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May 30, 2026

Clinicopathologic profile and comparative performance of limited next-generation sequencing and qPCR for detection of RAS/RAF mutations in stage IV colorectal cancer (mCRC).

Key Points

This study aims to compare the performance of limited next-generation sequencing and qPCR for detecting RAS/RAF mutations in stage IV colorectal cancer.
Prospective analysis of 90 stage IV colorectal cancer patients at a tertiary academic institute in Eastern India.
Data collected included age, gender, tumor characteristics, and genetic mutations detected by NGS and qPCR.
Statistical analysis was performed using SPSS Statistics (version 27).
Sensitivity, specificity, and concordance rates for BRAF mutation detection by qPCR were 100%, 97.29%, and 97.43%, respectively.
For KRAS, sensitivity was 100% with specificity at 87.5% and concordance at 85.29%; NRAS showed 100% sensitivity with 96.29% specificity and 96.42% concordance.
Lower specificity for KRAS and NRAS was due to additional variants uniquely identified by NGS.

Abstract

e15576 Background: The implications of limited next-generation sequencing (NGS) compared with quantitative/real-time PCR (qPCR) in metastatic colorectal cancer (mCRC) remain unclear in resource-constrained settings. Our study explores correlations between genetic alterations and patient demographics, and compares the sensitivity, specificity, and concordance rate of KRAS, NRAS, and BRAF mutation analysis detected by NGS with qPCR in mCRC. Methods: A prospective analysis was conducted on stage IV colorectal cancer patients diagnosed at a tertiary academic institute in Eastern India, from September 2023 to March 2024. Data on age, gender, tumour grade, tumour location, mismatch repair (MMR) status by immunohistochemistry, and mutations in TP53 , KRAS , NRAS , BRAF , PIK3CA , and HER2, detected by NGS on formalin-fixed, paraffin-embedded tumour tissue blocks, were collected. Post-NGS paraffin blocks with adequate tumour tissue remaining were taken up for qPCR analysis of KRAS, NRAS, and BRAF mutations. Statistical analysis was done using SPSS Statistics (version 27). Results: A total of 90 patients (male-50, 55.6%; female-40, 44.4%; median age 51 years) were included in the study. The Majority had left-sided tumours (63.3%), with rectum being the most common (MC) site (33.3%). MC mutation was TP53 more in left-sided tumours (p = 0.042). BRAF mutations were more common in males (p = 0.034) and grade 3 histology (p = 0.006). Younger patients (15–39 years) had higher-grade tumours (p = 0.007), KRAS mutations (p = 0.084) and deficient MMR (dMMR) (p = 0.047). Only 39 patients were eligible for evaluation of BRAF mutation by qPCR post-NGS, for which the sensitivity, specificity, and concordance rate were 100%, 97.29% KRAS G13C, G12V, R68M; NRAS G12D) uniquely identified by NGS. Conclusions: In low-and middle-income country settings, where access to comprehensive NGS remains limited by cost and infrastructure, our findings provide important evidence that targeted qPCR can reliably identify key actionable mutations required for standard-of-care treatment decisions in mCRC. A stepwise testing strategy, employing limited NGS or qPCR initially followed by comprehensive NGS only in highly selected patients, can optimize resource utilization, reduce diagnostic delays, and expand equitable access to precision oncology.

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