Phase II study of high-sensitivity genotyping of KRAS, NRAS, BRAF and PIK3CA to ultra-select metastatic colorectal cancer patients for panitumumab plus FOLFIRI: the ULTRA trial.

BACKGROUND: Several studies show the importance of accurately quantifying not only KRAS and other low-abundant mutations because benefits of anti-EGFR therapies may depend on certain sensitivity thresholds. We assessed whether ultra-selection of patients using a high-sensitive digital PCR (dPCR) to determine KRAS, NRAS, BRAF and PIK3CA status can improve clinical outcomes of panitumumab plus FOLFIRI. PATIENTS AND METHODS: This was a single-arm phase II trial that analysed 38 KRAS, NRAS, BRAF and PIK3CA hotspots in tumour tissues of irinotecan-resistant metastatic colorectal cancer patients who received panitumumab plus FOLFIRI until disease progression or early withdrawal. Mutation profiles were identified by nanofluidic dPCR and correlated with clinical outcomes (ORR, overall response rate; PFS, progression-free survival; OS, overall survival) using cut-offs from 0% to 5%. A quantitative PCR (qPCR) analysis was also performed. RESULTS: Seventy-two evaluable patients were enrolled. RAS (KRAS/NRAS) mutations were detected in 23 (32%) patients and RAS/BRAF mutations in 25 (35%) by dPCR, while they were detected in 7 (10%) and 11 (15%) patients, respectively, by qPCR. PIK3CA mutations were not considered in the analyses as they were only detected in 2 (3%) patients by dPCR and in 1 (1%) patient by qPCR. The use of different dPCR cut-offs for RAS (KRAS/NRAS) and RAS/BRAF analyses translated into differential clinical outcomes. The highest ORR, PFS and OS in wild-type patients with their lowest values in patients with mutations were achieved with a 5% cut-off. We observed similar outcomes in RAS/BRAF wild-type and mutant patients defined by qPCR. CONCLUSIONS: High-sensitive dPCR accurately identified patients with KRAS, NRAS, BRAF and PIK3CA mutations. The optimal RAS/BRAF mutational cut-off for outcome prediction is 5%, which explains that the predictive performance of qPCR was not improved by dPCR. The biological and clinical implications of low-frequent mutated alleles warrant further investigations. CLINICALTRIALS.GOV NUMBER: NCT01704703. EUDRACT NUMBER: 2012-001955-38.

Ultra-selection of metastatic colorectal cancer patients using next-generation sequencing to improve clinical efficacy of anti-EGFR therapy.

BACKGROUND: Extended RAS analysis is mandatory in metastatic colorectal cancer (mCRC) patients. The optimal threshold of RAS mutated subclones to identify patients most likely to benefit from antiepidermal growth factor receptor (EGFR) therapy is controversial. Our aim was to assess the clinical impact of detecting mutations in RAS, BRAF, PIK3CA and EGFRS492R in basal tissue tumour samples by using a highly sensitive next-generation sequencing (NGS) technology in mCRC patients treated with chemotherapy plus anti-EGFR or anti-vascular endothelial growth factor. PATIENTS AND METHODS: Five hundred and eighty-one tumour samples from untreated mCRC patients from 7 clinical studies were collected. Mutational analysis was carried out by standard-of-care (therascreen pyro) with a sensitivity detection of 5% mutant allele fraction (MAF), and compared with NGS technology using 454GS Junior platform (Roche Applied Science, Germany) with a sensitivity of 1%. Molecular results were correlated with clinical outcomes. RESULTS: After quality assessment, 380 samples were evaluable for molecular analysis. Standard-of-care mutational analysis detected RAS, BRAFV600E or PIK3CA mutations in 56.05% of samples compared with 69.21% by NGS (P = 0.00018). NGS identified coexistence of multiple low-frequency mutant alleles in 96 of the 263 mutated cases (36.5%; range 2-7). Response rate (RR), progression-free survival (PFS) and overall survival (OS) were increasingly improved in patients with RAS wild-type, RAS/BRAF wild-type or quadruple (KRAS/NRAS/BRAF/PIK3CA) wild-type tumours treated with anti-EGFR, assessed by standard-of-care. No additional benefit in RR, PFS or OS was observed by increasing the detection threshold to 1% by NGS. An inverse correlation between the MAF of the most prevalent mutation detected by NGS and anti-EGFR response was observed (P = 0.039). EGFRS492Rmutation was not detected in untreated samples. CONCLUSIONS: No improvement in the selection of patients for anti-EGFR therapy was obtained by adjusting the mutation detection threshold in tissue samples from 5% to 1% MAF. Response to anti-EGFR was significantly better in patients with quadruple wild-type tumours.

Prediction of pathological response to neoadjuvant treatment in rectal cancer with a two-protein immunohistochemical score derived from stromal gene-profiling.

BACKGROUND: Preoperative chemoradiotherapy followed by surgical mesorectal resection is the standard of care for locally advanced rectal carcinomas. Yet, predicting that patients will respond to treatment remains an unmet clinical challenge. EXPERIMENTAL DESIGN: Using laser-capture microdissection we isolated RNA from stroma and tumour glands from prospective pre-treatment samples (n = 15). Transcriptomic profiles were obtained hybridising PrimeView Affymetrix arrays. We modelled a carcinoma-associated fibroblast-specific genes filtering data using GSE39396. RESULTS: The analysis of differentially expressed genes of stroma/tumour glands from responder and non-responder patients shows that most changes were associated with the stromal compartment; codifying mainly for extracellular matrix and ribosomal components. We built a carcinoma-associated fibroblast (CAF) specific classifier with genes showing changes in expression according to the tumour regression grade (FN1, COL3A1, COL1A1, MMP2 and IGFBP5). We assessed these five genes at the protein level by means of immunohistochemical staining in a patient's cohort (n = 38). For predictive purposes we used a leave-one-out cross-validated model with a positive predictive value (PPV) of 83.3%. Random Forest identified FN1 and COL3A1 as the best predictors. Rebuilding the leave-one-out cross-validated regression model improved the classification performance with a PPV of 93.3%. An independent cohort was used for classifier validation (n = 36), achieving a PPV of 88.2%. In a multivariate analysis, the two-protein classifier proved to be the only independent predictor of response. CONCLUSION: We developed a two-protein immunohistochemical classifier that performs well at predicting the non-response to neoadjuvant treatment in rectal cancer.

Identification of a founder BRCA1 mutation in the Moroccan population.

Breast cancer (BC) is the most frequent cancer among women in Morocco. However, the role of the most prevalent BC-predisposing genes, BRCA1 and BRCA2, has been largely unexplored. To help define the role of BRCA1 in BC in Morocco, we characterized the first potential BRCA1 founder mutation in this population. Genetic testing of BRCA1 and BRCA2 in BC high-risk families identified mutation BRCA1 c.5309G>T, p.(Gly1770Val) or G1770V in five independent families from Morocco, suggesting a founder effect. To confirm this hypothesis, haplotype construction was performed using seven intragenic and flanking BRCA1 microsatellite markers. Clinical data were also compiled. Clinical data from carriers of mutation G1770V correspond to data from carriers of BRCA1 pathogenic mutations. Microsatellite analysis showed a common haplotype for the five families in a region comprising 1.54 Mb, confirming G1770V as the first specific founder BRCA1 mutation in the Moroccan population. Our findings contribute to a better understanding of BC genetics in the Moroccan population. Nevertheless, comprehensive studies of mutation G1770V in large series of BC patients from Morocco are needed to assess the real prevalence of this mutation and to improve genetic testing and risk assessment in this population.

Detailed characterization of MLH1 p.D41H and p.N710D variants coexisting in a Lynch syndrome family with conserved MLH1 expression tumors.

Lynch syndrome (LS) is an autosomal dominant cancer-susceptibility disease caused by inactivating germline mutations in mismatch repair (MMR) genes. Variants of unknown significance (VUS) are often detected in mutational analysis of MMR genes. Here we describe a large family fulfilling Amsterdam I criteria carrying two rare VUS in the MLH1 gene: c.121G > C (p.D41H) and c.2128A > G (p.N710D). Collection of clinico-pathological data, multifactorial analysis, in silico predictions, and functional analyses were used to elucidate the clinical significance of the identified MLH1 VUS. Only the c.121G > C variant cosegregated with LS-associated tumors in the family. Diagnosed colorectal tumors were microsatellite unstable although immunohistochemical staining revealed no loss of MMR proteins expression. Multifactorial likelihood analysis classified c.2128A > G as a non-pathogenic variant and c.121G > C as pathogenic. In vitro functional tests revealed impaired MMR activity and diminished expression of c.121G > C. Accordingly, the N710 residue is located in the unconserved MLH1 C-terminal domain, whereas D41 is highly conserved and located in the ATPase domain. The obtained results will enable adequate genetic counseling of c.121G > C and c.2128A > G variant carriers and their families. Furthermore, they exemplify how cumulative data and comprehensive analyses are mandatory to refine the classification of MMR variants.

Identification of a founder EPCAM deletion in Spanish Lynch syndrome families.

Germline deletions at the 3'-end of EPCAM have been involved in the etiology of Lynch syndrome (LS). The aim of this study was to characterize at the molecular level Spanish families harboring EPCAM deletions. Non-commercial multiplex ligation-dependent probe amplification (MLPA) probes and long-range polymerase chain reaction (PCR) amplification were used to characterize each deletion. Haplotyping was performed by analyzing eight microsatellite markers and five MSH2single nucleotide polymorphisms (SNPs). Methylation of MSH2 was analyzed by methylation specific-MLPA. Tumors diagnosed in seven Spanish families harboring EPCAM deletions were almost exclusively colorectal. Mosaicism in MSH2 methylation was observed in EPCAM deletion carrier samples, being average methylation levels higher in normal colon and colorectal tumors (27.6% and 31.1%), than in lymphocytes and oral mucosa (1.1% and 0.7%). Three families shared the deletion c.858 + 2568_*4596del, with a common haplotype comprising 9.9 Mb. In two families the novel EPCAM deletion c.858 + 2488_*7469del was identified. This study provides knowledge on the clinical and molecular characteristics of mosaic MSH2 epimutations. The identification of an EPCAM founder mutation has useful implications for the molecular diagnosis of LS in Spain.

Bacillus spp. toxicity against Haemonchus contortus larvae in sheep fecal cultures.

The gastrointestinal nematode Haemonchus contortus is a major productivity constraint in sheep. In this study, the nematicidal effects of Bacillus circulans, Bacillus cereus, Bacillus thuringiensis var. israelensis, Bt. var. osvaldocruzi, Bt. var. morrisoni, and Bt. var. kurstaki were assessed in free-living larval stages of H. contortus. A spore-crystal suspension containing approximately 2×10(8)UFC/mL of each strain was added to sheep feces that were naturally infected with H. contortus eggs, and the presence of larvae was then evaluated. We observed a significant (p>0.05) reduction in larval development when using B. circulans, B. thuringiensis var. israelensis, Bt. var. osvaldocruzi and Bt. var. kurstaki, and these effects were proportional with the amount of bacteria added to the feces. However, no effect was observed when Bt. var. morrisoni or B. cereus was added. These observations suggest that these bacteria might be effective as nematicides and may allow for the development of integrated biological control of zooparasitic nematodes.

Localized reactive erythemas of different types in a family.

"Reactive erythemas" is an umbrella term grouping several different conditions, all of which have in common the fact of being stereotypical inflammatory clinical patterns of the skin in response to disparate infectious, immune, or toxic factors. Typically, such eruptions are symmetrical or disseminated. The here reported cases are different. An elderly man underwent recurrent infections of an epidermoid cyst, accompanied by a typical erythema annulare centrifugum near the infectious focus. His grandson, aged ten months, presented with an infectious conjunctivitis, during the resolution of which two small annular lesions, compatible with annular erythema of infancy, appeared on the face. A man aged 42, respectively son and father of the two former patients, presented with an erythema multiforme target lesion proximally to an infected wound. There were no detectable predisposing factors in all cases. Familial cases of reactive erythemas have been reported. However, such limited distributions have not yet been described.

No association between germline allele-specific expression of TGFBR1 and colorectal cancer risk in Caucasian and Ashkenazi populations.

BACKGROUND: Germline allele-specific expression (ASE) of the TGFBR1 gene has been reported as a strong risk factor for colorectal cancer (CRC) with an odds ratio close to 9. Considering the potential implications of the finding, we undertook the task of validating the initial results in this study. METHODS: Allele-specific expression was measured using the highly quantitative and robust technique of pyrosequencing. Individuals from two different populations were studied, one Caucasian-dominated and the other of Ashkenazi Jewish descent, with different sources of non-tumoral genetic material in each. RESULTS: Our results showed no statistically significant differences in the degree of ASE between CRC patients and controls, considering ASE as either a quantitative or a binary trait. Using defined cutoff values to categorise ASE, 1.0% of blood lymphocytes from informative Israeli cases (total n=96) were ASE positive (median 1.00; range 0.76-1.31) and 2.2% of informative matched controls (total n=90) were ASE positive (median 1.00; range 0.76-1.87). Likewise, normal mucosae from Spanish patients (median 1.03; range: 0.68-1.43; n=75) did not show significant differences in the degree of ASE when compared with the Israeli patients or controls. CONCLUSIONS: Taken together, these results suggest that ASE of TGFBR1 does not confer an increased risk of CRC.

Clinical and genetic characterization of classical forms of familial adenomatous polyposis: a Spanish population study.

BACKGROUND: Classical familial adenomatous polyposis (FAP) is characterized by the appearance of >100 colorectal adenomas. PATIENTS AND METHODS: We screened the APC and MUTYH genes for mutations and evaluated the genotype-phenotype correlation in 136 Spanish classical FAP families. RESULTS: APC/MUTYH mutations were detected in 107 families. Sixty-four distinct APC point mutations were detected in 95 families of which all were truncating mutations. A significant proportion (39.6%) had not been previously reported. Mutations were spread over the entire coding region and great rearrangements were identified in six families. Another six families exhibited biallelic MUTYH mutations. No APC or MUTYH mutations were detected in 29 families. These APC/MUTYH-negative families showed clinical differences with the APC-positive families. A poor correlation between phenotype and mutation site was observed. CONCLUSIONS: Our results highlight that a broad approach in the genetic study must be considered for classical FAP due to involvement of both APC and MUTYH and the heterogeneous spectrum of APC mutations observed in this Spanish population. The scarcely consistent genotype-phenotype correlation does not allow making specific recommendations regarding screening and management. Differences observed in APC/MUTYH-negative families may reflect a genetic basis other than mutations in APC and MUTYH genes for FAP predisposition.

Helicobacter pylori cagA and vacA genotypes as predictors of progression of gastric preneoplastic lesions: a long-term follow-up in a high-risk area in Spain.

OBJECTIVES: There are no established predictive markers of progression of gastric preneoplastic lesions. The aim of this study was to analyze the relationship between Helicobacter pylori cagA and vacA genotypes and progression of gastric preneoplastic lesions. METHODS: This was a follow-up study that carried out in a province of Spain with a high risk of gastric cancer. A total of 312 patients who underwent upper endoscopy with gastric biopsy in 1988-1994 with diagnoses of normal mucosa, non-atrophic gastritis (NAG), non-metaplastic multifocal atrophic gastritis (MAG), and complete or incomplete intestinal metaplasia (IM), and who accepted to undergo a new biopsy during 2005-2007 or had an end point during follow-up, were included in this study. Detection and characterization of H. pylori cagA and vacA genotypes was performed directly in baseline paraffin-embedded gastric biopsy specimens by PCR followed by reverse hybridization onto a line probe assay. Inter- and intra-observer variability of histological diagnosis was assessed. Analysis was done using unconditional logistic regression. RESULTS: The mean age of patients was 48.5 years (45% males) and the mean of follow-up was 12.8 years. H. pylori strains harboring cagA, vacA s1, and vacA m1 genotypes were more frequently found in patients with more advanced gastric preneoplastic lesions. Infection with cagA-positive, vacA s1, and vacA m1 strains was associated with progression of gastric preneoplastic lesions (multivariate odds ratio (OR)=2.28, 95% confidence interval (CI) 1.13-4.58; OR=2.90, 95% CI 1.38-6.13; and OR=3.38, 95% CI 1.34-8.53, respectively). Infection with strains that are simultaneously cagA positive and vacA s1/m1 was associated with progression of gastric precancerous lesions with an OR of 4.80 (95% CI 1.71-13.5) in relation to those infected with cagA-negative/vacA s2/m2 strains. CONCLUSIONS: H. pylori genotyping may be useful for the identification of patients at high risk of progression of gastric preneoplastic lesions and who need more intensive surveillance.

Peutz-Jeghers syndrome: a systematic review and recommendations for management.

Peutz-Jeghers syndrome (PJS, MIM175200) is an autosomal dominant condition defined by the development of characteristic polyps throughout the gastrointestinal tract and mucocutaneous pigmentation. The majority of patients that meet the clinical diagnostic criteria have a causative mutation in the STK11 gene, which is located at 19p13.3. The cancer risks in this condition are substantial, particularly for breast and gastrointestinal cancer, although ascertainment and publication bias may have led to overestimates in some publications. Current surveillance protocols are controversial and not evidence-based, due to the relative rarity of the condition. Initially, endoscopies are more likely to be done to detect polyps that may be a risk for future intussusception or obstruction rather than cancers, but surveillance for the various cancers for which these patients are susceptible is an important part of their later management. This review assesses the current literature on the clinical features and management of the condition, genotype-phenotype studies, and suggested guidelines for surveillance and management of individuals with PJS. The proposed guidelines contained in this article have been produced as a consensus statement on behalf of a group of European experts who met in Mallorca in 2007 and who have produced guidelines on the clinical management of Lynch syndrome and familial adenomatous polyposis.

Antihelminthic action of the Anethum graveolens essential oil on Haemonchus contortus eggs and larvae.

Gastrointestinal nematodes are responsible for great economic losses in sheep raising, and their control has long been carried out almost exclusively by the administration of anthelmintics, which have led to serious resistance problems. In the search for alternative control measures, phytotherapic research is highlighted. The aim of this study was to evaluate the action of Anethum graveolens (dill) essential oil on different stages of Haemonchus contortus life cycle, as well its cytotoxicity MDBK (Madin-Darby bovine kidney) cells. H. contortus larvae and eggs were obtained from infected sheep feces, and essential oil extracted from plant seeds through the Clevenger apparatus. 9.4, 4.7, 2.35, 1.17. 0.58 and 0.29 mg/mL concentrations were evaluated. The Egg Hatch Inhibition (HI), Larval Development Inhibition (LDI) and Larval Migration Inhibition (LMI) techniques were used. Thybendazole 0.025 mg/mL in HI and Levamisole 0.02 mg/mL in the LDI and LMI tests were used as positive controls, while distilled water and a Tween 80 solution were used as positive negative controls. The inhibition results obtained for the highest oil concentration were: HI 100%, LDI 98.58% and LMI 63.7%, differing (𝑝 <0.05) from negative controls. Main A. graveolens oil components present in 95.93% of the total oil were Dihydrocarvone (39.1%), Carvone (22.24%), D-Limonene (16.84%), Apiol (10.49%) and Trans-dihydrocarvone (7.26%). Minimum A. graveolens essential oil concentrations required to inhibit 50% (IC50) of egg hatching, larval development and larval migration were 0.006 mg/mL, 2.536 mg/mL and 3.963 mg/mL, respectively. Cell viability in MDBK (Madin-Darby bovine kidney) cells, when incubated with A. graveolens essential oil, was 86% for the highest (9.4 mg/mL) and 99% for the lowest concentration (0.29 mg/mL). A. graveolens essential oil, according to the results obtained in this study, is a promising alternative in sheep gastrointestinal nematode control.

Exome sequencing of early-onset patients supports genetic heterogeneity in colorectal cancer.

Colorectal cancer (CRC) is a complex disease that can be caused by a spectrum of genetic variants ranging from low to high penetrance changes, that interact with the environment to determine which individuals will develop the disease. In this study, we sequenced 20 early-onset CRC patients to discover novel genetic variants that could be linked to the prompt disease development. Eight genes, CHAD, CHD1L, ERCC6, IGTB7, PTPN13, SPATA20, TDG and TGS1, were selected and re-sequenced in a further 304 early onset CRC patients to search for rare, high-impact variants. Although we found a recurring truncating variant in the TDG gene shared by two independent patients, the results obtained did not help consolidate any of the candidates as promising CRC predisposing genes. However, we found that potential risk alleles in our extended list of candidate variants have a tendency to appear at higher numbers in younger cases. This supports the idea that CRC onset may be oligogenic in nature and may show molecular heterogeneity. Further, larger and robust studies are thus needed to unravel the genetics behind early-onset CRC development, coupled with novel functional analyses and omic approaches that may offer complementary insight.

A large-scale meta-analysis to refine colorectal cancer risk estimates associated with MUTYH variants.

BACKGROUND: defective DNA repair has a causal role in hereditary colorectal cancer (CRC). Defects in the base excision repair gene MUTYH are responsible for MUTYH-associated polyposis and CRC predisposition as an autosomal recessive trait. Numerous reports have suggested MUTYH mono-allelic variants to be low penetrance risk alleles. We report a large collaborative meta-analysis to assess and refine CRC risk estimates associated with bi-allelic and mono-allelic MUTYH variants and investigate age and sex influence on risk. METHODS: MUTYH genotype data were included from 20 565 cases and 15 524 controls. Three logistic regression models were tested: a crude model; adjusted for age and sex; adjusted for age, sex and study. RESULTS: all three models produced very similar results. MUTYH bi-allelic carriers demonstrated a 28-fold increase in risk (95% confidence interval (CI): 6.95-115). Significant bi-allelic effects were also observed for G396D and Y179C/G396D compound heterozygotes and a marginal mono-allelic effect for variant Y179C (odds ratio (OR)=1.34; 95% CI: 1.00-1.80). A pooled meta-analysis of all published and unpublished datasets submitted showed bi-allelic effects for MUTYH, G396D and Y179C (OR=10.8, 95% CI: 5.02-23.2; OR=6.47, 95% CI: 2.33-18.0; OR=3.35, 95% CI: 1.14-9.89) and marginal mono-allelic effect for variants MUTYH (OR=1.16, 95% CI: 1.00-1.34) and Y179C alone (OR=1.34, 95% CI: 1.01-1.77). CONCLUSIONS: overall, this large study refines estimates of disease risk associated with mono-allelic and bi-allelic MUTYH carriers.

Founder effect of a pathogenic MSH2 mutation identified in Spanish families with Lynch syndrome.

Lynch syndrome is caused by germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. The novel MSH2 c.[2635-3T>C; 2635-5C>T] mutation was identified in 4 Lynch families, cosegregating with the disease. This mutation, located in intron 15, was predicted to alter the correct mRNA processing by in silico analysis. Our aim was to perform the c.[2635-3T>C; 2635-5C>T] mutation screening in high risk CRC cases and control populations, to evaluate the founder effect in our population by haplotype analysis and to confirm the pathogenic effect of the mutation by MSH2 expression studies. Mutation screening was performed by SSCP and CSCE in genomic DNA from 323 high risk CRC cases and 289 controls. Haplotyping was performed analysing 4 MSH2 extragenic microsatellite markers (D2S288, D2S2227, D2S1247 and D2S1248) in 50 controls and mutation carriers by using the PHASE program. We analysed the effect of the mutation in mRNA processing by RT-PCR and in MSH2 expression by qRT-PCR using RNA from 5 mutation carriers and 18 controls. None of the remaining high risk CRC cases or controls analysed harboured the mutation. We identified a common telomeric haplotype and two centromeric haplotypes, both rare in our population. Although we were not able to identify any abnormal transcript by RT-PCR with the design used, we observed a significant reduction of mRNA MSH2 expression in carriers when compared with controls. Haplotype analyses suggest a founder effect of the c.[2635-3T>C; 2635-5C>T] MSH2 mutation and expression studies support a pathogenic role of this mutation.

Saccharomyces cerevisiae (YT001) supplementation for the control of Haemonchus contortus and modulation of the immune response of sheep.

Studies aiming at the development and evaluation of alternative methods to minimise losses caused by the gastrointestinal nematode Haemonchus contortus are extremely important. Such research is essential, given the high morbidity rates among sheep and the significant mortality rates of lambs, allied to the low efficacy of commercial products for the control of this parasite. The purpose of this study was to evaluate the effect of the Saccharomyces cerevisiae (YT001 - YEASTECH) on the control of H. contortus and its modulation of the immune response in experimentally infected sheep. Eighteen sheep were divided into two groups. Group 1, the control group, comprised animals infected with H. contortus and supplemented with distilled water, while Group 2, the treated group, consisted of animals infected and supplemented with S. cerevisiae (400 million cfu/day of suspension for 49 days). The following parasitological parameters were evaluated: number of eggs per gram of faeces, number of infective larvae (L3) recovered per faecal culture, and parasitic load of the abomasum. The following immunological parameters were quantified: immunoglobulin (Ig)A in the mucous secretions and serum IgG; cytokines interleukin (IL)-4, IL-5 and IL-10; number of eosinophils in the abomasal mucosa and groups of cells positive for the markers: MHCII, CD4+CD25+, CD5+CD8+, WC4, CD5+CD4+, CD8+CD11b+ and CD5+WC1 by whole blood flow cytometry. The results revealed a significant decrease (P<0.05) in the number of larvae and significantly higher serum IgG levels (P<0.05) in the group supplemented with S. cerevisiae. The supplemented animals showed significantly larger numbers of eosinophils (P<0.05), as well as more cells positive for MHCII, CD4+CD25+, CD5+CD8+ than the control animals. This study confirmed the beneficial action of S. cerevisiae on the host immune response to H. contortus, as evidenced mainly by the smaller number of L3 recovered from the faeces of sheep supplemented with S. cerevisiae.

Long-range epigenetic silencing at 2q14.2 affects most human colorectal cancers and may have application as a non-invasive biomarker of disease.

Large chromosomal regions can be suppressed in cancer cells as denoted by hypermethylation of neighbouring CpG islands and downregulation of most genes within the region. We have analysed the extent and prevalence of long-range epigenetic silencing at 2q14.2 (the first and best characterised example of coordinated epigenetic remodelling) and investigated its possible applicability as a non-invasive diagnostic marker of human colorectal cancer using different approaches and biological samples. Hypermethylation of at least one of the CpG islands analysed (EN1, SCTR, INHBB) occurred in most carcinomas (90%), with EN1 methylated in 73 and 40% of carcinomas and adenomas, respectively. Gene suppression was a common phenomenon in all the tumours analysed and affected both methylated and unmethylated genes. Detection of methylated EN1 using bisulfite treatment and melting curve (MC) analysis from stool DNA in patients and controls resulted in a predictive capacity of, 44% sensitivity in positive patients (27% of overall sensitivity) and 97% specificity. We conclude that epigenetic suppression along 2q14.2 is common to most colorectal cancers and the presence of a methylated EN1 CpG island in stool DNA might be used as biomarker of neoplastic disease.

Non-Hodgkin lymphoma related to hereditary nonpolyposis colorectal cancer in a patient with a novel heterozygous complex deletion in the MSH2 gene.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal disorder caused by mutations in DNA mismatch repair (MMR) genes. Tumors of the HNPCC-spectrum are associated with microsatellite instability (MSI) and loss of MMR protein expression. Lymphomas are not considered to be HNPCC-related tumors. We report and analyze a case of an HNPCC patient with three colorectal cancers and a B-cell non-Hodgkin lymphoma. Quantitative multiplex PCR of short fluorescent fragments detected a novel MSH2 rearrangement involving exons 9 and 10, which proved to be the pathogenic cause of the disease in the family. Tumor tissues including the lymphoma showed MSI and loss of MSH2 expression. Multiplex ligation-dependent probe amplification analysis revealed a somatic loss of the wild-type MSH2 allele in the lymphoma. These results support the fact that the total loss of a MMR gene can lead to lymphomagenesis, as seen in biallelic MMR-deficient families and knockout mice. Moreover, this is the first report of a B-cell non-Hodgkin lymphoma with a loss of the MSH2 protein expression, linked to a heterozygous germline MSH2 mutation in an HNPCC family.