第一部分

错配修复缺陷的子宫内膜样癌中微卫星不稳定性分析

中文摘要

背景：

子宫内膜癌(Endometrial Cancer，EC)是女性生殖系统最常见的恶性肿瘤之一，主要的组织学类型为子宫内膜样癌（endometroid endometrial carinoma，）。DNA 错配修复（mismatch repair，MMR）系统是DNA损伤修复机制的主要类型之一，由于MMR基因的突变或MLH1启动子甲基化状态导致MMR蛋白功能缺陷，进而引起微卫星（Microsatellite,MS）长度发生改变，出现微卫星不稳定（microsatellite instability，MSI）。在不同组织学类型的EC中，DNA错配修复功能缺陷（deficient mismatch repair，dMMR）和微卫星高度不稳定（MSI-High,MSI-H）主要见于EEC。临床上主要采用免疫组化（immunohistochemistry，IHC）技术检测MMR蛋白的表达情况，采用下一代测序技术（Nest generation sequencing technology，NGS）和PCR技术检测MSI ，以评估肿瘤MMR/MSI状态。本研究选取了dMMR EEC作为研究对象，对比了IHC检测dMMR蛋白表达情况和NGS技术检测MSI状态的结果，探索不一致的原因，期望为EEC的MMR/MSI分子病理检测方法选择提供指导。

目的：

研究dMMR EEC的MSI状态，探讨MMR/MSI分子病理检测方法间的一致性和优缺点。

探索MMR/MSI检测结果不一致的机制。

方法：

选取北京协和医院病理科IHC检测结果为dMMR的EEC病例60例，并对IHC结果进行复核。利用NGS对60例样本进行MSI状态及MMR基因胚系/体细胞突变状态的检测。MLH1蛋白表达缺失的病例用甲基化特异性PCR(methylation-specific PCR, MSP)技术检测MLH1启动子甲基化状态。在MMR IHC和MSI NGS检测结果不相符的样本中，用PCR法再次检测MSI状态。结合MMR基因突变及MLH1启动子甲基化检测，探索结果不一致的原因。

结果：

在IHC结果为dMMR的 60例EEC样本中，3例经复核为MMR蛋白表达完整（proficient mismatch repair，pMMR），57例被确认为dMMR病例。57例dMMR EEC中，3例肿瘤细胞含量不足。其余54例dMMR EEC中，47例MSI NGS检测结果为MSI-H，7例结果为MSS，dMMR与MSI-H的符合率为87%（47/54）。7例不符合病例经PCR再次检测MSI状态：MSI-H 1例；MSS 5例；剩余样本不足1例。57例dMMR的EEC病例中：7例存在MMR基因胚系突变,确诊为林奇综合征（Lynch syndrome，LS）；35例为MLH1启动子高甲基化状态；剩余15例为林奇样综合征（Lynch-Like Syndrome，LLS）。

结论：

在IHC检测结果为dMMR 的EEC中，绝大多数病例NGS/PCR检测结果为MSI-H，一致性较高。

的确存在少数dMMR EEC病例表现为MSS，多见于MLH1启动子高甲基化状态造成的MLH1/PMS2表达共缺失病例。

dMMR EEC病例基于分子发生机制的不同可以分为LS、MLH1启动子高甲基化、LLS 3个亚组，其中MLH1启动子高甲基化组病例占主要部分。

MMR IHC和MSI NGS/PCR检测方法各有优缺点，必要时可以互为补充。

第二部分

不同分子机制的错配修复缺陷型子宫内膜样癌临床病理特征分析

中文摘要

背景：

子宫内膜癌(Endometrial Cancer，EC)是我国女性生殖系统中常见的恶性肿瘤，病理学类型主要为子宫内膜样癌（endometroid endometrial carinoma，EEC）。错配修复缺陷（deficient mismatch repair，dMMR）的EEC分子致病机制复杂多样，国内外研究中尚未发现明确的分类方法，对不同分子机制病例的临床病理特征研究成果不显著。

目的：

了解dMMR型EEC的分子致病机制，探讨根据不同分子机制进行分类的方法。

分析不同分子致病机制的dMMR型EEC的临床病理特征。

了解不同分子致病机制的dMMR型EEC的预后。

方法：

实验选取2017年11月—2019年02月北京协和医院病理科诊断为dMMR的EEC连续病例，复核免疫组化结果。收集临床病理资料，包括患者的年龄、肿瘤的分化程度、错配修复（mismatch repair，MMR）蛋白的表达情况、TNM分期、FIGO分期、有无脉管内瘤栓、手术时间。通过电话随访了解患者病情是否进展（复发或转移）、进展时间；是否死亡、死亡时间。采用下一代测序技术（Nest generation sequencing technology，NGS）检测病例样本的MMR基因胚系突变情况，未发生胚系突变且MLH1/PMS2蛋白表达共缺失的样本经甲基化特异性PCR（Methylation-Specific PCR，MSP）检测MLH1启动子甲基化状态。根据NGS和MSP结果将病例分为胚系突变/林奇综合征（Lynch syndrome，LS）组、MLH1启动子高甲基化组、林奇样综合征（Lynch-Like Syndrome，LLS）组。分析3组病例的临床病理特征及预后。

结果：

56例dMMR的EEC病例中：胚系突变/LS组7例（12.5%），平均发病年龄和中位发病年龄分为45.0岁和42.0岁，分期见于T1期（85.7%）和T3期 (14.3%)、N0期（100.0%)、M0期（100.0%）、FIGOⅠ期(85.7%)、FIGOⅢ期（14.3%），以高分化（71.4%）和中分化（14.2%）多见,14.3%的病例可见脉管内瘤栓，无肿瘤复发和（或）转移病例，无死亡病例；MLH1启动子高甲基化组34例（60.7%），平均发病年龄和中位发病年龄分为56.9岁和55.5岁，分期见于T1期（88.3%）、T3期（8.8%)、T4期 (2.9%)、FIGOⅠ期（82.4%）、FIGOⅢ期（14.7%）、FIGOⅣ期（2.9%）, 以高分化（35.3%）和中分化（44.1%）多见,23.5%的病例可见脉管内瘤栓，3例患者出现肿瘤复发和转移，1例患者死亡；LLS组15例（26.7%），平均发病年龄和中位发病年龄分为56.7岁和54.0岁，以高分化（46.7%）和中分化（33.3%）多见,分期见于T1期（93.3%）、T2期 (6.7%)、N0期（100.0%）、M0期（100.0%）、FIGOⅠ期（93.3%）、FIGOⅡ期（6.7%）,40.0%的病例可见脉管内瘤栓，无肿瘤复发和转移病例，无死亡病例。3组病例在发病年龄上具有统计学差异(P<0.05),在分化程度、T期、N期、M期、FIGO分期、有无脉管内瘤栓和无进展生存期（PFS）上无统计学差异（P>0.05）。

结论：

dMMR的EEC病例基于dMMR分子机制的不同可以分为胚系突变/LS（12.5%）、MLH1启动子高甲基化（60.7%）、LLS（26.7%）3个亚组。

胚系突变/LS相关的dMMR的EEC患者发病年龄要早于MLH1启动子高甲基化和LLS的患者。

3个分子亚组病例在分化程度、TNM分期、FIGO分期、脉管内瘤栓方面具有相似的临床病理特征。

dMMR的EEC患者在术后4年内较少出现肿瘤的复发和转移，较少出现死亡。胚系突变/LS、MLH1启动子高甲基化和LLS 3个亚组在PFS上无统计学差异。

Analysis of microsatellite instability in endometroid endometrial carcinoma with deficient mismatch repair

Background: 

The DNA mismatch repair (MMR) system is one of the major types of DNA damage repair mechanisms, where mutations in the MMR gene or the MLH1 promoter methylation status lead to defective MMR protein function, which in turn causes microsatellite (MS) length changes and microsatellite instability (MSI). In different histologic types of EC, deficient mismatch repair (dMMR) and microsatellite High (MSI-H) are mainly found in EEC. In clinical, the expression of MMR protein was detected by Immunohistochemistry (IHC), MSI status was detected by Nest Generation Sequencing technology (NGS) and PCR. This study compared the results of dMMR by IHC and MSI by NGS，and the reasons for the discrepancy were discussed with gene mutation and MLH1 promoter methylation results.The aim is to provide guidance for the selection of MMR/MSI molecular pathological detection methods in EEC.

Objective: 

To analyze the microsatellite instability (MSI) in endometrioid endometrial carcinoma (EEC) with deficient mismatch repair (dMMR)，and to explore the concordance between MSI next generation sequencing (NGS)/PCR and MMR immunohistochemistry(IHC).

To explore the reasons for discrepant results between MMR IHC and MSI NGS.

Methods:

Sixty dMMR EEC cases by IHC were selected from the Department of Pathology of Peking Union Medical College Hospital. Two pathologists reviewed the IHC results. The MSI status and the germline/somatic mutational status of MMR genes were analyzed by NGS. MLH1 promoter methylation status was determined by methylation-specific PCR (MSP) in cases with MLH1 protein deficiency. In cases with discrepant results between MMR IHC and MSI NGS, the MSI status was detected again by PCR, and the reasons for the discrepancy were discussed with gene mutation and MLH1 promoter methylation results.

Results:

Among the 60 EEC samples with IHC results of dMMR, 3 cases were reclassified and determined to have intact MMR protein expression (proficient mismatch repair, pMMR) after pathological review. And the remaining 57 cases were confirmed as dMMR cases, among which 3 contained insufficient tumor cells for subsequent molecular analyses. Among the remaining 54 dMMR EEC cases, 47 were MSI-H by NGS, 7 cases were MSS, with a concordance rate of 87% (47/54) between IHC and NGS methods. The 7 non-concordant cases were tested again by PCR for MSI status and revealed 1 case of MSI-H; 5 cases of MSS; the remaining case did not have sufficient specimen for the test. Of the 57 EEC cases with dMMR, 7 cases had germline mutations in the MMR gene and were diagnosed with Lynch syndrome (LS); 35 cases had MLH1 promoter hypermethylation; the remaining 15 cases had Lynch-Like syndrome (LLS).

Conclusion:

1. Among the EEC with dMMR results by IHC, the majority of cases were MSI-H by NGS/PCR, showing a good concordance.

2. A few dMMR EEC cases do exhibit MSS, mostly in cases with loss of MLH1/PMS2 expression due to MLH1 promoter hypermethylation status.

3. dMMR EEC cases can be divided into 3 subgroups based on the different molecular mechanisms of occurrence: LS, MLH1 promoter hypermethylation, and LLS, with cases in the MLH1 promoter hypermethylation group accounting for the major part.

4. The MMR IHC and MSI NGS/PCR assays have their advantages and disadvantages and can complement each other when necessary.

Clinicopathological characteristics of different molecular subtype in endometrioid carcinoma with mismatch repair deficiency

Background:

Endometrial carcinoma (EC) is a common malignant tumor in the female reproductive system in China. The main pathological type is Endometroid Endometrial carinoma (EEC). The EEC molecular mechanism of deficient mismatch repair (dMMR) is complex. There are no clear classification methods that have been found in domestic and overseas researches. The clinicopathological characteristics of cases with different molecular mechanisms are not clear.

Objective:

To know the molecular pathological mechanism of EEC with dMMR, and to explore the methods of classification according to different molecular pathological mechanisms.

To analyze the clinicopathological features of dMMR EEC with different molecular pathological mechanisms.

To know the prognosis of dMMR EEC with different molecular pathological mechanisms.

Methods:

Sixty dMMR EEC cases by IHC were selected from the Department of Pathology of Peking Union Medical College Hospital. Two pathologists reviewed the IHC results. Information about clinicopathologicalcharacteristics were collected, including age, pathologic grade, MMR protein expression, TNM stage, FIGO stage, lymph-vascular invasion, operative time，recurrence or metastasis，time of progression，death or not and time of death. The MSI status and the germline/somatic mutational status of MMR genes were analyzed by NGS. MLH1 promoter methylation status was determined by methylation-specific PCR (MSP) in cases with MLH1 protein deficiency. According to the results of NGS and MSP, the patients were divided into 3 groups (germline mutation /LS, MLH1 promoter hypermethylation , LLS). The clinicopathological characteristics and prognosis of the 3 groups of cases were analyzed.

Results:

Amongthe56 EEC cases of dMMR, 7 cases (12.5%) were in the germline mutation/LS group. In the 7 cases, the average age of onset and median age of onset were 45.0 years old and 42.0 years old. The stages were in T1 (85.7%) ,T3 (14.3) %), N0 stage (100.0%), M0 stage (100.0%), FIGO stage Ⅰ(85.7%) and FIGO stage Ⅲ(14.3%), well differentiated (71.4%) and moderately differentiated (14.2%) were more common, 14.3% of the cases were lymph-vascular invasion, no tumor recurrence and/or metastasis cases and no death caseswere found; 34 cases (60.7%) were in the MLH1 promoter hypermethylation group. In the 34 cases, the average age of onset and median age of onset were 56.9 years old and 55.5 years old .The stages were in T1 (88.3%), T3 (8.8%), T4 (2.9%), FIGO I (82.4%), FIGO III (14.7%) and FIGO IV (2.9%), well differentiated (35.3%) and moderately differentiated (44.1%) were more common, 23.5% of the cases were lymph-vascular invasion, 3 patients had tumor recurrence and metastasis, and one patient died; 15 cases (26.7%) were in the LLS group. In the 15 cases, the average age of onset and the moderate age of onset were 56.7 years and 54.0 years old, well differentiated (46.7%) and moderately differentiated (33.3%) were more common. The stages were in T1 (93.3%), T2 (6.7%), N0 (100.0%), M0 stage (100.0%), FIGO stage Ⅰ(93.3%), FIGO stage Ⅱ(6.7%). 40% of the cases were lymph-vascular invasion, no tumor recurrence and/or metastasis cases and no death caseswere found. The three groups of cases had statistical differences in age of onset (P<0.05), but no differences in degree of tumor differentiation, T stage, N stage, M stage, FIGO staging, lymph-vascular invasion, and progression-free survival (PFS) ( P>0.05).

Conclusion:

DMMR EEC cases can be divided into three subgroups based on the molecular pathogenicity mechanisms: germline mutation/LS (12.5%), MLH1 promoter hypermethylation (60.7%), and LLS (26.7%).

The age of onset of dMMR EEC patients with germline mutation/LS is earlier than patients with MLH1 promoter hypermethylation and LLS.

Three subgroups of germline mutation/LS, MLH1 promoter hypermethylation and LLS have similar clinicopathological characteristics in degree of tumor differentiation, T stage, N stage, M stage, FIGO staging, lymph-vascular invasion.

DMMR EEC patients have fewer tumor recurrences or/and metastases and fewer deaths within 4 years after surgery. The three subgroups of germline mutation/LS, MLH1 promoter hypermethylation and LLS had no statistical difference in PFS.