1p Deletions Research Articles

Overexpression of the A Disintegrin and Metalloproteinase ADAM15 is linked to a Small but Highly Aggressive Subset of Prostate Cancers

The A Disintegrin and Metalloproteinase (ADAM) family of endopeptidases plays a role in many solid cancers and includes promising targets for anticancer therapies. Deregulation of ADAM15 has been linked to tumor aggressiveness and cell line studies suggest that ADAM15 overexpression may also be implicated in prostate cancer. To evaluate the impact of ADAM15 expression and its relationship with key genomic alterations, a tissue microarray containing 12,427 prostate cancers was analyzed by immunohistochemistry. ADAM15 expression was compared to phenotype, prognosis and molecular features including TMPRSS2:ERG fusion and frequent deletions involving PTEN, 3p, 5q and 6q. Normal prostate epithelium did not show ADAM15 staining. In prostate cancers, negative, weak, moderate, and strong ADAM15 staining was found in 87.7%, 3.7%, 5.6%, and 3.0% of 9826 interpretable tumors. Strong ADAM15 staining was linked to high Gleason grade, advanced pathological tumor stage, positive nodal stage and resection margin. ADAM15 overexpression was also associated with TMPRSS2:ERG fusions and PTEN deletions (P<.0001) but unrelated to deletions of 3p, 5q and 6q. In univariate analysis, high ADAM15 expression was strongly linked to PSA recurrence (P<.0001). However, in multivariate analyses this association was only maintained if the analysis was limited to preoperatively available parameters in ERG-negative cancers. The results of our study demonstrate that ADAM15 is strongly up regulated in a small but highly aggressive fraction of prostate cancers. In these tumors, ADAM15 may represent a suitable drug target. In a preoperative scenario, ADAM15 expression measurement may assist prognosis assessment, either alone or in combination with other markers.

Genetic characterization of Polish ccRCC patients: somatic mutation analysis of PBRM1, BAP1 and KDMC5, genomic SNP array analysis in tumor biopsy and preliminary results of chromosome aberrations analysis in plasma cell free DNA

BackgroundMutation analysis and cytogenetic testing in clear cell renal cell carcinoma (ccRCC) is not yet implemented in a routine diagnostics of ccRCC.Material and methodsWe characterized the chromosomal alterations in 83 ccRCC tumors from Polish patients using whole genome SNP genotyping assay. Moreover, the utility of next generation sequencing of cell free DNA (cfDNA) in patients plasma as a potential tool for non-invasive cytogenetic analysis was tested. Additionally, tumor specific somatic mutations in PBRM1, BAP1 and KDM5C were determinedResultsWe confirmed a correlation between deletions at 9p and higher tumor size, and deletion of chromosome 20 and the survival time. In Fuhrman grade 1, only aberrations of 3p and 8p deletion, gain of 5q and 13q and gains of chromosome 7 and 16 were present. The number of aberrations increased with Fuhrman grade, all chromosomes displayed cytogenetic changes in G3 and G4. ccRCC specific chromosome aberrations were observed in cfDNA, although discrepancies were found between cfDNA and tumor samples. In total 12 common and 94 rare variants were detected in PBRM1, BAP1 and KDM5C, with four potentially pathogenic variants. We observed markedly lower mutation load in PBRM1.ConclusionsCytogenetic analysis of cfDNA may allow more accurate diagnosis of tumor aberrations and therefore the correlation between the chromosome aberrations in cfDNA and clinical outcome should be studied in larger cohorts. The functional studies on in BAP1, KDM5C, PBRM1 mutations in large, independent sample set would be necessary for the assessment of their prognostic and diagnostic potential.

Abstract P1-01-10: Exome sequencing of circulating tumor cells in metastatic breast cancer

Abstract Aim: We interrogated whether Circulating Tumor Cells (CTCs) can complement metastatic biopsies for genomic analyses. Patients and Methods: We compared single nucleotide variants (SNVs) and copy number aberrations (CNAs) identified using whole exome sequencing (WES) of DNA from frozen tumor tissue (primary/metastasis), amplified DNA from CTCs and normal DNA from 3 metastatic breast cancer (BC) patients (pts). All samples of the same patient were collected at the same timepoint. CTC isolation was performed using CellSearch and DEPArray systems followed by whole genome amplification (Ampli1 kit). WES was performed using the Illumina HiSeq2000 with 200X targeted coverage. Reads were aligned using bwa. SNVs had to be called by both Haplotype Caller (vs. reference genome) and Strelka (vs. paired normal). CNAs were determined by counting reads in 1MB windows and by comparing tumor/CTC samples with normal DNA. Pairwise concordance of CNAs profiles of different samples from the same patient was assessed using Spearman correlation (ρ). Significance of ρ differences between pts was obtained by Kruskal-Wallis test. Orthogonal validation for selected SNVs was performed. Results: We studied 3 patients from the 3 major BC subtypes, patient (pt)1 with ER-/HER2+ BC (samples collected at diagnosis, initially metastatic disease), pt2 with triple-negative BC (samples collected 2 years from diagnosis) and pt3 with ER+/HER2- BC (samples collected 8 years from diagnosis). We first compared tumor tissue and CTCs for SNVs. For pt1, of the 77 SNVs identified in the tumor, 51 were found on at least one of 12 CTCs samples. For pt2, of the 62 SNVs identified in the tumor, 19 were found on at least 1 of 11 CTCs samples. For pt3, of the 225 SNVs identified in the tumor, 48 were found on at least 1 of 3 CTCs samples. Interestingly, by increasing the number of CTCs analyzed, we increased the % of identified SNVs from synchronous tumor tissue. SNVs with high variant allele fraction (VAF) in tumor tissue were detected significantly more often in CTCs: 22% of the SNVs with VAFs &amp;lt;20% were found at least once, compared to 53% and 74% of SNVs with VAFs &amp;gt;20% and &amp;gt;40%, respectively (p=10-12, Fisher exact test). Then, we compared tumor tissue and CTCs for CNAs. As time from diagnosis of metastatic disease to samples collection increased, we observed significantly higher heterogeneity within CTCs from the same patient (median ρ between CTCs was 86% for pt1, 84% for pt2 and 28% for pt3, p&amp;lt;0.01) and between CTCs and tumor tissue from the same patient (median ρ was 78% for pt1, 67% for pt2 and 21% for pt3, p&amp;lt;10-4). Interestingly, in pt3 one CTC was more similar to the metastasis than the other 2 (ρ of 53%, 21% and 21%). When a phylogenetic tree was constructed for pt3 by combining SNVs and CNAs data, three clones were identified: one clone with an AKT1 (E17K) and a TP53 (R248W) mutation and a 8p deletion, a second clone with the above profile plus an 8q amplification and a third clone with an AKT1 and an ESR1 (Y537N) mutation and 1p deletion. The metastasis was similar with the first clone. Conclusions: These data suggest that tumor tissue and single CTC exome sequencing analyses provide complementary information to map tumor heterogeneity. Further validation for potential clinical applications is needed. Citation Format: Ignatiadis M, Rothé F, Peeters D, Rouas G, Smeets D, Haan J, Lambrechts D, Campbell P, Piccart M, Voet T, Dirix L, Venet D, Sotiriou C. Exome sequencing of circulating tumor cells in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-10.

Correction: Genomic deletion of chromosome 12p is an independent prognostic marker in prostate cancer.

Correction: Genomic deletion of chromosome 12p is an independent prognostic marker in prostate cancer.

Serum LDH level may predict outcome of chronic lymphocytic leukemia patients with a 17p deletion: a retrospective analysis of prognostic factors in China.

ObjectiveThis study aims to evaluate the natural history of patients with chronic lymphocytic leukemia (CLL) and a 17p deletion (17p-) and identify the predictive factors within this subgroup.MethodsThe sample of patients with CLL were analyzed by fluorescencein situ hybridization for deletions in chromosome bands 11q22, 13q14 and 17p13; trisomy of bands 12q13; and translocation involving band 14q32. The data from 456 patients with or without a 17p- were retrospectively collected and analyzed. ResultsThe overall response rate (ORR) in patients with a 17p- was 56.9%, and patients with a high percentage of 17p- (defined as more than 25% of cells harbouring a 17p-) had a lower ORR. The median overall survival (OS) in patients with a 17p- was 78.0 months, which was significantly shorter than the OS in patients without this genetic abnormality (median 162.0 months, P<0.001). Within the subgroup with a 17p-, the progression-free survival was significantly shorter in patients at Binet stage B-C and patients with elevated lactate dehydrogenase (LDH), B symptoms, unmutatedIGHV and a high percentage of 17p-. ConclusionsThese results indicated that patients with a 17p- CLL have a variable prognosis that might be predicted using simple clinical and laboratory characteristics.

Morphologic, Immunohistochemical and Molecular Analyses of a Huge Clear Cell Papillary Renal Cell Carcinoma

Among the tumors of the kidney, the clear cell papillary renal cell carcinoma (CCPRCC) is a recently described entity. This tumor is well circumscribed, with a fibrous capsule, showing mostly a prominent cystic component and a diameter less than 4 cm. Necrosis is not a feature. Tumor cells are clear with low grade nuclei with a predominantly tubule-papillary architecture and immunohistochemical staining strongly positive for CK7, and for CA IX and negative for RACEMASE, features which differentiate this tumor from clear cell renal cell carcinoma (CCRCC) and papillary renal cell carcinoma (papillary RCC). In these latter tumors, it has been reported, unlike the other two, no loss or gain of chromosomes 7 and Y, no chromosome 3p deletion, and no KRAS mutation. In this report, a huge clear cell papillary renal cell carcinoma case, 9 cm diameter, in a 42-year-old male is described. Pathologic diagnosis of the tumor was confirmed by immunohistochemical analysis including CD10, CA IX, CK7, RACEMASE, and 34 beta E12 stainings. Molecular detection of KRAS, BRAF, NRAS, PIK3CA, ALK, ERBB2, DDR2, MAP2K1, RET, and EGFR gene mutational analysis has also been performed. Molecular findings are in accordance with the speculation that the CCRCC is an indolent tumor which may be defined as low malignant potential. In fact, in the molecular analysis, none out of all genes evaluated showed mutation.

3p Deletion Syndrome and Congenital Heart Disease: Case Report

3p Deletion Syndrome and Congenital Heart Disease: Case Report

Validation of 34betaE12 immunoexpression in clear cell papillary renal cell carcinoma as a sensitive biomarker

Clear cell papillary renal cell carcinoma (CCPRCC) is a recently recognised neoplasm with a broad spectrum of morphological characteristics, thus representing a challenging differential diagnosis, especially with the low malignant potential multicystic renal cell neoplasms and clear cell renal cell carcinoma. We selected 14 cases of CCPRCC with a wide spectrum of morphological features diagnosed on morphology and CK7 immunoreactivity and analysed them using a panel of immunohistochemical markers, focusing on 34βE12 and related CKs 1,5,10 and 14 and several molecular analyses such as fluorescence in situ hybridisation (FISH), array comparative genomic hybridisation (aCGH), VHL methylation, VHL and TCEB1 sequencing and multiplex ligation-dependent probe amplification (MLPA). Twelve of 13 (92%) CCPRCC tumours were positive for 34βE12. One tumour without 3p alteration by FISH revealed VHL mutation and 3p deletion at aCGH; thus, it was re-classified as clear cell RCC. We concluded that: (1) immunohistochemical expression of CK7 is necessary for diagnostic purposes, but may not be sufficient to identify CCPRCC, while 34βE12, in part due to the presence of CK14 antigen expression, can be extremely useful for the recognition of this tumour; and (2) further molecular analysis of chromosome 3p should be considered to support of CCPRCC diagnosis, when FISH analysis does not evidence the common loss of chromosome 3p.

Impact of 1p Deletions in Myelodysplastic Syndromes and Secondary AML Arising from Myelodysplastic Syndromes

Background: Deletions in the short arm of chromosome 1 are rare, recurrent abnormalities in Myelodysplastic syndromes (MDS) and are observed as the sole abnormality in 0.2% (Schanz et al. JCO 2012). So far no comprehensive characterization of this subset has been performed. Aim: The aim of this study was to characterize MDS and secondary AML evolving from MDS harboring a 1p deletion with respect to 1) accompanying cytogenetic and molecular genetic abnormalities, 2) the size of the 1p deletion and the minimal deleted region. Patients and Methods: 50 cases with MDS (de novo MDS: n=38, t-MDS: n=8) and secondary AML evolving from MDS (n=4) harboring a 1p deletion were selected for analysis. All cases were evaluated by chromosome banding analysis. From 30 cases sufficient material was available to perform genomic array analysis (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) and amplicon sequencing to detect mutations in ASXL1, CBL, CSF3R, CSNK1A1, DNMT3A, ETNK1, ETV6, EZH2, GATA1, IDH1, IDH2, JAK, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, and U2AF1. Variants of unknown significance were excluded from statistical analysis. Results: 62% were male and median age was 75 years (range: 35 - 91). The 1p deletion was the sole chromosomal abnormality in 5/50 cases (10%) and was accompanied by one, two and more than two additional aberrations in 12 (24%), 15 (30%), and 18 (36%) cases, respectively. In total 129 chromosome abnormalities were observed in addition to the 1p deletion (median per patient: 2, range: 0-9). Of these only 10 were balanced, while 119 were unbalanced abnormalities leading to gain or loss of chromosomal material. Loss of 1p was most frequently accompanied by del(5q) (n=24; 48%), +8 (n=20; 39%), 7q-/-7 (n=11; 22%), del(17p) (n=5; 10%), and -Y (n=3; 6%). In 15 cases (29%) a duplication of the short arm of chromosome 1 harboring the 1p deletion was observed. Genomic array analyses revealed a median size of the 1p deletion of 25 MB (range: 13-34 MB). A minimal deleted region encountered in all 30 evaluable patients ranged from genomic position 17,872,935 to 24,285,861 encompassing 72 genes (e.g. E2F2, ID3, PAX7, UBR4, ZBTB40) and 10 micro RNAs. One, 2, 3, and 4 mutations were present in 10 (33%), 8 (26%), 5 (17%) and 2 (7%) cases, respectively. No mutations in any of the analyzed genes were observed in 5 cases (17%). Mutations were detected in SF3B1 (16%), TET2 (16%), ASXL1 (13%), DNMT3A (13%), EZH2 (13%), NRAS (13%), SRSF2 (13%), TP53 (13%), JAK2 (10%), KRAS (7%), U2AF1 (7%), CALR (3%), IDH2 (3%), MPL (3%), RUNX1 (3%), and SETBP1 (3%). No mutations were detected in CBL, CSF3R, CSNK1A1, ETNK1, ETV6, GATA1, IDH1, KIT, and NPM1. Compared to published data (Bejar et al. NEJM 2012, Papaemmanuil et al. Blood 2013, Haferlach et al. Leukemia 2014) SF3B1 (20-30%) and TET2 (20-30%) mutations seem to be less frequent and mutations in TP53 (6-8%), EZH2 (5-7%), NRAS (4-5%) and KRAS (1-3%) more frequent in MDS with 1p deletion compared to an unselected MDS cohort. Patients harboring either a 1p deletion as the sole abnormality or a duplication of the deleted chromosome 1 (n=20) had an excellent prognosis (3 year overall survival (OS): 100%), while patients with a 1p deletion accompanied by -7/7q- (n=11) had a very poor outcome (3 year OS: 0%; p&lt;0.001). Patients with 1p deletion and other accompanying cytogenetic abnormalities (n=19) had an intermediate outcome (3 year OS: 34%, p=0.03, p=0.003). Conclusions: 1) Interstitial deletions in the short arm of chromosome 1 are rare recurrent abnormalities in MDS. 2) 5q deletion, +8, -7/7q- are frequently observed in addition to 1p deletion. 3) Both MDS with a 1p deletion as the sole abnormality and MDS with a duplication of deleted chromosome 1 are associated with very favorable outcome. 4) Accompanying abnormalities, especially -7/7q- have a negative impact on outcome. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Complex Landscape of Rearrangements in Smoldering and Symptomatic Multiple Myeloma Revealed By Whole-Genome Sequencing

INTRODUCTION:Multiple myeloma is a heterogeneous disease featured by recurrent translocations involving the IgH region. Such cytogenetic events have a driver role in early transformation of a normal plasma cell into a MM cell. Although several studies have reported the presence of limited number of other structural chromosomal events using different approaches, including conventional cytogenetics, high-resolution genome mapping, interphase fluorescence in situ hybridization (FISH) and whole exome sequencing, the full catalogue of genomic rearrangements in MM samples has never been carried out systematically. Here, we have utilized whole-genome sequencing technologies to perform a systematic, genome-wide analysis to uncover the frequency and nature of rearrangements in MM.MATERIAL AND METHODS:We performed Whole genome sequencing (WGS) using the Illumina X10 platform in 68 serial samples from 30 patients including 11 patients with smoldering myeloma, 13 newly-diagnosed patients and 44 relapsed patient samples to provide further insight into evolution of rearrangements in MM. Structural variations (translocations, deletions, inversions, internal tandem duplications, fusions) and copy number changes were analyzed using the analysis pipeline at the Wellcome Trust Sanger Institute as recently described (Nik-Zainal Nature 2016).RESULTS:We observed a total of 1295 rearrangements for a median of 27 per sample (range 2-138) including a median of 6 (range 1-36) inversions, 5 (range 1-33) internal tandem duplications, 10 (range 1-40) deletions, 7 (range 1-32) translocations and 5 fusions (0-20). While the vast majority of events was non-recurrent, the high prevalence of rearrangements at smoldering stage and in myeloma at diagnosis and further increase at the time of relapse suggest a much more complex genomic landscape than previously thought. Translocations involving the IGH locus were identified including t(11;14) in 6 (20%), t(4;14) in 4 (13%) and t(8;14) in 3 (10%) of 30 unique patients. We also report frequent involvement by light chain loci in the rearrangements. The MYC locus was recurrently affected by non-IGH rearrangements in 11/30 (36%) patients. The other main MYC partners were IGL (4/30) and IGK (2/30), while about one-third of cases were involved by rearrangements not involving immunoglobulins or other obvious partners. MYC is therefore frequently involved by rearrangements through immunoglobulin-independent mechanisms.Interestingly, many regions affected by recurrent copy number abnormalities (CNAs) were associated with rearrangements. In particular 7/14 (50%) 1q gains and 6/8 (75%) 1p deletions were involved by translocations and inversions respectively (i.e Figure 1a). Overall 15/22 chromosome 1 CNAs were associated with a specific rearrangements. A similar association between copy number changes and rearrangement breakpoints was observed among other recurrent genomic aberrations such as 6q deletions (6/12, 50%), 8p deletions (4/7, 57%) and 16q deletions (7/13, 53%).In addition to deletions, inversions, internal tandem duplications (ITDs) and translocations, we observed at least one and often more regions of chromothripsis in 10/30 (33%) patients. Chromothripsis represents a complex event characterized by localized chromosome shattering and repair occurring in a one-off catastrophic event (Korbel J. et al. Cell 2013) (Figure 1b) and known to be associated with worse prognosis in MM. In our series, chromothriptic events were always conserved during every investigated evolution process: suggesting an early onset of this complex event in myelomagenesis.CONCLUSION:We report for the first time a comprehensive catalogue of rearrangements in MM based on whole-genome sequencing data. Our data provide evidence that the genomic landscape of rearrangements in MM is very complex and heterogeneous than speculated before and besides IgH involves number of other recurrent chromosomal alteration mechanisms. These diverse aberrations, in many cases acquired early, may deregulate oncogenes as illustrated by the MYC locus. [Display omitted] DisclosuresMoreau:Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Avet-Loiseau:sanofi: Consultancy; celgene: Consultancy; amgen: Consultancy; janssen: Consultancy.

Comparative Analysis of Japanese and European Typical CLL Patients

Background: Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in the Western countries but very rare in the East Asia including Japan. The previous reports from Japan showed that CLL patients in Japan have different characteristics including atypical morphology and immunophenotypes compared to those of Western countries. We started the cooperative study of CLL between Japan and Austria from 2012.Aim: The aim of this retrospective study is to compare the characteristics of typical CLL patients in Japan and Austria.Material and Methods: Diagnostic procedures were first harmonized between sites and laboratories. Samples were exchanged between Japan and Austria, and morphology, flow cytometry (FCM), FISH and IGVH gene sequence were independently assessed. After technical agreement, we selected 86 Austrian CLL patients from the database of the Medical University of Vienna, and 80 Japanese patients from the CLLRSG-01 prospective observational study. All CLL cases had typical immunophenotypes (Matutes score 4 or 5). The analyses of morphology, FCM and FISH including the deletion in 11q22, 13q14, and 17p13, and trisomy 12 were performed. Immunoglobulin heavy chain variable region (IGHV) gene mutation was also analysed.Results: Median age of CLL patients was 62.5 years old (range: 35-93) in Austria and 66.4 years (range: 34-93) in Japan. Japanese patients were significantly older at diagnosis than Austrian patients (P = 0.027). No differences were found for sex and the Binet stage. Also, there were no differences in laboratory findings (lymphocyte and platelet counts and serum LDH and beta-2-microglobulin levels) except for hemoglobin (Hb) level (median: 13.8g/dL in Austria vs. 13.1g/dL in Japan, P = 0.005) between two countries. Immunophenotypic study by FCM showed higher proportion of CD38 expression in Austrian CLL patients (34% in Austria vs. 10% in Japan, P < 0.001). No differences were found for chromosomal aberrations by FISH analyses between Austria and Japan. del(13q14) was found in 62% and 56% (P = 0.456), trisomy 12 in 7% and 13% (P = 0.315), del(11q22) in 13% and 10% (P = 0.513) and del(17p13) in 7% and 3% (P = 0.179), in Austria and Japan, respectively. The IGHV mutation rate was higher in Japan (P = 0.002). Mutated IGHV was found in 61% of Austrian and 84% of Japanese patients. IGHV family usage profile was significantly different. In Austrian patients, VH1, 2, 3, 4, 5, 6, and 7 were 24%, 1%, 54%, 19%, 1%, 1% and 0%, respectively. However, in Japanese patients, they were 2%, 0%, 75%, 20%, 2%, 0% and 2%, respectively. The proportion of VH1 was surprisingly lower in Japan (P = 0.006). Notably, none of the Japanese patients used VH1-69, in contrast to the use of 10% in Austrian patients.Conclusion: Our analysis indicates that there are clear differences between Japanese and European CLL patients, in age, Hb level, CD38 expression and IGHV usage and mutations. The reason for particular IGHV usage should further be investigated. DisclosuresTakizawa:Teijin: Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria; Chugai: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Janssen: Honoraria; Sumitomo Dainippon: Honoraria, Research Funding; MSD: Honoraria, Research Funding. Suzuki:Bristol-Myers Squibb: Honoraria; Kyowa Hakko kirin: Honoraria; Chugai: Honoraria. Hoermann:Gilead: Research Funding; Novartis: Honoraria; Amgen: Honoraria; Ariad: Honoraria. Aoki:SymBio Pharmaceuticals: Consultancy. Suzumiya:Kyowa Hakko kirin: Research Funding; Eisai: Honoraria, Research Funding; Astellas: Research Funding; Takeda: Honoraria; Toyama Chemical: Research Funding; Chugai: Honoraria, Research Funding. Jaeger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

NGS-Based Copy Number Analysis in 1,185 Patients with Myeloid Neoplasms

BackgroundCopy number alteration (CNA) is a hallmark of cancer genomes and has been implicated in the development of human cancers, including myeloid neoplasms. We developed a novel, next-generation sequencing-based platform for highly sensitive detection of CNAs with a single exon resolution, which was applied to sequencing data from 1,185 patients to delineate a comprehensive landscape of CNAs in myeloid neoplasms.Materials and MethodsWe enrolled 1,185 patients with different myeloid neoplasms including myelodysplastic syndromes (n = 607), myelodysplastic/myeloproliferative neoplasms (n = 80), de novo acute myeloid leukemia (AML) (n = 136), secondary AML (sAML) (n = 226), and unknown myeloid malignancies (n = 136). Whole-exome sequencing (WES) was performed on samples from 260 patients, while samples from 925 patients including pre-transplantation peripheral blood samples provided by Japan Marrow Donor Program were subjected to targeted deep sequencing. Eight cases were serially evaluated before and after progression tosAML. RNA baits for targeted deep sequencing were designed to cover 69 driver genes in myeloid neoplasms and 1,158 single-nucleotide polymorphisms (SNPs)for assessment of allelic imbalance. In WES, allelic imbalance was examined using allele frequencies of SNPs within coding regions. Focal CNAs were defined as CNAs whose lengths relative to the chromosomal arms were below 10%.ResultsTo obtain a landscape of CNAs in coding regions, a comprehensive copy number analysis was performed on 260 patients including 136 with de novo AML and 124 with myeloid neoplasms with myelodysplasia, all of whom were studied by WES. A total of 755 CNAs (502 deletions and 253 amplifications) were identified, where 52% of the patients harbored at least one alteration. Using GISTIC 2.0 algorism, we identified 21 significantly altered regions involving known or putative driver genes (Figure 1): losses of 7q22.1 (CUX1), 12p13.2 (ETV6), 13q14 (RB1),17p13.1(TP53), and 17q11.2 (NF1), and gains of 3q26-27 (EVI1), 8q24.21 (MYC), 11q13.5-14.1(PAK1), 11q23.3 (MLL),11q24-25 (ETS1), 13q12.2 (FLT3),21q22.2 (ETS2 and ERG). We next compared the frequencies of CNAs between de novo AML and myeloid neoplasms with myelodysplasia. While chromosomes 7, 12, and 17 were commonly affected, deletions of 13q14 were significantly enriched in myeloid neoplasms with myelodysplasia (Odds ratio [OR]: 5.07, P = 0.040), and amplifications of 11q24-25 (OR: 5.54, P = 0.028), and 21q22.2 (OR: 6.10, P = 0.020) in de novo AML, suggesting a specific role of these events in each disease entity. In addition, serial sampling revealed trisomy8, deletions of 7q and 12p were recurrently acquired during leukemic transformation in patients withmyelodysplasia. Taken together, many driver genes in myeloid neoplasms were frequently targeted by CNAs includingmicrodeletions.Based on these finding, we sought to obtain a more detailed landscape of CNAs in a larger cohort. We combined copy number profiles of patients studied by targeted deep sequencing and those by WES. Of total, 1,691 CNAs (1,096 deletions and 595 amplifications) were detected, where 39% of the cases harbored at least one alteration. Microdeletionsor focal amplifications were frequently found in the significantly altered regions revealed by WES: microdeletionsof ETV6 (n = 10), NF1 (n = 8), CUX1 (n = 5), TP53 (n = 5), and amplifications of FLT3 (n = 7), ETS1 (n = 3), ETS2 (n = 3), and ERG (n = 3), validating the result obtained from a cohort studied by WES. We also identified known driver genes in myeloid neoplasms were recurrently affected with focal CNAs: microdeletions of RUNX1, BCOR, ASXL2, DNMT3A, and ZRSR2, and amplifications of GNAS, RIT1, CSF3R, and BCL11A. Among them, DNMT3A and ASXL2, located within 500 kb in chromosome 2, tended to be co-deleted (3 out of 4 cases). Focal deletions of TP53 were often affected with homozygous deletions or were accompanied by gene mutations, implying bi-allelic inactivation. High amplifications were also observed in regions including ETS1, MLL, FLT3, MYC, and PAK1, which suggest a critical role in the pathogenesis of myeloid malignancy.ConclusionWe obtained the landscape of CNAs in myeloid neoplasms based on the sequencing data of 1,185 patients. Collectively, our results indicated that CNAs targeted a specific set genes including well-known drivers of myeloid malignancies, indicating a critical role inleukemogenesis. [Display omitted] DisclosuresKanda:Otsuka Pharmaceutical: Honoraria, Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

High fidelity of driver chromosomal alterations among primary and metastatic renal cell carcinomas: implications for tumor clonal evolution and treatment

AbstractRecent studies have demonstrated considerable genomic heterogeneity in both primary and metastatic renal cell carcinoma (RCC). This mutational diversity has serious implications for the development and implementation of targeted molecular therapies. We evaluated 39 cases of primary RCC tumors with their matched metastatic tumors to determine if the hallmark chromosomal anomalies of these tumors are preserved over the course of disease progression. Thirty-nine matched pairs of primary and metastatic RCCs (20 clear cell RCC, 16 papillary RCC, and 3 chromophobe RCC) were analyzed. All clear cell RCC and papillary RCC tumors were evaluated for chromosome 3p deletion, trisomy 7 and 17 using fluorescence in situ hybridization. Chromophobe RCC tumors were evaluated for genetic alterations in chromosomes 1, 2, 6, 10, and 17. Of the 20 clear cell RCC tumors, 18 primary tumors (90%) showed a deletion of chromosome 3p and were disomic for chromosomes 7 and 17. All molecular aberrations were conserved within the matched metastatic tumor. Of the 16 papillary RCC tumors, 10 primary tumors (62%) showed trisomy for both chromosomes 7 and 17 without 3p deletion. These molecular aberrations and others were conserved in the paired metastatic tumors. Of the three chromophobe RCC tumors, multiple genetic anomalies were identified in chromosomes 1, 2, 6, 10, and 17. These chromosomal aberrations were conserved in the matched metastatic tumors. Our results demonstrated genomic fidelity among the primary and metastatic lesions in RCCs. These findings may have important clinical and diagnostic implications.

Abstract PR03: HVEM (TNFRSF14) tumor suppressor in immune therapies of follicular lymphoma

Abstract Follicular lymphoma (FL) is among the most common and incurable forms of indolent B cell lymphoma. Somatic mutations that disrupt the HVEM (TNFRSF14) receptor gene in 28% of FL and include frequent homozygous lesions. The HVEM receptor interacts with the activating LIGHT and the inhibitory BTLA (B- and T- lymphocyte attenuator) receptors on B and T cells. Exactly, how HVEM mutations contribute to FL development is unknown. Here we show that the immune cell receptor HVEM (TNFRSF14) is a p53 response gene, and a frequent target of both somatic mutations, and recurrent chromosome 1p36 deletions in FL. Moreover, HVEM deficiency promotes FL development in vivo and establishes a favorable immune environment. Specifically, HVEM deficient lymphomas show an exaggerated activation of stromal components including follicular dendritic cells (FDCs) and increased recruitment of T follicular helper (TFH) cells that support B lymphocytes by secreting interleukins IL21 and IL4. Furthermore, HVEM loss stimulates B cell growth by disrupting an inhibitory BTLA (B and T Lymphocyte Attenuator) mediated signal. Notably, HVEM's tumor suppressor functions reside at the cell surface and extrinsic application of the HVEM ectodomain(P37-V202) can restore B cell growth control and block lymphoma growth in vivo. In addition, we demonstrate that CD19-specific, chimeric antigen receptor (CAR)-modified T cells that are further modified to constitutively secrete soluble HVEM ectodomain display significantly higher cytotoxicity against cancer B-cells with high BTLA expression in vivo. Citation Format: Darin Salloum. HVEM (TNFRSF14) tumor suppressor in immune therapies of follicular lymphoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR03.

The Molecular Landscape of Recurrent and Metastatic Head and Neck Cancers

ImportanceRecurrent and/or metastatic head and neck cancer is usually incurable. Implementation of precision oncology for these patients has been limited by incomplete understanding of the molecular alterations underlying advanced disease. At the same time, the molecular profiles of many rare head and neck cancer types are unknown. These significant gaps in knowledge need to be addressed to rationally devise new therapies.ObjectiveTo illuminate the distinct biology of recurrent and metastatic head and neck cancers and review implementation of precision oncology for patients with advanced disease.Design, Setting, and ParticipantsAfter exclusions, 151 patients with advanced, treatment-resistant head and neck tumors, including squamous cell carcinoma (HNSCC), adenoid cystic carcinoma (ACC), and other salivary and cutaneous cancers, whose tumors were sequenced between January 2014 and July 2015 at Memorial Sloan Kettering were recruited. Next-generation sequencing of tumors as part of clinical care included high-depth (median 600×) exonic coverage of 410 cancer genes and whole-genome copy number analysis.InterventionsNext-generation sequencing of tumors and matched normal DNA.Main Outcomes and MeasuresFeasibility, the frequency of actionable molecular alterations, the effect on decision making, and identification of alterations associated with recurrent and metastatic disease.ResultsOverall, 151 patients (95 men and 56 women; mean [range] age, 61.8 [17-100] years) were included in the study. Next-generation sequencing ultimately guided therapy in 21 of 151 patients (14%) (13 of 53 [25%] of patients with HNSCC) by refining diagnoses and matching patients to specific therapies, in some cases with dramatic responses on basket studies. Molecular alterations were potentially actionable in 28 of 135 patients (21%). The genetic profiles of recurrent and metastatic tumors were often distinct from primary tumors. Compared to primary human papillomavirus (HPV)-positive tumors, many recurrent and metastatic HPV-positive tumors exhibited a molecular profile more similar to HPV-negative tumors, including enriched frequencies of TP53 mutation (3 of 20 tumors [15%]), whole genome duplication (5 of 20 tumors [25%]), and 3p deletion (11 of 20 tumors [55%]). There were high rates of TERT promoter mutation in recurrent and metastatic HPV-negative HNSCC (13 of 30 tumors [43%]), cutaneous SCC (11 of 21 tumors [52%]), basal cell carcinoma (3 of 4 tumors [75%]), and ACC (5 of 36 tumors [14%]). Activating NOTCH1 mutations were enriched in metastatic ACCs (8 of 36 tumors [22%]).Conclusions and RelevanceThese findings reveal the molecular landscape of advanced disease and rare cancer subtypes, both predominant challenges in head and neck oncology. To understand the repertoire of targetable alterations in advanced cancers, it is necessary to sequence recurrent and metastatic tumors. These data are important first steps toward implementation of precision head and neck oncology.

Abstract 94: Genomic landscape and clonal expansions of upper urinary tract urothelial carcinoma

Abstract Backgrounds Upper urinary tract urothelial carcinoma (UUTUC) is a relatively rare and poor prognostic cancer which accounts for 5-10% of all urothelial malignancies. Despite the histological similarity, there are epidemiological and clinical differences between UUTUC and bladder urothelial carcinoma (BUC). Compared to BUC, the molecular pathogenesis of UUTUC is poorly understood. Urothelial carcinoma is also characterized by frequent multifocal lesions, suggesting field effects from mutagenic agents in urine and/or a dissemination from the primary tumor. To reveal the origin of the multifocality as well as the molecular pathogenesis of UUTUC, we performed comprehensive molecular analysis of UUTUC with regional multiple sampling. Materials &amp; methods Surgical specimens of UUTUC and matched normal samples were obtained from 57 patients with various stages who underwent nephroureterectomy. Morphologically normal urothelial epithelia were also obtained in 5 cases, all of which were confirmed to be pathologically intact by an expert pathologist. Genomic DNA was extracted from each specimen and was subjected to whole exome sequencing using Hiseq 2500 for the detection of both somatic mutations and copy number lesions. Results In copy number analysis, recurrent deletions in 8p, 9p and 9q were identified, where homo deletions of 9p21.3 (CDKN2A) were most frequent (40.7%) Focal amplifications in 11q13.3 (CCND1) and 12q15 (MDM2) were also found. On average, 230 somatic nonsynonymous mutations per sample were detected. The mutation signature was biased to age-related and APOBEC patterns. Mutations were most frequently observed in KMT2D (42% of cases), followed by TP53 (32%), FGFR3 (24%), KDM6A (24%), EP300 (21%) and CREBBP (13%), which were also reported to be significantly mutated in BUC. However, the frequencies of these genetic lesions were substantially different between UUTUC and BUC. For example, abnormalities in RB1, CDKN1A and PIK3CA were not detected or rare in UUTUC but have been reported to be common targets of genetic lesions in BUC. In the analysis of normal epithelia, an average of 12.7 somatic mutations per sample were found but with low variant allele frequencies between 0.05−0.1. Somatic mutations in adjacent epithelia and preoperative urine were frequently shared by primary tumor, supporting a possibility of tumor disseminations from the original sites through urinary flow. In contrast, in other cases, distant epithelia harbored driver gene mutations that were not shared by the primary tumors, suggesting the presence of a mutagenic field effect on urothelial carcinogenesis. Conclusions Despite the similarity in their mutation spectrum, UUTUC and BUC seemed to have distinct pathogenesis in terms of the difference in mutation frequencies. Our data suggested that both dissemination and field effect might contribute to multifocal occurrence of UUTUC. These findings will provide a new insight into the pathophysiology of UUTUC. Citation Format: Yoichi Fujii, Yusuke Sato, Shigekatsu Maekawa, Hiromichi Suzuki, Tetsuichi Yoshizato, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Tohru Nakagawa, Haruki Kume, Hiroaki Nishimatsu, Yoshikazu Hirano, Masashi Sanada, Hideki Makishima, Satoru Miyano, Yukio Homma, Seishi Ogawa. Genomic landscape and clonal expansions of upper urinary tract urothelial carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 94.

Abstract 5019: Impact of the ERG status on the prognostic relevance of prostate cancer biomarkers: a survey of 13,000 patients

Abstract The TMPPRS:ERG fusion is the most frequent genomic alteration in prostate cancer leading to androgen dependent up-regulation of the ERG transcription factor. ERG activation deregulates more than 1,600 ERG-responsive genes, and some molecular alterations have been linked to either the subset of ERG negative or ERG positive cancers. While studies in large cohorts of prostate cancers treated by radical prostatectomy have demonstrated that ERG fusion alone has no impact on patient outcome, there is growing evidence that the prognostic relevance of many other biomarkers may be different in cancers with and without ERG fusion. In order to better understand the impact of the ERG status on the prognostic relevance of prostate cancer biomarkers, we took advantage of our molecular database obtained from immunohistochemistry and fluorescence in situ hybridization (FISH) analyses of more than 50 markers with respect to expression levels and gene copy number changes in 13,665 prostate cancers that had been assembled in a tissue microarray (TMA) format. Markers with strongest associations to presence of ERG fusion included CRISP3, HOOK3, RBM3, LPCAT1, BAZ2A, and deletions of 3p and PTEN (p&amp;lt;0.0001 each). Markers with strongest associations to absence of ERG fusion included SPINK1 and deletions of 6q and 5q (p&amp;lt;0.0001 each). Alterations of 47 markers were linked to reduced time to biochemical recurrence if all cancers were analyzed regardless if the ERG status. However, subset analyses revealed, that the prognostic relevance of 18 (38%) of these 47 markers was driven either by the subset of ERG positive (n = 7) or ERG negative cancers only (n = 11). Combined analysis of these markers identified ERG status dependent molecular classifiers that provided prognostic information exclusively in ERG negative or in ERG positive prostate cancers. Multivariate modeling indicated that the classifiers were independent of established prognostic factors, including pre-surgical (PSA, Gleason grade on biopsy, clinical stage) and post-surgical (pT stage, Gleason of the radical prostatectomy, nodal stage, resection margin stage) parameters. In conclusion, the results of our study demonstrate that the impact of many candidate prognostic markers in prostate cancer strongly depends on the ERG status. Our growing molecular database attached to more than 13,000 prostate cancers allows for the continuous validation of candidate prognostic markers and their impact in molecularly defined subsets of the disease. Citation Format: Ronald Simon, Martina Kluth, Claudia Hube-Magg, Maria Christina Tsourlakis, Sarah Minner, Christoph Burdelski, Katharina Grupp, Nathaniel Melling, Asmus Heumann, Christina Koop, Thomas Steuber, Markus Graefen, Hartwig Huland, Guido Sauter, Thorsten Schlomm. Impact of the ERG status on the prognostic relevance of prostate cancer biomarkers: a survey of 13,000 patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5019.

Abstract LB-137: IGF2BP1 and miR-34a genetic instability found in deadly neuroblastoma

Abstract Chromosomal 17q21-ter gain in neuroblastoma is both a common and clinically significant occurrence. The oncogenic, insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) gene is located near the proximal edge of this region. We have previously reported that in 69 neuroblastoma samples, IGF2BP1 DNA copy number (increased in 84% of tumors), mRNA, and protein levels are significantly higher in stage 4 compared with stage 1 tumors. Moreover, clinical and experimental data pointed to a strong association of IGF2BP1 with the MYCN oncogene. From the late 1990s onwards, 17q gain and 1p deletions (either alone or both) have been speculated as essential precursors to the MYCN amplification. As the tumor suppressive, miR-34a is located within 1p and is known to directly target MYCN and putatively IGF2BP1 mRNAs (Targetminer website), in this study we analyzed the co-occurrence of IGF2BP1 gain, miR-34a loss and MYCN amplification) in 69 neuroblastomas. qPCR was utilized to assess copy number. Changes were found for IGF2BP1 (58 tumors), miR-34a (15) and MYCN (12). miR-34a was never completed deleted. Interestingly, in all (12/12) MYCN amplified tumors, miR-34a was partially lost. Whilst, IGF2BP1 was gained in 9/12 MYCN amplified tumors. IGF2BP1 and miR-34a were aberrant together only twice with the absence of MYCN amplification. Of the 17 patients that died, all but one had at least an IGF2BP1 or miR-34a change in copy number. This stage 4 patent also had low MYCN protein levels, comparable to the stage 1 patients, indicating an aggressive tumor not involving the MYCN pathway. may A second larger cohort is certainly required to confirm these important results. IGF2BP1 and miR-34a locus have aberrant copy numbers in the majority of neuroblastoma, most strikingly, in the patients that died. IGF2BP1 and miR-34a may just be representative DNA of the larger locus changes and not be the causative event in affected neuroblastoma, but because of their known roles in cancer, the above results are intriguing. Although further experimental data is required, we speculate that either IGF2BP1 or mir-34a oncogenic changes are required for the clonal expansion of MYCN amplified tumor cells in neuroblastoma, and that these factors are crucial in the ability for an increase in MYCN transcription to be oncogenic in neuroblastoma. Citation Format: Jessica L. Bell, Stefan Hüttelmaier. IGF2BP1 and miR-34a genetic instability found in deadly neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-137.

Prenatal Array Comparative Genomic Hybridization in Fetuses With Structural Cardiac Anomalies.

To examine the diagnostic performance of array comparative genomic hybridization (CGH) for fetal cardiac anomalies in two medium-sized Canadian prenatal genetics clinics. We prospectively recruited 22 pregnant women with fetal structural cardiac anomalies, normal rapid aneuploidy detection, and FISH for 22q11.2 testing for array CGH analysis. One case had an 8p deletion that was also visible on karyotype and included the GATA4 gene, which has been associated with congenital heart disease. Two cases had inherited pathogenic copy number variants (CNVs) of variable expressivity and penetrance: one was a duplication of 16p11.2 and the other a deletion of 15q11.2. One case had the incidental finding of being a carrier of a recessive disease unrelated to the cardiac anomaly. Of these prospectively recruited cases of fetal cardiac anomalies, 14% had a pathogenic result on array CGH. Pathogenic CNVs of variable penetrance and expressivity were a significant proportion of the positive results identified. These CNVs are generally associated with neurodevelopmental issues and may or may not have been associated with the fetus' underlying congenital heart disease. Array CGH increases the diagnostic yield in this group of patients; however, certain CNVs remain a challenge for counselling in the prenatal setting.

Coordinated Tumor Suppression by Chromosome 8p

In this issue of Cancer Cell, Cai etal. use genome editing to study 8p deletions in a mammary epithelial cellmodel and show that 8p loss of heterozygosity (LOH) attenuates the action of several genes that collectively promote cell invasion and enhance cellular sensitivity to autophagy inhibitors.