Yosuke Hirotsu1, Yuki Nagakubo2, Kenji Amemiya3, Toshio Oyama4, Hitoshi Mochizuki5, Masao Omata6. 1. Genome Analysis Center, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan. Electronic address: hirotsu-bdyu@ych.pref.yamanashi.jp. 2. Division of Genetics and Clinical Laboratory, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan. 3. Genome Analysis Center, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan. 4. Department of Pathology, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan. 5. Genome Analysis Center, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan; Department of Gastroenterology, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan. 6. Department of Gastroenterology, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan; The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
Abstract
BACKGROUND: Microsatellite instability (MSI) occurs in solid tumors and is a predictive biomarker for remarkable response to immune checkpoint inhibitors. Detection of MSI status has been conventionally conducted by PCR-electrophoresis-based assay (MSI-PCR) and immunohistochemistry (IHC) of mismatch repair proteins. However, these approaches require visual confirmation and involve some difficulties in determining MSI statuses from equivocal results. METHODS: We performed amplicon-based targeted sequencing of 76 microsatellite loci (MSI-NGS) in 184 formalin-fixed paraffin-embedded (FFPE) tumor tissues and baseline control samples. A bioinformatics tool, MSIcall, was used to calculate the quantitative values based on the aligned sequence reads and evaluated MSI status. Furthermore, we examined the concordance between the results from MSI-NGS and MSI-PCR/IHC. Diagnostic accuracy, sensitivity, and specificity were estimated by receiver operating characteristic (ROC) curve analysis. For validation cohort, we studied additional 50 tumor samples to determine the MSI status. RESULTS: Of 184 tumor samples, MSI-PCR and IHC analysis classified 161 tumors as MSS/pMMR and 23 as MSI-H/dMMR. Using MSI-NGS combined with MSIcall, we predicted MSI status with high accuracy (98.9%), specificity (91.3%), and sensitivity (100%) in 25 types of cancers. This method achieved an area under the ROC curve (AUC) value of 0.9986. Furthermore, we achieved the 100% concordant results using additional 50 samples for validation. CONCLUSION: We demonstrated newly developed MSI-NGS with MSIcall accurately determines the MSI status of FFPE tumor tissues thorough sequencing of tumor samples alone without patient-matched normal controls. This approach can be applied to all types of solid tumors to determine responders to immune-oncology therapy.
BACKGROUND: Microsatellite instability (MSI) occurs in solid tumors and is a predictive biomarker for remarkable response to immune checkpoint inhibitors. Detection of MSI status has been conventionally conducted by PCR-electrophoresis-based assay (MSI-PCR) and immunohistochemistry (IHC) of mismatch repair proteins. However, these approaches require visual confirmation and involve some difficulties in determining MSI statuses from equivocal results. METHODS: We performed amplicon-based targeted sequencing of 76 microsatellite loci (MSI-NGS) in 184 formalin-fixed paraffin-embedded (FFPE) tumor tissues and baseline control samples. A bioinformatics tool, MSIcall, was used to calculate the quantitative values based on the aligned sequence reads and evaluated MSI status. Furthermore, we examined the concordance between the results from MSI-NGS and MSI-PCR/IHC. Diagnostic accuracy, sensitivity, and specificity were estimated by receiver operating characteristic (ROC) curve analysis. For validation cohort, we studied additional 50 tumor samples to determine the MSI status. RESULTS: Of 184 tumor samples, MSI-PCR and IHC analysis classified 161 tumors as MSS/pMMR and 23 as MSI-H/dMMR. Using MSI-NGS combined with MSIcall, we predicted MSI status with high accuracy (98.9%), specificity (91.3%), and sensitivity (100%) in 25 types of cancers. This method achieved an area under the ROC curve (AUC) value of 0.9986. Furthermore, we achieved the 100% concordant results using additional 50 samples for validation. CONCLUSION: We demonstrated newly developed MSI-NGS with MSIcall accurately determines the MSI status of FFPE tumor tissues thorough sequencing of tumor samples alone without patient-matched normal controls. This approach can be applied to all types of solid tumors to determine responders to immune-oncology therapy.
Authors: Richard K Yang; Hui Chen; Sinchita Roy-Chowdhuri; Asif Rashid; Hector Alvarez; Mark Routbort; Keyur P Patel; Raja Luthra; L Jeffrey Medeiros; Gokce A Toruner Journal: Cancers (Basel) Date: 2022-09-20 Impact factor: 6.575