The best screening practice for Lynch syndrome (LS) in endometrial cancer (EC) remains unknown. We sought to determine whether tumor microsatellite instability (MSI) typing along with ...immunohistochemistry (IHC) and MLH1 methylation analysis can help identify women with LS.
ECs from GOG210 patients were assessed for MSI, MLH1 methylation, and mismatch repair (MMR) protein expression. Each tumor was classified as having normal MMR, defective MMR associated with MLH1 methylation, or probable MMR mutation (ie, defective MMR but no methylation). Cancer family history and demographic and clinical features were compared for the three groups. Lynch mutation testing was performed for a subset of women.
Analysis of 1,002 ECs suggested possible MMR mutation in 11.8% of tumors. The number of patients with a family history suggestive of LS was highest among women whose tumors were classified as probable MMR mutation (P = .001). Lynch mutations were identified in 41% of patient cases classified as probable mutation (21 of 51 tested). One of the MSH6 Lynch mutations was identified in a patient whose tumor had intact MSH6 expression. Age at diagnosis was younger for mutation carriers than noncarriers (54.3 v 62.3 years; P < .01), with five carriers diagnosed at age > 60 years.
Combined MSI, methylation, and IHC analysis may prove useful in Lynch screening in EC. Twenty-four percent of mutation carriers presented with ECs at age > 60 years, and one carrier had an MSI-positive tumor with no IHC defect. Restricting Lynch testing to women diagnosed at age < 60 years or to women with IHC defects could result in missing a substantial fraction of genetic disease.
•MLH1 methylated endometrial tumors have poor prognostic features including larger size.•Tumor volume and mismatch repair class are associated with lymph node involvement.•Women with MLH1 methylated ...tumors have reduced recurrence-free survival.•Recurrence rate by MMR class differed dramatically in advanced stage endometrial cancer.•MMR defective tumors with MLH1 methylation may exhibit chemoresistance.
To determine the relationship between mismatch repair (MMR) classification and clinicopathologic features including tumor volume, and explore outcomes by MMR class in a contemporary cohort.
Single institution cohort evaluating MMR classification for endometrial cancers (EC). MMR immunohistochemistry (IHC)±microsatellite instability (MSI) testing and reflex MLH1 methylation testing was performed. Tumors with MMR abnormalities by IHC or MSI and MLH1 methylation were classified as epigenetic MMR deficiency while those without MLH1 methylation were classified as probable MMR mutations. Clinicopathologic characteristics were analyzed.
466 endometrial cancers were classified; 75% as MMR proficient, 20% epigenetic MMR defects, and 5% as probable MMR mutations. Epigenetic MMR defects were associated with advanced stage, higher grade, presence of lymphovascular space invasion, and older age. MMR class was significantly associated with tumor volume, an association not previously reported. The epigenetic MMR defect tumors median volume was 10,220mm3 compared to 3321mm3 and 2,846mm3, for MMR proficient and probable MMR mutations respectively (P<0.0001). Higher tumor volume was associated with lymph node involvement. Endometrioid EC cases with epigenetic MMR defects had significantly reduced recurrence-free survival (RFS). Among advanced stage (III/IV) endometrioid EC the epigenetic MMR defect group was more likely to recur compared to the MMR proficient group (47.7% vs 3.4%) despite receiving similar adjuvant therapy. In contrast, there was no difference in the number of early stage recurrences for the different MMR classes.
MMR testing that includes MLH1 methylation analysis defines a subset of tumors that have worse prognostic features and reduced RFS.
Background
Mismatch‐repair (MMR)/microsatellite instability (MSI) status has therapeutic implications in endometrial cancer (EC). The authors evaluated the concordance of testing and factors ...contributing to MMR expression heterogeneity.
Methods
Six hundred sixty‐six ECs were characterized using immunohistochemistry (IHC), MSI testing, and mut‐L homolog 1 (MLH1) methylation. Select samples underwent whole‐transcriptome analysis and next‐generation sequencing. MMR expression of metastatic/recurrent sites was evaluated.
Results
MSI testing identified 27.3% of cases as MSI‐high (n = 182), MMR IHC identified 25.1% cases as MMR‐deficient (n = 167), and 3.8% of cases (n = 25) demonstrated discordant results. A review of IHC staining explained discordant results in 18 cases, revealing subclonal loss of MLH1/Pms 1 homolog 2 (PMS2) (n = 10) and heterogeneous MMR IHC (mut‐S homolog 6 MSH6, n = 7; MLH1/PMS2, n = 1). MSH6‐associated Lynch syndrome was diagnosed in three of six cases with heterogeneous expression. Subclonal or heterogeneous cases had a 38.9% recurrence rate (compared with 16.7% in complete MMR‐deficient cases and 9% in MMR‐proficient cases) and had abnormal MMR IHC results in all metastatic recurrent sites (n = 7). Tumors with subclonal MLH1/PMS2 demonstrated 74 differentially expressed genes (determined using digital spatial transcriptomics) when stratified by MLH1 expression, including many associated with epithelial–mesenchymal transition.
Conclusions
Subclonal/heterogeneous MMR IHC cases showed epigenetic loss in 66.7%, germline mutations in 16.7%, and somatic mutations in 16.7%. MMR IHC reported as intact/deficient missed 21% of cases of Lynch syndrome. EC with subclonal/heterogeneous MMR expression demonstrated a high recurrence rate, and metastatic/recurrent sites were MMR‐deficient. Transcriptional analysis indicated an increased risk for migration/metastasis, suggesting that clonal MMR deficiency may be a driver for tumor aggressiveness. Reporting MMR IHC only as intact/deficient, without reporting subclonal and heterogeneous staining, misses opportunities for biomarker‐directed therapy.
Plain Language Summary
Endometrial cancer is the most common gynecologic cancer, and 20%–40% of tumors have a defect in DNA proofreading known as mismatch‐repair (MMR) deficiency.
These results can be used to guide therapy.
Tests for this defect can yield differing results, revealing heterogeneous (mixed) proofreading capabilities.
Tumors with discordant testing results and mixed MMR findings can have germline or somatic defects in MMR genes.
Cells with deficient DNA proofreading in tumors with mixed MMR findings have DNA expression profiles linked to more aggressive characteristics and cancer spread.
These MMR‐deficient cells may drive tumor behavior and the risk of spreading cancer.
Intratumoral heterogeneity, including subclonal and heterogeneous mismatch‐repair (MMR) expression, can yield discordant results from diagnostic assays, such as immunohistochemistry and polymerase chain reaction‐based microsatellite instability testing. Within endometrial cancers, subclonal or heterogeneous MMR expression may be attributed to germline or somatic mutations in MMR proteins, and the authors demonstrate that these tumors are characterized by clonal selection favoring the propagation of MMR deficiency at sites of metastasis or recurrence.
Abstract only
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Background: The use of upfront germline genetic testing for cancer patients to identify hereditary syndromes and to aid in treatment decision making has increased dramatically. ...Recent evidence suggests that such testing should be considered for all solid tumors. In EC, mismatch repair deficiency (MMRd) has emerged as an important molecular marker for treatment with checkpoint inhibitors. MMRd is the hallmark of Lynch syndrome (LS), the most common hereditary cause of EC. Therefore, identifying LS not only affords opportunities for cancer prevention but also for making treatment decisions for women who already have EC. Although tumor-based screening is highly effective, some LS diagnoses will be missed. Upfront multi-gene panel testing (MGPT) for EC has been evaluated as an alternative approach to identifying LS with the potential to simultaneously find actionable germline variants in other cancer susceptibility genes (CSGs). Our objective was to determine the frequency and types of actionable germline variants in a large, unselected group of women with EC. Methods: Prospective germline MGPT for 47 CSGs was performed for 961 unselected EC cases. Patients diagnosed from 2017-2020 were enrolled at nine different institutions. Clinicopathologic data were abstracted from patients’ records. Results: 101 likely pathogenic (LP) or pathogenic variants (PV) were identified in 98 women (10.2%). LP/PVs in LS genes were most common: 29 LS cases were identified (3.02%, 95% CI 2.1 - 4.3%). MGPT found 9 cases (one-third of LS cases) that were not identified by tumor screening: 6 were from institutions that do not perform tumor screening and 3 had normal immunohistochemistry. There were 72 LP/PVs found in 17 different CSGs. 21 patients (2.1%) had LP/PVs in high penetrance CSGs other than the LS genes, 19 of which were in genes associated with breast and/or ovarian cancer (4 in BRCA1, 6 in BRCA2, 6 in BRIP1, 2 in PALB2, 1 in RAD51C). BRCA1/2 PVs (1.04% of the study population, 95% CI 0.6 - 1.9%) were significantly more frequent in women with type II cancers than the rest of the cohort (P =.005, HR 2.00, 95% CI 1.16 - 4.75). 21 additional LP/PVs were found in moderate risk CSGs ( ATM, CHEK2, NBN, NF1). Conclusions: Upfront MGPT in an unselected EC population improved LS diagnosis and identified an additional 2% of patients with LP/PVs in highly penetrant CSGs. The enrichment of germline BRCA1/2 PVs in type II cancers is consistent with prior reports that non-endometrioid tumors are frequently deficient in homologous recombination. Germline BRCA mutation is a known predictive biomarker in ovarian cancer and an attractive therapeutic target in EC. Knowing germline status at the time of diagnosis facilitates further delineation of germline/phenotype associations, and it defines a genetic syndrome allowing for cancer prevention. Upfront MGPT in EC provides clinically impactful information and should be adopted into routine clinical care. Clinical trial information: NCT03460483.
To determine the frequency and spectrum of LS pathogenic variants in EC patients using upfront germline testing for cancer susceptibility. Secondary objectives included determining the sensitivity ...and specificity of current LS screening.
Women diagnosed with EC were prospectively enrolled at 9 institutions. Clinical germline testing (panel testing for common cancer susceptibility genes) was performed for 840 unselected EC patients between 10/1/2017 and 10/1/2020. Mismatch repair (MMR) IHC, MLH1 methylation status and relevant clinicopathologic data were abstracted from patients’ records. Detailed family history data were obtained for all subjects.
Germline testing revealed 26 of 840 EC patients (3.1%) had a pathogenic/likely pathogenic MMR gene mutation. Mutations in PMS2 were most frequent (10/26, 38%), followed by MSH6 (8/26, 31%), MSH2 (6/26, 23%), and MLH1 (2/26, 8%). Mean BMI for PMS2 carriers was greater than other mutation carriers (31.3 vs 27.8). Age at diagnosis was also higher for PMS2 carriers (59 vs 52). IHC combined with MLH1 methylation correctly predicted LS for all MLH1 and MSH2 mutation carriers. For MSH6 and PMS2, IHC and germline mutation status concordance was lower. Only 4/9 (44%) PMS2 carriers had isolated PMS2 loss. IHC was not completed for one PMS2 carrier (insufficient tumor). IHC correctly predicted MSH6 mutations in 7/8 (88%) carriers. Family history did not predict LS for PMS2 carriers: using PREMM5 (LS prediction model), all had low (<5%) scores. None had prior knowledge of their mutation, or of LS in their families. Half of the MLH1 and MSH2 probands were aware of a LS diagnosis prior to study enrollment.
This is the largest cohort of prospectively enrolled EC patients to undergo germline genetic testing in an unselected manner, irrespective of patient age, family history or tumor results. It shows that a majority of LS mutations in EC are in the low-penetrance genes, MSH6 and PMS2. PMS2 mutations were the most common, 10/840 (1.2%). Universal screening with IHC would have missed three PMS2 carriers and one MSH6 carrier. Accurate identification of patients with MMR defects is critical not only for identification of LS, but potentially in consideration of treatment options. In the past, ascertainment of LS relied largely on a strong family cancer history, which is most often seen with MLH1 and MSH2 mutations. For all PMS2 carriers in our study, family history was not predictive of LS and the proband's EC diagnosis was the sentinel event leading to a LS diagnosis in the family. Our study may highlight an evolving LS phenotype in which gene/environment interactions are changing. The observation that EC patients with PMS2 mutations have higher BMIs leads us to hypothesize the existence of an obesity-mediated pathway important in PMS2-associated EC. It may be that in the general population, PMS2 mutation carriers face higher risks for EC than colon cancer.
Clinical utility of up-front multigene panel testing (MGPT) is directly related to the frequency of pathogenic variants (PVs) in the population screened and how genetic findings can be used to guide ...treatment decision making and cancer prevention efforts. The benefit of MGPT for many common malignancies remains to be determined. In this study, we evaluated up-front MGPT in unselected patients with endometrial cancer (EC) to determine the frequency of PVs in cancer susceptibility genes.
Patients with EC were prospectively enrolled at nine Ohio institutions from October 1, 2017, to December 31, 2020. Nine hundred and sixty-one patients with newly diagnosed EC underwent clinical germline MGPT for 47 cancer susceptibility genes. In addition to estimating the prevalence of germline PVs, the number of individuals identified with Lynch syndrome (LS) was compared between MGPT and tumor-based screening.
Likely pathogenic variants or PVs were identified in 97 of 961 women (10.1%). LS was diagnosed in 29 of 961 patients (3%; 95% CI, 2.1 to 4.3), with PVs in
most frequent. MGPT revealed nine patients with LS in addition to the 20 identified through routine tumor-based screening.
and
PVs were found in 1% (10 of 961; 95% CI, 0.6 to 1.9) of patients and that group was significantly enriched for type II ECs.
This prospective, multicenter study revealed potentially actionable germline variants in 10% of unselected women with newly diagnosed EC, supporting the use of up-front MGPT for all EC patients. The discovery that
or
heterozygotes frequently had type II cancers points to therapeutic opportunities for women with aggressive histologic EC subtypes.
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