Updated evaluation of clinicopathologic landscape in histopathologic subtypes of endometrial carcinoma with MMR gene mutation

Background: This study aimed to identify an optimal screening strategy to detect mismatch repair (MMR) mutation for each histologic subtype of endometrial carcinoma. Methods: We performed a comparative analysis of the demographic, clinical, pathologic, and molecular data for 562 patients from The Cancer Genome Atlas database, stratied by tumor histologic subtype.

There are numerous merits of detecting MMR de ciency among women with newly identi ed endometrial carcinoma. Firstly, surveillance testing for the patients with MMR mutation can detect Lynch Syndromeassociated cancers, like colorectal carcinoma and others. Second, relatives would have more chances of receiving surveillance testing, genetic counseling, as well as risk-reducing operations, including hysterectomy or colectomy. Finally, immunotherapy with PD-1/PD-L1 inhibitors have been a promising treatment for advanced/recurrent endometrial carcinoma with MMR mutations or microsatellite instability (MSI-H) [7,[15][16][17][18] and pembrolizumab was recommended by the National Comprehensive Cancer Network as a treatment option for recurrent MSI-H or MMR de cient endometrial cancer that has progressed after standard cytotoxic chemotherapy.
Currently, MLH1, MSH2, MSH6, PMS2, and EPCAM are routinely used in the screening strategy. MLH3, MSH3, and PMS1 associated with Lynch Syndrome are not routinely tested. This study aims to add them to the current gene panel and compare demographic, clinicopathologic, and molecular characteristics of patients with endometrial cancer-based mutation status and strati ed by histologic subtypes in order to provide evidence of optimal criteria for screening. These results may provide further evidence of the biology of MMR mutations, enhance clinical management of mutation carriers, and subsequently impact clinical trial design.

Methods
This study was approved by an institutional research ethics committee of Zhangjiagang TCM Hospital A liated to Nanjing University of Chinese Medicine (2019-023). The data released from the Cancer Genome Atlas database did not require informed consent of patients because cancers are reportable diseases in every state in the United States.

Study population
We evaluated data from the publicly available National Cancer Institute the Cancer Genome Atlas EC database [http://www.cbioportal.org]. The nal retrospective cohort was generated from 529 patients of the Pancancer Atlas and 548 patients from the Provisional database. All patients with endometrioid endometrial adenocarcinoma, or uterine serous carcinoma, or mixed endometroid and serous carcinoma or mixed clear and serous carcinoma were included in the study. After excluding duplicate patients from both databases (n = 515), the nal cohort was comprised of 562 patients. We strati ed the cohort into four groups of patients based on the MMR gene mutation status (MLH1, MLH3, PMS1, PMS2, MSH2, MSH3, MSH6, and EPCAM) and histology, including grade 1 endometroid endometrial carcinoma, grade 2 endometroid endometrial carcinoma, grade 3 endometroid endometrial carcinoma, and uterine serous carcinoma. Two experienced pathologists reviewed the pathologic diagnosis of all patients. We did not separate germline and somatic mutations. All patients were managed by National Comprehensive Cancer Loading [MathJax]/jax/output/CommonHTML/jax.js Network guidelines. The following variables were recorded for each case: age at diagnosis (categorized by <50, 50 -69, 70 years old), mean age at the diagnosis, myometrial invasion (categorized 50% myometrial invasion, < 50% myometrial invasion), lymph node involvement, and clinical stage (categorized by FIGO IA, IB, II, III, IV). De nition of overall survival (OS) was the interval from the initial surgery to con rmation of death. De nition of progression-free interval (PFI) was the interval from the date of last chemotherapy or radiotherapy to the date of recurrence.

Statistical Analysis
Statistical analysis was performed using the Mann-Whitney test for continuous variables, and Chi-Square analysis or Fisher exact test for categorical variables, unless otherwise speci ed. The Student's t test was performed in analyzing mean of body mass index and the age at diagnosis. Multivariate Cox proportional hazards models was used to analyze prognosis. OS or PFI was evaluated utilizing the log-rank test starting at the time of diagnosis, and the Kaplan-Meier method was used to generate survival curves. Statistical signi cance was evaluated at 0.05 level (exact 2-tailed). SPSS software (Version 23, IBM, USA) was applied in all calculations.   Of the 562 endometrial carcinomas, alterations in MSH2 (n = 55), MSH6 (n = 54), and MSH3 (n = 50) were the most frequent mutations. Among the USC, grade 3 endometroid endometrial carcinoma, grade 2 endometroid endometrial carcinoma, and grade 1 endometroid endometrial carcinoma, the most frequent alterations were MSH2, MSH3, PMS2, and MLH3, respectively (Table 1b). We analyzed MLH3, MSH,3 and PMS1 in this study since others have been well-studied. There were 30 patients with endometrial carcinoma with MLH3 mutation, including 9 patients with only MLH3 mutations and 21 women with MLH3 mutations cooccurrence with other MMR mutations (Table 1b). We found 50 patients with MSH3 mutation, including 17 patients with only MSH3 mutations and 33 women with MSH3 mutations cooccurrence with other MMR mutations (Table 1b). There were 20 patients with endometrial carcinoma with PMS1 mutation, including seven patients with only PMS1 mutations and 13 women with PMS1 mutations cooccurrence with other MMR mutations (Table 1b).

Results
Among eight genes, two genes expressed simultaneously, including ten combinations; co-occurrence of three genes had six combinations; expression of four genes simultaneously presented eight combinations; ve genes concurrently expressed two combinations; and cooccurrence of six genes had only 1 combination. (Table S1) Within grade 3 endometroid endometrial carcinoma, patients with MMR-mutated tumors were older and had lower body mass index than those with noncarriers (p = 0.024, p = 0.028, respectively) ( Table 2). Similarly, MMR de cient USC patients (Table 3) also had lower body mass index than MMR non carriers (p = 0.001). Lymph node metastasis was more frequent in patients with MMR mutations than noncarriers (p = 0.007). We found no differences in myometrial invasion, lymph node involvement, or FIGO stage between patients with MMR-mutated and nonmutated grade 3 endometroid endometrial carcinoma (Table 2). Similarly, patients with USC showed no association between age, body mass index, myometrial invasion, or FIGO stage and MMR status (   Favorable prognostic factors for all tumors included: myometrial invasion < 50% (OS and PFI), age < 50 years old (OS), minimal invasive surgery (PFI), and lymph nodes negative (OS) were good prognosis factors (Table 4). We found no signi cant differences in OS and PFI between MMR mutation carriers and nonmutation carriers (p = 0.647, p = 0.350, respectively) (Fig. 1A, 1B). In our sub-group analysis, minimally invasive surgery was a good prognosis factor in grade 2 endometroid endometrial carcinoma; myometrial invasion < 50% was a good prognosis factor in grade 3 endometroid endometrial carcinoma; minimal invasive surgery, lymph node negative, and early stage were prognosis factors in USC (  Fig. 1C, 1D). OS and PFI were not evaluated for grade 1 endometroid endometrial carcinoma due to limited cases with MMR mutation.  [19]. MLH1, MSH2, MSH6, PMS2, and EPCAM genes are most frequent and clinically related mutations in Lynch Syndrome [20,21]. One prospective study of over 3000 patients with Lynch Syndrome demonstrated that mutations occurred in MLH1, MSH2, and MSH6, which total incidences of endometrial carcinoma (less than age 75) to be 43%, 57%, and 46%, respectively [2]. However, our study demonstrated that MSH2 (n = 55), MSH3 (n = 50), and MSH6 (n = 54) mutations were the most frequent alterations among endometrial carcinomas. The total incidences of MSH2, MSH3, and MSH6 were much higher than previous literatures since we did not separate germline and somatic MMR mutation.
Limited data reported the role of MLH3, MSH3, and PMS1 mutation in colorectal or endometrial cancers [22][23][24][25] Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on request.

Competing interests
Authors declared no con icts of interests.

Funding
This study was not funded.
Author contribution AW and WH analyzed and interpreted the data and was a major contributor in writing the manuscript; AW, YC and QZ interpreted the data; QZ and JR reviewed all pathologic images and conformed pathologic diagnosis; ZZ and ZZ abstracted the data; AW, YC and QZ designed the work and interpreted the data. All authors read and approved the nal manuscript.