Mismatch Repair Immunohistochemistry Research Articles

Clinical patterns and genomic profiling of recurrent ‘ultra-low risk’ endometrial cancer

ObjectiveDespite good prognosis for patients with low-risk endometrial cancer, a small subset of women with low-grade/low-stage endometrial cancer experience disease recurrence and death. The aim of this study was to...

Evaluation of KRAS, NRAS and BRAF mutational status and microsatellite instability in early colorectal carcinomas invading the submucosa (pT1): towards an in-house molecular prognostication for pathologists?

AimWe aimed to study the prognostic value of KRAS, NRAS, BRAF mutations and microsatellite stable (MSS)/instable (MSI) in the field of colorectal cancer invading the submucosa (ie, pT1 colorectal cancer...

MMR Deficiency is Homogeneous in Pancreatic Carcinoma and Associated with High Density of Cd8-Positive Lymphocytes

BackgroundMicrosatellite instability (MSI) has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of predicitive biomarkers. MSI has been reported in pancreatic cancer, but data on the possible extent of intratumoral heterogeneity are lacking.MethodsTo study MSI heterogeneity in pancreatic cancer, a tissue microarray (TMA) comprising 597 tumors was screened by immunohistochemistry with antibodies for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6.ResultsIn six suspicious cases, large section immunohistochemistry and microsatellite analysis (Bethesda panel) resulted in the identification of 4 (0.8%) validated MSI cases out of 480 interpretable pancreatic ductal adenocarcinomas. MSI was absent in 55 adenocarcinomas of the ampulla of Vater and 7 acinar cell carcinomas. MMR deficiency always involved MSH6 loss, in three cases with additional loss of MSH2 expression. Three cancers were MSI-high and one case with isolated MSH6 loss was MSS in PCR analysis. The analysis of 44 cancer-containing tumor blocks revealed that the loss of MMR protein expression was always homogeneous in affected tumors. Automated digital image analysis of CD8 immunostaining demonstrated markedly higher CD8 + tumor infiltrating lymphocytes in tumors with (mean = 685, median = 626) than without (mean = 227; median = 124) MMR deficiency (p < 0.0001), suggesting a role of MSI for immune response.ConclusionsOur data suggest that MSI occurs early in a small subset of ductal adenocarcinomas of the pancreas and that immunohistochemical MMR analysis on limited biopsy or cytology material may be sufficient to estimate MMR status of the entire cancer mass.

Should you repeat mismatch repair testing in cases of tumour recurrence? An evaluation of repeat mismatch repair testing by the use of immunohistochemistry in recurrent tumours of the gastrointestinal and gynaecological tracts.

The role of mismatch repair (MMR) testing has evolved from identifying Lynch syndrome patients to predicting response to immune checkpoint inhibitors. This has led to requests from clinicians to retest recurrences of MMR-proficient primary tumours in the hope that the recurrence may show a different MMR status and qualify the patient for treatment. We aimed to determine whether repeat testing is warranted. We evaluated recurrent tumours (local recurrences or metastases) from 137 patients with MMR-proficient primary tumours of the gastrointestinal and gynaecological tracts. The local recurrences and metastases all occurred at least 30days after resection of the primary tumour. We used a combination of a tissue microarray and whole slide staining to perform immunohistochemistry (IHC) for PMS2, MLH1, MSH2, and MSH6, and compared the results with the MMR status of the primary tumour. Three of 137 (2%) initially showed a discordant staining pattern. However, further investigation showed that these discordances were attributable to some of the known pitfalls associated with MMR IHC interpretation - post-radiotherapy loss of MSH6 expression and subclonal loss of MLH1 staining. We did not identify any cases with a genuine discordance in MMR status. We conclude that repeat MMR IHC testing of recurrences is not warranted, as MMR status does not change relative to that of the primary tumour.

Clinicopathological significance of deficient DNA mismatch repair and MLH1 promoter methylation in endometrioid endometrial carcinoma.

The pathogenesis of DNA mismatch repair (MMR)-deficient endometrial carcinoma (EC) is driven by inactivating methylation or less frequently mutation of an MMR gene (MLH1, PMS2, MSH2, or MSH6). This study evaluated the prognostic and clinicopathologic differences between methylation-linked and nonmethylated MMR-deficient endometrioid ECs. We performed MMR immunohistochemistry and methylation-specific multiplex ligation-dependent probe amplification, and classified 682 unselected endometrioid ECs as MMR proficient (MMRp, n = 438) and MMR deficient (MMRd, n = 244), with the latter subcategorized as methylated (MMRd Met) and nonmethylated tumors. Loss of MMR protein expression was detected in 35.8% of the tumors as follows: MLH1 + PMS2 in 29.8%, PMS2 in 0.9%, MSH2 + MSH6 in 1.3%, MSH6 in 2.8%, and multiple abnormalities in 0.9%. Of the 244 MMRd cases, 76% were methylation-linked. MMR deficiency was associated with older age, high grade of differentiation (G3), advanced stage (II-IV), larger tumor size, abundant tumor-infiltrating lymphocytes, PD-L1 positivity in immune cells and combined positive score, wild-type p53, negative L1CAM, ARID1A loss, and type of adjuvant therapy. MMRd-Met phenotype correlated with older age and larger tumor size, and predicted diminished disease-specific survival in the whole cohort. In the MMRd subgroup, univariate analysis demonstrated an association between disease-specific survival and disease stage II-IV, high grade (G3), deep myometrial invasion, lymphovascular invasion, ER negativity, and L1CAM positivity. In conclusion, MMR methylation profile correlates with clinicopathologic characteristics of endometrioid EC, and MMRd-Met phenotype predicts lower disease-specific survival. MMR deficiency, but not MLH1 methylation status, correlates with T-cell inflammation and PD-L1 expression.

The efficacy of DNA mismatch repair enzyme immunohistochemistry as a screening test for hypermutated gliomas

A subset of gliomas has DNA repair defects that lead to hypermutated genomes. While such tumors are resistant to alkylating chemotherapies, they may also express more mutant neoantigens on their cell surfaces, and thus be more responsive to immunotherapies. A fast, inexpensive method of screening for hypermutated gliomas would therefore be of great clinical value. Since immunohistochemistry (IHC) for the DNA mismatch repair (MMR) proteins Msh2, Msh6, Mlh1, and Pms2 is already used to screen for hypermutated colorectal cancers, we sought to determine whether that panel might have similar utility in gliomas. MMR IHC was scored in 100 WHO grade I-IV gliomas (from 96 patients) with known tumor mutation burden (TMB), while blinded to TMB data. Cases included 70 grade IV GBMs, 13 grade III astrocytomas, 4 grade II astrocytomas (3 diffuse astrocytomas and 1 pleomorphic xanthoastrocytoma), 1 grade I pilocytic astrocytoma, 2 grade III oligodendrogliomas, 7 grade II oligodendrogliomas, and 3 grade I glioneuronal tumors. Eight of 100 tumors showed loss of one or more MMR proteins by IHC, and all 8 were hypermutated. Among the remaining 92 gliomas with intact MMR IHC, only one was hypermutated; that tumor had an inactivating mutation in another DNA repair gene, ATM. Overall accuracy, sensitivity, and specificity for DNA MMR IHC compared to the gold standard of TMB were 99, 89, and 100%, respectively. The strongest correlates with hypermutation were prior TMZ treatment, MGMT promoter methylation, and IDH1 mutation. Among the 8 MMR-deficient hypermutated gliomas, 4 (50%) contained both MMR-lost and MMR-retained tumor cells. Together, these data suggest that MMR IHC could be a viable front-line screening test for gliomas in which immunotherapy is being considered. They also suggest that not all cells in a hypermutated glioma may actually be MMR-deficient, a finding that might need to be considered when treating such tumors with immunotherapies.

Natural Language Processing for the Accurate Identification of Colorectal Cancer Mismatch Repair Status in Lynch Syndrome Screening

Lynch syndrome (LS) is the most common type of hereditary colorectal cancer (CRC) syndrome caused by pathogenic variants in mismatch repair (MMR) genes.1 Current multisociety guidelines recommend screening all CRC tumors for LS.2,3 The most widely adopted screening method is MMR immunohistochemistry (IHC) followed by germline analysis if indicated.2,3 However, the text-based nature of pathology and IHC reports used for LS screening results impedes creation of an efficient tracking system for identifying affected patients and screening outcomes.4 In this study, we developed and validated a natural language processing (NLP) tool for extracting MMR IHC results in LS screening in a large, diverse, multicenter, community-based setting.5.

An audit of 10 years’ experience of MMR immunohistochemistry

Aim: Canterbury Health Laboratories provides mismatch repair (MMR) immunohistochemical testing nationally for patients investigated by the New Zealand Familial Gastrointestinal Cancer Service for suspected Lynch Syndrome. We will review the rates and types of MMR deficiency in this cohort. Where available we will compare our results to those of the referring laboratory. Methods: Information was obtained from clinical databases and the NZ Cancer Register. Results: 534 colorectal adenocarcinoma cases were tested for MMR IHC. 49% were MMR IHC deficient. After excluding those with a BRAF mutation/positive MLH1 promoter hypermethylation status, the total number of dMMR was 67 (13%). Of these, 75% had MLH1/PMS2 loss, 10% had isolated PMS2 loss, and 7% had MSH2/MSH6 loss. 308 separate cases underwent BRAF mutation and/or MLHI promoter hypermethylation testing only. There was a high degree of discordance (28/96) in MMR interpretation between our laboratory and referring laboratories if testing had been performed previously. In the majority we interpreted the IHC as combined MLH1/PMS2 loss, while the referring laboratory interpreted their IHC as isolated PMS2 loss. This may be due to misinterpretation of punctate MLH1 staining as retained positivity. Discussion: In this cohort there was a much higher proportion of MLH1/PMS2 deficient tumours than has been reported elsewhere.1 Reference1.Moreira L, Balaguer F, Lindor N, et al. Identification of Lynch Syndrome among patients with colorectal cancer. JAMA 2012; 308: 1555–65.

Frequent loss of mutation-specific mismatch repair protein expression in nonneoplastic endometrium of Lynch syndrome patients.

Lynch syndrome is most often caused by a germline mutation in one of four DNA mismatch repair (MMR) genes (MLH1, PMS2, MSH2, or MSH6) or EPCAM and is associated with a significantly increased risk of endometrial cancer in affected women. Although universal screening of endometrial cancer for Lynch syndrome is becoming increasingly common by various algorithms using MMR immunohistochemistry and/or microsatellite instability testing by PCR, establishing the diagnosis of Lynch syndrome can be still challenging. MMR-deficient nonneoplastic colonic crypts have been recently described in Lynch syndrome patients with colorectal carcinoma, and have been proposed to be a novel indicator of Lynch syndrome. Presence of MMR-deficient nonneoplastic endometrial glands have not yet been systematically evaluated in Lynch syndrome patients. We performed MMR protein immunohistochemistry in prophylactic hysterectomies and endometrial curettings/biopsies from 27 patients with known Lynch syndrome confirmed by germline mutation analysis. A total of 56 control benign endometrial tissues were also analyzed, and included benign endometrium adjacent to MMR-deficient sporadic (MLH1 promoter hypermethylated) endometrial carcinoma (n = 9), adjacent to MMR-intact sporadic endometrial carcinoma (n = 27), and normal endometrium from hysterectomies performed for benign disease (n = 20). MMR protein deficient nonneoplastic endometrial glands were identified in 70% (19 of 27) of Lynch syndrome patients. In all 19 cases the MMR protein loss was specific for the patients' known germline mutation. None of the control cases showed loss of MMR protein expression in nonneoplastic endometrium. Our findings suggest that MMR-deficient nonneoplastic endometrial glands may be a unique, specific marker of Lynch syndrome, and may provide an important insight into the pathogenesis of Lynch syndrome-associated endometrial cancer. Evaluation of MMR protein expression of benign background endometrium in endometrial cancer patients may be further explored as a possible useful addition to the Lynch syndrome screening algorithm.

Mismatch Repair Deficiency in Uterine Carcinosarcoma: A Multi-institution Retrospective Review.

Immunohistochemistry (IHC) for mismatch repair (MMR) proteins is recommended in endometrial carcinomas as a screening test for Lynch syndrome, and mismatch repair deficiency (MMRd) is reported in ∼30% of cases. However, few studies have evaluated the rate of MMR loss in uterine carcinosarcomas. A 5-year retrospective database search of uterine carcinosarcomas was performed at 3 academic institutions. The histologic diagnoses, type of carcinoma present, and MMR IHC interpretations were confirmed by a gynecologic pathologist. One hundred three cases of uterine carcinosarcomas with available MMR IHC results were identified. Ninety-nine cases (96%) showed intact expression and 4 cases (4%) showed loss of MLH1/PMS2. All MMRd carcinosarcomas identified in this series had an endometrioid carcinomatous component and wild-type p53 expression. In contrast, the majority of MMR intact carcinosarcomas had a serous morphology and aberrant p53 expression. Three additional cases initially diagnosed as carcinosarcoma also revealed MMRd; however, given the lack of clear mesenchymal differentiation, these cases were reclassified as dedifferentiated endometrial carcinomas and were subsequently excluded from the carcinosarcoma category. No cases of Lynch syndrome were identified among carcinosarcoma patients, as all 4 MMRd cases were due to somatic MLH1 hypermethylation. In summary, we found that the rate of MMRd is markedly lower in uterine carcinosarcoma when compared with endometrial carcinoma. In the setting of MMR loss, a diagnosis of dedifferentiated carcinoma should be considered as almost half of the MMRd tumors which were called carcinosarcomas initially were reclassified as dedifferentiated on review. However, given the interobserver variability in the classification of carcinosarcoma versus dedifferentiated carcinoma a universal screening approach that includes uterine carcinosarcoma is still recommended.

Immunohistochemical mismatch repair deficiency versus PCR microsatellite instability: a tale of two methodologies in endometrial carcinomas

Immunohistochemical mismatch repair deficiency versus PCR microsatellite instability: a tale of two methodologies in endometrial carcinomas

Retained mismatch repair protein expression occurs in approximately 6% of microsatellite instability-high cancers and is associated with missense mutations in mismatch repair genes.

Immunohistochemistry for mismatch repair protein expression is widely used as a surrogate for microsatellite instability status- an important signature for immunotherapy and germline testing. There are no systematic analyses examining the sensitivity of immunohistochemistry for microsatellite instability-high status. Mismatch repair immunohistochemistry and microsatellite instability testing were performed routinely as clinically validated assays. We classified germline/somatic mutation types as truncating (nonsense, frameshift, in/del) versus missense and predicted pathogenicity of the latter. Discordant cases were compared to concordant groups: microsatellite instability-high/ mismatch repair-deficient for mutation comparison and microsatellite stable/ mismatch repair-proficient for immunohistochemical comparison. 32 of 443 (7%) microsatellite instability-high cases had immunohistochemistry. Four additional microsatellite instability-high research cases had discordant immunohistochemistry. Of 36 microsatellite instability-high cases with discordant immnohistochemistry, 30 were mismatch repair-proficient while 6 (5 MLH1 and 1 MSH2) retained expression of the defective mismatch repair protein and lost its partner. In microsatellite instability-high tumors with discordant immunohistochemistry, we observed an enrichment in deleterious missense mutations over truncating mutations, with nearly 70% (25/36) of cases having pathogenic germline or somatic missense mutations, as opposed to only 17% (6/36) in a matched microsatellite instability-high group with concordant immunohistochemistry (p=0.0007). In microsatellite instability-high cases with discordant immunohistochemistry and MLH1 or PMS2 abnormalities, less cells showed expression (p=0.015 and p=0.00095 respectively) compared to microsatellite stable/ mismatch repair-proficient cases. Tumor mutation burden, MSIsensor score, and truncating mismatch repair gene mutations were similar between microsatellite instability-high cases with concordant versus discordant immunohistochemical expression. Approximately 6% of microsatellite instability-high cases have retained mismatch repair protein expression and would be missed by immunohistochemistry-based testing, hindering patient access to immunotherapy. Another 1% of microsatellite instability-high cases show isolated loss of the defective gene’s dimerization partner, which may lead to germline testing of the wrong gene. These cases are enriched for pathogenic mismatch repair missense mutations.

Differences in PD-L1-Expressing Macrophages and Immune Microenvironment in Testicular Germ Cell Tumors.

To characterize the tumor microenvironment of testicular germ cell tumors (GCTs) using immunohistochemical markers. Seventy-seven orchiectomies, including 36 nonmetastatic (NM) seminomas, 15 metastatic (M) seminomas, 13 nonmetastatic nonseminomatous germ cell tumors (NSGCTs), and 13 metastatic NSGCTs, were studied with PD-1, PD-L1, FOXP3, CD68, CD163, and mismatch repair (MMR) immunohistochemistry. FOXP3+ and PD-1+ tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) expressing CD68 and CD163 were enumerated. PDL-1 expression was evaluated on tumor cells and macrophages. GCTs primarily express PD-L1 on TAMs, except choriocarcinoma, where true tumor cell positivity was noted. Seminomas reveal increased intratumoral PD-L1+ TAMs compared with NSGCTs (P < .05). Activated TILs are increased in NM-seminomas compared with M-seminomas (P < .05). All GCTs retained MMR expression. Robust PD-L1+ TAMs are significantly expanded in seminomas compared with NSGCTs. Among all GCTs, only choriocarcinoma cells reveal true positivity for PD-L1. These findings expand the realm of potentially targeted treatments for GCTs.

Microsatellite Instability assessment in Black South African Colorectal Cancer patients reveal an increased incidence of suspected Lynch syndrome

Microsatellite Instability (MSI) is a hallmark of colorectal cancer (CRC) and occurs in 15–16% of CRC. Molecular biological information of CRC in South Africa (SA) is largely unrecorded. This study was undertaken to determine the frequency of MSI, with particular reference to Lynch syndrome (LS) with a view to improve surveillance and prevention strategies. This was a retrospective study on CRC samples diagnosed between 2011–2015 at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). Samples diagnosed between 2011–2012 were screened for MSI by PCR and mismatch repair (MMR) immunohistochemistry (IHC), and additional BRAFV600E mutational analysis performed. T-tests, Fischer’s exact and Chi square statistical tests were applied. Twelve percent of patients displayed MSI, with increased frequency in black (15%) versus other ethnic group (OEG) (8%) patients. MSI patients were significantly younger than microsatellite stable (MSS) patients, however when stratified by ethnicity, black patients were predominantly younger (median age: 47), with increased MSH2/6 loss, and no BRAF mutations. These findings suggest a large proportion of young black SA CRC patients develop via the LS pathway due to earlier age onset and predominant MSH2/6 protein loss. SA patients of other ethnicities appear to follow the more well established sporadic MSI pathway.

Universal screening to identify Lynch syndrome: two years of experience in a Northern Italian Center.

Lynch syndrome is caused by germline mutations of genes affecting the mismatch repair proteins MLH1, MSH2, MSH6 or PMS2. Identification of Lynch syndrome patients using germline molecular testing in colorectal cancer (CRC) affected patients and in their healthy relatives is a cost-effective model of cancer prevention. Several studies demonstrate that universal tumor testing using immunohistochemical (IHC) analysis of CRC samples is the most efficient approach to identifying patients affected by Lynch syndrome. We studied a cohort of 352 consecutive CRCs for MSH2, MLH1, MSH6 and PMS2 protein expression using universal IHC screening. IHC mismatch repair (MMR) defects were identified in 70 out of 352 cases (19.8%) including six CRCs MSH2/MSH6 defective, two CRCs, respectively, MSH6 and PMS2 defective, 58 CRCs MLH1/PMS2 defective and four CRCs showing atypical MMR pattern. MLH1 promoter methylation and V600E BRAF mutation analysis were investigated on 61 CRCs. Cancer genetic counseling was offered to all 68 patients affected by MMR defective CRCs and 25 patients opted in to this service (36.8% compliance). Pathogenetic variants of MSH2 genes were identified in two cases (55 and 79 years old). Universal screening based on an IHC approach showed a Lynch syndrome incidence of 1/173. The protocol recommended by regional law improved patient compliance. This study demonstrates that the IHC approach for both MMR deficiency and V600E BRAF mutation detections is the most efficient approach for Lynch syndrome screening in the Italian population.

Mismatch Repair Deficiency in Endometrial Cancer: Immunohistochemistry Staining and Clinical Implications.

DNA mismatch repair (MMR) deficiency is associated with increased risk of developing several types of cancer and is the most common cause of hereditary endometrial cancer. Identification of the microsatellite instability (MSI) phenotype in endometrial carcinoma is important given that such tumors are frequent. The objective of this study was to assess the utility of immunohistochemistry (IHC), a simple and fast technique, in detecting MSI status in endometrial carcinoma and evaluate the correlation between the MSI phenotype and the various anatomo-clinical parameters. IHC expression of 4 markers (MLH1, MSH2, PMS2, and MSH6) was studied. For all IHC markers, a combined score based on the intensity of nuclear labeling and the percentage of labeled cells was defined to establish a score. Correlation between MSI phenotype and different clinicopathologic parameters was evaluated using statistical analysis (software STATA and the Fisher exact test). The mean age of the patients was 58.6 years. Positive staining was highly extended (score 3) with 79% to 100% of marked cells. Less than 10% of positive tumor cells were seen in 3% of cases for MSH6 and PMS2. Abnormal MMR IHC was detected in 10 cases (22.22%). Seven tumors showed loss of MLH1/PMS2. The loss of MSH2/MSH6 was observed in 1 case. The loss of MLH1 or PMS2 was seen only in 2 cases. The number of MSI positive status was 10 cases (22.7%). Correlation between clinicopathologic parameters showed MMR deficiency was significantly associated with low-grade tumor and localized stage. There was no positive correlation between age, histologic subtype, or myometrium invasion. In summary, detection of DNA MMR deficiencies by IHC can effectively diagnose the MSI phenotype in endometrial carcinoma. Correlation between clinicopathologic parameters showed MMR deficiency was significantly associated with low-grade tumor and localized stage.

Lynch syndrome screening in gynaecological cancers: results of an international survey with recommendations for uniform reporting terminology for mismatch repair immunohistochemistry results.

Lynch syndrome (LS) is associated with an increased risk of developing endometrial carcinoma (EC) and ovarian carcinoma (OC). There is considerable variability in current practices and opinions related to screening of newly diagnosed patients with EC/OC for LS. An online survey was undertaken to explore the extent of these differences. An online questionnaire was developed by a panel of experts and sent to all members of the British Association of Gynaecological Pathologists (BAGP) and the International Society of Gynecological Pathologists (ISGyP). Anonymised results were received and analysed. Thirty-six BAGP and 44 ISGyP members completed the survey. More than 90% of respondents were aware of the association of LS with both EC and OC, but 34% were not aware of specific guidelines for LS screening. Seventy-one per cent of respondents agreed that universal screening for LS should be carried out in all newly diagnosed EC cases, with immunohistochemistry (IHC) alone as the preferred approach. Only 36% of respondents currently performed IHC or microsatellite instability testing on all newly diagnosed EC cases, with most of the remaining respondents practising selective screening, based on clinical or pathological features or both. A significant minority of respondents (35%) believed that patient consent was required before performance of mismatch repair (MMR) protein IHC. Almost all respondents favoured the use of standardised terminology for reporting MMR protein staining results, and this is proposed herein. There is wide support for universal LS screening in patients with EC, but this survey highlights areas of considerable variation in practice.

Cost-effectiveness analysis of reflex testing for Lynch syndrome in women with endometrial cancer in the UK setting.

BackgroundLynch syndrome is a hereditary cancer syndrome caused by constitutional pathogenic variants in the DNA mismatch repair (MMR) system, leading to increased risk of colorectal, endometrial and other cancers. The study aimed to identify the incremental costs and consequences of strategies to identify Lynch syndrome in women with endometrial cancer.MethodsA decision-analytic model was developed to evaluate the relative cost-effectiveness of reflex testing strategies for identifying Lynch syndrome in women with endometrial cancer taking the NHS perspective and a lifetime horizon. Model input parameters were sourced from various published sources. Consequences were measured using quality-adjusted life years (QALYs). A cost-effectiveness threshold of £20 000/QALY was used.ResultsReflex testing for Lynch syndrome using MMR immunohistochemistry and MLH1 methylation testing was cost-effective versus no testing, costing £14 200 per QALY gained. There was uncertainty due to parameter imprecision, with an estimated 42% chance this strategy is not cost-effective compared with no testing. Age had a significant impact on cost-effectiveness, with testing not predicted to be cost-effective in patients aged 65 years and over.ConclusionsTesting for Lynch syndrome in younger women with endometrial cancer using MMR immunohistochemistry and MLH1 methylation testing may be cost-effective. Age cut-offs may be controversial and adversely affect implementation.

Full-length transcript amplification and sequencing as universal method to test mRNA integrity and biallelic expression in mismatch repair genes

In pathogenicity assessment, RNA-based analyses are important for the correct classification of variants, and require gene-specific cut-offs for allelic representation and alternative/aberrant splicing. Beside this, the diagnostic yield of RNA-based techniques capable to detect aberrant splicing or allelic loss due to intronic/regulatory variants has to be elaborated. We established a cDNA analysis for full-length transcripts (FLT) of the four DNA mismatch repair (MMR) genes to investigate the splicing pattern and transcript integrity with active/inhibited nonsense-mediated mRNA-decay (NMD). Validation was based on results from normal controls, samples with premature termination codons (PTC), samples with splice-site defects (SSD), and samples with pathogenic putative missense variants. The method was applied to patients with variants of uncertain significance (VUS) or unexplained immunohistochemical MMR deficiency. We categorized the allelic representation into biallelic (50 ± 10%) or allelic loss (≤10%), and >10% and <40% as unclear. We defined isoforms up to 10% and exon-specific exceptions as alternative splicing, set the cut-off for SSD in cDNA + P to 30-50%, and regard >10% and <30% as unclear. FLT cDNA analyses designated 16% of all putative missense variants and 12% of VUS as SSD, detected MMR-defects in 19% of the unsolved patients, and re-classified >30% of VUS. Our method allows a standardized, systematic cDNA analysis of the MMR FLTs to assess the pathogenicity mechanism of VUS on RNA level, which will gain relevance for precision medicine and gene therapy. Diagnostic accuracy will be enhanced by detecting MMR defects in hitherto unsolved patients. The data generated will help to calibrate a high-throughput NGS-based mRNA-analysis and optimize prediction programs.

Application of the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) to patients conservatively treated: Outcomes from an institutional series

ObjectiveTo test the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) and determine the frequency of specific/prognostic molecular alterations within a cohort of endometrial cancer (EC) women conservatively treated by combined hysteroscopic resection and progestin therapy. Study designWe used blocks of formalin-fixed paraffin-embedded tissue from the primary tumors of patients enrolled into the ECCo trial (EudraCT 2010-018581-23) between 2007 and 2016. In order to assign EC resectoscopic specimens to one of four ProMisE subgroups, testing involved sequential assessment of i) immunohistochemistry (IHC) for mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2; ii) sequencing for POLE/POLD1 exonuclease domain mutations (EDMs); iii) p53 IHC. ResultsMolecular analysis methods were used in 25 patients (stage IA, G1-2 endometrioid EC), of whom 15 (60%) represented fully evaluable cases. Seven cases (46.7%) had abnormal MMR IHC, POLE/POLD1 EDMs were found in 3 cases (20%), and abnormal p53 IHC in 1 case (6.6%). Three patients (20%) had more than one molecular feature. Among 10 (40%) ‘unclassifiable’ patients, six failures in achieving complete molecular categorization were due to the low tumor volume. Molecular classification of the 15 fully evaluable cases yielded the following ProMisE subtypes: 7 (46.7%) MMR IHC abnormal, 1 (6.6%) POLE EDM, 0 (0%) p53 IHC abnormal, 7 (46.7%) p53 IHC wild-type. ConclusionsAlthough larger series are needed to further assess the feasibility of a molecular categorization in a fertility-sparing setting, data presented are promising. In women with early stage low-volume disease, operative hysteroscopy could be advantageous to provide samples allowing complete genetic risk assessment.