Analysis Of Copy Number Aberrations Research Articles

Abstract 3362: Mice with a “deletor” phenotype allow high resolution detection of lymphoid leukemia tumor suppressor genes

Abstract Mice that are homozygous for a deficiency allele of the DNA replication factor minichromosome maintenance protein 2 (designated Mcm2def) are born viable and are healthy for the first 2 months of life. Beginning at three months, these mice develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Copy number aberration (CNA) analysis showed that these pre-T LBL samples had 8-14 small (100-1000 kb) interstitial deletions per sample. Remarkably, all mice had two or more deletions that encompassed genes known to be relevant for human pre-T LBL, including Pten, Cdkn1a, Tcf3, and Tcf12. Mice that express a NUP98-HOXD13 (NHD13) transgene develop a wide array of leukemias, most commonly myeloid, less commonly T-cell, and, rarely, B-lineage. To identify myeloid tumor suppressor genes, we crossed the NHD13 transgene onto the Mcm2def background. All Mcm2def:NHD13+ mice developed pre-T LBL by 3 months of age, reflecting the highly penetrant nature of the Mcm2def phenotype. None of the Mcm2def:NHD13+ mice developed myeloid leukemia. Surprisingly, approximately 30% of the Mcm2def:NHD13+ mice developed concurrent B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and pre-T LBL. The thymus was typically infiltrated with pre-T LBL cells, whereas the bone marrow and spleen were infiltrated with BCP-ALL cells, characterized by clonal VDJ rearrangement and CD19 staining. Parenchymal organs (lung, kidney, liver) were variably infiltrated with pre-T LBL, BCP-ALL, or both. CNA analysis showed that the pre-T LBL were characterized by Pten, CDkn1a, Tcf3, and Tcf12 deletions, similar to the Mcm2def pre-T LBL, whereas the BCP-ALL were characterized by homozygous or heterozygous deletions of Pax5 and a 400 kb region encompassing Cebpb and Ptpn1. There were no shared deletions present in both BCP-ALL and pre-T LBL from the same mouse, indicating that the BCP-ALL and pre-T LBL arose independently, and not from a common precursor. The high frequency (5/7 samples) of acquired Pax5 deletions in the BCP-ALL samples serves to validate the model; whereas the finding of Cebpb/Ptpn1 deletions (5/7 samples) suggests an unanticipated role of either Cebpb or Ptpn1 in BCP-ALL. Citation Format: Mianmian Yin, Timour Baslan, Amy Freeland, Steven C. Pruitt, Peter D. Aplan. Mice with a “deletor” phenotype allow high resolution detection of lymphoid leukemia tumor suppressor genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3362.

Genetic landscape of ultra-stable chronic lymphocytic leukemia patients

BackgroundChronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile. Patients and methodsPatients with absence of disease progression for over 10years (10–34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultra-deep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis. ResultsWES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients. ConclusionsThe genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.

Whole-Exome Sequencing of Acquired Nevi Identifies Mechanisms for Development and Maintenance of Benign Neoplasms

The melanoma transformation rate of an individual nevus is very low despite the detection of oncogenic BRAF or NRAS mutations in 100% of nevi. Acquired melanocytic nevi do, however, mimic melanoma, and approximately 30% of all melanomas arise within pre-existing nevi. Using whole-exome sequencing of 30 matched nevi, adjacent normal skin, and saliva we sought to identify the underlying genetic mechanisms for nevus development. All nevi were clinically, dermoscopically, and histopathologically documented. In addition to identifying somatic mutations, we found mutational signatures relating to UVR mirroring those found in cutaneous melanoma. In nevi we frequently observed the presence of the UVR mutation signature compared with adjacent normal skin (97% vs. 10%, respectively). Copy number aberration analysis showed that for nevi with copy number loss of tumor suppressor genes, this loss was balanced by loss of potent oncogenes. Moreover, reticular and nonspecific patterned nevi showed an increased (P < 0.0001) number of copy number aberrations compared with globular nevi. The mutation signature data generated in this study confirms that UVR strongly contributes to nevogenesis. Copy number changes reflect at a genomic level the dermoscopic differences of acquired melanocytic nevi. Finally, we propose that the balanced loss of tumor suppressor genes and oncogenes is a protective mechanism of acquired melanocytic nevi.

Abstract GS1-04: Copy number aberration analysis to predict response to neoadjuvant anti-HER2 therapy: Results from the NeoALTTO phase III trial

Abstract Background In the NeoALTTO trial, the combination of trastuzumab and lapatinib for the neoadjuvant treatment of HER2 positive early breast cancer patients (pts) nearly doubled the rate of pathological complete response (pCR) compared with either anti-HER2 treatment alone. In the same trial, pCR was shown to be a surrogate for long-term outcome. Expression of ERBB2, ESR1 and immune signatures were the main drivers of pCR (Fumagalli et al. JAMA Oncol 2016). The main aim of the current study was to investigate the relevance of copy number aberrations (CNAs) for pCR and event-free survival (EFS). Methods For 271 out of the 455 pts enrolled in the study with sufficient tumour content, DNA was hybridized on CytoScan HD arrays. The log2 ratio intensities and B allele frequencies were segmented jointly using the multitrack segmentation algorithm. Integer level estimates of total copy number and major allele were obtained using GAP. Recurrent CNAs were found with GISTIC2. The genome instability index (GII) was defined as the median absolute deviation of the normalized copy number. Tumor infiltrating lymphocytes (TILs) and gene expression were obtained as described previously. The correlations between differents parameters were assessed using Spearman correlations (ρ). The Mann-Whitney test was used to relate binary and numerical features. EFS analysis was performed using the Cox proportional hazard model. Results CNAs estimates were obtained for 185 out of 271 pts. CNA distribution was similar to the ones observed in HER2+ pts from the METABRIC and the TCGA datasets. There were 64% of diploid pts, 26% of triploid and 9% of tetraploid or more. Aneuploidy level was not associated with pCR or EFS. Of interest, there were many significant differences in CNA profiles between ER+ and ER- pts. Those differences mirror those observed between ER+ and ER- among HER2- pts in TCGA (ρ=63%) and METABRIC (ρ=56%). ERBB2 amplification was predictive of high pCR (p=0.0007), albeit less so than ERBB2 expression, and ceased to be significant correcting for ERBB2 expression. The pCR rate increased with the GII (p=0.03), independently of ERBB2 amplification. The effect was stronger in ER+ patients (p=0.01). GISTIC analysis revealed 159 recurrent CNA regions. Amplification of 2 regions on 6q23-24 was significantly associated with higher pCR (p=0.00005 and p=0.00087, FDR=0.006 and 0.05). The most significant segment of 6q23-24 contained 39 genes, some whose expression level also predicted pCR (e.g. MAP3K5, p=10&amp;lt;sup style="font-family: Calibri;"&amp;gt;-4). Gene ontology analysis of the genes correlated with this segment highlighted the category 'response to interferon-alpha' (p=4.3x10&amp;lt;sup style="font-family: Calibri;"&amp;gt;-7). No amplified region or gene was found to be predictive of EFS, after multiple testing correction. Conclusions The amplification of ERBB2 was shown to be predictive of pCR, however its expression was more predictive. High genomic instability was associated with higher rate of pCR in ER+ subgroup. Of interest, a novel amplified region, involving chromosome 6 was shown to be predictive of pCR, which may warrant further investigation. Citation Format: Sotiriou C, Rothé F, Maetens M, Fumagalli D, Brown DN, Salgado R, Bradbury I, Pusztai L, Harbeck N, Ignatiadis M, Sarp S, de la Pena L, de Azambuja E, Huober J, Nuciforo P, Baselga J, Piccart M, Loi S, Venet D. Copy number aberration analysis to predict response to neoadjuvant anti-HER2 therapy: Results from the NeoALTTO phase III trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-04.

Epstein-Barr virus-associated primary nodal T/NK-cell lymphoma shows a distinct molecular signature and copy number changes.

The molecular biology of primary nodal T- and NK-cell lymphoma and its relationship with extranodal NK/T-cell lymphoma, nasal type is poorly understood. In this study, we assessed the relationship between nodal and extranodal Epstein-Barr virus-positive T/NK-cell lymphomas using gene expression profiling and copy number aberration analyses. We performed gene expression profiling and copy number aberration analysis on 66 cases of Epstein-Barr virus-associated T/NK-cell lymphoma from nodal and extranodal sites, and correlated the molecular signatures with clinicopathological features. Three distinct molecular clusters were identified with one enriched for nodal presentation and loss of 14q11.2 (TCRA loci). T/NK-cell lymphomas with a nodal presentation (nodal-group) were significantly associated with older age, lack of nasal involvement, and T-cell lineage compared to those with an extranodal presentation (extranodal-group). On multivariate analysis, nodal presentation was an independent factor associated with short survival. Comparing the molecular signatures of the nodal and extranodal groups it was seen that the former was characterized by upregulation of PD-L1 and T-cell-related genes, including CD2 and CD8, and downregulation of CD56, consistent with the CD8+/CD56-immunophenotype. PD-L1 and CD2 protein expression levels were validated using multiplexed immunofluorescence. Interestingly, nodal group lymphomas were associated with 14q11.2 loss which correlated with loss of TCR loci and T-cell origin. Overall, our results suggest that T/NK-cell lymphoma with nodal presentation is distinct and deserves to be classified separately from T/NK-cell lymphoma with extranodal presentation. Upregulation of PD-L1 indicates that it may be possible to use anti-PD1 immunotherapy in this distinctive entity. In addition, loss of 14q11.2 may be a potentially useful diagnostic marker of T-cell lineage.

Exploring the Link between Radiation Exposure and Multifocal Basal Cell Carcinomas in a Former Chernobyl Clean-up Worker by Combining Different Molecular Biological Techniques.

Thirty years after the Chernobyl nuclear power plant accident we report on a patient who was a clean-up worker, who subsequently developed multiple cutaneous basal cell carcinomas (BCCs). We used several methods to assess the biological long-term effects related to low-dose external and internal radiation exposure. Specifically, because BCC risk may be increased with ionizing radiation exposure, we endeavored to determine whether the multifocal BCCs were related to the patient's past clean-up work. We assessed cytogenetic changes using peripheral blood, and internal incorporation was measured with a whole-body counter. Gene expression alterations were determined and array-based comparative genomic hybridization was performed for copy number aberration analysis of available BCC samples. In 1,053 metaphase cells, the dicentric yield of 0.005 dicentrics, with acentrics/cell, was significantly increased compared to the established calibration curve (P < 0.001). A 2.5-fold increase in total translocations was observed compared to the expected translocation rate. No internal contamination was detected with the whole-body counter. At the RNA level, two of seven genes (HNRNPA1, AGAP4/6/8) indicated internal plutonium exposure associated with the lowest dose category found in Mayak workers (>0-0.055 Gy). Relevant DNA copy number changes were only detected within the most aggressive BCC focus. Our results suggest that the examined worker had low and more recent radiation exposure with presumably internalized radionuclides that were below the detection level of a whole-body counter. The multifocal BCC could not be related to past occupational radiation exposure. The findings from our study suggest that integrating different methodologies potentially provides an improved overall assessment of individual health risks associated with or excluding occupational radiation exposure.

Comprehensive analysis of copy number aberrations in microsatellite stable colon cancer in view of stromal component

Background:Somatic copy number aberrations (CNAs) are common acquired changes in cancer cells having an important role in the progression of colon cancer (colorectal cancer, CRC). This study aimed to perform a characterisation of CNA and their impact in gene expression.Methods:Copy number aberrations were inferred from SNP array data in a series of 99 CRC. Copy number aberration events were calculated and used to assess the association between copy number dosage, clinical and molecular characteristics of the tumours, and gene expression changes. All analyses were adjusted for the quantity of stroma in each sample, which was inferred from gene expression data.Results:High heterogeneity among samples was observed; the proportion of altered genome ranged between 0.04 and 26.6%. Recurrent CNA regions with gains were frequent in chromosomes 7p, 8q, 13q, and 20, whereas 8p, 17p, and 18 cumulated losses. A significant positive correlation was observed between the number of somatic mutations and total CNA (Spearman’s r=0.42, P=0.006). Approximately 37% of genes located in CNA regions changed their level of expression and the average partial correlation (adjusted for stromal content) with copy number was 0.54 (interquartile range 0.20 to 0.81). Altered genes showed enrichment in pathways relevant for CRC. Tumours classified as CMS2 and CMS4 by the consensus molecular subtyping showed higher frequency of CNA. Losses of one small region in 1p36.33, with gene CDK11B, were associated with poor prognosis. More than 66% of the recurrent CNA were validated in the The Cancer Genome Atlas (TCGA) data when analysed with the same procedure. Furthermore, 79% of the genes with altered expression in our data were validated in the TCGA.Conclusions:Although CNA are frequent events in microsatellite stable CRC, few focal recurrent regions were found. These aberrations have strong effects on gene expression and contribute to deregulate relevant cancer pathways. Owing to the diploid nature of stromal cells, it is important to consider the purity of tumour samples to accurately calculate CNA events in CRC.

Rapid single-cell copy-number aberration analysis using high-throughput sequencing

Rapid single-cell copy-number aberration analysis using high-throughput sequencing

Inferring the Mutational History of a Tumor Using Multi-state Perfect Phylogeny Mixtures

Phylogenetic techniques are increasingly applied toinfer the somatic mutational history of a tumor from DNA sequencing data. However, standard phylogenetic tree reconstruction techniques do not account for the fact that bulk sequencing data measures mutations in a population of cells. We formulate and solve the multi-state perfect phylogeny mixture deconvolution problem of reconstructing a phylogenetic tree given mixtures of its leaves, under themulti-state perfect phylogeny, or infinite alleles model. Our somatic phylogeny reconstruction usingcombinatorial enumeration (SPRUCE) algorithm uses this model to construct phylogenetic trees jointly from single-nucleotide variants (SNVs) and copy-number aberrations (CNAs). We show that SPRUCE addresses complexities in simultaneous analysis of SNVs and CNAs. In particular, there are often many possible phylogenetic trees consistent with the data, but the ambiguity decreases considerably with an increasing number of samples. These findings have implications for tumor sequencing strategies, suggest caution in drawing strong conclusions based on a single tree reconstruction, and explain difficulties faced by applying existing phylogenetic techniques to tumor sequencing data.

Detection of Novel Genomic Markers for Predicting Prognosis in Hepatocellular Carcinoma Patients by Integrative Analysis of Copy Number Aberrations and Gene Expression Profiles: Results from a Long-Term Follow-Up.

The aim of this study was to explore novel genomic biomarkers predicting hepatocellular carcinoma (HCC) prognosis by integrative analysis of DNA copy number aberrations (CNAs) and gene expression profiles. Array comparative genomic hybridization and expression array were performed on 45 and 31 HCC samples, respectively. To identify functionally important genes, concordant results of DNA copy number and gene expression were retrieved by integrative analysis. Cox regression analysis indicated that the CNAs in 192 genomic regions were significantly associated with overall survival (OS; p < 0.05). Integrative analysis capturing concordant results demonstrated that the low expression of TLE4 (p = 0.041) and XPA (p = 0.006) was associated with poor OS. In the analysis of tumor recurrence, 514 genomic regions with CNAs were associated with recurrence. Integrative analysis revealed that the overexpression of 16 genes, including FGR (p = 0.003), RELA (p = 0.049), LTBP3 (p = 0.050), and RIN1 (p = 0.023), was significantly associated with shorter time to tumor recurrence. On multivariate analysis, FGR and XPA were independent risk factors of early recurrence and poor OS, respectively. Integrated analysis of CNAs and gene expression profiles correlated with long-term follow-up data successfully identified potential prognostic markers predicting survival and tumor recurrence in patients with HCC who underwent surgical resection.

GENO-27GENOMIC CHARACTERIZATION OF PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA REVEALS PREDOMINANT NONSYNONYMOUS SOMATIC MUTATIONS OF MYD88 AND PIM1 GENES

Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to CNS. Despite low tendency of systemic dissemination, its prognosis is poor with median overall survival of 4 years when treated with standard of care high-dose methotrexate-based chemoradiotherapy. Despite majority of PCNSL is diffuse large B-cell lymphoma (DLBCL), its response to therapy and biological behavior differ from the systemic disease, necessitating detailed investigation of its pathogenesis and genetic alterations. Here we performed whole-exome sequencing (WES) on 41 PCNSL and paired normal specimens as well as RNA sequencing. Seven cases were classified as germinal center B-cell-like (GCB) subtype (17%), while 34 cases were non-GCB. Progression-free survival was significantly longer in cases with GCB subtype as in systemic DLBCL (p = 0.027). WES yielded sequencing data with a mean coverage of 158Χ with 98% of the targeted bases covered by ≥20 independent reads. We identified a total of 17,385 somatic mutations, of which 62% were nonsynonymous single nucleotide variations. The predominant nucleotide substitution was a C > T transition (57.8%), especially in the context of the sequence GCG. WES revealed a substantial extent of aberrant somatic hypermutation (aSHM) with a high frequency of nonsynonymous somatic mutations especially in PIM1 (90.2%) and BTG2 (65.9%). Genes mutated other than aSHM include MYD88 (85.4%), most of which being L265P activating mutation. These mutations were not associated with GCB/non-GCB subtypes. Notably, mutations in a number of genes related to the NF-κB signaling pathway were also prominent. Copy number aberration analysis identified gains in chromosomes 1q, 7, 12, and 18q, and a loss of 6q. These results suggest that MYD88 and PIM1 mutations may play an important role in PCNSL development regardless of GCB subtypes, and NF-κB pathway could be the therapeutic targets of this intractable disease.

BIRC3 disruption and Copy Number Aberrations in Chronic Lymphocytic Leukemia (CLL) Patients with 11q Deletion

Introduction. Chronic lymphocytic leukemia (CLL) with 11q deletion has been associated to a poor prognosis, but the clinical course of patients carrying this lesion is variable. This aberration, most often monoallelic, is present in 10-17% of newly diagnosed CLL and in 20-30% of patients with progressive or chemorefractory disease. The minimal deleted region (MDR) (2-3 Mbp) is located on the 11q22.3-q23.1 region and includes ATM. Moreover, 30-40% of 11q- CLL have also an inactivating ATM mutation on the other allele. The deleted region on 11q can also include BIRC3, a gene that is often deleted or mutated in advanced/chemorefractory stages of the disease. Although BIRC3 disruption has been associated to a poor prognosis, its prognostic implications in addition to ATM deletion are not well defined. The aim of this study was to perform a copy number aberration (CNA) and gene sequencing analyses on a cohort of CLL patients with 11q- in order to identify subgroups with potential prognostic relevance based on: i) the inclusion of BIRC3 in the deleted region; ii) the presence of BIRC3 mutation; iii) the presence of other CNAs.Methods. The study has included 55 untreated CLL patients followed at our Institution or enrolled in GIMEMA clinical trials (2003-2013). Genomic DNA was extracted from peripheral blood samples. CNA analysis was performed by genomic hybridization on the CytoScan HD array (Affymetrix), which contains more than 2.6 x 106 markers for copy number analysis and 750.000 SNPs. Data were analyzed using both Partek Genomics Suite and ChAS (Chromosome Analysis Suite, Affymetrix) software. The resulting CNAs were verified by visual examination of the plotted copy number profiles. BIRC3 mutations (exons 6-9) were evaluated by Sanger sequencing. Time to first treatment (TFT) was calculated from the date of diagnosis to the date of first therapy or last follow-up; progression-free survival (PFS) from the date of first therapy to the date of progression, death or last follow-up.Results. Baseline characteristics of the 55 cases were as follows: median age at diagnosis 59 years (range 39-84), male gender in 81.8% of patients, progressive disease in 62%. All patients showed 11q- by FISH (median 80%, range 25-99% of nuclei); germline IGHV were present in 96.4% of cases; TP53 deletion in 1 case and TP53 mutation in none; NOTCH1 mutation in 4/40 cases; SF3B1 mutation in 5/40 (all mutually exclusive with only 1 case having both SF3B1 and BIRC3 mutations). By CytoScan HD array, the size of 11q- was very variable, ranging from 0.36 Mbp to 65.14 Mbp; the MDR was located on 11q22.3 region, encompassing 4 genes (ACAT1, ATM, CUL5, NPAT). BIRC3 was included in the deleted region in 45/55 cases (81.8%) and was mutated in 4/54 (7.5%), being always deleted on the other allele. Beside 11q-, 51 cases (92.7%) showed several additional CNAs (average 4.9, range 1-14 per patient), with 5 recurrent lesions: 2p gain in 11 cases, del4(p15.2) in 6, del19(p13.3) in 6, 8q gain in 5 and del4(q22.1) in 4. BIRC3 deletion was not associated to the number of additional CNAs nor to specific CNA. After a follow-up of 59.6 months (range 7.4-229.7), 40 of 47 evaluable patients have received treatment (median TFT 15.8 months, range 0-167). BIRC3 deleted cases (n=37) showed a TFT not significantly different from WT cases (n=10). Conversely, BIRC3 mutation was associated to a shorter TFT (p <0.0001), 0.5 vs 19.3 months of WT cases. Notably, 3/4 cases with the biallelic BIRC3 disruption had a marked hyperleukocytosis at diagnosis (212.4-302.8 x 109L). The presence of additional CNAs was associated to a shorter, though not significant, TFT considering either >3 CNAs larger than 5 Mb (n=14) or >10 CNAs (n=5), or the presence of 2p gain, del4(p15.2), del19(p13.3) or 8q gain. So far, 22 patients have been evaluated for PFS after first-line therapy (median 28.7 months). BIRC3 deleted cases (n=17) were not associated to a shorter PFS compared to WT cases (n=5), in line with the results from Rose-Zerilli et al (Haematologica 2014).Conclusions. Among CLL with 11q-: 1) BIRC3 deletion involves more than 80% of cases, whilst the mutation is rare (7.5%); 2) BIRC3 deletion is not associated to a higher genomic complexity; 3) BIRC3 deletion does not seem to influence TFT or PFS of 11q- CLL; 4) BIRC3 mutation is strongly associated to a short TFT; 5) BIRC3 biallelic lesions can be associated to a marked hyperleucocytosis at diagnosis and immediate need of treatment. DisclosuresNo relevant conflicts of interest to declare.

340P - Microarray Analysis of Dna Copy Number Aberrations in Breast Tumor: Correlation with the Efficacy of Neoadjuvant Chemotherapy

ABSTRACT Aim: Neoadjuvant chemotherapy (NAC) is intensively used for the treatment of primary breast cancer to downstage tumors allowing breast-conserving surgery to be performed and hence to improve breast cancer (BC) survival. To find novel effective markers to predict NAC efficacy, we performed a combined analysis of copy number aberration (CNA) and deletion of multidrug resistance (MDR) gene loci in tumor DNA in relation to clinical response to NAC in BC patients. Methods: CNA and deletions of MDR gene loci in biopsy specimens, collected before treatment were tested using high-density microarray platform CytoScanTM HD Array (Affymetrix, USA). 68 BC patients were treated with 4 courses of anthracycline-based neoadjuvant chemotherapy. The immediate tumor response to NAC was measured in accordance with WHO recommendations. Results: In breast tumors, structural CNA in 1q, 8p, 8q, 11q, 16q, 17p chromosomal regions were observed most frequently. 1q43 amplification correlated with good response to NAC. Partial tumor regression was observed in 83% (33/39) of patients with 1q43 amplification, whereas no response to NAC was found in 78% (18/23) of patients with normal 1q43 region (р = 1.18x10-6, Fisher's test,). Amplification in 1q43 along with deletion in 18р11.21 loci resulted in partial tumor regression in 92% (35/38) of patients. In the case of the normal status of these loci, 81% (21/26) of patients had no response to NAC (р = 2.26x10-9). All patients with deletions of АВСВ1, АВСВ3, ABCC5 gene loci –7q21.1, 6p21.32, 3q27 correspondingly, and most patients having deletions in АВСС1 (16p13.1), АВСС2 (10q24), ABCG1 (21q22.3), ABCG2 (4q22.1), responded favorably to NAC. Conclusions: The presence of unbalanced chromosome abnormalities in 1q, 18p loci and the loss of chromosomal loci containing the MDR gene within breast tumors correlates with good response to NAC and can be considered as a promising marker for predicting response to neoadjuvant chemotherapy with high confidence value. Acknowledgments: RFBR project 13-04-98111 р_sibir_а. Disclosure: All authors have declared no conflicts of interest.

O2–049 - Analysis of Copy Number Aberration in Breast Cancer

Background: Chromosomal aneuploidy and segmental copy number changes (deletions and duplications) are a source of human diversity. This is called copy number variation (CNV). CNVs are contained in genomes of healthy individuals. And it has also revealed that some CNVs are related to disease, including several cancers. A number of studies have revealed the association various types of CNVs and breast cancer. In this study, we aimed to identify true somatic copy number change in breast cancer patients by comparing to the germline DNA of corresponding patients.

Abstract B017: A targeted sequencing approach to characterize the mutational profiles of 178 genes in 2600 primary breast tumors

Abstract The heterogeneity of breast cancer has now been demonstrated in a number of large-scale genomics projects, and many recent efforts have focused on describing subgroups of patients by identifying distinguishing biological features that may also be relevant for use in the clinic. Recently, as part of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), we have performed an integrated analysis of copy number aberrations (CNA) and gene expression profiles for almost 2000 primary tumors, and have identified ten robust Integrative Clusters (IntClusts) with distinct genomic properties. To further refine these IntClusts, we have undertaken a targeted sequencing approach to investigate the mutational profiles of 178 breast cancer-related genes selected based on their appearance in previous sequencing projects as putative drivers. We have used Illumina's transposase-based Nextera method to prepare libraries for sequencing, with custom oligos used for in-solution capture of the exons within the targeted genes. Overall, targeted sequencing with an average coverage depth of 200X was performed for 2,600 primary tumors, of which over 550 had matched normal DNA extracted from either adjacent breast tissue or peripheral blood lymphocytes. The GATK pipeline was used for data preprocessing, with custom scripts used for calling single nucleotide variants and short indels, and to classify their somatic status. We have also determined the copy number status of the target genes in order to obtain a complete platform-consistent characterization of the tumors. Independent expression and SNP6.0 data are available for the majority of samples, while a minimum of 5 years of clinical follow-up data are available for all patients. We are also performing whole exome and targeted RNA sequencing for a subset of the tumors. Our findings so far have both corroborated and added to our knowledge of the breast cancer genome. For example, mutation frequencies of approximately 30% in PIK3CA and TP53 are observed in the overall population; however, the additional resolution provided by the IntClusts has allowed us to identify a subgroup of ER+ tumors that has a lower PIK3CA mutation rate, but a higher number of TP53 mutations (IntClust6; 11% with PIK3CA mutations, 47% with TP53 mutations), compared to other ER+ clusters (e.g. IntClust3; 64% with PIK3CA mutations, 10% with TP53 mutations). Furthermore, there is evidence to suggest that PIK3CA mutations may be associated with survival in this subgroup, even though the gene's prognostic power is otherwise mainly limited to ER- tumors. PIK3CA mutation status also appears to have prognostic value in IntClust4, a CNA-devoid subtype with low genomic instability. We have also investigated a number of other genes in terms of their mutation frequencies and mutation classes, and have identified other candidates that may potentially help refine classification according to the IntClusts. These initial findings illustrate the power of complete genomic characterization of a large number of tumors with detailed long-term clinical information, and demonstrate the utility of the IntClust system in distinguishing between tumors with what may be subtle biological differences. Moreover, we believe that this comprehensive resource will be of great use for the breast cancer field in answering questions that are of both biological and clinical relevance. Citation Format: Bernard Pereira, Suet-Feung Chin, Oscar M. Rueda, Michelle Osborne, Helen Northern, John Peden, Sarah-Jane Dawson, Jean Abraham, METABRIC Group, NeoTango Group, Mark Ross, David Bentley, Carlos Caldas. A targeted sequencing approach to characterize the mutational profiles of 178 genes in 2600 primary breast tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B017.

Abstract 5473: Integrative approach towards the functionality of CIAO1 gene

Abstract Previous genome-wide analysis of copy number aberrations in oral squamous cell carcinoma (SCC) and oral epithelial dysplasia, a pre-malignant lesion, by array CGH has revealed that the genomes of these cancers and pre-cancers frequently harbor narrow (∼3Mb) regions of amplification on chromosome 2q11 region. Overexpression of CIAO1, a putative cytosolic iron-sulfur protein within this region, has been observed in patients’ cancerous samples and oral SCC lines. Knockdown of CIAO1 protein by RNAi results in growth retardation in oral SCC lines. Therefore, three HaCaT cell clones with an inducible Tet-on CIAO1 gene have been established to unravel the nature of the CIAO1 signaling network. Through the triangulation of protein-protein interactions, quantitative PCR array and transcription factor reporter array, we have investigated the early effects of CIAO1 overexpression in immortalized HaCaT cells, which may add to the understanding of oral cancer tumorigenesis. 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 5473. doi:1538-7445.AM2012-5473

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization

BackgroundIt has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH).MethodsTumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested.ResultsThe mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers.ConclusionsThus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A-bomb radiation may affect the increased amount of CNA as a hallmark of GIN and, subsequently, be associated with a higher histologic grade in breast cancer found in A-bomb survivors.

High-Resolution Genomic Profiling of Adult and Pediatric Core Binding Factor Acute Myeloid Leukemia Reveals New Recurrent Genomic Aberrations

AbstractAbstract 849Core-binding-factor (CBF) acute myeloid leukemia (AML) defined by the presence of t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) is associated with favorable outcome. However, about 30–40% of patients are not cured by current treatment approaches. Secondary genetic changes are believed to cause clinical heterogeneity. To identify new secondary genetic lesions, we performed high-resolution, genome-wide analysis of copy number aberrations (CNA) and copy neutral loss of heterozygosity (CN-LOH) using Affymetrix 6.0 single nucleotide polymorphism (SNP) microarrays in 300 adult and pediatric CBF AMLs; t(8;21), n=157 (adult, n=114; pediatric, n=43); and inv(16), n=143 (adult, n=104; pediatric, n=39). Germline control DNA from remission bone marrow or peripheral blood was available for paired analysis in 175 patients. In addition, for 42 patients matched relapse samples were analyzed. Data were processed using reference alignment, dChipSNP and circular binary segmentation. Paired analysis revealed a median of 1.28 somatic CNAs per case [t(8;21): 1.14, range: 0–5; inv(16): 1.45, range: 0–9], with deletions more common than gains [t(8;21): 0.94 losses/case vs. 0.2 gains/case; inv(16): 0.95 vs. 0.5]. Recurrent deletions were detected at chromosomal bands 7q36.1 (n=23), 9q21.13 (n=15), 11p13 (n=7), 17q11.2 (n=6), 10q24.32 (n=2), and at the chromosomal breakpoints of t(8;21) and inv(16) on 8q21.3 (n=9), 21q22 (n=16), 16p13.11 (n=28), and 16q22.11 (n=21). Deletions at 7q, 11p and 17q were validated using FISH analysis. Minimally deleted regions (MDR) less than 1.5 Mb were identified at 7q36.1 (647 Kb, 4 genes), 9q21.13 (1125 Kb, 9 genes), 11p13 (130 Kb, 1 gene), and 17q11.2 (902 Kb, 11 genes), with each region containing a putative tumor suppressor (e.g., MLL3 on 7q, FRMD3 on 9q, WT1 on 11p, and NF1 on 17q). Sequence analysis of MLL3 in 23 cases with del(7q), 1 case with 7q CN-LOH, and 23 randomly selected cases identified a MLL3 truncating mutation leading to a premature stop codon in a case that lacked a 7q alteration. The del(11p13) contained only WT1 and primarily affected inv(16)-cases (5 of 7). Sequence analysis of WT1 in four cases with del(11p) revealed an additional frame shift mutation in the remaining allele in one case. Sequence analysis of WT1 in an additional 103 inv(16)-containing cases revealed mutations in 10 (9%) of the cases. The MDR at 17q11.2 was exclusively identified in inv(16)-containing cases (n=6) and included the tumor supressor NF1. Sequence analysis of all coding exons in NF1 in 4 additional inv(16)-containing AMLs revealed no additional mutation. Recurrent gains were identified at 22q11.21-q13.33 (n=20; 32 Mb), 8q24.21 (n=14; 138 Kb), 13q21.1-q34 (n=6; 14 Mb), and 11q25 (n=5; 368 Kb). The smallest gains were identified at 8q24.21 and 11q25, both containing only a single non-coding RNA gene (CCDC26 and LOC283177, respectively). Somatic CN-LOH were uncommon and only found at 1p36.33-p12 (n=2), 4q (n=2), and 19p (n=2). In a comparative analysis of paired diagnostic and relapse samples, novel CNAs at the time of relapse were identified at 3q13.31 (n=5), 5q (n=2), 17p (n=2), and 17q (n=2). The MDR at 3q was only 46 kb in size and contains a single transcript that has been connected to LSAMP, a putative tumor suppressor located 404 Kb upstream of the deletion. In summary, our data provide a comprehensive profiling of copy number alterations in pediatric and adult CBF AML. These data demonstrate a very low number of CNAs, with no significant differences noted between pediatric and adult cases. Interestingly, a number of novel recurrent secondary genetic alterations are identified. Exploring the biological role of these lesions in leukemogenesis and drug resistance should provide important insights into the CBF leukemias. Disclosures:No relevant conflicts of interest to declare.

Abstract 2133: Identification of driver genes for amplification of the narrow amplicon at 2q11 present in oral cancers and pre-cancers

Abstract Genome-wide analysis of copy number aberrations in oral squamous cell carcinoma (SCC) and oral epithelial dysplasia, a pre-malignant lesion, by array CGH has revealed that the genomes of these cancers and pre-cancers frequently harbor narrow (∼3 Mb) regions of amplification. These amplicons are recurrent, but rare, being present in &amp;lt;10% of cases. Nevertheless, candidate driver genes in the amplicon are often up-regulated in tumors without amplification. Thus, these narrow amplicons highlight genes/pathways that are de-regulated in oral cancer. A narrow amplicon at 2q11 has been observed in 3/44 oral dysplasia cases, in 1/89 oral SCC and one primary oral SCC cell line. In one patient with synchronous mild and moderate dysplasia lesions, the 2q11 amplicon was uniquely present in moderate dysplasia, suggesting that the amplified and overexpressed gene(s) at 2q11 contributed to progression. The 2q11 amplicon spans 3.5 Mb and includes 43 genes. Examination of two independent head and neck cancer gene expression datasets retrieved from the NCBI Gene Expression Omnibus database revealed up-regulation of 11 of these genes in one or both datasets. All 11 genes are overexpressed in oral SCC lines SCC4, SCC9 and HSC3. Knockdown of individual genes in a panel of eight oral SCC cell lines was performed with one or more siRNA's targeting each of the genes. These studies revealed that cellular proliferation and morphology were significantly disrupted in cells transfected with siRNA to two of the genes, suggesting there are two potential driver genes for the 2q11 amplicon. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2133.

Combfit: A Normalization Method for Array CGH Data

The recently developed array-based comparative genomic hybridization(array CGH) technique measures DNA copy number aberrations that occur as causes or consequences of cell diseases such as cancers. Conventional array CGH analysis classifies DNA copy number aberrations into three categories: no significant change, significant gain, and significant loss. However, recent improvements in microarray measurement precision enable more quantitative analysis of copy number aberrations. We propose a method, called comb fitting, that extracts a quantitative interpretation from array CGH data. We also propose modifications that allow us to apply comb fitting to cases featuring heterogeneity of local aberrations in DNA copy numbers. By using comb fitting, we can correct the baseline of the fluorescence ratio data measured by array CGH and simultaneously translate them into the amount of changed copy numbers for each small part of the chromosome, such as 0, ±1, ±2, ···. Comb fitting is applicable even when a considerable amount of contamination by normal cells exists and when heterogeneity in the ploidy number cannot be neglected.