Treatment of tailgut cysts by extended distal rectal segmental resection with rectoanal anastomosis.

PURPOSE: Complete surgical resection is the treatment of choice for tailgut cysts, because of their malignant potential and tendency to regrow if incompletely resected. We report our experience of treating patients with tailgut cysts, and discuss diagnostics, surgical approaches, and follow-up. METHODS: We performed extended distal rectal segmental resection of the tailgut cyst, with rectoanal anastomosis. We report the clinical, radiological, pathological, and surgical findings, describe the procedures performed, and summarize follow-up data. RESULTS: Two patients underwent en-bloc resection of a tailgut cyst, the adjacent part of the levator muscle, and the distal rectal segment, followed by an end-to-end rectoanal anastomosis. There was no evidence of anastomotic leakage postoperatively. At the time of writing, our patients were relapse-free with no, or non-limiting, symptoms of anal incontinence, respectively. CONCLUSIONS: This surgical approach appears to have a low complication rate and good recovery outcomes. Moreover, as the sphincter is preserved, so is the postoperative anorectal function. This approach could result in a low recurrence rate.

Prevalence of Lynch syndrome in unselected patients with endometrial or ovarian cancer.

INTRODUCTION: Lynch syndrome is known by healthcare providers mainly for patients with colorectal cancer. Awareness of other associated tumors, such as endometrial or ovarian cancer, is low. This study aimed to analyze the prevalence of Lynch syndrome in unselected patients with endometrial or ovarian cancer. In addition, the willingness of patients and family members to undergo germline mutation testing was investigated. METHODS: The medical records of all patients diagnosed with endometrial or ovarian cancer at the Department of Gynecology and Obsterics, University Hospital Dresden, between 1998 and 2012, were screened for a family history of HNPCC-associated cancer. Telephone interviews were used to screen, inform, and enroll patients in this genetic analysis study. Molecular analysis was performed by prescreening of tumor tissue, followed by germline mutation analysis. RESULTS: Two hundred and eighty-three patients were diagnosed with endometrial cancer, 291 with ovarian cancer, and 14 with both. A positive family history for tumors associated with Lynch syndrome was documented for 61 patients. Two pathogenic mutations in the genes MLH1 and MSH2 in nine genetic analyses had already been detected before. After genetic counseling, only 10 of 31 index patients (32.3 %) consented for mutation analysis. However, no additional pathogenic heterozygous mutations were found. CONCLUSION: In this retrospective cohort study in unselected patients with endometrial or ovarian cancer, only a small number of patients with suspected Lynch syndrome could be identified. Of those, acceptance of germline analyses was moderate, only. As a result, the rate of identified pathogenic germline mutations was lower than expected. Therefore, we are convinced that more information on cancer risks, options for predictive molecular testing and preventive procedures, needs to be provided to patients and gynecologists.

An unusual case of Cowden syndrome associated with ganglioneuromatous polyposis.

BACKGROUND: Ganglioneuromatous polyposis (GP) is a very rare disorder which may be associated with other clinical manifestations and syndromes, such as Cowden syndrome, multiple endocrine neoplasia (MEN) type II and neurofibromatosis (NF) 1. The risk for malignant transformation of ganglioneuromas is unknown, and the combination of GP with colon cancer has been only very seldom reported. METHODS AND RESULTS: We report the case of a 60-year old male patient with adenocarcinoma, adenomas and lipomas of the colon and multiple gastroduodenal lesions combined with generalised lipomatosis and macrocephaly. Based on the initial endoscopic and histological findings, a (restorative) proctocolectomy was recommended but declined by the patient. Instead, a colectomy was performed. The histological examination revealed an unforeseen GP in addition to the colon cancer. Extensive molecular diagnostics allowed for the differential diagnosis of the causes of the clinical manifestations, and the clinical suspicion of Cowden syndrome could not be confirmed using Sanger Sequencing and MLPA for the analysis of PTEN. Finally, a pathogenic germline mutation in PTEN (heterozygous stop mutation in exon 2: NM_000314 (PTEN):c.138C > A; p.Tyr46*) could be detected by next-generation sequencing (NGS), confirming an unusual presentation of Cowden syndrome with GP. CONCLUSIONS: Cowden syndrome should be considered in cases of GP with extracolonic manifestation and verified by combined clinical and molecular diagnostics. Because GP may represent a premalignant condition, a surgical-oncological prophylactic procedure should be considered. Based on our experience, we recommend early implementation of Panel NGS rather than classical Sanger sequencing for genetic diagnostics, especially if various diagnoses are considered.

ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma.

AIM OF THE STUDY: Small bowel adenocarcinoma (SBA) is a rare and aggressive tumour with poor outcomes. Because of its low incidence, the number prospective studies remains insufficient leading to poor knowledge and absence of standard of care. Aiming to better understand small bowel carcinogenesis we investigated the frequency of somatic mutations in a large data set of patients in more than 740 mutational hotspots among 46 genes. METHODS: In total, 83 SBA cases were selected from two European databases. The sequencing was performed using the Ion 316 Chip. Additionally we looked into ERBB2 expression and microsatellite instability (MSI) status. RESULTS: The tumours most frequently were duodenal (47%) and stage ⩾3 (63%). Eight genes were mutated with a frequency >5%: KRAS, TP53, APC, SMAD4, PIK3CA, ERBB2, BRAF and FBXW7. ERBB2 alterations are present in 10 patients (12%) through mutations (7 cases) or amplifications (3 cases). ERBB2 mutations were significantly associated with duodenal tumour location (P=0.04). In this group, there was a positive association with dMMR status (P=0.006) and APC mutation (P=0.02) but negative association with p53 mutations (P=0.038). CONCLUSIONS: This study describes the first large screening of somatic mutations in SBA using next generation sequencing. The ERBB2 mutation was revealed to be one of the most frequent alterations in SBA with a distribution dependent on tumour location. In most cases ERBB2 mutation was identical (p.L755S). In clinical practice, this may suggest that more than 10% of the patients with SBA could be treated using an anti-ERBB2-targeted agent.

Colorectal liver metastases: an update on palliative treatment options.

Only approximately 30% of patients with colorectal cancer liver metastasis qualify for curative therapy, which is in most cases liver lesion resection. Due primarily to the extent of the tumors and patient comorbidities, palliative therapy remains the only option in non-resection cases. Palliation enables local, symptomatic control and prolonged survival in some cases. As established methods are continuously improved, new palliative therapy methods are tested in clinical trials and subsequently introduced into clinical practice. The present review provides an overview of current colorectal liver metastasis treatment when resection is not an option. This review gives the basis for an interdisciplinary decision making process for the treatment of liver metastasis.

Is there still a place for adrenal venous sampling in the diagnostic localization of pheochromocytoma?

Our objective is to outline the utility of adrenal venous sampling (AVS) with measurements of metanephrine to normetanephrine ratios for diagnostic localization of phaeochromocytoma in a patient with normal plasma levels of catecholamines. A 53-year-old-woman was referred for evaluation of recurrent pheochromocytoma following a right adrenalectomy 14 years earlier. Diagnosis of recurrent disease was established from elevations in plasma metanephrines with normal levels of catecholamines. Magnetic resonance imaging indicated two 1-2 cm masses in the right surgical bed and another 1-1.5 cm mass in the left adrenal. These masses were negative on (123)I-metaiodobenzylguanidine scintigraphy. There was no evidence of a hereditary syndrome. We, therefore, carried out two investigational approaches to identify the tumor masses. Hybrid positron emission tomography/computed tomography with (68)Ga-DOTATOC ((68)Ga-DOTATOC-PET/CT) confirmed the presence of recurrent disease in the right surgical bed and also suggested additional left adrenal involvement. Normal plasma catecholamines precluded the use of adrenal venous sampling (AVS) with catecholamine measurements. Hence, we performed AVS with measurements of plasma metanephrines, which were 4- to 7-fold higher in the left adrenal vein than in central venous plasma. We observed a reversal of the normally high metanephrine to normetanephrine ratio (mean value ± SD 5.28 ± 1.86; range 3.36-8.84, n = 13) to 0.73, establishing the presence of a left adrenal pheochromocytoma. Surgical pathology confirmed bilateral disease. This case highlights a scenario where a combination of (68)Ga-DOTATOC-PET/CT and AVS with measurement of the metanephrine to normetanephrine ratio was crucial for the preoperative assessment of a patient with bilateral pheochromocytoma.

Mutational status of KIT and PDGFRA and expression of PDGFRA are not associated with prognosis after curative resection of primary gastrointestinal stromal tumors (GISTs).

BACKGROUND: The aim of this study was to investigate if immunohistochemical expression and mutational status of KIT and PDGFRA in GISTs are associated with the clinical course and disease-free survival after curative resection of the primary tumor without adjuvant systemic therapy. METHODS: Paraffin-embedded tumor sections of 95 GISTs were analyzed for KIT and PDGFRA expression by immunohistochemistry. PDGFRA expression was judged using a scoring system subdividing tumors in negative/weak and strong immunoreactivity groups. For mutation analysis, exons 9, 10, 11, 13, and 17 of KIT and exons 10, 12, 14, and 18 of PDGFRA were sequenced. RESULTS: Of 95 R0-resected GISTs, 69% showed strong PDGFRA immunoreactivity. Gastric GISTs revealed a significantly higher rate of strong PDGFRA immunoreactivity (P = 0.01) and longer DFS (P = 0.015) than GISTs of the small intestine. KIT mutations were detected in 43 of 63 (68.3%) completely sequenced cases while PDGFRA mutations were identified in 6 cases (10%). In multivariate analysis, neither KIT/PDGFRA expression nor mutational status of KIT or PDGFRA were independent prognostic factors. Only mitotic rate predicted recurrence independently. CONCLUSION: Our data do not support the notion that expression of PDGFRA or mutations in KIT or PDGFRA are independent prognostic factors after curative resection of primary GIST.

Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease with high penetrance, caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 and MLH3. Most reported pathogenic mutations are point mutations, comprising single base substitutions, small insertions and deletions. In addition, genomic rearrangements, such as large deletions and duplications not detectable by PCR and Sanger sequencing, have been identified in a significant proportion of HNPCC families, which do not carry a pathogenic MMR gene point mutation. To clarify whether genomic rearrangements in MLH1, MSH2 or MSH6 also occur in patients carrying a point mutation, we subjected normal tissue DNA of 137 colorectal cancer (CRC) patients to multiplex ligation-dependent probe amplification (MLPA) analysis. Patients fulfilled the following pre-requisites: all patients met at least one criterion of the Bethesda guidelines and their tumors exhibited high microsatellite instability (MSI-H) and/or showed loss of expression of MLH1, MSH2 or MSH6 proteins. PCR amplification and Sanger sequencing of all exons of at least one MMR gene, whose protein expression had been lost in the tumor tissue, identified 52 index patients without a point mutation (Group 1), 71 index patients with a pathogenic point mutation in MLH1 (n=38) or MSH2 (n=22) or MSH6 (n=11) (Group 2) and 14 patients with an unclassified variant in MLH1 (n=9) or MSH2 (n=3) or MSH6 (n=2) (Group 3). In 13 of 52 patients of group 1 deletions of at least one exon were identified. In addition, in group 3 one EX1_15del in MLH1 was found. No genomic rearrangement was identified in group 2 patients. Genomic rearrangements represent a significant proportion of pathogenic mutations of MMR genes in HNPCC patients. However, genomic rearrangements are rare in patients carrying point mutations in MMR genes. These findings suggest the use of genomic rearrangement tests in addition to Sanger sequencing in HNPCC patients.

N-Acetyltransferase (NAT) 2 acetylator status and age of tumour onset in patients with sporadic and familial, microsatellite stable (MSS) colorectal cancer.

INTRODUCTION: N-Acetyltransferase (NAT) 2 is an important enzyme involved in the metabolism of different xenobiotics, including potential carcinogens. Allelic variants of the NAT2 gene are determined by a pattern of single nucleotide polymorphisms (SNPs) resulting in slow (SA), intermediate (IA) or rapid acetylator (RA) phenotypes and causing the individual differences in the NAT2 metabolic capacity. To clarify the potential modifying role of the NAT2 acetylator status in microsatellite stable (MSS) colorectal cancer (CRC), we studied 140 patients with sporadic CRC (group 1) and 69 patients with CRC who met at least one criterion of the revised Bethesda guidelines (group 2). OBSERVATIONS: We did not observe any significant difference in the NAT2 acetylator status frequency between patients in both groups and 100 healthy controls (P=0.486). Regardless of a younger median age of tumour onset (AO) of 41 years in group 2 patients compared to 64 years in group 1 patients, no significant difference in AO was found between RA and SA status patients in both groups. The median AO in group 1 was 65 years in patients with RA and 63 years with SA status (P=0.065). The median AO in group 2 was 40 years in patients with RA and 42 years with SA status (P=0.814). Multivariate Cox regression analysis revealed that neither the NAT2 acetylator status (P=0.064 and 0.810, respectively) nor the gender (P=0.165 and 0.918, respectively) was a risk factor for the AO in both groups. These data do not support the hypothesis that the NAT2 acetylatorship acts as a modifying factor on the AO in sporadic and familial, microsatellite stable CRC.

Occult endometrial cancer and decision making for prophylactic hysterectomy in hereditary nonpolyposis colorectal cancer patients.

BACKGROUND AND OBJECTIVE: Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent form of hereditary colorectal cancer. In addition to the high lifetime risk for colorectal cancer in mutation carriers, there is also a remarkably increased risk for endometrial cancer (EC). METHODS: In this retrospective study, clinical and molecular approach to the individual decision making as to whether or not to perform a prophylactic hysterectomy in a subset of HNPCC patients is discussed. 147 female patients meeting at least one criterion of the Bethesda guidelines were included in this analysis between 1995 and 2003. After clinical and genetic counseling, patients gave informed written consent and microsatellite analysis, immunohistochemistry and sequencing of the mismatch repair genes MLH1, MSH2 and MSH6 was performed. RESULTS: 11 of the analyzed patients had a personal history of EC and had undergone previous hysterectomy at ages 26 to 62 years. Prophylactic hysterectomy with oophorectomy was considered in postmenopausal women meeting the Amsterdam criteria and/or carrying a disease causing mismatch repair gene mutation who were operated on because of diagnosed colorectal cancer in our center for hereditary cancer. This procedure was performed in 4 patients. None of them had shown any symptoms of a gynecologic malignancy. Preoperative gynecological examination showed no evidence for EC or ovarian cancer in these patients. Postoperative histological examination showed EC stage T1b N0 M0 in 2 patients. CONCLUSIONS: Since the efficiency of gynecological surveillance is uncertain, prophylactic hysterectomy could be an option for a subset of HNPCC patients and mutation carriers.

N-acetyltransferase (NAT) 2 acetylator status and age of onset in patients with hereditary nonpolyposis colorectal cancer (HNPCC).

N-acetyltransferase (NAT) 2 is an essential polymorphic enzyme involved in the metabolism of various xenobiotics, including potential carcinogens. The individual differences in the NAT2 metabolic capacity are caused by allelic variants of the NAT2 gene which are determined by a pattern of single nucleotide polymorphisms (SNPs) resulting in slow (SA), intermediate (IA) or rapid acetylator (RA) phenotypes. Highly penetrant germline mutations in mismatch repair (MMR) genes are the cause of the disease in hereditary nonpolyposis colorectal cancer (HNPCC). There is no strict correlation between the type of germline mutation in MMR genes and the HNPCC phenotype, but age of tumor onset (AO) in HNPCC has been associated at least in part with different variants in apoptosis-related genes. To clarify the potential modifying role of the NAT2 acetylator status in HNPCC, we performed a multicenter study in 226 individuals with colorectal cancer carrying exclusively pathogenic germline mutations in MSH2 or MLH1. We did not observe any significant difference in the NAT2 acetylator status frequency between HNPCC patients and 107 healthy controls (P=0.156), and between MLH1 and MSH2 mutation carriers (P=0.198). Multivariate Cox regression analysis revealed that male patients had a significantly increased risk to develop CRC compared to females during any interval (P=0.043), while the NAT2 acetylator status (P=0.447) and the mutated gene (MLH1 or MSH2) (P=0.236) were not risk factors for AO. The median AO in HNPCC patients was 39 years in patients with RA as well as with SA status (P=0.347). In MLH1 mutation carriers, the median AO was 38 years in RA and 36 years in SA status patients (P=0.901), whereas in MSH2 mutation carriers, the median AO was 39 years in RA and 42 years in SA status patients (P=0.163). Log-rank test revealed a significantly lower age of CRC onset in male compared to female HNPCC patients (P=0.0442). These data do not support the hypothesis that the NAT2 acetylatorship acts as a modifying factor on AO in HNPCC-associated CRC.

Ten novel MSH2 and MLH1 germline mutations in families with HNPCC.

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common hereditary cancer-susceptibility syndromes. Germline mutations in mismatch repair genes are associated with the clinical phenotype of HNPCC. We report ten novel germline mutations, three in MSH2 and seven in MLH1. All but one mutation have been found in families fulfilling criteria of the Bethesda guidelines; four of them additionally fulfilled the Amsterdam criteria I or II. Eight mutations were considered pathogenic and predictive diagnostics in healthy family members at risk shall be undertaken; these include five frameshift mutations leading to premature stop codons, in MSH2: c.1672delT (p.S558Xfs) and c.2466_2467delTG (p.C822X) and in MLH1: c.1023delG (p.R341Xfs), c.1127_1128dupAT (p.K377Xfs) and c.1310delC (p.P437Xfs); three mutations leading to splice aberrations, in MSH2: c.1661G>C (r.1511_1661del) and in MLH1: c.677+3A>C (r.589_677del) and c.1990-2A>G predicted to result in a splice site defect. The remaining two mutations are unclassified variants with assumed pathogenicity: one missense mutation in the highly conserved ATPase domain of MLH1 (c.122A>G [p.D41G]) and one in-frame insertion of twelve nucleotides in MLH1 (c.2155_2156insATGTGTTCCACA [p.I719delinsNVFHI]). These two mutations were not found in 102 alleles of healthy control individuals. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IHC) revealed complete loss of expression of the affected protein in the tumor cells from all but three patients. The tumors from the patients with the mutations c.1127_1128dupAT and c.1990-2A>G showed a reduction of expression of the MLH1-protein, rather than complete loss. In the tumor from the patient with the missense mutation c.122A>G [p.D41G] a normal expression of the proteins coded by MLH1 and MSH2 was noticed.

Loss of MSH3 protein expression is frequent in MLH1-deficient colorectal cancer and is associated with disease progression.

Mononucleotide repeat sequences are particularly prone to frameshift mutations in tumors with biallelic inactivation of the mismatch repair (MMR) genes MLH1 or MSH2. In these tumors, several genes harboring mononucleotide repeats in their coding region have been proposed as targets involved in tumor progression, among which are also the MMR genes MSH3 and MSH6. We have analyzed the expression of the MSH3 and MSH6 proteins by immunohistochemistry in 31 colorectal carcinomas in which MLH1 was inactivated. Loss of MSH3 expression was identified in 15 tumors (48.5%), whereas all tumors expressed MSH6. Frameshift mutations at coding microsatellites were more frequent in MSH3 (16 of 31) than in MSH6 (3 of 31; Fisher's exact test, P < 0.001). Frameshift mutations and allelic losses of MSH3 were more frequent in MSH3-negative tumors compared with those with normal expression (22 mutations in 30 alleles versus 8 mutations in 28 alleles; chi(2), P = 0.001). Biallelic inactivation was evident or inferred for 60% of MSH3-negative tumors but none of the tumors with normal MSH3 expression. In contrast, we did not identify frameshift mutations in the (A)8 tract of MSH3 in a control group of 18 colorectal carcinomas in which the MMR deficiency was based on the inactivation of MSH2. As it has been suggested that mutations of MSH3 might play a role in tumor progression, we studied the association between MSH3 expression and disease stage assessed by lymph node and distant metastases status. Dukes stages C and D were more frequent in primary tumors with loss of MSH3 expression (9 of 13), compared with tumors with retained expression (1 of 14; Fisher's exact test, P = 0.001), suggesting that MSH3 abrogation may be a predictor of metastatic disease or even favor tumor cell spread in MLH1-deficient colorectal cancers.

Preventive surgery for colon cancer in familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer syndrome.

BACKGROUND: A better understanding of the molecular basis of hereditary colorectal cancer syndromes such as hereditary nonpolyposis colorectal cancer syndrome (HNPCC) and familial adenomatous polyposis (FAP) has profound consequences for both the diagnosis and (prophylactic) treatment of (pre)malignant neoplastic lesions. DISCUSSION: Sequence analysis of the underlying genes for these conditions and the detection of disease-causing genetic alterations in an index patient enable predictive testing for individuals at risk within an affected family. However, the clinical implications of predictive molecular testing depend on the overall penetrance and variability in the expression of pathogenic mutations. The extent of these parameters differs considerably among the various known hereditary colorectal cancer syndromes. Hence the integration of genetic information into the daily surgical practice remains challenging. CONCLUSIONS: This review provides an update on the indications for family assessment, purpose and limitations of the genetic testing and resulting recommendations for prophylactic surgery in FAP and HNPCC.

Methylenetetrahydrofolate reductase polymorphisms and risk of sporadic and hereditary colorectal cancer with or without microsatellite instability.

Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in the folate metabolism, which affects DNA synthesis and methylation. Low enzyme activity may reduce the capacity of DNA methylation, and possibly reduce uracil misincorporation into DNA, which can result in double strand breaks. Both processes may be critical for the oncogenic transformation of human cells. Two common amino acid-changing and enzyme activity-reducing nucleotide polymorphisms (677C --> T/Ala222Val and 1298A --> C/Glu428Ala) have been described in MTHFR. We performed estimations of the relative risk associated with these two polymorphisms in samples from 287 colorectal cancer patients, compared to 346 healthy controls. Relative risk were further determined for subpopulations of cancer patients having sporadic (n = 227) or suspected/verified hereditary disease (n = 60) and tumours exhibiting high-level microsatellite instability (n = 41) or not (n = 246). No significant differences for the relative risk of colorectal cancer were observed for the MTHFR genotypes either alone or in combination in the analysed cohorts, although the frequency of the 1298AA + AC genotypes was increased among the 60 cases with hereditary disease. Whereas our results do not support an association of high enzyme activity and increased risk of colorectal cancer in general, we can not exclude an association of patients with hereditary disease and the MTHFR 1298A --> C variant.

Identification of six novel MSH2 and MLH1 germline mutations in HNPCC.

Germline mutations in mismatch repair genes are responsible for hereditary nonpolyposis colorectal cancer (HNPCC), the most common hereditary cancer-susceptibility syndrome. We report six novel germline mutations, three in MSH2 and three in MLH1. All but one mutation have been found in families fulfilling the criteria of the Bethesda guidelines; two of them additionally fulfilled the Amsterdam criteria. We identified two nonsense mutations in MSH2 (c.1764T>G [p.Y588X], c.2579C>A [p.S860X]), one duplication of four nucleotides causing premature stop codon (MLH1: c.821_824dupAAGC [p.A275fsX307]), one splice site mutation resulting in skipping of exon 8 from the MLH1 transcript (c.677+3A>G), one duplication of 18 nucleotides leading to duplication of six amino acids in the mismatch-binding domain of MSH2 (c.4_21dup [p.A2_E7dup) and one missense mutation in the PMS2 interaction domain of MLH1 (c.1756G>C [p.A586P]). The three latter mutations were not found in 73, 90 and 94 healthy control individuals, respectively. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IH) revealed complete loss of expression of the affected protein in the tumor cells from the patients with the nonsense, splice-site and missense mutation. The tumor from the patient with the c.821_824dupAAGC mutation showed a reduced, rather than lost, expression of the MLH1-protein.

Juvenile polyposis: massive gastric polyposis is more common in MADH4 mutation carriers than in BMPR1A mutation carriers.

Juvenile polyposis syndrome (JPS) is an autosomal dominant predisposition to multiple juvenile polyps in the gastrointestinal tract. Germline mutations in the MADH4 or BMPR1A genes have been found to be causative of the disease in a subset of JPS patients. So far, no genotype-phenotype correlation has been reported. We examined 29 patients with the clinical diagnosis of JPS for germline mutations in the MADH4 or BMPR1A genes and identified MADH4 mutations in seven (24%) and BMPR1A mutations in five patients (17%). A remarkable prevalence of massive gastric polyposis was observed in patients with MADH4 mutations when compared with patients with BMPR1A mutations or without identified mutations. This is the first genotype-phenotype correlation observed in JPS.

Involvement of hMSH6 in the development of hereditary and sporadic colorectal cancer revealed by immunostaining is based on germline mutations, but rarely on somatic inactivation.

Germline mutations in human mismatch repair (MMR) genes yield a predisposition for the hereditary nonpolyposis colon cancer (HNPCC) syndrome. In contrast to hMLH1 and hMSH2, little is known about the overall involvement of hMSH6 in colorectal cancer. We investigated 82 tumors from patients who fulfilled the Bethesda guidelines for HNPCC as well as 146 sporadic tumors, analyzing microsatellite instability and expression of the 4 MMR proteins hMSH6, hMSH2, hMLH1 and hPMS2. Four tumors with lost expression and 1 tumor with cytoplasmic expression of hMSH6 were identified. Sequence analysis revealed germline mutations in 4 of the 5 patients, including 1 patient with sporadic disease. The lost or reduced expression of hMSH2 and hMLH1 was always identical to its heterodimerization partners, hMSH6 and hPMS2, respectively. Furthermore, hMSH2 expression was reduced upon hMSH6 deficiency. Abnormal expression of 1 or more of the 4 proteins was always associated with a high level of microsatellite instability (MSI-H). Conversely, all but 1 of the 44 MSI-H tumors had abnormal expression of 1 or more of the proteins, basically excluding additional genes associated with the MSI-H phenotype. We conclude that the involvement of somatic or epigenetic hMSH6 inactivation in colorectal cancer is rare.

Seven novel MLH1 and MSH2 germline mutations in hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent hereditary form of colorectal cancer and is caused by germline mutations in mismatch repair (MMR) genes. The majority of mutations occur in MLH1 and MSH2. We report hereby seven novel germline mutations in these two genes (five in MLH1 and two in MSH2). All mutations have been found in families fulfilling criteria of the Bethesda guidelines and four of which also fulfilled the Amsterdam criteria. We identified three insertions or deletions of 1 bp leading to premature stop codons (MLH1: c.341delC, c.1413-1414insA; MSH2: c.1119delG) and three nonsense mutations (MLH1: c.67G>T [E23X], c.436C>T [Q146X]; MSH2: c.1857T>G [Y619X]). The corresponding tumors showed a high level of microsatellite instability (MSI-H) and a complete loss of expression of the affected protein. In addition, a missense mutation in MLH1 was identified (c.1984A>C [T662P]). The respective tumor also showed a high level of microsatellite instability but a reduced, rather then lost, expression of the MLH1-protein. This missense mutation was not found in 107 healthy control individuals and in 54 HNPCC patients.