Toky Ratovomanana1, Romain Cohen2, Magali Svrcek3, Florence Renaud4, Pascale Cervera5, Aurélie Siret1, Quentin Letourneur1, Olivier Buhard1, Pierre Bourgoin1, Erell Guillerm5, Coralie Dorard1, Remy Nicolle6, Mira Ayadi6, Mehdi Touat7, Franck Bielle8, Marc Sanson8, Philippe Le Rouzic5, Marie-Pierre Buisine9, Guillaume Piessen10, Ada Collura1, Jean-François Fléjou3, Aurélien de Reyniès6, Florence Coulet5, François Ghiringhelli11, Thierry André2, Vincent Jonchère5, Alex Duval12. 1. Sorbonne Université, INSERM, Unité Mixte de Recherche Scientifique 938 and Site de Recherche Intégrée sur le Cancer (SIRIC) Cancer United Research Associating Medicine, University & Society (CURAMUS), Centre de Recherche Saint-Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France. 2. Sorbonne Université, INSERM, Unité Mixte de Recherche Scientifique 938 and Site de Recherche Intégrée sur le Cancer (SIRIC) Cancer United Research Associating Medicine, University & Society (CURAMUS), Centre de Recherche Saint-Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France; Sorbonne Université, Department of Medical Oncology, AP-HP, Hôpital Saint-Antoine, Paris, France. 3. Sorbonne Université, INSERM, Unité Mixte de Recherche Scientifique 938 and Site de Recherche Intégrée sur le Cancer (SIRIC) Cancer United Research Associating Medicine, University & Society (CURAMUS), Centre de Recherche Saint-Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France; Sorbonne Université, Department of Pathology, AP-HP, Hôpital Saint-Antoine, Paris, France. 4. Sorbonne Université, INSERM, Unité Mixte de Recherche Scientifique 938 and Site de Recherche Intégrée sur le Cancer (SIRIC) Cancer United Research Associating Medicine, University & Society (CURAMUS), Centre de Recherche Saint-Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France; Université de Lille, CNRS, INSERM, CHU Lille, UMR9020-U1277 CANTHER-Cancer Heterogeneity Plasticity and Resistance to Therapies, Lille, France. 5. Sorbonne Université, INSERM, Unité Mixte de Recherche Scientifique 938 and Site de Recherche Intégrée sur le Cancer (SIRIC) Cancer United Research Associating Medicine, University & Society (CURAMUS), Centre de Recherche Saint-Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France; Sorbonne Université, Genetics Department, AP-HP, Hospital Pitié-Salpêtrière, Paris, France. 6. Programme "Cartes d'Identité des Tumeurs," Ligue Nationale Contre le Cancer, Paris, France. 7. Sorbonne Université, INSERM, Unité Mixte de Recherche Scientifique 938 and Site de Recherche Intégrée sur le Cancer (SIRIC) Cancer United Research Associating Medicine, University & Society (CURAMUS), Centre de Recherche Saint-Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France; Service de Neurologie 2-Mazarin, Sorbonne Université, Inserm, CNRS, UMR S 1127, Institut du Cerveau, ICM, AP-HP, Hôpitaux Universitaires La Pitié Salpêtrière-Charles Foix, Paris, France. 8. Service de Neurologie 2-Mazarin, Sorbonne Université, Inserm, CNRS, UMR S 1127, Institut du Cerveau, ICM, AP-HP, Hôpitaux Universitaires La Pitié Salpêtrière-Charles Foix, Paris, France; AP-HP, Hôpitaux Universitaires La Pitié Salpêtrière-Charles Foix, Département de Neuropathologie, Paris, France. 9. Université de Lille, CNRS, INSERM, CHU Lille, UMR9020-U1277 CANTHER-Cancer Heterogeneity Plasticity and Resistance to Therapies, Lille, France; Molecular Oncogenetics, Department of Biochemistry and Molecular Biology, Lille University Hospital, Lille, France. 10. Université de Lille, CNRS, INSERM, CHU Lille, UMR9020-U1277 CANTHER-Cancer Heterogeneity Plasticity and Resistance to Therapies, Lille, France; Department of Digestive and Oncological Surgery, Claude Huriez University Hospital, University Lille, Lille, France. 11. Department of Medical Oncology, Centre Georges-François Leclerc, Dijon, France; INSERM, UMR866, Burgundy University, Dijon, France. 12. Sorbonne Université, INSERM, Unité Mixte de Recherche Scientifique 938 and Site de Recherche Intégrée sur le Cancer (SIRIC) Cancer United Research Associating Medicine, University & Society (CURAMUS), Centre de Recherche Saint-Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France; Sorbonne Université, Genetics Department, AP-HP, Hospital Pitié-Salpêtrière, Paris, France. Electronic address: alex.duval@inserm.fr.
Abstract
BACKGROUND & AIMS: Next-generation sequencing (NGS) was recently approved by the United States Food and Drug Administration to detect microsatellite instability (MSI) arising from defective mismatch repair (dMMR) in patients with metastatic colorectal cancer (mCRC) before treatment with immune checkpoint inhibitors (ICI). In this study, we aimed to evaluate and improve the performance of NGS to identify MSI in CRC, especially dMMR mCRC treated with ICI. METHODS: CRC samples used in this post hoc study were reassessed centrally for MSI and dMMR status using the reference methods of pentaplex polymerase chain reaction and immunohistochemistry. Whole-exome sequencing (WES) was used to evaluate MSISensor, the Food and Drug Administration-approved and NGS-based method for assessment of MSI. This was performed in (1) a prospective, multicenter cohort of 102 patients with mCRC (C1; 25 dMMR/MSI, 24 treated with ICI) from clinical trials NCT02840604 and NCT033501260, (2) an independent retrospective, multicenter cohort of 113 patients (C2; 25 mCRC, 88 non-mCRC, all dMMR/MSI untreated with ICI), and (3) a publicly available series of 118 patients with CRC from The Cancer Genome Atlas (C3; 51 dMMR/MSI). A new NGS-based algorithm, namely MSICare, was developed. Its performance for assessment of MSI was compared with MSISensor in C1, C2, and C3 at the exome level or after downsampling sequencing data to the MSK-IMPACT gene panel. MSICare was validated in an additional retrospective, multicenter cohort (C4) of 152 patients with new CRC (137 dMMR/MSI) enriched in tumors deficient in MSH6 (n = 35) and PMS2 (n = 9) after targeted sequencing of samples with an optimized set of microsatellite markers (MSIDIAG). RESULTS: At the exome level, MSISensor was highly specific but failed to diagnose MSI in 16% of MSI/dMMR mCRC from C1 (4 of 25; sensitivity, 84%; 95% confidence interval [CI], 63.9%-95.5%), 32% of mCRC (8 of 25; sensitivity, 68%; 95% CI, 46.5%-85.1%), and 9.1% of non-mCRC from C2 (8 of 88; sensitivity, 90.9%; 95% CI, 82.9%-96%), and 9.8% of CRC from C3 (5 of 51; sensitivity, 90.2%; 95% CI, 78.6%-96.7%). Misdiagnosis included 4 mCRCs treated with ICI, of which 3 showed an overall response rate without progression at this date. At the exome level, reevaluation of the MSI genomic signal using MSICare detected 100% of cases with true MSI status among C1 and C2. Further validation of MSICare was obtained in CRC tumors from C3, with 96.1% concordance for MSI status. Whereas misdiagnosis with MSISensor even increased when analyzing downsampled WES data from C1 and C2 with microsatellite markers restricted to the MSK-IMPACT gene panel (sensitivity, 72.5%; 95% CI, 64.2%-79.7%), particularly in the MSH6-deficient setting, MSICare sensitivity and specificity remained optimal (100%). Similar results were obtained with MSICare after targeted NGS of tumors from C4 with the optimized microsatellite panel MSIDIAG (sensitivity, 99.3%; 95% CI, 96%-100%; specificity, 100%). CONCLUSIONS: In contrast to MSISensor, the new MSICare test we propose performs at least as efficiently as the reference method, MSI polymerase chain reaction, to detect MSI in CRC regardless of the defective MMR protein under both WES and targeted NGS conditions. We suggest MSICare may rapidly become a reference method for NGS-based testing of MSI in CRC, especially in mCRC, where accurate MSI status is required before the prescription of ICI.
BACKGROUND & AIMS: Next-generation sequencing (NGS) was recently approved by the United States Food and Drug Administration to detect microsatellite instability (MSI) arising from defective mismatch repair (dMMR) in patients with metastatic colorectal cancer (mCRC) before treatment with immune checkpoint inhibitors (ICI). In this study, we aimed to evaluate and improve the performance of NGS to identify MSI in CRC, especially dMMR mCRC treated with ICI. METHODS: CRC samples used in this post hoc study were reassessed centrally for MSI and dMMR status using the reference methods of pentaplex polymerase chain reaction and immunohistochemistry. Whole-exome sequencing (WES) was used to evaluate MSISensor, the Food and Drug Administration-approved and NGS-based method for assessment of MSI. This was performed in (1) a prospective, multicenter cohort of 102 patients with mCRC (C1; 25 dMMR/MSI, 24 treated with ICI) from clinical trials NCT02840604 and NCT033501260, (2) an independent retrospective, multicenter cohort of 113 patients (C2; 25 mCRC, 88 non-mCRC, all dMMR/MSI untreated with ICI), and (3) a publicly available series of 118 patients with CRC from The Cancer Genome Atlas (C3; 51 dMMR/MSI). A new NGS-based algorithm, namely MSICare, was developed. Its performance for assessment of MSI was compared with MSISensor in C1, C2, and C3 at the exome level or after downsampling sequencing data to the MSK-IMPACT gene panel. MSICare was validated in an additional retrospective, multicenter cohort (C4) of 152 patients with new CRC (137 dMMR/MSI) enriched in tumors deficient in MSH6 (n = 35) and PMS2 (n = 9) after targeted sequencing of samples with an optimized set of microsatellite markers (MSIDIAG). RESULTS: At the exome level, MSISensor was highly specific but failed to diagnose MSI in 16% of MSI/dMMR mCRC from C1 (4 of 25; sensitivity, 84%; 95% confidence interval [CI], 63.9%-95.5%), 32% of mCRC (8 of 25; sensitivity, 68%; 95% CI, 46.5%-85.1%), and 9.1% of non-mCRC from C2 (8 of 88; sensitivity, 90.9%; 95% CI, 82.9%-96%), and 9.8% of CRC from C3 (5 of 51; sensitivity, 90.2%; 95% CI, 78.6%-96.7%). Misdiagnosis included 4 mCRCs treated with ICI, of which 3 showed an overall response rate without progression at this date. At the exome level, reevaluation of the MSI genomic signal using MSICare detected 100% of cases with true MSI status among C1 and C2. Further validation of MSICare was obtained in CRC tumors from C3, with 96.1% concordance for MSI status. Whereas misdiagnosis with MSISensor even increased when analyzing downsampled WES data from C1 and C2 with microsatellite markers restricted to the MSK-IMPACT gene panel (sensitivity, 72.5%; 95% CI, 64.2%-79.7%), particularly in the MSH6-deficient setting, MSICare sensitivity and specificity remained optimal (100%). Similar results were obtained with MSICare after targeted NGS of tumors from C4 with the optimized microsatellite panel MSIDIAG (sensitivity, 99.3%; 95% CI, 96%-100%; specificity, 100%). CONCLUSIONS: In contrast to MSISensor, the new MSICare test we propose performs at least as efficiently as the reference method, MSI polymerase chain reaction, to detect MSI in CRC regardless of the defective MMR protein under both WES and targeted NGS conditions. We suggest MSICare may rapidly become a reference method for NGS-based testing of MSI in CRC, especially in mCRC, where accurate MSI status is required before the prescription of ICI.