Microsatellite instability-high endometrial cancers with MLH1 promoter hypermethylation have distinct molecular and clinical profiles

Abstract

Purpose:

Microsatellite instability-high (MSI-H) endometrial carcinomas (ECs) are underpinned by distinct mechanisms of DNA mismatch repair deficiency (MMR-D). We sought to characterize the clinical and genetic features of MSI-H ECs harboring germline or somatic mutations in MMR genes or MLH1 promoter hypermethylation (MLH1ph).

Design:

Of >1,100 EC patients that underwent clinical tumor-normal sequencing, 184 had MSI-H ECs due to somatic MMR mutations or MLH1ph, or harbored pathogenic germline MMR mutations. Clinicopathologic features, mutational landscape, and tumor infiltrating lymphocyte (TIL) scores were compared among MMR-D groups using non-parametric tests. Log-rank tests were used for categorical associations; Kaplan-Meier method and Wald-test based on Cox proportional-hazards models were employed for continuous variables and survival analyses.

Results:

Compared to patients with germline (n=25) and somatic (n=39) mutations, patients with MLH1ph ECs (n=120) were older (p<0.001), more obese (p=0.001) and had more advanced disease at diagnosis (p=0.025). MLH1ph ECs were enriched for JAK1 somatic mutations as opposed to germline MMR-D ECs which showed enrichment for pathogenic ERBB2 mutations. MLH1ph ECs exhibited lower tumor mutational burden and TIL scores compared to ECs harboring germline or somatic MMR mutations (p<0.01). MLH1ph EC patients had shorter progression-free survival (PFS) on univariate analysis, but in multivariable models stage at diagnosis remained the only predictor of survival. For stage I/II EC, two-year PFS was inferior for patients with MLH1ph ECs compared to germline and somatic MMR groups (70% vs. 100%, respectively).

Conclusions:

MLH1ph ECs likely constitute a distinct clinicopathologic entity compared to germline and somatic MMR-D ECs with potential treatment implications.

INTRODUCTION

DNA mismatch repair deficiency (MMR-D) or high microsatellite instability (MSI-H) (1) is present in 20-40% of endometrial carcinomas (ECs) and defines distinct molecular subclasses of EC (2). The molecular mechanisms of MMR-D/MSI-H ECs include epigenetic MLH1 promoter hypermethylation (MLH1ph; 70-75%), somatic (15-20%) and germline (5-10%) mutations in MLH1, MSH2, MSH6, PMS2 and/or EPCAM (3,4).

MMR-D/MSI-H ECs exhibit specific pathologic features, including a predominance of tumor-infiltrating lymphocytes (TILs) (3,5). Accordingly, immune checkpoint inhibitors (ICI) have been shown to be effective for patients with MSI-H ECs, and pembrolizumab and dostarlimab have been FDA-approved for the treatment of recurrent MMR-D/MSI-H EC (6,7).

Differences in immunogenicity of tumors and patient outcomes between various MMR-D/MSI-H mechanisms are poorly understood (8,9). Previously published data suggested that MMR-D ECs may have increased recurrence rates (10), in particular MLH1ph tumors (11). Data are conflicting, however, and other studies reported better outcomes for MSI-H EC patients compared to those with copy-number high ECs, suggesting heterogeneity among MMR-D/MSI-H tumors (2). The potential differences in outcomes of MSI-H ECs, if confirmed, could have implications on treatment selection, given that prior clinical trials and subsequent approvals for ICIs have included all MMR-D ECs regardless of the underlying mechanism of MMR-D (6,7). Whether the genomic landscapes vary according to MMR-D mechanism is currently unknown.

We sought to determine whether the clinicopathologic characteristics, genomic features (e.g. tumor mutational burden (TMB) and mutational signatures), and levels of immune infiltration would vary in ECs according to the mechanism of MMR-D (i.e., germline, somatic or MLH1ph). In addition, we investigated the impact of the MMR-D mechanism on oncologic outcomes of MSI-H EC patients, including response to ICI.

MATERIALS AND METHODS

Case selection

Of 1,157 consented EC patients who underwent clinical FDA-authorized tumor-normal sequencing using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) between 03/2015-07/2020, 202 had MSI-H tumors using MSIsensor or pathogenic germline mutations affecting MLH1, MSH2, MSH6, PMS2 or EPCAM. ECs with pathogenic POLE mutations (n=6) (12), non-uterine primary sites (n=2), or no MMR immunohistochemistry data (n=10) were excluded, yielding a final cohort of 184 patients (Figure 1). Molecular and clinicopathologic analyses were conducted using the entire cohort. To minimize referral biases, clinical outcomes were assessed only in patients who had their initial treatment at MSK and MSK-IMPACT sequencing within 6 months of diagnosis (n=119; Figure 1). Women who presented to MSK at time of recurrence were excluded from the analyses of clinical outcomes, as previously described (13). This study has been approved by the institutional review board (IRB) of MSK, and written informed consent was obtained from all patients. This study was conducted in accordance with the Declaration of Helsinki.

Figure 1. CONSORT diagram summarizing selection process and final series of endometrial cancer patients included in this study.

Abbreviations: EC, endometrial cancer; IHC, immunohistochemistry; MMR, mismatch repair; MSI-H, microsatellite instability-high; POLE, DNA polymerase epsilon, catalytic subunit.

Massively parallel sequencing analysis and genomic data extraction

Massively parallel sequencing was performed on primary (n=153) or recurrent (n=31) ECs and matched normal blood using the MSK-IMPACT assay targeting 341 (n=1), 410 (n=13) or 468 (n=170) cancer-related genes (14,15). The median sequencing depth was 637x (range 129x-1,368x). Relevant genomic data derived from MSK-IMPACT included, but was not limited to, somatic and germline mutations in MLH1, MSH2, MSH6, PMS2 and EPCAM, MSIsensor score (MSIsensor, RRID:SCR_006418) (16,17), and TMB (somatic mutations per Mb). MSIsensor scores of ≥10 were considered MSI-H, ≥3 to <10 MSI-indeterminate, and <3 microsatellite stable (MSS) (16,17). Mutational signatures (COSMIC, version 3.1) were defined using deconstructSigs at default parameters for ECs with ≥20 single nucleotide variants (SNVs) (18), as previously described (19). Germline sequencing was available for 179 (97%) patients, and for those with pathogenic germline variants, loss of heterozygosity (LOH) of the wild-type allele was assessed in tumors using FACETS (20).

Orthogonal assessment of MMR status

Immunohistochemistry (IHC) for MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed in all 184 ECs to confirm the underlying MMR alteration (21). An aberrant staining pattern was defined as loss of nuclear immunoreactivity in the tumor cells. Nuclei of adjacent non-neoplastic tissue (e.g., immune and/or stromal cells) served as a positive internal control. ECs exhibiting absent nuclear MLH1 and/or PMS2 staining by IHC underwent MLH1ph testing. MLH1ph status was determined by the bisulfite mediated detection of methylated cytosines, as previously described (22).

Classification of MMR alterations

ECs were classified by mechanism of MMR (i.e., germline, somatic or MLH1ph) and by affected gene (i.e., MLH1, MSH2, MSH6, PMS2); no pathogenic EPCAM alterations were identified. ECs from patients harboring pathogenic/likely pathogenic germline variants in MMR genes were classified as MMR germline-altered (Lynch) according to the American College of Medical Genetics (ACMG) guidelines (23). For ECs harboring pathogenic somatic MMR mutations, tumors were classified according to the gene harboring two pathogenic alterations or based on the presence of a pathogenic mutation associated with LOH of the respective wild-type allele.

TIL assessment

Hematoxylin and eosin-stained whole slide images were used to assess TILs within corresponding tumor regions sequenced by MSK-IMPACT. A semi-quantitative estimation of the density and distribution of TILs within the intratumoral, stromal, and peritumoral compartments was performed by two gynecologic pathologists (K.D. and L.H.E.) independently, blinded from the results of the molecular analysis and following a previously reported scoring framework (24,25). Intratumoral TILs were defined as lymphocytes admixed within malignant epithelium. A density (absent – 0, mild – 1, moderate – 2, marked – 3) and distribution (absent – 0, focal – 1, multifocal – 2, diffuse – 3) score was assigned. Stromal TILs were defined as lymphocytes within the stroma interdigitating and within the tumor, and an overall density percentage ((area occupied by stromal TILs/total area of stromal tissue) x 100) and distribution score (as described above) were assigned. Peritumoral TILs were defined as lymphocytes adjacent to the border of the tumor (<1mm), and density and distribution scores (as described above) were assigned. A ‘TIL score’ was generated by multiplying density and distribution scores (intratumoral range 0-9; stromal range 0-100; peritumoral range 0-9). Zones exhibiting crush artifact, poor preservation and/or necrosis were excluded, as were endometrial biopsies and lymph node metastases (n=11).

Clinicopathologic characteristics

Clinicopathologic data were abstracted from electronic medical records. Stage at diagnosis was assigned following the International Federation of Gynecology and Obstetrics (FIGO) 2014 staging system (26). Tumor grade, histology, lymphovascular space invasion (LVSI), and microcystic, elongated, and fragmented (MELF) pattern data were abstracted from pathology reports. Adjuvant therapy was defined as any additional therapy given following surgery and included observation, chemotherapy, and intravaginal radiation therapy (IVRT)/external beam radiation therapy (EBRT). Body mass index (BMI) was collected from diagnosis, and obesity was defined as BMI ≥30 kg/m2 (27).

Treatment and survival outcomes

To investigate the association of mechanism of MMR-D with clinical and treatment outcomes, patients who received their initial care at MSK were selected for this analysis, as described above (n=119; Figure 1). Progression-free survival (PFS) was measured from the date of diagnosis to date of progression, as determined by biopsy, surgical resection, or imaging. Overall survival (OS) was measured from the date of diagnosis to date of death. Patients alive and disease-free or alive with disease were censored for PFS and OS, respectively, at date of last-follow-up. A subset of patients receiving pembrolizumab for recurrence per FDA label were assessed for radiographic response following RECIST 1.1 criteria by independent, blinded radiology review (Y.L. and W.M.) (28). Those receiving ICI on clinical trials were excluded. Clinical benefit was defined as patients who achieved a complete response, partial response, or stable disease as best response on therapy, while overall response rate was defined as patients who achieved a complete response or partial response as best response on therapy. Percent change in tumor volume was quantified by dividing the summation of target lesion diameter at best response by the summation of target lesion diameter at baseline (28).

Statistical analyses

Associations between MMR-D mechanism and continuous and categorical clinicopathologic variables were compared using Kruskal-Wallis and Fisher’s exact tests, respectively. TMB, MSIsensor score, mutational signatures and TIL scores were compared between groups graphically and using appropriate non-parametric tests. For analyses of primary treatment after surgery, landmark analysis was applied with a 6-week landmark time, with 4 patients excluded due to early progression in PFS analyses (29). Survival curves were created using the Kaplan-Meier method. P-values were generated by applying log-rank test for categorical variables and Wald-test based on Cox Proportional Hazards (CoxPH) model for continuous variables. Hazard ratios (HR) with 95% confidence intervals (CI) were obtained based on CoxPH model. A p-value<0.05 was considered statistically significant. P-values with fewer than 3 events were considered unreliable but are reported in a hypothesis-generating fashion. All statistical analyses were performed using R version 4.1.1 (https://cran.r-project.org/) (CRAN, RRID:SCR_003005). In those with recurrent EC treated with ICI, RECIST-defined responses were tabulated and plotted using swimmer’s and waterfall plots.

Data availability

Targeted sequencing data that support the findings of this study are available at cBioPortal (www.cbioportal.org).

RESULTS

Clinicopathologic characteristics

Of 184 EC patients, 25 (14%) had germline MMR gene mutations, 39 (21%) had somatic MMR gene mutations and 120 (65%) had MLH1ph (Figure 1, Table 1). Compared to patients harboring germline or somatic mutations, those with MLH1ph ECs were older at diagnosis (median 64 years MLH1 vs. 54 years germline/somatic; p<0.001), more likely to be obese (58% MLH1ph vs. 42% germline and 26% somatic; p=0.001), diagnosed at higher stages (p=0.025) and had tumors with higher rates of LVSI (p=0.033). There were no significant differences in histology, MELF pattern, or primary treatment between groups (Table 1). Also, within the group of ECs harboring MLH1 alterations, patients with MLH1ph ECs compared to those with MLH1 mutations (germline or somatic) were older at diagnosis (median 64 vs. 50 years; p<0.001) and more likely to be obese (58% vs. 9%; p<0.001). No other significant differences were observed (Supplementary Table S1).

Table 1.

Clinicopathologic characteristics of endometrial cancer by mechanism of MMR-D

Characteristic	Germline (n=25)	Somatic (n=39)	MLH1ph (n=120)	p-value
Altered Gene				<0.001
MLH1	3 (12%)	10 (26%)	120 (100%)
MSH2	8 (32%)	19 (49%)	0 (0%)
MSH6	12 (48%)	7 (18%)	0 (0%)
PMS2	2 (8.0%)	3 (7.7%)	0 (0%)
Age at diagnosis (median, range)	54 (31-69)	54 (40-83)	64 (35-93)	<0.001
BMI at diagnosis*				0.001
Normal (<25)	8 (33%)	18 (53%)	18 (17%)
Overweight (25 to <30)	6 (25%)	7 (21%)	25 (24%)
Obese (≥30)	10 (42%)	9 (26%)	60 (58%)
Stage (FIGO 2014)				0.025
I	15 (60%)	25 (64%)	67 (56%)
II	4 (16%)	3 (7.7%)	3 (2.5%)
III	5 (20%)	10 (26%)	30 (25%)
IV	1 (4.0%)	1 (2.6%)	20 (17%)
Histology				0.67**
Endometrioid	23 (92%)	32 (82%)	101 (84%)
G1	14 (56%)	12 (32%)	30 (25%)
G2	6 (24%)	10 (25%)	47 (39%)
G3	3 (12%)	10 (25%)	24 (20%)
Clear Cell	0	1 (2%)	0 (0%)
Mixed	0	3 (8%)	1 (1%)
Carcinosarcoma	1 (4.0%)	0 (0%)	7 (6%)
Undifferentiated/Dedifferentiated	1 (4.0%)	3 (8%)	11 (9%)
MELF pattern				0.17
No	20 (80%)	30 (77%)	106 (88%)
Yes	5 (20%)	9 (23%)	14 (12%)
LVSI				0.033
No	17 (68%)	17 (44%)	47 (39%)
Yes	8 (32%)	22 (56%)	73 (61%)
Dominant mutational signature				<0.001
Aging	15 (62%)	20 (54%)	27 (23%)
MMR (signatures 6, 15, 20, 260	9 (38%)	17 (46%)	88 (77%)
Primary treatment after surgery				0.34
None	9 (41%)	10 (26%)	23 (20%)
Chemotherapy	9 (41%)	21 (54%)	62 (53%)
IVRT/ EBRT	4 (18%)	8 (21%)	31 (27%)

Statistical tests performed: Kruskal-Wallis test; Fisher’s exact test. MLH1ph, MLH1 promoter hypermethylation. BMI, Body Mass Index. MELF, Microcystic, Elongated, and Fragmented. LVSI, lymphovascular space invasion. MMR, mismatch repair. IVRT/EBRT, intravaginal radiation therapy/external beam radiation therapy.

^*Missing values: BMI (n=23)

^**P-values calculated for endometrioid vs carcinosarcoma vs un/dedifferentiated.

Somatic mutational landscapes

The landscape of somatic mutations varied across ECs by MMR-D mechanism. In the germline group, tumors were enriched in ERBB2 (29%), ERBB3 (25%) and FBXW7 (46%) hotspot mutations compared to the MLH1ph group (3%, 2%, and 10% respectively, p<0.05). Alterations in JAK1, predominantly frameshift-indels, were enriched in MLH1ph ECs compared to the germline group (45% vs. 4%, p<0.05). The frequencies of ARID1A, PTEN, and PIK3CA mutations were similar among the different MMR-D EC groups (Figure 2).

Figure 2. Landscape of somatic mutations in endometrial cancer by mechanism of MMR-D.

Heatmap displaying somatic mutations in endometrial cancers (ECs) with MLH1 promoter hypermethylation (left), germline MMR gene mutations (middle) and somatic MMR gene mutations (right). Given the high frequency of passenger mutations and variants of unknown significance in MMR-deficient ECs, only pathogenic mutations affecting cancer-related genes are shown. Cases are represented by columns and genes by rows. Genes altered in a least three ECs are shown. Mutation types are color-coded according to the legend, and genes statistically significantly altered between the different MMR-D mechanisms are in bold and color-coded according to the legend (Fisher’s exact test, *p<0.1, **p<0.05). Indel, insertion/deletion. SNV, single nucleotide variant.

Median TMB was significantly lower among ECs with MLH1ph (32 mt/Mb, range 13-302 mt/Mb) compared to germline (44 mt/Mb, range 1-74 mt/MB) and somatic MMR mutations (48 mt/Mb, range 25-89 mt/Mb; p<0.001; Supplementary Figure S1A). All ECs in the MLH1ph and somatic groups were MSI-H; however, 83% (10/12) of ECs from MSH6 germline mutation carriers were classified as MSS or MSI-indeterminate, and 31% (4/13) of ECs from MLH1, PMS2, or MSH2 germline mutated ECs were classified as MSS or MSI-indeterminate (p<0.02). Upon further review, in all MSS or MSI-indeterminate ECs from germline MMR mutation carriers, biallelic loss was observed in the tumor in 5/14 (36%) of cases. Low tumor content was detected in 9/14 (64%) of samples (Supplementary Table S2) potentially negatively impacting the accuracy of MSISensor and LOH assessments. Of note, among the 25 germline patients, there were 2 MMR-proficient and 3 equivocal MSH6-associated ECs as well as 2 equivocal MSH2-associated ECs (Supplementary Table S3).

Mutational signature analysis revealed a dominant aging-related signature 1 in 62% (15/25) of germline and 54% (20/39) of somatic MMR-D ECs compared to 23% (27/120) of MLH1ph ECs (p<0.001). Conversely, 77% (88/120) of MLH1ph ECs had dominant MMR-D mutational signatures 6, 15, 20 or 26 compared with 38% (9/25) and 46% (17/39) of germline and somatic MMR-D ECs, respectively (p<0.001; Table 1, Supplementary Figure S1B).

TILs

While TILs were present in all EC samples analyzed, TIL scores were significantly lower among ECs with MLH1ph compared to those with germline/somatic MMR mutations across all compartments (p=0.002 intratumoral, p=0.002 stromal, p=0.001 peritumoral; Figure 3; Supplementary Figure S2).

Figure 3. Tumor infiltrating lymphocytes in endometrial cancers by mechanism of MMR-D.

(A) Intratumoral (B) stromal and (C) peritumoral tumor infiltrating lymphocyte (TIL) scores in endometrial cancers (ECs) with germline MMR mutations (left), somatic MMR gene mutations (middle) and MLH1 promoter hypermethylation (MLH1ph; right). (D) Representative micrographs of ECs with intratumoral TILs exhibiting semi-quantitative mild (1+), moderate (2+) or marked (3+) density scores, respectively. MMR, mismatch repair.

Survival analyses

Among 119 patients included in the survival analyses, 29 patients experienced progression; no deaths occurred without progression. Median follow-up for PFS was 25 months (range 1.7-60.0 months). In univariate analyses of PFS (Supplementary Table S4), MLH1ph was associated with inferior PFS (p=0.005; Figure 4), as was older age at diagnosis (p=0.019), higher stage (p<0.001) and presence of LVSI (p=0.025). In contrast, TMB had borderline association with improved PFS (p=0.06). In multivariate models including MMR-D mechanism, age and stage at diagnosis, and presence of LVSI, only stage was negatively associated with PFS (p<0.001; Supplementary Table S4). Of note, an exploratory analysis revealed that two-year PFS was worse among patients with early-stage EC (stage I/II) and MLH1ph (70.3%) compared to germline (100%) and somatic mutations (100%, Figure 4C).

Figure 4. Survival analysis of patients with endometrial cancer by mechanism of MMR-D.

Kaplan-Meier curve comparing (A) progression-free survival, (B) overall survival and (C) progression-free survival in stage I/II (FIGO 2014 staging) in endometrial cancers (ECs) with germline MMR mutations (blue), somatic MMR gene mutations (red) and MLH1 promoter hypermethylation (MLH1ph; gray, dotted). Survival compared with log-rank test. MMR, mismatch repair.

In the same cohort, 11 deaths were observed, and median follow-up for OS was 25.7 months (range 1.7-63.1 months; Figure 4B). In univariate analyses of OS (Supplementary Table S5), higher stage was associated with worse OS (p=0.002), and there was a trend towards higher TMB being associated with improved OS (p=0.051). Multivariate analysis was not conducted due to the rarity of OS events.

Response to ICI in recurrent disease

In a subset of patients with recurrent disease treated with on-label pembrolizumab (n=18), clinical benefit defined by RECIST 1.1 criteria was evaluated in a hypothesis-generating exploratory analysis among patients with germline (n=1), somatic (n=1), and MLH1ph ECs (n=16). This subgroup included patients with grade 1 endometrioid (n=5), grade 2 endometrioid (n=4), grade 3 endometrioid (n=6), undifferentiated/dedifferentiated (n=2), and mixed (n=1) histologies. Overall, 7/18 (39%) and 11/18 (61%) and patients had stage I/II and stage III/IV disease at diagnosis, respectively. The median number of prior lines of cytotoxic therapy was 1 (range 0-2). One patient who was not a candidate for platinum-based chemotherapy received pembrolizumab for recurrence.

Clinical activity of pembrolizumab was consistent with published literature, and although limited by sample size, disease progression on pembrolizumab was observed in 4/16 (25%) patients with MLH1ph ECs with no progression observed for either the germline or somatic EC patients (Figures 5A/B). For the 16 patients with MLH1ph ECs, 2 had stable disease, 8 experienced partial response, and 2 had a complete response with a clinical benefit rate of 75% and overall response rate of 63%. The median TMB of MLH1ph ECs in patients receiving ICI was 33.8 mt/Mb (range 22.8-66.7 mt/Mb).

Figure 5. Response of patients with endometrial cancer to immune checkpoint inhibition by mechanism of MMR-D.

(A) Time on immune checkpoint inhibition (ICI) and radiographic responses (RECIST 1.1) to on-label pembrolizumab in patients with recurrent endometrial cancer (EC) and a germline MMR gene mutation (n=1), a somatic MMR gene mutation (n=1), or MLH1 promoter hypermethylation (n=16). Ongoing therapy indicated by arrows; bars represent cessation of treatment. Green dots represent start of response to ICI, and red dots represent disease progression. For those with durable response after cessation of ICI, black dots depict time of last follow-up. (B) Best response in target lesion to on-label pembrolizumab among EC patients with germline MMR gene mutation (n=1), somatic MMR gene mutation (n=1), or MLH1 promoter hypermethylation (n=16). MMR, mismatch repair. Dashed lines annotate RECIST-defined thresholds for partial response (−30%) and progression of disease (+20%). *0% change in target lesion. Note that patient 7 progression was in a non-target lesion and is therefore not depicted.

DISCUSSION

We demonstrate here that MLH1ph ECs have distinct clinicopathologic and molecular features as well as TIL densities when compared with germline and somatic MMR-mutated ECs, potentially affecting outcomes. MLH1ph ECs were observed more frequently in older and obese patients and were diagnosed at more advanced stages with higher rates of LVSI compared to EC patients with germline and somatic MMR gene mutations. MLH1ph tumors exhibited a distinct mutational profile compared to the germline and somatic groups. TMB and TIL scores were also lower in MLH1ph compared to germline and somatic mutated ECs. Although PFS and OS were not significantly different between the various mechanisms of MMR-D and stage was the main driver of survival in multivariate models, when examining early-stage EC only, MLH1ph was associated with worse PFS compared to germline and somatic MMR gene-mutated ECs.

Previous studies have reported conflicting findings regarding outcomes of MMR-D/MSI-H EC compared to ECs of copy number-low or POLE molecular subtypes (5,30–32), potentially reflecting the heterogeneity present in this group. Our findings corroborate and expand the results of previous studies, demonstrating that MLH1ph ECs are associated with high-risk features including older age, higher stage, obesity, and LVSI (3,5,10,33), which may portend worse outcomes (34). Historically, early-stage EC (stage I/II) is associated with 5-year survival rates of 87-96% (35); however, our study demonstrated a 2-year PFS rate of 70% in patients with MLH1ph EC compared to 100% in both MMR germline and somatically-mutated EC. Whilst these results are limited given the sample size, our findings warrant additional research to confirm these findings in larger cohorts and to define whether patients with early-stage ECs with MLH1ph may benefit from novel therapies, such as radiation combined with immune checkpoint inhibition in the upfront setting (as in NRG-GY020 or NCT04774419) to prevent recurrence.

Our study revealed that MLH1ph ECs may not only have distinct molecular and immune profiles but also an enrichment of JAK1 mutations and lower TMB and TIL scores. This is consistent with previous reports on decreased presence of TILs (36) and PD-L1 expression (37) in MLH1ph compared to other MMR-D ECs as well as variation in TMB by underlying cause of MMR-D (38), suggesting distinct immunological entities with implications for treatment (39). Our data further the notion of a direct relationship between magnitude of TMB and response to immunotherapy across multiple cancer types, including EC (40,41). These findings indicate that sub-stratification by MMR-D mechanism may be warranted in future trials, such as those investigating the impact of upfront ICI and radiation combinations for MMR-D ECs (NRG-GY020 and NCT04774419), those investigating the efficacy of dual immune checkpoint blockade (NRG-GY025), or those evaluating the efficacy of immune checkpoint blockade in combination with chemotherapy in the upfront or recurrent setting (NRG-GY018) (42). Previous studies have described ICI response and resistance mechanisms in JAK1/2 (MMR-D colon cancer and melanoma), PBRM1 (clear cell renal carcinoma), and PTEN (acquired ICI resistance in uterine leiomyosarcoma) (43–46). A recent phase II study of pembrolizumab in recurrent, MSI-H EC found worse responses in tumors with MLH1ph compared to somatic MMR mutations and hypothesized that alterations in the JAK pathway may be associated with ICI resistance (47). We have also observed that MLH1ph ECs display a significantly higher rate of JAK1 alterations compared to germline and somatic MMR-D tumors, and future studies and clinical trials should investigate the need for more aggressive treatments and combination immunotherapy to overcome potential resistance and augment response.

Of note, we found similar outcomes between germline and somatic MMR-D EC with comparable levels of TIL infiltration. The prevalence of mutations in genes commonly altered in ECs, including PTEN, PIK3CA, and ARID1A were similar across different MMR groups. However, MMR germline-mutated ECs were enriched in ERBB2 hotspot mutations, which have emerged as a target through irreversible HER2 tyrosine kinase inhibition in non-small cell lung and breast cancers (48). Additional investigation of this potentially targetable alteration in the setting of Lynch syndrome-associated ECs is still required, including determining the frequency of ERBB2 mutations and whether they represent early clonal/truncal versus late subclonal events.

Our study has several limitations beyond those inherent to retrospective research. The patient selection is restricted to those with ECs who underwent tumor-normal sequencing and may exclude patients who underwent germline only testing through other avenues, potentially underestimating the germline component of MMR-D ECs. The mutational analyses were restricted to 341-468 cancer-related genes, and it is possible that unexplored differences in genetic alterations may underpin differences observed between MMR-D mechanism. Although we utilized a comprehensive TIL score to correlate the immune microenvironment with the genomic findings, future assessments incorporating multiplex IHC to allow differentiation of CD4/CD8+ T-cell populations as well as other immune markers such as PD-L1 may be informative. Although independent radiologic review and RECIST classification were conducted to establish ICI response, imaging was obtained outside of a clinical trial setting and was thus subject to less standardized time intervals. Additionally, we noted discrepancies between IHC-based MMR and molecular MSI assessments among groups, particularly in the germline EC group. Of the MMR germline-mutated ECs 28% were MMR-proficient/equivocal on IHC, compared to none of either the somatic or MLH1ph groups, and 56% of germline MMR-D ECs had stable or indeterminate MSIsensor score. Together with previous reports showing that molecular MSI analysis has lower sensitivity for MMR-D detection in EC than in colorectal cancer, and that a subset of molecular MSI-H cancers have retained/distinct MMR IHC expression patterns (49,50), our work underpins the importance of conducting both IHC and MSI assessment in the diagnostic workup of ECs. Finally, given the sample size restrictions of this study, the exploratory analyses investigating the associations between MLH1ph ECs and PFS events in early-stage disease as well as the associations between subclassification of MMR-D ECs and response to ICI should be interpreted with caution and requires additional study using a larger cohort.

In summary, our findings suggest that subclassification of MMR-D EC is likely warranted, given the clinicopathologic, genetic and immunologic differences based on the mechanism of MMR-D reported in this study. These differences may influence clinical outcomes and response to treatment. Future therapeutic studies should account for these differences to design targeted interventions towards this high-risk group of MLH1ph ECs and explore biomarkers in addition to MMR-D/MSI and TMB for immune-based therapies.

Translational Relevance.

Epigenetic MLH1 promoter hypermethylation (MLH1ph), somatic, and germline mutations are separate DNA mismatch repair-deficiency (MMR-D) mechanisms that drive microsatellite instability-high (MSI-H) endometrial carcinomas (ECs). However, sub-stratification by MMR-D mechanism is not routinely used for treatment or prognostication of MMR-D/MSI-H ECs. We demonstrate that MLH1ph EC patients are clinically distinct as an older and more obese population with higher stage tumors harboring more lymphovascular space invasion. We further show that MLH1ph ECs are enriched for JAK1 somatic mutations, exhibit lower tumor mutational burden and tumor infiltrating lymphocyte scores, and that stage I/II MLH1ph EC patients have a shorter two-year progression-free survival compared to those with germline or somatic MMR-D ECs. Thus, subclassification of MMR-D ECs by mechanism should be considered in future clinical trials to account for high-risk MLH1ph ECs given potential implications for treatment and prognosis.