Loss Of MLH1 Expression Research Articles

Mismatch Repair Protein Deficiency in Triple Negative Breast Carcinomas: Clinicopathological Features

Abstract Introduction/Objective Female breast cancer has now surpassed lung cancer as the most commonly diagnosed cancer worldwide. Triple-negative breast cancer (TNBC) accounts for approximately 10–15% of all breast cancers. Chemotherapy remains the mainstay of systemic treatment for patients with TNBCs. Studies of mismatch repair (MMR) deficiency in TNBC reported variable prevalence ranging, from 0.2% to 18.6%. Recent studies described an excellent response in a patient with metastatic breast cancer treated with anti-PD1 illustrating the urgent need to test for MMRP deficiency as a predictive biomarkers to select those patients who could benefit from immunotherapy. The aim of the study is to detect MMRP deficiency in a cohort of TNBC Jordanian patients using the immunohistochemical methods. Methods/Case Report This study targeted cases of TNBC diagnosed and treated at King Hussein Cancer Center from 2015 until 2021. Clinicopathological data was obtained from 152 patients’ records. Immunohistochemistry for MLH1, MSH2, MSH6 and PMS2 proteins were performed on paraffin embedded tissue containing carcinoma. Results (if a Case Study enter NA) The median age of patients at diagnosis was 49 years. A total of 14 out of 152 (9.2%) cases showed deficient MMR (dMMR) expression. Loss of expression of PMS2 was seen in 13 cases, 5 of which showed also loss of MLH1 expression. Loss of expression of both MSH6 and MSH2 was seen in one case. Positive family history of cancer was present in 5 of 12 patients with available data, three of whom had isolated loss of PMS2 and two had combined loss of PMS2 and MLH1. One pt. with loss of MSH6 and MSH2, had a personal history of a second malignancy (Lung cancer). The median duration of follow-up was 27 months. The overall (OS) and event- free survival (EFS) were better for dMMR despite the lack of statistically significant differences. At 24 months EFS and OS were 77.9% and 85.7% for dMMR and 63.9% and 56.9% for pMMR respectively. Conclusion The prevalence of dMMR in TNBC is 9% with most of the cases apparently related to Lynch syndrome. dMMR tumors tend to be associated with better outcomes compared to pMMR cases. dMMR can serve as a biomarker for better prognosis. These results are of value in directing the clinical management.

Context-dependent environmental associations with endometrial cancer histotype and genotype

ObjectiveMLH1 loss due to MLH1 methylation, detected during Lynch syndrome screening, is one of the most common molecular changes in endometrial cancer. It is well established that environmental influences such...

Comprehensive Study of Microsatellite Instability Testing and Its Comparison With Immunohistochemistry in Gastric Cancers.

In this study, polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing was comprehensively analyzed and compared with immunohistochemistry (IHC) for mismatch repair (MMR) protein expression in patients with gastric cancer (GC). In 5,676 GC cases, PCR-based MSI testing using five microsatellites (BAT-26, BAT-25, D5S346, D2S123, and D17S250) and IHC for MLH1 were performed. Re-evaluation of MSI testing/MLH1 IHC and additional IHC for MSH2, MSH6, and PMS2 were performed in discordant/indeterminate cases. Of the 5,676 cases, microsatellite stable (MSS)/MSI-low and intact MLH1 were observed in 5,082 cases (89.5%), whereas MSI-high (MSI-H) and loss of MLH1 expression were observed in 502 cases (8.8%). We re-evaluated the remaining 92 cases (1.6%) with a discordant/indeterminate status. Re-evaluation showed 1) 37 concordant cases (0.7%) (18 and 19 cases of MSI-H/MMR-deficient (dMMR) and MSS/MMR-proficient (pMMR), respectively), 2) 6 discordant cases (0.1%) (3 cases each of MSI-H/pMMR and MSS/dMMR), 3) 14 MSI indeterminate cases (0.2%) (1 case of dMMR and 13 cases of pMMR), and 4) 35 IHC indeterminate cases (0.6%) (22 and 13 cases of MSI-H and MSS, respectively). Finally, MSI-H or dMMR was observed in 549 cases (9.7%), of which 47 (0.8%) were additionally confirmed as MSI-H or dMMR by re-evaluation. Sensitivity was 99.3% for MSI testing and 95.4% for MMR IHC. Considering the low incidence of MSI-H or dMMR, discordant/indeterminate results were occasionally identified in GCs, in which case complementary testing is required. These findings could help improve the accuracy of MSI/MMR testing in daily practice.

Expression of epigenetic pathway related genes in association with PD-L1, ER/PgR and MLH1 in endometrial carcinoma

The distribution of Endometrial Cancer (EC)-related deaths is uneven among the morphologic subtypes of EC. Serous Cancer (SC) makes 10% of all EC and accounts for 40% of EC-related deaths. We investigated expression of selected genes involved in epigenetic pathways by immunohistochemistry in a cohort of 106 EC patients and analyzed mRNA-based expression levels for the same set of genes in EC samples from The Cancer Genome Atlas (TCGA) dataset. A tissue microarray was constructed using low-grade (n = 30) and high-grade (n = 28) endometrioid, serous (n = 31) and clear cell carcinoma (n = 17) samples. Epigenetic marker levels were associated with PD-L1, ER/PgR, and MLH1 expression. Epigenetic markers were evaluated by H-score and PD-L1 expression was recorded by using Combined Positive Score. Results were correlated with disease stage and survival outcome. BRD4, KAT6a and HDAC9 levels were higher in SC compared to other histologic subtypes (p<0.001–0.038). After adjusting for multiple comparisons, DNMT3b expression was higher in SC compared to endometrioid-type but not between SC and CCC. The expression levels of BRD4 (p = 0.021) and KAT6a (p = 0.0027) were positively associated with PD-L abundance, while PgR (p = 0.029) and PD-L1 expression were negatively associated. In addition, BRD4 expression was low in specimens with loss of MLH1 expression (p = 0.02). More importantly, BRD4 abundance had a negative impact on disease outcome (p = 0.02). Transcriptionally, BRD4, KAT6a and DNMT3b expression levels were higher in SC in TCGA dataset. The median PD-L1 expression was marginally associated with BRD4, a transcriptional activator of CD274/PD-L1 (p = 0.069) and positively with KAT6a (p = 0.0095). In conclusion, the protein expression levels of epigenetic markers involved in cancer pathogenesis are increased by immunohistochemistry in SC. PD-L1 levels are associated with BRD4 and KAT6a in EC samples. A combination therapy with BRD4/PD-L1 or KAT6a/PD-L1 inhibitors might have a potential use in EC, in particular serous-type carcinoma.

Immunohistochemical Analysis of Mismatch Repair Gene Proteins in Early Gastric Cancer Based on Microsatellite Status

Background: Microsatellite instability (MSI) is a major pathway involved in gastric carcinogenesis and is observed in 10–20% of early gastric cancers (EGCs). Early detection of EGCs with an MSI-high phenotype would be useful for elucidating the mechanisms of gastric carcinogenesis and improving outcomes in patients with GC. Objective: We explored the usefulness of immunohistochemical expression of mismatch repair (MMR) proteins, including MLH1, PMS2, MSH2, and MSH6 in EGC. Methods: We examined the expression of 4 MMR proteins using immunohistochemistry in 119 patients with EGC based on MS status, as determined by polymerase chain reaction-microsatellite analysis. In addition, methylation of the MLH1 gene was quantified by pyrosequencing. Results: EGCs were classified into 46 MSI-high phenotypes and 73 microsatellite stable (MSS) phenotypes. Although loss of MLH1 expression was associated with loss of PMS2 expression in the MSI-high phenotype, discordant cases of loss of expression between MLH1 and PMS2 were found (MLH1 [−]/PMS2 [+], 3 cases). Loss of MLH1/PMS2 expression was observed in 2 of 73 MSS phenotypes. Loss of MSH2/MSH6 expression was found in 4 of 46 MSI-high phenotypes, whereas loss of MSH2/MSH6 expression was not detected in the MSS phenotype. In addition, loss of MLH1 expression was correlated with methylation of MLH1. However, there were discordant cases in which loss of MLH1 expression was not accompanied by methylation of MLH1. Conclusion: Although immunostaining of MMR proteins could help predict MSI in EGCs, immunostaining did not have the same value as genetic testing for determination of MSI.

Analysis of clinicopathological and molecular features of crawling-type gastric adenocarcinoma

BackgroundCrawling-type adenocarcinoma (CRA) is an important gastric cancer (GC) subtype that exhibits a specific histological pattern and has characteristic clinicopathological findings. Despite its characteristic histology, little is known about the molecular characteristics of CRA.MethodsWe examined 177 GC cases, including 51 cases of CRA and 126 cases having conventional differentiated adenocarcinomas (CDAs). Results for immunohistochemistry (mucin phenotype; Muc5AC, Muc6, Muc2 and CD10, CDX-2, MLH-1, p53 and β-catenin), mutation analysis (TP53, KRAS and BRAF), microsatellite instability (BAT25, BAT26, D2S123, D5S346 and D17S250), DNA methylation status by a two-panel method (RUNX3, MINT31, LOX, NEUROG1, ELMO1 and THBD), MLH-1 promoter methylation, and allelic imbalance (AI; 1p, 3p, 4p, 5q, 8p, 9p, 13q, TP53, 18q and 22q) were examined.ResultsCRAs were more likely to occur in the middle third of the stomach, in younger patients and to be macroscopically depressed. Nuclear accumulation of β-catenin and loss of MLH-1 expression were less frequent among CRA cases compared to CDA cases. At a molecular level, CRA is often characterized by the deletion mutation c.529_546 (18-base pair deletion at codon 177–182 in exon 5) in the TP53 gene (10 cases). Although the low methylation epigenotype was significantly more frequent for CRAs compared to CDAs, multiple AIs were more often seen in CRAs relative to CDAs.ConclusionsThe results demonstrated that TP53 mutations, particularly c.529_546del, and multiple AIs are closely associated with CRA carcinogenesis. Our results suggest that CRA is an independent entity of GC in terms of clinicopathologic and molecular findings.

Abstract IA35: Molecular characterization of sessile serrated adenoma/polyps: A risk factor for higher colorectal cancer that further associates with endometrial polyps in female African Americans

Abstract Colorectal cancer is the third leading cause of cancer-related deaths in the United States, and rates are highest among African Americans (AAs). Sessile serrated adenoma/polyps (SSA/Ps) may be precursors to up to 30% of all colorectal cancers. Flat and mucinous features make SSA/Ps difficult to detect and diagnose. As such, there is a need for specific sensitive molecular biomarkers for an accurate and reliable diagnosis. Our aim was to assess the diagnostic value of molecular biomarkers that may distinguish SSA/Ps from benign hyperplastic polyps (HPs) among AA SSA/P patients. We conducted a retrospective study of all colonoscopies (n=12,085) performed at Howard University Hospital (2010-2015), which confirmed 4,070 AA patients with polyps, including 252 with SSA/Ps. Gene expression and mutation frequency profiles were analyzed in a total of 47 patients (62 specimens: 29 SSA/Ps, 26 HPs, 3 tubular adenomas, and 4 normal tissues). We tested 4 transcripts (MUC6, FSCN1, SEMG1, and TRNP1) using qRT-PCR. MSI and BRAF mutations were analyzed. CIMP analysis was performed using CACNA1G, IGF2, NEUROG1, RUNX, SOCS, and MLH1. In a parallel study, we assessed the association between endometrial polyp occurrence in patients with different types of colorectal lesions. MUC6, SEMG1, TRNP1, and FSCN1 were significantly more expressed in SSA/Ps vs. HPs (P&amp;lt;0.05; fold differences of 37.2, 10.7, 5.8 and 2.5, respectively). BRAF mutation was found in 55.6% of SSA/Ps vs. 12.0% in HPs (P = 0.001). The frequency of CIMP was higher in SSA/Ps but not statistically significant, while MSI was more prevalent in HPs (P &amp;gt; 0.05). There was a higher loss of MLH1 expression in HPs than SSA/Ps (42.9% showing expression vs 70.3% IHC staining &amp;gt;=2 in HPs and SSA/Ps). The SSA/Ps in our AA study were primarily distal (67%). In female patients, SSA/Ps associated with the higher frequency of endometrial polyps (8% vs. 2% in controls, p=0.003). Our results show that MUC6-SEMG1-TRNP1 expression and BRAF mutation have the strongest correlation with SSA/Ps. The distal location might help explain why MSI and CIMP may not be optimal molecular biomarkers in African American patients with SSA/Ps. These markers may be of high relevance for the diagnosis of ambiguous lesions and will benefit patients’ management for scheduling follow-ups based on the nature of index lesions. Females with colon lesions of the SSA/Ps type might benefit from a screening for an endometrial polyp in an age-independent manner. Citation Format: Hassan Ashktorab, Saman Azam, Babak Shokrani, Edward Lee, Taraneh Arjomand, Priyanka Kanth, Don Delker, Adeyinka Laiyemo, John Caretheres, Mehdi Nouraie, Hassan Brim. Molecular characterization of sessile serrated adenoma/polyps: A risk factor for higher colorectal cancer that further associates with endometrial polyps in female African Americans [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr IA35.

Tumor lymphocytic infiltration impacts recurrence in endometrioid-type endometrial carcinoma.

e15239 Background: Endometrial cancers that have mismatch repair deficiency are associated with higher numbers of tumor-associated lymphocytes, but the clinical significance of this observation is unknown. Our objective was to quantify CD3+ and CD8+ tumor lymphocytes of MMR intact (MMRi) and MMRd endometrioid-type endometrial carcinomas and determine if there was an association with survival. Methods: MMRd was defined as endometrial carcinomas with loss of MLH1 expression due to MLH1 gene methylation and determined by immunohistochemistry. MMRi was defined as positive expression of MLH1, MSH2, MSH6, and PMS2. This was followed by Aperio image-based quantification was used to assess CD3+ and CD8+ lymphocyte populations in different regions of the primary endometrial carcinomas, including tumor periphery (tumor-myometrial interface), tumor center (bounded on all sides by tumor), and tumor hotspot (area with highest number of lymphocytes). Recurrence-free survival was estimated using Kaplan Meier and Cox regression. Median follow up time was 44 months. Results: 180 patients with endometrial carcinoma were analyzed of which 132 were MMRi and 48MMRd. The MMRd group had significantly higher levels of CD3+ and CD8+ lymphocytes regardless of which tumor region was assessed (Figure 1a, P &lt; 0.001). Lymphocyte counts in both MMRd and MMRi groups had wide standard deviations such that there was some overlap in counts between the groups (Figure 1). Both MMRd and higher CD3+ counts were associated with worse recurrence-free survival. CD3+ quantification in the tumor periphery captured 21/23 recurrences (Figure 1b, HR = 8.04; 95% CI: 1.88 -34.31; p = 0.005); this included all of the MMRd cases that recurred and 7 MMRi cases with higher numbers of CD3+ lymphocytes that also recurred. Conclusions: MMRd endometrial cancers have increased numbers of CD3+ lymphocytic infiltrates within the primary tumor. Higher CD3+ infiltration is associated with greater risk of recurrence regardless of tumor location. In predicting tumor recurrence, lymphocytic counts performed better than assessment of MMR. Thus, quantification of CD3+ lymphocytes should be explored as a predictive biomarker.

Abstract P4-05-07: Whole-exome and RNA-sequencing analyses of acinic cell carcinomas of the breast

Abstract Introduction: Acinic cell carcinoma of the breast (ACC) is an exceedingly rare histologic form of triple-negative breast cancer (TNBC) with a generally good clinical behavior. Despite its unique histology, targeted sequencing analysis of ACC has failed to identify recurrent genetic alterations other than those found in common forms of TNBC. Here, we subjected three breast ACCs to whole-exome and RNA-sequencing, seeking to define whether they would be underpinned by a pathognomonic genetic alteration that would distinguish them from common forms of TNBC. Materials and methods: Three breast ACCs were subjected to whole-exome sequencing (WES) and RNA-sequencing. Somatic mutations, copy number alterations, mutational signatures and fusion genes were determined using a validated bioinformatics pipeline. We also estimated DNA-based measures of genomic instability including mutational signatures (using SigMA), large-scale state transition (LST) and telomeric allelic imbalance (NtAI). Results: No oncogenic in-frame fusion transcript was identified in the breast ACCs analyzed. ACCs displayed a median of 173 (range, 92-230) non-synonymous somatic mutations as defined by WES. We detected clonal TP53 hotspot mutations associated with loss of heterozygosity (LOH) of the wild-type allele in two ACCs (ACC1 and ACC18). In contrast, the TP53 wild-type ACC (ACC12) harbored a pathogenic MLH1 germline mutation (p.Glu227_Ser295del) and a clonal hotspot mutation in CTNNB1 (p.Lys335Ile). No cancer gene other than TP53 was found to be mutated in &amp;gt;1 case. Copy number analysis revealed that ACC18 harbored a BRCA1 homozygous deletion and focal amplification of 20p12.3 encompassing PCNA. Moreover, the TP53-mutant cases, ACC1 and ACC18, displayed complex copy number profiles, with multiple gains and losses and focal high-level amplifications. Consistent with the BRCA1 homozygous deletion, ACC18 displayed genomic features of homologous recombination DNA repair deficiency (HRD), such as a dominant mutational signature 3, a high LST score (24), a high NtAI score (23) and a high number of ‘small deletions’ &amp;gt;5bps. In contrast, and consistent with its DNA mismatch repair (MMR) deficiency, ACC12 had a simple genome without detectable copy number alterations, displayed a dominant mutational signature 6 associated with defective MMR, microsatellite instability (MSI) as determined by MSIsensor and loss of MLH1 expression by immunohistochemical analysis. Conclusions: Our findings lend further support to the notion that ACCs of the breast are genetically heterogeneous and display genomic features overlapping with those of common forms of TNBCs. Although no pathognomonic genetic alterations were detected in these cases, our data suggest that at least some ACCs of the breast may arise in the setting of HRD or MSI through distinct molecular mechanisms. Our data also suggest the possibility that both BRCA1 and TP53 loss of function may not be sufficient for a TNBC to display high-grade features and that inactivation of these two genes may not necessarily result in the development of high-grade TNBC. Citation Format: Francisco Beca, Simon SK Lee, Fresia Pareja, Arnaud Da Cruz Paula, Pier Selenica, Lorenzo Ferrando, Hannah Y Wen, Hong Zhang, Elena Guerini-Rocco, Emad A Rakha, Britta Weigelt, Jorge S Reis-Filho. Whole-exome and RNA-sequencing analyses of acinic cell carcinomas of the breast [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-07.

Lynch-like syndrome is as frequent as Lynch syndrome in early-onset nonfamilial nonpolyposis colorectal cancer.

Early-onset (<50 years-old) nonpolyposis nonfamilial colorectal cancer (EO NP NF CRC) is a common clinical challenge. Although Lynch syndrome (LS) is associated with EO CRC, the frequency of this syndrome in the EO NF cases remains unknown. Besides, mismatch repair deficient (MMRd) CRCs with negative MMR gene testing have recently been described in up to 60% of cases and termed "Lynch-like syndrome" (LLS). Management and counseling decisions of these patients are complicated because of unconfirmed suspicions of hereditary cancer. To define the prevalence of MMR deficient CRCs, LS and LLS in patients with EO NP NF CRC, we recruited 102 patients with a first diagnosis of NP NF CRC ≤ 50 years old during 2003-2009 who underwent genetic counseling at our institution in Argentina. Tumor immunohistochemical (IHC) MMR for protein expression and microsatellite instability (MSI) status were evaluated, and in those with loss of MLH1 expression by IHC, somatic BRAF V600E mutation and both somatic and germline MLH1 methylation levels were studied. Tumors characterized as MMRd without somatic BRAF mutation nor MLH1 methylation were sent for germline analysis. Twenty one (20.6%) tumors were MMRd. Fourteen of 16 putative LS cases underwent germline testing: 6 pathogenic mutations were identified and 8 cases had no identifiable pathogenic mutations. The prevalence of LS and LLS in this cohort was 5.8% (6/102) and 7.8% (8/102), respectively. As a conclusion we found that 20% of patients with EO NP NF CRC have MMRd tumors, and at least half of these are likely to have LLS.

Streamlining universal screening for lynch syndrome (LS): Towards improved yield of genetic counseling (GC).

503 Background: LS is largely underdiagnosed although Universal Screening (US) in colorectal cancer (CRC) patients through MisMatch Repair deficiency (MMR-d) testing is widely endorsed. Low adherence to guidelines among oncologists may be partly due to a lack of consensus on whether all MMR-d patients should be referred to GC/Genetic Testing (GT). As BRAF mutation rules out LS, we estimated the increased yield of LS diagnosis from GC /GT which could be obtained by selecting candidates for GC through BRAF testing. Methods: From 2011 to 2016, 1447 consecutive stage I-IV CRC surgical patients at a single institution, underwent immunohistochemistry (IHC) for LS using anti MLH1, MSH2, MSH6 and PMS2 antibodies. Oncologists were invited to refer all MMR-d patients to GC/GT. BRAFV600E testing was carried out only in case of MLH1 protein loss at IHC. Results: MMR-d was found in 194 patients (13%), with 171 showing loss of MLH1 expression (88%). Oncologists referred 27 (16%) to GC. Among the 21 who underwent GC, BRAF testing and GT, 9 were BRAF wild type (wt) (43%) and none had LS. Among the 23 MMR-d patients with loss of expression of MSH2, MSH6 or PMS2 (≠MLH1), oncologists referred 9 to GC (39%): 7 underwent GC / GT and 3 carried LS (43%) at GT. Median age was 76 years (range 30-97) in the MMR-d group, 78 (range 41-97) in the MLH1 group and 63 (range 30-86) in the ≠MLH1 group. Overall, LS was diagnosed in 3 of the 28 MMR-d patients (11%) who underwent GC /GT, possibly an underestimate due to the advanced median age of our MLH1 loss patients. Had we only offered GC to the 9 BRAF wt patients among the 21 with MLH1 loss, we could have avoided 12 (57%) of the GC sessions conducted, increasing the yield of LS diagnosis from 3/28 (11%) to 3/16 (19%) (75% increase). Conclusions: When US for LS is adopted, a GC referral rate reduction of 57% among MLH1 loss patients, and an overall increase in the yield of GC of about 75% can be obtained by testing for BRAF mutation before oncologist referral to GC rather than after. As multistep selection of patients by oncologists may be unfeasible, CRC pathology reports with combined MMR-d and BRAF testing (for MLH1 loss at IHC) and an ‘LS suspicion alert’ could improve oncologists’ awareness of LS and compliance with guidelines.

Disruption of a -35 kb Enhancer Impairs CTCF Binding and MLH1 Expression in Colorectal Cells.

Purpose:MLH1 is a major tumor suppressor gene involved in the pathogenesis of Lynch syndrome and various sporadic cancers. Despite their potential pathogenic importance, genomic regions capable of regulating MLH1 expression over long distances have yet to be identified.Experimental Design: Here, we use chromosome conformation capture (3C) to screen a 650-kb region flanking the MLH1 locus to identify interactions between the MLH1 promoter and distal regions in MLH1-expressing and nonexpressing cells. Putative enhancers were functionally validated using luciferase reporter assays, chromatin immunoprecipitation, and CRISPR-Cas9-mediated deletion of endogenous regions. To evaluate whether germline variants in the enhancer might contribute to impaired MLH1 expression in patients with suspected Lynch syndrome, we also screened germline DNA from a cohort of 74 patients with no known coding mutations or epimutations at the MLH1 promoter.Results: A 1.8-kb DNA fragment, 35 kb upstream of the MLH1 transcription start site enhances MLH1 gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9-mediated deletion of a core binding region impairs endogenous MLH1 expression. A total of 5.4% of suspected Lynch syndrome patients have a rare single-nucleotide variant (G > A; rs143969848; 2.5% in gnomAD European, non-Finnish) within a highly conserved CTCF-binding motif, which disrupts enhancer activity in SW620 colorectal carcinoma cells.Conclusions: A CTCF-bound region within the MLH1-35 enhancer regulates MLH1 expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression. Clin Cancer Res; 24(18); 4602-11. ©2018 AACR.

Sessile serrated adenomas with dysplasia: morphological patterns and correlations with MLH1 immunohistochemistry

Sessile serrated adenomas are the precursor polyp of approximately 20% of colorectal carcinomas. Sessile serrated adenomas with dysplasia are rarely encountered and represent an intermediate step to malignant progression, frequently associated with loss of MLH1 expression. Accurate diagnosis of these lesions is important to facilitate appropriate surveillance, particularly because progression from dysplasia to carcinoma can be rapid. The current World Health Organization classification describes two main patterns of dysplasia occurring in sessile serrated adenomas, namely, serrated and conventional. However, this may not adequately reflect the spectrum of changes seen by pathologists in routine practice. Furthermore, subtle patterns of dysplasia that are nevertheless associated with loss of MLH1 expression are not encompassed in this classification. We performed a morphological analysis of 266 sessile serrated adenomas with dysplasia with concurrent MLH1 immunohistochemistry with the aims of better defining the spectrum of dysplasia occurring in these lesions and correlating dysplasia patterns with MLH1 expression. We found that dysplasia can be divided morphologically into four major patterns, comprising minimal deviation (19%), serrated (12%), adenomatous (8%) and not otherwise specified (79%) groups. Minimal deviation dysplasia is defined by minor architectural and cytological changes that typically requires loss of MLH1 immunohistochemical expression to support the diagnosis. Serrated dysplasia and adenomatous dysplasia have distinctive histological features and are less frequently associated with loss of MLH1 expression (13 and 5%, respectively). Finally, dysplasia not otherwise specified encompasses most cases and shows a diverse range of morphological changes that do not fall into the other subgroups and are frequently associated with loss of MLH1 expression (83%). This morphological classification of sessile serrated adenomas with dysplasia may represent an improvement on the current description as it correlates with the underlying mismatch repair protein status of the polyps and better highlights the range of morphologies seen by pathologists.

Contribution of MLH1 constitutional methylation for Lynch syndrome diagnosis in patients with tumor MLH1 downregulation.

Constitutional epimutation of the two major mismatch repair genes, MLH1 and MSH2, has been identified as an alternative mechanism that predisposes to the development of Lynch syndrome. In the present work, we aimed to investigate the prevalence of MLH1 constitutional methylation in colorectal cancer (CRC) patients with abnormal expression of the MLH1 protein in their tumors. In a series of 38 patients who met clinical criteria for Lynch syndrome genetic testing, with loss of MLH1 expression in the tumor and with no germline mutations in the MLH1 gene (35/38) or with tumors presenting the BRAF p.Val600Glu mutation (3/38), we screened for constitutional methylation of the MLH1 gene promoter using methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA) in various biological samples. We found four (4/38; 10.5%) patients with constitutional methylation in the MLH1 gene promoter. RNA studies demonstrated decreased MLH1 expression in the cases with constitutional methylation when compared with controls. We could infer the mosaic nature of MLH1 constitutional hypermethylation in tissues originated from different embryonic germ layers, and in one family we could show that it occurred de novo. We conclude that constitutional MLH1 methylation occurs in a significant proportion of patients who have loss of MLH1 protein expression in their tumors and no MLH1 pathogenic germline mutation. Furthermore, we provide evidence that MLH1 constitutional hypermethylation is the molecular mechanism behind about 3% of Lynch syndrome families diagnosed in our institution, especially in patients with early onset or multiple primary tumors without significant family history.

Sessile serrated adenomas with dysplasia: morphological patterns and correlations with MLH1 immunohistochemistry

Sessile serrated adenomas are the precursor polyp of approximately 20% of colorectal carcinomas. Sessile serrated adenomas with dysplasia are rarely encountered and represent an intermediate step to malignant progression, frequently associated with loss of MLH1 expression. Accurate diagnosis of these lesions is important to facilitate appropriate surveillance, particularly because progression from dysplasia to carcinoma can be rapid. The current World Health Organization classification describes two main patterns of dysplasia occurring in sessile serrated adenomas, namely, serrated and conventional. However, this may not adequately reflect the spectrum of changes seen by pathologists in routine practice. Furthermore, subtle patterns of dysplasia that are nevertheless associated with loss of MLH1 expression are not encompassed in this classification. We performed a morphological analysis of 266 sessile serrated adenomas with dysplasia with concurrent MLH1 immunohistochemistry with the aims of better defining the spectrum of dysplasia occurring in these lesions and correlating dysplasia patterns with MLH1 expression. We found that dysplasia can be divided morphologically into four major patterns, comprising minimal deviation (19%), serrated (12%), adenomatous (8%) and not otherwise specified (79%) groups. Minimal deviation dysplasia is defined by minor architectural and cytological changes that typically requires loss of MLH1 immunohistochemical expression to support the diagnosis. Serrated dysplasia and adenomatous dysplasia have distinctive histological features and are less frequently associated with loss of MLH1 expression (13 and 5%, respectively). Finally, dysplasia not otherwise specified encompasses most cases and shows a diverse range of morphological changes that do not fall into the other subgroups and are frequently associated with loss of MLH1 expression (83%). This morphological classification of sessile serrated adenomas with dysplasia may represent an improvement on the current description as it correlates with the underlying mismatch repair protein status of the polyps and better highlights the range of morphologies seen by pathologists.

Constitutional mismatch repair deficiency and Lynch syndrome among consecutive Arab Bedouins with colorectal cancer in Israel.

We assessed the molecular characteristics and the frequency of mutations in mismatch-repair genes among Bedouin patients with colorectal cancer (CRC) in Israel. Bedouin patients with a diagnosis of CRC at a major hospital in the southern part of Israel were deemed eligible for this study. The primary screening method was immunohistochemical staining for mismatch-repair proteins (MLH1, MSH2, MSH6, and PMS2). For subjects with abnormal immunohistochemical staining, we performed microsatellite instability (MSI) analyses, and for tumors with a loss of MLH1 expression we also performed BRAF testing. In MSI high cases we searched further for germline mutations. Of the 24 patients enrolled, four subjects (16.7%) had MSI high tumors: one subject was found to harbor a biallelic PMS2 mutation, one subject had Lynch syndrome (LS) with MSH6 mutation and two subjects had a loss of MLH1/PMS2 proteins/BRAF wild type/normal MLH1 sequence. Ten patients (41.7%) were younger than 50 at the time of diagnosis and none had first degree relatives with CRC. In conclusion, in this cohort of 24 consecutive Arab Bedouins with CRC, one patient was found to harbor a constitutional mismatch repair deficiency, one patient had LS with MSH6 mutation, and two patients had unresolved loss of MLH1/PMS2 proteins/BRAF wild type phenotype.

Finding the needle in a haystack: identification of cases of Lynch syndrome with MLH1 epimutation.

Constitutional epimutation of the DNA mismatch repair gene, MLH1, represents a minor cause of Lynch syndrome. MLH1 epimutations are characterized by the soma-wide distribution of methylation of a single allele of the MLH1 promoter accompanied by constitutive allelic loss of transcription. 'Primary' MLH1 epimutations, considered pure epigenetic defects, tend to arise de novo in patients without a family history or any apparent genetic mutation. These demonstrate non-Mendelian inheritance. 'Secondary' MLH1 epimutations have a genetic basis and have been linked to non-coding genetic alterations in the vicinity of MLH1. These demonstrate autosomal dominant inheritance. Despite convincing evidence of their role in causing Lynch-type cancers, routine screening for MLH1 epimutations has not been widely adopted. Complicating factors may include: the need to perform additional methylation-based testing beyond the standard genetic screening for a germline mutation; the lack of a consensus algorithm for the selection of patients warranting MLH1 epimutation testing; overlapping molecular pathology features of MLH1 methylation and loss of MLH1 expression with more prevalent sporadic MSI cancers; the rarity of MLH1 epimutation; the variable inter-generational inheritance patterns; and the cost-effectiveness of screening. Nevertheless, a positive molecular diagnosis of MLH1 epimutation is clinically important because carriers have a high personal risk of developing metachronous Lynch-type cancers, and their relatives may also be at risk of carriage. Extending existing universal and clinic-based screening algorithms for Lynch syndrome to include an additional arm of selection criteria for cases warranting MLH1 epimutation testing could provide a cost-effective means of diagnosing these cases.

Do serrated neoplasms of the small intestine represent a distinct entity? Pathological findings and molecular alterations in a series of 13 cases.

To characterize pathological, immunohistochemical and molecular features of small intestinal serrated neoplasms. We report 13 serrated neoplasms located predominantly in the duodenum (median age, 71 years; male to female ratio, 7:6). The serrated adenomas demonstrated prominent serration, ectopic crypt formations and cytological features reminiscent of colorectal traditional serrated adenomas. Almost half the serrated adenomas demonstrated high-grade dysplasia or were associated with an adenocarcinoma. Immunohistochemical and molecular analysis showed an intestinal (CDX2-positive) phenotype in all tumours, abnormal β-catenin staining in three cases (23%), abnormal p53 expression in four cases (31%), focal loss of MGMT expression in one case (8%), KRAS mutation in five cases (38%) and CpG island methylator phenotype in six cases (50%). A diffuse pattern of Ki67 expression was present in eight adenomas (62%) and was associated with high-grade dysplasia (P = 0.02). No BRAF(V600E) mutation or loss of MLH1 expression was observed. To our knowledge, this is the first series reporting serrated adenoma in the small intestine, a rare subtype of adenomas resembling traditional serrated adenoma with aggressive morphological features. The absence of the BRAF(V600E) mutation does not support a role for the serrated neoplasia pathway in the development of these lesions, as in colorectal serrated polyps.

194 Pancreatic ductal organoids as a new platform for drug discovery

stroma content and vascularization. At the transcriptomic level, we identified several PDX clusters similar to Goblet-like, Enterocyte, Transit-Amplifying, Inflammatory and Stem-like subtypes recently reported in CRC patients. In addition, well and moderately differentiated PDX showed a different gene expression profile than poorly and undifferentiated models. At the genomic level, the colon PDX models exhibited alteration profiles comparable to those reported for patient tumors regarding signatures of mutational processes (patient-age related or associated to mismatch repair gene deficiencies), chromosomal rearrangement and mutated genes (e.g. APC, TP53, KRAS and TP53). Moreover, we identified 6 PDX models with microsatellite instability (MSI), 5 of them being hyper-mutant with a loss of MLH1 expression. Interestingly, colon PDX displaying the MSI phenotype showed gene expression patterns of the Globlet-like or Inflammatory subtypes. To demonstrate the potential of our approach, we searched for molecular determinants of cetuximab sensitivity in colon PDX by combining in vivo drug sensitivity and molecular data. The study identified the Transit Amplifying subtype, AREG/EREG expression and KRAS mutation among the best predictors of response. Similarly, predictive biomarkers for oxaliplatin and irinotecan were found, and their associations with particular PDX subtypes are under investigation. The accuracy in predicting patient outcome following treatment with FOLFOX and FOLFIRI will be evaluated by assessing publicly available datasets. The comprehensive characterization of our colon PDX models confirmed their similarities with patient tumors and their inter-tumoral diversity. The combined use of PDX molecular and drug sensitivity data represents a promising approach for biomarker identification and tumor response prediction at a preclinical stage.

193 The identification of new therapies for ependymoma subgroups

stroma content and vascularization. At the transcriptomic level, we identified several PDX clusters similar to Goblet-like, Enterocyte, Transit-Amplifying, Inflammatory and Stem-like subtypes recently reported in CRC patients. In addition, well and moderately differentiated PDX showed a different gene expression profile than poorly and undifferentiated models. At the genomic level, the colon PDX models exhibited alteration profiles comparable to those reported for patient tumors regarding signatures of mutational processes (patient-age related or associated to mismatch repair gene deficiencies), chromosomal rearrangement and mutated genes (e.g. APC, TP53, KRAS and TP53). Moreover, we identified 6 PDX models with microsatellite instability (MSI), 5 of them being hyper-mutant with a loss of MLH1 expression. Interestingly, colon PDX displaying the MSI phenotype showed gene expression patterns of the Globlet-like or Inflammatory subtypes. To demonstrate the potential of our approach, we searched for molecular determinants of cetuximab sensitivity in colon PDX by combining in vivo drug sensitivity and molecular data. The study identified the Transit Amplifying subtype, AREG/EREG expression and KRAS mutation among the best predictors of response. Similarly, predictive biomarkers for oxaliplatin and irinotecan were found, and their associations with particular PDX subtypes are under investigation. The accuracy in predicting patient outcome following treatment with FOLFOX and FOLFIRI will be evaluated by assessing publicly available datasets. The comprehensive characterization of our colon PDX models confirmed their similarities with patient tumors and their inter-tumoral diversity. The combined use of PDX molecular and drug sensitivity data represents a promising approach for biomarker identification and tumor response prediction at a preclinical stage.