DNA Copy Number Changes Research Articles

Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma

The clinical significance of the detection of low copy numbers of cytomegalovirus (CMV) DNA in immune-suppressed patients remains unclear. In this study, we compared the artus CMV Rotor-Gene PCR, utilizing an automated nucleic acid extraction and assay setup (the artus CMV protocol), with the COBAS Amplicor CMV Monitor test (our reference protocol). We then analyzed the results of all CMV PCR tests ordered following the implementation of the artus CMV protocol at our institution and followed 91 adult patients with positive test results. The artus CMV protocol had a linear range extending from 2.0 to 7.0 log(10) copies/ml and had a lower limit of 95% detection of 57 copies/ml. With archived plasma samples, this protocol demonstrated 100% sensitivity and 94% specificity for the detection of CMV DNA. Following implementation of the artus CMV protocol, 320 of 1,403 (22.8%) plasma samples tested positive (compared with 323/3,579 [9.0%] samples in the preceding 6 months), and 227 (16.2%) samples had copy numbers of <400/ml. Ninety-one adult patients had at least one positive test. The data were analyzed using a threshold of 200 copies/ml, and in 22 episodes, the viral load increased from <200 copies/ml to ≥ 200 copies/ml on sequential tests. In 21 of these 22 episodes, either the viral load continued to increase or antiviral treatment was initiated in response to the repeat value. In summary, we evaluate the performance characteristics of a protocol utilizing the artus CMV PCR and identify clinically meaningful changes in CMV DNA copy numbers even when they are initially detected at a low level.

Abstract 3397: Patterns of DNA copy number changes in colorectal cancer metastasis are highly similar to their corresponding primaries with additional focal alterations that differ between individual patients

Abstract Objective: Metastasis is a complex process that remains an obstacle in the management of colorectal cancer (CRC). Over the years many models have been developed to explain the metastatic process in detail, however, the molecular basis is still poorly understood. To gain better insight into the biology of metastases we investigated genomic aberrations in matched triplets of normal, CRC primaries and metastases of various distant sites. Methods: In total, 63 primary colorectal tumours, 63 matched normal specimens, and 69 matched metastases (liver, lung, ovarian, omentum and distant lymph nodes) were analysed by high resolution array comparative genomic hybridization (array CGH). Our findings were validated in a publicly available dataset consisting of 21 matched primary tumours and liver metastases.1 Fluorescence in situ hybridization (FISH) was used to confirm our findings and to evaluate intra-tumour heterogeneity of the observed copy number differences. Results: The overall pattern of copy number aberrations was highly similar between primary tumours and their metastases. However, higher deflections were observed in the metastases compared to the primary tumours, which was confirmed in the validation set. Across all patients, 17 regions showed significantly lower DNA copy number ratio and 2 regions showed significantly higher DNA copy number ratio in the metastases compared to the primary tumour. None of these significant DNA copy number changes occurred in a substantial percentage of our patients. In the training set we did not detect a higher frequency of chromosome 11p15.5 gain in liver metastasis compared to the primary tumour, as previously reported in the validation set. In two different patients, two specific regions showed amplifications in the three metastases (one patient with two metastatic sites) which were absent in the primary tumours. One of these regions harboured the MYC oncogene. FISH analysis confirmed the extensive MYC amplification in the three metastases, but also showed tumour cell populations with smaller MYC amplifications within the primary tumour, which were not detected by array CGH. Conclusions: Highly similar patterns of DNA copy number profiles between metastatic and primary CRC were observed using array CGH. Focal differences that were observed in individual patients are probably explained by heterogeneity within the primary tumour in contrast to the metastatic sub clone. True additional copy number aberrations are rare and rather specific for individual patients, which is consistent with the hypothesis that the metastatic potential is already present in the primary tumour. Referentie: 1. Stange DE, Engel F, Longerich T et al. Expression of an ASCL2 related stem cell signature and IGF2 in colorectal cancer liver metastases with 11p15.5 gain. Gut 2011;59:1236-44. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3397. doi:1538-7445.AM2012-3397

Abstract 5100: Genomic profiling of triple negative breast cancer from South of Brazil

Abstract Triple negative breast cancer (TNBC) are defined by the lack of expression of ER, PR and HER2 receptors and characterized by an aggressive clinical history and poor outcome. Considering that these tumors do not present a good response to the available adjuvant target therapies (hormonal and HER-2 based therapies), there is a critical need to identify new markers to improve treatment options for these patients. The use of genome-wide methodologies is a progress towards this direction, through the identification of biomarkers that can be translated into novel target-based therapies. We report the genomic profiling performed by array-CGH (Agilent 4x44K) in 33 cases of TNBC obtained from Hospital N.S. das Gracas and Hospital das Clinicas (UFPR), Curitiba, South of Brazil. Copy number alterations in potentially therapeutic target genes for TNBC (involved in the PI3K-AKT pathway) were evaluated by TaqMan copy number assay (Applied Biosystems). The data was correlated with pathological parameters from the patients. Copy number changes were observed by array-CGH in 87% (25/30) of the TNBC cases analyzed, with a median of 9.97+12.37 alterations per case. Based on this value, the samples were divided into two categories (&amp;gt;10 and &amp;lt;10 alterations); no significantly difference was observed between these categories and tumor size (t=1.74;P&amp;gt;0.05) or the presence of lymph node (LN) metastasis (≥2=1.8;P&amp;gt;0.10) and tumor grade (≥2=1.18;P&amp;gt;0.20). Although a high degree of molecular heterogeneity was observed, amplification on 8q was observed in 50% (15/30) of the cases, mostly involving 8q24.3. Tumor size was not significantly different among the cases with or without 8q amplification (t=1,88; P&amp;gt;0.20). These cases were also equally distributed in relation to LN metastasis (≥2=2.67;P&amp;gt;0.10) and tumor grade (≥2=0.5;P&amp;gt;0.50). Finally, alterations in at least one gene involved in the PIK3/AKT pathway were observed in 88% (29/33) of the cases and in more than three genes in 45% (13/29) of the cases. PIK3CA and PTEN were the most frequently altered genes: gained and lost in 42% and 40% of the cases respectively. AKT2, AKT1, and FGFR2 genes were observed gained in 24%, 21% and 21% of the cases respectively. Alterations in those genes were also not significantly correlated with tumor size (t=0.95; P&amp;gt;0.30), LN metastasis (≥2=0.16; P&amp;gt;0.50) and tumor grade (≥2=3.23; P&amp;gt;0.05). Our results indicate a high level of DNA copy number changes in TNBC cases, involving mostly chromosome 8q. Alterations on genes involved in the PIK3/AKT pathway were also common; immunohistochemistry analyses are in progress to evaluate those alterations at the expression level. No significant correlations were observed among the genomic alterations and the pathological parameters from the patients. This lack of correlation can be due to the limited sample size or can indicate that in our set of TNBC cases tumor size, LN metastasis and tumor grade are not associated with an elevated genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5100. doi:1538-7445.AM2012-5100

Abstract 5073: Integrated genomics of high-resolution esophageal adenocarcinomas identify candidate driver genes in regions of large copy number aberrations

Abstract Introduction: Esophageal cancer occurs with two distinct histologies_ squamous cell carcinoma (SCC) and esophageal adenocarcinoma (EA). The incidence of EA has risen over the past few decades and is now the most common esophageal malignancy in the United States and Western Europe. The genome of esophageal adenocarcinoma is highly unstable due to the large number of amplification and deletion events. Presumably, such genomic events drive tumor formation and disease progression by altering the expression of genes within these regions. Several studies have reported large regions of genomic instability especially spanning chromosomal arms or in gene dense regions. Using a large patient cohort and high-resolution data for DNA copy number and expression, this study aims to identify potential driver genes and prognostic markers especially in whole chromosomal aberrations and gene dense regions. Materials &amp; Methods: DNA copy number aberrations were explored in 116 esophageal adenocarcinomas (EA) using Affymetrix SNP 6.0 microarrays. Additionally, gene expression data from a subset of 110 of these tumors using Affymetrix U133 plus 2.0 microarrays were analyzed. DNA copy number aberrations were analyzed using Nexus 5.0 and GISTIC software while the gene expression data was processed in Partek Genomic Suite. Copy number and expression correlations for each gene in the regions of copy number aberrations were calculated using t-test to compare samples with the changes versus samples without the genomic aberrations. Results: We identified at least 50 regions of gains and losses of which 12 regions are either whole chromosomal arm aberrations or aberrations in gene dense regions. Some regions with large number genes include gains at 1q (460 genes), 7q21-22 (80 genes), 10q21-22 (100 genes), and losses at 4q (98 genes), 5q (283 genes), 21q (82 genes). Copy number and expression correlations for each gene in these regions considerably reduced the number of genes that could be considered as candidate driver genes. Furthermore, scouring the literature helped identify potential candidate driver genes such as ANP32E (1q), VCL (10q), CDKN2AIP (4q), and ING2 (4q). Additionally, we report identifying copy number and expression correlations of potential prognostic markers such as MCM4 (8q), CA9 (9p), KLF5 (13q) that lie in focal/smaller regions of genomic instability but at high frequencies. Conclusion: Analysis of the DNA copy number changes identified several regions of frequent gains and losses. At least 12 regions span the chromosomal arm or are in the gene dense regions. Using an integrated genomics approach with high-resolution DNA copy number and expression data, we are able to identify potential driver genes and prognostic markers that lie in these large amplification/deletion regions in EA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5073. doi:1538-7445.AM2012-5073

Abstract 5067: Exome sequencing reveals both DNA sequence and copy number changes in AML: Potential driver changes and mechanisms of drug resistance revealed from serial samples from the same patients

Abstract Despite significant advances in characterizing the molecular genetics of AML, the clonal evolution of leukemic cells and the dynamic impact of genomic changes on the development of the disease and progression to drug resistance are not well understood. Here, we applied next-generation sequencing to quantify aberrant tumor subclones carrying specific mutant alleles of key cancer genes and developed a method to extract quantitative high-resolution copy number changes across the genome using exome sequencing data from matching cancer and normal DNA. Serial bone marrow (BM) samples collected from diagnosis to relapse to post-treatment drug resistance in a patient-centric manner made it possible to trace the clonal evolution of AML and to identify variants potentially involved in drug resistance. Exome sequencing from AML blast cells and normal skin biopsies was performed as part of the Finnish Hematology Registry and Biobanking (FHRB) effort. Consecutive paired samples from different patients revealed unique genetic patterns of clonal evolution and cancer progression in each patient. In a pre-resistant sample of one AML M5 patient, we identified four closely spaced insertions in the Wilm's Tumor (WT1) suppressor gene, none of which appear on the same sequence reads. This suggests the presence of multiple distinct leukemic subclones even before treatment resistance and underscores the strong selective advantage conferred by WT1 mutations. After relapse, one of the subclones was lost, and another one significantly increased suggesting that the relapse arose from the expansion of a pre-existing resistant subclone. In this patient, recurrent clones otherwise featured similar copy number changes and the same fusion genes as the primary diagnostic sample. In another AML patient developing recurrence an opposite pattern was observed: The relapsed, drug-resistant cells displayed an enormous increase of small microdeletions compared to the diagnostic, pre-treatment sample, while almost all sequence-level alterations in potential cancer genes were the same between the two samples. This suggests that a distinct type of DNA repair deficiency may have contributed to the drug resistant clone in this patient. Conclusions: Exome sequencing from paired samples of AML cells before and after relapse makes it possible to trace the clonal evolution of the disease and study the impact of therapy both at the level of sequence alterations of key cancer genes and simultaneously at the level of copy number changes inferred from exome sequence data. This analysis has highlighted multiple genomic patterns by which resistance may evolve in vivo during cancer treatment. Refined bioinformatic analysis and interpretation of exome-seq data provides a rich resource to identify genetic biomarkers of drug response and minimal residual disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5067. doi:1538-7445.AM2012-5067

Enhanced RAD21 cohesin expression confers poor prognosis in BRCA2 and BRCAX, but not BRCA1 familial breast cancers

IntroductionThe RAD21 gene encodes a key component of the cohesin complex, which is essential for chromosome segregation, and together with BRCA1 and BRCA2, for high-fidelity DNA repair by homologous recombination. Although its expression correlates with early relapse and treatment resistance in sporadic breast cancers, it is unclear whether familial breast cancers behave in a similar manner.MethodsWe performed an immunohistochemical analysis of RAD21 expression in a cohort of 94 familial breast cancers (28 BRCA1, 27 BRCA2, and 39 BRCAX) and correlated these data with genotype and clinicopathologic parameters, including survival. In these cancers, we also correlated RAD21 expression with genomic expression profiling and gene copy-number changes and miRNAs predicted to target RAD21.ResultsNo significant differences in nuclear RAD21 expression were observed between BRCA1 (12 (43%) of 28), BRCA2 (12 (44%) of 27), and BRCAX cancers (12 (33%) of 39 (p = 0.598). No correlation was found between RAD21 expression and grade, size, or lymph node, ER, or HER2 status (all P > 0.05). As for sporadic breast cancers, RAD21 expression correlated with shorter survival in grade 3 (P = 0.009) and but not in grade 1 (P = 0.065) or 2 cancers (P = 0.090). Expression of RAD21 correlated with poorer survival in patients treated with chemotherapy (P = 0.036) but not with hormonal therapy (P = 0.881). RAD21 expression correlated with shorter survival in BRCA2 (P = 0.006) and BRCAX (P = 0.008), but not BRCA1 cancers (P = 0.713). Changes in RAD21 mRNA were reflected by genomic changes in DNA copy number (P < 0.001) and by RAD21 protein expression, as assessed with immunohistochemistry (P = 0.047). High RAD21 expression was associated with genomic instability, as assessed by the total number of base pairs affected by genomic change (P = 0.048). Of 15 miRNAs predicted to target RAD21, mir-299-5p inversely correlated with RAD21 expression (P = 0.002).ConclusionsPotential use of RAD21 as a predictive and prognostic marker in familial breast cancers is hence feasible and may therefore take into account the patient's BRCA1/2 mutation status.

Analysis of molecular cytogenetic alterations in uterine leiomyosarcoma by array-based comparative genomic hybridization

The aim of this study was to identify novel genes following genomic DNA copy number changes using a genome-wide array-based comparative genomic hybridization (array-CGH) analysis in uterine leiomyosarcoma (ULMS). Genomic DNA copy number changes were analyzed in 15 cases of ULMS from St Mary's Hospital of the Catholic University of Korea. The paraffin-fixed tissue samples were micro-dissected under microscope, and DNA was extracted. Array-based CGH and genomic polymerase chain reaction were carried out with statistical analyses such as hierarchical clustering and Gene Ontology. All of 15 cases of ULMS showed specific gains and losses. The percentage of average gains and losses were 8.4 and 16.6%, respectively. The analysis limit of average gains and losses was 40%. The regions of high level of gain were 1q23.3, 7p14.2, 7q34, 7q35, 7q36.3, 13q34, and 16p13.3. And the regions of homozygous loss were 2q21.1, 2q22.1, 2p23.2, 12q23.3, 4q21.22, 4q34.3, 11q24.2, 12q23.3, 13q13.1, 13q21.33, and 14q24.3. In ULMS samples, recurrent regions of gain were 1p36.33, 1p36.32, 5q35.3, 7q36.3, and 8q24.3 and recurrent regions of loss were 1p31.1-p31.3, 1p32.1-p32.3, 2p12, 2p13.3, 2p14, 2p16.2-p16.3, 2q12.1-q12.3, 2q21.1-q21.2, 2q22.2-q22.3, 2q34, 2q36.1-q36.3, 5q21.3, 5q23.3, 5q31.1, 6p11.2, 6p12.1, 10q11.23, 10q21.2-q21.3, 10q23.2, 10q23.31, 10q25.1-q25.2, 10q25.3, 10q26.13, 10q26.2-q26.3, 11p11.2, 11p11.12, 11p12, 11p13, 11p15.4, 11q23.1-q23.2, 11q23.3, 13q14.12, 13q14.13-13q14.2, 13q14.2, 13q14.2, 13q14.3, 13q21.33, 13q22.1-q22.3, 14q24.2, 14q24.3, 14q31.1, 14q32.33, 15q11.2-q13, 15q14, 16q22.3, 16q23.1, 16q23.2, 16q24.1, 20p12.1, and 21q22.3. Representative frequently gained BAC clones encoded genes were HDAC9, CRR9, SOX18, PTPRN2, SKI, SOLH, and KIAA1199. The genes encoded by frequently lost BAC clones were LOC150516 and AMY2A. A subset of cellular processes from each gene were clustered by Gene Ontology database. The present study using array-CGH analyses sought a deeper elucidation of the specific genomic alterations related to ULMS. The high resolution of array-CGH combined with human genome database would give a chance at identifying relevant target genes.

Comparison of Whole Genome Amplification Methods for Analysis of DNA Extracted from Microdissected Early Breast Lesions in Formalin-Fixed Paraffin-Embedded Tissue

To understand cancer progression, it is desirable to study the earliest stages of its development, which are often microscopic lesions. Array comparative genomic hybridization (aCGH) is a valuable high-throughput molecular approach for discovering DNA copy number changes; however, it requires a relatively large amount of DNA, which is difficult to obtain from microdissected lesions. Whole genome amplification (WGA) methods were developed to increase DNA quantity; however their reproducibility, fidelity, and suitability for formalin-fixed paraffin-embedded (FFPE) samples are questioned. Using aCGH analysis, we compared two widely used approaches for WGA: single cell comparative genomic hybridization protocol (SCOMP) and degenerate oligonucleotide primed PCR (DOP-PCR). Cancer cell line and microdissected FFPE breast cancer DNA samples were amplified by the two WGA methods and subjected to aCGH. The genomic profiles of amplified DNA were compared with those of non-amplified controls by four analytic methods and validated by quantitative PCR (Q-PCR). We found that SCOMP-amplified samples had close similarity to non-amplified controls with concordance rates close to those of reference tests, while DOP-amplified samples had a statistically significant amount of changes. SCOMP is able to amplify small amounts of DNA extracted from FFPE samples and provides quality of aCGH data similar to non-amplified samples.

Association of copy number variation in the AHI1 gene with risk of obesity in the Chinese population

The prevalence of obesity has increased dramatically over the past decade. Gene copy number variants (CNVs) have been recognized as a hereditable source of susceptibility in human complex diseases including obesity. Recent studies have shown that Abelson helper integration site 1 (Ahi1) gene has a significant contribution in the homeostasis regulation in mouse models of obesity. A study was therefore carried out to investigate whether CNVs in AHI1 gene contribute to human obesity. We analyzed samples from 70 Chinese overweight adults and 74 healthy controls for DNA copy number change using the Affymetrix single-nucleotide polymorphism (SNP) 6.0 array. Validation of CNVs of AHI1 was achieved by real-time PCR using the ΔΔC(t) method. Copy number gain analysis revealed significant gains (P=0.0017) of AHI1 gene copy number in 17 of 70 (24.3%) samples but only four of 74 (5.4%) controls overall. Then we studied the frequency distribution of CNVs in AHI1 gene according to body mass index (BMI) grade. Five out of 28 (18.5%) at-risk obese, six out of 26 (26.9%) moderate obese, and six out of 17 (29.4%) severe obese subjects studied showed increased AHI1 gene copy number. The result suggested that there was a significant linear trend for increasing AHI1 gene copy number frequencies with increasing BMI.

Identification of TFG (TRK‐fused gene) as a putative metastatic melanoma tumor suppressor gene

High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation "hotspot" at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.

Integrated Genomic Analysis of Esophageal Adenocarcinoma Identifies DNA Copy Number Changes and Related Gene Expression Alterations That are Associated With Survival

Integrated Genomic Analysis of Esophageal Adenocarcinoma Identifies DNA Copy Number Changes and Related Gene Expression Alterations That are Associated With Survival

Detecting Changes in DNA Copy Number: Reviewing signal processing techniques

Alterations in the number of DNA copies of portions of a genome are both a process that contributes to natural genetic diversity in healthy individuals and also a driving cause for cancer and other genetic diseases. Even though recent advances in microarray technology and high throughput sequencing now allow very high resolution scans for very large cohorts of samples, small alterations remain particularly difficult to detect, especially under severe noise degradation conditions. Thus, estimation of the DNA copy number remains a challenging problem. Some of the core challenges in copy number estimation can be seen as essentially signal processing problems, where it is necessary to exploit fundamentally different characteristics between the desired underlying biological signal and the measurement noise. Indeed, some of the most successful methods used to address this problem have been inspired by well-known signal processing techniques.

Amplification of theUQCRFS1Gene in Gastric Cancers

PurposeThe specific aim of this study is to unravel a DNA copy number alterations, and to search for novel genes that are associated with the development of Korean gastric cancer.Materials and MethodsWe investigated a DNA copy number changes in 23 gastric adenocarcinomas by array-comparative genomic hybridization and quantitative real-time polymerase chain reaction analyses. Besides, the expression of UQCRFS1, which shows amplification in array-CGH, was examined in 186 gastric cancer tissues by an immunohistochemistry, and in 9 gastric cancer cell lines, as well as 24 gastric cancer tissues by immunoblotting.ResultsWe found common gains at 48 different loci, and a common loss at 19 different loci. Amplification of UQCRFS1 gene at 19q12 was found in 5 (21.7%) of the 23 gastric cancers in an array-comparative genomic hybridization and DNA copy number were increased in 5 (20.0%) out of the 25 gastric cancer in quantitative real-time polymerase chain reaction. In immunohistochemistry, the overexpression of the protein was detected in 105 (56.5%) out of the 186 gastric cancer tissues. Statistically, there was no significant relationship between the overexpression of UQCRFS1 and clinicopathologic parameters (P>0.05). In parallel, the overexpression of UQCRFS1 protein was confirmed in 6 (66.7%) of the 9 gastric cancer cell lines, and 12 (50.0%) of the 24 gastric cancer tissues by immunoblotting.ConclusionsThese results suggest that the overexpression of UQCRFS1 gene may contribute to the development and/or progression of gastric cancer, and further supported that mitochondrial change may serve as a potential cancer biomarker.

A Meta-Analysis of Array-CGH Studies Implicates Antiviral Immunity Pathways in the Development of Hepatocellular Carcinoma

BackgroundThe development and progression of hepatocellular carcinoma (HCC) is significantly correlated to the accumulation of genomic alterations. Array-based comparative genomic hybridization (array CGH) has been applied to a wide range of tumors including HCCs for the genome-wide high resolution screening of DNA copy number changes. However, the relevant chromosomal variations that play a central role in the development of HCC still are not fully elucidated.MethodsIn present study, in order to further characterize the copy number alterations (CNAs) important to HCC development, we conducted a meta-analysis of four published independent array-CGH datasets including total 159 samples.ResultsEighty five significant gains (frequency ≥25%) were mostly mapped to five broad chromosomal regions including 1q, 6p, 8q, 17q and 20p, as well as two narrow regions 5p15.33 and 9q34.2-34.3. Eighty eight significant losses (frequency ≥25%) were most frequently present in 4q, 6q, 8p, 9p, 13q, 14q, 16q, and 17p. Significant correlations existed between chromosomal aberrations either located on the same chromosome or the different chromosomes. HCCs with different etiologies largely exhibited surprisingly similar profiles of chromosomal aberrations with only a few exceptions. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the genes affected by these chromosomal aberrations were significantly enriched in 31 canonical pathways with the highest enrichment observed for antiviral immunity pathways.ConclusionsTaken together, our findings provide novel and important clues for the implications of antiviral immunity-related gene pathways in the pathogenesis and progression of HCC.

Integrated DNA copy number and methylation profiling of lymphoid neoplasms using a single array

Changes in DNA copy number (CN) and DNA methylation represent important aberrations for lymphomas and other cancers. Here, for the first time, we show that the Illumina Infinium Methylation (IIM) assay, although not originally designed for CN profiling, is able to estimate CN changes. We compared the IIM CN profiles to those obtained with a standard technique in a series of diffuse large B-cell lymphomas: the profiles showed a high degree of consensus. The demonstration of CN profiling as an additional function of the IIM assay may impact the choice of platform for methylation profiling of haematological and solid tumours.

DNA Copy Number Changes and Immunophenotype Pattern in Karyotypically Normal Acute Myeloid Leukemia Patients from an Indian Population

Chromosomal abnormalities are important in the diagnosis and prognosis of patients with acute myeloid leukemia (AML). The purpose of this study was to identify DNA copy number variations in karyotypically normal AML patients and their correlation with immunophenotypes. Conventional comparative genomic hybridization (CGH) and immunophenotyping were performed in 46 untreated AML patients aged 7-68 years. Among the 86 Indian patients who had AML, 40 (46.5%) showed an abnormal karyotype and 46 (53.4%) showed no chromosome aberrations. The karyotypically abnormal AML patients were excluded from the study. Out of the 46 patients without chromosomal aberrations, 24 (52.2%) showed DNA copy number variations including losses and gains. The DNA copy number variations involved chromosomes 1, 3, 6, 12, 15, 16, 17 (gains) and 1, 4, 2, 3, 5, 7, 8, 9, 10, 11, 13, 15, 18, 20, 21 (losses). The aberrant immunophenotype was noticed in 13 of these 24 (54%) cases. The hidden chromosome rearrangements in karyotypically normal AML, which could not be detected by conventional cytogenetics and fluorescence in situ hybridization, were detected by CGH. These genetic changes have an important role in the prognosis of the disease. The DNA copy number changes might also be involved in the aberrant immunophenotypes in our study.

SNP-Arrays Provide New Insights Into the Pathogenesis of Richter Syndrome (RS)

Abstract 263RS represents the development of a diffuse large B-cell lymphoma (DLBCL) in the context of chronic lymphocytic leukemia (CLL). The pathogenesis of RS is still largely unknown. Analysis of RS has been often focused on the study of lesions previously identified in de novo DLBCL. However, we have previously shown that RS lacks many of the typical genetic lesions shown in DLBCL (Rossi et al, Blood 2011). Here, we have applied an unbiased, genome-wide approach, searching for DNA copy number alterations in a large series of RS, comparing them with de novo DLBCL, CLL-phase of RS and untransformed CLL.Methods. The study included 50 RS, 30 CLL-phases preceding RS, 100 DLBCL, 318 CLL. All RS were classified as DLBCL. DNA was extracted from frozen biopsies and was processed using the Affymetrix GeneChip Human Mapping SNP6 arrays. Differences in frequencies between subgroups were evaluated using Fisher's exact test. Clinical data were available in half of the cases and genomic lesions were evaluated for their impact on clinical outcome with the log-rank test.Results. In RS, the most common gains were +12 (40%), +4q12 (14%), +2p25.3-25.2 (13%), +8q23.3-qter (12%), +1q32.1, +2p16.1-13.2 (BCL11A/REL), +15q22.31-26.3, +18q21.33 (BCL2) (10%), and +3q24.1-q26.3 (9%). Amplifications were observed at the BCL2 locus and at 1q32.1. The most common losses were at 17p13.1 (TP53; 29%), 8p21.3 (TNFRSF10A/B; 15%), 8p23.3 (16%), 11q22.3 (ATM) (15%), 14q23.3 (13%), 7q33–35 (11%), 14q32.11 (10%), 14q32.31-32.33 (TRAF3), and 9p21.3 (CDKN2A) (9%). The latter locus was also the target of recurrent homozygous deletions.RS appeared intermediate between CLL-phase and de novo DLBCL in terms of number of recurrent lesions. When compared with de novo DLBCL, RS presented less -6q23 (TNFAIP3; P=2.54E-05), -15q15.1-q21.1 (B2M) (P=5.64E-04), +7/7q (P=5.71E-04), -6q21 (PRDM1; P=6.53E-04), +2p16.1 (BCL11A; P=3.22E-03), -1p36 (TNFRSF14; P 9.57E-03), copy-neutral LOH at 6p25.3-21.32 (P =1.01E-02), +5p (P=1.39E-02), +11q14.1-q25 (P=1.63E-02), +1q22-q41 (P 2.13E-02), +6p21 (P 2.48E-02). Conversely, RS presented more often +4q12 (P=8.96E-03), -7q33-35 (P=2.04E-02), -11q22.3 (ATM; P=2.14E-02), -14q23.3 (P=2.69E-02). No statistical differences were observed for lesions such as +3/+3q, +8q, -8p, - 9p21.3 (CDKN2A), +12q, +15q, -17p (TP53). Compared to CLL-phase, RS had significantly more gains at 1q32.1 (P=2.28E-02), 11q23.3 (P=5.38E-03), 11q24 (P=1.10E-02) and 9p21.3 losses (CDKN2A; P=1.10E-02).Compared to the large series of CLL with no history of transformation, CLL-phase samples presented less frequently losses at 13q14.3 (DLEU2, MIR15A, MIR16-1a; 6.58E-03), more commonly gains at 12 (P=2.66E-02), 4q12 (P=1.103E-02), +2p (P=2.97E-02), and loss at TP53 locus (P=2.23E-03).The presence of losses of CDKN2A (P=4.6E-03), TNFRSF10A/B (P=3.2E-02) and BCL2 gains (P=4.0E-02) determined a poor overall survival in RS.Conclusions. DNA copy number changes in RS are intermediate between de novo DLBCL and CLL-phase. Recurrent lesions were identified that appeared more common in RS than in DLBCL, and that seem to be involved in the progression from CLL to an aggressive lymphoma. Also, the acquisition of mono- and bi-allelic losses of CDKN2A appears as one of the most important event in the transition from CLL-phase to RS. CDKN2A and TNFRSF10A/B losses and BCL2 gains were associated with a poor outcome.AR & EC equally contributed to the work. Disclosures:No relevant conflicts of interest to declare.

Time to Recurrence and Survival in Serous Ovarian Tumors Predicted from Integrated Genomic Profiles

BackgroundSerous ovarian cancer (SeOvCa) is an aggressive disease with differential and often inadequate therapeutic outcome after standard treatment. The Cancer Genome Atlas (TCGA) has provided rich molecular and genetic profiles from hundreds of primary surgical samples. These profiles confirm mutations of TP53 in ∼100% of patients and an extraordinarily complex profile of DNA copy number changes with considerable patient-to-patient diversity. This raises the joint challenge of exploiting all new available datasets and reducing their confounding complexity for the purpose of predicting clinical outcomes and identifying disease relevant pathway alterations. We therefore set out to use multi-data type genomic profiles (mRNA, DNA methylation, DNA copy-number alteration and microRNA) available from TCGA to identify prognostic signatures for the prediction of progression-free survival (PFS) and overall survival (OS).Methodology/Principal FindingsWe implemented a multivariate Cox Lasso model and median time-to-event prediction algorithm and applied it to two datasets integrated from the four genomic data types. We (1) selected features through cross-validation; (2) generated a prognostic index for patient risk stratification; and (3) directly predicted continuous clinical outcome measures, that is, the time to recurrence and survival time. We used Kaplan-Meier p-values, hazard ratios (HR), and concordance probability estimates (CPE) to assess prediction performance, comparing separate and integrated datasets. Data integration resulted in the best PFS signature (withheld data: p-value = 0.008; HR = 2.83; CPE = 0.72).Conclusions/SignificanceWe provide a prediction tool that inputs genomic profiles of primary surgical samples and generates patient-specific predictions for the time to recurrence and survival, along with outcome risk predictions. Using integrated genomic profiles resulted in information gain for prediction of outcomes. Pathway analysis provided potential insights into functional changes affecting disease progression. The prognostic signatures, if prospectively validated, may be useful for interpreting therapeutic outcomes for clinical trials that aim to improve the therapy for SeOvCa patients.

Nodular lesions arising in a large congenital melanocytic naevus in a newborn with eruptive disseminated Spitz naevi

Congenital malignant melanoma within a pre-existing large congenital melanocytic naevus (CMN) is exceedingly rare. Its incidence is difficult to determine due to the small number of reported cases and because of problems associated with diagnosis. Some benign nodular proliferations (called proliferative nodules) arising in CMN, while rare, are significantly more common and can mimic malignant melanoma clinically or histologically. There are no reported cases of congenital melanoma or benign proliferative nodules in CMN in patients who also had eruptive disseminated Spitz naevi. We describe a girl who was noted to have a dark-brown plaque with several large erythematous nodules affecting the scalp at delivery, in addition to multiple erythematous dome-shaped papules that developed in a disseminated manner over several months, beginning at 10 days of age. It was difficult, not only clinically but also histologically, to determine the benign or malignant nature of all of these lesions. As primary cutaneous melanoma, atypical proliferative nodules in CMN, bland CMN or CMN with foci of increased cellularity and Spitz naevi show clear differences in the genetic aberration patterns, comparative genomic hybridization (CGH) could be a diagnostic help in ambiguous cases such as this. CGH performed on this patient showed multiple DNA copy number changes in the most atypical nodule, but such alterations could not be found in the remainder of the lesions. CGH showed differences between the nodular lesions that occurred in the CMN and helped us in supporting the diagnosis of this unique case of benign proliferative nodules and a possible congenital melanoma arising in a large CMN, associated with multiple widespread eruptive Spitz naevi.

Utility of Real-Time Polymerase Chain Reaction in Diagnosing and Treating Acanthamoeba Keratitis

Using real-time polymerase chain reaction (PCR), we detected Acanthamoeba and monitored the changes in Acanthamoeba DNA copy number over the treatment course in patients suspected of Acanthamoeba keratitis (AK). Subjects were 6 patients (average age, 26.2 years) suspected of AK at the Kinki University Outpatient Clinic. For detection of Acanthamoeba, patients' corneal scrapings were collected for smear analysis, culture, and real-time PCR. After the diagnosis of AK was confirmed, treatment was initiated based on the quantitative result of the real-time PCR. Both the smear and culture were positive for Acanthamoeba in 4 cases and negative in 2 cases (agreement in 3 cases and disagreement in 2 cases). By real-time PCR, all 6 cases were positive for Acanthamoeba with an average DNA copy number of 4.8 ± 9.1 × 10 copies per sample. We further monitored the variation in the Acanthamoeba DNA copy number over the treatment course and successfully treated all the patients. DNA copy number provided a parallel with other clinical features of AK. Real-time PCR can be a useful method for a rapid and precise diagnosis of AK. Moreover, utility of the Acanthamoeba DNA copy number obtained by real-time PCR can help ophthalmologists in making the best treatment decision.