Specific Gene Expression Signature Research Articles

The role of microglia in ischemic preconditioning.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic stimulus confers protection against a subsequent prolonged ischemic event. Initially thought to be due to mechanistic changes in neurons, our understanding of IPC has evolved to encompass a global reprogramming of the Central Nervous System (CNS) after transient ischemia/reperfusion that requires innate immune signaling pathways including Toll-like receptors (TLRs) and Type I interferons. Microglia are the CNS resident neuroimmune cells that express these key innate immune receptors. Studies suggest that microglia are required for IPC-mediated neuronal and axonal protection. Multiple paradigms targeting TLRs have converged on a distinctive Type I interferon response in microglia that is critical for preconditioning-mediated protection against ischemia. These pathways can be targeted through administration of TLR agonists, cytokines including interferon-β, and pharmaceutical agents that induce preconditioning through cross-tolerance mechanisms. Transcriptomic analyses and single cell RNA studies point to specific gene expression signatures in microglia that functionally shift these mutable cells to an immunomodulatory or protective phenotype. Although there are technological challenges and gaps in knowledge to overcome, the targeting of specific molecular signaling pathways in microglia is a promising direction for development of novel and effective pharmacotherapies for stroke. Studies on preconditioning in animal models, including nonhuman primates, show promise as prophylactic preconditioning treatments for selected at risk patient populations. In addition, our growing understanding of the mechanisms of IPC-mediated protection is identifying novel cellular and molecular targets for therapeutic interventions that could apply broadly to both acute stroke and chronic vascular cognitive impairment patients.

Abstract 274: The ATR inhibitor BAY 1895344 shows strong preclinical activity in lymphomas and appears associated with specific gene expression signatures

Abstract Introduction. Targeting the DNA repairing pathways represents an intriguing new approach for cancer treatment. BAY 1895344 is a potent, highly selective and orally available ATR inhibitor (Luecking et al, AACR 2017) in its early clinical development in patients with advanced solid tumors and lymphomas (NCT03188965). We performed evaluated its antitumor activity in a large panel of lymphoma cell lines. Methods. IC50s and caspase 3/7 activation were obtained in cell lines derived from human B (n=50) or T cell (n=9), murine (n=2) and canine (n=1) lymphomas exposed to increasing doses of BAY 1895344 for 72h. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation vs controls. Transcriptome data (Illumina HumanHT 12 Expression BeadChips, HTG Biomarker Panel) were analyzed with GSEA (statistical significance: absolute NES >1.5, FDR < 0.01). In vivo efficacy was evaluated in xenograft studies of human lymphoma in mice. Results. BAY 1895344 showed anti-tumor activity with a median IC50 of 60 nM (95%C.I.; 3-500 nM) across the 62 cell lines, comprising mainly germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL, n.=17), activated B-cell DLBCL (8), mantle cell lymphoma (MCL, 10), marginal zone lymphoma (6), T cell lymphomas (9). BAY 1895344 was mostly cytotoxic with apoptosis induction in 38/62 (61%) of the cell lines. Sensitivity was not affected by lymphoma histology or DLBCL cell of origin, TP53/BCL2/MYC status or ATM/ATR loss. A xenograft experiment (MCL Rec-1 cell line) demonstrated complete tumor remission with BAY 1895344 (50 mg/kg, twice daily, 3 days on and 4 days off). Comparing the gene expression profile of 4 less (IC50>200nM) vs 3 very sensitive (IC50<10nM) GCB-DLBCL cell lines, cell cycle regulation and DNA repair genes were positively associated with higher sensitivity (E2F targets; G2M checkpoint; DNA repair), while survival and inflammation transcripts with lower (TNFA signaling; IL2/STAT5 signaling). Similar results for E2F targets, G2M checkpoint, TNFA signaling and IL2/STAT5 signaling were confirmed in MCL cell lines. Finally, we compared the transcriptome obtained using an NGS-based platform in 30 B cell lymphoma cell lines divided by the median IC50. Genes involved in BRCA1 and/or ATM networks, RB1 and/or TP53 targets, WNT signaling were higher in the most sensitive cell lines, while the opposite was true for genes taking part to apoptosis, cytokine interaction pathway, inflammatory response and TNFA signaling via NFκB, and MAPK signaling. Conclusion. A strong anti-tumor activity, associated with specific gene expression signatures, was seen with BAY 1895344 in lymphoma models, providing further support for the on-going phase I study. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Filippo Spriano, Luciano Cascione, Alberto Arribas, Emanuele Zucca, Anastasios Stathis, Antje Margret Wengner, Francesco Bertoni. The ATR inhibitor BAY 1895344 shows strong preclinical activity in lymphomas and appears associated with specific gene expression signatures [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 274.

Abstract LB-029: Identification of predictive genetic signatures of Cytarabine responsiveness in acute myeloid leukemia using a novel 3D translational culture model of primary bone marrow

Abstract Acute myeloid leukemia (AML) is a malignancy with complex genetics, poor survival rates and high rates of chemotherapy resistance. This study reports the establishment of a novel bone marrow mononuclear cell 3D-culture model and the application of this model to define sensitivity and resistance biomarkers as evident by DNA and RNA expression signatures in response to standard Cytarabine (Ara-C) chemotherapy. By simulating the bone marrow microenvironment, we optimized the growth conditions using frozen bone marrow mononuclear cells (BMMC) derived from healthy donors. We validated our platform by demonstrating that healthy BMMCs are capable of differentiating into all hematopoietic lineages and various types of stromal cells. Using this platform, we then characterized BMMC samples from 47 AML patients. Sixty percent of AML samples were capable of proliferating and 23 samples demonstrated robust growth. RNA sequencing was performed for 3D and 2D cultures to determine which conditions are superior at maintaining original gene expression signatures as compared to the uncultured bone marrow, To elucidate specific genetic mutations and/or gene expression signatures associated with the ability of ex vivo growth of these AML donors, whole exosome sequencing and RNA Sequencing were performed. The model identified unique gene expression signatures that correlated with sensitive as well as resistant donors in response to Ara-C treatment. These data demonstrate the translational value of our platform which should be widely applicable to evaluate other therapies in AML. Citation Format: Haiyan Xu, Eric S. Muise, Sarah Javaid, Lan Chen, Razvan Cristescu, My Sam Mansueto, Nicole Follmer, Jennifer Cho, Kimberley Kerr, Rachel Altura, Michelle Machacek, Benjamin Nicholson, George Addona, Ilona Kariv, Hongmin Chen. Identification of predictive genetic signatures of Cytarabine responsiveness in acute myeloid leukemia using a novel 3D translational culture model of primary bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-029.

Abstract 730: Genomic characterization of hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a rare and aggressive kidney cancer

Abstract Background Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) is a rare autosomal-dominant syndromic disorder associated with germline FH mutations. Affected individuals have a predisposition to cutaneous and uterine leiomyoma’s, and are at increased risk of developing a renal cell carcinoma (HLRCC- associated RCC) with aggressive clinical outcome. However, aside from FH bi-allelic loss, additional genomic features of HLRCC are currently poorly understood. Method Five cases of HLRCC-associated RCC were characterized using integrated next generation sequencing (NGS) technology. This included an index case where multiple tumor sites (kidney, lung, liver, lymph node, skin, and uterine) from a single patient were characterized. For integrative analysis, in-house data were combined with five RCC cases with FH bi-allelic loss from The Cancer Genome Atlas Study. Single cell sequencing was carried out on normal human kidney to study the cell of origin for HLRCC- associated RCC. Results: Integrative NGS profiling and analysis of HLRCC-associated RCC tumors (n=10) revealed several important observations such as 1) a high frequency loss of whole chromosomes, which is reminiscent of tumors arising from the distal nephron, 2) low mutational burden similar to that observed in other kidney tumors and, 3) a HLRCC specific gene expression signature and discovery of a novel therapeutic candidate (HLRCC-TC1). Importantly, among renal tumors, HLRCC-TC1 was specifically expressed in HLRCC and other RCCs with poor clinical outcome. Application of siRNAs and neutralizing antibodies to HLRCC-TC1 reduced cell proliferation of the HLRCC cell line UOK262, but did not for that of control cell lines. Conclusion: The observed FH bi-allelic mutation along with recurrent chromosome-wide losses and integrative analysis with human kidney single cell sequencing data support a distal nephron origin for HLRCC- associated RCC. In addition, our study provides evidence for nominating HLRCC-TC1 as a novel cancer-specific therapeutic target. Further studies are needed to conduct an in-depth characterization of HLRCC-TC1. Citation Format: Pankaj Vats, Yuping Zhang, Sarvana Mohan Dhanashekaran, Narayanan Sathiya Pandi, Xuhong Cao, Fengyun Su, Sudhanshu Shukla, Rui Wang, Marcin Cieslik, Jincheng Pan, Christopher J. Ricketts, Prem Kumar Kumpati, Ajjai Alva, Marston W. Linehan, Rohit Mehra, Arul M. Chinnaiyan. Genomic characterization of hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a rare and aggressive kidney 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 730.

Systems Biology Approaches to Investigate Genetic and Epigenetic Molecular Progression Mechanisms for Identifying Gene Expression Signatures in Papillary Thyroid Cancer.

Thyroid cancer is the most common endocrine cancer. Particularly, papillary thyroid cancer (PTC) accounts for the highest proportion of thyroid cancer. Up to now, there are few researches discussing the pathogenesis and progression mechanisms of PTC from the viewpoint of systems biology approaches. In this study, first we constructed the candidate genetic and epigenetic network (GEN) consisting of candidate protein–protein interaction network (PPIN) and candidate gene regulatory network (GRN) by big database mining. Secondly, system identification and system order detection methods were applied to prune candidate GEN via next-generation sequencing (NGS) and DNA methylation profiles to obtain the real GEN. After that, we extracted core GENs from real GENs by the principal network projection (PNP) method. To investigate the pathogenic and progression mechanisms in each stage of PTC, core GEN was denoted in respect of KEGG pathways. Finally, by comparing two successive core signaling pathways of PTC, we not only shed light on the causes of PTC progression, but also identified essential biomarkers with specific gene expression signature. Moreover, based on the identified gene expression signature, we suggested potential candidate drugs to prevent the progression of PTC with querying Connectivity Map (CMap).

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

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

A specific gene expression signature for visceral organ metastasis in breast cancer

BackgroundVisceral organ metastasis is associated with poor survival outcomes in terms of metastasis free- and overall survival in breast carcinomas. Identification of a gene expression profile in tumours that selects a subpopulation of patients that is more likely to develop visceral organ metastases will help elucidate mechanisms for the development of distant metastases and could be of clinical value. With this study we aimed to determine genomic predictors that would help to distinguish breast cancer patients with more likelihood to develop visceral metastasis.MethodsGene expression profiling data of 157 primary tumours from breast cancer patients who developed distant metastases were analyzed and differentially expressed genes between the group of tumours with visceral metastasis and the those without visceral metastases were identified. Published data were used to validate our findings. Multivariate logistic regression tests were applied to further investigate the association between the gene-expression-signature and clinical variables. Survival analyses were performed by the Kaplan-Meier method.ResultsFourteen differentially expressed genes (WDR6, CDYL, ATP6V0A4, CHAD, IDUA, MYL5, PREP, RTN4IP1, BTG2, TPRG1, ABHD14A, KIF18A, S100PBP and BEND3) were identified between the group of tumours with and without visceral metastatic disease. Five of these genes (CDYL, ATP6V0A4, PREP, RTN4IP1 and KIF18A) were up-regulated and the other genes were down-regulated. This gene expression signature was validated in the training and in the independent data set (p 2.13e− 08 and p 9.68e− 06, respectively). Multivariate analyses revealed that the 14-gene-expression-signature was associated with visceral metastatic disease (p 0.001, 95% CI 1.43–4.27), independent of other clinicopathologic features. This signature has been also found to be associated with survival status of the patients (p < .001).ConclusionWe have identified an unique gene expression signature which is specific to visceral metastasis. This 14-gene-expression-signature may play a role in identifying the subgroup of patients with potential to develop visceral metastasis.

Abstract PD2-07: mRNA signatures predict response to durvalumab therapy in triple negative breast cancer (TNBC)– Results of the translational biomarker programme of the neoadjuvant double-blind placebo controlled GeparNuevo trial

Abstract Background: The GeparNuevo trial showed a numerical increase in the pCR rate to 53% vs 44%; p=0.281 compared to placebo in TNBC with the addition of the anti-PD-L1 antibody durvalumab to a neoadjuvant anthracycline-taxane containing chemotherapy (Loibl S et al. ASCO 2018). In a predefined subgroup analysis, a significant increase of the pCR rate was observed for patients that received durvalumab for 2 weeks alone prior to the start of chemotherapy in a window phase (61% vs 41%, p interaction=0.048), while the pCR rate was not increased for the subset of patients that did start durvalumab together with chemotherapy. Here we report the main results of the translational programme for GeparNuevo with a focus on mRNA signatures predictive for pCR in pretherapeutic core biopsies. Methods: A total of 162 baseline FFPE core biopsies were evaluable for expression of 2560 genes using the HTG EdgeSeq® system that combines a modified nuclease protection assay with next generation sequencing. Data was processed as recommended by the HTG and median transformed for further analyses. For differential gene expression analyses, the data was scale-normalized (TMM normalization; EdgeR package) and linear models were fit (limma package). Prior to these analyses, genes were filtered based on minimal expression (&amp;gt; 4) and variability (IQR &amp;gt; 1). As a first step, predefined immune-genes signature (TILs signature) (Denkert et al. JCO 2016) as well as IFN-gamma signatures were evaluated for correlation with pCR in logistic regression models. Subsequently, we performed a differential gene expression analysis according to therapy response for the durvalumab-arm and the placebo arm using the pre-filtered candidate genes. Gene names are not included in this abstract to allow filing of IP, but full gene names will be presented at the SABCS meeting. Results: The predefined TIL- and IFN-gamma signatures were associated with increased pCR rates in the complete cohort (TIL-signature: OR 1.44, 95% CI 1.15-1.82, p=0.002; IFN-Gamma-signature: OR 1.63, 95% CI 1.22-2.24, p=0.002) as well as in the durvalumab arm (p=0.012 and 0.042) and the placebo arm (p=0.050 and 0.011). These signatures were general pCR predictors without specificity for durvalumab response. Additional 44 genes were significantly (p&amp;lt;0.05) correlated with pCR in the durvalumab arm. Of those, 21 genes were upregulated and 23 genes were downregulated in pCR patients. 14 of the 21 upregulated genes are related to tumorbiologically relevant immune cell functions. A total of 6 of the 44 genes had a positive test for interaction (interaction p&amp;lt;0.05) with the therapy arm (durvalumab + NACT vs. placebo + NACT), suggesting that these genes might specifically predict response to durvalumab. Additional analyses investigating the role of molecular tumor subtypes, additional immune gene signatures and other subgroup analyses will be presented at the meeting. Conclusion: Our results show that specific immune-related gene expression signatures predict response to durvalumab in primary triple negative breast cancer. The trial was financially supported by Astra Zeneca and Celgene Citation Format: Loibl S, Sinn BV, Karn T, Untch M, Treue D, Sinn H-P, Weber KE, Hanusch CA, Fasching PA, Huober J, Zahm D-M, Jackisch C, Thomalla J, Blohmer J-U, Marmé F, Klauschen F, Rhiem K, Felder B, von Minckwitz G, Burchardi N, Schneeweiss A, Denkert C. mRNA signatures predict response to durvalumab therapy in triple negative breast cancer (TNBC)– Results of the translational biomarker programme of the neoadjuvant double-blind placebo controlled GeparNuevo trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-07.

Abstract PD5-03: Characterization of high TIL breast cancers reveals a prognostic and functionally distinct tissue-resident memory subpopulation

Abstract Background: Tumor infiltrating lymphocytes (TILs) assessed via light microscopy are prognostic and predictive in the early stage and advanced triple negative and HER2-amplified breast cancer (BC). Higher TILs can also identify patients more likely to benefit from anti-PD-1 therapy. In this study we interrogated T cell subsets that comprise high TILs to determine if distinct subpopulations are key mediators of anti-tumor immunity. Methods: We characterised TILs with a focus on CD3+ T cells in 129 primary and metastatic BC samples using flow cytometry, bulk RNASeq on flow sorted T cell populations, multiplex immunohistochemistry and microdroplet based single cell 3' mRNA sequencing on the 10X Genomics Chromium platform. Cell type specific gene expression signatures were determined from differential expression between putative T cell subpopulations. These signatures were investigated in clinical cohorts, including trial cohorts treated with pembrolizumab. Results: High TIL Infiltrates consisted primarily of CD3+ T cells, with both CD8 and CD4 populations. Unsupervised clustering of single cell sequencing identified 9 CD8 and CD4 subpopulations with distinct gene expression profiles. In addition to Tregs and CD8 effector memory (TEM) T cells, we found a CD8+ tissue resident memory (TRM) population expressing greater levels of T-cell checkpoints and cytotoxic markers compared to effector memory cells. In 2 primary tumours and 1 liver metastasis, bulk RNASeq of flow sorted TEM and TRM corroborated the single cell mRNASeq results. T cell receptor profiling (TCR) in the 3 samples found non-overlapping repertoires in the 2 primary tumours, but overlap in one metastatic lesion, suggesting divergent developmental origins in the breast, but the potential for nascent TRM differentiation in a metastatic niche. Clustering of these TCRs suggested differing antigen specificities between TRM and non-TRM CD8 T cells. Using Metabric data, the CD8 TRM gene expression signature was prognostic for disease free survival (DFS) in primary TNBCs (n=329, log-rank p=0.003), and was able to further stratify cases with high and low CD8A expression for DFS (log-rank p = 0.03). The CD8 TRM signature was enriched in baseline tumour samples of responders (n = 9) compared with non-responders (n=36) in 45 patients with metastatic melanoma treated with T cell checkpoint blockade (p &amp;lt; 0.0001). Additional single cell sequencing data with TCR sequencing will be combined with these initial results, and an independent data set of single cell mRNASeq and TCR Seq on CD3+ BC TILs will be used to confirm our findings. Cell type specific signatures will be explored in additional clinical cohorts including KEYNOTE-086, and presented at the meeting. Conclusion: Using single cell profiling of the immune microenvironment in BC we demonstrate that high TIL BCs contain multiple T cell subpopulations with different functional and prognostic significance. Our approach identified a CD8 TRM population with a distinct gene expression profile and strong expression of key immune checkpoints likely representing the presence of true tumor specific immunity. This population may be a key target of immune checkpoint blockade. Citation Format: Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, Salgado R, Teo ZL, Dushyanthen S, Byrne A, Luen SJ, Fox SB, Speed TP, Mackay LK, Neeson PJ, Loi S. Characterization of high TIL breast cancers reveals a prognostic and functionally distinct tissue-resident memory subpopulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-03.

Abstract P2-01-04: Epithelial-mesenchymal transition promotes triple negative breast cancer immune suppression

Abstract Background: Epithelial-Mesenchymal Transition (EMT) is a developmental process co-opted by carcinomas to facilitate metastasis. Triple negative breast cancer (TNBC), which has often undergone at least partial EMT, has a poor prognosis due to frequent and rapid recurrence as metastatic disease when compared to other breast cancer subtypes. To identify drivers of TNBC progression microRNA miR-200c was restored to the human TNBC mesenchymal-like BT549 cell line, which decreased 80% of the pan-EMT mesenchymal signature and restored 60% of the epithelial signature. In addition to decreasing proteins involved in immune-suppression via contact-dependent mechanisms, such as CD274 (PD-L1) and (CD273) PD-L2, Ingenuity Pathway and GSEA analysis revealed reduced expression of genes encoding enzymes, including Tryptophan 2,3-Dioxygenase (TDO2) and Heme Oxygenase-1 (HO-1), that generate immune-suppressive metabolites that function in a non-contact dependent manner. TNBC utilizes these and other immune-suppressive factors made by fetal trophoblasts to suppress the maternal immune system to achieve fetal tolerance during pregnancy, suggesting that TNBC may mimic a physiologically relevant mechanism to promote immune evasion. Hypothesis: TNBC metabolism is altered as part of an EMT program resulting in generation of immune-suppressive metabolites that promote metastasis by facilitating contact-independent immune evasion. Methods: Expression and activity of TDO2 and HO-1 were determined by UHPLC-MS for kynurenine (generated by TDO2) and bilirubin (generated by HO-1) in multiple TNBC cell lines. Single cell RNAseq was performed to identify genes co-expressed with these enzymes. To develop an immune competent preclinical model, doxycycline inducible miR-200c (TripZ-200c) was stably introduced into the Met-1 mammary carcinoma cell line derived from the MMTV-PyMT mouse model developed in FVB/NJ mice. Results were confirmed via transient miR-200c expression in 66cl-4 mammary carcinoma cells (from a spontaneous BALB/c tumor). Results: Restoration of miR-200c decreased TDO2 and HMOX1 (HO-1) by 80% and 40% respectively in Met-1 and 66cl-4 mammary carcinoma cells and reduced secretion of kynurenine and bilirubin. HO-1 was increased in lung metastasis from Met-1 and 66cl-4 cells when compared to cells prior to tail vein injection. ScRNAseq revealed specific gene expression signatures co-expressed with TDO2 and are being analyzed to better detect TDO2 in tumor tissues. In vivo studies are underway to investigate the effects of inhibiting tumor TDO2 and HO-1 on infiltrating lymphocyte composition, the anti-tumor immune response, and tumor progression. Conclusions: Restoration of miR-200c revealed EMT-associated alterations in tumor metabolism and consequent secretion of immune-suppressive metabolites produced by TDO2 and HO-1. We propose that targeting these contact-independent immune-suppressive factors may complement current contact-dependent therapeutic strategies (checkpoint inhibitors) to boost anti-tumor immune cell function and thereby prevent or decrease TNBC metastasis. Citation Format: Williams MM, Christenson JL, O'Neill K, Rogers TJ, Greene LI, Slansky J, Richer JK. Epithelial-mesenchymal transition promotes triple negative breast cancer immune suppression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-04.

Genomic landscape of pediatric B-other acute lymphoblastic leukemia in a consecutive European cohort.

Novel biological subtypes and clinically important genetic aberrations (druggable lesions, prognostic factors) have been described in B-other acute lymphoblastic leukemia (ALL) during the last decade; however, due to a lack of studies on unselected cohorts, their population frequency and mutual associations still have to be established. We studied 110 consecutively diagnosed and uniformly treated childhood B-other patients using single nucleotide polymorphism arrays and whole exome/transcriptome sequencing. The frequency of DUX4-rearranged, BCR-ABL1-like, ZNF384-rearranged, ETV6-RUNX1-like, iAMP21 and MEF2D-rearranged subtypes was 27%, 15%, 5%, 5%, 4%, and 2%, respectively; 43% of cases were not classified into any of these subtypes (B-rest). We found worse early response to treatment in DUX4-rearranged leukemia and a strong association of ZNF384-rearranged leukemia with B-myeloid immunophenotype. Of the druggable lesions, JAK/STAT-class and RAS/RAF/MAPK-class aberrations were found in 21% and 43% of patients, respectively; an ABL-class aberration was found in one patient. A recently described negative prognostic factor, IKZF1plus, was found in 14% of patients and was enriched in (but not exclusive for) BCR-ABL1-like subtype. PAX5 fusions (including 4 novel), intragenic amplifications and P80R mutations were mutually exclusive and only occurred in the B-rest subset, altogether accounting for 20% of the B-other group. PAX5 P80R was associated with a specific gene expression signature, potentially defining a novel leukemia subtype. Our study shows unbiased European population-based frequencies of novel ALL subtypes, recurrent (cyto)genetic aberrations and their mutual associations. This study also strengthens and widens the current knowledge of B-other ALL and provides an objective basis for optimization of current genetic diagnostics.

Sex-Related Differences in Lactotroph Tumor Aggressiveness Are Associated With a Specific Gene-Expression Signature and Genome Instability

Sex-related differences have been reported in various cancers, in particular men with lactotroph tumors have a worse prognosis than women. While the underlying mechanism of this sexual dimorphism remains unclear, it has been suggested that a lower estrogen receptor alpha expression may drive the sex differences observed in aggressive and malignant lactotroph tumors that are resistant to dopamine agonists. Based on this observation, we aimed to explore the molecular importance of the estrogen pathway through a detailed analysis of the transcriptomic profile of lactotroph tumors from 20 men and 10 women. We undertook gene expression analysis of the selected lactotroph tumors following their pathological grading using the five-tiered classification. Chromosomic alterations were further determined in 13 tumors. Functional analysis showed that there were differences between tumors from men and women in gene signatures associated with cell morphology, cell growth, cell proliferation, development, and cell movement. Hundred-forty genes showed an increased or decreased expression with a minimum 2-fold change. A large subset of those genes belonged to the estrogen receptor signaling pathway, therefore confirming the potent role of this pathway in lactotroph tumor sex-associated aggressiveness. Genes belonging to the X chromosome, such as CTAG2, FGF13, and VEGF-D, were identified as appealing candidates with a sex-linked dysregulation in lactotroph tumors. Through our comparative genomic hybridization analyses (CGH), chromosomic gain, in particular chromosome 19p, was found only in tumors from men, while deletion of chromosome 11 was sex-independent, as it was found in most (5/6) of the aggressive and malignant tumors. Comparison of transcriptomic and CGH analysis revealed four genes (CRB3, FAM138F, MATK, and STAP2) located on gained regions of chromosome 19 and upregulated in lactotroph tumors from men. MATK and STAP2 are both implicated in cell growth and are reported to be associated with the estrogen signaling pathway. Our work confirms the proposed involvement of the estrogen signaling pathway in favoring the increased aggressiveness of lactotroph tumors in men. More importantly, we highlight a number of ER-related candidate genes and further identify a series of target molecules with sex-specific expression that could contribute to the aggressive behavior of lactotroph tumors in men.

The Inner Ear Heat Shock Transcriptional Signature Identifies Compounds That Protect Against Aminoglycoside Ototoxicity.

Mechanosensory hair cells of the inner ear transduce auditory and vestibular sensory input. Hair cells are susceptible to death from a variety of stressors, including treatment with therapeutic drugs that have ototoxic side effects. There is a need for co-therapies to mitigate drug-induced ototoxicity, and we showed previously that induction of heat shock proteins (HSPs) protects against hair cell death and hearing loss caused by aminoglycoside antibiotics in mouse. Here, we utilized the library of integrated cellular signatures (LINCS) to identify perturbagens that induce transcriptional profiles similar to that of heat shock. Massively parallel sequencing of RNA (RNA-Seq) of heat shocked and control mouse utricles provided a heat shock gene expression signature that was used in conjunction with LINCS to identify candidate perturbagens, several of which were known to protect the inner ear. Our data indicate that LINCS is a useful tool to screen for compounds that generate specific gene expression signatures in the inner ear. Forty-two LINCS-identified perturbagens were tested for otoprotection in zebrafish, and three of these were protective. These compounds also induced the heat shock gene expression signature in mouse utricles, and one compound protected against aminoglycoside-induced hair cell death in whole organ cultures of utricles from adult mice.

Biology of nonmuscle-invasive bladder cancer: pathology, genomic implications, and immunology.

Despite that nearly 75% of bladder cancer patients are diagnosed with nonmuscle-invasive disease, our understanding of the biological landscape in bladder cancer is primarily within the context of muscle-invasive bladder cancer. More recent studies addressing the genomic changes and immunology of nonmuscle-invasive bladder cancer (NMIBC) have helped to extend our understanding of this prevalent disease. Genomic studies reveal that NMIBC possesses complexity that can be defined by specific gene expression signatures and has helped to define subsets within this disease. These subsets possess different risk profiles that may impact treatment decisions. In addition, the baseline or posttreatment immunological response to the growing tumor may help to inform whether a specific NMIBC subset is likely to progress. Findings from studies addressing the molecular landscape of NMIBC may help to establish parameters for stratifying patient risk within this disease as well as establish novel or targeted treatment strategies. Inclusion of information about the immune response within tumors will likely contribute to defining the relative risk and treatment strategy for these patients.

Abstract 419: Deep Sequencing Provides Evidence Of Mitochondrial Abnormalities In Right Ventricular Failure In Pulmonary Arterial Hypertension

Introduction: Right ventricular (RV) function is a critical predictor of both morbidity and mortality in pulmonary arterial hypertension (PAH). In PAH patients present either with adaptive remodelling RV and compensatory RV hypertrophy (RVH), or maladaptive remodeling that rapidly deteriorates to RV failure (RVF). While the molecular mechanisms underlying RVF remain poorly understood, acquired mitochondrial abnormalities including impaired mitochondrial electron transport chain activity, altered metabolic substrate utilization, and aberrant mitochondrial dynamics likely play a crucial role. We assessed the role of mitochondria in RVF using an unbiased assessment of gene expression in the RV of rats with maladaptive vs adaptive RVH and PAH. Method and result: Male rats were treated with either sugen+hypoxia (SuHx) or Monocrotaline (MCT). We determined RV status using non-invasive echocardiography and right heart catheterisation. Compared to control (Ctrl; n=15) both MCT (n=8) and SuHx (n=5) rats displayed severe PAH with increased RVSP, mPAP, total pulmonary resistance (TPR) and decreased PA acceleration time (PAAT) (p&lt;0.05). MCT rats developed RVF characterized by increased RV end diastolic volume (EDV), decreased CO, TAPSE and ventricular-arterial decoupling (decreased Emax/Ea ratio) (p&lt;0.05). SuHx rats had adaptive RVH. RNAsequencing revealed 2,547 differentially regulated transcripts. Functional Annotation analysis revealed multiple enriched GOterms, the most significant of which described mitochondrion (GO: 0005739; p=1.6E-53 which describes 414 genes). Significant GOterms also include mitochondrial inner membrane (GO: 0005743; p=3.9E-20) and mitochondrial respiratory chain complex I (GO: 0005747 ; p=4.9E-10). We validated 15 key dysregulated mitochondrial genes in maladaptive RVH (MCT; n=7) compared to both adaptive RVH (SuHx; n=4) and Ctrl (n=12) by qPCR, Finally, we validated these findings in human samples from PAH patients with RVH compared to controls (n=5 per group; p&lt;0.05). Conclusion: We combined haemodynamic investigation and transcriptomics to identify specific mitochondrial-metabolic gene expression signatures associated with RVF in two different preclinical models and in human samples.

Embryonic atrazine exposure elicits proteomic, behavioral, and brain abnormalities with developmental time specific gene expression signatures

Atrazine (ATZ), the second most commonly used herbicide in the United States, is an endocrine disrupting chemical linked to cancer and a common drinking water contaminant. This study further investigates ATZ-related developmental toxicity by testing the following hypotheses in zebrafish: the effects of embryonic ATZ exposure are dependent on timing of exposure; embryonic ATZ exposure alters brain development and function; and embryonic ATZ exposure changes protein abundance in carcinogenesis-related pathways. After exposing embryos to 0, 0.3, 3, or 30 parts per billion (ppb) ATZ, we monitored the expression of cytochrome P450 family 17 subfamily A member 1 (cyp17a1), glyoxalase I (glo1), ring finger protein 14 (rnf14), salt inducible kinase 2 (sik2), tetratricopeptide domain 3 (ttc3), and tumor protein D52 like 1 (tpd52l1) at multiple embryonic time points to determine normal expression and if ATZ exposure altered expression. Only cyp17a1 had normal dynamic expression, but ttc3 and tpd52l1 had ATZ-related expression changes before 72 h. Larvae exposed to 0.3 ppb ATZ had increased brain length, while larvae exposed to 30 ppb ATZ were hypoactive. Proteomic analysis identified altered protein abundance in pathways related to cellular function, neurodevelopment, and genital-tract cancer. The results indicate embryonic ATZ toxicity involves interactions of multiple pathways. SignificanceThis is the first report of proteomic alterations following embryonic exposure to atrazine, an environmentally persistent pesticide and common water contaminant. Although the transcriptomic alterations in larval zebrafish with embryonic atrazine exposure have been reported, neither the time at which gene expression changes occur nor the resulting proteomic changes have been investigated. This study seeks to address these knowledge gaps by evaluating atrazine's effect on gene expression through multiple time points during embryogenesis, and correlating changes in gene expression to pathological alterations in brain length and functional changes in behavior. Finally, pathway analysis of the proteomic alterations identifies connections between the molecular changes and functional outcomes associated with embryonic atrazine exposure.

22 Luminal progenitor cells as a new target in castration-resistant prostate cancer (CRPC)

IntroductionCastration-resistant prostate cancer (CRPC) is a lethal disease. Therefore the identification and understanding of castration-tolerant cell(s) is mandatory. While there is strong evidence for the existence of castration-tolerant cells within the basal layer of the prostate epithelium, most of human prostate cancers (hormone-naïve and CRPC) exhibit a luminal phenotype devoid of basal cell features. The nature of the putative castration-resistant luminal cell(s) is unknown.We recently identified in the mouse prostate a population of luminal progenitor cells that we called LSCmed (Lin−/Sca-1+/CD49fmed). This population is rare (5%) in healthy prostates but significantly enriched (30%) in premalignant prostates. The aim of this study was to investigate the relevance of LSCmed in prostate cancer.Material and methodsWe compared 6–8 month-old wild type (WT), probasin-prolactin (Pb-PRL) and Pb-Cre4/PTEN floxed (Pten-KO) mice exhibiting healthy, premalignant or malignant prostates, respectively. We used cell sorting (Lin, CD49f, Sca-1) to quantify or enrich the various prostate cell subpopulations from intact versus castrated mice. Transcriptomic profiling of prostate cell subpopulations was performed using GeneChip Mouse Transcriptome Arrays 1.0 (Affymetrix). Tumor-initiating properties of LSCmed cells was determined using the in vivo regeneration assay.Results and discussionsTranscriptomic profiling showed that LSCmed is a distinct cell entity that exhibits a specific gene expression signature, among which cytokeratin 4 (CK4) was validated as a specific marker to track them on tissue sections. Using CK4 immunohistochemistry and cell sorting we discovered that LSCmed represent the major cell component (80%) of aggressive prostate tumours harboured by Pten-KO mice. LSCmed are castration-tolerant in the three genotypes, which correlates their intrinsically low androgen signalling. According to their progenitor properties, Pten-KO LSCmed exhibit cancer-initiating properties in transplantation assays, and generate more aggressive tumours than basal cells used in control. Finally, in Pten-KO prostate tumours, several clusters of LSCmed continue to proliferate after castration.ConclusionLSCmed represent a newly-identified luminal prostatic cell subpopulation. The combination of progenitor, castration-resistance and tumor-initiating properties makes them strong candidates for mediating prostate cancer recurrence under androgen deprivation therapy (CRPC).

Transcriptome-pathology correlation identifies interplay between TDP-43 and the expression of its kinase CK1E in sporadic ALS.

Sporadic amyotrophic lateral sclerosis (sALS) is the most common form of ALS, however, the molecular mechanisms underlying cellular damage and motor neuron degeneration remain elusive. To identify molecular signatures of sALS we performed genome-wide expression profiling in laser capture microdissection-enriched surviving motor neurons (MNs) from lumbar spinal cords of sALS patients with rostral onset and caudal progression. After correcting for immunological background, we discover a highly specific gene expression signature for sALS that is associated with phosphorylated TDP-43 (pTDP-43) pathology. Transcriptome–pathology correlation identified casein kinase 1ε (CSNK1E) mRNA as tightly correlated to levels of pTDP-43 in sALS patients. Enhanced crosslinking and immunoprecipitation in human sALS patient- and healthy control-derived frontal cortex, revealed that TDP-43 binds directly to and regulates the expression of CSNK1E mRNA. Additionally, we were able to show that pTDP-43 itself binds RNA. CK1E, the protein product of CSNK1E, in turn interacts with TDP-43 and promotes cytoplasmic accumulation of pTDP-43 in human stem-cell-derived MNs. Pathological TDP-43 phosphorylation is therefore, reciprocally regulated by CK1E activity and TDP-43 RNA binding. Our framework of transcriptome–pathology correlations identifies candidate genes with relevance to novel mechanisms of neurodegeneration.Electronic supplementary materialThe online version of this article (10.1007/s00401-018-1870-7) contains supplementary material, which is available to authorized users.

IDH mutation status is associated with distinct vascular gene expression signatures in lower-grade gliomas.

BackgroundVascular gene expression patterns in lower-grade gliomas (LGGs; diffuse World Health Organization [WHO] grades II–III gliomas) have not been thoroughly investigated. The aim of this study was to molecularly characterize LGG vessels and determine if tumor isocitrate dehydrogenase (IDH) mutation status affects vascular phenotype.MethodsGene expression was analyzed using an in-house dataset derived from microdissected vessels and total tumor samples from human glioma in combination with expression data from 289 LGG samples available in the database of The Cancer Genome Atlas. Vascular protein expression was examined by immunohistochemistry in human brain tumor tissue microarrays (TMAs) representing WHO grades II–IV gliomas and nonmalignant brain samples. Regulation of gene expression was examined in primary endothelial cells in vitro.ResultsGene expression analysis of WHO grade II glioma indicated an intermediate stage of vascular abnormality, less severe than that of glioblastoma vessels but distinct from normal vessels. Enhanced expression of laminin subunit alpha 4 (LAMA4) and angiopoietin 2 (ANGPT2) in WHO grade II glioma was confirmed by staining of human TMAs. IDH wild-type LGGs displayed a specific angiogenic gene expression signature, including upregulation of ANGPT2 and serpin family H (SERPINH1), connected to enhanced endothelial cell migration and matrix remodeling. Transcription factor analysis indicated increased transforming growth factor beta (TGFβ) and hypoxia signaling in IDH wild-type LGGs. A subset of genes specifically induced in IDH wild-type LGG vessels was upregulated by stimulation of endothelial cells with TGFβ2, vascular endothelial growth factor, or cobalt chloride in vitro.ConclusionIDH wild-type LGG vessels are molecularly distinct from the vasculature of IDH-mutated LGGs. TGFβ and hypoxia-related signaling pathways may be potential targets for anti-angiogenic therapy of IDH wild-type LGG.

The Reparative Abilities of Menstrual Stem Cells Modulate the Wound Matrix Signals and Improve Cutaneous Regeneration.

Considerable advances have been made toward understanding the cellular and molecular mechanism of wound healing, however, treatments for chronic wounds remain elusive. Emerging concepts utilizing mesenchymal stem cells (MSCs) from umbilical cord, adipose tissue and bone marrow have shown therapeutical advantages for wound healing. Based on this positive outcome, efforts to determine the optimal sources for MSCs are required in order to improve their migratory, angiogenic, immunomodulatory, and reparative abilities. An alternative source suitable for repetitive, non-invasive collection of MSCs is from the menstrual fluid (MenSCs), displaying a major practical advantage over other sources. This study aims to compare the biological functions and the transcriptomic pattern of MenSCs with umbilical cord MSCs in conditions resembling the wound microenvironment. Consequently, we correlate the specific gene expression signature from MenSCs with changes of the wound matrix signals in vivo. The direct comparison revealed a superior clonogenic and migratory potential of MenSCs as well as a beneficial effect of their secretome on human dermal fibroblast migration in vitro. Furthermore, MenSCs showed increased immunomodulatory properties, inhibiting T-cell proliferation in co-culture. We further, investigated the expression of selected genes involved in wound repair (growth factors, cytokines, chemokines, AMPs, MMPs) and found considerably higher expression levels in MenSCs (ANGPT1 1.5-fold; PDGFA 1.8-fold; PDGFB 791-fold; MMP3 21.6-fold; ELN 13.4-fold; and MMP10 9.2-fold). This difference became more pronounced under a pro-inflammatory stimulation, resembling wound bed conditions. Locally applied in a murine excisional wound splinting model, MenSCs showed a significantly improved wound closure after 14 days, as well as enhanced neovascularization, compared to the untreated group. Interestingly, analysis of excised wound tissue revealed a significantly higher expression of VEGF (1.42-fold) among other factors, translating an important conversion of the matrix signals in the wound site. Furthermore, histological analysis of the wound tissue from MenSCs-treated group displayed a more mature robust vascular network and a genuinely higher collagen content confirming the pro-angiogenic and reparative effect of MenSCs treatment. In conclusion, the superior clonogenicity, immunosuppressive and migration potential in combination with specific paracrine signature of MenSCs, resulted in an enhanced wound healing and cutaneous regeneration process.