Evaluating the performance of clinical criteria for predicting mismatch repair gene mutations in Lynch syndrome: A comprehensive analysis of 3,671 families

Carriers of mismatch repair (MMR) gene mutations have a high lifetime risk for colorectal and endometrial cancers, as well as other malignancies. As mutation analysis to detect these patients is expensive and time‐consuming, clinical criteria and tumor‐tissue analysis are widely used as pre‐screening methods. The aim of our study was to evaluate the performance of commonly applied clinical criteria (the Amsterdam I and II Criteria, and the original and revised Bethesda Guidelines) and the results of tumor‐tissue analysis in predicting MMR gene mutations. We analyzed 3,671 families from the German HNPCC Registry and divided them into nine mutually exclusive groups with different clinical criteria. A total of 680 families (18.5%) were found to have a pathogenic MMR gene mutation. Among all 1,284 families with microsatellite instability‐high (MSI‐H) colorectal cancer, the overall mutation detection rate was 53.0%. Mutation frequencies and their distribution between the four MMR genes differed significantly between clinical groups (p < 0.001). The highest frequencies were found in families fulfilling the Amsterdam Criteria (46.4%). Families with loss of MSH2 expression had higher mutation detection rates (69.5%) than families with loss of MLH1 expression (43.1%). MMR mutations were found significantly more often in families with at least one MSI‐H small‐bowel cancer (p < 0.001). No MMR mutations were found among patients under 40‐years‐old with only colorectal adenoma. Familial clustering of Lynch syndrome‐related tumors, early age of onset, and familial occurrence of small‐bowel cancer were clinically relevant predictors for Lynch syndrome.     

Exclusion of breast cancer as an integral tumor of hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant genetic predisposition syndrome that accounts for 2-7% of all colorectal cancers. Diagnosis of HNPCC is based on family history (defined by Amsterdam or Bethesda Criteria), which often includes a history of multiple synchronous or metachronous cancers. The majority of HNPCC results from germ-line mutations in the DNA mismatch repair (MMR) genes hMSH2 and hMLH1 with rare alterations in hMSH6 and hPMS2 in atypical families. Both HNPCC and sporadic MMR-deficient tumors invariably display high microsatellite instability (MSI-H). Two types of HNPCC families can be distinguished: type I (Lynch I) with tumors exclusively located in the colon; and type II (Lynch II) with tumors found in the endometrium, stomach, ovary, and upper urinary tract in addition to the colon. A proposed association of breast cancer with type II HNPCC is controversial. To address this important clinical question, we examined MSI in a series of 27 female patients who presented with synchronous or metachronous breast plus colorectal cancer. Although MSI-H was found in 5 of 27 (18.5%) of the colon cancers, in all cases the matched breast cancer was microsatellite stable. We also examined the breast tumors from three women who were carriers of MMR gene mutations from HNPCC families. None of these three breast tumors displayed MSI nor was the expression of MMR proteins altered in these tumors. We conclude that breast cancer largely arises sporadically in HNPCC patients and is rarely associated with the HNPCC syndrome.     

Analysis of mismatch repair gene mutations in Turkish HNPCC patients

Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) is caused by the inheritance of a mutant allele of a DNA mismatch repair gene. We aimed to investigate types and frequencies of mismatch repair (MMR) gene mutations in Turkish patients with HNPCC and to identify specific biomarkers for early diagnosis of their non-symptomatic kindred’s. The molecular characteristics of 28 Turkish colorectal cancer patients at high-risk for HNPCC were investigated by analysis of microsatellite instability (MSI), immunohistochemistry and methylation-specific PCR in order to select tumors for mutation analysis. Ten cases (35.7%) were classified as MSI (+). Lack of expression of the main MMR proteins was observed in MSI (+) tumors. Hypermethylation of the MLH1 promoter region was observed in one tumor. Nine Lynch syndrome cases showed novel germ-line alterations of the MMR gene: two frame-shifts (MLH1 c.1843dupC and MLH1 c.1743delG) and three missense mutations (MLH1 c.293G>C, MLH1 c.954_955delinsTA and MSH2 c.2210G>A). Unclassified variants were evaluated as likely to be pathogenic by using the in-silico analyses. In addition, the MSH2 c.2210G>A alteration could be considered as a founder mutation for the Turkish population due to its identification in five different Lynch syndrome families and absence in control group. The present study adds new information about MMR gene mutation types and their role in Lynch syndrome. This is the first detailed research on Turkish Lynch syndrome families.     

Influence of selection criteria on mutation detection in patients with hereditary nonpolyposis colorectal cancer.

BACKGROUND: Hereditary nonpolyposis colorectal cancer (HNPCC) is linked genetically to mutations in DNA mismatch repair (MMR) genes. Because a deficiency in MMR does not predict a specific phenotype, the original selection criteria may be too restrictive in identifying additional families. The current study was performed to determine whether a relaxation of the Amsterdam criteria (AC) could be applied to identify more families associated with DNA MMR. METHODS: Twenty-eight unrelated Swiss families (15 complying with the AC and 13 fulfilling extended criteria [EC] to include other tumors of the HNPCC spectrum as well) were screened for mutations in the MMR genes hMSH2 and hMLH1, using single-stranded conformation polymorphism and direct DNA sequencing. Microsatellite instability (MSI) was determined in 14 families. A comparison was made between the phenotypic characteristics of the mutation positive and mutation negative families. RESULTS: Ten AC families (67%) harbored germline mutations in hMLH1 (6 kindreds) or hMSH2 (4 kindreds). In none of the EC kindreds could an unambiguous disease-causing mutation be identified. Seven of eight AC families were found to display MSI whereas all colorectal carcinomas (CRC) in eight EC kindreds were MSI stable. CRC patients from mutation positive families had an earlier age at diagnosis (44 years vs. 49 years) and appeared to have a better survival (11.1 years vs. 7.7 years). CONCLUSIONS: Extending the AC to include extracolonic tumors of the HNPCC spectrum results in a very low mutation detection rate for hMSH2 and hMLH1. The EC families appear to represent an alternative genetic entity not necessarily related to DNA MMR gene mutations because they do not display MSI.     

Screening for Lynch Syndrome in Young Colorectal Cancer Patients from Saudi Arabia Using Microsatellite Instability as the Initial Test

Background: Lynch Syndrome (LS) is a familial cancer condition caused by germline mutations in DNA mismatch repair genes. Individuals with LS have a greatly increased risk of developing colorectal cancer (CRC) and it is therefore important to identify mutation carriers so they can undergo regular surveillance. Tumor DNA from LS patients characteristically shows microsatellite instability (MSI). Our aim here was to screen young CRC patients for MSI as a first step in the identification of unrecognized cases of LS in the Saudi population. Materials and Methods: Archival tumor tissue was obtained from 284 CRC patients treated at 4 institutes in Dammam and Riyadh between 2006 and 2015 and aged less than 60 years at diagnosis. MSI screening was performed using the BAT-26 microsatellite marker and positive cases confirmed using the pentaplex MSI analysis system. Positive cases were screened for BRAF mutations to exclude sporadic CRC and were evaluated for loss of expression of 4 DNA mismatch repair proteins using immunohistochemistry. Results: MSI was found in 33/284 (11.6%) cases, of which only one showed a BRAF mutation. Saudi MSI cases showed similar instability in the BAT-26 and BAT-25 markers to Australian MSI cases, but significantly lower frequencies of instability in 3 other microsatellite markers. Conclusions: MSI screening of young Saudi CRC patients reveals that approximately 1 in 9 are candidates for LS. Patients with MSI are strongly recommended to undergo genetic counselling and germline mutation testing for LS. Other affected family members can then be identified and offered regular surveillance for early detection of LS-associated cancers.     

Evaluating Lynch syndrome in very early onset colorectal cancer probands without apparent polyposis

To characterize the frequency of germline mutations associated with Lynch syndrome and review the potential expanded differential diagnoses in very early onset colorectal cancer (CRC) cases without apparent polyposis. Retrospectively reviewed medical records of 96 probands with CRC diagnosed prior to age 36 from three cancer centers. Determined the frequency of germline mutations in probands meeting different clinical criteria used to identify Lynch syndrome. Three of 46 (6.5%) single case indicators (probands without additional personal or family history suspicious for Lynch syndrome) were identified to carry a deleterious or suspected deleterious mismatch repair (MMR) mutation compared with 10 of 19 (52.6%) in the cases meeting at least one additional revised Bethesda guideline, and 11 of 15 (73.3%) in the cases meeting Amsterdam criteria. Two families without MMR mutations were documented to have a germline APC or TP53 mutation after additional clinical features were identified. Our results suggest that single cases of CRC (those without additional personal or family history suspicious of Lynch syndrome) diagnosed prior to age 36 infrequently have identifiable MMR mutations, especially when compared to cases meeting additional criteria. Careful attention to evolving or additional clinical features is warranted and may lead to an alternate genetic diagnosis in families with early onset CRC.     

Population‐based detection of Lynch syndrome in young colorectal cancer patients using microsatellite instability as the initial test

Approximately 1–2% of colorectal cancers (CRC) arise because of germline mutations in DNA mismatch repair genes, referred to as Lynch syndrome. These tumours show microsatellite instability (MSI) and loss of expression of mismatch repair proteins. Pre‐symptomatic identification of mutation carriers has been demonstrated to improve survival; however, there is concern that many are not being identified using current practices. We evaluated population‐based MSI screening of CRC in young patients as a means of ascertaining mutation carriers. CRC diagnosed in patients aged <60 years were identified from pathology records. No prior information was available on family history of cancer. PCR techniques were used to determine MSI in the BAT‐26 mononucleotide repeat and mutation in the BRAF oncogene. Loss of MLH1, MSH2, MSH6 and PMS2 protein expression was evaluated in MSI+ tumours by immunohistochemistry. MSI+ tumours were found in 105/1,344 (7.8%) patients, of which 7 were excluded as possible Lynch syndrome because of BRAF mutation. Of the 98 “red flag” cases that were followed up, 25 were already known as mutation carriers or members of mutation carrier families. Germline test results were obtained for 35 patients and revealed that 22 showed no apparent mutation, 11 showed likely pathogenic mutations and 2 had unclassified variants. The proportion of MSI+ cases in different age groups that were estimated to be mutation carriers was 89% (<30 years), 83% (30–39), 68% (40–49) and 17% (50–59). We recommend MSI as the initial test for population‐based screening of Lynch syndrome in younger CRC patients, regardless of family history. © 2008 Wiley‐Liss, Inc.     

Microsatellite instability and MLH1 and MSH2 germline defects are related to clinicopathological features in sporadic colorectal cancer

Clinical and pathological features were evaluated to predict tumor microsatellite instability (MSI) and germline mutations in MLH1 and MSH2 DNA mismatch repair genes in two patient groups with sporadic colorectal cancer (CRC): 38 young patients (age /=60 years). Nine (25.7%) young patients out of 35 and five (16%) old patients out of 31 exhibited MSI in their cancers. MSI+ cancers were related to proximal cancer and mucinous carcinoma independently of the age at cancer onset. Three (7.9%) out of 38 young patients had mutations in MLH1 and MSH2 genes that led to truncated protein products; they were all at age <35 years and showed MSI in their tumors, with mucinous histotype in two cases. In conclusion, histopathological and clinical features of CRC allow identification of cancers showing DNA microsatellite instability. MSI in CRC at very early onset (age <35 years) appears useful to predict germline MMR gene defects.     

Clinical features and mismatch repair genes analyses of Chinese suspected hereditary non-polyposis colorectal cancer: a cost-effective screening strategy proposal

China has the largest numbers of hereditary non-polyposis colorectal cancer (HNPCC) patients based on its population of 1.4 billion. However, the clinical data and mismatch repair (MMR) gene analyses have been limited. Here we performed microsatellite instability (MSI) and immunohistochemistry (IHC) analyses on a series of patients with a high-risk for HNPCC: 61 patients with family histories fulfilling Amsterdam criteria II (ACII-HNPCC) or suspected HNPCC criteria (S-HNPCC), and 106 early onset colorectal cancer (CRC) patients. Sixty late-onset CRC patients were used as control. Methylation of the hMLH1 promoter was analyzed on tumors lacking hMLH1 expression. MMR germ-line mutations were screened on patients with tumors classified as MSI-H/L or negative for IHC. We identified 27 germ-line MMR variants in the 167 patients with a high-risk for HNPCC while only one germ-line mutation in hMSH6 was found in the late-onset CRC group. Of those, 23 were pathogenic mutations. The high incidence of gastric and hepatobiliary cancers coupled with the increasing number of small families in China reduces the sensitivity (43.5%, 30.4%) and positive predictive value (PPV) (45.5%, 17.9%) of the ACII- or S-HNPCC criteria. MSI or IHC testing are highly sensitive in detecting pathogenic mutations (sensitivities = 91.3% and 95.6%, respectively), but the PPVs are quite low (25.6% and 27.8%, respectively). Considering that all 12 tumors with pathogenic mutations in hMLH1 also showed promoter unmethylation, the sensitivity of IHC in conjunction with hMLH1 promoter methylation analysis is not reduced, but the PPV was increased from 27.8% to 61.1%, and the total cost was greatly reduced. ( Cancer Sci 2008; 99: 770–780)     

When does MMR loss occur during HNPCC progression?

Inactivation of DNA mismatch repair (MMR) is the hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) and sporadic colorectal cancers with microsatellite instability (MSI+). MMR loss results in a markedly elevated mutation rate, and many MS mutations are found in MSI+ cancers. In theory, it is possible to estimate the interval between MMR loss and cancer removal by counting numbers of cancer MS mutations--the more MS mutations, the longer the intervals since MMR loss. Using this somatic molecular clock approach, MMR loss is estimated to precede transformation (clonal expansion) and likely occurs in normal appearing colon. Surprising, ages at MMR loss are more consistent with MMR loss as a relatively late event during progression to MSI+ cancer.     

Microsatellite instability in colorectal cancer

Approximately 20 percent of right-sided colon cancers and 5 percent of left-sided colon and rectal cancers have a deficient DNA mismatch repair system. This results in the widespread accumulation of mutations to nucleotide repeats, some of which occur within the coding regions of cancer-related genes such as TGFβRII and BAX. A standardized definition for microsatellite instability (MSI) based on the presence of deletions to mononucleotide repeats is gaining widespread acceptance in both research and the clinic. Colorectal cancer (CRC) with MSI are characterized histologically by an abundance of tumor-infiltrating lymphocytes, poor differentiation and a signet ring or mucinous phenotype. In younger patients these tumors usually develop along the chromosomal instability pathway, in which case the mismatch repair genes are inactivated by germline mutation, somatic mutation and loss of heterozygosity. In older patients MSI CRC usually develops against a background of widespread hypermethylation that includes methylation-induced silencing of the mismatch repair gene MLH1. The overall biological and clinical phenotype of MSI CRC that arise in these two pathways is likely to be different and may account for some of the discordant results reported in the literature relating to the clinical properties of these tumors. The available evidence indicates that MSI is unlikely to be a clinically useful marker for the prognostic stratification of early-stage CRC. The predictive value of MSI for response to 5-fluorouracil-based chemotherapy remains controversial, while for other agents the predictive value is difficult to assess because they are used in combination regimens. The MSI phenotype is being actively investigated for novel therapeutic approaches based on the principle of synthetic lethality. Finally, the MSI status of CRC is an extremely useful marker for population-based screening programs that aim to identify individuals and families with the hereditary cancer condition known as Lynch syndrome.     

Unusual presentation of Lynch Syndrome

Lynch Syndrome/HNPCC is a syndrome of cancer predisposition linked to inherited mutations of genes participating in post-replicative DNA mismatch repair (MMR). The spectrum of cancer associated with Lynch Syndrome includes tumours of the colorectum, endometrium, ovary, upper gastrointestinal tract and the urothelium although other cancers are rarely described. We describe a family of Lynch Syndrome with an hMLH1 mutation, that harbours an unusual tumour spectrum and its diagnostic and management challenges.     

Microsatellite instability and mutation analysis among southern Italian patients with colorectal carcinoma: detection of different alterations accounting for MLH1 and MSH2 inactivation in familial cases.

BACKGROUND: Microsatellite instability (MSI) is due to defective DNA mismatch repair (MMR) and has been detected at various rates in colorectal carcinoma (CRC). The role of MSI in colorectal tumorigenesis was assessed further in this study by both microsatellite analysis of two CRC subsets [unselected patients (n = 215) and patients <50 years of age (n = 95)], and mutation screening of the two major MMR genes MLH1 and MSH2 among familial CRC cases. PATIENTS AND METHODS: PCR-based microsatellite analysis was performed on paraffin-embedded tissues. In CRC families, MLH1/MSH2 mutation analysis and MLH1/MSH2 immunostaining were performed on germline DNA and MSI+ tumour tissues, respectively. RESULTS: The MSI+ phenotype was detected in 75 (24%) patients, with higher incidence in early-onset or proximally located tumours. Among 220 patients investigated for family cancer history, MSI frequency was markedly higher in familial [18/27 (67%)] than in sporadic [32/193 (17%)] cases. Three MLH1 and six MSH2 germline mutations were identified in 14 out of 36 (39%) CRC families. Prevalence of MLH1/MSH2 mutations in CRC families was significantly increased by the presence of: (i) fulfilled Amsterdam criteria; (ii) four or more CRCs; or (iii) one or more endometrial cancer. While MSH2 was found mostly mutated, almost all [8/9 (89%)] familial MSI+ cases with loss of the MLH1 protein were negative for MLH1 germline mutations. CONCLUSIONS: Both genetic (for MSH2) and gene-silencing (for MLH1) alterations seem to be involved in CRC pathogenesis.     

Immunohistochemistry and microsatellite instability testing for selecting MLH1, MSH2 and MSH6 mutation carriers in hereditary non-polyposis colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC) represents 1-3% of all colorectal cancers. HNPCC is caused by a constitutional defect in a mismatch repair (MMR) gene, most commonly affecting the genes MLH1, MSH2 and MSH6. The MMR defect results in an increased cancer risk, with the greatest lifetime risk for colorectal cancer and other cancers associated to HNPCC. The HNPCC-associated tumor phenotype is generally characterized by microsatellite instability (MSI) and immunohistochemical loss of expression of the affected MMR protein. The aim of this study was to determine the sensitivity of IHC for MLH1, MSH2 and MSH6, and MSI analysis in tumors from known MMR gene mutation carriers. Fifty-eight paired normal and tumor samples from HNPCC families enrolled in our high-risk colorectal cancer registry were studied for the presence of germline mutations in MLH1, MSH2 and MSH6 by DGGE and direct sequencing. MSI analysis and immunostaining for MLH1, MSH2 and MSH6 were evaluated. Of the 28 patients with a real pathogenic mutation, loss of immunohistochemical expression for at least 1 of these MMR proteins was found, and all except 1 have MSI-H. Sensitivity by MSI analysis was 96%. IHC analysis had a sensitivity of 100% in detecting MMR deficiency in carriers of a pathogenic MMR mutation, and can be used to predict which gene is expected to harbor the mutation for MLH1, MSH2 and MSH6. This study suggests that both analyses are useful for selecting high-risk patients because most MLH1, MSH2 and MSH6 gene carriers will be detected by this 2-step approach. This practical method should have immediate application in the clinical work of patients with inherited colorectal cancer syndromes.     

Evaluation of predictive models in daily practice for the identification of patients with Lynch syndrome

The optimal strategy for identifying patients with Lynch syndrome among patients with newly diagnosed colorectal cancer (CRC) is still debated. Several predictive models (e.g., MMRpredict, PREMM1,2 and MMRpro) combining personal and familial data have recently been developed to quantify the risk that a given patient with CRC carries a Lynch syndrome-causing mutation. Their clinical applicability to patients with CRC from the general population requires evaluation. We studied a consecutive series of 214 patients with newly diagnosed CRC characterized for tumor microsatellite instability (MSI), somatic BRAF mutation, MLH1 promoter methylation and mismatch repair (MMR) gene germline mutation status. The performances of the models for identifying MMR mutation carriers (8/214, 3.7%) were evaluated and compared to the revised Bethesda guidelines and a molecular strategy based on MSI testing in all patients followed by the exclusion of MSI-positive sporadic cases from mutational testing by screening for BRAF mutation and MLH1 promoter methylation. The sensitivities of the three models, at the lowest thresholds proposed, were identical (75%), with similar numbers of probands eligible for further MSI testing (almost half the patients). In our dataset, the prediction models gave no better discrimination than the revised Bethesda guidelines. Both approaches failed to identify two of the eight mutation carriers (the same two patients, aged 67 and 81 years, both with no family history). Thus, like the revised Bethesda guidelines, predictive models did not identify all patients with Lynch syndrome in our series of consecutive CRC. Our results support systematic screening for MMR deficiency in all new CRC cases.     

Association of low-risk MSH3 and MSH2 variant alleles with Lynch syndrome: probability of synergistic effects

Mutations in the MLH1 and MSH2 genes account for a majority of cases of families with Lynch Syndrome. Germ-line mutations in MSH6, PMS2 and MLH3 are responsible for disease in a minority of cases, usually associated with milder and variable phenotypes. No germ-line mutations in MSH3 have so far been associated with Lynch Syndrome, although it is known that impaired MSH3 activity leads to a partial defect in mismatch repair (MMR), with low levels of microsatellite instability at the loci with dinucleotide repeats in colorectal cancer (CRC), thus suggesting a role for MSH3 in carcinogenesis. To determine a possible role of MSH3 as predisposing to CRC in Lynch syndrome, we screened MSH3 for germ-line mutations in 79 unrelated Lynch patients who were negative for pathogenetic mutations in MLH1, MSH2 and MSH6. We found 13 mutant alleles, including silent, missense and intronic variants. These variants were identified through denaturing high performance liquid chromatography and subsequent DNA sequencing. In one Lynch family, the index case with early-onset colon cancer was a carrier of a polymorphism in the MSH2 gene and two variants in the MSH3 gene. These variants were associated with the disease in the family, thus suggesting the involvement of MSH3 in colon tumour progression. We hypothesise a model in which variants of the MSH3 gene behave as low-risk alleles that contribute to the risk of colon cancer in Lynch families, mostly with other low-risk alleles of MMR genes.     

Recent discoveries in the molecular genetics of Lynch syndrome

Lynch syndrome is the inherited predisposition to cancer caused by a germline mutation in a DNA mismatch repair gene. The consequent tumors have a characteristic microsatellite instability (MSI) phenotype. Genomic sequencing of Lynch syndrome-associated colorectal cancers (CRCs) has demonstrated that these tumors have a substantially greater number of mutations than non-MSI CRCs, and that the target mutations driving tumor behavior are also different from what occurs in sporadic tumors. There are multiple non-Lynch syndrome entities that can create clinical confusion with that disease, including the acquired methylation of MLH1, Lynch-like syndrome, and Familial CRC-Type X. Patients with Lynch syndrome-associated CRCs have a substantially better prognosis, and there is growing evidence that this is due to the generation of immunogenic frameshift peptides as a consequence of defective DNA mismatch repair, and an effective immune response to the tumor.     

修饰型微卫星不稳定性与散发结直肠癌p53基因点突变的相关性

目的 高频度微卫星不稳定性被认定为DNA错配修复缺陷的标志,但既往研究发现一个显著矛盾,即在高频度微卫星不稳定结直肠癌中,p53突变率较一般结直肠癌低.研究旨在确认该矛盾的存在并试图阐明其机制.方法 对180例散发结直肠癌采用高分辨率荧光标记微卫星分析法检测微卫星位点稳定性,PCR扩增直接测序检测p53突变.结果 微卫星不稳定性呈现修饰型和跳跃型两种变化.低频度微卫星不稳定性均呈现修饰型而无跳跃型变化;高频度微卫星不稳定性均检出了跳跃型变化,一部分也并存修饰型变化.微卫星不稳定与肿瘤部位及分化程度明显相关,p53突变与肿瘤分化明显相关.高频度微卫星不稳定肿瘤未检出p53突变,而低频度微卫星不稳定肿瘤p53突变率较高.结论 低频度微卫星不稳定性呈现的修饰型微卫星位点长度变化可能是DNA错配修复缺陷的表型;此表型与提高的碱基置换突变率有关.单纯DNA错配修复缺陷可能不足以导致微卫星不稳定性的跳跃型变化,高频度微卫星不稳定的真正原因仍有待阐明.     

Differential cancer predisposition in Lynch syndrome: insights from molecular analysis of brain and urinary tract tumors

Hereditary non-polyposis colorectal carcinoma (Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). Lynch syndrome patients are predisposed to different cancers in a non-random fashion, the basis of which is poorly understood. We addressed this issue by determining the molecular profiles for different tumors from a nationwide cohort of Lynch syndrome families (approximately 150 tumors in total). We focused on some less prevalent cancers, affecting the brain (n = 7) and urinary tract (five bladder and five ureter uroepithelial cancers and four kidney adenocarcinomas), and compared their molecular characteristics to those of the most common cancers, colorectal, gastric and endometrial adenocarcinomas, from the same families. Despite origin from verified MMR gene mutation carriers, the frequency of high-level microsatellite instability in tumors varied between high (100-96% for ureter, stomach and colon), intermediate (63-60% for endometrium and bladder) and low (25-0% for kidney and brain). In contrast to gastrointestinal and endometrial carcinomas, active (nuclear) beta-catenin was rare and KRAS mutations were absent in brain and urological tumors. Compared with other tumors, frequent stabilization of p53 protein characterized urinary tract cancers. Promoter methylation of tumor suppressor genes discriminated the tumors in an organ-specific manner. Our findings suggest that different Lynch syndrome tumors develop along different routes. Uroepithelial cancers of the ureter (and bladder to lesser extent) share many characteristics of MMR deficiency-driven tumorigenesis, whereas brain tumors and kidney adenocarcinomas follow separate pathways.     

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.