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Introduction: RET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions.Methods: Between January 2016 and November 2019, a total of 4873 patients with NSCLC were routinely screened for RET fusions using either FISH (n = 2858) or targeted RNA next-generation sequencing (NGS) (n = 2015). If sufficient material was available, positive cases were analyzed by both methods (n = 39) and multiple FISH assays (n = 17). In an independent cohort of 520 patients with NSCLC, whole-genome sequencing data were investigated for disruptive structural variations and functional fusions in the RET and compared with ALK and ROS1 loci.Results: FISH analysis revealed RET rearrangement in 48 of 2858 cases; of 30 rearranged cases double tested with NGS, only nine had a functional RET fusion. RNA NGS yielded RET fusions in 14 of 2015 cases; all nine cases double tested by FISH had RET locus rearrangement. Of these 18 verified RET fusion cases, 16 had a split signal and two a complex rearrangement by FISH. By whole-genome sequencing, the prevalence of functional fusions compared with all disruptive events was lower in the RET (4 of 9, 44%) than the ALK (27 of 34, 79%) and ROS1 (9 of 12, 75%) loci.Conclusions: FISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC. (C) 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc.

Basal cell nevus syndrome (BCNS, Gorlin syndrome) is a rare inherited disorder characterized by the development of multiple basal cell carcinomas (BCCs), odontogenic keratocysts, and palmar pits.1 BCC development is caused by sonic hedgehog pathway (SHH) activation caused by mutations in tumor suppressor gene patched 1 (PTCH1) or activating mutations in the oncogene smoothened (SMO).2 Because patients with BCNS carry a germ-line mutation in PTCH1, one additional somatic mutation (second hit) results in BCC development at a young age. In 2012, the US Food and Drug Administration approved vismodegib for treatment of locally advanced BCC (laBCC) or metastatic BCC (mBCC). Vismodegib prevents activation of the SHH pathway by binding and inhibiting the SMO protein.3 Vismodegib resistance, mainly caused by SMO mutations, is an important problem seen in laBCC or mBCC in patients with and without BCNS.4, 5 Vismodegib resistance in smaller BCCs, which are far more frequent in BCNS patients, is only described once.6 Here, vismodegib resistance of those smaller BCCs in a BCNS patient is genetically explained, and a clinical treatment approach is given.

Basal cell naevus syndrome (BCNS) is an autosomal dominant disorder most commonly caused by a germline mutation in the Drosophila homologue of patched-1 gene (PTCH1). Here we describe a patient with clinical signs of BCNS, caused by postzygotic mosaicism of a PTCH1 mutation. We performed restriction fragment length polymorphism analysis and Droplet Digital polymerase chain reaction to determine the degree of mosaicism in different tissues of this patient. Our case shows that a relatively low-grade mosaicism can lead to clinical signs reminiscent of those caused by a germline mutation. This finding has important implications for genetic counselling and therefore is pivotal to recognize for dermatologists, as well as for clinical geneticists and clinical laboratory geneticists.What's already known about this topic?Basal cell naevus syndrome (BCNS) is generally caused by a germline mutation in the patched-1 gene (PTCH1).Genetic mosaicism in BCNS has been described.What does this study add?A low-grade postzygotic mosaicism of a PTCH1 mutation can cause a clinical presentation fulfilling the diagnostic criteria of BCNS.It is important to determine the degree of mosaicism as accurately as possible with a quantitative technique, for example Droplet Digital polymerase chain reaction, to provide adequate genetic counselling.

Background Pulmonary large cell neuroendocrine carcinoma (LCNEC) can be subdivided in two types: the co-mutated TP53 and RB1 subtype, and the TP53 and STK11/KEAP1 mutated subtype (Derks et al. Clin Cancer Res 2018; J Thorac Oncol 2018). DLL3 is a member of the Notch ligand family and a possible treatment target for neuroendocrine carcinoma. We investigated DLL3 and NE marker expression in mutational subtypes of metastatic LCNEC. Methods Immunohistochemical (IHC) analysis for DLL3 (clone SC16.65) was performed on 94 pathological reviewed pretreatment stage IV LCNEC. Samples were scored positive if ≥1% tumor cells showed cytoplasmic or dotlike DLL3 immunostaining. Also an H-score was calculated by multiplying intensity by percentage of positive cells. Targeted next generation sequencing (TP53, RB1, STK11, KEAP1) could be performed in 66 patients. Results DLL3 was expressed in 70/94 (75%) LCNEC, 56 of which showed cytoplasmic immunostaining. 37/70 (53%) samples had an H-score>100. DLL3 staining was more often seen in STK11 and/or KEAP1 mutated LCNEC (13/14;93%) than in tumors with wildtype STK11/KEAP1 (36/52;69%) (trend;p=0.093). In addition, DLL3 positivity was associated with expression of ≥2 NE-markers (64/79 LCNEC (81%) vs 3/8 (37%) tumors with Conclusions A high percentage (75%) of stage IV LCNEC shows cytoplasmatic DLL3 epression in association with NE markers, half of which with H-scores>100. Interestingly, DLL3 positivity was observed in almost all STK11/KEAP1 mutated LCNEC, which is in agreement with recent data (George et al. Nat Commun 2018). It thus might be worthwhile to investigate the efficacy of DLL3 targeted therapy in LCNEC. Citation Format: Ernst-Jan M. Speel, Bregtje C. Hermans, Jules L. Derks, Erik Thunnissen, Robert Jan van Suylen, Michael A. den Bakker, Harry J. Groen, Egbert F. Smit, Ronald A. Damhuis, Esther C. van den Broek, Cecile M. Stallinga, Guido M. Roemen, Anne-Marie C. Dingemans. A high percentage of LCNEC shows DLL3 expression in association with molecular subtypes and neuroendocrine markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 487.

Contains fulltext : 168099.pdf (Publisher’s version ) (Open Access) Congenital nephrotic syndrome (CNS) caused by a mutation in the Wilms tumor 1 suppressor gene (WT1) is part of Denys Drash Syndrome or Frasier syndrome. In the framework of genetic counseling, the diagnosis of CNS can be refined with gene mutation studies on long-term stored formalin-fixed paraffin-embedded tissue from postmortem examination. We report a case of diffuse mesangial sclerosis with perinatal death caused by a de novo mutation in the WT1 gene in a girl with an XY-genotype. This is the first case of Denys Drash Syndrome with the uncommon missense c.1097G>A [p.(Arg366His)] mutation in the WT1 gene which has been diagnosed on long-term stored formalin-fixed paraffin-embedded tissue in 1993. This emphasizes the importance of retained and adequately stored tissue as a resource in the ongoing medical care and counseling.

Objective. To study alterations within the p53 pathway in relation to the development of recurrent stage I endometrioid endometrial carcinoma. Methods. Paraffin-embedded tumor tissue of both primary and recurrent tumors from 44 patients with and 44 without recurrence was used for immunohistochemical analysis of TP53, hMdm2, P21 Waf1/Cip1 and M30. DNA was extracted, and mutation analysis of p53 (exon 5–8, 11) was performed by direct sequencing. Results. TP53 overexpression was significantly associated with recurrent disease: Odds Ratio 3.8 (95% CI: 1.5–9.8). Overexpression of TP53 was associated with lower staining indices (SI:0–9) of both hMdm2 and P21 in tumors of patients with recurrence, compared to controls: 2.0 ± 0.4 vs. 4.0 ± 0.8 and 1.9 ± 0.8 vs. 3.6 ± 0.8, respectively. Eight p53 missense mutations were identified in six patients with recurrence and two controls. One nonsense mutation was found in a patient with recurrence and one deletion in a control patient. Only a minority of TP53 overexpression cases could be explained by the presence of these p53 mutations. Conclusion. TP53 overexpression was significantly predictive for recurrent endometrial carcinoma, and mostly not correlated with p53 mutations. Concomitant low hMdm2 and P21 Waf1/Cip1 expression in tumors with overexpressed TP53 suggests a dysfunctional TP53-P21 Waf1/Cip1 pathway.

Folate deficiency has been associated with colorectal cancer risk and may be involved in colorectal carcinogenesis through increased chromosome instability, gene mutations, and aberrant DNA methylation. Within the Netherlands Cohort Study on diet and cancer, we investigated the associations between dietary folate intake and colorectal cancer risk with (APC+) and without (APC-) truncating APC mutations, accounting for hMLH1 expression and K-ras mutations. In total, 528 cases and 4200 subcohort members were available for data analyses of the study cohort (n = 120,852) from a follow-up period between 2.3 and 7.3 y after baseline. Adjusted gender-specific incidence rate ratios (RR) over tertiles of folate intake were calculated in case-cohort analyses for colon and rectal cancer. Although relatively high folate intake was not associated with overall colorectal cancer risk, it reduced the risk of APC- colon tumors in men (RR 0.58, 95% CI 0.32-1.05, P trend = 0.06 for the highest vs. lowest tertile of folate intake). In contrast, it was positively associated with APC+ colon tumors in men (highest vs. lowest tertile: RR 2.77, 95% CI 1.29-5.95, Ptrend = 0.008) and was even stronger when the lack of hMLH1 expression and K-ras mutations were excluded (RR 3.99, 95% CI 1.43-11.14, Ptrend = 0.007). Such positive associations were not observed among women; nor was folate intake associated with rectal cancer when APC mutation status was taken into account. Relatively high folate consumption reduced the risk of APC- colon tumors, but folate intake was positively associated with APC+ colon tumors among men. These opposite results may indicate that folate enhances colorectal carcinogenesis through a distinct APC mutated pathway. © 2006 American Society for Nutrition.

Supplemental Digital Content is available in the text, Treatment of BRAFV600E-mutant melanoma by small molecule inhibitors that target BRAF or MEK kinases is increasingly used in clinical practice and significantly improve patient outcome. However, patients eventually become resistant and therapeutic improvement is required. Molecular diversity within individual tumors (intratumor heterogeneity) and between tumors within a single patient (intrapatient heterogeneity) poses a significant challenge to precision medicine. Using immunohistochemistry, we determined the extent of BRAFV600E intratumor and intrapatient heterogeneity and the influence of morphological heterogeneity in a large series of 171 melanomas of 81 patients. The BRAFV600E mutation rate found in our melanoma series is 44%, with none of 22 (0%) melanoma in situ, 23 of 56 (41%) primary tumors, 28 of 59 (48%) regional metastases, and 24 of 34 (71%) distant metastases harboring the mutation. In general, a diffuse homogeneous immunostaining was seen, even in tumors consisting of more than one cell type, that is, epithelioid, spindle, and/or small cell types. Nevertheless, BRAFV600E-mutant melanomas more often had a purely epithelioid cell population (P = 0.063), that is more evident among distant metastases (P = 0.014). Only two of 75 (3%) mutated specimens (one primary and one metastasis) displayed heterogeneous BRAFV600E expression. The primary tumor was also morphologically heterogeneous and exclusively displayed BRAFV600E in the epithelioid component, confirming an association between BRAFV600E and epithelioid cells. Twenty-eight of 30 patients (93%) had concordant BRAF mutation status between their tumors. Taken together, BRAFV600E intratumor and intrapatient heterogeneity in melanoma is diminutive, nevertheless, the identified exceptions will have important implications for the clinical management of this disease.

Objective: The aim of this study was to investigate the associations between meat and fish consumption and APC mutation status and hMLH1 expression in colon and rectal cancer. Methods: The associations were investigated in the Netherlands Cohort Study, and included 434 colon and 154 rectal cancer patients on whom case-cohort analyses (subcohort n = 2948) were performed. Results: Total meat consumption was not associated with the endpoints studied. Meat product (i.e. processed meat) consumption showed a positive association with colon tumours harbouring a truncating APC mutation, whereas beef consumption was associated with an increased risk of colon tumours without a truncating APC mutation (incidence rate ratio (RR) highest versus lowest quartile of intake 1.61, 95% confidence interval (CI) 0.96-2.71, p-trend = 0.04 and 1.58, 95% CI 1.10-2.25, p-trend = 0.01, respectively). Consumption of other meat (horsemeat, lamb, mutton, frankfurters and deep-fried meat rolls) was associated with an increased risk of rectal cancer without a truncating APC mutation (RR intake versus no intake 1.79, 95% CI 1.10-2.90). No associations were observed for meat consumption and tumours lacking hMLH1 expression. Conclusions: Our data indicate that several types of meat may contribute differently to the aetiology of colon and rectal cancer, depending on APC mutation status but not hMLH1 expression of the tumour. © Springer 2005.

Purpose Ovarian serous borderline tumors (SBT) are characterized by arborizing papillae lined by stratified epithelial cells, varying atypia, and absence of stromal invasion. Originally, these tumors have been classified as borderline because they behaved in a remarkably indolent manner, even with widespread tumor deposits called implants and the presence of lymph node involvement. The molecular biology of these lesions has just begun to be explored. High prevalence of B-RAF/K-RAS mutations in SBTs in contrast to serous carcinomas (SCAs) indicates that the mitogenic RAS-RAF-MEK-ERK-MAP kinase pathway is crucial for the pathogenesis of SBTs. The purpose of this study was to further unravel the genetic pathways through which SBTs develop, with a special focus on explaining the generally benign SBT behavior. Materials and Methods We generated RNA expression profiles of 38 ovarian serous neoplasms. Global Test pathway analysis and significance analysis of microarrays (SAM) of the expression profiles was performed. Results SAM and Global Testing showed that although the mitogenic pathway is activated in SBTs, activation of downstream genes involved in extracellular matrix (ECM) degradation is absent, suggesting an uncoupling of both events. In addition, we show that two genes involved in regulating this uncoupling, ERK-inhibitor Dusp 4 and uPA-inhibitor Serpina 5, are downregulated in SCAs in contrast to SBTs. In SCAs, this was associated with downstream MMP-9 activation at both mRNA and protein level. Conclusion We propose that the putative tumor suppressor genes Dusp 4 and Serpina 5 provide a major clue to the indolent behavior of SBTs.

The contribution of cigarette smoking to sporadic colorectal cancer may differ according to molecular aspects of the tumor or according to glutathione S-transferase M1 (GSTM1) or glutathione S-transferase T1 (GSTT1) genotype. In the prospective Netherlands Cohort Study on Diet and Cancer, adjusted incidence rate ratios for 1986-1993 were computed for overall colorectal cancer, tumors with and without adenomatous polyposis coli (APC) mutations, and tumors with and without human mut-L homologue 1 (hMLH1) expression, according to cigarette smoking characteristics (661 cases, 2,948 subcohort members). Case-only analyses were performed to estimate odds ratios for interaction between cigarette smoking and GSTM1 and GSTT1 genotypes. In comparison with never smokers, a high smoking frequency increased the risk of colorectal cancer (for a five-cigarette/day increment, incidence rate ratio (IRR) = 1.07, 95% confidence interval (CI): 1.03, 1.12), and this association was stronger in 371 tumors without a truncating APC mutation (IRR = 1.11, 95% CI: 1.05, 1.17). Long-term smoking was associated with lack of hMLH1 expression in 56 tumors (for a 10-year increment, IRR = 1.17, 95% CI: 1.00, 1.37). No statistically significant interactions between smoking and GSTM1 or GSTT1 genotype were observed. These results indicate that cigarette smoking is associated with risk of colorectal cancer, and this association may depend on molecular characteristics of the tumor as defined by APC mutation and hMLH1 expression status. Copyright © 2005 by the Johns Hopkins Bloomberg School of Public Health. All rights reserved. Chemicals / CAS: glutathione transferase, 50812-37-8; protein MLH1, 155577-96-1; Carrier Proteins; Glutathione Transferase, EC 2.5.1.18; MLH1 protein, human; Neoplasm Proteins; Nuclear Proteins

We studied the association between dietary folate and specific K-ras mutations in colon and rectal cancer in The Netherlands Cohort Study on diet and cancer. After 7.3 years of follow-up, 448 colon and 160 rectal cancer patients and 3,048 sub-cohort members (55-69 years at baseline) were available for data analyses. Mutation analysis of the K-ras gene was carried out on all archival adenocarcinoma specimens. Case-cohort analyses were used to compute adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) for colon and rectal cancer overall and for K-ras mutation status subgroups according to 100 mug/day increased intake in dietary folate. Dietary folate intake was not significantly associated with colon cancer risk for men or women, neither overall nor with K-ras mutation status. For rectal cancer, folate intake was associated with a decreased disease risk in men and was most pronounced for K-ras mutated tumors, whereas an increased association was observed for women. Regarding the K-ras mutation status in women, an increased association was observed for both wild-type and mutated K-ras tumors. Specifically, folate intake was associated with an increased risk of G>T and G>C transversions in rectal tumors (RR = 2.69, 95% CI = 1.43-5.09), but inversely associated with G>A transitions (RR = 0.08, 95% CI = 0.01-0.53). Our data suggest that the effect of folate on rectal cancer risk is different for men and women and depends on the K-ras mutation status of the tumor. (c) 2004 Wiley-Liss, Inc.

Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS/RAF/MEK/ERK/MAP-kinase pathway. Activating mutations in BRAF have recently been identified in melanomas, colorectal cancers, and thyroid and ovarian tumours. In the present study, an extensive characterization of BRAF and KRAS mutations has been performed in 264 epithelial and non-epithelial ovarian neoplasms. The epithelial tumours ranged from adenomas and borderline neoplasms to invasive carcinomas including serous, mucinous, clear cell, and endometrioid lesions. It is shown that BRAF mutations in ovarian tumours occur exclusively in low-grade serous neoplasms (33 of 91, 36%); these included serous borderline tumours (typical and micropapillary variants), an invasive micropapillary carcinoma and a psammocarcinoma. KRAS mutations were identified in 26 of 91 (29.5%) low-grade serous tumours, 7 of 49 (12%) high-grade serous carcinomas, 2 of 6 mucinous adenomas, 22 of 28 mucinous borderline tumours, and 10 of 18 mucinous carcinomas. Of note, two serous borderline tumours were found to harbour both BRAF and KRAS mutations. The finding that at least 60% of serous borderline tumours harbour mutations in two members of the ERK-MAP-kinase pathway (BRAF 36%, KRAS 30%) compared with 12% of high-grade serous carcinomas (BRAF 0%, KRAS 12%) indicates that the majority of serous borderline tumours do not progress to serous carcinomas. Furthermore, no BRAF mutations were detected in the other 173 ovarian tumours in this study.

Background & Aims: Current models of colorectal adenoma to carcinoma progression do not fully reflect the genetic heterogeneity and complexity of the disease. The aim of the present study was to identify genetic changes discriminating adenomas that have progressed to carcinoma from adenomas that have not progressed, and to refine the current genetic models of colorectal adenoma to carcinoma progression, based on a genome-wide analysis of chromosomal aberrations. Methods: Sixty-six nonprogressed colorectal adenomas, 46 progressed adenomas (malignant polyps), and 36 colorectal carcinomas were screened for chromosomal aberrations by comparative genomic hybridization, and for mutations in the adenomatous polyposis coli (APC) and K- ras gene. Data analysis focused on cancer-associated genetic changes in adenomas. Results: Accumulation of losses in 8p21-pter, 15q11-q21, 17p12-13, and 18q12-21, and gains in 8q23-qter, 13q14-31, and 20q13 were strongly associated with adenoma-to-carcinoma progression, independent of the degree of dysplasia. Hierarchic cluster analysis demonstrated the presence of 3 distinct subgroups of adenomas, characterized by unique combinations of genetic aberrations in the adenomas (17p loss and K- ras mutation, 8q and 13q gain, and 18q loss and 20q gain, respectively). Conclusions: The presence of 2 or more of the aforementioned 7 chromosomal changes was associated with progressed colorectal adenomas and colorectal cancer. In addition, evidence was found that these chromosomal abnormalities occurred in specific combinations of a few abnormalities rather than as a mere accumulation of events, indicating the existence of multiple independent chromosomal instability pathways of colorectal cancer progression. GASTROENTEROLOGY 2002;123:1109-1119

Human cancer is characterized by genetic and epigenetic alterations. In this study we provide evidence for the interruption of Ras signaling in sporadic colorectal cancer (CRC) by either genetic activation of the K-ras oncogene or epigenetic silencing of the putative tumor suppressor gene RASSF1A. Paraffin embedded tumor tissue samples from 222 sporadic CRC patients were analysed for K-ras codon 12 and codon 13 activating mutations and RASSF1A promoter hypermethylation. Overall, K-ras mutations were observed in 87 of 222 (39%) and RASSF1A methylation was observed in 45 of 222 (20%) of CRCs. Mutation of K-ras alone was detected in 76 of 222 (34%) CRCs. RASSF1A promoter methylation with wild-type K-ras was observed in 34 of 222 (15%) CRCs. In 101 of 222 (46%) CRCs neither K-ras mutations nor RASSF1A methylation was observed and 11 of 222 (5%) CRCs showed both K-ras mutations and RASSF1A methylation. These data show that the majority of the studied CRCs with K-ras mutations lack RASSF1A promoter methylation, an event which occurs predominantly in K-ras wild-type CRCs (P=0.023, Chi-square test).

Aims:To determine the incidence of activating v-raf murine sarcoma viral oncogene (BRAF) mutations in 30 serous borderline tumors (SBTs) of the ovary and the accompanying implants and to link BRAF mutation status to the clinical behavior of these tumors.Methods and Results:Serous borderline tumors and noninvasive implants of 30 patients were analyzed for the presence of the BRAF V599E mutation, and mutation status was correlated to 70 months of clinical follow-up. Mutation status could be assessed in 27 SBTs. Eleven (41%) showed a BRAF mulation. Four (80%) of 5 patients with bilateral SBT showed a BRAF mutation in both ovaries. From the 8 implants that were analyzed for BRAF, 2 (25%) were mutated together with their primary tumor. v-Raf murine sarcoma viral oncogene mutation positive SBTs tend to present with a lower International Federation of Gynecology and Obstetrics stage and a higher tumor volume and are less frequently aneuploid. Seventy months' follow-up indicated no significant recurrence-free survival difference between these groups.Conclusions:v-Raf murine sarcoma viral oncogene mutations are common in ovarian SBT, are strongly associated with bilateral tumors, and are also found in implants. A larger number of tumors should be investigated to assess clinical importance of BRAF mutation status in SBTs.

Desmoid-type fibromatosis is a locally aggressive deep soft tissue tumor. Some cases are associated with adenosis polyposis coli germline mutations whereas others harbor somatic beta-catenin point mutations mainly in exon 3, codons 41 and 45. These mutations result in stabilization of beta-catenin, and activation of the Wnt signaling pathway. The aim of this study was to determine the specificity and sensitivity of these 3 most common beta-catenin mutations in the diagnosis of desmoid-type fibromatosis using paraffin-embedded material. The results were compared with nuclear expression of beta-catenin. Mutation-specific restriction enzyme digestion methodology was employed to detect the 3 mutations. One hundred and thirty-three cases were analyzed, including 76 desmoid-type, and 18 superficial fibromatosis, in addition to a further 39 fibromatosis mimics. A restriction site was present for analysis of the codon 41 mutation. Mismatch primers were designed for the codon 45 mutations. Mutations were detected in 66 cases (87%) of 76 desmoid-type fibromatosis (71 extra-abdominal). Of these, 34 (45%) were in codon 45 (TCTTTT), 27 (35%) in codon 41 (ACCGCC), and 5 (7%) in codon 45 (TCTCCT). No mutations were detected in the other lesions studied. All desmoid-type fibromatosis cases and 72% of the mimics tested showed nuclear positivity for beta-catenin indicating immunohistochemistry is a sensitive but not a specific test for desmoid-type fibromatosis. In contrast, to date, beta-catenin mutations have not been detected in any lesions which mimic desmoid-type fibromatosis. Mutation-specific restriction enzyme digestion, a simple and efficient means of detecting the common beta-catenin mutations in desmoid-type fibromatosis, complements light microscopy in reaching a diagnosis.

Serous borderline tumours (SBTs) of the ovary were originally classified as such because the vast majority behave in a remarkably indolent manner, even in the presence of widespread tumour deposits, termed implants, and/or lymph node involvement. The pathogenesis of the implants is currently unknown. Two major hypotheses have been proposed: the first favours a monoclonal origin, arguing that the peritoneal lesions derive from neoplastic cells that are shed from the primary ovarian tumour. The second hypothesis favours a polyclonal origin as a result of a field defect of susceptible Mullerian cells from which multiple independent tumours arise. To test both hypotheses, genome-wide allelotyping and B-RAF/K-RAS mutation analyses were employed to assess clonality in 25 metachronous or synchronous tumours from ten SBT patients. Loss of heterozygosity (LOH) profiling and K-RAS/B-RAF mutation analysis showed concordance of the genetic changes in all sites in 21 tumours from eight patients who were informative. These results favour a common origin, underscored by a likelihood ratio (probability of common origin/probability of independent origin) ranging from 2.43 to 7 662 850. In conclusion, this study strongly supports the hypothesis that both non-invasive and invasive implants arise as a consequence of spread from a single ovarian site. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Case-cohort analyses were performed on meat and fish consumption in relation to K-ras mutations in 448 colon and 160 rectal cancers that occurred during 7.3 years of follow-up, excluding the first 2.3 years, and 2948 subcohort members of The Netherlands Cohort Study on diet and cancer. Adjusted incidence rate ratios and 95% confidence intervals were computed for colon and rectal cancer and for K-ras mutation status subgroups. Total fresh meat, most types of fresh meat and fish were not associated with colon or rectal cancer, neither overall nor with K-ras mutation status. However, several weak associations were observed for tumours with a wild-type K-ras, including beef and colon tumours, and an inverse association for pork with colon and rectal tumours; for meat products, an increased association was observed with wild-type K-ras tumours in the colon and possibly with G > A transitions in rectal tumours. © 2005 Cancer Research UK.

Associations between dietary intake of various fats and specific K-ras mutations in colorectal cancer (CRC) were investigated within the framework of The Netherlands Cohort Study on diet and cancer (NLCS). After 7.3 years of follow-up and with exclusion of the first 2.3 years, 448 colon and 160 rectal cancer patients and 2948 subcohort members (55-69 years at baseline) were available for data-analyses. Mutation analysis of the K-ras gene was performed on all archival colon and rectal adenocarcinoma specimens. Case-cohort analyses were used to compute adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) for colon and rectal cancer cases and for K-ras mutation subgroups. The intake of total, saturated and monounsaturated fat was not significantly associated with colon or rectal cancer. High intake of dietary polyunsaturated fat and, specifically, linoleic acid is associated with an increased risk of mutated K-ras colon tumours. The RRs for 1 SD of increase of polyunsaturated fat and linoleic acid were 1.21 (95% CI 1.05-1.41) and 1.22 (95% CI 1.05-1.42), respectively, and similar associations were observed for both G > A transitions and G >T or G > C transversions in the colon. In contrast, no significant associations were observed with rectal cancer risk, overall nor with specific K-ras mutation status. A high intake of polyunsaturated fat, in particular linoleic acid, may be an important dietary risk factor for K-ras mutated colon tumours, possibly by generating G > A transitions or G > T or G > C transversions in the K-ras oncogene. © Oxford University Press 2004; all rights reserved.

The adenomatous polyposis coli (APC) gene is considered to be a gatekeeper in colorectal tumourigenesis. Inactivating mutations in APC have been reported in 34-70% of sporadic colorectal cancer patients, the majority of which occur in the mutation cluster region (MCR). In this study, tumour tissue from 665 incident colorectal cancer patients, who originate from 120 852 men and women (55-69 years of age at baseline) participating in The Netherlands Cohort Study, was evaluated for the occurrence and type of APC mutations with regard to age at diagnosis, gender, family history of colorectal cancer, Dukes' stage, tumour differentiation and sub-localization. Mutation analysis of the MCR, which spans codons 1286-1513, was performed on archival adenocarcinoma samples using macrodissection, nested PCR and direct sequencing of purified PCR fragments. A large number of genetic aberrations (n = 978), including point mutations (n = 833), deletions (n = 126) and insertions (n = 19) was detected in the MCR in 72% of patients (479/665). In particular, we observed a large number of missense mutations, more than reported previously. This may indicate involvement in colorectal carcinogenesis, although their significance for APC functions is unclear. Truncating mutations were found in 37% of patients (248/665). Patients with rectosigmoid and rectum tumours relatively more frequently harboured C > T nonsense mutations and truncating frameshift mutations as compared with patients with proximal and distal colon tumours (P = 0.009 and P = 0.045, respectively). Differences in occurrence of truncating mutations with regard to tumour sublocalization suggest a different aetiology of tumourigenesis in colon and rectum. © Oxford University Press 2004; all rights reserved.

K-ras oncogene mutations in sporadic colorectal cancer in The Netherlands Cohort Study.Brink M, de Goeij AF, Weijenberg MP, Roemen GM, Lentjes MH, Pachen MM, Smits KM, de Bruine AP, Goldbohm RA, van den Brandt PA.Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Epidemiology, Maastricht University, PO Box 616, The Netherlands. m.brink@epid.unimaas.nlActivation of K-ras oncogene has been implicated in colorectal carcinogenesis, being mutated in 30-60% of the adenocarcinomas. In this study, 737 incident colorectal cancer (CRC) patients, originating from 120 852 men and women (55-69 years at baseline) participating in the Netherlands Cohort Study (NLCS), were studied in order to evaluate subgroups with respect to K-ras mutation status. Mutation analysis of the exon 1 fragment of the K-ras oncogene, spanning codons 8-29, was performed on archival colorectal adenocarcinoma samples of all patients using macrodissection, nested PCR and direct sequencing of purified fragments. The method of mutation detection was validated by the confirmation of reported K-ras status in CRC cell lines, a good correlation between fresh-frozen and routinely fixed, paraffin-embedded tissue, a detection limit of 5% mutated DNA and a good reproducibility. Various types of K-ras mutations were evaluated with respect to tumour sub-localization, Dukes' stage and tumour differentiation. In 37% (271/737) of the patients, the exon 1 fragment of K-ras gene was found to be mutated. The predominant mutations are G>A transitions and G>T transversions, and codons 12 and 13 are the most frequently affected codons. Patients with a rectal tumour were found to have the highest frequency of G>T transversions as compared with patients with a colon or rectosigmoid tumour. This difference appeared to be confined to women with a rectal tumour harbouring G>T transversions. No significant differences were observed for Dukes' stage with respect to types of K-ras mutation, which does not support direct involvement of the K-ras oncogene in adenocarcinoma progression. The equal distribution of K-ras mutations among cases with or without a family history of colorectal cancer argues against an important role for this mutation in familial colorectal cancer, and could imply that K-ras mutations are more probably involved in environmental mechanisms of colorectal carcinogenesis.

Background The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. Methods In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Results Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. Conclusion CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.