Immunohistochemical Screening of Upper Tract Urothelial Carcinomas for Lynch Syndrome Diagnostics: A Systematic Review.

OBJECTIVE: To review the effect of universal screening of newly diagnosed upper tract urothelial carcinomas (UTUC) for mismatch repair (MMR) protein loss to aid in Lynch syndrome diagnostics. MATERIALS AND METHODS: Studies were identified through PubMed on December 1, 2021. Eligibility criteria were universal immunohistochemical analyses for at least 2 MMR proteins in unselected, consecutively collected UTUC cohorts. Exclusion criteria included reviews, case-reports, non-English language, and non-humans. Risk of bias was assessed using a modified Newcastle-Ottawa scale. Meta-analyses were performed to compare the association between clinical criteria and Lynch syndrome diagnoses. RESULTS: From 12 included studies, 1628 surgically removed UTUC from 1626 patients were screened for MMR protein loss. In 11 studies, 140 of the 1559 patients had tumors with loss (9.0%) with 80.7% showing loss of MSH2, MSH6, or both. In 7 studies, genetic testing confirmed Lynch syndrome diagnosis for 20 of 970 patients (2.1%). In 8 studies, 31 patients were given a clinical Lynch syndrome diagnosis (2.6%). In total, 51 assumed or verified Lynch syndrome patients were identified among 1087 patients (4.7%). Meta-analyses of 3 studies showed significant association between previous cancer diagnosis and Lynch syndrome-associated UTUC (P = .038). CONCLUSION: Despite the few studies conducted and lack of genetic testing, current data suggests that universal screening for MMR protein loss in UTUC may result in Lynch syndrome diagnoses in 4.7%. However, for the screening to be effective for Lynch syndrome diagnostics, follow-up investigations, such as genetic testing for MMR variants, are needed.

Investigation of discrepant mismatch repair immunohistochemistry and microsatellite instability polymerase chain reaction test results for gynecologic cancers using next-generation sequencing.

Gynecologic cancers are routinely screened for DNA mismatch repair (MMR) gene mutations using immunohistochemistry (IHC) and/or polymerase chain reaction (PCR) for microsatellite instability (MSI) to enable selection of immune checkpoint inhibitor therapy and screen for Lynch syndrome. The limited data that compare IHC and MSI in endometrial tumors have shown discordance rates of 5-10%. We reviewed MMR/MSI results in gynecologic cancers and used next-generation sequencing (NGS) to interrogate discrepancies. Of the 328 cases with both IHC and MSI results, 256 (78.0%) were microsatellite stable (MSS) with preserved MMR (pMMR), 64 (19.5%) cases were MSI-High (MSI-H) with MMR deficient (dMMR), 2 cases showed subclonal loss of MLH1 and PMS2 with MSI-H, and 6 cases were discordant. Overall, there was a 98.2% (322/328) IHC/MSI concordance. Discordant cases were retested and/or subject to NGS. Of the six discrepant cases, five showed dMMR with MSS and one showed pMMR with MSI-H. One dMMR/MSI-L case showed loss of PMS2 with a germline pathogenic mutation. The pMMR/MSI-H case was found to harbor pathogenic variants in MLH1 and MSH6. One of the two cases with subclonal populations demonstrated MSI-H in the dMMR area and MSS in the pMMR area. These results emphasize the importance of selecting the appropriate tumor tissue for both IHC and molecular testing and demonstrate that NGS can help resolve discrepant MMR and MSI results.

Morphological predictors for microsatellite instability in urothelial carcinoma.

INTRODUCTION: Microsatellite instability occurs due to a series of mutations in the DNA pairing error repair (Mismatch repair; MMR) genes, which can affect germ cells as occurs in Lynch syndrome, whose patients are at high risk of developing multiple cancers. The loss of MMR protein is commonly determined by immunohistochemical studies. Although the relation between microsatellite instability and urothelial carcinomas has been widely studied, its evaluation is not currently performed in the analysis of urothelial carcinomas. METHODS: In this study, the microsatellite status of 139 urothelial carcinomas was analyzed and their clinicopathological characteristics were evaluated. We identified that 10.3% (13 patients) of urothelial carcinomas had loss of MMR protein expression (9 MLH1; 5 MSH2; 2 PMS2; 2 PSH6; n = 139). RESULTS: Results suggest that these tumors occur more frequently in males, are more frequently located in the bladder or ureters, and present a high tumor grade with a papillary histological pattern that does not infiltrate the lamina propria or, in the case of infiltrating tumors, that grows into perivesical tissues. CONCLUSIONS: We identified patients with the aforementioned tumor characteristics as patients with a high probability of presenting loss of MMR protein expression, and consider that only these patients should undergo further immunohistochemical and molecular techniques for proper diagnosis. Therefore, we propose that the clinicopathological characteristics found in the present study could become possible markers to determine which cases should undergo additional tests.

PD-L1, PARP1, and MMRs as potential therapeutic biomarkers for neuroendocrine cervical cancer.

OBJECTIVE: Neuroendocrine cervical cancer (NECC) is a rare cervical cancer with high aggressivity that causes poor prognosis even in the early stage. Given other neuroendocrine carcinomas and other types of cervical cancer have been proved to have expression of programmed cell death protein 1 ligand 1(PD-L1) and poly ADP-ribose polymerase-1(PARP1), we would measure and analyze these proteins in this invasive cancer. The purpose of this study is to investigate the application value of PD-1/PD-L1 and PARP1 inhibitors in NECC. METHODS: The NECC cases in our center with formalin-fixed paraffin-embedded tissue blocks were collected, and immunohistochemical (IHC) staining of PD-L1, PARP1, Mismatch repair proteins (MMRs), and P53 was performed. Chi-square test was used to analyze associations between various protein expressions. We analyzed the efficacy of immunotherapy in a recent patient with secondary recurrence after two courses of chemotherapy. RESULTS: After rigorous screening, 20 cases were finally included. Three cases did not undergo surgical treatment because of their advanced stage. Twelve (60%) developed distant metastases or relapsed within five years, and most of them within two years. The positive rate of PD-L1 and PARP1 were 70% and 75% respectively. Among all the cases, microsatellite instability (MSI) was seen in six cases (30%) and abnormal p53 expression was in 15 patients (75%). PD-L1 was associated with PARP1 expression in the MSI subgroup. The patient treated with chemotherapy + VEGF inhibitor (VEGFi) + programmed cell death protein 1(PD-1) inhibitor had an excellent improvement in clinical symptoms, tumor markers, and mass size. CONCLUSION: The IHC results of PD-L1, PARP1, and MMRs suggested that NECC was the target of immunotargeted therapy. Our case confirmed that immune checkpoint therapy was effective in patients with PD-L1 positive and MMRs loss. Considering the clinical practicability, more cases should be collected, and effective biomarkers still need to be further searched.

Mismatch repair deficiency is rare in bone and soft tissue tumors.

INTRODUCTION: There has been an increased demand for mismatch repair (MMR) status testing in sarcoma patients after the success of immune checkpoint inhibition (ICI) in MMR deficient tumors. However, data on MMR deficiency in bone and soft tissue tumors is sparse, rendering it unclear if routine screening should be applied. Hence, we aimed to study the frequency of MMR deficiency in bone and soft tissue tumors after we were prompted by two (potential) Lynch syndrome patients developing sarcomas. METHODS: Immunohistochemical expression of MLH1, PMS2, MSH2 and MSH6 was assessed on tissue micro arrays (TMAs), and included 353 bone and 539 soft tissue tumors. Molecular data was either retrieved from reports or microsatellite instability (MSI) analysis was performed. In MLH1 negative cases, additional MLH1 promoter hypermethylation analysis followed. Furthermore, a systematic literature review on MMR deficiency in bone and soft tissue tumors was conducted. RESULTS: Eight MMR deficient tumors were identified (1%), which included four leiomyosarcoma, two rhabdomyosarcoma, one malignant peripheral nerve sheath tumor and one radiation-associated sarcoma. Three patients were suspected for Lynch syndrome. Literature review revealed 30 MMR deficient sarcomas, of which 33% were undifferentiated/unclassifiable sarcomas. 57% of the patients were genetically predisposed. CONCLUSION: MMR deficiency is rare in bone and soft tissue tumors. Screening focusing on tumors with myogenic differentiation, undifferentiated/unclassifiable sarcomas and in patients with a genetic predisposition / co-occurrence of other malignancies can be helpful in identifying patients potentially eligible for ICI.

Clinicopathologic features and prognosis of synchronous and metachronous multiple primary colorectal cancer.

PURPOSE: Multiple primary colorectal cancers (MPCCs) are different from solitary colorectal cancers in many aspects, which are not well studied. The aim of this study was to clarify the clinicopathological features and prognosis of MPCCs. METHODS: The data of 64 patients with MPCCs out of 2300 patients with colorectal cancers (CRCs) from January 2009 to December 2017 were retrospectively analyzed. Stratified analysis was conducted based on subtypes and microsatellite status. RESULTS: The overall incidence of MPCC was 2.8% and the median follow-up duration was 51.5 (range 1-120) months. Metachronous CRCs (MCRCs) are more likely to appear in the right colon (p < 0.05). However, no significant differences regarding age, sex, BMI, tumor size, smoking/drinking history, TNM stage, family history of cancer, and 5-year survival rate were observed between synchronous CRC (SCRC) and MCRC. Advanced TNM stage (III) and the presence of polyps were found to be independent poor prognostic factors for MPCCs. The prevalence of mismatch repair deficiency (dMMR) in MPCCs was 28.1%. Deficient MMR is more likely to appear in younger, lighter MPCC patients with polyps (p < 0.05). Of four mismatch repair proteins, MLH-1, MSH-2, MSH-6, and PMS-2 were negative in nine, nine, five, and nine patients, respectively. The 5-year survival rate did not differ significantly between MMR-proficient (pMMR) and dMMR groups (p = 0.752). CONCLUSIONS: Synchronous CRC (SCRC) and MCRC might represent similar disease entities with different courses. Deficient MMR is more likely to appear in younger, lighter MPCC patients with polyps and it is an essential indicator for screening Lynch syndrome.

Universal Mismatch Repair Protein Screening in Upper Tract Urothelial Carcinoma.

OBJECTIVES: Universal screening of upper tract urothelial carcinoma (UTUC) for Lynch syndrome by mismatch repair (MMR) protein immunohistochemistry (IHC) has been recommended by some investigators. Herein, we assess this recommendation retrospectively by simulating its performance on a retrospective, unselected cohort of UTUCs, with comparison to the established setting of colorectal and endometrial adenocarcinoma. METHODS: We assessed for complete loss of MMR protein (MLH1, MSH2, MSH6, and PMS2) IHC in 74 consecutive cases of UTUC and then tabulated clinical and pathologic factors. MMR findings from same-institution colorectal and endometrial adenocarcinomas were tabulated for comparison. RESULTS: We observed loss of at least one MMR protein in 12% in our UTUC cohort (three MSH2/MSH6, three MSH6 only, one MLH1/PMS2, and two PMS2 only). Of these nine cases (seven males, two females, median age 67 years, five associated with colorectal adenocarcinoma), at least three (4% of the overall cohort) proved to be Lynch syndrome. Overall, MMR loss in UTUC was comparable to colorectal (11%; 50 of 471 cases) and endometrial (12%; 12 of 101 cases) adenocarcinomas. CONCLUSIONS: The rate of MMR loss observed in UTUC was comparable to that in the established setting of colorectal and endometrial adenocarcinomas, supporting universal UTUC screening at our institution and others.

Population bias in somatic measurement of microsatellite instability status.

Microsatellite instability (MSI) is a key secondary effect of a defective DNA mismatch repair mechanism resulting in incorrectly replicated microsatellites in many malignant tumors. Historically, MSI detection has been performed by fragment analysis (FA) on a panel of representative genomic markers. More recently, using next-generation sequencing (NGS) to analyze thousands of microsatellites has been shown to improve the robustness and sensitivity of MSI detection. However, NGS-based MSI tests can be prone to population biases if NGS results are aligned to a reference genome instead of patient-matched normal tissue. We observed an increased rate of false positives in patients of African ancestry with an NGS-based diagnostic for MSI status utilizing 7317 microsatellite loci. We then minimized this bias by training a modified calling model that utilized 2011 microsatellite loci. With these adjustments 100% (95% CI: 89.1% to 100%) of African ancestry patients in an independent validation test were called correctly using the updated model. This poses not only a significant technical improvement but also has an important clinical impact on directing immune checkpoint inhibitor therapy.

An Integrative Morphomolecular Classification System of Gastric Carcinoma With Distinct Clinical Outcomes.

A robust morphomolecular classification system for gastric carcinoma is required. A 4-tier morphologic classification is proposed, including diffuse, intestinal, tubular, and lymphoid types. A tissue microarray for mismatch repair immunohistochemistry and Epstein-Barr virus (EBV) in situ hybridization were performed in 329 gastric carcinomas. DNA flow cytometry was used to detect aneuploidy in formalin-fixed paraffin-embedded samples. Lymphoid histology was the third most common histologic pattern at our institute and strongly associated with EBV infection and PMS2/MLH1-deficiency (both P<0.001). HER2 overexpression and SATB2 expression more frequently occurred in intestinal histology (both P<0.001). Loss of ARID1A expression was strikingly associated with lymphoid histology (P<0.001) and negative E-cadherin expression was correlated with diffuse histology (P=0.001). Programmed death-ligand 1 expression was most frequently present in lymphoid-type gastric carcinoma than other histologic subtypes and correlated with the molecular features of PMS2/MLH1-deficiency and EBV infection (all P<0.001). Aneuploidy was detected in 53% of gastric carcinomas and was highly correlated with intestinal type and the least with the lymphoid type (P<0.001). Notably, lymphoid-type gastric carcinoma showed the best outcome, whereas tubular type showed the worst survival rate (P<0.001). We integrated aneuploidy with morphologic patterns to propose a morphomolecular classification scheme, which served as a successful and independent prognostic factor in multivariate 5-year disease-free survival analysis (P<0.001). Overall, we describe an integrated morphomolecular classification system for gastric carcinomas to effectively predict patient outcomes. This system is cost-effective and reliable and can help select target therapeutics and facilitate clinical management.

An Intrapatient Concordance Study of Mismatch Repair Protein Immunohistochemical Staining Patterns in Patients With Muir-Torre Syndrome.

Importance: Appropriate use criteria for Muir-Torre syndrome (MTS) screening suggest that mismatch repair protein (MMRP) immunohistochemical (IHC) testing is usually appropriate in patients with 2 or more sebaceous neoplasms (SNs). While MTS is known to be caused by a germline mutation in mismatch repair genes, data are limited as to whether individual sebaceous tumors in these patients with multiple lesions show identical MMRP IHC staining patterns. Objective: To determine concordance of MMRP IHC staining patterns in lesions of patients with MTS who have multiple SNs. Design, Setting, and Participants: This retrospective single-center case series evaluated 38 SNs in 11 patients with MTS confirmed by genetic testing for MMRP IHC staining patterns. Tumor sites were classified as either facial or extrafacial. Data were collected between January 1, 2007, and January 1, 2018. Main Outcomes and Measures: In each patient, MMRP IHC staining patterns for SNs were compared with one another to evaluate intrapatient concordance between lesions, and to the patient's known germline mutation. Results: A total of 11 patients (7 women and 4 men) with MTS, with a mean (SD) age of 59.3 (10.6) years at time of SN biopsy, were identified. There was high concordance between MMRP IHC staining results (2-4 lesions per patient) and the patient's mutation status, with 36 of 38 total lesions (95%) matching (sensitivity, 94.7%; 95% CI, 82.3%-99.4%). Extrafacial site tumors represented 16 of 38 total lesions (42%) and demonstrated 100% concordance of IHC results to germline mutation. Only 1 of 11 patients (9%) demonstrated discordant results, with both lesions in this patient occurring on a facial site. Conclusions and Relevance: In patients with known MTS, SNs present with highly concordant MMRP IHC staining profiles across multiple lesions. There is also a strong association with underlying germline mutations. A diagnosis of MTS might be supported by MMRP IHC when the pretest probability is high.

Homogeneous MMR Deficiency Throughout the Entire Tumor Mass Occurs in a Subset of Colorectal Neuroendocrine Carcinomas.

Neuroendocrine neoplasms comprise a heterogeneous group of tumors, categorized into neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) depending on tumor differentiation. NECs and high-grade NETs (G3) confer a poor prognosis, demanding novel treatment strategies such as immune checkpoint inhibition in tumors with microsatellite instability (MSI). To study any possible intratumoral heterogeneity of MSI, a tissue microarray (TMA) containing 199 NETs and 40 NECs was constructed to screen for MSI using immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. Four cases suspicious for MSI were identified. Validation of MSI by repeated IHC on large sections and polymerase chain reaction (PCR)-based analysis using the "Bethesda Panel" confirmed MSI in 3 cecal NECs. One pancreatic NET G3 with MSI-compatible TMA results was MMR intact on large section IHC and microsatellite stable (MSS). The remaining 235 tumors exhibited intact MMR. Protein loss of MLH1/PMS2 was found in two and MSH6 loss in one cancer with MSI. Large section IHC on all available tumor-containing tissue blocks in NECs with MSI did not identify aberrant tumor areas with intact MMR. Our data indicate that MSI is common in colorectal NECs (3 out of 10) but highly infrequent in neuroendocrine neoplasms from many other sites. The lack of intratumoral heterogeneity of MMR deficiency suggests early development of MSI during tumorigenesis in a subset of colorectal NECs and indicates that microsatellite status obtained from small biopsies may be representative for the entire cancer mass.

Switch/sucrose nonfermenting nucleosome complex-deficient colorectal carcinomas have distinct clinicopathologic features.

The switch/sucrose nonfermenting (SWI/SNF) nucleosome complex consists of several proteins that are involved in cellular proliferation and tumor suppression. The aim of this study was to correlate immunohistochemical expression of four SWI/SNF complex subunits, SMARCA2, SMARCB1, SMARCA4, and ARID1A, with clinicopathologic and molecular features and patient survival in 338 patients with colorectal adenocarcinoma using a tissue microarray approach. Twenty-three (7%) colorectal adenocarcinomas demonstrated deficient SWI/SNF expression: 7 had SMARCA2 deficiency, 12 had ARID1A deficiency, and 4 had both SMARCA2 and ARID1A deficiency. No cases were SMARCB1 or SMARCA4 deficient. Twelve (52%) SWI/SNF complex-deficient tumors demonstrated mismatch repair (MMR) deficiency (p = 0.02), 6 (26%) showed medullary differentiation (p = 0.001), and 9 were negative for CDX2 expression (p < 0.001). Among the MMR-deficient SWI/SNF complex-deficient tumors, 8 were sporadic MLH1 deficient, and 4 were seen in patients with Lynch syndrome. Compared with tumors with ARID1A deficiency alone, SMARCA2-deficient tumors were less likely to exhibit MMR deficiency (27% vs. 75%, p = 0.04), medullary differentiation (0% vs. 50%, p = 0.01), and mucinous differentiation (0% vs. 42%, p = 0.04). Conventional gland-forming histology was more often identified in SMARCA2-deficient tumors (11/11, 100%) than in tumors with ARID1A deficiency alone (4/12, 33%) (p = 0.001). There was no difference in KRAS mutation, BRAF mutation, stage, disease-specific survival, or disease-free survival for patients stratified by SWI/SNF expression (all with p > 0.05). In conclusion, SMARCA2-deficient and ARID1A-deficient colorectal carcinomas had distinctly different clinicopathologic features, with ARID1A-deficient tumors exhibiting medullary and mucinous differentiation and MMR deficiency and SMARCA2-deficient tumors demonstrating conventional gland-forming histologic growth with less frequent MMR deficiency.

Interpretation of Mismatch Repair Protein Immunohistochemistry in Endometrial Carcinoma Should Consider Both Lynch Syndrome Screening and Immunotherapy Susceptibility: An Illustrative Case Report.

We present an instructive case of FIGO grade 1 endometrioid endometrial carcinoma with a biphasic morphology, corresponding to subclonal loss of mismatch repair proteins (MMRP) MLH1 and PMS2 by immunohistochemistry and subclonal microsatellite instability. A pulmonary metastasis represented only the tumor component with retention of MMRPs. This case illustrates the need for pathologists to recognize and report heterogenous expression of MMRPs in endometrial carcinoma, to consider tumor heterogeneity when selecting foci for molecular studies, and to re-evaluate MMRP expression in tumor metastases, when clinically indicated. These considerations are particularly important as the relevance of MMRP expression in endometrial cancer expands beyond Lynch syndrome screening, into a new role as a predictive marker for immunotherapy.

Enhanced Tumoral MLH1-Expression in MLH1-/PMS2-Deficient Colon Cancer Is Indicative of Sporadic Colon Cancer and Not HNPCC.

Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is caused by germline mutations of mismatch-repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. MLH1-/PMS2-deficient colorectal carcinomas might be HNPCC-associated but also caused by MLH1-promoter methylation in sporadic colon carcinoma. This study analyzed semiquantitatively whether the MLH1 staining pattern might be indicative of sporadic or HNPCC-associated colorectal cancer. Using a semiquantitative score ranging from 0 (negative) to 12 (maximum immunopositivity) we analyzed MLH1 expression patterns in 130 MLH1-/PMS2-deficient colorectal cancers. The collective consisted of 70 HNPCC-associated colorectal cancers and 60 sporadic colon cancers. In tumor cells of 70 HNPCC-associated colorectal cancers, 64 cases (91.43%) showed no MLH1 staining, 5 cases weak (7.14%) and 1 case (1.43%) stronger staining intensity. In contrast, in tumor cells of 60 sporadic colorectal cancers 45 cases (75.0%) showed no MLH1 staining, 10 cases weak (16.67%) and 5 cases (8.33%) stronger staining intensity to a varying extent. In immunopositive cases, MLH1 showed a characteristic dot-like nuclear staining pattern in the tumor cells. We compared cases with absent to weak MLH1-staining (immunoscores 0 to 2) to cases with elevated immunoscores (3 to 12) detecting a statistically significant difference between HNPCC-associated and sporadic colon cancers (p value = 0.0031, Fisher's exact test). Taken together, enhanced tumoral MLH1 expression in MLH1-/PMS2-deficient colorectal carcinomas seems to be indicative of sporadic origin. In contrast, HNPCC-associated colorectal cancer showed absent or very weak MLH1 immunopositivity. Therefore, this semiquantitative and easy to exert MLH1 immunoscore might help to identify sporadic MLH1-/PMS2-deficient colorectal cancer cases prior to time-consuming methylation analysis.

[Comparison of the mismatch repair deficiency of colorectal cancers between African and European cohorts]. / Comparaison du « deficit mismatch repair ¼ des cancers colorectaux entre des cohortes africaines et européennes.

INTRODUCTION: According to European and American series, up to 20% of colorectal cancers are characterised by instability at microsatellites sites. MMR deficient colorectal cancers are predominantly found in the right colon. Although an increasing rate of colorectal cancer has been observed in many low-income countries including in West-Africa, data on epidemiology and biology of colorectal cancer in native Africans from this region are scarce. MATERIALS AND METHODS: We aimed to study the incidence of MMR deficiency in Côte d'Ivoire and to compare the data with those from a tertiary center in Belgium. Immunohistochemistry for MLH1, MSH2, MSH6 and PMS2 was performed on paraffin-embedded tissue samples from 83 colorectal cancers (46% males) operated in Abidjan and from 343 colorectal cancers (53% males) from Brussels. RESULTS: Colorectal cancer was occuring at a younger age in Côte d'Ivoire compared to Belgium (median age: 53 versus 66). MMR deficiency was detected in 11,7% of Belgian cases and in 13,3% of Ivorian cases. Whereas MMR deficient cancers in Brussels were mainly found in women (24/40 i.e. 60%), in Abidjan only 3/11 (27%) were female. Moreover, the predominant location of MMR deficient tumours was different between both series: in Brussels, mainly located in the right colon (24/40 i.e. 60%) whereas in Abidjan predominantly (10/11 i.e. 91%) in the left colon. In Brussels we observed in the majority of cases (67,5%) loss of expression of MLH1 and PMS2, in Abidjan loss of expression of MSH2 and MSH6 (54,5%). CONCLUSIONS: Our pilot study reveals differences in presentation of MMR deficient colorectal cancer between the two geographic regions suggesting differences in epidemiology and biology of colorectal cancer in native Africans.

Correlation of MLH1 polymorphisms, survival statistics, in silico assessment and gene downregulation with clinical outcomes among breast cancer cases.

This study aimed to investigate the role of MLH1 polymorphisms, respective protein structure prediction, survival analysis, related clinicopathological details and MLH1 expression in breast cancer (BC). Genotyping of selected SNPs in BC patients (493) and age matched controls (387) were performed by Tetra-ARMS PCR. Gene expression among breast tumors (127) and adjacent control tissues were analysed using reverse transcriptase PCR (RT-PCR) and immunohistochemistry. Statistical analysis was performed by SPSS and MedCalc. Conditional logistic regression analysis was applied to compute the odds ratio and confidence interval. Phyre2 and I-TASSER were used to generate MLH1 protein structures and verified by a variety of computational tools. Genotyping illustrated that MLH1 polymorphisms (rs63749795 and rs63749820) were significantly associated (P ≤ 0.05) with risk of developing BC. Down regulation of MLH1 gene expression/loss of the MLH1 protein (OR 12; CI 2.8-53.1) was observed in BC cases, illustrating its potential role in disease development. Moreover, loss of the MLH1 protein was found to be associated with higher grade cancer (P = 0.02) and lymph node positivity (P = 0.03), highlighting its essential role, as a component of the mismatch repair (MMR) machinery. Bioinformatics analysis confirmed that nonsense mutations produce a truncated MLH1 protein, causing a reduction in MMR efficiency. No association between MLH1 polymorphisms and overall and progression free survival statistics was observed among BC cases, possibly due to short follow-up study. Results at DNA, RNA and protein levels, along with in silico analysis, highlights the potential role of MLH1 in DNA repair mechanisms, within BC. Therefore, it was concluded that MLH1 may contribute towards BC development and progression.

Tumour mismatch repair protein loss is associated with advanced stage in oral cavity squamous cell carcinoma.

An unexplained increase in the incidence of oral cavity squamous cell carcinoma (oSCC) has been observed despite decreasing smoking rates, particularly in younger patients. Links to defects in the DNA mismatch repair (MMR) system are well established in early onset colorectal, urothelial and gynaecological malignancies. MMR deficient patients treated with immune checkpoint inhibitors have demonstrated improved response rates. Studies exploring MMR status in head and neck squamous cell carcinoma (HNSCC) demonstrate conflicting results. This study explores the incidence of MMR protein loss and its association with clinicopathological features and outcome in oSCC. Immunohistochemical staining using tissue microarrays to assess the expression of MMR proteins (hMLH1, hMSH2, hMSH6, and hPMS2) was performed on 285 consecutive oSCC cases between 2000 and 2016. Data on smoking, alcohol and metachronous malignancies were retrospectively collected. Proportional hazards regression models were used to compare survival in MMR intact and deficient patients. MMR deficiency was seen in 21 patients (7.4%). MMR deficient tumours were associated with bone invasion (52% vs 32%, p=0.05), higher pT stage (pT4 in 57% vs 35%, p<0.001) and a higher number of metachronous malignancies (p=0.05). MMR deficiency was not associated with younger age at presentation or absence of smoking or alcohol. There was no significant association between MMR status and survival (overall survival hazard ratio 1.36; p=0.32). The incidence of MMR loss in oSCC is low and is not associated with young age at presentation. MMR deficiency in oSCC is associated with an increase in the number of metachronous malignancies and more advanced primary tumours.

Immunoexpression of TS, p53, COX2, EGFR, MSH6 and MLH1 biomarkers and its correlation with degree of differentiation, tumor staging and prognostic factors in colorectal adenocarcinoma: a retrospective longitudinal study.

BACKGROUND: There are cases of colorectal tumors that, although small, show more aggressive evolution than large tumors. This motivated us to study whether there are any proteins capable of alerting about these changes. The aim here was to correlate the immunoexpression of the TS, p53, COX2, EGFR, MSH6 and MLH1 biomarkers in tumors in patients with colorectal adenocarcinoma, with the degree of cell differentiation, tumor staging and clinical-pathological prognostic factors. DESIGN AND SETTING: Retrospective observational study at a public tertiary-level hospital. METHODS: We analyzed tissue-microarray paraffin blocks of tumor tissues that had been resected from 107 patients. We used Fisher's exact test to study associations between tumor differentiation/staging and the immunoexpression of biomarkers. We also used Kaplan-Meier estimation, the log-rank test and the adjusted Cox regression model to investigate the patients' overall survival (in months) according to biomarkers and disease-free interval. RESULTS: The degree of tumor differentiation and tumor staging were not associated with the biomarkers, except in cases of patients in stages III or IV, in which there was a correlation with MLH1 expression (P=0.021). Patient survival and disease-free interval were not associated with the biomarkers. CONCLUSION: There were no associations between the degree of tumor differentiation, staging, length of survival or disease-free interval and the immunoexpression of the TS, p53, COX2, EGFR or MSH6 tumor markers. In advanced cases of colorectal adenocarcinoma (stages III and IV), there was a higher percentage of MLH1-negative results.