Clinical Significance of the Mutations
BRAF is one of three serine/threonine protein kinases (ARAF, BRAF and CRAF) that mediate intracellular signaling through the MAPK pathway and lie just downstream of the RAS proteins. RAF proteins are components of the mitogen-activated protein kinase (MAPK)/ERKs pathway, which transmits signals affecting such processes as cell division and differentiation from cell surface receptors to the nucleus. Activating mutations of BRAF are present in approximately 50% of malignant melanomas, 40% of thyroid carcinomas, 12% of colorectal and ovarian cancers, and a wide variety of other malignancies at lower frequencies . The 1799T>A point mutation, causing the missense substitution V600E, accounts for the majority (80-85%) of tumor-associated BRAF mutations. Mutant forms of BRAF harboring the V600E and V600K substitutions promote constitutive downstream signaling in the MAPK pathway .
Recently, selective inhibitors of mutant BRAF (e.g., PLX4032, Plexxikon, Inc.) have been developed. These drugs have shown remarkable effectiveness as therapy for BRAF V600E-positive, metastatic melanoma in clinical trials [3,4]. In metastatic colon cancer, BRAF mutation, like KRAS mutation, is associated with non-response to EGFR-targeted antibody therapy . In addition, mutation analysis of BRAF in breast cancer may be useful, in conjunction with analysis of KRAS, PIK3CA and PTEN, for prediction of response to the mTOR inhibitor everolimus .
The BRAF mutations assayed in the YNHH Tumor Profiling panel comprise 96% of BRAF mutations found in tumors .
Mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor gene (EGFR) are found in 10% and 30% of non-small cell lung cancers (NSCLC) from Caucasian and East-Asian patients, respectively . These mutations are restricted to exons 18-21, which encode the TK domain. Small, in-frame deletions in exon 19 account for 48% of all EGFR mutations in NSCLC; the L858R point mutation in exon 21 accounts for 43%; and mutations affecting residue G719 in exon 18 account for 3%. These alterations in exons 18, 19 and 21 are all activating mutations that are predictive of tumor sensitivity to the EGFR tyrosine kinase inhibitor drugs gefitinib and erlotinib [8, 9]. In contrast, the T790M point mutation in exon 20 is associated with resistance to these drugs and accounts for approximately 50% of cases of acquired resistance to gefitinib/erlotinib therapy. Small, in-frame insertions (usually duplications) in exon 20 account for 4% of EGFR mutations in NSCLC and are not associated with clinical response to gefitinib or erlotinib.
The EGFR mutations assayed in the YNHH Tumor Profiling panel comprise 77% of EGFR kinase domain (exons 18-21) mutations found in all tumors  and 83% of EGFR kinase domain mutations found in NSCLC . Additional EGFR mutation assays are currently under development; when these are added, the YNHH Tumor Profiling panel will be able to detect 90% of all EGFR kinase domain mutations occurring in NSCLC.
The ERBB2 gene encodes HER2/neu, a cell surface growth factor receptor belonging to the same class of receptor tyrosine kinases that includes EGFR. In up to 25% of breast carcinomas, ERBB2 gene amplification leads to overexpression of the HER2/neu receptor and drives tumor growth. A different mechanism of HER2/neu oncogenic activation, ERBB2 gene mutation, is found in approximately 2% of NSCLCs [11, 12]. These ERBB2 mutations are predominantly small, in-frame insertions in exon 20 and lead to constitutive activation of the mutant HER2/neu kinase. In NSCLC, activating mutations of EGFR and ERBB2 occur in a mutually exclusive manner, and tumors harboring ERBB2 mutations do not respond to treatment with EGFR-directed tyrosine kinase inhibitors. However, ERBB2 mutations in NSCLC do appear to be associated with tumor sensitivity to anti-HER2 antibody therapy [12, 13].
The ERBB2 mutations assayed in the YNHH Tumor Profiling panel comprise 70 to 85% of ERBB2 mutations found in lung cancer [1, 12].
RAS proteins (KRAS, NRAS and HRAS) are critical mediators of cellular proliferation signals initiated by a broad range of receptor tyrosine kinases and transduced to downstream effectors in the mitogen-activated protein kinase (MAPK) pathway . Activating mutations of KRAS are found most commonly in tumors of the pancreas (60%), colon (30-40%) and biliary tract (30%), as well as in approximately 17% of lung cancers [1,15]. Mutations are located most often in codon 12 or 13 and at a lower frequency in codon 61. KRAS mutation in colorectal cancer predicts non-response to anti-EGFR antibody (e.g., cetuximab) therapy . In adenocarcinomas of the lung, KRAS and EGFR TK domain mutations occur in a mutually exclusive manner, and only EGFR-mutant tumors respond to treatment with EGFR-targeted tyrosine kinase inhibitors . Mutation analysis of KRAS in breast cancer may be useful, in conjunction with analysis of BRAF, PIK3CA and PTEN, for prediction of response to the mTOR inhibitor everolimus .
The KRAS mutations assayed in the YNHH Tumor Profiling panel comprise 98% of KRAS mutations found in tumors .
NRAS:NRAS is a homolog of KRAS, and NRAS proteins have similar functions as mediators of cellular growth signals generated by upstream receptor tyrosine kinases and transduced to the nucleus through the MAPK pathway . Activating mutations of NRAS are found in approximately 20% of melanomas, 12% of acute myeloid leukemias, 8% of thyroid carcinomas, and 2-3% of colon cancers . The NRAS mutations in melanoma, thyroid cancer, and colon cancer occur predominantly in codon 61, whereas those found in hematopoietic neoplasms can involve codon 12, 13 or 61. In colon cancers without KRAS mutation, the presence of an NRAS mutation is correlated with resistance to anti-EGFR antibody therapy .
The NRAS mutations assayed in the YNHH Tumor Profiling panel comprise 95% of NRAS mutations found in all tumors, including 94% of those found in melanoma .
PIK3CA:The PIK3CA gene encodes the catalytic subunit of a lipid kinase involved in growth factor and insulin signaling . The activation of tyrosine kinase receptors generally leads to downstream signaling through both RAS and PI3K. Oncogenic somatic mutations of PIK3CA occur primarily in a few “hotspot” codons within the helical and kinase domains and lead to enhanced signaling through the PI3K/AKT/mTOR pathway . Activating mutations of PI3KCA are common in cancer, occurring in 30-40% of breast carcinomas, 20-30% of colon cancers, 20% of urinary tract malignancies, 20% of endometrial cancers, 5-10% of ovarian carcinomas, and lower percentages of many other tumor types .
Many agents that target the PI3K/AKT/mTOR pathway are in development for potential use as anti-cancer therapies . In general, tumor markers that indicate PI3K pathway activation, such as oncogenic mutations of PIK3CA, are expected to predict positive responses to treatment with drugs targeting the PI3K pathway, such as mTOR inhibitors. In contrast, tumors containing mutations that activate an alternate oncogenic pathway, such as the RAS/RAF/MAPK pathway, may be expected not to respond to mTOR inhibition. In accordance with these predictions, there is recent evidence that breast cancers with either activating mutations of PIK3CA or loss of PTEN function respond to mTOR inhibition by everolimus, provided that they do not have concurrent KRAS or BRAF activating mutations; breast tumors with both PI3K activation and KRAS or BRAF mutation appear resistant to everolimus .
The PIK3CA mutations assayed in the YNHH Tumor Profiling panel comprise 77% of PIK3CA mutations found in tumors, including 89% of those occurring in breast cancer .
 COSMIC (Catalog Of Somatic Mutations In Cancer) database, The Wellcome Trust Sanger Institute (www.sanger.ac.uk/genetics/CGP/cosmic/).
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