A next-generation sequencing-based strategy combining microsatellite instability and tumor mutation burden for comprehensive molecular diagnosis of advanced colorectal cancer

Abstract

Background Mismatch repair (MMR)/microsatellite instability (MSI) and tumor mutational burden (TMB) are independent biomarkers that complement each other for predicting immune checkpoint inhibitors (ICIs) efficacy. Here we aim to establish a strategy that integrates MSI and TMB determination for colorectal cancer (CRC) in one single assay. Methods Surgical or biopsy specimens retrospectively collected from CRC patients were subjected to NGS analysis. Immunohistochemistry (IHC) and polymerase chain reaction (PCR) were also used to determine MMR/MSI for those having enough tissues. The NGS-MSI method was validated against IHC and PCR. The MSI-high (MSI-H) or microsatellite stable (MSS) groups were further stratified based on tumor mutational burden, followed by validation using the The Cancer Genome Atlas (TCGA) CRC dataset. Immune microenvironment was evaluated for each subgroup be profiling the expression of immune signatures. Results Tissues from 430 CRC patients were analyzed using a 381-gene NGS panel. Alterations in KRAS, NRAS, BRAF, and HER2 occurred at a significantly higher incidence among MSI-H tumors than in MSS patients (83.6% vs. 58.4%, p = 0.0003). A subset comprising 98 tumors were tested for MSI/MMR using all three techniques, where NGS proved to be 99.0 and 93.9% concordant with PCR and IHC, respectively. Four of the 7 IHC-PCR discordant cases had low TMB (1.1–8.1 muts/Mb) and were confirmed to have been misdiagnosed by IHC. Intriguingly, 4 of the 66 MSS tumors (as determined by NGS) were defined as TMB-high (TMB-H) using a cut-off of 29 mut/Mb. Likewise, 15 of the 456 MSS tumors in the TCGA CRC cohort were also TMB-H with a cut-off of 9 muts/Mb. Expression of immune signatures across subgroups (MSS-TMB-H, MSI-H-TMB-H, and MSS-TMB-L) confirmed that the microenvironment of the MSS-TMB-H tumors was similar to that of the MSI-H-TMB-H tumors, but significantly more immune-responsive than that of the MSS-TMB-L tumors, indicating that MSI combined with TMB may be more precise than MSI alone for immune microenvironment prediction. Conclusion This study demonstrated that NGS panel-based method is both robust and tissue-efficient for comprehensive molecular diagnosis of CRC. It also underscores the importance of combining MSI and TMB information for discerning patients with different microenvironment.

Oncomine™ Comprehensive Assay v3 vs. Oncomine™ Comprehensive Assay Plus

“…OCA-Plus also include microsatellite instability (MSI) and tumor mutational burden (TMB) assays, providing a time-efficient single workflow. The addition of MSI and TMB may provide information of potential immunotherapies for patients with solid tumors [10]. However, high-grade serous adenocarcinoma is often not harboring a high number of mutations (TMB-high) reflected in low genomic instability.…”

Next-Generation Sequencing Targeted Panel in Routine Care for Metastatic Colon Cancers

“…However, these studies were only conducted in small CRC cohorts with mostly localized cancers [ 17 ], or in the context of clinical trials [ 18 ]. Moreover, despite more recent studies [ 19 , 20 ], clinical practice guidelines and recommendations for the use of NGS in this setting have not changed [ 21 ].…”

The Impact of Patient Characteristics and Tumor Biology on the Accuracy of Preoperative Staging of Colon Cancer in Denmark. A Nationwide Cohort Study

“…We found a significant and persistent association between dMMR and overestimation of the N-category, but we also found a significant impact of histopathology and tumor sidedness. Reactive lymph node enlargement in dMMR cancers is to be expected, because inability to repair mismatch results in accumulation of mutations, leading to the production of immune-response-eliciting proteins [18,19]. Additionally, there is a growing interest in regarding right-and left-sided cancers as distinct biological and clinical entities [20][21][22][23][24][25][26][27][28][29].…”