Loss Of 5q Research Articles

Title: SNP Microarray Reveals Predicted Outcomes of a Novel High Risk AML Subgroup with ERG Amplification

Increased expression of ERG (ETS-related gene), a proto-oncogene within the ETS (erythroblast transformation specific) family, is associated with an unfavorable clinical outcome in AML patients. A mechanism for ERG over-expression is ERG amplification. ERG amplification is present in 4-6% of adult AML. This study investigates the genetic profile of ERG-amplified AML by assessment of genomic copy number changes and mutational status of AML-related genes and how this correlates to treatment response and overall outcome. Currently copy number analysis is not recommended by ELN or WHO as a diagnostic standard in AML. An institutional retrospective review of 205 adult AML cases from 2015 to 2018 provided two cohorts: a discovery cohort and control cohort. The discovery cohort includes 11 cases (4%) with ERG amplification. All cases in the discovery cohort include either TP53 mutation and/or loss/LOH of 17p. Thus, we developed a control cohort of 39 cases with TP53 abnormalities without ERG amplifications to determine the role TP53 plays in the observed outcomes. The 179 adult AML cases from the TCGA public database comprises the validation cohort, which incorporates both copy number and molecular sequencing data. Approximately 4% of AML cases in the TCGA dataset show ERG amplification. For our discovery and control cohorts, genomic DNA was extracted (Maxwell, Promega, Madison, WI) from fresh bone marrow aspirates. Copy number/LOH analysis was performed using the CytoSNP microarray (Illumina, San Diego, CA). Sequence variants were evaluated in hotspot regions of 49 genes using the ThunderBolts Myeloid Panel library preparation (RainDance Technologies, Billerica, MA) and paired-end sequencing (MiSeq, Illumina, San Diego, CA). Alignment and variant calling was performed with MiSeq Reporter (Illumina) and NextGene (Soft Genetics, State College, PA) and sequences were annotated using VarSeq (Golden Helix, Bozman, MT). SNP microarray analysis of the discovery cohort revealed a distinct pattern of complex cytogenetics including chromothripsis, TP53 loss/LOH and loss of 5q. Molecular sequencing was available for 10 of 11 patients. All specimens analyzed for mutational status demonstrated TP53 loss of function mutations. The majority of cases (9/11) demonstrated complete loss of function of TP53 by mutation in conjunction with a "second hit" in the form of loss due to deletion or LOH of 17p. These nine patients failed to achieve any response with chemotherapy with a median survival time of 3.8 months. The two surviving patients had a single TP53 mutation with an intact second allele. Both of these patients are post-allogeneic stem cell transplant with no evidence of relapse. There is an association of ERG amplification with MDS as 8 of the 11 patients transformed from very-high risk MDS. The interaction between ERG and TP53 allows one to predict treatment response and outcomes based on the presence or absence of an intact TP53 allele. Within the control group, there were no significant differences in survival or response to treatment based on number of intact TP53 alleles. The validation cohort of TCGA patients confirmed the strong positive correlation between ERG amplification and TP53 mutations. There were 8 cases with ERG amplification. Of these, 6 cases had a similar genetic pattern found in the discovery cohort including TP53 mutations, loss of 5q and complex cytogenetics. The median overall survival (OS) of this cohort of ERG amplified + TP53 mutation was 2.5 months. ERG amplification classifies a high-risk population of AML detected by copy number/LOH analysis with a distinct genetic signature including loss of 5q, complex cytogenetics including chromothripsis and loss of function TP53 mutation. The successive loss of the second TP53 allele in the setting of ERG amplification identifies a subset of patients with resistance to cytarabine and a very poor prognosis. These patients are unlikely to benefit from cytotoxic chemotherapy and may have improved outcomes with alternative regimens and/or clinical trial. ERG amplification is associated with AML that transforms from very-high risk MDS. Recognizing these patients before transforming to AML could allow for early transplant. This high-risk subgroup is currently un-recognized by standard cytogenetics and genetic mutation profiling; therefore, we recommend that copy number/LOH analysis become part of the standard diagnostic testing for MDS and AML. Disclosures No relevant conflicts of interest to declare.

Deletions of 5q Promote Genome Instability in TP53-Mutant MDS/AML

Purpose TP53 mutations in myeloid neoplasms (MDS/AML) are associated with high-risk disease, poor outcome, and complex karyotype. The molecular mechanisms which lead to global chromosomal instability remain poorly understood. Loss of 5q [del(5q)] is the most frequent cytogenetic abnormality associated with TP53 mutations suggesting that haploinsufficiency of genes on 5q contributes to chromosomal instability. Methods We reprogrammed MDS/AML patient samples to establish genetically accurate iPSC lines from preleukemic subclones. We generated iPSCs with TP53 mutations and del(5q), differentiated them to hematopoietic progenitors (HPCs), and determined the contribution of del(5q) to genome instability. Results By reprogramming MDS/AML complex karyotype patient samples, we identified iPSCs with heterozygous TP53 mutations (TP53-only), as well as iPSCs with TP53 mutations and del5(q22-q31) (TP53;del5q), and an otherwise normal karyotype. HPCs derived from TP53;del5q iPSCs had decreased multilineage differentiation potential compared to the TP53-only HPCs. Gene expression analysis of TP53;del5q HPCs revealed downregulation of genes involved in chromosome segregation and DNA damage repair. Following irradiation TP53;del5q cells had significantly delayed DNA damage repair kinetics. In order to evaluate the effects of TP53 and del(5q) on chromosomal segregation during stress, we arrested the cells in mitosis by disrupting the mitotic spindle and quantified the induction of micronuclei, a marker of chromosomal instability that occurs due to lagging chromosomes. TP53;del5q cells had an increased frequency of micronuclei formation compared to TP53-only cells. We also detected micronuclei in primary AML patient samples. Micronuclei in iPSC-HPCs and primary patient cells had disrupted nuclear envelope and DNA damage marked by y-H2AX. Conclusions Our reprogramming approach revealed that TP53 mutations are disease-initiating and frequently followed by 5q loss. We propose that del(5q) cooperates with mutant TP53 to promote genome instability via two distinct mechanisms: classical double-stranded break repair and micronuclei formation. The latter is associated with global chromosomal instability, aneuploidy, and chromothripsis. We propose that loss of 5q accelerates genome instability in TP53-mutant cells which over time impedes normal hematopoietic differentiation and leads to complex karyotype. Disclosures Becker: The France Foundation: Honoraria; Accordant Health Services/Caremark: Consultancy; AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding.

RARE-32. POLYMORPHOUS LOW-GRADE NEUROEPITHELIAL TUMOR OF THE YOUNG (PLNTY): GENETIC ANALYSIS CONFIRMS FREQUENT MAPK PATHWAY ACTIVATION

Abstract Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and genetic MAPK pathway activation through alterations such as BRAF V600E mutation, and FGFR2-KIAA1598/CTNNA3 and FGFR3-TACC3 fusions. We report the molecular profiling of PLNTY in 9 patients, 7 female: 2 male, median age at diagnosis of 16 years (range, 5–34). All tumors were supratentorial with diagnostic morphological features of PLNTY including oligodendroglioma-like areas and strong diffuse CD34 immunostaining. Four (of 9; 44%) tumors were positive for BRAF V600E immunostain, indicating the presence of a BRAF V600E mutation. The 6 cases evaluated for IDH status were negative by IDH1-R132H immunostain (n=5; 3 BRAF V600E-negative and 2 BRAF V600E-positive) or by NGS (n=1; BRAF V600E-negative). Oncoscan chromosomal microarray performed in 5 BRAF V600E-negative tumors showed recurrent copy number changes including gain of whole chromosomes 5, 7, 8, 9, 12, 18, 19, 20, 21 and X, and loss of chromosomes 1, 2, 10q, 13, 22 and Xq. The 10q losses (n=2) were highly suggestive of an FGFR2-KIAA1598 and of a potentially novel FGFR2 underlying fusion event in one case each. Custom targeted neuro-oncology DNA and RNA NGS panel performed in 2 cases revealed a fusion similar to fusions previously reported in pilocytic astrocytoma: one with a KIAA1549-BRAF (exon 15-exon 9) fusion associated with a 7q34 ~785 kilobase deletion rather than the characteristic ~2 megabase duplication seen in pilocytic astrocytoma, and another with a QKI-NTRK2 (exon 6-exon 15) fusion associated with a 9q21 ~191 kilobase duplication disrupting NTRK2. The KIAA1549-BRAF fusion-positive case also had a TP53 mutation with loss of whole chromosome 17, suggesting TP53 complete inactivation. This study confirms that PLNTY is a low-grade neuroepithelial tumor with frequent MAPK pathway activation and expands the spectrum of MAPK activating alterations observed in PLNTY.

P04.10 IDH-wildtype low grade gliomas: overall survival and prognostic indicators

Abstract BACKGROUND IDH-wildtype WHO grade II diffuse gliomas represent a rare subgroup of low grade tumors characterized by poor prognosis. The clinical and molecular profile associated with these tumors has not been fully elucidated yet, and the ongoing uncertainties regarding their biological behavior hamper to establish a standard of treatment. The aim of this study is to define the median overall survival and the main prognostic factors associated with this rare tumor entity. MATERIALS AND METHODS We performed a retrospective research in our center for all patients diagnosed with diffuse WHO grade II and III gliomas from 1976 to 2018. WHO grade II and III gliomas were divided into three molecular subgroups according to the IDH1/2 mutation and the 1p/19q codeletion status (1: IDH-mutant, 1p/19q codeleted; 2: IDH-mutant, 1p/19q non codeleted; 3: IDH-wildtype). We analyzed the clinical and molecular characteristics of the three subgroups, and then the clinical, radiological, histological and molecular features of IDH-wildtype WHO grade II gliomas. RESULTS We identified 445 patients with diffuse WHO grade II gliomas, including 59 IDH1/2-wildtype tumors. IDH-wildtype grade II gliomas affected more frequently male (75% vs. 55%, p = 0.004) and older (mean age: 50.0 vs. 39.6 years, p<0.0001) patients, had frequent fronto-temporo-insular location (41%) and commonly underwent biopsy (53%) rather than resection. We found TERT promoter mutations (18/42, 43%), chromosome 7q gains (12/30, 40%), chromosome 10q losses (12/44, 27%), chromosome 9p losses (7/47, 15%), EGFR amplifications (5/51, 10%) and p16 deletions (4/50, 8%) but no P53 (0/16) mutations. Median overall survival was 46 months (vs. 98 for IDH-mutant non codeleted and 175 for IDH-mutant codeleted WHO grade II gliomas (p<0.0001); vs. 20 months for IDH-wildtype WHO grade III gliomas (p = 0.001)). Survival was not significantly influenced by age, preoperative KPS, tumor location, extent of resection or adjuvant treatment schemes. Chromosome 9p loss had a strong negative impact on overall survival (p=0.002). CONCLUSION The median overall survival associated with IDH-wildtype WHO grade II gliomas does not exceed 4 years from diagnosis. As some genetic alterations seem to have a strong prognostic impact, an exhaustive genetic assessment can be helpful in this rare tumor group for purposes of prognostic stratification and treatment decision.

Reprogramming identifies functionally distinct stages of clonal evolution in myelodysplastic syndromes

AbstractMyeloid neoplasms, including myelodysplastic syndromes (MDS), are genetically heterogeneous disorders driven by clonal acquisition of somatic mutations in hematopoietic stem and progenitor cells (HPCs). The order of premalignant mutations and their impact on HPC self-renewal and differentiation remain poorly understood. We show that episomal reprogramming of MDS patient samples generates induced pluripotent stem cells from single premalignant cells with a partial complement of mutations, directly informing the temporal order of mutations in the individual patient. Reprogramming preferentially captured early subclones with fewer mutations, which were rare among single patient cells. To evaluate the functional impact of clonal evolution in individual patients, we differentiated isogenic MDS induced pluripotent stem cells harboring up to 4 successive clonal abnormalities recapitulating a progressive decrease in hematopoietic differentiation potential. SF3B1, in concert with epigenetic mutations, perturbed mitochondrial function leading to accumulation of damaged mitochondria during disease progression, resulting in apoptosis and ineffective erythropoiesis. Reprogramming also informed the order of premalignant mutations in patients with complex karyotype and identified 5q deletion as an early cytogenetic anomaly. The loss of chromosome 5q cooperated with TP53 mutations to perturb genome stability, promoting acquisition of structural and karyotypic abnormalities. Reprogramming thus enables molecular and functional interrogation of preleukemic clonal evolution, identifying mitochondrial function and chromosome stability as key pathways affected by acquisition of somatic mutations in MDS.

Abstract LB-300: The life history of breast cancer in Nigerian women: Evidence for ethnic differences in tumor evolution based on whole-genome sequencing

Abstract Breast cancer is a heterogeneous disease and the incidence to mortality ratio is highest for women of African ancestry. Paucity of data from non-European ancestry groups limits our understanding of the underlying etiological differences or alternative routes to progression that may explain differential outcomes. To examine the contribution of genomic differences, we performed high-depth whole-genome sequencing on 100 breast tumors (90X) and their paired normal samples (30X) from indigenous African women with breast cancer in Southwest Nigeria and performed comparative analysis with 84 TCGA whole genome sequences of breast tumor/normal pairs (46 White, 30 African American and 8 samples representing other ethnicities). High confidence somatic single nucleotide variants (SNVs) were obtained by using MuTect and Strelka. To analyse intra-tumoral heterogeneity (ITH), Battenberg and DPClust were used to call copy number aberrations (CNA) and cluster somatic SNVs based on cancer cell fraction (CCF) respectively. We find that in the HR-/HER2+ subtype, clonal losses of chromosome 14q are highly enriched in Nigerians (41%) but absent in the Whites even though the proportion of this subtype was comparable between the two groups (42% and 33% respectively). This is an interesting observation since not only 14q loss is known to be an event associated with breast cancer aggressiveness, but HR-/HER2+ has also been reported to be enriched within the relatively younger Nigerian patients with breast cancer. This may in part explain inter-ethnic disparity in survival. Also, somatic SNV clustering analysis showed that Nigerian cancers have a higher level of ITH than Whites, which may explain the pronounced aggressiveness of breast cancer in women of African ancestry. In contrast, early drivers (e.g. TP53 and PIK3CA) and whole genome duplication rates were mostly similar between the groups. Our observations suggest differences in the underlying evolutionary trajectories of breast cancer across ethnic backgrounds. These data underscore the need for larger and more in-depth studies of diverse cancer genomes and, if validated, may translate into clinical intervention opportunities tailored to women of African ancestry and accelerate progress in precision cancer care. Note: This abstract was not presented at the meeting. Citation Format: Naser Ansari-Pour, Jason J. Pitt, Stefan Dentro, Dominic Fitzgerald, Yonglan Zheng, Toshio F. Yoshimatsu, Padma Rajagopal, Andreas Gruber, Ayodele Sanni, Olayiwola Oluwasola, Mustapha Ajani, Abayomi Odetunde, Abiodun Popoola, Adeyinka Falusi, Temidayo Ogundiran, John Obafunwa, Oladosu Ojengbede, Nasiru Ibrahim, Kevin P. White, Dezheng Huo, Peter Van Loo, David C. Wedge, Olufunmilayo Olopade. The life history of breast cancer in Nigerian women: Evidence for ethnic differences in tumor evolution based on whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-300.

Micropapillary variant of mucinous carcinoma of the breast shows genetic alterations intermediate between those of mucinous carcinoma and micropapillary carcinoma.

Micropapillary variant of mucinous carcinoma of the breast (MPMC) is a rare histological form of oestrogen receptor (ER)-positive invasive carcinoma that is characterised by micropapillary clusters of tumour cells in lakes of extracellular mucin. The aims of this study were to determine the genetic alterations underpinning MPMCs, and to determine whether they overlap with those of mucinous carcinomas and/or invasive micropapillary carcinomas. DNA from five MPMCs was subjected to whole-exome sequencing. Somatic mutations, copy number alterations and mutational signatures were determined with state-of-the-art bioinformatics methods. No mutations in genes significantly mutated in breast cancer, including TP53, PIK3CA, GATA3, and MAP3K1, were detected. We identified copy number alterations that have been reported in invasive micropapillary carcinomas, such as recurrent gains in 1q, 6p, 8q, and 10q, and recurrent losses in 16q, 11q, and 13q, as well as a recurrent 8p12-8p11.2 amplification encompassing FGFR1. Like mucinous carcinomas, three of the five MPMCs analysed lacked PIK3CA mutations, 1q gains, and 16q losses, which are the hallmark genetic alterations of ER-positive breast cancers, whereas two MPMCs harboured 16q losses and/or a complex pattern of copy number alterations similar to those found in breast-invasive micropapillary carcinomas. MPMCs are heterogeneous at the genetic level; some tumours show a pattern of somatic genetic alterations similar to those of mucinous carcinomas, whereas others resemble invasive micropapillary carcinomas at the genetic level. These findings suggest that MPMCs may not constitute one histological subtype, but rather a convergent phenotype that can stem from mucinous carcinomas or invasive micropapillary carcinomas.

Subgroup-specific prognostic signaling and metabolic pathways in pediatric medulloblastoma

BackgroundUsing a pathway-focused approach, we aimed to provide a subgroup-specific basis for finding novel therapeutic strategies and further refinement of the risk stratification in pediatric medulloblastoma.MethodBased on genome-wide Cox regression and Gene Set Enrichment Analysis, we investigated prognosis-related signaling pathways and core genes in pediatric medulloblastoma subgroups using 530 patient data from Medulloblastoma Advanced Genomic International Consortium (MAGIC) project. We further examined the relationship between expression of the prognostic core genes and frequent chromosome aberrations using broad range copy number change data.ResultsIn SHH subgroup, relatively high expression of the core genes involved in p53, PLK1, FOXM1, and Aurora B signaling pathways are associated with poor prognosis, and their average expression synergistically increases with co-occurrence of losses of 17p, 14q, or 10q, or gain of 17q. In Group 3, in addition to high MYC expression, relatively elevated expression of PDGFRA, IGF1R, and FGF2 and their downstream genes in PI3K/AKT and MAPK/ERK pathways are related to poor survival outcome, and their average expression is increased with the presence of isochromosome 17q [i(17q)] and synergistically down-regulated with simultaneous losses of 16p, 8q, or 4q. In Group 4, up-regulation of the genes encoding various immune receptors and those involved in NOTCH, NF-κB, PI3K/AKT, or RHOA signaling pathways are associated with worse prognosis. Additionally, the expressions of Notch genes correlate with those of the prognostic immune receptors. Besides the Group 4 patients with previously known prognostic aberration, loss of chromosome 11, those with loss of 8q but without i(17q) show excellent survival outcomes and low average expression of the prognostic core genes whereas those harboring 10q loss, 1q gain, or 12q gain accompanied by i(17q) show bad outcomes. Finally, several metabolic pathways known to be reprogrammed in cancer cells are detected as prognostic pathways including glutamate metabolism in SHH subgroup, pentose phosphate pathway and TCA cycle in Group 3, and folate-mediated one carbon-metabolism in Group 4.ConclusionsThe results underscore several subgroup-specific pathways for potential therapeutic interventions: SHH-GLI-FOXM1 pathway in SHH subgroup, receptor tyrosine kinases and their downstream pathways in Group 3, and immune and inflammatory pathways in Group 4.

Unraveling the molecular profile underpinning pancreatic tropisms in metastatic clear cell renal cell carcinoma.

e16096 Background: The tropism of cancer metastases is poorly understood yet holds prognostic value. Clear cell renal cell carcinoma (ccRCC) exhibits a broad pattern of metastases, making it an optimal model to study organotropism. Notably, when ccRCC metastasizes to the pancreas (PM) independently of other sites, it is associated with favorable outcomes in patients for unclear reasons. Here, we comprehensively analyzed the clinical and molecular profile of patients with PM. Methods: RCC patients with PM from UTSW and Cleveland Clinic were identified. Clinicopathologic data and oncologic outcomes were analyzed. Whole exome sequencing (WES), RNAseq, and histologic assessment of primary and metastatic tumors from PM patients were conducted. Results: 31 RCC patients with PM were identified. We observed remarkably favorable outcomes in our PM cohort, with a median overall survival (OS) of 10.7 years from metastatic diagnosis and a long latency between initial diagnosis and development of metastasis (median 69 months in patients who were non-metastatic at diagnosis). OS was independent of both metastatic tumor burden and known IMDC prognostic factors. We discovered that tumors from PM patients were markedly uniform and clustered together by gene expression analysis. WES and DNA copy number analyses revealed a high frequency of VHL and PBRM1 mutations, 3p loss, and 5q amplification, along with a lower frequency of 9p, 14q and 4q losses and BAP1 mutations, characteristic of indolent ccRCC. Furthermore, the genomic and histologic features of tumors from patients with PM can be recapitulated in patient-derived xenograft models. Conclusions: To our knowledge, this is the first report to unravel molecular determinants of organotropism, and we highlight that organotropism can be an independent prognostic factor. Understanding tumor heterogeneity may help refine prognostic models for metastatic RCC and hold implications for improved personalization of therapy.

Genome-wide methylation profiling and copy number analysis in atypical fibroxanthomas and pleomorphic dermal sarcomas indicate a similar molecular phenotype

BackgroundAtypical fibroxanthomas (AFX) and pleomorphic dermal sarcomas (PDS) are lesions of the skin with overlapping histologic features and unspecific molecular traits. PDS behaves aggressive compared to AFX. Thus, a precise delineation, although challenging in some instances, is relevant.MethodsWe examined the value of DNA-methylation profiling and copy number analysis for separating these tumors. DNA-methylation data were generated from 17 AFX and 15 PDS using the Illumina EPIC array. These were compared with DNA-methylation data generated from 196 tumors encompassing potential histologic mimics like cutaneous squamous carcinomas (cSCC; n = 19), basal cell carcinomas (n = 10), melanoma metastases originating from the skin (n = 11), leiomyosarcomas (n = 11), angiosarcomas of the skin and soft tissue (n = 11), malignant peripheral nerve sheath tumors (n = 19), dermatofibrosarcomas protuberans (n = 13), extraskeletal myxoid chondrosarcomas (n = 9), myxoid liposarcomas (n = 14), schwannomas (n = 10), neurofibromas (n = 21), alveolar (n = 19) and embryonal (n = 17) rhabdomyosarcomas as well as undifferentiated pleomorphic sarcomas (n = 12).ResultsDNA-methylation profiling did not separate AFX from PDS. The DNA-methylation profiles of the other cases, however, were distinct from AFX/PDS. They reliably assigned to subtype-specific DNA-methylation clusters, although overlap occurred between some AFX/PDS and cSCC. Copy number profiling revealed alterations in a similar frequency and distribution between AFX and PDS. They involved losses of 9p (22/32) and 13q (25/32). Gains frequently involved 8q (8/32). Notably, a homozygous deletion of CDKN2A was more frequent in PDS (6/15) than in AFX (2/17), whereas amplifications were non-recurrent and overall rare (5/32).ConclusionsOur findings support the concept that AFX and PDS belong to a common tumor spectrum. We could demonstrate the diagnostic value of DNA-methylation profiling to delineating AFX/PDS from potential mimics. However, the assessment of certain histologic features remains crucial for separating PDS from AFX.

Genomic dissection of gastrointestinal and lung neuroendocrine neoplasm.

ObjectiveNeuroendocrine neoplasms (NENs) are relatively rare and heterogeneous malignancies with two major subtypes: low-grade neuroendocrine tumor (NET) and high-grade neuroendocrine carcinoma (NEC). Comprehensive molecular characterization of NENs is needed to refine our understanding of the biological underpinnings of different NEN subtypes and to predict disease progression more accurately.MethodsWe performed whole-exome sequencing (WES) of NEN samples from 49 patients (25 NETs and 24 NECs) arising from the stomach, intestines or lung. Clinicopathologic features were assessed and associated with molecular events.ResultsNENs generally harbor a low mutation burden, with TP53 being the top mutated gene found in 31% of patients. Consistent with other studies, p53 signaling pathway dysfunction is significantly enriched in NECs compared to NETs (P<0.01). Other thanTP53, tissue type-specific mutation profiles of NENs were observed in our cohort compared to those reported in pancreatic NETs. Importantly, we observed significant genomic instability, with increased copy number alterations observed across the NEN genome, which was more profound in NECs and independently correlated with poor overall survival (OS) (P<0.001). NECs could be further stratified into two molecular subtypes based on OS (P<0.001) and the chromosomal instability score (CIS). Interestingly, we discovered that the gain of whole chromosome 5 occurred at the early stage of NEN development, followed by the loss of 5q exclusively in NECs (P<0.001). ConclusionsThese findings provide novel insights into the molecular characteristics of NENs and highlight the association of genomic stability with clinical outcomes.

Role of columnar cell lesions in breast carcinogenesis: analysis of chromosome 16 copy number changes by multiplex ligation-dependent probe amplification

Columnar cell lesions have been proposed as precursor lesions of low-grade breast cancer. The molecular characteristic of low-grade breast neoplasia is whole-arm loss of chromosome 16q. Copy number changes of 6 genes on 16p and 20 genes on 16q were analysed by multiplex ligation-dependent probe amplification in 165 lesions of 103 patients. Twenty-three columnar cell lesions and 19 atypical ducal hyperplasia lesions arising in columnar cell lesions were included, as well as cases of usual ductal hyperplasia, blunt duct adenosis, ductal carcinoma in situ, lobular neoplasia and invasive carcinoma. Usual ductal hyperplasia and blunt duct adenosis lacked whole-arm losses of 16q. In contrast, columnar cell lesions without atypia, columnar cell lesions with atypia, atypical ductal hyperplasia, low-grade ductal carcinoma in situ and low-grade invasive carcinomas increasingly harboured whole-arm losses of 16q (17%, 27%, 47% and 57%, respectively). However, no recurrent losses in specific genes could be identified. In several patients, columnar cell lesions and atypical ductal hyperplasia harboured similar losses as related ductal carcinoma in situ or invasive carcinomas within the same breast. There were indications for 16q breakpoints near the centromere. Whole-arm gains on 16p were relatively scarce and there was no relation between whole-arm gains of 16p and progression of lesions of the low-grade breast neoplasia family. In conclusion, columnar cell lesions (with and without atypia) often harbour whole-arm losses of 16q, which underlines their role as precursors in low-grade breast carcinogenesis, in contrast with usual ductal hyperplasia and blunt duct adenosis. However, no recurrent losses in specific genes could be identified, pointing to minor events in multiple tumour suppressor genes rather than major events in a single 16q gene contributing to low-grade breast carcinogenesis.

Unraveling the Landscape of Copy Number Aberrations in Hodgkin Lymphoma: A Joint KU Leuven and Lysa Study on Circulating Cell Free DNA

Introduction. For decades, the study of genomic alterations in Hodgkin lymphoma (HL) has been hampered by the low abundance of the malignant Hodgkin Reed-Sternberg (HRS) cells in HL lymph node biopsies. Laser microdissection or flow cytometric cell sorting allow the study of purified HRS cells, but the use of these applications is restricted to specialized research centers. We have recently demonstrated proof-of-principle that copy number aberrations (CNA) in HRS cells can be retrieved in circulating cell-free DNA (cfDNA) of HL patients (Vandenberghe et al., Lancet Haematol. 2015). Taking advantage of the presence of HRS cell-derived DNA (ctDNA) in plasma, the aim of this study was to catalogue CNA in HL in a large series of prospectively recruited HL patients.Methods. We analyzed plasma collected from 177 patients (median age 29, range 3-86) with newly diagnosed HL. 60 cases were diagnosed at our institution, including all disease subtypes and stages, with a majority of nodular sclerosis. 118 patients were recruited in the context of the BREACH study, a multicentric Phase 2B study for unfavorable early classical HL (NCT02292979). After cfDNA extraction, samples were low-pass sequenced (0.1x coverage) and analyzed using ichorCNA, an algorithm that produces read depth-based log2 CNA profiles and quantifies the cfDNA tumor content.Results. At diagnosis, 86 % (152/177) of patients showed obvious genomic imbalances in cfDNA, in early-stage (85 % (131/155)) as well as in advanced cases (95 % (21/22)). Among the abnormal profiles, gains encompassing 2p16, 5p15, 9p24, 12q13 and 19q13 were the most frequent aberrations, occurring in 79 %, 53 %, 57 %, 63 % and 80 % respectively. Losses most frequently affected regions 4q34, 6q23, 11q22 and 13q13, in 53 %, 63 %, 49 % and 59 % of abnormal profiles respectively (fig. 1A). All these CNAs have been previously described with varying frequencies in smaller series of 10-53 patients, using arrayCGH or whole exome sequencing on microdissected HRS cells or even HRS cell-derived cell lines as input material. The observed pattern of CNAs is distinct from the pattern we observed in cfDNA obtained from other hematological malignances, e.g. multiple myeloma and diffuse large B-cell lymphoma (fig. 1B, C).Analysis of follow-up cfDNA samples revealed that the majority of patients rapidly clear their profiles during the first two treatment cycles. Of 123 samples analysed at d15 of cycle 1, 89 samples (72 %) showed a normal profile without CNAs.This suggests a relationship between the fraction of ctDNA in cfDNA and the disease burden. However, we could not find a correlation between the ctDNA fraction as calculated by ichorCNA and the metabolic tumor volume as computed from PET/CT scans. This could be due to several factors, e.g. the small fraction of HRS cells in the metabolic tumor volume. We are currently investigating whether a correlation does exist between the HRS content in the lymph node biopsy and the ctDNA load.Conclusions. In this largest study of CNA in HL to date, we provide a comprehensive catalogue of the types of CNAs, as well as their frequencies and patterns in HL. In this series of 177 patients, gains affecting 2p, 12q and 19q and loss of 6q and 13q emerge as the most commonly recurrent CNAs in HL, across all HL subtypes and stages. The data confirm and extend our previous findings that the majority of HL patients, including those with early-stage disease, have detectable CNAs in their cfDNA at diagnosis. We demonstrate a broad spectrum of aberrations, gains and losses, some of which recur at higher frequencies than gains of 9p24, harboring PD-L1. This warrants further studies on how these CNAs are implicated in the pathogenesis of HL. It further endorses the use of ctDNA as an alternative gateway to the genome of HRS cells, and as a substrate for the evaluation of early disease response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genomic Analysis of Myelodysplastic Syndromes Among Nagasaki Atomic Bomb Survivors

Exposure to ionizing radiation is a well-established risk of cancer development. Atomic bomb (A-bomb) survivors were exposed to A-bomb radiation, then developed acute myeloid leukemia (AML), acute lymphoblastic leukemia, and chronic myeloid leukemia with the highest incidence around five to ten years after the bombing. On the other hand, the risk of myelodysplastic syndromes (MDS) were significantly high among the survivors even after 40 to 60 years from the bombing (Iwanaga et al. J Clin Oncol 2011), indicating a long-lasting influence of A-bomb radiation on human hematopoiesis. In general, genetic mutations are found in almost all MDS, however, the landscape of genetic alterations in MDS among A-bomb survivors remains to be elucidated.To study the role of A-bomb radiation on the pathogenesis of MDS and related conditions, we analyzed 32 patients with MDS and 3 cases diagnosed as idiopathic cytopenia of undetermined significance (ICUS) among A-bomb survivors using next generation sequencing technologies and SNP array karyotyping. Patients were categorized into two groups by the exposure distance; proximally exposed group (PE-group, 18 patients, < 2.7 km from the hypocenter) and distally exposed group (DE-group, 17 patients, directly exposed in 2.7-10 km, or those who entered the bombing area [inside of 2 km from the hypocenter] within two weeks after the bombing [no acute radiation syndromes]). Median exposure distance was 1.1 and 3.4 km in PE- and DE-group, respectively (P < .001). There was no significant difference in sex, subtype of MDS, and ages at bombing and the diagnosis, between the two groups, whereas frequencies of abnormal karyotype (78% vs. 59%, P = .29) and complex karyotype (33% vs. 12%, P = .23) were higher in PE-group without statistical significance.Whole genome sequencing of three patients in PE-group revealed that most frequent nucleotide substitution was cytosine-to-thymine (C to T), which is a hallmark of age-related mutational changes. Using whole exome sequencing and 154 genes-targeted capture sequencing (T-S), we identified significant differences in mutated genes between the two groups. No patients of PE-group had mutations in TET2 although it was most frequently affected in DE-group (29% [5 out of 17 patients], P = .019), and the frequency of gene mutations in DNA methylation pathway was 5.6% (1 out of 18 patients) and 41% (7 out of 17) in PE- and DE-group, respectively (P = .018). TP53 and the genes coding RNA splicing factors (SFs) were mutated equally in both groups (TP53, 11% and 12% [P = 1]; SFs, 33% and 35% [P = 1] in PE- and DE-group, respectively).Copy number alterations were analyzed using SNP-array and T-S data. We found that loss of long arm of chromosome 11 (11q loss) was significantly accumulated in PE-group (33% [6 out of 18 patients] vs. 0%, P = .019), whereas loss of chromosome 5q and chromosome 7 were almost equally identified in both groups (chromosome 5q, 22% and 12% [P = .66]; chromosome 7, 22% and 29% [P = .71] in PE- and DE-group, respectively). On the commonly affected region of 11q in this study, there were three genes recurrently altered in MDS and AML; ATM, KMT2A, and CBL. Copy number loss was found in these three genes, and the alteration of ATM was significantly increased in PE-group (28% [5 out of 18 patients] vs. 0%, P = .046) than DE-group. The remaining allele of ATM also possessed a mutation in two out of five patients in PE-group, indicating a deleterious effect on the function of ATM.In summary, we revealed two unique genetic features in MDS among A-bomb survivors who were proximally exposed to A-bomb radiation; significantly less frequent mutations in DNA methylation pathway and significant accumulation of 11q loss, suggesting a long-lasting effect of ionizing radiation and an important role of 11q loss in the initiation of MDS. DisclosuresNo relevant conflicts of interest to declare.

Genomic Analysis of Ibrutinib Resistance in Waldenstrom Macroglobulinemia

Introduction: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is the first approved therapy for Waldenström's macroglobulinemia (WM), and is highly active in both treatment-naive and relapsing or refractory patients. Although ibrutinib is highly active in WM patients, disease progression can occur. Acquired mutations in BTK at the binding site of ibrutinib (Cys481), or in the protein immediately downstream of BTK, the phospholipase PLCG2, have been identified in half of progressing WM patients on ibrutinib (Xu et al, Blood 2017). However, not all ibrutinib resistant patients harbor these alterations, suggesting that there are other causes of disease progression on ibrutinib. The aim of this study was to identify alternative molecular mechanisms that can drive ibrutinib resistance.Patients and Methods: Five WM patients who progressed while on ibrutinib were studied. Tumor DNA samples at diagnosis, relapse, and germline DNA were available in three patients. For the remaining two, relapse and germline samples were sequenced. DNA was extracted from CD19-selected bone marrow mononuclear cells from patients. Non-CD19 cells from peripheral blood were used as germline controls. Samples were sequenced using whole exome sequencing. Data were analyzed following the Broad Institute's GATK Best Practice Guidelines. Small variants were analyzed using Strelka and MuTect2. Somatic structural variants were detected using Manta, and copy number alterations were called using Control-FREEC.Results: Copy number analysis identified deletions in chromosome 6q in all patients, becoming homozygous in two of them at relapse. In another patient, the homozygous deletion was already present at baseline in a third of the tumor population, and increased at relapse. No other recurrent copy number alterations were detected, though in two patients multiple deletions or gains involving large chromosomal regions were observed. Regarding small variants, relapse samples showed a high proportion of acquired mutations detected at relapse only (median 85%, range 79%-88%) compared to persistent mutations detected at both baseline and relapse (median 15%, range 12%-21%). Three out of the five patients harbored mutations in BTK (two with p.Cys481Ser in the kinase domain and the other a p.Thr62Asn in the PH domain). In patients with a BTK mutation, other alterations were observed in genes related to the B-cell receptor pathway including PLCG2 p.Y495H; CD79B p.D33Y; LYN p.A2Stop and LYN p.A139T.In patients without BTK mutations, we identified several mutations with a putative role in ibrutinib resistance that emerged at relapse including AIP4, an E3 protein-ubiquitin ligase whose substrates are CXCR4, LYN or SYK, in which a recurring p.A646S mutation was observed in two patients; RNF19B, an E3 protein-ubiquitin ligase involved in the cytolytic activity of natural killer cells and cytotoxic T-cells, in which a p.R30G mutation was observed in two patients; FCRL3, an Fc receptor-like 3 that differentially modulates innate immune signaling in B cells, in which a p.E694Q was observed in one patient; Mutations in the negative regulators of Toll-like receptors signaling were observed that included DOK2 p.Y345Stop, and TOLLIP p.M242R and p.R228H which were each observed once in separate patients. Mutant allele frequency clustering analysis by k-means reflected a linear pattern of evolution from baseline to relapse, in which most persistent alterations maintained the same allele frequency (median 18%), and acquired mutations were present in a small proportion of tumor cells (median 9.3%, p<0.01).Conclusions: Our findings depict uniform deletion of 6q, including homozygous loss of 6q, which encompass key regulators of BTK, BCL2, and NFKB; as well as emergence of novel gene mutations, including recurring mutations in E3 ubiquitin ligases, innate immune signaling, and Toll receptor/MYD88 pathway regulators as significant genomic alterations that accompany disease progression on ibrutinib in WM patients. DisclosuresCastillo:Genentech: Consultancy; Beigene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Consultancy, Research Funding. Treon:Johnson & Johnson: Consultancy; BMS: Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Hunter:Pharmacyclics: Consultancy.

Correlation between genomic index lesions and mpMRI and 68Ga-PSMA-PET/CT imaging features in primary prostate cancer

Magnetic resonance imaging (MRI) and prostate specific membrane antigen (PSMA)- positron emission tomography (PET)/computed tomography (CT)-imaging of prostate cancer (PCa) are emerging techniques to assess the presence of significant disease and tumor progression. It is not known, however, whether and to what extent lesions detected by these imaging techniques correlate with genomic features of PCa. The aim of this study was therefore to define a genomic index lesion based on chromosomal copy number alterations (CNAs) as marker for tumor aggressiveness in prostate biopsies in direct correlation to multiparametric (mp) MRI and 68Ga-PSMA-PET/CT imaging features. CNA profiles of 46 biopsies from five consecutive patients with clinically high-risk PCa were obtained from radiologically suspicious and unsuspicious areas. All patients underwent mpMRI, MRI/TRUS-fusion biopsy, 68Ga-PSMA-PET/CT and a radical prostatectomy. CNAs were directly correlated to imaging features and radiogenomic analyses were performed. Highly significant CNAs (≥10 Mbp) were found in 22 of 46 biopsies. Chromosome 8p, 13q and 5q losses were the most common findings. There was an strong correspondence between the radiologic and the genomic index lesions. The radiogenomic analyses suggest the feasibility of developing radiologic signatures that can distinguish between genomically more or less aggressive lesions. In conclusion, imaging features of mpMRI and 68Ga-PSMA-PET/CT can guide to the genomically most aggressive lesion of a PCa. Radiogenomics may help to better differentiate between indolent and aggressive PCa in the future.

Childhood supratentorial ependymomas with YAP1-MAMLD1 fusion: an entity with characteristic clinical, radiological, cytogenetic and histopathological features.

Ependymoma with YAP1‐MAMLD1 fusion is a rare, recently described supratentorial neoplasm of childhood, with few cases published so far. We report on 15 pediatric patients with ependymomas carrying YAP1‐MAMLD1 fusions, with their characteristic histopathology, immunophenotype and molecular/cytogenetic, radiological and clinical features. The YAP1‐MAMLD1 fusion was documented by RT‐PCR/Sanger sequencing, and tumor genomes were studied by molecular inversion probe (MIP) analysis. Significant copy number alterations were identified by GISTIC (Genomic Identification of Significant Targets in Cancer) analysis. All cases showed similar histopathological features including areas of high cellularity, presence of perivascular pseudo‐rosettes, small to medium‐sized nuclei with characteristic granular chromatin and strikingly abundant cells with dot‐like cytoplasmic expression of epithelial membrane antigen. Eleven cases presented features of anaplasia, corresponding to WHO grade III. MRI showed large supratentorial multinodular tumors with cystic components, heterogeneous contrast enhancement, located in the ventricular or periventricular region. One of two variants of YAP1‐MAMLD1 fusions was detected in all cases. The MIP genome profiles showed balanced profiles, with focal alterations of the YAP1 locus at 11q22.1–11q21.2 (7/14), MAMLD1 locus (Xp28) (10/14) and losses of chromosome arm 22q (5/14). Most patients were female (13/15) and younger than 3 years at diagnosis (12/15; median age, 8.2 months). Apart from one patient who died during surgery, all patients are alive without evidence of disease progression after receiving different treatment protocols, three without postoperative further treatment (median follow‐up, 4.84 years). In this to date, largest series of ependymomas with YAP1‐MAMLD1 fusions we show that they harbor characteristic histopathological, cytogenetic and imaging features, occur mostly in young girls under 3 years and are associated with good outcome. Therefore, this genetically defined neoplasm should be considered a distinct disease entity. The diagnosis should be confirmed by demonstration of the specific fusion. Further studies on large collaborative series are warranted to confirm our findings.

Seminoma component of mixed testicular germ cell tumor shows a higher incidence of loss of heterozygosity than pure-type seminoma

Using analysis of allelic loss (loss of heterozygosity [LOH]), we previously reported a putative progression pathway from germ cell neoplasia in situ (GCNIS) to seminoma and then to embryonal carcinoma in mixed-type testicular germ cell tumors. To identify the genetic backgrounds related to the progression of nonseminomatous germ cell tumor, patterns of LOH were studied in seminoma components in mixed tumors (18 cases), pure seminomas (20 cases), and coexisting GCNIS lesions. Each tumor was assessed for LOH at 22 polymorphic loci located on 12 chromosomal arms: 3q, 5q, 6p, 9p, 10q, 11p, 12p, 12q, 13q, 17p, 17q, and 18q. For all informative loci, the frequency of LOH in seminoma components in mixed tumors was significantly higher than that in pure seminomas (32% [96/302 loci] versus 19% [60/323 loci], P < .0001). The frequency of LOH in GCNIS lesions was not significantly different between the 2 tumor groups. The frequencies of LOH at chromosomes 6p and 10q were significantly higher in seminoma components in mixed tumors than in pure seminomas (P = .020 and P = .0041, respectively). Immunohistochemical analysis demonstrated a close association between the allelic status of the 10q23 locus and levels of phosphatase and tensin homolog deleted from chromosome 10 protein expression in seminoma (P = .00051). These data indicate that the seminoma, which has a potential to progress to nonseminomatous germ cell tumor, already exhibits several genetic changes including allelic losses of 6p and 10q, unlike pure seminoma.

Abstract B058: Genetic contribution of MYC to the development of primary prostate cancer

Abstract Introduction: The single best predictor of prostate cancer (PCa) outcome is Gleason score (Gs); however, the molecular characteristics that underpin specific Gleason patterns (Gp) and the biology associated with the aggressiveness of higher grades remain understudied. In a series of Gs 7 cancers, we recently observed increased activation of MYC and increased MYC protein levels in Gp4 prostate foci as compared to adjacent Gp3. While MYC is often associated with proliferative potential in other cancer types, subsets of prostate cancers show dramatically elevated MYC levels despite no increase in proliferation. These data suggest that increased MYC activity may drive Gp3 progression to Gp4 in a subset of cancers. Thus, the objective of this study is to examine the genetic contribution of MYC to locally advanced PCa development. Approach: To test the hypothesis that increased MYC activity drives the progression of Gp3 to Gp4 in a subset of cases, we screened a new series of Gs7 tumors in which the Gp3 and Gp4 components are adjacent and represent contributing elements of the index lesion. Guided by anti-MYC immunohistochemistry, tumor foci were collected by laser capture microdissection based on MYC staining, focusing in particular where MYC staining was markedly increased in Gp4 versus Gp3. We then copurified DNA and RNA; whole-exome sequencing and transcriptome profiling were performed. To further guide our bioinformatic analyses, we separately generated two prostate cell culture models with altered MYC levels: RWPE-1 cells to overexpress MYC and LNCAP cells to knockdown MYC. Total RNA was extracted and whole-transcriptome sequencing was used to assess differential gene expression. Results: Preliminary results from our first case suggest distinct histology and biology based on MYC tumor status from whole-exome sequencing analyses. Using somatic mutations and somatic copy number alterations, we observed a clonal relationship between two MYC-positive foci: a region of intraductal carcinoma and MYC-positive Gp4 adenocarcinoma. These regions shared identical single copy arm-level losses in chromosomes 6q and 8p, single-copy arm-level gains of 8q, 9p, and 9q, and 14 high-confidence point mutations that were not observed in the MYC-negative Gp3 component, suggesting that MYC activation may have driven evolution of the Gp4 component. When we cross-referenced differentially expressed genes between the Gp3 and Gp4 foci with our engineered cell lines and performed gene set enrichment analysis, we found recurrent enrichment for a JAK2 gene set, correlating with MYC activity. Conclusions: Our results demonstrate the feasibility of performing laser capture microdissection and integrated genomic analyses on tissue with differential MYC status. Although further cases are needed to determine the frequency at which JAK/STAT signaling drives MYC-driven prostate cancer, our improved understanding of its biology may lend itself to new strategies for managing newly diagnosed patients or delaying disease progression. Citation Format: Nichelle C. Whitlock, Rayann Atway, Qi Yang, Huihui Ye, Adam Sowalsky. Genetic contribution of MYC to the development of primary prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B058.

Combined somatic mutation and copy number analysis in the survival of familial CLL.

Recurrent large-scale somatic copy number alterations (SCNAs), and somatic point mutations can be analysed to stratify patients with chronic lymphocytic leukaemia (CLL) into distinct prognostic groups. To investigate the relationship between SCNAs and somatic mutations, we performed whole-exome sequencing and single nucleotide polymorphism microarray analyses on 98 CLL patients from 40 families with a high burden of CLL. Overall, 69 somatic mutations in 29 CLL driver genes were detected among 45 subjects (46%), with the most frequently mutated genes being TP53 (8·2%), NOTCH1 (8·2%) and ATM (5·1%). Additionally, 142 SCNAs from 54 subjects (57%) were detected, including losses of chromosome 13q14 (28·9%), 11q (5·6%), 17p (2·1%), and gain of chromosome 12 (4·2%). We found that patients having both an adverse point mutation in a CLL driver gene and an unfavourable SCNA tended to have poorer survival (Hazard ratio [HR]=3·17, 95% confidence interval [CI]=0·97-10·35; P=0·056) than patients having either a point mutation (HR=1·34, 95%CI=0·66-2·71; P=0·42) or SCNAs (HR=2·65, 95%CI=0·77-9·13; P=0·12). TP53 mutation carriers were associated with the poorest overall survival (HR=4·39, 95%CI=1·28-15·04; P=0·018). Our study suggests that combining SCNA and mutational data could contribute to predicting outcome in familial CLL.