Co-amplification Of MYC Research Articles

Establishment of a Novel DLBCL Cell Line: AMU-ML2, Derived from a Primary Refractory Patient Shows Homogeneous Staining Region of 8q24 Inducing High Expression of Long Non-Coding RNAs Encoded By PVT1 and Resistance to Vincristine

Introduction: Primary refractory DLBCL is an extremely difficult condition to treat and represent an unmet medical need. The clarification of the molecular pathogenesis of this disease can contribute the development of new therapeutic possibilities. PVT1 is located adjacent to MYC at 8q24 and a non-protein coding gene. PVT1 produces a variety of long non-coding RNAs (lncRNAs) and has now emerged as a potential regulator in the pathogenesis of a lot of cancers. However, the precise biological and clinical significance of PVT1 remains largely unknown. In this study we established a novel human DLBCL cell line with hsr (homogeneous staining region) of 8q24 inducing high expression of PVT1 lncRNAs, named AMU-ML2. The cell line was established from a patient with primary refractory DLBCL before initiation of chemotherapy. We analyzed the genetic characteristics and investigated the drug sensitivity of the cell line in comparison with other B-cell lymphoma cell lines which had 8q24 abnormalities.Case: A 64-year-old man was diagnosed as DLBCL involved with bone marrow and pleural effusion. He was refractory to initial R-CHOP and subsequent R-hyper-CVAD/MA therapy and died of Trichosporon asahiisepsis 6 months after diagnosis. His lymphoma cells at diagnosis were positive for CD19, CD20, BCL6 and HLA-DR, and negative for CD3, CD5, CD10, cyclinD1, BCL2, MUM1, TP53 and EBER by flow cytometry and/or immunohistochemical staining, indicative of germinal center B-cell-like (GCB) DLBCL. The representative karyotype of cells was 46,XY, including add(6)(p11), add(8)(q24), hsr 8q24, add(9)(p13) and add(17)(p11.2).Establishment of the cell line: After 2 weeks of culture, the cells in pleural effusion collected before chemotherapy started to grow in suspension. The cell line was designated as AMU-ML2 after confirmation that the cells started growing again after the conventional freeze-thaw procedure.Materials and methods: B-cell lymphoma cell lines; AMU-ML2, SU-DHL-10, Raji, P3HR-1 and VAL were used in the present study. These cell lines had t(8;14)(q24;q32) or 8q24 amplification. The genomic aberration of AMU-ML2 was analyzed by cytogenetics including G-banding and FISH (fluorescence in situ hybridization) combined with array-CGH (SurePrint G3, Agilent). The TaqMan real time RT-PCR was used for measurement of expression levels of MYC and PVT1. MTT assay was used for the cell proliferation to analyze the drug sensitivities of cyclophosphamide, doxorubicin, vincristine (VCR) and prednisolone.Results: AMU-ML2 cells showed same immunophenotypic feature and karyotype as the primary sample from the patient. FISH using a MYC/IGH probe set showed no fusion signal for IGH and MYC; however, the MYC copy number was extremely increased, corresponding to hsr on chromosome 8q24. Array-CGH revealed that a 1,462 kb region, containing both the entire MYC and PVT1 genes at 8q24.21, was amplified, with greater than 20 copies present in cells (Figure 1). In addition to amplification of the MYC/PVT1 region at 8q24, 14 additional copy number alterations were detected. These included segment losses on 6p22.1-6p21.31 and 17p13 led to the LOH of human leukocyte antigen (HLA) loci and TP53, respectively (Figure 1). Using the real time RT-PCR, the expression level of the PVT1 lncRNAs were highest in AMU-ML2 among other B-cell lymphoma cell lines that we used, while the expression level of MYC in AMU-ML2 was relatively low. The proliferation rate of AMU-ML2 was significantly higher after 72-h exposure to VCR (100, 500 and 1,000 nM) compared with other cell lines (Figure 2).Discussion: AMU-ML2 was established in 2-week culture from a refractory patient before starting the chemotherapy, therefore this cell line seems to reflect the real nature of primary refractory DLBCL, not related to chemotherapy and/or cell culture. MYC amplification and the LOH of TP53 and HLA may contribute to the development of lymphoma in our case. High expression of PVT1 lncRNAs was likely to be more related with drug resistance to VCR than MYC expression, although AMU-ML2 had co-amplification of MYC and PVT1. Thus, AMU-ML2 can provide insight into the genetic and biological features and the therapeutic approaches in primary refractory DLBCL. [Display omitted] [Display omitted] DisclosuresUeda:Mundipharma KK: Consultancy; Kyowa Hakko Kirin: Research Funding.

Concurrent Amplification of MYC and 1q21 in Multiple Myeloma: Focal and Segmental Jumping Translocations of MYC

IntroductionMultiple myeloma (MM) is characterized by an accumulation of secondary chromosome aberrations which lead to cytogenetically heterogeneous subpopulations of cells in the subclonal progression of this disease. Copy number aberrations (CNAs) represent a large proportion of these aberrations and include both numerical and structural chromosome aberrations which involve unbalanced translocations, amplifications, insertions, and deletions.Secondary MYC translocations are thought to play a role in driving the transition from MGUS to MM, and during tumor progression additional secondary CNAs of both MYC (8q24) and 1q21 are found in relapsed patients. When CNAs are taken into account the percentage of myeloma cases where MYC is deregulated is 55%, making it the most common genetic event in MM (Walker B. et al., Nature Comm, 2015). In fact, high resolution SNP arrays have shown that somatic CNAs of MYC and 1q21 are two of the top ten CNAs in all cancers (Beroukhim R. et al., Nature, 2010). Two types of amplification predominate by SNPs array, either large arm-length amplifications (25% of the genome) or small focal CNAs (10% of the genome). Unfortunately, the origins of the complex structural chromosomal aberrations which underlie these types CNAs have not been well characterized in MM.To better understand the possible relationship between MYC and 1q21 CNAs we undertook a comprehensive metaphase cytogenetic analysis of 26 diagnostic specimens showing concurrent MYC and 1q21 CNAs by G-banding.MethodsLocus specific FISH probes and Spectral karyotyping were used to define and follow the progression of aberrations occurring in these two regions. Locus specific FISH probes for selected regions of chromosomes 1 and 8 pericentromeric regions and the regions surrounding 1q21 and 8q24 were utilized.ResultsThree primary aberration patterns were observed:The most frequent pattern involved copy number gains in both regions, apparently occurring independently of each other (sixteen patients).The second most frequent pattern showed unbalanced segmental jumping translocations (SJTs) of 8q and the MYC locus to different receptor chromosomes. These unbalanced translocations of MYC were found in seven patients involving varying sized segments of 8q, and included both intra and inter-chromosomal translocations. Surprisingly, three patients in this group showed clones with the co-amplification of MYC and 1q21 on the same derivative chromosome, where 8q and MYC were in essence piggybacking on jumping translocations of 1q12 (JT1q12) to receptor chromosomes.Finally, the third and most complex structural aberration pattern involved the breakage-fusion-bridge cycle mechanism, which results in focal amplifications of genes. This mechanism typically results from telomere shortening and loss and subsequent formation of dicentric chromosomes. Evidence of dicentric chromosomes 8 and the breakage fusion break amplification of MYC was identified in three patients and, strikingly, in one case was found to be initiated by a JT1q12 to 8qter. Therefore, this case identifies a novel alternative mechanism for the focal amplification of the MYC locus whereby the JT1q12 initiates sister chromatid fusion at 8q24.ConclusionWe demonstrate for the first time the mechanistic coupling of MYC and 1q21 amplifications involving sequential jumping translocations of 8q and JT1q12s. These findings provide evidence for the origin of both arm-length and focal amplifications of MYC, and that these aberrations can co-evolve in the same clones. The concurrent amplification of MYC and 1q21 suggests pericentromeric instability, and to a lesser extent telomeric instability, play a role in driving CNAs of both of these genomic lesions in MM. DisclosuresDavies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding.

Activity of sorafenib in radiation-associated breast angiosarcomas harboring MYC and FLT4 amplifications.

10019 Background: Angiosarcomas (ASs) are rare, aggressive vascular malignancies of endothelial cell differentiation which arise de novo or secondary to radiation therapy (RAS.) A subset of patients(pts) with AS harbor mutations in KDR (VEGFR-2) or overexpression of MYC and/or FLT4(VEGFR-3). MYC & FLT4 gene amplifications occur almost exclusively in RAS. These alterations provide a rationale for investigating agents that target angiogenesis in RAS. In a phase II trial of sorafenib, AS pts had a partial response (PR) of 14%; targeting the appropriate pt population may be essential. We hypothesize that co-amplification of MYC & FLT4 may be predictive of response to sorafenib. Methods: Using our institutional sarcoma database & data query system, we identified AS patients treated with sorafenib at Memorial Sloan-Kettering Cancer Center between 1992-2011. Molecular analysis performed on available tissue. With IRB approval, clinical information was collected. Results: We identified 10 women with RAS that received sorafenib. Average age 68 (range 51-84.) 7 pts had distant metastatic disease. Median lines of systemic therapy prior to sorafenib: 2 (range 1-4.) Sorafenib was first line in 60% of pts, administered at 400mg daily and adjusted for toxicity. Best responses included: complete response (CR) 2/9(22%), PR 1/9(11%), stable disease (SD) 5/9(56%) range 5-23m, progressive disease 1/9(11%) for a clinical benefit rate of (CR+PR+SD) 89%. Responses were seen in patients with lung metastases (n=2) and locally advanced disease (n=1) and were durable for 17, 42 and 26 months. Median duration on therapy was 15 months (range 0-42 months.) 7 pts underwent molecular analysis: co-amplification of MYC & FLT4 3 pts (30%); MYC amplification 4 pts (40%); KDR mutation 0 patients. Of the 3 responders, MYC & FLT4 co-amplification were observed in 2 patients and not evaluated in one. Conclusions: Sorafenib is active against RAS of the breast. In this small series, complete and partial responses were seen in patients with co-amplification of MYC & FLT4. This observation requires further study. Performing molecular studies on all AS pts will help define the pathophysiology of this deadly disease to guide rationale clinical trials.

The PVT gene frequently amplifies with MYC in tumor cells.

The line of human colon carcinoma cells known as COLO320-DM contains an amplified and abnormal allele of the proto-oncogene MYC (DMMYC). Exon 1 and most of intron 1 of MYC have been displaced from DMMYC by a rearrangement of DNA. The RNA transcribed from DMMYC is a chimera that begins with an ectopic sequence of 176 nucleotides and then continues with exons 2 and 3 of MYC. The template for the ectopic sequence represents exon 1 of a gene known as PVT, which lies 50 kilobase pairs downstream of MYC. We encountered three abnormal configurations of MYC and PVT in the cell lines analyzed here: (i) amplification of the genes, accompanied by insertion of exon 1 and an undetermined additional portion of PVT within intron 1 of MYC to create DMMYC; (ii) selective deletion of exon 1 of PVT from amplified DNA that contains downstream portions of PVT and an intact allele of MYC; and (iii) coamplification of MYC and exon 1 of PVT, but not of downstream portions of PVT. We conclude that part or all of PVT is frequently amplified with MYC and that intron 1 of PVT represents a preferred boundary for amplification affecting MYC.

The PVT gene frequently amplifies with MYC in tumor cells.

The line of human colon carcinoma cells known as COLO320-DM contains an amplified and abnormal allele of the proto-oncogene MYC (DMMYC). Exon 1 and most of intron 1 of MYC have been displaced from DMMYC by a rearrangement of DNA. The RNA transcribed from DMMYC is a chimera that begins with an ectopic sequence of 176 nucleotides and then continues with exons 2 and 3 of MYC. The template for the ectopic sequence represents exon 1 of a gene known as PVT, which lies 50 kilobase pairs downstream of MYC. We encountered three abnormal configurations of MYC and PVT in the cell lines analyzed here: (i) amplification of the genes, accompanied by insertion of exon 1 and an undetermined additional portion of PVT within intron 1 of MYC to create DMMYC; (ii) selective deletion of exon 1 of PVT from amplified DNA that contains downstream portions of PVT and an intact allele of MYC; and (iii) coamplification of MYC and exon 1 of PVT, but not of downstream portions of PVT. We conclude that part or all of PVT is frequently amplified with MYC and that intron 1 of PVT represents a preferred boundary for amplification affecting MYC.