Loss Of MSH6 Research Articles

Early Onset Colorectal Cancer in Arabs, Are We Dealing with a Distinct Disease?

Early-onset colorectal cancer (EOCRC) incidence is increasing worldwide. Efforts are directed to understand the biological and clinical signatures of EOCRC compared to late-onset colorectal cancer (LOCRC). EOCRC is thought to present differently across different ethnic groups and geographical regions. This study was an attempt to contribute with data from the Arab world toward the understanding of the clinicopathological parameters of EOCRC compared to LOCRC. Data from 254 CRC patients diagnosed at Sultan Qaboos University Hospital from the period 2015-2020 were studied. About 32.6% of all diagnosed CRC patients are below 50 years old, with no differences in gender distribution between EOCRC and LOCRC (p-value 0.417). Rectal involvement and tumor laterality were comparable among the two groups. Adenocarcinoma accounts for 83.3% and 94.2% of EOCRC and LOCRC, respectively. More mucinous and signet ring adenocarcinoma (8.3% each) were reported in EOCRC than LOCRC (2.9% and 2.2%, respectively). MLH1 and PMS2 loss are more common among LOCRC, but MSH6 loss is more frequent in EOCRC. The overall survival of EOCRC and LOCRC was comparable (median survival 64.88 and 67.24 months, respectively). This study showed comparable clinicopathological parameters between EOCRC and LOCRC from Arabs, which adds to the bigger picture of understand the disease.

Status ofPD-1 and PD-L1 expression in invasive urothelial carcinoma ofthebladder with mismatch repair protein deficiency.

It has been reported that mismatch repair deficient (d-MMR) tumors show sensitivity to immune checkpoint inhibitors. We aimed to evaluate thecorrelation ofd-MMR and PD-1/PD-L1 expression in invasive urothelial carcinoma ofthebladder. Tissue microarray (TMA) tissues were stained PD-1/PD-L1 and MMR proteins. Theexpression ratio ofthese markers has been compared with histopathologic parameters. d-MMR tumors were more superficial muscle invasive (p=0.012). When thed-MMR, and PD-1/PD-L1 expression ratios were examined, asignificant correlation was obtained between thed-MMR and PD-L1 expression ratio of> 5% in both thetumor and immune cells (p=0.02 and p=0.004, respectively). Theexpression ratio was higher in thepatients without MMR loss. PD-1 and PD-L1expression in those with MSH6 loss was one or none. When PD1/PDL1 expression was compared with histopathological parameters, asignificant relationship was observed between tumor grade and depth ofmuscle invasion. PD-L1 expression was not observed in thesuperficial muscle invasive tumors. This study was shown thestatus ofd-MMR and PD-1/PD-L1 in invasive urothelial cancers and their correlation with prognostic markers. PD-1/PD-L1 expression may contribute to theprogression and poor prognosis ofbladder cancer. However, further studies are required to research theclinical utility.

Abstract P015: Mechanisms of mitochondrial dysfunction in development of mismatch repair-deficient endometrial cancer

Abstract Background: Lynch syndrome (LS) is a hereditary cancer syndrome carrying roughly 60% lifetime risk of endometrial cancer (EC) development. LS-EC is caused by mutations in mismatch repair genes (most commonly MSH2), which lead to DNA repair deficiency and hypermutability. Identifying mechanisms that impact cancer development could provide novel opportunities for EC prevention. Our recent studies in Msh2-deficient mice identified mitochondrial dysfunction in EC pathogenesis. The objective of this study is to define the impact of MSH2 loss on mitochondrial function and oxidative DNA damage to identify targets for EC prevention. We hypothesized that MSH2 loss disrupts mitochondrial function and increases oxidative DNA damage in EC cells. Methods: The effects of MSH2 loss on EC pathogenesis were evaluated using our novel mouse model for EC development (PR-Cre+Msh2flox/flox, abbreviated Msh2KO) and primary cell lines derived from this model. Mitochondrial stress tests (MSTs) compared mitochondrial function in murine and human MSH2-deficient and -intact EC cell lines. Western blots compared mitochondrial protein expression between cell lines. Transcriptomic data of human ECs from The Cancer Genome Atlas (TCGA) were probed using cBioPortal. To examine direct effects of MSH2 loss on oxidative stress and mitochondrial function, we performed lentiviral shRNA knockdown (KD) of MSH2 in two human EC cell lines (KLE, Hec1a) and MSH2 overexpression (MSH2++) in MFE280 cells. MSTs and Western blots were utilized as above. Immunofluorescent staining compared oxidative DNA damage in MSH2-KD, MSH2++, and control cell lines after pharmacologically induced oxidative stress via H2O2. RESULTS Transcriptomic signatures of histologically normal endometrium and EC from Msh2KO mice versus control mice reveal mitochondrial dysfunction as a key aberrant pathway. MSTs show reduced baseline and induced mitochondrial function in murine and human MSH2-deficient EC cells compared to MSH2-intact (p<0.0001). Western blots show that MSH2-deficient EC cells exhibit loss of mitochondrial complex II compared to MSH2-intact cells (p<0.05). TCGA data confirm MSH2-deficient human ECs have reduced complex II subunits (p<0.05). Isogenic in vitro studies show MSH2-KD reduces complex II expression yet has variable effects on mitochondrial function. MSH2 overexpression increases complex II expression and mitochondrial function. Oxidative DNA damage did not differ at baseline, but H2O2-induced DNA damage increased after MSH2-KD in one of two cell lines. Conclusions: MSH2 and complex II loss are strongly associated in EC. Further studies on the downstream effects of complex II loss are necessary to determine the contribution to LS-EC development. Complex II loss could result in disruptions to oxidative phosphorylation, the citric acid cycle, metabolic reprogramming, and oncometabolite production. Defining direct effects of MSH2 loss on these potential cancer susceptibility mechanisms will open new avenues for LS-associated EC prevention. Citation Format: Diana L. Moreno, Mikayla S. Borthwick, Leah Peralta, Wai Kin Chan, Brenda Melendez, Qian Zhang, Nisha Gokul, Melinda S. Yates. Mechanisms of mitochondrial dysfunction in development of mismatch repair-deficient endometrial cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P015.

Cervical Adenocarcinoma: A Still Under-investigated Malignancy.

The aim of the study was to correlate the expression of mismatch repairs proteins (MMR), programmed-death-ligand1 (PDL-1), and estro-progestinic receptors (ER/PgR) in tissue samples from a series of cervical adenocarcinoma (ADC) patients with their clinicopathological features. Thirty-nine ADC specimens were retrospectively retrieved from the Division of Pathology of the University Hospital of Pisa from 2015 to 2021. Histological subtype, grade (G), Silva pattern, presence of lymph vascular space invasion (LVI), and perineural invasion (PNI) were annotated. On representative samples, immunostaining for ER/PgR, MLH1, PMS2, MSH2, MSH6, and PDL-1(sp142) was performed. Thirty-five ADCs were HPV-associated usual type (24 invasive and 11 in situ), 2 were clear cell type, one was a minimal deviation adenocarcinoma (MDA), and one was an invasive stratified mucin-producing carcinoma (iSMC). ADC associated with LVI were mostly G2-3, whereas those associated also with PNI were G3 with Silva pattern C. No difference in the expression of ER/PgR was observed with a dichotomic age stratification (51 years) of patients. Only 6 ADCs were MMR-deficient, all of them were of the usual type (4 invasive and 2 in situ). The heterodimer MLH-1/PMS2 was the one most frequently altered (5/6), whereas only one case had MSH6 loss. None of ADCs express PDL-1, except iSMC which showed PDL-1 expression >1% in neoplastic cells. Both invasive and in situ usual type ADCs indicate MMR deficiency, highlighting how this could be an early event in tumorigenesis. None of the cases, except for iSMC, express PDL-1.

Mismatch repair markers in preoperative and operative endometrial cancer samples; expression concordance and prognostic value

BackgroundThe endometrial cancer mismatch repair (MMR) deficient subgroup is defined by loss of MSH6, MSH2, PMS2 or MLH1. We compare MMR status in paired preoperative and operative samples and investigate the prognostic impact of differential MMR protein expression levels.MethodsTumour lesions from 1058 endometrial cancer patients were immunohistochemically stained for MSH6, MSH2, PMS2 and MLH1. MMR protein expression was evaluated as loss or intact to determine MMR status, or by staining index to evaluate the prognostic potential of differential expression. Gene expression data from a local (n = 235) and the TCGA (n = 524) endometrial cancer cohorts was used for validation.ResultsWe identified a substantial agreement in MMR status between paired curettage and hysterectomy samples. Individual high expression of all four MMR markers associated with non-endometrioid subtype, and high MSH6 or MSH2 strongly associated with several aggressive disease characteristics including high tumour grade and FIGO stage, and for MSH6, with lymph node metastasis. In multivariate Cox analysis, MSH6 remained an independent prognostic marker, also within the endometrioid low-grade subgroup (P < 0.001).ConclusionWe demonstrate that in addition to determine MMR status, MMR protein expression levels, particularly MSH6, may add prognostic information in endometrial cancer.

Validity of a two-antibody testing algorithm for mismatch repair deficiency testing in cancer; a systematic literature review and meta-analysis

Reflex mismatch repair immunohistochemistry (MMR IHC) testing for MLH1, PMS2, MSH2 and MSH6 is used to screen for Lynch syndrome. Recently MMR-deficiency (MMRd) has been approved as a pan-cancer predictive biomarker for checkpoint inhibitor therapy, leading to a vast increase in the use of MMR IHC in clinical practice. We explored whether immunohistochemical staining with PMS2 and MSH6 can be used as a reliable substitute. This two-antibody testing algorithm has the benefit of saving tissue, cutting costs and saving time. PubMed, Embase and Cochrane library were systematically searched for articles reporting on MMR IHC. The weighed percentage of cases with isolated MLH1 or MSH2 loss or combined MLH1/MSH2 loss alone was analyzed using a random effects model meta-analysis in R. The search yielded 1704 unique citations, of which 131 studies were included, describing 9014 patients. A weighed percentage of 1.1% (95% CI 0.53–18.87, I = 87%) of cases with isolated MLH1 or MSH2 loss or combined MLH1/MSH2 loss alone was observed. In the six articles with the main aim of investigating the two-antibody testing algorithm all MMRd cases were detected with the two-antibody testing algorithm, there were no cases with isolated MLH1 or MSH2 loss or combined MLH1/MSH2 loss alone. This high detection rate of MMRd of the two-antibody testing algorithm supports its use in clinical practice by specialized pathologists. Staining of all four antibodies should remain the standard in cases with equivocal results of the two-antibody testing algorithm. Finally, educational sessions in which staining pattern pitfalls are discussed will continue to be important.

Diagnostic Utility of Whole Genome Sequencing in Adults with B-Other Acute Lymphoblastic Leukaemia

Background B-other ALL represents a working definition for patients with B cell precursor (BCP) ALL without a known primary chromosomal abnormality at diagnosis. In this study we use whole genome sequencing (WGS) to characterize the molecular pathogenesis of adult B-other cases (age ≥ 25yrs) from the UKALL14 trial (NCT01085617). Methods Of 652 patients aged 25-65yrs enrolled onto UKALL14, 333 (51%) had B-other ALL. Sufficient material was available to screen 156/333 B-other cases for recurrent Ph-like fusion events (CRLF2, JAK2, ABL1, ABL2 & PDGFRB) using FISH and MLPA (kit P335). This identified 28 (18%) "Ph-like" fusion events (21 CRLF2, 5 ABL-class and 2 JAK2). Of the remaining 128 B-other cases 57 had available samples for tumor normal paired WGS (read depth 60x and 30x respectively). Diagnostic RNA was available for 33 of the 57 cases Bioinformatic analysis was performed to determine small somatic mutations; single nucleotide variants (SNVs) and insertion/deletions (INDELs) as well as copy number aberrations (CNA) and structural variants (SV). We developed WGS approaches to identify IGH enhancer hijack and DUX4 rearrangements, utilizing gGnome and GRIDSS respectively. For 33 cases RNA-seq data was analyzed to validate fusion genes and genetic classification into one of 18 ALL subtypes using the ALLSorts v2 classifier. Results 52/57 cases passed QC. In 5 (10%) cases an established WHO2016 genetic subtype was identified that was missed by standard of care including three high-risk (2 low hypodiploid and a near haploid) and 2 standard risk (high hyperdiploid and TCF3::PBX1) cases. Cytogenetic analysis for these 5 cases either failed (n=2) or produced a normal karyotype (n=3). The WGS inferred tumor purity for these 5 cases was variable with a median of 46% and a range of 33% to 96% compared to 89% (range 13%-99%) for the remaining cases (n=47) in the dataset. Among the remaining 47 B-other cases, WGS analysis identified abnormalities enabling 41 (87%) cases to be classified into a genetic ALL subtype: DUX4 rearrangements (DUX4r, n=8), PAX5alt (n=7), PAX5 P80R (n=4), ZNF384 rearrangements (ZNF384r, n=5), ZEB2/CEBP (n=4), MEF2D rearrangements (MEF2Dr, n=3), UBTF::ATXN7L3 fusions (n=2), IDH1/2 mutations (n=2), JAK-STAT abnormalities (n=2), along with single cases of IGK::BCL2, IGH::CEBPA, IGH::ID4 and IGH::MIR125B1. WGS resolves 8 cases with complex karyotype into one of 6 different genomic subtypes: 2xDUX4r, 2xMEF2Dr, ZEB2/CEBP, UBTF::ATXN7L3, IGK::BCL2 & IGH::ID4. The MEF2Dr subtype (n=3), is enriched in chromothripsis (2/3), and complex SVs targeting the MEF2D gene. Lastly, in all cases with PAX5 mutations (PAX5 P80R (n=4) and PAX5alt (n=4)) we observe bi-allelic targeting of locus by mutations, deletions or LOH. The remaining 6 (13%) B-other cases could not be classified into a recognized B-other subtype. A MYO18A::FGFR1 fusion (t(8;17)(p11;q23)), which most likely maps to the WHO2016 "Lymphoid neoplasms with FGFR1 rearrangement" classification. Two cases had clonal frameshifts in genes associated with T-ALL (PTPN2 p.A108fs*5 and WT1 p.V371fs*14 respectively). A case with a somatic ETV6 p.R399C previously reported in germline risk with a ZNF292::KDM6A fusion. Lastly a case with a clonal mutation typically associated with myeloid disease ASXL1 p.R417*. Among the 52 cases studied, WGS only failed to identify a driver or putative driver in one case, which had the lowest burden of SNVs (861) and an estimated tumor purity, 28%. For the 31 cases with both DNA and RNA, WGS and RNA analysis were concordant for 100% of cases, with RNA identifying an additional KDM6A::CGA fusion. In 3 cases WGS provides evidence that would have challenged diagnosis and/or treatment; IGK::BCL2 with BCL2 and MYC overexpression, IGH::DUX4 with hypermutation (MSH6 loss) and IGH::ID4 with homozygous loss of CD58 and concurrent LOH of HLA-B. Conclusions WGS assigned 88% (46/52) of cases called B-other to an established genetic subtype of ALL. This includes 5 cases with WHO2016 defined subtypes, 3 were high-risk ploidy subtypes that would have changed their UKALL14 risk group and post-induction treatment in the absence of other risk factors. Among the true B-other cases, 87% (41/47) were assigned to one of the newly described genetic subtypes of ALL. Taken together our findings demonstrate the clinical utility of WGS as a diagnostic assay to inform and improve the management of adult B-Other ALL patients in the future.

MODL-20. REDUCTION OR LOSS OF MSH6 CONFERS RESISTANCE TO TEMOZOLOMIDE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The only intervention that has improved the survival rate of GBM patients over the past several decades has been combining temozolomide (TMZ) with radiotherapy (RT), which increased median survival by only ~2.5 months to where it currently stands at ~15 months. Unfortunately, all GBM patients eventually die due to tumor recurrence. Intrinsic or acquired resistance to TMZ is a significant contributing factor to tumor progression, and various mechanisms have been suggested, including deficiencies in DNA mismatch repair (MMR) genes such as MSH6. To test the biological significance of Msh6 in GBM growth and resistance to TMZ and immunotherapy, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline loss of Msh6. To generate tumors with reduced or abrogated Msh6, we utilized GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh6. When PDGFB was overexpressed in combination with the silencing of Tp53 to induce tumors, tumor-bearing mice with reduced or deficient Msh6 demonstrated increased tumor growth and shorter survival time compared to wild-type tumor-bearing mice. Our data demonstrate that two weeks of TMZ treatment at a clinically relevant dose of 25 mg/kg provides a significant survival advantage in WT-tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh6. We are currently evaluating the mechanism by which of Msh6 confers TMZ-resistance.

Unifocal Peritoneal Malignant Mesothelioma as Initial Presentation of Lynch Syndrome: A Case Report and Brief Review

Abstract Introduction/Objective Peritoneal malignant mesothelioma (pMM) has scarcely been reported in the context of Lynch Syndrome (LS). No cases have directly led to a diagnosis of LS. We report an unusual case of pMM presenting as a localized mass, leading to the diagnosis of LS. Methods/Case Report A 55 year old female with no prior oncologic history presented with a solitary epigastric mass. Her mother and maternal relatives had colonic, gastric and ovarian cancers. Imaging studies revealed a perigastric mass abutting the left liver. The resected 9.8 cm mass showed a variegated histology with tubulopapillary formations and solid sheets. The neoplastic cells also showed variable cytologic features including anisocytosis, pleomorphic nuclei, multinucleation, prominent nucleoli, intranuclear pseudoinclusions, and rhabdoid features. A dense intratumoral and peritumoral lymphoplasmacytic infiltrate was present. Epithelioid pMM was confirmed with expression of cytokeratin 5/6, calretinin, WT-1, and thrombomodulin and loss of MOC31. Abnormal loss of MSH2 and MSH6 was seen (retained expression MLH1/PMS2 expression), suggesting high-level microsatellite instability. A germline pathogenic variant of MSH2 was identified. Results (if a Case Study enter NA) NA. Conclusion We report the first case of LS presenting as an atypical (unifocal) pMM showing variegated histology and lymphocytic infiltrate. Microsatellite instability in pMM is rare and has only been reported in known LS patients. Awareness of the pMM within LS is evolving and atypical features may prompt evaluation of DNA microsatellite repair system, with implications for the patient (treatment/screening) and their family.

Microsatellite instability profiles of gastrointestinal cancers: comparison between non-colorectal and colorectal origin.

Microsatellite instability (MSI) is a major carcinogenic pathway with prognostic and predictive implications. The validity of polymerase chain reaction (PCR)-based MSI testing is well established in colorectal cancer; however, the data are limited in non-colorectal gastrointestinal cancers. The aim of this study is to clarify the detailed MSI profiles of non-colorectal gastrointestinal cancers and to investigate the differences from those of colorectal cancers. MSI testing was performed using paired tumour/normal tissues of 123 mismatch repair-deficient cancers detected by immunohistochemistry including 80 non-colorectal cancers (eight oesophagogastric junction (EGJ), 57 gastric and 15 small intestine) and 43 colorectal cancers. Fragment size analysis revealed that the mean nucleotide shifts of five markers (Promega panel) were the highest in the stomach (6.4), followed by colorectum (5.7), small intestine (5.0) and EGJ cancers (mean=4.0; P= 0.015, versus stomach). All cases showed ≥ 1 nucleotide shift in ≥ 2 markers and were considered as MSI-high. However, when the cut-off was set to ≥ 3 nucleotide shifts in ≥ 2 markers, three EGJ (37.5%), two small intestine (13.3%) and two gastric (3.5%) cancers showed false-negative results. In addition, cases with isolated loss of MSH6 or PMS2 showed smaller nucleotide shifts than those in others. MSI testing is applicable to non-colorectal gastrointestinal cancers; however, a subset can yield false-negative results due to subtle nucleotide shift in multiple markers. Analysis of paired tumour/normal tissues and careful interpretation is necessary to avoid false-negative results and ensure appropriate treatment.

Clinicopathological characteristics and loss of mismatch repair protein expression in Chinese upper tract urothelial carcinomas.

Expression of DNA mismatch repair (MMR) protein (MLH1, PMS2, MSH2, and MSH6) in upper tract urothelial carcinoma (UTUC) has been explored in Western cohorts, but it is rarely reported in Eastern cohorts. We aimed to assess the loss of MMR protein expression among Chinese UTUC patients and study its clinicopathological implications. We enrolled 175 UTUC patients at our center and tested the expression of MMR proteins by immunohistochemistry. Then, we explored these patients' clinicopathological characteristics. We found loss of MMR proteins in 19 (10.9%) of 175 patients in our cohort (6 MSH2 and MSH6, 2 MSH6 alone, 6 MSH2 alone, 3 MLH1 and PMS2, and 2 PMS2 alone). Loss of MMR proteins was not a significant prognostic factor of relapse-free survival for these patients. In addition, patients with lower T stage or with bladder cancer history were more likely to have loss of MMR protein expression. At last, two metastatic patients (MSH2 and MSH6 loss; MSH2 loss) with loss of MMR protein experienced tumor recession after several cycles of anti-PD-1 immunotherapy. In conclusion, this is the largest Chinese UTUC cohort study to date that explores the loss of MMR protein expression. The rate of MMR loss observed was comparable to that in the Western UTUC cohort, supporting universal UTUC screening in China. Furthermore, a subset of advanced UTUCs with MMR protein loss are probably immunogenic, for whom single or combined immunotherapy may be potential therapeutic options in the future.

Histopathological Predictors of Microsatellite Instability in Colorectal Cancer-a Tertiary Care Center Experience.

Microsatellite instability (MSI) has a therapeutic and prognostic implication in colorectal carcinomas (CRCs). It can be detected either by immunohistochemistry (IHC) or molecular studies. In developing countries, a significant proportion of the patients experience financial constraints limiting the utilization of healthcare facilities. We aimed to identify the possible clinicopathological variables which can be used as predictors of microsatellite instability in such patients. CRC cases received for MSI detection by IHC (for 1 and 1/2years) were included. A panel of four IHC markers (anti-MLH1, anti-PMS2, anti-MSH2, and anti-MSH6) was used. Confirmation by the molecular study was recommended in all the IHC-proven MSI cases. Various clinicopathological parameters were evaluated as predictors of MSI. Microsatellite instability was detected in 40.6% (30/74) cases with MLH1 and PMS2 dual loss in 27% cases, MSH2 and MSH6 dual loss in 6.8%, loss of all four MMR proteins in 2.7%, and isolated PMS2 loss in 4.1%. MSI-H expression was shown by 36.5% cases with only 4.1% cases showing MSI-L expression. The age cut-off value to differentiate both the study groups (MSI vs MSS) was 63years with a sensitivity of 47.7% and specificity of 86.7%. ROC curve showed an area under the curve of 0.65 (95% CI, 0.515-0.776; p-value = 0.03). On univariate analysis, age < 63years, colon site, and absence of nodal metastasis were significantly higher in the MSI group. However, on multivariate analysis, only the age < 63years was found to be significantly higher in the MSI group. Confirmation was molecular study could only be obtained in 12 cases and was completely concordant with MSI detection by IHC. MSI detection can be performed either by IHC or by molecular study. In this study, no histological parameter appeared to be the independent predictor of MSI status. The age < 63years might predict the microsatellite instability, yet larger studies are needed for its validation. Thus, we recommend that IHC testing should be performed in all CRC cases.

Feasibility and clinical applicability of genomic profiling based on cervical smear samples in patients with endometrial cancer.

PurposeCervical smear samples are easily obtainable and may effectively reflect the tumor microenvironment in gynecological cancers. Therefore, we investigated the feasibility of genomic profiling based on tumor DNA analysis from cervical smear samples from endometrial cancer patients.Materials and methodsPreoperative cervical smear samples were obtained via vaginal sampling in 50 patients, including 39 with endometrial cancer and 11 with benign uterine disease. Matched blood samples were obtained simultaneously. Genomic DNA (gDNA) from cervical smear and/or cell-free DNA from whole blood were extracted and sequenced using the Pan100 panel covering 100 endometrial cancer-related genes.ResultsCervical swab-based gDNA analysis detected cancer with 67% sensitivity and 100% specificity, showing a superior performance compared to that of the matched blood or Pap smear tests. Cervical swab-based gDNA effectively identified patients with loss of MSH2 or MSH6 and aberrant p53 expression based on immunohistochemistry. Genomic landscape analysis of cervical swab-based gDNA identified PTEN, PIK3CA, TP53, and ARID1A as the most frequently altered genes. Furthermore, 26 endometrial cancer patients could be classified according to the Proactive Molecular Risk Classifier for Endometrial Cancer.ConclusionCervical swab-based gDNA test showed an improved detection potential and allowed the classification of patients, which has both predictive and prognostic implications.

PMS2 Expression With Combination of PD-L1 and TILs for Predicting Survival of Esophageal Squamous Cell Carcinoma.

BackgroundDNA mismatch repair (MMR) deficiency (dMMR) has been recognized as an important biomarker for immunotherapy in esophageal squamous cell carcinoma (ESCC), along with programmed death ligand 1 (PD-L1) expression and/or tumor-infiltrated lymphocytes (TILs). However, in ESCC, MMR protein assessment has not been well studied at present.MethodsA total of 484 ESCC tissues treated between 2007 and 2010, in our hospital, were enrolled. Immunohistochemical expression of MLH1, MSH2, MSH6, PMS2, and PD-L1 on tissue microarray specimens and clinicopathological features, including TILs, were analyzed retrospectively.ResultsOut of the 484 studied cases, loss of MLH1, MSH2, MSH6, and PMS2 expression were found in 6.8%, 2.1%, 8.7%, and 4.8% patients, respectively. dMMR was found in 65 patients, 37 cases involved in one MMR protein, 17 cases involved in two proteins, 7 cases involved in three proteins, and 4 cases involved in four proteins. There was no significant survival difference between pMMR (MMR-proficient) and dMMR patients (P>0.05). However, 224 patients with low PMS2 expression had better DFS and OS than 260 patients with high PMS2 expression (P=0.006 for DFS and 0.008 for OS), which was identified as an independent prognostic factor in multivariate analyses. Positive PD-L1 expression was detected in 341 (70.5%) samples. In stage I-II disease, patients with PD-L1 expression had better DFS and OS than those without PD-L1 expression(P<0.05), which was not found in stage III-IV disease. With the ITWG system, 40.1% of cases were classified as high TILs. Patients in the high-TILs group tended to have better DFS (P=0.055) and OS (P=0.070) than those in the low-TILs group and the differences were statistically significant in pMMR, high MSH6, or PMS2 expression cases (P<0.05). Also, high PMS2 expression patients with both PD-L1 expression and high TILs, had similar DFS and OS compared with low PMS2 expression patients (P>0.05), which were much better than other high PMS2 expression patients.ConclusionThe expression level of MMR proteins could also be used as a prognostic factor in ESCC and PMS2 expression outperformed other MMR proteins for predicting survival. The combination of PD-L1 expression and TILs may lead to more efficient risk stratification of ESCC.

Abstract 1634: Reduction or loss of Msh2 confers resistance to temozolomide in glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The only intervention that has improved the survival rate of GBM patients over the past several decades has been combining temozolomide (TMZ) with radiotherapy (RT), which increased median survival by only ~2.5 months to where it currently stands at ~15 months. Unfortunately, all GBM patients eventually die due to tumor recurrence. Intrinsic or acquired resistance to TMZ is a significant contributing factor to tumor progression, and various mechanisms have been suggested, including deficiencies in DNA mismatch repair (MMR) genes such as MSH2. Utilizing patient GBM samples from the TCGA, we have identified a correlation between low MSH2 expression and shorter patient survival. To test the biological significance of Msh2 in GBM growth and resistance to TMZ and immunotherapy, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline or somatic loss of Msh2. To generate tumors with reduced or abrogated Msh2, we utilized GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh2. When PDGFB was overexpressed in combination with the silencing of Tp53 to induce tumors, tumor-bearing mice with reduced or deficient Msh2 demonstrated shorter survival time compared to wild-type tumor-bearing mice. In addition, our results indicate Msh2 deficiency increases the malignancy of gliomas. Our data demonstrate that two weeks of TMZ treatment at a clinically relevant dose of 25 mg/kg provides a significant survival advantage in WT-tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh2. We are currently evaluating the role of Msh2 in the efficacy of immune checkpoint inhibitors in glioblastoma. Citation Format: Montserrat Puigdelloses Vallcorba, Alice Buonfiglioli, Gonzalo Piñero, Zhihong Chen, Dolores Hambardzumyan. Reduction or loss of Msh2 confers resistance to temozolomide in glioblastoma [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 1634.

DAXX, ATRX, and MSI in PanNET and Their Metastases: Correlation with Histopathological Data and Prognosis

Introduction: Studies on pancreatic neuroendocrine tumors (PanNETs) regarding loss of ATRX, DAXX, or frequency of microsatellite instability (MSI) show inconclusive results. So far, data on corresponding metastaseshave not been published. Methods: We performed immunohistochemistry (IHC) of ATRX, DAXX, MSH2, MSH6, MLH1, and PMS2 on 74 PanNETs and 19 metastases. ATRX- and DAXX-negative PanNETs were further sequenced for mutations. We used polymerase chain reaction for MSI on cases with IHC loss of MSH2, MSH6, MLH1, and PMS2. Results: Immunohistochemical loss of DAXX and ATRX was observed in 8/74 (11%) and 6/74 (8%) PanNETs. Loss of DAXX immunoreactivity was statistically associated with higher tumor grade and showed a tendency toward a decreased overall survival. Sequencing of DAXX- (7/11 [64%]) and ATRX-negative (5/11 [45%]) PanNETs revealed a mutation in 6/7 (86%) and 2/5 (40%). The specificity of immunohistochemical loss of DAXX and ATRX for mutation was 80% and 67%, respectively. The expression status of DAXX compared to primary tumor differs in 2/12 (17%) lymph node metastases. We further identified 3/74 (4%) tumors as MSI, associated with a poor prognosis. Discussion/Conclusion: Our study supports the hypothesis that a loss of DAXX immunoreactivity can identify a more aggressive subtype of PanNET with high confidence, while ATRX loss is a weaker indicator. Our results also strengthen the role of DAXX immunolabeling as a prognostic marker. We could show that ATRX might be less suitable as a surrogate for sequencing. Our results indicate that IHC of DAXX and ATRX may identify PanNET subtypes as targets for more aggressive therapy.

Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.

DNA repair protein MSH2 binds and regulates cell adhesion genes by enabling enhancer-promoter interactions, and loss of MSH2 causes deficient cell adhesion and bromodomain and extraterminal motif inhibitor synthetic lethality in gastric cancer.

Prevalence of mismatch repair mutations in colorectal carcinoma patients in Mangalore

Colorectal cancer (CRC) is highly prevalent throughout the world and represents the 3rd most common cancer in men and the 2nd in women worldwide. Microsatellite instability (MSI) is a term used to denote a hypermutable phenotype caused by the loss of DNA mismatch repair (MMR) activity, and is a phenomenon now linked to the pathways of colorectal carcinogenesis. Compounding its importance is its integral association with Lynch syndrome, the most common cause for CRCs in young individuals. In the present study, we aimed to analyse the proportion of patients with risk of microsatellite instability by checking for loss of immunostaining for mismatch repair (MMR) proteins.From January 2016 to December 2016 and May 2017 to October 2017, 40 consecutive newly diagnosed cases of colorectal cancer were included in the study. The expression of MMR proteins in the tumour tissue using IHC for MSH2, MSH6, MLH1 and PMS2 was studied.Among the 40 cases, 3 (7.5%) demonstrated loss of MMR proteins and 37 (92.5%) cases had intact nuclear expression. Out of the three cases with MMR loss, one showed concurrent loss of MLH1 and PMS2, the second showed concurrent loss of MSH2 and MSH6 and the third showed an isolated loss of MSH6.Colorectal carcinomas showing MMR mutations are seen in the Mangalorean population. However, the incidence in our study was relatively low compared to most other studies, probably due to a variation in ethnicity.

An unusual phenotype occurs in 15% of mismatch repair-deficient tumors and is associated with non-colorectal cancers and genetic syndromes

Immunohistochemistry (IHC) and/or MSI-PCR (microsatellite instability-polymerase chain reaction) tests are performed routinely to detect mismatch repair deficiency (MMR-D). Classical MMR-D tumors present a loss of MLH1/PMS2 or MSH2/MSH6 with MSI-High. Other profiles of MMR-D tumors have been described but have been rarely studied. In this study, we established a classification of unusual MMR-D tumors and determined their frequency and clinical impact. All MMR-D tumors identified between 2007 and 2017 were selected. Any profile besides the classical MMR-D phenotype was defined as unusual. For patients with unusual MMR-D tumors, IHC, and PCR data were reviewed, the tumor mutation burden (TMB) was evaluated and clinical and genetic features were collected. Of the 4948 cases of MMR testing, 3800 had both the available IHC and MSI-PCR results and 585 of these had MMR-D. After reviewing the IHC and PCR, 21% of the cases initially identified as unusual MMR-D were reclassified, which resulted in a final identification of 89 unusual MMR-D tumors (15%). Unusual MMR-D tumors were more often associated with non-CRC than classical MMR-D tumors. Unusual MMR-D tumors were classified into four sub-groups: i) isolated loss of PMS2 or MSH6, ii) classical loss of MLH1/PMS2 or MSH2/MSH6 without MSI, iii) four MMR proteins retained with MSI and, iv) complex loss of MMR proteins, with clinical characteristics for each sub-group. TMB-high or -intermediate was shown in 96% of the cancers studied (24/25), which confirmed MMR deficiency. Genetic syndromes were identified in 44.9% (40/89) and 21.4% (106/496) of patients with unusual and classical MMR-D tumors, respectively (P < 0.001). Five patients treated with an immune checkpoint inhibitor (ICI) had a prolonged clinical benefit. Our classification of unusual MMR-D phenotype helps to identify MMR deficiency. Unusual MMR-D phenotype occurs in 15% of MMR-D tumors. A high frequency of genetic syndromes was noted in these patients who could benefit from ICI.

A16 ESOPHAGEAL ORGANOID PROLIFERATION AND DIFFERENTIATION ARE ALTERED BY LOSS OF MSH2

BackgroundThe stratified epithelium of the esophagus includes Krt15+ basal stem cells that display self-renewing and regenerative capacity, and multipotency. However, the mechanisms that specifically control their functions remain unknown. Interestingly, RNA sequencing and GSEA revealed an enrichment of a gene set associated with DNA repair in Krt15+ cells vs Krt15- cells. We also observed that Msh2 (DNA mismatch repair pathway) is the most significantly upregulated gene in Krt15+ stem cells.AimsTo determine the effect of Msh2 loss on self-renewal and differentiation of esophageal organoids.MethodsEsophageal epithelial cells were isolated from a wild-type mouse. Using flow cytometry, esophageal Krt15+ (GFP+) and Krt15- (GFP-) cells were sorted from Krt15-CrePR1 (R26mT/mG) mice. All cell populations were grown as organoids and Msh2 was depleted using a CRISPR/Cas9 approach. Impact of Msh2 loss on self-renewal and differentiation in esophageal epithelial organoids was evaluated through organoid formation assays, WST-1 proliferation assays and histological analysis.ResultsAt baseline, organoids depleted for Msh2 formed more poorly differentiated and less well-differentiated organoids than controls. Lower expression of differentiation gene Krt13 was also observed in Msh2-depleted organoids, confirming an altered differentiation pattern. Furthermore, these organoids showed a higher organoid formation rate and proliferation by WST-1 assay, suggesting that self-renewal capacity and viability are increased when Msh2 is depleted. Interestingly, following radiation, organoids depleted for Msh2 showed higher residual levels of p-H2AX (DNA damage marker), suggesting that their capacity to cope with DNA damages is altered. As mentioned above, we previously reported that Msh2 is the most upregulated gene in Krt15+ vs Krt15- cells. Therefore, to determine if Msh2 role is distinct in both populations, we depleted Msh2 in Krt15+ and Krt15- cells-derived organoids. Interestingly, our preliminary results suggest that Msh2 deletion led to increased p-H2AX and decreased Krt13 levels in Krt15+ organoids but not in Krt15- organoids.ConclusionsOur results show that Msh2 is potentially a key contributor of esophageal stemness in homeostatic and injured conditions.Funding AgenciesCIHRCanada Research Chair