1p Loss Research Articles

Genomic analysis reveals secondary glioblastoma after radiotherapy in a subset of recurrent medulloblastomas.

Despite great advances in understanding of molecular pathogenesis and achievement of a high cure rate in medulloblastoma, recurrent medulloblastomas are still dismal. Additionally, misidentification of secondary malignancies due to histological ambiguity leads to misdiagnosis and eventually to inappropriate treatment. Nevertheless, the genomic characteristics of recurrent medulloblastomas are poorly understood, largely due to a lack of matched primary and recurrent tumor tissues. We performed a genomic analysis of recurrent tumors from 17 pediatric medulloblastoma patients. Whole transcriptome sequencing revealed that a subset of recurrent tumors initially diagnosed as locally recurrent medulloblastomas are secondary glioblastomas after radiotherapy, showing high similarity to the non-G-CIMP proneural subtype of glioblastoma. Further analysis, including whole exome sequencing, revealed missense mutations or complex gene fusion events in PDGFRA with augmented expression in the secondary glioblastomas after radiotherapy, implicating PDGFRA as a putative driver in the development of secondary glioblastomas after treatment exposure. This result provides insight into the possible application of PDGFRA-targeted therapy in these second malignancies. Furthermore, genomic alterations of TP53 including 17p loss or germline/somatic mutations were also found in most of the secondary glioblastomas after radiotherapy, indicating a crucial role of TP53 alteration in the process. On the other hand, analysis of recurrent medulloblastomas revealed that the most prevalent alterations are the loss of 17p region including TP53 and gain of 7q region containing EZH2 which already exist in primary tumors. The 7q gain events are frequently accompanied by high expression levels of EZH2 in both primary and recurrent medulloblastomas, which provides a clue to a new therapeutic target to prevent recurrence. Considering the fact that it is often challenging to differentiate between recurrent medulloblastomas and secondary glioblastomas after radiotherapy, our findings have major clinical implications both for correct diagnosis and for potential therapeutic interventions in these devastating diseases.

PD46-09 COMPREHENSIVE GENETIC ANALYSIS OF TFE3-OVEREXPRESSED RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2018PD46-09 COMPREHENSIVE GENETIC ANALYSIS OF TFE3-OVEREXPRESSED RENAL CELL CARCINOMA Chang Wook Jeong, Jae So Cho, Moses Lee, Murim Choi, Cheryn Song, Ja Hyeon Ku, Cheol Kwak, and Hyeon Hoe Kim Chang Wook JeongChang Wook Jeong More articles by this author , Jae So ChoJae So Cho More articles by this author , Moses LeeMoses Lee More articles by this author , Murim ChoiMurim Choi More articles by this author , Cheryn SongCheryn Song More articles by this author , Ja Hyeon KuJa Hyeon Ku More articles by this author , Cheol KwakCheol Kwak More articles by this author , and Hyeon Hoe KimHyeon Hoe Kim More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2157AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES MiT family translocation renal cell carcinoma (RCC) is very rare variant which has translocation of Xp11.2/TFE3 gene or t(6;11)/TFEB gene. Xp11.2 translocation RCC is predominantly reported in younger age and has aggressive features. However, its pathophysiology or genetic characteristics are rarely understood. The aim of the study is to describe the comprehensive genetic characteristics of TFE3-positive RCC diagnosed by immunohistochemistry. METHODS We identified patients who were clinically diagnosed as Xp11.2 translocation RCC by immunostaining positive of TFE3 from two tertiary referral hospitals. Among them, a total of 19 patients whose fresh frozen tissue and normal DNA were achieved were included in the analysis. Whole exome sequencing (WES) was performed to evaluate somatic mutations and copy-number variation (CNV). We also performed RNAseq on the TFE3-positive RCC, 7 clear cell RCC, and 4 normal kidney tissue for comparison. Fusion partners were identified and clustering analysis was done by RNAseq. RESULTS Mutational landscape of TFE3-positive RCC is unique by low somatic mutation rate and very rare COSMIC variants. High prevalence of CNV with loss of 3p, 6q, 9, gain of 5, 7, and 12 were most frequent. We identified NONO, RBM10, SFPQ, ASPSCR1 and PRCC as fusion partner as previously described. We could not find TFE3 fusion in 4 out of 19 patients by RNAseq, and they were older and had more mutations than the others. They were also genetically clustered with clear cell RCC. Oxidative reducatase pathway was upregulated and immune response and cell adhesion were downregulated by gene ontology results from heatmap clusters. These may promote metastasis, resistant to immunity and cell survival in poorer condition which could be potential mechanism of Xp11.2 translocation RCC aggressiveness. CONCLUSIONS TFE3-positive RCC showed distinctive mutation pattern with low somatic mutation and rare common driver gene. A quarter of clinically diagnosed Xp11.2 translocation RCC (4/19) did not have real gene fusion even in TFE3 overexpression. They were older and had more somatic mutations than the others. Furthermore, they were clustered with clear cell RCC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e894 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Chang Wook Jeong More articles by this author Jae So Cho More articles by this author Moses Lee More articles by this author Murim Choi More articles by this author Cheryn Song More articles by this author Ja Hyeon Ku More articles by this author Cheol Kwak More articles by this author Hyeon Hoe Kim More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Genomic and Functional Approaches to Understanding Cancer Aneuploidy

Aneuploidy, whole chromosome or chromosome arm imbalance, is a near-universal characteristic of human cancers. In 10,522 cancer genomes from The Cancer Genome Atlas, aneuploidy was correlated with TP53 mutation, somatic mutation rate, and expression of proliferation genes. Aneuploidy was anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. Chromosome arm-level alterations show cancer-specific patterns, including loss of chromosome arm 3p in squamous cancers. We applied genome engineering to delete 3p in lung cells, causing decreased proliferation rescued in part by chromosome 3 duplication. This study defines genomic and phenotypic correlates of cancer aneuploidy and provides an experimental approach to study chromosome arm aneuploidy.

Medulloblastoma, WNT-activated/SHH-activated: clinical impact of molecular analysis and histogenetic evaluation.

Medulloblastoma (MDB) is a small cell poorly differentiated embryonal tumor of the cerebellum, which more frequently compromises children. Overall prognosis is favorable, but dependent of stage, histopathological pattern and molecular group. Approximately 30% of the affected patients will die from the disease. WHO 2016 Classification of Tumors of the Central Nervous System (CNS) has been classified MDB into four principal groups: WNT-activated MDB, SHH-activated MDB, group 3 MDB, and group 4 MDB. WNT-activated MDB is associated to monosomy 6, CTNNB1, DDX3X and TP53 mutations, beta-catenin nuclear immunoexpression, and a better prognosis than SHH-activated MDB. WNT-activated tumors account approximately for 10% of cases of MDBs, and are thought to arise from cells in the dorsal brain stem/lower rhombic lip progenitor cells. SHH-activated MDB more frequently arises in the lateral hemispheres of the cerebellum, and clinical outcome in this group is variable. TP53-mutant SHHactivated MDB usually shows the large cell/anaplastic pattern, and can be related to MYCN amplification, GLI2 amplification and 17p loss. TP53-wildtype SHH-activated MDB is more commonly of desmoplastic/nodular morphology, and can be related to PTCH1 deletion and 10q loss. Gene expression and methylation profiling is the gold standard for defining molecular groups of MDB. In immunohistochemistry assays, anti-GAB1 antibody expression is positive in tumors showing SHH pathway activation or PTCH mutation, while positive immunoexpression for YAP1 antibody can be only found in WNT-activated and SHH-activated MDB.

Comprehensive genetic characterization of TFE3-positive renal cell carcinoma.

635 Background: MiT family translocation renal cell carcinoma (RCC) is very rare variant which has translocation of Xp11.2/TFE3 gene or t(6;11)/TFEB gene. Xp11.2 translocation RCC is predominantly reported in younger age and has aggressive features. However, its pathophysiology or genetic characteristics are rarely understood. The aim of the study is to describe the comprehensive genetic characteristics of TFE3-positive RCC diagnosed by immunohistochemistry. Methods: We identified patients who were clinically diagnosed as Xp11.2 translocation RCC by immunostaining positive of TFE3 from two tertiary referral hospitals. Among them, a total of 19 patients whose fresh frozen tissue and normal DNA were achieved were included in the analysis. Whole exome sequencing (WES) was performed to evaluate somatic mutations and copy-number variation (CNV). We also performed RNAseq on the TFE3-positive RCC, 7 clear cell RCC, and 4 normal kidney tissue for comparison. Fusion partners were identified and clustering analysis was done by RNAseq. Results: Mutational landscape of TFE3-positive RCC is unique by low somatic mutation rate and very rare COSMIC variants. High prevalence of CNV with loss of 3p, 6q, 9, gain of 5, 7, and 12 were most frequent. We identified NONO, RBM10, SFPQ, ASPSCR1 and PRCC as fusion partner as previously described. We could not find TFE3 fusion in 6 out of 19 patients by RNAseq, and they were older and had more mutations than the others. They were also genetically clustered with clear cell RCC. Oxidative reducatase pathway was upregulated and immune response and cell adhesion were downregulated by gene ontology results from heatmap clusters. These may promote metastasis, resistant to immunity and cell survival in poorer condition which could be potential mechanism of Xp11.2 translocation RCC aggressiveness. Conclusions: TFE3-positive RCC showed distinctive mutation pattern with low somatic mutation and rare common driver gene. One-third clinically diagnosed Xp11.2 translocation RCC (6/19) did not have real gene fusion even in TFE3 overexpression. They were older and had more somatic mutations than the others. Furthermore, they were clustered with clear cell RCC.

PTEN loss affects the immune and inflammatory response in prostate cancer

Loss of the PTEN tumor suppressor gene occurs in 20-30% of prostate cancer (PCa) cases and is associated with worse disease outcome. Recent evidence highlights the role of inflammation in the tumor microenvironment (TME) and its association with PCa disease progression. The aim of this study is to determine if PTEN genomic loss is associated with the inflammatory response mediated by IFN regulated pathways in the TME. We performed an in silico analysis of genomic and corresponding transcriptomic profiles PCa tumors (n=493) from the Genomic Data Commons (GDC) cohort to identify significant alterations in immune response pathways when PTEN was lost. We identified 449 genes of interest using the Gene ontology (GO) Biological Function in Nexus Expression 3.0 with the keywords “Immune” and “Inflammatory”. We detected that 104 tumors (21%) had either homozygous or hemizygous loss of PTEN. We also found significant associations between PTEN loss, 17p loss and 21q loss (TMPRSS2-ERG fusion gene). PTEN loss was directly associated with worse PCa outcome. Of the 449 selected immune genes, 124 (28%) were differentially expressed when the PTEN loss group was compared to the PTEN intact group. Different cytokines and chemokines presented differential expression by comparing PTEN loss vs. PTEN intact PCa samples. IL13RA1, CXCL9, CXCL10, CXCL11 and CXCL14 showed upregulation in PTEN loss group (P<0.0001), while CXCL12 was found to be downregulated in PTEN loss group. DAVID enrichment analysis showed upregulation of 16 pathways, including RIG-I-like signaling pathway (P<0.0001), chemokine signaling pathway (P<0.0001), and toll-like receptor signaling pathway (P<0.0001). In addition, we identified ten downregulated pathways, including cytokine-cytokine receptor interaction pathway (P<0.003) and intestinal immune network for IgA production pathway (P<0.006). The 25 most differentially expressed genes were associated directly with type I and II IFN response. Collectively, our findings based on in silico studies suggest that PCa with PTEN loss exhibits dysregulated Type I and II IFN response that permits progression to an aggressive disease phenotype. Future investigations will be required to define the mechanisms underlying these correlations, and their role in PCa disease progression so that inflammation biomarker can be therapeutically exploited using immunotherapies.

Abstract P3-16-01: Using the new pan-cancer clinical data resource (TCGA-CDR) to identify breast cancer genomic correlates associating with different survival outcome endpoints

Abstract Introduction The Cancer Genome Atlas (TCGA) generated abundant high quality molecular data, however its relatively short-term patient follow-up limited its immediate clinical utility. We led a PanCanAtlas effort to systematically collate, integrate, and quality check the large body of acquired clinicopathologic data, generated 4 primary clinical outcome endpoints for each case, and created a new Pan-Cancer Clinical Data Resource (TCGA-CDR) for public use. We report here on the utility and validity of this TCGA-CDR in relating breast cancer (BC) genomic information to survival endpoints. Methods Clinicopathologic data from all data files were integrated and processed. Overall survival (OS), disease-specific survival (DSS, an approximation), progression-free interval (PFI), and disease-free interval (DFI)were derived.Tests of the adequacy of the follow-up intervals for each endpoint were performed, and quality evaluation of these endpoints was established by their comparison with different clinical features. As a case study we compared each survival endpoint for significant association (FDR <0.2) with chromosomal aneuploidy. Results The 4 endpoints were derived for 1097 TCGA BC cases having a median follow-up time of 27.7 months. Median times to events/censorship for OS, DSS, PFI, and DFI were 41.8/25.0, 32.6/26.0, 26.0/25.0, and 25.4/25.0 months respectively. PFI and DFI passed tests for adequate follow-up times; OS and DSS partially passed the same tests signaling some caution with their use in genomic associations. Using the endpoints, outcomes of patients with ER+ and ER- tumors were compared, along with those of patients with low (I&II) and high (III&IV) stage breast tumors. Univariate analyses suggested patients with ER+ tumors had significantly better survival than patients with ER- tumors when using PFI (p=0.005), DFI (p=0.001), and DSS (p=0.009), with OS not reaching significance (p=0.09). Patients with low stage tumors showed significantly better outcomes than patients with high stage tumors for each endpoint (p<0.001). The 4 endpoints were also evaluated for their significant associations with chromosomal arm aneuploidy. Adjusted for patient age and AJCC stage, tumors with a loss of 8q and 8p (p=0.019, FDR=0.37) had worse PFI; and those with loss of 8q, 20q, and 8p had worse DFI. Tumors with gain of 11q or loss of 14, 7q, 12q, 18q, 20q, 3p, 7p, 8p, 18p, and 20p had worse OS. In contrast, tumors with loss of 16q had better DSS, while those with loss of 3q, 12q, 17q, 18q, 19q, 20q, 3p, 8p, 12p, 18p, 19p, and 20p had worse DSS. The finding that 8p loss associated with worse survival for all 4 endpoints, while 18p loss associated with worse OS and DSS, agrees with literature reports. Conclusion These findings confirm that PFI and DFI, as extracted from the TCGA-CDR, are valid and appropriate BC survival endpoints, while OS and DSS may be recommended with some caution when employing TCGA data to evaluate new relationships between breast cancer genomic abnormalities and clinical outcomes. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Army/Navy/Air Force, the Department of Defense, or U.S. Government. Citation Format: Liu J, Lichtenberg T, Hoadley KA, Cherniack A, Poisson L, Kovatich AJ, Benz C, Thorsson V, TCGA PanCanAtlas Research Network, Shriver CD, Hu H. Using the new pan-cancer clinical data resource (TCGA-CDR) to identify breast cancer genomic correlates associating with different survival outcome endpoints [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-16-01.

Detection of copy number aberrations in cholangiocarcinoma using shallow whole genome sequencing of plasma DNA.

293 Background: Cholangiocarcinoma (CCA) is a deadly cancer with ~20% five year survival. Obtaining tissue to confirm diagnosis of CCA is often challenging. This also limits the use of tumor genotyping that could guide potential molecularly targeted treatment (such as FGFR inhibitors for a subset of cases). We investigated whether circulating tumor DNA (ctDNA) analysis in plasma is feasible for noninvasive tumor genotyping in patients with advanced CCA. Methods: We collected and processed plasma samples for ctDNA analysis from 17 patients with CCA at presentation and from 11 healthy volunteers. Following cell-free DNA extraction and quality assessment using digital PCR, we performed low-pass, shallow whole genome sequencing (sWGS). We analyzed sequencing data to identify copy number aberrations and quantify tumor fraction in plasma using recently published computational approaches. Results: Median total cfDNA concentration in patients with CCA was 7,003 genome equivalents (GEs)/mL plasma (range: 1,512- 107,707 GEs/mL) and significantly higher than healthy volunteers (two tailed t-test p < 0.04, median: 1,658 GEs/mL, range: 661-4,938 GEs/mL). We successfully sequenced all tumor libraries, generating a median of 173 million paired-end reads. ctDNA was detectable in 10/17 samples using sWGS. In these 10 samples, median tumor fraction in plasma DNA was 7.1% (range: 3.9%-44.6%). We found recurrent copy number aberrations expected in CCA including 1q gain in 80%, 7p gain in 50%, 1p loss in 70% and 3p loss in 50% of cases. Conclusions: ctDNA analysis for noninvasive tumor genotyping is feasible in patients with advanced cholangiocarcinoma. On-going work is focused on expanding these results using targeted and whole-exome sequencing.

Targeted Genomic Profiling Reveals Recurrent KRAS Mutations in Mesonephric-like Adenocarcinomas of the Female Genital Tract.

Mesonephric adenocarcinoma most commonly arises in the cervix and is presumed to be derived from normal or hyperplastic mesonephric remnants. It is characterized by recurrent KRAS mutations and lack of PIK3CA/PTEN alterations. Adenocarcinomas of the uterine corpus and ovary characterized by morphologic and immunophenotypic similarities to mesonephric adenocarcinoma have been reported. The pathogenesis of these tumors, which have been designated "mesonephric-like adenocarcinomas" is unknown, and it has been debated whether these represent mesonephric adenocarcinomas that arise in the endometrium/ovary or endometrioid adenocarcinomas that closely mimic mesonephric adenocarcinoma. The relationship at the molecular level between mesonephric adenocarcinomas and mesonephric-like adenocarcinomas is unknown. The aim of this study was to examine the molecular alterations in mesonephric-like adenocarcinomas to identify driver mutations and potential therapeutically targetable mutations, and to determine the relationship between mesonephric-like adenocarcinomas and mesonephric adenocarcinomas using targeted next-generation sequencing. Seven mesonephric-like adenocarcinomas (4 ovarian, 3 uterine corpus) underwent targeted next-generation sequencing to detect mutations, copy number variations and structural variants in exonic regions of 300 cancer genes, and 113 selected intronic regions across 35 genes. All 7 tumors (100%) harbored canonical activating KRAS mutations (4 G12D, 3 G12V). PIK3CA activating mutations were identified in 3 of 7 (43%) cases. There were no alterations in PTEN, ARID1A, or TP53 in any of the tumors. In copy number analysis, 5 of 7 (71%) tumors exhibited 1q gain, which was accompanied by 1p loss in 2 cases. In addition, 4 of 7 (57%) tumors had chromosome 10 gain, which was accompanied by gain of chromosome 12 in 3 cases. Mesonephric-like adenocarcinomas, similar to mesonephric adenocarcinomas, are characterized by recurrent KRAS mutations, gain of 1q, lack of PTEN mutations, and gains of chromosomes 10 and 12. PIK3CA mutations, which have not previously been identified in mesonephric adenocarcinoma, were found in 3 of 7 (43%) mesonephric-like adenocarcinomas in our study. Mesonephric-like adenocarcinomas exhibit strikingly similar molecular aberrations to mesonephric adenocarcinomas, but also frequently harbor PIK3CA mutations, demonstrating biological overlap with carcinomas of both mesonephric and Mullerian (endometrioid) differentiation. Given the previously documented association with endometriosis (ovarian neoplasms) and the prominent endometrial involvement (uterine corpus neoplasms), we believe these are best regarded as of Mullerian origin and representing adenocarcinomas which differentiate along mesonephric lines; as such, we propose the term mesonephric-like Mullerian adenocarcinoma.

De novo acute lymphoblastic leukemia-like disease of high grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements: a case report and literature review

BackgroundB-cell lymphomas harboring the 8q24/MYC plus 18q21/BCL2 translocations are now referred to as high grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-MBR). Although HGBL-MBR is frequently found in cases with diffuse large B-cell lymphoma or Burkitt lymphoma-like B-cell lymphoma, acute lymphoblastic leukemia (ALL)-like disease of HGBL-MBR (AL-HGBL-MBR) has been reported incidentally.Case presentationA 69-year-old Japanese woman developed remittent fever and increasing systemic bone pain. The bone marrow examination revealed that more than 90% of nuclear cells were blastoid cells, which were positive for CD10, CD19, CD20, and surface IgMκ and negative for terminal deoxynucleotidyl transferase (TdT). Cytogenetic studies confirmed that the patient had de novo AL-HGBL-MBR with the extra copies of MYC and loss of chromosome 17p. She showed resistance to chemoimmunotherapy and died seven months after the diagnosis. The literature review identified further 47 de novo AL-HGBL-MBR cases within the last 32 years. The median age was 61 years (range, 27 − 86); the male/female ratio was 2.0. Thirty-eight cases (79%) presented a clinical picture of ALL at diagnosis; 14 (36%) of 39 available cases showed central nervous system involvement. Loss of 17p and translocations at 2p12–13, 3q27, 9p13 were frequently observed as additional cytogenetic abnormalities. Although the median survival of 46 available cases was only five months (range, 0.1–18), rituximab use significantly improved the survival of AL-HGBL-MBR (log-rank test, P = 0.0294).ConclusionOur patient and most reported de novo AL-HGBL-MBR cases showed resistance to conventional chemoimmunotherapy and disastrous consequences. AL-HGBL-MBL is a rare, but should be considered a distinct clinical condition in HGBL-MBR. Other therapeutic strategies, such as using inhibitors of MYC and BCL2, are needed to overcome the chemoresistance of AL-HGBL-MBR.

Mutations of candidate tumor suppressor genes at chromosome 3p in intrahepatic cholangiocarcinoma

The genetic status of candidate tumor suppressor genes (TSGs) at chromosome 3p has not yet been elucidated in intrahepatic cholangiocarcinoma (iCCA). Herein, we retrospectively investigated 32 fresh iCCA case samples from a single medical institution to clarify mutations of 11 TSGs by next-generation sequencing. Validation of the mutations was performed on the MassARRAY platform or by high-resolution melting curve analysis. We then integrated the gene mutations into copy number alterations at chromosome 3p that had been generated in a previous study using the same fresh iCCA samples, and correlated the integration results with the clinicopathologic features. Nine of the 32 (28.1%) iCCA patients had gene mutations at chromosome 3p, totaling 11 mutations across five genes. Those included five (15.6%) BAP1 mutations, two each (6.3%) of CACNA2D3 and RASSF1 mutations, and one each (3.1%) of ATG7 and PLCD1 mutations. Six (18.8%) cases had concurrent loss of chromosome 3p and gene mutations. Patients with TSG mutations had shorter disease-free and survival times than those without the mutations. Our data showed that iCCA patients with TSG mutations at chromosome 3p faced an adverse prognosis. BAP1 was the common target of mutational inactivation and may be a principal driver of 3p21 losses.

154 Genomic Landscape of Radiation-Induced Meningiomas

Abstract INTRODUCTION Majority of pediatric cancers require the irradiation of the central nervous system (CNS), and as more patients survive into adulthood from improved oncological therapy the sequelae of brain radiation are increasing in prevalence. Radiation-induced meningiomas (RIMs), one such secondary effect, demonstrate a clinically more aggressive behaviour than sporadic meningiomas (SMs). We aimed to describe the genomic mutational landscape of RIMs METHODS We analyzed a principal cohort of 18 RIMs, with 31 RIMs overall, from patients who received childhood radiation therapy and 30 SMs, as a comparator population. We performed a multiplatform integrative genomic analysis; including methylation, whole exome and RNA sequencing. RESULTS >RIMs exhibited a five-fold increase in copy number alterations, commonly the loss of chromosome 1p (17/18 RIMs) and 22q (17/18 RIMs), which was significantly more than observed in sporadic meningiomas. Furthermore, RNA sequencing data revealed an NF2 gene fusion event in 35.3% of RIMs In all 6 cases, there was a complete NF2 exon spliced into a complete exon of a reciprocal gene, suggesting that the breakpoints of genomic rearrangement are intronic. All tumours with the NF2 fusion also possessed monosomy of chromosome 22q, rendering the cells with homozygous disruption of NF2. Clinically, RIMs with the NF2 fusion exhibited ill-defined borders and a tendency to develop in anatomic frontal location. The NF2 fusion RIMs, also, had a significantly faster growth rate compared to non-fusion RIMS (P < 0.05). Also, targeted sequencing panel confirmed that RIMs had fewer nonsynonymous NF2 mutations (6.5% vs. 30% in SM) and absence of mutations in TRAF7, SMO, KLF4, PIK3CA and AKT1, genes traditionally involved in SMs. CONCLUSION Our study demonstrates that RIMs have distinct genomic drivers of oncogenesis as compared to SMs, specifically NF2 inactivation through fusion event. Radiation therapy possibly triggers genomic structural rearrangements through error-prone repair of double-stranded DNA breaks.

Interim results from the CATNON trial (EORTC study 26053-22054) of treatment with concurrent and adjuvant temozolomide for 1p/19q non-co-deleted anaplastic glioma: a phase 3, randomised, open-label intergroup study

The role of temozolomide chemotherapy in newly diagnosed 1p/19q non-co-deleted anaplastic gliomas, which are associated with lower sensitivity to chemotherapy and worse prognosis than 1p/19q co-deleted tumours, is unclear. We assessed the use of radiotherapy with concurrent and adjuvant temozolomide in adults with non-co-deleted anaplastic gliomas. This was a phase 3, randomised, open-label study with a 2 × 2 factorial design. Eligible patients were aged 18 years or older and had newly diagnosed non-co-deleted anaplastic glioma with WHO performance status scores of 0-2. The randomisation schedule was generated with the electronic EORTC web-based ORTA system. Patients were assigned in equal numbers (1:1:1:1), using the minimisation technique, to receive radiotherapy (59·4 Gy in 33 fractions of 1·8 Gy) alone or with adjuvant temozolomide (12 4-week cycles of 150-200 mg/m2 temozolomide given on days 1-5); or to receive radiotherapy with concurrent temozolomide 75 mg/m2 per day, with or without adjuvant temozolomide. The primary endpoint was overall survival adjusted for performance status score, age, 1p loss of heterozygosity, presence of oligodendroglial elements, and MGMT promoter methylation status, analysed by intention to treat. We did a planned interim analysis after 219 (41%) deaths had occurred to test the null hypothesis of no efficacy (threshold for rejection p<0·0084). This trial is registered with ClinicalTrials.gov, number NCT00626990. At the time of the interim analysis, 745 (99%) of the planned 748 patients had been enrolled. The hazard ratio for overall survival with use of adjuvant temozolomide was 0·65 (99·145% CI 0·45-0·93). Overall survival at 5 years was 55·9% (95% CI 47·2-63·8) with and 44·1% (36·3-51·6) without adjuvant temozolomide. Grade 3-4 adverse events were seen in 8-12% of 549 patients assigned temozolomide, and were mainly haematological and reversible. Adjuvant temozolomide chemotherapy was associated with a significant survival benefit in patients with newly diagnosed non-co-deleted anaplastic glioma. Further analysis of the role of concurrent temozolomide treatment and molecular factors is needed. Schering Plough and MSD.

Therapeutic radiation for childhood cancer drives structural aberrations of NF2 in meningiomas

Cranial radiotherapy improves survival of the most common childhood cancers, including brain tumors and leukemia. Unfortunately, long-term survivors are faced with consequences of secondary neoplasia, including radiation-induced meningiomas (RIMs). We characterized 31 RIMs with exome/NF2 intronic sequencing, RNA sequencing and methylation profiling, and found NF2 gene rearrangements in 12/31 of RIMs, an observation previously unreported in sporadic meningioma (SM). Additionally, known recurrent mutations characteristic of SM, including AKT1, KLF4, TRAF7 and SMO, were not observed in RIMs. Combined losses of chromosomes 1p and 22q were common in RIMs (16/18 cases) and overall, chromosomal aberrations were more complex than that observed in SM. Patterns of DNA methylation profiling supported similar cell of origin between RIMs and SMs. The findings indicate that the mutational landscape of RIMs is distinct from SMs, and have significant therapeutic implications for survivors of childhood cranial radiation and the elucidation of the molecular pathogenesis of meningiomas.

Combined deletion and DNA methylation result in silencing of FAM107A gene in laryngeal tumors

Larynx squamous cell carcinoma (LSCC) is characterized by complex genotypes, with numerous abnormalities in various genes. Despite the progress in diagnosis and treatment of this disease, 5-year survival rates remain unsatisfactory. Therefore, the extended studies are conducted, with the aim to find genes, potentially implicated in this cancer. In this study, we focus on the FAM107A (3p14.3) gene, since we found its significantly reduced expression in LSCC by microarray profiling (Affymetrix U133 Plus 2.0 array). By RT-PCR we have confirmed complete FAM107A downregulation in laryngeal cancer cell lines (15/15) and primary tumors (21/21) and this finding was further supported by FAM107A protein immunohistochemistry (15/15). We further demonstrate that a combined two hit mechanism including loss of 3p and hypermethylation of FAM107A promoter region (in 9/15 cell lines (p < 0.0001) and in 15/21 primary tumors (p < 0.0001)) prevails in the gene transcriptional loss. As a proof of principle, we show that Decitabine - a hypomethylating agent – restores FAM107A expression (5 to 6 fold increase) in the UT-SCC-29 cell line, characterized by high DNA methylation. Therefore, we report the recurrent inactivation of FAM107A in LSCC, what may suggest that the gene is a promising tumor suppressor candidate involved in LSCC development.

Abstract 3429: Genome and epigenome profiling of high stage neuroblastoma

Abstract Neuroblastoma (NB) is the most common pediatric extracranial solid tumor with a wide range of clinical phenotypes from spontaneous regression to highly resistant to chemotherapy. Loss of 1p and 11q and genomic alterations of MYCN, ALK, ATRX and TERT have been known to be involved in oncogenesis and unfavorable phenotype of NB, however, only small numbers of recurrent driver mutations have been observed by NGS-based studies. This implies epigenetic alterations also influence the pathogenesis of NB. In this study, to elucidate the molecular basis of malignant subtype of NB, we conducted methylome analysis for 145 primary NBs (stage 1:3; stage 2:8; stage 3:16; stage 4:105; stage 4s:13) by Infinium Human Methylation 450 BeadChip. By hierarchical clustering of methylome signature, the tumors were classified into four clusters. Cluster-1 shows good similarity with nerve cell controls and, together with cluster-3 (very young, early tumor stage), exhibited favorable prognosis. Unfavorable cluster-2 and -4 exhibited hyper-methylation phenotypes in CpG islands (CGI) and the former was enriched by MYCN-amplified tumors. These two clusters (n=73) showed strong correlation with poor patient prognosis (p=0.0055). 1400 hyper-methylated CGI locus which corresponded 550 genes were picked up and annotated by GO database. The term “cell adhesion”, “cell-cell signaling”, “transmission of nerve impulse” and “cellular component morphogenesis” were enriched. No 1p or 11q genes were enriched in hyper-methylated genes but several genes such as ANKK1 on chr11q whose lower expression levels are strongly correlated with unfavorable prognosis of the patient (p=4.0e-07) were included. Further analyses by comparing the influences in gene expression profiles by methylation and by histone modification of PRC2 are ongoing. Our recent data indicated that NTRK1, whose higher expression is a strong favorable marker, is transcriptionally downregulated in MYCN-amplified tumors by both methylation and H3K27me3 on distinct promoter regions (Li Z et al). Since application of epigenetic drugs are one of the possible therapeutic choices for NB, transcriptional regulation by the epigenetic alterations are needed to be intensively studied. Citation Format: Miki Ohira-Ichikawa, Ryuichi P. Sugino, Hisanori Takenobu, Zhenghao Li, Masayuki Haruta, Genta Nagae, Claire Renard-Guillet, Akira Nakagawara, Hiroyuki Aburatani, Takehiko Kamijo. Genome and epigenome profiling of high stage neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3429.

Abstract 2717: Identification of three distinct molecular subtypes in meningioma samples using microarrays for copy-number variants

Abstract Background: Meningioma represents one of the most common intracranial tumors. They are generally thought to progress from low to high-grade lesions. However, the molecular mechanisms underlying their pathogenesis remain still uncertain. Identification of meningioma molecular subgroups may have significant potential to improve clinical management, through molecular disease risk stratification strategies and the identification of patients who could benefit from targeted molecular therapeutics. Methods and patients: Formalin-fixed paraffin embedded tumor samples were obtained from 45 meningioma patients and 5 healthy controls (dura mater). Comprehensive clinical-pathological data were mined. There were 15 males and 30 females; median age was 54 years, range 28 - 99 years. Total DNA was purified from FFPE samples after pathological verification using proteinase K treatment followed by QIAmp DNA FFPE Tissue Kit (Qiagen). Microarray analysis was performed using the OncoScan FFPE Assay Kit (Affymetrix), raw data were obtained using Chromosome Analysis Suite (Affymetrix) in default manner. The data were analyzed using “R” software, version 3.2.3. Results: Our results confirm that chromosome 22 deletion and del(1p) are the most common (55%, resp. 47% of cases) deletions in meningioma. We revealed chromosomal gains not as rare as was published previously while the dup(3q) was present in 31% of cases. Three meningioma molecular patterns were identified based on CNV profiling - normal-like profile, deletion profile and complex profile. Deletion profile was characterized by loss of 1p and monosomy of chromosomes 6, 10, 14, 18 and 22. The complex profiles combined gains (3q, 8q, chromosomes 12, 15 and 20) and losses (1p, chromosomes 13, 21 and 22). The primary tumor samples of patients with meningioma recurrence (20 patients) had different CNV profile in comparison with patients without recurrence (25 patients). Recurrent tumors had statistically significant higher frequencies of losses on chromosomes 1, 9, 10, 11, 13 and 21 and gains on chromosomes 12, 19 and 20. Moreover, we analyzed CNV profiles of paired tumor samples of 15 recurrent meningioma patients (primary tumor and recurrent tumor in each patient). After extraction, it was found, that the most common alteration in recurrent tumors is chromosome 7q31 and 1p36 gains and chromosome 15q21 losses. Conclusion: We have identified three distinct meningioma molecular patterns - normal-like profile, deletion profile and complex profile. Chromosome 22 deletion, del(1p) and dup(3q) are the most common CNV (55%, 47%, resp. 31% of cases) in meningioma. Potential CNV changes in recurrent meningioma were identified. However, it will require further validation using FISH. Acknowledgment: This work was financially supported by Ministry of Health of the Czech Republic, grant nr. 15-29021A, IGA UP LF 2016_010 and NPU LO1304. Citation Format: Josef Srovnal, Vladimir Balik, Magdalena Houdova Megova, Jiri Ehrmann, Miroslav Vaverka, Lumir Hrabalek, Radek Trojanec, Katerina Staffova, Jana Vrbkova, Marian Hajduch. Identification of three distinct molecular subtypes in meningioma samples using microarrays for copy-number variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2717. doi:10.1158/1538-7445.AM2017-2717

Abstract 1434: Mutational landscape in the normal-appearing airway cancerization field of early-stage non-small cell lung cancer

Abstract Lung cancer, of which non-small cell lung cancer (NSCLC) is the most common form, is the second most prevalent cancer in the U.S. and the leading cause of cancer mortality. Field cancerization phenomenon establishes that normal appearing tissues surrounding a tumor will exhibit a field effect – particularly in smokers – with the tissues proximal to the tumor showing more, and even sharing, alterations with the tumor. Previous studies in NSCLC provide evidence for both complex progression trajectories, yet, precise mechanisms remain unknown. To investigate the field cancerization phenomenon, we conducted a genome-wide survey of acquired DNA alterations in normal-appearing tissues including small airways adjacent to NSCLC, mainstem bronchi (large airways), nasal epithelium, distant normal lung and blood, along with multiple samples from their surgically resected paired tumors, for a total of 500 samples from 48 patients with early-stage NSCLC (11 squamous cell carcinomas and 37 adenocarcinomas). Since we expect low mutation burdens and mutant cell fractions in pathologically normal tissues, we assessed somatic point mutations via deep targeted sequencing of 409 genes and paired these data with our recent study of acquired copy number alterations (CNAs) inferred from high-density whole-genome SNP microarrays, thus offering a deep and wide survey of somatic DNA alterations. After aggregating results from multiple mutation callers, we observed somatic mutations (exonic, splicing and UTRs) in 257 samples of which 65 (in 35 patients) were from normal-appearing field (non-tumor, non-blood) samples. Tumor and field samples in smokers showed a higher mutation burden and large proportion of C:G&amp;gt;A:T changes. We also observed concordance of mutations in the airway and corresponding tumor profiles. Further, a statistically significant field effect was established with mutational burden (measured by the variant allele frequency) increasing with proximity to the tumor. Among the 35 NSCLC cases with field mutations, the small adjacent (to tumor) airways in six patients exhibited mutations in lung cancer drivers such as KRAS, STK11, TP53 and KEAP1. We also identified mutations in the large airway (TP53, SETD2, CDKN2A), distant normal lung parenchyma (RB1, RET) and nasal epithelium (AKT1). We then correlated point mutation and CNA mutation profiles. Of eight cases showing a nonsynonymous or stopgain variants in established lung cancer drivers in the airways, four exhibited putative “two-hit” progression models, e.g. both KEAP1 and STK11 mutations with 19p loss, overlapping TP53 mutation/17p loss, or KRAS mutation/12p gain. Our findings in normal-appearing tissues of the respiratory epithelium offer insights into the earliest mutational events in their progression to NSCLC and, possibly, in tumor relapse and that may represent suitable targets for early detection and chemoprevention. Citation Format: Smruthy Sivakumar, Yasminka Jakubek, Wenhua Lang, Tina McDowell, Melinda M. Garcia, Chi-Wan Chow, Zachary Weber, Carmen Behrens, Neda Kalhor, Cesar Moran, Randa El-Zein, Gareth Davies, Junya Fujimoto, Reza Mehran, Stephen G. Swisher, Jing Wang, Avrum E. Spira, Jerry Fowler, F Anthony San Lucas, Ignacio I. Wistuba, Erik Ehli, Paul Scheet, Humam Kadara. Mutational landscape in the normal-appearing airway cancerization field of early-stage non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1434. doi:10.1158/1538-7445.AM2017-1434

Abstract 4879: Genomic and epigenomic profiling of high-stage neuroblastoma

Abstract Neuroblastoma (NB) is the most common pediatric extracranial solid tumor with a wide range of clinical phenotypes from spontaneous regression to highly resistant to chemotherapy. Despite intense multi-modal therapy, NB remains the second most common cause of cancer deaths among children. MYCN amplification is the most prominent poor prognosis factor for NB, however, certain subgroup of high stage tumor without MYCN amplification also shows low survival rate in terms of long-term follow-up (10 years survival rate was 51%, n=161). To elucidate the molecular basis of malignant subtype of NB, integrated genomic and epigenomic analyses were performed. Array CGH of 537 tumors indicated that genomic subgroups of 17q gain, with 1p loss and/or 11q loss, exhibited aggressive characteristics even without MYCN amplification. Whole exome sequencing of 92 high stage, MYCN-non-amplified primary tumors (SureSelect XT Human All Exon V4, mean depth CDS:138, x20 coverage:96%) identified 1,735 non-silent somatic mutations, among which 53 were significant mutations observed more than once in the sample set. Aggressive tumors (died of disease, n=48) had several notable features, with mutations in chromatin remodeling/modulating genes (27%, cf. ATRX), in DNA-damage response (30%), in TP53 (28%) and RB (22%)-related gene networks, as well as with high TERT expression (38%). By hierarchical clustering of methylome signature (Infinium HumanMethylation450 BeadChip), the 92 tumors were subdivided into four clusters. Unfavorable cluster-2 and -4 exhibited hyper-methylation phenotypes in CpG islands and the former was enriched by MYCN-amplified tumors. These two clusters (53%) showed strong correlation with patient prognosis (p=0.0055). Thus, these prognosis-related genomic features could be useful markers for risk classification and help understanding molecular mechanism for aggressive phenotypes of MYCN-non-amplified NBs. Citation Format: Miki Ohira, Yasutoshi Tatsumi, Yohko Nakamura, Kenji Tatsuno, Shuichi Tsutsumi, Shogo Yamamoto, Genta Nagae, Claire Renard-Guillet, Ryuichi Sugino, Hiroki Nagase, Takehiko Kamijo, Hiroyuki Aburatani, Akira Nakagawara. Genomic and epigenomic profiling of high-stage neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4879. doi:10.1158/1538-7445.AM2017-4879

Abstract 4882: Genomic mechanisms of disease progression in pediatric medullary thyroid cancer (MTC)

Abstract Background: Multiple Endocrine Neoplasia (MEN) 2B is a rare hereditary disorder characterized by medullary thyroid cancer (MTC) in early childhood, pheochromocytoma, and mucosal neuromas. Patients with advanced MTC are treated with rearranged during transfection (RET) targeting tyrosine kinase inhibitors (TKIs) such as vandetanib. Despite initial responses, many patients progress on TKI therapy and mechanisms of resistance are yet to be elucidated. We analyzed tumor samples from seven children with MEN2B and MTC enrolled in a natural history study (NCT01660984). Methods: DNA samples from tumor and adjacent normal tissue from paraffin embedded blocks or unstained slides were analyzed by a custom capture next-generation sequencing panel. Bioinformatics analyses identified point mutations, insertions, deletions and copy number variations. Results: Seven patients (median age at study enrollment 14 years, range 11-17 years) with the RET p.Met918Thr germline mutation were included in analysis. Tumor samples were available for three patients pre-TKI (four samples), two patients at progression on TKI (three samples), and two patients both, pre-TKI and at progression (five samples). Pre-TKI samples exhibited few tumor specific mutations or copy number variations and 4/5 patients had loss of chromosome 1p. Progression for one patient was associated with acquisition of a previously unidentified p.Leu790Phe mutation within the kinase domain of the RET gene. Loss of heterozygosity and increase in copy number variations were noted in 4/5 samples at tumor progression. Two patients had copy number loss of chromosome 14 and three had copy number gain of chromosome 1q. Recurrent, somatic, non-synonymous mutations were not identified in the sample set. Conclusion: In children with MEN2B and MTC, we identified increase in copy number variations and a somatic mutation within the RET gene as potential mechanisms of drug resistance. Our data imply that a common genetic mechanism for progression on TKI may not exist within this small sample set and highlight the need for serial collection of tumor tissue. Further, whole genome aneuploidy may provide rationale for the evaluation of cytotoxic chemotherapy in patients who experience progressive disease on TKI therapy. Analysis of additional samples and whole exome DNA and RNA sequencing are ongoing. Citation Format: Ira L. Kraft, Srivandana Akshintala, Keith J. Killian, Robert B. Hufnagel, John W. Glod, Claudia Derse-Anthony, Yuelin Zhu, Holly S. Stevenson, Diana Bradford, Maria J. Merino, Frank M. Balis, Elizabeth Fox, Brigitte C. Widemann, Jack F. Shern, Paul S. Meltzer. Genomic mechanisms of disease progression in pediatric medullary thyroid cancer (MTC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4882. doi:10.1158/1538-7445.AM2017-4882