Mismatch Repair Proteins MLH1 Research Articles

Initiation of molecular testing of endometrial carcinomas in a population-based setting: practical considerations and pitfalls.

Since the publication of The Cancer Genome Atlas (TCGA) molecular Classification of endometrial carcinomas in 2013, multiple studies have demonstrated the prognostic and therapeutic importance of this. However, there is great variability on whether and how this is undertaken in different institutions, and this is often dependent on resources and availability of molecular testing. Points of controversy include whether molecular classification is needed on all endometrial carcinomas and whether pure molecular testing is undertaken or a surrogate such as the ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) Classifier. Herein we report our experience instigating molecular classification of endometrial carcinomas in Northern Ireland. From 1st March 2023, all endometrial carcinomas diagnosed on biopsy in the four pathology laboratories in Northern Ireland were referred to the central molecular pathology laboratory for genomic analysis using a custom next-generation sequencing (NGS) panel; the NGS panel included the entire coding regions of polymerase epsilon (POLE) and TP53 genes, as well as microsatellite instability (MSI) analysis. All cases also underwent immunohistochemical staining with oestrogen receptor (ER), p53, and the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. The molecular results were available by the time of surgery (if a hysterectomy was performed) allowing integration into the final pathology report where a TCGA molecular type was assigned. Two hundred and sixty-seven endometrial carcinomas underwent molecular testing; in five cases, there was insufficient material for testing, leaving 262 cases. The TCGA groups were POLEmut (19; 7.3%), MMRd (63; 24%), p53abn (62; 23.7%), and no specific molecular profile (NSMP) 118 (45%). Seventeen tumours (6.5%) were "multiple-classifiers": five POLEmut-p53abn, two POLEmut-MMRd, one POLE-MMRd-p53abn (all included in the POLEmut TCGA group), and nine MMRd-p53abn (included in the MMRd group). This represents one of the first population-based studies investigating the prevalence of the different TCGA molecular groups of endometrial carcinomas in an unselected population. Performing molecular testing on biopsies enables management to be tailored to the molecular group and allows integration of the TCGA group into the report of the final resection specimen. We hope our experience will facilitate other laboratories in undertaking TCGA molecular classification.

Universal testing in endometrial cancer in Sweden

BackgroundThe aim of the study was to test a universal screening strategy on endometrial cancer to evaluate its effectiveness to find Lynch Syndrome (LS) cases to two established clinical criteria: Amsterdam II criteria, and the revised Bethesda criteria to select cases for prescreening with immunohistochemistry (IHC). Cases were subsequently screened for germline disease causing variants regarding the DNA mismatch repair (MMR) genes.MethodsIHC was performed on 221 endometrial cancer (EC) cases, using antibodies against the DNA mismatch repair proteins MLH1, PMS2, MSH2, and MSH6. MMR loss was found in 54 cases, and gene mutation screening was undertaken in 52 of those.ResultsIn this set of patients, the use of Amsterdam II criteria detected two (0.9%), the Bethesda criteria two (0.9%), and universal testing five (2.3%) cases of LS. The combination of universal testing and family history criteria resulted in detection of five patients (2.3%) with LS.ConclusionsBased on our results and other similar studies to date we propose a screening protocol for LS on EC tumors with prescreening using IHC for the four MMR proteins on all new EC cases diagnosed before 70 years of age, followed by mutation screening of all tumors with loss of MSH2 and/or MSH6 or only PMS2, plus consideration for mutation screening of all LS genes in cases fulfilling the clinical Amsterdam II criteria regardless of MMR status on IHC.

Erk Inhibition as a Promising Therapeutic Strategy for High IL-8-Secreting and Low SPTAN1-Expressing Colorectal Cancer.

Tumor recurrence and drug resistance are responsible for poor prognosis in colorectal cancer (CRC). DNA mismatch repair (MMR) deficiency or elevated interleukin-8 (IL-8) levels are characteristics of CRCs, which have been independently correlated with treatment resistance to common therapies. We recently demonstrated significantly impaired therapeutical response and increased IL-8 release of CRC cell lines with reduced expression of MMR protein MLH1 as well as cytoskeletal non-erythrocytic spectrin alpha II (SPTAN1). In the present study, decreased intratumoral MLH1 and SPTAN1 expression in CRCs could be significantly correlated with enhanced serum IL-8. Furthermore, using stably reduced SPTAN1-expressing SW480, SW620 or HT-29 cell lines, the RAS-mediated RAF/MEK/ERK pathway was analyzed. Here, a close connection between low SPTAN1 expression, increased IL-8 secretion, enhanced extracellular-signal-regulated kinase (ERK) phosphorylation and a mesenchymal phenotype were detected. The inhibition of ERK by U0126 led to a significant reduction in IL-8 secretion, and the combination therapy of U0126 with FOLFOX optimizes the response of corresponding cancer cell lines. Therefore, we hypothesize that the combination therapy of FOLFOX and U0126 may have great potential to improve drug efficacy on this subgroup of CRCs, showing decreased MLH1 and SPTAN1 accompanied with high serum IL-8 in affected patients.

TDP43 Interacts with MLH1 and MSH6 Proteins in A DNA Damage-Inducible Manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complimentary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 hr treatment of 10μM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS affected neurons.

O09 Microsatellite-unstable sporadic sebaceous and duodenal tumours are associated with loss of mismatch protein expression

Abstract Introduction and aims Lynch syndrome (LS) is a common heritable cancer predisposition syndrome. Tumours from patients with LS demonstrate microsatellite instability (MSI-high), a genetic signature detectable by low-cost DNA sequencing assays that is currently utilized in colorectal cancer screening. The utility of MSI assays for LS detection has not been extensively studied in unselected sebaceous skin tumours or duodenal tumours, where currently immunohistochemical (IHC) expression of mismatch repair (MMR) proteins is used. In addition, it has recently been suggested that cutaneous squamous cell carcinoma (cSCC) could be linked to LS; however MSI status has not been studied in patients with early-onset cSCC. Methods We investigated MSI status in archival formalin-fixed paraffin-embedded cases with ethical approval. We examined 110 sebaceous tumours (STs) (45 female patients, 65 male patients; median age 77 years), 50 duodenal tumours (DTs) (28 female patients, 22 male patients; median age 64 years), and 148 cSCCs selected from patients under 50 years of age (69 female patients, 79 male patients; median age 49 years). Immunohistochemistry (IHC) was performed on each MSI-high sample to determine the expression of MMR proteins MLH1, MSH2, MSH6 and PMS2. Samples that passed quality control were included in the analysis. Results A total of 42 of 106 STs (39.6%) were MSI-high. All STs had loss of at least one MMR protein. Overall, 10 of 41 (24%) DTs were MSI-high. All DTs had loss of at least one MMR protein. We found that 12 of 148 (8.1%) cSCCs were MSI-high and 5 of 12 SCCs exhibited loss of at least one MMR protein expression (41%). Conclusions MSI-high status correlated well with loss of MMR protein expression in STs and DTs samples. Future studies will help establish MSI status in tumours initially selected by MMR loss on IHC, informing its use as an adjunct to MMR IHC in LS detection in patients with STs and DTs.

Expression of EGFR, PD-L1, and the mismatch repair proteins before and following therapy in malignant serous effusions with metastatic high-grade serous tubo-ovarian carcinoma.

To compare the immunochemical expression of EGFR, PD-L1, and the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6 between matched malignant effusions obtained before and following the administration of chemotherapy in patients with high-grade serous tubo-ovarian carcinoma (HGSC). In the enrolled HGSCs, matched formalin-fixed and paraffin-embedded cell blocks (CBs) from effusions sampled before (treatment-naïve patients) and during recurrence (following chemotherapy administration), in addition to their matched HGSC tissues obtained from the ovaries at initial diagnosis (treatment-naïve patients), were subjected to EGFR, PD-L1, and MMR immunochemical analysis. EGFR was more often overexpressed in effusions obtained after chemotherapy administration compared to both effusions (100% vs. 57.1%) and their matched tubo-ovarian tumors (100% vs. 7.1%) from treatment-naïve patients, respectively. EGFR immunochemistry was concordant in just 9.1% of the effusions sampled during recurrence and their paired ovarian samples before recurrence. Whereas all HGSC treatment-naïve samples (ovarian lesions and effusions) were PD-L1 negative, 3/11 (27.3%) malignant effusions obtained during recurrence showed PD-L1 overexpression. Lastly, none of the tested HGSC samples exhibited MMR deficiency. Measuring biomarkers using CBs from malignant effusions may provide clinicians with significant information related to HGSC prognosis and therapy selection, especially in patients with resistance to chemotherapy.

The prevalence of lynch syndrome (DNA mismatch repair protein deficiency) in patients with primary localized prostate cancer using immunohistochemistry screening

BackgroundProstate cancer is one of the most heritable human cancers. Lynch syndrome is an autosomal dominant inheritance caused by germline mutations in DNA mismatch repair (MMR) genes, which are also associated with an increased incidence of prostate cancer. However, prostate cancer has not been defined as a Lynch syndrome-associated cancer. The proportion of Lynch syndrome patients in primary prostate cancers is unclear. In this study, we investigated MMR protein loss using universal immunohistochemical screening to determine the prevalence of Lynch syndrome in patients with localized prostate cancer who underwent radical prostatectomy.MethodsOne hundred twenty-nine surgical specimens from radical prostatectomy performed at Toranomon Hospital between 2012 and 2015 were retrospectively tested using universal screening with immunohistochemistry staining for expression of the MMR proteins MLH1, PMS2, MSH2, and MSH6. For all suspected MMR-deficient patients, germline genetic tests focusing on MMR genes were performed.ResultsMMR protein loss was found in only one patient (0.8%) who showed dual MSH2/MSH6 loss. This patient showed a single nucleotide pathogenic germline mutation from c.1129 C to T (p.Gln377*) at exon 7 in the MSH2 gene. He was diagnosed with a primary prostate cancer at 66 years of age. He had a documented history of Lynch syndrome (Muir–Torre syndrome) with previous colon cancer, sebaceous tumor, and keratoacanthoma as well as subsequent bladder cancer, all of which also showed dual MSH2/MSH6 loss. He also had a strong family history of colorectal and other Lynch syndrome-associated cancers. The pathological stage was pT3aN0M0, and the pathological grade was Gleason 7(4 + 3) with tertiary pattern 5.ConclusionsIn this study, immunohistochemical screening of MMR proteins for Lynch syndrome was performed in a series of prostate cancer cases. The prevalence of Lynch syndrome in localized prostate cancer was 0.8%, which is low compared with other Lynch syndrome-associated cancers.

Mismatch Repair Deficiency Is a Prognostic Factor Predicting Good Survival of Opisthorchis viverrini-Associated Cholangiocarcinoma at Early Cancer Stage.

The mismatch repair (MMR) system prevents DNA mutation; therefore, deficient MMR protein (dMMR) expression causes genetic alterations and microsatellite instability (MSI). dMMR is correlated with a good outcome and treatment response in various cancers; however, the situation remains ambiguous in cholangiocarcinoma (CCA). This study aims to evaluate the prevalence of dMMR and investigate the correlation with clinicopathological features and the survival of CCA patients after resection. Serum and tissues were collected from CCA patients who underwent resection from January 2005 to December 2017. Serum OV IgG was examined using ELISA. The expression of MMR proteins MLH1, MSH2, MSH6 and PMS2 was investigated by immunohistochemistry; subsequently, MMR assessment was evaluated as either proficient or as deficient by pathologists. The clinicopathological features and MMR status were compared using the Chi-square test. Univariate and multivariate analyses were conducted to identify prognostic factors. Among the 102 CCA patients, dMMR was detected in 22.5%. Survival analysis revealed that dMMR patients had better survival than pMMR (HR = 0.50, p = 0.008). In multivariate analysis, dMMR was an independent factor for a good prognosis in CCA patients (HR = 0.58, p = 0.041), especially at an early stage (HR = 0.18, p = 0.027). Moreover, subgroup analysis showed dMMR patients who received adjuvant chemotherapy had better survival than surgery alone (HR = 0.28, p = 0.012). This study showed a high prevalence of dMMR in cholangiocarcinoma with dMMR being the independent prognostic factor for good survival, especially in early-stage CCA and for patients who received adjuvant chemotherapy. dMMR should be the marker for selecting patients to receive a specific adjuvant treatment after resection for CCA.

Prevalence of mismatch repair deficiency in a large prospective prostate cancer cohort.

5031 Background: Mismatch repair (MMR) deficiency and microsatellite instability (MSI) are predictive biomarkers for immunotherapy response. The best approach to identify patients with such tumors is unclear in prostate cancer. Methods: This study included men diagnosed with primary prostate cancer during prospective follow-up of the Health Professionals Follow-up Study (HPFS) and Physicians’ Health Study (PHS). The highest-grade/index lesions of tumor tissue from radical prostatectomy (95%) or transurethral resections of the prostate were mounted in triplicate on tissue microarrays. Immunohistochemistry for the MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed, with scoring as MMR-deficient requiring a visible staining in a non-tumor internal positive control of the same case. For validation, a polymerase chain reaction-based MSI assay (Idylla MSI Test, Biocartis) was performed on tumor DNA of MMR-deficient cases and a selection of MMR-intact cases. Results: The study included 1016 men with prostate cancer. Tumor stage was predominantly pathologically localized (71% stage pT1/2, 18% T3a/b) with a full distribution of Gleason scores, including 20% Gleason score 6 (grade group 1), 36% 3+4 (grade group 2), 23% 4+3 (grade group 3), 8% 8 (grade group 4) and 13% 9-10 (grade group 5). MMR tumor scoring could be performed for MLH1 in 747 cases (75% of those with tumor tissue), for MSH2 in 903 cases (90%), for MSH6 in 708 cases (74%), and for PMS2 in 703 cases (72%). The remaining tumors were unevaluable due to lack of non-tumor tissue necessary as an internal positive control. Of the 903 tumors evaluable for MSH2 protein loss, 4 tumors had loss of MSH2 (prevalence 0.4%, 95% confidence interval [CI] 0.2–1.1%), and 3 of 708 evaluable tumors had concomitant loss of MSH6 (prevalence 0.4%, 95% CI 0.1–1.2%). No tumor had loss of MLH1 or PMS2. The MMR-deficient tumors had Gleason scores of 3+4, 8 (two cases), and 9–10. Tumor DNA of 1 of the 4 MMR-deficient tumors met the manufacturer’s cut-off for MSI-high; two additional MMR-deficient cases had non-zero repeats, with good reproducibility between tumor cores and technical replicates. One MSH2-deficient tumor and 6 DNA samples from 3 MMR-intact cases had repeat scores of zero. Conclusions: In this nationwide prospective study, MMR deficiency was rare in primary, surgically treated prostate cancer. This low prevalence contrasts with hospital-based studies of MMR deficiency and MSI that may have represented tumors with certain clinical features. The low prevalence and the need for an internal positive control for reliable scoring calls into question to what extent immunohistochemistry-based screening for MMR deficiency on limited tissue specimens, such as prostate biopsies, is routinely feasible.

Endometrium Kanseri Alt Tiplerinde Mikrosatellit İnstabilite (MSI) ve p16/p53 Gen Durumu: Tümör Saldırganlığı ile İlişkisi

Aim: Microsatellite instability (MSI) has recently emerged as one of the most important pathways in endometrial carcinogenesis, especially in endometrioid subtype (type I). However, non-endometrioid subtypes, like serous, papillary or mucinous tumors, have long been known to overexpress p16 and/or p53 genes. In the present study, we investigated the immunohistochemical (IHC) panel in all subtypes of endometrial cancer (EC), and correlated the results with nuclear mitosis index (Ki-67) as a marker of tumoral mitosis rate. Methods: Medical records of patients admitted with EC and underwent surgery between 2010 and 2022 were reviewed. IHC panel results of estrogen and progesterone receptors (ER, PR), mismatch repair (MMR) proteins MLH1, PMS2, MSH2, MSH6, Ki-67 and p16/p53 status were recorded. Chi square test was used for statistical analysis. Results: Total of 44 female patients (with pathology reports including all of IHC panel markers) were included. Mean age was 64.1±12.51 years. Type I EC was the most common pathology (72%). ER and PR positivity were very prominent in type I tumors in comparison with non-endometrioid (type II) tumors (84% vs 16%, respectively; p

Universal screening for Lynch syndrome in operated colorectal cancer by immunohistochemistry: a cohort of patients in Liaoning province, China.

Lynch syndrome (LS) is the most common hereditary colorectal cancer syndrome worldwide. Due to the decreasing family size in Liaoning province. The Bethesda and Amsterdam II criteria have lower sensitivity and specificity and are not suitable for the local population. Immunohistochemistry screening for mutations in DNA mismatch repair (MMR) in newly diagnosed colorectal cancer can improve the detection rate of LS. All newly diagnosed colorectal cancer patients who underwent surgery between January 2018 and June 2020 at Cancer Hospital of China Medical University and Shengjing Hospital of China Medical University from Liaoning China were included retrospectively, and the ratio of universal LS screening by immunohistochemistry, MMR protein deficiency (dMMR) ratio, MLH1 loss, MSH2 loss, MSH6 loss, and PMS2 loss was analyzed. The clinicopathological characteristics of patients with pMMR and dMMR were analyzed. A total of 7019 colorectal cancer patients underwent surgery and 4802 (68.41%) patients were screened by immunohistochemistry for MMR, 258 (5.37%) cases were reported to have a loss of MMR expression. In the dMMR group, a higher number of patients were under 50 years old, more tumors were located at the right colon, less patients have lymph node metastasis, more tumors were stage II, and histological types of mucinous carcinoma or signet ring carcinoma were more common, compared with the pMMR group. Only 2.71% dMMR patients meet Amsterdam criteria II, 2.71% of patients meet Revised Bethesda guidelines, and 17.83% meet Chinese LS criteria. Twenty-five dMMR patients were confirmed by next-generation sequencing and five families were confirmed as Lynch family. These data imply that universal screening for LS by immunohistochemistry may be effective in Liaoning province.

Mis-Match Repair (MMR) Protein Expression and Microsatellite Instability (MSI) Analysis in Sporadic Adenocarcinoma Diagnosed on Colonoscopic Biopsy

Background and aim Colorectal adenocarcinoma is a major cause of morbidity and mortality worldwide. Mismatch repair MMR protein expression plays a crucial role in the development and progression of colorectal adenocarcinomas. Immunohistochemistry IHC is a widely used technique to assess MMR protein expression. However there is limited data on the prospective evaluation of MMR protein expression by IHC on endoscopic biopsies of colorectal adenocarcinomas. In this study we aimed to evaluate the MMR protein expression by IHC on endoscopic biopsies of colorectal adenocarcinomas at a tertiary care center.Methods Forty-Two colonoscopic endoscopic biopsies pathologically diagnosed as colorectal adenocarcinoma were analyzed for IHC expression of MMR proteins MLH1 PMS2 MSH2 and MSH6.Results Out of the 42 cases 22 52.3 were mismatch repair proficient pMMR while the remaining 20 47.6 were mismatch repair deficient dMMR. MLH1PMS2- was the most common pattern accounting for 20 of dMMR cases and MLH1-PMS2-MSH2-MSH6- or the ldquoNull Patternrdquo was the least common with 10 of dMMR cases.Conclusion Evaluation of MMR proteins using immunohistochemistry is relatively easy in routine testing and is a useful tool in detecting MSI in CRC.nbspnbsp

Case report: Undifferentiated sarcoma with multiple tumors involved in Lynch syndrome: Unexpected favorable outcome to sintilimab combined with chemotherapy

Patients with Lynch syndrome are at an increased risk of developing simultaneous or metachronous tumors, while sarcomas have been occasionally reported. Sarcomas are generally not considered part of the common Lynch syndrome tumor spectrum. However, more and more studies and case reports suggested that sarcoma could be a rare clinical manifestation of Lynch syndrome, leading to new treatment strategies for sarcoma. We report the case of a 74-year-old male patient with Lynch syndrome who had rectal mucinous adenocarcinoma and prostate adenocarcinoma and then developed undifferentiated sarcoma of the left neck two years later. Mismatch repair deficiency (dMMR) was confirmed by immunohistochemical staining for the mismatch repair proteins MSH2, MSH6, MLH1 and PMS2. The result of polymerase chain reaction (PCR) microsatellite instability (MSI) testing of sarcoma showed high-level microsatellite instability (MSI-H). Additionally, a pathogenic germline mutation in MSH2 (c.2459-12A>G) was detected by next-generation sequencing (NGS). Taking into account HE morphology, immunohistochemical phenotype, MSI status, NGS result, medical history and germline MSH2 gene mutation, the pathological diagnosis of left neck biopsy tissue was Lynch syndrome related undifferentiated sarcoma with epithelioid morphology. The patient has been receiving immunotherapy (sintilimab) combined with chemotherapy (tegafur, gimeracil and oteracil potassium capsules) and currently has stable disease. We also reviewed the literature to understand the association between sarcoma and Lynch syndrome. Sarcoma may now be considered a rare clinical manifestation of Lynch syndrome. Attention and awareness about the association between Lynch syndrome and sarcoma need to be increased. Therefore, timely detection of MMR proteins and validation at the gene level for suspicious patients are the keys to avoiding missed or delayed diagnosis and to identifying patients suited for immunotherapy, which may also help to provide appropriate genetic counseling and follow-up management for patients.

128. Clinical testing of mismatch repair in neoplasms using multiple laboratory methods

<h3>Background</h3> Deficiency of DNA mismatch repair function can be assessed by immunohistochemical (IHC) analysis for loss of expression of the mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 or microsatellite instability (MSI) analysis. In some cases, however, there is a discrepancy between IHC and MSI analysis. Our aim was to explain with molecular mutation analysis these discordant cases between IHC and MSI results. <h3>Methods</h3> We reviewed 706 neoplasms accessioned between 2015-2021 (345 gastrointestinal, 142 genitourinary, 192 gynecologic, 27 miscellaneous). All neoplasms were assessed by IHC testing, MSI analysis and tumor-normal paired next generation sequencing (NGS) analysis, and 80% of cases with MLH1 loss had concurrent MLH1 methylation analysis. <h3>Results</h3> Fifty-eight (8.2%) of neoplasms had deficient MMR. Forty-four (76%) of these patients had both dMMR and MSI-high status. Ten of 14 discordant cases had a major discordance between IHC and MSI analysis: [MSI-High/intact MMR expression (n=3), microsatellite stable (MSS)/loss of MLH1&PMS2 (n=1), MSS/loss of MSH2&MSH6 (n=3), MSS/loss of MSH6 (n=3), MSS/loss of MSH1 &MSH2 (n=1)]. Remaining 4 neoplasms are either MSI-low or with equivocal IHC results. Methylation and NGS analysis revealed a molecular etiology in 3 of 10 (30%) neoplasms with major discordance, whereas in neoplasms without major discordance, this figure was 77%. (p=0.007) <h3>Conclusion</h3> For most neoplasms, IHC and MSI testing are concordant, and a molecular etiology that explains dMMR or MSI-High results can be identified. For neoplasms with major discordance, the addition and integration of NGS results and MLH1 methylation analysis can be beneficial for resolving borderline cases.

Delineating the intra-patient heterogeneity of molecular alterations in treatment-naïve colorectal cancer with peritoneal carcinomatosis

AbstractIn a non-negligible number of patients with metastatic colorectal cancer (mCRC), the peritoneum is the predominant site of dissemination. Cure can be achieved by cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC), but this procedure is associated with long-term morbidity and high relapse rates. Thus, there is a pressing need for improved therapeutic strategies and complementary biomarkers. The present study explored the molecular heterogeneity in mCRC with peritoneal carcinomatosis (PC), and the potential clinical implications thereof. Multi-region immunohistochemical profiling and deep targeted DNA-sequencing was performed on chemotherapy-naïve tumours from seven patients with synchronous colorectal PC who underwent CRS and HIPEC. In total, 88 samples (5-19 per patient) were analysed, representing primary tumour, lymph node metastases, tumour deposits, PC and liver metastases. Expression of special AT-rich sequence-binding protein 2 (SATB2), a marker of colorectal lineage, was lacking in the majority of cases, and a conspicuous intra-patient heterogeneity was denoted for expression of the proposed prognostic and predictive biomarker RNA-binding motif protein 3 (RBM3). Loss of mismatch repair proteins MLH1 and PSM2, observed in one case, was concordant with microsatellite instability and the highest tumour mutational burden. When present in a patient, mutations in key CRC driver genes, i.e., KRAS, APC and TP53, were homogenously distributed across all samples, while less common mutations were more heterogenous. On the same note, copy number variations showed intra-patient as well inter-patient heterogeneity. In two out of seven cases, hierarchical clustering revealed that samples from the PC and lymph node metastases were more similar to each other than to the primary tumour. In summary, these findings should encourage additional studies addressing the potential distinctiveness of mCRC with PC, which might pave the way for improved personalized treatment of these patients.

Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair.

DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.

Abstract 1325: Naturally occurring colorectal cancer in nonhuman primates used to study human immunotherapeutic agents confirms a link between DNA methylation and mismatch repair deficiency

Abstract Background Non-human primates (NHP) such as rhesus macaques with naturally occurring cancers are a proposed model for translational cancer immunotherapy (CIT) research and have generated relevant proof-of-mechanism evidence for 3 different CIT agents. NHP spontaneously develop cancers with progression patterns, histology, and clinical symptoms similar to humans. Gene suppression by DNA hypermethylation in the promoter region is the major characteristics of the CpG-island-methylator-phenotype (CIMP) described in human CRC patients but information in rhesus macaques is scarce. To further validate these animals as translational models for CIT, we conducted a deep molecular characterization of NHP colorectal cancers and established novel qPCR panels to assess DNA methylation of marker genes published for humans. Methods Our cohort (n=16) consisted of Indian-origin rhesus macaques (Macaca mulatta) with naturally occurring CRC (n=16, female=11). Clinical examination, imaging (contrast-enhanced CT, FDG-PET) and biopsy to confirm cancer histology were performed. Molecular characterization was done by IHC for CRC-associated mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 and by PCR/electrophoresis for microsatellite instability. Ultimately, we designed DNA methylation- and rhesus-specific qPCR probes (Methylight) targeting corresponding regions as published in human patients, including CACNA1G, CDKN2A, CRABP1, MLH1, and NEUROG1 as parts of the CIMP panel and BMP3, NDRG4, and SEPTIN9 as probed for human CRC-screening. Results MLH1 deficiency by IHC, in conjunction with PMS2 absence, is observed in all NHP CRC cases, clearly exceeding frequencies reported in human CRCs (ranging from 2-15%). Moreover, we have documented microsatellite instable cases in some NHP CRCs, analog to human CRCs. DNA methylation of the MLH1 promoter region was significantly elevated in CRCs (100% of CRCs &amp;gt;2-fold, p&amp;lt;0.0001) compared to healthy colon. We hypothesized that this elevation would suppress MLH1 mRNA expression. This hypothesis of epigenetic suppression is corroborated by both qPCR and RNA sequencing which demonstrate significantly downregulated levels of MLH1 mRNA. DNA methylation of the other markers is less consistent but revealed CIMP positive and CIMP negative cases in our NHP CRC cohort. Conclusions Transcriptional suppression of MLH1 by promoter hypermethylation is a major and widespread driver of genetic instability and carcinogenesis in rhesus macaque colorectal cancer. Differential DNA methylation in the promoter regions as observed in NHP CRCs can provide a screening target for liquid biopsies. This work highlights the possible translatability of naturally occurring NHP cancers for human cancer immunotherapy research and can be further explored in future tumor-bearing monkey trials. Citation Format: Simon Deycmar, Brendan Johnson, Declan Ryan, Shane Sills, David Caudell, Greg Dugan, George Schaaf, Christopher Whitlow, Kiran Kumar Solingapuram Sai, Betsy Ferguson, Benjamin Bimber, Karina Ray, Cassandra Cullin, Brandy Dozier, Armando Burgos, Michael Hettich, Bruno Gomes, Jehad Charo, Maurizio Ceppi, Mark Cline. Naturally occurring colorectal cancer in nonhuman primates used to study human immunotherapeutic agents confirms a link between DNA methylation and mismatch repair deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1325.

Predictive functional assay-based classification of PMS2 variants in Lynch syndrome.

The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low‐penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain significance (VUS). The inability to classify most VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis‐based quantitative tool to improve the classification of VUS in MMR proteins. To derive a formula that translates CIMRA assay results into the odds of pathogenicity (OddsPath) for VUS in PMS2 we used a set of clinically classified PMS2 variants supplemented by inactivating variants that were generated by an in cellulo genetic screen, as proxies for cancer‐predisposing variants. Validation of this OddsPath revealed high predictive values for benign and predisposing PMS2 VUS. We conclude that the OddsPath provides an integral metric that, following the other, higher penetrance, MMR proteins MSH2, MSH6 and MLH1 can be incorporated as strong evidence type into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP).

Expression of DNA mismatch repair proteins in melanoma patients treated with immune checkpoint inhibitors

PurposeTo investigate the protein expression of DNA mismatch repair (MMR) proteins in patients with cutaneous melanoma (CM) under immune checkpoint inhibitor (ICI) therapy.MethodsImmunohistochemistry was performed on tumor tissue for MMR proteins MLH1, MSH2, MSH6, and PMS2 in 50 metastatic CM patients treated with ICI (ipilimumab, nivolumab, pembrolizumab).ResultsBest overall response (BOR) rate was 48% (24/50). Reduced MMR protein expression (nuclear expression in < 80% of tumor cells) was observed in 8 patients (16%). Compared to other clinical parameters, baseline neutrophil/lymphocyte ratio and reduced intratumoral MMR protein expression (P = 0.0033) were determined as the only parameters significantly associated with favorable BOR. However, in this small study population, reduced MMR protein expression did not reach statistical significance in multivariate analysis.ConclusionReduced MMR protein expression is observed in CM and might predict favorable BOR in patients treated with ICI, as was observed for other entities. However, these findings need to be substantiated in larger patient cohorts.

High Expression of Casein Kinase 2 Alpha Is Responsible for Enhanced Phosphorylation of DNA Mismatch Repair Protein MLH1 and Increased Tumor Mutation Rates in Colorectal Cancer.

Simple SummaryColorectal cancer (CRC) is associated with DNA mismatch repair (MMR) deficiency. The serine/threonine casein kinase 2 alpha (CK2α) is able to phosphorylate and inhibit MMR protein MLH1 in vitro. This study aimed to analyze the relevance of CK2α for MLH1 phosphorylation in vivo. Around 50% of CRCs were identified to express significantly increased nuclear/cytoplasmic CK2α. High nuclear/cytoplasmic CK2α level could be significantly correlated with reduced 5-year survival outcome of patients, increased MLH1 phosphorylation, and enriched somatic tumor mutation rates. Overall, our study demonstrated, in vivo, that enhanced CK2α leads to an increase of MLH1 phosphorylation, higher tumor mutation rates, and is an unfavorable prognosis for patients.DNA mismatch repair (MMR) deficiency plays an essential role in the development of colorectal cancer (CRC). We recently demonstrated in vitro that the serine/threonine casein kinase 2 alpha (CK2α) causes phosphorylation of the MMR protein MLH1 at position serine 477, which significantly inhibits the MMR. In the present study, CK2α-dependent MLH1 phosphorylation was analyzed in vivo. Using a cohort of 165 patients, we identified 88 CRCs showing significantly increased nuclear/cytoplasmic CK2α expression, 28 tumors with high nuclear CK2α expression and 49 cases showing a general low CK2α expression. Patients with high nuclear/cytoplasmic CK2α expression demonstrated significantly reduced 5-year survival outcome. By immunoprecipitation and Western blot analysis, we showed that high nuclear/cytoplasmic CK2α expression significantly correlates with increased MLH1 phosphorylation and enriched somatic tumor mutation rates. The CK2α mRNA levels tended to be enhanced in high nuclear/cytoplasmic and high nuclear CK2α-expressing tumors. Furthermore, we identified various SNPs in the promotor region of CK2α, which might cause differential CK2α expression. In summary, we demonstrated that high nuclear/cytoplasmic CK2α expression in CRCs correlates with enhanced MLH1 phosphorylation in vivo and seems to be causative for increased mutation rates, presumably induced by reduced MMR. These observations could provide important new therapeutic targets.