Characteristics of Mismatch Repair-Deficient Colon Cancer in Relation to Mismatch Repair Protein Loss, Hypermethylation Silencing, and Constitutional and Biallelic Somatic Mismatch Repair Gene Pathogenic Variants.

BACKGROUND: Mismatch repair-deficient colon cancer is heterogeneous. Differentiating inherited constitutional variants from somatic genetic alterations and gene silencing is important for surveillance and genetic counseling. OBJECTIVE: This study aimed to determine the extent to which the underlying mechanism of loss of mismatch repair influences molecular and clinicopathologic features of microsatellite instability-high colon cancer. DESIGN: This is a retrospective analysis. SETTINGS: This study was conducted at a comprehensive cancer center. PATIENTS: Patients with microsatellite instability-high colon cancer of stage I, II, or III were included. INTERVENTION: Patients underwent a curative surgical resection. MAIN OUTCOME MEASURES: The main outcome measures were hypermethylation of the MLH1 promoter, biallelic inactivation, constitutional pathogenic variants, and loss of specific mismatch repair proteins. RESULTS: Of the 157 identified tumors with complete genetic analysis, 66% had hypermethylation of the MLH1 promoter, 18% had constitutional pathogenic variants, (Lynch syndrome), 11% had biallelic somatic mismatch repair gene pathogenic variants, and 6% had unexplained high microsatellite instability. The distribution of mismatch repair loss was as follows: MLH1 and PMS2 co-loss, 79% of the tumors; MSH2 and MSH6 co-loss, 10%; MSH6 alone, 3%; PMS2 alone, 2%; other combinations, 2%; no loss, 2%. Tumor mutational burden was lowest in MLH1- and PMS2-deficient tumors. MSH6-deficient tumors had the lowest levels of tumor-infiltrating lymphocytes, lowest MSI scores, and fewest frameshift deletions. Patients with MLH1 promoter hypermethylation were significantly more likely to be older and female and to have right-sided colon lesions than patients with biallelic inactivation. Mutation was the most prevalent second hit in tumors with biallelic inactivation and tumors of patients with Lynch syndrome. LIMITATIONS: This study was limited by potential selection or referral bias, missing data for some patients, and relatively small sizes of some subgroups. CONCLUSIONS: Clinical characteristics of mismatch repair-deficient colon cancer vary with the etiology of microsatellite instability, and its molecular characteristics vary with the affected mismatch repair protein. See Video Abstract at http://links.lww.com/DCR/B984 . CARACTERSTICAS DEL CNCER DE COLON CON DEFICIENCIA EN LA REPARACIN DE ERRORES DE EMPAREJAMIENTO EN RELACIN CON LA PRDIDA DE PROTENAS MMR, SILENCIAMIENTO DE LA HIPERMETILACIN Y LAS VARIANTES PATGENAS SOMTICAS DE GENES MMR CONSTITUCIONAL Y BIALLICO: ANTECEDENTES:El cáncer de colon deficiente en la reparación de errores de emparejamiento es heterogéneo. La diferenciación de las variantes constitucionales heredadas de las alteraciones genéticas somáticas y el silenciamiento de genes es importante para la vigilancia y el asesoramiento genético.OBJETIVO:Determinar hasta qué punto el mecanismo subyacente de pérdida de reparación de desajustes influye en las características moleculares y clinicopatológicas del cáncer de colon con alta inestabilidad de microsatélites.DISEÑO:Análisis retrospectivo.ESCENARIO:Centro integral de cáncer.PACIENTES:Pacientes con cáncer de colon con inestabilidad de microsatélites alta en estadio I, II, o III.INTERVENCIÓN:Resección quirúrgica con intención curativa.PRINCIPALES RESULTADOS Y MEDIDAS:Hipermetilación del promotor MLH1, inactivación bialélica, variante patógena constitucional y pérdida de proteínas específicas reparadoras de desajustes.RESULTADOS:De los 157 tumores identificados con un análisis genético completo, el 66 % tenía hipermetilación del promotor MLH1, el 18 % tenía una variante patogénica constitucional (síndrome de Lynch), el 11 % tenía variantes patogénicas somáticas bialélicas de algún gen MMR y el 6 % tenía una alta inestabilidad de microsatélites sin explicación. La distribución de la pérdida según la proteína de reparación del desajuste fue la siguiente: pérdida conjunta de MLH1 y PMS2, 79 % de los tumores; co-pérdida de MSH2 y MSH6, 10%; MSH6 solo, 3%; PMS2 solo, 2%; otras combinaciones, 2%; sin pérdida, 2%. La carga mutacional del tumor fue más baja en los tumores deficientes en MLH1 y PMS2. Los tumores con deficiencia de MSH6 tenían los niveles más bajos de linfocitos infiltrantes de tumores, las puntuaciones más bajas del sensor de IMS y la menor cantidad de deleciones por cambio de marco. Los pacientes con hipermetilación del promotor MLH1 tenían significativamente más probabilidades de ser mayores y mujeres y de tener lesiones en el colon derecho que los pacientes con inactivación bialélica. La mutación fue el segundo golpe más frecuente en tumores con inactivación bialélica y tumores de pacientes con síndrome de Lynch.LIMITACIONES:Sesgo potencial de selección o referencia, datos faltantes para algunos pacientes y tamaños relativamente pequeños de algunos subgrupos.CONCLUSIONES:Las características clínicas del cáncer de colon deficiente en reparación de desajustes varían con la etiología de la inestabilidad de microsatélites, y sus características moleculares varían con la proteína de reparación de desajustes afectada. Vea Resumen de video en http://links.lww.com/DCR/B984 . (Traducción-Dr. Felipe Bellolio ).

BAT26 Only Microsatellite Instability with High Tumor Mutation Burden-A Rare Entity Associated with PTEN Protein Loss and High PD-L1 Expression.

Detecting microsatellite instability (MSI) in advanced cancers is crucial for clinical decision-making, as it helps in identifying patients with differential treatment responses and prognoses. BAT26 is a highly sensitive MSI marker that defines the mismatch repair (MMR) status with high sensitivity and specificity. However, isolated BAT26-only instability is rare and has not been previously reported. Of the 6476 cases tested using pentaplex MSI polymerase chain reaction, we identified two BAT26-only instability cases (0.03%) in this study. The case #1 patient was diagnosed with endometrial adenocarcinoma without MMR germline mutations. The endometrial tumor showed BAT26-only instability, partial loss of MLH1/PMS2 protein expression, and a high programmed cell death ligand 1 (PD-L1) combined positive score (CPS = 8). The tumor exhibited a somatic phosphatase and tensin homolog (PTEN) R303P missense mutation and loss of the PTEN protein. On a comprehensive cancer panel sequencing with ≥500 genes, the tumor showed an MSI score of 11.38% and high tumor mutation burden (TMB) (19.5 mt/mb). The case #2 patient was diagnosed with colorectal carcinoma with proficient MMR and PTEN protein loss without PTEN alteration, as well as a high PD-L1 CPS (CPS = 10). A pathogenic KRAS A146T mutation was detected with an MSI score of 3.36% and high TMB (13 mt/mb). In conclusion, BAT26-only instability is very rare and associated with PTEN protein loss, high TMB, and a high PD-L1 score. Our results suggest that patients with BAT26-only instability may show good responses to immunotherapy.

Prognostic significance of BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, DARC, MSH2 and PMS2 in colorectal cancer treated with FOLFOX chemotherapy with or without aspirin.

Evasion of apoptosis is associated with treatment resistance and metastasis in colorectal cancer (CRC). Various cellular processes are associated with evasion of apoptosis. These include overexpression of pro-apoptotic proteins (including p53 and PD-L1), anti-apoptotic proteins (BIRC7/Livin and Bcl-2), chemokine receptors (including DARC), and dysregulation of DNA mismatch repair proteins (including MSH2 and PMS2). The aim of this study was to determine the effect of folinic acid, 5-FU and oxaliplatin (FOLFOX) as a single agent and aspirin plus FOLFOX in various combinations on the aforementioned proteins in human CRC, SW480 cell line and rat models of N-Methyl-N-Nitrosourea (NMU)-induced CRC. In addition, effects of the NMU-induced CRC and chemotherapeutic regimens on haematological and biochemical parameters in the rat models were studied. Immunohistochemistry, immunofluorescence and immunoblot techniques were used to study the expression pattern of the related proteins in the human CRC cells pre- and post-treatment. Double contrast barium enema, post-mortem examination and histological analyses were used to confirm tumour growth and the effect of the treatment in vivo in rat models. Notably, we found in human mucinous CRC, a significant increase in expression of the BIRC7/Livin post-FOLFOX treatment compared with pre-treatment (p = 0.0001). This increase provides new insights into the prognostic role of BIRC7/Livin in evasion of apoptosis and facilitation of treatment resistance, local recurrence and metastasis particularly among mucinous CRCs post-FOLFOX chemotherapy. These poor prognostic features in the CRC may be further compounded by the significant suppression of DARC, PD-L1, PMS2 and overexpression of MSH2 and anti-apoptotic Bcl-2 and p53 proteins observed in our study (p < 0.05). Importantly, we found a significant reduction in expression of BIRC7/Livin and reactivation of DARC and PD-L1 with a surge in Annexin V expression in rat models of CRC cells post-treatment with a sequential dose of aspirin plus FOLFOX compared with other treatments in vivo (p <0.05). The mechanistic rational of these effects underscores the importance of expanded concept of possible aspirin combination therapy with FOLFOX sequentially in future CRC management. Validation of our findings through randomized clinical trials of aspirin plus FOLFOX sequentially in patients with CRC is therefore warranted.

Efficacy of paired tumor and germline testing in evaluation of patients with Lynch-like syndrome in a large integrated healthcare setting.

Patients with mismatch repair (MMR) deficient colorectal cancer (CRC) without detectable germline pathogenic variants (PVs) or likely pathogenic variants (LPVs) in MMR genes are often labeled as Lynch-like syndrome (LLS). We sought to evaluate the efficacy of paired tumor and germline testing in risk stratification of patients with LLS in a large, community-based, integrated healthcare setting. Through the universal screening program for Lynch syndrome at Kaiser Permanente Northern California, we identified all patients with MMR deficient colorectal tumors without detectable germline PVs or LPVs between April 2011 and October 2018. These patients were categorized as LLS and were offered paired tumor and germline testing. Risk stratification and patient management were assessed upon completion of all testing. Of the 50 patients with LLS who underwent paired tumor and germline testing, 62% (n = 31) were categorized as sporadic, 6% (n = 3) had Lynch syndrome, and 32% (n = 16) remained inconclusive. Among the sporadic cases, 65% (n = 20) had a PV (n = 18) or LPV (n = 2) in combination with loss of heterozygosity while 35% (n = 11) had two somatic PVs/LPVs involving the same MMR gene. Our findings showed paired tumor and germline testing resolved the etiology in the majority of patients and is a valuable strategy in risk stratification and management of patients with LLS. Further studies are needed to assess the optimal application of paired testing in different practice settings, particularly with evolving technology and decreasing cost of molecular sequencing.

Prognostic Role of Mismatch Repair Status, Histotype and High-Risk Pathologic Features in Stage II Small Bowel Adenocarcinomas.

BACKGROUND: Small bowel adenocarcinoma is a relatively rare cancer, often diagnosed in an advanced stage. In localized and resectable disease, surgery alone or in combination with adjuvant chemotherapy is the mainstay of treatment. In the recently published National Comprehensive Cancer Network Clinical Practice guidelines, criteria for selecting patients with stage II small bowel adenocarcinoma to receive adjuvant chemotherapy are provided, and they are mainly extrapolated from studies on colorectal cancer. PATIENTS AND METHODS: In the present study, we aimed to verify whether mismatch repair deficiency phenotype, high-risk pathologic features (including T4, positive resection margins and a low number of lymph nodes harvested), as well as tumor histologic subtype, were associated with cancer-specific survival in 66 stage II non-ampullary small bowel adenocarcinoma patients, collected through the Small Bowel Cancer Italian Consortium. A central histopathology review was performed. Mismatch repair deficiency was tested by immunohistochemistry for MLH1, MSH2, MSH6 and PMS2, and confirmed by polymerase chain reaction for microsatellite instability. RESULTS: We identified mismatch repair deficiency, glandular/medullary histologic subtype, and celiac disease as significant predictors of favorable cancer-specific survival using univariable analysis with retained significance in bivariable models adjusted for pT stage. Among the high-risk features, only T4 showed a significant association with an increased risk of death; however, its prognostic value was not independent of mismatch repair status. CONCLUSIONS: Mismatch repair protein expression, histologic subtype, association with celiac disease, and, in the mismatch repair proficient subset only, T stage, may help identify patients who may benefit from adjuvant chemotherapy.

A Novel Germline MLH1 In-Frame Deletion in a Slovenian Lynch Syndrome Family Associated with Uncommon Isolated PMS2 Loss in Tumor Tissue.

The diagnostics of Lynch syndrome (LS) is focused on the detection of DNA mismatch repair (MMR) system deficiency. MMR deficiency can be detected on tumor tissue by microsatellite instability (MSI) using molecular genetic test or by loss of expression of one of the four proteins (MLH1, MSH2, MSH6, and PMS2) involved in the MMR system using immunohistochemistry (IHC) staining. According to the National Comprehensive Cancer Network (NCCN) guidelines, definitive diagnosis of LS requires the identification of the germline pathogenic variant in one of the MMR genes. In the report, we are presenting interesting novel MLH1 in-frame deletion LRG_216t1:c.2236_2247delCTGCCTGATCTA p.(Leu746_Leu749del) associated with LS. The variant appears to be associated with uncommon isolated loss of PMS2 immunohistochemistry protein staining (expression) in tumor tissue instead of MLH1 and PMS2 protein loss, which is commonly seen with pathogenic variants in MLH1. The variant was classified as likely pathogenic, based on segregation analysis and molecular characterization of blood and tumor samples. According to the American College of Medical Genetics (ACMG) guidelines, the following evidence categories of PM1, PM2, PM4, and PP1 moderate have been used for classification of the novel variant. By detecting and classifying the novel MLH1 variant as likely pathogenic, we confirmed the LS in this family.

Implementation of a Systematic Tumor Screening Program for Lynch Syndrome in an Integrated Health Care Setting.

A subset of colorectal cancer (CRC) cases are attributable to Lynch syndrome (LS), a hereditary form of CRC. Effective evaluation for LS can be done on CRC tumors to guide diagnostic testing. Increased diagnosis of LS allows for surveillance and risk reduction, which can mitigate CRC-related burden and prevent cancer-related deaths. We evaluated participation in LS screening among newly diagnosed adult CRC patients. Some cases were referred for genetics evaluation prior to study recruitment (selective screening). Those not referred directly were randomized to the intervention or control (usual care) arms. Control cases were observed for one year, then given information about LS screening. Patients who declined participation were followed through the medical record. Of 601 cases of CRC, 194 (32%) enrolled in our study and were offered LS screening, 43 (7%) were followed as a control group, 148 (25%) declined participation and 216 (36%) were ineligible [63 (10%) of which received prior selective screening]. Six and nine cases of LS were identified through the intervention and selective screening groups, respectively. Overall, a higher proportion of PMS2 variants were identified in the intervention (3/6, 50%) versus selective screening groups (2/9, 22%) (not statistically significant). Eighty-eight percent and 23% of intervention and control patients, respectively, received LS screening. No control patients were found to have LS. Systems-based approaches are needed to ensure we fully identify LS cases. The proportion of LS cases from this program was 4% of newly diagnosed cases of CRC, similar to other programs.

Mismatch Repair Deficiency, Microsatellite Instability, and Survival: An Exploratory Analysis of the Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) Trial.

IMPORTANCE: Mismatch repair (MMR) deficiency (MMRD) and microsatellite instability (MSI) are prognostic for survival in many cancers and for resistance to fluoropyrimidines in early colon cancer. However, the effect of MMRD and MSI in curatively resected gastric cancer treated with perioperative chemotherapy is unknown. OBJECTIVE: To examine the association among MMRD, MSI, and survival in patients with resectable gastroesophageal cancer randomized to surgery alone or perioperative epirubicin, cisplatin, and fluorouracil chemotherapy in the Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) trial. DESIGN, SETTING, AND PARTICIPANTS: This secondary post hoc analysis of the MAGIC trial included participants who were treated with surgery alone or perioperative chemotherapy plus surgery for operable gastroesophageal cancer from July 1, 1994, through April 30, 2002. Tumor sections were assessed for expression of the MMR proteins mutL homologue 1, mutS homologue 2, mutS homologue 6, and PMS1 homologue 2. The association among MSI, MMRD, and survival was assessed. MAIN OUTCOMES AND MEASURES: Interaction between MMRD and MSI status and overall survival (OS). RESULTS: Of the 503 study participants, MSI results were available for 303 patients (283 with microsatellite stability or low MSI [median age, 62 years; 219 males (77.4%)] and 20 with high MSI [median age, 66 years; 14 males (70.0%)]). A total of 254 patients had MSI and MMR results available. Patients treated with surgery alone who had high MSI or MMRD had a median OS that was not reached (95% CI, 11.5 months to not reached) compared with a median OS among those who had neither high MSI nor MMRD of 20.5 months (95% CI, 16.7-27.8 months; hazard ratio, 0.42; 95% CI, 0.15-1.15; P = .09). In contrast, patients treated with chemotherapy plus surgery who had either high MSI or MMRD had a median OS of 9.6 months (95% CI, 0.1-22.5 months) compared with a median OS among those who were neither high MSI nor MMRD of 19.5 months (95% CI, 15.4-35.2 months; hazard ratio, 2.18; 95% CI, 1.08-4.42; P = .03). CONCLUSIONS AND RELEVANCE: In the MAGIC trial, MMRD and high MSI were associated with a positive prognostic effect in patients treated with surgery alone and a differentially negative prognostic effect in patients treated with chemotherapy. If independently validated, MSI or MMRD determined by preoperative biopsies could be used to select patients for perioperative chemotherapy.

Selenium compounds activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in colorectal cancer cells.

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G(2)/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.

Epigenetic repression of DNA mismatch repair by inflammation and hypoxia in inflammatory bowel disease-associated colorectal cancer.

Sporadic human mismatch repair (MMR)-deficient colorectal cancers account for approximately 12.5% of all cases of colorectal cancer. MMR-deficient colorectal cancers are classically characterized by right-sided location, multifocality, mucinous histology, and lymphocytic infiltration. However, tumors in germ-line MMR-deficient mouse models lack these histopathologic features. Mice lacking the heterotrimeric G protein alpha subunit Gialpha2 develop chronic colitis and multifocal, right-sided cancers with mucinous histopathology, similar to human MMR-deficient colorectal cancer. Young Gialpha2-/- colonic epithelium has normal MMR expression but selectively loses MLH1 and consequently PMS2 expression following inflammation. Gialpha2-/- cancers have microsatellite instability. Mlh1 is epigenetically silenced not by promoter hypermethylation but by decreased histone acetylation. Chronically inflamed Gialpha2-/- colonic mucosa contains patchy hypoxia, with increased crypt expression of the hypoxia markers DEC-1 and BNIP3. Chromatin immunoprecipitation identified increased binding of the transcriptional repressor DEC-1 to the proximal Mlh1 promoter in hypoxic YAMC cells and colitic Gialpha2-/- crypts. Treating Gialpha2-/- mice with the histone deacetylase inhibitor suberoylanilide hydroxamic acid significantly decreased colitis activity and rescued MLH1 expression in crypt epithelial cells, which was associated with increased acetyl histone H3 levels and decreased DEC-1 binding at the proximal Mlh1 promoter, consistent with a histone deacetylase-dependent mechanism. These data link chronic hypoxic inflammation, epigenetic MMR protein down-regulation, development of MMR-deficient colorectal cancer, and the firstmouse model of somatically acquired MMR-deficient colorectal cancer.

Contribution of human mlh1 and pms2 ATPase activities to DNA mismatch repair.

MutLalpha, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLalpha and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLalpha proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLalpha demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLalpha activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.