Deletion Of Tumor Suppressor Gene Research Articles

Quantitative Measurement of HER2/neu Oncogene Amplification and p53 Tumor Suppressor Gene Deletion by RT-PCR in Breast Cancer

Quantitative Measurement of HER2/neu Oncogene Amplification and p53 Tumor Suppressor Gene Deletion by RT-PCR in Breast Cancer

PTEN Loss Expands the Histopathologic Diversity and Lineage Plasticity of Lung Cancers Initiated by Rb1/Trp53 Deletion

IntroductionHigh-grade neuroendocrine tumors of the lung such as SCLC are recalcitrant cancers for which more effective systemic therapies are needed. Despite their histopathologic and molecular heterogeneity, they are generally treated as a single disease entity with similar chemotherapy regimens. Whereas marked clinical responses can be observed, they are short-lived. Inter- and intratumoral heterogeneity is considered a confounding factor in these unsatisfactory clinical outcomes, yet the origin of this heterogeneity and its impact on therapeutic responses is not well understood. MethodsNew genetically engineered mouse models are used to test the effects of PTEN loss on the development of lung tumors initiated by Rb1 and Trp53 tumor suppressor gene deletion. ResultsComplete PTEN loss drives more rapid tumor development with a greater diversity of tumor histopathology ranging from adenocarcinoma to SCLC. PTEN loss also drives transcriptional heterogeneity as marked lineage plasticity is observed within histopathologic subtypes. Spatial profiling indicates transcriptional heterogeneity exists both within and among tumor foci with transcriptional patterns correlating with spatial position, implying that the growth environment influences gene expression. ConclusionsThese results identify PTEN loss as a clinically relevant genetic alteration driving the molecular and histopathologic heterogeneity of neuroendocrine lung tumors initiated by Rb1/Trp53 mutations.

Trisomy-6: The Ying and Yang of Chromosome 6 Aberrations

Karyotypic aberrations of chromosome 6 (chr.6), include duplications (trisomy 6; +6), deletion of its short arm (del6p), uniparental disomy 6 (UPD6p), as well as various microduplications and microdeletions. Among many important genes, the HLA locus is located on 6p, and loss of heterozygosity and allelic loss have been described as means of immune escape in immune-mediated aplastic anemia (AA) and in relapse of myeloid neoplasia following allogeneic bone marrow transplant. The pathogenesis of duplication of chr.6 appears more complex and might involve deletion of a putative tumor suppressor gene (TSG) causing duplication of the HLA locus with increased HLA expression. We have encountered several patients with +6 who inspired us to investigate the clinical and molecular phenotypes of +6. A total of 29 +6 MDS patients were identified, of whom 16 had isolated +6 (Iso +6). When compared to normal karyotype (NK) MDS (n=420), median age was younger in Iso +6 (70 vs 42 years, P<.0001). While no differences were noted for white blood counts, Iso +6 patients had lower platelets (median 27 vs. 112 x 103/uL, P=.19), were more frequently anemic (median hemoglobin 8.4 vs. 9.5 g/dL, P=.04) and showed higher frequency of hypocellularity at marrow evaluation (62.5% vs. 9.5%, P=0.0016) as opposed to NK MDS. Indeed, 67% of Iso +6 patients had a clinical picture reminiscent of AA or MDS phenotype which might have evolved from an antecedent AA phase. However, 67% of Iso +6 MDS had high risk MDS (IPSS-R ≥3.5) and 37% transformed to AML. Thus, our cohort indicated a phenotypic dichotomy which include hypocellular MDS/AA and high-risk disease with a poor prognosis. Of all +6 MDS, mutations most commonly affected TP53 (69%) followed by EZH2 (19%). Recurrent copy number variations (CNV) such as micro-duplications and microdeletions may be used to identify culprit genes. Indeed, microdeletions involving HLA have previously hinted towards the pathogenic role of HLA deletions. We obtained CNV data and compared them to conventional germline chromosome 6 CNV gains publicly available in the Database of Genomic Variants (DGV). Genes pertaining to significantly duplicated segments were then identified using the UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgGateway). In our cohort, in addition to 833 cases with del6p, we have identified microduplications involving 6p locus in 115 (11.5%) NK MDS cases. Overall, a total of 1198 6p copy gains were found in these patients, ranging in size 0.09-7.8Mb and with a median of 0.12Mb. The genomic architecture of each segment was investigated using the UCSC Genome Browser, from which only complete gene duplications that mirror an extra chromosomal copy were considered. Complete duplications were most commonly seen in loci containing genes of DUSP22 (12.2%) and most importantly HLA-DRB1 (10.4%), HLA-DRB5 (10.4%), HLA-DQA1 (9.6%) and MICA (9.6%). All of these five genes were significantly enriched in NK MDS as compared to healthy controls (DUSP22 3% P<0.0001, HLA-DRB1 0.4% P<0.0001, HLA-DRB5 0.5% P<0.0001, HLA-DQA1 0.5% P<0.0001 and MICA 0.2% P<0.0001) (Fig 1). Interestingly, while checking the Cosmic Cancer Browser database (https://cancer.sanger.ac.uk), we noticed that copy gains involving HLA-DRB1 and HLA-DRB5 were also more common in primary AML than in healthy controls from DGV (3.6 vs 0.4%, P<0.0001 and 3.6 vs 0.5%, P<0.0001, respectively). While the involvement of the HLA genes hints towards an immune-mediated mechanism underlying the pathogenesis of +6 MDS, further involvement in primary AML may suggest a common mechanism through which CN gains of the HLA locus promote myelo-neoplasia. We stipulate that while clonal deletion or UPD of HLA locus can be a result of immune pressure as seen in AA and during GvL, clonal duplication may lead to increased autoantigens presentation beyond the TCR activation threshold, triggering immune reactions ultimately resulting in hypocellular marrow and AA-like presentation. Alternatively, +6 and related lesions may lead to risk allele duplication and consequential more efficient presentation of a specific antigenic peptide. In more advanced diseases, deletion of TSG may also play a role, with progression reflecting a somatic pressure to overcome immune inhibition. HLA constellation and mutational studies will be presented in the context of the findings of 6p duplications and microdeletion in MDS and AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 1594: Reversal of lineage plasticity in RB1/TP53-deleted prostate cancer through FGFR and Janus kinase inhibition

Abstract The inherent plasticity of tumor cells provides a mechanism of resistance to many molecularly targeted therapies, exemplified by adeno-to-neuroendocrine lineage transitions seen in prostate and lung cancer. Here we investigate the root cause of this lineage plasticity in a primary murine prostate organoid model that mirrors the lineage transition seen in patients. These cells lose luminal identity within weeks following deletion of Trp53 and Rb1, ultimately acquiring an Ar-negative, Syp+ phenotype after orthotopic in vivo transplantation. We performed single-cell transcriptomic analysis of a time-course experiment on the prostate organoid following Trp53 and Rb1 deletion. Critical to this study, we developed SEACells, a method that enumerates distinct, highly granular cell states, allowing for robust transcriptomic quantification. Leveraging the SEACell platform, we developed several graph-based computational approaches based on Markov absorption, diffusion maps, and attributed stochastic block models to quantify dynamic changes in plasticity. These quantitative models independently confirmed rapid collapse of cell-type fidelity in the form of a mixed luminal-basal phenotype following tumor suppressor gene deletion. These methods compute metrics for plasticity that we correlated to candidate driver gene programs. Among the strongest plasticity correlates, Jak-Stat and Fgfr signaling stood out as gene programs activated early in the time-course prior to any corresponding morphological changes. We further developed a regression-based approach to nominate ligand-receptor interactions that activate downstream Jak-Stat signaling, which identified Fgf-Fgfr interactions that were functionally validated with growth factor addition and pharmacological inhibition. Most strikingly, genetic or pharmacologic inhibition of Jak1/2 in combination with Fgfr blockade not only reversed the plastic state and restored organoids to their wild-type morphology, but also re-sensitized drug-resistant cells to antiandrogen therapy in models with residual AR expression. We additionally confirm early activation of Jak/Stat transcriptional programs in an Rb1/Trp53/Pten-deleted genetically engineered mouse model undergoing substantial cell-type diversification under plasticity in the context of the tumor microenvironment. Collectively, we show that lineage plasticity initiates quickly as a largely cell-autonomous process that is further increased in the in vivo setting, and through newly developed computational approaches, we identify a pharmacological strategy that restores lineage identity using clinical grade inhibitors. Citation Format: Joseph M. Chan, Wouter R. Karthaus, Manu Setty, Jillian R. Love, Samir Zaidi, Jimmy Zhao, Zi-ning Choo, Sitara Persad, Justin LaClair, Kayla E. Lawrence, Ojasvi Chaudhary, Ignas Masilionis, Linas Mazutis, Ronan Chaligne, Dana Pe'er, Charles Sawyers. Reversal of lineage plasticity in RB1/TP53-deleted prostate cancer through FGFR and Janus kinase inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1594.

918 Doxycycline regulatable ß-catenin demonstrates inducible immune evasion in a melanoma GEM model

BackgroundLack of response to checkpoint blockade immunotherapy has been linked to a deficiency of immune cell infiltration within the tumor microenvironment (TME). One demonstrated mechanism sufficient for the non-T cell inflamed TME is tumor cell-intrinsic activation of the β-catenin signaling pathway. Using genetically engineered mouse models (GEMMs), tumors constitutively expressing active β-catenin lack a robust endogenous T cell infiltrate and fail to respond to immunotherapies. In support of these mouse studies, human melanoma metastases with increased active β-catenin signaling exhibit decreased numbers of tumor infiltrating Batf3-driven cDC1 and CD3+ T cells. However, whether temporal activation and inactivation of β-catenin within the same developing tumor would alter immune cell infiltration is not known.MethodsA model was created in which tamoxifen-regulated Cre-recombinase mediates BRAFV600E oncogene activation and PTEN tumor suppressor gene deletion as well as expression of a doxycycline regulatable reverse transactivator. Upon administration of doxycycline via the drinking water to these animals, a non-degradable form of nuclear β-catenin becomes expressed. Immunofluorescence assays were performed assessing the β-catenin expression status in the tumor cells as well as immune cell infiltration within the TME. Additionally, immunotherapy efficacy experiments were performed.ResultsWe observed that administration of doxycycline to these animals drove expression of an active form of nuclear β-catenin. Activation of nuclear β-catenin resulted in a 2-fold decrease in the overall CD3+ T cells infiltration into the TME. Moreover, this decrease in immune infiltration also resulted in loss of anti-PD-L1 + anti-CTLA-4 therapy efficacy. We next performed studies assessing the kinetics with which β-catenin levels diminish upon doxycycline removal. Switching animals to regular drinking water resulted in rapid reduction of nuclear β-catenin levels, including 50 percent reduction after two days of doxycycline removal and almost complete reduction of nuclear β-catenin after four days.ConclusionsWe describe a novel mouse model in which we induce autochthonous melanoma tumors in mice along with inducible expression of a non-degradable, nuclear β-catenin modulated by doxycycline in the drinking water. Activation of β-catenin signaling in melanoma tumors resulted in reduction of immune cells in the TME as well as loss of checkpoint blockade immunotherapy efficacy. This activation can be rapidly reversed by removing doxycycline, allowing for future studies evaluating the consequences of turning off β-catenin once it has already driven a non-T cell-inflamed TME.AcknowledgementsThis work was supported by the Wissler Fellowship from the University of Chicago (SS) K99/R00 (NCI; SS), and R35CA210098 (TG).

Circulating Cell-Free DNA-Based Detection of Tumor Suppressor Gene Copy Number Loss and Its Clinical Implication in Metastatic Prostate Cancer.

Current liquid biopsy assays lack sufficient sensitivity to detect copy number loss, which limits the interrogation of critical tumor suppressor gene deletions during cancer progression and treatment. Here we describe a liquid biopsy assay with improved sensitivity for detection of copy number loss in blood samples with low levels of circulating tumor DNA, and demonstrate its utility by profiling PTEN, RB1, and TP53 genetic loss in metastatic prostate cancer patients.

Predictive Simulation Modeling of Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) Patient Genomics Identifies Targeted Combination Treatment Options

ETP-ALL, an orphan disease, is a subtype of T-ALL with poor prognosis and high risk of relapse after standard of care (SOC) chemotherapy. ETP-ALL is a genomically heterogeneous disease with many cases harboring complex karyotype and genetic mutations typically seen in myeloid neoplasias, such as acute myeloid leukemia (AML). Targeted therapies for ETP-ALL are limited because identified gene mutations are not actionable by currently available drugs. Unfortunately, empirical chemotherapy trials have yet to show significant improvements in clinical outcomes of ETP-ALL patients (pts), which makes treating ETP-ALL challenging. Ideally, selecting treatment for ETP-ALL is rationally designed from the panoply of unique molecular abnormalities observed in ETP-ALL cases. Recently, we developed a predictive simulation modeling method that projects protein network maps from the hundreds of leukemia genomic mutations observed in individual pts (PMID 27855285). Our computational modeling enables multi-gene/multi-drug simulations. We hypothesized that ETP-ALL is comprised of protein network disturbances distinct from other T-ALL cases. We furthermore hypothesized these unique ETP-ALL protein networks would lead to the discovery of new treatment combinations for pts with ETP-ALL.Aim: Identify novel combinations of drugs for ETP-ALL pts who have failed SOC therapy by using an integrated genomics and computational approach.Methods: 66 ETP-ALL pts genomic data (whole exome sequencing, copy number variations, cytogenetics) were collected from various sources including NCBI-GEO dataset (GSE28703) (N=12), Moffitt Cancer Center (N=6) (IRB201600284) and published literature (PMID 22237106, 19147408) (N=48). The genomic data for each profile were entered into a computational biology modeling (CBM) software (Cellworks Group), which generated a disease-specific protein network map using PubMed and other online resources. Disease-specific biomarkers were identified within the protein network maps using CBM technology. A digital library of FDA-approved drugs, along with investigational agents, was simulated on every pt model both as single agent and combinations at varying doses. Drug impact was assessed by quantitatively measuring drug effect on a cell growth score (CGS), which is a composite of cell proliferation, viability and apoptosis. Unique combinations that reduced the CGS by interacting with the pt specific disease-biomarkers were selected. Synergy of drug combinations was calculated using the Co-efficient of Drug Interaction (CDI) formula. Each novel therapeutic agent or drug combination was mapped to the pts genomics, and supported by a PubMed referenced scientific mechanism of action based on disease biology.Results: Of the 66 ETP-ALL profiles, 22/66 (33%) harbored actionable mutations, including NRAS, TET2, FLT3 and various deletions such as del(5q) or deletions in tumor suppressor genes including CDKN2A / 2B, TSC1 and others. 44/66 (67%) had many deleterious mutations that were not directly actionable by currently available drugs. The integrated genomics and CBM workflow identified targeted combination therapies for 59/66 (89.4%) of cases. Eighty-seven unique targeted combinations were identified across the 66 ETP-ALL pts in this study (Fig 1). Eleven of the identified combinations were synergistic, as calculated by CDI, and 5 of the combinations included idarubicin as one of the agents, while the most synergistic combination was lenalidomide + nelfinavir (CDI = 3.34). Analysis of CBM identified pt-specific biomarkers for 66/66 (100%) pts. Dysregulated protein networks unique to ETP-ALL pt cases included pathways involving TP53, MYC, NFKB1, FOXM1, RUNX3, STAT3, CTNNB1, E2F1 and CEBPA. Some of the key kinase biomarkers identified in the ETP-ALL profiles were AURKB, CSNK2A1, MAP3K14, RAF1, ERK and mTOR (Fig 2).Conclusions: Computational biology analysis of complex ETP-ALL genomics found a unique protein network that may be amenable to several new treatment combinations. This CBM approach identified personalized drug regimens in all cases, including those who had no actionable genetic mutations present. This predictive technology improves the utility of cancer genetic profiling and may enhance clinical decision-making to identify treatments for ETP-ALL pts who would otherwise have no options based on limited SOC alternatives. [Display omitted] DisclosuresKumar:Cellworks: Employment. K:Cellworks: Employment. Bhargav:Cellworks: Employment. Choudhary:Cellworks: Employment. L:Cellworks: Employment. Birajdar:Cellworks: Employment. Roy:Cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

FaNDOM: Fast Nested Distance-Based Seeding of Optical Maps

Optical mapping provides single-molecule readouts of the locations of fluorescently labeled sequence motifs on long fragments of DNA, resolved to nucleotide-level coordinates. With the advent of microfluidic technologies for manipulating large numbers of individual molecules, and advances in the imaging of fluorescent DNA tags, it is possible to inexpensively generate high coverage and long OM molecules (>150Kbp). In addition to its use in scaffolding for de novo assembly, optical map data can also be mapped to a reference for identifying genetic structural variants implicated in Mendelian diseases and cancer. To address the computational challenge, we introduce FaNDOM (Fast Nested Distance seeding of Optical Maps) - an optical map alignment tool which greatly reduces the search space of the alignment process. On 4 benchmark human data-sets, FaNDOM was significantly (4-14X) faster than competing tools while maintaining comparable sensitivity and specificity. We used FaNDOM to map variants in 3 cancer cell-lines and identified many structural variations, including deletion of tumor-suppressor genes, duplications, gene fusions and gene-disrupting rearrangements. FaNDOM is publicly available at (https://github.com/jluebeck/FaNDOM).

NFATc2-rearranged sarcomas: clinicopathologic, molecular, and cytogenetic study of 7 cases with evidence of AGGRECAN as a novel diagnostic marker

NFATc2-rearranged sarcomas (NFATc2-Sarcomas) are infrequent round cell tumors characterized by EWSR1-NFATc2 fusions and FUS-NFATc2 fusions. Although our knowledge on these neoplasms has increased recently, novel diagnostic tools and more comprehensive series are still needed. Here, we describe the features of a series of seven molecularly confirmed NFATc2-Sarcomas (EWSR1-NFATc2, n = 4; FUS-NFATc2, n = 3) and demonstrate the utility of AGGRECAN immunohistochemistry for their identification. Patients were four males and three females, ranging in age from 19 to 66 years (median: 33). All were primary bone tumors (femur, n = 4; tibia, n = 2; ilium, n = 1), frequently infiltrating the surrounding soft tissues. Treatment often consisted of neoadjuvant chemotherapy and surgery. Follow-up was available for six patients (median 18 months, range 5–102 months), three patients died of disease and four patients are currently alive. Histologically, tumors consisted of monotonous round cells growing in lobules and sheets in variable amounts of fibrous to myxoid stroma. Other findings included spindle cells, corded and trabecular architecture, nuclear pleomorphism, cartilaginous differentiation, and osteoid-like matrix. Histological response to neoadjuvant chemotherapy was poor in all resection specimens available for review (n = 4). Tumors were diffusely positive for AGGRECAN and CD99 (7/7), and a subset expressed Pan-Keratin (AE1-AE3; 3/6), S100 (2/6), BCOR (2/6), ETV-4 (2/5), WT1 (2/6), and ERG (2/5). Desmin, NKX3-1, and SATB2 were negative (0/6). Diffuse AGGRECAN staining was also seen in 8/129 round cell sarcomas used for comparison, including mesenchymal chondrosarcoma (7/26) and CIC-sarcoma (1/26). Array-CGH showed complex karyotypes with recurrent deletions of tumor suppressor genes (CDKN2A/B, TUSC7, and DMD) in three FUS-NFATC2 cases and a simpler profile without homozygous losses in one EWSR1-NFATc2 case. Segmental chromosomal gains covering the loci of the fusion genes were detected in both variants. Overall, our study confirms and expands previous observations on NFATc2-sarcomas and supports that AGGRECAN is a useful biomarker of these tumors.

Abstract PR09: Frequency and etiology of ctDNA-positive metastatic prostate cancer with BRCA2, ATM, or CDK12 mutations

Abstract Recent sequencing efforts have shown that genomic alterations within DNA damage repair (DDR) pathways are relatively common in metastatic prostate cancer (mPCa). Patients harboring DDR gene aberrations can respond poorly to standard-of-care androgen receptor (AR)-targeted therapy, potentially because defective DNA repair enables rapid tumor evolution. Research has shown that DDR-deficient mPCa is often genetically unstable due to its high mutation frequency; however, the specific differences in mutations and copy number alterations between DDR mutant and nonmutant tumors have not been established. We performed targeted next-generation sequencing on 1,214 cell-free DNA samples from 718 patients with mPCa. In 30 patients with disruption of BRCA2, ATM, or CDK12, we also sequenced archival primary tumor tissue. 118 patients had a deleterious germline or somatic mutation in at least one of the 22 DDR genes on our targeted panel. The most commonly altered were BRCA2 (n=41), ATM (n=20), and CDK12 (n=21), together present in ~14% of patients. Disruption of the intact second allele was detected in 96%, 82%, and 84% of patients with mutations in BRCA2, ATM, and CDK12, respectively. Patients with a mutation in any one of these genes had fewer mutations in TP53 than a control cohort of DDR-intact patients (p&amp;lt;0.05). However, BRCA2-defective patients had frequent compound deletions of tumor-suppressor genes (TP53, PTEN, RB1). In patients with CDK12 mutations, PTEN or RB1 deletion was rare and samples instead exhibited copy number gains of MDM2 and/or CCND1. ATM-deficient cases did not demonstrate the same degree of genomic instability as either BRCA2- or CDK12-defective disease. In summary, patients with BRCA2, ATM, or CDK12 gene disruption possess distinct patterns of genomic alterations. These findings might help clarify why some DDR-deficient mPCa cases represent a more aggressive disease subtype with a poor response to AR-targeted therapy. This abstract is also being presented as Poster A36. Citation Format: Evan Warner, Steven Yip, Matti Annala, Gang Wang, Arkhjamil Angeles, Arshia Beigi, Elena Schönlau, Amanda Wong, Sinja Taavitsainen, Gillian Vandekerkhove, Kevin Beja, Matti Nykter, Daniel Khalaf, Kim Chi, Alexander Wyatt. Frequency and etiology of ctDNA-positive metastatic prostate cancer with BRCA2, ATM, or CDK12 mutations [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR09.

Transcriptomic profiling of gamma ray induced mutants from the CGL1 human hybrid cell system reveals novel insights into the mechanisms of radiation-induced carcinogenesis

BackgroundSomatic cell hybrid systems generated by combining cancerous with non-cancerous cells provide useful model systems to study neoplastic transformation. Combined with recent advances in omics-based technologies, novel molecular signatures that drive radiation-induced carcinogenesis can be analyzed at an exceptional global level. MethodsHere, we present a complete whole-transcriptome analysis of gamma-induced mutants (GIM) and gamma irradiated control (CON) segregants isolated from the CGL1 (HeLa x normal fibroblast) human hybrid cell-system exposed to high doses of radiation.Using the Human Transcriptome Array 2.0 microarray technology and conservative discrimination parameters, we have elucidated 1067 differentially expressed genes (DEGs) between tumorigenic and non-tumorigenic cells. ResultsGene ontology enrichment analysis revealed that tumorigenic cells demonstrated shifts in extracellular matrix (ECM) and cellular adhesion profiles, dysregulation of cyclic AMP (cAMP) signaling, and alterations in nutrient transport and cellular energetics. Furthermore, putative upstream master regulator analysis demonstrated that loss of TGFβ1 signaling due to reduced SMAD3 expression is involved in radiation-induced carcinogenesis. ConclusionsTaken together, this study presents novel insights into specific gene expression and pathway level differences that contribute to radiation-induced carcinogenesis in a human cell-based model. This global transcriptomic analysis and our published tumor suppressor gene deletion loci analyses will allow us to identify and functionally test candidate nexus upstream tumor suppressor genes that are deleted or silenced after exposure to radiation.

Integrated Analysis of TP53 Gene and Pathway Alterations in The Cancer Genome Atlas

The TP53 tumor suppressor gene is frequently mutated in human cancers. An analysis of five data platforms in 10,225 patient samples from 32 cancers reported by The Cancer Genome Atlas (TCGA) enables comprehensive assessment of p53 pathway involvement in these cancers. More than 91% of TP53-mutant cancers exhibit second allele loss by mutation, chromosomal deletion, or copy-neutral loss of heterozygosity. TP53 mutations are associated with enhanced chromosomal instability, including increased amplification of oncogenes and deep deletion of tumor suppressor genes. Tumors with TP53 mutations differ from their non-mutated counterparts in RNA, miRNA, and protein expression patterns, with mutant TP53 tumors displaying enhanced expression of cell cycle progression genes and proteins. A mutant TP53 RNA expression signature shows significant correlation with reduced survival in 11 cancer types. Thus, TP53 mutation has profound effects on tumor cell genomic structure, expression, and clinical outlook.

Promoter hypermethylation-mediated downregulation of tumor suppressor gene SEMA3B and lncRNA SEMA3B-AS1 correlates with progression and prognosis of esophageal squamous cell carcinoma.

Frequent deletions of tumor-suppressor genes at chromosome 3p21.3 have been detected in esophageal squamous cell carcinoma (ESCC). As a candidate tumor suppressor gene, semaphorin 3B (SEMA3B) is located at 3p21.3 and is frequently inactivated in several tumors. However, the role and inactivation mechanisms of SEMA3B and its antisense long non-coding RNA (lncRNA) SEMA3B-AS1 in the carcinogenesis of ESCC have not been fully elucidated. The present study was conducted to investigate the role, epigenetic inactivation mechanisms, and prognostic value of SEMA3B and SEMA3B-AS1 in ESCC tumorigenesis and prognosis. Frequent downregulation of SEMA3B and SEMA3B-AS1 was detected in esophageal cancer cells and ESCC tissues, and the expression level of SEMA3B and SEMA3B-AS1 in ESCC tissues was correlated with TNM stage and lymph node metastasis. SEMA3B and SEMA3B-AS1 shared the same CpG island in the promoter region and the expression of both genes might be regulated by the promoter methylation status. Furthermore, transcription factor Sp1 activated SEMA3B or SEMA3B-AS1 transcription and the promoter hypermethylation of SEMA3B and SEMA3B-AS1 influenced Sp1 binding ability. Moreover, over-expression of SEMA3B and SEMA3B-AS1 suppressed the viability and invasion of esophageal cancer cells in vitro. SEMA3B-AS1 influenced the protein expression of SEMA3B. SEMA3B or SEMA3B-AS1 expression and promoter methylation status were correlated with ESCC patients' survival. Thus, these findings suggest that SEMA3B and SEMA3B-AS1 may act as tumor suppressors and may serve as potential targets for antitumor therapy.

Loss of Heterozygosity and Possible Loss of Allele Amplification in Homozygous Short Tandem Repeats Loci in Two ALL Patients with Central Nervous System and Bone Marrow Relapses

Introduction: Loss of heterozygosity (LOH) was described for many forms of cancer including leukemia. LOH may contribute to oncogenesis through the deletion of tumor suppressor genes due to appropriate allelic loss. LOH in short tandem repeats loci (STR) has been reported for solid tumors and is widely used as malignant cells population marker. It should be noted that little is known yet about possible loss of one of certain homozygous alleles in tumour cells and STR analysis might be a promising approach to investigate this phenomenon. STR analysis is used as a routine test for chimerism monitoring after bone marrow transplantation. It could not also be excluded that recipient's chimerism estimation may be distorted in some cases due to LOH issues.The aim: To investigate possible allele loss in homozygous loci in tumour cells and to assess the contribution of LOH to the error in mixed chimerism estimation.Patients and Methods: This study includes analysis of STR DNA-profiles of two ALL patients after allogeneic stem cells transplantation. Patient 1 was 54 years old female with B-ALL, she underwent allogenic BM transplantation but after it she had four central nervous system (CNS) relapses and three bone marrow relapses. Patient 2 was 23 years old male with preT-ALL, two CNS relapses: first before transplantation and second after it. The DNAs were isolated from bone marrow samples of patients in remission before transplantation, from donors blood samples, from cerebrospinal fluid during neuro relapse and from bone marrow samples. The presence of blast cells in bone marrow samples and cerebrospinal fluid was confirmed morphologically and cytogenetically. STR-profiles were assessed by polymerase chain reaction with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software.Results: The STR profile analysis for Patient 1 showed complete recipient chimerism in cerebrospinal fluid with LOH in 10 from 15 heterozygous loci (Fig.1a,b) comparative to control sample of DNA (bone marrow before transplantation). The same LOH pattern was detected in archival DNA sample from first spinal relapse. Mixed chimerism in bone marrow samples estimated by STR analysis was equal to blast cell percentage assessed by cytogenetics. Loss of non-informative allele in D16S539 (Fig.1b) didn't distort chimerism estimation. However decreased levels of mixed chimerism were calculated in two homozygous loci (Fig.1a,c). We suspected that this decrease could be caused by the loss of one allele amplification in homozygous locus. Therefore, we proposed the mathematical model f(x), where x - is donor percentage in chimera, f(x) - difference between real recipient percentage (100-x) and calculated for locus with loss of allele amplification ((100-x)/2:((100-x)/2+x))*100% (Fig.1c) to test this hypothesis. The highest difference in chimerism calculation (about 17%) is expected in about 40% of donor and 60% recipient percentage. For Patient 2 the STR analysis detected only one LOH locus in DNA samples from cerebrospinal fluid during CNS relapse before and after BMT. However this patient has eight homozygous STR loci and complete donor chimerism in bone marrow. To find possible “loss of allele amplification” we used the artificial chimera, the mix of Patient 2 and unrelated random healthy donor DNA (Fig.1d). We used control DNA of Patient 2 (bone marrow before BMT) to check chimerism level deviations in loci of interest. The decreased level of mixed chimerism was detected in two homozygous loci from cerebrospinal fluid DNA-sample. The decreased estimate for chimerism correlated with proposed math model. For the same STR loci in control DNA no chimerism deviations were detected in mixed artificial chimera (Fig.1e).Conclusions: The LOH loci pattern might be preserved during long period of disease for certain malignant clone. Mixed chimerism or artificial mixed chimera can be useful in finding of homozygous loci with loss of one allele amplification. We suppose homozygous loci are the preferable subject for investigating LOH mechanisms. Loci with “loss of allele amplification” might be the used as malignant cells population marker.This study was supported by the Russian Foundation for Basic Research (RFBR) grant 18-015-00399a. DisclosuresRisinskaya:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding. Chabaeva:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding. Kulikov:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding.

Whole genome duplication is an early event leading to aneuploidy in IDH-wild type glioblastoma.

Glioblastoma, the most frequent and lethal form of glioma, displays chromosome instability and recurrent somatic copy number alterations (SCNA). Chromothripsis and whole genome duplication (WGD) have been recently identified in cancer. In the present study, we analyzed SCNA and determine the ploidy pattern in 123 IDH-wild-type glioblastomas, using SNP array data. WGD and chromothripsis events were validated using, respectively, FISH and CTLPScanner. WGD was detected in 11.4% glioblastomas (14/123) and was associated with TP53 mutation (p = 0.0068). It was an early event occurring after the recurrent SCNA observed in diffuse high-grade gliomas. Glioblastomas with WGD were more aneuploid compared to glioblastomas without WGD (p < 0.0001). Chromothripsis occurred in 29.3% glioblastomas (36/123) and mostly affected chromosomes 7, 9 and 12, with amplification of oncogenes (EGFR, MDM2/CDK4), and homozygous deletion of tumor suppressor genes (CDKN2A). There was a significant association between chromothripsis and gene rearrangement at a given locus. WGD is an early genetic event significantly associated to TP53 mutation and leading to chromosome instability and aneuploidy in IDH-wild-type glioblastoma. Chromothripsis recurrently targets oncogenes and tumor suppressor genes that are key players in gliomagenesis and tumor progression. The occurrence of chromothripsis points to underlying gene rearrangements (including gene fusions), potential therapeutic targets in glioblastoma.

Concise Review: Molecular Cytogenetics and Quality Control: Clinical Guardians for Pluripotent Stem Cells.

Now that induced pluripotent stem cell (iPSC)‐based transplants have been performed in humans and organizations have begun producing clinical‐grade iPSCs, it is imperative that strict quality control standards are agreed upon. This is essential as both ESCs and iPSCs have been shown to accumulate genomic aberrations during long‐term culturing. These aberrations can include copy number variations, trisomy, amplifications of chromosomal regions, deletions of chromosomal regions, loss of heterozygosity, and epigenetic abnormalities. Moreover, although the differences between iPSCs and ESCs appear largely negligible when a high enough n number is used for comparison, the reprogramming process can generate further aberrations in iPSCs, including copy number variations and deletions in tumor‐suppressor genes. If mutations or epigenetic signatures are present in parental cells, these can also be carried over into iPSCs. To maximize patient safety, we recommend a set of standards to be utilized when preparing iPSCs for clinical use. Reprogramming methods that do not involve genomic integration should be used. Cultured cells should be grown using feeder‐free and serum‐free systems to avoid animal contamination. Karyotyping, whole‐genome sequencing, gene expression analyses, and standard sterility tests should all become routine quality control tests. Analysis of mitochondrial DNA integrity, whole‐epigenome analyses, as well as single‐cell genome sequencing of large cell populations may also prove beneficial. Furthermore, clinical‐grade stem cells need to be produced under accepted regulatory good manufacturing process standards. The creation of haplobanks that provide major histocompatibility complex matching is also recommended to improve allogeneic stem cell engraftment. Stem Cells Translational Medicine 2018;7:867–875

Abstract 3427: Gatekeeper inactivation drives glioma progression into secondary glioblastoma

Abstract &amp;lt;Glioblastoma (GBM) is the most common and lethal form of brain cancer in humans. Median survival is 15 months with best available treatment. Most GBMs arise de novo (primary GBM), but 5 - 10% progress from lower grade gliomas (secondary GBM). As progression of low grade glioma into secondary GBM significantly impacts prognosis, a better understanding of this process is paramount for treatment and monitoring of affected patients. In this study, we applied whole genome and transcriptome sequencing to primary glioma and relapsed secondary GBM tissue from seven patients with progression. All primary gliomas carried IDH1 mutations, and in all cases the mutation was inherited by the secondary GBM. ATRX alterations in all five astrocytomas and TERT promoter mutations in both 1p19q-codeleted oligoastrocytomas were also inherited in progressed tumors. In five patients, progression was associated with increased genomic instability, whereas mutation load was significantly increased in two other patients. One of them exhibited a hypermutation signature caused by a mutation in the proofreading domain of DNA polymerase epsilon, while the second had lost both copies of the DNA mismatch protein MSH2. In addition, both 1p19q-codeleted tumors had acquired focal inactivating deletions of the protein tyrosine phosphatase PTPRD at progression, suggesting a novel driver mechanism for GBM progression. The most common progression-related genomic alterations were CDKN2A deletions, TP53 mutations, RB1 deletions, PTEN deletions, and deletions of genes crucial to the double strand break repair pathway. Taken together, progression into secondary GBM was significantly related to deletions in tumor suppressor genes as well as TP53 mutations. Disruption of these gatekeepers appears to be a significant mechanism for glioma progression.&amp;gt; Citation Format: Kirsi Johanna Granberg, Matti Annala, Serafiina Jaatinen, Joonas Haapasalo, Olli Yli-Harja, Hannu Haapasalo, Wei Zhang, Matti Nykter. Gatekeeper inactivation drives glioma progression into secondary glioblastoma [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 3427.

Abstract 388: p16/MTAP status as a model for investigating gene-environment interactions in metabolism

Abstract While the consequences of tumor suppressor gene deletions have been extensively characterized, little is known about how concurrent deletions of neighboring genes can influence the interaction of cancer cells with the environment. Using a panel of tissue-matched cancer cell lines exhibiting either wild-type or homozygous deletions of the p16 locus, metabolomics analysis showed that concurrent deletion of the proximal gene encoding the methionine salvage enzyme methylthioadenosine phosphorylase (MTAP) is associated with a subtle but reproducible global shift in metabolite levels. However, MTAP status does not appear to be predictive of metabolic responsiveness to restriction of key nutrients that feed into and out of the methionine cycle. Of particular interest is the finding that cell lines exhibit remarkable heterogeneity in responsiveness to nutrient availability, seemingly independent of MTAP status or tissue of origin. Similarly, MTAP-deleted cell lines exhibit variable global metabolic reprogramming in response to acute ectopic expression of MTAP, and maintain their heterogeneous responsiveness to nutrient restriction. These findings illustrate the exceptional metabolic diversity across cancer cell lines despite similar culture conditions, demonstrating the usefulness of examining a variety of cell lines in studies of cancer cell metabolism. Citation Format: Sydney M. Sanderson, Peter Mikhael, Ziwei Dai, Jason W. Locasale. p16/MTAP status as a model for investigating gene-environment interactions in metabolism [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 388.

GENE-44. CHROMOTHRIPSIS LEADS TO CHROMOSOMAL REARRANGEMENTS AND GENE FUSIONS IN PRIMARY GLIOBLASTOMAS

AbstractBACKGROUNDGlioblastoma (GB) is the most frequent and lethal form of diffuse glioma. GB displays chromosome instability and recurrent somatic copy number alterations. Recently, chromothripsis (CT) has been identified in cancer patients. It is a cataclysmic event in which a single or a few chromosomes (chr) are shattered and then randomly reassembled in a derivative chr, sometimes with additional double-minute chr and gene amplification. It may also disrupt tumor suppressor genes or lead to oncogenic fusion genes. Such genes may represent potential therapeutic targets. CT and consequent gene fusions have not been well described in large GB cohorts.METHODSWe used pan-genomic single nucleotide polymorphism (SNP) arrays (CytoSNP 850K, Illumina) to identify CT patterns in 183 primary GB. We performed RNA sequencing in 19 cases with CT. We validated the gene fusions detected by RT-PCR and Sanger sequencing.RESULTSCT occurred in 38.3 % of GB (70/183 cases) and mostly affected chrs 7, 9 and 12, with amplification of oncogenes (e.g., EGFR, MDM2/CDK4), and homozygous deletion of tumor suppressor genes (e.g., CDKN2A). We detected and validated 26 gene fusions (17 in-frame and 9 out-of-frame) in 15 of the 19 cases RNA-sequenced. The 17 in-frame fusions (from 11 patients) were all located in CT regions. They were intrachromosomal events involving nearby genes. We identified novel gene fusions implicating (potential) oncogenes such as ACVR1, MDM2, HMGA2, EGFR, MET, and MAPK4. Some of these fusions may be recurring events like the FGFR3-TACC3 fusion. Additional GB cases with CT are being RNA-sequenced.CONCLUSIONCT is a frequent event in GB. It recurrently targets key genes in gliomagenesis/tumor progression and leads to gene fusions in GB. Thorough analysis of genomic regions affected by CT will most likely lead to the identification of novel fusion genes, potential therapeutic targets in this fatal disease.