下一代测序在结直肠癌中检测微卫星不稳定性的性能，该肿瘤存在缺陷的 DNA 错配修复。

Performance of Next-Generation Sequencing for the Detection of Microsatellite Instability in Colorectal Cancer With Deficient DNA Mismatch Repair.

机构信息

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.

出版信息

Gastroenterology. 2021 Sep;161(3):814-826.e7. doi: 10.1053/j.gastro.2021.05.007. Epub 2021 May 13.

DOI:10.1053/j.gastro.2021.05.007

PMID:33992635

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.

摘要

背景与目的

新一代测序（NGS）最近获得美国食品和药物管理局批准，可在转移性结直肠癌（mCRC）患者接受免疫检查点抑制剂（ICI）治疗前，检测由缺陷错配修复（dMMR）引起的微卫星不稳定（MSI）。本研究旨在评估和改进 NGS 识别 CRC 中 MSI 的性能，特别是针对接受 ICI 治疗的 dMMR mCRC。

方法

本回顾性研究使用参考方法五重聚合酶链反应和免疫组化，对 CRC 样本的 MSI 和 dMMR 状态进行重新评估。全外显子组测序（WES）用于评估 MSISensor，这是一种经美国食品和药物管理局批准的、基于 NGS 的 MSI 评估方法。该方法在以下三个方面进行了评估：（1）一项前瞻性、多中心的临床试验 NCT02840604 和 NCT033501260 中的 102 例 mCRC 患者（C1；25 例 dMMR/MSI，24 例接受 ICI 治疗）的队列研究；（2）一项独立的回顾性、多中心的 113 例患者（C2；25 例 mCRC，88 例非 mCRC，均未接受 ICI 治疗）的队列研究；（3）来自癌症基因组图谱（C3）的 118 例 CRC 患者的公开可用系列研究。开发了一种新的基于 NGS 的算法，即 MSICare。在 C1、C2 和 C3 中，我们将其在全外显子组水平或对 WES 数据进行下采样至 MSK-IMPACT 基因面板后，用于评估 MSI 的性能，并与 MSISensor 进行比较。在另一个多中心、回顾性队列研究（C4）中，我们对新的 CRC 患者（152 例，其中 137 例 dMMR/MSI）的样本进行靶向测序，优化了微卫星标记物的集合（MSIDIAG），验证了 MSICare 的性能。该队列中，MSH6（n=35）和 PMS2（n=9）缺失的肿瘤患者比例较高。

结果

在外显子组水平上，MSISensor 具有很高的特异性，但在 C1 中未能诊断出 16%的 MSI/dMMR mCRC（4/25；敏感性，84%；95%置信区间[CI]，63.9%-95.5%）、32%的 mCRC（8/25；敏感性，68%；95% CI，46.5%-85.1%）和 9.1%的非 mCRC（8/88；敏感性，90.9%；95% CI，82.9%-96%），以及 C3 中 9.8%的 CRC（5/51；敏感性，90.2%；95% CI，78.6%-96.7%）。误诊包括 4 例接受 ICI 治疗的 mCRC，其中 3 例在该日期时无进展且整体反应率较高。在外显子组水平上，使用 MSICare 重新评估 MSI 基因组信号可检测出 C1 和 C2 中所有具有真实 MSI 状态的病例。在 C3 的 CRC 肿瘤中进一步验证了 MSICare，MSI 状态的一致性为 96.1%。在外显子组水平上，即使对 C1 和 C2 的 WES 数据进行下采样并限制在 MSK-IMPACT 基因面板上分析微卫星标记物（敏感性，72.5%；95% CI，64.2%-79.7%）时，MSISensor 的误诊率甚至更高，特别是在 MSH6 缺失的情况下，MSICare 的敏感性和特异性仍然最佳（100%）。使用优化后的微卫星面板 MSIDIAG 对 C4 中肿瘤进行靶向 NGS 后，也获得了类似的结果（敏感性，99.3%；95% CI，96%-100%；特异性，100%）。

结论

与 MSISensor 相比，我们提出的新 MSICare 测试至少与参考方法、MSI 聚合酶链反应一样高效，可在 WES 和靶向 NGS 条件下检测 CRC 中的 MSI，无论缺陷错配修复蛋白如何。我们建议 MSICare 可能很快成为 CRC 中基于 NGS 的 MSI 检测的参考方法，特别是在 mCRC 中，在开具 ICI 处方之前需要准确的 MSI 状态。