Gene Amplification In Cancer Research Articles

Gene amplifications and extrachromosomal circular DNAs: function and biogenesis.

Gene amplification is an increase in the copy number of restricted chromosomal segments that contain gene(s) and frequently results in the over-expression of the corresponding gene(s). Amplification may be found in the form of extrachromosomal circular DNAs (eccDNAs) or as linear repetitive amplicon regions that are integrated into chromosomes, which may form cytogenetically observable homogeneously staining regions or may be scattered throughout the genome. eccDNAs are structurally circular and in terms of their function and content, they can be classified into various subtypes. They play pivotal roles in many physiological and pathological phenomena such as tumor pathogenesis, aging, maintenance of telomere length and ribosomalDNAs (rDNAs), and gain of resistance against chemotherapeutic agents. Amplification of oncogenes is consistently seen in various types of cancers and can be associated with prognostic factors. eccDNAs are known to be originated from chromosomes as a consequence of various cellular events such as repair processes of damaged DNA or DNA replication errors. In this review, we highlighted the role of gene amplification in cancer, the functional aspects of eccDNAs subtypes, the proposed biogenesis mechanisms, and their role in gene or segmental-DNA amplification.

Chromothripsis drives the evolution of gene amplification in cancer

Focal chromosomal amplification is an important route to generating cancer through mediating over-expression of oncogenes1–3 or to developing cancer therapy resistance by increasing expression of a gene whose action diminishes efficacy of an anti-cancer drug. Here we used whole-genome sequencing of clonal isolates developing chemotherapeutic resistance to identify chromothripsis as a major driver of extrachromosomal DNA (ecDNA) amplification into circular double minutes (DMs) through PARP- and DNA-PKcs-dependent mechanisms. Longitudinal analyses revealed that DMs undergo continuing structural evolution to promote increased drug tolerance through additional chromothriptic events. In-situ Hi-C sequencing is used to demonstrate that DMs preferentially tether near chromosome ends where they re-integrate when DNA damage is present. Intrachromosomal amplifications formed initially under low-level drug selection undergo continuing breakage-fusion-bridge cycles, generating >100 megabase-long amplicons that we show become trapped within interphase bridges and then shattered, producing micronuclei that mediate DM formation. Similar genome rearrangement profiles linked to localized gene amplification are identified in human cancers with acquired drug resistance or with oncogene amplifications. We propose that chromothripsis is a primary mechanism accelerating genomic DNA amplification and which enables rapid acquisition of tolerance to altered growth conditions.

MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences

Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres. Surprisingly, amplified genes (mainly PVT1) frequently participated in fusion transcripts lacking a corresponding DNA template. We also detected a significant overexpression of the circular RNA of PVT1 (circPVT1) in AML-amp cases versus AML with a normal karyotype. Our results show that 8q24 amplicons in AML are surprisingly plastic DNA structures with an unexpected association to novel fusion transcripts and circular RNAs.

MYC-containing amplicons in acute myeloid leukemia: Genomic structures, evolution, and transcriptional consequences

Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres. Surprisingly, amplified genes (mainly PVT1) frequently participated in fusion transcripts lacking a corresponding DNA template. We also detected a significant overexpression of the circular RNA of PVT1 (circPVT1) in AML-amp cases versus AML with a normal karyotype. Our results show that 8q24 amplicons in AML are surprisingly plastic DNA structures with an unexpected association to novel fusion transcripts and circular RNAs.Leukemia accepted article preview online, 28 November 2017. doi:10.1038/leu.2017.337.

Abstract 625: The frequency of gene amplifications in cancer revealed by a droplet digital PCR (ddPCR) based pan-cancer gene panel test

Abstract The development of molecular assays designed to detect gene amplifications has largely been hampered by technical challenges such as limited DNA quantity and tumor heterogeneity, which demand methods of very high precision and sensitivity. The recent introduction of affordable digital PCR platforms, such as droplet digital PCR (ddPCR), that are capable of providing single molecule resolution of target abundances thus provides a unique opportunity to address this gap in molecular diagnostics. A ddPCR-based test was therefore developed under CLIA-CAP guidelines to determine the amplification status of 12 commonly amplified genes targeted by FDA approved drugs. Termed the Amplinome Test, this test was applied to 49 clinical samples received over a period of 6 months and compared to the results obtained from a commercially available clinical sequencing test applied that also reports copy number alterations (CNAs). An overall concordance rate of 90% (532/588 calls) was observed between the two tests across the entire gene set, 5 of which were identified as amplified. There were 56 discordant copy number amplifications between the two testing modalities. The vast majority (55/56) of discordant calls arose from gene amplifications identified by the Amplinome test, but not detected by sequencing, indicating an 11-fold increase in sensitivity in detecting amplifications. One discordant call (HER3) was identified as amplified via sequencing, but confirmed to be unamplified via FISH. At the patient level, the Amplinome test identified 5-fold more patients as having actionable amplifications in at least one of the 12 assayed genes versus clinical sequencing (29 vs. 6). Clinical management was altered in 14 of the 29 patients with an actionable CNA identified on the Amplinome test; those patients received targeted therapy directed against the amplified gene. The ddPCR-based Amplinome test thus provides a highly sensitive method for measuring gene amplifications in cancer that alters patient management, and suggests that the prevalence of actionable amplifications may be significantly underestimated by standard clinical next-generation sequencing tests. Citation Format: Austin P. So, Amy Wong, Jennifer Pecson, Girish Putcha, Gregory Jensen, Michael Lucero, Gary Stone, Jason Gillman, Pravin Mishra, David Loughmiller, Derrick S. Haslem, Lincoln Nadauld. The frequency of gene amplifications in cancer revealed by a droplet digital PCR (ddPCR) based pan-cancer gene panel test. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2015-625

Abstract LB-284: Toward clinical sequencing: analysis of FFPE samples of 121 gastric cancer by a benchtop sequencer

Abstract Background: Clinical sequencing is becoming more important in cancer therapy, and analysis of formalin-fixed, paraffin-embedded (FFPE) samples is often required. In this study, we optimized pipeline in sequencing analysis of FFPE samples using a benchtop sequencer and profiled mutations and amplifications of cancer-related genes in advanced gastric cancer. Methods: FFPE samples of cancer and corresponding non-cancer were collected from patients with stage III/IV gastric cancer who underwent gastrectomy. Sequence libraries for almost all the exons of 409 cancer-related genes were prepared using the Ion AmpliSeq Comprehensive Cancer Panel. Sequence variations were analyzed using the Ion Proton Sequencer and the CLC Genomic Workbench software. Gene amplification in cancer was evaluated by ratio between reading depth of an individual gene divided by total reading depth in a cancer sample and that in a corresponding non-cancer sample. Results: We selected 129 pairs of samples whose template DNA had >32 copies in cancer samples and >5 copies in non-cancer samples, and sequence data were successfully obtained for 121 of the 129 pairs. If sequence variations with >10% frequency of reads were counted, a large number of variations was observed even in non-cancer samples (mean = 6.5). Therefore, the unfiltered results using FFPE samples were likely to contain false positive variants. Selection of variation (i) observed on both forward and reverse read, count >5; ii) total variant count >40) markedly reduced the variation in non-cancer samples (mean = 0.8). After the filtering, the mean number of variation in cancer samples was 4.9 and it was similar to the result of frozen samples (mean = 4.8). Next, profiling of mutation and amplification in the 121 gastric cancer was performed. The most frequently mutated genes were TP53 (36.4%). Mutations in oncogenes such as PIK3CA (7.4%), ROS1 (5.0%), ERBB2 (4.1%), MET (1.7%) and ALK (1.7%) were also detected. In addition, frequent mutations in genes not reported as gastric cancer-related genes such as SYNE1 (10.7%), CSMD3 (9.1%) and LRP1B (9.1%) were found. Amplification was detected for oncogenes such as SRC (20.7%, max 3.3-fold), ERBB2 (14.9%, 54-fold), CCNE1 (13.2%, 10-fold), CCND1 (9.1%, 8.6-fold), EGFR (7.4%, 22-fold), KRAS (7.4%, 22-fold) and MET (6.6%, 7.7-fold). Conclusion: We successfully optimized the pipeline for sequencing analysis of FFPE samples, and profiled gene mutations and amplifications in advanced gastric cancer. The pipeline will help us to drive clinical sequencing using FFPE samples. Citation Format: Satoshi Yamashita, Yasutoshi Kuboki, Tohru Niwa, Akiko Nagatsuma, Kohei Shitara, Nozomu Fuse, Toshihiko Doi, Atsushi Ochiai, Atsushi Ohtsu, Toshikazu Ushijima. Toward clinical sequencing: analysis of FFPE samples of 121 gastric cancer by a benchtop sequencer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-284. doi:10.1158/1538-7445.AM2014-LB-284

Potential Significance of EMP3 in Patients with Upper Urinary Tract Urothelial Carcinoma: Crosstalk with ErbB2-PI3K-Akt Pathway

Upper urinary tract (pyelocalyceal cavities and ureter) urothelial carcinoma is a relatively rare neoplastic disease. Although diagnosis and treatment of this tumor variant have improved significantly, accurate risk stratification remains a challenge. To identify the putative oncogene involved in urothelial carcinoma progression we performed bioinformatics guided experimental investigation targeting chromosome 19q13. We investigated the effects of EMP3 on cancer cell growth, migration and adhesion in transfection and siRNA experiments in vitro. Crosstalk of integrins or ErbB2 with EMP3 was examined by reverse transcriptase-polymerase chain reaction and immunoblot. The potential involvement of epigenetic alterations of EMP3 in vitro and in vivo was analyzed by methylation specific polymerase chain reaction. To validate clinical relevance we measured EMP3 expression at the mRNA and protein levels in a cohort of 77 patients with upper urinary tract urothelial carcinoma and compared prognostic significance in relation to that of ErbB2 expression. We noted functional crosstalk between ErbB2 and EMP3 in vitro. EMP3 over expression promoted cancer cell proliferation and migration but suppressed cell adhesion in vitro. EMP3 activated the ErbB2-PI3K-AKT pathway to increase cell growth in vitro. In the clinical cohort Kaplan-Meier survival estimates showed that ErbB2 and EMP3 co-expression was the most important indicator of progression-free and metastasis-free survival in patients with upper urinary tract urothelial carcinoma (log rank test p = 0.018 and 0.04, respectively). EMP3 is an important prognostic indicator for selecting patients with upper urinary tract urothelial carcinoma for more intensive therapy. EMP3 is an innovative co-targeting candidate for designing ErbB2 based cancer therapy.

Abstract LB-233: Identification of DNA palindromes in the MCF-7 breast cancer cell line.

Abstract DNA palindromes are implicated as a causative agent in cancer formation due to their association with genomic instability and DNA amplification. However, the unique properties of palindromes render them refractory to current methods of analyses including PCR amplification and cloning into bacterial vectors. Consequently, sequencing protocols that are dependent on PCR amplification fail on DNA palindromes. New tools are required for their discovery and characterization. Here we report our analysis of DNA palindromes from MCF-7, a widely used cell line model for estrogen receptor positive breast cancer. We developed a new strategy to identify and clone palindrome junctions by high throughput sequence analysis of DNA isolated by the Genome-wide Analysis of Palindrome Formation technique. We identified 35 palindrome candidates from MCF-7 and have cloned and sequenced 4 of the centers of the palindromes. None of these identified palindromes are present in other published data sets, despite the fact that MCF-7 DNA has been extensively sequenced and breakpoints analyzed, confirming that conventional methods fail on DNA palindromes. Our results provide further support for a correlation between palindromes and gene amplification in cancer, and underscore the importance of developing new tools for identifying and characterizing palindromes. Citation Format: Hui Yang, Natalia Volfovsky, Alison Rattray, Xiongfong Chen, Hisashi Tanaka, Strathern Jeffrey. Identification of DNA palindromes in the MCF-7 breast cancer cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-233. doi:10.1158/1538-7445.AM2013-LB-233

Recombination-restarted replication makes inverted chromosome fusions at inverted repeats.

SummaryImpediments to DNA replication are known to induce gross chromosomal rearrangements (GCR) and copy number variations (CNV). GCRs/CNVs underlie human genomic disorders1 and are a feature of cancer2. During cancer development environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs/CNVs are proposed to contribute to cancer development and therapy resistance3. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination (HR) or microhomology-primed re-initiation (FoSTeS/MMBIR)4,5. Here we ascertain the consequences of replication with a fork restarted by HR. We identify a new mechanism of chromosomal rearrangement: recombination-restarted forks have an exceptionally high propensity to execute a U-turn at small inverted repeats (up to 1:40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer and to non-recurrent CNVs in genomic disorders.

Combinatorics of the Breakage-Fusion-Bridge Mechanism

The breakage-fusion-bridge (BFB) mechanism was proposed over seven decades ago and is a source of genomic variability and gene amplification in cancer. Here we formally model and analyze the BFB mechanism, to our knowledge the first time this has been undertaken. We show that BFB can be modeled as successive inverted prefix duplications of a string. Using this model, we show that BFB can achieve a surprisingly broad range of amplification patterns. We find that a sequence of BFB operations can be found that nearly fits most patterns of copy number increases along a chromosome. We conclude that this limits the usefulness of methods like array CGH for detecting BFB.

Initial validation of a highly sensitive RT-qPCR assay for detecting all fusions and wild-type upregulation of ALK.

e21070 Background: Anaplastic lymphoma kinase (ALK) fusions cause a variety of malignancies (e.g. NSCLC and NHL). Patients with ALK fusion-driven cancers treated with the ALK inhibitor crizotinib, currently in clinical trials, have had marked antitumor responses. There are 15+ ALK fusions reported in NSCLC and other cancers, and testing by fluorescence in situ hybridization (FISH) has shown ALK gene amplification in cancers as well. The clinical significance of amplification is currently undetermined. We report results from a novel RT-qPCR assay (Insight ALK Screen) that is unbiased toward fusion partners and also detects wild-type upregulation. Results were compared to FISH and immunohistochemistry (IHC) to assess the accuracy of the qPCR assay and to correlate mRNA expression with gene amplification. Methods: Twenty-three NSCLC FFPE specimens were blinded and analyzed by the qPCR assay. ALK mutation status was independently determined by FISH and IHC. FISH studies used the ALK break-apart probes (Abbott); IHC used the D5F3 antibody (Cell Signaling). The qPCR method amplifies two regions corresponding to the ALK extracellular and intracellular kinase domains. EML4-ALK and other ALK fusion events result in a change in threshold cycle increase in transcripts encoding the ALK kinase domain over wild-type ALK expression levels. Results: The qPCR assay was positive for 11 samples from the enriched NSCLC cohort. The assay was 100% concordant with FISH in detecting the 4 samples with ALK fusions. The qPCR assay was also positive for 3 cases that were below (13%, 10% and 8%) the widely accepted criterion for being FISH-positive (>15% of tumor cells with split ALK 5’ and 3’ probe signals or with isolated 3’ signals) for fusions. Four other samples were both positive by qPCR and showed some level of gene amplification by FISH. Conclusions: Insight ALK Screen is a highly sensitive assay for detecting any ALK fusion regardless of the fusion partner. Detecting ALK mRNA expression by qPCR in cases below the FISH criterion for fusion positivity or that showed gene amplification suggests that more NSCLC patients could potentially qualify as candidates for ALK inhibitor therapy; further work is investigating this possibility.

Extrachromosomal amplification mechanisms in a glioma with amplified sequences from multiple chromosome loci

Accumulation of extrachromosomal DNA molecules (double minute) is often responsible for gene amplification in cancers, but the mechanisms leading to their formation are still largely unknown. By using quantitative PCR, chromosome walking, in situ hybridization on metaphase chromosomes and whole genome analysis, we studied a glioma containing four extrachromosomally amplified loci (7p11, 1q32.1, 5p15 and 9p2). Complex extrachromosomal DNA molecules were formed by the fusion of several syntenic or non-syntenic DNA fragments from 7p11, 5p15 to 9p2. Fragments ranged from a few base pairs to megabase pairs. Scars of the amplification process remained at the original locus in the form of deletions or chromosome rearrangements. Chromosome fragmentation, due to replication stress, could explain this complex situation. In contrast, at 1q32.1, the initial extrachromosomal DNA molecule resulted from the circularization of a single fragment associated with an intrachromosomal deletion including, but larger than, the amplified sequence. The nature of the sequences involved in these rearrangements suggests that a V(D)J-like illegitimate recombination contributes to its formation.

Tumor Protein D52 Overexpression and Gene Amplification in Cancers from a Mosaic of Microarrays

The tumor protein D52 gene is increasingly recognized to be overexpressed in different cancer types, frequently through gain of the corresponding locus at chromosome 8q21.13. This review summarizes the literature identifying D52 overexpression and/or gene amplification in different cancers, as well as recent publications directly analyzing D52 functions. Since D52 overexpression is increasingly being identified in tumors of different cellular origins, this is likely to perturb fundamental cell properties common to different cell types. Furthermore,since increased D52 expression occurs at different stages of tumorigenesis and progression, this could contribute to these processes through multiple mechanisms. Although functions are beginning to emerge from targeted studies, the pathways through which D52 overexpression contributes to cell transformation and the molecular interactions required remain undefined. In summary, while targeting D52 overexpression could provide therapeutic benefits in many cancer types, this will require an improved understanding of D52's cellular and molecular functions.

O VIII.4 - O VIII.4 Biological significance of gene amplification in cancer

O VIII.4 - O VIII.4 Biological significance of gene amplification in cancer

Gene amplification in cancer: A molecular cytogenetic approach

Many human tumors have been shown to exhibit cytologic evidence of gene amplification. In several instances the amplified genes have proven to be cellular oncogenes. We present an approach for combining cytogenetic and molecular biological techniques to systematically investigate this phenomenon.