WO2011104694A2 - Detection of braf v600e mutation by allele specific real time quantitative pcr (as-qpcr) using locked nucleic acids primers and beacon probes - Google Patents

Detection of braf v600e mutation by allele specific real time quantitative pcr (as-qpcr) using locked nucleic acids primers and beacon probes Download PDF

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WO2011104694A2
WO2011104694A2 PCT/IB2011/050814 IB2011050814W WO2011104694A2 WO 2011104694 A2 WO2011104694 A2 WO 2011104694A2 IB 2011050814 W IB2011050814 W IB 2011050814W WO 2011104694 A2 WO2011104694 A2 WO 2011104694A2
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braf
allele
mutation
primer
molecular beacon
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WO2011104694A3 (en
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Luca Morandi
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GAMMAGENETICS Sàrl
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Definitions

  • the -raf (BRAF) gene encodes a serine-threonine-specific protein kinase that acts as an intermediary in the mitogen- activated protein kinase (MAPK) signaling pathway 1 ' 2 .
  • MAPK mitogen- activated protein kinase
  • Activation of this pathway in response to binding of extracellular signals to receptor tyrosine kinase and G- protein-coupled receptors affects proliferation, differentiation, survival, and invasion.
  • Such a positive growth effect selects for mutant proteins that produce high-level and/or persistent activation of the MAPK pathway 2 ' 3.
  • RAS the upstream activator of BRAF, has been studied intensely, with reports indicating that 17% to 25% of all human tumors contain activating mutations of a ras gene 4 .
  • V600E valine with aspartic acid
  • BRAF mutations may help exclude a diagnosis of Spitz nevus, thus leading to a more accurate diagnosis and appropriate management of a melanocytic neoplasm.
  • diagnosis of PTC is potentially complicated, as it can be difficult to detect among a large background of benign thyroid nodules.
  • BRAF mutations have been reported to occur in 30% to 80% of PTCs, but not to any significant degree in any other malignant or benign thyroid tumors. 7 ' 14 ' 23—25 . Although the prevalence of BRAF mutations appears to be lower in follicular variant of papillary carcinoma (FV-
  • BRAF mutations do not seem to occur in follicular carcinomas of thyroid, although these tumors may up-regulate BRAF through increased gene copy number rather than mutation. 30 Furthermore, the presence of a BRAF mutation has been associated with a more aggressive tumor behavior and, subsequently, a less favorable prognosis than those PTCs harboring an unmutated BRAF. 31 ' 32 . The presence of a BRAF mutation may, therefore, justify a more aggressive course of treatment. Preoperative decisions regarding PTC revolve around cytologic analysis of fine-needle aspirates (FNAs) of thyroid nodules.
  • FNAs fine-needle aspirates
  • BRAF mutational analysis of FNA samples suspicious for PTC may be advantageous for diagnosis, prognosis, and treatment. Indeed, several recent reports have described BRAF mutation detection using FNA samples 14 ' 34 36 Recently, BRAF mutations have been identified in morphologically benign thyroid inclusions in lymph nodes, suggesting that these lesions were likely malignant. 37 BRAF and RET mutations, the two most common genetic abnormalities in spontaneous, adult PTCs, both result in activation of the RAS signaling pathway. As both RET and BRAF are targets for designer therapeutics, it is likely that identification of mutations in these genes will eventually serve a therapeutic purpose in the future. 38 There are numerous methods available for the detection of BRAF mutations that include, but are not limited to, single- stranded conformation polymorphism analysis, ligase detection, the Mutector assay, and melting curve analysis. 36 ' 39 ⁇ 2
  • BRAF wild-type was found to be required for response to panitumumab or cetuximab in colorectal cancer and could be used to select patients who are eligible for the treatment 47 .
  • mutated BRAF kinase contributes to carcinogenesis by increasing resistance to apoptotic stimuli and promoting development of invasive phenotype.
  • BRAF mutations have been associated with a poor survival in colon cancer 48-49 . Given that the BRAF V600E mutation is exclusively found in malignant cells, this alteration provides a novel target for anticancer therapeutics, and BRAF inhibitors are currently under clinical investigation 50"51 .
  • BRAF V600E In addition to the use of BRAF V600E as a potential predictive factor, detection of BRAF V600E mutation has been proposed as a diagnostic marker to distinguish sporadic micro satellite instable colon cancer from hereditary non-polyposis colorectal cancer (Lynch syndrome).
  • the presence of BRAF V600E suggests a sporadic origin of micro satellite instable colorectal cancer and detection of this mutation, therefore has a potential to be used before time-consuming and expensive hereditary non-polyposis colorectal cancer testing 52- " 53.
  • the present invention is related to a novel method based on allele specific quantitative real time PCR (AS-qPCR) using molecular beacon probes to detect the V600E mutation of exon 15 of BRAF gene.
  • AS-qPCR allele specific quantitative real time PCR
  • the assay revealed increased allelic specificity and maintained high sensitivity (0.01% mutated cells detected in a background of 40 ng of wild type DNA) compared with other techniques. Fluorescent detection of newly amplified amplicons in real time were detected using molecular beacon chemistry using short probes with internal LNA modifications.
  • AS-qPCR real-time polymerase chain reaction
  • SEQ ID 1 TTAATCAGTGGAAAAATAGCCTCA for reverse primers use for both AS-qPCR
  • SEQ ID 2 TAGGTGATTTTGGTCTAGCTACAG [ +T ] for wild V600E allele
  • Figure 1 represents the nucleic acid folding and hybridization prediction of the PCR target by MFOLD prediction software (www. http://mfold.bioinfo.rpi.edu).
  • the picture shows a hairpin loop which does not affect primers and beacon probe. Primer regions are not affected by secondary structures and this allows high PCR efficiency.
  • the whole amplicon was tested by repeat masker and no repetitive sequences were detected in.
  • Sensitivity of the assay was tested using dilutions of ARO cell line (ATCC) which is mutated for V600E(heterozygote for this mutation), spiked in with TPCl cell line (ATCC), which is wild type for V600E.
  • ARO cell line ARO cell line
  • ATCC TPCl cell line
  • DNA mixtures were tested: 70 ng of ARO; 1% dilution: 70 ng of TPCl with 700 picogram of ARO; 0.1% dilution: 70 nanogram of TPCl with 70 picogram of ARO; 0.01% dilution: 70 nanogram of TPCl with 7 picogram of ARO; 0.001% dilution: 70 nanogram of TPCl with 700 femptogram of ARO; 0.0001% dilution: 70 nanogram of TPCl with 70 femptogram of ARO.
  • a detection sensitivity of 0.01% of ARO cells spiked in 70 ng of TPCl cells was achieved according to CEIVD guidelines: 24 replicates of this dilution (0.01%) gave positive signal.
  • the assay showed high PCR efficiency (slope: - 3.18; R2: 0.9976) and good specificity (12 healthy blood donors were tested finding no V600E mutations).
  • Basto D Trovisco V, Lopes JM, et al. Mutation analysis of B-RAF gene in human gliomas. Acta Neuropathol (Berl). 2005;109:207-210.
  • Soares P, Trovisco V, Rocha AS, et al. BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC. ⁇ %cogeiie.2003;22:4578 ⁇ 1580.
  • Ciampi R Nikiforov YE. RET/PTC rearrangements and BRAF mutations in thyroid tumorigenesis. Endocrinology. 2007; 148:936-941.

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Abstract

Method for conducting an allele specific polymerase chain reaction which comprises subjecting DNA which may contain either or both of first and second alleles to a polymerase chain reaction utilizing a primer pair in which one of the primers is complementary to said first allele, but which primer forms a mismatch with said second allele at the 3'-terminal nucleotide of the primer, and utilizing a DNA polymerase wherein said first allele is specifically amplified but no amplification of said second allele occurs.

Description

Detection of BRAF V600E mutation by allele specific real time quantitative PCR (AS-qPCR) using locked nucleic acids primers and beacon probes
FIELD OF INVENTION
The -raf (BRAF) gene encodes a serine-threonine-specific protein kinase that acts as an intermediary in the mitogen- activated protein kinase (MAPK) signaling pathway 1 ' 2 . Activation of this pathway in response to binding of extracellular signals to receptor tyrosine kinase and G- protein-coupled receptors affects proliferation, differentiation, survival, and invasion. Such a positive growth effect selects for mutant proteins that produce high-level and/or persistent activation of the MAPK pathway 2 ' 3. Since 1982, RAS, the upstream activator of BRAF, has been studied intensely, with reports indicating that 17% to 25% of all human tumors contain activating mutations of a ras gene4. More recently, identification of somatic-activating mutations of BRAF in various cancers have been reported. Forty different mutations have been identified, with the T1799A mutation leading to substitution of valine with aspartic acid (V600E), accounting for more than 90% of the mutations 3 ' 5—11 The V600E mutation is within the activation segment of the BRAF kinase and is thought to function as a phospho-mimetic, resulting in elevated kinase activity 12. Studies have shown that up to 15% of all human tumors harbor BRAF mutations with melanoma (25%-80%) and papillary thyroid carcinoma (PTC; 30%-80%), representing the highest incidences of mutation13'14. The association of BRAF mutations with melanocytic lesions has been well documented. In addition to malignant lesions, up to 80% of benign melanocytic lesions, including congenital, intradermal, compound, and dysplastic nevi, harbor BRAF mutations3'15'16 . Conspicuously absent from this group are Spitz nevi which, as several studies have shown, do not exhibit to BRAF mutations' 17—20 although two series recently have reported BRAF mutations in these lesions.21,22 Spitz nevi can be notoriously difficult to distinguish from melanoma, and it is not uncommon for such neoplasms to later be diagnosed as melanoma. Identification of a BRAF mutation may help exclude a diagnosis of Spitz nevus, thus leading to a more accurate diagnosis and appropriate management of a melanocytic neoplasm. Similarly to melanoma, diagnosis of PTC is potentially complicated, as it can be difficult to detect among a large background of benign thyroid nodules. BRAF mutations have been reported to occur in 30% to 80% of PTCs, but not to any significant degree in any other malignant or benign thyroid tumors. 7 ' 14 ' 23—25 . Although the prevalence of BRAF mutations appears to be lower in follicular variant of papillary carcinoma (FV-
PTC), they still occur 26 ' 27 but they may be manifested as a BRAF K60 IE mutation.28 Interestingly, BRAF mutations do not seem to occur in follicular carcinomas of thyroid, although these tumors may up-regulate BRAF through increased gene copy number rather than mutation. 30 Furthermore, the presence of a BRAF mutation has been associated with a more aggressive tumor behavior and, subsequently, a less favorable prognosis than those PTCs harboring an unmutated BRAF. 31 ' 32 . The presence of a BRAF mutation may, therefore, justify a more aggressive course of treatment. Preoperative decisions regarding PTC revolve around cytologic analysis of fine-needle aspirates (FNAs) of thyroid nodules. Unfortunately, between 15% and 20% of FNA cytologic results are indeterminate or classified as suspicious, but cannot be confidently diagnosed as malignant. 33 As such, BRAF mutational analysis of FNA samples suspicious for PTC may be advantageous for diagnosis, prognosis, and treatment. Indeed, several recent reports have described BRAF mutation detection using FNA samples 14'34 36 Recently, BRAF mutations have been identified in morphologically benign thyroid inclusions in lymph nodes, suggesting that these lesions were likely malignant. 37 BRAF and RET mutations, the two most common genetic abnormalities in spontaneous, adult PTCs, both result in activation of the RAS signaling pathway. As both RET and BRAF are targets for designer therapeutics, it is likely that identification of mutations in these genes will eventually serve a therapeutic purpose in the future. 38 There are numerous methods available for the detection of BRAF mutations that include, but are not limited to, single- stranded conformation polymorphism analysis, ligase detection, the Mutector assay, and melting curve analysis.36'39^2
Recently, BRAF wild-type was found to be required for response to panitumumab or cetuximab in colorectal cancer and could be used to select patients who are eligible for the treatment47. In colorectal cancer, mutated BRAF kinase contributes to carcinogenesis by increasing resistance to apoptotic stimuli and promoting development of invasive phenotype. BRAF mutations have been associated with a poor survival in colon cancer 48-49. Given that the BRAF V600E mutation is exclusively found in malignant cells, this alteration provides a novel target for anticancer therapeutics, and BRAF inhibitors are currently under clinical investigation50"51. In addition to the use of BRAF V600E as a potential predictive factor, detection of BRAF V600E mutation has been proposed as a diagnostic marker to distinguish sporadic micro satellite instable colon cancer from hereditary non-polyposis colorectal cancer (Lynch syndrome). The presence of BRAF V600E suggests a sporadic origin of micro satellite instable colorectal cancer and detection of this mutation, therefore has a potential to be used before time-consuming and expensive hereditary non-polyposis colorectal cancer testing 52-"53. DESCRIPTION OF THE RELATED ART
Until now, a variety of methods for detection of this mutation has been described, including single- strand conformation analysis, DNA sequencing, TaqMan-based real-time PCR, real-time allele- specific PCR, pyrosequencing, and oligonucleotide microarray54"59. DNA sequencing, currently the gold standard for mutational analysis, is limited by high cost and low sensitivity. Furthermore, most of the mentioned assays are time consuming and require manipulation of amplified PCR products, which is a common source of sample contamination. Pyrosequencing offers a sensitive alternative method, but the equipment is expensive and may not be economical for low-throughput laboratories. For Taqman- based real-time PCR analysis the major disadvantage is the low sensitivity respect to allele specific PCR. High resolution melting (HRM) analysis is a recently developed molecular technique proved to be applicable for detection of various clinically relevant human mutations60, but it does not give information about the locus implicated.
BRIEF SUMMARY OF THE INVENTION
The present invention is related to a novel method based on allele specific quantitative real time PCR (AS-qPCR) using molecular beacon probes to detect the V600E mutation of exon 15 of BRAF gene. Using 3' LNA (Locked nucleic acids) residues in AS-PCR primers for T1796A locus, the assay revealed increased allelic specificity and maintained high sensitivity (0.01% mutated cells detected in a background of 40 ng of wild type DNA) compared with other techniques. Fluorescent detection of newly amplified amplicons in real time were detected using molecular beacon chemistry using short probes with internal LNA modifications. The specificity of the assay was assessed testing a series of DNA from whole blood of 12 healthy donors without detecting any positive signal for the mutated T allele. The total failure rate of wild type allele which should be considered as an internal control was 0%. DETAILED DESCRIPTION OF THE INVENTION
We chose to develop our own allele specific real-time polymerase chain reaction (AS-qPCR) assay for detection of the BRAF V600E mutation. Our approach was chosen for its potential sensitivity, relative ease of use, and quick assay time.
The sequence involved of BRAF gene (Exon 15) is the following:
caaaaattta atcagtggaa aaatagcctc aattcttacc atccacaaaa tggatccaga caactgttca aactgatggg acccactcca tcgagatttc
w
ctgtagcta gaccaaaatc acctattttt actgtgaggt cttcatgaag aaatatatct gaggtgtagt aagtaaagga aaacagtaga tctcattttc
Primers for AS-qPCR:
SEQ ID 1: TTAATCAGTGGAAAAATAGCCTCA for reverse primers use for both AS-qPCR SEQ ID 2: TAGGTGATTTTGGTCTAGCTACAG [ +T ] for wild V600E allele
SEQ ID 3: TAGGTGATTTTGGTCTAGCTACAG [+A] for wild type allele
SEQ ID 4: 5 ' -FAM-CCGAAGGGGATC [ +C ] AGACAA [ +C ] TGTTCAAACTGCCTTCGG-BHQ-1-3 ' for molecular beacon
Figure 1 represents the nucleic acid folding and hybridization prediction of the PCR target by MFOLD prediction software (www. http://mfold.bioinfo.rpi.edu). The picture shows a hairpin loop which does not affect primers and beacon probe. Primer regions are not affected by secondary structures and this allows high PCR efficiency. The whole amplicon was tested by repeat masker and no repetitive sequences were detected in.
AS-qPCR: Allele specific quantitative PCR was performed in 25 μΐ volume containing, 10 pmol (2 ul of the 5 pmol/ul) of each of the forward (TTAATCAGTGGAAAAATAGCCTCA) and reverse primers (TAG GTG ATTTTG GTCT AG CT AC AG +T for V600E allele), (TAG GTG ATTTTG GTCTAG CT AC AG + A for wild type allele )(bases inside [+] are Locked nucleic acid nucleotides), 5 pmol of beacon probe (1 ul of 5 pmol/ul)(FAM-CCGGTGTGGATC+CAGACAACTGTTCAAA+CTGATGCACCGG-BHl), 1 x (12.5 ul) FastStart Universal Probe Master (including Rox, Roche Applied Science), additional 2 ul of MgC12 25 mM (final concentration 4 mM), 5 ul of Ultrapure water (Invitrogen), and 2.5 ul of DNA. Cycling conditions consisted of 10 minutes at 95°C to activate the enzyme, 30 seconds at 95°C, 30 seconds at 60°C with reading plate, and 30 seconds at 72°C for a total of 38 cycles.
Sensitivity of the assay was tested using dilutions of ARO cell line (ATCC) which is mutated for V600E(heterozygote for this mutation), spiked in with TPCl cell line (ATCC), which is wild type for V600E. The following DNA mixtures were tested: 70 ng of ARO; 1% dilution: 70 ng of TPCl with 700 picogram of ARO; 0.1% dilution: 70 nanogram of TPCl with 70 picogram of ARO; 0.01% dilution: 70 nanogram of TPCl with 7 picogram of ARO; 0.001% dilution: 70 nanogram of TPCl with 700 femptogram of ARO; 0.0001% dilution: 70 nanogram of TPCl with 70 femptogram of ARO.
A detection sensitivity of 0.01% of ARO cells spiked in 70 ng of TPCl cells was achieved according to CEIVD guidelines: 24 replicates of this dilution (0.01%) gave positive signal. The assay showed high PCR efficiency (slope: - 3.18; R2: 0.9976) and good specificity (12 healthy blood donors were tested finding no V600E mutations).
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Claims

Claims
1. A method for conducting an allele specific polymerase chain reaction which comprises subjecting DNA which may contain either or both of first and second alleles to a polymerase chain reaction utilizing a primer pair in which one of the primers is complementary to said first allele, but which primer forms a mismatch with said second allele at the 3'-terminal nucleotide of the primer, and utilizing a DNA polymerase wherein said first allele is specifically amplified but no amplification of said second allele occurs.
2. A method for diagnosing a single point mutation of T1799A of exon 15 of BRAF gene. The T 1799 A BRAF mutation causes a V600E amino acid change in the BRAF protein, resulting in the constitutive and oncogenic activation of the mutated BRAF kinase. This method comprises:
(a) obtaining a genomic DNA sample from a tumor tissue specimen which was formalin fixed and paraffin embedded, or was fresh/frozen;
(b) subjecting said genomic DNA sample to separate first and second polymerase chain reactions,
(c) utilizing in said first polymerase chain reaction a first primer set specific to direct amplification of the T→A transversion mutation of the V600E allele, wherein one primer of said first primer set has a 3'-terminal nucleotide complementary to said transversion mutation using, and wherein one primer of said first primer set is modified at its 3'-end with a locked nucleic acid,
(d) utilizing in said second polymerase chain reaction a second primer set which is specific to direct amplification of the normal allele, wherein one primer of said second set has a 3'-terminal nucleotide which is complementary to the normal nucleotide, and said 3'-terminal nucleotide forms a mismatch with, said T A transversion mutation of the V600E allele, and wherein on primer of said second primer set is modified at its 3'-end with a locked nucleic acid
4. The method of claim 3 wherein said first and second polymerase chain reactions are performed simultaneously by real time PCR
5. The method of claim 3 wherein said a molecular beacon comprising: an oligonucleotide (SEQ ID NO.4) comprising a stem and a loop structure and having a photoluminescent dye at one of the 5' or 3' ends and a quenching agent at an opposite 3' or 5' ends, wherein said loop consists of about 13-25 bases and has a melting temperature (Tm) of 60-68° C. recognizing the inner parto of the amplification product, and wherein the stem consists of two non-contiguous stretches of base pairs having sequences comprising 5'-CCG AAG G (SEQ ID NO.4) and CC TTC GG" (SEQ ID NO.4) [respectively] .
6. The molecular beacon of claim 5, wherein said photoluminescent dye is fluorescein, phycoerythrin, CY3, CY5, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, 6-FAM,
JOE, TAMRA, TET, VIC, or a combination thereof.
7. The molecular beacon of claim 5, wherein said photoluminescent dye is 6-FAM. 8. The molecular beacon of claim 5, wherein said quenching agent acts via proximal quenching or fluorescence resonance energy transfer (FRET).
9. The molecular beacon of claim 5, wherein said quenching agent is EDANS, BHQ1. 10. The molecular beacon of claim 5, further comprising a nuclease resistant backbone.
11. The molecular beacon of claim 5, wherein said loop has a sequence complementary to a coding, or non coding region of BRAF gene. 12. The molecular beacon of claim 5, wherein said region is the 15th exon of BRAF gene.
13. The molecular beacon of claim 12, wherein said sequence comprises 5'-FAM-CCG AAG GGG ATC [+C]AG ACA A[+C]T GTT CAA ACT GCC TTC GG-3BHQ-1 -3' (SEQ ID NO.4) where + precedes locked nucleic acid
14. A method of evaluating the efficiency of a cancer chemotherapy in a subject comprising: testing the cancer cells for V600E mutation
15. The method of claim 14, wherein said cancer is colon cancer, melanoma, thyroid cancer
16. A kit for testing the presence of V600E mutation of BRAF gene comprising the method of claim 5
17. The kit of claim 16, further comprising: a DNA polymerase; detection reagents, buffers or a combination thereof. 18. The kit of claim 17, further comprising one or more of: packaging materials, instructions for using the components, one or more containers for holding the components, standards for calibrating any real time allele specific PCR
19. The kit of claim 18, further comprising a method to quantify the number of mutated cells in a population of tumor cells 20. The kit of claim 19, using an external calibrator cell line heterozygote for wt/V600E
21. The kit of claim 20, comprising ARO cell line (heterozygote for wt/V600E) as external calibrator
22. The kit of claim 21, comprising the following calculation to determine the ratio between wild type and mutated allele: ACt test = [Ct(WT - V600E]
ACt DNA mutant for BRAF V600E (Calibrator) = [Ct(WT - BRAF V600E]
AACt = ACt(test) - ACt(DNA mutant for BRAF V600E)
2~AACt = ratio between mutant and wild type allele
PCT/IB2011/050814 2010-02-26 2011-02-25 Detection of braf v600e mutation by allele specific real time quantitative pcr (as-qpcr) using locked nucleic acids primers and beacon probes WO2011104694A2 (en)

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WO2013181125A2 (en) 2012-05-29 2013-12-05 Abbott Laboratories, Inc. Method of designing primers, method of detecting single nucleotide polymorphisms (snps), method of distinguishing snps, and related primers, detectable oligonucleotides, and kits
WO2014027056A1 (en) * 2012-08-17 2014-02-20 F. Hoffmann-La Roche Ag Combination therapies for melanoma comprising administering cobimetinib and vemurafinib
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