Number Of Copy Number Alterations Research Articles

Profiling of Copy Number Alterations Using Low-Coverage Whole-Genome Sequencing Informs Differential Diagnosis and Prognosis in Primary Cutaneous Follicle Center Lymphoma

Primary cutaneous follicle center lymphoma (PCFCL) has an excellent prognosis using local treatment, whereas nodal follicular lymphoma (nFL), occasionally presenting with cutaneous spread, often requires systemic therapy. Distinction of the 2 diseases based on histopathology alone might be challenging. Copy number alterations (CNAs) have scarcely been explored on a genome-wide scale in PCFCL; however, they might serve as potential biomarkers during differential diagnosis and risk stratification. Low-coverage whole-genome sequencing is a robust, high-throughput method for genome-wide copy number profiling. In this study, we analyzed 28 PCFCL samples from 20 patients and compared the copy number profiles with a cohort of diagnostic samples of 64 nFL patients. Although the copy number profile of PCFCL was similar to that of nFL, PCFCL lacked amplifications of 18q, with the frequency peaking at 18q21.33 in nFL cases involving the BCL2 locus (PCFCL: 5.0% vs nFL: 31.3%, P = .018, Fisher exact test). Development of distant cutaneous spread was significantly associated with higher genomic instability including the proportion of genome altered (0.02 vs 0.13, P = .033) and number of CNAs (2 vs 9 P = .017), as well as the enrichment of 2p22.2-p15 amplification involving REL and XPO1 (6.3% vs 60.0%, P = .005), 3q23-q24 amplification (0.0% vs 50.0%, P = .004), 6q16.1-q23.3 deletion (6.3% vs 50.0%, P = .018), and 9p21.3 deletion covering CDKN2A and CDKN2B loci (0.0% vs 40.0%, P = .014, all Fisher exact test) in PCFCL. Analysis of sequential tumor samples in 2 cases harboring an unfavorable clinical course pointed to the acquisition of 2p amplification in the earliest common progenitor underlining its pivotal role in malignant transformation. By performing genome-wide copy number profiling on the largest patient cohort to date, we identified distinctive CNA alterations conceivably facilitating the differential diagnosis of PCFCL and secondary cutaneous involvement of nFL and potentially aiding the risk stratification of patients with PCFCL in the future.

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.

TESTICULAR LARGE B‐CELL LYMPHOMA IS GENETICALLY SIMILAR TO PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA AND DISTINCT FROM NODAL DIFFUSE LARGE B‐CELL LYMPHOMA

Introduction: Testicular large B-cell lymphoma (TLBCL) is an infrequent and aggressive lymphoma presenting in an immune-privileged site, recently recognized as a distinct entity from diffuse large B-cell lymphoma (DLBCL). Here, we performed the genetic characterization of TLBCL and compare it with a published series of nodal DLBCL and primary central nervous system lymphoma (PCNSL). Patients and Methods: We collected 61 patients with TLBCL diagnosed between 2002 and 2021. Cell-of-origin was determined by immunochemistry and Lymph2Cx assay. To characterize the genomic profile of TLBCL we performed targeted next-generation sequencing, copy number arrays, and fluorescent in situ hybridization, and compared them to those of DLBCL and PCNSL. Results: The median age was 70 years and 30% of the patients had disseminated disease at diagnosis, including six cases with CNS involvement. Using Hans' algorithm, 83% (44/53) were identified as a non-germinal center while the Lymph2Cx assay categorized 71% (30/42) as activated B-cell phenotype and 12% (5/42) as unclassified. BCL6 rearrangements were detected in 36% (17/47) of cases, and no concomitant BCL2 and MYC rearrangements were found. Integrative analysis of 40 cases with single nucleotide variants, indels, and copy number alterations (CNA) data showed that the most frequent alterations (>50%) in TLBCL were PRDM1, CDKN2A/B, TNFAIP3, SGK1, ARID1B, MYD88L265P, SPIB, PIM1, and CD79B (Figure). Concomitant variants in MYD88L265P, CD79B, and PIM1 were detected in twelve (29%) cases. Using the LymphGen tool, 71% (30/42) of the cases could be classified into a molecular subgroup, with MCD being the most frequent group (66%, 20/30). Interestingly, patients with localized or disseminated disease displayed similar genomic complexity based on the number of CNAs and the number of mutated genes. Compared with nodal DLBCL, localized and disseminated TLBCL have less CNA complexity (P = 0.01 and P < 0.04, respectively) but showed a higher number of variants (P = 0.01 and P < 0.001, respectively). TLBCL also presented more frequently 18q21.32-q23 (BCL2) gains and 6q and 9p21.3 (CDKN2A/B) deletions. PIM1, MYD88L265P, CD79B, TBL1XR1, MEF2B, CIITA, EP300, and ETV6 variants were enriched in TLBCL, and BCL10 mutations in nodal DLBCL. There were no genetic differences between TLBCL and PCNSL. Encore Abstract - previously submitted to regional or national meetings (up to <1’000 attendees). The research was funded by: Asociación Española Contra el Cancer AECC/CIBER: PROYE18020BEA (SB); Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III Instituto de Salud Carlos III, “Cofinanciado por la Unión Europea” and Fondos FEDER: European Regional Development Fund “Una manera de hacer Europa”: PI17/01061 (SB), PI19/00887 (ALG and EG), and INT20/00050 (ALG); Marató TV3 TV3-Cancer/ 201904-30 (SB). ARD is supported by a grant from Sociedad Española de Hematología y Hemoterapia. CL is supported by postdoctoral Beatriu de Pinós grant from Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and by Marie Sklodowska-Curie COFUND program from H2020 (2018-BP-00055). Keywords: Extranodal non-Hodgkin lymphoma, Pathology and Classification of Lymphomas Conflicts of interests pertinent to the abstract. F. Nadeu Honoraria: Janssen, AbbVie E. Campo Consultant or advisory role: Takeda, NanoString, AbbVie, Illumina Honoraria: Janssen, EUSPharma, Roche Research funding: AstraZeneca A. López-Guillermo Consultant or advisory role: Roche, Gilead, Kite Pharma, Celgene, Bristol Myers Squibb, Incyte, Takeda, Kern Pharma, Pfizer, Janssen Honoraria: Janssen, Roche, Kite Research funding: Gilead, Janssen, Celgene, Bristol Myers Squibb Educational grants: Roche, Kite/Gilead

Genomic arrays identify high-risk chronic lymphocytic leukemia with genomic complexity: a multi-center study.

Complex karyotype identified by chromosome-banding analysis has been shown to have prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genomewide detection of copy-number alterations (CNA) and could therefore be well equipped to detect the presence of a complex karyotype. Current knowledge on genomic arrays in CLL is based on outcomes of single-center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2,293 arrays from 13 diagnostic laboratories according to established standards. CNA were found outside regions captured by CLL fluorescence in situ hybridization probes in 34% of patients, and several of them, including gains of 8q, deletions of 9p and 18p (P<0.01), were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided into three distinct prognostic subgroups based on the number of CNA. In multivariable analysis only high genomic complexity, defined as ≥5 CNA, emerged as an independent adverse prognosticator for time to first treatment (hazard ratio: 2.15; 95% confidence interval: 1.36-3.41; P=0.001) and overall survival (hazard ratio: 2.54, 95% confidence interval: 1.54-4.17; P<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and, in terms of risk stratification, performed at least as well as simultaneous chromosome banding analysis as carried out in 122 patients. Our findings indicate that genomic array is an accurate tool for CLL risk stratification.

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

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

Abstract A14: Copy number profiling in South African breast cancer patients

Abstract Introduction: Breast cancer is the most common cancer among South African women, where as in many other Sub-Saharan countries, is the leading cause of female death. Although several risk factors can be attributed to their observed high mortality rate, the biology of their tumors are not extensively investigated; consequently the molecular profiles associated with their disease is poorly characterized. Main goal: In this study our main goal was to characterize the wide-genomic copy number profile of a group of patients with breast cancer diagnosed at the Groote Schuur Hospital, Cape Town, South Africa and associate with their corresponding clinical-pathological data. Material and Methods: Twenty-eight specimens of formalin fixed paraffin embedded (FFPE) material from twenty-eight patients with invasive breast cancer were acquired prior to any treatment and with complete clinical annotation from the archives of the Division of Anatomical Pathology, National Health Laboratory Service-Groote Schuur Hospital. The mean age and tumor size of the patients at diagnosis was 48.2±12.9 years and 38.2±14.0 mm, respectively. Most of the tumors were of grade 2 and 3 (39.2% and 37.7%, respectively) and stage III (67.8%). ER and ERBB2 positivity was observed in 57.1% and 53.8% of the patients, respectively. PR status was available for only 21.4% of the patients, out of which 33% (2/6) were positive. Three patients were negative for ER, PR and ERBB2 receptors. Race information was self-reported; 61.4% of the patients were from the Colored group, followed by 19.2% from the White and Black group, which are the three most predominant ethnic groups in South Africa. Array-CGH analysis was performed in microdissected tumor areas using the Agilent 8x60K oligo nucleotide array platform. Results: The average number of copy number alterations (CNAs) observed in these patients was 8.3±1.2 which was significantly associated with patients' tumor grade (P&amp;lt;0.01). The most frequent cytobands affected were Xp22.33-p21.1 (86.7% of the cases), 6p25.3-p21 (83.3%), 19p13.3-p13.11 (56.7%), 21p11.2-p11.1 (50%) and 16p13.3-p11.2 (43.3%), that except for the 21p region, were observed gained and/or amplified. From these commonly affected cytobands, gains on 6p was significantly associated with tumor grade (P&amp;lt;0.05). No significant association was observed among the average number of CNAs and/or the most frequent cytobands with other clinical-histopathological parameters and/or ethnic groups. Conclusions: Our results indicated that CNAs occur in South African breast cancer patients, in association with advanced tumor grades, but irrespectively of their ethnic descendance and other factors at diagnosis that are known to influence prognosis. Additional molecular studies, such as ours, that aim to determine the biologic contributions to breast-cancer risk in women in South Africa and other African countries are imperative. The understanding of the genetic determinants that are associated with the etiology of their tumors is a direction towards the reduction of their observed high mortality rates. Citation Format: Kamil Lupicki, Selene Elifio-Esposito, Aline S. Fonseca, Akanksha Mahajan, Silma R. Pereira, B Langa, Dhirendra Govender, Eugenio Panieri, Donavon Hiss, S Abdul-Rasool, Luciane R. Cavalli. Copy number profiling in South African breast cancer patients [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A14.

Abstract 716: Genomic classification of longitudinally observed small colorectal polyps

Abstract Introduction: Colorectal polyps are very common in elderly people with a prevalence of 15-30% reported in screening series. However, only a minority of 5% of these lesions is estimated to develop into invasive cancers, whereas others will remain stable or regress over time. In standard practice all polyps observed during colonoscopy are removed by polypectomy and therefore, knowledge about their natural history is limited. In this study, small (6-9mm) colorectal polyps were followed over time and subsequently resected during colonoscopy. Aim: The prevalence of DNA copy number alterations (CNAs), a feature associated with adenoma to carcinoma progression, was compared in polyps that morphologically progressed versus those that did not. Methods: In the CT-colonography (CTC) arm of the COCOS-trial1 small 6-9mm polyps were left in situ. After a three-year surveillance interval, growth according to CTC of 95 small polyps was measured. Based on overall volumetric change on CTC, polyps were classified as either progressed (&amp;gt;30% growth), stable (30% to -30% growth) or regressed (&amp;lt;-30% growth). From 65 resected polyps, formalin-fixed paraffin-embedded material was retrieved and reviewed by an expert pathologist (GAM). Of these polyps, 48% (31/65) progressed, 41% (27/65) remained stable and 11% (7/65) regressed. Seventy-two percent (47/65) were tubular adenomas, 14% (9/65) tubulovillous adenomas, 3% (2/65) sessile serrated lesions and 11% (7/65) hyperplastic polyps. Subsequently, the samples that yielded sufficient quality DNA were analyzed with low-coverage whole genome sequencing (HiSeq, Illumina, San Diego, CA, USA). DNA copy number status was called with the R-package QDNAseq. Results: From 50/68 resected polyps, originating from 38 individuals (mean age of 66.8 years (s.d. 7.0); 63% male), sufficient DNA was available. The mean surveillance interval of these polyps was 3.27 years (s.d. 0.29). The mean number of CNAs per sample was 0.8 (range 0-8), the most frequently observed was 13q gain (20%). No significant differences in number, or regions of CNAs were observed between polyps that progressed and the ones that did not change or diminished in size (stable or regressed). Larger polyps showed more CNAs, of which specifically 13q gain was more often present (43% in ≥10mm and 8% in &amp;lt;10mm polyps). Of note, no serrated lesions were ≥10mm. No differences in CNAs were found between the different histological subtypes. Conclusions: In a series of colorectal polyps left in situ for an average 3.27 years, no difference in prevalence of DNA copy number alterations was observed between polyps that volumetrically progressed and those that did not. Of the CNAs observed, 13q gain was most prevalent and found in 43% of the ≥10mm polyps. 1. Stoop, E. M. et al. Participation and yield of colonoscopy versus non-cathartic CT colonography in population-based screening for colorectal cancer: A randomised controlled trial. Lancet Oncol. 13, 55-64 (2012). Citation Format: Meta C. van Lanschot, Beatriz Carvalho, Charlotte J. Tutein Nolthenius, Christian R. Rausch, Ernst J. Kuipers, Jaap Stoker, Evelien Dekker, Gerrit A. Meijer. Genomic classification of longitudinally observed small colorectal polyps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 716. doi:10.1158/1538-7445.AM2017-716

Clincial and Genetic Landscapes Differ Between IGH-CRLF2 and P2RY8-CRLF2 Acute Lymphoblastic Leukaemia

Deregulated expression of the type I cytokine receptor, cytokine receptor-like factor 2 (CRLF2 -d), is observed in 5% of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). It occurs via three known aberrations; a cryptic reciprocal translocation with the immunoglobulin heavy chain locus (IGH); an interstitial deletion within PAR1, resulting in the P2RY8-CRLF2 fusion; rare but recurrent CRLF2 mutation (F323C). All three result in overexpression of CRLF2, however alone it is insufficient to cause overt leukaemia. Mutations of the Janus kinase (JAK) family genes and the IL7 receptor, are recurrent (50% of CRLF2-d) and together with CRLF2 -d result in transformation of mouse BaF3 cells. We noted that outcome data from different study groups were inconsistent, with patients being classified as either high or intermediate risk. Thus, in this largest study to date, our aim was to acertain whether differences in clinical and/or genetic features between patients with CRLF2-d and involvement of either IGH or P2RY8 may define them as different disease entities and partly explain the outcome heterogeneity.Among 160 CRLF2-d patients, chromosomal analysis confirmed a high incidence of additional somatic copies of chromosomes X (39/88, 44%) and 21 (23/88, 26%) and identified that aneuploidy of chromosomes 9 (5/88, 6%) and 17 (7/88, 8%) were recurrent in both groups. From comparison of patients with IGH-CRLF2 and P2RY8-CRLF2, we noted a higher frequency of IKZF1 (79% v 37% p<0.001) and BTG1 (50% v 8%, p<0.001) deletions and greater complexity (>3 additional copy number alterations (CNA)) by MLPA (SALSA MLPA P335 kit, MRC Holland, The Netherlands) (p=0.037) among IGH-CRLF2 patients. We verified that (48/160) 30% of CRLF2 -d patients were Down syndrome (DS).In addition to CNA within the immunoglobulin loci and the genes investigated by MLPA, SNP6.0 arrays (Affymetrix, Santa Clara, USA) (n=25) and subsequent FISH screening identified recurrent CNA in the histone cluster at 6p22.2 (7/25, 28%), VPREB1 (6/25, 24%), ADD3 (14/58, 24%), BTLA (4/25, 16%), SLX4IP (12/43, 28%), SERP2 and TSC22D1 (6/53, 11%), PBX3 (16/54, 30%). A novel finding was an interstitial deletion at Xp11.4 fusing USP9X to DDX3X (9/50, 18%).A total of 137 somatic structural variants (SV) were detected by whole genome sequencing of 11 patients [tandem duplication- (n=6), deletion- (n=104), inversion- (n=14) and translocation-type (n=13)] creating an average of 12.7 validated SV per patient. Mutations were identified at an incidence of 12.8 per patient, including mutations in JAK1 and JAK2 (40%) and IL7R, CACNA1D and USH2A (n=2). Mutations in kinase genes were common and unique to all patients, a number have not been previously reported (e.g. ERBB4, TTBK1 and STK38L). Of interest, 7 patients had ≥1 point mutation in gene(s) involved in cell adhesion (e.g. NPNT, ADAM29, ITGB7, COL3A1 and USH2A).There was a significant difference in the median age and white blood cell count (WBC) between P2RY8-CRLF2 and IGH-CRLF2 (4yrs v 14yrs, p<0.001 and WBC >50x106 /L - 25% v 44%, p=0.021). Overall IGH-CRLF2 patients had a worse outcome compared to P2RY8-CRLF2 (p=0.191) with outcome heterogeneity in relation to gender (IGH females, HR 3.75, p=0.003), number of CNA (IGH <3 genes deleted, HR 3.79, p=0.047) and treatment trial (IGH have worse outcome on ALL97, HR 3.15, p=0.014) being observed. There was no heterogeneity for age (p=0.458), WBC (p=0.331), cytogenetic risk (p=1.0), MRD (p=1.0) or DS-ALL (p=0.877).In summary we have confirmed differences in age and WBC, involvement of kinase gene mutations and chromosomal gains in CRLF2-d ALL. Other findings included recurrent deletions involving TSC22D1 and PBX3, transcription factors involved in other human cancers; SLX4IP, an uncharacterized gene, frequently deleted in childhood ALL; the novel involvement of USP9X, a deubiquitinase involved in cancer development. We also identified recurrent mutations in cell adhesion genes. We showed that IGH-CRLF2 patients had a worse outcome, with heterogeneity seen between gender, number of gene deletions and treatment trial. We have shown that there are genetic and clinical differences dependent on whether CRLF2 is deregulated by IGH or P2RY8, suggesting that they should be regarded as different clincial entities in future studies. DisclosuresCampbell:14M genomics: Other: Co-founder and consultant.

Abstract B1-54: Genomic findings in the UCEC study: Identifying diagnostically relevant changes in early-stage endometrial cancer

Abstract Background: Endometrial cancer (EC) encompasses the common endometroid histologic subtype, with variable clinical outcomes, and the less common papillary serous/clear cell carcinoma (PSC), with uniformly adverse prognosis. The primary unmet diagnostic need in EC is to identify cases of low-stage endometriod histology that have risk of recurrence and would benefit from adjuvant chemotherapy. The multicenter Uterine Corpus Endometrial Carcinoma (UCEC) study (Nature 2013;497:67–73) recently employed a multiplatform investigation to address this need and identified four distinct molecular clusters of EC. These included a group of POLE-mutated cases with an extremely high mutation rate and favorable prognosis and a group of mostly but not exclusively PSC cases with TP53 mutations, frequent genomic copy-number (CN) changes and poor prognosis. However, most of the cases presented with more variable outcomes (155/232, 66.8%) including endometriod EC with unmutated TP53 and few CN changes or microsatellite instability (MSI). We examined here in more detail how mutation pattern, MSI status, total number of CN alterations, and mutation load interact in the UCEC data set to predict outcome and recurrence in the clinically relevant stage I/II endometriod cases. Methods and Findings: We performed data analysis for the full 232 UCEC tumor set and, separately, for the 155 cases in the endometriod subset and of those, the 127 cases in the stage I/II set. Prognostic information provided in the UCEC data set included recurrence and outcome; the overall recurrence rate was 19% (45/232), with 23 deaths (10%) reported. In the entire data set, recurrence was more common in the CN-high group (22/60; 37%) and did not occur in the POLE-mutated group. A model filtered for the 5 most significantly mutated (chi-squared) genes and MSI status could predict the four outcome clusters reported in the UCEC study with 96% accuracy. However, Kaplan-Meier analysis showed no significant outcome prediction power for binary MSI status and CN class when analysis was restricted to the stage I/II endometriod subgroup (p=0.41). The number of mutations per case was significantly lower in low-stage cases (p&amp;lt;0.01) but did not correlate with recurrence. Commonly mutated oncogenes in EC, including PTEN and PIK3CA, were not significantly differentially mutated by clinical stage or recurrence status in endometriod cases. In contrast, several other genes not previously well-studied in EC were differentially mutated in the stage I/II endometroid subgroup. These included the estrogen receptor-α gene (ESR1), in which mutations were differentially associated with recurrence (p &amp;lt; 0.01, Fisher's exact). Higher CN alteration scores on microarray analysis were also significantly associated with recurrence in that subgroup (p &amp;lt; 0.01, Student's t-test). Genomic complexity in low-stage endometriod cases was not associated with TP53 mutation (124/127 unmutated) or TP53 loss (126/127 with no deletion), implicating other genome maintenance alterations. Conclusions: We were able to accurately replicate the previously identified prognostic groups from the UCEC data using the mutation status of just a small number of genes and MSI status. However, the outcome prediction power of this model in the stage I/II endometriod subset is limited. In contrast, genomic complexity and mutation status of a small set of genes, including ESR1, show promise as recurrence risk predictors in low-stage endometriod tumors. ESR1 mutation was previously identified to be a marker of aggressive disease in metastatic breast cancer (Nature Genetics 2013;45;1446–1451). Independent studies with adequate statistical power and an extended outcome analysis of the UCEC study are needed to expand on these findings. Note: The results shown here are in whole based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov. Citation Format: Kevin J. Arvai, Shere S. Billouin-Frazier, Yongbao Wang, Dan Jones. Genomic findings in the UCEC study: Identifying diagnostically relevant changes in early-stage endometrial cancer. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-54.

Identification of novel genomic aberrations in AML-M5 in a level of array CGH.

To assess the possible existence of unbalanced chromosomal abnormalities and delineate the characterization of copy number alterations (CNAs) of acute myeloid leukemia-M5 (AML-M5), R-banding karyotype, oligonucelotide array CGH and FISH were performed in 24 patients with AML-M5. A total of 117 CNAs with size ranging from 0.004 to 146.263 Mb was recognized in 12 of 24 cases, involving all chromosomes other than chromosome 1, 4, X and Y. Cryptic CNAs with size less than 5 Mb accounted for 59.8% of all the CNAs. 12 recurrent chromosomal alterations were mapped. Seven out of them were described in the previous AML studies and five were new candidate AML-M5 associated CNAs, including gains of 3q26.2-qter and 13q31.3 as well as losses of 2q24.2, 8p12 and 14q32. Amplication of 3q26.2-qter was the sole large recurrent chromosomal anomaly and the pathogenic mechanism in AML-M5 was possibly different from the classical recurrent 3q21q26 abnormality in AML. As a tumor suppressor gene, FOXN3, was singled out from the small recurrent CNA of 14q32, however, it is proved that deletion of FOXN3 is a common marker of myeloid leukemia rather than a specific marker for AML-M5 subtype. Moreover, the concurrent amplication of MLL and deletion of CDKN2A were noted and it might be associated with AML-M5. The number of CNA did not show a significant association with clinico-biological parameters and CR number of the 22 patients received chemotherapy. This study provided the evidence that array CGH served as a complementary platform for routine cytogenetic analysis to identify those cryptic alterations in the patients with AML-M5. As a subtype of AML, AML-M5 carries both common recurrent CNAs and unique CNAs, which may harbor novel oncogenes or tumor suppressor genes. Clarifying the role of these genes will contribute to the understanding of leukemogenic network of AML-M5.

Use of DNA copy number analysis of grade 2-4 gliomas to reveal differences in molecular ontogeny associated with IDH mutation status.

2026 Background: The genetic alterations of glioma have been studied extensively. IDH1 mutation is associated with younger age and better survival. However, differences in molecular ontogeny within glioma related to IDH1 mutation remain unknown. Here we describe a detailed analysis of copy number alterations (CNA) between IDH1mut vs IDH1wt gliomas of grade 2-3 and 4. Methods: CNA were detected by molecular inversion probes (Affymetrix) and analyzed with Nexus Copy Number Software (BioDiscovery). DNA was extracted from 94 patient FFPE samples including grade 2-3: IDH1wt (n = 17) and IDH1mut (n = 28), and grade IV: IDH1wt ( n = 25) and IDH1mut(n = 24). Chromothripsis was detected using a stringent criteria of at least ten switches of CNA in individual chromosomes. Results: We validated prior findings that IDH1wt GBM have higher frequency of Chr7 amplification (including EGFR) and loss of Chr10 (including PTEN). Other CNA across all grades were: gain of 19q12 and loss of 14q11 in IDH1wt, and gain of 11q21, 10p11, 8q21 and loss of 11p15, 19q13 in IDH1mut. Within grade 2-3 samples, few CNA were associated with mutation status: 2-3wt demonstrated higher frequencies of gain of 7q and loss of 10q, 14q11, and 22q13, while 2-3mut demonstrated higher frequencies of 11q21 gain and 19q13 loss. Grade 4 tumors demonstrated more CNA that differed by mutation status, with 4wt tumors demonstrating gain of 7 and loss of 10 and 14q11, while 4mut demonstrated gains of 8q, 10p, 12p13, 1q23, and loss of 11p15, 3p, 19q13, among others. Comparison of grade 2-3mut vs grade 4mut tumors demonstrated larger number of CNA in the grade 4mut tumors including gain of 1p, 14q, 13q33, 9p, 8q and loss of 22q, 11p15, 10q, and 3p, among others. A significantly higher incidence of chromothripsis events was observed in grade 4mut compared to grade 4wt (p = 0.0374). Conclusions: CNA analysis showed significant differences in molecular ontogeny between IDH1wt and IDH1mut, some of which may further elucidate pathogenesis. Significant CNA increases and increased chromthripsis in grade 4mut support malignant transformation of low grade gliomas through accumulation of genomic instability and genomic catastrophe.

Identification of Recurrent Submicroscopic Deletions of chromosome1q31.3 As Possible Molecular Prognostic Markers in Acute Promyelocytic Leukemia (APL)

Abstract 420▪▪This icon denotes a clinically relevant abstract Introduction:The characteristic hallmark of acute promyelocytic leukemia (APL) is a balanced reciprocal chromosomal translocation between chromosomes 15 and 17 leading to a fusion product consisting of the promyelocytic leukemia gene (PML) and the retinoic acid receptor alpha (RARA). The PML-RARA fusion product is necessary but not sufficient for the generation of leukemia and it is hypothesized that additional genomic lesions play a role in the pathogenesis of APL. Therefore, we have performed a high density SNP array analysis on 101 APL patient samples to identify new copy number alterations (CNAs) which may be relevant for the biology and prognosis of APL. Patients and Methods: Molecular and clinical outcome analyses were carried out retrospectively on patients diagnosed with APL, whose samples were referred to the molecular laboratory of the Department of Hematology and Oncology of the Medical Faculty Mannheim, University of Heidelberg, Germany between 1997 and 2010. 500 ng of genomic DNA from leukemic blasts per sample were processed according to the Genome Wide Human SNP 6.0 Array protocol (Affymetrix, Santa Clara, CA). The CNAG 3.3 software was used to perform allele-specific copy number analysis with anonymous references. CNAs of special interest were validated to be acquired in leukemia cells by performing allele-specific copy number analysis in matched pair SNP 6.0 array analysis, quantitative real time PCR and direct sequencing of genomic DNA from initial diagnosis and molecular remission samples. Results: We identified 279 acquired CNAs consisting of 185 heterozygous deletions, 87 amplifications and 7 regions of copy number neutral loss of heterozygosity (CNLOH). Besides common chromosomal aberrations such as trisomy 8 or duplications of the long arm of chromosome 8, deletions of 7q or isochromosome ider(17)(q10)t(15;17), numerous novel recurrent micro-deletions were discovered. The most common was a somatically acquired ∼100 kilobase deletion of chromosome 1q31.3 in 13 of 101 (13%) patients. These deletions encompassed the coding regions for the microRNAs mir181a1/b1. In univariate analyses of overall survival (OS) and relapse free survival (RFS) using Logrank tests, we found that patients carrying 2 or more CNAs as compared to 0 or 1 CNA as detected by SNP array had a significantly increased risk of death (p=0.016) and relapse (p=0.019). Patients carrying the recurrent deletion of chromosome 1q31.3 as compared to patients not carrying this deletion had a significantly increased risk of relapse (p=0.005), a markedly higher number of CNAs (median 8 vs. 2, p < 0.0001) and significantly higher white blood cell counts (WBC) at initial diagnosis (median 2550/μl vs. 16900/μl, p=0.009). We performed a multivariable analysis using Cox proportional hazards models to evaluate power of CNAs detected by SNP-array and deletions of chr. 1q31.3 as possible independent prognostic markers in APL as compared to age, WBC, platelet count (PC) and FLT3 mutational status. For the full model of OS only age and the number of CNAs detected by SNP-A met a 0.10 level of entry into the model and confirmed that the group of patients with 2 or more CNAs represent a subgroup with inferior outcome (“2 or more lesions”: hazard ratio = 5.942, p = 0.0015, age: hazard ratio = 1.08, p < 0.0001). Of note, for the analysis of RFS the presence of a chr. 1q31.3 deletion was the only effect, which met the entry level into the model of RFS and therefore was identified as a new strong predictor for an increased risk of relapse (“presence of del1q31.3”: hazard ratio = 28.9, p = 0.0031). Conclusion: The profiles of submicroscopic CNAs in APL patients are heterogeneic and may serve as strong independent prognostic markers for disease risk definition. Recurrent submicroscopic deletions of chr. 1q31.3 in leukemia cells of APL patients were associated with an increased number and characteristic pattern of further newly identified CNAs, unfavorable laboratory parameters and a higher risk of relapse. The number of CNAs was shown to be predictive for early death rate and overall survival. The further pursuit of these new potential molecular markers is highly warranted as they could refine the current risk stratification of APL by identifying new subgroups of patients, who could possibly gain from adapted treatment strategies. Disclosures:No relevant conflicts of interest to declare.

Abstract PL02-02: The molecular pathology of acute leukemia

Abstract PL02-02: The molecular pathology of acute leukemia

Genome-Wide DNA Copy Number Analysis by SNP Arrays of B-Cell Chronic Lymphocytic Leukemia: Correlation with Known Biological and Molecular Prognostic Markers.

B-cell chronic lymphocytic leukemia (B-CLL) is a genetically heterogeneous disease with a variable clinical course. Chromosomal changes have been identified by FISH in approximately 80% of patients, and the presence of specific lesions, such as trisomy 12 and 13q14, 11q23, 17p13.1 and 6q23 deletions represent prognostic markers for disease progression and survival. In order to characterize further the complexity of B-CLL genomic lesions, we performed high density, single nucleotide polymorphism (SNP) array analysis in highly purified neoplastic cells (>92%) from a panel of 100 untreated, newly diagnosed patients (57 males and 43 females; age, median 63 years, range 30–87) in Binet stage A. All patients were investigated by FISH for the presence of trisomy 12 (21 cases); 13q14 deletion (44 cases, 34 as the sole abnormality); 11q22.3, 17p13.1 and 6q23 (15, 7 and 2 patients, respectively). In addition, ZAP-70 and CD38 expression resulted positive in 42 and 46 patients, whereas IgVH genes were mutated in 45 patients. Genome-wide DNA profiling data were generated on GeneChip® Human Mapping 250K NspI arrays (Affymetrix); copy number alterations (CNA) were calculated using the DNA copy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS) (Olshen et al, 2004). A total of 782 CNAs (ranging from 1 to 31 per sample, mean and median values 7.82 and 7, respectively) were detected; DNA losses (365/782=46.67% loss; 194/782=24.81% biallelic deletion) were found to be more frequent than gains (148/782=18.93% gain; 75/782=9.59% amplification). The most recurrent alterations detected by FISH were all confirmed by SNP array analysis, strengthening further the good reliability of such high-resolution technology. We identified 12 minimally altered regions (MARs) larger than 100 kb with a frequency higher than 5%. Among well known alterations, the largest was represented by chromosome 12 trisomy, followed by 6q, 17p and 11q23 deletions (32.87, 19.09 and 10.43 Mb, respectively) and 13q14 deletion (635 kb). Gain of 2p25.3 involves a common region of 4.39 Mb region in 7 patients, although it was extended to the whole short arm of chromosome 2 in 3 cases. Among those alterations previously described in B-CLL, we found losses at 14q32.33 (12 pts) and 22q11.2 (5 pts) involving the IGH and IGLλ loci, respectively. With regard to novel regions, we identified losses at 4q35.2 (5 pts) and 11q25 (6 pts). In addition we found a high frequency of losses/gains at 14q11.2 (42 pts) and 15q11.2 (33 pts), two genomic regions reported to be affected by DNA copy number variations in normal individuals. As regards correlations between CNAs and biological markers, we found that the number of CNAs is significantly higher in cases with unmutated IgVH (9.4; range 2–31) as compared with mutated IgVH (6; range 1–13) (p=0.002), while neither CD38 nor ZAP-70 expression showed significant correlation. In addition, a significant higher number of either CNAs (p=0.001), total MARs (p<0.0001) or even only novel MARs (p=0.009) was significantly associated with cases with 17p deletion or multiple cytogenetic aberrations as evaluated by FISH analysis. Our data indicate that genetic abnormalities involving chromosomal gains and losses are very common in early-stage B-CLL and further support the application of high resolution SNP array platforms in the characterization of genetic changes in the disease. In addition, we detected novel altered chromosomal regions that warrant further investigations to better define their pathogenetic and prognostic role in B-CLL.

High Resolution Array-CGH in Splenic Marginal Zone B-Cell Lymphoma: Correlation of Copy Number Imbalances with HCV Status and Prognostic Categories.

In splenic marginal zone B-cell lymphoma (SMZL) no specific genetic alterations are known. Abnormalities of chromosome 7p and of p53 are reported as adverse prognostic factors. In a recent multicentre study (Arcaini et al Blood 2006), a prognostic model based on hemoglobin, albumin and LDH identified 3 risk categories. HCV infection was present in nearly 20% of patients (pts). At now, no data are available on genetic alterations in the HCV-positive subset of SMZL and in the different prognostic categories. The aims of the study were: a) to analyze copy number alterations (CNAs) by means of array comparative genomic hybridization (array-CGH) with a resolution of ∼100 kb; b) to compare CNAs in HCV-positive and HCV-negative pts; c) to identify potential genetic alterations related to the clinical features and to the prognostic categories. We analyzed marrow and blood samples from 34 pts with SMZL: 22 were HCV-negative (serology and HCV-RNA) and 12 were HCV-positive (genotype 2a/2c in 10 pts, genotype 1 in 2). DNA was extracted from bone marrow (16) and peripheral blood lymphocytes (18) and was hybridized with pooled blood lymphocyte reference DNA on Agilent's 44K oligonucleotide microarray (kit 44B). Images and data were analyzed using Agilent's Feature Extraction (v9.1) and CGH analytics (v3.4.27) softwares. Ten cases (4 HCV+ and 6 HCV-) did not show CNAs. A single alteration was present in 7 pts, 2 to 5 alterations in 11 and >5 in 6. All CNAs were detected in mosaicism (from 20% to 90%). A median of 5.6 (range 1 to 20) and 3.8 (range 1 to 13) CNAs were detected in HCV+ and in HCV- cases, respectively. The most frequent CNAs were hetereogeneous in size with the following common regions: losses of 1p36.21-p35.3 (3 pts), 7q31.1-q32.3 (7 pts), 8p21.3-p12 (6 pts), 13q14.2-q14.3 (6 pts), 14q32.12-q32.13 (4 pts) and 17pter-p12 (8 pts); gains of 3q21.1-q29 (5 pts), 12q13.1-q21.31 (5 pts), 17q24.1-qter (4 pts), Xpter-p11.23 (4pts). A homozygous 13q14.2 deletion, overlapping that found in CLL and including Rb1 gene, was found in one HCV- pt. The del(7)(q31.1-q32.3) was the more frequent and it ranges from 14,1Mb to 34Mb. No difference in number of CNAs and in specific common regions alterations was found between HCV+ and HCV- cases except for dup(X)(pter-p11.23) (p=0.01, 4 HCV+ pts and none HCV- pt). High-risk prognostic category was significantly associated with del(7)(q31.1-q32.3) (p=0.01) and del(17)(pter-p12) (p=0.02). Mutational status of immunoglobulin variable heavy-chain gene was related to del(7)(q31.1-q32.3) (p=0.04) and dup(12)(q13.1-q21.31) (p=0.03). The presence of villous lymphocytes was associated with del(1)(p36.21-p35.3) (p=0.02); del(8)(p21.3–p12) was related to an autoimmune background in the HCV+ subset (p=0.04). The number of CNAs was associated to leukemic disease (p=0.02) and to the presence of villous lymphocytes (p=0.04). In conclusion, array-CGH in SMZL does not show specific genetic abnormalities for pts with HCV-positive or HCV-negative SMZL. 7q and 17p deletions are significantly associated with the high-risk prognostic category, clinically and biologically identifying a group of pts with aggressive disease.

Genomic imbalances in T-cell acute lymphoblastic leukemia cell lines

Genomic imbalances were investigated in 15 T-cell acute lymphoblastic leukemia cell lines using the comparative genomic hybridization (CGH) technique. In addition, in vitro response to the cytostatic drug doxorubicin was evaluated by means of a growth inhibition assay. The number of significant DNA copy number alterations (CNAs) varied from 0 to 16 per cell line and the number of additional alterations with borderline significance was in a range of 0-7. Three of the cell lines had a total number of genomic changes of >/=20, five had 11-19, and eight had </=10 CNAs. One cell line did not show any imbalance at all. Among the significant CNAs, losses of genomic material were slightly more frequent than gains (60:49). Gains dominated among the borderline alterations compared to losses (45:9). CNAs common to all cell lines examined were not found. The most frequent genomic imbalance (gain of 6q23) was shared by 9 of the 15 cell lines. A significant loss of 18q23 was observed in eight lines, however, often close to borderline significance. Seven of the cell lines were characterized by a loss of the entire short arm of chromosome 9 or parts of it with 9p21 as minimal band of overlap. Interestingly, cell lines with a 9p21 deletion exhibited twice the number of gains and 1.6 times the number of losses per line as compared with the cell lines without this deletion. A consistent pattern of the CNAs accompanying the 9p deletion could not be detected, although some associations were more obvious than others, e.g., gains of 6q22 in five cell lines with del(9p21), of 6p21 in four lines combined with a gain of 6q22, and of 20q in four cell lines, of 1p32 in three of these seven lines, a loss of 14q32 in three of them. Three of the lines with 9p deletion had gains of 6p21, 6q22 and 20q in common. Enh(6q22) or dim(14q32), respectively, were found in only one of the nine cell lines without the 9p deletion. Based on the dose response curves of the cell lines for doxorubicin, eight doxorubicin-sensitive cell lines had an inhibition concentration 50% (IC50) <10 nM (CCRF-CEM2, JURKAT, KE-37, MOLT-3, MOLT-4, P12-Ichikawa, PEER, and RPMI-8402) and seven doxorubicin-resistant cell lines had an IC50 >10 nM (BE-13, CCRF-CEM1, HUT-78, J-Jhan, Karpas-45, MOLT-17, and PF-382). The average number of CNAs per cell line was higher in the sensitive than in the resistant group (total 13.1:8.5; significant CNAs 9.1:5.8).

Pattern of genomic imbalances in oral squamous cell carcinomas with and without an increased copy number of 11q13.

Among 23 squamous cell carcinomas (SCC) of the oral cavity which were screened for DNA copy number alterations (CNAs) using comparative genomic hybridization, 14 showed a gain of, and 5 of these 14 even an amplification of band 11q13. Amplification of 11q13 was also detected in three of the four studied SCC cell lines and was confirmed by interphase FISH. The number of CNAs in addition to 11q13 varied from 14 to 47 in these carcinomas. All these tumors had seven other specific CNAs in common, i.e. gain on 1p36.3-36.6, 5p15, 9q34, 12p12-13, 14q32, 19 and 20q, all but one showed also an increase of copy number in 7p22, 8q24, 10q26, 12q26, 15q24-25, 16p, 16q23-24, 17q and 22q12-qter. These imbalances were distinctly rarer in the tumors without CNA in 11q13. Loss of material apparently played a minor role in these tumors with gain of 11q13, the most frequent losses (3p12-14 and 5q21) being present in 10 of the 14 cases and loss of 9p13-21 in 5/14 tumors. The three tumors with the highest number of CNAs in addition to 11q13, were histologically classified as pT4, three of the five tumors with 11q13 amplification were highly node-positive (pN 2b-2c). Two of the pT4 tumors shared as many as 23 specific chromosomal segments affected by CNA. Thus, gain of 11q13, though being found at different stages of karyotypic evolution, is apparently associated with a rather specific pattern of other CNAs and involved in progressed stages of malignancy in oral squamous cell carcinoma. In addition, the proportion of patients deceased within one year after diagnosis was clearly higher in the group whose tumors showed an increased 11q13 copy number as compared to the group without this increase. This could point to an association of gain in 11q13 and aggressiveness of the respective tumor.