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WO2011107939A1 - Méthodes de prédiction de l'efficacité d'un traitement par anti-vegfa pour des tumeurs solides - Google Patents

Méthodes de prédiction de l'efficacité d'un traitement par anti-vegfa pour des tumeurs solides Download PDF

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WO2011107939A1
WO2011107939A1 PCT/IB2011/050868 IB2011050868W WO2011107939A1 WO 2011107939 A1 WO2011107939 A1 WO 2011107939A1 IB 2011050868 W IB2011050868 W IB 2011050868W WO 2011107939 A1 WO2011107939 A1 WO 2011107939A1
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vegfa
treatment
solid tumor
vegf
seq
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Yinon Ben-Neriah
Eli Pikarsky
Ilan Stein
Peter Angel
Julia Nemeth
Jochen Hess
Jorn Hendrik Reuter
Elad Horwitz
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Deutsches Krebsforschungszentrum DKFZ
Hadasit Medical Research Services and Development Co
Yissum Research Development Co of Hebrew University of Jerusalem
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Deutsches Krebsforschungszentrum DKFZ
Hadasit Medical Research Services and Development Co
Yissum Research Development Co of Hebrew University of Jerusalem
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    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • 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).

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Abstract

On divulgue des méthodes de prédiction de l'efficacité d'un traitement par un facteur de croissance endothélial anti-vasculaire A (VEGF-A) chez un sujet diagnostiqué comme atteint d'une tumeur solide telle qu'un carcinome, par exemple un carcinome hépatocellulaire, en déterminant la présence ou l'absence d'une amplification génomique comprenant un gène du VEGF-A dans un échantillon de la tumeur solide, où la présence ou l'absence de l'amplification génomique prédit l'efficacité du traitement par anti-VEGFA chez le sujet diagnostiqué comme atteint d'une tumeur solide. On divulgue également des méthodes de traitement d'un sujet diagnostiqué comme atteint d'une tumeur solide par la prédiction de l'efficacité du traitement par anti-VEGFA selon la méthode de l'invention et le choix d'un régime de traitement basé sur la prédiction.
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