Detection of PTCH1 Copy-Number Variants in Mosaic Basal Cell Nevus Syndrome.

Basal cell nevus syndrome (BCNS) is an inherited disorder characterized mainly by the development of basal cell carcinomas (BCCs) at an early age. BCNS is caused by heterozygous small-nucleotide variants (SNVs) and copy-number variants (CNVs) in the Patched1 (PTCH1) gene. Genetic diagnosis may be complicated in mosaic BCNS patients, as accurate SNV and CNV analysis requires high-sensitivity methods due to possible low variant allele frequencies. We compared test outcomes for PTCH1 CNV detection using multiplex ligation-probe amplification (MLPA) and digital droplet PCR (ddPCR) with samples from a BCNS patient heterozygous for a PTCH1 CNV duplication and the patient's father, suspected to have a mosaic form of BCNS. ddPCR detected a significantly increased PTCH1 copy-number ratio in the index patient's blood, and the father's blood and tissues, indicating that the father was postzygotic mosaic and the index patient inherited the CNV from him. MLPA only detected the PTCH1 duplication in the index patient's blood and in hair and saliva from the mosaic father. Our data indicate that ddPCR more accurately detects CNVs, even in low-grade mosaic BCNS patients, which may be missed by MLPA. In general, quantitative ddPCR can be of added value in the genetic diagnosis of mosaic BCNS patients and in estimating the recurrence risk for offspring.

Association of RANKL and EGFR gene expression with bone metastases in patients with metastatic non-small cell lung cancer.

Introduction: Bone metastases are frequent in patients with non-small cell lung cancer (NSCLC). The receptor activator of Nuclear Factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) pathway is important in bone metastases development. Furthermore, epidermal growth factor receptor (EGFR) signaling promotes osteoclast formation and stimulation. The understanding of the biological mechanism of bone metastases development might have implications for treatment strategies. Therefore, we studied whether there is an association between EGFR, RANKL, RANK and OPG gene expression in the tumor and presence of bone metastases in patients with NSCLC. Methods: From an updated multicenter study, including patients with EGFR mutated (EGFR+), Kirsten rat sarcoma (KRAS+) and EGFR/KRAS wildtype metastatic NSCLC, all patients with available formalin-fixed paraffin-embedded (FFPE) tumor samples were selected. Ribonucleic Acid (RNA) was isolated from these samples and gene expressions of EGFR, RANKL, OPG and RANKL were determined via quantitative Polymerase Chain Reaction (qPCR). Data on demographics, histology and molecular subtyping, sample origin, presence of bone metastasis, SREs and bone progression were collected. Primary endpoint was relation between EGFR, RANK, RANKL, OPG gene expression, RANKL: OPG ratio and bone metastases. Results: In 73/335 (32% EGFR+, 49% KRAS+, 19% EGFR/KRAS wildtype) samples from unique patients, gene expression analysis could be performed. Of these 73 patients, 46 (63%) had bone metastases at diagnosis or developed bone metastases during the disease course. No association was found between EGFR expression and presence of bone metastases. Patients with bone metastases had a significantly higher RANKL expression and RANKL: OPG ratio compared to those without. An increased RANKL: OPG ratio resulted in a 1.65x increased risk to develop bone metastases, especially in the first 450 days after diagnosis of metastatic NSCLC. Conclusion: Increased RANKL gene expression and RANKL: OPG ratio, but not EGFR expression, was associated with presence of bone metastases. Additionally, an increased RANKL: OPG gene ratio was associated with a higher incidence of bone metastases development.

Cutaneous metastases of internal malignancies: a single-institution experience.

AIMS: Cutaneous metastases of internal malignancies occur in 1-10% of cancer patients. The diagnosis can sometimes be challenging, especially in cases with an unknown primary cancer. MATERIALS AND METHODS: A retrospective case review was performed including all cases of skin metastases from primary internal malignancies diagnosed at the Department of Pathology at the Maastricht University Medical Centre+ from 2007 to 2021. The clinicopathological data were collected and immunohistochemical and molecular diagnostic tests were performed to confirm the primary origin of the metastases. RESULTS: We identified 152 cases (71 female; 31 male patients) of cutaneous metastases of internal malignancies. 28 patients (20 women and 8 men) were diagnosed with multiple cutaneous metastases. Among the female patients, the most common primary tumour was breast cancer (50% of the cases), followed by lung (13.6%), gynaecological (7.3%), and gastrointestinal origin (7.3%). Among the male patients, the most common primary sites were gastrointestinal and lung origin (altogether, 50% of the cases). In 19 patients, the cutaneous metastasis was the first presentation of a clinically silent internal malignancy (18.6%), of which most (78.9%) represented metastatic lung carcinomas. Finally, metastasizing patterns were different across tumour types and gender. CONCLUSION: Breast, lung, gastrointestinal, and gynaecologic cancers are the most common primary tumours demonstrating skin metastases. Infrequently, cutaneous metastases can be the first clinically visual manifestation of an underlying not yet diagnosed internal malignancy; therefore, occasional broad immunohistochemical profiling, molecular clonal analysis, and a continuous high level of awareness are necessary for a precise diagnosis of cutaneous metastases of internal malignancies.

RET Fluorescence In Situ Hybridization Analysis Is a Sensitive but Highly Unspecific Screening Method for RET Fusions in Lung Cancer.

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.

Multicenter Evaluation of the Fully Automated PCR-Based Idylla EGFR Mutation Assay on Formalin-Fixed, Paraffin-Embedded Tissue of Human Lung Cancer.

Before initiating treatment of advanced non-small-cell lung cancer with tyrosine kinase inhibitors (eg, erlotinib, gefitinib, osimertinib, and afatinib), which inhibit the catalytic activity of epidermal growth factor receptor (EGFR), clinical guidelines require determining the EGFR mutational status for activating (EGFR exons 18, 19, 20, or 21) and resistance (EGFR exon 20) mutations. The EGFR resistance mutation T790M should be monitored at cancer progression. The Idylla EGFR Mutation Assay, performed on the Idylla molecular diagnostics platform, is a fully automated (<2.5 hours turnaround time) sample-to-result molecular test to qualitatively detect 51 EGFR oncogene point mutations, deletions, or insertions. In a 15-center evaluation, Idylla results on 449 archived formalin-fixed, paraffin-embedded tissue sections, originating from non-small-cell lung cancer biopsies and resection specimens, were compared with data obtained earlier with routine reference methods, including next-generation sequencing, Sanger sequencing, pyrosequencing, mass spectrometry, and PCR-based assays. When results were discordant, a third method of analysis was performed, when possible, to confirm test results. After confirmation testing and excluding invalids/errors and discordant results by design, a concordance of 97.6% was obtained between Idylla and routine test results. Even with <10 mm2 of tissue area, a valid Idylla result was obtained in 98.9% of the cases. The Idylla EGFR Mutation Assay enables sensitive detection of most relevant EGFR mutations in concordance with current guidelines, with minimal molecular expertise or infrastructure.

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

Treatment of BRAFV600E-mutant melanoma by small molecule inhibitors that target BRAFV600E 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 BRAFV600E 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.

v-Raf murine sarcoma viral oncogene mutation status in serous borderline ovarian tumors and the effect on clinical behavior.

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.

Dietary folate and APC mutations in sporadic colorectal cancer.

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, P(trend) = 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, P(trend) = 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.

TP53 overexpression in recurrent endometrial carcinoma.

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.

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study.

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.

Meat and fish consumption, APC gene mutations and hMLH1 expression in colon and rectal cancer: a prospective cohort study (The Netherlands).

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.

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.

Cigarette smoking and colorectal cancer: APC mutations, hMLH1 expression, and GSTM1 and GSTT1 polymorphisms.

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.

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 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.

Fat and K-ras mutations in sporadic colorectal cancer in The Netherlands Cohort Study.

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.

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.

APC mutations in sporadic colorectal carcinomas from The Netherlands Cohort Study.

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 sub-localization suggest a different aetiology of tumourigenesis in colon and rectum.

Effects of dietary folate and alcohol intake on promoter methylation in sporadic colorectal cancer: the Netherlands cohort study on diet and cancer.

Sporadic colorectal cancer (CRC) is characterized by genetic and epigenetic changes such as regional DNA hypermethylation and global DNA hypomethylation. Epidemiological and animal studies suggest that aberrant DNA methylation is associated with low dietary folate intake, which is aggravated by high alcohol intake. The relationship between promoter methylation of genes involved in CRC carcinogenesis and folate and alcohol intake was investigated. Methylation of the APC-1A, p14(ARF), p16(INK4A), hMLH1, O(6)-MGMT, and RASSF1A promoters was studied using methylation-specific PCR in 122 sporadic CRCs, derived from patients with folate and alcohol intake at either the lower or the higher quintiles of the distribution. Overall, promoter hypermethylation frequencies observed were: 39% for APC; 33% for p14(ARF); 31% for p16(INK4A); 29% for hMLH1; 41% for O(6)-MGMT; and 20% for RASSF1A. For each of the tested genes, the prevalence of promoter hypermethylation was higher in CRCs derived from patients with low folate/high alcohol intake (n = 61) when compared with CRCs from patients with high folate/low alcohol intake (n = 61), but the differences were not statistically significant. The number of CRCs with at least one gene methylated was higher (84%) in the low folate intake/high alcohol intake group when compared with the high folate intake/low alcohol intake group (70%; P = 0.085). Despite the size limitations of this study, these data suggest that folate and alcohol intake may be associated with changes in promoter hypermethylation in CRC.

Differences between the CD34+ and CD34- blast compartments in apoptosis resistance in acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Altered expression of members of the Bcl-2 family might account for the observed apoptosis resistance to chemotherapy in acute myeloid leukemia (AML). Given the poor prognosis associated with CD34+ expression in AML, we studied the role of spontaneous apoptosis and apoptosis regulatory proteins in sorted CD34+ and CD34- primary AML fractions. DESIGN AND METHODS: The expression levels of apoptosis regulatory proteins and spontaneous apoptosis were measured in primary AML samples by Western blot analysis and flow cytometry. To determine the role of CD34+ cells in apoptosis resistance, spontaneous apoptosis in serum-free conditions and apoptosis regulatory protein levels were measured in CD34+ and CD34- sorted cells from CD34+ primary AML samples. RESULTS: We show that CD34+ AML fractions are more resistant to apoptosis than are corresponding CD34- AML fractions, and that this is paralleled by higher Bcl-2, Bcl-xL, Mcl-1, Pgp and lower Bax expression levels. Interestingly, as the percentage of CD34 cells increased in the primary AML sample, so too did the apoptosis resistance in the corresponding CD34- fraction, which was reflected by an increasing anti-apoptosis protein profile. INTERPRETATION AND CONCLUSIONS: The data show that the CD34+ fraction is more resistant to apoptosis than is the corresponding CD34- fraction and secondly that the AML as a whole is more apoptosis resistant with increasing CD34 percentage.