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Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients.

Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAF (p.V600E) mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAF (p.V600E) mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAF (p.V600E) with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10(-9)). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAF (p.V600E) and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1 D27 , which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAF (p.V600E) tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAF (p.V600E) stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAF (p.V600E) colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.

Germline variants affecting the exonuclease domains of POLE and POLD1 predispose to multiple colorectal adenomas and/or colorectal cancer (CRC). The aim of this study was to estimate the prevalence of previously described heterozygous germline variants POLE c.1270C>G, p.(Leu424Val) and POLD1 c.1433G>A, p.(Ser478Asn) in a Dutch series of unexplained familial, early onset CRC and polyposis index cases. We examined 1188 familial CRC and polyposis index patients for POLE p.(Leu424Val) and POLD1 p.(Ser478Asn) variants using competitive allele-specific PCR. In addition, protein expression of the POLE and DNA mismatch repair genes was studied by immunohistochemistry in tumours from POLE carriers. Somatic mutations were screened using semiconductor sequencing. We detected three index patients (0.25%) with a POLE p.(Leu424Val) variant. In one patient, the variant was found to be de-novo. Tumours from three patients from two families were microsatellite instable, and immunohistochemistry showed MSH6/MSH2 deficiency suggestive of Lynch syndrome. Somatic mutations but no germline MSH6 and MSH2 variants were subsequently found, and one tumour displayed a hypermutator phenotype. None of the 1188 patients carried the POLD1 p.(Ser478Asn) variant. POLE germline variant carriers are also associated with a microsatellite instable CRC. POLE DNA analysis now seems warranted in microsatellite instable CRC, especially in the absence of a causative DNA mismatch repair gene germline variant.

Background Colorectal adenomatous polyposis is associated with a high risk of colorectal cancer (CRC) and is frequently caused by germline mutations in APC or MUTYH. However, in about 20–30% of patients no underlying gene defect can be identified. In this study, we tested if recently identified CRC risk variants play a role in patients with >10 adenomas. Methods We analysed a total of 16 SNPs with a reported association with CRC in a cohort of 252 genetically unexplained index patients with >10 colorectal adenomas and 745 controls. In addition, we collected detailed clinical information from index patients and their first-degree relatives (FDRs). Results We found a statistically significant association with two of the variants tested: rs3802842 (at chromosome 11q23, OR=1.60, 95% CI 1.3 to 2.0) and rs4779584 (at chromosome 15q13, OR=1.50, 95% CI 1.2 to 1.9). The majority of index patients (84%) had between 10 and 100 adenomas and 15% had >100 adenomas. Only two index patients (1%), both with >100 adenomas, had FDRs with polyposis. Forty-one per cent of the index patients had one or more FDRs with CRC. Conclusions These SNPs are the first common, low-penetrant variants reported to be associated with adenomatous polyposis not caused by a defect in the APC, MUTYH, POLD1 and POLE genes. Even though familial occurrence of polyposis was very rare, CRC was over-represented in FDRs of polyposis patients and, if confirmed, these relatives will therefore benefit from surveillance.

Whilst common genetic variation in many non-coding genomic regulatory regions are known to impart risk of colorectal cancer (CRC), much of the heritability of CRC remains unexplained. To examine the role of recurrent coding sequence variation in CRC aetiology, we genotyped 12,638 CRCs cases and 29,045 controls from six European populations. Single-variant analysis identified a coding variant (rs3184504) in SH2B3 (12q24) associated with CRC risk (OR = 1.08, P = 3.9 × 10 −7 ) and novel damaging coding variants in 3 genes previously tagged by GWAS efforts; rs16888728 (8q24) in UTP23 (OR = 1.15, P = 1.4 × 10 −7 ); rs6580742 and rs12303082 (12q13) in FAM186A (OR = 1.11, P = 1.2 × 10 −7 and OR = 1.09, P = 7.4 × 10 −8 ); rs1129406 (12q13) in ATF1 (OR = 1.11, P = 8.3 × 10 −9 ), all reaching exome-wide significance levels. Gene based tests identified associations between CRC and PCDHGA genes (P < 2.90 × 10 −6 ). We found an excess of rare, damaging variants in base-excision (P = 2.4 × 10 −4 ) and DNA mismatch repair genes (P = 6.1 × 10 −4 ) consistent with a recessive mode of inheritance. This study comprehensively explores the contribution of coding sequence variation to CRC risk, identifying associations with coding variation in 4 genes and PCDHG gene cluster and several candidate recessive alleles. However, these findings suggest that recurrent, low-frequency coding variants account for a minority of the unexplained heritability of CRC.

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.

Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAFp.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAFp.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAFp.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAFp.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAFp.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAFp.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAFp.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.

Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAF p.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAF p.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAF p.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAF p.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1 D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAF p.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAF p.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAF p.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.

Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAFp.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAFp.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAFp.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAFp.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAFp.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAFp.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAFp.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.

Very few principles have been unraveled that explain the relationship between soil properties and soil biota across large spatial scales and different land-use types. Here, we seek these general relationships using data from 52 differently managed grassland and forest soils in three study regions spanning a latitudinal gradient in Germany. We hypothesize that, after extraction of variation that is explained by location and land-use type, soil properties still explain significant proportions of variation in the abundance and diversity of soil biota. If the relationships between predictors and soil organisms were analyzed individually for each predictor group, soil properties explained the highest amount of variation in soil biota abundance and diversity, followed by land-use type and sampling location. After extraction of variation that originated from location or land-use, abiotic soil properties explained significant amounts of variation in fungal, meso- and macrofauna, but not in yeast or bacterial biomass or diversity. Nitrate or nitrogen concentration and fungal biomass were positively related, but nitrate concentration was negatively related to the abundances of Collembola and mites and to the myriapod species richness across a range of forest and grassland soils. The species richness of earthworms was positively correlated with clay content of soils independent of sample location and land-use type. Our study indicates that after accounting for heterogeneity resulting from large scale differences among sampling locations and land-use types, soil properties still explain significant proportions of variation in fungal and soil fauna abundance or diversity. However, soil biota was also related to processes that act at larger spatial scales and bacteria or soil yeasts only showed weak relationships to soil properties. We therefore argue that more general relationships between soil properties and soil biota can only be derived from future studies that consider larger spatial scales and different land-use types.