Frequent Copy Number Alterations Research Articles

Genomic Copy Number Signatures Uncovered a Genetically Distinct Group from Adenocarcinoma and Squamous Cell Carcinoma in Non-Small Cell Lung Cancer.

Adenocarcinoma (AC) and squamous cell carcinoma (SCC) of non-small cell lung carcinoma (NSCLC) have different clinical presentations, morphologies, treatments, and prognoses. Recent studies suggested that fundamental genetic alterations related to carcinogenesis of each tumor type may be different. In this study, we investigated the genomic alterations of 47 primary NSCLC samples (22 ACs and 25 SCCs) as well as the corresponding normal tissue using array comparative genomic hybridization. Frequent copy number alterations (CNAs), which were identified in more than 68% of all of the cases, were evaluated in each subtype (SCC and AC), and a CNA signature was established. Among these CNAs, 37 genes from the SCCs and 15 genes from the ACs were located in a region of gain, and 4 genes from the SCCs and 13 genes from the ACs were located in a region of loss. The most frequent gain was located on 3q26-29 including the gene TP63 in SCCs and 7q11.23 and 7q36.3 in ACs. Moreover, we identified 3 genetically distinct groups (group I [16 SCC] with CNA signature of SCC; group II [7 SCC + 8 AC], which has a genetically distinctive CNA signature from SCC and AC; and group III [2 SCC + 14 AC] with CNA signature of AC) by gene clustering extracted from CNAs, which are associated with a prognosis. The present study contributed to the molecular characterization of AC and SCC of NSCLC and showed a subtype of tumor that has a unique genetic CNA signature. However, further study about the significance of these 3 distinct groups and their usefulness as a diagnostic marker of identified CNAs is necessary.

Integrated copy number and gene expression profiling analysis of Epstein-Barr virus-positive diffuse large B-cell lymphoma.

Viral oncogenes and host immunosenescence have been suggested as causes of Epstein-Barr virus-positive diffuse large B-cell lymphoma (EBV + DLBCL) of the elderly. To investigate the molecular genetic basis of immune evasion and tumor outgrowth, we analyzed copy number alterations (CNAs) and gene expression profiles in EBV + DLBCL samples compared with EBV - DLBCL. There were relatively few genomic alterations in EBV + DLBCL compared with those detected in EBV-negative DLBCL. The most frequent CNAs (>30%) in EBV + DLBCLs were gains at 1q23.2-23.3, 1q23.3, 1q32.1, 5p15.3, 8q22.3, 8q24.1-24.2, and 9p24.1; losses at 6q27, 7q11.2, and 7q36.2-36.3 were also recurrent. A gene expression profile analysis identified the host immune response as a key molecular signature in EBV + DLBCL. Antiviral response genes, proinflammatory cytokines, and chemokines associated with the innate immune response were overexpressed, indicating the presence of a virusinduced inflammatory microenvironment. Genes associated with the B-cell receptor signaling pathway were downregulated. An integrated analysis indicated that SLAMF1 and PDL2 were key targets of the gains detected at 1q23.2-23.3 and 9p24.1. The chromosomal gain at 9p24.1 was associated with poor overall survival. Taken together, our results led to the identification of recurrent copy number alterations and distinct gene expression associated with the host immune response in EBV + DLBCL. We suggest that the upregulation of PDL2 on 9p24.1 promotes immune evasion and is associated with poor prognosis in EBV + DLBCL.

Clonal heterogeneity in platinum-resistant metastatic urothelial cancer.

290 Background: Beyond platinum-based chemotherapy, there are currently no approved therapies for advanced PRUC. Our objective was to generate the first detailed genomic profile of metastatic PRUC to identify molecular changes critical to platinum-resistance and metastasis development. Methods: Following informed consent, we collected 50 urothelial cancer (UC) tissue samples, from 23 patients. Metastatic tumor samples were obtained from biopsies or metastatectomy. Germline samples were prospectively collected and matched archival formalin-fixed paraffin-embedded primary tumors from the same patients were retrieved. Our cohort comprises 23 metastatic samples, 37 PRUC samples and 18 trios of matched primary, metastatic and germline samples including 2 rapid autopsies yielding tumor samples from multiple sites. We performed whole exome sequencing of all tumor and germline samples followed by integrated analysis of somatic single nucleotide variants and copy-number alterations and to analyze clonality. We utilized DAVID bioinformatic tool for pathway analysis. Results: Advanced PRUC samples were enriched for several molecular alterations, we identified 414 recurrently mutated genes including common genes such as TP53 (45%) as well as actionable alterations in PIK3CA (11%) and TSC1 (19%). We also identified frequent alterations in novel genes such as DPCR1 (38%) and NIN (28%). Frequent copy number alterations included CDKN2A deletion (33%), E2F3 amplification (10%) and ERBB2 amplification (7%). Pathway analysis showed enrichment of mutations in the apoptosis (p=1.0E-7) and cell cycle regulation (p=1.6E-2) pathways compared to TCGA primary UC dataset. We reconstructed phylogenetic trees from matched primary, metastatic and germline trios. Variant allele frequencies of certain shared mutations increased from primary tumors to lymph node and visceral metastases revealing clonal selection of mutations present in primary tumors. Conclusions: This study generates a detailed profile of the genomic landscape of advanced PRUC revealing extensive heterogeneity and clonal selection underlying platinum-resistance and metastatic spread.

Cooperative genetic changes in pediatric B-cell precursor acute lymphoblastic leukemia with deletions or mutations of IKZF1.

In contrast to IKZF1 deletions (ΔIKZF1), IKZF1 sequence mutations (mutIKZF1) have been reported to be rare in B-cell precursor acute lymphoblastic leukemia and their clinical implications are unknown. We performed targeted deep sequencing of all exons of IKZF1 in 140 pediatric cases, eight (5.7%) of which harbored a mutIKZF1. The probabilities of relapse (pRel) and event-free survival (pEFS) did not differ between cases with or without mutIKZF1, whereas pEFS was decreased and pRel increased in ΔIKZF1-positive case. Coexisting microdeletions, mutations (FLT3, JAK2, SH2B3, and SPRED1), and rearrangements (ABL1, CRLF2, JAK2, and PDGFRB) in 35 ΔIKZF1 and/or mutIKZF1-positive cases were ascertained using fluorescence in situ hybridization, single nucleotide polymorphism array, Sanger, and targeted deep sequencing analyses. The overall frequencies of copy number alterations did not differ between cases with our without ΔIKZF1/mutIKZF1. Deletions of HIST1, SH2B3, and the pseudoautosomal region (PAR1), associated with deregulation of CRLF2, were more common in ΔIKZF1-positive cases, whereas PAR1 deletions and JAK2 mutations were overrepresented in the combined ΔIKZF1/mutIKZF1 group. There was no significant impact on pRel of the deletions in ΔIKZF1-positive cases or of JAK2 mutations in cases with ΔIKZF1/mutIKZF1. In contrast, the pRel was higher (P = 0.005) in ΔIKZF1/mutIKZF1-positive cases with PAR1 deletions.

Comprehensive genomic characterization of head and neck squamous cell carcinomas.

The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.Supplementary informationThe online version of this article (doi:10.1038/nature14129) contains supplementary material, which is available to authorized users.

Amplification of Chromosome 8 Genes in Lung Cancer

Chromosomal alterations are frequent events in lung carcinogenesis and usually display regions of focal amplification containing several overexpressed oncogenes. Although gains and losses of chromosomal loci have been reported copy number changes of the individual genes have not been analyzed in lung cancer. In this study 22 genes were analyzed by MLPA in tumors and matched normal tissue samples from 82 patients with non-small cell lung cancer. Gene amplifications were observed in 84% of the samples. Chromosome 8 was found to harbor the most frequent copy number alterations. The most frequently amplified genes were ZNF703, PRDM14 and MYC on chromosome 8 and the BIRC5 gene on chromosome 17. The frequency of deletions were much lower and the most frequently deleted gene was ADAM9. Amplification of the ZNF703, PRDM14 and MYC genes were highly correlated suggesting that the genes displaying high copy number changes on chromosome 8 collaborate during lung carcinogenesis.

Targeting Genomic Heterogeneity of Adult Acute Lymphoblastic Leukemia

Introduction: In acute lymphoblastic leukemia (ALL) genetic diversity at diagnosis has been reported to be correlated with an increased likelihood of emerging drug resistance and an increased relapse risk.Aim: To evaluate 1. the frequency of chromosome abnormalities at initial diagnosis and their acquisition during the course of the disease (clonal evolution), 2. the pattern of acquired genetic abnormalities.Patients: We investigated 11 adult ALL patients (age 18.9-76.7 years, median 42.6 years) with paired samples at initial diagnosis and at relapse by chromosome banding analysis (CBA), array-based comparative genomic hybridization (aCGH) and molecular analysis. The cohort consisted of 7 B-ALL and 4 T-ALL cases. CBA was performed in both diagnostic and relapse samples in 10 cases. aCGH was performed in 6 cases (12x270 K microarray slides, Roche Nimblegen, Madison, Wl) both at initial diagnosis and at relapse. Molecular analysis was performed in all cases by 454 amplicon deep-sequencing (Roche, Branford, CT) in the following genes: AKT1, FBXW7, IL7R, JAK1, NOTCH1, PHF6, PTEN and WT1 in T-ALL cases, and in CRLF2, IL7R, IKZF1, JAK2, LEF1 and PAX5 in B-ALL cases at diagnosis and relapse.Results: Median duration from diagnosis to relapse was 10 months (range 4-18 months). Regarding cytogenetics of B-ALL, 2 cases had t(9;22)(q34;q11), 1 case a MYC-rearrangement, 3 cases complex aberrations, 1 case a translocation t(5;16)(p15;q13) and 1 case had a normal karyotype (NK). In T-ALL, 2 cases had NK, 1 case had t(10;14) (q24;q11) and in 1 case no cytogenetic data was available.At initial diagnosis, 8/10 cases (80.0%) with available CBA had one cytogenetic clone, while 2/10 (20.0%) cases had 2 cytogenetically related subclones. At relapse, one case retained both clones already detected at diagnosis. The second case showed loss of genetic aberrations in one subclone (clonal regression), while the second clone was stable. Evolution of genetically related clones was detected by CBA in 5/10 cases. The acquired abnormalities comprized unbalanced (n=8) or balanced (n=3) translocations, gains (n=3) or deletions (n=2) of whole or partial chromosomes. 3 of these 5 cases in addition showed clonal regression which may, however, be due to impaired in vitro proliferation of the subclone at relapse. The lost abnormalities comprized unbalanced (n=6) or balanced (n=1) translocations, gains (n=1) or deletions (n=1) of whole or partial chromosomes.In all 6 cases analyzed by aCGH, clonal evolution was detected. In one case, without clonal evolution detected by CBA, aCGH revealed clonal evolution on the submicroscopic level. In detail, 92 copy number alterations (CNA) were observed (mean 8 per case, range: -33) at initial diagnosis and 105 CNA at relapse (mean 9 per case; range 2-36). Losses were more frequent than gains (79 vs 13 at initial diagnosis; 87 vs 18 at relapse). 53 CNA of initial diagnosis and 33 of relapse were larger than 10 Mbp and were already detected by CBA accordingly. 43 CNA at initial diagnosis (mean: 7 per case; range 4-11) and 40 CNA at relapse (mean 7 per case; range 3-11) were smaller than 10 Mbp and thus below the resolution of CBA. The most frequent CNA were loss of 9p21 (CDKN2A, n=5) and 7p12 (IKZF1, n=3). These stayed stable from initial diagnosis to relapse. Apart from these, no specific pattern of gains or losses of distinct chromosomal regions was observed. In 4/6 cases, clonal regression of 5 CNA was detected on submicroscopic level. By sequencing 17 mutations and 2 variants were observed in 9/11 patients without major changes between initial diagnosis and relapse. In B-ALL cases, mutations were detected in JAK2 (n=2) and IKZF1 (n=3). In 2 cases a variant in CRLF2 was observed, which cannot be assigned as mutation or SNP according to current knowledge. In T-ALL cases, mutations were detected in NOTCH1 (n=3), PHF6 (n=1), PTEN (n=1), WT1 (n=1) and IL7R (n=1). The pattern of mutations was stable in 7/9 cases. One case lost a mutation in NOTCH1 at relapse, while another patient gained a mutation in IKZF1. Two patients without mutations at diagnosis also showed no mutations at relapse.Conclusions: 1. Adult ALL is characterized by multiple genetic aberrations a subset of which can be detected by aCGH only. 2. The genomic heterogeneity of adult ALL increases at relapse. 3. Based on this limited set of patients gene mutations seem to be more stable between diagnosis and relapse compared to cytogenetic aberrations. DisclosuresFasan:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Ulke:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

TP53 Alterations Are Frequent in Patients over 60 Years with B-Precursor Acute Lymphoblastic Leukemia (ALL) and Low Hypodiploid/Near Triploid (HoTr) Karyotype; They Correlate with RB1 Deletion and Leukemic Telomere Gain

IntroductionTP53 is the most frequently altered gene in cancer. Until recently data on its frequency and prognostic impact in ALL has been scant, particularly in the elderly. TP53 alteration in the context of telomere shortening (the hallmark of aging) results in telomere attrition and genomic instability with resultant overexpression of telomerase to overcome the genomic crisis.ObjectiveTo determine TP53 alteration frequency and its association with cytogenetic subgroups, key B cell differentiation/cell cycle genes abnormalities and induction therapy response in ALL patients aged ≥60 and its impact on leukemic telomere stateMethodsPatients enrolled in the UK NCRI UKALL14 and UKALL60+ trials aged ≥60 years were assessed at diagnosis for 17p deletions by cytogenetics, TP53 alterations by FISH and/or direct sequencing of hot spots exons 5-8. Data were correlated with IKZF1, CDKN2A/B, PAR1, BTG, RB1, ETV6, EBF and PAX5 gene copy number status, assessed by MLPA (P335-B1). MYC-rearrangement was an exclusion criterion. Minimal residual disease (MRD) was assessed by quantitative PCR (qPCR) for Ig/TCR rearrangements or BCR-ABL1, where applicable. Leukemic DNA telomere length relative to that of remission DNA was assessed by monochrome multiplex (MM) qPCR and expressed as a log2 ratio. Using expectation maximization mixture model a log2 ratio >3.18 defined gain (reflecting telomerase overexpression) and values below this were defined as loss.ResultsThe cohort included 63 patients; characteristics are presented in table 1. Median age was 63 years (range 60–83). TP53 state by cytogenetics and/or sequencing was available in all. Eleven had TP53 alteration (17%); TP53 mutation only, n=3; TP53 deletion only, n=6; TP53 deletion and mutation, n=2. Cytogenetics was available in 60 patients. Nine patients had HoTr (14%). Seven of 9 patients with HoTr had TP53 alteration (78%) versus 4/51 without HoTr (4%); p<0.001. MLPA was done in 57 patients. Those with TP53 alteration had more copy number alterations (CNA)(median 4 versus 1; p<0.01). Twelve (21%) had RB1 deletion; 3 were due to cytogenetically visible 13q abnormalities rather than ‘true’ intragenic aberration. Four of 9 patients with TP53 alteration had RB1 deletion versus 8/51 without TP53 alteration, p=0.07. All other microaberrations were not significantly associated with TP53 alteration. Sixty-two achieved complete remission after 2 cycles of induction. MRD state (n=32) was not impacted by TP53 (2/5 with TP53 alteration and 13/27 without were MRD positive after 2 cycles of induction; p=0.563). Fourteen of 59 (24%) patients relapsed; median of four months from diagnosis (range 2–21), three had TP53 alteration versus 11 without TP53 alteration; p=0.65. Relapse occurred significantly earlier in patients with TP53 alteration (median two months versus eight months; p=0.03). Leukemic telomere state was assessed in 28 patients. Three of 4 patients with telomere gain had TP53 alteration versus 3/24 without; p=0.02ConclusionsThis is one of the largest reports of TP53 frequency in older patients with non-MYC-rearranged ALL. TP53 was deleted and/or mutated in 17%, with highest frequency in those with HoTr (7/9). RB1 deletion was the commonest associated microaberration, noted in over a third of patients. TP53 alteration was associated with earlier relapse but had no impact on induction response. TP53 alteration was notably associated with frequent CNA and telomere gain suggesting a putative potential role of TP53 in genomic instability though sample size for the latter limits assertion of this hypothesis. Mechanistic work to shed insight into this model is currently underway.Table 1: Patient characteristicsTP53 deleted and/or mutated n=11TP53 wild type n=52Age at Diagnosis, median(range)63(60-75)63(60-83)Gender; M:F06:0523:29Phenotype, n (%)· Precursor B cell11 (100)51 (98)· Precursor T cell0 (0)1 (2)Cytogenetics (risk stratification as per Moorman et al. Blood 2007; 109:3189–97), n (%)Standard Risk0 (0)23 (44)t(9;22)1 (9)21 (40)t(4;11)0 (0)2(4)Ho/Tri7 (64)2(4)Complex2 (18)2 (4)Unknown1 (9)2 (4)Morphological Remission after Cycle 2 Induction, n (%)11 (100)51(98)Induction Deaths, n (%)2 (18)15 (29)CNA by MLPA, median (range)4 (2-7)1 (0-2)RB1 deletion, n (%)· Present4 (36)8 (15)· Absent5 (45)43 (83)· Unknown2 (18)1 (2)Leukemic Telomere State· Gain3 (27)3 (6)· Loss1 (9)21 (40)· Unknown7 (64)28 (54) DisclosuresNo relevant conflicts of interest to declare.

Genetic Markers Add Significant Prognostic Information to Age and WBC Count in High-Risk, Ph-Negative, B-Precursor Adult Acute Lymphoblastic Leukemia (ALL): Study of 96 Patients Treated According to Risk-Adapted Protocols from the Pethema Group

IntroductionRecurrent Copy Number Alterations (CNA) in genes potentially involved in the pathogenesis of ALL have been identified in genes involved in B-cell development, cell cycle regulation, proliferation, apoptosis and drug resistance. Their independent prognostic significance in adult ALL patients is controversial. The aim of this study was to analyze the prognostic significance of CNA in a series of 96 high-risk, Ph-negative, B-precursor adult ALL patients treated according to risk-adapted protocols from the Spanish PETHEMA Group.MethodsMLPA assays (MRC-Holland) were performed for the following genes: IKZF1, IKZF2, IKZF3, EBF1, CDKN2A/B, PAX5, ETV6, BTG1, RB1, hsa-miR-31, X/Y PAR1 region genes (CRLF2, CSF2RA, IL3RA) and 14q32.33 region genes (IGH D, MTA1, KIAA0284). Fragment analysis was made by Genescan in an ABI-3130 sequencer (Applied Biosystems). Data normalization provided a value indicative of the presence or absence of CNA: 0-0.20 homozygous deletion, 0.21-0.70 heterozygous deletion, 0.71-1.30 normal, 1.31-1.70 heterozygous duplication and 1.71-2.20 homozygous duplication. Univariable and multivariable analyses including the most relevant clinical parameters (age, WBC count, phenotype, cytogenetics, CNS involvement) were performed for CR attainment, CR duration and OS.ResultsThe median age [range] of the 96 patients was 39 [15-72] years, 50 (52%) patients were males, with a median WBC count 14.3 x109/L [0.4-388]. Phenotype: early pre-B 19 (20%), common 51 (54%), pre-B 22 (24%), unknown 2 (2%). Cytogenetics: normal 18 (19%), hyperdiploid 5 (5%), hypodiploid 2 (2%), near haploid 6 (6%), t(1;19) 7 (8%), 11q23/MLL 11 (12%), complex 1 (1%), other 27 (29%), no growth 17 (18%). The most frequent CNA deletions involved CDKN2A/B (43/96, 45%), PAX5 (34/94, 36%), IKZF1 (32/95, 34%), hsa-miR-31 (25/96, 26%), 14q32.33 region (18/96, 19%), RB1 (17/96, 18%), EBF1(12/91, 13%) and X/Y PAR (10/96, 10%). The most frequent duplications involved X/Y PAR (11/96, 12%) and 14q32.33 region (7/96, 7%).The CR rate was 83% (80/96), the median (95%CI) of CR duration was 2.7 years (0-5.9) and the median (95%CI) of OS was 2.1 (1.0-3.2), being the median (range) follow-up of the series of 3.8 (0.6-8.0) years. Table 1 shows the results of univariable and multivariable analyses. By multivariable analyses advanced age and EBF1 deletions were significantly associated with less CR rate, WBC count and X/Y PAR duplication were associated with shorter CR duration, and advanced age and CDKN2A/Bdeletion were associated with shorter OS.ConclusionsThe CNA of EBF1, X/Y PAR1 genes and CDKN2A/Bhave independent prognostic significance in adult patients with high-risk, Ph-negative, B-precursor ALL. This study suggests that these genetic studies should be added to the initial work-up of these patients for more accurate prognostic assessmentSupported by grants PI10/01417, RD12-0036-0029 from Instituto Carlos III, 2014 SGR225 (GRE) from Generalitat de Catalunya and a grant from the Spanish Society of Hematology and Hemotherapy (2012).Abstract 3798. Table 1Results of the univariable and multivariable studies.VariableCR rateCR durationOSP (univ)OR (95%CI)P (univ)HR (95%CI)P (univ)HR (95%CI)Age0.0110.93 (0.89 - 0.98)NS-0.0051.03 (1.01 - 1.05)WBCNS-<0.0011.01 (1.00 - 1.01)NS-IKZF1*NS-0.048-NS-EBF1*0.0250.11 (0.02 - 0.54)NS-NS-CDKN2A/B*NS-NS-0.0142.32 (1.35 - 4.00)X/Y PAR**NS-0.0134.26 (1.64 - 11.09)NS-*Normal versus deleted; ** Normal versus duplicated; NS: not significant DisclosuresNo relevant conflicts of interest to declare.

Role of BC040587 as a predictor of poor outcome in breast cancer.

BackgroundAccumulating studies have focused on the oncogenic and tumor suppressive roles of the newly identified lncRNAs. A novel lncRNA BC040587 in 3q13.31 locus which exists frequent copy number alterations was found to be associated with poor survival of osteosarcoma patients. However, its role in breast cancer (BC) remains unknown. The aim of this study was to examine the expression pattern of BC040587 in BC and to evaluate its biological role and clinical significance in prediction of prognosis.MethodsExpression of BC040587 was analyzed in 20 pairs of BC cancer tissues and adjacent noncancerous tissues (ANCT), also in 151 BC tissues, 9 BC cell lines and one normal breast cell line by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Differences between groups were tested for significance using Student’s t-test (two-tailed). Then we analyzed the potential relationship between BC040587 expression and clinic pathological features of BC patients. The correlation was analyzed by SPSS software.ResultsIt showed that BC040587 expression was down regulated both in BC samples and in BC cell lines compared with corresponding normal control. BC040587 expression was correlated with menopausal status (p = 0.040) and tumor differentiation (p = 0.035). The Kaplan-Meier survival curves indicated that the overall survival (OS) was significantly poor in low BC040587 expression BC patients (p = 0.023). Furthermore, expression of BC040587 was significantly associated with worse prognosis and was shown to be an independent prognostic marker breast cancer (p = 0.032). Our studies indicate that BC040587 may represent a new marker of prognosis in breast cancer.ConclusionOur studies indicate that BC040587 is significantly down-regulated in BC tissues and BC cell lines. BC040587 may represent a new marker of prognosis in breast cancer.

Abstract LB-205: Genome-wide copy number analysis of pediatric hepatocellular carcinomas: Identification of characteristic aberrations

Abstract Hepatocellular carcinomas (HCC) of childhood are rare neoplams. Their pathogenesis is believed to be different from HCC in adulthood. The differential diagnosis from hepatoblastoma (HBL)is important because of worse prognosis of pediatric HCC (pHCC) and different treatment approaches. However, the diagnosis by conventional histological analysis may be difficult in some cases. Informations on chromosomal alterations in pHCC are lacking. The aim of this study was to identify recurrent chromosomal aberrations in a larger cohort of pHCC and compare it to a previously published cohort of HBL (Weber et al., American Journal of Pathology, 2000) to identify characteristic chromosomal alterations of pHCC. Genomic DNA was extracted from FFPE samples of a cohort of 34 pHCC including 4 cases of the fibrolamellar subtype and 3 cases with partial fibrolamellar differentiation from the archives of the German Pediatric Tumor Registry, University of Kiel. Genome-wide copy analysis was performed by molecular inversion profiling. GISTIC analysis was done to identify significant large and focal aberrations. Most frequent copy number alterations were gains of chromosome 1q (65%), 2q (35%), 6p (35%), 20 (35%), 19q (32%) and 8q (29%) and losses of chromosomal regions 1p (56%) and 4q (38%). In addition, GISTIC analysis identified several significant fokal alterations including losses of the AXIN1 tumor suppressor locus on 15q26 in 53% of cases. 6/34 pHCC lacked any larger chromosomal imbalances. Of note, 5 of these 6 genomically stable tumors showed fibrolamellar differentiation. When pHCC with fibrolamellar differentiation were compared to the other pHCC, loss of 1p and 4q and gains of 1q, 2q, 6q and 20 occurred significantly more frequent in the latter. When pHCC were compared to HBL, both entities shared similar alterations including gains if 1q and 2q, but losses of 1p (56% vs. 3%), and gains of 6p (35% vs. 3%) and 19q (32% vs. 0%) were significantly more frequent in pHCC. In this genome-wide copy number analysis of a large cohort of pHCC we were able to identify for the first time characteristic alterations which may indicate a specific pathogenesis of these neoplasms, may help to subclassify pHCC and to differentiate these tumors from HBL. Supported by the German Childhood Cancer Foundation. Citation Format: Torsten Pietsch, A. Carmichael, Christian Vokuhl, Ivo Leuschner. Genome-wide copy number analysis of pediatric hepatocellular carcinomas: Identification of characteristic aberrations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-205. doi:10.1158/1538-7445.AM2014-LB-205

Gene copy number alterations in the azoospermia-associated AZFc region and their effect on spermatogenic impairment.

The azoospermia factor c (AZFc) region in the long arm of human Y chromosome is characterized by massive palindromes. It harbors eight multi-copy gene families that are expressed exclusively or predominantly in testis. To assess systematically the role of the AZFc region and these eight gene families in spermatogenesis, we conducted a comprehensive molecular analysis (including Y chromosome haplogrouping, AZFc deletion typing and gene copy quantification) in 654 idiopathic infertile men and 781 healthy controls in a Han Chinese population. The b2/b3 partial deletion (including both deletion-only and deletion-duplication) was consistently associated with spermatogenic impairment. In the subjects without partial AZFc deletions, a notable finding was that the frequency of DAZ and/or BPY2 copy number alterations in the infertile group was significantly higher than in the controls. Combined patterns of DAZ and/or BPY2 copy number abnormality were associated with spermatogenic impairment when compared with the pattern of all AZFc genes with common level copies. In addition, in Y chromosome haplogroup O1 (Y-hg O1), the frequency of copy number alterations of all eight gene families was significantly higher in the case group than that in the control group. Our findings indicate that the DAZ, BPY2 genes may be prominent players in spermatogenesis, and genomic rearrangements may be enriched in individuals belonging to Y-hg O1. Our findings emphasize the necessity of routine molecular analysis of AZFc structural variation during the workup of azoospermia and/or oligozoospermia, which may diminish the genetic risk of assisted reproduction.

Copy number alterations and neoplasia‐specific mutations in MELK, PDCD1LG2, TLN1, and PAX5 at 9p in different neoplasias

Genetic alterations affecting 9p are commonly present in many cancer types and many cancer-related genes are located in this chromosomal region. We sequenced all of the genes located in a 32Mb region of 9p by targeted next generation sequencing (NGS) in 96 patients with different cancer types, including acute lymphoblastic leukemia, bone malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma, fibrosarcoma, Ewing's sarcoma, and lung carcinoma. Copy number alterations (CNA), and mutations were studied from the NGS data. We detected a deletion at the CDKN2A locus as being the most frequent genetic alteration in all cancer types. In addition to this locus, NGS also identified other small regions of copy number loss and gain. However, different cancer types did not reveal any statistically significant differences with regard to CNA frequency or type. Of the 191 genes within the target region, two novel recurrent mutations were found in the MELK and PDCD1LG2 genes. The most commonly mutated gene in sarcomas was TLN1 (8%) and PAX5 in ALL (9%). Mutations in PAX5, and RUSC2, were seen exclusively in ALL patients and those in KIAA1432, CA9, TLN1, and MELK only in sarcomas (MFH, FS, EFT). Thus using targeted NGS of the 9p region, in addition to commonly deleted CDKN2A locus, we were able to identify a number of small deletions and gains, as well as novel recurrent mutations in different cancer types. © 2014 Wiley Periodicals, Inc.

Exposure to arsenic and intra-chromosomal instability in blood.

The 450k Chip Analysis Methylation Pipeline (ChAMP) is a novel Illumina Infinium HumanMethylation450 BeadChip data processing algorithm that allows the analysis of copy number alterations (CNAs). With this pipeline we evaluated the prevalence of CNAs in peripheral blood leukocyte samples from healthy Argentinean Andean women with varying exposure to inorganic arsenic in drinking water. Arsenic exposure was assessed based on the sum concentrations of metabolites of inorganic arsenic in urine (U-As), which ranged from 10-663 μg L(-1) with a median of 185 μg L(-1). We used linear regression analysis to elucidate the association between U-As and the prevalence of CNAs. We found that increasing arsenic exposure was positively associated with the frequency of CNAs (p = 0.002), possibly in a dose-response relationship. Adjustment of the regression model for age and BMI of the subjects did not significantly change the effect estimate, although both covariates were significant predictors. Our results suggest that exposure to arsenic increases genomic instability in the form of CNAs.

Genomic landscape of pancreatic neuroendocrine tumors

To investigate the prognostic role of genomic stability and copy number alterations (CNAs) pancreatic neuroendocrine tumors (PanNETs). A high-resolution array-based comparative genomic hybridization approach was utilized in order to investigate and quantify chromosomal aberrations in a panel of 37 primary PanNET and 11 metastatic samples. DNA samples were extracted from formalin-fixed and paraffin-embedded tumor specimen. Genomic findings were correlated with histopathological and immunohistochemical data. Moreover, the dataset was subjected to employing an unsupervised hierarchical clustering analysis approach utilizing Euclidean distance and average linkage and associations between genomically defined tumor groups and recurrent CNAs or clinicopathological features of the study group were assessed. Numerous chromosomal aberrations were recurrently detected in both, primary tumor samples and metastases. Copy number gains were most frequently observed at 06p22.2-p22.1 (27.1%), 17p13.1 (20.8%), 07p21.3-p21.2 (18.8%), 09q34.11 (18.8%). Genomic losses were significantly less frequent and the only recurrent aberration affected 08q24.3 (6.3%). Moreover, we detected a high degree of genomic heterogeneity between primary tumors and metastatic lesions. Unsupervised hierarchical clustering of loci affected by CNAs in more than 3 primary tumor samples revealed two genetically distinct tumor groups as well as two chromosomal clusters of genomic imbalances indicating a small subset of tumors with common molecular features (13.5%). Aberrations affecting 6p22.2-22.1, 8q24.3, 9q34.11 and 17p13.1 (P = 0.011; 0.003; 0.003; 0.001), were significantly associated with a poorer survival prognosis. This study suggests that several frequent CNAs in numerous candidate regions are involved in the pathogenesis and metastatic progression of PanNET.

Molecular Characterization Of Myelofibrosis Patients With Cytopenia Treated With Pomalidomide: Results From The Mpnsg 01-09 Study

BackgroundIn recent phase-I/II studies, pomalidomide (POM) has shown activity in myelofibrosis (MF) patients (pts) with anemia and/or thrombocytopenia. In the first cohort of the German multicenter MPNSG 01-09 phase-II trial, 38 MF pts were treated with POM 2 mg QD +/- prednisolone (PRED); 34% responded according to IWG-MRT criteria or became RBC-transfusion independent (Schlenk et al., ASH 2012). Additional 58 MF pts were treated in the second study cohort with POM 0.5 mg QD +/- PRED. Main inclusion criteria were age >50y, RBC-transfusion-dependency or Hb <10 g/dL, and/or PLT <50 /nL. The study population (n=96) represents advanced disease: 24% high-risk pts, 65% intermediate-II risk pts, and 11% intermediate-I risk pts according to the DIPS score (DIPSS). AimsTo explore the presence of copy number alterations (CNAs), uniparental disomies (UPDs) and MPN-related gene mutations in the 01-09 study cohort and to correlate genetic parameters with treatment response. MethodsAffymetrix 6.0 SNP arrays were used for genome-wide screening of CNAs and UPDs in 91% (87/96) of pts. Frequently mutated genes were analyzed in all pts using PCR-based techniques and sequence analysis of coding exons: JAK2 V617F, MPL W515L, ASXL1 (exon 12), EZH2 (exon 2-20), IDH1/2 (exons 4), SRSF2 (exon 1-2), and TP53(exon 4-10). ResultsIn total, 78 CNAs were identified by SNP array analysis. The overall frequency of CNAs was 52% (45/87 pts); 24% (21 pts) showed ≥2 CNAs, while 7% (6 pts) had a complex genotype defined by ≥3 genomic aberrations. Recurrent large (>5 Mb) gains were trisomy 8 (7%; n=6), gain 1q, and trisomy 9 (5% each; n=4), whereas recurrent large losses were identified in 20q (11%; n=10), 5q (8%; n=7), 13q (7%; n=6), 7q/7 (6%; n=5), and 12p/12 (2%; n=2). Moreover, 20% (17/87) of pts showed 19 informative micro-deletions (<5 Mb).Large UPDs (>10 Mb) affecting a terminal end of the chromosome were present in 32% (28/87) of pts. The most frequent UPDs occurred in 9p including JAK2 (17%; n=15). Other recurrent UPDs mapped to 1p (5%; n=4), 4q, and 7q (2% each; n=2). UPDs in 9p were associated with JAK2 V617F in all pts, whereas 75% (3/4) of pts with UPD in 1p were MPLW515L mutated.Mutations in JAK2 (V617F) and MPL (W515L) were present in 55% (53/96) and 6% (6/96) of pts, respectively. The mutation frequencies for the remaining genes were 30% (ASXL1; n=29/96), 9% (SRSF2; n=9/96), 5% (EZH2; n=5/96), 2% (TP53; n=2/96), and 1% (IDH2; n=1/96). Taken together, at least one mutation was found in 79% (76/96) of pts, whereas 21% (20/96) of pts lacked any of these mutations; 28% (27/96) of pts showed ≥2 concurrent mutations.To evaluate genetic differences and predictive factors, pts were separated into a responder (R, n=23) and a non-responder (Non-R, n=73) group. Regarding DIPSS, Non-R pts were not associated with higher risk compared to R pts: 27% (20/73) high risk vs. 17% (4/23) (p=0.334), 61% (44/73) intermediate-II risk vs. 79% (18/23), and 12% (9/73) intermediate-I vs. 4% (1/23) (p=0.275). However, the overall frequency of CNAs was in trend higher in the Non-R group: 56% vs. 39% (p=0.159). Since the total number of genomic losses was similarly distributed (27% vs. 29%), the difference was mainly due to the higher frequency of large genomic gains/trisomies: 4% (R) vs. 22% (Non-R) (p=0.083). UPDs were similar frequent in R and Non-R pts (26% vs. 34%) (p=0.466).The total number of gene mutations was not different between both groups (n=25 in 23 R pts vs. n=77 in 73 Non-R pts) (p=0.475). However, genetic alterations being associated with unfavourable outcome were enriched in the Non-R group: ASXL1 mutation (39% vs. 22%), EZH2 mutation (5% vs. 0%), loss in 5q (30% vs. 4%), trisomy 8 (9% vs. 0%), 12p / ETV6 deletion (6% vs. 0%), 17p deletion / TP53 mutation (3% vs. 0%), and complex genotype (7% vs. 0%). Taken together, adverse genetic alterations were significantly more frequent in Non-R pts compared to R pts: 48% (35/73) vs. 22% (5/23) (p=0.026). Of note, adverse genetic alterations indicated high risk according to DIPSS in no more than 50% (12/24) of the pts. ConclusionOur study on a well-defined patient cohort with advanced MF revealed a high frequency of genetic alterations reflecting the molecular complexity of the disease. Pts with adverse genetic alterations identified by SNP-array and mutation analyses were not sufficiently represented by DIPSS and showed inferior response to POM +/- PRED. Disclosures:Reiter:Sanofi: Honoraria. Gattermann:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Platzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Schlenk:Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Chugai: Research Funding; Ambit: Honoraria.

Genetic and Epigenetic Profile Of Early Relapsed Childhood ALL

Relapsed acute lymphoblastic leukemia (ALL) is one of the leading causes of death among children with cancer. While children who relapse late (≥ 3 years from initial diagnosis) fare better than those who relapse early in treatment, the prognosis for these children remains poor, even with aggressive treatment. Thus new approaches to prevent and treat relapsed ALL are needed. To discover the underlying biological pathways that may play a role in drug resistance and relapse, we integrated three high-throughput assays.Using matched diagnosis/relapse bone marrow samples from children with early relapsed Pre B-ALL (n=10) and T-ALL (n=6) we evaluated gene expression, DNA methylation and DNA copy number alterations (CNAs) analysis. All diagnosis and relapsed samples had more than 80% bone marrow blast account and were initially treated on BFM protocol. As controls, we used flow-sorted normal B cell progenitor subpopulations and CD4+CD8+ T cell samples purified from thymus. Gene expression profile of RNA samples were detected by Illumina HumanHT-12v4 ExpressionChip and bisulfite converted DNA materials were hybridized to the Infinium HumanMethylation450K BeadChip. DNA copy number and LOH data were obtained using Illumina HumanCytoSNP-12. Analysis variant CpG sites in an unsupervised manner, we identified three clearly distinct DNA methylome profiles in samples. T-ALL cases clustered separately from B-ALL samples and B-ALL samples clustered two branch according to their structural variations. We performed supervised comparisons of methylation data between B-ALL and normal B cells, as well as comparing B-ALL with T-ALL cases and T-ALL cases with normal T cells. Differentially methylated regions were identified using limma and lumi packages. ALL cases did not show significant differences between paired diagnose/ relapse samples though they had hyper DNA methylation than control samples. Aberrant DNA methylation had been detected in negative regulator of cell cycle genes and DNA damage repair genes in ALL. Unsupervised expression analysis of the pairs, T-ALL cases clustered separately from B-ALL samples. We defined a signature of 5,762 probe sets that differentially expressed between samples and control. Matched-pair analyses revealed significant differences in the expression of genes involved in cell-cycle regulation, and apoptosis between diagnostic and early relapse samples. Copy number analysis of patients revealed varying numbers of genetic lesions ranging from 0 to 45 CNAs per sample. The vast majority of CNAs observed were shared between diagnosis and relapse in the same patients. For the B-ALL majority of the copy number changes were gross, beside that T-ALL samples mostly had cryptic lesions. One of the most frequent CNAs involved deletions of CDKN2A/B at 9p21.3, occurring in 8 patients, 2 of 8 patients lost the CDKN2A/B at relapse time. CDKN2A, a benchmark gene and had been found hypermethylated at relapse parallel to recent studies.Early relapse samples were more likely to be similar to their respective diagnostic sample that suggests early-relapse results from the emergence of a related clone. Differential expression profile had been observed between relapse and diagnose samples that was independent from the methylation profile. By combining three high-throughput platforms, we demonstrated that both methylation and genomic alterations contribute to evolution of relapse and drug resistance. Disclosures:Karakas:Novartis: Honoraria, Research Funding. Sayitoglu:Roche Diagnostics: Research Support Other.

HMCan: a method for detecting chromatin modifications in cancer samples using ChIP-seq data

Motivation: Cancer cells are often characterized by epigenetic changes, which include aberrant histone modifications. In particular, local or regional epigenetic silencing is a common mechanism in cancer for silencing expression of tumor suppressor genes. Though several tools have been created to enable detection of histone marks in ChIP-seq data from normal samples, it is unclear whether these tools can be efficiently applied to ChIP-seq data generated from cancer samples. Indeed, cancer genomes are often characterized by frequent copy number alterations: gains and losses of large regions of chromosomal material. Copy number alterations may create a substantial statistical bias in the evaluation of histone mark signal enrichment and result in underdetection of the signal in the regions of loss and overdetection of the signal in the regions of gain.Results: We present HMCan (Histone modifications in cancer), a tool specially designed to analyze histone modification ChIP-seq data produced from cancer genomes. HMCan corrects for the GC-content and copy number bias and then applies Hidden Markov Models to detect the signal from the corrected data. On simulated data, HMCan outperformed several commonly used tools developed to analyze histone modification data produced from genomes without copy number alterations. HMCan also showed superior results on a ChIP-seq dataset generated for the repressive histone mark H3K27me3 in a bladder cancer cell line. HMCan predictions matched well with experimental data (qPCR validated regions) and included, for example, the previously detected H3K27me3 mark in the promoter of the DLEC1 gene, missed by other tools we tested.Availability: Source code and binaries can be downloaded at http://www.cbrc.kaust.edu.sa/hmcan/, implemented in C++.Contact: haitham.ashoor@kaust.edu.saSupplementary information: Supplementary data are available at Bioinformatics online.

Molecular Screening for a Personalized Treatment Approach in Advanced Adrenocortical Cancer

Adrenocortical cancer (ACC) is a rare cancer with poor prognosis and scant treatment options. In ACC, no personalized approach has emerged but no extensive molecular screening has been performed to date. The objective of the study was to evaluate the presence of a large number of potentially targetable molecular events in a large cohort of advanced ACC. We used hot spot gene sequencing (Ion Torrent, 40 patients) and comparative genomic hybridization (CGH; 28 patients; a subset of the entire cohort) in adult stage III-IV ACC samples to screen for mutations and copy number abnormalities of potential interest for therapeutic use in 46 and 130 genes, respectively. At least one copy number alteration or mutation was found in 19 patients (47.5%). The most frequent mutations were detected on TP53, ATM, and CTNNB1 [6 of 40 (15%), 5 of 40 (12.5%), and 4 of 40 (10%), respectively]. The most frequent copy number alterations identified were: amplification of the CDK4 oncogene (5 of 28; 17.9%) and deletion of the CDKN2A (4 of 28; 14.3%) and CDKN2B (3 of 28; 10.7%) tumor suppressor genes. Amplifications of FGFR1, FGF9, or FRS2 were discovered in three subjects (10.7%). Associated alterations were: deletions of CDKN2A, CDKN2B with ATM mutations, and TP53 mutations with CTNNB1 mutations. No simple targetable molecular event emerged. Drugs targeting the cell cycle could be the most relevant new therapeutic approach for patients with advanced ACC. Inhibitors of the fibroblast growth factor receptor pathway could also be a therapeutic option in a subset of patients, whereas other targeted therapies should be considered on a case-by-case basis.

Abstract A08: Chromothripsis and gene-specific copy number aberrations associated with mutation of IDH1 in glioma

Abstract Background: IDH gene mutation in gliomas is associated with younger age and better survival. IDH1 mutation has also been demonstrated to cause global hypermethylation and G-CIMP phenotype in astrocytes. However, the effects of IDH mutation and hypermethylation on molecular ontogeny and DNA copy number alterations in gliomas remain unknown. Here we describe a detailed analysis of copy number alterations (CNA) comparing IDH mutated (IDHmut) and IDH wild-type (IDHwt) gliomas of different grades. Method: CNAs were detected by molecular inversion probes (OncoScan FFPE Express, Affymetrix) and analyzed with Nexus Copy Number Software (BioDiscovery). DNA was extracted from 94 patient FFPE glioma samples including Grade II-III IDHwt (n=17) and IDHmut (n=28), and Grade IV: IDHwt (n=25) and IDHmut (n=24). Chromothripsis was determined in individual samples using stringent criteria of at least ten distinct CNAs within a single chromosome. Results: General analysis of CNAs between IDHmut versus IDHwt grade IV (glioblastoma) samples validated prior observations that IDHwt GBM have high frequencies of Chr7 amplification (including EGFR) and loss of Chr10 (including PTEN). The frequency of these specific alterations in IDHmut grade IV tumors was significantly lower. Other CNAs that were significantly different were gain of 19q12, and loss of 14q11 and 22q13 in IDHwt, and gain of 11q21, 10p11, 8q21, 12p13, 1q23, 11q and loss of 11p15, 3p, and 19q13 in IDHmut. Overall, both IDHwt and IDHmut grade IV tumors demonstrated more CNAs compared with lower grade tumors. Inspection of data from individual samples suggested that a higher number of samples from the grade IV IDHmut demonstrated frequent CNAs within individual chromosomes, indicative of chromothripsis. Using stringent criteria of ten distinct CNAs per chromosome, we found a significantly high incidence of chromothripsis events were observed in Grade IV IDHmut compared to IDHwt (p=0.0374). There was also a trend towards higher incidence of chromothripsis in grade IV versus grade I-III IDHmut tumors, but this did not reach statistical significance. Analysis of CNAs at the p53 locus at 17p13 demonstrated that cases with chromothripsis across all tumor grades had a significantly higher incidence of copy number loss or LOH at this locus compared to cases without chromothripsis (p=0.0368). Conclusions: CNA analysis showed significant differences in CNAs between IDHwt and IDHmut, indicating significant differences in molecular ontogeny. Significant CNA increases and increased chromothripsis in Grade IV IDHmut gliomas suggests that malignant transformation of low grade gliomas may occur through accumulation of genomic instability and genomic catastrophe. Since IDH mutation is thought to be an early event in gliomagenesis, it is possible that the global hypermethylation and other epigenetic alterations induced by IDH mutation in these tumors is mechanistically related to chromosomal instability and chromothripsis. In addition, higher incidence of LOH and copy number loss at the p53 loci in the cases with chromothripsis suggests a potential cooperative role between IDH1 and p53 in this process. Ongoing studies are aimed at further determining the contribution of p53 and ATRX mutation to this phenomenon. Citation Format: Howard Colman, Mariko Sato, Clinton C. Mason, Kristin Diefes, Lindsey Heathcock, Lisa Abegglen, Ken Aldape, Joshua D. Schiffman. Chromothripsis and gene-specific copy number aberrations associated with mutation of IDH1 in glioma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A08.