Table 1_Evaluation of microsatellite instability patterns in mismatch repair deficiency: a retrospective analysis of 285 endometrial cancers.docx

ObjectiveIn this study, we systematically compared the microsatellite shift patterns detected by PCR-based microsatellite instability analysis (PCR-MSI) in mismatch repair (MMR)-deficient ECs and analyzed the clinicopathological features associated with minimal versus major shifts.
MethodWe evaluated the microsatellite shift patterns using five NCI-recommended loci in 285 MMR-deficient ECs identified through immunohistochemistry (IHC). A minimal shift was defined as a one-to-three nucleotide repeat shift observed at least at one locus. Then, clinicopathological characteristics were analyzed for two distinct groups: the minimal shift group and the major shift group. Finally, further analysis of MMR/MSI discordant cases was performed through MLH1 promoter methylation detection and MMR gene germline mutation detection.
ResultOf the 285 MMR-deficiency ECs, 169 (59.3%) had combined loss of MLH1 and PMS2, 54 (18.9%) had combined loss of MSH2 and MSH6, 39 (13.7%) had isolated loss of MSH6 and 23 (8.1%) had isolated loss of PMS2 by IHC. The rate of inconsistency between MMR-IHC and PCR-MSI was 12.3% (35/285). However, based on the minimal shifting criteria, 13 cases with MSI-L were reassessed as MSI-H because of the occurrence of minimal microsatellite shifts, and the inconsistency rate between MMR-IHC and MSI-PCR decreased to 7.7% (22/285). Additionally, discordant cases showed a higher frequency (91%, 20/22 cases) of minimal shift involving the mononucleotide locus. Among the 7 MLH1/PMS2-deficient cases, 3 were successfully detected and showed MLH1 promoter methylation. A total of 13 of 22 patients were successfully completed MMR gene germline testing, 11 cases had germline mutations in MSH6 and 3 cases harbored frameshift deletions (p.F1088Lfs*). Overall, the frequency of minimal shift was 100% (39/39) at isolated loss of MSH6, 85.8% (145/169) at the loss of MLH1 and PMS2, 66.7% (36/54) at the loss of MSH2 and MSH6, and 47.9% (11/23) at the isolated loss of PMS2, respectively. There is no correlation between minimal shift group or major shift group and clinicopathological features.
ConclusionMMR-deficient ECs exhibit a high frequency of minimal microsatellite shifts, particularly in cases with isolated loss of MSH6. The combination of MMR-IHC and MSI-PCR assays could enhance the accuracy of MSI detection, thereby facilitating more precise treatment strategies of ECs.

**研究目的**：本研究系统比较了错配修复（MMR）缺陷型子宫内膜癌（endometrial carcinomas, ECs）中基于聚合酶链反应的微卫星不稳定性分析（PCR-MSI）所检测到的微卫星偏移模式，并分析了与轻度偏移和重度偏移相关的临床病理特征。
**方法**：本研究通过免疫组化（immunohistochemistry, IHC）鉴定出285例MMR缺陷型ECs，采用美国国家癌症研究所（National Cancer Institute, NCI）推荐的5个微卫星位点评估微卫星偏移模式。本研究将轻度偏移定义为至少在1个位点上出现1~3个核苷酸重复序列的偏移。随后，针对轻度偏移组和重度偏移组这两个独立队列，分析其临床病理特征。最后，通过MLH1启动子甲基化检测及MMR基因生殖系突变检测，对MMR/MSI不一致病例开展进一步分析。
**结果**：经IHC检测，285例MMR缺陷型ECs中，169例（59.3%）表现为MLH1与PMS2联合缺失，54例（18.9%）表现为MSH2与MSH6联合缺失，39例（13.7%）为MSH6单独缺失，23例（8.1%）为PMS2单独缺失。MMR-IHC与PCR-MSI的不一致率为12.3%（35/285）。然而，基于轻度偏移判定标准，13例原归类为微卫星低度不稳定性（MSI-L）的病例因出现微卫星轻度偏移被重新判定为微卫星高度不稳定性（MSI-H），此时MMR-IHC与MSI-PCR的不一致率降至7.7%（22/285）。此外，不一致病例中涉及单核苷酸位点的轻度偏移发生率更高，达91%（20/22例）。在7例MLH1/PMS2缺陷型病例中，3例成功检出MLH1启动子甲基化。22例患者中共13例完成了MMR基因生殖系检测，其中11例存在MSH6生殖系突变，3例携带移码缺失突变（p.F1088Lfs*）。总体而言，MSH6单独缺失病例的轻度偏移发生率为100%（39/39），MLH1与PMS2联合缺失病例为85.8%（145/169），MSH2与MSH6联合缺失病例为66.7%（36/54），PMS2单独缺失病例为47.9%（11/23）。轻度偏移组与重度偏移组的临床病理特征均无显著相关性。
**结论**：MMR缺陷型ECs存在较高的微卫星轻度偏移发生率，尤以MSH6单独缺失病例为著。联合应用MMR-IHC与MSI-PCR检测可提升MSI检测的准确性，从而为ECs制定更精准的治疗策略提供依据。