Hendrik F. van Essen

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.

CGH Arrays Compared for DNA Isolated from Formalin-Fixed, Paraffin-Embedded Material

Formalin‐fixed, paraffin‐embedded (FFPE) archival tissue is an important source of DNA material. The most commonly used technique to identify copy number aberrations from chromosomal DNA in tumorigenesis is array comparative genomic hybridization (aCGH). Although copy number analysis using DNA from FFPE archival tissue is challenging, several research groups have reported high quality and reproducible DNA copy number results using aCGH. Aim of this study is to compare the commercially available aCGH platforms suitable for high‐resolution copy number analysis using FFPE‐derived DNA. Two dual channel aCGH platforms (Agilent and NimbleGen) and a single channel SNP‐based platform (Affymetrix) were evaluated using seven FFPE colon cancer samples, and median absolute deviation (MAD), deflection, signal‐to‐noise ratio, and DNA input requirements were used as quality criteria. Large differences were observed between platforms; Agilent and NimbleGen showed better MAD values (0.13 for both) compared with Affymetrix (0.22). On the contrary, Affymetrix showed a better deflection of 0.94, followed by 0.71 for Agilent and 0.51 for NimbleGen. This resulted in signal‐to‐nose ratios that were comparable between the three commercially available platforms. Interestingly, DNA input amounts from FFPE material lower than recommended still yielded high quality profiles on all platforms. Copy number analysis using DNA derived from FFPE archival material is feasible using all three high‐resolution copy number platforms and shows reproducible results, also with DNA input amounts lower than recommended.

Genomic landscape of metastatic colorectal cancer

Response to drug therapy in individual colorectal cancer (CRC) patients is associated with tumour biology. Here we describe the genomic landscape of tumour samples of a homogeneous well-annotated series of patients with metastatic CRC (mCRC) of two phase III clinical trials, CAIRO and CAIRO2. DNA copy number aberrations of 349 patients are determined. Within three treatment arms, 194 chromosomal subregions are associated with progression-free survival (PFS; uncorrected single-test P-values <0.005). These subregions are filtered for effect on messenger RNA expression, using an independent data set from The Cancer Genome Atlas which returned 171 genes. Three chromosomal regions are associated with a significant difference in PFS between treatment arms with or without irinotecan. One of these regions, 6q16.1–q21, correlates in vitro with sensitivity to SN-38, the active metabolite of irinotecan. This genomic landscape of mCRC reveals a number of DNA copy number aberrations associated with response to drug therapy.

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.

Breast Cancers with a BRCA1-like DNA Copy Number Profile Recur Less Often Than Expected after High-Dose Alkylating Chemotherapy

Purpose: Breast cancers in carriers of inactivating mutations of the BRCA1 gene carry a specific DNA copy-number signature (“BRCA1-like”). This signature is shared with cancers that inactivate BRCA1 through other mechanisms. Because BRCA1 is important in repair of DNA double-strand breaks through error-free homologous recombination, patients with a BRCA1-like tumor may benefit from high-dose alkylating (HD) chemotherapy, which induces DNA double-strand breaks. Experimental Design: We investigated a single institution cohort of high-risk patients that received tandem HD chemotherapy schedule comprising ifosfamide, epirubicin, and carboplatin or conventional chemotherapy. We classified copy-number profiles to be BRCA1-like or non–BRCA1-like and analyzed clinical associations and performed survival analysis with a treatment by biomarker interaction design. Results: BRCA1-like status associated with high-grade and triple-negative breast cancers. BRCA1-like cases benefitted from the HD compared with a conventional regimen on disease-free survival (DFS): [hazard ratio (HR), 0.05; 95% confidence interval (CI), 0.01–0.38; P = 0.003]; distant DFS (DDFS): (HR, 0.06; 95% CI, 0.01–0.43; P = 0.01); and overall survival (OS; HR, 0.15; 95% CI, 0.03–0.83; P = 0.03) after correction for prognostic factors. No such benefit was observed in the non–BRCA1-like cases on DFS (HR, 0.74; 95% CI, 0.38–1.46; P = 0.39), DDFS (HR, 0.79; 95% CI, 0.41–1.52; P = 0.47), and OS (HR, 0.93; 95% CI, 0.52–1.64; P = 0.79). The P values for interaction were 0.01 (DFS), 0.01 (DDFS), and 0.045 (OS). Conclusions: BRCA1-like tumors recurred significantly less often after HD than conventional chemotherapy. BRCA1-like copy-number profile classification may be a predictive marker for HD alkylating chemotherapy. Clin Cancer Res; 21(4); 763–70. ©2014 AACR.

Chromosomal copy number aberrations in colorectal metastases resemble their primary counterparts and differences are typically non-recurrent

The metastatic process is complex and remains a major obstacle in the management of colorectal cancer. To gain a better insight into the pathology of metastasis, we investigated genomic aberrations in a large cohort of matched colorectal cancer primaries and distant metastases from various sites by high resolution array comparative genomic hybridization. In total, 62 primary colorectal cancers, and 68 matched metastases (22 liver, 11 lung, 12 ovary, 12 omentum, and 11 distant lymph nodes) were analyzed. Public datasets were used for validation purposes. Metastases resemble their matched primary tumors in the majority of the patients. This validates the significant overlap in chromosomal aberrations between primary tumors and corresponding metastases observed previously. We observed 15 statistically significant different regions between the primary tumors and their matched metastases, of which only one recurrent event in metastases was observed. We conclude, based on detailed analysis and large independent datasets, that chromosomal copy number aberrations in colorectal metastases resemble their primary counterparts, and differences are typically non-recurrent.

Reduced genomic tumor heterogeneity after neoadjuvant chemotherapy is related to favorable outcome in patients with esophageal adenocarcinoma

Neoadjuvant chemo(radio)therapy followed by surgery is the standard of care for patients with locally advanced resectable esophageal adenocarcinoma (EAC). There is increasing evidence that drug resistance might be related to genomic heterogeneity. We investigated whether genomic tumor heterogeneity is different after cytotoxic chemotherapy and is associated with EAC patient survival. We used arrayCGH and a quantitative assessment of the whole genome DNA copy number aberration patterns (‘DNA copy number entropy’) to establish the level of genomic tumor heterogeneity in 80 EAC treated with neoadjuvant chemotherapy followed by surgery (CS group) or surgery alone (S group). The association between DNA copy number entropy, clinicopathological variables and survival was investigated. DNA copy number entropy was reduced after chemotherapy, even if there was no morphological evidence of response to therapy (p<0.001). Low DNA copy number entropy was associated with improved survival in the CS group (p=0.011) but not in the S group (p=0.396). Our results suggest that cytotoxic chemotherapy reduces DNA copy number entropy, which might be a more sensitive tumor response marker than changes in the morphological tumor phenotype. The use of DNA copy number entropy in clinical practice will require validation of our results in a prospective study.

Abstract 3426: A novel approach to copy number assessment by whole genome sequencing reveals extensive spatial heterogeneity in diffuse low-grade glioma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: The disease course of patients with diffuse low-grade gliomas (LGGs) is notoriously unpredictable. Detailed analysis of the genetic make-up is therefore key to understand this wide variety in overall survival of patients diagnosed with LGGs. Reports on the prognostic value of CNAs other than 1p/19q co-deletion are conflicting, which may be explained by spatial copy number heterogeneity. Aim: Prognostic implications of spatial and temporal copy number aberrations in LGGs by whole genome sequencing. Methods: Approximately 25% of LGG patients have a life expectation of more than 20 years following diagnosis, which necessitates collection of samples with long clinical follow up information. Therefore archival material was selected for this project and WGS was developed to access these samples without the requirement for a normal reference. We collected 156 formalin-fixed and paraffin-embedded (FFPE) samples in a discovery cohort of 98 LGG patients with extensive clinical follow-up data, which included recurrent tumours and spatially distinct regions. An independent cohort of 126 samples was obtained from a recently published study (Alentorn et al, Neuro-oncology 2014). Technical challenges for genome-wide inference of copy number aberrations include repetitive and common DNA sequences in the genome and sequence variation across the general population, as well as the compromised and variable quality of DNA obtained from archived tissues. We developed a robust and cost effective method that infers copy number aberrations from WGS data of approximately 0.1x coverage, without the need for a reference signal. This method implements (1) a combined LOESS correction for mappability and GC content, and improves on previous methods by (2) comprehensive filtering based on public genome project data, 1000-Genomes project and ENCODE blacklists. Results: Both prognostic value, temporal evolution and spatial heterogeneity of CNA were assessed by WGS. We confirmed prognostic favourable value of 1p/19q co-deletion, and demonstrated loss of 10q to be an unfavourable marker. In paired recurrences 10q loss was invariably maintained and surfaced in 4 additional recurrences of the discovery cohort. In spatial regions of LGGs we recognized extensive copy number heterogeneity; 15 of 17 LGGs show spatial variability of CNAs. 1p/19q co-deletion is homogeneous, while loss of 10q is heterogeneously present. Conclusions: we present clinically relevant CNAs, but also demonstrate extensive spatial copy number heterogeneity in diffuse LGGs that might complicate unequivocal biomarker discovery. Citation Format: Ilari Scheinin, Hinke F. van Thuijl, Daoud Sie, Hendrik F. van Essen, Paul P. Eijk, Francois Rustenburg, Ahmed Idbaih, Agusti Alentorn, Gerrit A. Meijer, Mark A. van der Wiel, Henrik Bengtsson, Adam Olshen, Eleonora Aronica, Jan J. Heimans, Jaap C. Reijneveld, Pieter Wesseling, Donna G. Albertson, Dan Pinkel, Bauke Ylstra. A novel approach to copy number assessment by whole genome sequencing reveals extensive spatial heterogeneity in diffuse low-grade glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3426. doi:10.1158/1538-7445.AM2014-3426