Diets high in red meat have been consistently associated with colorectal cancer (CRC) risk and may result in exposure to carcinogens that cause DNA damage [i.e polycyclic aromatic hydrocarbons, heterocyclic amines (HCAs) and N-nitroso compounds]. Using a family-based study, we investigated whether polymorphisms in the nucleotide excision repair (NER) (ERCC1 3' untranslated region (UTR) G/T, XPD Asp312Asn and Lys751Gln, XPC intron 11 C/A, XPA 5' UTR C/T, XPF Arg415Gln and XPG Asp1104His) and mismatch repair (MLH1 Ile219Val and MSH2 Gly322Asp) pathways modified the association with red meat and poultry intake. We tested for gene-environment interactions using case-only analyses (n = 577) and compared the results using case-unaffected sibling comparisons (n = 307 sibships). Increased risk of CRC was observed for intake of more than or equal to three servings per week of red meat [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.3-2.5)] or high-temperature cooked red meat (OR = 1.6, 95% CI = 1.1-2.2). Intake of red meat heavily brown on the outside or inside increased CRC risk only among subjects who carried the XPD codon 751 Lys/Lys genotype (case-only interaction P = 0.006 and P = 0.001, respectively, for doneness outside or inside) or the XPD codon 312 Asp/Asp genotype (case-only interaction P = 0.090 and P < 0.001, respectively). These interactions were stronger for rectal cancer cases (heterogeneity test P = 0.002 for XPD Asp312Asn and P = 0.03 for XPD Lys751Gln) and remained statistically significant after accounting for multiple testing. Case-unaffected sibling analyses were generally supportive of the case-only results. These findings highlight the possible contribution of diets high in red meat to the formation of lesions that elicit the NER pathway, such as carcinogen-induced bulky adducts.

The mismatch repair (MMR) system, highly conserved throughout evolution, corrects nucleotide mispairing that arise during cellular DNA replication. We report here that proliferating cell nuclear antigen (PCNA), the clamp loader complex (RF-C), and a series of MMR proteins like MSH-2, MSH-6, MLH1, and hPSM2 can be assembled to Epstein-Barr virus replication compartments, the sites of viral DNA synthesis. Levels of the DNA-bound form of PCNA increased with progression of viral productive replication. Bromodeoxyuridine-labeled chromatin immunodepletion analyses confirmed that PCNA is loaded onto newly synthesized viral DNA as well as BALF2 and BMRF1 viral proteins during lytic replication. Furthermore, the anti-PCNA, -MSH2, -MSH3, or -MSH6 antibodies could immunoprecipitate BMRF1 replication protein probably via the viral DNA genome. PCNA loading might trigger transfer of a series of host MMR proteins to the sites of viral DNA synthesis. The MMR factors might function for the repair of mismatches that arise during viral replication or act to inhibit recombination between moderately divergent (homologous) sequences.

Hereditary nonpolyposis colon cancer (HNPCC) is the most common hereditary colon cancer syndrome. It is characterized by multiple colon as well as extracolonic cancers such as endometrial, ovarian and urinary tract cancers. In addition, it is well known that some cases of HNPCC can present with unique tumor spectrums such as sebaceous tumors, which is often referred to as the 'Muir-Torre' syndrome. In recent years there have been a few reports of families presenting with early onset of colon tumors along with café-au-lait spots and/or hematologic malignancies often associated with homozygous mutations involving one of the mismatch repair genes. In this article we have performed a comprehensive review of the entire medical literature to identify all cases with similar presentations reported in the literature and have summarized the clinical features and genetic test results of the same. The available data clearly highlight such presentations as a distinct clinical entity characterized by early onset of gastrointestinal tumors, hematologic malignancies as well as features of neurofibromatosis (easily remembered by the acronym ;CoLoN'; Colon tumors or/and Leukemia/Lymphoma or/and Neurofibromatosis features). Furthermore, there has also been some evidence that the neurofibromatosis type-1 gene is a mutational target of the mismatch repair deficiency that is seen in families with HNPCC, and that mlh1 deficiency can accelerate the development of leukemia in neurofibromatosis (Nf1) heterozygous mice. Recognition of this syndrome has significant importance in terms of earlier detection of cancers, cancer screening recommendations as well as genetic counseling offered to such families.

Genetic epidemiology studies of colorectal cancer (CRC) can identify persons who are at inordinately high risk and who thereby might benefit from targeted early detection and primary prevention programs, inclusive of prophylactic surgery in selected cases. The discipline of molecular genetics has identified germline mutations that include APC in familial adenomatous polyposis (FAP) and mutator genes, namely MSH2, MLH1, PMS1, and PMS2 in hereditary nonpolyposis colorectal cancer (HNPCC). These discoveries have significantly enhanced our ability to identify individuals whose cancer destiny can literally be determined at birth. This review updates HNPCC's differential diagnosis, heterogeneity, tumor spectrum, newly found evidence of accelerated colonic adenoma to CRC, survival advantage, and currently available surveillance and management programs. Emphasis has been on how knowledge of the genetics and natural history of HNPCC can be used effectively to promote early diagnosis or prevention of cancer.

OBJECTIVE

Ovarian cancer has one of the highest fractions of hereditary cases. The hereditary breast and ovarian cancer syndrome, primarily due to mutations in BRCA1 and BRCA2, is the main cause of heredity, but also the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome confers an increased risk of ovarian cancer. In order to clarify the contribution of HNPCC to the development of ovarian cancer, we collected data on family history of cancer and characterized MMR function in a consecutive series of 128 tumors unselected for age at diagnosis and previously characterized for BRCA gene mutations.

METHODS

Expression of the MMR proteins MLH1, PMS2, MSH2, and MSH6 was analyzed by immunohistochemistry using tissue microarray sections. Tumors with reduced staining or loss of staining were also analyzed for microsatellite instability (MSI).

RESULTS

Loss of MMR protein expression was identified in 3 ovarian cancers, all of which had a MSI-high phenotype. DNA sequence analysis revealed disease-causing germline mutations (deletions of exons 4-6 in MLH1 and a 1-nucleotide deletion in exon 5 of MSH6) in two patients diagnosed at ages 40 and 49 years, both of whom had family histories suggestive of HNPCC. The genetic defect in the third case, which was a 47-year old woman without knowledge about her family history with loss of MLH1/PMS2 expression in the tumor tissue, remains elusive. A family history suggestive of HNPCC was identified in an additional case, but this tumor showed normal, retained MMR protein expression and a microsatellite stable phenotype.

CONCLUSIONS

About 2% of ovarian cancer is caused by germline mutations in the MMR-genes, a minor proportion as compared to the contribution of the BRCA-genes (11% in the present series). However, identification of HNPCC patients is important since it allows inclusion of high-risk individuals into control programs aimed at preventing the more frequent colorectal and endometrial cancers. Tumors within the HNPCC-spectrum should therefore be included when recording a family history of cancer among patients diagnosed with ovarian cancer.

BACKGROUND

The incidence of hereditary nonpolyposis colon cancer (HNPCC) in the general population is not well defined because of the lack of large population-based studies. We characterized the incidence of HNPCC in a large, population-based cohort of colorectal cancer probands and analyzed the location of colorectal tumors.

METHODS

Of the participating 1134 probands from three counties in Southern California, 907 had a negative family history of colorectal cancer and 227 had a positive family history of colorectal cancer. In addition, 11 referral case subjects with HNPCC were used to study mutation frequencies in two mismatch repair genes (MSH2 and MLH1) and microsatellite instability. All statistical tests were two-sided.

RESULTS

Among the probands diagnosed in Orange County during 1994 (population-based sample, all ages), five were consistent with the Amsterdam criteria for HNPCC (0.9%; 95% confidence interval [CI] = 0. 3%-2.1%). Among probands diagnosed at less than 65 years of age-from the wider three-county area and a longer time span-16 (2.1%; 95% CI = 1.2%-3.4%) had a clinical history consistent with the Amsterdam criteria for HNPCC. Five (approximately 45%) of 11 of the referral HNPCC case subjects had a mutation in MSH2 or MLH1 and also showed microsatellite instability. The family members of case subjects with mutations tended to show an earlier age at diagnosis of HNPCC and more multiple primary cancers than those of case subjects without detectable mutations. Many of the known characteristics of HNPCC, including the presence of ureteral and endometrial cancers, were seen in both sets of families. The previously reported proximal location of colorectal tumors in HNPCC kindreds was not seen in the population-based dataset but was similar to the location reported in the referral cases.

CONCLUSIONS

On the basis of our data, we believe that the prevalence of HNPCC in the general population is likely to be closer to 1% than to 5%. Furthermore, our study suggests that some previously reported characteristics of HNPCC, such as the proximal location of tumors in the syndrome, may not always hold true in a population-based sample.

OBJECTIVE

Hereditary Nonpolyposis Colorectal Cancer (HNPCC) has been defined clinically and genetically. The disorder has traditionally been recognized in kindreds with a clustering of related cancers in association with mutations in DNA mismatch repair genes. HNPCC is associated with a substantially increased risk for several forms of malignancy but particularly colorectal and endometrial cancer. There were three main objectives of this report: (1) to assess the sensitivity, specificity, and reliability of laboratory and genetic tests commonly used in evaluating patients for HNPCC (analytic validity); (2) to summarize the accuracy of commonly used clinical and laboratory characteristics for predicting the presence of HNPCC in patients with colorectal cancer (clinical validity) and use these estimates to describe the efficiency of various strategies for identifying patients with a mismatch repair mutation; (3) to describe the benefits and harms related to screening and testing patients with colorectal cancer and their family members for HNPCC.

METHODS

Published literature identified through an electronic search (through April 2006), review of relevant bibliographies, and suggestions from technical experts.

METHODS

We evaluated studies critically and summarized the data qualitatively or by meta-analysis when studies used similar methodology and endpoints. We used decision trees to describe the efficiency of various strategies for identifying patients with HNPCC from a hypothetical population of patients with colorectal cancer.

RESULTS

We included a total of 104 studies of which 40 addressed issues related to clinical validity, 3 to analytic validity, and 61 to benefits and harms. We identified only three studies on analytic validity and thus there exists a major gap in the published literature with regard to the accuracy and reliability of specific tests used in the evaluation of HNPCC. Among unselected patients with colorectal cancer who fulfilled the Amsterdam I criteria, 44% (95% CI: 35, 52%) carried pathogenic mismatch repair mutations (mainly in the MLH1 and MSH2 genes). The proportion was somewhat higher (51% [95% CI: 35, 66%]) among studies that performed sequencing on all available samples. The prevalence of MMR mutation carriers may be higher when genetic testing includes evaluation for large genomic deletions/rearrangements and when testing is also performed on MSH6 and PMS2. Approximately 71% (95% CI 63, 78%) of colorectal cancers from patients who fulfilled the Amsterdam I criteria demonstrated microsatellite instability while 40% (95% CI: 28, 53%) demonstrated loss of protein expression by immunohistochemistry. Of nine clinical strategies considered for detecting the presence of mismatch repair mutations in patients with colorectal cancer, the combination of three clinical predictors (age less than 50 years old at diagnosis; or a history of colorectal or endometrial cancer in a first degree family member; or the presence of multiple, synchronous or metachronous colorectal or endometrial cancers in the proband) combined with either immunohistochemistry (IHC) or MSI testing of tumor tissue identified a similar number of patients with mismatch repair mutations as other more complex strategies. There was little published information regarding potential harms associated with screening individuals with HNPCC-related cancers using clinical criteria (e.g. the Amsterdam criteria), MSI or IHC testing. Limited data suggested that testing probands for MMR mutations was not associated with severe psychological impact following formal counseling. Pre-test genetic counseling had good efficacy in improving knowledge about HNPCC and resulted in a high likelihood of proceeding with genetic testing, satisfaction in the decision to undergo genetic testing, and decreasing depression and distress levels among family members of HNPCC probands with cancer and among asymptomatic individuals from HNPCC families. Identification of HNPCC mutations was associated with an increase in the likelihood that family members of probands with CRC would undergo cancer-screening procedures. HNPCC family members who underwent cancer-screening procedures had a lower risk of developing HNPCC-related cancers and lower mortality rates than those who did not take actions. However, all of the relevant studies suggesting these benefits had important limitations. Survival was increased among asymptomatic HNPCC family members who received colonoscopy screening, regardless of their mutation status. There was limited direct evidence related to harms of the cancer-screening procedures in family members of probands with HNPCC. However, complication rates associated with these procedures in other settings are probably similar.

CONCLUSIONS

This report characterizes the accuracy of clinical and laboratory predictors of MMR mutations that can be used to identify patients with an increased risk of having MMR mutations. However, the sensitivity, specificity, and reliability of the tests used to evaluate individuals for suspected HNPCC is not known confidently. Data regarding the net benefits and harms associated with predictive genetic testing in patients with HNPCC-related cancers and their families members is incomplete but suggest that such testing improves compliance with screening procedures. At-risk family members who undergo screening colonoscopy have a reduced risk of developing HNPCC-related cancers and lower mortality. However, all studies supporting these benefits had important limitations.

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.

The extracolonic tumor spectrum of hereditary nonpolyposis colorectal cancer (HNPCC) includes cancer of the endometrium, ovaries, stomach, biliary tract, and urinary tract. This study was designed to determine the penetrance of colorectal and extracolonic tumors in HNPCC mutation carriers. Forty-nine patients (22 females and 27 males) were identified with an MSH2 germline mutation, and 56 patients (28 females and 28 males) were identified with an MLH1 I mutation. Cumulative incidence by age 60 (lifetime risk) and mean age of cancer diagnosis were compared. The lifetime risk of extracolonic cancers in MSH2 and MLH1 carriers was 48% and 11%, respectively (P = 0.016). Extracolonic cancer risk in MSH2 females and males was 69% and 34%, respectively (P = 0.042). Mean age of extracolonic cancer diagnosis was significantly older for MSH2 males than females (55.4 vs. 39.0, P = 0.013). No difference was observed in colorectal cancer risk between MLH1 and MSH2 carriers (84% vs. 71%). Colorectal cancer risk was 96% in MSH2 males compared to 39% in MSH2 females (P = 0.034). No differences in colorectal and extracolonic cancer risks between MLH1 females and males were identified. The risk of extracolonic cancer by age 60 was greater in MSH2 mutation carriers than in MLH1 carriers. Gender differences in colorectal and extracolonic cancer risk were observed for MSH2 carriers only. These phenotypic features of HNPCC genotypes may have clinical significance in the design of genotype-specific screening, surveillance, and follow-up for affected individuals.

Autosomal dominant cancer predisposition genes for common cancers such as breast cancer and colorectal cancer have been well recognized for over a decade. Monoallelic mutations in these genes are associated with high risks of adult-onset cancer. In recent years, it has become apparent that biallelic mutations in some of these genes, such as BRCA2, MSH2 and MLH1, result in distinctive phenotypes, including childhood cancer predisposition. Conversely, it has also become evident that some genes which cause autosomal recessive cancer predisposition syndromes such as Fanconi anaemia and ataxia-telangiectasia are associated with modestly increased risks of adult cancers in monoallelic mutation carriers. These observations raise interesting implications with respect to the identification and phenotypic characterization of cancer predisposition genes.

We have recently shown that hypomorphic Mre11 complex mouse mutants exhibit defects in the repair of meiotic double strand breaks (DSBs). This is associated with perturbation of synaptonemal complex morphogenesis, repair and regulation of crossover formation. To further assess the Mre11 complex's role in meiotic progression, we identified testis-specific NBS1-interacting proteins via two-hybrid screening in yeast. In this screen, Zip4h (Tex11), a male germ cell specific X-linked gene was isolated. Based on sequence and predicted structural similarity to the S. cerevisiae and A. thaliana Zip4 orthologs, ZIP4H appears to be the mammalian ortholog. In S. cerevisiae and A. thaliana, Zip4 is a meiosis-specific protein that regulates the level of meiotic crossovers, thus influencing homologous chromosome segregation in these organisms. As is true for hypomorphic Nbs1 (Nbs1(DeltaB/DeltaB)) mice, Zip4h(-/Y) mutant mice were fertile. Analysis of spermatocytes revealed a delay in meiotic double strand break repair and decreased crossover formation as inferred from DMC1 and MLH1 staining patterns, respectively. Achiasmate chromosomes at the first meiotic division were also observed in Zip4h(-/Y) mutants, consistent with the observed reduction in MLH1 focus formation. These results indicate that meiotic functions of Zip4 family members are conserved and support the view that the Mre11 complex and ZIP4H interact functionally during the execution of the meiotic program in mammals.

The Mre11 complex (consisting of MRE11, RAD50, and NBS1/Xrs2) is required for double-strand break (DSB) formation, processing, and checkpoint signaling during meiotic cell division in S. cerevisiae. Whereas studies of Mre11 complex mutants in S. pombe and A. thaliana indicate that the complex has other essential meiotic roles , relatively little is known regarding the functions of the complex downstream of meiotic break formation and processing or its role in meiosis in higher eukaryotes. We analyzed meiotic events in mice harboring hypomorphic Mre11 and Nbs1 mutations which, unlike null mutants, support viability . Our studies revealed defects in the temporal progression of meiotic prophase, incomplete and aberrant synapsis of homologous chromosomes, persistence of strand exchange proteins, and alterations in both the frequency and placement of MLH1 foci, a marker of crossovers. A unique sex-dependent effect on MLH1 foci and chiasmata numbers was observed: males exhibited an increase and females a decrease in recombination levels. Thus, our findings implicate the Mre11 complex in meiotic DNA repair and synapsis in mammals and indicate that the complex may contribute to the establishment of normal sex-specific differences in meiosis.

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.

OBJECTIVE

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths in the Western world, but our understanding of this disease is incomplete. The recent advent of new technologies has provided novel insights into the pathogenesis of CRC.

RESULTS

Genome-wide association studies have recently linked CRC to 10 common genetic variants or single-nucleotide polymorphisms that map to chromosomes 8q23, 8q24, 10p14, 11q23, 14q22, 15q13, 16q22, 18q21, 19q13 and 20p1. However, the causal significance of these variants is not understood, and some are located in poorly characterized genomic regions or gene deserts. Recent studies indicate that the single-nucleotide polymorphism rs6983267, which maps to 8q24, serves as an enhancer of MYC expression by binding T cell factor 4 (TCF4) and influencing Wnt signaling. In addition, several microRNAs interact with genes such as K-RAS, APC, p53, PTEN, TCF4, COX-2, DNMT3a and DNMT3b. Germline hypermethylation of the DNA mismatch repair genes MLH1 and MSH2 may serve as predisposing events in some CRC patients.

CONCLUSIONS

Recent studies have elucidated novel mechanisms involved in CRC, including the involvement of single-nucleotide polymorphisms not located within traditional genes, the role of microRNAs and epimutations in DNA mismatch repair genes. Interestingly, most of this progress has been made by understanding DNA that does not encode genes.

In most eutherian mammals, sex chromosomes synapse and recombine during male meiosis in a small region called pseudoautosomal region. However in some species sex chromosomes do not synapse, and how these chromosomes manage to ensure their proper segregation is under discussion. Here we present a study of the meiotic structure and behavior of sex chromosomes in one of these species, the Mongolian gerbil (Meriones unguiculatus). We have analyzed the location of synaptonemal complex (SC) proteins SYCP1 and SYCP3, as well as three proteins involved in the process of meiotic recombination (RAD51, MLH1, and gamma-H2AX). Our results show that although X and Y chromosomes are associated at pachytene and form a sex body, their axial elements (AEs) do not contact, and they never assemble a SC central element. Furthermore, MLH1 is not detected on the AEs of the sex chromosomes, indicating the absence of reciprocal recombination. At diplotene the organization of sex chromosomes changes strikingly, their AEs associate end to end, and SYCP3 forms an intricate network that occupies the Y chromosome and the distal region of the X chromosome long arm. Both the association of sex chromosomes and the SYCP3 structure are maintained until metaphase I. In anaphase I sex chromosomes migrate to opposite poles, but SYCP3 filaments connecting both chromosomes are observed. Hence, one can assume that SYCP3 modifications detected from diplotene onwards are correlated with the maintenance of sex chromosome association. These results demonstrate that some components of the SC may participate in the segregation of achiasmate sex chromosomes in eutherian mammals.

Microsatellite (MS) instability occurs in tumors with DNA mismatch repair (MMR) deficiencies but is typically absent in adjacent normal tissue. However, MS mutations have been observed in normal tissues from rare individuals with congenital MMR deficiencies. Autopsy tissues from a 4-year-old with congenital MMR deficiency (MLH1-/-) were examined for MS mutations. Insertions and deletions were observed in CA-repeat MS loci. Approximately 0.26 to 1.4 mutations per MS locus per cell were estimated to be present in normal heart, lymph node, kidney, and bladder epithelium. These findings illustrate that phenotypically normal MMR-deficient cells commonly accumulate MS mutations. Loss of MMR and the accumulation of some MS mutations may occur early in MMR-deficient tumor progression, even before a gatekeeper mutation.

The aim of this study was to estimate the contribution of deleterious mutations in BRCA1, BRCA2, MLH1, MSH2, MSH6 and PMS2 to invasive epithelial ovarian cancer (EOC) in the population. The coding sequence and splice site boundaries of all six genes were amplified in germline DNA from 2240 invasive EOC cases and 1535 controls. Barcoded fragment libraries were sequenced using the Illumina GAII or HiSeq and sequence data for each subject de-multiplexed prior to interpretation. GATK and Annovar were used for variant detection and annotation. After quality control 2222 cases (99.2%) and 1528 controls (99.5%) were included in the final analysis. We identified 193 EOC cases (8.7%) carrying a deleterious mutation in at least one gene compared with 10 controls (0.65%). Mutations were most frequent in BRCA1 and BRCA2, with 84 EOC cases (3.8%) carrying a BRCA1 mutation and 94 EOC cases (4.2%) carrying a BRCA2 mutation. The combined BRCA1 and BRCA2 mutation prevalence was 11% in high-grade serous disease. Seventeen EOC cases carried a mutation in a mismatch repair gene, including 10 MSH6 mutation carriers (0.45%) and 4 MSH2 mutation carriers (0.18%). At least 1 in 10 women with high-grade serous EOC has a BRCA1 or BRCA2 mutation. The development of next generation sequencing technologies enables rapid mutation screening for multiple susceptibility genes at once, suggesting that routine clinical testing of all incidence cases should be considered.

BACKGROUND

Lynch syndrome is an inherited tumour predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes. Mutation carriers have a high risk of developing colorectal cancer, but do not present with polyposis, a typical feature of other colorectal cancer syndromes such as familial adenomatous polyposis, in which polyposis reflects the high frequency of biallelic APC gene inactivation. We asked whether in Lynch syndrome biallelic inactivation of MMR genes occurred at a similar frequency to that of APC gene, and whether MMR inactivation resulted in detectable lesions within the intestinal mucosa.

METHODS

Resections done for small and large bowel cancer between January, 2002, and January, 2011, were retrieved. We systematically analysed non-tumorous mucosa from carriers of a Lynch syndrome mutation (set 1: ten patients) and control patients without Lynch syndrome (set 1: nine patients) for MMR protein expression (MLH1, MSH2, and EPCAM) with immunohistochemistry. We validated the findings in an independent sample set (set 2: 30 Lynch syndrome patients, 79 controls). We did an analysis of microsatellite instability by PCR analysis to test lesions for mismatch repair deficiency. We applied a Poisson regression model to analyse the distribution of MMR-deficient crypt foci counts and a Fisher's exact test to compare the prevalence of these foci between mutation carriers and control patients.

RESULTS

20 crypt foci with no MMR protein expression were detected in 20·1 cm(2) of non-tumorous mucosa from Lynch syndrome patients (set 1), an additional five were detected upon resectioning of two samples. In an independent validation set (set 2), two MMR-deficient crypt foci were noted in 2·2 cm(2) of mucosa. No MMR-deficient crypt foci were noted in non-tumorous mucosa from control patients without evidence for Lynch syndrome (set 1: 3·7 cm(2), set 2: 4·8 cm(2)). Microsatellite instability was detected in all seven MMR-deficient crypt foci analysed. A subset of these foci displayed unusual architectural and cytological abnormalities, although they had no polypous or adenomatous appearance.

CONCLUSIONS

We identified a novel type of lesion, the MMR-deficient crypt focus, as the manifestation of biallelic MMR gene inactivation in Lynch syndrome. The abundance of MMR-deficient crypt foci indicates a high frequency of biallelic MMR gene inactivation, which is in sharp contrast with the low number of clinically manifest cancers in Lynch syndrome. This discrepancy suggests that most MMR-deficient crypt foci do not progress to cancer. We propose Lynch syndrome as a unique model syndrome for studying initial steps of MMR deficiency, tumour initiation and, possibly, elimination.

BACKGROUND

German Cancer Aid and German Research Foundation.

Hereditary non-polyposis colorectal cancer (HNPCC) was previously synonymous with Lynch syndrome; however, identification of the role of germline mutations in the DNA mismatch repair (MMR) genes has made it possible to differentiate Lynch syndrome from other conditions associated with familial colorectal cancer (CRC). Broadly, HNPCC may be dichotomized into conditions that demonstrate defective DNA MMR and microsatellite instability (MSI) vs those conditions that demonstrate intact DNA MMR. Conditions characterized by MMR deficient CRCs include Lynch syndrome (germline MMR mutation), Lynch-like syndrome (biallelic somatic MMR mutations), constitutional MMR deficiency syndrome (biallelic germline MMR mutations), and sporadic MSI CRC (somatic biallelic methylation of MLH1). HNPCC conditions with intact DNA MMR associated with familial CRC include polymerase proofreading associated polyposis and familial colorectal cancer type X. Although next generation sequencing technologies have elucidated the genetic cause for some HNPCC conditions, others remain genetically undefined. Differentiating between Lynch syndrome and the other HNPCC disorders has profound implications for cancer risk assessment and surveillance of affected patients and their at-risk relatives. Clinical suspicion coupled with molecular tumor analysis and testing for germline mutations can help differentiate the clinical mimicry within HNPCC and facilitate diagnosis and management.

Carcinomas of the endometrium and ovary with undifferentiated components are uncommon neoplasms that are likely underdiagnosed. They are important to recognize as they have been shown to be clinically aggressive. We identified 32 carcinomas with undifferentiated components as defined by Silva and co-workers, 26 endometrial and 6 of ovarian origin. The patient age ranged from 21 to 76 years (median 55); 40% of patients were <or=50 years of age. Most patients (58% of endometrial and 83% of ovarian carcinomas with undifferentiated components) presented at advanced stages (FIGO III-IV). Pelvic and para-aortic lymph nodes were the most frequent sites of metastases. Twenty tumors, entirely undifferentiated, consisted of sheets of dyshesive, ovoid cells with uniform, large vesicular nuclei, whereas 12 tumors contained combinations of differentiated endometrioid adenocarcinoma with undifferentiated components. Although most undifferentiated tumors had a monotonous cytologic appearance without prominent stroma, six showed focal nuclear pleomorphism and eight cases had variably sized zones of rhabdoid cells in a background of myxoid stroma. The tumors were frequently misdiagnosed; they received a wide range of diagnoses, including FIGO grade 2 or 3 endometrioid carcinoma, carcinosarcoma, high-grade sarcoma including endometrial stromal sarcoma, neuroendocrine carcinoma, lymphoma, granulosa cell tumor and epithelioid sarcoma. Up to 86% of the cases showed focal, but strong keratin and/or epithelial membrane antigen staining, with CK18 being the most frequently positive keratin stain. They were predominantly negative for neuroendocrine markers, smooth muscle markers and estrogen receptor/progesterone receptor. Mismatch repair protein expression by immunohistochemistry was evaluated in 17 cases, and 8 (47%) were abnormal (7 with loss of <em>MLH1</em>/PMS2 and 1 with MSH6 loss). Follow-up was available for 27 patients, although it was very short in many cases, ranging from 0.5 to 89 months (median 9 months). Eleven patients (41%) died of the disease in 0.5-20 months, four are alive with disease and twelve patients have no evidence of disease. Endometrial and ovarian carcinomas with undifferentiated components have a broad histologic differential diagnosis, but they show specific histologic features that should enable accurate diagnosis. These tumors can occur in young women, may be associated with microsatellite instability and behave in a clinically aggressive manner.

Genomic sequences encoding the 3' exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms.

OBJECTIVE

Locally aggressive pituitary tumors (LAPT) and pituitary carcinomas respond poorly to conventional therapy and cytotoxic drugs. Temozolomide (TMZ) is an oral alkylating drug with good tolerability, approved for treatment of malignant gliomas. The experience of its use in pituitary tumors is limited.

METHODS

We report on 24 patients with aggressive pituitary tumors (16 LAPTs, 8 carcinomas) treated with TMZ for a median of 6 months (range 1-23). Follow-up ranged from 4 to 91 months with a median of 32.5 months. 19/24 tumors were hormone secreting (PRL 9, ACTH 4, GH 4, GH/PRL 2). Ki-67 was 2-50% in LAPTs, and 5-80% in carcinomas.

RESULTS

Response to TMZ and the association with tumor expression of O6-methylguanine DNA methyltransferase (MGMT), MLH1, MSH2, and MSH6, examined by immunohistochemistry.

RESULTS

Complete tumor regression occurred in two carcinomas and persisted at follow-up after 48 and 91 months, respectively. Partial regress of tumor mass ranging from 35% to 80% occurred in 5 LAPTs and 2 carcinomas. Another patient with LAPT had a 71% decrease in prolactin levels without change in tumor volume. Three LAPTs could not be evaluated. Median MGMT staining was 9% (5-20%) in responders vs 93% (50-100%) in nonresponders. Loss of MSH2 and MSH 6 was observed in a single patient who had a rapid development of resistance to TMZ.

CONCLUSIONS

This study shows that TMZ is a valuable treatment option for patients with uncontrolled pituitary tumors. The data suggest that tumoral MGMT staining below 50% is associated with a high likelihood of treatment response.

BACKGROUND

DNA polymerase ɛ (POLE) exonuclease domain mutations characterize a subtype of endometrial cancer (EC) with a markedly increased somatic mutational burden. POLE-mutant tumors were described as a molecular subtype with improved progression-free survival by The Cancer Genome Atlas. In this study, the frequency, spectrum, prognostic significance, and potential clinical application of POLE mutations were investigated in patients with endometrioid EC.

METHODS

Polymerase chain reaction amplification and Sanger sequencing were used to test for POLE mutations in 544 tumors. Correlations between demographic, survival, clinicopathologic, and molecular features were investigated. Statistical tests were 2-sided.

RESULTS

Thirty POLE mutations (5.6%) were identified. Mutations were associated with younger age (<60 years; P=.001). POLE mutations were detected in tumors with microsatellite stability (MSS) and microsatellite instability (MSI) at similar frequencies (5.9% and 5.2%, respectively) and were most common in tumors with MSI that lacked mutL homolog 1 (MLH1) methylation (P<.001). There was no association with progression-free survival (hazard ratio, 0.22; P=.127).

CONCLUSIONS

The discovery that mutations occur with equal frequency in MSS and MSI tumors and are most frequent in MSI tumors lacking MLH1 methylation has implications for Lynch syndrome screening and mutation testing. The current results indicate that POLE mutations are associated with somatic mutation in DNA mismatch repair genes in a subset of tumors. The absence of an association between POLE mutation and progression-free survival indicates that POLE mutation status is unlikely to be a clinically useful prognostic marker. However, POLE testing in MSI ECs could serve as a marker of somatic disease origin. Therefore, POLE tumor testing may be a valuable exclusionary criterion for Lynch syndrome gene testing.

OBJECTIVE

Mismatch repair (MMR) deficiencies are the hallmark of tumors arising in Lynch syndrome, however, in approximately 15% of sporadic colorectal cancers (CRC) these deficiencies most often are associated with somatic methylation of the MMR gene MLH1. Recently, an oncogenic mutation in the BRAF gene has been involved in sporadic CRC showing MMR deficiencies as a result of MLH1 promoter methylation. The aim of this study was to evaluate the contribution of BRAF V600E mutation analysis in the identification of MSH2/MLH1 gene mutation carriers in newly diagnosed CRC patients.

METHODS

BRAF V600E mutation was analyzed in CRC patients with MMR deficiencies (microsatellite instability and/or lack of MLH1/MSH2 protein expression) in the EPICOLON population-based study. The effectiveness and efficiency of different strategies were evaluated with respect to the presence of MSH2/MLH1 germline mutations.

RESULTS

MMR deficiencies were detected in 119 of the 1222 CRC patients with tumors showing either microsatellite instability (n = 111) or loss of protein expression (n = 81). BRAF mutation was detected in 22 (18.5%) of the patients. None of the patients with unambiguous germline mutation had BRAF mutation. Regardless of the strategy used to identify MSH2/MLH1 gene carriers, the introduction of BRAF analysis in these patients slightly improves their effectiveness. The introduction of BRAF mutation analysis as a step before germline genetic testing in patients with MMR deficiencies achieved a significant reduction in costs per mutation detected.

CONCLUSIONS

Detection of BRAF V600E mutation could simplify and improve the cost effectiveness of genetic testing for hereditary nonpolyposis colorectal cancer, especially in patients whose family history is incomplete or unknown.