Complex Genomic Changes Research Articles

MAPT haplotype-associated transcriptomic changes in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a neurodegenerative movement and cognitive disorder characterized by abnormal accumulation of the microtubule-associated protein tau in the brain. Biochemically, inclusions in PSP are enriched for tau proteoforms with four microtubule-binding domain repeats (4R), an isoform that arises from alternative tau pre-mRNA splicing. While preferential aggregation and reduced degradation of 4R tau protein is thought to play a role in inclusion formation and toxicity, an alternative hypothesis is that altered expression of tau mRNA isoforms plays a causal role. This stems from the observation that PSP is associated with common variation in the tau gene (MAPT) at the 17q21.31 locus which contains low copy number repeats flanking a large recurrent genomic inversion. The complex genomic structural changes at the locus give rise to two dominant haplotypes, termed H1 and H2, that have the potential to markedly influence gene expression. Here, we explored haplotype-dependent differences in gene expression using a bulk RNA-seq dataset derived from human post-mortem brain tissue from PSP (n = 84) and controls (n = 77) using a rigorous computational pipeline, including alternative pre-mRNA splicing. We found 3579 differentially expressed genes in the temporal cortex and 10,011 in the cerebellum. We also found 7214 differential splicing events in the temporal cortex and 18,802 in the cerebellum. In the cerebellum, total tau mRNA levels and the proportion of transcripts encoding 4R tau were significantly increased in PSP compared to controls. In the temporal cortex, the proportion of reads that expressed 4R tau was increased in cases compared to controls. 4R tau mRNA levels were significantly associated with the H1 haplotype in the temporal cortex. Further, we observed a marked haplotype-dependent difference in KANSL1 expression that was strongly associated with H1 in both brain regions. These findings support the hypothesis that sporadic PSP is associated with haplotype-dependent increases in 4R tau mRNA that might play a causal role in this disorder.

Array CGH + SNP in ALL Enhances Detection of Complex Cytogenetic Profiles and Suggests That PRSS1 Loss May Play a Role in Leukemogenesis

Introduction Cytogenetic alterations are essential prognostic biomarkers in patients (pts) with acute lymphoblastic leukemia/lymphoma (ALL), but are limited by the propensity of leukemic blasts to undergo apoptosis in culture, poor-quality chromosomes, and overgrowth by normal cells. Array CGH + SNP has 10,000-fold higher resolution than conventional cytogenetics and can identify submicroscopic gains, losses and copy number neutral loss of heterozygosity (cnLOH) irrespective of mitotic rate. Few studies outside of clinical trials have investigated microarray findings in adults with ALL or correlated microarray findings with clinical outcomes. Methods We retrospectively reviewed pts with ALL and mixed phenotype acute leukemia (MPAL) aged ≥18 years at diagnosis at our institution between 2017 and 2022. Array analysis was performed on diagnostic bone marrow or peripheral blood samples using high resolution CGH + SNP Genetisure cancer array platform (Agilent). Statistical analysis was done using R software (R core team, 2022). Results Baseline characteristics of included pts (n = 60) are shown in Table 1. CGH + SNP analysis identified 93.3% (56/60) of pts with genomic changes with a mean of 7 (range 0 - 28) aberrations per pt. Among pts with a normal karyotype, 81.8% (9/11) had new genomic abnormalities revealed by CGH + SNP, but significantly fewer aberrations were detected per pt compared to those with abnormal karyotypes (mean 3.09 v. 7.67, p = .008). Pts with Ph-like B-ALL/MPAL had significantly more aberrations on array than pts without Ph-like phenotype (mean 12.0 v. 5.85, p.= .002). Among the 78.3% of pts without complex cytogenetics on chromosome analysis, 44.7% (21/47) were found to have a complex genomic profile (≥5 aberrations) with the addition of CGH + SNP. However, there was no difference in CR rates or OS in pts with <5 or ≥5 microarray aberrations (p = .515 and p = .970, respectively). Quantity of copy number alterations (CNAs) as detected on array was not predictive of achieving end of induction CR/CRi/CRh (mean 6.08 in pts who achieved CR/CRi/CRh v. 7.02 in pts with primary refractory disease, p = .310). Hispanic pts had the same number of CNAs as non-Hispanic pts (mean 6.69 v. 6.94, p = .435). On linear regression analysis, there was no correlation between number of CNAs and age (r 2 = -.01, F = 0.1941, p = .661) or BMI (r 2 = 5.21 x10 -5, F = 0.0029, p = .957). The most frequent focal aberrations were detected in CDKN2A/B (n = 19), IKZF1 (n = 17), PRSS1 (n = 11), PAX5 (n = 8), BTG1 (n = 8) and RB1 (n = 8) (Figure 1). RB1 loss was enriched in pts with T-ALL (45.5% v. 6%, p = .003),and occurred at significantly higher rates than previously reported in T-ALL (Schraders, 2009). Loss of PRSS1 was detected in 18.3% (11/60) of the cohort. Homozygous PRSS1 deletions were identified in 3/11 (27.3%) pts. We identified the minimal overlapping deleted region on 7q34 in all 11 pts, which included the PRSS1 gene within the 124 kb region. Five of 6 pts who had repeat arrays performed at the end of induction had resolution of this aberration. The one pt who retained PRSS1 loss after induction had primary refractory disease, but after further treatment and attainment of CR1, this pt cleared the PRSS1 loss. Asparaginase associated pancreatitis, which has been linked to PRSS1 variations, occurred in only 1 pt with PRSS1 loss. Pts harboring PRSS1 loss were significantly less likely to achieve MRD negativity after induction (50% vs. 82%, P = .041) and had numerically more relapses (37.5% vs. 19%, p = .300), but no OS difference was observed (p = .290). Conclusions In our institutional cohort, the addition of CGH + SNP array reclassified almost half (44.7%) of pts without complex cytogenetics to harbor complex genomic changes (≥5 aberrations). Pts with Ph-like ALL had significantly more aberrations on array than those with Ph-positive or -negative B-ALL. Focal PRSS1 loss was associated with reduced rates of MRD clearance at the end of induction. This aberration resolved upon disease remission, raising the possibility that both homozygous and/or heterozygous deletions of 124 kb from the 7q34 chromosomal region of the PRSS1 gene may potentially have a role in ALL pathogenesis. Further studies with larger cohorts utilizing CGH + SNP analyses at additional time points during treatment are needed to explore the potential integration of microarray data for refined ALL risk stratification as well as to elucidate the functional role of PRSS1 in leukemogenesis.

A Methodologically Updated De-Novo Extraction of Copy Number Signatures in Multiple Myeloma: Clinical Significance and Putative Aetiologies

BACKGROUND: The tumor genomes of most Multiple Myeloma (MM) patients are heavily burdened with highly heterogenous copy number (CN) alterations, as detected by multiple molecular methods including whole genome sequencing (WGS), and have been shown to have a strong prognostic and predictive significance for patients' survival. Cutting-edge computational developments have made it possible to analyze complex genomic CN changes by identifying CN alterations' recurrent patterns among large cohorts of patients, named CN signatures (CNS), that are the result of cumulative chromosomic instability (CIN) processes throughout the course of cancer cells evolution. In fact, a robust methodological framework for CNS computation, along with a large compendium of CNS aetiologies have been recently published for many cancer types, notably not including MM (Drews 2022). To our knowledge, the only study that analyzes CNS in MM focused on the role of CNS in predicting chromothripsis events, but remarkably it did not include neither CNS aetiologies nor complete CNS characterization for all the discovered CNS (Maclachlan 2021). AIM: To define a novel methodological framework, based on previous studies, aimed at the identification of CNS in MM, in order to assess both the aetiologies and the biological significance of MM CNS and to evaluate their prognostic impact on MM clinical outcome. The newly extracted CNS defined at diagnosis will be also used as novel disease biomarkers, to develop an improved, aetiology-based MM patients' stratification in different molecular subtypes. METHODS: We calculated the MM genomic distributions of the six essential CN features (segment length, breakpoint per 10 Mb, breakpoint per chromosome arm, segment CN change, CN value, length of oscillating CN states) that have been previously shown to encode patterns of CN alterations, underlying the observed tumor CIN. Starting from 886 WGS generated CN profiles, included in the CoMMpass study, the above mentioned features were computed. Features were then categorized into components, by using mixture models' decomposition and CNS were finally extracted from the components, by using both a Hierarchical Dirichlet Process (HDP) and a Non-Negative Matrix Factorization (NNMF) approach. RESULTS: The main novel characteristics of the developed methodological framework aimed at CNS assessment were 1) the use of a "continuous CN value" feature, which enabled the evaluation of sub-clonal events and 2) the use of a logarithmic scale in "segment length" feature, which favored a higher resolution for categorizing focal and/or gene level CN events, that are very common in MM. Thanks to these implementations, 33 Gaussian mixture components were identified (as compared to 28 detected in Maclachlan 2021). After deriving a Sample x Component - Sum of Posteriors Matrix, the signatures were extracted by applying two parallel state-of-art approaches, namely HDP and NNMF. This allowed the extraction of 9 CNS that were characterized by their component's composition. The signature's exposure levels were correlated to well-known MM biomarkers (e.g. TP53 mut and/or del, 1q CN gain, 13q CN loss, t-IgH, hyperdiploidy), showing that all signatures correlated to at least one of the well known MM biomarkers; in particular, CN.SIG5 exposures was found to correlate to high-risk MM biomarkers (TP53 p<0.001, 1q CN gain p<0.001, t(4;14) p<0.001), thus suggesting its possible involvement in the aetiology of this peculiar genomic configuration. Finally, a survival analysis was performed in patients characterized by high exposure (4th quartile) to the CN.SIG5 (75 patients), as compared to the others (811 patients), showing a significant negative impact of this CNS on both overall (OS p<0.001)) and progression free survivals (PFS p<0.001). Cox-analysis revealed an OS HR= 1.37 p<0.001, PFS HR= 1.16, p<0.001, per 5% increase in exposure. CONCLUSION: By employing a novel bio-informatic approach, based on the use of continuous CN data for CNS extraction, 33 feature's components were identified. We observed that CN.SIG5 significantly affected patients carrying well-known high-risk genomic features, and patients highly exposed to this CNS had decreased PFS and OS. Additional characterizations are needed to unveil the biological meaning of CNS exposure; however, MM CNS, while informing on disease outcome, might be considered as new comprehensive biomarkers in this disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Adaptation to a new environment with pre-adaptive genomic features - Evidence from woody plants colonizing the land-sea interface.

Adaptation to new environments is a key evolutionary process which presumably involves complex genomic changes. Mangroves, a collection of approximately 80 woody plants that have independently invaded intertidal zones >20 times, are ideal for studying this process. We assembled near-chromosome-scale genomes of three Xylocarpus species as well as an outgroup species using single-molecule real-time sequencing. Phylogenomic analysis reveals two separate lineages, one with the mangrove Xylocarpus granatum and the other comprising a mangrove Xylocarpus moluccensis and a terrestrial Xylocarpus rumphii. In conjunction with previous studies, we identified several genomic features associated with mangroves: (i) signals of positive selection in genes related to salt tolerance and root development; (ii) genome-wide elevated ratios of non-synonymous to synonymous substitution relative to terrestrial relatives; and (iii) active elimination of long terminal repeats. These features are found in the terrestrial X. rumphii in addition to the two mangroves. These genomic features, not being strictly mangrove-specific, are hence considered pre-adaptive. We infer that the coastal but non-intertidal habitat of X. rumphii may have predisposed the common ancestor to invasion of true mangrove habitats. Other features including the preferential retention of duplicated genes and intolerance to pseudogenization are not found in X. rumphii and are likely true adaptive features in mangroves. In conclusion, by studying adaptive shift and partial shifts among closely related species, we set up a framework to study genomic features that are acquired at different stages of the pre-adaptation and adaptation to new environments.

A novel heptasomy 21 associated with complete loss of heterozygosity and loss of function RUNX1 mutation in acute myeloid leukemia

Chromosomal aberrations are among the most important prognostic parameters in AML, and conventional cytogenetic analysis remains essential for risk stratification. In this report, we describe an adult male patient with a high percentage of circulating blasts, pathologically confirmed as AML with maturation. Cytogenetic analysis of a bone marrow sample revealed heptasomy 21 and trisomy 13 within a complex karyotype of 52,XY,der(2)t(2;13)(q33.3;q32.1),+13,+21,+21,+21,+21,+21 in all 20 cells examined, which was confirmed by metaphase FISH. Chromosomal microarray analysis (CMA) revealed complete loss of heterozygosity (LOH) of chromosome 21, supporting a common origin. In addition, LOH of chromosome 1p, trisomy 13, and partial tetrasomy of 13q and partial monosomy of 2q as a result of an unbalanced translocation between chromosomes 2 and 13 were observed. Molecular analysis identified two pathogenic missense variants: RUNX1 p.D198Y and SRSF2 p.P95R. The clonal allele ratio of RUNX1 p.D198Y was consistent with all copies of chromosome 21 in the leukemic clone carrying the mutation. Within the medical literature, there are no reports of heptasomy 21 for comparison; however, there are reports of AML with either polysomy 21 or trisomy 13. Our results suggest that even relatively ‘common’ AML aneuploidies may be associated with much more complex genomic changes, including loss of heterozygosity, which impact prognosis.

Abstract AP09: COPY-NUMBER SIGNATURES AND MUTATIONAL PROCESSES IN HIGH GRADE SEROUS OVARIAN CARCINOMA

Abstract BACKGROUND: The genomic complexity of profound copy-number aberration has prevented effective molecular stratification of high grade serous ovarian carcinoma (HGSOC). Recent algorithmic advances have enabled interpretation of complex genomic changes by identifying mutational signatures—genomic patterns that are the imprint of mutagenic processes accumulated over the lifetime of a cancer cell. We hypothesized that specific features of copy-number (CN) abnormalities could represent the imprints of distinct mutational processes, and developed methods to identify signatures from copy-number features in HGSOC. METHODS: We derived copy-number signatures from absolute copy number profiles from 253 primary and relapsed HGSOC samples from 132 patients in the BriTROC-1 cohort using low-cost shallow whole-genome sequencing (sWGS; 0.1×). A subset of 56 of these cases had deep whole-genome sequencing (dWGS) performed for mutation analysis and comparison with sWGS data. Independent validation was performed using 112 dWGS HGSOC cases from PCAWG and 415 HGSOC cases with SNP array and whole exome sequence from TCGA. CN signature exposures were correlated with mutation data, SNV signatures, and other measures derived from deep WGS and exome sequencing to identify statistically significant genomic associations using a false discovery rate &amp;lt;0.05. RESULTS: We identified 7 CN signatures that provided a molecular framework to rederive the major defining elements of HGSOC genomes, including defective homologous recombination (HRD), tandem duplication, amplification of CCNE1 and amplification-associated fold-back inversions. Almost all patients with HGSOC demonstrated a mixture of signatures indicative of combinations of mutational processes, including those with early driver events such as BRCA2 mutation (in addition to HRD signatures). High exposure to CN signature 3, characterised by BRCA1/2-related HRD, was associated with improved overall survival. Conversely, high exposure to signature 1, which was characterised by oncogenic RAS signaling (including NF1, KRAS and NRAS mutation), predicted platinum-resistant relapse and poor survival. CONCLUSIONS: HGSOC lacks clinically-relevant patient stratification, which is reflected in poor survival and is a significant barrier to precision medicine. Copy-number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Our results suggest that early TP53 mutation, the ubiquitous initiating event in HGSOC, may permit multiple mutational processes to co-evolve, potentially simultaneously and that additional signature exposures may alter the risk of developing therapeutic resistance. Thus, our results suggest that HGSOC is a continuum of genomes. We derived signatures using inexpensive sWGS of DNA from core biopsies. These approaches are rapid and cost effective, thus providing a clear path to clinical implementation. By dissecting the mutational forces shaping HGSOC genomes, our study paves the way to understanding extreme genomic complexity, as well as revealing the evolution of tumors as they relapse and acquire resistance to therapy. Citation Format: Geoff Macintyre, Teodora E. Goranova, Dilrini De Silva, Darren Ennis, Anna M. Piskorz, Matthew Eldridge, Daoud Sie, Liz-Anne Lewsley, Aishah Hanif, Cheryl Wilson, Suzanne Dowson, Rosalind M. Glasspool, Michelle Lockley, Elly Brockbank, Ana Montes, Axel Walther, Sudha Sundar, Richard Edmondson, Geoff D. Hall, Andrew Clamp, Charlie Gourley, Marcia Hall, Christina Fotopoulou, Hani Gabra, James Paul, Anna Supernat, David Millan, Aoisha Hoyle, Gareth Bryson, Craig Nourse, Laura Mincarelli, Luis Navarro Sanchez, Bauke Ylstra, Mercedes Jimenez-Linan, Luiza Moore, Oliver Hofmann, Florian Markowetz, Iain A. McNeish, James D. Brenton. COPY-NUMBER SIGNATURES AND MUTATIONAL PROCESSES IN HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP09.

Overview of liposarcomas and their genomic landscape

Liposarcoma (LPS) is among the most common soft tissue sarcoma affecting adults. LPS is divided into three biologic subtypes characterized by specific genetic alterations. The most common LPS subtypes, well-differentiated and dedifferentiated LPS, are nearly uniformly characterized by ring chromosomes and giant markers with chromosomal amplification of 12q13-15 and resulting amplification of oncogenes MDM2, CDK4, and HMGA2. Myxoid/round cell LPS commonly exhibits a distinctive (12; 16) translocation resulting in the FUS-DDIT3 fusion gene. Finally, pleomorphic LPS harbors diverse complex genomic changes and chromosomal rearrangements and frequent mutations in TP53, RB1, and NF1 leading to dysregulation of tumor suppressor pathways. In this review, we summarize the currently available knowledge on the genomics and genetics of LPS subtypes as well as recent advances in the multimodality management of LPS.

Abstract B04: Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules

Abstract Acute myeloid leukemia (AML) is a disease that can affect both children and adults and is characterized by complex and heterogeneous genomic changes, including MYC overexpression driven in part by the bromodomain (BRD) and extraterminal (BET) protein family of epigenetic readers. Current limitations in treatment approaches, including protein overexpression and mutations that confer resistance to therapy, lead to a wide range of response variability to conventional therapy, excessive treatment-related toxicity, and an overall poor outcome. A new therapeutic strategy involves stabilizing the MYC promoter G-quadruplex to downregulate expression. We hypothesize that direct downregulation of MYC in AML cells using MYC-promoter G-quadruplex interacting drugs (GQID) is a feasible novel approach in targeted therapy of AML. Treatment of AML cell lines KG-1a and UT-7epo with the MYC-promoter GQID, GQC-05 resulted in decreased MYC expression and induced a more significant decrease in c-Myc protein than treatment with the BET inhibitor (+)-JQ1. GQC-05 also decreased cell viability and increased apoptosis. RNA-seq of GQC-05-treated KG-1a cells identified 947 significantly differentially expressed genes compared to DMSO. The expression of several c-MYC target genes (including E2F1, GNL3, HSPA8, MAT2A, RCC1, SHMT1, and TFRC) was significantly decreased concomitant with MYC downregulation. The 947 genes were compared with genes with putative quadruplex sequences (PQS) from the literature. Of the 17 genes in common, MYC, NOTCH1, PIM1, and RHOU are significantly downregulated following GQC-05 treatment. These findings were validated by Q-RT-PCR in KG-1a and several other AML cell lines. Drug sensitivity and resistance (DSR) screening on a panel of AML cell lines using a library of 55 anticancer and targeted agents identified a range of sensitivities to GQC-05 (AUC range: 1.65-2.99; DMSO AUC ~ 4). These were compared to microarray gene-expression profiles and, as expected, GQC-05 sensitivity was not correlated with MYC expression. Nevertheless, ANOVA identified 1,049 significantly differentially expressed probe sets between the most sensitive vs. the least sensitive cell lines. Functional annotation revealed over-representation of cadherin, Wnt, and p53 signaling pathways as well over genes involved in hemostasis and hematopoietic cell lineage. In conclusion, directly targeting the MYC promoter G-quadruplex in AML cells leads to knockdown of MYC expression and induces apoptosis. These results further support the development of GQID for targeting key genetic drivers in AML, and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. Citation Format: Megan A. Turnidge, Daniel H. Wai, Apurvi Patel, Justin J. Montoya, David W. Lee, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B04.

Abstract 4397: Tak-1 inhibition re-sensitizes cancer cells to TNFα

Abstract Identifying new pharmaceutical targets in cancer has been a long lasting goal of academia. Complex genomic changes coupled with a cancer cell's ability to adapt make tumors particularly difficult to target. To this end, we have identified the TAK1 kinase in the TNFα pathway as a potential mediator of cell survival or apoptosis. Here we evaluate the apoptotic effects of a novel TAK1 inhibitor, Takinib. The selectivity of Takinib was established with an IC50 of 9nm for TAK1, followed by an IC50 for IRAK4 and IRAK1 at 120nm and 390nm respectively. Treatment of MDA-MB-231 cells with Takinib on its own is not sufficient to induce consequential cell death. However, combination therapy with TNFα induces caspase 3/7 activity and subsequent cell death in a dose-dependent manner. Genetic knock out of TAK1 expression in MDA-MB-231 cells with the CRISPR/Cas9 system significantly sensitized previous resistant cells to TNFα. Furthermore, we have shown that activation of human THP-1 cells with LPS is sufficient to produce TNFα levels necessary to facilitate Takinib mediated cell death, indicating that potential immunotherapy applications may be possible. Together these findings support the idea that TAK1 may be an effective target in the TNFα pathway, shifting the cellular mechanisms from pro survival to pro apoptosis. Citation Format: Scott Scarneo, Madeline Sell, Juliane Totzke, David Carlson, Phillip Hughes, Tim Haystead. Tak-1 inhibition re-sensitizes cancer cells to TNFα [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 4397.

Chromothripsis 18 in multiple myeloma patient with rapid extramedullary relapse

BackgroundCatastrophic chromosomal event known as chromothripsis was proven to be a significant hallmark of poor prognosis in several cancer diseases. While this phenomenon is very rare in among multiple myeloma (MM) patients, its presence in karyotype is associated with very poor prognosis.Case presentationIn our case, we report a 62 year female patient with rapid progression of multiple myeloma (MM) into extramedullary disease and short overall survival (OS = 23 months). I-FISH investigation revealed presence of gain 1q21 and hyperdiploidy (+ 5,+ 9,+ 15) in 82% and 86%, respectively, while IgH rearrangements, del(17)(p13) and del(13)(q14) were evaluated as negative.Whole-genome profiling using array-CGH showed complex genomic changes including hyperdiploidy (+ 3,+ 5,+ 9,+ 11, + 15,+ 19), monosomy X, structural gains (1q21-1q23.1, 1q32-1q44, 16p13.13-16p11.2) and losses (1q23.1-1q32.1; 8p23.3-8p11.21) of genetic material and chromothripsis in chromosome 18 with 6 breakpoint areas. Next-generation sequencing showed a total of 338 variants with 1.8% (6/338) of pathological mutations in NRAS (c.181C > A; p.Gln61Lys) or variants of unknown significance in TP53, CUX1 and POU4F1.ConclusionsOur findings suggest that presence of chromothripsis should be considered as another important genetic hallmark of poor prognosis in MM patients and utilization of genome-wide screening techniques such as array-CGH and NGS improves the clinical diagnostics of the disease.

The role of recombination in evolutionary adaptation of Escherichia coli to a novel nutrient.

The benefits and detriments of recombination for adaptive evolution have been studied both theoretically and experimentally, with conflicting predictions and observations. Most pertinent experiments examine recombination's effects in an unchanging environment and do not study its genomewide effects. Here, we evolved six replicate populations of either highly recombining R+ or lowly recombining R- E.coli strains in a changing environment, by introducing the novel nutrients L-arabinose or indole into the environment. The experiment's ancestral strains are not viable on these nutrients, but 130 generations of adaptive evolution were sufficient to render them viable. Recombination conferred a more pronounced advantage to populations adapting to indole. To study the genomic changes associated with this advantage, we sequenced the genomes of 384 clones isolated from selected replicates at the end of the experiment. These genomes harbour complex changes that range from point mutations to large-scale DNA amplifications. Among several candidate adaptive mutations, those in the tryptophanase regulator tnaC stand out, because the tna operon in which it resides has a known role in indole metabolism. One of the highly recombining populations also shows a significant excess of large-scale segmental DNA amplifications that include the tna operon. This lineage also shows a unique and potentially adaptive combination of point mutations and DNA amplifications that may have originated independently from one another, to be joined later by recombination. Our data illustrate that the advantages of recombination for adaptive evolution strongly depend on the environment and that they can be associated with complex genomic changes.

Oncogenic Properties of Candidate Oncogenes in Chromosome Region 17p11.2p12 in Human Osteosarcoma

Osteosarcomas are primary tumors of bone that most often develop in adolescents. They are characterized by complex genomic changes including amplifications, deletions, and translocations. The chromosome region 17p11.2p12 is frequently amplified in human high grade osteosarcomas (25% of cases), suggesting the presence of one or more oncogenes. In previous studies, we identified 9 candidate oncogenes in this region (GID4, ARGHAP44, LRRC75A-AS1, TOP3A, COPS3, SHMT1, PRPSAP2, PMP22, and RASD1). The aim of the present study was to determine their oncogenic properties. Therefore, we generated osteosarcoma cell lines overexpressing these genes, except for LRRC75A-AS1 and PRPSAP2, and subjected these to functional oncogenic assays. We found that TOP3A, SHMT1, and RASD1 overexpression provided increased proliferation and that ARGHAP44, COPS3, and PMP22 overexpression had a stimulatory effect on migration and invasion of the cells. COPS3 and PMP22 overexpression additionally improved the ability of the cells to form new colonies. No oncogenic effect could be demonstrated for GID4 overexpression. We conclude that the concerted amplification-mediated overexpression of these genes in 17p11.2p12 may contribute to the oncogenic process in malignant osteosarcoma.

Abstract 3937: Comparative genomic hybridization reveals complex genomic changes in plasmacytoid urothelial carcinoma

Abstract Introduction The WHO classification of 2004 defined several histologic subtypes of urothelial carcinoma (UC) such as plasmacytoid UC (PUC). Since this definition, molecular characterization of these different tumor types has gained increasing attention. For the majority of histologic variants, it seems that they are associated with an even worse clinical outcome than conventional UC. However, PUC in particular is reported to be associated with an aggressive clinical course and advanced disease at the time of diagnosis. Nevertheless, genomic characterization of PUC, which examined genomic imbalances and chromosomal aberrations, is still missing. Material and Methods We analyzed formalin-fixed archival tissue of 25 PUCs using metaphase comparative genomic hybridization (CGH) for detecting genomic imbalances. Aberrant chromosomal regions were defined as characteristic when they were found in at least 20% of the tumors analyzed. Representative genes in aberrant chromosomal regions were additionally analyzed for copy number variations (CNV) using quantitative PCR to explore their possible roles in PUC carcinogenesis. Results Chromosomal aberrations were detected in every PUC analyzed and the average number of aberrations was 10.24 (ranging from 1-15). Characteristic aberrations were gains on 1q (48%), 3p (20%), 6p (32%), 11q (72%), 15q (36%), 16q (44%), 17p (76%), 17q (88%), and 20q (88%) and losses on 2q (24%) 4p (36%), 4q (84%), 5q (44%), 6q (68%), 13q (20%), and Xq (52%). PCR based analysis of CNV for CCND1 (11q13) showed a deletion in 73% of the cases. CDH1 (16q22) was deleted in 72% and amplified in 5%. ERBB2 (17q12) displayed remarkably few copy number alterations, with only 14% showing an amplification. SNAI1 (20q13) showed reduced gene copy numbers in 59.1% of the cases, while no copy number gains were detected. FOXO3 (6q21) exhibited the lowest number of copy number alterations, with 9% of all cases showing an amplification. Conclusions In PUCs, the frequency of aneuploidy and the complexity of genomic changes per tumor are greater than those described in conventional UC. The aberrations described in PUC involve the same regions that are associated with aggressive biological behavior in conventional UC. Gains on 11q, 17q, 17p and 20q and losses on 4q and 6q affect the majority of PUCs and seem to harbor important chromosomal regions for PUC carcinogenesis. Large-scale deletions on chromosome 9 were not detected. CNV analysis indicates heterozygous deletion of CDH1 as one underlying mechanism of loss of membranous E-cadherin in PUC. Loss of CCND1 and SNAI1 are common molecular features and could contribute to the aggressive biological behavior of PUC. Acknowledgements This study was supported by a grant from the ELAN Funds of the University Hospital Erlangen to BK and RS. Citation Format: Sven Wach, Christina Ellmann, Robert Stoehr, Katrin Weigelt, Peter J. Goebell, Frank Kunath, Helge Taubert, Arndt Hartmann, Bernd Wullich, Bastian Keck. Comparative genomic hybridization reveals complex genomic changes in plasmacytoid urothelial carcinoma. [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 3937. doi:10.1158/1538-7445.AM2015-3937

Abstract 3864: A transition zone showing highly discontinuous or rapidly oscillating levels of stem cell and proliferation markers characterizes the development of colorectal cancer

Abstract Background: Stepwise acquisition of genetic changes characterize the development of colorectal cancer (CRC). These mutational events are correlated with discrete morphologic transitions from hyperplastic to adenomatous areas followed by in situ transformation and finally invasive carcinoma. The genetic drivers for each stage of this process, however, have not been fully established and can be confounded by tumor heterogeneity. The goal of this study was to identify easily assessed biomarkers of key morphogenetic transitions in CRC. Methods and Findings: We analyzed the pattern of proliferation and expression of growth regulatory and stem cell markers across distinct morphologic transition zones in 726 cases of CRC. In cases with preserved adenoma-carcinoma morphologic transitions, we identified a characteristic zone of adenomatous epithelium, often located immediately adjacent to and extending into the invasive component, that showed rapidly oscillating intraglandular stretches of Ki-67+ and Ki-67- cells. This pattern correlated with oscillating expression of other cell cycle mediators and the growth regulators PTEN and SMAD4. These multifocal stretches of adenomatous epithelium showed alternating abrupt positive/negative expression boundaries. These zones also demonstrated similar abrupt intragland oscillations in the levels and/or subcellular localization of multiple cancer stem cell (CSC) markers including beta-catenin/CTNNB1, MGMT and CD44. In contrast, the expression levels of most of these markers were largely homogenous in the proximal adenomatous and deeper invasive carcinoma surrounding the transitional region. This CSC-like transitional zone, as detected by PTEN IHC, was prominent in 50/726 of all CRC (6.9%) but at least focally present in 97/201 (48.2%) cases with intact adenoma-carcinoma junctions. Genomic microarray (OncoScan HD) and mutation analysis (Ampliseq) on CRC with prominent CSC-like expansions demonstrated complex genomic changes in 12/18 (66.7%) with a similar frequency of KRAS, BRAF and CTNNB1 mutations as expected in unselected CRC cases. The transition zones in these cases also frequently demonstrated unstable genomes from cell-to-cell (as assessed by FISH) indicating high genetic instability in these areas. Conclusions: We show that multiple immunohistochemical markers, including PTEN, SMAD4, CD44 and CTNNB1, highlight a localized CSC-like transition zone of rapidly alternating quiescent/proliferative adenomatous epithelium in primary CRC tumors. This transition zone often begins in pre-invasive adenomatous epithelium adjacent to invasive tumor areas that have more uniform proliferation and more homogeneous genetic and expression profiles. This easily assessed phenomenon appears to represent a commonly occurring genetically unstable forerunner or transitional stage in CRC evolution. Citation Format: Kevin J. Arvai, Ya-Hsuan Hsu, Lobin A. Lee, Dan Jones. A transition zone showing highly discontinuous or rapidly oscillating levels of stem cell and proliferation markers characterizes the development of colorectal cancer. [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 3864. doi:10.1158/1538-7445.AM2014-3864

Comparative genomic hybridization shows complex genomic changes of plasmacytoid urothelial carcinoma

ObjectivesTo describe genomic imbalances in plasmacytoid urothelial carcinoma (PUC), which is a rare and aggressive variant of urothelial carcinoma (UC). Methods and materialsIn total, 25 formalin-fixed paraffin-embedded PUCs were analyzed by metaphase comparative genomic hybridization. Genomic imbalances were considered to be characteristic if they were detected in≥20% of the cases. Chromosome regions deviating by≥3 standard deviations from the average chromosome profile were scored as chromosomal gains or losses. Copy-number variations (CNVs) of CDH1 (16q 22.1), SNAI1 (20q 13.1), CCND1 (11q13.3), ERBB2 (17q12), and FOXO3 (6q21) were validated using quantitative polymerase chain reaction. ResultsChromosomal aberrations were detected in every PUC analyzed, and the average number of aberrations was 10.24 (ranging from 1–15). Characteristic aberrations were gains on 1q (48%), 3p (20%), 6p (32%), 11q (72%), 15q (36%), 16q (44%), 17p (76%), 17q (88%), and 20q (88%) and losses on 2q (24%) 4p (36%), 4q (84%), 5q (44%), 6q (68%), 13q (20%), and Xq (52%). polymerase chain reaction-based analysis of CNV for CCND1 (11q13) showed a deletion in 73% of the cases. CDH1 (16q22) was deleted in 72% and amplified in 5%. ERBB2 (17q12) displayed remarkably few copy-number alterations, with only 14% showing an amplification. SNAI1 (20q13) showed reduced gene copy numbers in 59.1% of the cases, whereas no copy-number gains were detected. FOXO3 (6q21) exhibited the lowest number of copy-number alterations, with 9% of all cases showing an amplification. ConclusionsIn PUCs, the frequency of aneuploidy and the complexity of genomic changes per tumor are greater than those described in conventional UC. The aberrations described in PUC involve the same regions that are associated with aggressive biological behavior in conventional UC. Gains on 11q, 17q, 17p, and 20q and losses on 4q and 6q affect most PUCs and seem to harbor important chromosomal regions for PUC carcinogenesis. Large-scale deletions on chromosome 9 were not detected. CNV analysis indicates heterozygous deletion of CDH1 as one underlying mechanism of loss of membranous E-cadherin in PUC. Loss of CCND1 and SNAI1 is a common molecular feature and could contribute to the aggressive biological behavior of PUC.

Performance Comparison of Affymetrix SNP6.0 and Cytogenetic 2.7M Whole-Genome Microarrays in Complex Cancer Samples

The Affymetrix cytogenetic 2.7M whole-genome microarray (Cyto2.7M) detects genomic aberrations. The Cyto2.7M array has increased coverage in regions with cancer-related genes, ∼4-fold reduced processing time, and 5-fold reduced input requirements (100 ng) compared to the commonly used Affymetrix SNP6.0 genome-wide microarray (SNP6.0). We set out to compare the performance of these microarrays on cancer samples containing complex genomic changes. We analyzed genomic DNA from 8 lymphoma samples and 1 blood sample using both SNP6.0 and Cyto2.7M microarrays. We compared the arrays with respect to 4 parameters, including detection of copy number variations (CNV), CNV boundaries, the actual copy number (CN) assigned to the aberrations, and loss of heterozygosity. The CN state of selected regions was validated by quantitative PCR. Very high consistency between arrays on all parameters tested was observed, hence only 30 of 224 aberrations disagreed on the CN state, corresponding to a total of ∼12 Mb or 0.06% of the analyzed base pairs. Thus, the SNP6.0 and Cyto2.7M arrays are equally well suited to detect genomic aberrations in complex samples such as cancer samples. With reduced processing time and lower input requirements, the Cyto2.7M array enables genomic analysis of samples where only limited DNA is available.

Older patients with normal karyotype acute myeloid leukemia have a higher rate of genomic changes compared to young patients as determined by SNP array analysis

Older patients with AML have a worse outcome compared to young patients. To study for potential contributors to their poor prognosis, we compared two NK-AML cohorts, young (<60 years old) and old (≥60 years old), via high density SNP array analysis. Older patients had more genomic changes (1.83±0.23 vs. 1.16±0.2, p=0.037) and a trend for a higher number of copy number neutral loss of heterozygosity (0.5±0.2 vs. 0.24±0.08, p=0.088) compared to young patients. We speculate that complex genomic changes in NK-AML may be a sign of an increase in genomic instability and an indicator of a worse prognosis.

Genomic and phenotypic analysis of BRCA2mutated breast cancers reveals co-occurring changes linked to progression

BackgroundInherited mutations in the BRCA2 gene greatly increase the risk of developing breast cancer. Consistent with an important role for BRCA2 in error-free DNA repair, complex genomic changes are frequently observed in tumors derived from BRCA2 mutation carriers. Here, we explore the impact of DNA copy-number changes in BRCA2 tumors with respect to phenotype and clinical staging of the disease.MethodsBreast tumors (n = 33) derived from BRCA2 999del5 mutation carriers were examined in terms of copy-number changes with high-resolution aCGH (array comparative genomic hybridization) containing 385 thousand probes (about one for each 7 kbp) and expression of phenotypic markers on TMAs (tissue microarrays). The data were examined with respect to clinical parameters including TNM staging, histologic grade, S phase, and ploidy.ResultsTumors from BRCA2 carriers of luminal and basal/triple-negative phenotypes (TNPs) differ with respect to patterns of DNA copy-number changes. The basal/TNP subtype was characterized by lack of pRb (RB1) coupled with high/intense expression of p16 (CDKN2A) gene products. We found increased proportions of Ki-67-positive cells to be significantly associated with loss of the wild-type (wt) BRCA2 allele in luminal types, whereas BRCA2wt loss was less frequent in BRCA2 tumors displaying basal/TNP phenotypes. Furthermore, we show that deletions at 13q13.1, involving the BRCA2wt allele, represents a part of a larger network of co-occurring genetic changes, including deletions at 6q22.32-q22.33, 11q14.2-q24.1, and gains at 17q24.1. Importantly, copy-number changes at these BRCA2-linked networking regions coincide with those associated with advanced progression, involving the capacity to metastasize to the nodes or more-distant sites at diagnosis.ConclusionsThe results presented here demonstrate divergent paths of tumor evolution in BRCA2 carriers and that deletion of the wild-type BRCA2 allele, together with co-occurring changes at 6 q, 11 q, and 17 q, are important events in progression toward advanced disease.

Comprehensive Ultra High Resolution Analysis of Copy Number and Allele Status Including Novel Microdeletions Spanning NF1 in Acute Myelogenous Leukemia

Karyotypic abnormalities are of dominant importance in AML risk prognostication and therapy selection. A comprehensive description of subchromosomal genomic copy number changes and allele status together with gene mutation analysis and identification of chromosomal translocations is needed to fully harvest the prognostic and biological power of genomic changes in AML.We have analyzed DNA from 96 AML-derived pure blast populations (purified using column-based multi parameter negative selection followed by multi-gated FACS sorting) compared with paired buccal DNA using the Affymetrix 6.0 SNP platform. To support data analysis and display, we have developed the software tools PLUT and LOH tool version 2 and have refined dChipSNP. Data were supplemented with blast karyotypes and mutation status of Flt3, NPM1, p53, N-ras and K-ras.Results: AML cases carried between zero and thirty-four subchromosomal losses and gains. Approximately 23% of all cases had ≥3 subchromosomal lesions and approximately 50% of all cases had no such lesions. Of the 22 cases with complex and hypercomplex genomic changes by SNP profiling, ~50% had mutations in p53 exons 5–9 and ~50% were p53 wild-type by sequence analysis. Monoallelic deletion of p53 as part of various deletions at 17p was found in 7 of 96 (7%) cases. Importantly, 7/96=7% additional cases demonstrated UPD at 17p which spanned the p53 locus. Of all 14/96=15% of cases with LOH at 17p (with and without copy loss) 9/14=64% carried p53 mutations and 11/14=79% had complex karyotypes. Of the AML cases with NPM1 mutations (19 of 96 cases or 20%), 75% carried no detectable subchromosomal lesions, while 25% carried between 1 and 4 such lesions.Previously unidentified microdeletions resulting in monoallelic NF1 loss were identified on 17p in a total of 10 of 96 (10%) cases, thus suggesting activation of the Ras pathway independent of Ras mutations (all affected cases had wild-type N-ras and K-ras) in a substantial subset of AML cases. Mutational analysis of all coding exons of NF1 on the retained allele in the affected cases is ongoing.Additional recurrent microdeletions were identified on chromosomes 3p and 3q as well as 12p, the latter encompassing the genes ETV6 and p27, as previously reported.Finally, analysis of the prognostically and therapeutically important deletions 5q and 7q identified large deletions without recurrent microdeletions. Regions of minimal loss on 5q have been delineated.

Effects of aneuploidy on genome structure, expression, and interphase organization in Arabidopsis thaliana.

Aneuploidy refers to losses and/or gains of individual chromosomes from the normal chromosome set. The resulting gene dosage imbalance has a noticeable affect on the phenotype, as illustrated by aneuploid syndromes, including Down syndrome in humans, and by human solid tumor cells, which are highly aneuploid. Although the phenotypic manifestations of aneuploidy are usually apparent, information about the underlying alterations in structure, expression, and interphase organization of unbalanced chromosome sets is still sparse. Plants generally tolerate aneuploidy better than animals, and, through colchicine treatment and breeding strategies, it is possible to obtain inbred sibling plants with different numbers of chromosomes. This possibility, combined with the genetic and genomics tools available for Arabidopsis thaliana, provides a powerful means to assess systematically the molecular and cytological consequences of aberrant numbers of specific chromosomes. Here, we report on the generation of Arabidopsis plants in which chromosome 5 is present in triplicate. We compare the global transcript profiles of normal diploids and chromosome 5 trisomics, and assess genome integrity using array comparative genome hybridization. We use live cell imaging to determine the interphase 3D arrangement of transgene-encoded fluorescent tags on chromosome 5 in trisomic and triploid plants. The results indicate that trisomy 5 disrupts gene expression throughout the genome and supports the production and/or retention of truncated copies of chromosome 5. Although trisomy 5 does not grossly distort the interphase arrangement of fluorescent-tagged sites on chromosome 5, it may somewhat enhance associations between transgene alleles. Our analysis reveals the complex genomic changes that can occur in aneuploids and underscores the importance of using multiple experimental approaches to investigate how chromosome numerical changes condition abnormal phenotypes and progressive genome instability.