Dina Ruano

Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-Small-Cell Lung Cancer Using Allele-Specific qPCR

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.

Functional Gene Group Analysis Reveals a Role of Synaptic Heterotrimeric G Proteins in Cognitive Ability

Although cognitive ability is a highly heritable complex trait, only a few genes have been identified, explaining relatively low proportions of the observed trait variation. This implies that hundreds of genes of small effect may be of importance for cognitive ability. We applied an innovative method in which we tested for the effect of groups of genes defined according to cellular function (functional gene group analysis). Using an initial sample of 627 subjects, this functional gene group analysis detected that synaptic heterotrimeric guanine nucleotide binding proteins (G proteins) play an important role in cognitive ability (PEMP = 1.9 × 10−4). The association with heterotrimeric G proteins was validated in an independent population sample of 1507 subjects. Heterotrimeric G proteins are central relay factors between the activation of plasma membrane receptors by extracellular ligands and the cellular responses that these induce, and they can be considered a point of convergence, or a “signaling bottleneck.” Although alterations in synaptic signaling processes may not be the exclusive explanation for the association of heterotrimeric G proteins with cognitive ability, such alterations may prominently affect the properties of neuronal networks in the brain in such a manner that impaired cognitive ability and lower intelligence are observed. The reported association of synaptic heterotrimeric G proteins with cognitive ability clearly points to a new direction in the study of the genetic basis of cognitive ability.

Germline variants in POLE are associated with early onset mismatch repair deficient colorectal cancer

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.

Combined mismatch repair and POLE/POLD1 defects explain unresolved suspected Lynch syndrome cancers

Many suspected Lynch Syndrome (sLS) patients who lack mismatch repair (MMR) germline gene variants and MLH1 or MSH2 hypermethylation are currently explained by somatic MMR gene variants or, occasionally, by germline POLE variants. To further investigate unexplained sLS patients, we analyzed leukocyte and tumor DNA of 62 sLS patients using gene panel sequencing including the POLE, POLD1 and MMR genes. Forty tumors showed either one, two or more somatic MMR variants predicted to affect function. Nine sLS tumors showed a likely ultramutated phenotype and were found to carry germline (n=2) or somatic variants (n=7) in the POLE/POLD1 exonuclease domain (EDM). Six of these POLE/POLD1-EDM mutated tumors also carried somatic MMR variants. Our findings suggest that faulty proofreading may result in loss of MMR and thereby in microsatellite instability.

Increased frequency of 20q gain and copy-neutral loss of heterozygosity in mismatch repair proficient familial colorectal carcinomas.

Many hereditary nonpolyposis colorectal cancers (CRCs) cannot be explained by Lynch syndrome. Other high penetrance genetic risk factors are likely to play a role in these mismatch repair (MMR)‐proficient CRC families. Because genomic profiles of CRC tend to vary with CRC susceptibility syndromes, our aim is to analyze the genomic profile of MMR‐proficient familial CRC to obtain insight into the biological basis of MMR‐proficient familial CRC. We studied 30 MMR‐proficient familial colorectal carcinomas, from 15 families, for genomic aberrations, including gains, physical losses, and copy‐neutral loss of heterozygosity LOH (cnLOH) using SNP array comparative genomic hybridization. In addition, we performed somatic mutation analysis for KRAS, BRAF, PIK3CA and GNAS. The frequency of 20q gain (77%) is remarkably increased when compared with sporadic CRC, suggesting that 20q gain is involved in tumor progression of familial CRC. There is also a significant increase in the frequency of cnLOH and, as a consequence, a reduced frequency of physical loss compared with sporadic CRC. The most frequent aberrations observed included gains of 7p, 7q, 8q, 13q, 20p and 20q as well as physical losses of 17p, 18p and 18q. Most of these changes are also observed in sporadic CRC. Mutations in KRAS were identified in 37% of the MMR‐proficient CRCs, and mutations in BRAF were identified in 16%. No mutations were identified in PIK3CA or chromosome 20 candidate gene GNAS. We show that the patterns of chromosomal instability of MMR‐proficient familial CRC are clearly distinct from those from sporadic CRC. Both the increased gain on chromosome 20 and the increased levels of cnLOH suggest the presence of yet undiscovered germline defects that can, in part, underlie the cancer risk in these families.

Whole-Exome Sequencing Studies of Parathyroid Carcinomas Reveal Novel PRUNE2 Mutations, Distinctive Mutational Spectra Related to APOBEC-Catalyzed DNA Mutagenesis and Mutational Enrichment in Kinases Associated With Cell Migration and Invasion

CONTEXT Cell division cycle 73 (CDC73), encoding the protein parafibromin, is the most prevalent mutated gene in familial and sporadic parathyroid carcinoma (PC). OBJECTIVE To identify additional genetic abnormalities in PCs. DESIGN Whole-exome sequencing was performed using DNA from seven pairs of matched PCs and one triplet containing double primary tumor and normal leukocyte. Somatic variants were confirmed using Sanger sequencing and recurrently mutated genes were assessed in 13 additional PCs as well as 40 parathyroid adenomas (PA). RESULTS PC had an average of 51 somatic variants/tumor (range 3-176) with approximately 58% of variants occurring as nonsynonymous single nucleotide variants. The importance of CDC73 in PC is reinforced with a remarkable preferential amplification of the mutant CDC73 allele. Furthermore, recurrent germ line and somatic mutations in prune homolog 2 [Drosophila] (PRUNE2) were found in PC and computationally predicted to be deleterious; in addition, recurrent mutations in kinase genes related to cell migration and invasion were found. PRUNE2 showed recurrent mutations in 18% (4/22) of PCs with additional screening in 40 PAs revealing only one rare missense polymorphism (Asp1677Asn). For the first time, the mutational signature associated with apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-catalyzed cytosine-to-uracil deamination is found in a subset of PC. CONCLUSION This study outlines the genetic landscape of PC and attempts to characterize the mutational processes shaping the PC genome.

Integral analysis of p53 and its value as prognostic factor in sporadic colon cancer

Backgroundp53 (encoded by TP53) is involved in DNA damage repair, cell cycle regulation, apoptosis, aging and cellular senescence. TP53 is mutated in around 50% of human cancers. Nevertheless, the consequences of p53 inactivation in colon cancer outcome remain unclear. Recently, a new role of p53 together with CSNK1A1 in colon cancer invasiveness has been described in mice.MethodsBy combining data on different levels of p53 inactivation, we aimed to predict p53 functionality and to determine its effects on colon cancer outcome. Moreover, survival effects of CSNK1A1 together with p53 were also studied.Eighty-three formalin fixed paraffin embedded colon tumors were enriched for tumor cells using flow sorting, the extracted DNA was used in a custom SNP array to determine chr17p13-11 allelic state; p53 immunostaining, TP53 exons 5, 6, 7 and 8 mutations were determined in combination with mRNA expression analysis on frozen tissue.ResultsPatients with a predicted functional p53 had a better prognosis than patients with non functional p53 (Log Rank p=0.009). Expression of CSNK1A1 modified p53 survival effects. Patients with low CSNK1A1 expression and non-functional p53 had a very poor survival both in the univariate (Log Rank p<0.001) and in the multivariate survival analysis (HR=4.74 95% CI 1.45 – 15.3 p=0.009).ConclusionThe combination of mutational, genomic, protein and downstream transcriptional activity data predicted p53 functionality which is shown to have a prognostic effect on colon cancer patients. This effect was specifically modified by CSKN1A1 expression.

Genome haploidisation with chromosome 7 retention in oncocytic follicular thyroid carcinoma.

Background Recurrent non-medullary thyroid carcinoma (NMTC) is a rare disease. We initially characterized 27 recurrent NMTC: 13 papillary thyroid cancers (PTC), 10 oncocytic follicular carcinomas (FTC-OV), and 4 non-oncocytic follicular carcinomas (FTC). A validation cohort composed of benign and malignant (both recurrent and non-recurrent) thyroid tumours was subsequently analysed (n = 20). Methods Data from genome-wide SNP arrays and flow cytometry were combined to determine the chromosomal dosage (allelic state) in these tumours, including mutation analysis of components of PIK3CA/AKT and MAPK pathways. Results All FTC-OVs showed a very distinct pattern of genomic alterations. Ten out of 10 FTC-OV cases showed near-haploidisation with or without subsequent genome endoreduplication. Near-haploidisation was seen in 5/10 as extensive chromosome-wide monosomy (allelic state [A]) with near-haploid DNA indices and retention of especially chromosome 7 (seen as a heterozygous allelic state [AB]). In the remaining 5/10 chromosomal allelic states AA with near diploid DNA indices were seen with allelic state AABB of chromosome 7, suggesting endoreduplication after preceding haploidisation. The latter was supported by the presence of both near-haploid and endoreduplicated tumour fractions in some of the cases. Results were confirmed using FISH analysis. Relatively to FTC-OV limited numbers of genomic alterations were identified in other types of recurrent NMTC studied, except for chromosome 22q which showed alterations in 6 of 13 PTCs. Only two HRAS, but no mutations of EGFR or BRAF were found in FTC-OV. The validation cohort showed two additional tumours with the distinct pattern of genomic alterations (both with oncocytic features and recurrent). Conclusions We demonstrate that recurrent FTC-OV is frequently characterised by genome-wide DNA haploidisation, heterozygous retention of chromosome 7, and endoreduplication of a near-haploid genome. Whether normal gene dosage on especially chromosome 7 (containing EGFR, BRAF, cMET) is crucial for FTC-OV tumour survival is an important topic for future research. Microarrays Data are made available at GEO (GSE31828).

Colorectal cancer risk variants on 11q23 and 15q13 are associated with unexplained adenomatous polyposis

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.

The ATXN1 and TRIM31 Genes Are Related to Intelligence in an ADHD Background: Evidence From a Large Collaborative Study Totaling 4,963 Subjects

Intelligence is a highly heritable trait for which it has proven difficult to identify the actual genes. In the past decade, five whole‐genome linkage scans have suggested genomic regions important to human intelligence; however, so far none of the responsible genes or variants in those regions have been identified. Apart from these regions, a handful of candidate genes have been identified, although most of these are in need of replication. The recent growth in publicly available data sets that contain both whole genome association data and a wealth of phenotypic data, serves as an excellent resource for fine mapping and candidate gene replication. We used the publicly available data of 947 families participating in the International Multi‐Centre ADHD Genetics (IMAGE) study to conduct an in silico fine mapping study of previously associated genomic locations, and to attempt replication of previously reported candidate genes for intelligence. Although this sample was ascertained for attention deficit/hyperactivity disorder (ADHD), intelligence quotient (IQ) scores were distributed normally. We tested 667 single nucleotide polymorphisms (SNPs) within 15 previously reported candidate genes for intelligence and 29451 SNPs in five genomic loci previously identified through whole genome linkage and association analyses. Significant SNPs were tested in four independent samples (4,357 subjects), one ascertained for ADHD, and three population‐based samples. Associations between intelligence and SNPs in the ATXN1 and TRIM31 genes and in three genomic locations showed replicated association, but only in the samples ascertained for ADHD, suggesting that these genetic variants become particularly relevant to IQ on the background of a psychiatric disorder.