Droplet digital PCR revealed high concordance between primary tumors and lymph node metastases in multiplex screening of KRAS mutations in colorectal cancer.

The proto-oncogene KRAS belongs among the most frequently mutated genes in all types of cancer and is also very important oncogene related to colorectal tumors. The detection of mutations in this gene in primary tumor is a predictive biomarker for the anti-EGFR therapy in metastatic CRC (mCRC); however, the patients with wild-type KRAS can also show resistance to the personalized medicine. The droplet-based digital PCR technology has improved the analytical sensitivity of the mutations detection, which led us to the idea about the optimization of this approach for KRAS testing. In this study, we report the application of ddPCR technology in order to analyze the presence of KRAS mutations in primary tumor and matched metastasis in lymph nodes (LNs) from patients with mCRC and address the question, whether the improvement in the detection method can lower the discrepancies of KRAS mutations detection between the primary tumor and regional LNs. Genomic DNA with wtKRAS and commercial DNA with mtKRAS (G12D) were used to set up the ddPCR reaction. Formalin-fixed paraffin-embedded tissues from primary tumor and positive lymph node from 31 patients with mCRC were analyzed using ddPCR and Sanger sequencing. KRAS status of primary tumors was known; however, the mutation status of lymph nodes was not detected previously. From 31 samples of primary tumors, our results corresponded to results from IVD kit in 30 cases. For one patient, ddPCR detected KRAS mutation in comparison with negative result of the IVD kit. In the samples of metastatic infiltrated LNs, ddPCR detected 16 samples as a WT KRAS and 15 lymph nodes showed positivity for KRAS mutation, whereby Sanger sequencing found KRAS mutations in 8 cases only. We also found two cases where genetic conditions of KRAS gene differed between primary tumor and infiltrated lymph node, both "low-grade" adenocarcinoma. Our study approved that ddPCR method is adequate technique with high sensitivity and in the future may be used as a diagnostic tool for evaluation of KRAS mutations, especially in infiltrated LNs of patients with mCRC.

Targeted data-independent acquisition for mass spectrometric detection of RAS mutations in formalin-fixed, paraffin-embedded tumor biopsies.

Genomic testing for KRAS and NRAS mutations in clinical biopsies of various cancers is routinely performed to predict futility of anti-epidermal growth factor receptor (anti-EGFR) therapies. We hypothesized that RAS mutations could be detected and quantified at the protein level for diagnostic purposes using data-independent acquisition (DIA)-based mass spectrometry in formalin-fixed, paraffin-embedded (FFPE) tumor samples. We developed a targeted DIA assay that surveys the specific mass range of all possible peptides harboring activating mutations in KRAS exon 2. When the assay was applied to tumor samples with known KRAS or NRAS mutations (G12A, G12D, G12V, and G13D), RAS-mutant and wild-type peptides were successfully detected in 11 of 13 biopsy samples. Mutation statuses obtained by DIA were concordant with those obtained by DNA sequencing, and yields of mutant peptide (mutant peptide/[mutant + wild-type peptides]) exhibited linear correlation with yields of RAS-mutant mRNA. When applied to biopsy samples with failed DNA testing results, the DIA assay identified an additional RAS-mutated sample. SIGNIFICANCE: Proteomic detection of RAS mutations by DIA in tumor biopsies can provide solid evidence of mutant RAS protein regardless of the mutation types and sites in exon 2. This robust method could rescue samples that fail genomic testing due to insufficient tumor tissue or lack of sequenceable DNA. It can be used to explore the relationship between protein expression level of mutant RAS and therapeutic outcome.

Multiplex detection of KRAS mutations by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay.

OBJECTIVES: Mutations of the Kirsten rat sarcoma viral oncogene (KRAS) gene are known to be important in the pathogenesis of a variety of cancers. Patients with mutant KRAS tumors do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Testing for KRAS mutations is now being recommended as a tailored therapeutic strategy prior to anti-EGFR treatment; however, the low sensitivity of direct sequencing frequently leads to failure of detection of KRAS mutations in clinical samples. DESIGN AND METHODS: We developed restriction fragment mass polymorphism (RFMP) assays, to detect KRAS mutations in codons 12, 13, and 61. We performed RFMP analysis for KRAS on DNA isolated from eight different KRAS mutant cell lines and 34 formalin-fixed paraffin-embedded (FFPE) lung cancer tissues. RESULTS: Overall, the RFMP assay was in good concordance with direct sequencing analysis in the detection of KRAS mutations. By using dilutions of KRAS mutant DNA in wild type DNA from mutation cell lines with a known KRAS status, we confirmed that the RFMP assay has a higher analytical sensitivity, requiring only 3% of cells in a sample to have mutant alleles, compared to direct sequencing, which had a detection threshold of ~25%. In the FFPE samples, RFMP successfully detected KRAS genotypes in codons 12, 13, and 61. CONCLUSION: The RFMP might be efficiently applicable for the detection of KRAS mutations in a clinical setting, particularly for tumor samples containing abundant non-neoplastic cells.