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The mutational profiles of primary colorectal cancers (CRCs) and corresponding ovarian metastases were compared. Using a custom‐made next generation sequencing panel, 115 cancer‐driving genes were analyzed in a cohort of 26 primary CRCs and 30 matching ovarian metastases (four with bilateral metastases). To obtain a complete overview of the mutational profile, low thresholds were used in bioinformatics analysis to prevent low frequency passenger mutations from being filtered out. A subset of variants was validated using Sanger and/or hydrolysis probe assays. The mutational landscape of CRC that metastasized to the ovary was not strikingly different from CRC in consecutive series. When comparing primary CRCs and their matching ovarian metastases, there was considerable overlap in the mutations of early affected genes. A subset of mutations demonstrated less overlap, presumably being passenger mutations. In particular, primary CRCs showed a substantially high number of passenger mutations. We also compared the primary CRCs and matching metastases for stratifying variants of six genes (KRAS, NRAS, BRAF, FBXW7, PTEN and PIK3CA) that select for established (EGFR directed) or future targeted therapies. In a total of 31 variants 12 were not found in either of the two locations. Tumours thus differed in the number of discordant variants between the primary tumours and matching metastases. Half of these discordant variants were definitive class 4/5 pathogenic variants. However, in terms of temporal heterogeneity, no clear relationship was observed between the number of discordant variants and the time interval between primary CRCs and the detection of ovarian metastases. This suggests that dormant metastases may be present from the early days of the primary tumours.

The Human Variome Project (HVP) has established a pilot program with the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) to compile all inherited variation affecting colon cancer susceptibility genes. An HVP-InSiGHT Workshop was held on May 10, 2010, prior to the HVP Integration and Implementation Meeting at UNESCO in Paris, to review the progress of this pilot program. A wide range of topics were covered, including issues relating to genotype-phenotype data submission to the InSiGHT Colon Cancer Gene Variant Databases (chromium.liacs.nl/LOVD2/colon_cancer/home.php). The meeting also canvassed the recent exciting developments in models to evaluate the pathogenicity of unclassified variants using in silico data, tumor pathology information, and functional assays, and made further plans for the future progress and sustainability of the pilot program. Hum Mutat 32: 491-494, 2011. (C) 2011 Wiley-Liss, Inc.

// Annemarie Boleij 1 , Bastiaan B.J. Tops 2 , Paul D.M. Rombout 1 , Elizabeth M. Dequeker 3 , Marjolijn J.L. Ligtenberg 1,2 , J. Han van Krieken 1 and Dutch RAS EQA Initiative 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 1 Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands 2 Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands 3 Department of Public Health and Primary Care, Biomedical Quality Assurance Research Unit, KU Leuven - University of Leuven, Leuven, Belgium 4 C.J.M. van Noesel, Academic Medical Center (AMC), Department of Pathology, Amsterdam 5 C.C. Scheidel-Jacobse (Technical specialist), J.A. Kummer (Pathologist/KMBP), P. Roepman (KMBP in training), St. Antonius Ziekenhuis, Department of Pathology, Nieuwegein 6 C.F.M. Prinsen (KMBP), S.H.M. van den Berg-van Erp (Pathologist), Canisius Wilhelmina Ziekenhuis (CWZ), Department of Pathology, Nijmegen 7 J.M.H.H. van Gorp, Diakonessenhuis, Laboratory for Pathology, Utrecht 8 P.M. Nederlof, Dutch Cancer Institute (NKI), Amsterdam. 9 E. Caspers, St. Elisabeth Ziekenhuis, Department of Molecular Pathology, Tilburg 10 W.N.M. Dinjens, E.C.W. Beerens, Erasmus MC, Department of Pathology, Molecular Diagnostics, Rotterdam 11 N.A. ‘t Hart, Isala, Department of Pathology, Zwolle 12 A.J.C. van den Brule, Jeroen Bosch Ziekenhuis, Molecular Diagnostics, ‘s-Hertogenbosch 13 R. van der Geize (KMBP), S.A. Riemersma (Pathologist), Laboratory for Pathology Oost-Nederland (LABPON), Hengelo 14 T. van Wezel (KMBP), H. Morreau (Pathologist), R. van Eijk (Technical specialist), Leiden University Medical Center (LUMC), Department of Pathology, Leiden 15 J.W.M. Jeuken, Laboratory for Pathology and Medical Microbiology (PAMM), Eindhoven 16 A. Dirkx, Pathan B.V., Molecular Diagnostics, Rotterdam 17 M. Klomp, Rijnstate Ziekenhuis, Department of Pathology, Arnhem 18 W.T.M van Blokland, University Medical Center (UMC) Utrecht, Molecular Pathology, Utrecht 19 A. ter Elst (Technical specialist/KMBP in training), E. Schuuring (KMBP), A. Diepstra (Pathologist), University Medical Center Groningen (UMCG), Department of Pathology, Groningen 20 D.A.M. Heideman (KMBP), N.C.T. van Grieken (Pathologist), D. Sie (Technical specialist), VU-University Medical Center (VUMC), Department of Pathology, Amsterdam Correspondence to: J.Han van Krieken, email: // Keywords : RAS, colorectal cancer, metastasis, quality control, next generation sequencing Received : February 09, 2015 Accepted : March 18, 2015 Published : April 12, 2015 Abstract In 2013 the European Medicine Agency (EMA) restricted the indication for anti-EGFR targeted therapy to metastatic colorectal cancer (mCRC) with a wild-type RAS gene, increasing the need for reliable RAS mutation testing. We evaluated the completeness and reproducibility of RAS -testing in the Netherlands. From 17 laboratories, tumor DNA of the first 10 CRC cases tested in 2014 in routine clinical practice was re-tested by a reference laboratory using a custom next generation sequencing panel. In total, 171 CRC cases were re-evaluated for hotspot mutations in KRAS , NRAS and BRAF . Most laboratories had introduced complete RAS -testing (65%) and BRAF -testing (71%) by January 2014. The most employed method for all hotspot regions was Sanger sequencing (range 35.7 – 49.2%). The reference laboratory detected all mutations that had been found in the participating laboratories ( n = 92), plus 10 additional mutations. This concerned three RAS and seven BRAF mutations that were missed due to incomplete testing of the participating laboratory. Overall, the concordance of tests performed by both the reference and participating laboratory was 100% (163/163; κ-static 1.0) for RAS and 100% (144/144; κ-static 1.0) for BRAF . Our study shows that RAS and BRAF mutations can be reproducibly assessed using a variety of testing methods.

Members of the EPICOLON Consortium (Gastrointestinal Oncology Group of the Spanish Gastroenterological Association) are: Hospital 12 de Octubre, Madrid: Juan Diego Morillas (local coordinator), Raquel Muñoz, Marisa Manzano, Francisco Colina, Jose Díaz, Carolina Ibarrola, Guadalupe López, Alberto Ibáñez; Hospital Clínic, Barcelona: Antoni Castells (local coordinator), Virgínia Piñol, Sergi Castellví-Bel, Francesc Balaguer, Victoria Gonzalo, Teresa Ocaña, María Dolores Giráldez, Maria Pellisé, Anna Serradesanferm, Leticia Moreira, Miriam Cuatrecasas, Josep M. Piqué; Hospital Clínico Universitario, Zaragoza: Ángel Lanas (local coordinator), Javier Alcedo, Javier Ortego; Hospital Cristal-Piñor, Complexo Hospitalario de Ourense: Joaquin Cubiella (local coordinator), Ma Soledad Díez, Mercedes Salgado, Eloy Sánchez, Mariano Vega; Parc de Salut Mar, Barcelona: Montserrat Andreu (local coordinator), Anna Abuli, Xavier Bessa, Mar Iglesias, Agustín Seoane, Felipe Bory, Gemma Navarro, Beatriz Bellosillo; Josep Ma Dedeu, Cristina Álvarez, Marc Puigvehí; Hospital San Eloy, Baracaldo and Hospital Donostia, CIBERehd, University of Basque Country, San Sebastián: Luis Bujanda (local coordinator) Ángel Cosme, Inés Gil, Mikel Larzabal, Carlos Placer, María del Mar Ramírez, Elisabeth Hijona, Jose M. Enríquez-Navascués, Jose L. Elosegui; Hospital General Universitario de Alicante: Artemio Payá (EPICOLON I local coordinator), Rodrigo Jover (EPICOLON II local coordinator), Cristina Alenda, Laura Sempere, Nuria Acame, Estefanía Rojas, Lucía Pérez-Carbonell; Hospital General de Granollers: Joaquim Rigau (local coordinator), Ángel Serrano, Anna Giménez; Hospital General de Vic: Joan Saló (local coordinator), Eduard Batiste-Alentorn, Josefina Autonell, Ramon Barniol; Hospital General Universitario de Guadalajara and Fundación para la Formación e Investigación Sanitarias Murcia: Ana María García (local coordinator), Fernando Carballo, Antonio Bienvenido, Eduardo Sanz, Fernando González, Jaime Sánchez, Akiko Ono; Hospital General Universitario de Valencia: Mercedes Latorre (local coordinator), Enrique Medina, Jaime Cuquerella, Pilar Canelles, Miguel Martorell, José Ángel García, Francisco Quiles, Elisa Orti; CHUVI-Hospital Meixoeiro, Vigo: EPICOLON I: Juan Clofent (local coordinator), Jaime Seoane, Antoni Tardío, Eugenia Sanchez. EPICOLON II Ma Luisa de Castro (local coordinator), Antoni Tardío, Juan Clofent, Vicent Hernández; Hospital Universitari Germans Trias i Pujol, Badalona and Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, IL, USA: Xavier Llor (local coordinator), Rosa M. Xicola, Marta Piñol, Mercè Rosinach, Anna Roca, Elisenda Pons, José M. Hernández, Miquel A. Gassull; Hospital Universitari Mútua de Terrassa: Fernando Fernández-Bañares (local coordinator), Josep M. Viver, Antonio Salas, Jorge Espinós, Montserrat Forné, Maria Esteve; Hospital Universitari Arnau de Vilanova, Lleida: Josep M. Reñé (local coordinator), Carmen Piñol, Juan Buenestado, Joan Viñas; Hospital Universitario de Canarias: Enrique Quintero (local coordinator), David Nicolás, Adolfo Parra, Antonio Martín; Hospital Universitario La Fe, Valencia: Lidia Argüello (local coordinator), Vicente Pons, Virginia Pertejo, Teresa Sala; Hospital Sant Pau, Barcelona: Dolors Gonzalez (local coordinator) Eva Roman, Teresa Ramon, Maria Poca, Ma Mar Concepción, Marta Martin, Lourdes Pétriz; Hospital Xeral Cies, Vigo: Daniel Martinez (local coordinator); Fundacion Publica Galega de Medicina Xenomica (FPGMX), CIBERER, Genomic Medicine Group-University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain: Ángel Carracedo (local coordinator), Clara Ruiz-Ponte, Ceres Fernández-Rozadilla, Ma Magdalena Castro; Hospital Universitario Central de Asturias: Sabino Riestra (local coordinator), Luis Rodrigo; Hospital de Galdácano, Vizcaya: Javier Fernández (local coordinator), Jose Luis Cabriada; Fundación Hospital de Calahorra (La Rioja) La Rioja: Luis Carreño (local coordinator), Susana Oquiñena, Federico Bolado; Hospital Royo Villanova, Zaragoza: Elena Peña (local coordinator), José Manuel Blas, Gloria Ceña, Juan José Sebastián; Hospital Universitario Reina Sofía, Córdoba: Antonio Naranjo (local coordinator). The common −652 6N del variant in the CASP8 promoter (rs3834129) has been described as a putative low-penetrance risk factor for different cancer types. In particular, some studies suggested that the deleted allele (del) was inversely associated with CRC risk while other analyses failed to confirm this. Hence, to better understand the role of this variant in the risk of developing CRC, we performed a multi-centric case-control study. In the study, the variant −652 6N del was genotyped in a total of 6,733 CRC cases and 7,576 controls recruited by six different centers located in Spain, Italy, USA, England, Czech Republic and the Netherlands collaborating to the international consortium COGENT (COlorectal cancer GENeTics). Our analysis indicated that rs3834129 was not associated with CRC risk in the full data set. However, the del allele was under-represented in one set of cases with a family history of CRC (per allele model OR = 0.79, 95% CI = 0.69–0.90) suggesting this allele might be a protective factor versus familial CRC. Since this multi-centric case-control study was performed on a very large sample size, it provided robust clarification of the effect of rs3834129 on the risk of developing CRC in Caucasians. For all cohorts: This work was supported by COST Action BM1206. Spanish cohort: SCB is supported by a contract from the Fondo de Investigación Sanitaria (CP 03-0070 to SCB). Networked Biomedical Research Centre for Hepatic and Digestive Diseases and Centro de Investigación Biomédica en Red de Enfermedades Raras are funded by the Instituto de Salud Carlos III. This work was supported by grants from the Fondo de Investigación Sanitaria/FEDER (08/0024, 08/1276, PS09/02368, 11/00219, 11/00681), Instituto de Salud Carlos III (Acción Transversal de Cáncer), Xunta de Galicia (07PXIB9101209PR), Ministerio de Ciencia e Innovación (SAF2010-19273), Asociación Española contra el Cáncer (Fundación Científica y Junta de Barcelona), Fundació Olga Torres (SCB and CRP), FP7 CHIBCHA Consortium (SCB and A. Carracedo). American cohort: Kentucky Colon Cancer Genetic Epidemiology Study is supported by National Institutes of Health grant R01CA136726 to LL. English cohort: Core funding to the Wellcome Trust Centre for Human Genetics was provided by the Wellcome Trust (090532/Z/09/Z). Czech cohort: Grant agency of the Czech Republic (GACR) [CZ:GACR:GA P304/10/1286 and P304/12/1585] and by Prvouk-P27/LF1/1 from Ministry of Education, Youth and Sport, Czech Republic (First Medical Faculty, Charles University, Prague, Czech Republic as a recipient). Dutch cohort: Dutch Cancer Society, grant KWF-UL-2010-4656.

Colorectal tumours with high stromal content have a poor prognosis. In part 1, I investigated, at the protein and gene expression levels, the composition of these tumours in comparison to stroma-low tumours. In part 2, KRAS mutant colorectal cancer cell lines had a different metabolism compared to KRAS wild type after inhibition of mTORC1. This may contribute to therapeutic resistance.

An estimated 15-25% of patients with colorectal cancer have a positive family history, but no known underlying genetic cause. In this thesis we aimed to detect the underlying genetic cause in patients with suspected Lynch Syndrome, a familial disorder caused by mutations in the mismatch repair (MMR) genes. We hypothesized that these patients could be explained by missed MMR variants, somatic inactivation of the MMR genes, or variants in other genes, leading to secondary MMR-deficiency. In our cohort we found 10 patients with two somatic MMR variants in the tumor and 9 patients with a germline or somatic mutation in the POLE or POLD1 genes. Variants in the exonuclease domain of these genes results in highly mutated tumors. Additionally, we describe how to assess the effect of splice variants, and how to sequence the complex PMS2 gene with next generation sequencing. In the second part we aimed to detect the underlying genetic cause in patients with unexplained adenomatous polyposis. By testing multiple adenomas, we found that if two or more adenomas carry the same variant in the APC gene, this was indicative of an underlying mosaic genetic cause. Nine patients with 21-100 adenomas could be explained by APC mosaicism.

The first study described the colon tumor-specific methylation of a low CG-dense CpG island that is located in the first intron of the PTPRG gene (PTPRGint1). High levels of specificity and sensitivity of this region were observed in sporadic and familial colon cancer which makes this region interesting for application in epigenetic screening panels. In the second study the relation between mutations and DNA methylation was investigated in an attempt to identify initiating factors for DNA methylation in colon cancer. In this study, we were unable to identify an intrinsic tendency towards CpG island hypermethylation other than aberrant accumulation of CpG island methylation via a somatic mutation of BRAF. The relationship between BRAF mutations and DNA methylation was explored further in Chapter 4, which describes an improved study of DNA methylation in BRAF mutationassociated colon cancer. We describe BRAF mutation-specific promoter methylation of the FOX transcription factor genes FOXB1, FOXB2 and FOXD3 and speculate that this methylation might help these tumors escape BRAF-induced senescence. In the final study the DMH technique was combined with oligonucleotide microarrays that contained high CpG island coverage to determine the methylation patterns of infant B-ALL patients. The majority of MLL-rearranged infant ALL cases (i.e., those who are characterized by a t(4;11) or t(11;19) translocation) represent hypermethylated leukemias. In contrast, infant ALL patients with a t(9;11) translocation and those without any MLL translocation (wild-type MLL) displayed DNA methylation patterns that closely resembled the pattern seen in normal bone marrow. This study indicates that patients with a t(4;11) or t(11;19) translocation who have high levels of DNA methylation might be promising candidates for therapies that inhibit DNA methylation.

Colorectal cancer (CRC) is one of the most common cancers in the Western world and in about 30% hereditary factors play a role. Although several genetic factors that predispose families to CRC are known, in many families affected with CRC the underlying genetics remain elusive. The work described in this thesis aimed to identify novel genetic factors that lead to an increased risk for CRC in these families. Several approaches were applied, including both germ line genetic analysis and the study of genomic aberrations in colorectal carcinomas. Linkage analysis did not provide evidence for a novel high risk factor, but provided supportive evidence for a previously identified region on 3q. Enrichment of common low risk variants was observed in a cohort of familial CRC patients but not in early-onset solitary patients (without a family history of CRC). Profiling of genomic aberrations in colorectal carcinomas showed distinct profiles for different hereditary CRC syndromes: MUTYH-associated carcinomas showed high frequencies of copy-neutral LOH. Mismatch repair proficient familial carcinomas appeared to resemble the genomic profile of sporadic CRC, but with a remarkably increased frequency of 20q gain and genome-wide cnLOH.