Gene Expression Array Data Research Articles

Genome-Wide DNA Methylation in Early-Onset-Dementia Patients Brain Tissue and Lymphoblastoid Cell Lines.

Epigenetics, a potential underlying pathogenic mechanism of neurodegenerative diseases, has been in the scope of several studies performed so far. However, there is a gap in regard to analyzing different forms of early-onset dementia and the use of Lymphoblastoid cell lines (LCLs). We performed a genome-wide DNA methylation analysis on sixty-four samples (from the prefrontal cortex and LCLs) including those taken from patients with early-onset forms of Alzheimer's disease (AD) and frontotemporal dementia (FTD) and healthy controls. A beta regression model and adjusted p-values were used to obtain differentially methylated positions (DMPs) via pairwise comparisons. A correlation analysis of DMP levels with Clariom D array gene expression data from the same cohort was also performed. The results showed hypermethylation as the most frequent finding in both tissues studied in the patient groups. Biological significance analysis revealed common pathways altered in AD and FTD patients, affecting neuron development, metabolism, signal transduction, and immune system pathways. These alterations were also found in LCL samples, suggesting the epigenetic changes might not be limited to the central nervous system. In the brain, CpG methylation presented an inverse correlation with gene expression, while in LCLs, we observed mainly a positive correlation. This study enhances our understanding of the biological pathways that are associated with neurodegeneration, describes differential methylation patterns, and suggests LCLs are a potential cell model for studying neurodegenerative diseases in earlier clinical phases than brain tissue.

Genome-wide profiling of dysregulated piRNAs and their target genes implicated in oncogenicity of tongue squamous cell carcinoma

PIWI-interacting RNAs (piRNAs) are single-stranded, 23–36 nucleotide long RNAs that regulate gene expression in the germline but are also detected in some cancers. However, there are no reports yet on piRNA expression in tongue squamous cell carcinoma (TSCC), the most common oral cancer (80–90% percent of all oral cancers). We performed small RNA and whole transcriptome sequencing in H357 tongue cancer and HOK cells (GEO database accession numbers: GSE196674 and GSE196688). We also examined nine published sets of gene expression array data of TSCC tissues from the GEO database to decode piRNAs and their putative targets that may be involved in tumorigenesis. We identified a pool of 16,058 and 25,677 piRNAs in H357 and HOK, respectively, among which 406 are differentially expressed. We also found that 2094 protein-coding genes are differentially expressed in either TSCC tissues or cell lines. We performed target predictions for these piRNAs, pathway and disease function (DF) analyses, as well as qRT-PCR validation of piRNA-target pairs. These experiments revealed one up-regulated (FDFT1) and four down-regulated (OGA, BDH1, TAT, HYAL4) target genes that are enriched in 11 canonical pathways (CPs), with postulated roles in the initiation and progression of TSCC. Downregulation of piR-33422 is predicted to upregulate the FDFT1 gene, which encodes a mevalonate/cholesterol-pathway related farnesyl-diphosphate farnesyltransferase. The FDFT1 appears to be involved in the largest number of oncogenesis-related processes and is interacting with statins, which is a classical cancer drug. This study provides the first evidence of the piRNome of TSCC, which could be investigated further to decode piRNA-mediated gene regulations in malignancy and potential drug targets, such as FDFT1.

A Bioinformatics Perspective on the Dysregulation of Ferroptosis and Ferroptosis-related Immune Cell Infiltration in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent dementia worldwide, but its pathophysiology and molecular events remain unknown. Herein, we first analyzed the differential expression pattern of patients' AD hippocampus through gene expression array data from the GEO database. Notch2nl, TGFB1I1, and LTF were up-regulated in AD patients, while ARPC1A, CHGB, and MPV17 down-regulated. Second, dysregulation of ferroptosis related genes was demonstrated from our data: PCBP2 and FTL significantly up-significant in AD hippocampus, while VDAC2, LPCAT3, GSS, ACSL4, and ACSL6 significantly down-regulated. The protein-protein interactions (PPI) network revealed that FTL was involved in iron metabolism and utilization, while ACSL4 and ACSL6 were involved in a polyunsaturated fatty acids metabolism network. Gene correlation analysis on differential expressed genes (DEGs) indicated that ferroptosis regulates a series of biological processes and pathways related to AD pathogenesis. Third, ferroptosis-related DEGs regulated the immune cell infiltration pattern in the AD hippocampus, characterized by decreased memory B cells, increased memory resting CD4+ T cells, memory activated CD4+ T cells, and resting NK cells. The altered expression of ferroptosis-related DEGs affected the infiltration of specific immune cell types. The model constructed by the seven ferroptosis-related differential genes may accurately predict the outcome of AD occurrence. Finally, qPCR validation on these ferroptosis-related DEGs in APPswe/PSEN1dE9 mice confirmed the dysregulated expression of Pcbp2, FTL, GSS, and ACSL4 in the AD hippocampus and forebrain. In conclusion, our results supported the conception that the AD brain revealed dysregulated ferroptosis and immune cell infiltration.

The Effect of Cytomegalovirus on Pediatric Acute Lymphoblastic Leukemia

Background: Recent evidence supports the role of cytomegalovirus (CMV) in the development of childhood ALL. The underlying mechanism and CMV's role in the leukemic cell phenotype is unknown, but CMV typically interacts with the host immune system allowing the virus to survive in a latent state; it may be that this immune dysregulation affects the risk of ALL. This study aims to explore the association of CMV and ALL at the time of leukemia diagnosis, using AML cases as a control and further, to determine whether CMV affects certain subgroups of ALL patients such as specific ethnicities, age groups, or cytogenetic subtypes.Methods: Pediatric diagnostic leukemia bone marrow samples obtained from the California Childhood Leukemia Study were screened for the presence of CMV DNA using a custom-designed droplet digital PCR assay. A total of 869 cases were analyzed including 125 AML cases and 744 ALL cases. Demographic and clinical features were compared between patients found to be CMV positive (cases with any detectable CMV positive droplets) and those who were CMV negative (cases with no detectable CMV positive droplets). The effect of the level of CMV viral DNA load was also assessed. For a subset of cases (n=61), Affymetrix Array gene expression data were available and differential gene expression performed to compare CMV positive cases with high viral load to CMV negative cases. Odds ratios and confidence intervals were estimated using logistic regression.Results: ALL cases were more likely to be CMV positive compared to AML (OR: 2.50; CI 1.00, 5.47, p = 0.039 for CMV highest quintile vs. CMV negative). Within ALL cases, B-cell ALL (B-ALL) was significantly associated with CMV positivity compared to T-cell ALL (T-ALL) (OR: 2.93; CI: 1.01, 8.52, p = 0.048 for CMV highest tertile vs. CMV negative). Further subtype analysis of B-ALL cases revealed CMV positivity to be significantly associated with high hyperdiploidy, one of the most common ALL subtypes, when compared to ETV6-RUNX1 (OR: 2.52; CI:1.34, 4.73, p = 0.004 for highest CMV tertile vs. CMV negative). CMV positive B-ALL cases were also more likely to harbor deletions of EBF1, a B-cell development gene, compared to CMV negative cases (OR: 6.10; CI: 1.09, 34.06, p = 0.04 for CMV 2 nd tertile vs. CMV negative; OR: 5.54; CI: 1.13, 27.18, p = 0.03 for CMV highest tertile vs. CMV negative). Differential gene expression analysis revealed 830 genes to be significantly differentially expressed between the highest quintile of CMV positive cases and CMV negative cases, and gene ontology analysis revealed upregulation of processes involved in viral infection and replication. Specifically, cytokine signaling pathways including IL-1, IL-8, and IL-7 were upregulated in CMV positive cases while Th1 and the pathway facilitating crosstalk between dendritic cells and natural killer cells were downregulated. Interestingly, B-cell receptor signaling was also upregulated in CMV positive cases.Conclusion: Our results support the hypothesis that CMV plays an enhanced role in leukemia development in specific subtypes of ALL, and not in AML development. The ability of CMV to interact with the host immune system, highlights immune dysregulation as a potential mechanism by which CMV contributes to risk of ALL. Gene expression analysis on a subset of cases revealed differentially expressed genes to be enriched in pathways involved in immune response, suggesting a potential role for active CMV infection in the leukemic phenotype. The patterns of up- and downregulation in these pathways were consistent with the host response to CMV. Additionally, acute CMV infection has been shown to promote B-cell activation and proliferation. Further, CMV seropositive individuals have been reported to have altered immune responses even with the virus in a latent state highlighting the virus's effect on B-cells. In our study we also found B-cell receptor signaling to be upregulated in CMV positive cases. This is consistent with the known effects of CMV in a typical host, but in patients with leukemia it provides an interesting potential link between CMV infection and development of pediatric ALL. These intriguing results require validation and warrant continued investigation of the role of CMV in pediatric leukemia development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Based on bioinformatics analysis lncrna SNHG5 modulates the function of vascular smooth muscle cells through mir-205-5p/SMAD4 in abdominal aortic aneurysm.

ObjectiveThe aim of this study was to explore expression profiles of long noncoding RNA (lncRNA)‐messenger RNA (mRNA) in abdominal aortic aneurysm (AAA) patients. Further, we explored the mechanisms by which lncRNA SNHG5 modulates the function of vascular smooth muscle cells (VSMC) in AAA.MethodsHuman gene expression profile GSE57691 dataset, was retrieved from Gene Expression Omnibus database. The dataset included gene expression array data of 49 AAA patients and 10 control aortic specimens from organ donors. To explore the main roles of the biological network, differentially expressed lncRNA and mRNAs in the aortic aneurysm (AAA) and normal aortic specimens were determined. Differentially expressed lncRNA and mRNAs were then used to construct a competing endogenous RNA (ceRNA) network using Cytoscape software, and the five key lncRNA were identified. SNHG5 which was significantly downregulated in the AAA was chosen and analysis showed that it regulates mir‐205‐5p and SMAD4 by binding to mir‐205‐5p. Double luciferase reporter gene assays, RNA immunoprecipitation, and RNA knockdown studies were used to establish the relationship between SNHG5 and mir‐205‐5p. Apoptosis rate was determined using flow cytometry, whereas cell proliferation was evaluated using Edu, and 24 well Transwell assay. Western blot analysis was used to determine protein expression levels.ResultsThe five differentially expressed lncRNAs were significantly correlated with 34 microRNAs and 112 mRNAs. mRNAs in the ceRNA network are implicated in protein binding, signal transduction, DNA and RNA transcription, development, and cell differentiation. SNHG5 was downregulated in the AAA and acts as a molecular sponge for mir‐205. Downregulation of SNHG5 induces expression of mir‐205‐5p. Increased mir‐205‐5p expression level inhibits SMAD4 production, thus inhibiting proliferation and migration and promotes apoptosis of smooth muscle cells.ConclusionBioinformatics were used to explore molecular mechanism of AAA progression. The findings of this study show that lncRNA SNHG5 regulates proliferation and apoptosis of VSMC cells through modulation of the mir‐205‐5p/SMAD4 axis. Therefore, SNHG5 is a potential therapeutic target for AAA disease.

Comprehensive Profiling of Somatic Mutations Which Define Primary Disease and Relapse in Various Acute Leukemia Subtypes

AbstractAbstract 783Acute leukemia comprises several distinct subtypes. Apart from certain specific cytogenetic abnormalities associated with each of these subtypes, acquisition of additional somatic mutations is crucial to disease development. The present study is a comprehensive exploration of somatic mutations associated with several leukemia subtypes to catalog pattern of genetic changes associated with each category of leukemia. Moreover, the focus of the study is on identifying relapse specific mutations, thereby enabling us to understand the evolutionary relationship between clones present in primary leukemia and relapse.Whole exome sequencing was performed on 5–10 matched diagnosis, relapse and remission samples from each AML subclass characterized by either t(8;21), t(15;17), FLT3-ITD, MLL-PTD or normal cytogenetics (without FLT3-ITD and MLL-PTD) AML. Identification of somatic mutations and small indels was carried out using an automatic analysis pipeline which included 1) genome mapping using BWA, 2) calling for variant sites using SAMTOOLS, 3) annotation using ANNOVAR, and 4) filtering using Fisher exact test and sequence quality information. Candidate mutations were validated using Sanger sequencing and 5–10 nonsynonymous mutations per sample were found in each subtype, except the normal cytogenetics samples where 25–30 mutations were found, signifying a higher mutational load. In all subtypes, excluding APL, a large proportion of the mutations present at diagnosis were retained in relapse, suggesting a similar pattern of disease progression. In t(8;21) AML, we sequenced 10 paired diagnosis and relapse cases and identified 47 mutations shared between the two stages, while 10 mutations were diagnosis-specific and 35 were relapse-specific. Recurrent mutations were observed in KIT, TET2, TTN, and MGA genes in this subtype. In FLT3-ITD AML, 60 mutations in 50 different genes were found in 10 relapse and diagnosis cases. Amongst 50 mutations present at diagnosis, 38 were shared with the relapse samples. We observe recurrent mutations in several known genes including DNMT3A, WT1, RUNX1 and IDH1. In 5 cases of MLL-PTD AML, 42 genes were mutated, 24 of which were shared at both diagnosis and relapse. Similarly, a high prevalence of primary disease-associated mutations was also detected at relapse in normal cytogenetics AML, indicating persistence of disease clones during chemotherapy. In 8 cases of APL, 63 mutations in 61 genes occurred, with mutations in WT1 and PML appearing twice. Interestingly, while 4 cases shared at least one mutation between diagnosis and relapse stages, in the remaining 4 cases, the relapse resulted from independent clones.In acute lymphocytic leukemia exomes from 7 matched diagnosis and relapse pediatric cases, which should have done well but did poorly (normal cytogenetic or hyperdiploid cases), a similar rate of somatic mutations occurred as observed in myeloid malignancies. Interestingly, no non-synonymous mutations were detected in primary disease samples in 4 out of 7 cases, but each accumulated multiple mutations at relapse.The frequency of mutations in the newly identified mutant genes is currently being examined in a large cohort of diagnosis/relapse samples using custom target capture probes. We are integrating the exome sequencing data with gene expression and SNP array data for further selection of potential driver mutations. Pathway analysis is also being performed using the validated gene list from each leukemia subtype to explore affected biological processes. Finally, clonal evolution analysis using density-based clustering algorithm (DBSCAN) is being carried out to study the emergence of disease clones and its progression from primary disease to relapse stage. To our knowledge, this is the first large scale analysis of relapse leukemia in a variety of subtypes. Disclosures:Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Analysis of Monoamine and Organic Cation Transporter Expression in Neuroblastoma and Relationship with Overall Survival

Neuroblastoma (NB) is a neuroendocrine cancer with great clinical heterogeneity and most prevalent in children. Despite recent improvements in therapy, long term survival of high-risk NB patients is still below 50%. Although amplification of the proto-oncogene MYCN is a well-established risk factor in NB, little is known about prognosis markers for high-risk NB patients without MYCN amplification. On account of being a cancer originated from early sympathetic nerve cells, the majority of NB tumors express the norepinephrine transporter (NET). NET is a solute carrier (SLC) monoamine transporter known to play an important role in targeting radiopharmaceuticals (e.g. 123I- or 131I-mIBG) for NB diagnosis and treatment. However, little is currently known about the expression of other functionally related SLC transporters in NB. Here, we analyzed the expression of MYCN, NET and other monoamine and organic cation transporters in NB patient samples and explored their association with overall survival of those patients. In order to perform this analysis, we mined the data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program database. The TARGET database contains gene expression data of tumor samples from multiple NB clinical trials consisting of predominantly high-risk or relapsed NB patients with predetermined MYCN amplification status. To compare transporter expression in NB, we analyzed mRNAseq data of NET, the dopamine and serotonin transporters (DAT and SERT), the plasma membrane monoamine transporter (PMAT), vesicular monoamine transporters (VMAT1/2), organic cation transporters (OCT1-3), and multidrug and toxin extrusion proteins (MATE1/2-K) in NB tumor samples and normalized their expression to GAPDH. The association between transporter expression and the overall survival of patients was then explored using Gene Expression Array data from 243 NB patient samples matched to clinical outcomes. Patient samples were divided into high and low expression for each transporter and survival analysis was performed using Kaplan-Meier curves. Our data revealed that NET, PMAT and VMAT1/2 are highly expressed in NB tumors especially in MYCN non-amplified samples. In contrast, DAT, SERT, OCT1-3, and MATE1/2-K are minimally or not expressed in NB tumor samples. Interestingly, survival analysis using Kaplan-Meier curves and Log-rank test indicates a significant association between low expression of PMAT and VMAT1/2 and poor overall survival, especially in patients without MYCN amplification. Our study suggests that besides NET, PMAT and VMAT1/2 are highly expressed in NB tumor samples and can be explored for targeting therapeutics in NB tumor cells. The apparent association of PMAT and VMAT1/2 with overall survival suggests that these transporters could be further explored as biomarkers for disease progression and risk prediction for NB patients without MYCN amplification.

Revealing Epigenetic Factors of circRNA Expression by Machine Learning in Various Cellular Contexts.

SummaryCircular RNAs (circRNAs) have been identified as naturally occurring RNAs that are highly represented in the eukaryotic transcriptome. Although a large number of circRNAs have been reported, the underlying regulatory mechanism of circRNAs biogenesis remains largely unknown. Here, we integrated in-depth multi-omics data including epigenome, transcriptome, and non-coding RNA and identified candidate circRNAs in six cellular contexts. Next, circRNAs were divided into two classes (high versus low) with different expression levels. Machine learning models were constructed that predicted circRNA expression levels based on 11 different histone modifications and host gene expression. We found that the models achieve great accuracy in predicting high versus low expressed circRNAs. Furthermore, the expression levels of host genes of circRNAs, H3k36me3, H3k79me2, and H4k20me1 contributed greatly to the classification models in six cellular contexts. In summary, all these results suggest that epigenetic modifications, particularly histone modifications, can effectively predict expression levels of circRNAs.

Abstract A28: Intratumor heterogeneity and homologous recombination deficiency of high-grade serous ovarian cancer are associated with prognosis and molecular subtypes and change in treatment course

Abstract Background: High-grade serous ovarian cancers (HGSOC) are genomically characterized by homologous recombination deficiency (HRD) and TP53 mutations, which lead to intratumor heterogeneity (ITH). High degree of loss of heterozygosity (LOH) indicates HRD, a characteristic associated with sensitivity to platinum agents and poly-(ADP ribose) polymerase (PARP) inhibitors. This study aimed to reveal the relationship between HRD, ITH, and prognosis and analyze their changes during treatment. Methods: We obtained 573 single-nucleotide polymorphism (SNP) array and gene expression array data from The Cancer Genome Atlas. SNP array data were processed to calculate the Clonality Index (CI) and LOH scores. Gene expression array data were used for classifying molecular subtypes. Pathway analysis was performed by Ingenuity Pathway Analysis (IPA). Additionally, we obtained 33 samples from 20 HGSOC patients, including 4 samples from interval debulking surgery (IDS) and 9 samples from recurrent surgery. DNA extracted from FFPE specimens was analyzed with an OncoScan FFPE Assay Kit and the number of clones and the LOH score was calculated. GISTIC 2.0 was used to identify copy number aberrations in tumors. Results: We divided HGSOC samples into 2 groups by the CI and LOH scores, respectively. The high CI group (CI≧3) showed statistically shorter disease-free survival (DFS) and progression-free survival (PFS), but there was no statistically significant difference about overall survival (OS) (p<0.001, 0.01, p=0.419, respectively). The high LOH group (LOH score≧16) showed statistically longer DFS, PFS, and OS (p<0.001, 0.01, 0.001, respectively). Combining the two factors, the high LOH/low CI group showed a statistically good prognosis. In terms of molecular subtypes, the mesenchymal subtype, which had a poor prognosis, showed a high CI with statistically significant difference (p=0.0403) and the immunoreactive subtype, which had a good prognosis, showed a tendency to have a high LOH score (p=0.0762). With IPA analysis, it was predicted that the activation, migration, and adhesion of immune cells were activated in the high LOH group and high LOH/low CI group. Throughout treatment, the CI at primary surgery was 3 in 3 cases and 2 in 1 case, and at IDS, the CI decreased to 1 in all 4 cases, and then the CI increased at the secondary surgery in all 3 recurrent cases. The LOH score did not change in 2 cases and greatly decreased in 2 cases from primary surgery to IDS and then increased at secondary surgery. With GISTIC analysis, a pattern of the copy number variant compared between primary and recurrent surgery had few differences, but amplification of 8q24 was found at IDS with statistical significance. Conclusions: ITH and HRD were associated with prognosis in HGSOC. ITH of remaining tumors at IDS decreased compared with that of primary tumors. This study indicated that it is important to analyze tumors that remain after chemotherapy for investigating the mechanism of the development of chemoresistance. Citation Format: Hisamitsu Takaya, Hidekatsu Nakai, Kosuke Murakami, Noriomi Matsumura. Intratumor heterogeneity and homologous recombination deficiency of high-grade serous ovarian cancer are associated with prognosis and molecular subtypes and change in treatment course [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A28.

Abstract A03: PrOTYPE (Predictor of high-grade-serous Ovarian carcinoma molecular subTYPE): The development and validation of a clinical-grade consensus classifier for the molecular subtypes of high-grade serous tubo-ovarian cancer

Abstract Background: Gene expression-based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC) are distinguished by differential immune and stromal infiltration and may provide opportunities for targeted therapies. Integration of molecular subtypes into clinical trials has been hindered by inconsistent subtyping methodology. Methods: Adopting two independent approaches, we derived and internally validated algorithms for molecular subtype prediction from gene-expression array data in 1,650 tumors. We applied resulting models to assign labels to 3829 HGSOCs from the Ovarian Tumor Tissue Analysis (OTTA) consortium evaluated on NanoString. Using the labeled NanoString data, we developed, confirmed, and validated a minimal gene set, clinical-grade test and prediction tool. We also used the OTTA dataset to evaluate associations between molecular subtype, biologic, and clinical features. Findings: The locked-down test included a model with 55 genes that predicted HGSOC molecular subtype with >95% accuracy. Subtype varied between primary and metastatic site taken at the time of primary surgery, and was significantly associated with age, stage, CD8+ lymphocyte infiltration, residual disease, and outcome. In multivariable models, molecular subtypes lose their prognostic significance in the presence of risk factors such as residual disease and BRCA1/2 germline mutations. Interpretation: We validated the Predictor of high-grade-serous Ovarian carcinoma molecular subTYPE, or PrOTYPE, following the Institute of Medicine guidelines for the development of omics-based tests. This simple-to-use, cost-effective, fully defined, and locked-down clinical-grade assay will facilitate molecular subtype stratification into clinical trial design. PrOTYPE will allow for objective assessment of HGSOC molecular subtype predictive value in precision medicine applications. Citation Format: Aline Talhouk, Joshy George, Chen Wang, Ellen Goode, Susan Ramus, Jennifer Doherty, David Bowtell, Michael Anglesio, OTTA Consortium. PrOTYPE (Predictor of high-grade-serous Ovarian carcinoma molecular subTYPE): The development and validation of a clinical-grade consensus classifier for the molecular subtypes of high-grade serous tubo-ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A03.

Network Pharmacology Analysis of Anticonvulsant Effect of Xiao Chai Hu Tang

Epilepsy is the second largest disease after headache in the neurology department of the hospital. Previous studies have shown that Xiao Chai Hu Tang (XCHT) has a certain therapeutic effect on epilepsy. We used the gene expression array data of epilepsy patients, and the relevant data of the Chinese medicine components in the TCMSP database and DrugBank database, combined with bioinformatics analysis methods, to construct herbs-compounds-targets (HCT) network and protein-protein interaction (PPI) core network of XCHT. The result suggests that Caveolin 1 (CAV1) may be a key target in the treatment of epilepsy, and β-carotene may have potential antiepileptic activity. In addition, the PPI core function network is enriched in the process of transcription factor regulation, and the related protein is located on the nuclear chromosome and closely related to the immune process, which provides a reference for other researchers.

Abstract C103: ERRβ: Validating a novel target for triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC)—defined as estrogen receptor- (ER), progesterone receptor- (PR), and human epidermal growth factor receptor 2- (HER2) negative—is a highly aggressive form of breast cancer prevalent in African-American (AA) women. Current treatment strategies rely on cytotoxic chemotherapy because ER- and HER2-targeted therapies are ineffective in TNBC, leaving a tremendous need for new effective therapies with less toxicity. Nuclear receptors are highly druggable targets. Classical examples include ligand-regulated receptors like ER, but there is opportunity for orphan nuclear receptors (ONR) to be studied as potential targets for cancer therapy. Our lab has published that increased mRNA expression of ONR estrogen-related receptor beta (ERRβ, gene symbol ESRRB) correlates with better recurrence- and distant metastasis-free survival in women with TNBC/basal-like breast cancer. We also showed that treatment with a small-molecule agonist ligand for ERRβ (DY131) has growth-inhibitory and antimitotic activity in TNBC cell lines. The goal of our current work is to comprehensively characterize ERRβ copy number, mRNA, protein expression, and ERRβ function in TNBC. ESRRB copy number was determined in 106 primary breast tumors (TNBC n=56, nonTNBC n=50) by Agilent SurePrint G3 Human CGH Microarray in a cohort AA and Caucasian (CA) women. ESRRB mRNA expression was determined in The Cancer Genome Atlas (TCGA) Breast Cancer RNAseq data. Association of ESRRB mRNA with overall survival was determined in Illumina gene expression array data from METABRIC. ERR protein expression is being assessed by immunohistochemistry (IHC) in a tissue microarray of 150 primary breast tumors (50 TNBC, 50 ER+, 50 HER2+). Ongoing studies are focusing on genetic modification of cell lines, specifically the overexpression of ERRβ in nontransformed mammary epithelial and TNBC cell lines. DNA: In both TNBC and nonTNBC, AA patients had a markedly higher frequency of ESRRB copy number loss than CA patients (2 *p=0.012 for TNBC, p=0.052 for nonTNBC). RNA: In TCGA breast cancer patients by PAM50 subtype, TNBC/basal-like patients have significantly lower ESRRB mRNA expression than Luminal A patients (*p=0.0015). Among systemically untreated patients in the METABRIC dataset, low ESRRB mRNA is significantly associated with poor overall survival in TNBC (hazard ratio 0.24, 95% confidence interval 0.07-0.85, *p=0.016). Protein: Analysis of ERR protein expression by IHC is ongoing. Function: Focusing on overexpression of ERRβ in cell lines, we aim to analyze multiple phenotypes including cell proliferation, cell invasion and migration, and differential gene expression. Conclusions: Breast tumors from AA women are significantly more likely to have reduced ESRRB copy number vs. CA women. TNBC patients have lower ESRRB mRNA expression and low ESRRB mRNA expression predicts for poor overall survival in TNBC. These data suggest that ERRβ expression has prognostic value in breast cancer, particularly in TNBC. Citation Format: Aileen I. Fernandez, Garrett Graham, Bálazs Györffy, Luciane R. Cavalli, Rebecca B. Riggins. ERRβ: Validating a novel target for triple-negative breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C103.

Gene X environment: the cellular environment governs the transcriptional response to environmental chemicals

BackgroundAn individual’s response to environmental exposures varies depending on their genotype, which has been termed the gene-environment interaction. The phenotype of cell exposed can also be a key determinant in the response to physiological cues, indicating that a cell-gene-environment interaction may exist. We investigated whether the cellular environment could alter the transcriptional response to environmental chemicals. Publicly available gene expression array data permitted a targeted comparison of the transcriptional response to a unique subclass of environmental chemicals that alter the activity of the estrogen receptor, xenoestrogens.ResultsThirty xenoestrogens were included in the analysis, for which 426 human gene expression studies were identified. Comparisons were made for studies that met the predefined criteria for exposure length, concentration, and experimental replicates. The cellular response to the phytoestrogen genistein resulted in remarkably unique transcriptional profiles in breast, liver, and uterine cell-types. Analysis of gene regulatory networks and molecular pathways revealed that the cellular context mediated the activation or repression of functions important to cellular organization and survival, including opposing effects by genistein in breast vs. liver and uterine cell-types. When controlling for cell-type, xenoestrogens regulate unique gene networks and biological functions, despite belonging to the same class of environmental chemicals. Interestingly, the genetic sex of the cell-type also strongly influenced the transcriptional response to xenoestrogens in the liver, with only 22% of the genes significantly regulated by genistein common between male and female cells.ConclusionsOur results demonstrate that the transcriptional response to environmental chemicals depends on a variety of factors, including the cellular context, the genetic sex of a cell, and the individual chemical. These findings highlight the importance of evaluating the impact of exposure across cell-types, as the effect is responsive to the cellular environment. These comparative genetic results support the concept of a cell-gene-environment interaction.

Intratumor heterogeneity and homologous recombination deficiency of high-grade serous ovarian cancer are associated with prognosis and molecular subtype and change in treatment course

ObjectiveHigh-grade serous ovarian cancers (HGSOC) are genomically characterized by homologous recombination deficiency (HRD) and TP53 mutations, which lead to intratumor heterogeneity (ITH). This study aimed to reveal the relationship between HRD, ITH and prognosis and analyze their changes during treatment. MethodsWe obtained 573 SNP array and gene expression array data from The Cancer Genome Atlas. SNP array data were processed to calculate the Clonality Index (CI) and loss of heterozygosity (LOH) scores. Gene expression array data were used for classifying molecular subtypes. Additionally, we obtained 33 samples from 20 HGSOC patients, including 4 samples from interval debulking surgery (IDS) and 9 samples from recurrent surgery. ResultsWe divided HGSOC samples into 2 groups. The high CI group showed a high recurrent risk, and the high LOH group showed a statistically good prognosis. Combining the two factors, the high LOH/low CI group showed a statistically good prognosis. In terms of molecular subtypes, the mesenchymal subtype, which had a poor prognosis, showed a high CI with statisitically significant difference and the immunoreactive subtype, which had a good prognosis, showed a tendency to have a high LOH score. Throughout treatment, the CI decreased to one at the IDS (n = 4) and then increased at recurrence (n = 3). LOH scores greatly decreased in two cases at the IDS. ConclusionsITH and HRD were associated with prognosis in HGSOC. ITH decreased after neoadjuvant chemotherapy, suggesting that the chemo-resistant cancer clone remains after chemotherapy.

Inhibition of the stem cell factor 248 isoform attenuates the development of pulmonary remodeling disease.

Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.

Abstract 475: Clinical significance of eif5-mimic protein 1 in pancreatic cancer

Abstract Background: Pancreatic cancer (PC) is the most lethal cancer in humans, with a 5-year survival rate of less than 10%. Thus, identification of prognostic biomarkers as well as molecular therapeutic targets should be critical. Translational reprogramming in cancer has been attracting attention. Several studies have reported that genomic alternations of a translational regulatory gene contributing to malignancy. eIF5-mimic protein 1 (5MP1), previously termed BZW2 is a member of the bZIP superfamily of transcription factors. 5MP1 is reported to be upregulated and has a pro-tumor effect via activation of the Akt/mTOR signaling pathway in osteosarcoma. However, the role and mechanisms of 5MP1 in PC remains unknown. The aim of this study is to clarify the clinical significance of 5MP1 expression in PC. Material and method: Firstly, we analyzed public datasets of PC (178 RNA-sequencing data from The Cancer Genome Atlas, 36 gene expression array data from GSE 15471, 45 gene expression array data from GSE 28735, 69 gene expression array data from GSE 62452, 145 primary PC and 46 normal pancreas gene expression array data from GSE 71729, 45 Copy number variation and expression array data from Broad-Novartis Cancer Cell Line Encyclopedia(CCLE)). Then we performed Gene set enrichment analysis (GSEA) to identify the gene sets which were associated with the expression of 5MP1 in PC. Finally, we investigated the localization of 5MP1 by immunohistochemical staining data of THE HUMAN PROTEIN ATLAS and also confirmed with staining our histological specimens. Results: In tumor tissues, mRNA expression of 5MP1 was higher than normal tissues (t-test. p<0.05). Survival analysis showed high expression of 5MP1 was significantly associated with poor prognosis (Kaplan-Meier method, Log-rank test. p<0.05). Analysis of pancreas cancer cell line CCLE indicated a significant positive correlation between Copy number variation and expression of 5MP1 ( r = 0.41 ). Moreover, GSEA revealed the relationship between enhanced 5MP1 expression and cycle-related gene set. Immunohistochemistry staining showed prominent expression of 5MP1 in the plasma membranes of PC cells. Conclusion and Significance: We demonstrated that 5MP1 was overexpressed in PC cells, and the high expression was associated with poor prognosis. These results suggest that 5MP1 may be associated with tumor progression and can be a promising biomarker for a patient with a poor prognosis of PC. Now we are performing a knockdown experiment for 5MP1 to clarify its biological significance in PC. Citation Format: Yushi Motomura, Takaaki Masuda, Kuniaki Sato, Atsushi Fujii, Akihiro Kitagawa, Hiroaki Wakiyama, Kensuke Koike, Junichi Takahashi, Dai Shimizu, Shotaro Kuramitsu, Miwa Noda, Yusuke Tsuruda, Hajime Otsu, Yosuke Kuroda, Hidetoshi Eguchi, Katsumi Sakamoto, Masakazu Hirakawa, Hiroshi Honda, Koshi Mimori. Clinical significance of eif5-mimic protein 1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 475.

The Transcription Factor Deaf1 Modulates Engrailed-1 Expression to Regulate Skin Appendage Fate

The Transcription Factor Deaf1 Modulates Engrailed-1 Expression to Regulate Skin Appendage Fate

Induction of APOBEC3B cytidine deaminase in HTLV-1-infected humanized mice.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL). Following viral infection with HTLV-1, certain infected cells exhibit clonal proliferation. Additional genetic and epigenetic changes in these clonally proliferating cells provide them with the selective advantage of growth, which eventually results in ATL. The precise mechanism, however, has yet to be completely elucidated. It has previously been established that APOBEC3 enzymes are potent host-antiviral restriction factors. Conversely, previous studies have reported that the A3B level is increased in tumor virus infections, such as those caused by HBV and HPV, suggesting that A3B exerts a function as a mutagen. Therefore, the present study analyzed the expression of APOBEC3 family members in various HTLV-1 infection states. No significant differences were observed in the expression between healthy donors and patients with HTLV-1-associated myelopathy. Although no significant changes in the expressions of A3C, A3D, A3F and A3G between uninfected and HTLV-1-infected mice were observed, an increased A3B expression was observed in a short-term humanized mouse model following HTLV-1 infection. In a long-term humanized mouse model following HTLV-1 infection, the gene expression array data exhibited an apparent increase in A3B and CADM1, which are indicators of ATL. Collectively, the results of the present study suggest that A3B is likely involved in the development of ATL in HTLV-1-infected humanized mice.

The long-term prognostic and predictive capacity of cyclin D1 gene amplification in 2305 breast tumours

BackgroundUse of cyclin D1 (CCND1) gene amplification as a breast cancer biomarker has been hampered by conflicting assessments of the relationship between cyclin D1 protein levels and patient survival. Here, we aimed to clarify its prognostic and treatment predictive potential through comprehensive long-term survival analyses.MethodsCCND1 amplification was assessed using SNP arrays from two cohorts of 1965 and 340 patients with matching gene expression array and clinical follow-up data of over 15 years. Kaplan-Meier and multivariable Cox regression analyses were used to determine survival differences between CCND1 amplified vs. non-amplified tumours in clinically relevant patient sets, within PAM50 subtypes and within treatment-specific subgroups. Boxplots and differential gene expression analyses were performed to assess differences between amplified vs. non-amplified tumours within PAM50 subtypes.ResultsWhen combining both cohorts, worse survival was found for patients with CCND1-amplified tumours in luminal A (HR = 1.68; 95% CI, 1.15–2.46), luminal B (1.37; 1.01–1.86) and ER+/LN−/HER2− (1.66; 1.14–2.41) subgroups. In gene expression analysis, CCND1-amplified luminal A tumours showed increased proliferation (P < 0.001) and decreased progesterone (P = 0.002) levels along with a large overlap in differentially expressed genes when comparing luminal A and B-amplified vs. non-amplified tumours.ConclusionsOur results indicate that CCND1 amplification is associated with worse 15-year survival in ER+/LN−/HER2−, luminal A and luminal B patients. Moreover, luminal A CCND1-amplified tumours display gene expression changes consistent with a more aggressive phenotype. These novel findings highlight the potential of CCND1 to identify patients that could benefit from long-term treatment strategies.

Poor Overall Survival in Hyperhaploid Multiple Myeloma Is Defined By Double-Hit Bi-Allelic Inactivation of TP53

Introduction Hyperhaploid multiple myeloma is a rare numerical aberration group defined by a range of 24-34 chromosomes. We have previously shown that hyperhaploid myeloma is associated with a poor prognosis with a 5-year survival rate of 23.1%, compared to 64% for hyperdiploid myeloma, and 80.4% for those with a normal karyotype. It is known that hyperhaploid myeloma frequently has monosomy of chromosome 17, making it a high risk group, but no data are currently available on the mutational status of this interesting sub-group, or how the copy number difference arises.Methods We analyzed data from whole genome, whole exome, and targeted panel sequencing from 1141 newly diagnosed myeloma patients. Internal samples were selected for whole exome or targeted sequencing based on previous karyotype information, or were identified in the process of other sequencing studies. The CoMMpass dataset was screened for the presence of hyperhaploidy. Hyperhaploid samples without prior karyotype information were identified by conflicting copy number profile and B allele frequency information, where the samples had incorrectly been normalized to a diploid copy number. These samples were re-normalized to a haploid copy number. Copy number, B allele frequency, and mutations of key genes were examined.Results In the entire dataset 9 hyperhaploid samples were identified, of which 2 came from the CoMMpass dataset. From those with gene expression array data, 5/7 were GEP70 high risk and all belonged to the D1 hyperdiploid gene expression subgroup. Samples had a median of 13 monosomies (range 9-14), which in general were those not associated with trisomies in hyperdiploid samples. The chromosomes traditionally trisomic in hyperdiploid myeloma were disomic in hyperhaploid myeloma. We examined the B allele frequency of these disomic chromosomes and saw that they all retained heterodisomy. Retention of heterodisomy indicates that the method of generating hyperhaploidy is through deletion of the monosomic chromosomes, rather than reverting to a haploid genome followed by duplication of some chromosomes. Retention of heterodisomy was also seen on chromosome 18, which is not normally trisomic in hyperdiploid samples, indicating that heterodisomy of chromosome 18 may be essential for a viable plasma cell clone.We examined the hyperhaploid samples for frequently mutated genes and found that 8/9 (88.8%) of hyperhaploid samples had a mutation in TP53. This rate of mutation far exceeds the overall rate of mutation in newly diagnosed patients (5.5%), indicating an oncogenic dependency in this group. The sample without mutation of TP53 had only 9 monosomies, fewer than the other samples (12-14 monosomies), indicating there may be a prognostic difference that is dependent on the total chromosome count. All samples with TP53 mutation also had monosomy of chromosome 17, indicating bi-allelic inactivation of TP53. The variant allele frequency of the TP53 mutations was high (median=0.94), indicating that bi-allelic inactivation was a clonal event. No other significant mutations were found, including those that encode chromosome segregation or kinetochore proteins.Conclusions We have previously described bi-allelic inactivation of TP53 as Double Hit myeloma, and here we identify that hyperhaploid myeloma belongs to this poor prognosis group. The method of generating the hyperhaploid clone is through deletion of chromosomes, which may happen in a way that is similar to gain of chromosomes in hyperdiploid myeloma. These Double Hit patients may be good candidates for new therapies, but using next generation sequencing techniques researchers must be careful when normalizing data to correctly identify them as hyperhaploid rather than hyperdiploid, using copy number and B allele frequency data. DisclosuresDavies:MMRF: Honoraria; TRM Oncology: Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; ASH: Honoraria. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding.