TW201309806A - Single nucleotide polymorphisms in the promoter of VEGFA gene and their use as predictive markers for anti-VEGF treatments - Google Patents

Abstract

The invention relates to the treatment and the diagnosis of a group of patients bearing specific alleles of single nucleotide polymorphisms in the promoter region of the VEGFA gene. These patients are more responsive to Aflibercept and more likely to be efficiently treated by anti-VEGF therapy.

Description

Single nucleotide polymorphism in the VEGFA gene promoter and its use as a predictive marker for anti-VEGF therapy

The present invention relates to anti-angiogenic therapy, and more particularly to a patient population carrying a specific nucleotide polymorphism (SNP) specific dual gene in the VEGFA gene promoter sequence, which has been found to be resistant to -VEGF The treatment response is stronger.

Anti-angiogenic therapy has improved the clinical outcome of selected tumors by inhibiting tumor growth by limiting neovascularization. As the tumor grows, the lack of sufficient blood supply causes an oxygen-deficient environment, stimulating the release of factors such as hypoxia inducible factor (HIF)-1α, which activates vascular endothelial growth factor (VEGF). Transcription-induced angiogenesis [Ferrara, N. et al. (2002) VEGF and the quest for tumor angiogenesis factors. Nat. Rev. Cancer . 2: 795-803].

VEGFA and its receptors are often overexpressed in human tumors including breast cancer, non-small cell lung cancer, colorectal cancer, and prostate cancer. More specifically, VEGFA is associated with a poor prognosis in breast cancer. Numerous studies have shown that the overall survival and disease-free survival of these tumors overexpressing VEGFA are reduced. Although the exact mechanism of VEGFA's role in tumor progression remains unclear, the performance of VEGFA may play an important role in tumor angiogenesis.

The broad term "VEGFA" encompasses a number of proteins from two families that result from alternative splicing of mRNA from a single, 8-exon VEGFA gene. The VEGFA gene is located on chromosome 6 at position 6p21.1. Its coding region spans approximately 14 kilobases and consists of eight exons [Tischer, E. et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exeon splicing (1991) J. Biol. Chem. 266: 11947-954]. Many SNPs are present in the 5' and 3' untranslated regions (UTR) of the promoter of VEGFA .

VEGFA and related signaling pathways have become targets for many novel anticancer drugs. In particular, optimized ligands for VEGFA that inhibit or reduce the biological activity of VEGFA are currently being developed, and such ligands are referred to as anti-VEGF proteins. Such anti-VEGF proteins may be derived from natural or artificial proteins that exhibit certain VEGFA affinities, such as anti-VEGFA antibodies or VEGFA receptors. An example of such anti-VEGF proteins that have been available to date for commercialization or clinical trials is Bevacizumab (Avastin) [Ferrara N. et al. (2004) Discovery and development Of Bevacizumab, an anti-VEGF antibody for treating cancer, Nature Reviews 3:391-400], Ranibizumab (Lucentis) and Abcecept (VEGF-trap) [Holash, J. (2002) VEGF-Trap: A VEGF blocker with potent antitumor effects, PNAS , 99(17): 11393-98].

Abecept is a VEGFA polypeptide ligand comprising an anti-angiogenic activity of the human vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2) extracellular domain fused to the constant region (Fc) of human IgG1 (extracellular domain) Section of ). This protein acts as a soluble decoy receptor. It binds to pro-angiogenic vascular endothelial growth factor (VEGFA), thereby preventing it VEGFA binds to its cellular receptors. Destruction of VEGFA binding to its cellular receptor results in inhibition of tumor angiogenesis, metastasis and ultimately tumor regression.

Anti-VEGF molecules have also been found to be useful in the treatment of macular degeneration as the effect of counteracting VEGFA can provide significant therapeutic effects in patients suffering from the disease. Age-related Macular Degeneration (AMD) is the leading cause of acquired blindness. In AMD, new blood vessels grow below the retina and leak blood and fluid. This leakage causes destruction and dysfunction of the retina, causing blind spots in the central field of view, and it can explain blindness in such patients.

Anti-VEGF molecules have shown promising effects in preclinical and clinical studies [Rakesh KJ et al. (2006) Lessons from Phase III clinical trials on anti-VEGF therapy for cancer, Nature Clinical Practice Oncology , 3:24-40] . However, it is often expensive to manufacture and some patients experience drug resistance, limited activity, and severe toxicity. Therefore, there is a strong need to identify markers that enable a priori selection of patients who may benefit from such agents.

Several studies have examined the role of SNPs in the VEGFA gene as predictive and prognostic markers for major solid tumors. However, such studies have led to conflicting reporting [Jain L. et al. (2009) Mol. Cancer. Ther. 8(9): 2496-2508]. In addition to recognizing that -1154G>A SNP is associated with a significant reduction in the risk of prostate cancer, it has not been determined that other VEGFA SNPs are significantly associated with a lower or higher incidence of cancer.

Another study by Schneider et al . [ J. Clin. Oncol. (2008) 26: 4672-4678] found that in patients with metastatic breast cancer treated with bevacizumab, compared with patients carrying the -2578C dual gene, The VEGFA-2578AA genotype was associated with overall survival in the upper middle.

Therefore, it has been difficult to predict whether patients with cancer or macular degeneration may respond to anti-VEGF therapy.

The studies disclosed herein unexpectedly show that patients carrying certain specific mutations in the VEGFA gene promoter sequence are more potent than other patients in anti-VEGF protein-based therapy, particularly aboxicept. These mutations corresponding to polymorphism (SNP) naturally exist in the human world population corresponding to the dual genes called -2578C, -2549del, -1498T and -1190G.

These patients appear to form a stronger group of anti-VEGF therapeutic responses and may therefore have better therapeutic outcomes.

Based on these findings, the present invention provides a diagnostic method consisting of genetic testing that can be performed on a blood sample to determine which patients carry the SNPs and should be treated with anti-VEGF molecules.

The present invention also proposes anti-VEGF treatment primarily for patients carrying at least one of the dual genes -2578C, -2549del, -1498T and -1190G.

The present invention therefore targets a general diagnostic method for determining whether a patient is likely to have a positive response to anti-VEGF therapy, wherein the method comprises detecting a promoter sequence encoding a VEGFA gene isolated from the patient (such as SEQ ID NO. a step of the presence of at least one nucleotide polymorphism. Compared with the wild type sequence SEQ ID NO. 1, the nucleotide polymorphism can be Should be a single mutation (SNP), sequence deletion (del), insertion (ins), repeat. Such polymorphisms can be detected by any standard method known in the art.

More specifically, the diagnostic method of the present invention proposes detecting at least one of polymorphisms -2578C, -2549del, -1498T and -1190G in the promoter polynucleotide sequence encoding the VEGFA gene.

The number of polymorphisms corresponds to the position of the first nucleotide of the mutation or deletion relative to the translation initiation codon of the VEGFA gene. It is preceded by a minus sign indicating that it is located before the first nucleotide. The letters A, T, C, and G correspond to the nitrogen-containing adenine, thymine, cytosine, and guanine, respectively. With regard to -2549del, the abbreviation "del" means that one or several nucleotide bases of position -2549 originally present before the "ATG" site of transcription are deleted. In one embodiment, as indicated by the bold in Figure 1, the deletion corresponds to one or several bases from positions 1435 to 1452 of SEQ ID NO.

SEQ ID NO. 1 represents the major portion of the VEGFA gene promoter sequence. The complete VEGFA gene sequence was obtained from the human genome database (Glycosome contig GRCh37.p2 (see NCBI: NT_007592)). In the context of the present invention, this sequence is considered to be a reference to the VEGFA promoter or a wild-type polynucleotide sequence.

SEQ ID NO. 2 represents the promoter sequence of VEGFA gene (identical region of SEQ ID NO. 1) as the dual gene sequence of the present invention, which includes all four polymorphic -2578C, -2549del, -1498T and -1,190G .

When the polymorphisms are detected independently of each other, the corresponding dual genes detected are simply referred to as -2578C, -2549del, -1498T, and -1190G.

Table 1 shows that the symbols for the dual gene mutations used in this patent application are consistent with other identical tokens that take into account the base position of the transcription initiation site of VEGFA . Changes induced by polymorphism were also indicated as compared to the reference numbers for each SNP in SEQ ID NO. 1 and the NCBI database (www.ncbi.nlm.nih.gov/snp).

To carry out the diagnostic method of the invention as an in vitro method, the polynucleotide DNA sequence is isolated from the patient primarily by conventional procedures. It can be separated from the blood sample.

The patient to be diagnosed is usually a patient who is primarily diagnosed with cancer, macular degeneration or any other disease that is sensitive to treatment with anti-VEGF therapy. Breast cancer, non-small cell lung cancer, colorectal cancer, and prostate cancer are known to be more sensitive to treatment by anti-VEGF therapy to date.

According to the present invention, anti-VEGF therapy can be carried out using an anti-VEGF protein which is a protein derived from a VEGF receptor or an antibody against a VEGF protein, more specifically an anti-VEGFA protein.

It should be noted that according to the present invention, the detectable dual genes -2578C, -2549del, -1498T and -1190G are usually found simultaneously in the genome of the same patient, Because the dual genes are statistically related (non-equilibrium dual gene distribution). Thus, as shown, for example, in SEQ ID NO. 2, when one of the dual genes is found in the genome of a patient, other dual genes may also be present in the patient's VEGFA promoter sequence.

Notably, the above SNPs have an embryonic origin and are therefore generally not produced by mutations that occur during the tumor transformation process. The inventors of the present invention have demonstrated that among 99% of the tumors analyzed, the dual gene carrying the SNP is present in non-tumor cells surrounding the tumor cells. Since these SNPs are derived from embryonic origin, the diagnostic methods of the present invention can be performed directly from blood samples rather than from tumor samples, which constitutes a much less invasive diagnostic method.

It has also been found that patients carrying one of the dual genes -2578C, -2549del, -1498T and -1190G in the VEGFA gene promoter sequence constitute a patient population that is more responsive to anti-VEGF therapy.

The detection of the dual gene -2578C gave particularly significant results.

According to an embodiment of the present invention, all four of the above mentioned dual genes can be searched for in the genome of a patient as SNP tags. The detection of two, three or four of these dual genes allows us to obtain a more reliable diagnosis than an individual dual gene. For example, the inventors of the present invention have observed that in non-small cell lung cancer patients, the presence of dual genes -2578C, -2549del, and 1498T is significantly associated with a stable stability of the disease for more than 120 days after anti-VEGF treatment.

The present invention can be carried out using specific probes or primers designed to detect the aforementioned dual genes in the patient's gene. Amplification techniques are well known in the art and can be applied to biological samples from patients, especially from patients. Crude DNA preparation. For example, PCR can be performed using a primer that distinguishes between the sequence of the dual gene as SEQ ID NO. 2 and the VEGFA promoter reference sequence of SEQ ID NO. 1 in the absence of any of the dual genes.

The probe or primer of the present invention may be a polynucleotide sequence comprising the following polynucleotide sequence: SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO.

According to an embodiment of the invention, detection is performed by PCR using the following PCR sequences: - SEQ ID NO. 3 (forward primer): CTGGGGAGCCAAGTGG; - SEQ ID NO. 4 (reverse primer): CCAGCACTAAGGAACGTCTGTAGGC.

The above sequences are particularly useful for amplification and/or sequencing of the dual gene-2578C and -2549del sequences.

- SEQ ID NO. 5 (forward primer): GCGAGCGTCTTCGAGAG; - SEQ ID NO. 6 (reverse primer): CCGGTCCACCTAACCGCTGC.

The above sequences are particularly useful for amplification and/or sequencing of the dual gene-1498T and -1190G sequences.

Another object of the invention is a method of treating a human subject suffering from or at risk of developing an angiogenic disease or condition, wherein the method comprises the steps of: (a) detecting a gene encoding a VEGFA in a nucleic acid sample from an individual The presence of at least one single nucleotide polymorphism (SNP) in the promoter sequence (SEQ ID NO. 1); and (b) if the SNP is present in the individual, the patient is administered the anti- VEGF therapy.

Furthermore, the invention relates to a method for selecting a therapy for a human subject suffering from or at risk of an angiogenic disease or condition, wherein the method comprises the steps of: (a) detecting a nucleic acid sample from the individual The presence of at least one single nucleotide polymorphism (SNP) in the promoter sequence encoding VEGFA (SEQ ID NO. 1); and (b) if the SNP is present in the individual, the anti-VEGF is selected for the patient therapy.

In view of detecting a patient who is more responsive to anti-VEGF therapy, another object of the present invention is a diagnostic kit that allows us to perform a diagnostic method as previously described.

The diagnostic kit can comprise a set of primers, at least one of which comprises one of the sequences SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO.

The kit may additionally comprise an enzyme having polymerase activity, a restriction enzyme or a labeling reagent that facilitates detection of the presence of the above polymorphism in the genetic DNA of the patient.

The above methods and kits are intended to detect patients carrying the above polymorphic or dual genes in the genome.

According to the experimental data collected by the inventors of the present invention, a patient carrying at least one of the above-mentioned dual genes forms a patient population different from the general population, and the patient population is particularly responsive to anti-VEGF therapy. Therefore, the health status of such patients may be improved by anti-VEGF therapy.

Thus, the diagnostic methods of the invention are particularly intended to select patients who can benefit from anti-VEGF therapy for the treatment of cancer, more particularly breast cancer, non-small cell lung cancer, colorectal cancer or prostate cancer. It is also intended to select patients who can benefit from anti-VEGF therapy for the treatment of macular degeneration.

Anti-VEGF therapy means any therapeutic treatment that targets the VEGF protein to reduce or inhibit its biological activity.

When anti-VEGF therapy improves the health of a patient receiving treatment for the disease, the patient is considered to be responsive to the treatment.

For example, in the case of cancer, it may include reducing tumor growth. In terms of macular degeneration, it stabilizes retinal tissue.

In the context of the present invention, reactivity was observed after treatment of this patient population with aboxicept, a VEGF-trapping molecule belonging to a class of molecules that act as anti-VEGFA ligands, ie, inhibition of binding to VEGFA. Agent molecule. Therefore, the newly identified patient population is likely to also be derived from other anti-VEGFA ligands, more specifically from anti-VEGFA antibodies or receptor protein ligands (such as bevacizumab (Avastin) and blue Nimone (Le Mingqing) responded.

Without being bound by theory, the inventors of the present invention have hypothesized that mutations in the VEGFA promoter may reduce the expression of VEGFA, thus increasing the ratio of VEGFA/anti-VEGFA molecules, which will make anti-VEGF treatment even more effective. However, more experimental work will be required to fully determine the potential precise mechanism of SNP effects on anti-VEGF treatment.

In this context, the invention also provides a method of treating a patient comprising at least one dual gene comprising -5778C, -2549del, -1498T or -1190G carrying a VEGFA gene promoter sequence using an anti-VEGF ligand according to the method of treatment Cancer in the group, especially breast cancer and lung cancer treatment. The ligand can be an anti-VEGFA protein. In one embodiment is Abspir.

The patient population is defined as a diagnostic method according to the present invention in which the above-mentioned polymorphic or dual gene patients can be detected. These patients respond better to anti-VEGF therapy than the general population.

Similarly, an anti-VEGF ligand as defined above and more particularly ranibizumab (Lemingqing) is also suitable for treating at least -2578C, -2549del, -1498T or -1190G carrying the VEGFA gene promoter sequence. Macular degeneration in a patient population of dual genes.

The following examples are provided in a non-limiting manner to illustrate the invention.

Instance Materials and methods

Crude DNA was extracted from whole blood of patients treated with aboxicept during clinical analysis at Sanofi-Aventis to assess the efficacy of this product against different cancers (lung cancer, ovarian cancer, and breast cancer). This DNA extraction was carried out by Sambrook, J. using a general method [Molecular Cloning (Third Edition) (2001) Cold Spring Harbour Laboratory Press, N.Y.].

The following SNP-2578C/A was sequenced by Sanger et al. by classical methods:

‧ 92 Caucasian individuals from the Coriell Institute to determine the frequency of these SNPs in the general population of healthy patients.

‧ 131 DNA tumor extracts (lung, ovary, breast) and 131 DNA samples extracted from the same tumor edge to confirm gene variants The origin of the embryo.

• 31 samples from pre-clinical patients treated with aboxicept to determine the possible association between these SNPs and albeccept activity.

‧ 47 DNA extracted from blood from patients with lung cancer.

‧ From 145 DNA extracted from blood from ovarian cancer patients, the latter two analyses were designed to verify the association between SNP and increased clinical activity of aboxicept. Different VEGFA polytypes -2578C/A, -2549del/ins, -1498T/C and -1190G/A are described according to the reference rs699947, rs34357231, rs833061 and rs13207351 (see Table 1) in the NCBI database dbSNP. Figure 1 illustrates the dual gene including -2578C, -2549del, -1498T, and -1190G (SEQ ID NO. 2).

The following primers were used for PCR and sequencing: amplification and sequencing by PCR for the dual genes -2578C and -2549del: - SEQ ID NO. 3 (forward primer): CTGGGGAGCCAAGTGG; - SEQ ID NO. 4 (reverse) Primer): CCAGCACTAAGGAACGTCTGTAGGC.

For amplification of the dual genes -1498T and -1190G by PCR and sequencing: - SEQ ID NO. 5 (forward primer): GCGAGCGTCTTCGAGAG; - SEQ ID NO. 6 (reverse primer): CCGGTCCACCTAACCGCTGC.

Genomic DNA (2 μL, concentration 10 ng/μL) was used as a template for PCR amplification. Add 1 μL of 10 μM forward primer to 1 μL of 10 μM reverse primer and Promega 2× mixture (12 μL; Promega, Madison, Wisconsin, USA) to give a final reaction volume of 20 μL. The PCR conditions were as follows: maintained at 95 ° C for 3 min, followed by 2 cycles at 95 ° C for 30 sec and 70 ° C for 30 sec, 2 cycles at 95 ° C for 30 sec and 65 ° C for 30 sec, 2 cycles 95 The temperature was maintained at 30 ° C for 30 sec and 60 ° C for 30 sec, 40 cycles at 95 ° C for 30 sec, 56 ° C for 30 sec and 72 ° C for 30 sec, and 72 ° C for 10 min to terminate.

The resulting purified PCR product (2 μL) was used as a template for the sequencing reaction. 1 μL of 10 μM primer was added to the large dye terminator sequencing mixture (1.5 μL; Applied Biosystems, Foster City, USA) to give a final reaction volume of 10 μL. The cycle conditions were as described by the manufacturer. The sequencing reaction was purified on a G50 Sephadex column (Amersham). The reaction on ABI 3730 was analyzed as recommended by the manufacturer. Combination and SNP recognition using PHRED, PHRAP and POLYPHRED software.

result:

1. The C-pair gene of SNP-2578C/A tends to be associated with preclinical responses to aboxicept.

2. The frequency of -2578C/A in the population considered to be composed of healthy patients was found to be approximately 51% for the C-pair gene (corresponding to SEQ ID NO. 2) and 49% for the A-pair gene (corresponding to SEQ ID NO. 1) .

3. SNP-2578C was recognized in both tumor and marginal tissues, demonstrating that the dual gene is present in blast cells and is not due to tumor transformation. This is the case in 37 of 38 breast tumors, 46 ovarian tumors, and 47 lung tumors.

4. As shown in Table 2, it was found that there was a significant correlation between -2578C and positive response to albsicept in terms of lung cancer. In these patients, -2578C was completely associated with the other dual genes -2549del and -1498T (SNP-carrying patients compared to non-carrying patients (χ ² ): p=0.028; uniformity test for dual gene distribution (χ ² ): p=0.044).

discuss:

Significant results were obtained when testing the correlation of SNP marker -2578C with the response to aboxicept treatment. Responsive patients were those who carried one or two C-pair genes at the -2578 position, while patients with weaker response to abecept were those who carried two A-pair genes at this position.

Detecting the presence of such C-pair genes in patients can improve resistance by treating only those patients who are truly responsive to the treatment and by avoiding the toxic effects of other patients being unnecessarily exposed to the anti-VEGF therapeutic - Benefit/risk balance for VEGF treatment.

The detection of -2578C appears to be sufficient to distinguish between patients who responded to anti-VEGF therapy. However, it can be combined with several other SNP markers that are strongly associated with the -2578C dual gene, such as -2549del and -1498T, and -1190G. Thus, this correlation forms a SNP tag that helps to more easily identify patients who may benefit from anti-VEGF therapy. This label should apply not only to Aberdeen Xipu, and is applicable to any VEGF inhibitor, especially a VEGFA ligand, such as avastin (a protein derived from the anti-VEGFA antibody bevacizumab).

Furthermore, since the -2578C dual gene has an embryonic origin, it can be easily detected in blood tests by using standard genetic tests.

Figure 1: shows the VEGFA gene promoter sequence SEQ ID NO. 2, showing the corresponding polymorphisms -2578C, -2549del, -1498T and -1190G in bold.

<110> French company Sanofi

<120> Single nucleotide polymorphism in the VEGFA gene promoter and its use as a predictive marker for anti-VEGF therapy

<130> FR2011-011

<140> 101106075

<141> 2012-02-23

<150> 11305188.2

<151> 2011-02-23

<160> 6

<170> PatentIn version 3.3

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<211> 2961

<212> DNA

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<223> Promoter region of VEGFA gene (wild-type dual gene)

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<213> Homo sapiens

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<221> Promoter

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<223> VEGFA gene promoter, including polymorphic -2578C (position 1406), -2549del (position 1434), -1498T (position 2485) and -1190G (position 2793)

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<223> Inverse PCR primers were used to amplify the VEGFA promoter region and detect the -2578C and -2549del dual genes.

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<223> Reverse PCR primers were used to amplify the VEGFA promoter region and detect the -1498T and -1190G dual genes.

<400> 6

Claims (21)

A diagnostic method for determining whether a patient is likely to have a positive response to anti-VEGF therapy, wherein the method comprises detecting at least one nucleotide of a VEGFA-derived gene promoter sequence (SEQ ID NO. 1) isolated from the patient The step of the existence of polymorphism. The diagnostic method of claim 1, wherein the promoter sequence is isolated from the blood of the patient. The diagnostic method of claim 1, wherein the polymorphism corresponds to a allele selected from -2578C, -2549del, -1498T or -1190G. The diagnostic method of claim 2, wherein the dual gene -2578C is detected. The diagnostic method of claim 2 or 3, wherein all of the dual genes -2578C, -2549del, -1498T or -1190G are detected. The diagnostic method of any one of claims 1 to 4, wherein the method selects a patient who can benefit from anti-VEGF therapy for treating cancer. The diagnostic method of claim 6, wherein the cancer to be treated is breast cancer, non-small cell lung cancer, colorectal cancer or prostate cancer. The diagnostic method of claim 7, wherein the cancer is breast cancer. The diagnostic method of any one of claims 1 to 4, wherein the method selects a patient who can benefit from anti-VEGF therapy for treating macular degeneration. The diagnostic method according to any one of claims 1 to 4, wherein the nucleotide polymorphism or the dual gene is detected by hybridization of at least one primer comprising SEQ ID NO. 3, SEQ ID NO .4, SEQ ID NO. 5 or SEQ ID NO. 6. The diagnostic method according to any one of claims 1 to 4, wherein the anti-VEGF therapy The system is operated using an anti-VEGFA protein ligand protein such as Aflibercept, Bevacizumab or Ranibizumab. The diagnostic method of any one of claims 1 to 4, wherein the anti-VEGF therapy is performed using an abesicept molecule. A diagnostic kit for determining whether a patient is likely to respond positively to anti-VEGF therapy, comprising at least one primer that hybridizes to SEQ ID NO. 1 under standard hybridization conditions. The diagnostic kit of claim 13, wherein the sequence of the primer that hybridizes to SEQ ID NO. 1 comprises SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. An anti-VEGFA ligand for use in treating cancer in a population of patients carrying at least one of the genes selected from the group consisting of -2578C, -2549del, -1498T or -1190G in a promoter sequence encoding a VEGFA gene. An anti-VEGFA ligand for use in the treatment of breast cancer in a population of patients carrying at least one of the genes selected from the group consisting of -2578C, -2549del, -1498T or -1190G in a promoter sequence encoding a VEGFA gene. An anti-VEGFA ligand for use in the treatment of macular degeneration in a population of patients carrying at least one of the genes selected from the group consisting of -2578C, -2549del, -1498T or -1190G in a promoter sequence encoding a VEGFA gene. A method for determining the clinical responsiveness of a human subject suffering from or at risk of an angiogenic disease or condition to an anti-VEGF therapy, wherein the method comprises detecting initiation of a gene encoding a VEGFA in a nucleic acid sample from the individual At least one of the subsequences (SEQ ID NO. 1) A step of the presence of a single nucleotide polymorphism (SNP), wherein the presence of the SNP indicates clinical responsiveness to the anti-VEGF therapy. Use of an anti-VEGAFA ligand for the manufacture of a medicament for treating a human subject suffering from or at risk of an angiogenic disease or condition, wherein a nucleic acid sample from the individual is detected prior to administration of the medicament The presence of at least one single nucleotide polymorphism (SNP) in the gene promoter sequence (SEQ ID NO. 1) encoding VEGFA, and if the SNP is present in the individual, the drug is administered to the individual. A method for selecting a therapy for a human subject suffering from or at risk of developing an angiogenic disease or condition, wherein the method comprises the steps of: (a) detecting a gene promoter encoding a VEGFA in a nucleic acid sample from the individual The presence of at least one single nucleotide polymorphism (SNP) in the sequence (SEQ ID NO. 1); and (b) if the SNP is present in the individual, the patient is selected for anti-VEGF therapy. A diagnostic kit for determining the clinical responsiveness of a human subject suffering from or at risk of an angiogenic disease or condition to an anti-VEGF therapy, wherein the kit comprises at least one nucleic acid sample capable of detecting the individual from the individual A primer for the presence of at least one single nucleotide polymorphism (SNP) in the promoter sequence of VEGFA (SEQ ID NO. 1).