Daoud Sie

DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly

Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraffin-embedded (FFPE) archival material, the analysis of which is important for studies of cancer, presents particular analytical difficulties due to degradation of the DNA and frequent lack of matched reference samples. We present a robust, cost-effective WGS method for DNA copy number analysis that addresses these challenges more successfully than currently available procedures. In practice, very useful profiles can be obtained with ∼0.1× genome coverage. We improve on previous methods by first implementing a combined correction for sequence mappability and GC content, and second, by applying this procedure to sequence data from the 1000 Genomes Project in order to develop a blacklist of problematic genome regions. A small subset of these blacklisted regions was previously identified by ENCODE, but the vast majority are novel unappreciated problematic regions. Our procedures are implemented in a pipeline called QDNAseq. We have analyzed over 1000 samples, most of which were obtained from the fixed tissue archives of more than 25 institutions. We demonstrate that for most samples our sequencing and analysis procedures yield genome profiles with noise levels near the statistical limit imposed by read counting. The described procedures also provide better correction of artifacts introduced by low DNA quality than prior approaches and better copy number data than high-resolution microarrays at a substantially lower cost.

Somatic mutations found in the healthy blood compartment of a 115-yr-old woman demonstrate oligoclonal hematopoiesis

The somatic mutation burden in healthy white blood cells (WBCs) is not well known. Based on deep whole-genome sequencing, we estimate that approximately 450 somatic mutations accumulated in the nonrepetitive genome within the healthy blood compartment of a 115-yr-old woman. The detected mutations appear to have been harmless passenger mutations: They were enriched in noncoding, AT-rich regions that are not evolutionarily conserved, and they were depleted for genomic elements where mutations might have favorable or adverse effects on cellular fitness, such as regions with actively transcribed genes. The distribution of variant allele frequencies of these mutations suggests that the majority of the peripheral white blood cells were offspring of two related hematopoietic stem cell (HSC) clones. Moreover, telomere lengths of the WBCs were significantly shorter than telomere lengths from other tissues. Together, this suggests that the finite lifespan of HSCs, rather than somatic mutation effects, may lead to hematopoietic clonal evolution at extreme ages.

Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology

PurposeNext generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling.MethodsTSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (nu2009=u200958/nu2009=u200945 FFPE-tumor DNA samples) against ‘gold standard’ high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (nu2009=u200975).ResultsIn the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82xa0% (37/45) compliance to the quality criteria and 95xa0% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappau2009=u20090.92; 95xa0% CIu2009=u20090.81–1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84xa0% (63/75), and a high concordance with HRM-sequencing (95xa0% (60/63); kappau2009=u20090.92; 95xa0% CIu2009=u20090.84–1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes.ConclusionTSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.

Diagnosis of Fanconi Anemia: Mutation Analysis by Next-Generation Sequencing

Fanconi anemia (FA) is a rare genetic instability syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. Fifteen genetic subtypes have been distinguished. The majority of patients (≈85%) belong to the subtypes A (≈60%), C (≈15%) or G (≈10%), while a minority (≈15%) is distributed over the remaining 12 subtypes. All subtypes seem to fit within the “classical” FA phenotype, except for D1 and N patients, who have more severe clinical symptoms. Since FA patients need special clinical management, the diagnosis should be firmly established, to exclude conditions with overlapping phenotypes. A valid FA diagnosis requires the detection of pathogenic mutations in a FA gene and/or a positive result from a chromosomal breakage test. Identification of the pathogenic mutations is also important for adequate genetic counselling and to facilitate prenatal or preimplantation genetic diagnosis. Here we describe and validate a comprehensive protocol for the molecular diagnosis of FA, based on massively parallel sequencing. We used this approach to identify BRCA2, FANCD2, FANCI and FANCL mutations in novel unclassified FA patients.

No evidence for active human papillomavirus (HPV) in fields surrounding HPV-positive oropharyngeal tumors

BACKGROUNDnPatients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis than patients with HPV-negative OPSCCs. Important factors contributing to this better prognosis are relatively low numbers of local/regional recurrences (LRRs) and second primary tumors (SPTs) in patients with HPV-positive OPSCC. These low numbers may be explained in addition by the absence of a field cancerization effect, which is a cause of LRRs and SPTs in patients with HPV-negative OPSCC. We aimed to detect a possible field effect in patients with HPV-positive OPSCC. As HPV is involved in the early stage of carcinogenesis in OPSCCs, its presence is considered a reliable marker for the detection of such a field effect. Therefore, the presence of transcriptionally active HPV was analyzed in the mucosa surrounding HPV-positive OPSCCs.nnnMETHODSnWe included 20 patients who were surgically treated for an HPV-positive OPSCC in the period 2000-2006. Of each patient, the formalin-fixed paraffin-embedded tumor sample and all available resection margins were collected. In total, 97 resection margins were investigated with an average of five resection margins per tumor. All samples were analyzed for the presence of tumor and the presence of transcriptionally active HPV by HPV16-E6-mRNA detection.nnnRESULTSnAll tumors were HPV16-E6-mRNA positive. HPV16-E6-mRNA could be detected in the resection margins that contained tumor (n = 6). All tumor-negative resection margins (n = 91) scored negative for HPV16-E6-mRNA.nnnCONCLUSIONSnIn conclusion, transcriptional active HPV could not be detected in the mucosa surrounding an HPV-positive OPSCC, which suggests the absence of field effect. This observation may explain the lower number of LRRs and SPTs in HPV-positive patients.

Spatial and temporal evolution of distal 10q deletion, a prognostically unfavorable event in diffuse low-grade gliomas

BackgroundThe disease course of patients with diffuse low-grade glioma is notoriously unpredictable. Temporal and spatially distinct samples may provide insight into the evolution of clinically relevant copy number aberrations (CNAs). The purpose of this study is to identify CNAs that are indicative of aggressive tumor behavior and can thereby complement the prognostically favorable 1p/19q co-deletion.ResultsGenome-wide, 50 base pair single-end sequencing was performed to detect CNAs in a clinically well-characterized cohort of 98 formalin-fixed paraffin-embedded low-grade gliomas. CNAs are correlated with overall survival as an endpoint. Seventy-five additional samples from spatially distinct regions and paired recurrent tumors of the discovery cohort were analyzed to interrogate the intratumoral heterogeneity and spatial evolution. Loss of 10q25.2-qter is a frequent subclonal event and significantly correlates with an unfavorable prognosis. A significant correlation is furthermore observed in a validation set of 126 and confirmation set of 184 patients. Loss of 10q25.2-qter arises in a longitudinal manner in paired recurrent tumor specimens, whereas the prognostically favorable 1p/19q co-deletion is the only CNA that is stable across spatial regions and recurrent tumors.ConclusionsCNAs in low-grade gliomas display extensive intratumoral heterogeneity. Distal loss of 10q is a late onset event and a marker for reduced overall survival in low-grade glioma patients. Intratumoral heterogeneity and higher frequencies of distal 10q loss in recurrences suggest this event is involved in outgrowth to the recurrent tumor.

Landscape of chromosomal copy number aberrations in gangliogliomas and dysembryoplastic neuroepithelial tumours

Gangliogliomas (GGs) and dysembryoplastic neuroepithelial tumours (DNTs) represent the most common histological entities within the spectrum of glioneuronal tumours (GNTs). The wide variability of morphological features complicates histological classification, including discrimination from prognostically distinct diffuse low‐grade astrocytomas (AIIs). This study was performed to increase our understanding of these tumours.

EGFR mutation analysis in sputum of lung cancer patients: A multitechnique study

OBJECTIVESnEpidermal growth factor receptor (EGFR) mutations have been identified in lung adenocarcinomas and are associated with high response chance to EGFR tyrosine kinase inhibitors. EGFR mutations can be detected in tumour tissue, cytology specimens and blood from lung cancer patients. Thus far, EGFR mutation analysis has not been systematically demonstrated for sputum samples. The aim of the present study was to determine whether EGFR mutation analysis is attainable on sputum samples, employing different assays in a multicenter study.nnnMATERIALS AND METHODSnSputum DNA from 10 lung cancer patients with confirmed EGFR mutation in their tumour tissue, 10 lung cancer patients without evidence of an EGFR mutation, and 10 patients with chronic obstructive pulmonary disease (COPD) was used for mutation analysis by Cycleave PCR, COLD-PCR, PangaeaBiotech SL Technology (PST), and High Resolution Melting, respectively. Targeted resequencing (TruSeq Amplicon Cancer Panel) and droplet digital PCR were additionally performed on the 10 samples with EGFR mutation.nnnRESULTSnDependent on the assay, EGFR mutations could be detected in 30-50% of the sputum samples of patients with EGFR mutations. The different techniques revealed consistent results, with slightly higher sensitivity for PST. Neither the lung cancer patients without EGFR mutation nor the COPD controls tested positive for EGFR mutations in their sputum samples, indicating high clinical specificity of all assays.nnnCONCLUSIONnEGFR mutations can be detected in sputum samples from patients with EGFR-mutated non-small cell lung cancer, which may replace biopsy procedure for some patients.

Proper genomic profiling of (BRCA1‐mutated) basal‐like breast carcinomas requires prior removal of tumor infiltrating lymphocytes

BRCA1‐mutated breast carcinomas may have distinct biological features, suggesting the involvement of specific oncogenic pathways in tumor development. The identification of genomic aberrations characteristic for BRCA1‐mutated breast carcinomas could lead to a better understanding of BRCA1‐associated oncogenic events and could prove valuable in clinical testing for BRCA1‐involvement in patients.

ShrinkBayes: a versatile R-package for analysis of count-based sequencing data in complex study designs

BackgroundComplex designs are common in (observational) clinical studies. Sequencing data for such studies are produced more and more often, implying challenges for the analysis, such as excess of zeros, presence of random effects and multi-parameter inference. Moreover, when sample sizes are small, inference is likely to be too liberal when, in a Bayesian setting, applying a non-appropriate prior or to lack power when not carefully borrowing information across features.ResultsWe show on microRNA sequencing data from a clinical cancer study how our software ShrinkBayes tackles the aforementioned challenges. In addition, we illustrate its comparatively good performance on multi-parameter inference for groups using a data-based simulation. Finally, in the small sample size setting, we demonstrate its high power and improved FDR estimation by use of Gaussian mixture priors that include a point mass.ConclusionShrinkBayes is a versatile software package for the analysis of count-based sequencing data, which is particularly useful for studies with small sample sizes or complex designs.