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Background: Mismatch‐repair (MMR)/microsatellite instability (MSI) status has therapeutic implications in endometrial cancer (EC). The authors evaluated the concordance of testing and factors contributing to MMR expression heterogeneity. Methods: Six hundred sixty‐six ECs were characterized using immunohistochemistry (IHC), MSI testing, and mut‐L homolog 1 (MLH1) methylation. Select samples underwent whole‐transcriptome analysis and next‐generation sequencing. MMR expression of metastatic/recurrent sites was evaluated. Results: MSI testing identified 27.3% of cases as MSI‐high (n = 182), MMR IHC identified 25.1% cases as MMR‐deficient (n = 167), and 3.8% of cases (n = 25) demonstrated discordant results. A review of IHC staining explained discordant results in 18 cases, revealing subclonal loss of MLH1/Pms 1 homolog 2 (PMS2) (n = 10) and heterogeneous MMR IHC (mut‐S homolog 6 [MSH6], n = 7; MLH1/PMS2, n = 1). MSH6‐associated Lynch syndrome was diagnosed in three of six cases with heterogeneous expression. Subclonal or heterogeneous cases had a 38.9% recurrence rate (compared with 16.7% in complete MMR‐deficient cases and 9% in MMR‐proficient cases) and had abnormal MMR IHC results in all metastatic recurrent sites (n = 7). Tumors with subclonal MLH1/PMS2 demonstrated 74 differentially expressed genes (determined using digital spatial transcriptomics) when stratified by MLH1 expression, including many associated with epithelial–mesenchymal transition. Conclusions: Subclonal/heterogeneous MMR IHC cases showed epigenetic loss in 66.7%, germline mutations in 16.7%, and somatic mutations in 16.7%. MMR IHC reported as intact/deficient missed 21% of cases of Lynch syndrome. EC with subclonal/heterogeneous MMR expression demonstrated a high recurrence rate, and metastatic/recurrent sites were MMR‐deficient. Transcriptional analysis indicated an increased risk for migration/metastasis, suggesting that clonal MMR deficiency may be a driver for tumor aggressiveness. Reporting MMR IHC only as intact/deficient, without reporting subclonal and heterogeneous staining, misses opportunities for biomarker‐directed therapy. Plain Language Summary: Endometrial cancer is the most common gynecologic cancer, and 20%–40% of tumors have a defect in DNA proofreading known as mismatch‐repair (MMR) deficiency.These results can be used to guide therapy.Tests for this defect can yield differing results, revealing heterogeneous (mixed) proofreading capabilities.Tumors with discordant testing results and mixed MMR findings can have germline or somatic defects in MMR genes.Cells with deficient DNA proofreading in tumors with mixed MMR findings have DNA expression profiles linked to more aggressive characteristics and cancer spread.These MMR‐deficient cells may drive tumor behavior and the risk of spreading cancer. Intratumoral heterogeneity, including subclonal and heterogeneous mismatch‐repair (MMR) expression, can yield discordant results from diagnostic assays, such as immunohistochemistry and polymerase chain reaction‐based microsatellite instability testing. Within endometrial cancers, subclonal or heterogeneous MMR expression may be attributed to germline or somatic mutations in MMR proteins, and the authors demonstrate that these tumors are characterized by clonal selection favoring the propagation of MMR deficiency at sites of metastasis or recurrence. [ABSTRACT FROM AUTHOR]

This document is a reply to a previous article discussing the significance of tumors with "unusual staining" from mismatch repair (MMR) immunohistochemistry (IHC) in endometrial cancer. The authors agree that ongoing conversation and multiple testing strategies are needed to improve outcomes for patients. They also emphasize the importance of quality assurance in performing and interpreting MMR IHC, including uniform fixation and grossing protocols. The authors acknowledge the potential benefits of primary assessment of MMR IHC staining on biopsy specimens but note that in their cohort, preoperative biopsies were performed outside their institution and MMR IHC was not performed on these specimens. They also discuss the interpretation of MSI testing in endometrial cancer and the use of the Promega panel for clarifying cases that are MSI-low on the National Cancer Institute panel. Further research is needed to define optimal testing for MMR-deficient/MSI endometrial cancer and predict response to immunotherapy. [Extracted from the article]

BACKGROUND Exome and targeted sequencing studies have identified potential driver mutations for a variety of tumor types. Cutaneous squamous cell carcinoma (cSCC) is one of the most highly mutated cancers but typically is associated with low rates of metastasis and high survival rates. Nevertheless, metastatic cSCC is a significant health threat; up to 8800 individuals die each year of this disease. METHODS Because it is difficult to predict which cSCCs are more likely to metastasize, and because to the best of the authors' knowledge there are no targeted therapies specifically designated for patients with metastatic cSCC, exome and/or targeted sequencing of 18 metastatic and 10 primary cSCCs was performed to identify mutations that were more frequent in metastatic tumors and might be targeted for therapeutic benefit. The authors compared their results with published sequencing results of an additional 223 primary tumors and 68 metastatic cSCCs. RESULTS The authors identified genes demonstrating higher mutation frequencies in metastatic cSCC compared with primary tumors, including the chromatin remodeling gene lysine methyltransferase 2D ( KMT2D) and the classic skin tumor suppressor tumor protein p53 ( TP53), which was found to be mutated in 54% of primary tumors compared with 85% of metastatic tumors ( P<.0001). CONCLUSIONS These studies appear to uncover potential pathways that are important in metastatic cSCC and that broaden understanding of the biology contributing to aggressive tumor behavior. These results may lead to new therapeutic strategies. Cancer 2017;123:1184-1193. © 2016 American Cancer Society. [ABSTRACT FROM AUTHOR]

ABSTRACT Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty-three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance. [ABSTRACT FROM AUTHOR]

Preclinical Research Phenylmethimazole (C10) is an inhibitor of Toll ‐ like receptor (TLR3 and TLR4) expression and signaling. In this study, we carried out a detailed investigation of the effect of C10 on TLR4 and its molecular signaling products in RAW 264.7 macrophages using quantitative real ‐ time polymerase chain reaction (PCR), ELISA and cell toxicity assays, a set of in vitro assays that may be used to screen future C10 analogs. C10 exhibited an inhibitory effect on TLR4 MyD88 ‐ dependent and MyD88 ‐ independent pathways. Within the TLR4 pathway, C10 inhibited the expression of cytokines, cytokine receptors, kinases, adapter molecules and transcription factors, suggesting a pathway ‐ wide inhibitory effect. We also found that C10 dose ‐ dependently inhibited the expression of TLR4 signaling products, specifically IL ‐ 6, inducible nitric oxide (NO) synthase and IFNβ. Additionally, pre ‐ treatment of RAW 264.7 cells with C10 resulted in protection from lipopolysaccharide (LPS) insults, suggesting C10 may be bound to the target thus exhibiting activity during/following LPS stimulation. Also, dimethyl sulfoxide, the solvent for C10 exhibited inhibitory effect on TLR4 signaling products independent from the effects of C10. Combined, this study enhances understanding of the actions of action on TLR4 signaling pathway providing a path for the development of new C10 analogs for inhibiting TLR expression and signaling. [ABSTRACT FROM AUTHOR]

Background: Exome and targeted sequencing studies have identified potential driver mutations for a variety of tumor types. Cutaneous squamous cell carcinoma (cSCC) is one of the most highly mutated cancers but typically is associated with low rates of metastasis and high survival rates. Nevertheless, metastatic cSCC is a significant health threat; up to 8800 individuals die each year of this disease.Methods: Because it is difficult to predict which cSCCs are more likely to metastasize, and because to the best of the authors' knowledge there are no targeted therapies specifically designated for patients with metastatic cSCC, exome and/or targeted sequencing of 18 metastatic and 10 primary cSCCs was performed to identify mutations that were more frequent in metastatic tumors and might be targeted for therapeutic benefit. The authors compared their results with published sequencing results of an additional 223 primary tumors and 68 metastatic cSCCs.Results: The authors identified genes demonstrating higher mutation frequencies in metastatic cSCC compared with primary tumors, including the chromatin remodeling gene lysine methyltransferase 2D (KMT2D) and the classic skin tumor suppressor tumor protein p53 (TP53), which was found to be mutated in 54% of primary tumors compared with 85% of metastatic tumors (P<.0001).Conclusions: These studies appear to uncover potential pathways that are important in metastatic cSCC and that broaden understanding of the biology contributing to aggressive tumor behavior. These results may lead to new therapeutic strategies. Cancer 2017;123:1184-1193. © 2016 American Cancer Society. [ABSTRACT FROM AUTHOR]