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US 2010/0167273 A1Published application
Method for Selectively Quantifying Vegf Isoforms in a Biological Sample and Uses Thereof
AssigneesInserm (Inst.Nat. De La Sante Et De La Rech. Med.), Assistance Publique-Hopitaux de Paris
InventorsFabien Calvo, Samia Mourah, Benyoussef Naimi
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Abstract
The present invention pertains to a novel method for quantifying VEGF various isoforms in a biological sample, with a very high selectivity and sensitivity. It also concerns a method for establishing a diagnostic and/or a prognosis concerning a patient potentially suffering from cancer, diabetes, or cardiovascular disease, comprising a step of determining the level of at least one of the VEGF isoforms, in a biological sample from said patient.
Claims26
1:
- A method for selectively quantifying transcripts encoding VEGF isoforms selected amongst VEGF165, VEGF121, VEGF189, VEGF145 and VEGF206 in a biological sample, comprising a step of performing a real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR), wherein the first primer used for amplification comprises at least 15 consecutive nucleotides from exon 4, and wherein the second primer and/or the probe are as follows:
- for quantifying VEGF165 transcripts, the second primer comprises at least 15 consecutive nucleotides from exon 7, and the probe spans the junction between exons 5 and 7 and comprises at least the sequence 5′-gAAAATCCCTg-3′ (SEQ ID No:
- 19);
- for quantifying VEGF121 transcripts, the second primer spans the junction between exons 5 and 8 and comprises at least the sequence 5′-GAAAAATGTGAC-3′ (SEQ ID No:
- 8);
- for quantifying VEGF189 transcripts, the second primer spans the junction between exons 6a and 7 and comprises at least the sequence 5′-CAgggAACgC-3′ (SEQ ID No:
- 20);
- for quantifying VEGF145 transcripts, the second primer spans the junction between exons 6a and 8 and comprises at least the sequence 5′-CACATACgC-3′ (SEQ ID No:
- 21);
- for quantifying VEGF206 transcripts, the second primer spans the junction between exons 6a and 6b and comprises at least the sequence 5′-CgTACACgC-3′ (SEQ ID No:22).
7 more elements
2:
- The method according to claim 1, wherein the primer used for amplification comprising at least 15 consecutive nucleotides from exon 4 is a forward primer, and the other primer is a reverse primer.
3:
- The method according to claim 1, wherein, for at least one of the isoforms, the pair of primers used for the specific amplification of the mRNA encoding said isoform is selected amongst the following:
- for quantifying VEGF165 transcripts:
- primers of SEQ ID Nos:
- 3 and 9;
- for quantifying VEGF121 transcripts:
- primers of SEQ ID Nos:
- 1 and 2 or primers of SEQ ID Nos:
- 3 and 2;
- for quantifying VEGF189 transcripts:
- primers of SEQ ID Nos:
- 3 and 13;
- for quantifying VEGF145 transcripts:
- primers of SEQ ID Nos:
- 3 and 11;
- for quantifying VEGF206 transcripts:
- primers of SEQ ID Nos:
- 3 and 15.
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4:
- The method according to claim 1, wherein, for at least one of the isoforms VEGF121, VEGF189, VEGF145 and VEGF206, the probe used for real-time quantification of the mRNA encoding said isoform is as follows:
- for quantifying VEGF121 transcripts:
- the probe spans the junction between exons 4 and 5 and comprises at least the sequence 5′-CAGACC-3′;
- for quantifying VEGF189 and/or VEGF206 transcripts:
- the probe spans the junction between exons 5 and 6a and comprises at least the sequence 5′-AAAAAA-3′.
- for quantifying VEGF145 transcripts:
- the probe comprises at least 15 consecutive nucleotides from exon 6a.
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5:
- The method according to claim 4, wherein for at least one of the isoforms, the nucleotide sequence of the probe is the following:
- for quantifying VEGF165 transcripts:
- 5′-AGCAAGACAAGAAAATCCCTGTGGGCC-3′;
- (SEQ ID No:
- 10)
- for quantifying VEGF121 transcripts:
- 5′-TGCAGACCAAAGAAAGATAGAGCAAGACA-3′;
- (SEQ ID No:
- 4)
- for quantifying VEGF189 and/or VEGF206 transcripts:
- (SEQ ID No:
- 14)
- 5′-AGCAAGACAAGAAAAAAAATCAGTTCGAGGAAA-3′;
- for quantifying VEGF145 transcripts:
- 5′-AAACGAAAGCGCAAGAAATCCCGGTA-3′.
- (SEQ ID No:
- 12)
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6:
- The method of using a method according to claim 1, for establishing a diagnostic and/or a prognosis concerning a patient potentially suffering from a disease related to angiogenesis.
7:
- The method of using according to claim 6, wherein said disease related to angiogenesis is cancer, hemopathy, diabetes, or cardiovascular disease.
8:
- A method for in vitro establishing a prognosis concerning a patient suffering from acute myeloid leukaemia, comprising the following steps:
- isolating peripheral blood mononucleated cells from a blood sample from said patient;
- extracting RNA from said peripheral blood mononucleated cells;
- and
- measuring the level of VEGF121 transcript in said peripheral blood mononucleated cells.
2 more elements
9:
- The method according to claim 8, further comprising a step of measuring the level of VEGF 165 and/or VEGF189 transcripts in the isolated peripheral blood mononucleated cells.
10:
- A method for in vitro establishing a prognosis concerning a patient having a solid tumor, comprising a step of measuring the level of VEGF121 and VEGF165 transcripts in a biopsy from said tumor, and calculating the VEGF165/VEGF121 ratio.
11:
- A method for determining if a patient having a solid tumor needs an antiangiogenic treatment, comprising a step of in vitro measuring the level of VEGF121 and VEGF165 transcripts in a biopsy from said tumor, and calculating the VEGF165/VEGF121 ratio, wherein a VEGF165/VEGF121 ratio superior to 2 indicates that the patient needs an antiangiogenic treatment, and a VEGF165/VEGF121 ratio superior to 3 indicates that the patient is in strong need of such a treatment.
12:
- The method according to claim 10, wherein said tumor is a breast tumor.
13:
- The method according to claim 12, wherein a VEGF165/VEGF121 ratio superior to 3 is indicative of a poor prognosis.
14:
- The method according to claim 8, wherein the measure is performed by a method according to claim 1.
15:
- The method of using a method according to claim 1, for monitoring the antiangiogenic treatment of a patient.
16:
- The method of using a method according to claim 1, for obtaining information for orientating the treatment regimen of a patient suffering from cancer.
17:
- A set of oligonucleotides for performing the method according to claim 1, comprising at least one of the following pairs of primers:
- pair of primers specific for VEGF165:
- 5′-GAGCTTCCTACAGCACAACAAA-3′
- (SEQ ID No:
- 3)
- and
- 5′-GCTTTCTCCGCTCTGAGCA-3′;
- (SEQ ID No:
- 9)
- pairs of primers specific for VEGF121:
- 5′-CTCGGCTTGTCACATTTTTC-3′
- (SEQ ID No:
- 2)
- coupled to either
- 5′-AGGCCAGCACATAGGAGAGAT-3′
- (SEQ ID No:
- 1)
- or
- 5′-GAGCTTCCTACAGCACAACAAA-3′;
- (SEQ ID No:
- 3)
- pair of primers specific for VEGF189:
- 5′-GAGCTTCCTACAGCACAACAAA-3′
- (SEQ ID No:
- 3)
- and
- 5′-CCACAGGGAACGCTCCAGGAC-3′;
- (SEQ ID No:
- 13)
- pair of primers specific for VEGF145:
- 5′-GAGCTTCCTACAGCACAACAAA-3′
- (SEQ ID No:
- 3)
- and
- 5′-CTTGTCACATACGCTCCAGGAC-3′;
- (SEQ ID No:
- 11)
- pair of primers specific for VEGF206:
- 5′-GAGCTTCCTACAGCACAACAAA-3′
- (SEQ ID No:
- 3)
- and
- 5′-CACCAACGTACACGCTCCAGG-3′.
- (SEQ ID No:
- 15)
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18:
- The set of oligonucleotides according to claim 17, comprising at least one of said pairs of primers specific for VEGF121.
19:
- The set of oligonucleotides according to claim 18, further comprising a probe spanning the junction of exons 4 and 5 of VEGF mRNA, wherein said probe comprises at least the sequence 5′-CAGACC-3′.
20:
- The set of oligonucleotides according to claim 19, wherein the nucleotide sequence of the probe hybridizing to the junction of exons 4 and 5 of VEGF mRNA is 5′-TGCAGACCAAAGAAAGATAGAGCAAGACA-3′ (SEQ ID No:
- 4).
21:
- The set of oligonucleotides according to claim 18, further comprising the pair of primers of SEQ ID Nos:
- 3 and 9, specific for VEGF165.
22:
- The set of oligonucleotides according to claim 21, further comprising a probe spanning the junction of exons 5 and 7 of VEGF mRNA, wherein said second probe comprises at least the sequence 5′-AAATCC-3′.
23:
- The set of oligonucleotides according to claim 22, wherein the nucleotide sequence of the probe hybridizing to the junction of exons 5 and 7 of VEGF mRNA is 5′-AGCAAGACAAGAAAATCCCTGTGGGCC-3′ (SEQ ID No:
- 10).
24:
- The set of oligonucleotides according to claim 17, further comprising a pair of primers and a probe which are specific for a human housekeeping gene.
25:
- The set of oligonucleotides according to claim 24, wherein said housekeeping gene is β2 microglobulin or TBP, and wherein said pair of primers and said probe are the following:
- primers and probe specific for β2 microglobulin:
- (SEQ ID No:
- 5)
- β2m forward:
- 5′-CGCTCCGTGGCCTTAGC-3′;
- (SEQ ID No:
- 6)
- β2m reverse:
- 5′-GAGTACGCTGGATAGCCTCCA-3′;
- (SEQ ID No:
- 7)
- β2m probe:
- 5′-FAM-TGCTCGCGCTACTCTCTCTTTCTGGC-3′-TAMRA;
- or
- primers and probe specific for TBP:
- (SEQ ID No:
- 16)
- TBP forward:
- 5′-CACGAACCACGGCACTGATT-3′;
- (SEQ ID No:
- 17)
- TBP reverse:
- 5′-TTTTCTTGCTGCCAGTCTGGAC-3′;
- (SEQ ID No:
- 18)
- TBP probe:
- 5′-FAM-TGTCGACAGGAGCCAAGATTTCTGGC-3′-TAMRA.
25 more elements
26:
- A kit for selectively quantifying at least one of VEGF transcripts in a biological sample by quantitative RT-PCR, comprising at least a set of oligonucleotides according to claim 17, and random hexamers and/or Uracyl DNA glycosylase.