Inherited mutations affecting the SRCAP complex are central in moderate-penetrance predisposition to uterine leiomyomas.

Uterine leiomyomas (ULs) are benign smooth muscle tumors that are common in premenopausal women. Somatic alterations in MED12, HMGA2, FH, genes encoding subunits of the SRCAP complex, and genes involved in Cullin 3-RING E3 ligase neddylation are mutually exclusive UL drivers. Established predisposition genes explain only partially the estimated heritability of leiomyomas. Here, we examined loss-of-function variants across 18,899 genes in a cohort of 233,614 White European women, revealing variants in four genes encoding SRCAP complex subunits (YEATS4, ZNHIT1, DMAP1, and ACTL6A) with a significant association to ULs, and YEATS4 and ZNHIT1 strikingly rank first and second, respectively. Positive mutation status was also associated with younger age at diagnosis and hysterectomy. Moderate-penetrance UL risk was largely attributed to rare non-synonymous mutations affecting the SRCAP complex. To examine this disease phenotype more closely, we set out to identify inherited mutations affecting the SRCAP complex in our in-house sample collection of Finnish individuals with ULs (n = 860). We detected one individual with an ACTL6A splice-site mutation, two individuals with a YEATS4 missense mutation, and four individuals with DMAP1 mutations: one splice-site, one nonsense, and two missense variants. These individuals had large and/or multiple ULs, were often diagnosed at an early age, and many had family history of ULs. When a somatic second hit was found, ACTL6A and DMAP1 were silenced in tumors by somatic mutation and YEATS4 by promoter hypermethylation. Decreased H2A.Z staining was observed in the tumors, providing further evidence for the pathogenic nature of the germline mutations. Our results establish inactivation of genes encoding SRCAP complex subunits as a central contributor to moderate-penetrance UL predisposition.

3'RNA and whole-genome sequencing of archival uterine leiomyomas reveal a tumor subtype with chromosomal rearrangements affecting either HMGA2, HMGA1, or PLAG1.

Uterine leiomyomas, or fibroids, are very common smooth muscle tumors that arise from the myometrium. They can be divided into distinct molecular subtypes. We have previously shown that 3'RNA-sequencing is highly effective in classifying archival formalin-fixed paraffin-embedded (FFPE) leiomyomas according to the underlying mutation. In this study, we performed 3'RNA-sequencing with 111 FFPE leiomyomas previously classified as negative for driver alterations in mediator complex subunit 12 (MED12), high mobility group AT-hook 2 (HMGA2), and fumarate hydratase (FH) by Sanger sequencing and immunohistochemistry. This revealed 43 tumors that displayed expression features typically seen in HMGA2-positive tumors, including overexpression of PLAG1. We explored 12 such leiomyomas by whole-genome sequencing to identify their underlying genomic drivers and to evaluate the feasibility of detecting chromosomal driver alterations from FFPE material. Four tumors with significant HMGA2 overexpression at the protein-level served as controls. We identified chromosomal rearrangements targeting either HMGA2, HMGA1, or PLAG1 in all 16 tumors, demonstrating that it is possible to detect chromosomal driver alterations in archival leiomyoma specimens as old as 18 years. Furthermore, two tumors displayed biallelic loss of DEPDC5 and one tumor harbored a COL4A5-COL4A6 deletion. These observations suggest that instead of only HMGA2-positive leiomyomas, a distinct leiomyoma subtype is characterized by rearrangements targeting either HMGA2, HMGA1, or PLAG1. The results indicate that the frequency of HMGA2-positive leiomyomas may be higher than estimated in previous studies where immunohistochemistry has been used. This study also demonstrates the feasibility of detecting chromosomal driver alterations from archival FFPE material.

Molecular subclass of uterine fibroids predicts tumor shrinkage in response to ulipristal acetate.

Precision medicine carries great potential for management of all tumor types. The aim of this retrospective study was to investigate if the two most common genetically distinct uterine fibroid subclasses, driven by aberrations in MED12 and HMGA2 genes, respectively, influence response to treatment with the progesterone receptor modulator ulipristal acetate. Changes in diameter and mutation status were derived for 101 uterine fibroids surgically removed after ulipristal acetate treatment. A significant difference in treatment response between the two major subclasses was detected. MED12 mutant fibroids had 4.4 times higher odds of shrinking in response to ulipristal acetate treatment as compared to HMGA2 driven fibroids (95% confidence interval 1.37-13.9; P = 0.013), and in a multivariate analysis molecular subclassification was an independent predictive factor. Compatible with this finding, gene expression and DNA methylation analyses revealed subclass specific differences in progesterone receptor signaling. The work provides a proof-of-principle that uterine fibroid treatment response is influenced by molecular subclass and that the genetic subclasses should be taken into account when evaluating current and future uterine fibroid therapies.

Interpretable prognostic modeling of endometrial cancer.

Endometrial carcinoma (EC) is one of the most common gynecological cancers in the world. In this work we apply Cox proportional hazards (CPH) and optimal survival tree (OST) algorithms to the retrospective prognostic modeling of disease-specific survival in 842 EC patients. We demonstrate that linear CPH models are preferred for the EC risk assessment based on clinical features alone, while interpretable, non-linear OST models are favored when patient profiles can be supplemented with additional biomarker data. We show how visually interpretable tree models can help generate and explore novel research hypotheses by studying the OST decision path structure, in which L1 cell adhesion molecule expression and estrogen receptor status are correctly indicated as important risk factors in the p53 abnormal EC subgroup. To aid further clinical adoption of advanced machine learning techniques, we stress the importance of quantifying model discrimination and calibration performance in the development of explainable clinical prediction models.

A novel uterine leiomyoma subtype exhibits NRF2 activation and mutations in genes associated with neddylation of the Cullin 3-RING E3 ligase.

Uterine leiomyomas, or fibroids, are the most common tumors in women of reproductive age. Uterine leiomyomas can be classified into at least three main molecular subtypes according to mutations affecting MED12, HMGA2, or FH. FH-deficient leiomyomas are characterized by activation of the NRF2 pathway, including upregulation of the NRF2 target gene AKR1B10. Here, we have identified a novel leiomyoma subtype showing AKR1B10 expression but no alterations in FH or other known driver genes. Whole-exome and whole-genome sequencing revealed biallelic mutations in key genes involved in neddylation of the Cullin 3-RING E3 ligase, including UBE2M, NEDD8, CUL3, and NAE1. 3'RNA sequencing confirmed a distinct molecular subtype with activation of the NRF2 pathway. Most tumors displayed cellular histopathology, perivascular hypercellularity, and characteristics typically seen in FH-deficient leiomyomas. These results suggest a novel leiomyoma subtype that is characterized by distinct morphological features, genetic alterations disrupting neddylation of the Cullin 3-RING E3 ligase, and oncogenic NRF2 activation. They also present defective neddylation as a novel mechanism leading to aberrant NRF2 signaling. Molecular characterization of uterine leiomyomas provides novel opportunities for targeted treatment options.

Molecular classification of endometrial carcinoma: a clinically oriented review.

The Cancer Genome Atlas research network performed a genome-wide analysis of endometrial carcinomas in 2013 and classified tumours into four distinct subgroups: polymerase-ϵ ultramutated, microsatellite unstable hypermutated, copy-number low and copy-number high. These molecular alterations are mostly mutually exclusive as only about 3% of tumours exhibit more than one molecular signature. Apart from the polymerase-ϵ ultramutated subgroup, molecular classification can be reproduced by using surrogate markers. This has facilitated the implementation of molecular diagnostics into routine patient care. Molecular subgroups are associated with different prognoses; thus, improved risk assessment is their most obvious clinical application. However, based on their unique molecular architectures, molecular subgroups should not be regarded simply as risk groups but rather as distinct diseases. This has prompted us and others to examine the role of molecular subgroups in modifying the prognostic effect of traditional risk factors, including clinical factors, uterine factors and tissue biomarkers, and in predicting the response to adjuvant therapies. In the following review, we summarise the current knowledge of molecularly classified endometrial carcinoma and present, based on our own experience, a proposal for implementing molecular classification into daily practice in pathology laboratories.

Histopathologic and Molecular Characterization of Uterine Leiomyoma-like Inflammatory Myofibroblastic Tumor: Comparison to Molecular Subtypes of Uterine Leiomyoma.

Uterine leiomyoma (UL) is a common benign neoplasm which can sometimes be difficult to differentiate from the uterine inflammatory myofibroblastic tumor (IMT) based on morphology alone. IMT is a myofibroblastic/fibroblastic neoplasm which has typically been considered to be rare in the uterus. Its clinical behavior is usually indolent although aggressive variants exist. The majority of IMTs harbor genomic rearrangement of anaplastic lymphoma kinase ( ALK ), while ALK fusion has not been thus far detected in ULs. We analyzed 2263 ULs of which 9 (0.4%) had tyrosine-kinase activation. Seven of the samples were ALK immunopositive: 6 had an ALK fusion gene and 1 overexpressed an ALK transcript skipping exons 2 to 3, Moreover, 1 sample had a RET , and 1 a PDGFRB fusion gene. While no recurrent somatic mutations were found, 1 patient had an ALK germline mutation. Seven tumors showed leiomyoma-like morphology, 1 tumor had slightly loose, and 1 fibrous growth pattern. Six tumors had mild to moderate lymphocyte infiltration, while no immune cell infiltration was detected in 3 cases. None of the tumors showed aggressive behavior. Except for strong ALK positivity (7/9 tumors) the protein expression profile of the tumors was identical to ULs and distinct from other mesenchymal uterine tumors. In gene expression level, these tumors and the known UL subclasses did not separate perfectly. However, vitamin C metabolism and epithelial-mesenchymal transition pathways were uniquely enriched in these lesions. The overall similarity of the analyzed tumors to UL raises the question whether an UL diagnosis would be more proper for a subset of uterine IMTs.

TCGA molecular classification in endometriosis-associated ovarian carcinomas: Novel data on clear cell carcinoma.

BACKGROUND: Clear cell and endometrioid ovarian carcinomas (OCC and OEC, respectively) have a presumed origin in endometriosis and share molecular alterations with each other and with their endometrial counterparts. The Cancer Genome Atlas (TCGA)-based molecular classification stratifies endometrial carcinomas into four categories: POLE mutated (POLEmut), mismatch repair deficient (MMRd), p53 abnormal (p53abn) and no specific molecular profile (NSMP) with divergent prognoses. The subsequent studies are indicative that this TCGA classification has some value in OEC, but the knowledge related to OCC is limited. METHODS: Endometrial carcinoma molecular classification was evaluated and compared in a large series of OCCs (n = 115) and OECs (n = 158). POLE mutation analysis and tissue microarray-based immunohistochemistry for mismatch repair and p53 proteins were performed. RESULTS: The distribution to the molecular groups was as follows: POLEmut 0.9%/3.2%, MMRd 3.5%/6.3%, p53abn 20%/30%, and NSMP 76%/60% in OCCs/OECs, respectively. The proportion of NSMP tumors was the largest in both histological types and significantly higher in OCC than OEC (p = 0.009). The molecular classification correlated significantly with DSS in both OCCs and OECs (p < 0.001 and p = 0.009, respectively), and with DFS in OCCs (p = 0.001). POLEmut and MMRd OCCs carried excellent prognosis, whereas MMRd OECs presented with poorer outcome. The p53abn group was associated with the poorest prognosis in both tumor types, particularly emphasized in OCCs. CONCLUSIONS: TCGA molecular classification associated with patient outcome in both histotypes, and the difference in prognosis between the molecular groups was even more marked in OCC. The large amount of NSMP tumors highlights the need for further studies.

Testing for Lynch Syndrome in Endometrial Carcinoma: From Universal to Age-Selective MLH1 Methylation Analysis.

International guidelines recommend universal screening of endometrial carcinoma (EC) patients for Lynch syndrome (LS). This screening is generally based on mismatch repair (MMR) protein immunohistochemistry followed by MLH1 methylation analysis of MLH1-negative cases to exclude the likely sporadic cases from germline testing. As LS-associated EC is uncommon in the elderly, age-selective methylation testing could improve cost-efficiency. We performed MMR immunohistochemistry on 821 unselected ECs (clinic-based cohort) followed by a MLH1 promoter methylation test of all MLH1/PMS2-negative tumors. Non-methylated MLH1-deficient cases underwent NGS and MLPA-based germline analyses to identify MLH1 mutation carriers. A reduction in the test burden and corresponding false negative rates for LS screening were investigated for various age cut-offs. In addition, the age distribution of 132 MLH1 mutation carriers diagnosed with EC (registry-based cohort) was examined. A germline MLH1 mutation was found in 2/14 patients with non-methylated MLH1-deficient EC. When compared to a universal methylation analysis, selective testing with a cut-off age of 65 years, would have reduced the testing effort by 70.7% with a false negative rate for LS detection of 0% and 3% in the clinic and registry-based cohorts, respectively. The use of age-selective methylation analysis is a feasible way of reducing the costs and laboratory burden in LS screening for EC patients.

Clinicopathologic vs. Molecular Integrated Prognostication of Endometrial Carcinoma by European Guidelines.

This was a retrospective study of 604 patients with endometrial carcinoma, classified into ESGO-ESTRO-ESP 2021 clinicopathologic and molecular integrated risk groups. The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) and Leiden classifier were employed for molecular classification. Median follow-up time was 81 months. Clinicopathologic and molecular integrated risk groups were similarly associated with distinct prognoses (p < 0.001). Disease-specific survival was similar for all molecular subgroups within clinicopathologic intermediate-risk, high-risk, and advanced/metastatic groups. In contrast, the p53 abnormal subgroup (hazard ratio 9.1, 95% confidence interval 2.0-41; p = 0.004) and mismatch repair deficient subgroup (hazard ratio 3.5, 95% confidence interval 1.2-10; p = 0.024) were associated with disease-related death within clinicopathologic low-risk and high-intermediate-risk carcinomas, respectively. A risk-group shift occurred in 6.0% (36/604) and 7.4% (38/515) of patients classified by ProMisE and Leiden, respectively (p = 0.341). Of the 36 patients shifted in the ProMisE cohort, 27 were upshifted and 9 downshifted. Based on the Leiden classifier, polymerase-ϵ sequencing could be omitted in 60% (311/515) of patients without affecting the risk-group assessment. ESGO-ESTRO-ESP 2021 guidelines provide a platform for risk classification in future trials on molecularly directed treatment of endometrial carcinoma.

Clinical factors and biomarker profiles associated with patient outcome in endometrioid ovarian carcinoma - Emphasis on tumor grade.

OBJECTIVE: The role of clinicopathological factors and molecular markers in prognostic classification of endometrioid ovarian carcinoma (EnOC) is not established. Tumor grade is used in risk assessment, but the role of current 3-tier grading system has been challenged. METHODS: Clinicopathological factors and 12 immunohistochemical biomarkers (PR, ER, ß-catenin, vimentin, ARID1A, HNF1-ß, p53, p16, MIB-1, E-cadherin, c-erb-B2 and L1CAM) were analyzed as regards patient outcome in 215 contemporarily classified EnOCs. RESULTS: Of clinical parameters, grade and stage appeared as strong independent prognostic factors both for disease-free and disease-specific overall survival. Grades 1-3 distinguished clearly from each other in the survival analysis, whereas stages I-II and stages III-IV clustered with each other. PR, ER, nuclear ß-catenin and vimentin positivity were associated with favorable overall outcome and clinical parameters, whereas abnormal expression of p53, overexpression of p16 and L1CAM positivity were associated with aggressive disease characteristics and poor survival. The frequency of good-prognosis markers PR and ß-catenin gradually decreased and poor-prognosis markers p53, p16 and L1CAM gradually increased from grade 1-3. However, vimentin and ER were expressed at similar frequencies across different grades and presented with independent prognostic significance. CONCLUSIONS: We found histological grade and disease stage, but not residual tumor, to be independent clinical prognostic factors in EnOC. A set of good-prognosis markers (PR, ER, ß-catenin and vimentin) and poor-prognosis markers (p53, p16 and L1CAM) were identified. Our findings support continuation of the use of the 3-tier grading system for EnOC and provide clinically feasible IHC biomarkers for prognostic profiling.

Low Expression of Stanniocalcin 1 (STC-1) Protein Is Associated With Poor Clinicopathologic Features of Endometrial Cancer.

Stanniocalcin-1 (STC-1) is a glycoprotein hormone involved in diverse biological processes, including regulation of calcium phosphate homeostasis, cell proliferation, apoptosis, inflammation, oxidative stress responses, and cancer development. The role of STC-1 in endometrial cancer (EC) is yet to be elucidated. In this study, we investigated the protein expression pattern of STC-1 in a tissue microarray (TMA) cohort of hysterectomy specimens from 832 patients with EC. We then evaluated the prognostic value of STC-1 expression regarding the clinicopathologic features and patients survival over a period of 140 months. Our results revealed that in EC tissue samples, STC-1 is mainly localized in the endometrial epithelium, although some expression was also observed in the stroma. Decreased STC-1 expression was associated with factors relating to a worse prognosis, such as grade 3 endometrioid tumors (p = 0.030), deep myometrial invasion (p = 0.003), lymphovascular space invasion (p = 0.050), and large tumor size (p = 0.001). Moreover, STC-1 expression was decreased in tumors obtained from obese women (p = 0.014) and in women with diabetes mellitus type 2 (DMT2; p = 0.001). Interestingly, the data also showed an association between DNA mismatch repair (MMR) deficiency and weak STC-1 expression, specifically in the endometrial epithelium (p = 0.048). No association was observed between STC-1 expression and disease-specific survival. As STC-1 expression was particularly low in cases with obesity and DMT2 in the TMA cohort, we also evaluated the correlation between metformin use and STC-1 expression in an additional EC cohort that only included women with DMT2 (n = 111). The analysis showed no difference in STC-1 expression in either the epithelium or the stroma in women undergoing metformin therapy compared to metformin non-users. Overall, our data may suggest a favorable role for STC-1 in EC behavior; however, further studies are required to elucidate the detailed mechanism and possible applications to cancer treatment.

Differential impact of clinicopathological risk factors within the 2 largest ProMisE molecular subgroups of endometrial carcinoma.

OBJECTIVE: To assess whether the prognostic impact of conventional risk factors and ancillary biomarkers differs across the 2 largest ProMisE molecular subgroups of endometrial carcinoma (EC). METHODS: Direct sequencing of POLE exonuclease domain hot spots and immunohistochemistry for MLH1, PMS2, MSH2, MSH6 and p53 were performed on 745 unselected endometrioid ECs to identify mismatch repair deficient (MMR-D, n = 264) and no specific molecular profile (NSMP, n = 206) ECs. Molecular group-specific survival analyses and interaction analyses were performed to determine the prognostic relevance of clinicopathological factors and various biomarkers (L1 cell adhesion molecule, estrogen and progesterone receptor, beta-catenin, p16, E-cadherin, KRAS) within the subgroups. RESULTS: Molecular subgroup did not have an independent effect on disease-specific survival after adjustment for conventional risk factors (P = 0.101). High grade (G3) and p16 hyperexpression remained significant predictors of survival in NSMP. Stage II-IV, ≥50% myometrial invasion, lymphovascular space invasion and loss of E-cadherin were independent predictors in the MMR-D group. In the interaction analysis, molecular subclass significantly modified the prognostic effect of high grade and p16 hyperexpression, which showed a stronger negative effect on survival in NSMP as compared to MMR-D (P for interaction = 0.016 for grade and 0.033 for p16). CONCLUSIONS: Grade of differentiation and p16 hyperexpression appear to have a stronger prognostic impact in NSMP as compared to MMR-D EC. While these results need to be confirmed in a larger study population, they indicate that differential impact of risk factors needs to be taken into account when developing new molecular class-integrated risk stratification algorithms for EC.

Parity associates with chromosomal damage in uterine leiomyomas.

Mechanical forces in a constrained cellular environment were recently established as a facilitator of chromosomal damage. Whether this could contribute to tumorigenesis is not known. Uterine leiomyomas are common neoplasms that display relatively few chromosomal aberrations. We hypothesized that if mechanical forces contribute to chromosomal damage, signs of this could be seen in uterine leiomyomas from parous women. We examined the karyotypes of 1946 tumors, and found a striking overrepresentation of chromosomal damage associated with parity. We then subjected myometrial cells to physiological forces similar to those encountered during pregnancy, and found this to cause DNA breaks and a DNA repair response. While mechanical forces acting in constrained cellular environments may thus contribute to neoplastic degeneration, and genesis of uterine leiomyoma, further studies are needed to prove possible causality of the observed association. No evidence for progression to malignancy was found.

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma.

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility1, and are a common cause of hysterectomy2. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA23. Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex4, and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice5. Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.

Mismatch Repair Deficiency as a Predictive and Prognostic Biomarker in Molecularly Classified Endometrial Carcinoma.

The aggressiveness of mismatch repair (MMR) deficient endometrial carcinomas was examined in a single institution retrospective study. Outcomes were similar for MMR proficient (n = 508) and deficient (n = 287) carcinomas, identified by immunohistochemistry. In accordance with molecular classification based on The Cancer Genome Atlas (TCGA), tumors with abnormal p53 staining or polymerase-ϵ exonuclease domain mutation were excluded from the MMR proficient subgroup, termed as "no specific molecular profile" (NSMP). Compared with NSMP (n = 218), MMR deficiency (n = 191) was associated with poor disease-specific survival (p = 0.001). MMR deficiency was associated with an increased risk of cancer-related death when controlling for confounders (hazard ratio 2.0). In the absence of established clinicopathologic risk factors, MMR deficiency was invariably associated with an increased risk of cancer-related death in univariable analyses (hazard ratios ≥ 2.0). In contrast, outcomes for MMR deficient and NSMP subgroups did not differ when risk factors were present. Lymphatic dissemination was more common (p = 0.008) and the proportion of pelvic relapses was higher (p = 0.029) in the MMR deficient subgroup. Our findings emphasize the need for improved triage to adjuvant therapy and new therapeutic approaches in MMR deficient endometrial carcinomas.

Detection of microsatellite instability with Idylla MSI assay in colorectal and endometrial cancer.

Universal testing of microsatellite instability (MSI) is recommended for colorectal cancer (CRC) and endometrial cancer (EC) to screen for Lynch syndrome and to aid in assessing prognosis and optimal treatment. We compared the performance of Idylla MSI test to immunohistochemistry (IHC) of mismatch repair (MMR) proteins in consecutive series of 100 CRC and 108 EC samples, as well as in retrospective series of 28 CRC and 33 EC specimens with known deficient MMR protein expression. The concordance between the Idylla test and IHC was 100% in all CRC samples (n=128) but lower in EC samples (87.2%; n=141). In the EC samples, sensitivity of Idylla test was 72.7% and specificity 100%. EC MSI/dMMR agreement was 85.4% for MLH1, 87.5% for MSH2, and only 35.3% for MSH6. When we analyzed 14 EC samples that were discrepant, i.e., dMMR using IHC and microsatellite stable using Idylla, with microsatellite markers BAT25 and BAT26, we found four cases to be replication error (RER) positive. All RER positive cases were deficient for MSH6 protein expression. We also re-analyzed EC samples with variable tumor cellularity to determine the limit of detection of the Idylla test and found that a 30% or higher tumor cellularity is required. We conclude that Idylla MSI test offers a sensitive and specific method for CRC diagnostics but is less sensitive in EC samples especially in the case of MSH6 deficiency.

Mismatch repair protein and MLH1 methylation status as predictors of response to adjuvant therapy in endometrial cancer.

BACKGROUND: Mismatch repair (MMR) system has been implicated in the response of mammalian cells to ionizing radiation and DNA damaging agents. We investigated the value of the MMR system in predicting response to adjuvant therapy in endometrial cancer. METHODS: This was a single institution retrospective study. MMR protein status of endometrial carcinomas was assessed by immunohistochemistry. MMR deficient (MMR-D) tumors were identified as MLH1 methylated or nonmethylated by methylation-specific multiplex ligation-dependent probe amplification. Tumors with abnormal p53 staining or polymerase ϵ exonuclease domain mutation were excluded from the MMR proficient subgroup, which was termed as "no specific molecular profile" (NSMP). Disease-specific survival analyses were adjusted for age, stage, histology and grade, depth of myometrial invasion, and lymphovascular space invasion. RESULTS: A total of 505 patients were included in the study. Median follow-up time was 81 months (range 1-136). Whole pelvic radiotherapy (adjusted hazard ratio [HR] 0.092 vs. no adjuvant therapy) and chemotherapy combined with radiotherapy (adjusted HR 0.18) were associated with improved disease-specific survival in the NSMP subgroup (n = 218). In contrast, adjuvant therapies showed no effect on disease-specific survival in the full MMR-D cohort (n = 287) or in MLH1 methylated tumors (n = 154). Whole pelvic radiotherapy (adjusted HR 25 vs. no adjuvant therapy/vaginal brachytherapy) and chemotherapy combined with whole pelvic radiotherapy (adjusted HR 32) were associated with poor disease-specific survival in MMR-D nonmethylated tumors (n = 70). CONCLUSION: MMR protein and MLH1 methylation status predict the response to adjuvant therapy in endometrial cancer. The MMR system could be utilized for selection of patients who most likely benefit from adjuvant therapy.

Molecular characterization in the prediction of disease extent in endometrial carcinoma.

OBJECTIVE: Patients with endometrial carcinoma are usually triaged to staging lymphadenectomy selectively based on estimated risk of lymphatic spread. The risk is generally assessed by the presence of uterine risk factors, but their preoperative and intraoperative identification remain a challenge. The objective of this study was to assess the capability of molecular classification, described by The Cancer Genome Atlas (TCGA), to predict the stage of endometrial carcinoma. STUDY DESIGN: Sequencing of polymerase-ε (POLE) and immunohistochemistry of mismatch repair (MMR) proteins and p53 were performed to stratify endometrial carcinomas into subgroups of POLE exonuclease domain mutation (EDM), MMR deficiency, abnormal p53 (p53 abn) and 'no specific molecular profile' (NSMP). NSMP was the reference subgroup for comparisons. Associations of molecular subgroups and uterine risk factors with stage were examined in univariable and multivariable analyses. RESULTS: Six hundred and four patients were included in the study. None of the POLE EDM tumours extended beyond the uterine cervix. In an unadjusted analysis, p53 abn was associated with increased risk for stage IIIC-IV disease [odds ratio (OR) 4.6, 95% confidence interval (CI) 2.3-9.2; p < 0.0005]. When controlling for uterine risk factors (histotype and grade, depth of myometrial invasion, tumour size, lymphovascular space invasion), p53 was not an independent predictor of advanced disease. In contrast, POLE EDM independently predicted local disease (OR 0.12, 95% CI 0.015-0.99; p = 0.049 for stage II-IV cancer). Of the molecular subgroups, p53 abn was most strongly associated with the presence of high-risk uterine factors (ORs between 2.2 and 19; p ≤ 0.010). CONCLUSION: Of the TCGA-based molecular subgroups, POLE EDM independently predicted early-stage endometrial carcinoma. Although p53 abn was not an independent predictor of advanced disease, its association with uterine risk factors could allow utilization of molecular data in deciding the type of staging surgery if knowledge of uterine factors is deficient.

3'RNA Sequencing Accurately Classifies Formalin-Fixed Paraffin-Embedded Uterine Leiomyomas.

Uterine leiomyomas are benign smooth muscle tumors occurring in 70% of women of reproductive age. The majority of leiomyomas harbor one of three well-established genetic changes: a hotspot mutation in MED12, overexpression of HMGA2, or biallelic loss of FH. The majority of studies have classified leiomyomas by complex and costly methods, such as whole-genome sequencing, or by combining multiple traditional methods, such as immunohistochemistry and Sanger sequencing. The type of specimens and the amount of resources available often determine the choice. A more universal, cost-effective, and scalable method for classifying leiomyomas is needed. The aim of this study was to evaluate whether RNA sequencing can accurately classify formalin-fixed paraffin-embedded (FFPE) leiomyomas. We performed 3'RNA sequencing with 44 leiomyoma and 5 myometrium FFPE samples, revealing that the samples clustered according to the mutation status of MED12, HMGA2, and FH. Furthermore, we confirmed each subtype in a publicly available fresh frozen dataset. These results indicate that a targeted 3'RNA sequencing panel could serve as a cost-effective and robust tool for stratifying both fresh frozen and FFPE leiomyomas. This study also highlights 3'RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.