Integrated molecular profiling of RAS, BRAF mutations, and mismatch repair status in advanced colorectal carcinoma: insights from gender and tumor laterality.

Background: Colorectal carcinoma (CRC) is one of the most frequently diagnosed forms of cancer worldwide. The RAS (KRAS, NRAS) and BRAF genes encode proteins that are important therapeutic targets for the treatment of CRC and, together with the mismatch repair (MMR) system, are closely related to patient prognosis and survival in advanced CRC. Here we evaluate the mutational profile and the frequency of mutations in the KRAS, NRAS and BRAF genes, along with the expression of MMR in advanced CRC, at a tertiary hospital in southern Brazil. Methods: A cross-sectional retrospective study was carried out, where molecular analysis of mutations in the KRAS, NRAS and BRAF genes was carried out, as well as immunohistochemistry for MMR proteins. Results: Next-generation sequencing (NGS) analysis of 310 tumors revealed that 202 patients (65.2%) had mutations. The KRAS gene (53.2%) was the most frequently mutated in our sample, with G12D being the most frequent, representing 30.5% of the mutations in this gene. The most frequent mutation found in BRAF was V600E (n=25; 89.3%) and differed significantly in women and in the right colon in patients with MMR deficiency. Among the 283 patients tested for MMR, the rate of loss of expression was 8.8% (25/283). Conclusions: Deficiency in the MMR system is associated with the presence of the BRAF V600E mutation, tumors located in the right colon, and the female sex. In our case series, more than 60% of patients had at least one mutation in KRAS, NRAS, or BRAF. The presence of mutations in these genes is closely related to CRC prognosis and helps define the best therapeutic approach in patients with metastatic CRC.

Association between Helicobacter pylori infection, mismatch repair, HER2 and tumor-infiltrating lymphocytes in gastric cancer.

BACKGROUND: The influence of Helicobacter-pylori (H. pylori) infection and the characteristics of gastric cancer (GC) on tumor-infiltrating lymphocyte (TIL) levels has not been extensively studied. Analysis of infiltrating-immune-cell subtypes as well as survival is necessary to obtain comprehensive information. AIM: To determine the rates of deficient mismatch-repair (dMMR), HER2-status and H. pylori infection and their association with TIL levels in GC. METHODS: Samples from 503 resected GC tumors were included and TIL levels were evaluated following the international-TILs-working-group recommendations with assessment of the intratumoral (IT), stromal (ST) and invasive-border (IB) compartments. The density of CD3, CD8 and CD163 immune cells, and dMMR and HER2-status were determined by immunohistochemistry (IHC). H. pylori infection was evaluated by routine histology and quantitative PCR (qPCR) in a subset of samples. RESULTS: dMMR was found in 34.4%, HER2+ in 5% and H. pylori-positive in 55.7% of samples. High IT-TIL was associated with grade-3 (P = 0.038), while ST-TIL with grade-1 (P < 0.001), intestinal-histology (P < 0.001) and no-recurrence (P = 0.003). dMMR was associated with high TIL levels in the ST (P = 0.019) and IB (P = 0.01) compartments, and ST-CD3 (P = 0.049) and ST-CD8 (P = 0.05) densities. HER2- was associated with high IT-CD8 (P = 0.009). H. pylori-negative was associated with high IT-TIL levels (P = 0.009) when assessed by routine-histology, and with high TIL levels in the 3 compartments (P = 0.002-0.047) and CD8 density in the IT and ST compartments (P = 0.001) when assessed by qPCR. A longer overall survival was associated with low IT-CD163 (P = 0.003) and CD8/CD3 (P = 0.001 in IT and P = 0.002 in ST) and high IT-CD3 (P = 0.021), ST-CD3 (P = 0.003) and CD3/CD163 (P = 0.002). CONCLUSION: TIL levels were related to dMMR and H. pylori-negativity. Low CD8/CD3 and high CD163/CD3 were associated with lower recurrence and longer survival.

Perfil molecular de cáncer de endometrio en mujeres de Bogotá DC, Colombia / Molecular profile of endometrial cancer in women from Bogota DC, Colombia

Introducción: El cáncer de endometrio ocupa el sexto lugar en incidencia del cáncer en mujeres. La caracterización molecular de este cáncer permite optimizar la estratificación de riesgo para mejorar el tratamiento de las pacientes. Objetivo: Determinar el perfil molecular TCGA de pacientes con cáncer de endometrio en Bogotá, D.C., Colombia. Método: Estudio descriptivo en una cohorte de pacientes con cáncer de endometrio. Las mutaciones en los exones 9 a 14 del gen POLE fueron identificadas mediante amplificación por reacción en cadena de la polimerasa, seguida de secuenciación Sanger y análisis bioinformático. La expresión de las proteínas MMR y p53 se identificó mediante inmunohistoquímica. Resultados: Se incluyeron 40 pacientes con una mediana de edad de 66 años. El 15% presentaron mutaciones en el dominio exonucleasa de POLE. El 32% de las pacientes que no presentaron mutaciones manifestaron deficiencia en el sistema MMR. El 43,47% de las pacientes sin mutaciones en POLE ni alteración del sistema MMR presentaron alteración de la proteína p53. Conclusiones: La población de cáncer de endometrio analizada presenta un perfil molecular TCGA similar a lo reportado para otras poblaciones.

Introduction: Endometrial cancer ranks sixth in cancer incidence among women. Its molecular characterization allows for a more precise risk stratification with the aim of improving patient treatment. Objective: To determine the TCGA molecular profile of patients with endometrial cancer in Bogota, Colombia. Method: A descriptive study of a cohort of patients with endometrial cancer. The expression of MMR proteins and p53 was identified through immunohistochemistry. Mutations in exons 9 to 14 of the POLE gene were identified through polymerase chain reaction amplification, followed by Sanger sequencing and bioinformatic analysis. Results: Forty patients were included in the study, with a median age of 66 years, 15% of them exhibited mutations in the exonuclease domain of POLE, while 32% of patients without mutations showed deficiency in the MMR system. Forty three percent of patients without mutations in POLE or MMR alterations showed aberrant p53 protein expression. Conclusions: The analyzed population of endometrial cancer presents a TCGA molecular profile similar to that reported for other populations.

Real-world study on microsatellite instability and mismatch repair deficiency testing patterns among patients with metastatic colorectal cancer in Spain.

PURPOSE: Clinical practice guidelines recommend that all patients with metastatic colorectal cancer (mCRC) should be tested for mismatch repair deficiency (dMMR) or microsatellite instability-high (MSI-H). We aimed to describe the dMMR/MSI-H testing practice in patients with mCRC in Spanish centers. METHODS: Multicenter, observational retrospective study that included patients newly diagnosed with mCRC or who progressed to a metastatic stage from early/localized stages. RESULTS: Three hundred patients were included in the study from May 2020 through May 2021, with a median age of 68 years, and two hundred twenty-five (75%) had stage IV disease at initial diagnosis; two hundred eighty-four patients received first-line treatment, and dMMR/MSI-H testing was performed in two hundred fifty-one (84%) patients. The results of the dMMR/MSI-H tests were available in 61 (24%) of 251 patients before the diagnosis of metastatic disease and in 191 (81%) of 236 evaluable patients for this outcome before the initiation of first-line treatment. Among the 244 patients who were tested for dMMR/MSI-H with IHC or PCR, 14 (6%) were MMR deficient. The most frequent type of first-line treatment was the combination of chemotherapy and biological agent, that was received by 71% and 50% of patients with MMR proficient and deficient tumors, respectively, followed by chemotherapy alone, received in over 20% of patients in each subgroup. Only 29% of dMMR/MSI-H tumors received first-line immunotherapy. CONCLUSION: Our study suggests that a high proportion of patients with mCRC are currently tested for dMMR/MSI-H in tertiary hospitals across Spain. However, there is still room for improvement until universal testing is achieved. TRIAL REGISTRATION: Not applicable.

PD-1/PD-L1 inhibitors plus carboplatin and paclitaxel compared with carboplatin and paclitaxel in primary advanced or recurrent endometrial cancer: a systematic review and meta-analysis of randomized clinical trials.

BACKGROUND: Paclitaxel and carboplatin is the standard chemotherapy for the treatment of advanced or recurrent endometrial cancer. However, the benefit of adding programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors to chemotherapy is still unclear. METHOD: We searched PubMed, Scopus, Cochrane, and Web of Science databases for randomized controlled trials that investigated PD-1/PD-L1 inhibitors plus carboplatin and paclitaxel compared with carboplatin and paclitaxel in primary advanced or recurrent endometrial cancer. We computed hazard ratios (HRs) or risk ratios (RRs) for binary endpoints, with 95% confidence intervals (CIs). We used DerSimonian and Laird random-effect models for all endpoints. Heterogeneity was assessed using I2 statistics. R, version 4.2.3, was used for statistical analyses. RESULTS: A total of three studies and 1,431 patients were included. Compared with carboplatin plus paclitaxel-based chemotherapy, progression-free survival (PFS) rate (HR 0.32; 95% CI 0.23-0.44; p < 0.001) and overall survival (OS) at 30 months (RR 3.13; 95% CI 1.26-7.78; p = 0.01) were significant in favor of the PD-1/PD-L1 inhibitors plus carboplatin and paclitaxel group in the mismatch repair-deficient subgroup. However, there were no significant differences in the mismatch repair-proficient subgroup for PFS (HR 0.74; 95% CI 0.50-1.08; p = 0.117) or OS at 30 months (RR 2.24; 95% CI 0.79-6.39; p = 0.13). CONCLUSION: Immunotherapy plus carboplatin-paclitaxel increased significantly PFS and OS among patients with advanced or recurrent endometrial cancer, with a significant benefit in the mismatch repair-deficient and high microsatellite instability population.

PREMM5 distinguishes sporadic from Lynch syndrome-associated MMR-deficient/MSI-high colorectal cancer.

Current algorithms for diagnosing Lynch syndrome (LS) include multistep molecular tumor tests to distinguish LS-associated from sporadic colorectal cancer (CRC), which add cost and complexity to the evaluation. We hypothesized that PREMM5, a clinical LS prediction tool, could be an alternative approach to screen for LS, thereby lessening the need for specialized molecular diagnostics. We reviewed a consecutively ascertained institutional cohort of 1058 CRC patients on whom pathologic and clinical data were available, including prior LS germline testing. Data from MMR-D/MSI-H CRC patients were reviewed and PREMM5 scores were calculated for each individual. Using a PREMM5 score cutoff ≥ 2.5% to characterize the need for germline testing, we determined the rate of pathogenic/likely pathogenic germline variants (PGVs) in LS genes in patients with PREMM5 scores ≥ 2.5% versus < 2.5%. Sensitivity and negative predictive values (NPV) of PREMM5 were calculated for all MMR-D/MSI-H CRC patients, and those with MLH1-deficient CRC. MMR IHC and/or MSI results were available on 572/1058 cases. We identified 74/572 (12.9%) cases as MMR-D/MSI-H, of which 28/74 (37.8%) harbored a LS PGV. 11/49 (22.4%) patients with MLH1-deficient CRC harbored a LS PGV. PREMM5 had 100% sensitivity (95% CI: 87.7-100 for any MMR-D/MSI-H; 95% CI: 71.5-100 for MLH1-deficient CRC) and 100% NPV (95% CI: 83.2-100 for any MMR-D/MSI-H; 95% CI: 82.4-100 for MLH1-deficient CRC) for identifying LS PGVs in these cohorts. PREMM5 accurately distinguishes LS- from non-LS-associated MMR-D/MSI-H CRC without additional somatic molecular testing. These findings are particularly relevant for limited-resource settings where advanced molecular diagnostics may be unavailable.

Clinicopathological characteristics of endometrial carcinomas according to DNA mismatch repair protein status.

DNA mismatch repair protein deficiency (MMRd) in endometrial carcinoma is associated with the risk of Lynch syndrome and response to immune checkpoint inhibitors. It is also related to microsatellite instability and corresponds to a molecular subtype of endometrial tumor with an unclear prognosis. Here, we evaluated the clinicopathological characteristics and prognosis of 312 consecutive endometrial carcinoma cases submitted to complete surgical staging at a single institution. We compared MMRd and mismatch repair protein-proficient (MMRp) tumors and examined the effects of the MMR protein loss type (MLH1/PMS2 vs. MSH2/MSH6) and influence of L1CAM and p53 expression. The median follow-up period was 54.5 (range, 0-120.5) months. No difference was observed between MMRd [n = 166 (37.2%)] and MMRp [n = 196 (62.8%)] cases in terms of age, body mass index, FIGO stage, tumor grade, tumor size, depth of myometrial infiltration, or lymph node metastasis. More MMRd than MMRp tumors had endometrioid histology (87.9% vs. 75.5%) and despite MMRd had more lymphovascular space invasion (LVSI; 27.2% vs. 16.9%), they presented fewer recurrences and no difference in lymph node metastasis and disease-related death. Relative to those with MLH1/MSH6 loss, tumors with MSH2/MSH6 loss were diagnosed at earlier FIGO stages, were smaller, and had less ≥50% myometrial invasion, LVSI and lymph node metastasis. Outcomes, however, did not differ between these groups. L1CAM positivity and mutation-type p53 expression were more common in MMRp than in MMRd tumors and did not differ between the MLH1/PMS2 and MSH2/MSH6 loss groups. In the entire cohort, L1CAM and mutation p53 expression were associated with worse prognosis, but only non-endometrioid histology, FIGO stage III/IV, and deep myometrial infiltration were significant predictors. In the subgroup of endometrioid carcinomas, only FIGO stage III/IV was associated with poor outcomes. The risk of lymph node metastasis was associated with tumor size, non-endometrioid histology, and multifocal LVSI. For MMRd tumors, only tumor size and myometrial invasion depth were predictive of lymph node involvement. In our cohort, MMRd tumors were associated with greater recurrence-free, but not overall, survival. The precise identification of MMRd status, present in a substantial proportion of endometrial cancer cases, is a challenge to be overcome for proper patient management. MMRd status serves as a marker for Lynch syndrome, and a significant number of these tumors are high risk and candidate to immunotherapy.

Amplicon-based NGS test for assessing MLH1 promoter methylation and its correlation with BRAF mutation in colorectal cancer patients.

Detecting MLH1 promoter methylation is highly relevant to differentiate between possible Lynch syndrome patients or patients with sporadic causes of MLH1/PMS2 deficiency in colorectal (CRC) and endometrial cancers. Here, we aimed to develop a test for assessing MLH1 promoter methylation based in next generation sequencing (NGS), and to evaluate the concordance of MLH1 methylation and BRAF-V600 mutation status in CRC. For that, we performed a series of experiments with DNA from tumor, saliva and commercial control samples and our in house developed amplicon-based NGS test. In patients' samples, MLH1 methylation above 10% was only observed in tumors with MLH1/PMS2 loss. We confirmed the reproducibility and accuracy of MLH1 promoter analysis performing a serial dilution experiment with completely methylated and unmethylated control DNAs and a comparison between two NGS platforms (Ion Proton and Illumina). In MLH1/PMS2 deficient tumors, the MLH1 methylation status was concordant with the BRAF mutation status in 90% (18/20) of the cases. Our amplicon-based NGS test showed a great sensitivity and specificity for detecting MLH1 methylation in CRC samples, with a high agreement with the evaluation of BRAF mutation. This simple and affordable test could be used as a reflex test to identify patients with sporadic causes of MLH1/PMS2 deficiency in CRC, aiding to genetic test referral and identification of Lynch syndrome patients.

Can mismatch repair status be added to sentinel lymph node mapping algorithm in endometrioid endometrial cancer?

OBJECTIVE: To evaluate the relation between mismatch repair (MMR) status and the risk of lymph node metastasis in endometrial cancer, and whether this additional data can be incorporated to current SLN (sentinel lymph node) algorithm. METHODS: We included a series of 332 women that underwent SLN mapping ± systematic lymphadenectomy from January 2013 to December 2021. Protein expressions of MLH1, MSH2, MSH6, PMS2 were examined by immuno-histochemistry and considered MMRd (deficient) when at least one protein was not expressed. RESULTS: MMRd was noted in 20.8% of cases and correlated to grade 3 (p = 0.018) and presence of lymphovascular space invasion (p = 0.032). Moreover, MMRd was an independent risk factor for lymph node metastasis (OR 2.76, 95% CI 1.36-5.62). Notably, 21.7% (15/69) cases with MMRd had lymph node metastasis compared to 9.5% (25/263) of cases with MMRp (proficient) (p = 0.005). The overall and bilateral SLN detection rates were 91.9% and 75.9%, respectively. Of the 80 (24%) cases of non-bilateral SLN detection, 66.2% had low-grade tumors (G1/G2) and myometrial invasion <50%. Considering MMR status an independent prognostic factor for lymph node metastasis, a systematic lymphadenectomy (side specific or bilateral) would forgo in 53.7% (43/80) of cases with non-bilateral detection, representing 13% (43/332) of all endometroid tumors. CONCLUSION: MMR status was independently related to lymph node metastasis in endometrioid EC. Moreover, MMR status may help to select patients that can forgo systematic lymphadenectomy in case of undetected SLN.

DNA ploidy and stroma predicted the risk of recurrence in low-risk stage III colorectal cancer.

BACKGROUND: For clinically low-risk stage III colorectal cancer, the decision on cycles of adjuvant chemotherapy after surgery is disputed. The present study investigates the use of additional biomarkers of ploidy and stroma-ratio(PS) to stratify patients with low-risk stage III colorectal cancer, providing a basis for individualized treatment in the future. METHODS: This study retrospectively enrolled 198 patients with clinical-low-risk stage III colorectal cancer (T1-3N1M0) and analyzed the DNA ploidy and stroma ratio of FFPE tumor tissues. The patients were divided into PS-low-risk group (Diploidy or Low-stroma) and PS-high-risk group (Non-diploid and High-stroma). For survival analyses, Kaplan-Meier and Cox regression models were used. RESULTS: The results showed that the 5-year DFS of the PS-high-risk group was significantly lower than that in the PS-low-risk group (78.6 vs. 91.2%, HR = 2.606 [95% CI: 1.011-6.717], P = 0.039). Besides, in the PS-low-risk group, the 5 year OS (98.2 vs. 86.7%, P = 0.022; HR = 5.762 [95% CI: 1.281-25.920]) and DFS (95.6, vs 79.9%, P = 0.019; HR = 3.7 [95% CI: 1.24-11.04]) of patients received adjuvant chemotherapy for > 3 months were significantly higher than those received adjuvant chemotherapy for < 3 months. We also found that the PS could stratify the prognosis of patients with dMMR tumors. The 5-year OS (96.3 vs 71.4%, P = 0.037) and DFS (92.6 vs 57.1%, P = 0.015) were higher in the PS-low-risk dMMR patients than those in the PS-high-risk dMMR patients. CONCLUSION: In this study, we found that PS can predict the prognosis of patients with stage III low-risk CRC. Besides, it may guide the decision on postoperative adjuvant chemotherapy.

SOMATIC DEFICIENT MISMATCH REPAIR ASSESSED BY IMMUNOHISTOCHEMISTRY AND CLINICAL FEATURES IN BRAZILIAN GLIOBLASTOMA PATIENTS.

BACKGROUND: Glioblastoma (GBM) is the most frequent primary malignant CNS tumor. Deficient mismatch repair (dMMR) is associated with better prognosis and is a biomarker for immunotherapy. Evaluation of MMR by immunohistochemistry (IHC) is accessible, cost effective, sensitive, and specific. AIM: Our objective was to investigate MMR proteins in adult GBM patients. MATERIALS AND METHODS: We retrospectively analyzed 68 GBM samples to evaluate the proficiency of MMR genes expression assessed by IHC. Clinicopathologic and molecular features were compared in proficient (pMMR) or dMMR. RESULTS: 10 (14.7%) samples showed dMMR, and the most frequent was MSH6 (100%) followed by MSH2, PMS2, and MLH1. We observed heterogeneous expression of dMMR in 5 GBMs. The median overall survival did not differ between pMMR (19.8 months; 0.2-30) and dMMR (16.9 months; 6.4-27.5) (p = 0.31). We observed a significantly higher overall survival associated with gross total resection compared to subtotal resection or biopsy (30.7 vs. 13.6 months, p = 0.02) and MGMT methylated status (29.6 vs. 19.8 months, p = 0.049). At the analysis time, 10 patients were still alive, all in the pMMR group. CONCLUSIONS: Our data demonstrated dMMR phenotype assessed by IHC in an expressive portion of GBM patients, however without significant impact on overall survival.

Noncanonical Mismatch Repair Protein NucS Modulates the Emergence of Antibiotic Resistance in Mycobacterium abscessus.

NucS/EndoMS-dependent noncanonical mismatch repair (MMR) ensures the stability of genomic DNA in mycobacteria and acts as a guardian of the genome by preventing the accumulation of point mutations. In order to address whether the inactivation of noncanonical MMR could increase the acquisition of drug resistance by mutation, a ΔnucS strain was constructed and explored in the emerging pathogen Mycobacterium abscessus. Deletion of nucS resulted in a mutator phenotype with increased acquisition of resistance to macrolides and aminoglycosides, the two main groups of antimycobacterial agents for M. abscessus treatment, and also to second-line drugs such as fluoroquinolones. Inactivation of the noncanonical MMR in M. abscessus led to increases of 10- to 22-fold in the appearance of spontaneous mutants resistant to the macrolide clarithromycin and the aminoglycosides amikacin, gentamicin, and apramycin, compared with the wild-type strain. Furthermore, emergence of fluoroquinolone (ciprofloxacin) resistance was detected in a nucS-deficient strain but not in a wild-type M. abscessus strain. Acquired drug resistance to macrolides and aminoglycosides was analyzed through sequencing of the 23S rRNA gene rrl and the 16S rRNA gene rrs from independent drug-resistant colonies of both strains. When the acquisition of clarithromycin resistance was examined, a different mutational profile was detected in the M. abscessus ΔnucS strain compared with the wild-type one. To summarize, M. abscessus requires the NucS-dependent noncanonical MMR pathway to prevent the emergence of drug-resistant isolates by mutation. To our knowledge, this is the first report that reveals the role of NucS in a human pathogen, and these findings have potential implications for the treatment of M. abscessus infections. IMPORTANCE Chronic infections caused by M. abscessus are an emerging challenge in public health, posing a substantial health and economic burden, especially in patients with cystic fibrosis. Treatment of M. abscessus infections with antibiotics is particularly challenging, as its complex drug resistance mechanisms, including constitutive resistance through DNA mutation, lead to high rates of treatment failure. To decipher the evolution of antibiotic resistance in M. abscessus, we studied NucS-dependent noncanonical MMR, a unique DNA repair pathway involved in genomic maintenance. Inactivation of NucS is linked to the increase of DNA mutations (hypermutation), which can confer drug resistance. Our analysis detected increased acquisition of mutations conferring resistance to first-line and second-line antibiotics. We believe that this study will improve the knowledge of how this pathogen could evolve into an untreatable infectious agent, and it uncovers a role for hypermutators in chronic infectious diseases under antibiotic pressure.

MutS recognition of mismatches within primed DNA replication intermediates.

MutS initiates mismatch repair by recognizing mismatches in newly replicated DNA. Specific interactions between MutS and mismatches within double-stranded DNA promote ADP-ATP exchange and a conformational change into a sliding clamp. Here, we demonstrated that MutS from Pseudomonas aeruginosa associates with primed DNA replication intermediates. The predicted structure of this MutS-DNA complex revealed a new DNA binding site, in which Asn 279 and Arg 272 appeared to directly interact with the 3'-OH terminus of primed DNA. Mutation of these residues resulted in a noticeable defect in the interaction of MutS with primed DNA substrates. Remarkably, MutS interaction with a mismatch within primed DNA induced a compaction of the protein structure and impaired the formation of an ATP-bound sliding clamp. Our findings reveal a novel DNA binding mode, conformational change and intramolecular signaling for MutS recognition of mismatches within primed DNA structures.

Recorrência do ameloblastoma: análise de fatores clínicoradiográficos e imunoexpressão de hMSH2, hMLH1 e KI-67 / Ameloblastoma recurrence: analysis of clinical-radiographic factors and immunoexpression of hMSH2, hMLH1 and KI-67

Introdução: O ameloblastoma é uma neoplasia odontogênica benigna, que apresenta altas taxas de recorrência pós-operatória. Diversos estudos mostram a relação entre as características clínico-patológicas e as modalidades de tratamento na recorrência do ameloblastoma. Os mecanismos moleculares envolvidos com a etiopatogenia deste tumor são pouco conhecidos, e apesar de alterações no Sistema Mismatch favorecerem o desenvolvimento de diferentes neoplasias humanas, a importância destes no desenvolvimento do ameloblastoma ainda permanece pouco compreendido. Objetivo: Identificar os fatores clínico-patológicos associados à recorrência do ameloblastoma, bem como investigar o papel da imunoexpressão das proteínas hMLH1, hMSH2 e Ki-67 na recidiva desses tumores odontogênicos. Metodologia: Tratou-se de um estudo descritivo, transversal e restrospectivo, com uma amostra constituída por 22 casos de ameloblastomas recidivantes e 22 casos não-recidivantes. A análise imunoistoquímica foi realizada de forma quantitativa, considerando a localização celular (nuclear) das proteínas estudadas. O teste de McNemar foi utilizado para comparar as variáveis entre lesões da 1ª biópsia e recorrentes de AMB. A sobrevida livre de recorrência foi analisada pelo método de Kaplan-Meier e as funções de sobrevida foram comparadas de acordo com as variáveis pelo teste log-rank. Resultados: O gênero mais acometido foi o feminino (n=24; 54,5%), com média de idade de acometimento de 39,1 ± 19,8 anos, sendo 45,5% (n=20) leucodermas. A região posterior de mandíbula foi a mais frequente no grupo recidivante (n=18, 81,8%) e também para os casos que não apresentaram recidivas (n=16, 72,8%). O tempo livre de recorrência foi de 50,0 (34,5 ­ 63,6) meses. Foram fatores significativamente associadas à recorrência dos ameloblastomas: presença de expansão da cortical (p=0,0089), ausência de reconstrução óssea (p=0,018), tratamento conservador (p=0,021), perda de imunoexpressão de hMSH2 (p=0,006) e hMLH1 (p=0,038) e forte imunoexpressão de Ki-67 (p=0,029). Conclusão: Baseado nos achados desta pesquisa, aspecto radiográfico, modalidade do tratamento e imunoexpressão de proteínas do Sistema Mismatch e Ki-67 podem ser utilizados como indicadores para a recorrência em ameloblastomas (AU).

Introduction: Ameloblastoma is a benign odontogenic neoplasm, which has high rates of postoperative recurrence. Several studies show the relationship between clinicopathological characteristics and treatment modalities in ameloblastoma recurrence. The molecular mechanisms involved in the etiopathogenesis of this tumor are little known, and although changes in the Mismatch System favor the development of different human neoplasms, their importance in the development of ameloblastoma still remains poorly understood. Objective: To identify clinical and pathological factors associated with ameloblastoma recurrence, as well as to investigate the role of immunoexpression of hMLH1, hMSH2 and Ki-67 proteins in the recurrence of these odontogenic tumors. Methodology: This was a descriptive, cross-sectional and retrospective study, with a sample consisting of 22 cases of recurrent ameloblastoma and 22 non-recurrent cases. Immunohistochemical analysis was performed quantitatively, considering the cellular (nuclear) location of the studied proteins. McNemar's test was used to compare variables between 1st biopsy and recurrent AMB lesions. Recurrence-free survival was analyzed using the Kaplan-Meier method and survival functions were compared according to variables using the log-rank test. Results: The most affected gender was female (n=24; 54.5%), with a mean age of involvement of 39.1 ± 19.8 years, 45.5% (n=20) being white. The posterior region of the mandible was the most frequent in the relapsed group (n=18, 81.8%) and also for the cases that did not present recurrences (n=16, 72.8%). Recurrence-free time was 50.0 (34.5 ­ 63.6) months. Factors significantly associated with recurrence of AMBs were: presence of cortical expansion (p=0.0089), absence of bone reconstruction (p=0.018), conservative treatment (p=0.021), loss of hMSH2 immunoexpression (p=0.006) and hMLH1 (p=0.038) and strong Ki-67 immunoexpression (p=0.029). Conclusion: Based on the findings of this research, radiographic appearance, treatment modality and immunoexpression of proteins from the Mismatch System and Ki-67 can be used as indicators for recurrence in ameloblastomas (AU).

Dynamics of Mismatch and Alternative Excision-Dependent Repair in Replicating Bacillus subtilis DNA Examined Under Conditions of Neutral Selection.

Spontaneous DNA deamination is a potential source of transition mutations. In Bacillus subtilis, EndoV, a component of the alternative excision repair pathway (AER), counteracts the mutagenicity of base deamination-induced mispairs. Here, we report that the mismatch repair (MMR) system, MutSL, prevents the harmful effects of HNO2, a deaminating agent of Cytosine (C), Adenine (A), and Guanine (G). Using Maximum Depth Sequencing (MDS), which measures mutagenesis under conditions of neutral selection, in B. subtilis strains proficient or deficient in MutSL and/or EndoV, revealed asymmetric and heterogeneous patterns of mutations in both DNA template strands. While the lagging template strand showed a higher frequency of C → T substitutions; G → A mutations, occurred more frequently in the leading template strand in different genetic backgrounds. In summary, our results unveiled a role for MutSL in preventing the deleterious effects of base deamination and uncovered differential patterns of base deamination processing by the AER and MMR systems that are influenced by the sequence context and the replicating DNA strand.

Mismatch repair system in colorectal cancer. Frequency, cancer phenotype, and follow-up.

INTRODUCTION AND AIMS: A frequent task in the study of colorectal carcinomas (CRC) is to identify tumors harboring deficient DNA mismatch repair systems (dMMR), which are associated with microsatellite instability. Given that there is scant information on those tumors in Mexican patients, our aim was to describe their frequency, clinical and pathologic characteristics, and results, which are necessary for future trials. MATERIALS AND METHODS: A consecutive series of CRC patients, treated and followed at a tertiary care center was performed. The clinical and pathologic variables and the risk of hereditary or familial cancer syndrome were retrieved. The original slides and hMLH1, hPMS2, hMSH2, hMSH6 immunohistochemistry were evaluated. Tumors with an absence of at least one protein were considered dMMR. Differences were contrasted, utilizing non-parametric tests. RESULTS: One hundred and forty-four patients were included, with a median age of 65 years. A total of 134/93% patients presented with sporadic CRC, 8/5.6% had a family history of CRC, and 2/1.4% met the diagnostic criteria for hereditary non-polyposis colon cancer, according to the Amsterdam and Bethesda criteria. dMMR tumors were found in 39 patients, distributed among the three groups. They were locally advanced (p<0.001), right-sided, had the mucinous phenotype, and harbored a Crohn's-like lymphoid reaction (all three features, p<0.04). Adjuvant or palliative chemotherapy was administered to 57 (39.6%), concomitant chemoradiotherapy to 24 (16.7%), but 63 (43.8%) patients received no additional treatment to surgery. Five-year follow-up was completed in 131 of the patients and the outcomes alive-with-disease or died-of-disease were more frequently observed in the proficient (pMMR) lesions. CONCLUSIONS: In the present pre-FOLFOX case series, outcomes were better in dMMR CRC than in proficient lesions.

MiR-6511b-5p suppresses metastasis of pMMR colorectal cancer through methylation of CD44 by directly targeting BRG1.

PURPOSE: Distal metastases are a major cause of poor prognosis in colorectal cancer patients. Approximately 95% of metastatic colorectal cancers are defined as DNA mismatch repair proficient (pMMR). Our previous study found that miR-6511b-5p was downregulated in pMMR colorectal cancer. However, the mechanism of miR-6511b-5p in pMMR colorectal cancer metastases remain unclear. METHODS: We first used quantitative real-time PCR to evaluate the role of miR-6511b-5p in colorectal cancer. Second, we conducted invasion assays and wound healing assays to investigate the role of miR-6511b-5p and CD44 in colorectal cancer cells metastases. Third, luciferase reporter assay, in situ hybridization (ISH), and immunohistochemistry assays were performed to study the relationship between miR-6511b-5p and BRG1. Finally, real-time quantitative PCR, immunohistochemistry, and chromatin immunoprecipitation (ChIP) assays were performed to analyze the relationship between BRG1 and CD44 in colorectal cancer. RESULTS: We found that lower expression of miR-6511b-5p appeared more often in pMMR colorectal cancer patients compared with dMMR (mismatch repair deficient) cases, and was positively correlated with metastases. In vitro, overexpression of miR-6511b-5p inhibited metastasis by decreasing CD44 expression via directly targeting BRG1 in colorectal cancer. Furthermore, BRG1 knockdown decreased the expression of CD44 by promoting CD44 methylation in colorectal cancer cells. CONCLUSION: Our data suggest that miR-6511b-5p may act as a promising biomarker and treatment target for pMMR colorectal cancer, particularly in metastatic patients. Mechanistically, miR-6511b-5p suppresses invasion and migration of colorectal cancer cells through methylation of CD44 via directly targeting BRG1.

An effective algorithm to detect the possibility of being MSI phenotype in endometrial cancer given the BMI status and histological subtype: a statistical study.

PURPOSE: In endometrial cancer, the incidence of mutations in mismatch repair genes (MMR) is estimated at 17-30%. Patients with alterations at this level (MSI) are known to have different clinical and anatomopathological characteristics than those without this genetic alteration (MSS). In this study, we aim to identify the MSI phenotype in patients who underwent hysterectomy for endometrial cancer. We assessed the correlation of this phenotype with anatomoclinical parameters such as obesity and histological subtype. METHODS/PATIENTS: Clinical and anatomopathological data were collected from 147 patients diagnosed with endometrial cancer and an immunohistochemical study of MMR system proteins was performed. PMS2 and MSH6 proteins were evaluated as primary screening and subsequent evaluation of MLH1 and MSH6, respectively, if the former were negative. Statistical association between the anatomopathological data and the immunohistochemical result was analyzed. RESULTS AND CONCLUSIONS: 22.4% of our patients were MSI phenotype. We obtained statistically significant differences by multivariate analysis between endometrioid subtype and higher FIGO classification grade with MSI phenotype and obesity with MSS phenotype. Given these statistical results, we propose a function for predicting the probability of being MSI phenotype taking into account the histological subtype (endometrioid/non-endometrioid carcinoma) and FIGO grade as well as obesity. This prediction may be useful prior to hysterectomy, for genetic study of the MLH1 promoter and subsequent genetic counseling.

The Barley Chloroplast Mutator (cpm) Mutant: All Roads Lead to the Msh1 Gene.

The barley chloroplast mutator (cpm) is a nuclear gene mutant that induces a wide spectrum of cytoplasmically inherited chlorophyll deficiencies. Plastome instability of cpm seedlings was determined by identification of a particular landscape of polymorphisms that suggests failures in a plastome mismatch repair (MMR) protein. In Arabidopsis, MSH genes encode proteins that are in charge of mismatch repair and have anti-recombination activity. In this work, barley homologs of these genes were identified, and their sequences were analyzed in control and cpm mutant seedlings. A substitution, leading to a premature stop codon and a truncated MSH1 protein, was identified in the Msh1 gene of cpm plants. The relationship between this mutation and the presence of chlorophyll deficiencies was established in progenies from crosses and backcrosses. These results strongly suggest that the mutation identified in the Msh1 gene of the cpm mutant is responsible for the observed plastome instabilities. Interestingly, comparison of mutant phenotypes and molecular changes induced by the barley cpm mutant with those of Arabidopsis MSH1 mutants revealed marked differences.