The mutational spectrum of FOXA2 in endometrioid endometrial cancer points to a tumor suppressor role.

BACKGROUND: Forkhead box protein A2 (FOXA2) plays an important in development, cellular metabolism and tumorigenesis. The Cancer Genome Atlas (TCGA) identified a modest frequency of FOXA2 mutations in endometrioid endometrial cancers (EEC). The current study sought to determine the relationship between FOXA2 mutation and clinicopathologic features in EEC and FOXA2 expression. METHODS: Polymerase chain reaction (PCR) amplification and sequencing were used to identify mutations in 542 EEC. Western blot, quantitative reverse transcriptase PCR (qRT-PCR) and immunohistochemistry (IHC) were used to assess expression. Methylation analysis was performed using combined bisulfite restriction analysis (COBRA) and sequencing. Chi-squared, Fisher's exact, Student's t- and log-rank tests were performed. RESULTS: Fifty-one mutations were identified in 49 tumors (9.4% mutation rate). The majority of mutations were novel, loss of function (LOF) (78.4%) mutations, and most disrupted the DNA-binding domain (58.8%). Six recurrent mutations were identified. Only two tumors had two mutations and there was no evidence for FOXA2 allelic loss. Mutation status was associated with tumor grade and not associated with survival outcomes. Methylation of the FOXA2 promoter region was highly variable. Most tumors expressed FOXA2 at both the mRNA and protein level. In those tumors with mutations, the majority of cases expressed both alleles. CONCLUSION: FOXA2 is frequently mutated in EEC. The pattern of FOXA2 mutations and expression in tumors suggests complex regulation and a haploinsufficient or dominant-negative tumor suppressor function. In vitro studies may shed light on how mutations in FOXA2 affect FOXA2 pioneer and/or transcription factor functions in EEC.

Preferences for return of incidental findings from genome sequencing among women diagnosed with breast cancer at a young age.

While experts have made recommendations, information is needed regarding what genome sequencing results patients would want returned. We investigated what results women diagnosed with breast cancer at a young age would want returned and why. We conducted 60 semi-structured, in-person individual interviews with women diagnosed with breast cancer at age 40 or younger. We examined interest in six types of incidental findings and reasons for interest or disinterest in each type. Two coders independently coded interview transcripts; analysis was conducted using NVivo 10. Most participants were at least somewhat interested in all six result types, but strongest interest was in actionable results (i.e. variants affecting risk of a preventable or treatable disease and treatment response). Reasons for interest varied between different result types. Some participants were not interested or ambivalent about results not seen as currently actionable. Participants wanted to be able to choose what results are returned. Participants distinguished between types of individual genome sequencing results, with different reasons for wanting different types of information. The findings suggest that a focus on actionable results can be a common ground for all stakeholders in developing a policy for returning individual genome sequencing results.

Frequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes.

Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We have identified a total of 11 different FGFR2 mutations in 3/10 (30%) of endometrial cell lines and 19/187 (10%) of primary uterine tumors. Mutations were seen primarily in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). The majority of the somatic mutations identified were identical to germline activating mutations in FGFR2 and FGFR3 that cause Apert Syndrome, Beare-Stevenson Syndrome, hypochondroplasia, achondroplasia and SADDAN syndrome. The two most common somatic mutations identified were S252W (in eight tumors) and N550K (in five samples). Four novel mutations were identified, three of which are also likely to result in receptor gain-of-function. Extensive functional analyses have already been performed on many of these mutations, demonstrating they result in receptor activation through a variety of mechanisms. The discovery of activating FGFR2 mutations in endometrial carcinoma raises the possibility of employing anti-FGFR molecularly targeted therapies in patients with advanced or recurrent endometrial carcinoma.

Increased risk for abnormalities on perioperative colon screening in patients with microsatellite instability-positive endometrial carcinoma.

Microsatellite instability (MSI) is a feature of certain hereditary and sporadic endometrial and colon cancers. We set out to determine whether molecular stratification of endometrial cancers based on tumor MSI status could help identify patients at increased risk for abnormalities found on perioperative colon screening. From a prospectively accrued series of 413 patients, medical records were reviewed from 94 patients with MSI positive (MSI+) and 94 patients with MSI negative (MSI-) endometrial cancers, matched by year of diagnosis. We reviewed clinicopathologic data and results of perioperative colon screening. Differences were analyzed using Fisher exact test and logistic regression analysis. There were no significant clinicopathologic differences between the two cohorts. Sixty-five percent of patients in each group underwent perioperative colon screening. However, patients with MSI+ cancers had a twofold increase in the frequency of colonic abnormalities (30% versus 14.8%, P = 0.044) over those with MSI- cancers. Furthermore, the only primary colon cancers (N = 2) were found in women with MSI+ endometrial cancers that were unmethylated at the MLH1 promoter. Our data suggest that patients with MSI+ endometrial cancers are at increased risk for abnormalities on perioperative colon screening. Those with MSI+MLH1 unmethylated cancers appear to be at highest risk.

A randomised controlled trial of transverse skin crease vs. vertical midline incision for right hemicolectomy.

BACKGROUND: A transverse skin crease incision for right hemicolectomy may result in more rapid recovery than traditional vertical midline incision. This hypothesis was tested with a prospective randomised trial. METHODS: Patients from 2 centres undergoing right hemicolectomy were randomised to received a midline or transverse incision. Incision lengths were sufficient to enable unrestricted resection of the right colon. Patients and carers were blinded to the incisions using strategically placed dressings. Analgesia and oral intake were controlled by the patient. Operative details and recovery parameters were compared. RESULTS: A total of 28 patients were randomised. Demographic data and tumour characteristics of the two treatment groups were similar. The transverse incision group had a slightly shorter median wound (10 cm vs. 11 cm, p<0.05). Operative time, analgesia requirements, recovery parameters (time to discharge, 6.5 vs. 6.5 days) and frequency of complications were otherwise comparable. CONCLUSIONS: A transverse skin crease incision for right hemicolectomy results in a slightly smaller wound but no other advantages were demonstrated compared with a traditional vertical midline incision.

Mlh1 deficiency accelerates myeloid leukemogenesis in neurofibromatosis 1 (Nf1) heterozygous mice.

Defects in DNA mismatch repair (MMR) have been implicated in the genesis of a diverse set of human cancers. Recent studies have suggested that one of the targets of MMR is the neurofibromatosis 1 (NF1) gene. To evaluate the contribution of Mlh1 MMR deficiency to Nf1 tumorigenesis, Mlh1-/-;Nf1+/- mice were generated. All Mlh1-/-;Nf1+/- mice (n=21) were dead by 260 days compared to none of the Nf1+/- mice. In all, 50% of the Mlh1-/-;Nf1+/- mice were dead at 150 days compared to 252 days for Mlh1-/- mice. Nine of the Mlh1-/-;Nf1+/- mice were found to harbor intrathoracic NOS2-immunoreactive myeloid leukemias similar to the hematopoietic malignancies observed in older Nf1+/- mice. As expected, significant microsatellite instability was observed in six of six tumors and neurofibromin expression was lost in all tumors analysed. These results suggest that MMR deficiency can accelerate myeloid leukemogenesis in Nf1+/- mice, presumably by inactivating Nf1 gene expression.

Microsatellite instability, MLH1 promoter methylation, and loss of mismatch repair in endometrial cancer and concomitant atypical hyperplasia.

OBJECTIVE: MLH1 methylation is associated with the microsatellite instability (MSI) phenotype in endometrial cancer and atypical endometrial hyperplasia, a premalignant precursor to carcinoma. The observation that methylation is also seen in atypical endometrial hyperplasia without MSI suggests that methylation is an early event in endometrial tumorigenesis. Our objective was to determine if methylation is always present in MSI-positive atypical hyperplasia concomitant with MSI-positive, methylation-positive carcinoma. METHODS: We used laser capture microdissection to study MLH1 methylation and MSI in a large series of endometrial cancer cases that had previously been shown to have methylation and the MSI-high (MSI-H) phenotype. We resampled areas of carcinoma from 27 patients along with 51 foci of concomitant atypical endometrial hyperplasia. RESULTS: Consistent with previous reports, we saw MLH1 methylation in areas of atypical endometrial hyperplasia that did not show MSI. In addition, we noted that 18% of the MSI-H atypical endometrial hyperplasia DNAs lacked methylation of critical cytosines in the MLH1 promoter. Immunohistochemistry studies showed that these MSI-H unmethylated foci of atypical endometrial hyperplasia failed to express MLH1, as did regions of simple hyperplasia. CONCLUSION: Methylation of the MLH1 promoter is an early event in endometrial tumorigenesis. Given that not all MSI-positive tissues had methylation at cytosines -229 and -231, it appears that methylation may not be required for MLH1 silencing and loss of mismatch repair.

Genotypic and phenotypic progression in endometrial tumorigenesis: determining when defects in DNA mismatch repair and KRAS2 occur.

We set out to determine the relative timing of loss of DNA mismatch repair and KRAS2 mutation in endometrial tumorigenesis. We studied endometrial carcinoma (CA) and synchronous atypical endometrial hyperplasia (AEH), the premalignant precursor of endometrial cancer. Carcinoma and hyperplasia were investigated for loss of mismatch repair as evidenced by microsatellite instability (MSI) and for KRAS2 mutations. Endometrial cancers previously shown to be MSI-positive were evaluated for KRAS2 codon 12 and 13 mutations. DNA was isolated from foci of AEH concomitant with, but physically remote from, the cancers by use of tissues prepared by laser capture microdissection (LCM). The AEH DNAs were then assessed for MSI and KRAS2 mutations. Of 210 endometrial CAs investigated, 51 (26%) were MSI-positive, and among those, 21 (41%) arose concomitantly with AEH. Of 41 foci of AEH (mean, two foci per patient) investigated, 34 (83%) were MSI-positive. KRAS2 mutations were seen in 5/51 (10%) MSI-positive carcinomas. From the five patients informative for both KRAS2 mutation and MSI, 10 foci of AEH were available for investigation. All 10 AEH specimens (100%) were MSI-positive, and six (60%) had the KRAS2 mutation present in the coexisting CA. The observation that some MSI-positive AEH specimens lack the KRAS2 mutation seen in the coexisting CA supports a model in which loss of DNA mismatch repair precedes KRAS2 mutation. However, in addition to the absence of KRAS2 mutations in AEH, we discovered mutations in LCM hyperplasia and carcinoma specimens that were not present in the portion of the cancers originally investigated. These discordant genotypes suggest genetic heterogeneity in endometrial hyperplasia and concomitant cancer.

Characterization of the homeodomain gene EMX2: sequence conservation, expression analysis, and a search for mutations in endometrial cancers.

Previous loss-of-heterozygosity studies in endometrial carcinoma mapped a putative tumor suppressor gene to 10q25.3-26.1. An analysis of genomic sequences for the deletion interval showed several expressed sequence tags and the homeodomain gene EMX2, a homologue of Drosophila melanogaster empty spiracles. Expression studies showed that EMX2 transcripts are abundant in the adult uterus and that message levels seem to be inversely correlated with endometrial proliferation. EMX2 RNA was more abundant in quiescent postmenopausal endometrium than in premenopausal endometrium. We found decreased EMX2 expression in a subset of primary endometrial tumors, and four of six endometrial cancer cell lines investigated failed to express EMX2. The predicted protein showed extensive amino acid conservation with EMX2 sequences from several vertebrates. There was also considerable evolutionary conservation in the 3' untranslated region. To examine the potential function of EMX2 in endometrial tumorigenesis, we investigated 20 primary tumors and 6 endometrial cancer cell lines for mutations. Two primary tumors had mutations. Inactivation or reduced expression of EMX2 in cancers, coupled with increased expression in the quiescent endometrium, indicate that this homeodomain gene is involved in maintenance of the differentiated state.

The T-box transcription factor gene TBX22 is mutated in X-linked cleft palate and ankyloglossia.

Formation of the secondary palate is a complex step during craniofacial development. Disturbance of the events affecting palatogenesis results in a failure of the palate to close. As a consequence of deformity, an affected child will have problems with feeding, speech, hearing, dentition and psychological development. Cleft palate occurs frequently, affecting approximately 1 in 1,500 births; it is usually considered a sporadic occurrence resulting from an interaction between genetic and environmental factors. Although several susceptibility loci have been implicated, attempts to link genetic variation to functional effects have met with little success. Cleft palate with ankyloglossia (CPX; MIM 303400) is inherited as a semidominant X-linked disorder previously described in several large families of different ethnic origins and has been the subject of several studies that localized the causative gene to Xq21 (refs. 10-13). Here we show that CPX is caused by mutations in the gene encoding the recently described T-box transcription factor TBX22 (ref. 14). Members of the T-box gene family are known to play essential roles in early vertebrate development, especially in mesoderm specification. We demonstrate that TBX22 is a major gene determinant crucial to human palatogenesis. The spectrum of nonsense, splice-site, frameshift and missense mutations we have identified in this study indicates that the cleft phenotype results from a complete loss of TBX22 function.

Methylation and mutational analysis of p27(kip1) in prostate carcinoma.

BACKGROUND: We have previously identified 12p12-13 as a region of frequent genetic loss in prostate carcinoma. A candidate tumor suppressor gene at this locus is the cyclin dependent kinase inhibitor p27(kip1), which has been implicated as a marker of aggressive prostate carcinoma. Herein, we examine metastatic prostate tumors, xenografts, and cell lines for gene inactivation via mutational inactivation or promoter hypermethylation. METHODS: Mutation analysis was performed on metastatic prostate tumors of 18 patients, eight prostate carcinoma cell lines, and 18 xenografts by PCR amplification of the entire open reading frame of p27(kip1). PCR products were sequenced directly using internal primers. Methylation analysis was performed on four cell lines and nine xenografts using direct sequencing of cloned PCR products of bisulfite treated DNA. Presence of a CpG was consistent with methylation of that cytosine in the original sample. RESULTS: With the exception of the previously reported homozygous deletion, no additional mutations were identified. Methylated CpG residues were identified in three xenografts (LuCAP23, LuCAP35, and PC82) and the methylated residues clustered at six sites; the cytosines 69, 149, 191, 286, 349, and 487 base pairs 5' of the ATG start codon. However, no sample demonstrated promotor methylation in all sequenced clones and the number of methylated base pairs ranged from seven to three, not the level usually associated with gene silencing. CONCLUSIONS: Mutational inactivation of p27(kip1) is a rare event in metastatic prostate carcinoma. While CpG methylation does occur, it is an infrequent event and does not appear to be the mechanism of p27(kip1) down regulation in prostate carcinoma.

Clinical significance of microsatellite instability in endometrial carcinoma.

BACKGROUND: The purpose of this study was to compare the clinical characteristics of endometrial carcinomas with and without microsatellite instability (MSI). METHODS: The authors prospectively acquired DNA from patients with endometrial carcinomas at Washington University Medical Center. Tumors were assigned MSI (+) status when two or more of five microsatellite repeat markers revealed novel bands in tumor DNA not present in the corresponding normal DNA. Clinical characteristics and survival data of patients with and without MSI were abstracted from patient charts. Statistical significance was calculated with the chi-square test, and survival was assessed with Kaplan-Meier methods. RESULTS: The authors found 65 of 70 (93%) patients with MSI (+) tumors to be of white race, whereas only 124 of 159 (78%) patients with MSI (-) tumors were white (P = 0.012). Advanced disease (International Federation of Gynecology and Obstetrics Stage III-IV) was observed in 9 of 70 (13%) MSI (+) patients and 44 of 159 (28%) MSI (-) patients (P = 0.017). In addition, aggressive histologic subtypes were observed less frequently in MSI (+) tumors (6/70 [8%]) than in MSI (-) tumors (30 of 159 [19%]) (P = 0.034). Race and stage were shown by multivariate analysis to be different in MSI (+) and MSI (-) patients. Recurrence and overall survival were similar in the two groups. CONCLUSIONS: Patients with MSI (+) tumors were more likely to be of white race and to present with early stage disease. Further investigation is needed to explain why patients with MSI (+) tumors have similar survival to patients with MSI (-) tumors, despite presenting at earlier stages, being of white race, and being less likely to be associated with virulent histologic subtypes.

Absence of PTEN repeat tract mutation in endometrial cancers with microsatellite instability.

OBJECTIVE: PTEN, a tumor suppressor gene shown to be frequently mutated in endometrial cancers, has been suggested to be a target of microsatellite instability (MSI)-driven mutagenesis. We set out to investigate the relationship between MSI and PTEN mutation in a large series of primary endometrial carcinomas. METHODS: Thirty-nine MSI-positive endometrial cancers were evaluated by single-strand conformational variant analysis and direct sequencing to screen all nine PTEN exons for mutation. RESULTS: Fifteen specimens (38%) demonstrated 16 PTEN mutations. We observed only one alteration in the poly-adenine repeat of exon 8 that is suggested to be a target for mutation in endometrial cancers with MSI. Seven of 16 (44%) mutations in our series were deletions of >/=3 bp, a class of mutation not usually associated with tumors with defective DNA mismatch repair. To determine the significance of this high frequency of deletion, 26 additional endometrial cancers without MSI were matched with the 39 MSI-positive cancers for the prognostic factors of tumor histology, stage, grade, and patient race. The MSI-positive tumors had a significantly higher frequency of deletions involving >/=3 bp when compared with the MSI-negative group (5/11 versus 0/10, P = 0.035). CONCLUSIONS: Repeat tract mutation in PTEN is an uncommon event in MSI-positive cancers. Deletion of >/=3 bp in this gene is more common in MSI-positive cancers when compared with tumors without MSI.

16q loss of heterozygosity and microsatellite instability in Wilms' tumor.

BACKGROUND/PURPOSE: Wilms' tumor is the most common renal malignancy of childhood. Loss of heterozygosity (LOH) at 16q is seen in about 17% of cases and has been associated with a poor prognosis. To more precisely define the pattern of 16q deletion exhibited by Wilms' tumor, the authors performed a detailed LOH analysis of 96 specimens using polymorphic microsatellite repeat markers. The authors also evaluated the neoplasms for the presence of microsatellite instability (MSI). METHODS: A total of 96 DNA samples were studied using polymerase chain reaction-based LOH analyses amplifying polymorphic microsatellite repeat markers. Screening for MSI using 2 additional genetic markers also was carried out. RESULTS: The authors found 16q LOH in 14 of the specimens evaluated. Comprehensive analysis of these LOH-positive specimens showed a region of loss spanning 16p11.2-q22.1 and a separate distal region of LOH at 16q23.2-24.2. The distal region of deletion is very small, estimated to be approximately 2.4 megabases. In addition to the observed LOH, 2 specimens were found to consistently exhibit MSI, which has not been reported previously in Wilms' tumor. CONCLUSIONS: The smallest consensus region of deletion in our analysis of Wilms' tumor 16q LOH measures 2.4 megabases at 16q23.2-q24.2. Additionally, MSI was present in a subset of tumor specimens suggesting that defects in DNA mismatch repair may contribute to the pathogenesis of Wilms' tumor.

Atypical clustering of gynecologic malignancies: A family study including molecular analysis of candidate genes.

OBJECTIVE: We set out to determine whether hereditary nonpolyposis colorectal cancer (HNPCC) was responsible for cancer susceptibility in a family with gynecologic malignancies in three consecutive generations. METHODS: A detailed family history study, including review of medical records, was undertaken. Tumor DNAs from affected family members were evaluated for microsatellite instability (MSI). Linkage between cancer susceptibility and the candidate DNA mismatch repair genes MLH1, MSH2, MSH3, and MSH6 (GTBP) was investigated. MLH1 and MSH2 protein expression was evaluated by immunohistochemistry and MSH2 was investigated for mutation. RESULTS: Four gynecologic malignancies in the core family were confirmed. MSI was seen in six of seven cancers studied. The only MSI-negative tumor was an ovarian cancer from the proband's maternal grandmother, which arose at the age of 92. Haplotype analysis using chromosome 2p markers implicated the MSH2 gene in this family's cancer susceptibility. MSH2 protein expression was absent in an MSI-positive colon cancer from an affected family member. CONCLUSIONS: The inability to exclude linkage of MSH2 with the disease susceptibility, the presence of the MSI phenotype in cancers from family members sharing the same region of chromosome 2p, and the lack of immunodetectable MSH2 point to MSH2-associated HNPCC as a cause for this family's cancer susceptibility. Continued efforts to increase awareness of the heritability of endometrial cancer should improve our understanding of the disease, with resultant improved surveillance strategies, recommendations for surgical and chemoprophylaxis, and identification of patients at risk for malignancy as a result of HNPCC.

Mutational analysis of the PMS2 gene in sporadic endometrial cancers with microsatellite instability.

OBJECTIVE: Approximately 20% of endometrial tumors have a defect in DNA mismatch repair and exhibit microsatellite instability (MSI). We assessed the role of the PMS2 DNA mismatch repair gene in MSI-positive sporadic endometrial tumors. METHODS: We examined 40 sporadic endometrial tumor specimens with MSI. All 15 exons of the PMS2 gene were investigated for sequence alterations by single-strand conformational variant analysis. RESULTS: Twelve polymorphisms were identified, 8 of which were in the coding sequence. Four specimens revealed mutations in intronic sequences that are not predicted to affect the PMS2 mRNA. No mutations were detected within the coding region of the PMS2 gene. CONCLUSION: We conclude that structural mutations in the PMS2 gene are not responsible for defective DNA mismatch repair in sporadic endometrial cancers with MSI. The identification of single nucleotide polymorphisms in the PMS2 locus may aid in the mapping and characterization of genetic diseases.

A 1-Mb bacterial clone contig spanning the endometrial cancer deletion region at 1p32-p33.

Our laboratory previously reported a high frequency of loss of heterozygosity (LOH) at 1p32-p33 in endometrial cancers. LOH at 1p32-p33 is a specific feature of one of the most aggressive forms of endometrial carcinoma, uterine papillary serous cancer (UPSC). The minimum region of allelic loss in UPSC is defined by D1S190 and D1S447, an interval corresponding to less than 1 cM. Here we describe the construction and characterization of a sequence-ready clone contig that spans the deletion region. The contig consists of 24 bacterial artificial chromosome clones and 18 P1 artificial chromosome clones and spans approximately 1050 kb. The consensus region of allelic loss between D1S190 and D1S447 represents approximately 792 kb. Eight previously described ESTs have been positioned within the contig.

A specific CpG methylation pattern of the MGMT promoter region associated with reduced MGMT expression in primary colorectal cancers.

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation-sensitive restriction enzymes (BssHII, SacII, Eagl, Nael, and Smal) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of Eagl and Nael sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P<0.01). Methylation-sensitive single-nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by Eagl and Nael digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the Eagl and Nael sites and nt -128 than in the 13 tumors lacking the methylation pattern (P<0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression.

MLH1 promoter methylation and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers.

Defective DNA mismatch repair in human tumors leads to genome-wide instability of microsatellite repeats and a molecular phenotype referred to as microsatellite instability (MSI). MSI has been reported in a variety of cancers and is a consistent feature of tumors from patients with hereditary non-polyposis colorectal cancer. Approximately 20% of cancers of the uterine endometrium, the fifth most common cancer of women world-wide, exhibit MSI. Although the frequency of MSI is higher in endometrial cancers than in any other common malignancy, the genetic basis of MSI in these tumors has remained elusive. We investigated the role that methylation of the MLH1 DNA mismatch repair gene plays in the genesis of MSI in a large series of sporadic endometrial cancers. The MLH1 promoter was methylated in 41 of 53 (77%) MSI-positive cancers investigated. In MSI-negative tumors on the other hand, there was evidence for limited methylation in only one of 11 tumors studied. Immunohistochemical investigation of a subset of the tumors revealed that methylation of the MLH1 promoter in MSI-positive tumors was associated with loss of MLH1 expression. Immunohistochemistry proved that two MSI-positive tumors lacking MLH1 methylation failed to express the MSH2 mismatch repair gene. Both of these cancers came from women who had family and medical histories suggestive of inherited cancer susceptibility. These observations suggest that epigenetic changes in the MLH1 locus account for MSI in most cases of sporadic endometrial cancers and provide additional evidence that the MSH2 gene may contribute substantially to inherited forms of endometrial cancer.

Germline mutations in the multiple endocrine neoplasia type 1 gene: evidence for frequent splicing defects.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial cancer syndrome characterized by parathyroid hyperplasia, pituitary adenomas, and neuroendocrine tumors of the pancreas and duodenum. In 1997, the MEN1 tumor suppressor gene was identified, and numerous germline mutations have been reported to be distributed throughout the gene. We used single strand conformational variant (SSCV) analysis to search for germline mutations in the members of 33 kindreds with a confirmed diagnosis of MEN 1. SSCV analysis revealed 25 conformational variants representing germline mutations that are predicted to result in loss of normal menin function. Twenty different disease-associated mutations were identified: five resulting in potential abnormal RNA splicing, two missense mutations, seven nonsense mutations, and six frameshift mutations. The aberrant splice products were identified and confirmed by RT-PCR and direct sequence analysis for two of the five splice mutations. Sixteen of the 20 (80%) mutations identified have not been previously reported. Mutations were not identified in eight kindreds with signs and symptoms consistent with MEN 1. The SSCV analysis revealed mutations in 76% (25 of 33) of the kindreds investigated, thus showing SSCV analysis to be a reliable mutation detection strategy. One-fifth of the mutations identified in this study involve intron sequences, therefore, highlighting the importance of including intron sequences in the search for germline mutations in the MEN1 gene. The need to investigate the entire gene when characterizing new MEN 1 families presents challenges in the translation of genetic studies to efficient clinical diagnostic tests.