Search

Purpose: The present study aimed to determine the prevalence of MUTYH mutations in patients with multiple colonic polyps and to explore the best strategy for diagnosing MUTYH-associated polyposis (MAP) in these patients.Experimental Design: This study included 405 patients with at least 10 colonic polyps each. All cases were genetically tested for the three most frequent MUTYH mutations. Whole-gene analysis was performed in heterozygous patients and in 216 patients lacking the three most frequent mutations. Polyps from 56 patients were analyzed for the KRAS-Gly12Cys and BRAF V600E somatic mutations.Results: Twenty-seven (6.7%) patients were diagnosed with MAP, of which 40.8% showed serrated polyps. The sensitivity of studying only the three common variants was 74.1%. Of 216 patients without any monoallelic mutation in common variants, whole-gene analysis revealed biallelic pathogenic mutation in only one. G396D mutation was associated with serrated lesions and older age at diagnosis. There was a strong association between germinal MUTYH mutation and KRAS Gly12Cys somatic mutation in polyps. BRAF V600E mutation was found in 74% of serrated polyps in MUTYH-negative patients and in none of the polyps of MAP patients.Conclusions: We observed a low frequency of MUTYH mutations among patients with multiple adenomatous and serrated polyps. The MAP phenotype frequently included patients with serrated polyps, especially when G396D mutation was involved. Our results show that somatic molecular markers of polyps can be useful in identifying MAP cases and support the need for the complete MUTYH gene analysis only in patients heterozygous for recurrent variants. Clin Cancer Res; 20(5); 1158–68. ©2014 AACR.

CCR Translation for This Article from Prevalence and Characteristics of MUTYH-Associated Polyposis in Patients with Multiple Adenomatous and Serrated Polyps

An increased risk of developing colorectal cancer (CRC) and other types of tumor is associated to Lynch syndrome (LS), an inherited condition caused by germline mutations in mismatch repair genes. We selected a cohort of LS patients that had developed CRC and had undergone surgical resection. Formalin-fixed paraffin embedded (FFPE) tissue blocks from matched colorectal and normal mucosa were used for genomic DNA extraction with a commercial kit and sequenced by high-throughput sequencing. A metagenomic approach enabled the taxonomic and functional identification of the microbial community and associated genes detected in the specimens. Slightly lower taxonomic diversity was observed in the tumor compared to the non-tumor tissue. Furthermore, the most remarkable differences between tumors and healthy tissue was the significant increase in the genus Fusobacterium in the former, in particular the species F. nucleatum, as well as Camplylobacter or Bacteroides fragilis, in accordance with previous studies of CRC. However, unlike prior studies, the present work is not based on directed detection by qPCR but instead uses a metagenomic approach to retrieve the whole bacterial community, and addresses the additional difficulty of using long-term stored FFPE samples., We want to particularly acknowledge the patients and the Biobank IBSP-CV (PT17/0015/0017) integrated in the Spanish National Biobanks Network and in the Valencian Biobanking Network, especially A. Ahicart, D. Molina and J. Martínez, for their collaboration. This research was funded by grants to AM from the Fundación Científica de la Asociación Española contra el Cancer (project AECC 2017-1485), including a post-doctoral contract to VPB first and to SRR later. GD is recipient of a PhD fellowship from the Junta Asociada Provincial de Valencia AECC. Action co-financed by the European Union through the Operational Program of European Regional Development Fund (ERDF) of Valencia Region (Spain) 2014-2020.

Background Lynch syndrome (LS) is a hereditary condition characterized by a high risk of colorectal cancer, endometrial cancer, and other neoplasia associated with germline alterations in DNA mismatch repair genes. The classical genetic diagnostic strategy for LS consists of the Sanger sequencing of genes associated with the suspected syndrome. Next‐generation sequencing (NGS) enables the simultaneous sequencing of a large number of hereditary cancer genes. Here, we aimed to study whether other germline pathogenic variants of hereditary cancer genes are present in patients with LS. Methods A cohort of 84 probands with a previous genetic diagnosis of LS by Sanger sequencing was reanalyzed using NGS via a commercial panel of 94 hereditary cancer genes by hybrid capture. The American College of Medical Genetics and Genomics criteria were used to classify the clinical significance of the variants. The findings of NGS were confirmed by Sanger sequencing. When possible, genetic analyses of the new findings in the proband's relatives were also performed by Sanger sequencing. Results We identified five families (6%), out of 84, with at least two germline pathogenic variants conferring to high or moderate risk in different dominant cancer‐predisposing genes: [MLH1‐BRCA2‐NBN], [MLH1‐BRCA1], [MSH2‐ATM], [MSH6‐NF1], and [MLH1‐FANCA]. Interestingly, only one out of these five families exhibited a clinical phenotype associated with the new pathogenic variants. The family with three pathogenic variants of the [MLH1‐BRCA2‐NBN] genes showed a high aggregation of tumors associated with LS and breast and ovarian cancer syndrome. Conclusions Our results showed that the co‐occurrence of more than one pathogenic variant in cancer‐predisposing genes was remarkable among cases of LS. In most cases, no clinicial manifestations were associated with the secondary pathogenic variants. Further studies are needed to confirm these findings and elucidate their clinical impact. Reanalysis of LS families should be considered only in families with mixed clinical phenotypes., We aimed to explore whether other pathogenic variants for hereditary cancer genes are present in Lynch syndrome patients, to have a more comprehensive view of the genetic architecture for cancer risk. We found five families (6%) with at least two pathogenic variant in high‐ and/or moderate‐risk‐associated cancer genes. However, in most cases, no clinical manifestations associated with the secondary pathogenic variants were evidenced.

1580 Background: HLRCC is a hereditary condition with autosomal dominant inheritance due to germline mutations in the fumarate-hydratase gene ( FH). It is characterized by skin leiomyomas (SLM) in 48-84% of individuals, uterine leiomyomas (ULM) in 30-72%, renal cysts (RCy) and renal cell cancer (RCC) in 15-34%. We aimed to describe the genetics, the clinical features and the potential genotype-phenotype associations in the largest cohort of FH mutation carriers from Spain. Methods: We performed a multicenter, observational, retrospective study of individuals with genetic or clinical diagnosis of HLRCC. We collected clinical information from medical records. We analyzed genetic variants and looked for genotype-phenotype associations. Statistical analyses were performed by IBM-SPSS Statistics-v.22. Results: We included 197 individuals (113 women, 84 men), 74 index cases and 123 relatives. Twenty-seven different variants were detected, 26 pathogenic (12 missense, 5 frameshift, 4 large-deletions, 3 splice-site and 2 nonsense) and 1 variant of unknown significance (missense). Of 182 patients with full skin examination, 64.8% presented SLM (median age 36 years; range 8-85). ULM were diagnosed in 90.3% of 103 women with gynecologic exam (median age 30 years; range 17- 55). Hysterectomy was performed in 62.9% (median age 34 years; range 21-54). Of 153 patients with radiological records, 37.3 % presented RCy. Nineteen patients (10.9%) presented RCC, 11 males and 8 females (median age 37 years; range 10-67). The histological diagnoses were: 14 papillary, of which 10 were type 2; 3 clear cell carcinoma and 2 unclassified carcinoma. Six tumors had stage I, 2 stage II, 3 stage III, 4 stage IV, and 4 not available. The median overall survival among patients at stages 3-4 was 2.9 years [1.3-4.5]. Patients with missense pathogenic variants showed higher risk of developing SLM (p = 0.043) and ULM (p = 0.002) than those with loss of function variants. Conclusions: In our cohort, the frequency of RCC (10.9%) is lower than that published in cohorts of similar sample size. The most frequent histology was the papillary type-2; however, other histological patterns do not exclude HLRCC. Individuals with missense pathogenic variants show higher incidence of SLM and ULM.

Simple Summary Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is a very rare hereditary disorder characterized by cutaneous leiomyomas (CLMs), uterine leiomyomas (ULMs), renal cysts (RCys) and renal cell cancer (RCC), with no data on its prevalence worldwide. No genotype-phenotype associations have been described. The aim of our study was to describe the genotypic and phenotypic features of the largest series of patients with HLRCC from Spain reported to date. Of 27 FH germline pathogenic variants, 12 were not previously reported in databases. Patients with missense pathogenic variants showed higher frequencies of CLMs, ULMs and RCys, than those with loss-of-function variants. The frequency of RCCs (10.9%) was lower than those reported in the previously published series. Hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) is a very rare hereditary disorder characterized by cutaneous leiomyomas (CLMs), uterine leiomyomas (ULMs), renal cysts (RCys) and renal cell cancers (RCCs). We aimed to describe the genetics, clinical features and potential genotype-phenotype associations in the largest cohort of fumarate hydratase enzyme mutation carriers known from Spain using a multicentre, retrospective study of individuals with a genetic or clinical diagnosis of HLRCC. We collected clinical information from medical records, analysed genetic variants and looked for genotype-phenotype associations. Analyses were performed using R 3.6.0. software. We included 197 individuals: 74 index cases and 123 relatives. CLMs were diagnosed in 65% of patients, ULMs in 90% of women, RCys in 37% and RCC in 10.9%. Twenty-seven different pathogenic variants were detected, 12 (44%) of them not reported previously. Patients with missense pathogenic variants showed higher frequencies of CLMs, ULMs and RCys, than those with loss-of-function variants (p = 0.0380, p = 0.0015 and p = 0.024, respectively). This is the first report of patients with HLRCC from Spain. The frequency of RCCs was lower than those reported in the previously published series. Individuals with missense pathogenic variants had higher frequencies of CLMs, ULMs and RCys.

Lynch syndrome is an autosomal dominant disorder caused by germline mutations in DNA mismatch repair (MMR) system genes, such as MLH1, MSH2, MSH6, or PMS2. It is the most common hereditary colorectal cancer syndrome. Screening is regularly performed by using microsatellite instability (MSI) or immunohistochemistry for the MMR proteins in tumor samples. However, in a proportion of cases, MSI is found or MMR immunohistochemistry is impaired in the absence of a germline mutation in MMR genes, BRAF mutation, or MLH1 hypermethylation. These cases are defined as Lynch-like syndrome. Patients with Lynch-like syndrome represent a mixture of truly hereditary and sporadic cases, with a risk of colorectal cancer in first-degree relatives that is between the risk of Lynch syndrome in families and relatives of sporadic colon cancer cases. Although multiple approaches have been suggested to distinguish between hereditary and sporadic cases, a homogeneous testing protocol and consensus on the adequate classification of these patients is still lacking. For this reason, management of Lynch-like syndrome and prevention of cancer in these families is clinically challenging. This review explains the concept of Lynch-like syndrome, potential mechanisms for its development, and methods for adequately distinguishing between sporadic and hereditary cases of this entity.

Constitutional MLH1 epimutations are characterised by monoallelic methylation of the MLH1 promoter throughout normal tissues, accompanied by allele-specific silencing. The mechanism underlying primary MLH1 epimutations is currently unknown. The aim of this study was to perform an in-depth characterisation of constitutional MLH1 epimutations targeting the aberrantly methylated region around MLH1 and other genomic loci. Twelve MLH1 epimutation carriers, 61 Lynch syndrome patients, and 41 healthy controls, were analysed by Infinium 450 K array. Targeted molecular techniques were used to characterise the MLH1 epimutation carriers and their inheritance pattern. No nucleotide or structural variants were identified in-cis on the epimutated allele in 10 carriers, in which inter-generational methylation erasure was demonstrated in two, suggesting primary type of epimutation. CNVs outside the MLH1 locus were found in two cases. EPM2AIP1-MLH1 CpG island was identified as the sole differentially methylated region in MLH1 epimutation carriers compared to controls. Primary constitutional MLH1 epimutations arise as a focal epigenetic event at the EPM2AIP1-MLH1 CpG island in the absence of cis-acting genetic variants. Further molecular characterisation is needed to elucidate the mechanistic basis of MLH1 epimutations and their heritability/reversibility.

Background Approximately 5 to 10% of all cancers are caused by inherited germline mutations, many of which are associated with different Hereditary Cancer Syndromes (HCS). In the context of the Program of Hereditary Cancer of the Valencia Community, individuals belonging to specific HCS and their families receive genetic counselling and genetic testing according to internationally established guidelines. The current diagnostic approach is based on sequencing a few high-risk genes related to each HCS; however, this method is time-consuming, expensive and does not achieve a confirmatory genetic diagnosis in many cases. This study aims to test the level of improvement offered by a Next Generation Sequencing (NGS) gene-panel compared to the standard approach in a diagnostic reference laboratory setting. Methods A multi-gene NGS panel was used to test a total of 91 probands, previously classified as non-informative by analysing the high-risk genes defined in our guidelines. Results Nineteen deleterious mutations were detected in 16% of patients, some mutations were found in already-tested high-risk genes (BRCA1, BRCA2, MSH2) and others in non-prevalent genes (RAD51D, PALB2, ATM, TP53, MUTYH, BRIP1). Conclusions Overall, our findings reclassify several index cases into different HCS, and change the mutational status of 14 cases from non-informative to gene mutation carriers. In conclusion, we highlight the necessity of incorporating validated multi-gene NGS panels into the HCSs diagnostic routine to increase the performance of genetic diagnosis. Electronic supplementary material The online version of this article (10.1186/s13053-019-0104-x) contains supplementary material, which is available to authorized users.

Lynch syndrome (LS) is a common cause of hereditary colorectal cancer (CRC). Some CRC patients develop mismatch repair deficiency without germline pathogenic mutation, known as Lynch-like syndrome (LLS). We compared the risk of CRC in first-degree relatives (FDRs) in LLS and LS patients. LLS was diagnosed when tumors showed immunohistochemical loss of MSH2, MSH6, and PMS2, or loss of MLH1 with BRAF wild type, and/or no MLH1 methylation and absence of pathogenic mutation in these genes. CRC and other LS-related neoplasms were followed in patients diagnosed with LS and LLS and among their FDRs. Standardized incidence ratios (SIRs) were calculated for CRC and other neoplasms associated with LS among FDRs of LS and LLS patients. In total, 205 LS (1205 FDRs) and 131 LLS families (698 FDRs) had complete pedigrees. FDRs of patients with LLS had a high incidence of CRC (SIR, 2.08, 95% confidence interval (CI), 1.56&ndash, 2.71), which was significantly lower than that in FDRs of patients with LS (SIR, 4.25, 95% CI, 3.67&ndash, 4.90, p &lt, 0.001). The risk of developing other neoplasms associated with LS also increased among FDR of LLS patients (SIR, 2.04, 95% CI, 1.44&ndash, 2.80) but was lower than that among FDR of patients with LS (SIR, 5.01, 95% CI, 4.26&ndash, 5.84, 0.001). FDRs with LLS have an increased risk of developing CRC as well as LS-related neoplasms, although this risk is lower than that of families with LS. Thus, their management should take into account this increased risk.

BACKGROUND & AIMS: Lynch syndrome is characterized by DNA mismatch repair (MMR) deficiency. Some patients with suspected Lynch syndrome have DNA MMR deficiencies but no detectable mutations in genes that encode MMR proteins-this is called Lynch-like syndrome (LLS). There is no consensus on management of patients with LLS. We collected data from a large series of patients with LLS to identify clinical and pathology features. METHODS: We collected data from a nationwide-registry of patients with colorectal cancer (CRC) in Spain. We identified patients whose colorectal tumors had loss of MSH2, MSH6, PMS2, or MLH1 (based on immunohistochemistry), without the mutation encoding V600E in BRAF (detected by real-time PCR), and/or no methylation at MLH1 (determined by methylation-specific multiplex ligation-dependent probe amplification), and no pathogenic mutations in MMR genes, BRAF, or EPCAM (determined by DNA sequencing). These patients were considered to have LLS. We collected data on demographic, clinical, and pathology features and family history of neoplasms. The chi(2) test was used to analyze the association between qualitative variables, followed by the Fisher exact test and the Student t test or the Mann-Whitney test for quantitative variables. RESULTS: We identified 160 patients with LLS; their mean age at diagnosis of CRC was 55 years and 66 patients were female (41%). The Amsterdam I and II criteria for Lynch syndrome were fulfilled by 11% of cases and the revised Bethesda guideline criteria by 65% of cases. Of the patients with LLS, 24% were identified in universal screening. There were no proportional differences in sex, indication for colonoscopy, immunohistochemistry, pathology findings, or personal history of CRC or other Lynch syndrome-related tumors between patients who met the Amsterdam and/or Bethesda criteria for Lynch syndrome and patients identified in universal screening for Lynch syndrome, without a family history of CRC. CONCLUSIONS: Patients with LLS have homogeneous clinical, demographic, and pathology characteristics, regardless of family history of CRC.

Objective The aim of this study was to validate a molecular classification of colorectal cancer (CRC) based on microsatellite instability (MSI), CpG island methylator phenotype (CIMP) status, BRAF, and KRAS and investigate each subtype's response to chemotherapy. Design This retrospective observational study included a population-based cohort of 878 CRC patients. We classified tumours into five different subtypes based on BRAF and KRAS mutation, CIMP status, and MSI. Patients with advanced stage II (T4N0M0) and stage III tumours received 5-fluoruracil (5-FU)-based chemotherapy or no adjuvant treatment based on clinical criteria. The main outcome was disease-free survival (DFS). Results Patients with the combination of microsatellite stable (MSS) tumours, BRAF mutation and CIMP positive exhibited the worst prognosis in univariate (log rank P < 0.0001) and multivariate analyses (hazard ratio 1.75, 95% CI 1.05-2.93, P = 0.03) after adjusting for age, sex, chemotherapy, and TNM stage. Treatment with 5-FU-based regimens improved prognosis in patients with the combination of MSS tumours, KRAS mutation and CIMP negative (log rank P = 0.003) as well as in patients with MSS tumours plus BRAF and KRAS wild-type and CIMP negative (log-rank P < 0.001). After adjusting for age, sex, and TNM stage in the multivariate analysis, only patients with the latter molecular combination had independently improved prognosis after adjuvant chemotherapy (hazard ratio 2.06, 95% CI 1.24-3.44, P = 0.005). Conclusion We confirmed the prognostic value of stratifying CRC according to molecular subtypes using MSI, CIMP status, and somatic KRAS and BRAF mutation. Patients with traditional chromosomally unstable tumours obtained the best benefit from adjuvant 5-FU-based chemotherapy. This work was supported by the Instituto de Salud Carlos III (PI08/0726, INT-09/208, PI11/2630, INT-12-078, INT13-196, PI14/01386), FISABIO-ISABIAL foundation (UGP-13-221, UGP-14-265), and the Asociación Española contra el Cáncer (Fundación Científica GCB13131592CAST). Eva Hernández-Illán received a grant from Instituto de Salud Carlos III (FI12/00233). Mar Giner-Calabuig received a grant from VALi+d. EXP ACIF/2016/002. Miren Alustiza received a predoctoral grant from ISABIAL (UGP-16-138). Other financial support was obtained from Asociación Española de Gastroenterología grants (Gonzalo Miño 2014, Tamarite 2015, Grupo de endoscopia 2016).

The use of blood samples as liquid biopsy is a label-free method for cancer diagnosis that offers benefits over traditional invasive biopsy techniques. Cell sorting by acoustic waves offers a means to separate rare cells from blood samples based on their physical properties in a label-free, contactless and biocompatible manner. Herein, we describe a flow-through separation approach that provides an efficient separation of tumor cells (TCs) from white blood cells (WBCs) in a microfluidic device, "THINUS-Chip" (Thin-Ultrasonic-Separator-Chip), actuated by ultrasounds. We introduce for the first time the concept of plate acoustic waves (PAW) applied to acoustophoresis as a new strategy. It lies in the geometrical chip design: different to other microseparators based on either bulk acoustic waves (BAW) or surface waves (SAW, SSAW and tSAW), it allows the use of polymeric materials without restrictions in the frequency of work. We demonstrate its ability to perform high-throughput isolation of TCs from WBCs, allowing a recovery rate of 84%±8% of TCs with a purity higher than 80% and combined viability of 85% at a flow rate of 80 μL/min (4.8 mL/h). The THINUS-Chip performs cell fractionation with low-cost manufacturing processes, opening the door to possible easy printing fabrication., This work was partially supported by the Ministry of Economy and Competence of Spain (MINECO) under two projects: BIO2011-30535-C04-01: Development of a high-throughput ultrasonic technology for the isolation of circulating tumor cells from peripheral blood samples; and DPI2017-90147-R: Low intensity a Low Intensity ultrasounds for early diagnosis and modulation of tumor and stroma

Purpose: The present study aimed to determine the prevalence of MUTYH mutations in patients with multiple colonic polyps and to explore the best strategy for diagnosing MUTYH-associated polyposis (MAP) in these patients. Experimental Design: This study included 405 patients with at least 10 colonic polyps each. All cases were genetically tested for the three most frequent MUTYH mutations. Whole-gene analysis was performed in heterozygous patients and in 216 patients lacking the three most frequent mutations. Polyps from 56 patients were analyzed for the KRAS-Gly12Cys and BRAF V600E somatic mutations. Results: Twenty-seven (6.7%) patients were diagnosed with MAP, of which 40.8% showed serrated polyps. The sensitivity of studying only the three common variants was 74.1%. Of 216 patients without any monoallelic mutation in common variants, whole-gene analysis revealed biallelic pathogenic mutation in only one. G396D mutation was associated with serrated lesions and older age at diagnosis. There was a strong association between germinal MUTYH mutation and KRAS Gly12Cys somatic mutation in polyps. BRAF V600E mutation was found in 74% of serrated polyps in MUTYH-negative patients and in none of the polyps of MAP patients. Conclusions: We observed a low frequency of MUTYH mutations among patients with multiple adenomatous and serrated polyps. The MAP phenotype frequently included patients with serrated polyps, especially when G396D mutation was involved. Our results show that somatic molecular markers of polyps can be useful in identifying MAP cases and support the need for the complete MUTYH gene analysis only in patients heterozygous for recurrent variants. Clin Cancer Res; 20(5); 1158–68. ©2014 AACR.

Background & Aims We investigated whether patients with multiple serrated polyps, but not meeting the World Health Organization criteria for serrated polyposis syndrome, and their relatives have similar risks for colorectal cancer (CRC) as those diagnosed with serrated polyposis. Methods We collected data from patients with more than 10 colonic polyps, recruited in 2008–2009 from 24 hospitals in Spain for a study of causes of multiple colonic polyps. We analyzed data from 53 patients who met the criteria for serrated polyposis and 145 patients who did not meet these criteria, but who had more than 10 polyps throughout the colon, of which more than 50% were serrated. We calculated age- and sex-adjusted standardized incidence ratios (SIRs) for CRC in both groups, as well as in their first-degree relatives. Results The prevalence of CRC was similar between patients with confirmed serrated polyposis and multiple serrated polyps (odds ratio, 1.35; 95% confidence interval [CI], 0.64–2.82; P = .40). The SIR for CRC in patients with serrated polyposis (0.51; 95% CI, 0.01–2.82) did not differ significantly from the SIR for CRC in patients with multiple serrated polyps (0.74; 95% CI, 0.20–1.90; P = .70). The SIR for CRC also did not differ significantly between first-degree relatives of these groups (serrated polyposis: 3.28, 95% CI, 2.16–4.77; multiple serrated polyps: 2.79, 95% CI, 2.10–3.63; P = .50). Kaplan–Meier analysis showed no differences in the incidence of CRC between groups during the follow-up period (log-rank, 0.6). Conclusions The risk of CRC in patients with multiple serrated polyps who do not meet the criteria for serrated polyposis, and in their first-degree relatives, is similar to that of patients diagnosed with serrated polyposis.

DNA methylation (DNAm) has been linked to changes in chromatin structure, gene expression and disease. The DNAm level can be affected by genetic variation; although, how this differs by CpG dinucleotide density and genic location of the DNAm site is not well understood. Moreover, the effect of disease causing variants on the DNAm level in a tissue relevant to disease has yet to be fully elucidated. To this end, we investigated the phenotypic profiles, genetic effects and regional genomic heritability for 196080 DNAm sites in healthy colorectum tissue from 132 unrelated Colombian individuals. DNAm sites in regions of low-CpG density were more variable, on average more methylated and were more likely to be significantly heritable when compared with DNAm sites in regions of high-CpG density. DNAm sites located in intergenic regions had a higher mean DNAm level and were more likely to be heritable when compared with DNAm sites in the transcription start site (TSS) of a gene expressed in colon tissue. Within CpG-dense regions, the propensity of the DNAm level to be heritable was lower in the TSS of genes expressed in colon tissue than in the TSS of genes not expressed in colon tissue. In addition, regional genetic variation was associated with variation in local DNAm level no more frequently for DNAm sites within colorectal cancer risk regions than it was for DNAm sites outside such regions. Overall, DNAm sites located in different genomic contexts exhibited distinguishable profiles and may have a different biological function.

Background: The selection of patients for genetic testing to rule out Lynch syndrome is currently based on fulfilment of at least one of the revised Bethesda criteria followed by mismatch repair (MMR) status analysis. A study was undertaken to compare the present approach with universal MMR study-based strategies to detect Lynch syndrome in a large series of patients with colorectal cancer (CRC). Methods: 2093 patients with CRC from the EPICOLON I and II cohorts were included. Immunohistochemistry for MMR proteins and/or microsatellite instability (MSI) analysis was performed in tumour tissue. Germline MLH1 and MSH2 mutation analysis was performed in patients whose tumours showed loss of MLH1 or MSH2 staining, respectively. MSH6 genetic testing was done in patients whose tumours showed lack of MSH6 expression or a combined lack of MSH2 and MSH6 expression but did not have MSH2 mutations. PMS2 genetic testing was performed in patients showing isolated loss of PMS2 expression. In patients with MSI tumours and normal or not available MMR protein expression, all four MMR genes were studied. Results: A total of 180 patients (8.6%) showed loss of expression of some of the MMR proteins and/or MSI. Four hundred and eighty-six patients (23.2%) met some of the revised Bethesda criteria. Of the 14 (0.7%) patients who had a MMR gene mutation, 12 fulfilled at least one of the revised Bethesda criteria and two (14.3%) did not. Conclusions: Routine molecular screening of patients with CRC for Lynch syndrome using immunohistochemistry or MSI has better sensitivity for detecting mutation carriers than the Bethesda guidelines. Instituto de Salud Carlos III (INT09/208 and PI08/0726). Fundación de la CV para la Investigación en el Hospital General Universitario de Alicante. Grant SAF 07-64873 from the Ministerio de Educación y Ciencia. Funds from the AGAUR (2009 SGR 849) Instituto de Salud Carlos III (FI07/00303) CIBERehd is funded by the Instituto de Salud Carlos III. Grant from Conselleria d’Educació de la Generalitat Valenciana (VALi+d).

Resumen de la comunicacion presentada en 4th Biennial Meeting : International Society for Gastrointestinal Hereditary Tumours San Antonio, Texas, March 31-April 2, 2011.

Introduction Recent studies have identified both the prognostic and predictive utility of determining the number of circulating tumour cells (CTC) in patients with solid cancers. Material and methods In the present pilot study we evaluated the ability of two different methods to isolate CTC in combination with two strategies to enumerate CTC from patients with stages II and III surgically treated colorectal cancer (CRC). First, we used two systems for tumour cell enrichment (differential centrifugation and immunomagnetic beads), combined with two methods to enumerate CTC (real-time PCR and flow cytometry), to determine the most efficient combination. These experiments were performed in a model system using serial dilutions of HT29 tumour cell lines with lymphocytes. Then, CTC analysis using the technical approach selected before was performed in 109 blood samples from 16 stage II and III CRC patients during chemotherapy treatment and follow-up. Results Immunomagnetic beads followed by flow cytometry was the most efficient combination (ED=60.53; p=0.5). Two cases out of 16 patients analysed had clinical tumour relapse. In both, we detected a significant increase of CTC five and six months, respectively, before the relapse was clinically evidenced. An increase of CTC was also observed in another case without clinical evidence of relapse. The remaining cases (13) had very few or no detectable CTC and no clinical evidence of relapse (p=0.029). Conclusions Changes in CTC numbers during follow-up might predict tumour relapse. Further evaluation of CTC prognostic and predictive value in patients with early CRC is warranted.

The transforming growth factor-beta (TGF-β) signalling pathway plays an important role in tumor development and progression. We aimed at analyzing whether 7 different common variants in genes coding for 2 key members of the TGF-β signalling pathway (TGFB1 and TGFBR1) are associated with bladder cancer risk and prognosis. A total of 1,157 cases with urothelial cell carcinoma of the bladder and 1,157 matched controls where genotyped for 3 single nucleotide polymorphisms (SNPs) in TGFB1 (rs1982073, rs1800472, rs1800471) and an additional 3 SNPs and 1 indel polymorphism in TGFBR1 (rs868, rs928180, rs334358 and rs11466445, respectively). In the case-control study, we estimated odds ratios and 95% confidence intervals for each individual genetic variant using unconditional logistic regression adjusting for age, gender, study area and smoking status. Survival analysis was performed using the Kaplan-Meier method and Cox models. The endpoints of interest were tumor relapse, progression and death from bladder cancer. All the SNPs analyzed showed a similar distribution among cases and controls. The distribution of the TGFBR1*6A allele (rs11466445) was also similar among cases and controls, indicating no association with bladder cancer risk. Similarly, none of the haplotypes was significantly associated with bladder cancer risk. Among patients with muscle-invasive tumors, we found a significant association between TGFBR1-rs868 and disease-specific mortality with an allele dosage effect (p-trend = 0.003). In conclusion, the genetic variants analyzed were not associated with an increased risk of bladder cancer. The association of TGFBR1-rs868 with outcome should be validated in independent patient series. © 2008 Wiley-Liss, Inc.

Background The prevalence of MLH1 constitutional epimutations in the general population is unknown. We sought to analyse the prevalence of MLH1 constitutional epimutations in unselected and selected series of patients with colorectal cancer (CRC). Methods Patients with diagnoses of CRC (n=2123) were included in the unselected group. For comparison, a group of 847 selected patients with CRC who fulfilled the revised Bethesda guidelines (rBG) were also included. Somatic and constitutional MLH1 methylation was assayed via methylation-specific multiplex ligation-dependent probe amplification of cases lacking MLH1 expression. Germline alterations in mismatch-repair (MMR) genes were assessed via Sanger sequencing and methylation-specific multiplex ligation-dependent probe amplification. Results Loss of MLH1 expression occurred in 5.5% of the unselected series and 12.5% of the selected series (p

The choice of substrate material in a chip that combines ultrasound with microfluidics for handling biological and synthetic microparticles can have a profound effect on the performance of the device. This is due to the high surface-to-volume ratio that exists within such small structures and acquires particular relevance in polymer-based resonators with 3D standing waves. This paper presents three chips developed to perform particle flow-through separation by ultrasound based on a polymeric SU-8 layer containing channelization over three different substrates: Polymethyl methacrylate (PMMA); Pyrex; and a cracked PMMA composite-like structure. Through direct observations of polystyrene microbeads inside the channel, the three checked chips exhibit their potential as disposable continuous concentration devices with different spatial pressure patterns at frequencies of resonance close to 1 Mhz. Chips with Pyrex and cracked PMMA substrates show restrictions on the number of pressure nodes established in the channel associated with the inhibition of 3D modes in the solid structure. The glass-substrate chip presents some advantages associated with lower energy requirements to collect particles. According to the results, the use of polymer-based chips with rigid substrates can be advantageous for applications that require short treatment times (clinical tests handling human samples) and low-cost fabrication. © 2015 by the authors; licensee MDPI, Basel, Switzerland., The study has been performed in the framework of two Spanish National Research Project BIO2011-30535-C04-01,02,03, “Development of a high throughput for isolation of tumor cells circulating in peripheral blood”., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Background: We identified a new and a recurrent POLD1 mutation associated with predisposition to colorectal cancer (CRC). We characterized the molecular and clinical nature of the potential POLD1 founder mutation in families from Valencia (Spain). Methods: Clinical and molecular data were collected from four independent families known to have a POLD1 Leu474Pro mutation. To establish its founder effect, haplotype construction was performed using 14 flanking POLD1 polymorphic markers. We calculated penetrance estimates and clinical expressivity, globally and stratified by age and sex. Results: We included 32 individuals from the four families: 20 carriers and 12 noncarriers. A common haplotype was identified in these families in a region comprising 2,995 Mb, confirming L474P as the first founder POLD1 mutation identified. Thirteen tumors diagnosed in 10 POLD1 carriers: eight CRC, three endometrial and two other tumors were considered. The median age of cancer onset for POLD1 mutation carriers was 48 years. The observed penetrance was 50% and the cumulative risk at age of 50 years was 30%. Conclusions: The findings of the present study contribute to a better understanding of CRC genetics in the Spanish population. The clinical phenotype for this mutation is similar to that in Lynch syndrome. Future studies using next generation sequencing with large gene panels for any hereditary cancer condition will offer the possibility of detecting POLE/POLD1 mutations in unsuspected clinical situations, demonstrating a more real and unbiased picture of the associated phenotype. This work was supported by the Institute for Health and Biomedical Research of Alicante (ISABIAL, UGP‐16‐146). RFA is recipient of a Fellowship from the Consellería Educación of the Valencian Community. ACA is funded by the Acción Juvenil from the Spanish Ministry of Economy and Competitiveness. VDO is recipient of a Fellowship from the Spanish Association Against Cancer (AECC). AC and MIC are funded by Health and Biomedical Research Foundation from the Valencian Region (FISABIO). EHI is recipient of a fellowship from the Fondo Investigación Sanitaria ISCIII (FI12/00233).

There is an unambiguous association of Streptococcus gallolyticus infection with colorectal cancer, although there is limited information about epidemiology or interaction between molecular and environmental factors. We performed an original quantitative analysis of S. gallolyticus in unselected colorectal cancer patients (n = 190) and their association with clinical, pathological tumor molecular profiles (microsatellite instability, hypermethylator phenotype and chromosomal instability pathways), and other biological factors in colorectal tumor and normal tissues (cytomegalovirus and Epstein-Barr virus infection). We developed a new quantitative method to assess bacterial load. Analytical validation was reached with a very high sensitivity and specificity. Our results showed a 3.2% prevalence of S. gallolyticus infection in our unselected cohort of colorectal cancer cases (6/190). The average S. gallolyticus copy number was 7,018 (range 44–34,585). No previous reports relating to S. gallolyticus infection have been published for unselected cohorts of patients. Finally, and despite a low prevalence of S. gallolyticus in this study, we were able to define a specific association with tumor tissue (p = 0.03) and with coinfection with Epstein-Barr virus (p = 0.042; OR: 9.49; 95% IC: 1.1–82.9). The prevalence data provided will be very useful in the design of future studies, and will make it possible to estimate the sample size needed to assess precise objectives. In conclusion, our results show a low prevalence of S. gallolyticus infection in unselected colorectal cancer patients and an association of positive S. gallolyticus infection with tumor tissue and Epstein-Barr virus coinfection. Further studies will be needed to definitively assess the prevalence of S. gallolyticus in colorectal cancer and the associated clinicopathological and molecular profiles. This work was supported by Conselleria d’Educació Generalitat Valenciana, Spain (GV/2016/175).

Germline mutations in DNA polymerase E > (POLE) and delta (POLD1) have been recently identified in families with multiple colorectal adenomas and colorectal cancer (CRC). All reported cases carried POLE c.1270C > G (p.Leu424Val) or POLD1 c.1433G > A (p.Ser478Asn) mutations. Due to the scarcity of cases reported so far, an accurate clinical phenotype has not been defined. We aimed to assess the prevalence of these recurrent mutations in unexplained familial and early-onset CRC and polyposis, and to add additional information to define the clinical characteristics of mutated cases. A total of 858 familial/early onset CRC and polyposis patients were studied: 581 familial and early-onset CRC cases without mismatch repair (MMR) deficiency, 86 cases with MMR deficiency and 191 polyposis cases. Mutation screening was performed by KASPar genotyping assays and/or Sanger sequencing of the involved exons. POLE p.L424V was identified in a 28-year-old polyposis and CRC patient, as a de novo mutation. None of the 858 cases studied carried POLD1 p.S478N. A new mutation, POLD1 c.1421T > C (p.Leu474Pro), was identified in a mismatch repair proficient Amsterdam II family. Its pathogenicity was supported by cosegregation in the family, in silico predictions, and previously published yeast assays. POLE and POLD1 mutations explain a fraction of familial CRC and polyposis. Sequencing the proofreading domains of POLE and POLD1 should be considered in routine genetic diagnostics. Until additional evidence is gathered, POLE and POLD1 genetic testing should not be restricted to polyposis cases, and the presence of de novo mutations, considered.

Background and aims: Individuals with tumours showing mismatch repair (MMR) deficiency not linked to germline mutations or somatic methylation of MMR genes have been recently referred as having lynch-like syndrome' (LLS). The genetic basis of these LLS cases is unknown. MUTYH-associated polyposis patients show some phenotypic similarities to Lynch syndrome patients. The aim of this study was to investigate the prevalence of germline MUTYH mutations in a large series of LLS patients. Methods: Two hundred and twenty-five probands fulfilling LLS criteria were included in this study. Screening of MUTYH recurrent mutations, whole coding sequencing and a large rearrangement analysis were undertaken. Age, sex, clinical, pathological and molecular characteristics of tumours including KRAS mutations were assessed. Results: We found a prevalence of 3.1% of MAP syndrome in the whole series of LLS (7/225) and 3.9% when only cases fulfilling clinical criteria were considered (7/178). Patients with MUTYH biallelic mutations had more adenomas than monoallelic (P = 0.02) and wildtype patients (P < 0.0001). Six out of nine analysed tumours from six biallelic MUTYH carriers harboured KRAS-p.G12C mutation. This mutation was found to be associated with biallelic MUTYH germline mutation when compared with reported series of unselected colorectal cancer cohorts (P < 0.0001). Conclusions: A proportion of unexplained LLS cases is caused by biallelic MUTYH mutations. The obtained results further justify the inclusion of MUTYH in the diagnostic strategy for Lynch syndrome-suspected patients. (C) 2014 Elsevier Ltd. All rights reserved.

Background: Lynch syndrome (LS) is a hereditary condition that increases the risk for endometrial and other cancers. The identification of endometrial cancer (EC) patients with LS has the potential to influence life-saving interventions. We aimed to study the prevalence of LS among EC patients in our population. Methods: Universal screening for LS was applied for a consecutive series EC. Tumor testing using microsatellite instability (MSI), immunohistochemistry (IHC) for mismatch-repair (MMR) protein expression and MLH1-methylation analysis, when required, was used to select LS-suspicious cases. Sequencing of corresponding MMR genes was performed. Results: One hundred and seventy-three EC (average age, 63 years) were screened. Sixty-one patients (35%) had abnormal IHC or MSI results. After MLH1 methylation analysis, 27 cases were considered suspicious of LS. From these, 22 were contacted and referred for genetic counseling. Nineteen pursued genetic testing and eight were diagnosed of LS. Mutations were more frequent in younger patients (

A low-cost technological approach based on the strategic application of ultrasonic waves on polymeric chips has been developed to detect and extract circulating tumor cells from peripheral blood samples flowing through the microfluidic device, which are delivered viable for later biomolecular analyses. This free-label technique can be used as an early detection system to identify metastasis in the initial stages, assuming a very low number of rare cells present in the blood sample, less than 5 cells/7.5mL. We have demonstrated the feasibility of separation of the tumor cells utilizing hydrodynamic forces induced by ultrasounds in polymeric chips delivering viable single cells and multicellular clusters which differentially experience a drift motion as they flow through a microchannel in a sample containing white blood cells.

This work presents a study of polymeric microdevices implemented by piezoelectric actuators to carry out processes of sorting/separation on flowing suspensions, acting as acoustic tweezers to extract separately target particles or cells. They provide a low-cost and label-free platform, exploiting the difference of size, density and compressibility. Their ability to perform isolation of tumor cells from white blood cells makes them strategic tools for medical applications such as early detection or future tools for monitoring personalized chemotherapy. Polymer-based microdevices implemented by ultrasounds have been developed to isolate these cells with an efficiency level up to 70%. This technology is currently being optimized to reach efficiencies higher than 90% for its clinical application.

Background & Aims: Colorectal cancers (CRCs) with microsatellite instability (MSI) and a mismatch repair (MMR) immunohistochemical deficit without hypermethylation of the MLH1 promoter are likely to be caused by Lynch syndrome. Some patients with these cancers have not been found to have pathogenic germline mutations and are considered to have Lynch-like syndrome (LLS). The aim of this study was to determine the risk of cancer in families of patients with LLS. Methods: We studied a population-based cohort of 1705 consecutive patients, performing MSI tests and immunohistochemical analyses of MMR proteins. Patients were diagnosed with Lynch syndrome when they were found to have pathogenic germline mutations. Patients with MSI and loss of MSH2 and/or MSH6 expression, isolated loss of PMS2 or loss of MLH1 without MLH1 promoter hypermethylation, and no pathogenic mutation were considered to have LLS. The clinical characteristics of patients and the age- and sex-adjusted standardized incidence ratios (SIRs) of cancer in families were compared between groups. Results: The incidence of CRC was significantly lower in families of patients with LLS than in families with confirmed cases of Lynch syndrome (SIR for Lynch syndrome, 6.04; 95% confidence interval [CI], 3.58–9.54; SIR for LLS, 2.12; 95% CI, 1.16–3.56; P < .001). However, the incidence of CRC was higher in families of patients with LLS than in families with sporadic CRC (SIR for sporadic CRC, 0.48; 95% CI, 0.27–0.79; P < .001). Conclusions: The risk of cancer in families with LLS is lower that of families with Lynch syndrome but higher than that of families with sporadic CRC. These results confirm the need for special screening and surveillance strategies for these patients and their relatives. This work was supported by grants from Instituto de Salud Carlos III (PI-080726, INT-09/208, and PI11/026030), the Fondo de Investigación Sanitaria/FEDER (PS09/02368, 10/00384, 10/00918, 11/00219, and 11/00681), Fundació Olga Torres (CRP) and FP7 CHIBCHA Consortium (SCB and ACar), the Ministerio de Economía y Competitividad (SAF2010-19273), and Agència de Gestió d’Ajuts Universitaris i de Recerca (2009 SGR 849). SCB is supported by a contract from the Fondo de Investigación Sanitaria (CP03-0070). CIBERER and CIBERehd are funded by the Instituto de Salud Carlos III.

Circulating tumor cell (CTC) enumeration is important clinically for identifying prognostic and predictive factors in patients with solid cancers. The CellSearch device (Veridex) is an immunomagnetic CTC selection and enumeration system used in clinical practice. The ImageStream (Amnis) combines the strengths of flow cytometry and fluorescent microscopy in a single platform and has potential application for CTC counting. The performance in CTC enumeration was compared between the ImageStream and CellSearch systems. Various numbers of PANC-1 tumor cells were spiked into 7.5 mL of peripheral blood from a healthy donor. Before cell analysis by the ImageStream, tumor cell enrichment was performed by immunomagnetic selection with anti-EpPCAM. Anti-CD45 and anti-CK markers were used to discriminate between tumor cells and leukocytes. The ratios of tumor cells recovered from each dilution were calculated for both methods. The Wilcoxon rank test was applied to compare the results of the two methods and the reference value. The results of the two tested methods differed significantly from the reference value, but did not differ between them. Nevertheless, lower level of trueness and precision was observed in ImageStream when fewer numbers of CTCs were analyzed. Our results suggest that ImageStream platform for CTC enumeration has a potential value for the early diagnosis of disseminated disease, but needs an improvement of precision for the enumeration of low number of CTC.

In colorectal cancer (CRC), an inherited susceptibility risk affects about 35% of patients, whereas high-penetrance germline mutations account for

Background Lynch syndrome (LS) is an autosomal dominant inherited cancer syndrome characterized by early onset cancers of the colorectum, endometrium and other tumours. A significant proportion of DNA variants in LS patients are unclassified. Reports on the pathogenicity of the c.1852_1853AA>GC (p.Lys618Ala) variant of the MLH1 gene are conflicting. In this study, we provide new evidence indicating that this variant has no significant implications for LS. Methods The following approach was used to assess the clinical significance of the p.Lys618Ala variant: frequency in a control population, case-control comparison, co-occurrence of the p.Lys618Ala variant with a pathogenic mutation, co-segregation with the disease and microsatellite instability in tumours from carriers of the variant. We genotyped p.Lys618Ala in 1034 individuals (373 sporadic colorectal cancer [CRC] patients, 250 index subjects from families suspected of having LS [revised Bethesda guidelines] and 411 controls). Three well-characterized LS families that fulfilled the Amsterdam II Criteria and consisted of members with the p.Lys618Ala variant were included to assess co-occurrence and co-segregation. A subset of colorectal tumour DNA samples from 17 patients carrying the p.Lys618Ala variant was screened for microsatellite instability using five mononucleotide markers. Results Twenty-seven individuals were heterozygous for the p.Lys618Ala variant; nine had sporadic CRC (2.41%), seven were suspected of having hereditary CRC (2.8%) and 11 were controls (2.68%). There were no significant associations in the case-control and case-case studies. The p.Lys618Ala variant was co-existent with pathogenic mutations in two unrelated LS families. In one family, the allele distribution of the pathogenic and unclassified variant was in trans, in the other family the pathogenic variant was detected in the MSH6 gene and only the deleterious variant co-segregated with the disease in both families. Only two positive cases of microsatellite instability (2/17, 11.8%) were detected in tumours from p.Lys618Ala carriers, indicating that this variant does not play a role in functional inactivation of MLH1 in CRC patients. Conclusions The p.Lys618Ala variant should be considered a neutral variant for LS. These findings have implications for the clinical management of CRC probands and their relatives. This action has been supported in part by grants from the Generalitat Valenciana in Spain (AP140/08) and the Biomedical Research Foundation from the Hospital of Elche, Spain (FIBElx0902). CG, AMC, CEl and LPC are recipients of fellowships from the Conselleria de Educació (Generalitat Valenciana); Fundacion Juan Peran-Pikolinos; Fundacion Carolina-BBVA and Fondo Investigación Sanitaria (FI07/00303), respectively. RJ is receptor of a grant from Instituto de Salud Carlos III (INT09/208) SI

Summary Background The incidence of colorectal cancer (CRC) in Peru has been increasing, and no data have been published on the molecular features. We explored the most relevant genetic events involved in colorectal carcinogenesis, with clinical implications. Methods Using immunohistochemistry for mismatch-repair (MMR) proteins (MLH1, MSH2, MSH6, and PMS2) and microsatellite instability analysis, we evaluated the status of 90 non-selected CRC Peruvian patients followed in a nationwide reference hospital for cancer (INEN, Lima). Tumours with loss of hMLH1 were evaluated further for hMLH1 promoter hypermethylation and all cases were evaluated for the presence of KRAS and BRAF-V600E mutations. Results MMR deficiency was found in 35 (38.8%) patients. We identified an unexpected association between MMR deficiency and older age. Among the 14 cases with loss of MLH1, 10 samples exhibited hypermethylation. Of the 90 cases evaluated, 15 (16.7%) carried KRAS mutations; we found one previously unreported mutation (G13R). Conclusions Peruvian CRC tumours exhibited the highest prevalence of MMR deficiency reported to date. The expected hereditary component was also high. The age of onset of these MMR deficient tumours was greater than that observed for non-MMR deficient cases, suggesting the ineffectiveness of the Bethesda criteria for Lynch syndrome screening in Peru. Prospective studies are warranted to define the molecular characteristics of CRC in this population.

The standard genetic test for Lynch syndrome (LS) frequently reveals an absence of pathogenic mutations in DNA mismatch repair genes known to be associated with LS. It was recently shown that germ line deletions in the last exons of EPCAM are involved in the etiology of LS. The aim of this study was to evaluate the prevalence of EPCAM deletions in a Spanish population and the clinical implications of deletion. Probands from 501 families suspected of having LS were enrolled in the study. Twenty-five cases with MSH2 loss were identified: 10 had mutations of MSH2, five had mutations of MSH6, and 10 did not show MSH2/MSH6 mutations. These 25 cases were analyzed for EPCAM deletions using multiplex ligation-dependent probe amplification, and deletions were mapped using long-range PCR analysis. One subject with no MSH2/MSH6 mutations had a large deletion in the EPCAM locus that extended for 8.7 kb and included exons 8 and 9. The tumor exhibited MSH2 promoter hypermethylation. EPCAM deletion analysis followed by MSH2 methylation testing of the tumor is a fast low-cost procedure that can be used to identify mutations that cause LS. We propose that this procedure be incorporated into clinical genetic analysis strategies and present a decision-support flow diagram for the diagnosis of LS. Transversal Cancer Action (ISCIII) and the Biomedical Research Foundation from the Elche University Hospital. Carolina-BBVA Foundation and Juan Peran-Pikolinos Foundation.