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  • 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
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to methods, primers and probes for detecting the presence of mutant BRAF sequences in a sample, specifically for detecting the presence of the BRAF V600E, V600D, V600K, V600M, and V600A mutations.
• Cancer arises when a normal cell undergoes neoplastic transformation and becomes a malignant cell.
• Transformed (malignant) cells escape normal physiologic controls specifying cell phenotype and restraining cell proliferation.
• Transformed cells in an individual's body thus proliferate, forming a tumor.
• the clinical objective is to destroy malignant cells selectively while mitigating any harm caused to normal cells in the individual undergoing treatment.
• B-raf (or BRAF) encodes a protein that belongs to the Serine/Threonine protein kinases.
• BRAF is a part of the Ras/Raf/MEK/MAP signal transduction pathway and plays a role in regulating the MAP Kinse/ERK signaling pathway. Mutations in this gene have been associated with various cancers such as colorectal cancer (CRC), non small cell lung cancer (NSCLC), malignant melanomas and adenocarcinomas.
• CRC colorectal cancer
• NSCLC non small cell lung cancer
• Oncogenic mutations in BRAF nearly all of which are the V600E mutation, have been reported in colon cancer (Davies H, et al. Nature 2002;417:949-54; Rajagopalan H, et al., Nature 2002;418:934.). The V600E mutation has been observed in over half of all cancer
• V600E (formerly V599E) mutation is located on exon 15 of the B-raf gene (Accession number NM 04333.4) at position 1860 (1799 of the coding sequence).
• a thymidine is changed to an adenosine, which results in the change from a valine (V) in the wildtype/non mutant B-rag gene to a Glutamine (E) in the mutated gene.
• V valine
• E Glutamine
• a rare ( ⁇ 1%) V600K (1798-1799 GT>AA) mutation also exists.
• the V600D mutation exists in 4.6% of cases
• the V600A mutation exists in ⁇ 1% of cases
• the V600M mutation exists in ⁇ 1% of cases.
• the V600E BRAF mutation is found in a number of tissue/tumor types including: nervous system, thyroid, skin, gastrointestinal tract, large intestine, biliary tract, ovary, eye, prostate, central nervous system, liver, small intestine, breast, pancreas, soft tissue, upper, aerodigestive tract, adrenal gland, autonomic ganglia, haematopoietic and lymphoid tissue, lung, esophagus, pituitary, and stomach. DNA or RNA extracted from samples of any of these types of tissues can be utilized in assays of the present invention.
• CIMP CpG island methylator phenotype
• the V600E BRAF mutation was seen in 5% of microsatellite-stable tumors and 51.8% of microsatellite -unstable tumors. In microsatellite-stable tumors, this mutation was related to poor survival, CIMP high, advanced American Joint Committee on Cancer (AJCC) stage, and family history of colorectal cancer. The poor survival was observed in a univariate analysis of 5-year survival (16.7% versus 60.0%); in an analysis adjusted for age, stage, and tumor site; in stage-specific, age-adjusted analyses for AJCC stages 2 to 4 (HRR, 4.88, 3.60, and 2.04, respectively); and in Kaplan-Meier survival estimates for AJCC stages 2 to 4.
• Microsatellite-unstable tumors were associated with an excellent 5-year survival whether the V600E mutation was present or absent (76.2% and 75.0%, respectively).
• Samowitz has concluded that the BRAF V600E mutation in microsatellite-stable colon cancer is associated with a significantly poorer survival in stages 2 to 4 colon cancer but has no effect on the excellent prognosis of microsatellite- unstable tumors.
• HNPCC hereditary nonpolyposis colorectal cancer syndrome
• BRAF mutations have also been detected in tumor tissues from other types of cancer.
• Experimental studies have demonstrated that several BRAF mutations, especially the T 1799 A (formerly designated T 1796A) hotspot mutation, which accounts for 90% of BRAF mutations in melanoma, can transform fibroblasts in culture.
• T 1799 A now designated T 1796A hotspot mutation
• the present invention discloses methods of detecting BRAF V600E, V600D, V600K, V600M, and V600A mutations in a sample.
• the present invention discloses compositions comprising primer and probe sequences used in the amplification and detection of V600E, V600D, V600K, V600M, or V600A mutant BRAF sequences present in samples.
• Particular primer combinations as disclosed herein are used in amplifying particular BRAF mutations. It will be appreciated by those skilled in the art, one may also design primers specific to the 1798-1799 GT>AA double mutation.
• Figure 1 shows the primers and probes used in the amplification and detection of BRAF mutations.
• the present invention provides methods, primers, probes and kits useful for the detection of BRAF mutations.
• the methods, primers, probes and kits of the present invention can be used for detecting the BRAF V600E, V600D, V600K, V600M, and V600A mutations in many different cell types and thus can be used for the diagnosis of many different cancers, such as, but not limited to, melanoma, colorectal cancer, lung cancer and thyroid cancer.
• the methods of the invention may be useful as a predictor of outcome for cancer patients.
• the method of the present invention addresses the desperate need for a rapid, non-invasive, and accurate screening assay for detecting mutant BRAF sequences, the presence of which is a positive indicator of metastasizing disease. As such, it identifies those patients who need to be treated with more aggressive treatment regimens. Moreover, since the invention can be used for either DNA or RNA, sample preparation is facile, thereby reducing assay variability that can result from differences in the expertise level of laboratory technicians involved in sample
• the method of the present invention may be used to monitor patients with advanced, metastatic melanoma (Stages III/IV). These patients are at the highest risk for disease progression, and early detection of an increase in disease activity would lead to earlier treatment and improvement in outcome.
• the method of the present invention may also be directed to testing patients with earlier stages of disease (Stages I/II), who are at risk for metastatic spread of their disease. Again, early intervention with additional diagnostic tests and treatments would lead to improved patient survival.
• the present invention provides a method for detecting the presence of a BRAF mutation in a sample, said method comprising: (a) isolating nucleic acid from said sample wherein the sample comprises nucleic acid sequences; (b) performing an amplification reaction of said nucleic acid sequences of said sample, wherein said amplification reaction comprises a first primer capable of annealing specifically to a BRAF mutant sequence at a first position in a BRAF sequence wherein said first primer is SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 and a second primer capable of annealing specifically at a second position in a BRAF sequence wherein said second primer is SEQ ID NO: 10, wherein said first and second primers anneal to different strands of double stranded BRAF sequence, wherein the amplification reaction is capable of producing a BRAF mutant specific
• the present invention also provides a method for detecting the presence of metastatic melanoma in a sample, said method comprising: (a) isolating nucleic acid from said sample wherein the sample comprises nucleic acid sequences; (b) performing an amplification reaction of said nucleic acid sequences of said sample, wherein said amplification reaction comprises a first primer capable of annealing specifically to a BRAF mutant sequence at a first position in a BRAF sequence wherein said first primer is SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 and a second primer capable of annealing specifically at a second position in a BRAF sequence wherein said second primer is SEQ ID NO: 10, wherein said first and second primers anneal to different strands of double stranded BRAF sequence, wherein the amplification reaction is capable of producing a BRAF
• Embodiments of the present invention comprise BRAF V600E, V600D, V600K, V600M, and V600A mutant specific primers.
• Exemplary BRAF V600 E mutant specific primer pairs include SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 9 and SEQ ID NO: 10.
• Exemplary BRAF V600D mutant specific primer pairs include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7 and SEQ ID NO: 10.
• Exemplary BRAF V600K mutant specific primer pairs include SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 and SEQ ID NO: 10.
• Exemplary BRAF V600M mutant specific primer pairs include SEQ ID NO: 6 or SEQ ID NO: 8 and SEQ ID NO: 10.
• Exemplary BRAF V600A specific primers include SEQ ID NO: 3 and SEQ ID NO: 10. These primers were designed to avoid any known BRAF polymorphisms. As described herein, such oligonucleotides can be detectably labeled.
• BRAF V600 mutant specific primers (SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10) or appropriate BRAF mutant specific primer pairs may be components of compositions comprising biologically compatible salt solutions and/or other buffers or components.
• Embodiments of the present invention comprise oligonucleotide probe sequences, SEQ ID NO: 11 and SEQ ID NO: 12, wherein the oligonucleotide is used as a probe for the detection of BRAF mutant sequences.
• This probe was designed to avoid any known BRAF polymorphisms.
• the oligonucleotide is detectably labeled.
• the present invention also provides a kit comprising at least one of SEQ ID NO: 1-12.
• Embodiments of the present invention can be utilized to detect the V600E, V600D, V600K, V600M, and V600A BRAF mutations.
• the method comprises obtaining a sample of a tissue or a body fluid from the subject (e.g., a mammal) wherein the sample contains nucleic acid.
• tissue or body fluids that can be used include blood, plasma, lymph, tumor biopsies, and body tissue.
• the tissue sample comprises paraffin embedded tissue specimens.
• the nucleic acid is deoxyribonucleic acid (DNA).
• the nucleic acid is ribonucleic acid (RNA).
• the present method can be applied to any type of tissue from a patient.
• Sources of such tissue include but are not limited to nervous system, thyroid, skin, gastrointestinal tract, large intestine, biliary tract, ovary, eye, prostate, central nervous system, liver, small intestine, breast, pancreas, soft tissue, upper, aerodigestive tract, adrenal gland, autonomic ganglia, haematopoietic and lymphoid tissue, lung, esophagus, pituitary, and stomach.
• For examination of resistance of tumor tissue it is preferable to examine the tumor tissue.
• a portion of normal tissue from the patient from which the tumor is obtained is also examined.
• the methods of the present invention can be applied over a wide range of tumor types. This allows for the preparation of individual "tumor expression profiles" whereby expression levels of BRAF V600E, V600D, V600K, V600M, or V600A mutant sequences are determined in individual patient samples and response to various chemotherapeutics is predicted. In certain embodiments, the methods of the invention are applied to colon cancer or melanoma tumors.
• Embodiments of the present invention utilize methods of DNA isolation known to those skilled in the art.
• the aim is to separate DNA present in the nucleus of the cell from other cellular components.
• the isolation of DNA usually begins with lysis, or breakdown, of tissue or cells. This process is essential for the destruction of protein structures and allows for release of nucleic acids from the nucleus. Lysis is carried out in a salt solution, containing detergents to denature proteins or proteases (enzymes digesting proteins), such as Proteinase K, or in some cases both. It results in the breakdown of cells and dissolving of membranes.
• Methods of DNA isolation include, but are not limited to, phenol: chloroform extraction, high salt precipitation, alkaline denaturation, ion exchange column chromatography, resin binding, and paramagnetic bead binding.
• Embodiments of the present invention utilize methods of RNA isolation known to those skilled in the art.
• RNA may be isolated and prepared for hybridization by a variety of methods including, but not limited to, Trizol ® and Guanidinium thiocyanate -phenol- chloroform extraction.
• the principle of RNA isolation is based on cell/tissue lysis, followed by extraction, precipitation, and washing. It will be understood by those skilled in the art the selection of RNA isolation will depend on sample type. Incorporated by reference is US 12/144,388 directed to a method of RNA isolation from paraffin embedded tissue, a common source for oncogene marker testing.
• amplification methods including, but not limited to, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), helicase dependent amplification (HDA) and Nucleic Acid Sequence Based
• NASBA Amplification Refractory Mutation System
• ARMS Amplification Refractory Mutation System
• the primers and probes are used in ARMS.
• Embodiments of the present invention utilize detection methods including, but not limited to, labeling primers used during the amplification step such that the amplification products are labeled with a detectable marker and hybridizing the amplification product to oligonucleotide probes labeled with a detectable marker.
• Detectable markers include but are not limited to chemiluminescent tags, fluorescent tags, and radioactive tags.
• Labeled amplification product can be directly measured using methods corresponding to the type of label used according to methods would be known to one skilled in the art.
• Labeled probe can be hybridized to the amplification product according to methods known to one skilled in the art.
• BRAF V600E mutant expression levels are assayed in patient tumor samples to prognosticate the efficacy a treatment regimen.
• BRAF V600E mutant expression levels are assayed in patient tumor samples to predict the efficacy a treatment regimen.
• BRAF V600D mutant expression levels are assayed in patient tumor samples to predict the efficacy a treatment regimen.
• BRAF V600K mutant expression levels are assayed in patient tumor samples to predict the efficacy a treatment regimen.
• BRAF V600M mutant expression levels are assayed in patient tumor samples to predict the efficacy a treatment regimen.
• BRAF V600A mutant expression levels are assayed in patient tumor samples to predict the efficacy a treatment regimen.
• tumor cells are preferably isolated from the patient.
• Solid or lymphoid tumors or portions thereof are surgically resected from the patient or obtained by routine biopsy.
• RNA isolated from frozen or fresh samples is extracted from the cells by any of the methods typical in the art, for example, Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.), Cold Spring Harbor Laboratory Press, New York, (1989). Preferably, care is taken to avoid degradation of the RNA during the extraction process.
• Table 1
• Example 1 Testing of the primers of the present invention
• a synthetic V600E construct was made to test the ability of the primers and the probes of the present invention to specifically amplify a nucleic acid containing a BRAF V600E mutation.
• Two set of Primers/probes for BRAF V600E mutation were used for the validation.
• the V600E synthetic construct was serially diluted (1 :2) 17 times in a background of gDNA (0.67ng/uL, 5ng/PCR). The mutation concentration ranged from 1OfM to 0.15aM. Each diluted sample was assayed 6x in duplicate (12 total) for the control (Exonl3) and the V600E mutation.
• the rare V600K BRAF mutation can be detected utilizing the same pair of primers designed for the V600E mutation.
• the V600K mutation is a 1798-1799 GT> AA double mutation.
• SEQ ID NO:2 comprises a highly specific primer that will only result in amplified product in the presence of the single 1799 T>A mutation.
• V600D 2GA (specific for V600D)
• V600D 2GA (specific for V600D) This primer/probe set has been
• V600K 2AT (specific for V600K) This primer/probe set has been
• Results The following table describes fragments that were successfully amplified with specific primer probe sets. A plus (+) signifies that a specific fragment was amplified.
• V600 D fragment as the template amplification was performed with V600D 2GA primer/probes
• V600 D fragment as the template amplification was performed with 1799A 1GT primer/probes
• V600 D fragment as the template amplification was performed with V600K 2AT primer/probes
• primer/probe 1799 1GT would detect a T to A base pair change at 1799 and, therefore, would detect all mutations.
• V600K 2AT is exclusive for V600K mutation
• V600D 2GA is exclusive for V600D mutation
• 1799A 1GT is exclusive for 1799 T to A change which is contained in all three mutations (V600E, V600E and V600K)

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