

212 33553883 33561 1 15 824 825 7 1455

ITPR3 213 33589161 33664351 826 827 1456

LOCI 00

214 33599021 33601413 828 829 507563 1457

C6orfl2

215 33665346 33679504 830 831 5 1458

IP6K3 216 33689443 33714762 832 833 1459

LEMD2 217 33738990 33756906 834 835 1460

MLN 218 33762449 33771793 836 837 1461

LOCI 00

219 33857288 33864684 838 839 507584 1462

MIR 127

220 33967749 33967828 840 841 5 1463

GRM4 221 33989628 34101443 842 843 1464

KRT18P

222 34157509 34158918 844 845 9 1465

CYCSL

223 34187149 34188714 846 847 1 1466

HMGA1 224 34204577 34214008 848 849 1467

C6orfl 225 34214157 34216904 850 851 1468

RPL35P

226 34231047 34231500 852 853 2 1469

LOCI 00

227 34247456 34250571 854 855 507620 1470

NUDT3 228 34255997 34360441 856 857 1471

RPS10 229 34385231 34393876 858 859 1472

PACSIN

230 34433905 34503000 860 861 1 1473

SPDEF 231 34505580 34524091 864 865 1474

LOCI 00

232 34543878 34544534 866 867 101247 1475

C6orfl0

233 34555065 34664625 868 869 6 1476

RPL7P2

234 34584299 34585101 870 871 5 1477

LOCI 00

235 34664094 34665247 872 873 131607 1478

RPS 10P

236 3471 1917 34712495 874 875 13 1479

SNRPC 237 34724871 34741634 876 877 1480

UHRFl

238 34759794 34845291 878 879 BPl 1481

TAF1 1 239 34845555 34855819 880 881 1482

ANKS1

240 34857038 35059190 882 883 A 1483 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

TCP 11 241 35085849 35109187 884 885 1484

SCUBE

242 35182190 35218609 886 887 3 1485

ZNF76 243 35227510 35263760 888 889 1486

DEF6 244 35265595 35289548 890 891 1487

PPARD 245 35310335 35395968 892 893 1488

LOCI 00

246 35316761 35324633 894 895 507672 1489

MKR P

247 35413987 35417642 896 897 2 1490

FANCE 248 35420138 35434881 898 899 1491

RPL10A 249 35436178 35438558 900 901 1492

TEAD3 250 35441374 35464861 902 903 1493

TULP1 251 35465651 35480647 904 905 1494

RPS 15A

252 35523623 35524081 906 907 P19 1495

FKBP5 253 35541362 35696360 908 909 1496

RPL36P

254 35575370 35575757 910 91 1 9 1497

LOC285

255 35694539 35704724 912 913 847 1498

C6orf81 256 35704859 35716685 914 915 1499

C6orfl2

257 35744392 35747329 916 917 6 1500

C6orfl2

258 35748831 35755841 918 919 7 1501

CLPS 259 35762760 35765102 920 921 1502

LHFPL5 260 35773071 35791852 922 923 1503

SRPK1 261 3580081 1 35888957 924 925 1504

SLC26A

262 3591 1291 35992413 926 927 8 1505

DPRXP

263 35957267 35958269 928 929 2 1506

MAPK1

264 35995454 36079013 930 931 4 1507

LOCI 00

265 36059260 36060417 932 933 505482 1508

MAPK1

266 36098262 36107842 934 935 3 1509

BRPF3 267 36164550 36200567 936 937 1510

PNPLA

268 36210945 36276372 938 939 1 151 1

C6orf22

269 36283534 36304662 940 941 2 1512

ETV7 270 36333971 36355467 942 943 1513

PXT1 271 36358328 36410666 944 945 #N/A

KCTD2

272 36410544 36458319 946 947 0 1514 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

STK38 273 36461669 36515247 948 949 1515

SFRS3 274 36562090 36572244 950 951 1516

LOC389

275 36641527 36643222 952 953 386 1517

CDKN1

276 36646459 36655109 954 955 A 1518

GPR166

277 36704782 36705483 956 957 P 1519

LOCI 00

278 36704887 36705660 958 959 420942 1520

CPNE5 279 36708555 36807220 960 961 1521

LOCI 00

280 36808556 36812209 962 963 127961 1522

PPIL1 281 36822605 36842800 964 965 1523

C6orf89 282 36853640 36896740 966 967 1524

LOCI 00

283 36891451 36901406 968 969 505509 1525

PI16 284 36922209 36932613 970 971 1526

MTCH1 285 36935917 36953949 972 973 1527

FGD2 286 36973423 36996845 974 975 1528

COX6A

287 37012610 37013157 976 977 1P2 1529

RPL12P

288 37059003 37059621 978 979 2 1530

PIM1 289 37137922 37143204 980 981 1531

TMEM2

290 37179954 37225931 982 983 17 1532

TBC1D

291 37225548 37300746 984 985 22B 1533

RNF8 292 37321748 37362514 986 987 1534

FTSJD2 293 37400907 37449284 988 989 1535

C6orfl2

294 37450696 37467700 990 991 9 1536

LOCI 00

295 37506128 37509166 992 993 505530 1537

LOCI 00

296 3751 1329 37514538 994 995 505550 1538

MDGA1 297 37600284 37665766 996 997 1539

ZFAND

298 37787307 38122400 998 999 3 1540

BTBD9 299 38136227 38607924 1000 1001 1541

LOCI 00

300 38449372 38450303 1002 1003 505567 1542

LOCI 00

301 38555099 38556305 1004 1005 128379 1543

GLOl 302 38643701 38670952 1006 1007 1544

DNAH8 303 38690552 38998567 1008 1009 1545

ZRF1PS 304 38730632 38734659 1010 101 1 1546 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

LOCI 00

305 39007444 39010968 1012 1013 128655 1547

GLP1R 306 39016557 39055520 1014 1015 1548

C6orf64 307 39071840 39082865 1016 1017 1549

KCNK5 308 39156747 39197251 1018 1019 1550

KCNK1

309 39266777 39282237 1020 1021 7 1551

KCNK1

310 39282474 39290330 1022 1023 6 #N/A

KIF6 31 1 39302876 39693181 1024 1025 1552

LOCI 00

312 39321621 39322274 1026 1027 124373 1553

LOCI 00

313 39521593 39522716 1028 1029 131899 1554

DAAM2 314 39760793 39872641 1030 1031 1555

LOCI 00

315 39849580 39865028 1032 1033 505635 1556

MOCS1 316 39872034 39902290 1034 1035 1557

RPL23P

317 39926090 39926598 1036 1037 6 1558

LOC442

318 39960554 39967943 1038 1039 210 1559

FLJ4164

319 40312084 40323745 1040 1041 9 1560

TDRG1 320 40346163 40347631 1042 1043 1561

LRFN2 321 40359373 40555126 1044 1045 1562

LOCI 00

322 40484082 40491672 1046 1047 505697 1563

U C5C

323 40994640 41006938 1048 1049 L 1564

TSP02 324 41010237 41012076 1050 1051 1565

APOBE

325 41020940 41032630 1052 1053 C2 1566

C6orfl3

326 41034531 41040188 1054 1055 0 1567

NFYA 327 41040707 41070146 1056 1057 1568

LOC221

328 41068773 41 108573 1058 1059 442 1569

TREML

329 41 1 17342 41 122070 1060 1061 1 1570

TREM2 330 41 126246 41 130922 1062 1063 1571

TREML

331 41 157552 41 168925 1064 1065 2 1572

TREML

332 41 176292 41 185685 1066 1067 3 1573

TREML

333 41 196062 41206120 1068 1069 4 1574 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

TREML

334 41217115 41217327 1070 1071 2P 1575

TREM1 335 41243712 41254457 1072 1073 1576

RPL32P

336 41275696 41278043 1074 1075 15 1577

NCR2 337 41303528 41318625 1076 1077 1578

LOCI 00

338 41349130 41350823 1078 1079 505711 #N/A

LOCI 00

339 41491633 41516359 1080 1081 505730 1579

FOXP4 340 41514164 41570122 1082 1083 1580

MDFI 341 41606195 41621982 1084 1085 1581

LOCI 00

342 41634644 41635393 1086 1087 130606 1582

TFEB 343 41651716 41703997 1088 1089 1583

PGC 344 41704449 41715139 1090 1091 #N/A

FRS3 345 41737914 41747630 1092 1093 1584

PRICKL

346 41748500 41755110 1094 1095 E4 1585

TOMM

347 41755181 41757634 1096 1097 6 1586

USP49 348 41765383 41863099 1098 1099 1587

MED20 349 41873092 41888877 1100 1101 1588

BYSL 350 41888965 41900784 1102 1103 1589

CCND3 351 41902671 42016610 1104 1105 1590

TAF8 352 42018251 42048644 1106 1107 1591

C6orfl3

353 42068857 42110715 1108 1109 2 1592

GUCA1

354 42123144 42147794 1110 1111 A 1593

GUCA1

355 42151022 42162694 1112 1113 B 1594

MRPS1

356 42174539 42185633 1114 1115 0 1595

TRERF

357 42192669 42419783 1116 1117 1 1596

RPL36A

358 42467416 42467817 1118 1119 P5 1597

UBR2 359 42531760 42661243 1120 1121 1598

PRPH2 360 42664333 42690358 1122 1123 1599

LOC442

361 42694972 42695932 1124 1125 211 1600

TBCC 362 42712234 42713884 1126 1127 1601

FLJ3871

363 42750665 42753299 1128 1129 7 1602

KIAA02

364 42788794 42836296 1130 1131 40 1603

RPL7L1 365 42847671 42854731 1132 1133 1604 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

C6orf22

366 42858003 42858554 1134 1135 6 1605

PTCRA 367 42883727 42893576 1136 1137 1606

CNPY3 368 42896860 42907008 1138 1139 1607

RPL24P

369 42924074 42924504 1140 1141 4 1608

GNMT 370 42928500 42931618 1142 1143 1609

PEX6 371 42931611 42946981 1144 1145 1610

PPP2R5

372 42952330 42980080 1146 1147 D 1611

MEA1 373 42979965 42981618 1148 1149 1612

KLHDC

374 42981977 42989032 1150 1151 3 1613

C6orfl5

375 42989385 42997337 1152 1153 3 1614

LOCI 00

376 43000252 43002143 1154 1155 505808 1615

CUL7 377 43005355 43021683 1156 1157 1616

MRPL2 378 43021767 43027242 1158 1159 1617

KLC4 379 43027372 43042833 1160 1161 1618

PTK7 380 43044029 43129457 1162 1163 1619

SRF 381 43138920 43149244 1164 1165 1620

CUL9 382 43149922 43192325 1166 1167 1621

C6orfl0

383 43193367 43197211 1168 1169 8 1622

TTBK1 384 43211222 43255997 1170 1171 1623

SLC22A

385 43265998 43273276 1172 1173 7 1624

CRIP3 386 43273211 43276530 1174 1175 1625

RPL34P

387 43295590 43296214 1176 1177 14 1626

ZNF318 388 43303808 43337181 1178 1179 1627

RPS2P2

389 43331153 43331957 1180 1181 8 1628

ABCCl

390 43399489 43418163 1182 1183 0 1629

DLK2 391 43418090 43423786 1184 1185 1630

TJAP1 392 43445261 43474294 1186 1187 1631

C6orfl5

393 43474707 43478424 1188 1189 4 1632

YIPF3 394 43479565 43484728 1190 1191 1633

POLRl

395 43484791 43497114 1192 1193 C 1634

XP05 396 43490068 43543812 1194 1195 1635

RPS2P2

397 43506522 43507463 1196 1197 9 1636

POLH 398 43543878 43588260 1198 1199 1637 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

GTPBP

399 43588218 43596936 1200 1201 2 1638

MAD2L

400 43597279 43608689 1202 1203 IBP 1639

RSPH9 401 43612767 43638748 1204 1205 1640

MRPS1

402 43638934 43655549 1206 1207 8A 1641

LOCI 00

403 43673593 43674494 1208 1209 132242 1642

VEGFA 1 43737946 43754224 1210 121 1 1643

LOCI 00

405 43858765 43905944 1212 1213 132354 1644

C6orf22

406 43968337 43973695 1214 1215 3 1645

RPL29P

407 44056862 44057500 1216 1217 16 1646

MRPL1

408 44081372 44095191 1218 1219 4 1647

TMEM6

409 44095376 44123256 1220 1221 3B 1648

CAPN1

410 44126548 44152139 1222 1223 1 1649

SLC29A

41 1 44187242 44201888 1224 1225 1 1650

LOCI 00

412 44209727 44221620 1226 1227 505819 1651

HSP90A

413 44214849 44221614 1228 1229 Bl 1652

SLC35B

414 44221838 44225283 1230 1231 2 1653

NFKBI

415 44225903 44233525 1232 1233 E 1654

TMEM1

416 44238480 44247182 1234 1235 51B 1655

TCTE1 417 44247897 44265458 1236 1237 1656

AARS2 418 44266463 44281063 1238 1239 1657

SPATS 1 419 44310397 44344904 1240 1241 1658

CDC5L 420 44355302 44414780 1242 1243 1659

LOCI 00

421 44440641 44445639 1244 1245 128935 1660

LOCI 00

422 44513903 44519618 1246 1247 505862 1661

SUPT3

423 44796469 45345670 1248 1249 H 1662

LOCI 00

424 44866370 44867032 1250 1251 422452 1663

LOCI 00

425 45065696 45067086 1252 1253 271870 1664 Gene Probe

Gene For. SEQ Rev. SEQ

SEQ ID Start End SEQ ID Symbol ID NO: ID NO:

NO: NO:

LOCI 00

426 45126583 45127268 1254 1255 420052 1665

MIR586 427 4516541 1 45165507 1256 1257 1666

RU X2 428 45296054 45518819 1258 1259 1667

CLIC5 429 45866188 46048085 1260 1261 1668

ENPP4 430 46097701 461 14436 1262 1263 1669

ENPP5 431 46127762 46138717 1264 1265 1670

ACTG1

432 46172649 46174298 1266 1267 P9 1671

RCAN2 433 46188469 46293531 1268 1269 1672

Table 3. Provided are the genes encompassed within the human chromosome 6p21 genomic amplification region [shown by gene symbol, sequence identifier and the start and end nucleotide positions on human chromosome 6 according to UCSC on Human GRCh37 Assembly (human genome 19 (hgl9)], and means to quantifying the copy number of the genomic amplification in 6p21 using PCR primers for qDNA PCR (provided by sequence identifiers) and DNA probes for quantitative Southern blot (provided by sequence identifiers). "Gene SEQ ID NO:" - Genomic sequence SEQ ID NO:; "Start" - The first nucleotide corresponding to the gene as indicated by position on human Chr.6; "End" - The last nucleotide corresponding to the gene as indicated by position on human Chr. 6; "For. SEQ ID NO:" - Forward primer (5'→3') for qDNA PCR (SEQ ID NO:); "Rev. SEQ ID NO:" - Reverse primer for qDNA PCR (SEQ ID NO:); "probe SEQ ID NO:" - Probe for qSouthern blot. #N/A - not available.

Methods of performing quantitative Southern blot are known to the ordinary skilled in the art. Once the DNA is extracted from a test and a reference sample, the concentration of the DNA can be determined with a spectrophotometer, following which the DNA is digested with restriction enzyme endonucleases such as Hindlll and/or with combination of a few restriction enzymes (e.g., Hindlll and Bglll). Once digested, equal amounts of DNA (e.g., 5-10 μg per lane) from both the "test" sample(s) and the reference sample(s) are loaded onto an agarose gel (e.g., 0.6-1.3% agarose which contains ethidium bromide) and are subject to gel electrophoresis. Prior to blotting, the amount in each lane is equalized based on the intensity of DNA smear stained with ethidium bromide. After blotting to a membrane (e.g., Hybond N+; Amersham) the blotted DNA is hybridized with a labeled DNA probe. The probe can be labeled using methods well known in the art with a radioactive isotope such as ³²P, or with a nonradioactive labeling such as using Digoxigenin labeling.

Suitable DNA probes for quantitative Southern blot can be prepared from the genomic region of-interest, and/or from a complementary DNA (cDNA, complementary DNA probes, which are in the antisense direction with respect to the mRNA sense sequence) of any of the coding sequences comprised in the sequence of the genomic amplification which comprises the VEGF-A gene. The sequence of the cDNA can be obtained from the NCBI web site [Hypertext Transfer Protocol ://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/] by searching "GENE" with the "Gene Symbol" listed in Table 3 above, or by performing a sequence alignment (e.g., BLASTN) using as a "Query Sequence" any of the genomic sequences provided in SEQ ID NOs: l , 26-403 and 405-433 (Table 3 above). The length of the probe for Southern blot analysis can vary from few tens of nucleotides to several kilobases of nucleotides.

Suitable polynucleotide probes are those referred to as "unique probes" which specifically hybridize to the target DNA sequence but not to other DNA sequences in the sample under the same hybridization conditions. By way of example, hybridization of short polynucleotide probes (below 200 nucleotides in length) can be effected by the following hybridization protocols depending on the desired stringency; By way of example, hybridization of short polynucleotide probes (below 200 nucleotides in length) can be effected by the following hybridization protocols depending on the desired stringency; (i) an hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 °C below the T_m, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the T_m; (ii) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 2 - 2.5 °C below the T_m, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the T_m, final wash solution of 6 x SSC, and final wash at 22 °C; (Hi) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 37 °C, final wash solution of 6 x SSC and final wash at 22 °C. Variations in the hybridization conditions (e.g., hybridization solution, wash solutions and temperatures) can be made by one skilled in the art according to the probe length and its G/C content. For example, for polynucleotide probes larger than 200 nucleotides, the hybridization/washes temperatures can be about 55-80 °C (e.g., 65 °C) and the hybridization/wash solutions can include formamide (e.g., 50 %).

By way of example, hybridization with longer DNA probes (e.g., about 1-2 kb) can be performed using the following hybridization solution (i) 5X Denhardt's Solution [5 OX Denhardt's Solution consists of 1% BSA, 1% Polyvinylpyrrolidone, 1% Ficoll], 100 mg/ml Salmon or Herring Sperm DNA, 0.1% SDS, 5XSSPE [SSPE (20X) consists of: 3M NaCl, 0.2M Sodium Phosphate, pH 7.4, 25 mM EDTA], 50% formamide; hybridization temperature of 20°C below the calculated Tm (melting temperature); and washes 1 x 20 minutes in IX SSC, 0.1% SDS at 45° C, followed by 3 x 20 minutes in 0.2X SSC, 0.1 % SDS at 65° C.

According to some embodiments of the invention, the probe has a nucleotide sequence having as a 5' nucleotide the nucleotide at position "X" in SEQ ID NO:23 and as a 3' nucleotide the nucleotide at position "Y" in SEQ ID NO:23; wherein X is a numerator selected from nucleotide position 1-15,799,979 in SEQ ID NO:23, wherein Y is a numerator selected from nucleotide position 21-15,799,999 in SEQ ID NO:23, and wherein numerator Y is larger than the numerator X by at least about 20 nucleotides, e.g., by at least about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700 nucleotides or more.

According to some embodiments of the invention, the probe has a nucleotide sequence having as a 5' nucleotide the nucleotide at position "X" in SEQ ID NO:24 and as a 3' nucleotide the nucleotide at position "Y" in SEQ ID NO:24; wherein X is a numerator selected from nucleotide position 1-5,699,979 in SEQ ID NO:24, wherein Y is a numerator selected from nucleotide position 21-5,699,999 in SEQ ID NO:24, and wherein numerator Y is larger than the numerator X by at least about 20 nucleotides, e.g., by at least about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700 nucleotides or more.

According to some embodiments of the invention, the probe has a nucleotide sequence having as a 5' nucleotide the nucleotide at position "X" in SEQ ID NO:25 and as a 3' nucleotide the nucleotide at position "Y" in SEQ ID NO:25; wherein X is a numerator selected from nucleotide position 1-317,980 in SEQ ID NO:25, wherein Y is a numerator selected from nucleotide position 21-318,000 in SEQ ID NO:25, and wherein numerator Y is larger than the numerator X by at least about 20 nucleotides, e.g., by at least about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700 nucleotides or more.

According to some embodiments of the invention, the length of the probe varies from about 20 bases to about 10 kilobase (kb), e.g., from about 20 bases to about 7 kb, e.g., 100 bases to about 5 kb, e.g., from about 200 bases to about 2 kb, e.g., from about 300 bases to about 1.5 kb, e.g., from about 500-1300 bases. Various software are available to design appropriate DNA probes for genomic hybridization using Southern blot analysis. Examples include, but are not limited to G2C:: Software [Hypertext Transfer Protocol ://World Wide Web (dot) genes2cognition (dot) org/software/southern blot/probe tiling method (dot) html].

It should be noted that a probe for Southern blot analysis should be preferably devoid of repetitive DNA sequences, such that when hybridized to genomic DNA will result in a single or a few distinct bands (see e.g., determination of gene copy number by quantitative Southern blot analysis in the Dystrophin gene, Hiraishi Y., et al., 1992, J. Med. Genet. 29: 897-901, which is hereby incorporated by reference in its entirety).

Bioinformatic tools for removing repetitive sequences from nucleotide sequences are known in the art. For example, the RepeatMasker tool (Institute for System Biology, available via the Hypertext Transfer Protocol://World Wide Web (dot) repeatmasker (dot) org/ web site can be used. Another convenient tool for excluding repetitive sequences from a DNA sequence is via the UCSC Genome Browser [Hypertext Transfer Protocol ://genome (dot) ucsc (dot) edu/cgi-bin/hgGateway] in which one can insert the genomic region of interest (e.g., human chromosome 6p21) and in the "DNA" tool select the option of "Mask Repeats". The outcome of the repeat masking tools can be such that all repetitive sequences are converted to "NNN..." an can be further excluded from the sequence. Non-limiting examples of DNA probes (devoid of repetitive sequences) from the genomic region encompassed by the amplification (6p21) are provided in SEQ ID NOs: 1270-1672 as described in the "Probe" column in Table 3 above.

Qualifying of suitable probes for quantitative Southern blot can be done by Southern blot analysis using the identified probe (according to the teachings provided herein) using known reference DNA samples derived from a positive control biological sample (which is known to include the genomic amplification based on other methods such as FISH) and/or a negative control biological sample (which is known to be devoid of the genomic amplification based on other methods such as FISH).

According to some embodiments of the invention the method further comprising comparing an expression level of the VEGFA in the sample of the carcinoma to a reference expression data obtained from at least one sample devoid of cancer.

According to some embodiments of the invention an increase above a predetermined threshold in the expression level of the VEGFA in the sample of the carcinoma relative to the reference expression data predicts the efficacy of the anti- VEGFA treatment on the carcinoma (i.e., that the anti-VEGFA treatment is efficient in treating the solid tumor).

According to some embodiments of the invention the sample devoid of cancer is a liver sample.

The expression level of VEGFA can be detected using various RNA detection methods such as Northern Blot analysis, RT-PCR analysis, RNA in situ hybridization stain, In situ RT-PCR stain, DNA microarrays/DNA chips, Oligonucleotide microarray.

The expression and/or activity level of the VEGFA protein can be determined using methods known in the arts such as Enzyme linked immunosorbent assay (ELISA), Western blot, radio-immunoassay (RIA), fluorescence activated cell sorting (FACS), immunohistochemical analysis, in situ activity assay, in vitro activity assays.

As used herein the phrase "anti-VEGFA" refers to an agent (e.g., drug) which reduces, inhibits, or suppresses VEGFA levels (expression level and/or activity) in cells or tissue.

According to some embodiments of the invention, the anti-VEGF agent inhibits

VEGF-A activity by blocking the VEGF-A tyrosine kinase receptor. According to some embodiments of the invention, the anti-VEGF agent is a small molecule, which blocks the VEGF-A tyrosine kinase receptor.

According to some embodiments of the invention, the anti-VEGF agent reduces, inhibits or suppresses VEGFA levels in the tumor cells of a subject in need thereof.

As used herein the phrase "anti- VEGFA treatment" refers to administration of an anti-VEGF drug into a subject in need thereof. It should be noted that administration of an anti-VEGF drug may comprise a single or multiple dosages, as well as a continuous administration, depending on the pathology to be treated and the subject receiving the treatment.

Non-limiting examples of anti-VEGF agents which can be used according to the method of the invention include an anti-VEGF antibody (e.g., the monoclonal antibody bevacizumab such as AVASTIN™; ranibizumab), an anti- VEGFA RNA silencing agents (e.g., antisense, siRNA, shRNA), an anti- VEGFA Ribozyme, an anti- VEGFA DNAzyme, a soluble form of the VEGF-receptor (e.g., GenBank Accession No. AAC50060; SEQ ID NO: 16), a small molecule, thalidomide or an analogue thereof (e.g., as described in Miguel Fernandez Brana et al., European Journal of Medicinal Chemistry, Volume 44, Issue 9, 2009, Pages 3533-3542; Magdy A.-H. Zahran et al, Bioorganic & Medicinal Chemistry, Volume 16, Issue 22, 15 November 2008, Pages 9708-971; all of which are incorporated by reference in their entirety), anti- VEGFA aptamers (e.g., Pegaptanib, a pegylated anti-VEGF aptamer which specifically binds to VEGF 165; pegaptanib sodium), small molecules which are anti-VEGF -A tyrosine kinase inhibitors such as Sorafenib (Nexavar®, Bayer HealthCare Pharmaceuticals), ABT-869 (Linifanib; N-[4-(3 -Amino- lH-indazol-4-yl)phenyl]-N^*-(2-fluoro-5- methylphenyl)urea), Axitinib (also known as AGO 13736, a small molecule tyrosine kinase inhibitor under development by Pfizer), BIBF1120 (oral potent triple angiokinase inhibitor targeting VEGFR, PDGFR, FGFR kinases), Brivanib (BMS-582664), Brivanib alaninate, Cediranib (AZD2171), CHIR-258, E7080, Motesanib Diphosphate (AMG- 706), Pazopanib Hydrochloride, Sunitinib Malate (Sutent), Vandetanib, Vatalanib, Dihydrochloride Salt, XL 184, and/or XL880 (GSK1363089,EXEL-2880). Additional anti-VEGFA agents, which can be used according to the method of the invention, include those described in Murukesh N., et al, 2010, which is fully incorporated herein in its entirety. According to some embodiments of the invention, the anti- VEGFA comprises Sorafenib.

According to some embodiments of the invention, the anti- VEGFA comprises bevacizumab.

According to some embodiments of the invention, the anti- VEGFA comprises a soluble form of the VEGF-receptor.

According to some embodiments of the invention, the anti- VEGFA comprises thalidomide.

According to some embodiments of the invention, the anti- VEGFA comprises a combination of at least two-anti VEGFA drugs selected from the group consisting of Sorafenib, bevacizumab, a soluble form of the VEGF-receptor and thalidomide.

Non-limiting examples of such combination therapy with anti- VEGFA drugs comprises Sorafenib and bevacizumab; Sorafenib and a soluble form of the VEGF- receptor; Sorafenib and thalidomide; bevacizumab and a soluble form of the VEGF- receptor; and bevacizumab and thalidomide; a soluble form of the VEGF-receptor and thalidomide.

According to some embodiments of the invention, the anti-VEGFA antibody comprises an antigen binding region capable of specifically binding VEGFA. Preferably, the antigen binding region specifically binds at least one epitope of VEGFA. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.

Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al, Science 242:423-426 (1988); Pack et al, Bio/Technology 11 : 1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and coworkers [Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature 332:323- 327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology 10,: 779-783 (1992); Lonberg et al, Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

According to some embodiments of the invention the anti-VEGFA agent is an anti-VEGFA RNA silencing agent. As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

As used herein, the term "RNA silencing agent" refers to an RNA which is capable of inhibiting or "silencing" the expression of a target gene (e.g., VEGFA). In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla. Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.

Accordingly, the present invention contemplates use of dsRNA to downregulate protein expression from mRNA.

According to one embodiment, the dsRNA is greater than 30 bp. The use of long dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited owing to the belief that these longer regions of double stranded RNA will result in the induction of the interferon and PKR response. However, the use of long dsRNAs can provide numerous advantages in that the cell can select the optimal silencing sequence alleviating the need to test numerous siRNAs; long dsRNAs will allow for silencing libraries to have less complexity than would be necessary for siRNAs; and, perhaps most importantly, long dsRNA could prevent viral escape mutations when used as therapeutics.

Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects - see for example [Strat et al, Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004;13: 115-125; Diallo M., et al, Oligonucleotides. 2003;13:381-392; Paddison P.J., et al, Proc. Natl Acad. Sci. USA. 2002;99: 1443-1448; Tran N., et al, FEBS Lett. 2004;573: 127-134].

In particular, the present invention also contemplates introduction of long dsRNA (over 30 base transcripts) for gene silencing in cells where the interferon pathway is not activated (e.g. embryonic cells and oocytes) see for example Billy et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides, October 1, 2003, 13(5): 381-392. doi: 10.1089/154545703322617069.

The present invention also contemplates introduction of long dsRNA specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression. For example, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5 '-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.

Another method of evading the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNAs (siRNAs) either via transfection or endogenous expression.

The term "siRNA" refers to small inhibitory RNA duplexes (generally between 18-30 basepairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3 '-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100- fold increase in potency compared with 21mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27mer) instead of a product (21mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.

It has been found that position of the 3 '-overhang influences potency of an siRNA and asymmetric duplexes having a 3 '-overhang on the antisense strand are generally more potent than those with the 3'-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.

The strands of a double-stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). Thus, as mentioned the RNA silencing agent of the present invention may also be a short hairpin RNA (shRNA).

The term "shRNA", as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5'-UUCAAGAGA-3' (Brummelkamp, T. R. et al. (2002) Science 296: 550) and 5^*-UUUGUGUAG-3^* (Castanotto, D. et al. (2002) RNA 8: 1454). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.

According to another embodiment the RNA silencing agent may be a miRNA. miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants. The primary transcript (termed the "pri-miRNA") is processed through various nucleolytic steps to a shorter precursor miRNA, or "pre-miRNA." The pre -miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) The pre -miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17: 1376- 1386).

Unlike, siRNAs, miRNAs bind to transcript sequences with only partial complementarity (Zeng et al., 2002, Molec. Cell 9: 1327-1333) and repress translation without affecting steady-state RNA levels (Lee et al, 1993, Cell 75:843-854; Wightman et al, 1993, Cell 75:855-862). Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (Hutvagner et al., 2001, Science 293:834-838; Grishok et al, 2001, Cell 106: 23-34; Ketting et al, 2001, Genes Dev. 15:2654-2659; Williams et al, 2002, Proc. Natl. Acad. Sci. USA 99:6889- 6894; Hammond et al, 2001, Science 293: 1146-1150; Mourlatos et al, 2002, Genes Dev. 16:720-728). A recent report (Hutvagner et al, 2002, Sciencexpress 297:2056- 2060) hypothesizes that gene regulation through the miRNA pathway versus the siRNA pathway is determined solely by the degree of complementarity to the target transcript. It is speculated that siR As with only partial identity to the mRNA target will function in translational repression, similar to an miRNA, rather than triggering RNA degradation.

Synthesis of RNA silencing agents suitable for use with the present invention can be effected as follows. First, the VEGFA mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).

Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (world wide web (dot) ncbi (dot) nlm (dot) nih (dot) gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.

It will be appreciated that the RNA silencing agent of the present invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.

In some embodiments, the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide." As used herein, a "cell- penetrating peptide" is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non- endocytotic) translocation properties associated with transport of the membrane- permeable complex across the plasma and/or nuclear membranes of a cell. The cell- penetrating peptide used in the membrane-permeable complex of the present invention preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double-stranded ribonucleic acid that has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell-penetrating peptides of the present invention preferably include, but are not limited to, penetratin, transportan, plsl, TAT(48-60), pVEC, MTS, and MAP.

mRNAs to be targeted using RNA silencing agents include, but are not limited to, those whose expression is correlated with an undesired phenotypic trait. Exemplary mRNAs that may be targeted are those that encode truncated proteins i.e. comprise deletions. Accordingly the RNA silencing agent of the present invention may be targeted to a bridging region on either side of the deletion. Introduction of such RNA silencing agents into a cell would cause a down-regulation of the mutated protein while leaving the non-mutated protein unaffected.

Another anti-VEGFA agent is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the VEGFA. DNAzymes are single- stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 1997;943:4262) A general model (the " 10-23" model) for the DNAzyme has been proposed. "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate- recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 2002, Abstract 409, Ann Meeting Am Soc Gen Ther world wide web(dot) asgt (dot) org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.

Another anti- VEGFA agent is an antisense polynucleotide capable of specifically hybridizing with an m NA transcript encoding the VEGFA.

Design of antisense molecules which can be used to efficiently downregulate a

VEGFA must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.

The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett et al. Blood 91 : 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40 (1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al. (1997) Biochem Biophys Res Commun 231 : 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9 (1999)].

Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al. enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat g l30) in cell culture as evaluated by a kinetic PCR technique proved effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al, Nature Biotechnology 16: 1374 - 1375 (1998)].

Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin Mol Ther 1 :372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin Mol Ther 1 :297-306 (1999)].

More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al, Cancer Res 61 :7855-60 (2001)].

Thus, the current consensus is that recent developments in the field of antisense technology which, as described above, have led to the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for downregulating expression of known sequences without having to resort to undue trial and error experimentation.

Another anti-VEGFA agent is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding a VEGFA. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al, Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al, Clin Diagn Virol. 10: 163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page).

Qualifying agents which can reduce, inhibit or suppress the expression level and/or activity of VEGF can be performed using various in vitro (e.g., using a biological sample of the subject), ex vivo (e.g., in cells of a subject which are treated by the agent and are then injected into an animal model) or in vivo (e.g., by testing the expression level and/or activity of VEGF A in cells of a subject following treatment of the subject with the anti-VEGFA agent) methods.

Each of the anti-VEGFA agents described hereinabove or the expression vector encoding same can be administered to the individual per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the anti-VEGFA agent accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method. Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen- free water based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (anti-VEGFA agent) effective to prevent, alleviate or ameliorate symptoms of the cancer (e.g., carcinoma) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Dosage amount and interval may be adjusted individually to provide tissue levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

According to some embodiments of the invention, the method further comprising informing the subject on the results of the prediction of anti-VEGFA treatment efficacy test. For example, informing the subject that based on the presence of the genomic amplification (which comprises a VEGFA gene) in the solid tumor the anti- VEGFA treatment is predicted to be efficient in treating the cancer. On the other hand, if the solid tumor is devoid of the genomic amplification (which comprises a VEGFA gene), then informing the subject that, based on the absence of the genomic amplification, the anti- VEGFA treatment is likely not to be efficient for treating the cancer, and that alternative therapeutic approaches should be explored.

According to an aspect of some embodiments of the invention there is provided a method of treating a subject diagnosed with a solid tumor, the method comprising: (a) predicting the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the prediction; thereby treating of the subject diagnosed with the solid tumor.

The term "treating" refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition, e.g., the cancer, e.g., the carcinoma) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

As used herein, the term "subject" includes mammals, preferably human beings at any age which suffer from the pathology.

According to an aspect of some embodiments of the present invention there is provided a method of selecting or designing a treatment regimen for treating a subject diagnosed with a solid tumor, the method comprising: (a) predicting the efficacy of the anti-VEGFA treatment on the subject diagnosed with the solid tumor according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the prediction; thereby selecting or designing the treatment regimen for treating the subject diagnosed with a solid tumor.

As used herein the phrase "treatment regimen" refers to a treatment plan that specifies the type of treatment, dosage, schedule and/or duration of a treatment provided to a subject in need thereof (e.g., a subject diagnosed with a pathology). The selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the pathology) or a more moderate one which may relief symptoms of the pathology yet results in incomplete cure of the pathology. It will be appreciated that in certain cases the more aggressive treatment regimen may be associated with some discomfort to the subject or adverse side effects (e.g., a damage to healthy cells or tissue). The type of treatment can include the anti-VEGFA agent of the invention alone of in combination with other chemotherapeutic drugs, a surgical intervention (e.g., removal of lesion, diseased cells, tissue, or organ), a cell replacement therapy, an administration of a therapeutic drug (e.g., receptor agonists, antagonists, hormones, chemotherapy agents) in a local or a systemic mode, an exposure to radiation therapy using an external source (e.g., external beam) and/or an internal source (e.g., brachytherapy) and/or any combination thereof. The dosage, schedule and duration of treatment can vary, depending on the severity of pathology and the selected type of treatment, and those of skills in the art are capable of adjusting the type of treatment with the dosage, schedule and duration of treatment.

Non-limiting examples of chemotherapy agents which can be administered in combination with the anti-VEGFA agent of some embodiments of the invention include Mechlorethamine, (FiN 2), Cyclophosphamide, Ifosfamide, Melphalan, Chlorambucil, Estramustine, Hexamethyl-melamine, Thiotepa, Busulfan, Carmustine, Lomustine, Semustine, Streptozocin, Dacarbazine, Procarbazine, Aziridine, Methotrexate, Trimetrexate, Fluorouracil, Floxuridine, Cytarabine, Azacitidine, Mercaptopurine, Thioguanine, Pentostatin, Fludarabine, Vinblastine (VLB), Vincristine, Vindesine, Etoposide, Teniposide, Dactinomycin, Daunorubicin, Doxorubicin, 4'-, Deoxydoxorubicin, Bleomycin, Plicamycin, Mitomycin, L-Asparaginase, Docetaxel, Paclitaxel, Interferon Alfa, Tumor Necrosis Factor, Cisplatin, Carboplatin, Mitoxantrone, Hydroxyurea, Procarbazine, Mitotane, Aminoglutethimide, Hydroxy- progesterone, caproate, Medroxy-progesterone, acetate, Megestrol acetate, Diethylstil-, bestrol, Ethinyl estradiol, Tamoxifen, Testosterone, propionate, Fluoxymesterone, Flutamide, Leuprolide, Goserelin.

Non-limiting examples of approved oncology drugs which can be administered in combination with the anti-VEGFA agent of some embodiments of the invention include Aldesleukin, Alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, Asparaginase, BCG Live, bexarotene capsules, bexarotene gel, bleomycin, busulfan intravenous, busulfan oral, calusterone, capecitabine, carboplatin, carmustine, carmustine with Polifeprosan 20 Implant, celecoxib, chlorambucil, cisplatin, cladribine, cyclophosphamide, cyclophosphamide, cytarabine, cytarabine liposomal, dacarbazine, dactinomycin, actinomycin D, dactinomycin, actinomycin D, Darbepoetin alfa, Darbepoetin alfa, daunorubicin liposomal, daunorubicin, daunomycin, Denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, doxorubicin liposomal, DROMOSTANOLONE PROPIONATE, Elliott's B Solution, epirubicin, Epoetin alfa, estramustine, etoposide phosphate, etoposide VP- 16, exemestane, Filgrastim, floxuridine (intraarterial), fludarabine, fluorouracil, 5-FU, fulvestrant, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, hydroxyurea, Ibritumomab Tiuxetan, idarubicin, ifosfamide, imatinib mesylate, imatinib mesylate, Interferon alfa-2a, Interferon alfa-2b, irinotecan, letrozole, leucovorin, levamisole, lomustine, CCNU, meclorethamine, nitrogen mustard, megestrol acetate, melphalan, L- PAM, mercaptopurine, 6-MP, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, Nofetumomab, Oprelvekin, oxalip latin, paclitaxel, pamidronate, pegademase, Pegaspargase, Pegfilgrastim, pentostatin, pipobroman, plicamycin, mithramycin, porfimer sodium, porfimer sodium, procarbazine, quinacrine, Rasburicase, Rituximab, Sargramostim, streptozocin, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, Tositumomab, Trastuzumab, tretinoin, ATRA, Uracil Mustard, valrubicin, vinblastine, vincristine, vinorelbine, and zoledronate.

According to some embodiments of the invention the treatment regimen further comprises administering an anti-VEGFA agent in combination with a drug selected from the group consisting of erlotinib, oxaliplatin, cisplatin, platinum, rIFNa-2b, doxorubicin, fluorouracil, DX-8951f, thalidomide, doxorubicin, epirubicin, and taxol.

According to some embodiments of the invention the treatment regimen further comprises administering an anti-VEGFA agent in combination with a treatment selected from the group consisting of percutaneous ethanol injection, radio frequency ablation, transcatheter arterial chemoembolization. As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The term "consisting of means "including and limited to".

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al, "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al, "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

GENERAL MATERIALS AND EXPERIMENTAL METHODS

Mice - Mdr2^_/~ mice on FVB background were held in specific pathogen free conditions. Wild-type (WT) control mice were aged matched FVB mice. Two hours prior to sacrifice, mice were injected with 100 μΐ BrdU (Cell Proliferation labeling reagent, Amersham; Catalogue number: RPN201) per 10 gram body weight. Mice were anesthesized using Ketamine and Xylazine and were perfused via the heart with PBS- Heparin solution followed by perfusion with 4% formaldehyde. All animal experiments were performed in accordance with the guidelines of the institutional committee for the use of animals for research.

Adenoviral vectors and Sorafenib - Adenoviral vectors encoding green fluorescence protein (GFP) or GFP and sFLT (Soluble fms-like tyrosine kinase- 1) were prepared in GH354 cells using standard procedures. A titer of 109 transducing units was injected to mice tail veins. Sorafenib (Xingcheng Chempharm Co., Ltd Taizhou, China) was administered daily (50 mg/kg) by oral gavage. Cremophor EL/ethanol/water; (12.5: 12.5:75) was used as vehicle solution.

Immunohistochemistry (IHC) and ELISA - Antibodies used for IHC were vWF (DAKO Corp, Carpinteria, CA, USA, Catalogue number A0082), Cleaved Caspase 3 [Cell Signaling, USA, Catalogue number: 9661 (Rabbit polyclonal)], F4/80 (Serotec Raleigh, NC, USA; Catalogue number MCA497GA), HGF (R&D Systems Inc, Minneapolis, MN, USA; Catalogue number: AF2207), BrdU (NeoMarkers, Thermo Scientific, Fremont, USA, BrdU Ab-3, Catalogue number: MS-1058-P), Ki67 (NeoMarkers, Thermo Scientific, Fremont, USA, Catalogue number: RM-9106), pHH3 (phospho-Histone H2A.X, MILLIPORE, Catalogue number: 05-636). IHC was performed on 5 μιη paraffin sections. Antigen retrieval was performed in a Decloaking Chamber™ (Biocare Medical, Concord, CA, USA) in Citrate buffer for all antibodies except vWF and F4/80 for which retrieval was performed with Pronase (Sigma, St Louis, MO, USA, Catalogue number: P8038). VEGF-A ELISA was performed using Quantikine® mouse ELISA kit (R&D Systems Inc, Minneapolis, MN, USA). Secondary antibodies for all antibodies used were Histofme® (Nichirei Biosciences, Chuo-ku, Tokyo 104-8402 Japan), except for mouse derived antibodies that were detected with Envision™ (Corp, Carpenteria, CA, USA).

In-Situ hybridization - Probes for CISH analysis were prepared from the BAC clones RP24-215A3 for Chromosome 17 and RP23-174D11 for the pericentromeric region (BACPAC resources center). BAC clones were labeled with Digoxigenin (DIG) using Nick-Translation mix (Roche, Indianapolis, IN, USA, Catalogue No. 11745808910). Mouse Cot-1 DNA (Gibco-Invitrogen Corporation products, Grand Island, NY, USA) and sonicated murine genomic DNA were added to the probe for background block. Tissues were prepared by boiling in pretreatment buffer and digestion with Pepsin (Zymed® Catalog Number - 00-3009). Hybridization was performed overnight in 37°C after 5 minutes of denaturation in 95° C. The Spot-Light® detection kit (Invitrogen) was used for anti DIG antibody and secondary antibody.

qPCR and array-based Comparative Genomic Hybridization (aCGH): aCGH - Genomic DNA was isolated using the QIAGEN DNAeasy Tissue kit. Samples were hybridized to mouse CGH 60-mer oligonucleotides microarrays (Agilent Technologies, Santa Clara, CA, United States), washed and scanned according to Agilent Technologies instructions.

RNA qPCR - RNA was extracted from tissues by mechanical grinding in TriReagent® (Sigma, St Louis, MO, USA) with a Polytron tissue homogenizer (Kinematica, Bohemia, NY, USA). cDNA was prepared with MMLV reverse transcriptase (Invitrogen by Life Technologies). RNA qPCR analyses were carried out with SYBR® Green qPCR Detection (Invitrogen by Life Technologies) in 7900HT Fast Real-Time PCR System (Applied BioSystems). Results were analyzed using the qBase vl .3.5 software. Primer sequences are shown in Table 4 below. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) and PPIA [peptidylprolyl isomerase A (cyclophilin A)] together were used as reference genes in all analyses.

DNA Real-Time PCR (qPCR) Method - Genomic DNA was isolated using the QIAGEN DNAeasy Tissue kit. Primers sets were designed using the Oligo Design and Analysis Tools provided by Integrated DNA Technologies (IDT). qPCR analyses were carried out with SYBR green (Invitrogen) in 7900HT Fast Real-Time PCR System (Applied BioSystems), and the results were analyzed using the qBase vl .3.5 software. Primer sequences are shown in Table 4 below.

Table 4: Primers for quantitative PCR

SEQ SEQ

Gene Forward primer ID Reverse primer ID

NO: NO:

CCAAGCTGCATGTGGT CTGCACAGGTGTTGGTGAA

Clic5 1675 1676

CAAG CTC

CGACCTCTTCTCTCCAC TGTCTATCCGTCCCTCATC

Aars2 1677 1678

TGCTC TGC

ACTCGAGGCGCTCACA CAGGAAAGCCAAGCAACA

Nfkbie 1679 1680

TACATC GAAT

ACCGGTATGAGCGTCT GCTGTCAGCCAGGAACAG

Tmem63b 1681 1682

CACCTC AAG

CGGGAGATTCCCAGGC CTGGCTGAATGCTCTGCTG

Mrpll4 1683 1684

TGTTA AC

TGCTAAGGCTCCTCAA CTCGCACCAGCATGTTATG

Capnl 1 1685 1686

GAAAGC TGT

ATTGAAGGCCGAATCA ATAAGGCCGGATGAACTG

Mrpsl 8a 1687 1688

CAGAAA ACTG

ATTGAAGGCCGAATCA ATAAGGCCGGATGAACTG

Mrpsl 8a 1689 1690

CAGAAA ACTG

CCCACATGGTAACCCT AGACGCCAAACAGTGTGG

Gnmt 1691 1692

GGACTA GTAG

CCT CAC AAT CAC CCT CTG CTC ACA AGG TCC

EGF19 1693 1694

GCC AGC

CCAACCCACGAATGCA TAGTGAGTGGTGGCGGAC

Runx2 1695 1696

CTATC ATAC

Table 4. Provided are the primer sequences 5'— >3' (with sequence identifiers) which were used in the qPCR analysis. Unless indicated otherwise, the provided primers were used for qRNA-PCR. The primers from VEGFA-3'-UTR and VEGFA-promoter regions were used for DNA qPCR.

IHC quantification and statistics - Immunohistochemical stainings were quantitated when indicated using an Ariol SL-50 system (Applied Imaging, Grand Rapids, MI, USA). Data was analyzed using a paired two tails Student's T-Test at p < 0.05. Histological differences were analyzed using Pearson's χ² test at p < 0.05. Data was processed using Microsoft Excel 2007. Graphs were generated using either GraphPad Prism 5.0 or excel softwares.

EXAMPLE 1

CHARACTERIZATION OF A GENOMIC AMPLIFICATION WHICH INCLUDES

THE VEGF-A GENE

Experimental Results

Identification of a chromosomal gain in the genomic region encompassing the VEGF-A gene - In search of new candidate targets for tailored therapy, the present inventors have applied both array Comparative Genomic Hybridization (aCGH) and cDNA expression array to samples produced from ten HCCs obtained from 16 months old Mdr2^_/~ mice. The aCGH revealed several recurring genomic amplifications in the qB3 band of murine chromosome 17 (Chrl7qB3, data not shown) encoding among others the VEGF-A (GenelD: 7422), Mrpsl8a (human GenelD: 55168), Pare (CCL18, human GenelD: 6362) and Exportin 5 (XP05, human GenelD: 57510) (Figure 1).

The incidence of the genomic amplification on murine chromosome Chrl7qB3 is about 14% in Mdr2^'/' tumors - Out of the 93 successfully tested HCC tumors, 13 were found to bear this amplification (13.97%). To determine the rate of this amplification, the present inventors tested a larger cohort of Mdr2^_/~ tumors by quantitative PCR (qPCR) of tumor DNA (Figure 7) and chromogenic in situ hybridization (CISH, Figures 2A-H).

Mapping of the minimal genomic region on murine chromosome 17qB3 which is amplified in the HCC tumors - To map the minimally amplified region of this amplicon, DNA qPCR of several loci along the murine chromosome 17 was performed in a cohort of tumors bearing this amplification (Figure 21). The present inventors found that the common proximal border lies between 43.3 and 43.8 mega base pairs (mbp) from the chromosome start and the common distal border lies between 46.9 and 48.5 mbp. This region encodes 63 genes as listed in Table 5, hereinbelow.

Table 5

Genes found in the genomic amplification on murine chromosome 17 (17qB3)

Gene Start End Description

1 Clic5 44325521 444171 17 chloride intracellular channel 5

2 unx2 44740950 44951746 rant related transcription factor 2

3 4930564C03Rik 44325521 45505103 RIKEN cDNA 4930564C03 gene suppressor of Ty 3 homolog (S.

4 Supt3h 44914120 45256233

cerevisiae)

5 EG210562 44740950 45570656 predicted gene, EG210562

6 EG668314 44914120 45611887 predicted gene, EG668314

cell division cycle 5-like (S.

7 Cdc51 45528836 45570656

pombe)

spermatogenesis associated,

8 Spats 1 45585886 4561 1887

serine-rich 1

9 EG668319 45459310 45686626 predicted gene, EG668319

alanyl-tRNA synthetase 2,

10 Aars2 45643790 45657792

mitochondrial (putative)

Table 5. Provided is a list of 63 genes found in the genomic amplification on murine Chrl7qB3 (which is the syntenic region of chromosome 6p21 of the human) along with the full gene names and the location of their genomic sequences on murine chromosome 17. The start and end numbers relate to nucleotide position on mouse chromosome 17 according to accepted chromosomal nucleotide positions.

The genomic amplification matched increases in the mRNA levels of VEGF- A, Exportin 5 and Pare - These results were cross compared with the mRNA analysis looking for concomitant amplification and mRNA overexpression. The mRNA levels of several of these genes was analyzed in a set of Chrl7qB3 amplicon positive (herein denoted Amp^pos) and negative (Amp^pos) by qPCR. This analysis revealed amplification matched increases in the mRNA levels of several genes such as VEGF-A, Exportin 5, Pare, Aars2, Mrpll4, Tmem63b, Mrpsl 8a, and Tjapl [Figures 2J-0 and Figures 10A- K]. Some genes were not differentially elevated (e.g. Calpain 1 1 and IkBe) while others were elevated only in some of the Amp^pos tumors regardless of the copy number (e.g. HSP90abl and EGFL9). Thus, the amplification of the murine genomic region Chrl7qB3 is a recurrent event in HCC related with a unique expression signature of several of its residing genes. EXAMPLE 2

AMPLIFICATION OF VEGF-A CORRELATES WITH A SPECIFIC VEGF-A

RELATED TUMOR CHARACTERISTICS

Experimental Results

The present inventors checked whether the genomic amplification which includes the VEGF-A gene is necessary in order to elevate VEGF-A protein levels. A tight correlation was found between the changes in VEGF-A mR A and protein levels in Mdr2^_/~ derived tumors (Figure 3), suggesting that in these tumors, VEGF-A protein levels are dictated by mRNA change. Concordantly, the present inventors also found a 6-fold higher vessel density in Amp^pos compared to Amp^neg HCCs as measured by immunostaining for the endothelial marker vWF (Figure 4A-B and 4G).

The Amp^pos tumor group as a subgroup of HCCs characterized by specific histological changes and an increased rate of proliferation - Analysis of hematoxylin and eosin (H&E) stained sections of Amp^pos and Amp^neg HCCs revealed that the murine Amp^pos tumors are characterized by larger tumor cells, and extensive steatosis (Figures 11 A-E). BrdU immunostaining revealed that Amp^pos HCCs displayed a 2-fold increase in tumor cell proliferation (Figures 4C-D and 4H). Of note, proliferation is one of the strongest and most consistent markers of poor prognosis in human HCC (Ouchi, K., et al. Mitotic index is the best predictive factor for survival of patients with resected hepatocellular carcinoma. Dig Surg 17, 42-48, 2000). Cleaved Caspase 3 staining revealed no difference in the rate of apoptosis (data not shown). Altogether, these data signify the Amp^pos tumor group as a subgroup of HCCs characterized by specific histological changes and an increased rate of proliferation. EXAMPLE 3

AMPLIFICATION OF VEGF-A INDUCES A UNIQUE TUMOR MICROENVIRONMENT VIA A HEPATOCYTE-MACROPHAGE CROSSTALK Experimental Results

The Amp^pos HCC tumors have a distinct micro environment content with enhanced numbers of macrophages - VEGF-A was shown to exert its proangiogenic effects by attracting naive myeloid cells that in turn modulate the microenvironment (Grunewald, M., et al. VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells. Cell 124, 175-189, 2006). Immunostaining for the macrophage marker F4/80 showed a 4-fold higher macrophage content in Amp^pos compared with Amp^neg HCCs (Figures 4E-F and 41) while no differences in the neutrophil content between tumor groups were detected (data not shown). Furthermore, levels of several known markers of tumor associated macrophages including Arginase 1, TGF And Yml were elevated in the Amp^pos group while markers of classically activated macrophages, including TNFa, iNOS and CXCL10 were not changed (data not shown). Other markers of these macrophage subsets such as TNFa, INOS, CXCL10, Arginase 1, TGF and Yml did not show any significant differences (data not shown). Thus, among the Mdr2^_/~ HCCs tumors, those harboring the genomic amplification (Amp^pos) have a distinct microenvironment content with enhanced numbers of both macrophages and endothelial cells.

The AmpP™ HCC tumors express higher levels of hepatocyte growth factor (HGF) as compared to Amp^neg HCC tumors - The present inventors have detected a nearly 3 -fold elevation of HGF mRNA levels in Amp^pos versus Amp^neg HCCs (Figures 5N and 6A). Furthermore, immunostaining for HGF showed HGF expression in the Amp^pos tumors alone (Figures 6B-C). Interestingly, HGF staining in these tumors was restricted to non-neoplatsic intratumoral cells (Figure 6C).

EXAMPLE 4

INHIBITION OF VEGF-A ATTENUATES PROLIFERATION SELECTIVELY IN

AMP^POS TUMORS

Experimental Results

The VEGF-A inhibitor, soluble FLT receptor (sFLT), inhibits proliferation of hepatocytes in Amp^pos HCC tumors - To test the functional role of VEGF in Amp^pos and Amp^neg tumors, the present inventors injected adenoviral vectors encoding GFP alone or GFP and a soluble VEGF-A receptor 1 (sFLT), a well known potent inhibitor of VEGF-A (Mahasreshti, P. J., et al. 2001. Adenovirus-mediated soluble FLT-1 gene therapy for ovarian carcinoma. Clin Cancer Res 7, 2057-2066). Mice were sacrificed at ten days post injection and tumors were collected and examined. VEGF-A amplification status was determined after sacrifice by both DNA qPCR (data not shown) and VEGF- A mRNA expression (Figure 5M). The efficiency of adenovirus transduction was assessed by immunostaining for GFP (data not shown). Immunostaining for markers of proliferation such as Ki67, BrdU and phospho-histone 3 (pHH3) revealed that blocking VEGF markedly inhibited tumor cell proliferation in Amp^pos tumors, yet did not affect proliferation in Amp^neg tumors (Figures 5A-L). The decrease in proliferation was accompanied with a nearly 3 fold reduction in HGF m NA levels in sFLT treated tumors (Figure 5N). Notably, HGF levels in Amp^neg tumors were unaffected by VEGF blockade (Figures 5N), suggesting that the increase in HGF levels in the Amp^pos tumors can be attributed to VEGF-A, expression of which is driven by the amplicon. This effect was irrelevant of tumor size as the distribution of tumor diameter was similar between all four groups (data not shown). Treatment with adenovirus encoding GFP alone did not induce any change in either of the groups. Other Amp^pos associated traits like macrophage infiltration and steatosis were not affected significantly by VEGF-A inhibition (FIGs 12A-J and data not shown).

The anti-VEGF-A treatment resulted in a specific increase in the HIFla target genes Glutl and PGK1 - Macroscopic inspection and histological analysis of H&E stained sections revealed multiple foci of coagulative necrosis in three out of the six sFLT treated Amp^pos tumors (Figures 5Q-R). In these three specific tumors, the present inventors also found an elevation of the HIFla target genes Glutl and PGK1 (Figures 50-P), indicative of tissue hypoxia. No evidence of necrosis was detected in any of the other treatment groups (data not shown). Immunostaining for vWF revealed a trend (yet, statistically insignificant) towards decrease in vasculature only in the VEGF- A blocked Amp^pos tumors, mainly in the hypoxic tumors (Figures 8A-D, Figure 12A-D, 121), possibly explaining the induction of hypoxia in these tumors. Treatment with GFP alone did not induce any change in either of the groups. In addition, macrophage infiltration was not affected after 10 days of VEGF-A inhibition (Figure 12 E-H, 12J). Thus, 10 days inhibition of VEGF-A does not alter significantly the microenvironmental content. EXAMPLE 5

PROFILING OF PROANGIOGENIC FACTORS EXPRESSION IN MDR2^'1' HCC Experimental Results

In addition to VEGF-A, Angiopoietin 1 & 2, FGF 1 & 2, PDGF A, B & C, PLGF and VEGF-B are known to regulate angiogenesis. Analysis of their mRNA levels revealed that several of these factors are overexpressed in HCCs compared with normal livers, irrespective of amplicon status (data not shown). Notably, PDGF-C levels are significantly higher (2.4 fold) in Amp^neg compared with Amp^pos tumors (data not shown). This stands in line with previous reports showing that PDGF-C can promote angiogenesis in a VEGF-A independent manner (Crawford, Y., et al. PDGF-C mediates the angiogenic and tumorigenic properties of fibroblasts associated with tumors refractory to anti-VEGF treatment. Cancer Cell 15, 21-34, 2009) and could explain the lack of decrease in vessel density in the Amp^neg tumors in response to VEGF-A inhibition. Furthermore, the levels of Angiopoeitin 2, a known inhibitor of vessel maturation along with VEGF-A, were higher in the Amp^pos tumors accounting for the decrease in Amp^pos tumors vascularity upon treatment (data not shown).

EXAMPLE 6

AMP^POS TUMORS ARE UNIQUELY SENSITIVE TO SORAFENIB

Experimental Results

The present inventors have identified a sub-group of liver tumors that bears a specific genomic amplification and is distinguished by several traits from other tumors. This tumor sub population is extremely sensitive to treatment with an anti VEGF-A agent (sFLT). As this amplification was previously found in aCGH analyses performed on human HCCs, the present inventors suggest that detection of this amplification by either in situ hybridization or qPCR in biopsies taken from HCC patients could serve as a prognostic marker that would predict the tumor responsiveness to anti- VEGF-A treatments such as Bevacizumab, soluble VEGF-receptor or other antibody driven or pharmacological inhibitors of this pathway.

The multi-tyrosine kinase receptor inhibitor Sorafenib is the current preferred treatment for patients with unresectable HCC (Llovet, J.M., et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359, 378-390, 2008). Sorafenib inhibits the VEGF receptors and B-Raf, a downstream effector of both the VEGF receptors and c- Met (Wilhelm, S.M., et al. Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol Cancer Ther 7, 3129-3140, 2008). The present inventors tested whether Sorafenib could have a selective advantage towards Amp^pos tumors, as follows.

Mdr2^_/~ mice (age 1+ year) were treated with Sorafenib or vehicle alone for three days, after which they were sacrificed and tumor tissue was analyzed. Amplification status was assessed after sacrifice by DNA qPCR and VEGF-A mRNA expression (Figure 9 J and data not shown). Immunostaining for BrdU demonstrated decreased proliferation in the Amp^pos tumor group only (Figures 9A-D, Figure 9N). Similarly to VEGF-A inhibition, HGF levels were decreased only in the treated Amp^pos tumors (Figure 9K). At the same time, the non-amplified group was not different from the vehicle treated control in either proliferation (Figure 9N) or HGF expression (Figure 9K). No decrease in blood vessel density (Figures 9E-H, Figure 91) or signs of hypoxia (Figures 9L-M) were observed, possibly due to the short duration of treatment (only 3 days), implying that the effect of VEGF-A amplification on proliferation could be independent of angiogenesis.

These data suggest that the VEGF-A amplification distinguishes a subgroup of Mdr2^_/~ HCCs that are sensitive to direct VEGF-A blocking, as well as to Sorafenib treatment.

Analysis and Discussion

Using a mouse model of inflammation-induced HCC, the present inventors identified and characterized a specific subtype of HCCs. These tumors are defined by an amplification of a genomic region encompassing the VEGF-A gene. The present inventors show that tumors bearing this amplification (Amp^pos) differ from Amp^neg tumors in several aspects: histological appearance, rate of proliferation and microenvironmental interactions. A cytokine-based crosstalk between malignant Amp^pos hepatocytes and tumor associated macrophages was delineated. Most importantly, the present inventors show that Amp^pos tumors are uniquely sensitive to VEGF-A inhibition, suggesting that VEGF-A copy number could serve as a predictive biomarker for response to VEGF blocking agents in human HCC. The amplicon spanning the VEGF-A locus was previously reported in human HCC, lung and colorectal cancers. Cumulative analysis of human HCC [Chiang, D.Y., et al. Cancer Res 68, 6779-6788 (2008); Chochi, Y., et al. A copy number gain of the 6p arm is linked with advanced hepatocellular carcinoma: an array-based comparative genomic hybridization study. J Pathol 217, 677-684 (2009); Moinzadeh, P., et al, Chromosome alterations in human hepatocellular carcinomas correlate with aetiology and histological grade—results of an explorative CGH meta-analysis. Br J Cancer 92, 935-941 (2005); Patil, M.A., et al. Array-based comparative genomic hybridization reveals recurrent chromosomal aberrations and Jabl as a potential target for 8q gain in hepatocellular carcinoma. Carcinogenesis 26, 2050-2057 (2005); Weir, B.A., et al. Characterizing the cancer genome in lung adenocarcinoma. Nature 450, 893-898 (2007); Tsafrir, D., et al. Relationship of gene expression and chromosomal abnormalities in colorectal cancer. Cancer Res 66, 2129-2137 (2006)] shows that the VEGF-A specific region (VEGF-A amplicon) is amplified in between 7-24% of human HCCs and the whole chromosome arm is amplified in nearly 30% of human HCCs. In the Mdr2^_/~ mouse model, the present inventors found the VEGF-A amplicon in 14% of HCCs, irrespective of tumor size, suggesting that this could be an early event.

Inhibition of VEGF-A was already tested in human HCC. Treatment with Bevacizumab alone or in combination with Erlotinib was tested and yielded positive results in clinical experiments (Siegel, A.B., et al. 2008, J. Clin. Oncol. 26, 2992-2998; Thomas, M.B., et al. 2009, J. Clin. Oncol. 27, 843-850).

Importantly however, to date, there are no clinically reliable biomarkers for personalizing HCC treatment, identifying the most suitable patients and reducing the overall treatment morbidity. The findings described herein suggest that response to anti VEGF-A treatment or Sorafenib can be predicted by quantifying VEGF-A copy number in the tumor tissue from HCC patients. If this is the case, it could lead to substantial improvement in treatment selection for this highly aggressive tumor. Notably, the same amplification was found also in lung and colorectal cancers (Weir, B.A., et al. 2007, Nature 450, 893-898; Tsafrir, D., et al. 2006, Cancer Res. 66, 2129-2137), suggesting that similar treatment guidelines could be applied to all VEGF-A Amp^pos tumors. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

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