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 ).

A case of microsatellite instability-high clinically advanced castration-resistant prostate cancer showing a remarkable response to pembrolizumab sustained over at least 18 months.

Defective DNA mismatch repair genes can lead to microsatellite instability (MSI)-high status in prostate cancer (PC). Accumulation of replication errors in DNA leads to the production of abundant neoantigens, which could be targets for immune checkpoint inhibitors (CPIs). However, the incidence of MSI-high PC is low, and not all patients show a satisfactory therapeutic response to CPIs. Here, we present the case of a patient with MSI-high castration-resistant PC who showed a remarkable and durable response to pembrolizumab. The patient was resistant to abiraterone, docetaxel, and cabazitaxel and was suffering from multiple tumor-associated or treatment-related complications, such as urinary tract infection, infective endocarditis, and uncontrollable prostatic hemorrhage. Soon after the start of pembrolizumab therapy, the patient showed a dramatic decrease in prostate-specific antigen from 35.67 ng/mL to an undetectable level and a remarkable reduction in the size of a massive prostate mass and lymph node metastases, with an absence of treatment-related complications. Specimens from the transurethral resection of prostate cancer during cabazitaxel treatment for control of prostate bleeding and also that from the prostate biopsy at initial diagnosis revealed MSI-high status. Immunohistochemistry showed loss of MSH2 and MSH6, and whole-exome sequencing revealed an approximate tumor mutation burden of 61 mutations/Mb as well as biallelic loss of MSH2 Pembrolizumab could show a significant effect even in a heavily treated patient with MSI-high advanced PC. Accumulation of detailed clinical and genomic information of cases of MSI-high PC treated with pembrolizumab is necessary for optimal patient selection.

Racial and ethnic variation in multigene panel testing in a cohort of BRCA1/2-negative individuals who had genetic testing in a large urban comprehensive cancer center.

BACKGROUND: There is sparse clinical information on the racial and ethnic distribution of results of multigene panel testing among individuals at high risk for hereditary cancer. METHODS: We evaluated the results of multigene panel testing across eight clinical sites across the state of Michigan for individuals seen for genetic counseling from May 13, 2013 to October 31, 2019 at the Karmanos Cancer Institute's cancer genetics clinic. We estimated the prevalence of pathogenic variants and variants of uncertain significance (VUS) from genes other than BRCA1/2 among individuals of non-Hispanic White (NHW), Black or African American (AA), Ashkenazi Jewish (AJ), Arab, Hispanic, and other ancestry. RESULTS: The racial and ethnic distribution of 2419 individuals who had panel testing included 68.8% NHW, 22.1% AA, 2.3% Arab, 2.2% AJ, 1.0% Hispanic, and 3.6% other. Of these, 11.2% had pathogenic variants and 17.5% had VUS. After multivariable analyses, compared to NHW, AA were less likely to have pathogenic variants (OR 95% CI, 0.38, 0.24-0.59, p < 0.001). Both AA and Arabs were more likely to have VUS (OR 95% CI, 1.53, 1.18-1.98, p = 0.001 and OR 95% CI, 2.28, 1.17-4.43, p = 0.015, respectively). There were no significant differences for other groups. The most common pathogenic variants were CHEK2 (n = 65), MUTYH (n = 45), ATM (n = 28), and MSH2 (n = 22); the most common pathogenic variants by race and ethnicity were CHEK2 (NHW), MSH2 and MUTYH (AA), MSH2 (Arab), MSH6 and CHEK2 (AJ), and MLH1 (Hispanic); the most common pathogenic variants by primary cancer site were CHEK2 (breast), MSH2 (colon), BRIP1 and MUTYH (ovarian), and MSH2 and MSH6 (endometrial). CONCLUSIONS: Understanding the racial and ethnic distribution of pathogenic variants in multi-gene panels has the potential to lead to better identification of individuals at risk for hereditary cancer.

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.

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.

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.

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.

Effects of vitamin D and calcium on expression of MSH2 and transforming growth factors in normal-appearing colorectal mucosa of sporadic colorectal adenoma patients: A randomized clinical trial.

Abnormal expression of the DNA mismatch repair protein MSH2 and autocrine/paracrine transforming growth factors TGFα (growth promoter) and TGFß1 (growth inhibitor) is common during colorectal carcinogenesis. To estimate vitamin D and calcium effects on these biomarkers in the normal-appearing colorectal mucosa of sporadic colorectal adenoma patients, we conducted a pilot, randomized, double-blinded, placebo-controlled, modified 2 × 2 factorial chemoprevention clinical trial (N = 104) of supplemental vitamin D3 (1000 IU daily) and calcium (1200 mg daily), alone and in combination, versus placebo over 1 year. The expression of the three biomarkers and Ki-67/mib-1 in colorectal crypts in biopsies of normal-appearing rectal mucosa were detected using automated immunohistochemistry and quantified using image analysis. In the vitamin D3 and vitamin D3 plus calcium groups, relative to their reference groups, in the upper 40% (differentiation zone) of crypts, it was estimated that, respectively, the MSH2/mib-1 ratio increased by 47% (P = 0.14) and 62% (P = 0.08), TGFß1 expression increased by 41% (P = 0.25) and 78% (P = 0.14), and the TGFα/TGFß1 ratio decreased by 25% (P = 0.31) and 44% (P = 0.13). Although not statistically significant, these results support further research into (i) whether supplemental vitamin D3 , alone or in combination with calcium, may increase DNA mismatch repair relative to proliferation, increase TGFß1 expression, and decrease autocrine/paracrine growth promotion relative to growth inhibition in the colorectal epithelium, all hypothesized to reduce risk for colorectal carcinogenesis; and (ii) the expression of MSH2 relative to mib-1, TGFß1 alone, and TGFα relative to TGFß1 in the normal-appearing rectal mucosa as potential modifiable, pre-neoplastic markers of risk for colorectal neoplasms.

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.

Characterization of complementary determinant region 3δ in human MutS homologue 2-specific γδ T cells.

γδT cells function as sentinels in early host responses to infections and malignancies. Previously, we found ectopically expressed human MutS homologue 2 (hMSH2), recognized by γδT cells, triggered a γδT cell-mediated cytolysis to tumor cells. However, the characteristics of hMSH2-specific γδ Τ cells are not fully understood. In this study, we investigated the complementary determinant region (CDR) 3δ diversity of hMSH2-specific γδ T cells. We found that the CDR3δ sequences of hMSH2-specific γδ T cells displayed limited diversity, while the length and germline gene usage showed no differences compared with whole CDR3δ immune repertoire. There are more hydrophilic amino acids in P/N insert of hMSH2-specific γδ T cells including the more conserved amino acid at the position 97. Our results offer clues to understanding antigen recognition pattern of γδ T cells to stress-induced hMSH2 of tumor cells and also the mechanism of γδT cell-mediated tumor immune surveillance.

Hereditary cancer-associated mutations in women diagnosed with two primary cancers: an opportunity to identify hereditary cancer syndromes after the first cancer diagnosis.

OBJECTIVES: Patients with hereditary cancer syndromes are at high risk for a second primary cancer. Early identification of these patients after an initial cancer diagnosis is the key to implementing cancer risk-reducing strategies. METHODS: A commercial laboratory database was searched for women with a history of both breast and ovarian or colorectal and endometrial cancer who underwent genetic testing for hereditary breast and ovarian cancer (HBOC) or Lynch syndrome (LS). RESULTS: Among women with both breast and ovarian cancer, 22.4% (2,237/9,982) had a BRCA1 or BRCA2 mutation. Among women with both colorectal and ovarian cancer, 28.1% (264/941) had a mutation associated with LS. In 66.6% of BRCA1 or BRCA2 mutation carriers and in 58.3% of LS mutation carriers, >5 years passed between the cancer diagnoses. Of patients with HBOC and LS, 56 and 65.2%, respectively, met the National Comprehensive Cancer Network guidelines for hereditary cancer testing after their initial diagnosis based on their personal cancer history alone. CONCLUSIONS: A substantial number of women tested for LS or HBOC after being diagnosed with two successive primary cancers were diagnosed with a hereditary cancer syndrome. In many cases, the time interval between the diagnoses was long enough to allow for the implementation of surveillance and/or prophylactic measures.

Methionine concentration in the diet has a tissue-specific effect on chromosomal stability in female mice.

Inadequate nutrient intake can influence the genome. Since methionine is an essential amino acid that may influence DNA integrity due to its role in the one-carbon metabolism pathway, we were interested in whether methionine imbalance can lead to genotoxic events. Adult female Swiss mice were fed a control (0.3% dl-methionine), methionine-supplemented (2.0% DL-methionine) or methionine-deficient (0% DL-methionine) diet over a 10-week period. Chromosomal damage was assessed in peripheral blood using a micronucleus test, and DNA damage was assessed in the liver, heart and peripheral blood tissues using a comet assay. The mRNA expression of the mismatch repair genes Mlh1 and Msh2 was analyzed in the liver. The frequency of micronucleus in peripheral blood was increased by 122% in the methionine-supplemented group (p<0.05). The methionine-supplemented diet did not induce DNA damage in the heart and liver tissues, but it increased DNA damage in the peripheral blood. The methionine-deficient diet reduced basal DNA damage in liver tissue. This reduction was correlated with decreased mRNA expression of Msh2. Our results demonstrate that methionine has a tissue-specific effect because methionine-supplemented diet induced both chromosomal and DNA damage in peripheral blood while the methionine-deficient diet reduced basal DNA damage in the liver.

Frameshift-derived neoantigens constitute immunotherapeutic targets for patients with microsatellite-instable haematological malignancies: frameshift peptides for treating MSI+ blood cancers.

PURPOSE: Microsatellite instability (MSI) resulting from loss of functional DNA mismatch repair was recently found in various haematological disorders. In coding sequences, MSI leads to frameshift mutations (FSMs) and the production of C-terminally altered proteins which are foreign to the immune system. Here, we wondered whether these frame-shifted peptide (FSP) sequences represent tumour-specific antigens also for MSI(+) leukaemia and lymphomas (L/L). MATERIAL AND METHODS: A total of 33 coding region microsatellites were examined in MSI(+) L/L cell lines for the presence of FSMs. Thereafter, recognition of MSI(+) cells by established FSP-specific CD8(+) T cell lines was quantified using interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assays. In each experiment, MSI(+) L/L cell lines and T2 targets exogenously loaded with the cognate peptide (=internal control) were employed. Supplementary, lytic activity towards tumour cells was analysed by standard chromium release assay ((51)Cr). RESULTS: Mutational profiling of 33 coding microsatellite loci in nine MSI(+) L/L cell lines revealed instability in at least nine microsatellites. In each cell line, a distinct mutational profile was observed. Only three of the 33 loci were stable. FSP-specific and human leukocyte antigen-A2 (HLA-A2)-restricted T cells specifically recognised MSI(+) L/L cells endogenously expressing TGFßRII(-1), Caspase 5 (-1) and MSH3 (-1) in ELISpot assays. Moreover, specific killing of Caspase 5 (-1) and MSH3 (-1) expressing L/L cell lines was achieved in functional cytotoxicity assays. CONCLUSION: Data presented here expand the importance of FSPs as shared and general tumour-specific antigens. Consequently, they open new avenues for specific immunotherapies not only for solid but also for MSI(+) haematological malignancies.

Effects of hyperthermia on Hsp27 (HSPB1), Hsp72 (HSPA1A) and DNA repair proteins hMLH1 and hMSH2 in human colorectal cancer hMLH1-deficient and hMLH1-proficient cell lines.

PURPOSE: The objective of the present study was to examine the consequences of a mild hyperthermia in human tumour cell lines deficient and proficient in the DNA mismatch repair system (MMR) to advance our understanding on the relationship between MMR and heat shock proteins (HSPs). MATERIALS AND METHODS: The human colon carcinoma cell lines HCT116 (parent cells), HCT116 + ch2 (MMR-deficient), and HCT116 + ch3 (MMR-proficient) were used. Cells were incubated at 41°C and 42°C for 1 h and then at 37°C for 4 and 24 h. The expression of Hsp27 and Hsp72 was evaluated by immunocytochemistry. Hsp27, Hsp72, hMLH1 and hMSH2 levels were assessed by western blotting in nuclear and cytoplasmic fractions. The alkaline comet assay was used to evaluate the DNA damage. RESULTS: The mild hyperthermia significantly increased the protein expression levels of Hsp27 and Hsp72 in all cell lines, which was higher in the cytoplasm and nucleus of HCT116 + ch3 cells. We also observed that heat induced translocation of hMLH1 and hMSH2 proteins from the nucleus to the cytoplasm in HCT116 + ch3 cells. The comet assay revealed that HCT116 parent cells were more resistant to heat-induced DNA damage. However, the MMR-proficient and deficient cell lines repaired the DNA damage at the same rate. CONCLUSIONS: The present study demonstrates that hyperthermia induced the nuclear accumulation of Hsp27 and Hsp72 and affected the subcellular localisation of hMLH1 and hMSH2 in HCT116 + ch3 cells. Our findings suggest that the MMR system is not a direct determining factor for the different heat shock response in HCT116 cells.

DNA mismatch repair status and colon cancer recurrence and survival in clinical trials of 5-fluorouracil-based adjuvant therapy.

BACKGROUND: Approximately 15% of colorectal cancers develop because of defective function of the DNA mismatch repair (MMR) system. We determined the association of MMR status with colon cancer recurrence and examined the impact of 5-fluorouracil (FU)-based adjuvant therapy on recurrence variables. METHODS: We included stage II and III colon carcinoma patients (n = 2141) who were treated in randomized trials of 5-FU-based adjuvant therapy. Tumors were analyzed for microsatellite instability by polymerase chain reaction and/or for MMR protein expression by immunohistochemistry to determine deficient MMR (dMMR) or proficient MMR (pMMR) status. Associations of MMR status and/or 5-FU-based treatment with clinicopathologic and recurrence covariates were determined using χ(2) or Fisher Exact or Wilcoxon rank-sum tests. Time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS) were analyzed using univariate and multivariable Cox models, with the latter adjusted for covariates. Tumors showing dMMR were categorized by presumed germline vs sporadic origin and were assessed for their prognostic and predictive impact. All statistical tests were two-sided. RESULTS: In this study population, dMMR was detected in 344 of 2141 (16.1%) tumors. Compared with pMMR tumors, dMMR was associated with reduced 5-year recurrence rates (33% vs 22%; P < .001), delayed TTR (P < .001), and fewer distant recurrences (22% vs 12%; P < .001). In multivariable models, dMMR was independently associated with delayed TTR (hazard ratio = 0.72, 95% confidence interval = 0.56 to 0.91, P = .005) and improved DFS (P = .035) and OS (P = .031). In stage III cancers, 5-FU-based treatment vs surgery alone or no 5-FU was associated with reduced distant recurrence for dMMR tumors (11% vs 29%; P = .011) and reduced recurrence to all sites for pMMR tumors (P < .001). The dMMR tumors with suspected germline mutations were associated with improved DFS after 5-FU-based treatment compared with sporadic tumors where no benefit was observed (P = .006). CONCLUSIONS: Patients with dMMR colon cancers have reduced rates of tumor recurrence, delayed TTR, and improved survival rates, compared with pMMR colon cancers. Distant recurrences were reduced by 5-FU-based adjuvant treatment in dMMR stage III tumors, and a subset analysis suggested that any treatment benefit was restricted to suspected germline vs sporadic tumors.

SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK are prognosis-related in colorectal cancer.

AIM: To investigate the expression of markers that are correlated with the prognosis of colorectal cancer (CRC) patients. METHODS: One hundred and fifty-six CRC patients were followed up for more than 3 years after radical surgery. Immunohistochemical (IHC) analysis was performed to detect the expression of 14 pathway-related markers (p53, APC, p21ras, E-cadherin, endothelin-B receptor, Shp2, ADCY-2, SPARCL1, neuroligin1, hsp27, mmp-9, MAPK, MSH2 and rho) in specimens from these patients. Bioinformatics analysis involving a Support Vector Machine (SVM) was used to determine the best prognostic model from combinations of these markers. RESULTS: Seven markers (SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK) were significantly related to the prognosis and clinical pathological features of the CRC patients (P < 0.05). Prognostic models were established through SVM from combinations of these 7 markers and proved able to differentiate patients with dissimilar survival, especially in stage II/III patients. According to the best prognostic model, the p53/SPARCL1 model, patients having high p53 and low SPARCL1 expression had about 50% lower 3-year survival than others (P < 0.001). CONCLUSION: SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK are potential prognostic markers in CRC. A p53/SPARCL1 bioinformatics model may be used as a supplement to tumor-nodes-metastasis staging.

Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer.

PURPOSE: It is uncertain whether modest benefits from adjuvant chemotherapy in stage II colorectal cancer justify the toxicity, cost, and inconvenience. We investigated the usefulness of defective mismatch repair (dMMR), BRAF, and KRAS mutations in predicting tumor recurrence and sensitivity to chemotherapy. PATIENTS AND METHODS: Immunohistochemistry for dMMR and pyrosequencing for KRAS/BRAF were performed for 1,913 patients randomly assigned between fluorouracil and folinic acid chemotherapy and no chemotherapy in the Quick and Simple and Reliable (QUASAR) trial. RESULTS: Twenty-six percent of 695 right-sided colon, 3% of 685 left-sided colon, and 1% of 407 rectal tumors were dMMR. Similarly, 17% of right colon, 2% of left colon, and 2% of rectal tumors were BRAF mutant. KRAS mutant tumors were more evenly distributed: 40% right colon, 28% left colon, and 36% rectal tumors. Recurrence rate for dMMR tumors was half that for MMR-proficient tumors (11% [25 of 218] v 26% [438 of 1,695] recurred; risk ratio [RR], 0.53; 95% CI, 0.40 to 0.70; P < .001). Risk of recurrence was also significantly higher for KRAS mutant than KRAS wild-type tumors (28% [150 of 542] v 21% [219 of 1,041]; RR, 1.40; 95% CI, 1.12 to 1.74; P = .002) but did not differ significantly between BRAF mutant and wild-type tumors (P = .36). No marker predicted benefit from chemotherapy with efficacy not differing significantly by MMR, KRAS, or BRAF status. The prognostic value of MMR and KRAS was similar in the presence and absence of chemotherapy. CONCLUSION: MMR assays identify patients with a low risk of recurrence. KRAS mutational analysis provides useful additional risk stratification to guide use of chemotherapy.

The NSAID sulindac is chemopreventive in the mouse distal colon but carcinogenic in the proximal colon.

BACKGROUND AND AIMS: The non-steroidal anti-inflammatory drug sulindac is an effective chemopreventive agent in sporadic colorectal cancer but its potential benefit in mismatch repair deficient cancers remains to be defined. We wanted to determine whether genetic defects that are relevant for colorectal cancer, such as Msh2 or p53 deficiency, would influence the efficiency of sulindac chemoprevention or increase the side effects. METHODS: Msh2 or p53 deficient and wild-type mice received feed containing 160-320 ppm sulindac for up to 25 weeks with or without a concurrent treatment with the carcinogen azoxymethane. Colon tissue was analysed by histopathology and molecular biology methods. RESULTS: We show that sulindac prevented azoxymethane-induced distal colon tumours in all mice. In the proximal colon, however, sulindac induced new inflammatory lesions on the mucosal folds, which further developed into adenocarcinoma in up to 18-25% of the p53 or Msh2 deficient mice but rarely in wild-type mice. This region in the proximal colon was characterised by a distinct profile of pro- and anti-inflammatory factors, which were modulated by the sulindac diet, including upregulation of hypoxia inducible factor 1α and macrophage inflammatory protein 2. CONCLUSIONS: These data show that the sulindac diet promotes carcinogenesis in the mouse proximal colon possibly through chronic inflammation. Sulindac has both beneficial and harmful effects in vivo, which are associated with different microenvironments within the colon of experimental mice. Deficiency for the Msh2 or p53 tumour suppressor genes increases the harmful side effects of long-term sulindac treatment in the mouse colon.

Mismatch repair protein expression is an independent prognostic factor in sporadic colorectal cancer.

Abstract Background. Mismatch repair (MMR) status has been reported as a prognostic and predictive factor in sporadic colorectal cancer (CRC). The purpose of this study was to determine the prognostic and predictive value of MMR protein expression in the adjuvant setting. Patients and methods. The MMR status in the primary tumor was retrospectively assessed on paraffin-embedded formalin-fixed samples from 1 006 patients with sporadic CRC (488 stage II and 518 stage III) using immunohistochemical analysis (IHC) of MLH1 and MSH2 expression. The patients were included in adjuvant Nordic trials between 1991 and 1996 randomly assigned to surgery alone or surgery plus adjuvant 5-fluorouracil (5-FU)-based chemotherapy. Data was censored at 120 months after surgery. Results. One hundred fifty-seven patients (15.6%) showed a loss of MMR protein expression (139 MLH1 negative, 15 MSH2 negative and 3 MLH1 and MSH2 negative) and were classified as MMR protein negative. A normal MMR protein expression was found in 849 patients who were defined as MMR protein positive. MMR protein expression was a significant prognostic marker in the entire study group with a better overall survival (OS) among patients with MMR protein negative tumors compared to patients with MMR protein positive tumors (p=0.01). In a multivariate analysis the MMR protein expression was significantly associated with OS, (HR 0.70 [95% CI, 0.40 to 0.99]; p=0.01). The MMR status did not predict survival benefit from adjuvant 5-FU-based chemotherapy. Conclusion. This study reveals that IHC of MLH1 and MSH2 expression can yield important prognostic information but is not a predictive factor for adjuvant 5-FU-based chemotherapy in sporadic CRC.

Risk of pancreatic cancer in families with Lynch syndrome.

CONTEXT: Lynch syndrome is an inherited cause of colorectal cancer caused by mutations of DNA mismatch repair (MMR) genes. A number of extracolonic tumors have been associated with the disorder, including pancreatic cancer; however, the risk of pancreatic cancer in Lynch syndrome is uncertain and not quantified. OBJECTIVE: To estimate pancreatic cancer risk in families with germline MMR gene mutations. DESIGN, SETTING, AND PATIENTS: Cancer histories of probands and their relatives were evaluated in MMR gene mutation carriers in the familial cancer registries of the Dana-Farber Cancer Institute (n = 80), Boston, Massachusetts, and University of Michigan Comprehensive Cancer Center (n = 67), Ann Arbor, Michigan. Families enrolled before the study start date (June 2008) were eligible. Age-specific cumulative risks and hazard ratio estimates of pancreatic cancer risk were calculated and compared with the general population using modified segregation analysis, with correction for ascertainment. MAIN OUTCOME MEASURES: Age-specific cumulative risks and hazard ratio estimates of pancreatic cancer risk. RESULTS: Data on 6342 individuals from 147 families with MMR gene mutations were analyzed. Thirty-one families (21.1%) reported at least 1 case of pancreatic cancer. Forty-seven pancreatic cancers were reported (21 men and 26 women), with no sex-related difference in age of diagnosis (51.5 vs 56.5 years for men and women, respectively). The cumulative risk of pancreatic cancer in these families with gene mutations was 1.31% (95% confidence interval [CI], 0.31%-2.32%) up to age 50 years and 3.68% (95% CI, 1.45%-5.88%) up to age 70 years, which represents an 8.6-fold increase (95% CI, 4.7-15.7) compared with the general population. CONCLUSIONS: Among 147 families with germline MMR gene mutations, the risk of pancreatic cancer was increased compared with the US population. Individuals with MMR gene mutations and a family history of pancreatic cancer are appropriate to include in studies to further define the risk of premalignant and malignant pancreatic neoplasms and potential benefits and limitations of surveillance.