Namibian spitting cobra, Naja nigricincta nigricincta (Zebra snake): Antibiotic profile of bacteria cultured from the oral pharynx, venom and snakebite wounds.

This was a cross-sectional study with the aim of characterising Naja nigricincta nigricincta's oral bacterial flora as well as accompanying sensitivities and resistance towards antibiotics. Naja nigricincta nigricincta (zebra snake) is a spitting cobra indigenous to Namibia. Nasopharyngeal and venom swabs for bacterial culture and antibiotic sensitivity were taken from 37 native zebra snakes originating from the Khomas region that were captured for removal and relocation. Enterococcus faecalis, Proteus spp., Morganella morganii and Pseudomonas spp. were the organisms most often cultured. The antibiotic sensitivity profiles of these organisms suggest ciprofloxacin or a third-generation cephalosporin plus gentamicin or piperacillin-tazobactam as prophylactic antibiotics in case of Naja nigricincta nigricincta bites.

Identification of mismatch repair gene mutations in young patients with colorectal cancer and in patients with multiple tumours associated with hereditary non-polyposis colorectal cancer.

BACKGROUND: Patients with early-onset colorectal cancer (CRC) or those with multiple tumours associated with hereditary non-polyposis colorectal cancer (HNPCC) raise suspicion of the presence of germline DNA mismatch repair (MMR) gene mutations. AIM: To analyse the value of family history, microsatellite instability (MSI) analysis and MMR protein staining in the tumour to predict the presence of an MMR gene mutation in such patients. METHODS: In 281 patients diagnosed with CRC before the age of 50 years or with CRC and at least one additional HNPCC-associated cancer, germline mutation analysis in MLH1, MSH2 and MSH6 was carried out with denaturing gradient gel electrophoresis and multiplex ligation-dependent probe amplification. MSI analysis with five consensus markers and MMR protein staining for MLH1, MSH2 and MSH6 were carried out in the tumours. RESULTS: 25 pathogenic mutations (8 in MLH1, 9 in MSH2 and 8 in MSH6) were found. MSI analysis missed three and immunohistochemistry (IHC) missed two mutation carriers. Sensitivities of family history, MSI analysis and IHC for the presence of a mutation were 76%, 82% and 88%, specificities were 64%, 70% and 84%, and positive predictive values were 19%, 23% and 38%, respectively. Multivariate analysis showed the highest odds ratio for IHC (38.3, 95% confidence interval 9.0 to 184). Prevalence of pathogenic germline MMR gene mutations in patients with CRC before the age of 50 years was 6% and in those with > or =2 HNPCC-associated tumours was 22%. In the second group, no mutation carriers were found among the 29 patients who were diagnosed with their first tumour after the age of 60 years. CONCLUSION: Family history, MSI analysis and IHC are indicative parameters to select patients with CRC for MMR gene mutation analysis. The data show that IHC is the best single selection criterion.

Predictive value of thymidylate synthase and dihydropyrimidine dehydrogenase protein expression on survival in adjuvantly treated stage III colon cancer patients.

BACKGROUND: The predictive value of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression on long-term survival by influencing 5-fluorouracil (5-FU) effect were determined in primary tumours and node metastases of stage III colon cancer patients treated adjuvantly with 5-FU regimens (n=391). The effect of TP 53 mutation status, which is thought to be functionally linked to TS inhibition, was also examined. PATIENTS AND METHODS: TS and DPD protein expression was determined by immunohistochemical analysis using tissue microarrays of these colon tumours. Two hundred and twenty tumours had already been screened in a previous study for TP 53 mutations. RESULTS: Low TS protein levels in primary stage III colon tumours appeared to be associated with mucinous histology and low DPD protein levels with young age at time of randomisation. Concordance between TS and DPD expression in primary and metastatic tumours was low. No associations were found between disease-free survival (DFS) and TS or DPD protein levels. When stratified by TP 53 mutation status DFS did not differ with TS expression. CONCLUSIONS: Expression of TS and DPD proteins is not predictive for survival in patients with stage III colon cancer treated adjuvantly with 5-FU regimens. TS protein levels did not alter the effect of TP 53 mutation status.

RFP2, c13ORF1, and FAM10A4 are the most likely tumor suppressor gene candidates for B-cell chronic lymphocytic leukemia.

Occurrence of 13q14 deletions between D13S273 and D13S25 in B-cell chronic lymphocytic leukemia (B-CLL) suggests that the region contains a tumor suppressor gene. We constructed a PAC/cosmid contig largely corresponding to a 380-kb 13q14 YAC insert that we found deleted in a high proportion of B-CLL patients. We found seven genes by exon trapping, cDNA screening and analysis/cDNA extension of known expressed sequence tags. One appeared to originate from another region of 13q. Recent publications have focused on two of the genes that most likely do not have a tumor suppressor role. This study evaluates the remaining four genes in the region by mutation scanning and theoretical analysis of putative encoded products. No mutations suggestive of a pathogenic effect were found. The 13q14 deletions may be a consequence of an inherent instability of the region, an idea supported by our finding of a considerable proportion of AluY repeats. Deletion of putative enhancer sequences and/or genes in the region may result in an inactivation of tumor suppression by a haploinsufficiency mechanism. We conclude that RFP2, c13ORF1, and a chromosome 13-specific ST13-like gene, FAM10A4, are the most likely candidates for such a type of B-CLL TSG.

Novel expressed sequences obtained by means of a suppression subtractive hybridisation analysis from the 6q21 region that is frequently deleted in gastric cancer.

In our search for genomic regions that are involved in the development of gastric cancer, we recently identified a 2-cM minimal region of overlapping heterozygous deletions in 6q16.3-q23.1. Here, we describe an application of the suppression subtraction method (SSH) to search for genes in this small region of the genome, taking advantage of the fact that many human genes present on yeast artificial chromosomes (YACs) are expressed in yeast. Subtraction was performed with two virtually contiguous YACs that cover a region of approximately 2.5 Mb. Combined forward and reversed subtractions resulted in the identification of 12 clones of human origin, all of which could be confirmed by sequence analysis as originating from the 6q21 region. Expression in human tissues could be confirmed by Northern analysis for two of the clones, one of them showing a high level of expression in stomach tissue.

Allelic gains and losses in distinct regions of chromosome 6 in gastric carcinoma.

In gastric cancer, alterations in the long arm of chromosome 6 are a frequent event. Two regions of heterozygous loss have been described: 6q16.3-6q23 and 6q26-6q27. We have evaluated by microsatellite and FISH analyses the 6q status of three cell lines that we established from primary gastric carcinomas xenografted in nude mice, in order to elucidate the underlying mechanisms of 6q alterations. Alterations of the long arm of chromosome 6 were found in all three xenografts and corresponding cell lines. Allelic imbalance (AI) was found in the three cases, by microsatellite analysis. Fluorescence in situ hybridization analyses clearly demonstrated a duplication of the larger part of the 6q arm in all three cell lines. Two of the cell lines and the corresponding xenografts showed, in addition, complete loss of one of the parental alleles at the terminal part of 6q. Thus, the AI observed along the long arm of chromosome 6 is represented by gain of alleles in one distinct chromosomal segment and loss of alleles in another distinct segment.

Mapping of a susceptibility gene for multiple sclerosis to the 51 kb interval between G511525 and D6S1666 using a new method of haplotype sharing analysis.

Multiple sclerosis (MS) is a complex disease that is partly genetic in origin. Although an association of MS with specific human leukocyte antigen (HLA) types has been known for almost 30 years, the nature of this relationship has remained unclear. Furthermore, genetic resolution sufficient to implicate a specific gene in the HLA region has not been achieved. Many loci in the HLA region have been found to be significantly associated with MS, which is largely explained by the extended haplotype sharing and varying marker informativity of the region. We have determined 248 haplotypes of MS patients from the population of the northern Netherlands and 226 haplotypes of their relatives as controls using a set of 22 microsatellite markers covering the HLA region. The data were analyzed using standard association methods and a new statistical method, haplotype sharing statistics (HSS). Haplotype sharing statistics determines the extent of haplotype sharing for all pairs of haplotypes of patients and of controls and calculates the difference in mean haplotype sharing between patients and controls. Haplotype sharing was found to be significantly greater among patients than among controls in a region of 1.1 Mb between markers G511525 and TNFalpha. The involvement of this region is also supported by association analysis and the transmission/disequilibrium test (TDT). Within this region, HSS, which is largely independent of association and TDT, indicated the interval of 51 kb between G511525 and D6S1666 as that most likely to contain a susceptibility gene for MS. As DQB1 is the sole gene known in this interval at present, the results of our analysis suggest that this gene plays a role in the pathogenesis of MS.

A role for MLH3 in hereditary nonpolyposis colorectal cancer.

We investigated a possible role of the mismatch-repair gene MLH3 in hereditary nonpolyposis colorectal cancer by scanning for mutations in 39 HNPCC families and in 288 patients suspected of having HNPCC. We identified ten different germline MLH3 variants, one frameshift and nine missense mutations, in 12 patients suspected of HNPCC. Three of the 12 also carried a mutation in MSH6.

MEN2A-RET-induced cellular transformation by activation of STAT3.

The MEN2A oncogene encodes for a constitutive active member of the RET receptor tyrosine kinase family. Here, we report that MEN2A-RET activates Signal Transducer and Activator of Transcription 3 (STAT3) via two YxxV/Q STAT3 docking sites, Tyr752 and Tyr928. MEN2A-RET induces both Tyr705 and Ser727 phosphorylation of STAT3, and STAT3 serine phosphorylation is required for its maximal transcriptional activity. Stable NIH3T3 cell lines expressing both MEN2A-RET and STAT3alpha but not STAT3beta, are characterized by enhanced proliferation and cyclin-D1 promoter activity, and enhanced growth in soft agar. These data indicate that malignant cell growth induced by MEN2A-RET involves its activation of STAT3.

Germline mutations of EXO1 gene in patients with hereditary nonpolyposis colorectal cancer (HNPCC) and atypical HNPCC forms.

BACKGROUND & AIMS: Germline mutations in one of four mismatch repair genes have been found in the majority of families with hereditary nonpolyposis colorectal cancer (HNPCC), but only in a small part of families with atypical HNPCC. The recently cloned EXO1 gene might be involved in the pathogenesis of HNPCC because the EXO1 protein strongly interacts with the MSH2 protein. To determine its role in HNPCC, EXO1 was scanned for germline mutations. METHODS: All 14 exons of EXO1 were scanned for mutations in index patients from 33 families with HNPCC fulfilling the Amsterdam criteria and in 225 index patients suspected of HNPCC. RESULTS: Germline variants of EXO1 were detected in 14 patients, including one splice-site mutation in a family with HNPCC and 13 missense mutations in patients with atypical HNPCC. These variants did not occur in more than 200 control individuals. From 13 of these 14 patients, tumors were available for analysis of microsatellite instability and loss of heterozygosity. Six of the tumors showed microsatellite instability. Heterozygosity analysis showed one case without EXO1 allelic loss and 12 tumors with loss of the mutant allele and retention of the normal one. CONCLUSIONS: The results indicate a possible association of germline EXO1 variants with HNPCC and atypical HNPCC.

The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a new model to study carcinoma-directed immunotherapy.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M(r) 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia. To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with approximately 10-kb-upstream and approximately 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c. in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mouse tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.

MLH1 and MSH2 protein expression as a pre-screening marker in hereditary and non-hereditary endometrial hyperplasia and cancer.

The predictive value of MLH1 or MSH2 protein expression for the presence of truncating germline mutations was examined in benign and (pre)malignant endometrial samples from 3 patient groups: (I) 10 endometrial cancer patients from hereditary non-polyposis colorectal cancer (HNPCC) families with (n = 6) or without (n = 4) a known germline mutation; (II) 15 women from HNPCC families with (n = 7) or without (n = 8) a known germline mutation, who underwent endometrial sampling for non-malignant reasons; (III) 38 endometrial cancer patients <50 years of age, without HNPCC family history. Immunostaining for MLH1 and MSH2 was performed on paraffin-embedded sections. In group III, tumor DNA was examined for microsatellite instability (MSI) and MLH1, MSH2 and MSH6 mutation analysis was carried out. In 6/6 MLH1/MSH2 mutation carriers with endometrial cancer (group I), concordance was found between protein loss in the tumor and the corresponding mutation. In 3 MLH1 mutation carriers, MLH1 protein loss was also observed in concurrent endometrial hyperplasia. In group II, no protein loss was detected in normal endometrial tissue samples; in 3/4 patients with endometrial hyperplasia, MLH1/MSH2 protein loss was observed. In group III, protein loss was detected in 12/38 patients (9 MLH1, 3 MSH2), while in 3/11 patients with concurrent endometrial hyperplasia protein loss was also observed in the hyperplasia. MSI analysis in group III revealed 26 MSI-low and 12 MSI-high tumors. Mutation analysis in 28/38 patients showed only 1 missense MSH6 and no MLH1 or MSH2 germline mutations. In group III, loss of MLH1/MSH2 protein expression was not related to the presence of MSI or MLH1/MSH2 germline mutations. In conclusion, MLH1 or MSH2 protein loss in HNPCC-related endometrial neoplasia is strongly related to corresponding germline mutations. This relation was not clearly present in young sporadic endometrial cancer patients. Immunohistochemical pre-screening of the MLH1 and MSH2 proteins in endometrial hyperplasia or cancer can thus be helpful in HNPCC families. Frequent loss of MLH1 or MSH2 protein in endometrial hyperplasia indicates that this loss is an early event in endometrial carcinogenesis.

Prognostic significance of K-ras and TP53 mutations in the role of adjuvant chemotherapy on survival in patients with Dukes C colon cancer.

PURPOSE: Mutations in K-ras and TP53 genes are common in colorectal cancer. They affect biologic behavior and might influence chemotherapy susceptibility in these tumors. We investigated whether the survival of patients with Dukes C colon cancer treated with adjuvant chemotherapy is influenced by K-ras and TP53 mutations. METHODS: Mutation screening of the hot spots of the K-ras gene and of the evolutionarily conserved regions of the TP53 gene was performed by denaturing gradient gel electrophoresis technique in formalin-fixed paraffin-embedded specimens of 55 consecutive patients with Dukes C colon cancer treated with adjuvant 5-fluorouracil-based chemotherapy. The median follow-up was 47 (range, 32-66) months. RESULTS: Alterations in the mutation hot spots of K-ras were found at codon 12 (n = 11) and 13 (n = 4) in 15 of the 55 carcinomas (27 percent). No mutation was found at codon 61. Mutations of a probably causative nature in the evolutionarily conserved regions (exons 5-8) of the TP53 gene were found in 24 tumors (44 percent). K-ras and TP53 mutations were found equally in the group with recurrent disease (7/26 (26 percent) and 12/27 (44 percent), respectively) and in the group without recurrences (8/28 (24 percent) and 12/28 (43 percent), respectively). Cancer-specific survival did not differ significantly between patients with K-ras or TP53 or both mutated and nonmutated tumors, respectively (log-rank test: K-ras, P = 0.72 and TP53, P = 0.77; K-ras and TP53, P = 0.8). Also, potentially aggressive K-ras codon 12 and 13 mutations had the same survival as tumors without these mutations (log-rank test; P = 0.73). CONCLUSIONS: Patients with K-ras or TP53 or both mutated Dukes C colon tumors have the same survival as nonmutated tumors when treated with adjuvant chemotherapy. These data suggest that mutations in K-ras or TP53 alone are not prognostic indicators in patients with Dukes C colon cancer receiving adjuvant 5-Fluorouracil-based therapy.

Clinical definition of hereditary non-polyposis colorectal cancer: a search for the impossible?

Hereditary non-polyposis colorectal cancer is an autosomal dominant inherited disorder that predisposes its carriers to an almost 100% lifetime risk of cancer, in particular colorectal and endometrial cancer. Germline mutations, resulting in a deficient DNA mismatch repair system, are responsible for the disease. Because of the lack of specific phenotypical features, clinical diagnosis in an individual patient is impossible and relies heavily on family history. Genetic diagnosis by mismatch detection is now possible in a substantial proportion of families. Thus there is a great need for reliable but simple criteria that will help clinicians to recognize patients and families who can be referred for genetic diagnostics. In this article the different criteria that have been formulated and published in recent years are reviewed and the results, in terms of the proportions of subjects satisfying the criteria who were found to have a germline mutation, are discussed. In most studies the criteria were evaluated in only a small number of subjects. A population-based study is currently being carried out in the north of The Netherlands that aims to include 400 patients fulfilling one of a few simple criteria. Mutation analysis will be performed in all patients. The results of this study will help in the formulation of accurate and simple criteria for use in clinical practice.

New comprehensive denaturing-gradient-gel- electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours.

A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within two lanes of a DGGE gel under a single set of experimental conditions. Six known codon 12 mutations in genomic DNA from different paraffin-embedded tumours could readily be detected. When testing 35 paraffin-embedded Dukes' C colorectal carcinomas for unknown mutations, 12 tumours were found with mutations in codons 12 or 13. None of the tumours appeared to have a codon 61 mutation. In nine tumours, however, 11 additional sequence variations were found outside the hot-spot codons. None of these occurred in germline DNA from 57 individuals. Of these variations, three are considered as significant mutations, as they result in a non-conservative substitution of amino acid residues essential for the functioning of the KRAS protein. Thus, in total, 15 of the 35 Dukes' C tumours (43%) had a KRAS mutation of functional significance. Moreover, a novel exon 4B polymorphism was found to occur in 10 of the 35 tumours. The results of this study suggest that in restricting analysis of KRAS to hot-spot mutation sites only, significant information may be missed.

Inclusion of malignant fibrous histiocytoma in the tumour spectrum associated with hereditary non-polyposis colorectal cancer.

Sarcomas, including the malignant fibrous histiocytomas (MFHs), are not known to be part of the tumour spectrum of hereditary non-polyposis colorectal cancer (HNPCC) as epidemiologically established. Therefore, occurrence of MFH in an HNPCC family may very well be coincidental. HNPCC is associated with germline mutations in DNA mismatch repair genes, including the MSH2 gene. We analysed an MFH diagnosed in a 45-year-old male HNPCC patient carrying a germline MSH2 mutation for HNPCC-associated molecular characteristics, to investigate a possible relationship between the tumour and that mutation. DNA analysis revealed microsatellite instability and loss of one MSH2 copy, and immunohistochemistry showed absence of nuclear MSH2 protein staining. To investigate whether this is a common finding in MFH, microsatellite instability and nuclear MSH2 protein staining was tested for in 5 and 6 sporadic MFHs, respectively. None showed microsatellite instability and all stained positively for MSH2. Together, these findings show that in rare cases, MFH may be part of the HNPCC tumour spectrum.

Impact of KRAS and TP53 mutations on survival in patients with left- and right-sided Dukes' C colon cancer.

OBJECTIVE: It has been suggested that KRAS and TP53 mutated tumors might influence the phenotypic behavior of left- and right-sided colon tumors. We investigated the incidence of these mutations in left- and right-sided colon tumors and their possible influence on survival in a homogeneous group of patients with Dukes' C colon cancers. METHODS: The primary tumors of 55 patients with a sporadic Dukes' C colon cancer, all treated with adjuvant chemotherapy were analyzed for the presence of KRAS and TP53 mutations. Mutation detection of the KRAS and TP53 genes was performed on paraffin-embedded tumor material, using denaturating gradient gel electrophoresis. The 5-yr survival rates of KRAS and TP53 mutated tumors were analyzed regarding right-sided tumors (defined as tumors up to the splenic flexure) and left-sided tumors (defined as tumors from the splenic flexure to the rectosigmoid peritoneal reflection). RESULTS: KRAS mutations occurred more frequently in the right colon compared to the left colon (R = 38% (10/26); L = 10% (3/29); chi2 test: p = 0.014). KRAS mutations did not influence survival in patients with right-sided colon tumors. Patients with KRAS mutation-negative tumors in the right colon, however, had a significantly worse survival than patients with left-sided KRAS mutation-negative tumors (5-yr survival; R: 34% vs L: 65%, log-rank test: p = 0.007). TP53 mutations of a possible causative nature were found in 24 tumors (44%). Neither the incidence (R = 42% (11/26); L = 45% (13/29)) nor the survival of TP53 mutated tumors differed significantly between left- and right-sided tumors. Furthermore, survival of patients with TP53 mutation-negative tumors did not differ significantly between left- and right-sided tumors. CONCLUSIONS: There seems to be no difference in survival rate between patients with KRAS mutated and KRAS negative Dukes' C colon tumors; however, KRAS mutations are more frequently found in the right colon compared to the left colon. TP53 mutations do not have predominance for either side of the colon, and there are no differences in survival in patients with left-sided versus right-sided tumors. Patients with KRAS-nonmutated tumors in the right colon did have a worse survival compared to those with such tumors in the left colon. This suggests that other genetic factors may play a role in tumor genesis in this subgroup of patients.