Frequent Chromosomal Losses Research Articles

Papillary tumor of the pineal region: analysis of DNA methylation profiles and clinical outcomes in 76 cases

Papillary tumor of the pineal region (PTPR) is an uncommon tumor of the pineal region with distinctive histopathologic and molecular characteristics. Experience is limited with respect to its molecular heterogeneity and clinical characteristics. Here, we describe 39 new cases and combine these with 37 previously published cases for a cohort of 76 PTPR’s, all confirmed by methylation profiling. As previously reported, two main methylation groups were identified (PTPR-A and PTPR-B). In our analysis we extended the subtyping into three subtypes: PTPR-A, PTPR-B1 and PTPR-B2 supported by DNA methylation profile and genomic copy number variations. Frequent loss of chromosome 3 or 14 was found in PTPR-B1 tumors but not in PTPR-B2. Examination of clinical outcome showed that nearly half (14/30, 47%) of examined patients experienced tumor progression with significant difference among the subtypes (p value = 0.046). Our analysis extends the understanding of this uncommon but distinct neuroepithelial tumor by describing its molecular heterogeneity and clinical outcomes, including its tendency towards tumor recurrence.

Myeloid Neoplasms with MYC-Positive Double Minutes, a Specific Subgroup?

Background: Double minutes (dmin) are a rare cytogenetic finding in hematologic malignancies, mostly associated with acute myeloid leukemias. The acentric chromosomal structures usually represent the cytogenetic equivalent of the amplification of an oncogene, such as MYC. Due to their small size dmin are difficult to detect in chromosome banding analysis (CBA) and FISH analysis is required to identify the corresponding gene amplification. In literature several case reports described an association of the presence of MYC-positive dmin with an APL-like cytomorphology, however, larger studies comprehensively characterizing these cases are missing so far. Aim: Characterization of 68 cases of myeloid neoplasms harboring MYC dmin with respect to cytogenetics, morphology, gene expression (GE) and gene mutations. Patients and methods: The study enrolled 68 patients (pts) with myeloid neoplasia and MYC dmin. The diagnosis was established following WHO guidelines. Dmin and MYC amplification were assessed via CBA and FISH. Cases were screened for morphological features indicating a similarity to APL and/or suspected diagnosis of APL (APL-like). NGS of a 72 gene panel and WTS (50 Mio reads) were performed on NovaSeq instruments. Variant calling was performed with Pisces (minimum sensitivity: 3 %). Results: The majority of pts were diagnosed with AML (52/68, 77 %, 7 of these secondary AML) or MDS/AML (3/68, 4 %). Other diagnoses comprised CMML (4/68, 6 %), MPN in acceleration or blast crisis, MDS/MPN overlap or MDS (3/68, 4 % each). APL-like cases amounted to 17/68 (25 %). Auer rods were detected in 24/68 pts (35 %) with a higher frequency in APL-like cases (p=0.007). Myeloperoxidase level was >75 % in 46/54 pts (85 %). CBA revealed a complex karyotype (ck, ≥3 aberrations in addition to MYC dmin) in 21/68 pts (31 %), whereas 19/68 pts (28 %) presented with MYC dmin as the only cytogenetic aberration (MYC dmin only). Most common additional chromosomal gains were +4 (11/68, 16 %), +6 (9/68, 13 %) and +22 (8/68, 12 %), while most frequent chromosomal losses comprised 5q deletions (19/68, 28 %), 17p deletions (18/68, 27 %) and 9q deletions or -9 (16/68, 24 %). Gene mutations (mut) most frequently occurred in TET2 (48/68, 71 %), TP53 (22/67, 33 %), U2AF1 (17/67, 25 %) and NRAS (12/66, 18 %). Almost all pts (66/68, 97 %) harbored at least one mutation in TET2, TP53 or U2AF1. TP53 mut were associated with ck (p<0.001) and were not detected in MYC dmin only pts (p<0.001). EZH2 mut were exclusively observed in cases with non-ck and 5/7 mut occurred in MYC dmin only pts (p=0.014). TET2 mut were observed more frequently in cases with non-ck (89 % vs. 29 %, p<0.001). Similarly, U2AF1 mut co-occurred with ck only in one case (p=0.013). In addition, U2AF1 showed significantly higher mutation frequency in APL-like cases (47 % vs. 18 %, p=0.025). A comparison of the mutation frequencies of all MYC dmin cases to a cohort of AML from routine diagnostics (707 pts) revealed overrepresentation of mut in TP53 (33 % vs. 11 %, p=0.003), TET2 (71 % vs. 19 %, p<0.001), U2AF1 (25 % vs. 4 %, p<0.001) and EZH2 (11 % vs. 2 %, p<0.001) in the cohort of MYC dmin cases, whereas CEBPA, NPM1, FLT3, FLT3-ITD and WT1 mut were only detected in the AML cohort (10 %, p=0.004; 22 %, p<0.001; 8 %, p=0.011, 16 %, p<0.001 %; 8 %, p=0.017 respectively) and IDH2 mut occurred less frequently in MYC dmin cases (2 % vs. 9 %, p=0.022). GE analysis of 40 MYC dmin pts revealed high expression of MPO compared to various AML subtypes, similar to APL (Figure (A)). A tSNE plot based on the top 5 % most variable genes demonstrated similar GE profiles within the MYC dmin cases compared to other AML cases (857 pts) (Figure (B)). A comparison of GE profiles within the MYC dmin cohort revealed differential GE between APL-like and all other MYC dmin cases (46 differentially expressed genes (DEG), adjusted p-value <0.05), as well as between ck and non-ck MYC dmin cases (106 DEG, adjusted p-value <0.05). Conclusions:MYC dmin cases often show an APL-like cytomorphology, Auer rods and high levels of MPO expression. They are characterized by a high frequency of TET2, TP53 and U2AF1 mut as well as a relatively uniform GE profile. Mutation patterns and GE profiles allow further differentiation within MYC dmin cases. In cases with suspected APL after exclusion of a PML::RARA or other RARA fusions CBA and FISH for the detection of MYC dmin is helpful to identify this rare myeloid subtype which is described to be associated with an unfavorable prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Somatic Mutation Profiling in Head and Neck Paragangliomas.

ContextHead and neck paragangliomas (HNPGLs) are rare neoplasms with a high degree of heritability. Paragangliomas present as polygenic diseases caused by combined alterations in multiple genes; however, many driver changes remain unknown.ObjectiveThe objective of the study was to analyze somatic mutation profiles in HNPGLs.MethodsWhole-exome sequencing of 42 tumors and matched normal tissues obtained from Russian patients with HNPGLs was carried out. Somatic mutation profiling included variant calling and utilizing MutSig and SigProfiler packages.Results57% of patients harbored germline and somatic variants in paraganglioma (PGL) susceptibility genes or potentially related genes. Somatic variants in novel genes were found in 17% of patients without mutations in any known PGL-related genes. The studied cohort was characterized by 6 significantly mutated genes: SDHD, BCAS4, SLC25A14, RBM3, TP53, and ASCC1, as well as 4 COSMIC single base substitutions (SBS)-96 mutational signatures (SBS5, SBS29, SBS1, and SBS7b). Tumors with germline variants specifically displayed SBS11 and SBS19, when an SBS33-specific mutational signature was identified for cases without those. Beta allele frequency analysis of copy number variations revealed loss of heterozygosity of the wild-type allele in 1 patient with germline mutation c.287-2A>G in the SDHB gene. In patients with germline mutation c.A305G in the SDHD gene, frequent potential loss of chromosome 11 was observed.ConclusionThese results give an understanding of somatic changes and the mutational landscape associated with HNPGLs and are important for the identification of molecular mechanisms involved in tumor development.

Microsatellite (D9S905) Instability in Two Urogenital Schistosomiasis Patients with Abnormal Squamous Cells in the Urine

Background: Schistosomiasis ranks second to malaria among parasitic diseases of socio-economic and public health importance. Schistosoma haematobium infection-causing urinary schistosomiasis is widespread in Nigeria. Literature has shown that bladder cancer of squamous cell carcinoma type could be associated with long-term S. haematobium infection. Many studies have also reported genomic alterations in cancers and have detected identical microsatellite instability in bladder cancer and corresponding urine sediment from the same patients. Therefore, this study aimed to detect microsatellite instability in genomic DNA obtained from exfoliated urine cells of S. haematobium infected participants using microsatellite marker D9S905, which is one of the genetic markers located around regions of frequent chromosomal loss in bladder cancer. Methods: Genomic DNA was extracted from urine and blood of 24 S. haematobium infected study participants. Microsatellite marker D9S905 located on locus 9q34.2 with allele size 294 base pair (294bp) was analyzed to identify genetic variation between exfoliated urine cells and corresponding blood samples from the infected participants. Result: Microsatellite alterations due to allelic loss were seen in the DNA of exfoliated urine cells of 2 of the participants. Also, previous cytopathological examinations of exfoliated urine cells of these 2 participants revealed squamous cell abnormalities. Conclusion: Bladder cancer is one of the severe complications of chronic S. haematobium infection. Since not all cases of the infection are chronic, this might have accounted for the low number of cases of microsatellite instability seen among participants in this study

GCT-15. INTEGRATED CLINICAL, HISTOPATHOLOGICAL, AND MOLECULAR DATA ANALYSIS OF 190 CENTRAL NERVOUS SYSTEM GERM CELL TUMORS FROM THE IGCT CONSORTIUM

BACKGROUNDWe integrated clinical, histopathological, and molecular data of central nervous system germ cell tumors to provide insights into their management.METHODSData from the Intracranial Germ Cell Tumor Genome Analysis Consortium were reviewed. A total of 190 cases were classified as primary GCTs based on central pathological reviews.RESULTSAll but one of the cases that were bifocal (neurohypophysis and pineal glands) and cases with multiple lesions including neurohypophysis or pineal gland were germinomas (34 of 35). Age was significantly higher in patients with germinoma than other histologies. Comparison between tumor marker and histopathological diagnoses showed that 18.2% of histopathologically diagnosed germinomas were marker-positive and 6.1% of non-germinomatous GCTs were marker-negative, suggesting a limitation in the utility of markers or histopathology alone using small specimens for diagnosis. Comparison between local and central histopathological diagnoses revealed a discordance of 12.7%. Discordance was significantly less frequent in biopsy cases, implying difficulty in detecting all histopathological components of heterogeneous GCTs. Germinomas at the typical sites (neurohypophysis or pineal gland) showed a better PFS than those at atypical sites (p=0.03). A molecular-clinical association study revealed frequent MAPK pathway mutations in males (51.4 vs 14.3 %, p=0.007), and PI3K/mTOR pathway mutations in basal ganglia cases (p=0.004). Basal ganglia cases also had frequent chromosomal losses. Some chromosomal aberrations (2q, 8q gain, 5q, 9p/q, 13q, 15q loss) showed potential prognostic significance.CONCLUSIONSThese in-depth findings of this study regarding the clinical and molecular heterogeneity will increase our understanding of the pathogenesis of this enigmatic tumor.

MODL-30. DISSECTING THE ROLE OF MULTI-CILIOGENESIS NETWORK IN CHOROID PLEXUS TUMOR

The choroid plexus (CP) in brain ventricles consists of a fibro-vascular core encapsulated by epithelial cells that possess clusters of primary cilia on cell surface. CP tumors are rare primary brain neoplasms that most commonly occur in young children. Compared to the benign CP papilloma, choroid plexus carcinoma (CPC) is poorly understood and highly lethal with few treatments available. Molecular, cytogenetics and genomics studies uncovered complex alterations in CPC including frequent chromosomal loss and recurrent focal aberrations, whereas abnormal NOTCH signaling is observed in many CP tumors. We showed that activation of both NOTCH and Sonic Hedgehog (SHH) signaling in mice drives the formation of aggressive CP tumor. Molecular and histology analyses demonstrated that these murine CP tumors closely resemble their human counterparts, which also display aberrant SHH and NOTCH signaling, suggesting they may represent potential therapeutic avenues. Indeed, treatment with vismodegib, an FDA-approved SHH pathway inhibitor, suppresses CP tumor growth. Unlike multi-ciliated CP epithelial cells, tumor cells in these animal models are characterized by a solitary primary cilium. Though key genes of the multi-ciliogenesis circuit driven by Geminin coiled-coil domain-containing protein 1 (GEMC1) are expressed in CP epithelium, GEMC1-dependent transcriptional program is suppressed in NOTCH-driven CP tumors. Importantly, CPCs in humans consist of tumor cells with a solitary primary cilium and exhibit profound defects multi-ciliogenesis program. Together, these results indicate that a solitary primary cilium is crucial for CPC development, whereas multi-ciliogenesis circuit possesses tumor suppressive functions and may represent a novel therapeutic target in CPC.

Abstract P1-05-13: Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer

Abstract Background and Aims: Triple-negative breast cancer (TNBC) is the most aggressive subtype, and is often associated with mutations in the breast cancer susceptibility protein (BRCA) genes, which are known tumor-suppressor genes involved in DNA damage repair. Chromosomal instability (CIN) has been added to the hallmarks of cancer and is characterized by a frequent gain or loss of chromosomes during mitosis (i.e. chromosomal aberrations). BRCA1 and BRCA2 breast tumors develop by specific and distinct evolutionary paths, as their gene profiles and genome aberration spectra differ from each other and from those in sporadic BC. The lncRNA Non-Coding RNA Activated by DNA Damage (NORAD) has been demonstrated to contribute to the progression of several cancer entities by regulating genomic stability. Therefore, we wanted to explore a potential additive anti-cancerous effect by treating TNBC cells with a combination of NORAD silencing and PARP inhibition. Material and Methods: To clarify the potential clinical significance of NORAD in human TNBC, patient data from publicly available databases were analyzed. In order to further characterize NORAD, we performed phenotypic experiments after establishing an siRNA-mediated knock-down approach. Expression levels of NORAD as well as DNA-damage repair proteins were analyzed by qRT-PCR and Western Blot after combined cell treatment with NORAD silencing and PARP inhibition. To further unravel the molecular mechanism behind, we performed immunofluorescent experiments against markers for DNA-double strand breaks and homologous recombination efficiency (i.e. Rad51 and yH2AX) after TNBC treatment with NORAD siRNAs and PARP inhibitors. Results: High NORAD levels were identified to be a negative prognostic factor for disease-free survival of TNBC patients (HR with 95% CI: 2.4 (1.35-4.25); n=161; p=0.002). Furthermore, the NORAD gene is amplified in up to 3% of BC cases and mRNA levels are higher in tumor tissue when compared to healthy tissue. A qPCR based screening of several breast cancer cell lines showed an at least 4-times increased expression of NORAD when compared to normal mammary cells (i.e. MCF 12A). Knock-down of NORAD in TNBC cells resulted in a slightly reduced proliferation as well as to a decreased expression of several DNA repair proteins. Immunofluorescent experiments of TNBC cells treated with the PARP inhibitor olaparib and/or after NORAD silencing revealed increased yH2AX-dependent nuclear foci formation when compared to control conditions, indicating augmented DNA double-strand breaks after treatment. Conclusion: Our first data let us hypothesize that a combination of NORAD knock-down and PARPi treatment increases inhibition of DNA repair mechanisms and leads to a more pronounced “anti-tumorigenic” phenotype in TNBC cells. This approach could lead to the optimization of current treatment concepts. Furthermore, as increased NORAD expression is associated with an unfavorable patient survival, this lncRNA could serve as potential prognostic biomarker. Citation Format: Christiane Klec, Anita Kapeller, Felix Prinz, Christoph Trenk, Herbert Stöger, Martin Pichler. Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-13.

CS-04 INTEGRATED CLINICAL, HISTOPATHOLOGICAL, AND MOLECULAR DATA ANALYSIS OF 190 CENTRAL NERVOUS SYSTEM GERM CELL TUMORS FROM THE IGCT CONSORTIUM

BACKGROUNDWe integrated clinical, histopathological, and molecular data of central nervous system germ cell tumors to provide insights into their management.METHODSData from the Intracranial Germ Cell Tumor Genome Analysis Consortium were reviewed. A total of 190 cases were classified as primary GCTs based on central pathological reviews.RESULTSAll but one of the cases that were bifocal (neurohypophysis and pineal glands) and cases with multiple lesions including neurohypophysis or pineal gland were germinomas (34 of 35). Age was significantly higher in patients with germinoma than other histologies. Comparison between tumor marker and histopathological diagnoses showed that 18.2% of histopathologically diagnosed germinomas were marker-positive and 6.1% of non-germinomatous GCTs were marker-negative, suggesting a limitation in the utility of markers or histopathology alone using small specimens for diagnosis. Comparison between local and central histopathological diagnoses revealed a discordance of 12.7%. Discordance was significantly less frequent in biopsy cases, implying difficulty in detecting all histopathological components of heterogeneous GCTs. Germinomas at the typical sites (neurohypophysis or pineal gland) showed a better PFS than those at atypical sites (p=0.03). A molecular-clinical association study revealed frequent MAPK pathway mutations in males (51.4 vs 14.3 %, p=0.007), and PI3K/mTOR pathway mutations in basal ganglia cases (p=0.004). Basal ganglia cases also had frequent chromosomal losses. Some chromosomal aberrations (2q, 8q gain, 5q, 9p/q, 13q, 15q loss) showed potential prognostic significance.CONCLUSIONSThese in-depth findings of this study regarding the clinical and molecular heterogeneity will increase our understanding of the pathogenesis of this enigmatic tumor.

Integrated clinical, histopathological, and molecular data analysis of 190 central nervous system germ cell tumors from the iGCT Consortium.

We integrated clinical, histopathological, and molecular data of central nervous system germ cell tumors to provide insights into their management. Data from the Intracranial Germ Cell Tumor Genome Analysis (iGCT) Consortium were reviewed. A total of 190 cases were classified as primary germ cell tumors (GCTs) based on central pathological reviews. All but one of the cases that were bifocal (neurohypophysis and pineal glands) and cases with multiple lesions including neurohypophysis or pineal gland were germinomas (34 of 35). Age was significantly higher in patients with germinoma than other histologies. Comparison between tumor marker and histopathological diagnoses showed that 18.2% of histopathologically diagnosed germinomas were marker positive and 6.1% of non-germinomatous GCTs were marker negative, suggesting a limitation in the utility of markers or histopathology alone using small specimens for diagnosis. Comparison between local and central histopathological diagnoses revealed a discordance of 12.7%. Discordance was significantly less frequent in biopsy cases, implying difficulty in detecting all histopathological components of heterogeneous GCTs. Germinomas at the typical sites (neurohypophysis or pineal gland) showed a better progression-free survival than those at atypical sites (P = 0.03). A molecular clinical association study revealed frequent mitogen-activated protein kinase (MAPK) pathway mutations in males (51.4% vs 14.3%, P = 0.007), and phosphatidylinositol-3 kinase/mammalian target of rapamycin (PI3K/mTOR) pathway mutations in basal ganglia cases (P = 0.004). Basal ganglia cases also had frequent chromosomal losses. Some chromosomal aberrations (2q, 8q gain, 5q, 9p/q, 13q, 15q loss) showed potential prognostic significance. The in-depth findings of this study regarding clinical and molecular heterogeneity will increase our understanding of the pathogenesis of this enigmatic tumor.

Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

Renal cell carcinoma (RCC) accounts for 2%–3% of all adultmalignancies, representing the seventh most common cancer inmen and the ninth most common cancer in women [1].Worldwide, there are ∼209000 new cases and 102000 deathsper year. The incidence of all stages of RCC has increased overthe past several years, contributing to a steadily increasing mor-tality rate per unit population. Active and passive cigarettesmoking is an established risk factor for RCC as well as hyper-tension. However, anti-hypertensive medications such as diure-tics are not independently associated with RCC development.RCC also appears to be more common in patients with obesity,end-stage renal failure, acquired renal cystic disease and tuber-ous sclerosis.Approximately 2%–3% of RCC are hereditary and severalautosomal dominant syndromes are described, each with a dis-tinct genetic basis and phenotype, the most common one beingVon Hippel Lindau disease.In recent years, many new genes associated with RCC havebeen reported (such as PBRM1, SETD2, BAP1). Their roles inpathogenesis and as prognostic biomarkers are currently underinvestigation.

Blast Fungal Genomes Show Frequent Chromosomal Changes, Gene Gains and Losses, and Effector Gene Turnover.

Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance.

Abstract 2653: Cell proliferation and Cathepsin L expression are inversely correlated in bladder cancer progression and patient survival

Abstract In 2017 there will be an estimated 79,000 new cases of bladder cancer (UBC) resulting in 16,870 deaths. A better understanding of UBC progression and prognostic factors could improve patient management. UBC anatomic stage is determined by depth of invasion. Stage 0 is non-invasive, Stage 1 invades the sub-mucosa, Stage 2 invades the detrusor muscle, Stage 3 invades perivesical fat, and Stage 4 invades local or distant organs. 70-80% of UBCs are superficial, but can recur locally and be multifocal. Muscle invasive bladder cancers (MIBCs) can metastasize to pelvic lymph nodes or distant organs. To better understand the molecular mechanisms of UBC progression we measured cell proliferation utilizing the molecular marker of proliferating cells Ki 67 (Dako) and immunohistochemically detected expression of the lysosomal cysteine proteinase Cathepsin L (Abcam) in archived surgical specimens from 145 UBC patients. A board certified pathologist diagnosed and graded each histologic specimen and marked three areas for photography and image analysis. Clinical information and follow-up was obtained from existing medical records and the Kentucky Cancer Registry. IRB approval for the study was obtained from St. Elizabeth Healthcare who also donated the FFPE surgical specimens. We hypothesized that the % of proliferating tumor cells would increase with loss of cell differentiation and that Cathepsin L expression would increase with depth of tumor invasion into and beyond the detrusor muscle. Patients with high grade tumors (Grades 3 and 4) had a poor survival compared to patients with low grade tumors (p &amp;lt; 0.0001) and patients with MIBC (Stages 2-4) had a poorer survival that patients with superficial tumors (Stages 0 - 1) (p &amp;lt; 0.0001, HR = 18.63). Cell proliferation as determined by % of Ki 67 labeled nuclei was highly correlated with stage and grade (p &amp;lt;0.0001). In contrast Cathepsin L expression measured either semi-quantitatively by histoscore (area X intensity of stain) or quantitatively by computer assisted image analysis was negatively correlated with stage, grade, and Ki 67 labeling (p &amp;lt;0.0001). Recently multiple subtypes of MIBC have been identified, including basal tumors with squamous differentiation and luminal tumors. The highest rate of cell proliferation and the lowest expression of Cathepsin L in our data set were found in MIBC with squamous differentiation. Ki 67 labeling in UBC was highly prognostic of poor patient survival (p = 0.0003), whereas Cathepsin L expression as measured either by histoscore or image analysis was highly prognostic of good patient survival (p &amp;lt; 0.0001). Since Cathepsin L is encoded by the CTSL gene located on chromosome 9, these data are consistent with published reports of frequent genetic loss and mutations of chromosome 9 in UBC. These data also strongly support the value of UBC cell proliferation as a prognostic marker for stratifying UBC patients. Citation Format: Larry E. Douglass, Michelle C. Robillard, Dan Stelzer, Mariah Dooley, James A. Deddens, Julia H. Carter. Cell proliferation and Cathepsin L expression are inversely correlated in bladder cancer progression and patient survival [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 2653.

Unstable Allotetraploid Tobacco Genome due to Frequent Homeologous Recombination, Segmental Deletion, and Chromosome Loss

The types of mutations and their corresponding frequencies are difficult to measure in complex genomes. In this study, a high-throughput method was developed to identify spontaneous loss-of-function alleles for the resistance gene N and the transgenic avirulence gene P50 in allotetraploid tobacco. A total of 2134 loss-of-function alleles of the N gene were identified after screening 14 million F1 hybrids. Analysis of these mutants revealed striking evolutionary patterns for genes in polyploids. Only 14 of the loss-of-function mutations were caused by spontaneous point mutations or indels, while the others were caused by homeologous recombination (with a frequency of ∼1/12 000) or chromosome loss (∼1/15 000). Loss of the chromosome with the P50 insertion occurred at a similar frequency (∼1/13 000), and the frequency of spontaneous segmental deletion in this chromosome was ∼1/16 000. Both homeologous recombination and chromosome loss considerably decreased the viability of the mutants. Our data suggest that the high mutation rate in polyploids is probably due to the occurrence of homeologous recombination and the tolerance of large mutations such as chromosome loss in polyploid genomes. Frequent mutations tend to drive polyploids to extinction unless a novel mutation helps the polyploid to effectively compete with diploids or find a new ecological niche.

Tumor CDKN2A-Associated JAK2 Loss and Susceptibility to Immunotherapy Resistance

Poor clinical responses to checkpoint blockade with anti-CTLA-4 and anti-PD-1 antibodies in melanoma have recently been associated with acquired IFNγ resistance that protects tumor cells from the antiproliferative and pro-apoptotic cytokine activity. IFNγ-resistant melanoma cells very often lack functional expression of the IFNγ signaling pathway gene JAK2 due to gene deletions or inactivating gene mutations. Analyzing melanoma cell lines (n = 46, applying next-generation targeted sequencing and single nucleotide polymorphism arrays) as well as available genomic data sets from The Cancer Genome Atlas (TCGA) tumor tissue samples (cutaneous melanoma n = 367, lung squamous cell carcinoma n = 501, bladder urothelial carcinoma n = 408, breast invasive carcinoma n = 768, colorectal adenocarcinoma n = 257), we demonstrate that the frequent chromosomal losses of the tumor suppressor CDKN2A in melanoma and other tumor entities enhance the susceptibility to IFNγ resistance by concomitant deletion of the JAK2 gene (odds ratio = 223.17, 95% confidence interval = 66.91 to 1487.38, two-sided P = 7.6×10-46). Tumors with JAK2 mutations or homozygous JAK2 deletions demonstrate allelic losses covering both CDKN2A and JAK2. This suggests that patients with tumor chromosomal CDKN2A losses are susceptible to developing immunotherapy resistance and should be screened for JAK2 deficiency prior to and under immune checkpoint blocking therapy.

GATA4 loss of function in liver cancer impedes precursor to hepatocyte transition.

The most frequent chromosomal structural loss in hepatocellular carcinoma (HCC) is of the short arm of chromosome 8 (8p). Genes on the remaining homologous chromosome, however, are not recurrently mutated, and the identity of key 8p tumor-suppressor genes (TSG) is unknown. In this work, analysis of minimal commonly deleted 8p segments to identify candidate TSG implicated GATA4, a master transcription factor driver of hepatocyte epithelial lineage fate. In a murine model, liver-conditional deletion of 1 Gata4 allele to model the haploinsufficiency seen in HCC produced enlarged livers with a gene expression profile of persistent precursor proliferation and failed hepatocyte epithelial differentiation. HCC mimicked this gene expression profile, even in cases that were morphologically classified as well differentiated. HCC with intact chromosome 8p also featured GATA4 loss of function via GATA4 germline mutations that abrogated GATA4 interactions with a coactivator, MED12, or by inactivating mutations directly in GATA4 coactivators, including ARID1A. GATA4 reintroduction into GATA4-haploinsufficient HCC cells or ARID1A reintroduction into ARID1A-mutant/GATA4-intact HCC cells activated hundreds of hepatocyte genes and quenched the proliferative precursor program. Thus, disruption of GATA4-mediated transactivation in HCC suppresses hepatocyte epithelial differentiation to sustain replicative precursor phenotype.

MicroRNA-383 located in frequently deleted chromosomal locus 8p22 regulates CD44 in prostate cancer.

A major genomic alteration in prostate cancer (PCa) is frequent loss of chromosome (chr) 8p with a common region of loss of heterozygosity (LOH) at chr8p22 locus. Genomic studies implicate this locus in the initiation of clinically significant PCa and with progression to metastatic disease. However, the genes within this region have not been fully characterized to date. Here we demonstrate for the first time that a microRNA component of this region –miR-383- is frequently downregulated in prostate cancer, plays a critical role in determining tumor initiating potential and is involved in prostate cancer metastasis via direct regulation of CD44, a ubiquitous marker of PCa tumor initiating cells (TICs)/ stem cells. Expression analyses of miR-383 in PCa clinical tissues established that low miR-383 expression is associated with poor prognosis. Functional data suggests that miR-383 regulates PCa tumor initiating/ stem-like cells via CD44 regulation. Ectopic expression of miR-383 inhibited tumor initiating capacity of CD44+ PCa cells. Also, ‘anti-metastatic’ effects of ectopic miR-383 expression were observed in a PCa experimental metastasis model. In view of our results, we propose that frequent loss of miR-383 at chr8p22 region leads to tumor initiation and prostate cancer metastasis. Thus, we have identified a novel finding that associates a long observed genomic alteration to PCa stemness and metastasis. Our data suggests that restoration of miR-383 expression may be an effective therapeutic modality against PCa. Importantly, we identified miR-383 as a novel PCa tissue diagnostic biomarker with a potential that outperforms that of serum PSA.

Abstract 1934: MicroRNA-383 located in frequently deleted chromosomal locus 8p22 regulates prostate cancer stem cell marker CD44

Abstract A major genomic alteration in prostate cancer (PCa) is frequent loss of chromosome (chr) 8p with a common region of loss of heterozygosity (LOH) at chr8p22 locus. Genomic studies implicate this locus in the initiation of clinically significant PCa and with progression to metastatic disease. However, the genes within this region have not been fully characterized to date. Here we demonstrate for the first time that a microRNA component of this region -miR-383- is frequently downregulated in prostate cancer, plays a critical role in determining tumor initiating potential and is involved in prostate cancer metastasis via direct regulation of CD44, a ubiquitous marker of PCa tumor initiating cells (TICs)/ stem cells. Expression analyses of miR-383 in PCa clinical tissues established that low miR-383 expression is associated with poor prognosis. Functional data suggests that miR-383 regulates PCa tumor initiating/ stem-like cells via CD44 regulation. Ectopic expression of miR-383 inhibited tumor initiating capacity of CD44+ PCa cells. Also, ‘anti-metastatic’ effects of ectopic miR-383 expression were observed in a PCa experimental metastasis model. In view of our results, we propose that frequent loss of miR-383 at chr8p22 region leads to tumor initiation and prostate cancer metastasis. Thus, we have identified a novel finding that associates a long observed genomic alteration to PCa stemness and metastasis. Our data suggests that restoration of miR-383 expression may be an effective therapeutic modality against PCa relapse and metastasis. Importantly, we identified miR-383 as a novel PCa diagnostic biomarker with potential that outperforms that of serum PSA. Citation Format: Nathan Bucay, Kiran Sekhon, Varahram Shahryari, Yozo Mitsui, Guoren Deng, Z. Laura Tabatabai, Shahana Majid, Soichiro Yamamura, Rajvir Dahiya, Yuichiro Tanaka, Sharanjot Saini. MicroRNA-383 located in frequently deleted chromosomal locus 8p22 regulates prostate cancer stem cell marker CD44. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1934.

GENO-09LANDSCAPE OF GENOMIC ALTERATIONS IN PITUITARY ADENOMAS

BACKGROUND: The genetic underpinnings of sporadic pituitary adenomas remain incompletely understood, despite their prevalence in adults. We describe a comprehensive genetic characterization of the largest mixed cohort of pituitary macroadenomas, including hormonally active and atypical adenomas. METHODS: 42 pituitary adenomas were selected to represent a spectrum of histopathologic subtypes, de novo and recurrent tumors, and MIB-1 proliferative indices to interrogate the heterogeneity intrinsic to pituitary tumors. Whole-exome sequencing was performed on tumor and paired blood to identify somatic copy-number alterations, mutations, insertions and deletions. RESULTS: We appreciated two classes of pituitary tumors based upon the fraction of the genome disrupted. The first class (71% of tumors) had somatic copy-number alterations involving less than 6% of the genome; the other class (29%) had significantly greater levels of genomic disruption, up to 99% of the genome involved. The widespread genomic disruption observed in the second class was largely accounted for by arm-level events, with minimal involvement of focal events, which is rare among tumor types. Levels of genomic disruption were associated with phenotype. Among the less disrupted group, 87% (26/30) were clinically non-functional adenomas, whereas the majority of the disrupted group were functional or atypical null-cell adenomas. The high and low genomic disruption classes did not differ in mean MIB-1 proliferative index. Frequent loss of chromosomes 11 and 1p were observed, as well as significant recurrent amplifications of chromosome 5, 7, 9, 12, and 14q. On the whole, the tumors harbored a low rate of mutations, similar to other benign brain tumors. CONCLUSIONS: Genomic characterization of a large series of sporadic pituitary adenomas reveals two distinct classes of tumor, based on genomic disruption from broad copy-number alterations, and associates with tumor subtype.

ZBTB7A Suppresses Melanoma Metastasis by Transcriptionally Repressing MCAM.

The excessive metastatic propensity of melanoma makes it the most deadly form of skin cancer, yet the underlying mechanism of metastasis remains elusive. Here, mining of cancer genome datasets discovered a frequent loss of chromosome 19p13.3 and associated downregulation of the zinc finger transcription factor ZBTB7A in metastatic melanoma. Functional assessment of ZBTB7A-regulated genes identified MCAM, which encodes an adhesion protein key to melanoma metastasis. Using an integrated approach, it is demonstrated that ZBTB7A directly binds to the promoter and transcriptionally represses the expression of MCAM, establishing ZBTB7A as a bona fide transcriptional repressor of MCAM. Consistently, downregulation of ZBTB7A results in marked upregulation of MCAM and enhanced melanoma cell invasion and metastasis. An inverse correlation of ZBTB7A and MCAM expression in association with melanoma metastasis is further validated with data from analysis of human melanoma specimens. Together, these results uncover a previously unrecognized role of ZBTB7A in negative regulation of melanoma metastasis and have important clinical implications.

The functional BRCA1 rs799917 genetic polymorphism is associated with gastric cancer risk in a Chinese Han population.

BRCA1 is a crucial tumor suppressor which plays an essential role in maintaining genomic stability and integrity. Accumulated evidences demonstrated that there is frequent chromosome loss of BRCA1 or significant BRCA1 down-regulation via hypermethylation of its promoter in human gastric cancer specimens, highlighting the tumor-suppressing function of BRCA1 in gastric carcinogenesis. There is an rs799917 T>C single nucleotide polymorphism (SNP) located in the BRCA1 coding sequence (CDS). This SNP can disturb the interaction between BRCA1 mRNA and miR-638 and result in significantly decreased BRCA1 expression among carriers of rs799917C allele. In this study, we investigated the association between rs799917 and gastric cancer risk in a Chinese Han population using a case-control design. A total of 660 gastric cancer patients and 800 controls were enrolled and genotyped. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. We found that individuals with the rs799917 CT genotype was significantly associated with gastric cancer risk (OR = 1.81, 95% CI = 1.28-2.56; P = 0.001). Individuals having the rs799917 CC genotype had an OR of 1.40 (95% CI = 1.17-1.68; P = 2.2 × 10(-4)) for developing gastric cancer, compared with individual having the rs799917 TT genotype. However, stratified analyses did not find any evident gene-covariates interaction. Our results for the first time indicate that the functional BRCA1 rs799917 polymorphism contributes to gastric cancer susceptibility.