Study Of Genomic Alterations Research Articles

P02.11.A INTEGRATED GENOMIC PROfiLING OF LONG-TERM GLIOBLASTOMA SURVIVORS

Abstract BACKGROUND Glioblastoma (GBM) is a fatal brain tumor, and few patients survive beyond 5 years. Therefore, a study of genomic alterations in GBM long-term survivors (LTS) is essential for insights into tumor biology and biomarker identification. MATERIAL AND METHODS In our study, we reviewed medical records from Sun Yat-sen University Cancer Center for GBM patients received initial surgery between the years 2000 and 2018. Thirty patients with GBM had an overall survival of more than 5 years. In addition to established prognostic factors such as radiochemotherapy and total surgical excision, we sought to identify genomic alterations associated with LTS by collecting surgically resected tumor tissues and normal blood controls from 13 GBM LTS (overall survival > 60 months) and 19 short-term survivors (STS, overall survival < 24 months). We performed whole exome sequencing and transcriptome sequencing on these samples. RESULTS Our findings revealed no significant differences in the composition of driver genes between the two groups. However, mutation signature analysis indicated that the proportion of mutation signature 19 was higher in the LTS than in the STS (P=0.059). Analysis of copy number variants showed that the longer survivors group had higher copy number variants at the chromosomal level (P=0.049). At the chromosome arm level, the proportion of 19p amplification and 19q amplification in the longer survivors group was significantly higher than in the short survival group (P=0.001/0.002). And in the TCGA GBM dataset, GBM patients with 19p amplification also had a better prognosis (log-rank P=0.04). CONCLUSION In conclusion, our study identified the proportion of mutation signature 19 and amplification of 19p as potential genomic alterations that could be used as prognostic biomarkers for GBM. This study could pave the way for new therapeutic approaches to this challenging disease.

BIOM-62. INTEGRATED GENOMIC PROfiLING OF LONG-TERM GLIOBLASTOMA SURVIVORS

Abstract Glioblastoma (GBM) is a fatal brain tumor, and few patients survive beyond 5 years. Therefore, a study of genomic alterations in GBM long-term survivors is essential for insights into tumor biology and biomarker identification. In our study, 13 GBM long-term survivors (overall survival >60 months) and 19 short-term survivors (overall survival < 24 months) were retrospectively collected, and surgically resected tumor tissues and normal blood controls were extracted and were performed whole exome sequencing and transcriptome sequencing. Our results revealed that although no significant differences were found in the composition of driver genes between the two groups, mutation signature analysis of two GBM groups revealed that the proportion of mutation signature 19 was higher in the longer survivors than in the shorter survivors (P=0.059). Analysis of copy number variants showed that the longer survivors group had higher copy number variants at the chromosomal level (P=0.049). At the chromosome Arm level, the proportion of 19p amplification and 19q amplification in the longer survivors group was significantly higher than in the short survival group (P=0.001/0.002). And in the TCGA GBM dataset, GBM patients with 19p amplification also had a better prognosis (log-rank P=0.04). In conclusion, the proportion of mutation signature 19 and amplification of 19p was potential prognostic molecular features for GBM.

Unraveling the Landscape of Copy Number Aberrations in Hodgkin Lymphoma: A Joint KU Leuven and Lysa Study on Circulating Cell Free DNA

Introduction. For decades, the study of genomic alterations in Hodgkin lymphoma (HL) has been hampered by the low abundance of the malignant Hodgkin Reed-Sternberg (HRS) cells in HL lymph node biopsies. Laser microdissection or flow cytometric cell sorting allow the study of purified HRS cells, but the use of these applications is restricted to specialized research centers. We have recently demonstrated proof-of-principle that copy number aberrations (CNA) in HRS cells can be retrieved in circulating cell-free DNA (cfDNA) of HL patients (Vandenberghe et al., Lancet Haematol. 2015). Taking advantage of the presence of HRS cell-derived DNA (ctDNA) in plasma, the aim of this study was to catalogue CNA in HL in a large series of prospectively recruited HL patients.Methods. We analyzed plasma collected from 177 patients (median age 29, range 3-86) with newly diagnosed HL. 60 cases were diagnosed at our institution, including all disease subtypes and stages, with a majority of nodular sclerosis. 118 patients were recruited in the context of the BREACH study, a multicentric Phase 2B study for unfavorable early classical HL (NCT02292979). After cfDNA extraction, samples were low-pass sequenced (0.1x coverage) and analyzed using ichorCNA, an algorithm that produces read depth-based log2 CNA profiles and quantifies the cfDNA tumor content.Results. At diagnosis, 86 % (152/177) of patients showed obvious genomic imbalances in cfDNA, in early-stage (85 % (131/155)) as well as in advanced cases (95 % (21/22)). Among the abnormal profiles, gains encompassing 2p16, 5p15, 9p24, 12q13 and 19q13 were the most frequent aberrations, occurring in 79 %, 53 %, 57 %, 63 % and 80 % respectively. Losses most frequently affected regions 4q34, 6q23, 11q22 and 13q13, in 53 %, 63 %, 49 % and 59 % of abnormal profiles respectively (fig. 1A). All these CNAs have been previously described with varying frequencies in smaller series of 10-53 patients, using arrayCGH or whole exome sequencing on microdissected HRS cells or even HRS cell-derived cell lines as input material. The observed pattern of CNAs is distinct from the pattern we observed in cfDNA obtained from other hematological malignances, e.g. multiple myeloma and diffuse large B-cell lymphoma (fig. 1B, C).Analysis of follow-up cfDNA samples revealed that the majority of patients rapidly clear their profiles during the first two treatment cycles. Of 123 samples analysed at d15 of cycle 1, 89 samples (72 %) showed a normal profile without CNAs.This suggests a relationship between the fraction of ctDNA in cfDNA and the disease burden. However, we could not find a correlation between the ctDNA fraction as calculated by ichorCNA and the metabolic tumor volume as computed from PET/CT scans. This could be due to several factors, e.g. the small fraction of HRS cells in the metabolic tumor volume. We are currently investigating whether a correlation does exist between the HRS content in the lymph node biopsy and the ctDNA load.Conclusions. In this largest study of CNA in HL to date, we provide a comprehensive catalogue of the types of CNAs, as well as their frequencies and patterns in HL. In this series of 177 patients, gains affecting 2p, 12q and 19q and loss of 6q and 13q emerge as the most commonly recurrent CNAs in HL, across all HL subtypes and stages. The data confirm and extend our previous findings that the majority of HL patients, including those with early-stage disease, have detectable CNAs in their cfDNA at diagnosis. We demonstrate a broad spectrum of aberrations, gains and losses, some of which recur at higher frequencies than gains of 9p24, harboring PD-L1. This warrants further studies on how these CNAs are implicated in the pathogenesis of HL. It further endorses the use of ctDNA as an alternative gateway to the genome of HRS cells, and as a substrate for the evaluation of early disease response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The experimental study of genomic alterations of hepatocellular carcinoma by panel-based sequencing

Objective To explore the value of high-throughput capture sequencing technology of tumor gene in detection of somatic mutation and mutation patterns of hepatocellular carcinoma cells. Methods Based on the target gene sequencing technology of the second generation sequencing technology, a custom sequence capture system containing 325 tumor related genes panel, NimbleGen, was used, and the target region of 7 hepatoma cell lines (HuH-7, Hep3B, SK-HEP-1, MHCC97H, MHCC97L, HepG2, HepG2.2.15) DNA gene sequencing were captured and sequenced. The tumor-related mutant gene and mutation load rate, as well as the difference between the gene mutation trend were analysed. Results The results showed that the average coverage rate of the target area was 99.7%, and the average sequencing depth was about 1 000×, and 100 genes(mutation frequency≥5%)were detected, including 344 non synonymous mutations. There are 38 gene mutations closely related to cancer enriching in 5 oncogenic signaling pathways: chromatin remodeling, Notch, mitogen-activated protein kinase (MAPK), p53, and Wnt/beta-catenin pathways. Compared with the previous reports of somatic mutations in hepatocellular carcinoma (HCC) patients, the chromatin remodeling and gene mutations in the Notch signaling pathway were higher in HCC cell lines. Three mutant genes not reported in HCC patients ever were identified, including two tumor suppressor genes [MSH6, sex determining region Y box 9 (SOX9)] and one oncogene (ALK).44 mutant genes were identified in MHCC97 cell lines (MHCC97H and MHCC97L), of which 30 (68%) were co-mutations. Mutation genes are enriched in oxidative stress pathways (KEAP1), chromatin remodeling (NCOR1, KMT2D, ARID1A), MAPK (MAP3K1, MAP2K3) and Wnt/beta-catenin (AXIN1, SOX9) pathways. ALK and ERBB2 mutations were detected only in MHCC97L, APOB mutations only in MHCC97H. Notch pathway (Notch1, Notch2) had higher mutation frequency in MHCC97L, TERT in MHCC97H.37 mutant genes were identified in HepG2 and HepG2.2.15, of which 19 (51%) were co-mutations. HepG2 and HepG2.2.15 had the same mutation sites and similar mutation frequency in Notch pathway (Notch1, Notch2) and chromatin remodeling pathway (KMT2D, ZFHX4). Tp53 mutations were detected only in HepG2, adenomatous polyposis coli (APC) and ataxia-telangiectasia mutated (ATM) mutations only in HepG2.2.15. The four HCC cell lines (HuH-7, Hep3B, SK-HEP-1, HepG2) from different HCC patients had different mutation modes, but the same clone derived HCC cell lines showed a large similarity. Conclusion High throughput sequencing technology of tumor gene has high application value in the detection of cell lines and mutation patterns in hepatocellular carcinoma cell lines. Key words: Hepatocellular carcinoma; Gene mutation; Panel-based Sequencing

Molecular Biomarkers for Prediction of Targeted Therapy Response in Metastatic Breast Cancer: Trick or Treat?

In recent years, the study of genomic alterations and protein expression involved in the pathways of breast cancer carcinogenesis has provided an increasing number of targets for drugs development in the setting of metastatic breast cancer (i.e., trastuzumab, everolimus, palbociclib, etc.) significantly improving the prognosis of this disease. These drugs target specific molecular abnormalities that confer a survival advantage to cancer cells. On these bases, emerging evidence from clinical trials provided increasing proof that the genetic landscape of any tumor may dictate its sensitivity or resistance profile to specific agents and some studies have already showed that tumors treated with therapies matched with their molecular alterations obtain higher objective response rates and longer survival. Predictive molecular biomarkers may optimize the selection of effective therapies, thus reducing treatment costs and side effects. This review offers an overview of the main molecular pathways involved in breast carcinogenesis, the targeted therapies developed to inhibit these pathways, the principal mechanisms of resistance and, finally, the molecular biomarkers that, to date, are demonstrated in clinical trials to predict response/resistance to targeted treatments in metastatic breast cancer.

Abstract A2-20: Integrative study of genomic alterations in liposarcoma

Abstract Liposarcoma (LPS) is the most common type of soft tissue sarcoma and accounts for approximately 20% of all adult sarcomas. Due to absence of clinically effective treatment options with a high rate of recurrence and resistance to conventional therapeutics agent, a critical need exists to identify the therapeutic targets for treating this deadly disease. Our study aims to further understanding of LPS pathobiology by constructing a detailed genetic and molecular landscape of LPS through a series of integrated comprehensive genomic and transcriptomic analysis. We performed SNP CHIP array analysis on 92 LPS cases and 13 LPS cell lines, whole-exome sequencing analysis of 12 LPS-normal paired samples, transcriptome sequencing of 5 cases, targeted whole-exome sequencing of 86 LPS cases and 13 LPS cell lines and intra-tumor heterogeneity analysis in LPS. We found a variety of genomic aberrations including point mutations, copy number changes, genomic rearrangements, and fusion genes in different subtypes of LPS using next generation sequencing technology. SNP CHIP array analysis revealed significant recurrent copy number amplifications and deletions involving many important well-known cancer genes. One of the important potentially druggable alteration of carboxypeptidase M gene was emerged as new therapeutic strategy in SNP array analysis. The biological and functional characterization of carboxypeptidase M as therapeutic target was done in detail using LPS cell lines and mouse xenograft model system. Using whole exome sequencing, a total 377 potential somatic changes were identified and validated by Sanger sequencing in Discovery cohort. We analyzed the spectrum of mutations detected by whole exome sequencing and found presence of two distinct mutational signatures. Next, a Prevalence Set of additional 86 LPS patients' samples was examined by targeted exome sequencing and numerous non-synonymous mutations identified were validated using Sanger sequencing. Targeted exome sequencing revealed significant recurrent alterations in cell adhesion [60% of cases], DNA damage repair [65% of cases] and kinase [36% of cases] signalling pathways leading to identification of genes that can be potentially targeted using currently available drugs. A class of genes with regulatory roles in axon guidance and cancer cell growth, survival, invasion and angiogenesis were also found to be mutated. RNA sequencing was performed on 5 LPS samples of different histotypes to explore fusion genes. We identified various fusion genes and validated 29 chimeric fusion transcripts using RT-PCR followed by Sanger sequencing across the fusion junctions. We also found the well-known and established classical fusion in LPS cases. Intra-tumor heterogeneity analysis indicates ongoing regional clonal evolution due to the presence of unique mutations in different tumor regions of the same patient tumor sample. Mutations confined to only one of the three tumor regions indicate that interrogation of a single tumor site is not representative of the entire mutational landscape of a patient's tumor. A phylogenetic tree of different tumor regions by clonal ordering shows branching tumor evolution indicating that the majority of genetic events occurred after tumors diverged. The present investigation provides insights into the underlying mechanism driving liposarcomagenesis and also identifies new therapeutic targets which need to be further investigated. Comprehensive genomic characterization will improve our understanding of the LPS molecular genetics and lay the foundation to refine diagnostic classifications, determine prognosis and develop new therapeutics for treating LPS. Note: This abstract was not presented at the conference. Citation Format: Deepika Kanojia, Manoj Garg, Yasunobu Nagata, Dhong Hyun Lee, Hans-Ulrich Klein, Christoph Bartenhagen, Anand M T, Ngan B Doan, Jonathan W Said, Henry Yang, Charles Forscher, Martin Dugas, Seishi Ogawa, H Phillip Koeffler. Integrative study of genomic alterations in liposarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-20.

Abstract C03: A comprehensive study of genomic alterations reveals deregulation of the cell cycle in most esophageal squamous cell carcinomas

Abstract Objective: Esophageal squamous cell carcinoma (ESCC) is a major cause of cancer related mortality. To determine the molecular basis of ESCC development, this investigation sought to comprehensively identify the characteristic genome-wide changes in ESCC, including exonic mutations and structural alterations. The clinical implications of these genetic changes are also analyzed. Methods: Exome sequencing was applied to detect somatic mutations in nine pairs of ESCC and matched blood samples, followed by validation with MassARRAY genotyping and Sanger sequencing. Whole-genome SNP arrays were employed to study copy number changes and the loss of heterozygosity in 55 ESCCs. Results: 108 non-synonymous mutations in 102 genes were identified and validated in nine patients, suggesting a low somatic mutation rate (average 12 per tumor) in our ESCC cases. The chromatin modification process was found to be enriched in gene ontology analysis. Mutated TP53 was seen in five of the nine tumors (55.6%) undergone exome sequencing and in 23/46 tumors in the validation set. Integrating with the SNP array data, we found that tumor genomes with TP53 mutations were much more unstable than those without these mutations. In the landscape of genomic alterations, deletion of 9p21.3 covering CDKN2A (30.9%), amplification of 11q13.3 covering CCND1 (30.9%), and point mutation of TP53 (50.9%) occurred in two-thirds of ESCCs, suggesting deregulation of G1 in cell cycle is the key event in ESCC. Furthermore, six minimal common regions were found to be significantly altered in ESCC and three (9p21.3, 7p11.2, 3p12.1) were associated with lymph node metastasis. Conclusions: Frequent TP53 mutation and genomic instability are highly correlated in ESCC. The amplification of CCND1, deletion of CDKN2A/B, and somatic mutation of TP53 play pivotal roles via G1 deregulation and therefore classify this cancer into major groups. These findings provide clinical significance for future molecular diagnosis and therapeutic targeting. Citation Format: Jian Bai, Qiyan Wang, Kenan Gong, Hong Cai, Yang Ke, Changqing Zeng. A comprehensive study of genomic alterations reveals deregulation of the cell cycle in most esophageal squamous cell carcinomas. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C03.

Characterizations of the murine mesenchymal cell line expressing GFP

We established and characterized a murine mesenchymal stem cell line from the bone marrow of a transgenic C57BL mouse that ubiquitously expressed green fluorescent protein (GFP). Immunostaining revealed the presence of several markers common for mesenchymal stem cells (MSCs). The cells expressed specific fibroblast proteins, such as smooth muscle actin, which is localized in stress fibrils, and vimentin, a major protein of intermediate filaments in connective tissue cells. These proteins are responsible for the ability to differentiate into adipocytes or osteoblasts under appropriate conditions. The MSC karyotype was unstable. At the 6th passage cells, were aneuploid and genetically heterogeneous. The number of chromosomes ranged from near 2n to 8n. 80% of cells had chromosome numbers between 50 and 85 without a well-defined modal class. Differential G-staining of metaphase spreads showed variability in the copy numbers of individual chromosomes and presence of random chromosome rearrangements, such as ectopic associations of nonhomologous chromosomes. All cells analyzed contained a single dicentric marker chromosome. Some cells also had mini-chromosomes regarded as indicators of gene amplification. We suppose that the karyotypic instability of MSCs that express GFP is provoked by the insertion of foreign GFP transgenes into the murine genome. These cells could be useful for the study of genomic alterations during the spontaneous oncogenic transformation of stem cells.

Oligonucleotide array‐based comparative genomic hybridization (aCGH) of 90 neuroblastomas reveals aberration patterns closely associated with relapse pattern and outcome

The study of genomic alterations in neuroblastoma is of particular importance since several cytogenetic markers proved to be closely associated with the clinical phenotype. To disclose patterns of gains and losses, we performed high-resolution oligonucleotide array-based comparative genomic hybridization (aCGH). A total cohort of 90 patients was classified into 6 subsets according to tumor stage and outcome: Stages 1-3+ (with event), Stage 1-3- (no event), Stage 4+/-, and Stage 4S+/-. The aberration patterns in Stages 1-3- and 4S- tumors differed from all other groups as they were predominantly characterized by losses (3, 4, 14, X) and gains (7, 17) of whole chromosomes. However, 59/65 (91%) tumors of Stages 1-3+ or Stage 4 revealed numerous structural copy number alterations (sCNA). While deletions in chromosomes 1, 3, and 11 discriminated outcome in Stage 4, there were no specific sCNA that distinguished tumor stage within the subgroup of unfavorable tumors. sCNA in 1p, 3p, 11q, 17q, or MYCN amplification (MNA) was seen among 22/24 patients who died, 10/12 with metastatic relapses, and 5/9 with local recurrences. Detailed breakpoint analyses on chromosomes 1, 3, 11, and 17 disclosed preferred breaking areas, although breakpoints were not identical. Amplifications were found in 18 patients and involved 2p24 (MYCN) and other segments of chromosome 2, as well as regions on chromosome arms 6q, 12q, and 17q. One single feature in 21q21.1 (BU678720, without known function yet) attracted particular attention since five patients showed a homozygous loss of this sequence.