Cyto-Histological Profile of MicroRNAs as Diagnostic Biomarkers in Differentiated Thyroid Carcinomas.

INTRODUCTION: The repertoire of microRNAs (miRNAs) in thyroid carcinomas starts to be elucidated. Among differentiated thyroid carcinomas (DTCs), papillary thyroid carcinoma (PTC) is the most frequent. The assessment of miRNAs expression may contribute to refine the pre-surgical diagnosis in order to obtain a personalized and more effective treatment for patients. AIMS: This study aims to evaluate (1) the miRNAs in a series of DTCs, and their association with the presence of selected genetic mutations in order to improve diagnosis and predict the biologic behavior of DTC/PTC. (2) The reliability of molecular tests in Ultrasound-guided Fine Needle Aspiration Cytology (US-FNAC) for a more precise preoperative diagnosis. MATERIAL AND METHODS: This series includes 176 samples (98 cytology and 78 histology samples) obtained from 106 patients submitted to surgery, including 13 benign lesions (controls) and 93 DTCs (cases). The microRNA expression was assessed for miR-146b, miR-221, miR-222, and miR-15a through quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The results were analyzed by the 2-ΔΔCT method, using miR16 as an endogenous control. Regarding PTC diagnosis, the discriminative ability of miRNAs expression was assessed by the area under the Receiver Operating Characteristic Curve (AUC). In PTCs, the association of miRNAs expression, clinicopathological features, and genetic mutations (BRAF, RAS, and TERTp) was evaluated. RESULTS/DISCUSSION: All the analyzed miRNAs presented a tendency to be overexpressed in DTCs/PTCs when compared with benign lesions, both in cytology and histology samples. In cytology, miRNAs expression levels were higher in malignant tumors than in benign tumors. In histology, the discriminative abilities regarding PTC diagnosis were as follows: miR-146b (AUC 0.94, 95% CI 0.87-1), miR-221 (AUC 0.79, 95% CI 0.68-0.9), miR-222 (AUC 0.76, 95% CI 0.63-0.89), and miR-15a (AUC 0.85, 95% CI 0.74-0.97). miR-146b showed 89% sensitivity (se) and 87% specificity (sp); miR-221 se = 68.4, sp = 90; miR-222 se = 73, sp = 70; and mi-R15a se = 72, sp = 80. MicroRNAs were associated with worst-prognosis clinicopathological characteristics in PTCs (p < 0.05), particularly for miR-222. Our data reveal a significant association between higher expression levels of miR-146b, miR-221, and miR-222 in the presence of the BRAF mutation (p < 0.001) and miR-146b (p = 0.016) and miR-221 (p = 0.010) with the RAS mutation, suggesting an interplay of these mutations with miRNAs expression. Despite this study having a relatively small sample size, overexpression of miRNAs in cytology may contribute to a more precise preoperative diagnosis. The miRNAs presented a good discriminative ability in PTC diagnosis. The association between the miRNAs expression profile and genetic alterations can be advantageous for an accurate diagnosis of DTCs/PTCs in FNAC.

Comparative Cyto-Histological Genetic Profile in a Series of Differentiated Thyroid Carcinomas.

INTRODUCTION: Molecular tests can contribute to improve the preoperative diagnosis of thyroid nodules. Tests available are expensive and not adapted to different populations. AIM: This study aimed to compare the cyto-histological genetic profile and to evaluate the reliability of molecular tests using ultrasound-guided fine needle aspiration cytology (US-FNAC) in accurately diagnosing differentiated thyroid carcinomas (DTCs) and predicting biologic behavior of papillary thyroid carcinomas (PTCs). MATERIALS AND METHODS: The series included 259 patients with paired cyto-histological samples totaling 518 samples. The genetic alterations were analyzed via PCR/Sanger sequencing. The association with clinicopathologic features was evaluated in PTCs. RESULTS/DISCUSSION: From the 259 patients included, histologies were 50 (19.3%) benign controls and 209 (80.7%) DTC cases, from which 182 were PTCs; cytologies were 5.8% non-diagnostic, 18.2% benign, 39% indeterminate, and 37.1% malignant. In histology, indeterminate nodules (n = 101) were 22.8% benign and 77.2% malignant. Mutation frequencies in cytology and histology specimens were, respectively, TERTp: 3.7% vs. 7.9%; BRAF: 19.5% vs. 25.1%; and RAS: 11% vs. 17.5%. The overall cyto-histological agreement of the genetic mutations was 94.9%, with Cohen's k = 0.67, and in indeterminate nodules agreement was 95.7%, k = 0.64. The identified mutations exhibited a discriminative ability in diagnosing DTC with a specificity of 100% for TERTp and BRAF, and of 94% for RAS, albeit with low sensitivity. TERTp and BRAF mutations were associated with aggressive clinicopathological features and tumor progression in PTCs (p < 0.001). The obtained good cyto-histological agreement suggests that molecular analysis via US-FNAC may anticipate the genetic profile and the behavior of thyroid tumors, confirming malignancy and contributing to referring patients to surgery.

Eucalyptus globulus Leaf Aqueous Extract Differentially Inhibits the Growth of Three Bacterial Tomato Pathogens.

As available tools for crop disease management are scarce, new, effective, and eco-friendly solutions are needed. So, this study aimed at assessing the antibacterial activity of a dried leaf Eucalyptus globulus Labill. aqueous extract (DLE) against Pseudomonas syringae pv. tomato (Pst), Xanthomonas euvesicatoria (Xeu), and Clavibacter michiganensis michiganensis (Cmm). For this, the inhibitory activity of different concentrations of DLE (0, 15, 30, 45, 60, 75, 90, 105, 120, 135, and 250 g L-1) was monitored against the type strains of Pst, Xeu, and Cmm through the obtention of their growth curves. After 48 h, results showed that the pathogen growth was strongly inhibited by DLE, with Xeu the most susceptible species (15 g L-1 MIC and IC50), followed by Pst (30 g L-1 MIC and IC50), and Cmm (45 and 35 g L-1 MIC and IC50, respectively). Additionally, using the resazurin assay, it was possible to verify that DLE considerably impaired cell viability by more than 86%, 85%, and 69% after Pst, Xeu, and Cmm were incubated with DLE concentrations equal to or higher than their MIC, respectively. However, only the treatment with DLE at 120 g L-1 did not induce any hypersensitive response in all pathogens when treated bacterial suspensions were infiltrated onto tobacco leaves. Overall, DLE can represent a great strategy for the prophylactic treatment of tomato-associated bacterial diseases or reduce the application of environmentally toxic approaches.

Untargeted metabolomic profiling of fresh and dried leaf extracts of young and mature Eucalyptus globulus trees indicates differences in the presence of specialized metabolites.

Aqueous extracts from Eucalyptus globulus leaves contain a wide variety of specialized metabolites, mainly polyphenols and appreciable amounts of volatile compounds, which are responsible for their diverse biological activities, such as antioxidant, antimicrobial, and allelopathic features. For this reason, several studies have been conducted to explore the composition of E. globulus leaf extracts for multiple therapeutic and commercial applications. However, so far, the available bibliographic reports only refer to the chemical composition of extracts prepared with leaves from mature trees, leaving much to clarify about the composition of juvenile eucalyptus leaf extracts. Furthermore, there is no consensus regarding the type of leaves, fresh or dried ones, to be used in the extraction procedure, considering the highest recovery of biologically active compounds. In this sense, this study aimed to characterize the chemical composition of aqueous extracts prepared with fresh and dried leaves from young and mature E. globulus trees. For this, leaf biomass from young and mature E. globulus trees was collected in three distinct places from a forest area, and after oven-drying a portion of the leaves, an extraction in hot water was carried out, followed by GC-MS and HPLC-MS/MS analyses. The results revealed that the maturity of eucalyptus trees and biomass drying significantly influenced the volatile and non-volatile composition of the aqueous extracts. Accordingly, while fresh leaf extracts of young trees had great levels of hydrolysable tannins, extracts prepared with fresh leaves from mature trees presented a wide range of terpenes. When dried leaf material was used, extracts had notorious contents of amino acids derivatives, C13 norisoprenoids, fatty and other organic acids. Overall, this study showed, for the first time, that plant maturity (young vs mature) and pre-processing (fresh vs dried) of foliar biomass of E. globulus trees need to be considered in the preparation of leaf aqueous extracts depending on the desired purposes, since major changes in what regards biologically active compounds were found.

Epigenomics in Hurthle Cell Neoplasms: Filling in the Gaps Towards Clinical Application.

It has been widely described that cancer genomes have frequent alterations to the epigenome, including epigenetic silencing of various tumor suppressor genes with functions in almost all cancer-relevant signalling pathways, such as apoptosis, cell proliferation, cell migration and DNA repair. Epigenetic alterations comprise DNA methylation, histone modification, and microRNAs dysregulated expression and they play a significant role in the differentiation and proliferation properties of TC. In this review, our group assessed the published evidence on the tumorigenic role of epigenomics in Hurthle cell neoplasms (HCN), highlighting the yet limited, heteregeneous and non-validated data preventing its current use in clinical practice, despite the well developed assessment techniques available. The identified evidence gaps call for a joint endeavour by the medical community towards a deeper and more systematic study of HCN, aiming at defining epigenetic markers in early diagnose, allowing for accurate stratification of maligancy and disease risk and for effective systemic treatment.

TERTp mutations and p53 expression in head and neck cutaneous basal cell carcinomas with different aggressive features.

Cutaneous basal cell carcinoma (cBCC) is an economic burden to health services, due to its great morbidity and increasing incidence in old people. Infiltrative cBCCs and cBCCs with micronodular pattern are considered as more aggressive. The role of p53 expression and TERTp mutation on cBCC behavior remains to be clarified. We aimed to assess TERTp mutations and p53 expression in relation to the cBCC histological subtype in a cohort of patients referred to an ENT Department of a tertiary Hospital of Northern Portugal. We performed a retrospective clinicopathological and histological review of the head and neck cBCCs followed-up at the otorhinolaryngology department of Trás-os-Montes e Alto Douro hospital (January 2007-June 2018). We assessed TERTp mutations in 142 cBCCs and p53 protein expression, through immunohistochemistry, in 157 cBCCs. We detected TERTp mutations in 43.7% of cBCCs and p53 overexpression in 60.5% of cBCCs. We spotted association of p53 overexpression and TERTp mutation with necrosis. In the infitrative-growth pattern cBCCs, there was no significant association with the clinical and histological features evaluated, except for necrosis. In the indolent-growth cBCCs, we identified a significant association of TERTp mutation status with female sex, necrosis, multiple cBCCs, and p53 positive expression. Our results suggest that TERTp mutation may be useful to identify more aggressive features in the indolent-growth pattern cBCCs (nodular and superficial subtypes). Further studies with larger cohorts are warranted to clarify the relevance of TERTp mutation in cBCCs.

Genetic Determinants for Prediction of Outcome of Patients with Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) usually presents an excellent prognosis, but some patients present with aggressive metastatic disease. BRAF, RAS, and TERT promoter (TERTp) genes are altered in PTC, and their impact on patient outcomes remains controversial. We aimed to determine the role of genetic alterations in PTC patient outcomes (recurrent/persistent disease, structural disease, and disease-specific mortality (DSM)). The series included 241 PTC patients submitted to surgery, between 2002-2015, in a single hospital. DNA was extracted from tissue samples of 287 lesions (primary tumors and metastases). Molecular alterations were detected by Sanger sequencing. Primary tumors presented 143 BRAF, 16 TERTp, and 13 RAS mutations. Isolated TERTpmut showed increased risk of structural disease (HR = 7.0, p < 0.001) and DSM (HR = 10.1, p = 0.001). Combined genotypes, BRAFwt/TERTpmut (HR = 6.8, p = 0.003), BRAFmut/TERTpmut (HR = 3.2, p = 0.056) and BRAFmut/TERTpwt (HR = 2.2, p = 0.023) showed increased risk of recurrent/persistent disease. Patients with tumors BRAFwt/TERTpmut (HR = 24.2, p < 0.001) and BRAFmut/TERTpmut (HR = 11.5, p = 0.002) showed increased risk of structural disease. DSM was significantly increased in patients with TERTpmut regardless of BRAF status (BRAFmut/TERTpmut, log-rank p < 0.001; BRAFwt/TERTpmut, log-rank p < 0.001). Our results indicate that molecular markers may have a role in predicting PTC patients' outcome. BRAFmut/TERTpwt tumors were prone to associate with local aggressiveness (recurrent/persistent disease), whereas TERTpmut tumors were predisposed to recurrent structural disease and DSM.

Translational Potential of Epigenetic-Based Markers on Fine-Needle Aspiration Thyroid Specimens.

The awareness of epigenetic alterations leading to neoplasia attracted the attention of researchers toward its potential use in the management of cancer, from diagnosis to prognosis and prediction of response to therapies. Our group has focused its attention on the epigenomics of thyroid neoplasms. Although most of the epigenetic studies have been applied on histological samples, the fact is that cytology, through fine-needle aspiration, is a primary diagnostic method for many pathologies, of which thyroid nodules are one of the most paradigmatic examples. This has led to an increasing literature report of epigenetic studies using these biological samples over the past decade. In this review, our group aimed to document recent research of epigenetic alterations and its associated assessment techniques, based on cytology material. Our review covers the main epigenetic categories-DNA methylation, histone modification, and RNA-silencing-whose evidence in thyroid cytology samples may represent solid soil for future prospectively designed studies aiming at validating patterns of epigenetic alterations and their potential use in the clinical management of thyroid neoplasms.

Phytotoxic effects of bulk and nano-sized Ni on Lycium barbarum L. grown in vitro - Oxidative damage and antioxidant response.

This study aimed to evaluate the effects of nickel oxide nanomaterial (nano-NiO) on goji berry (Lycium barbarum L.) shoots grown under in vitro conditions and to determine if the nanomaterial was more harmful than its bulk counterpart, nickel (II) sulphate (NiSO4). For this purpose, in vitro shoots of L. barbarum were cultured on MS medium supplemented with 15 mg L-1 of NiSO4 or nano-NiO. Nano-NiO was more harmful for shoots growth and photosynthetic pigments than NiSO4, with reductions up to 82% in comparison to the control. Shoots treated with nano-NiO presented an overproduction of hydrogen peroxide (H2O2; 130% increase) and superoxide anion (O2-; 110% increase), which led to higher levels of lipid peroxidation (LP; 57% increase) and the occurrence of oxidative stress. In opposition, bulk Ni seemed not to induce oxidative stress, once LP and reactive oxygen species content decreased in comparison with the control. The evaluation of the non-enzymatic antioxidant (AOX) system revealed that, under nano-NiO excess, proline, ascorbate, glutathione and phenols levels increased up to 4-fold, but did not change in response to the bulk treatment. With respect to the enzymatic AOX system, nano-NiO enhanced the activities of superoxide dismutase (203%) and ascorbate peroxidase (62%), though catalase activity was negatively affected, while bulk Ni did not majorly affect these enzymes' behavior. Overall, the data showed that Ni phytotoxicity in L. barbarum shoots depends on the metal source and that, in this case, nano-NiO seemed to be more deleterious to goji shoots grown under in vitro conditions than NiSO4.

CSF1R copy number changes, point mutations, and RNA and protein overexpression in renal cell carcinomas.

Renal cell carcinomas comprise a heterogeneous group of tumors. Of these, 80% are clear cell renal cell carcinomas, which are characterized by loss of 3p, often with concomitant gain of 5q22qter. Although VHL is considered the main target gene of the 3p deletions, none has been identified as the relevant target gene for the 5q gain. We have studied 75 consecutive kidney tumors and paired normal kidney samples to evaluate at the genomic and expression levels the tyrosine kinase genes CSF1R and PDGFRB as potential targets in this region. Our findings show that RNA expression of CSF1R, but not of PDGFRB, was significantly higher in clear cell renal cell carcinomas than in normal tissue samples, something that was corroborated at the protein level by immunohistochemistry. The CSF1R staining pattern in clear cell renal cell carcinomas was clearly different from that observed in other renal cell carcinomas, suggesting its potential usefulness in differential diagnosis. FISH analysis demonstrated whole chromosomal gain and relative CSF1R/PDGFRB copy number gain in clear cell renal cell carcinomas, which might contribute to CSF1R overexpression. Finally, one polymorphism and two novel mutations were identified in CSF1R in clear cell renal cell carcinoma patients. Our data allow us to conclude that CSF1R plays a relevant role in clear cell renal cell carcinoma carcinogenesis and raise the possibility that CSF1R may represent a future valuable therapeutic target in these patients.

Overexpression of the mitotic checkpoint genes BUB1 and BUBR1 is associated with genomic complexity in clear cell kidney carcinomas.

BACKGROUND: A defective mitotic checkpoint has been proposed to contribute to chromosomal instability (CIN). We have previously shown that expression changes of the mitotic arrest deficiency (MAD) gene family plays a role in renal cell cancer (RCC) characterized by numerical chromosomal changes, namely papillary and chromophobe carcinomas, but nothing is known about the expression of mitotic checkpoint genes in the clear cell histotype (ccRCC). METHODS: We analyzed the mRNA expression levels of the major mitotic checkpoint genes of the budding uninhibited by benzimidazole family (BUB1, BUBR1, BUB3) and of the MAD gene family (MAD1, MAD2L1, MAD2L2) by real-time quantitative PCR in 39 ccRCC and in 36 normal kidney tissue samples. We have additionally analyzed these tumors by comparative genomic hybridization (CGH) in order to evaluate the relationship between mitotic checkpoint defects and the pattern of chromosome changes in this subset of RCC. RESULTS: BUB1, BUBR1, MAD1 and MAD2L1 showed significant expression differences in tumor tissue compared to controls (BUB1, BUBR1 and MAD2L1 were overexpressed, whereas MAD1 was underexpressed). Overexpression of BUB1 and BUBR1 was significantly correlated with the number of genomic copy number changes (p<0.001 for both genes) and with Furhman grade of the tumors (p=0.006 and p=0.005, respectively). CONCLUSIONS: We conclude that BUB1 and BUBR1 overexpression plays a role in cytogenetic and morphologic progression of ccRCC.

Somatic mutations in mismatch repair genes in sporadic gastric carcinomas are not a cause but a consequence of the mutator phenotype.

In hereditary nonpolyposis colorectal cancer (HNPCC), patients' mismatch repair (MMR) gene mutations cause MMR deficiency, leading to microsatellite instability (MSI-H). MSI-H is also found in a substantial fraction of sporadic gastric carcinomas (SGC), mainly due to MLH1 promoter hypermethylation, although somatic mutations in MMR genes have been described. We aimed to investigate which MMR defects are present in SGC. Twenty-nine MSI-H SGC investigated previously for MLH1 promoter hypermethylation were screened for somatic mutations in MLH1, MSH2, MSH6, MLH3, and MBD4 by denaturing gradient gel electrophoresis and sequencing. Five truncating mutations (three in MSH6, one in MLH3, and one in MBD4) and one missense mutation (MLH1) were identified. Of these, three truncating mutations were in MSI-H cases that lack MLH1 hypermethylation. As all truncating mutations were found in the coding poly-A tracts, it seems likely that they result from the MSI phenotype rather than cause it. In summary, somatic mutations in MMR genes are rare in SGC and do not explain the development of these tumors reflecting, rather than causing, the mutator phenotype. Other MMR genes are probably involved in MSI-H gastric cancer without MLH1 hypermethylation.

High promoter methylation levels of APC predict poor prognosis in sextant biopsies from prostate cancer patients.

PURPOSE: Prostate cancer is a highly prevalent malignancy and constitutes a major cause of cancer-related morbidity and mortality. Owing to the limitations of current clinical, serologic, and pathologic parameters in predicting disease progression, we sought to investigate the prognostic value of promoter methylation of a small panel of genes by quantitative methylation-specific PCR (QMSP) in prostate biopsies. EXPERIMENTAL DESIGN: Promoter methylation levels of APC, CCND2, GSTP1, RARB2, and RASSF1A were determined by QMSP in a prospective series of 83 prostate cancer patients submitted to sextant biopsy. Clinicopathologic data [age, serum prostate-specific antigen (PSA), stage, and Gleason score] and time to progression and/or death from prostate cancer were correlated with methylation findings. Log-rank test and Cox regression model were used to identify which epigenetic markers were independent predictors of prognosis. RESULTS: At a median follow-up time of 45 months, 15 (18%) patients died from prostate cancer, and 37 (45%) patients had recurrent disease. In univariate analysis, stage and hypermethylation of APC were significantly associated with worse disease-specific survival, whereas stage, Gleason score, high diagnostic serum PSA levels, and hypermethylation of APC, GSTP1, and RASSF1A were significantly associated with poor disease-free survival. However, in the final multivariate analysis, only clinical stage and high methylation of APC were significantly and independently associated with unfavorable prognosis, i.e., decreased disease-free and disease-specific survival. CONCLUSIONS: High-level APC promoter methylation is an independent predictor of poor prognosis in prostate biopsy samples and might provide relevant prognostic information for patient management.

Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors.

BACKGROUND: Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. METHODS: A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. RESULTS: Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively. CONCLUSION: The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

Expression changes of the MAD mitotic checkpoint gene family in renal cell carcinomas characterized by numerical chromosome changes.

Papillary and chromophobe renal cell carcinomas are characterized by multiple trisomies and monosomies, respectively, but the molecular mechanisms behind the acquisition of these numerical chromosome changes are unknown. To evaluate the role of mitotic checkpoint defects for the karyotypic patterns characteristic of these two renal cell cancer subtypes, we analyzed the messenger RNA expression levels of the major mitotic checkpoint genes of the budding uninhibited by benzimidazole family (BUB1, BUBR1, BUB3) and of the mitotic arrest deficiency family (MAD1, MAD2L1, MAD2L2) by real-time quantitative polymerase chain reaction in 30 renal cell cancer samples (11 chromophobe and 19 papillary) and 36 normal kidney tissue samples. MAD1, MAD2L1, and MAD2L2 showed significant expression differences in tumor tissue compared to controls. Chromophobe tumors presented underexpression of MAD1, and MAD2L2, whereas papillary tumors showed overexpression of MAD2L1. The expression level of the BUB gene family did not differ significantly from that of normal kidney. We conclude that expression changes in mitotic arrest deficiency genes (MAD1, MAD2L1, and MAD2L2) play a role in renal carcinogenesis characterized by multiple numerical chromosome abnormalities.

Activated BRAF targets proximal colon tumors with mismatch repair deficiency and MLH1 inactivation.

BRAF, a serine/threonine kinase of the RAF family, is a downstream transducer of the RAS-regulated MAPK pathway and signals upstream of MEK1/2 kinases. Recently, activating mutations within BRAF have been reported in a high percentage of melanomas and colorectal carcinomas and shown to have oncogenic capabilities. Further, their association to mismatch-repair-deficient tumors has suggested the involvement of the RAS/RAF pathway in the tumorigenesis of microsatellite-unstable colon cancers, and that RAS and RAF mutations are alternative genetic events. We determined whether colorectal mismatch-repair-deficient tumors with BRAF mutations show a specific genotype when compared with tumors with wild-type BRAF, and whether they can be associated with a particular clinicopathological feature. Here, we report a striking association of BRAF, but not of APC, KRAS2, AXIN2, and TP53 mutations, with proximal mismatch-repair-deficient colon tumors and MLH1 hypermethylation. Our results support the hypothesis that proximal and distal colorectal tumors with mismatch repair deficiency harbor different genetic alterations, and we suggest that the involvement of the RAS/RAF pathway in colorectal tumorigenesis is differentially modulated according to tumor location and MLH1 inactivation.

BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency.

Genes from the RAF family are Ras-regulated kinases involved in growth cellular responses. Recently, a V599E hotspot mutation within the BRAF gene was reported in a high percentage of colorectal tumors and significantly associated to defective mismatch repair (MMR). Additionally, BRAF mutations were described only in K-Ras-negative colon carcinomas, suggesting that BRAF/K-Ras activating mutations might be alternative genetic events in colon cancer. We have addressed to what extent the tumorigenic-positive selection exerted by BRAF mutations seen in colorectal MMR-deficient tumors was also involved in the tumorigenesis of gastric cancer. Accordingly, BRAF mutations were detected in 34% (25/74) of colorectal MMR-deficient tumors and in 5% (7/142) of MMR-proficient colorectal cases (P=0.0001). All mutations found in the MSI cases corresponded to the previously reported hotspot V599E. Two D593K and a K600E additional mutations were also detected in three MSS cases. However, only one mutation of BRAF was found within 124 MSS gastric tumors and none in 37 MSI gastric tumors, clearly suggesting that BRAF mutations are not involved in gastric tumorigenesis. Nonetheless, a high incidence of mutations of K-Ras was found within the MSI gastric group of tumors (P=0.0005), suggesting that the activation of K-Ras-dependent pathways contributes to the tumorigenesis of gastric cancers with MMR deficiency. Accordingly, our results show evidences that BRAF mutations characterize colon but not gastric tumors with MMR deficiency and are not involved in the tumorigenesis of gastric cancer of the mutator phenotype pathway.

MBD4 mutations are rare in gastric carcinomas with microsatellite instability.

MBD4 encodes a protein that interacts with the mismatch repair protein hMLH1. Therefore, it has been postulated that mutations in MBD4 may result in mismatch repair deficiency. Furthermore, it was shown that MBD4 is a target gene in mismatch repair-deficient tumors. We searched for mutations of MBD4 in sporadic gastric carcinomas (SGC) and compared them with the MSI status of the tumors by screening a series of 42 SGC [22 high microsatellte instability (MSI-H) and 20 microsatellite stable (MSS)] for mutations in both the poly(A)10 and (A)6 tracts of the gene as well as in its entire coding sequence. Mutations of the poly(A)10 tract of MBD4 were also screened in 126 MSI-H colon carcinomas. One of the 22 MSI-H gastric carcinomas and 12 of 126 MSI-H colon carcinomas showed MBD4 mutations in the poly(A)10 tract of exon 3. In addition, seven sequence variants were found in SGC, all of them having a polymorphic nature. We conclude that MBD4 mutations in the entire coding sequence, including the two poly(A) tracts, are rare in MSI gastric carcinomas. MBD4 is likely to be a bystander target gene in MSI-H tumors.

Promoter methylation of TGFbeta receptor I and mutation of TGFbeta receptor II are frequent events in MSI sporadic gastric carcinomas.

Transforming growth factor beta (TGFbeta) is a potent inhibitor of cell growth, whose action is transduced through interaction between type I (RI) and type II (RII) receptors. Abnormal expression of these receptors has been identified in several human cancers and was found to be associated with resistance to TGFbeta. TGFbeta RII mutations occur in many types of malignancy. TGFbeta RI hypermethylation has been suggested as a cause of abnormal or absent expression of this receptor in cancer. This study has analysed the methylation status of the promoter region of the TGFbeta RI gene using a methylation-sensitive enzyme followed by polymerase chain reaction (PCR), and TGFbeta RII mutations (BAT-RII and a GT(3)) in order to determine the frequency of alteration of the TGFbeta receptors in a series of 40 sporadic gastric carcinomas (SGCs), 25 of which showed microsatellite instability (MSI) and 15 of which were microsatellite stable (MSS). Methylation in the promoter region of the TGFbeta RI gene was detected in 20 of the 40 (50%) SGCs (64% of the MSI cases and 26.7% of the MSS); 17 of the 40 (42.5%) cases had mutations in the BAT-RII region of the TGFbeta RII gene (68% in the MSI cases; 0% in the MSS). In total, 25 of the 40 (62.5%) SGCs had alterations in at least one of the TGFbeta receptors (84% of the cases in the MSI group, in contrast with 16% of the MSS cases) (p = 0.0003). The clinicopathological features of the cases were also studied and significant associations were found between the presence of alterations in TGFbeta receptors and the age of the patients (p = 0.046), size (p = 0.011), and proliferative rate of the tumours (p = 0.048). In conclusion, alterations in the receptors of TGFbeta (TGFbeta RI promoter hypermethylation and TGFbeta RII mutations) are frequent events in MSI SGC and are associated with large size and high proliferative activity of the tumours, in keeping with loss of the growth inhibitory effects of TGFbeta in this setting.