Patterns Of Copy Number Alterations Research Articles

Differential copy number alteration profiles among gynecological malignancies.

e14744 Background: Copy number alterations (CNA) constitutes a genomic hallmark that defines specific outcomes in gynecological neoplasms, as evidenced by the integrated analysis of the high-grade serous ovarian carcinoma (HGSOC) and endometrial carcinomas (EC) performed by the TCGA Consortium. The purpose of this study is to set up an NGS-based technology to obtain CNA profiles on formalin-fixed paraffin-embedded (FFPE) samples with clinical utility. Methods: FFPE blocks from 14 EC and 12 HGSOC were analyzed using a SureSelectXT custom panel plus OneSeq backbone, interrogating 147.000 SNPs (Agilent). CNA groups were defined in EC with a RF algorithm based on genotyping data as previously reported (Romero, ASCO 2018) and in HGSOC Homologous recombination deficiency (HRD) was determined by sequencing a serie of 35 HR genes. saasCNV pipeline was used to perform CNA calling. Results: Ten (38.50 %) of our cases were classified as CN Low (CNL), 8 as CN high (CNH)-RF (30.75 %) and 8 CNH-HRD (30.75 %). CNL profiles from EC and OC are homogenous in terms of genomic stability. CNH-RF and CNH-HRD cases harbor the highest percentage of genomic instability (p = 0.012) with distinct patterns of CNA. CNH-HRD is characterized by a higher ratio of genomic losses (p = 0.023). However, CNH-RF is mainly defined by gains (p = 0.011) (Table). Conclusions: Our NGS-based approach represents a FFPE clinically available procedure to define CNA alterations in gynecological cancer with distinct CNH patterns with prognostic potential.[Table: see text]

Genomic analysis of Korean patients with advanced prostate cancer by use of a comprehensive next-generation sequencing panel and low-coverage, whole-genome sequencing

PurposeTo analyze the characteristics of somatic mutations and copy number alterations (CNAs) in Korean patients with advanced prostate cancer (PCa) by use of the Oncomine Comprehensive Panel (ThermoFisher Scientific) and low-coverage, whole-genome sequencing (LC-WGS).Materials and MethodsWe retrospectively analyzed PCa tissues obtained from 14 patients with advanced PCa (metastatic tumor, 12 [85.7%]; nonmetastatic castration-resistant PCa, 1 [7.1%]; pT3b, 1 [7.1%]) from 2009 to 2017. The Oncomine Comprehensive Panel included a total of 143 genes. Moreover, LC-WGS was performed to detect CNAs of the entire genome. Two plasma samples matched with tumor tissues were analyzed using LC-WGS to compare the chromosomal aberration patterns between circulating tumor DNA and tumor tissue.ResultsGenetic alterations were most frequently observed in the androgen receptor (AR) (42.9%, n=6/14), TP53 (14.3%, n=2/14), and PTEN (14.3%, n=2/14) genes in the Oncomine panel. AR amplification was the most common CNA (35.7%, n=5/14). As a result of LC-WGS, CNAs were confirmed in about 92.9% (n=13/14) of the samples in regions Xq12, 8q24.21, and 11q13.3 (gains) and in regions 6q16.1, 8p23.1, 10q25.1, 16q24.2, 18q12.3, Xq25, and Xq26.3 (losses). All CNAs identified in the Oncomine panel matched the results of LC-WGS. Additionally, LC-WGS of two plasma samples that matched tumor tissues revealed that CNA patterns of plasma samples (circulating tumor DNA) were very similar to those detected in tumor samples.ConclusionsOur data showed that the characteristics of mutations and CNAs in Korean patients with advanced PCa were similar to those observed in previous studies.

The FRA14B common fragile site maps to a region prone to somatic and germline rearrangements within the large GPHN gene.

Common fragile sites (cFSs) represent parts of the normal chromosome structure susceptible to breakage under replication stress. Although only a small number of cFSs have been molecularly characterized, genomic damage of cFS genes appears to be critical for the development of various human diseases. In this study, we fine mapped the location of FRA14B and showed that the fragile region spans 765 kb at 14q23.3, containing the large gephyrin (GPHN) gene. The FRA14B sequence is enriched in perfect A/T>24 stretches and R-loop forming sequences (RLFS), and harbors a large palindromic motif in the core region. FRA14B instability is not only limited to lymphocytes, but also occurs in neuroblastoma and breast epithelial cells. Using array comparative genomic hybridization (CGH), we examined copy number alteration patterns within FRA14B in a panel of 180 cancer cell lines and primary tumors. Our CGH data and a survey of 1046 Cancer Cell Line Encyclopedia profiles demonstrate that focal deletions cluster within FRA14B and disrupt the genomic integrity of GPHN in approximately 5% of cancer cells. Moreover, germline CNVs (copy number variants) profiles provided by the Database of Genomic Variants and available literature suggest that germline CNVs and rare pathogenic deletions associated with neurodevelopmental disorders cluster within the core fragile region of GPHN. Overall, our data provide insight into the molecular structure of FRA14B, and identify GPHN, as a large cFS gene in the human genome, whose disruption appears to trigger various neurodevelopmental diseases.

Patterns of copy number alterations in primary breast tumors of South African patients and their impact on functional cellular pathways.

Breast cancer is the most common and the leading cause of female mortality among South African (SA) women. Several non‑biological and biological risk factors may be attributed to their observed high mortality rate; however, the molecular profiles associated with their breast tumors are poorly characterized. The present study examined the patterns of genome-wide copy number alterations (CNAs) and their potential impact on functional cellular pathways targeted by cancer driver genes in patients with breast cancer from the Western Cape region of SA. Array-comparative genomic hybridization analysis, performed in 28 cases of invasive breast cancer, revealed a mean number of 8.68±6.18 CNAs per case, affecting primarily the Xp22.3 and 6p21-p25 cytobands (57.14% of the cases), followed by 19p13.3-p13.11 (35.7%), 2p25.3-p24.3, 4p16.3-p15.3, 8q11.1-q24.3 and 16 p13.3-p11.2 (32.14%). Functional enrichment analysis of genes and microRNA targets mapped in these affected cytobands revealed critical cancer-associated pathways, including fatty acid biosynthesis and metabolism, extracellular matrix-receptor interaction, hippo and tumor protein p53 signaling pathways, which are regulated by known cancer genes, including CCND1, CDKN1A, MAPK1, MDM2, TP53 and SMAD2. An inverse correlation was observed among the number of CNAs and tumor size and grade; CNAs on the 4p and 6p cytobands were also inversely correlated with tumor grade. No association was observed in the number of CNAs and/or the affected cytobands and the different ethnic groups of the SA patients, indicating that their tumor genome is affected by CNAs, irrespectively of their genetic descent. Additional genomic tumor profiling in SA and other Sub-Saharan African patients with breast cancer is required to determine the associations of the CNAs observed with prognosis and clinical outcome.

MS05.01 Staging and Pathology of Multiple Lung Nodules

MS05.01 Staging and Pathology of Multiple Lung Nodules

Molecular heterogeneity in human papillomavirus-dependent and -independent vulvar carcinogenesis.

Vulvar squamous cell carcinoma (VSCC) and precancerous vulvar intraepithelial neoplasia (VIN) can develop through human papillomavirus (HPV)‐dependent and ‐independent pathways, indicating a heterogeneous disease. Only a minority of VIN progress, but current clinicopathological classifications are insufficient to predict the cancer risk. Here we analyzed copy number alterations (CNA) to assess the molecular heterogeneity of vulvar lesions in relation to HPV and cancer risk. HPV‐status and CNA by means of whole‐genome next‐generation shallow‐sequencing were assessed in VSCC and VIN. The latter included VIN of women with associated VSCC (VINVSCC) and women who did not develop VSCC during follow‐up (VIN no VSCC). HPV‐testing resulted in 41 HPV‐positive (16 VINVSCC, 14 VIN no VSCC, and 11 VSCC) and 24 HPV‐negative (11 VINVSCC and 13 VSCC) lesions. HPV‐positive and ‐negative VSCC showed a partially overlapping pattern of recurrent CNA, including frequent gains of 3q and 8q. In contrast, amplification of 11q13/cyclinD1 was exclusively found in HPV‐negative lesions. HPV‐negative VINVSCC had less CNA than HPV‐negative VSCC (P = .009), but shared chromosome 8 alterations. HPV‐positive VIN no VSCC had less CNA than VINVSCC (P = .022). Interestingly, 1pq gain was detected in 81% of HPV‐positive VINVSCC and only in 21% of VIN no VSCC (P = .001). In conclusion, HPV‐dependent and ‐independent vulvar carcinogenesis is characterized by distinct CNA patterns at the VIN stage, while more comparable patterns are present at the cancer stage. Cancer risk in VIN seems to be reflected by the extent of CNA, in particular chromosome 1 gain in HPV‐positive cases.

Abstract 4267: Patterns of DNA copy-number alterations revealed by the GSVD and tensor GSVD encode for cell transformation and predict survival and response to platinum in adenocarcinomas

Abstract A quarter or more of lung, uterine, and ovarian adenocarcinoma (LUAD, USEC, and OV) tumors are resistant to platinum, the first-line systemic treatment for decades. Only recently, however, patterns of copy-number alterations (CNAs) were discovered that are predictive of OV survival, both in general and in response to platinum, and only by using a comparative spectral decomposition, the tensor generalized singular value decomposition (GSVD). We defined the tensor GSVD to extend the GSVD from two matrices to two tensors. Like the GSVD, the tensor GSVD can simultaneously identify the similar and dissimilar between two datasets recording different aspects of a single phenomenon (1, 2). In a comparison of Agilent microarray platform-matched profiles of patient-matched normal and primary, mostly high-grade OV DNA, the tensor GSVD revealed the tumor-exclusive and platform-consistent patterns across the chromosome arms 7p and Xq and the combination of 6p+12p. No other diagnostic can distinguish a shorter from a longer survival in response to platinum throughout the course of an adenocarcinoma. Here we show that the 6p+12p genotype predicts a survival phenotype in LUAD and USEC in addition to OV. We use the GSVD to compare whole-genome sequencing (WGS) and Affymetrix microarray profiles of patient-matched normal and primary LUAD and primary, mostly high-grade USEC and OV DNA. The GSVD reveals tumor-exclusive patterns of CNAs across the combination of the chromosome arms 6p+12p. We find that, first, like the Agilent OV pattern, the WGS LUAD and Affymetrix LUAD, USEC, and OV patterns identify a biologically consistent shorter survival phenotype, of approximately one and a half years median survival time in LUAD and USEC and three years in OV. Like the Agilent pattern, the WGS and Affymetrix patterns describe a biologically consistent genotype, where loss of the p21-encoding CDKN1A and p38-encoding MAPK14 on 6p, and gain of KRAS on 12p, together, but not separately, encode for human cell transformation. Second, by classifying the WGS and Affymetrix profiles, the Agilent pattern proves to be a technology-independent predictor of a LUAD, USEC, or OV patient's survival, both in general and in response to platinum. The pattern is independent of the tumor's stage, the best indicator of these adenocarcinomas at the time of initial diagnosis. Third, the pattern is a predictor of OV survival and response to platinum beyond the time of initial diagnosis and throughout the disease, even in patients experiencing remission after treatment of the primary tumor, and independent of the time to recurrence or progression. We conclude that comparative spectral decompositions, such as the GSVD and tensor GSVD, underlie a mathematically universal description of the relations between a tumor's genome and a patient's survival and response to treatment, which other methods miss.

Abstract 5050: Molecular heterogeneity in HPV-dependent and -independent vulvar carcinogenesis: Chromosome 1 gain reflects the cancer risk in vulvar intraepithelial neoplasia

Abstract Vulvar squamous cell carcinoma (VSCC) and precancerous high-grade vulvar intraepithelial neoplasia (VIN) can develop through human papillomavirus (HPV)-dependent and -independent pathways, indicating a heterogeneous disease. Only a minority of VIN progress, but current clinical and histological classifications are insufficient to predict the cancer risk. Here we analyzed copy number alterations (CNA) to assess the molecular heterogeneity of vulvar lesions in relation to HPV and cancer risk. HPV-status and CNA by means of whole-genome next-generation shallow-sequencing were assessed in 24 VSCC and 41 VIN lesions. The latter included VIN of women with associated VSCC (VINVSCC) and women who did not develop VSCC during >10 year follow-up (VINnoVSCC). HPV-testing resulted in 41 HPV-positive lesions (16 VINVSCC, 14 VINnoVSCC and 11 VSCC) and 24 HPV-negative lesions (11 VINVSCC and 13 VSCC). HPV-positive and -negative VSCC showed a partially overlapping pattern of recurrent CNA, including frequent gains of 3q and 8q. In contrast, amplification of 11q13/cyclinD1 was exclusively found in HPV-negative lesions. HPV-negative VINVSCC had less CNA than VSCC (P = 0.009), mainly 8q gains and 8p losses. In HPV-positive lesions, CNA frequencies were lower in VINnoVSCC than in VINVSCC (P = 0.022) and VSCC. Interestingly, a 1pq gain was detected in 81% of VINVSCC and only 29% of VINnoVSCC. In conclusion, HPV-dependent and -independent vulvar carcinogenesis is characterized by distinct CNA patterns at the VIN stage, while more comparable patterns are present at the cancer stage. Progression risk in VIN is reflected by the extent of CNA, in particularly chromosome 1 gain. Citation Format: Maaike CG Bleeker, Dorian RA Swarts, Quirinus JM Voorham, Saskia M. Wilting, Marc van Beurden, Renske DM Steenbergen. Molecular heterogeneity in HPV-dependent and -independent vulvar carcinogenesis: Chromosome 1 gain reflects the cancer risk in vulvar intraepithelial neoplasia [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 5050.

Abstract 3112: Distinct oncogenic events induce different DNA methylation and copy number changes in human mammary epithelial cells

Abstract Gene expression differences, combined with distinct patterns of genomic rearrangements and epigenetic modifications constitute the bases of molecular classification of breast cancer. Molecular subtypes may originate from different cell lineages in the mammary gland, but also from the early activation of oncogenes that may drive the establishment of these molecular subtypes. However, in the natural history of human cancer, it is difficult to discriminate between these two factors : cell lineage and initial oncogenic alterations. In this work, we designed an experimental strategy aiming at determining whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. We show that initial activation of CCNE1, WNT1 and RASv12 in shp53 immortalized HMECs results in different and reproducible profiles of mRNA and miR expression, copy number alterations (CNA) and DNA methylation modifications. Interestingly, first the extend of CNA measured as fraction of the genome altered was lower in HMECs transformed by RAS than CCNE1 and WNT1 transformed HMECs revealing a lower genetic instability. This was confirmed by less numerous γH2Ax and 53BP1 nuclear foci in HMECs transformed by RAS. Second, HMECs transformed by RAS bore specific profiles of CNAs and DNA methylation, clearly distinct of those shown by CCNE1 and WNT1 transformed HMECs. Genes differentially expressed in the RAS and the CCNE1/WNT1 clusters and included in CNAs or with variable CpG methylation were mostly different, depicting the activation of distinct signaling pathways in these two clusters. These data indicate that early activation of distinct oncogenic pathways may leads to adaptive events resulting in specific pattern of CNAs and DNA methylation changes. We postulated that the early activation of oncogenes may be an important determinant of the establishment of breast cancer molecular subtypes along with cell lineage origin. Citation Format: Stan P. du manoir, Claire Fonti, Anne Saumet, Amanda Abi-Khalil, Béatrice Orsetti, Cleroux Elouan, Ambre Bender, Michael Dumas, Jacques Colinge, Michael Weber, Charles Theillet. Distinct oncogenic events induce different DNA methylation and copy number changes in human mammary epithelial cells [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 3112.

Abstract 4262: Mathematically universal and biologically consistent astrocytoma genotype encodes for transformation and predicts survival phenotype

Abstract Recurring DNA alterations have been recognized as a hallmark of cancer for over a century, and observed in astrocytoma genomes for decades. Only recently, however, a copy-number genotype predictive of an astrocytoma survival phenotype was discovered, and only by using the generalized singular value decomposition (GSVD). We formulated the GSVD as a comparative spectral decomposition that can simultaneously identify the similar and dissimilar between two column-matched matrices, and, therefore, create a single coherent model from two datasets recording different aspects of a single phenomenon (1, 2). In recent comparisons of microarray-profiled patient-matched glioblastoma (GBM) and, separately, lower-grade astrocytoma tumor and normal genomes, the GSVD invariably separated the tumor-exclusive genotype and phenotype from those that occur in the normal genomes and from experimental batch effects. The tumor-exclusive genotype invariably predicted the survival phenotype statistically better than any other indicator of astrocytoma (3). Here we use the GSVD to compare whole-genome sequencing (WGS) read-count profiles of patient-matched astrocytoma tumor and normal DNA. The GSVD uncovers a tumor-exclusive genome-wide pattern of copy-number alterations (CNAs). We find that, first, this WGS astrocytoma pattern is bounded by the microarray GBM and lower-grade astrocytoma patterns. Like the microarray patterns, the WGS pattern is correlated with an approximately one-year median survival time. By filling in gaps in the genome, the WGS pattern reveals that this biologically consistent genotype encodes for transformation via the Notch together with the Ras and Shh signaling pathways. Second, like the GSVDs of the microarray profiles, the GSVD of the WGS profiles separates the tumor-exclusive pattern from normal copy-number variations (CNVs) and from experimental sources of variation. These include the WGS technology-specific effects of guanine-cytosine (GC) content variations across the tumor and normal genomes, which are correlated with experimental batches. Third, by identifying the biologically consistent phenotype among the WGS-profiled tumors, the GBM pattern proves to be a technology-independent predictor of astrocytoma survival and responses to chemotherapy and radiation, statistically better than the patient's age and tumor's grade, the best other indicators of astrocytoma, and existing tests for MGMT promoter methylation and IDH1 mutation. We conclude that by using the complex structure of the data, rather than simplifying them as is commonly done, comparative spectral decompositions, such as the GSVD, underlie a mathematically universal description of the genotype-phenotype relations in cancer that other methods miss.

Comparison of EpCAMhighCD44+ cancer stem cells with EpCAMhighCD44− tumor cells in colon cancer by single-cell sequencing

ABSTRACTCancer stem cells (CSCs) are considered to be responsible for tumorigenesis and cancer relapse. EpCAMhighCD44+ tumor cells are putative colorectal CSCs that express high levels of stem cell genes, while the EpCAMhighCD44− population mostly contains differentiated tumor cells (DTCs). This study aims to determine whether single CSC (EpCAMhighCD44+) and DTC (EpCAMhighCD44−) can be distinguished in terms of somatic copy number alterations (SCNAs). We applied fluorescence-activated cell sorting to isolate the CD45−EpCAMhighCD44+ and CD45−EpCAMhighCD44− populations from two primary colon tumors, on which low-coverage single-cell whole-genome sequencing (WGS) was then performed ∼0.1x depth. We compared the SCNAs of the CSCs and DTCs at single-cell resolution. In total, 47 qualified single cells of the two populations underwent WGS. The single-cell SCNA profiles showed that there were obvious SCNAs in both the CSCs and DTCs of each patient, and each patient had a specific copy number alteration pattern. Hierarchical clustering and correlation analysis both showed that the SCNA profiles of CSCs and DTCs from the same patient had similar SCNA pattern, while there were regional differences in the CSCs and DTCs in certain patient. SCNAs of CSCs in the same patient were highly reproducible. Our data suggest that major SCNAs occurred at an early stage and were inherited steadily. The similarity of ubiquitous SCNAs between the CSCs and DTCs might have arisen from lineage differentiation. CSCs from the same patient had reproducible SCNA profiles, indicating that gain or loss in certain chromosome is required for colon cancer development.

Abstract P2-05-08: Mucinous breast carcinomas: A genomically distinct subtype of estrogen receptor-positive invasive breast cancers

Abstract Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive invasive carcinoma, accounting for up to 2% of breast cancers. MCBs are characterized by clusters of tumor cells floating in lakes of extracellular mucin, and are classified into mucinous A (paucicellular) and mucinous B (hypercellular) subtypes. Some MCBs are found admixed with invasive ductal carcinoma components, and then classified as mixed MCBs. The aims of this study were to determine the repertoire of somatic mutations of MCBs and to ascertain whether these genetic alterations are distinct from those identified in common forms of ER+/HER2- invasive breast cancers (IBCs). We also sought to determine whether the mucinous and ductal components of mixed MCBs would be clonally related. Materials and methods: Thirty MCBs including 25 pure MCBs (n=13 mucinous A, n=12 mucinous B) and five mixed MCBs were microdissected and subjected to whole exome sequencing. Each tumor component of mixed cases was microdissected and analyzed separately. Somatic mutations, copy number alterations and mutational signatures were defined using state-of-the-art bioinformatics methods. The mutational repertoire of MCBs was compared with that of ER+/HER2- IBCs (n = 240) from The Cancer Genome Atlas (TCGA) breast cancer study. Results: The genes most frequently mutated in MCBs were GATA3 (27%, 8/30, all frameshift mutations), KMT2C (13%, 4/30) and MAP3K1 (10%, 3/30). No significant differences were identified in single gene comparisons between mucinous A and mucinous B MCBs or between pure MCBs and the mucinous component of mixed MCBs (Fisher's exact tests, p>0.05). As compared to common forms of ER+/HER2- IBC, MCBs had a lower frequency of PIK3CA mutations (7% vs 42%, p<0.001) and a higher frequency of GATA3 mutations (27% vs 12%, p=0.04). Mucinous B MCBs had a higher frequency of KMT2C mutations than ER+/HER2- IBCs (25% vs 6%, p=0.04). Most MCBs displayed the mutational signature 1 (aging-related; 20/30, 67%), and no differences in the frequency of specific mutational signatures according to the type of MCBs were observed. Concurrent 1q gains and 16q losses, which are the hallmark genetic alterations of low-grade ER+/HER2- breast cancers, were not observed in pure MCBs, but were found in three of the five mixed MCBs analyzed. The mucinous and ductal components of all five mixed MCBs shared a median of 58% of somatic mutations (range 42%-64%), including clonal GATA3 frameshift mutations in two of them, as well as a similar pattern of copy number alterations, supporting their clonal relatedness. Additional somatic mutations found to be restricted to the ductal or mucinous components of all mixed MCBs analyzed were identified, including clonal missense mutations in PIK3C2B and PIK3R2 in the ductal component of one case, and a PIK3R5 missense mutation in the mucinous component of another case. Conclusions: The repertoire of somatic mutations in MCBs is distinct from that of common forms of ER+/HER2- IBCs. These differences include the lack of concurrent 1q gains/16q losses, a lower frequency of PIK3CA mutations and a higher frequency of GATA3 mutations in pure MCBs. Citation Format: Pareja F, Geyer FC, Piscuoglio S, Selenica P, Kumar R, Lim RS, Guerini-Rocco E, Marchio C, Mariani O, Ng CKY, Brogi E, Norton L, Vincent-Salomon A, Weigelt B, Reis-Filho JS. Mucinous breast carcinomas: A genomically distinct subtype of estrogen receptor-positive invasive breast cancers [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 P2-05-08.

MethCNA: a database for integrating genomic and epigenomic data in human cancer

BackgroundThe integration of DNA methylation and copy number alteration data promises to provide valuable insight into the underlying molecular mechanisms responsible for cancer initiation and progression. However, the generation and processing of these datasets are costly and time-consuming if carried out separately. The Illumina Infinium HumanMethylation450 BeadChip, initially designed for the evaluation of DNA methylation levels, allows copy number variant calling using bioinformatics tools.ResultsA substantial amount of Infinium HumanMethylation450 data across various cancer types has been accumulated in recent years and is a valuable resource for large-scale data analysis. Here we present MethCNA, a comprehensive database for genomic and epigenomic data integration in human cancer. In the current release, MethCNA contains about 10,000 tumor samples representing 37 cancer types. All raw array data were collected from The Cancer Genome Atlas and NCBI Gene Expression Omnibus database and analyzed using a pipeline that integrated multiple computational resources and tools. The normalized copy number aberration data and DNA methylation alterations were obtained. We provide a user-friendly web-interface for data mining and visualization.ConclusionsThe Illumina Infinium HumanMethylation450 BeadChip enables the interrogation and integration of both genomic and epigenomic data from exactly the same DNA specimen, and thus can aid in distinguishing driver from passenger mutations in cancer. We expect MethCNA will enable researchers to explore DNA methylation and copy number alteration patterns, identify key oncogenic drivers in cancer, and assist in the development of targeted therapies. MethCNA is publicly available online at http://cgma.scu.edu.cn/MethCNA.

Abstract A182: Apobec-mutagenesis drives mutational heterogeneity while copy number alterations drive transcriptomic and proteogenomic heterogeneity in metastatic lung adenocarcinoma and thymic carcinoma

Abstract Background: Molecular characterization of metastatic tumors to investigate intra- and intertumor heterogeneity has proven more difficult than early stage disease as opportunities for surgical removal are limited. Rapid/warm autopsy programs that are conducted soon after death minimize tissue ischemia, thereby enabling procurement of high-quality viable tissue. To our knowledge, integrated proteo-genomics studies to characterize tumor heterogeneity of metastatic lung adenocarcinoma and thymic carcinoma are lacking. Methods: We sought to characterize and decipher mechanisms of intra- and intertumor heterogeneity of Stage IV lung adenocarcinoma and thymic carcinoma through whole exome sequencing, RNA sequencing, copy number estimation, and mass spectrometry-based proteomics of 38 tumors from five rapid/warm autopsy patients. The autopsy procedure was initiated between 2-4 hours of death. Results: One lung adenocarcinoma (RA003) and one thymic carcinoma (RA006) patient had high mutation burden and extreme mutational (SNV) heterogeneity. This was likely driven by APOBEC-mutagenesis. We constructed phylogenetic trees showing truncal, shared, and private SNVs and copy number alterations (CNAs). Interestingly, we discovered a pattern of recurrent, parallel but opposite arm level CNAs in different metastatic branches within a subset of patients (RA000, RA004, and RA006). In contrast, RA003 had only truncal CNAs and the least heterogeneity at the CNA level. Assessment of intra- and intertumor heterogeneity at the transcriptomic and proteomic levels revealed high concordance in gene expression and protein abundance within tumors from patient RA003 compared to patient RA004, potentially reflecting the downstream effect of CNA vs. APOBEC-induced mutations. Further analysis of cis vs. trans effects of CNA on gene expression and protein abundance in all the metastatic tumors revealed many trans-acting hot spots and limited cis effects, which is contrary to copy number effects on gene expression in primary lung adenocarcinoma tumors. Trans-acting hot spots differed between two major metastatic lineage of patient RA004, suggesting copy number changes may be responsible for high intermetastatic proteomic heterogeneity in this patient. Conclusions: Our results demonstrate ongoing mutational and chromosomal instability throughout the metastatic process of lung adenocarcinoma and thymic carcinoma. APOBEC mutagenesis was likely the primary driver of SNV heterogeneity. However, intertumor heterogeneity of metastatic disease at the level of the transcriptome and proteome occurred in association with CNA rather than mutational heterogeneity. APOBEC mutagenesis and CNA are complementary mechanisms of tumor evolution and heterogeneity affecting tumor metastasis. Citation Format: Nitin Roper, James Gao, Rouf Banday, Tapan Maity, Xu Zhang, Abhilash Venugopalan, Rajesh Patidar, Sivasish Sindiri, Constance Cultraro, Alexandr Goncearenco, Anna Panchenko, Romi Biswas, Jing Wang, Stephen Hewitt, David Kleiner, Bing Zhang, Javed Khan, Ludmila Prokunina-Olsson, Udayan Guha. Apobec-mutagenesis drives mutational heterogeneity while copy number alterations drive transcriptomic and proteogenomic heterogeneity in metastatic lung adenocarcinoma and thymic carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A182.

Abstract A28: Copy number and miRNA profiling in triple-negative breast cancer patients from Latin America

Abstract Epidemiologic and molecular studies have shown that breast cancer subtypes are distributed unevenly among racial/ethnic groups. The incidence of the triple-negative breast cancer (TNBC) subtype in particular, one of the most clinically aggressive breast cancer subtypes, affects around 20% of women from Latin America, 24-28% of African-Americans, 6-8% of Asians, and around 12% of non-Hispanic Whites. In general Latin American patients with TNBC are more often diagnosed with earlier age, advanced stage, are likely to experience metastasis and be refractory to treatment. Although the characterization of the genomic profiles in each breast cancer subtype has been extensively performed, few studies have characterized them in specific ethnic groups. This translates to a deficiency in the understanding of the intrinsic characteristics of their tumors' genome, which can differentially impact their tumor phenotypes and clinical behavior. In this study we performed genome-wide array-CGH and miRNA profiling in TNBC (n=29) and non-TNBC cases (n=27) of patients from Latin America, Brazil (Curitiba-PR), to characterize their patterns of copy number and miRNA expression. These data were integrated to determine whether the location of miRNAs in genomic regions rich in copy number alterations (CNAs) could impact their expression levels and whether there were common miRNA target genes affected by both CNAs and miRNA deregulation. Our findings showed a distinct pattern of CNAs and miRNA expression between the TNBC and non-TNBC cases. A total number of 309 and 124 CNAs were identified in the TNBC and non-TNBC cases, with an average of 10.65 and 7.75 CNAs per case, respectively. In the TNBC cases, the most frequent (>30% of the cases) cytobands affected by CNAs were 8q11.21-q24.3, 1q21.1-q44, 7q11.23-q36.3, 12p13.33-p11.1, 3q11.2-q29, and 6p25.3-p11.2. MiRNA profiling analysis showed 209 miRNAs with significant differentially expression between these two groups (P<0.05; FDR<0.05): 108 and 101 with up- and downregulated expression, respectively. The mapping of these 209 miRNAs in regions with CNAs in the same TNBC cases led to the identification of a panel of 27 miRNAs, 12 of which (miRs-544b, 567, 568, 586, 592, 610, 634, 668, 802, 944, 1260, and 3161) with miRNAs expression concordant with CNAs. Area Under the Curve (AUC) value >0.7 was observed for 75% of these miRNAs; miRs-567, 610, 802, 944 and 1260a presented the highest discriminatory power, with AUC values >0.8. A number of 1312 genes were identified mapped in the most common CNAs and targeted by these miRNAs. Subsequent pathway analysis of the 12 miRNAs showed their involvement in cancer-related pathways, including the Hippo, TGF-beta, and Ras signaling pathways; a close hierarchical cluster was observed with the miRs-592, 634, 802, 944, and 1260 in regulating these pathways. In conclusion, the TNBC and non-TNBC cases of the patients of this study presented a distinct pattern of CNAs and miRNA expression levels. The copy number and miRNA integrated analysis performed delineated the boundaries of genomic instability regions and the mapping of the most relevant miRNA targets in these TNBC cases, that can act as potential oncogenes and/or tumor suppressor genes in these regions. These findings contributed to the identification of unique ethnic miRNA/mRNA pairings and their corresponding cancer signaling pathways that are of relevance to the TNBC of Latin American patients and that can be directly and more efficiently targeted for therapy. Support: Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI U01FD004319), a collaborative effort between the university and the U.S. Food and Drug Administration. This research does not necessarily reflect the views of the FDA. The authors thank CNPq and CAPES, Brazil, for scholarships (B.S., S.R.P.). Note: This abstract was not presented at the conference. Citation Format: Bruna Sugita, Silma R. Pereira, Rodrigo Almeida, Rubens S. Lima, Cicero A. Urban, Simina Boca, Yuriy Gusev, Iglenir J. Cavalli, E.M.S.F. Ribeiro, Luciane R. Cavalli. Copy number and miRNA profiling in triple-negative breast cancer patients from Latin America [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A28.

MTE 05.01 Neuroendocrine Tumors: Pathology and Genetic Update

MTE 05.01 Neuroendocrine Tumors: Pathology and Genetic Update

Recent advances in genomic profiling of adenosquamous carcinoma of the pancreas.

Adenosquamous carcinoma of the pancreas (ASCP) is a mixed tumor type which contains squamous cell carcinoma and also ductal adenocarcinoma components. Due to the rarity of this malignancy, only very limited genomic profiling has been performed. A recent paper by Fang et al. published in The Journal of Pathology contributed to our knowledge of genomic alterations by performing whole-genome and -exome sequencing of 17 ASCP tumors. They found major genomic similarities to pancreatic ductal adenocarcinoma; however, the p53 pathway was altered in a greater proportion of cases, while a high frequency of 3p loss was a distinct copy number alteration pattern observed in ASCP. Laser capture microdissection revealed that adenocarcinoma and squamous carcinoma components of ASCP harbor similar genomic variations, indicating that the origin of tumor components is the same or similar. Although the study published by Fang et al. increases our knowledge of this rare mixed tumor type, further investigation, including RNA sequencing, will be needed to fully characterize this malignancy and to aid the development of novel treatment approaches. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract P1-05-18: Genomic copy number alterations (CNA) associated with pCR in HER2-positive (HER2+) early-stage breast cancer (BrCa) patients receiving neoadjuvant trastuzumab (T)

Abstract Introduction: Genetic alterations such as amplifications and deletions frequently contribute to tumorigenesis. These alterations can change gene expression which alters the normal cell growth and survival regulatory mechanisms. Characterisation of DNA copy number alterations (CNA) is important to understand cancer progression and response to therapy. The aim of this study is to determine patterns of CNAs in HER2+ early-stage BrCa patients achieving pathological complete response (pCR) to neoadjuvant T therapy. Methods: Retrospective analysis of our database of 95 HER2+ BrCa (stages I-III) who received T neoadjuvantly revealed 46 % (44/95) achieved pCR compared with 53 % (51/95) who did not respond (NR). DNA from pre-treatment tumour biopsy specimens from neoadjuvant T therapy patients was extracted, and array-based comparative genomic hybridization (aCGH, n = 8; 6 pCR:2 NR) was used to identify CNAs, which correlated with pCR. Pathway analysis was then used to identify functionally relevant genes in aberrant regions. Results: aCGH analysis of DNA from pCR and NR identified distinct patterns of CNAs. HER2 amplicon was confirmed by IHC and aCGH in all samples. Although there was no significant difference in the average CNAs between groups (20±17 vs 17±2), there was greater variation in the range of CNAs in pCR (8-56 CNA) compared to NR (15-19 CNA). More gains and amplifications were observed in pCR patients with more deletions in the NR group. The most common chromosomal amplification region included chr8q12.1-q24 with 87.5% of all cases displaying gains. Of the 6 patients who achieved pCR, 50% displayed a deletion in chr9 spanning p24.3-p21.3, consistent with a deletion of tumour suppressor CDKN2A. No aberrations in chr9 were observed in NR cohort. The deleted genomic region contained 65 common protein-coding genes, with the interferon biological pathway as the most significant (p=1.03E-36). Conclusions: Distinct genomic CNAs were observed between patients achieving pCR compared to NR. However, of the 8 pts characterised here, none have relapsed. Follow-up data revealed a relapse rate of 6.8 % (3/44) vs 11.8 % (6/51) in the pCR and NR groups, respectively. To further elucidate the immunological response, we will present CNA data patterns on relapse and response and compare the impact of CNAs, immune-related proteins and pCR as surrogate predictors for outcome. Citation Format: Walsh N, Gullo G, Maguire A, O'Donovan N, Quinn C, Crown J. Genomic copy number alterations (CNA) associated with pCR in HER2-positive (HER2+) early-stage breast cancer (BrCa) patients receiving neoadjuvant trastuzumab (T) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-18.

Establishment and characterization of hypomethylating agent-resistant cell lines, MOLM/AZA-1 and MOLM/DEC-5.

Two hypomethylating agents (HMAs), azacitidine and decitabine, have demonstrated clinical activities in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML); however, potential problems include development of acquired resistance. HMA-resistant patients have very poor prognosis and this cohort of patients constitutes an important area of research. To understand the mechanisms underlying HMA-resistance and to overcome it, we established an azacitidine-resistant cell line, MOLM/AZA-1 and a decitabine-resistant cell line, MOLM/DEC-5 using MOLM-13. For cytogenetic characterization, we performed microarray-based comparative genomic hybridization (array-CGH), which identified a total of 15 copy number alterations (CNAs). Among these CNAs, eight regions in HMA-resistant cell lines showed CNA patterns distinct from the parental MOLM-13 genome. Single nucleotide polymorphism (SNP) microarray was also performed to obtain a more reliable interpretation of the identified CNAs, and all HMA-resistance-specific CNAs except one detected by array-CGH were successfully validated. In addition to CNAs, copy neutral loss of heterozygosity and mosaic loss events were identified in HMA-resistant cell lines. In our resistant cell lines, MDR-1 was not overexpressed, while DNMT3b was upregulated. Azacitidine and decitabine did not inhibit DNMT1, DNMT3a, or DNMT3b in both HMA-resistant cell lines, while they inhibited the enzymes in parental MOLM-13. We also developed mouse xenograft models using MOLM/AZA-1 and MOLM/DEC-5. Our in vitro and in vivo models of HMA-resistant cell lines will provide clues for the elucidation of molecular mechanisms related to the development of resistance to HMA and tools for the application of novel therapeutics for AML and MDS.

Genome-wide analysis of DNA copy number alterations in early and advanced gastric cancers.

To better understand progressive changes in gastric cancer (GC), early and advanced GCs (EGC and AGC, respectively) were examined for copy number alterations (CNAs). A crypt isolation method was used to isolate DNA from tumors and normal glands in 20 AGCs, and fresh tumor samples were obtained from 45 EGCs. We assessed CNAs for differentiated-type GCs using an Infinium HumanCytoSNP-12v2.1 BeadChip in EGCs and AGCs. The most frequent aberrations in EGC were gains at 8q23.3 (42.2%) and 8q23.2 (40%), and loss of heterozygosity (LOH) at 3p14.2 (24.2%), suggesting that these CNAs were involved in the development of EGC. On the other hand, the highest frequencies of gains in AGC were found at 8q24.21 (65%) and 8q24.3 (60%). The most frequent LOHs in AGC were at 11q24.3-25, 11q23.2-24.1, 11q14.1, and 12p11.21-13.33, whereas that in EGC was at 3p14.2. In addition, regions of copy-neutral LOHs in AGC were detected at 11q21, 11q13.3-14.3, 11q11, 11p13-15.3, 12q21.1, 12q12-13.3 and 5q33.3-35.1. Comparisons of gains in EGC and AGC showed significant differences at 12q22-q23.2, 12q21.33, 11p12, 11p14.1, 12q21.31-32.32, 3p12.3, 3p14.1, 10p15.1, 1q24.2 and 2q12.1. Copy neutral LOHs were significantly higher in AGC than in EGC at 14q32.11-32.33, 14q21.3, 14q11.2, 5q11.2, 5q 13.3, 14q21.1-23.2, 14q13.2-13.3, 5q12.1-12.3, 5q11.1, and 17p13.3. The total lengths of the CNAs were significantly greater in AGC than in EGC. We found that the pattern of CNAs in AGC was quite different from that in EGC. We suggest that increasing numbers of CNAs are associated with disease progression from EGC to AGC. © 2016 Wiley Periodicals, Inc.