A comparison of DNA copy number profiling platforms

The accurate mapping of recurring DNA copy number aberrations (CNAs), a hallmark feature of the cancer genome, has facilitated the discovery of tumor suppressor genes and oncogenes. Microarray-based assays designed to detect these chromosomal copy number alterations on a genome-wide and high-resolution scale have emerged as a cornerstone technology in the genomic era. The diversity of commercially available platforms prompted a systematic comparison of five copy number profiling assays for their ability to detect 2-fold copy number gain and loss (4n or 1n, respectively) as well as focal high-amplitude CNAs. Here, using a collection of established human melanoma cell lines, we defined the reproducibility, absolute signals, signal to noise, and false-positive and false-negative rates for each of the five assays against ground truth defined by spectral karyotyping, in addition to comparing the concordance of CNA detection by two high-resolution Agilent and Affymetrix microarray platforms. Our analyses concluded that the Agilent's 60-mer oligonucleotide microarray with probe design optimized for genomic hybridization offers the highest sensitivity and specificity (area under receiver operator characteristic curve >0.99), whereas Affymetrix's single nucleotide polymorphism microarray seems to offer better detection of CNAs in gene-poor regions. Availability of these comparison results should guide study design decisions and facilitate further computational development.     

High-resolution DNA copy number profiling of malignant peripheral nerve sheath tumors using targeted microarray-based comparative genomic hybridization

PURPOSE: Neurofibromatosis type 1 (NF1) is an autosomal dominant condition that predisposes to benign and malignant tumors. The lifetime risk of a malignant peripheral nerve sheath tumor (MPNST) in NF1 is approximately 10%. These tumors have a poor survival rate and their molecular basis remains unclear. We report the first comprehensive investigation of DNA copy number across multitude of genes in NF1 tumors using high-resolution array comparative genomic hybridization (CGH), with the aim to identify molecular signatures that delineate malignant from benign NF1 tumors. EXPERIMENTAL DESIGN: We constructed an exon-level resolution microarray encompassing 57 selected genes and profiled DNA from 35 MPNSTs, 16 plexiform, and 8 dermal neurofibromas. Bioinformatic analysis was done on array CGH data to identify concurrent aberrations in malignant tumors. RESULTS: The array CGH profiles of MPNSTs and neurofibromas were markedly different. A number of MPNST-specific alterations were identified, including amplifications of ITGB4, PDGFRA, MET, TP73, and HGF plus deletions in NF1, HMMR/RHAMM, MMP13, L1CAM2, p16INK4A/CDKN2A, and TP53. Copy number changes of HMMR/RHAMM, MMP13, p16INK4A/CDKN2A, and ITGB4 were observed in 46%, 43%, 39%, and 32%, respectively of the malignant tumors, implicating these genes in MPNST pathogenesis. Concomitant amplifications of HGF, MET, and PDGFRA genes were also revealed in MPNSTs, suggesting the putative role of p70S6K pathway in NF1 tumor progression. CONCLUSIONS: This study highlights the potential of array CGH in identifying novel diagnostic markers for MPNSTs.     

DNA拷贝数变化与肿瘤

 DNA拷贝数变化（CNV）是一种介于1 kb至3 Mb的DNA片段的变异, 在人类基因组中广泛分布,极大地丰富了基因组遗传变异的多样性。研究发现,大部分CNV都与复杂疾病密切相关。CNV如果发生在肿瘤相关基因序列内部或周围可能引起癌基因激活、抑癌基因失活,最终导致肿瘤的发生。CNV通过改变基因剂量、调节基因活性影响基因表达、表型差异和表型适应,从而引起肿瘤以及其他遗传疾病。研究CNV对于探索染色体重组、基因组进化、基因表达以及多种复杂疾病尤其是肿瘤的发病机制有很大帮助。     

DNA copy number alterations in radiation-induced thyroid cancer

For many years, gene alterations of the mitogen activated protein kinase pathway have been investigated in papillary thyroid carcinomas (PTC) and the radiation-specific induction of Ret/PTC rearrangements has been discussed in the published literature. According to recent studies, these alterations are now considered as age-related changes rather than radiation-specific changes in PTC. Thus, there is a strong motivation to search for novel alterations that might represent radiation-specific markers in PTC. DNA copy number alterations (CNAs) are frequent in human cancers and are also prevalent in PTC. However, the only way to tease out radiation-specific CNAs is a comparative analysis of CNAs from closely matched tumour cohorts, as various factors such as the age of patients or the histology of tumours also influence the type and frequency of CNA. Therefore, this review focuses on the current knowledge on CNAs in PTC and on future strategies to identify radiation-specific changes in these tumours.     

肝细胞癌组织相关拷贝数扩增区域的鉴定

目的 确定与肝细胞癌(HCC)发生相关的基因组拷贝数变异区域.方法 采用GeneChip Human Mapping 500K单核苷酸多态性(SNP)芯片分析肝癌细胞系TJ3ZX-01中全基因组拷贝数的变异情况.根据SNP芯片分析的结果 ,利用荧光定量聚合酶链反应(FQ-PCR),将筛选得到的4个候选扩增区域在41例HCC组织中进行验证.结果 SNP芯片分析结果 显示,在1q21.2、1q22～23.1、7p22.1和22q13.1上存在着4个拷贝数扩增的候选区域.对序列标签位点(STS)的FQ-PCR检测结果 显示,位于1q21.2的STS RH69823位点在56.1%(23/41)的HCC组织中扩增,位于1q22～23.1的STS RH38210位点在80.5%(33/41)的HCC组织中扩增,位于7p22.1的STS RH75535位点在75.6%(31/41)的HCC组织中扩增,而位于22q13.1的STS RH57885位点仅在31.7%(13/41)的HCC组织中扩增.结论 经SNP芯片分析筛选与FQ-PCR验证的4个扩增区域中,有3个区域在超过50.0%的HCC组织中显示扩增,该区域的扩增可能与HCC的发生存在着某种联系.     

DNA copy number changes in cervical adenocarcinoma.

PURPOSE: There is evidence that specific genetic events are involved in the initiation and progression of squamous cell carcinoma of the uterine cervix. The genotype-phenotype correlations in cervical adenocarcinoma (AC) are unclear. Experimental Design: Comparative genomic hybridization was applied to screen for DNA copy number gains and losses in 22 cervical ACs of clinical stage IB. IHC was performed in all of the samples to determine HER-2/neu expression (HercepTest). RESULTS: The most frequent copy number alterations were DNA sequence gains of chromosome 17q (54%). HER-2/neu expression (score 2+) was immunohistochemically detected in 2 of 22 tumors. DNA sequence losses were most prevalent on chromosomes Xq (50%), Xp (36%), 18q (36%), and 4q (36%). DNA sequence losses of chromosome 18q were associated significantly with poor prognosis in cervical AC (P < 0.01). CONCLUSIONS: DNA sequence copy number gains of chromosome 17q are frequent events in ACs of the cervix. However, gains on 17q are not associated with HER-2/neu expression in cervical ACs. The inactivation of tumor suppressor genes on chromosome 18q might be responsible for the progression of both cervical AC and cervical squamous cell carcinoma.     

Alteration of the copy number and deletion of mitochondrial DNA in human hepatocellular carcinoma

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in hepatocellular carcinoma (HCC). However, it remains unclear whether mtDNA copy number and mitochondrial biogenesis are altered in HCC. In this study, we found that mtDNA copy number and the content of mitochondrial respiratory proteins were reduced in HCCs as compared with the corresponding non-tumorous livers. MtDNA copy number was significantly reduced in female HCC but not in male HCC. Expression of the peroxisome proliferator-activated receptor gamma coactivator-1 was significantly repressed in HCCs (P<0.005), while the expression of the mitochondrial single-strand DNA-binding protein was upregulated, indicating that the regulation of mitochondria biogenesis is disturbed in HCC. Moreover, 22% of HCCs carried a somatic mutation in the mtDNA D-loop region. The non-tumorous liver of the HCC patients with a long-term alcohol-drinking history contained reduced mtDNA copy number (P<0.05) and higher level of the 4977 bp-deleted mtDNA (P<0.05) as compared with non-alcohol patients. Our results suggest that reduced mtDNA copy number, impaired mitochondrial biogenesis and somatic mutations in mtDNA are important events during carcinogenesis of HCC, and the differential alterations in mtDNA of male and female HCC may contribute to the differences in the clinical manifestation between female and male HCC patients.     

Subtypes of familial breast tumours revealed by expression and copy number profiling

Extensive expression profiling studies have shown that sporadic breast cancer is composed of five clinically relevant molecular subtypes. However, although BRCA1-related tumours are known to be predominantly basal-like, there are few published data on other classes of familial breast tumours. We analysed a cohort of 75 BRCA1, BRCA2 and non-BRCA1/2 breast tumours by gene expression profiling and found that 74% BRCA1 tumours were basal-like, 73% of BRCA2 tumours were luminal A or B, and 52% non-BRCA1/2 tumours were luminal A. Thirty-four tumours were also analysed by single nucleotide polymorphism–comparative genomic hybridization (SNP-CGH) arrays. Copy number data could predict whether a tumour was basal-like or luminal with high accuracy, but could not predict its mutation class. Basal-like BRCA1 and basal-like non-BRCA1 tumours were very similar, and contained the highest number of chromosome aberrations. We identified regions of frequent gain containing potential driver genes in the basal (8q and 12p) and luminal A tumours (1q and 17q). Regions of homozygous loss associated with decreased expression of potential tumour suppressor genes were also detected, including in basal tumours (5q and 9p), and basal and luminal tumours (10q). This study highlights the heterogeneity of familial tumours and the clinical consequences for treatment and prognosis.     

Clinical implementation of integrated whole-genome copy number and mutation profiling for glioblastoma

Background

Multidimensional genotyping of formalin-fixed paraffin-embedded (FFPE) samples has the potential to improve diagnostics and clinical trials for brain tumors, but prospective use in the clinical setting is not yet routine. We report our experience with implementing a multiplexed copy number and mutation-testing program in a diagnostic laboratory certified by the Clinical Laboratory Improvement Amendments.

Methods

We collected and analyzed clinical testing results from whole-genome array comparative genomic hybridization (OncoCopy) of 420 brain tumors, including 148 glioblastomas. Mass spectrometry–based mutation genotyping (OncoMap, 471 mutations) was performed on 86 glioblastomas.

Results

OncoCopy was successful in 99% of samples for which sufficient DNA was obtained (n = 415). All clinically relevant loci for glioblastomas were detected, including amplifications (EGFR, PDGFRA, MET) and deletions (EGFRvIII, PTEN, 1p/19q). Glioblastoma patients ≤40 years old had distinct profiles compared with patients >40 years. OncoMap testing reliably identified mutations in IDH1, TP53, and PTEN. Seventy-seven glioblastoma patients enrolled on trials, of whom 51% participated in targeted therapeutic trials where multiplex data informed eligibility or outcomes. Data integration identified patients with complete tumor suppressor inactivation, albeit rarely (5% of patients) due to lack of whole-gene coverage in OncoMap.

Conclusions

Combined use of multiplexed copy number and mutation detection from FFPE samples in the clinical setting can efficiently replace singleton tests for clinical diagnosis and prognosis in most settings. Our results support incorporation of these assays into clinical trials as integral biomarkers and their potential to impact interpretation of results. Limited tumor suppressor variant capture by targeted genotyping highlights the need for whole-gene sequencing in glioblastoma.     

Studies of genomic copy number changes in human cancers reveal signatures of DNA replication stress

Human cancers are characterized by the presence of genomic instability. Recently, two studies have catalogued the presence of a specific class of genomic aberrations, large deletions and insertions, in a few thousand human cancers and reported that most of the prevalent recurrent focal deletions targeted common fragile sites and large genes. In various experimental systems, deletions in common fragile sites and large genes have been linked to the presence of DNA replication stress. Thus, taken together, these results suggest the presence of DNA replication stress in human cancers, consistent with the recently proposed oncogene-induced DNA damage model for cancer development.     

Comprehensive DNA copy number profiling of meningioma using a chromosome 1 tiling path microarray identifies novel candidate tumor suppressor loci

Meningiomas are common neoplasms of the meninges lining of the central nervous system. Deletions of 1p have been established as important for the initiation and/or progression of meningioma. The rationale of this array-CGH study was to characterize copy number imbalances of chromosome 1 in meningioma, using a full-coverage genomic microarray containing 2,118 distinct measurement points. In total, 82 meningiomas were analyzed, making this the most detailed analysis of chromosome 1 in a comprehensive series of tumors. We detected a broad range of aberrations, such as deletions and/or gains of various sizes. Deletions were the predominant finding and ranged from monosomy to a 3.5-Mb terminal 1p homozygous deletion. Although multiple aberrations were observed across chromosome 1, every meningioma in which imbalances were detected harbored 1p deletions. Tumor heterogeneity was also observed in three recurrent meningiomas, which most likely reflects a progressive loss of chromosomal segments at different stages of tumor development. The distribution of aberrations supports the existence of at least four candidate loci on chromosome 1, which are important for meningioma tumorigenesis. In one of these regions, our results already allow the analysis of a number of candidate genes. In a large series of cases, we observed an association between the presence of segmental duplications and deletion breakpoints, which suggests their role in the generation of these tumor-specific aberrations. As 1p is the site of the genome most frequently affected by tumor-specific aberrations, our results indicate loci of general importance for cancer development and progression.     

Genomic copy number changes of DNA repair genes ERCC1 and ERCC2 in human gliomas

Abnormalities of the genomic region of chromosome 19q13.2–13.4 are a common occurrence in brain malignancies and contain a possible tumor suppressor gene involved in gliomas. Since abnormalities of DNA repair are associated with malignancy, we assessed DNA status of the nucleotide excision repair genes located in this area, viz. ERCC1 and ERCC2.Radiodensitometry was used to assess gene copy number in samples obtained from brain tumor specimens from 24 patients. Nine tumors were of lower grade histology (3 pilocytic astrocytomas, 2 gangliogliomas, 4 astrocytomas); 15 tumors were pathologiclly higher grade (4 anaplastic astrocytomas, 11 glioblastomas). Tumor samples were obtained prior to radiation or chemotherapy. Abnormalities of gene copy number of ERCC1 and ERCC2 were observed in 11/24 specimens (46%). Whereas increased and decreased copy numbers were observed for ERCC1, only decreases in copy number of ERCC2 were seen. Three tumors (all lower grade) showed concurrent allelic loss of ERCC1 and ERCC2. Abnormalities of copy number for these genes were not associated with response to subsequent therapy nor survival. However, allelic loss of ERCC2 was associated with younger age at diagnosis when compared to those specimens which did not show loss. There were no significant differences between lower grade and higher grade tumors with respect to these investigations.Abnormalities in copy number of ERCC1 and ERCC2 are common in glial tumors. Further study of this genomic region is necessary to define the importance of these observations in tumor pathophysiology and treatment.     

DNA copy number changes in malignant ovarian germ cell tumors

Malignant ovarian germ cell tumors (OGCTs) include immature teratomas (ITs), dysgerminomas (DGs), endodermal sinus tumors (ESTs), choriocarcinomas, and embryonal carcinomas. Knowledge about the genetic changes associated with malignant OGCT development is sparse. We therefore analyzed 25 OGCTs (12 DGs, 4 ESTs, and 9 ITs) for gains and losses by comparative genomic hybridization. In total, more gains than losses were observed, and the number of alterations ranged from 0-20 per tumor. The average number of changes among DGs, ESTs, and ITs was 10, 6, and 1.4, respectively. The most common changes in DGs were gains from chromosome arms 1p (33%), 6p (33%), 12p (67%), 12q (75%), 15q (42%), 20q (50%), 21q (67%), and 22q (58%); gains of the whole of chromosomes 7 (42%), 8 (42%), 17 (42%), and 19 (50%); and losses from 13q (58%). Two of three DGs with a gonadoblastoma component showed gains of 3p21 and loss of 5p, whereas none of the nine pure DGs had these changes, suggesting that they might be characteristic either of gonadoblastoma or of DG developing from a gonadoblastoma. Gain of 12p and gain from 1q were seen in three of four ESTs, whereas gains from 3p, 11q, and Xp and loss from 18q were each found in two tumors. Five of the ITs revealed changes (range, 1-4 changes/tumor), with gains from 1p, 16p, 19, and 22q each being found in two tumors. We conclude that ovarian DGs and ESTs seem to develop via the same genetic pathways that are already known for testicular germ cell tumors. On the other hand, ITs do not exhibit gain of 12p and also typically show fewer changes than other malignant OGCTs, indicating that they arise via different pathogenetic mechanisms.