e14147 Background: In patients with metastatic colorectal cancer (mCRC), activating mutations within KRAS, which result in EGFR-independent intracellular signal transduction activation, are found in approximately 35-40% of patients with mCRC have been significantly associated with lack of response to cetuximab or panitumumab therapy. Although current guidelines recommend testing for frequent KRAS codons 12/13 mutations, emerging data indicate that additional KRAS and BRAF mutations are also predictive of non-responsiveness to anti-EGFR antibodies in mCRC. This study is aimed to analyze the prevalence of low-penetrance KRAS and BRAF V600 mutations in caucasian mCRC population. Methods: A two-institution retrospective cohort of 1,238 consecutive KRAS wild type mCRC patients previously studied for 7 mutations in codons 12/13 (G12D, G12A, G12V, G12S, G12R, G12C and G13D) by the CE-IVD marked ARMS-scorpion real-time polymerase chain reaction PCR (Therascreen, Qiagen) was assayed by the diagnostic TaqMelt PCR assay cobas KRAS mutation and cobas BRAF V600 mutation tests (Roche), which are designed to detect 19 mutations in KRAS codons 12, 13 and 61 (including G12F, G13C, G13R, G13S, G13A, G13V, G13I, Q61H, Q61K, Q61R, Q61L, Q61E and Q61P) and BRAF V600 (V600E, V600K and V600D) mutations. An additional cohort of 146 KRAS mutated patients by ARMS-scorpion PCR was studied. DNA was obtained by cobas DNA preparation kit from one single 5µm formalin-fixed paraffin-embedded tissue section. Results: In all samples, sufficient DNA was obtained for KRAS and BRAF mutational studies. Among 1238 KRAS codons 12/13 wild-type patients by ARMS-scorpion PCR,166 (13.4%) showed KRAS mutations, 117 (9.5%) in codons 12/13, and 49 (4%) in codon 61. BRAF V600 mutations were detected in 9% cases. In ARMS-scorpion PCR KRAS mutated patients, mutations were confirmed by cobas in all cases. Conclusions: The cobas mutation tests are robust and reproducible assays that, 1) detects a higher incidence (13.4%) of mutations in codons 12, 13, and 61 of the KRAS gene in wild-type mCRC population, 2) a relevant rate of BRAF mutations is present in the same population, and 3) requires a very small amount of tissue.
Improvement of DNA Extraction for Human Papillomavirus Genotyping from Formalin-Fixed Paraffin-Embedded Tissues