Germline DNA Copy Number Aberrations Identified as Potential Prognostic Factors for Breast Cancer Recurrence

Breast cancer recurrence (BCR) is a common treatment outcome despite curative-intent primary treatment of non-metastatic breast cancer. Currently used prognostic and predictive factors utilize tumor-based markers, and are not optimal determinants of risk of BCR. Germline-based copy number aberrations (CNAs) have not been evaluated as determinants of predisposition to experience BCR. In this study, we accessed germline DNA from 369 female breast cancer subjects who received curative-intent primary treatment following diagnosis. Of these, 155 experienced BCR and 214 did not, after a median duration of follow up after breast cancer diagnosis of 6.35 years (range = 0.60–21.78) and 8.60 years (range = 3.08–13.57), respectively. Whole genome CNA genotyping was performed on the Affymetrix SNP array 6.0 platform. CNAs were identified using the SNP-Fast Adaptive States Segmentation Technique 2 algorithm implemented in Nexus Copy Number 6.0. Six samples were removed due to poor quality scores, leaving 363 samples for further analysis. We identified 18,561 CNAs with ≥1 kb as a predefined cut-off for observed aberrations. Univariate survival analyses (log-rank tests) identified seven CNAs (two copy number gains and five copy neutral-loss of heterozygosities, CN-LOHs) showing significant differences (P<2.01×10⁻⁵) in recurrence-free survival (RFS) probabilities with and without CNAs.We also observed three additional but distinct CN-LOHs showing significant differences in RFS probabilities (P<2.86×10⁻⁵) when analyses were restricted to stratified cases (luminal A, n = 208) only. After adjusting for tumor stage and grade in multivariate analyses (Cox proportional hazards models), all the CNAs remained strongly associated with the phenotype of BCR. Of these, we confirmed three CNAs at 17q11.2, 11q13.1 and 6q24.1 in representative samples using independent genotyping platforms. Our results suggest further investigations on the potential use of germline DNA variations as prognostic markers in cancer-associated phenotypes.     

Micro-Scale Genomic DNA Copy Number Aberrations as Another Means of Mutagenesis in Breast Cancer

Introduction

In breast cancer, the basal-like subtype has high levels of genomic instability relative to other breast cancer subtypes with many basal-like-specific regions of aberration. There is evidence that this genomic instability extends to smaller scale genomic aberrations, as shown by a previously described micro-deletion event in the PTEN gene in the Basal-like SUM149 breast cancer cell line.

Methods

We sought to identify if small regions of genomic DNA copy number changes exist by using a high density, gene-centric Comparative Genomic Hybridizations (CGH) array on cell lines and primary tumors. A custom tiling array for CGH (244,000 probes, 200 bp tiling resolution) was created to identify small regions of genomic change, which was focused on previously identified basal-like-specific, and general cancer genes. Tumor genomic DNA from 94 patients and 2 breast cancer cell lines was labeled and hybridized to these arrays. Aberrations were called using SWITCHdna and the smallest 25% of SWITCHdna-defined genomic segments were called micro-aberrations (<64 contiguous probes, ∼ 15 kb).

Results

Our data showed that primary tumor breast cancer genomes frequently contained many small-scale copy number gains and losses, termed micro-aberrations, most of which are undetectable using typical-density genome-wide aCGH arrays. The basal-like subtype exhibited the highest incidence of these events. These micro-aberrations sometimes altered expression of the involved gene. We confirmed the presence of the PTEN micro-amplification in SUM149 and by mRNA-seq showed that this resulted in loss of expression of all exons downstream of this event. Micro-aberrations disproportionately affected the 5′ regions of the affected genes, including the promoter region, and high frequency of micro-aberrations was associated with poor survival.

Conclusion

Using a high-probe-density, gene-centric aCGH microarray, we present evidence of small-scale genomic aberrations that can contribute to gene inactivation. These events may contribute to tumor formation through mechanisms not detected using conventional DNA copy number analyses.     

Genomic DNA Copy Number Aberrations,Histological Diagnosis,Oral Subsite and Aneuploidy in OPMDs/OSCCs

Oral potentially malignant disorders (OPMDs) characterized by the presence of dysplasia and DNA copy number aberrations (CNAs), may reflect chromosomal instability (CIN) and predispose to oral squamous cell carcinoma (OSCC). Early detection of OPMDs with such characteristics may play a crucial role in OSCC prevention. The aim of this study was to explore the relationship between CNAs, histological diagnosis, oral subsite and aneuploidy in OPMDs/OSCCs. Samples from OPMDs and OSCCs were processed by high-resolution DNA flow cytometry (hr DNA-FCM) to determine the relative nuclear DNA content. Additionally, CNAs were obtained for a subset of these samples by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. Our study shows that: i) aneuploidy, global genomic imbalance (measured as the total number of CNAs) and specific focal CNAs occur early in the development of oral cancer and become more frequent at later stages; ii) OPMDs limited to tongue (TNG) mucosa display a higher frequency of aneuploidy compared to OPMDs confined to buccal mucosa (BM) as measured by DNA-FCM; iii) TNG OPMDs/OSCCs show peculiar features of CIN compared to BM OPMDs/OSCCs given the preferential association with total broad and specific focal CNA gains. Follow-up studies are warranted to establish whether the presence of DNA aneuploidy and specific focal or broad CNAs may predict cancer development in non-dysplastic OPMDs.     

Copy Number of the Transposon,Pokey, in rDNA Is Positively Correlated with rDNA Copy Number in Daphnia obtusa

Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species.     

Focal Chromosomal Copy Number Aberrations Identify CMTM8 and GPR177 as New Candidate Driver Genes in Osteosarcoma

Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, <3 Mb). For this purpose, we subjected 26 primary tumors of osteosarcoma patients to high-resolution single nucleotide polymorphism array analyses and identified 139 somatic focal CNAs. Of these, 72 had at least one gene located within or overlapping the focal CNA, with a total of 94 genes. For 84 of these genes, the expression status in 31 osteosarcoma samples was determined by expression microarray analysis. This enabled us to identify the genes of which the over- or underexpression was in more than 35% of cases in accordance to their copy number status (gain or loss). These candidate genes were subsequently validated in an independent set and furthermore corroborated as driver genes by verifying their role in other tumor types. We identified CMTM8 as a new candidate tumor suppressor gene and GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesis.     

DNA Copy Number Aberrations,and Human Papillomavirus Status in Penile Carcinoma. Clinico-Pathological Correlations and Potential Driver Genes

Penile squamous cell carcinoma is a rare disease, in which somatic genetic aberrations have yet to be characterized. We hypothesized that gene copy aberrations might correlate with human papillomavirus status and clinico-pathological features. We sought to determine the spectrum of gene copy number aberrations in a large series of PSCCs and to define their correlations with human papillomavirus, histopathological subtype, and tumor grade, stage and lymph node status. Seventy formalin-fixed, paraffin embedded penile squamous cell carcinomas were centrally reviewed by expert uropathologists. DNA was extracted from micro-dissected samples, subjected to PCR-based human papillomavirus assessment and genotyping (INNO-LiPA human papillomavirus Genotyping Extra Assay) and microarray-based comparative genomic hybridization using a 32K Bacterial Artificial Chromosome array platform. Sixty-four samples yielded interpretable results. Recurrent gains were observed in chromosomes 1p13.3-q44 (88%), 3p12.3-q29 (86%), 5p15.33-p11 (67%) and 8p12-q24.3 (84%). Amplifications of 5p15.33-p11 and 11p14.1-p12 were found in seven (11%) and four (6%) cases, respectively. Losses were observed in chromosomes 2q33-q37.3 (86%), 3p26.3-q11.1 (83%) and 11q12.2-q25 (81%). Although many losses and gains were similar throughout the cohort, there were small significant differences observed at specific loci, between human papillomavirus positive and negative tumors, between tumor types, and tumor grade and nodal status. These results demonstrate that despite the diversity of genetic aberrations in penile squamous cell carcinomas, there are significant correlations between the clinico-pathological data and the genetic changes that may play a role in disease natural history and progression and highlight potential driver genes, which may feature in molecular pathways for existing therapeutic agents.     

Canine Mammary Tumours Are Affected by Frequent Copy Number Aberrations,including Amplification of MYC and Loss of PTEN

BackgroundCopy number aberrations frequently occur during the development of many cancers. Such events affect dosage of involved genes and may cause further genomic instability and progression of cancer. In this survey, canine SNP microarrays were used to study 117 canine mammary tumours from 69 dogs.ResultsWe found a high occurrence of copy number aberrations in canine mammary tumours, losses being more frequent than gains. Increased frequency of aberrations and loss of heterozygosity were positively correlated with increased malignancy in terms of histopathological diagnosis. One of the most highly recurrently amplified regions harbored the MYC gene. PTEN was located to a frequently lost region and also homozygously deleted in five tumours. Thus, deregulation of these genes due to copy number aberrations appears to be an important event in canine mammary tumour development. Other potential contributors to canine mammary tumour pathogenesis are COL9A3, INPP5A, CYP2E1 and RB1. The present study also shows that a more detailed analysis of chromosomal aberrations associated with histopathological parameters may aid in identifying specific genes associated with canine mammary tumour progression.ConclusionsThe high frequency of copy number aberrations is a prominent feature of canine mammary tumours as seen in other canine and human cancers. Our findings share several features with corresponding studies in human breast tumours and strengthen the dog as a suitable model organism for this disease.     

Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs.     

脑胶质瘤IDH1基因突变HRM检测方法的初步建立

在大多数弥漫性人脑胶质瘤中常伴随着异柠檬酸脱氢酶基因(isocitrate dehydrogenase gene)1(IDH1)的突变.有着此突变的胶质瘤患者在临床上有良好的预后效果,因此可以作为胶质瘤患者的分子标记.通过提取石蜡切片中的胶质瘤组织DNA,优化条件以初步建立IDH1基因突变高分辨率熔解曲线分析法(high resolution melting,HRM)检测方法.用HRM对胶质瘤石蜡包埋组织标本中IDH1基因检测突变,并用直接测序法对比,观察反应体系和反应条件的可行性.正交试验结果证明引物最适退火温度为53℃,20μL HRM反应体系中,0.6μmol/L引物,2.5 mmol/L Mg2+,60 ng DNA模板为最佳.HRM IDH1基因突变的结果和直接测序法结果一致,但灵敏度更高.说明此HRM方法简单,特异性强,准确可靠,可为IDH1突变检测的临床应用提供借鉴依据.     

Proto-Oncogenic Role of Mutant IDH2 in Leukemia Initiation and Maintenance

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Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.     

Spectrum of EGFR Gene Copy Number Changes and KRAS Gene Mutation Status in Korean Triple Negative Breast Cancer Patients

Anti-epidermal growth factor receptor (EGFR) therapy has been tried in triple negative breast cancer (TNBC) patients without evaluation of molecular and clinical predictors in several randomized clinical studies. Only fewer than 20% of metastatic TNBCs showed response to anti-EGFR therapy. In order to increase the overall response rate, first step would be to classify TNBC into good or poor responders according to oncogenic mutation profiles. This study provides the molecular characteristics of TNBCs including EGFR gene copy number changes and mutation status of EGFR and KRAS gene in Korean TNBC patients. Mutation analysis for EGFR, KRAS, BRAF and TP53 from a total of 105 TNBC tissue samples was performed by direct sequencing, peptide nucleic acid-mediated PCR clamping method and real-time PCR. Copy number changes of EGFR gene were evaluated using multiplex ligation-dependent probe amplification. Out of all 105 TNBCs, 15.2% (16/105) showed EGFR copy number changes. Among them, increased or decreased EGFR copy number was detected in 13 (5 single copy gain, 2 amplification and 4 high-copy number amplification) and 3 cases (3 hemizygous deletion), respectively. The mutation frequencies of KRAS, EGFR and TP53 gene were 1.9% (G12V and G12D), 1.0% (exon 19 del) and 31.4%, respectively. There was no BRAF V600E mutation found. Future studies are needed to evaluate the clinical outcomes of TNBC patients who undergo anti-EGFR therapy according to the genetic status of EGFR.     

Molecular Diagnostic and Prognostic Subtyping of Gliomas in Tunisian Population

It has become increasingly evident that morphologically similar gliomas may have distinct clinical phenotypes arising from diverse genetic signatures. To date, glial tumours from the Tunisian population have not been investigated. To address this, we correlated the clinico-pathology with molecular data of 110 gliomas by a combination of HM450K array, MLPA and TMA-IHC. PTEN loss and EGFR amplification were distributed in different glioma histological groups. However, 1p19q co-deletion and KIAA1549:BRAF fusion were, respectively, restricted to Oligodendroglioma and Pilocytic Astrocytoma. CDKN2A loss and EGFR overexpression were more common within high-grade gliomas. Furthermore, survival statistical correlations led us to identify Glioblastoma (GB) prognosis subtypes. In fact, significant lower overall survival (OS) was detected within GB that overexpressed EGFR and Cox2. In addition, IDH1R132H mutation seemed to provide a markedly survival advantage. Interestingly, the association of IDHR132H mutation and EGFR normal status, as well as the association of differentiation markers, defined GB subtypes with good prognosis. By contrast, poor survival GB subtypes were defined by the combination of PTEN loss with PDGFRa expression and/or EGFR amplification. Additionally, GB presenting p53-negative staining associated with CDKN2A loss or p21 positivity represented a subtype with short survival. Thus, distinct molecular subtypes with individualised prognosis were identified. Interestingly, we found a unique histone mutation in a poor survival young adult GB case. This tumour exceptionally associated the H3F3A G34R mutation and MYCN amplification as well as 1p36 loss and 10q loss. Furthermore, by exhibiting a remarkable methylation profile, it emphasised the oncogenic power of G34R mutation connecting gliomagenesis and chromatin regulation.     

Negative Node Count Improvement Prognostic Prediction of the Seventh Edition of the TNM Classification for Gastric Cancer

Objective

To demonstrate that the seventh edition of the tumor-node-metastasis (TNM) classification for gastric cancer (GC) should be updated with the number of negative lymph nodes for the improvement of its prognostic prediction accuracy.

Methods

Clinicopathological data of 769 GC patients who underwent curative gastrectomy with lymphadenectomy between 1997 and 2006 were retrospectively analyzed to demonstrate the superiority of prognostic efficiency of the seventh edition of the TNM classification, which can be improved by combining the number of negative lymph nodes.

Results

With the Cox regression multivariate analysis, the seventh edition of the TNM classification, the number of negative nodes, the type of gastrectomy, and the depth of tumor invasion (T stage) were identified as independent factors for predicting the overall survival of GC patients. Furthermore, we confirmed that the T stage-N stage–number of negative lymph nodes–metastasis (TNnM) classification is the most appropriate prognostic predictor of GC patients by using case-control matched fashion and multinominal logistic regression. Finally, we were able to clarify that TNnM classification may provide more precise survival differences among the different TNM sub-stages of GC by using the measure of agreement (Kappa coefficient), the McNemar value, the Akaike information criterion, and the Bayesian Information Criterion compared with the seventh edition of the TNM classification.

Conclusion

The number of negative nodes, as an important prognostic predictor of GC, can improve the prognostic prediction efficiency of the seventh edition of the TNM classification for GC, which should be recommended for conventional clinical applications.     

Identification of Copy Number Variations in Xiang and Kele Pigs

Xiang and Kele pigs are two well-known local Chinese pig breeds that possess rich genetic resources and have enormous economic and scientific value. We performed a comprehensive genomic analysis of the copy number variations (CNVs) in these breeds. CNVs are one of the most important forms of genomic variation and have profound effects on phenotypic variation. In this study, PorcineSNP60 genotyping data from 98 Xiang pigs and 22 Kele pigs were used to identify CNVs. In total, 172 candidate CNV regions (CNVRs) were identified, ranging from 3.19 kb to 8175.26 kb and covering 80.41 Mb of the pig genome. Approximately 56.40% (97/172) of the CNVRs overlapped with those identified in seven previous studies, and 43.60% (75/172) of the identified CNVRs were novel. Of the identified CNVRs, 82 (47 gain, 33 loss, and two gain-loss events that covered 4.58 Mb of the pig genome) were found only in a Xiang population with a large litter size. In contrast, 13 CNVRs (8 gain and 5 loss events) were unique to a Xiang population with small litter sizes, and 30 CNVRs (14 loss and 16 gain events) were unique to Kele pigs. The CNVRs span approximately 660 annotated Sus scrofa genes that are significantly enriched for specific biological functions, such as sensory perception, cognition, reproduction, ATP biosynthetic processes, and neurological processes. Many CNVR-associated genes, particularly the genes involved in reproductive traits, differed between the Xiang populations with large and small litter sizes, and these genes warrant further investigation due to their importance in determining the reproductive performance of Xiang pigs. Our results provide meaningful information about genomic variation, which may be useful in future assessments of the associations between CNVs and important phenotypes in Xiang and Kele pigs to ultimately help protect these rare breeds.