[Show abstract][Hide abstract] ABSTRACT: Background
The 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 behaviour 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 analysed 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.
[Show abstract][Hide abstract] ABSTRACT: 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 ~0.1x genome coverage. We improve on previous methods by; first, implementing a combined correction for sequence mappability and GC content, and second, 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 were 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 1,000 samples, most of which were obtained from the fixed tissue archives of over 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 substantially lower cost.
[Show abstract][Hide abstract] ABSTRACT: Next 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2014; 9(2):e86833. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Array Comparative Genomic Hybridization (aCGH) is a widely used technique to assess chromosomal copy number alterations. Chromosomal content, however, is often not uniform throughout cell populations. Here we evaluated to what extent aCGH can detect DNA copy number alterations in heterogeneous cell populations. A systematic evaluation is currently lacking, despite its importance in diagnostics and research. The detection limits reported are a compound of analytical software and laboratory techniques and do not account for the number of probes in relation to sample homogeneity. METHODS: Detection limits were explored with DNA isolated from a patient with intellectual disability (ID) and from tumor cell line BT474. Both were diluted with increasing amounts of normal DNA to simulate different levels of cellularity. Samples were hybridized on microarrays containing 180,880 oligonucleotides evenly distributed over the genome (spacing ~17 kb). RESULTS: Single copy number alterations, represented by down to 249 probes (4 Mb) and present in 10 % of a cell population, could be detected. Alterations encompassing as few as 14 probes (~238 Kb) could also be detected, but for this a 35 % mosaic level was required. CONCLUSIONS: DNA copy number alterations can be detected in cell populations containing 10 % abnormal cells. Detection of sub-megabase alterations requires a higher percentage of abnormal cells or microarrays with a higher probe density.
[Show abstract][Hide abstract] ABSTRACT: We describe protocols to acquire high-quality DNA from formalin-fixed, paraffin-embedded (FFPE) tissues for the use in array comparative genome hybridization (CGH). Formalin fixation combined with paraffin embedding is routine procedure for solid malignancies in the diagnostic practice of the pathologist. As a consequence, large archives of FFPE tissues are available in pathology institutes across the globe. This archival material is for many research questions an invaluable resource, with long-term clinical follow-up and survival data available. FFPE is, thus, highly attractive for large genomics studies, including experiments requiring samples for test/learning and validation. Most larger array CGH studies have, therefore, made use of FFPE material and show that CNAs have tumor- and tissue-specific traits (Chin et al. Cancer Cell 10: 529-541, 2006; Fridlyand et al. BMC Cancer 6: 96, 2006; Weiss et al. Oncogene 22: 1872-1879, 2003; Jong et al. Oncogene 26: 1499-1506, 2007). The protocols described are tailored to array CGH of FFPE solid malignancies: from sectioning FFPE blocks to specific cynosures for pathological revisions of sections, DNA isolation, quality testing, and amplification. The protocols are technical in character and elaborate up to the labeling of isolated DNA while further processes and interpretation and data analysis are beyond the scope.
[Show abstract][Hide abstract] ABSTRACT: 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.
Genes Chromosomes and Cancer 12/2011; 51(4):344-52. · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lymph node (LN) yield in colon cancer resection specimens is an important indicator of treatment quality and has especially in early-stage patients therapeutic implications. However, underlying disease mechanisms, such as microsatellite instability (MSI), may also influence LN yield, as MSI tumors are known to exhibit more prominent lymphocytic antitumor reactions. The aim of the present study was to investigate the association of LN yield, MSI status, and recurrence rate in colon cancer.
Clinicopathological data and tumor samples were collected from 332 stage II and III colon cancer patients. DNA was isolated and PCR-based MSI analysis performed. LN yield was defined as "high" when 10 or more LNs were retrieved and "low" in case of fewer than 10 LNs.
Tumors with high LN yield were significantly associated with the MSI phenotype (high LN yield: 26.3% MSI tumors vs low LN yield: 15.1% MSI tumors; P=.01), mainly in stage III disease. Stage II patients with high LN yield had a lower recurrence rate compared with those with low LN yield. Patients with MSI tumors tended to develop fewer recurrences compared with those with MSS tumors, mainly in stage II disease.
In the present study, high LN yield was associated with MSI tumors, mainly in stage III patients. Besides adequate surgery and pathology, high LN yield is possibly a feature caused by biologic behavior of MSI tumors.
Annals of Surgical Oncology 10/2011; 19(4):1222-30. · 4.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Loss of the nuclear lamina protein lamin A/C (LMNA) has been observed in several human malignancies. The present study aimed to investigate associations between LMNA expression and clinical outcome in colon cancer patients.
Clinicopathological data and formalin-fixed paraffin embedded tissues were collected from 370 stage II and III colon cancer patients. Tissue microarrays were constructed, stained for lamin A/C and evaluated microscopically. Microsatellite instability status was determined for 318 tumours.
Low levels of LMNA expression were observed in 17.8% of colon tumours, with disease recurrence occurring in 45.5% of stage II and III colon cancer patients with LMNA-low expressing tumours compared to 29.6% of patients with LMNA-high expressing tumours (p=0.01). For stage II patients, disease recurrence was observed for 35.7% of LMNA-low compared to 20.3% of LMNA-high expressing tumours (p=0.03). Microsatellite stable (MSS) tumours exhibited more frequently low LMNA expression than microsatellite instable (MSI) tumours (21% versus 9.8%; p=0.05). Interestingly, disease recurrence among LMNA-low and LMNA-high expressing MSS tumours varied significantly for stage III patients who had not received adjuvant chemotherapy (100% versus 37.8%; p<0.01) while no such difference was observed for patients who received adjuvant chemotherapy (46.7% versus 46.0%; p=0.96).
These data indicate that low expression of LMNA is associated with an increased disease recurrence in stage II and III colon cancer patients, and suggest that these patients in particular may benefit from adjuvant chemotherapy.
European journal of cancer (Oxford, England: 1990) 05/2011; 47(12):1837-45. · 4.12 Impact Factor