Guido M. J. M. Roemen

In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low-grade serous tumours

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. Copyright

Differential Gene Expression in Ovarian Tumors Reveals Dusp 4 and Serpina 5 As Key Regulators for Benign Behavior of Serous Borderline Tumors

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.

Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): An ancillary diagnostic tool

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 β-catenin point mutations mainly in exon 3, codons 41 and 45. These mutations result in stabilization of β-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 β-catenin mutations in the diagnosis of desmoid-type fibromatosis using paraffin-embedded material. The results were compared with nuclear expression of β-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 (TCT>TTT), 27 (35%) in codon 41 (ACC>GCC), and 5 (7%) in codon 45 (TCT>CCT). No mutations were detected in the other lesions studied. All desmoid-type fibromatosis cases and 72% of the mimics tested showed nuclear positivity for β-catenin indicating immunohistochemistry is a sensitive but not a specific test for desmoid-type fibromatosis. In contrast, to date, β-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 β-catenin mutations in desmoid-type fibromatosis, complements light microscopy in reaching a diagnosis.

Meat consumption and K-ras mutations in sporadic colon and rectal cancer in The Netherlands Cohort Study

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.

Clonal analysis favours a monoclonal origin for serous borderline tumours with peritoneal implants

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 Müllerian 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

Dietary folate intake and k-ras mutations in sporadic colon and rectal cancer in The Netherlands Cohort Study.

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 μg/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.

BRAFV600E immunopositive melanomas show low frequency of heterogeneity and association with epithelioid tumor cells: a STROBE-compliant article.

AbstractTreatment 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.

Refining the Diagnosis of Congenital Nephrotic Syndrome on Long-term Stored Tissue: c.1097G>A (p.(Arg366His)) WT1 Mutation Causing Denys Drash Syndrome

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.