NanoString nCounter Assay Research Articles

Abstract LB-C18: An integrated multiplexing approach for the immunoprofiling of the tumor microenvironment of ovarian granulosa cell tumors

Abstract Background: Granulosa cell tumors (GCTs) are rare tumor in ovaries accounting for 2-5% of all ovarian cancers. The main current treatment for GCT is surgery, however a subset require chemotherapy for residual and recurrent disease. GCT malignancies are often low-grade with a five-year survival rate up to 90%, however a clinical characteristic of these tumors is a tendency for late recurrence and a high recurrent rate is the most critical factor for GCT death. As the onset of recurrence is unpredictable, future research should focus on identifying both biomarkers for prognosis prediction, as well as targets that could help guide clinical trials in the development of targeted therapies for this rare indication. As GCTs are rare tumors and tissue availability is very limited, we used a dual multiplexing approach in order to maximize the data output from a total of 14 FFPE tumor samples (6 primary tumors, and 8 recurrent tumors). With this approach we used a single 4 μm section to detect 15 markers in an IF multiplexing assay, and an adjacent 10 μm section to analyze expression of 770 immuno-related cancer genes with the aim to spatially profile T cell subsets, tumor-associated macrophages (TAMs), B cells, tumor proliferation, angiogenic vessels, as well as markers differentially expressed between primary and recurrent tumors. Methods: For protein multiplexing we have used MultiOmyx™, an immunofluorescence (IF) multiplexing assay utilizing a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by dye inactivation, and deep learning based cell classification algorithms identify positive cells for each biomarker to identify a 15-marker panel consisting of CD3, CD4, CD8, FoxP3, CD68, CD163, HLA-DR, CD34, CTLA-4, PD-1, PD-L1, Ki67, vimentin, S100, and Pan Cytokeratin. Gene expression analysis was done using the Nanostring nCounter assay, specifically the 770 gene PanCancer Immune Panel. RNA was extracted from the adjacent 10μm section and then proceeded with hybridization, purification and immobilization and count based on manufacturer’s protocol. Results: On protein level we confirmed previous findings that ovarian GCTs are so-called “cold” tumors, with a very low density of T cell infiltration. Likewise, levels of both CTLA-4, PD-1, and PD-L1 were very low in all GCTs. We are currently analyzing gene expression signatures for tumor antigens, defects in antigen presentation, and absence of T cell activation in order to detect some of the mechanisms behind the low T cell infiltration in this tumor type. When we analyzed the presence of TAMs in the tumor microenvironment (TME) however, we found a 113% increase in TAM density in recurrent tumors compared to primary tumors, and when doing a nearest neighbor analysis of the proximity between M2-type TAMs and angiogenic vessels in the TME, we found M2 TAMS to be much closer associated with the vessels. Interestingly, on gene level we found SPP1 (the gene coding for osteopontin) to have a 9-fold higher expression in recurrent versus primary GCT tumors, while VEGFA was found to have a 3-fold higher expression. As a pro-inflammatory cytokine osteopontin is thought to both facilitate the recruitment of TAMs and to stimulate angiogenesis by inducing VEGF expression in endothelial cells, suggesting that osteopontin could be a therapeutic target in patients with recurrent GCT. Citation Format: Anna Juncker-Jensen, Tyvette S Hilliard, Nicholas Stavrou, Mate Nagy, Erinn Parnell, Judy Kuo, Eric Leones, Sharon Stack. An integrated multiplexing approach for the immunoprofiling of the tumor microenvironment of ovarian granulosa cell tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C18. doi:10.1158/1535-7163.TARG-19-LB-C18

Abstract 2896: Impact of intratumoral heterogeneity on gene expression profiles and known biomarkers in FFPE tumor biopsies from early stage breast cancer patients

Abstract Background: Cancer is a dynamic disease that becomes more heterogeneous during its course, with cellular subpopulations with distinct molecular profiles across different regions. Because this intratumoral heterogeneity may result in varying levels of gene expression patterns and sensitivity to treatment across different regions of the tumor, it is important to understand patterns of heterogeneity to develop effective biomarkers. We conducted multiregion sampling within single breast tumor biopsies to evaluate the effect of intratumoral heterogeneity on expression of known and novel biomarkers. Methods: We sampled three sequential regions of breast tumor biopsies to compare gene expression patterns determined by a novel biomarker panel (BA355), 5 known biomarkers (ERS1, ERBB2, PGR, AR, and MKi67) and a novel predictive classifier (BA100). RNA expression levels were measured using a proprietary custom designed nanoString nCounter assay. We investigated formalin-fixed paraffin-embedded (FFPE) breast tumor biopsies from a cohort of 40 early stage breast cancer patients (T1 N0 M0) collected prospectively. RNA was extracted from upper, middle, and lower regions of each biopsy, resulting in a total of 120 samples quantified and QC using standard procedures. Each layer consisted of 40 micrometers, with an interval of 50 micrometers of tissue. BA100 linear regression models were used to generate prediction scores. Analyses were done in R. Results: Eighteen of the 120 samples had poor data quality (sample median = 0 or inter quantile range > mean + 2*sd). Further analyses only included 102 good quality samples corresponding to 34 FFPE biopsies. Among 33 of 34 samples, gene expression patterns between the three regions of the biopsies were highly correlated, with correlation coefficients ranging from 0.90-0.99 (p-values ranged from 2.07e-19 to 8.45e-319). Among 31 of 34 samples, the three regions were clustered together in cluster analysis. The coefficient of variation (CV) for ESR1 ranged from 0.37%-15.5%, with a majority (32 of 34) of the samples having CV <10%. While the CVs of ERBB2, PGR, and AR were within the same range as ESR1, MKI67 had much larger variation (CV ranged from 0.99%-173%). BA100 scores yielded consistent predictions for the three regions in 33 of 34 samples. Conclusion: Examination of gene expression patterns across multiple regions of breast tumor biopsies did not reveal a significant impact of intratumoral heterogeneity on expression patterns using a novel gene signature panel. However, at the individual gene level, while a number of genes including ESR1, ERBB2, PGR, and ARdisplayed consistent gene expression, genes such as MKI67 showed significant variation, indicating that tumor heterogeneity may impact individual gene biomarkers. Further studies are ongoing to evaluate intratumoral heterogeneity. Citation Format: Lucas Delmonico, Joan Chen, Trushna Desai, Lisa Barnett, Aladdin Shadyab, John Obenauer, Gilda Alves Brown, Mauricio Magalhaes Costa, Marcia Fournier. Impact of intratumoral heterogeneity on gene expression profiles and known biomarkers in FFPE tumor biopsies from early stage breast cancer patients [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 2896.

Abstract PD5-10: Immune characterization of matched primary and multiple metastatic samples issued from an institutional autopsy cohort

Abstract Introduction While immune infiltrates have already been extensively characterized in primary tumors (P), data on breast cancer metastases (M) remains limited. To this end we quantified and qualified the immune cells in a unique cohort of multiple matched P and M samples selected from an institutional breast cancer autopsy cohort. Patients and methods Twenty-three patients were selected from an institutional autopsy program (Semmelweis University, Budapest, Hungary) based on matched P and M sample availability (124 samples). All samples were centrally characterized for estrogen (ER), progesterone (PR) and HER2 receptors. The primary molecular subtypes were as follows: 9 ER+/PR+/HER2-, 8 triple negative and 6 HER2+. Ten patients relapsed ≤1 year after diagnosis and were further referred to as “early relapsers”, as opposed to the remaining qualified as “late relapsers”. Immunohistochemistry (IHC) was carried out against CD3/CD20 and CD4/CD8 in 21 patients (119 samples). Tumor infiltrating lymphocytes (TILs) were assessed on hematoxylin and eosin (H&E) and CD3-stained slides. Gene expression data were generated using the NanoString nCounter assay (PanCancer Immune Profiling Panel) for 11 patients (35 samples) and analyzedusing the R package NanoStringQCPro. The scores from published immune gene signatures were calculated as a weighted sum of the expressions of their genes. All samples were analyzed for 22 immune cell subtypes relative abundance using CIBERSORT. Results TILs assessed on H&E and CD3-stained slides were weakly correlated (Rho= 0.38, p<.001). TIL levels as well as the number of tertiary lymphoid structures (TLS) were significantly lower in Ms as compared to Ps (p<.001). Among the different metastatic sites, the lung was more infiltrated when considering CD3+ and CD4+ cells (p=.01 and .02, respectively). We further observed significantly higher levels of TILs, CD3+, CD4+ and CD8+ cells in the Ms but not in the Ps from late relapsers as opposed to those from early relapsers. Gene expression analyses further confirmed these observations as several immune gene signatures displayed significantly higher scores in the Ms from late compared to early relapsers. An unsupervised analysis identified 13 genes significantly differentially expressed between Ps and Ms: CSF1R, CXCL14, CYBB, IL21R, IL2RB, TNF and TNFSF15 were upregulated in Ps while BCL2L1, C7, HSD11B1, and PSMB7 were upregulated in Ms. The matched P/M CIBERSORT analyses revealed a distinct composition of immune cell types between P and M of a same patient. Apart from a potential increase in M0 macrophages, no common trait was observed in immune cell composition between the Ms from the different patients. Conclusion This is to the best of our knowledge the first study characterizing the immune infiltration in patients with multiple matched P and M samples. The results suggest that Ms have not only a globally lower immune infiltration as compared to Ps, but also a different immune composition. Additionally, Ms from late relapsers are more infiltrated as compared to early relapsers. The present data also uncovers not only important inter-patient but also intra-patient immune heterogeneity, which should be taken into consideration for optimal treatment decision. Citation Format: Szekely B, Bareche Y, Van den Eynden G, Salgado R, Buisseret L, Garaud S, Willard-Gallo K, Hatzis C, Szasz M, Kulka J, Larsimont D, Sotiriou C, Pusztai L, Desmedt C. Immune characterization of matched primary and multiple metastatic samples issued from an institutional autopsy cohort [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 PD5-10.

Genetic Modulation of Adenosine-to-Inosine RNA Editing Selectively Disrupts Inflammasome and Extracellular Matrix Genes in Multiple Myeloma

IntroductionAs the second most common blood cancer in the U.S., multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to therapy-resistant plasma cell leukemia (PCL). Despite many novel therapies, relapse rates remain high as a result of malignant regeneration (self-renewal) of MM cells in inflammatory microenvironments. In addition to recurrent DNA mutations and epigenetic deregulation, inflammatory cytokine-responsive adenosine deaminase associated with RNA (ADAR1) mediated adenosine to inosine (A-to-I) RNA editing has emerged as a key driver of cancer relapse and progression. In MM, copy number amplification of chromosome 1q21, which contains both ADAR1 and interleukin-6 receptor (IL-6R) gene loci, portends a poor prognosis. Thus, we hypothesized that ADAR1 copy number amplification combined with inflammatory cytokine activation of ADAR1 stimulate malignant regeneration of MM and therapeutic resistance.Methods and ResultsTo evaluate the contribution of ADAR1 modulation to global RNA editing changes and differential gene expression, we performed whole transcriptome sequencing of primary MM and PCL samples that were lentivirally transduced with shRNA targeting human ADAR1 compared to vector control shRNA. Analysis at the single nucleotide level revealed that 20-50% knockdown of ADAR1 was sufficient to induce substantial downregulation of A-to-I editing rates throughout the transcriptome, particularly at 3'UTR loci, along with specific modulation of extracellular matrix and inflammasome-associated gene expression patterns. Moreover, in contrast to lentiviral ADAR1 shRNA knockdown and overexpression of an editase defective ADAR1 mutant (ADAR1E912A), lentiviral wild-type ADAR1 overexpression enhanced editing of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist. Editing of GLI1 transcripts enhanced GLI transcriptional activity in luciferase reporter assays, and promoted lenalidomide resistance in vitro. Finally, lentiviral shRNA ADAR1 knockdown reduced regeneration of high-risk MM in humanized serial transplantation mouse models, indicative of reduced malignant self-renewal capacity. Notably, in human myeloma cells analyzed using NanoString nCounter assays, overexpression of wild-type versus edited GLI1 elicited distinct gene expression changes also in extracellular matrix and immune response genes. These data demonstrate that ADAR1 promotes malignant self-renewal of MM and if selectively inhibited may prevent progression and relapse through modulation of global A-to-I RNA editing and extracellular and immune response gene expression.ConclusionsDeregulated RNA editing, driven by aberrant ADAR1 activation, represents a unique source of transcriptomic and proteomic diversity, resulting in self-renewal of MM cells in inflammatory microenvironments. In summary, both genetic (1q21 amplification) and microenvironmental factors (inflammatory cytokines, immunomodulatory drugs) combine to drive A-to-I RNA editing-dependent malignant regeneration in MM through inflammasome and extracellular matrix gene pathway remodeling. These effects can be down-modulated even with small reductions in ADAR1 expression and activity. Thus, ADAR1 represents both a vital prognostic biomarker and therapeutic target in MM. DisclosuresCostello:Celgene: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Takeda: Consultancy.

Activation of MET signaling by HDAC6 offers a rationale for a novel ricolinostat and crizotinib combinatorial therapeutic strategy in diffuse large B-cell lymphoma.

Some histone deacetylases (HDACs) promote tumor cell growth and pan- or selective HDAC inhibitors are active in some cancers; however, the pivotal HDAC enzyme and its functions in human diffuse large B-cell lymphoma (DLBCL) remain largely unknown. Using NanoString nCounter assays, we profiled HDAC mRNA expression and identified HDAC6 as an upregulated HDAC family member in DLBCL tissue samples. We then found that HDAC6 plays an oncogenic role in DLBCL, as evidenced by its promotion of cell proliferation in vitro and tumor xenograft growth in vivo. Mechanistically, the interaction between HDAC6 and HR23B downregulated HR23B expression, thereby reducing the levels of casitas B-lineage lymphoma (c-Cbl), an E3 ubiquitin ligase for hepatocyte growth factor receptor (MET), which resulted in the inhibition of MET ubiquitination-dependent degradation. In addition, enhanced HDAC6 expression and decreased HR23B expression were correlated with poor overall survival rates among patients with DLBCL. Taken together, these results establish an HDAC6-HR23B-MET axis and indicate that HDAC6 is a potent promoter of lymphomagenesis in DLBCL. Thus, a therapeutic strategy based on HDAC6 inhibitors in combination with MET inhibitors is promising. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 4437: Down-modulation of ADAR1-mediated GLI1 editing alters extracellular and immune response genes in multiple myeloma

Abstract Introduction: Representing 10% of hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to therapy-resistant plasma cell leukemia (PCL). Despite many novel therapies, relapse rates remain high as a result of malignant regeneration (self-renewal) of MM cells in inflammatory microenvironments. In addition to recurrent DNA mutations and epigenetic deregulation, inflammatory cytokine-responsive adenosine deaminase associated with RNA (ADAR1)-mediated adenosine to inosine (A-to-I) RNA editing has emerged as a key driver of cancer relapse and progression. In MM, copy number amplification of chromosome 1q21, which contains both ADAR1 and interleukin-6 receptor (IL-6R) gene loci, portends a poor prognosis. Thus, we hypothesized that ADAR1 copy number amplification combined with inflammatory cytokine activation of ADAR1 stimulates malignant regeneration of MM and therapeutic resistance. Methods and Results: Analysis of MMRF CoMMpass RNA sequencing (RNA-seq) data revealed that high ADAR1 expression (n=162 patients) correlated with significantly reduced progression-free and overall survival compared with a low ADAR1 subset (n=159 patients). In contrast to lentiviral ADAR1 shRNA knockdown and overexpression of an editase defective ADAR1 mutant (ADAR1E912A), lentiviral wild-type ADAR1 overexpression enhanced editing of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist. Editing of GLI1 transcripts enhanced GLI transcriptional activity in luciferase reporter assays, and promoted lenalidomide resistance in vitro. Finally, lentiviral shRNA ADAR1 knockdown reduced regeneration of high-risk MM in humanized serial transplantation mouse models, indicative of reduced malignant self-renewal capacity. Whole-transcriptome RNA-sequencing of primary samples after lentiviral shRNA knockdown of ADAR1 revealed specific modulation of extracellular and immune response genes, while overexpression of wild-type versus edited GLI1 elicited distinct gene expression changes in human myeloma cells analyzed using NanoString nCounter assays. These data demonstrate that ADAR1 promotes malignant self-renewal of MM and, if selectively inhibited, may prevent progression and relapse through modulation of extracellular and immune response genes. Conclusions: Deregulated RNA editing, driven by aberrant ADAR1 activation, represents a unique source of transcriptomic and proteomic diversity, resulting in self-renewal of MM cells in inflammatory microenvironments. In summary, both genetic (1q21 amplification) and microenvironmental factors (inflammatory cytokines, IMiDs) combine to drive GLI1-dependent malignant regeneration in MM. Thus, ADAR1 represents both a vital prognostic biomarker and therapeutic target in MM. Citation Format: Leslie A. Crews, Elisa Lazzari, Phoebe K. Mondala, Nathaniel Delos Santos, Amber Miller, Gabriel Pineda, Qingfei Jiang, Anusha-Preethi Ganesan, Christina Wu, Caitlin Costello, Mark Minden, Raffaella Chiaramonte, A. Keith Stewart, Catriona H. M. Jamieson. Down-modulation of ADAR1-mediated GLI1 editing alters extracellular and immune response genes in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4437.

Abstract 4549: Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms

Abstract Background: Recently, pemetrexed and carboplatin in combination with PD–1 antibody (pembrolizumab) demonstrated markedly improved clinical outcome in NSCLC patients (KEYNOTE–021G trial) suggesting a positive interaction between pemetrexed–based chemotherapy and immunotherapy. However, the role of pemetrexed in modulating antitumor immune response is largely unknown. The objective of this study was to characterize the effects of pemetrexed on intra–tumor immune response in monotherapy and combination with carboplatin or PD–1 pathway blockade in preclinical models. Methods: Mice bearing syngeneic MC38 or Colon26 tumors were treated with pemetrexed with or without carboplatin or anti–mouse PD–L1 antibody (178G7). Immune cell subsets and immune–related changes in mouse tumor tissue and T cells were assessed by flow cytometry and gene expression analysis (Quantigene Plex and nanoString nCounter assays). Effects of pemetrexed on immunogenic cell death in tumor cells and mitochondrial respiration in T cells were evaluated by HMGB1 ELISA and Agilent Seahorse XF analysis, respectively. Results: In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intratumoral leukocytes and total and cycling (Ki67+) T cells accompanied by immune–related gene expression changes indicative of enhanced antigen presentation, T cell infiltration and/or activation and down–modulation of the myeloid cell compartment. Immune gene expression signature induced by pemetrexed was largely unaffected by carboplatin. In both MC38 and Colon26 tumor cells, in vitro treatment with pemetrexed induced a robust release of HMGB1 indicative of immunogenic cell death. Proliferation of primary human T cells stimulated with CD3/CD28 was inhibited by pemetrexed in a dose–dependent manner. At clinically relevant concentrations pemetrexed also enhanced T cell activation phenotype exemplified by an increased frequency of CD137+, GITR+ and PD–L1+ T cells as well as upregulation of multiple interferon gamma–induced genes, and increased mitochondrial respiration. In both MC38 and Colon26 models, treatment with pemetrexed and 178G7 demonstrated a combination benefit compared to either monotherapy, and nCounter profiling of Colon26 tumors followed by Ingenuity Pathway Analysis revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell–mediated immunity might underlie this combination effect. Conclusions: Pemetrexed promotes intra–tumor T cell–mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells leading to an enhanced anti–tumor efficacy in combination with PD–L1 antibody. Citation Format: Ruslan Novosiadly, David Schaer, Nelusha Amaladas, Erik Rasmussen, Zhao Hai Lu, Andreas Sonyi, Carmine Carpenito, Yanxia Li, Shuang Luo, Andrew Capen, Catalina Meyer, Xiaodong Huang, Jason Manro, Gregory Donoho, Thompson Doman, Gerald Hall, Sandaruwan Geeganage, Michael Kalos. Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4549.

Altered expression level of inflammation-related genes and long-term changes in ocular surface after trabeculectomy, a prospective cohort study

PurposeWe aim to evaluate changes in the ocular surface in a cohort of post-trabeculectomy patients and whether these were associated with conjunctival inflammatory gene expression. MethodsThis is a single-arm interventional cohort performed in a tertiary referral center. These were assessed: dry eye symptom questionnaire, tear osmolarity, Schirmer's test, non-invasive tear break up time (BUT), conjunctival redness and corneal fluorescein staining evaluation. Conjunctival impressions were performed using Eyeprim, and after RNA extraction, transcripts of 255 inflammatory genes were analysed using the Nanostring nCounter assay. ResultsThirty three patients were recruited with age 66.88 ± 9.76 at baseline, with a predominance of men. There was a significant decrease in inferior corneal staining at 6 months (p < 0.05) (n = 22) and significant decrease in tear osmolarity at 12 months (p < 0.01) (n = 27). No patient required glaucoma eyedrops post-surgery up to 3 years. At baseline 31/33 transcript profiles passed the quality control, and after normalization, 249 transcripts were subsequently analysed. Increased discomfort was associated with higher Protein Tyrosine Kinase-2 at the cross-sectional analysis at baseline. Lower baseline complement factor-D and higher levels of Mitogen associated kinase-8, MAP3K1 and MyD88, were associated with presence of corneal staining at 6 months. Nine genes, including the proinflammatory lipo-oxygenase (ALOX5) showed a significantly reduced level at 3 years (n = 5). ConclusionsGlaucoma surgery may confer long term beneficial effect on the ocular surface, if anti-glaucoma eyedrops are no longer necessary. This may be due to reduced expression of conjunctival proinflammatory genes and immune-related genes.

Selection and Application of Tissue microRNAs for Nonendoscopic Diagnosis of Barrett’s Esophagus

Background & AimsMicroRNA (miRNA) is highly stable in biospecimens and provides tissue-specific profiles, making it a useful biomarker of carcinogenesis. We aimed to discover a set of miRNAs that could accurately discriminate Barrett’s esophagus (BE) from normal esophageal tissue and to test its diagnostic accuracy when applied to samples collected by a noninvasive esophageal cell sampling device.MethodsWe analyzed miRNA expression profiles of 2 independent sets of esophageal biopsy tissues collected during endoscopy from 38 patients with BE and 26 patients with normal esophagus (controls) using Agilent microarray and Nanostring nCounter assays. Consistently up-regulated miRNAs were quantified by real-time polymerase chain reaction in esophageal tissues collected by Cytosponge from patients with BE vs without BE. miRNAs were expressed from plasmids and antisense oligonucleotides were expressed in normal esophageal squamous cells; effects on proliferation and gene expression patterns were analyzed.ResultsWe identified 15 miRNAs that were significantly up-regulated in BE vs control tissues. Of these, 11 (MIR215, MIR194, MIR 192, MIR196a, MIR199b, MIR10a, MIR145, MIR181a, MIR30a, MIR7, and MIR199a) were validated in Cytosponge samples. The miRNAs with the greatest increases in BE tissues (7.9-fold increase in expression or more, P < .0001: MIR196a, MIR192, MIR194, and MIR215) each identified BE vs control tissues with area under the curve (AUC) values of 0.82 or more. We developed an optimized multivariable logistic regression model, based on expression levels of 6 miRNAs (MIR7, MIR30a, MIR181a, MIR192, MIR196a, and MIR199a), that identified patients with BE with an AUC value of 0.89, 86.2% sensitivity, and 91.6% specificity. Expression level of MIR192, MIR196a, MIR199a, combined that of trefoil factor 3, identified patients with BE with an AUC of 0.93, 93.1% sensitivity, and 93.7% specificity. Hypomethylation was observed in the promoter region of the highly up-regulated cluster MIR192–MIR194. Overexpression of these miRNAs in normal esophageal squamous cells increased their proliferation, via GRHL3 and PTEN signaling.ConclusionsIn analyses of miRNA expression patterns of BE vs non-BE tissues, we identified a profile that can identify Cytosponge samples from patients with BE with an AUC of 0.93. Expression of MIR194 is increased in BE samples via epigenetic mechanisms that might be involved in BE pathogenesis.

Gene expression in triple-negative breast cancer in relation to survival

The identification of biomarkers related to the prognosis of triple-negative breast cancer (TNBC) is critically important for improved understanding of the biology that drives TNBC progression. We evaluated gene expression in total RNA isolated from formalin-fixed paraffin-embedded tumor samples using the NanoString nCounter assay for 469 TNBC cases from the Shanghai Breast Cancer Survival Study. We used Cox regression to quantify Hazard Ratios (HR) and corresponding confidence intervals (CI) for overall survival (OS) and disease-free survival (DFS) in models that included adjustment for breast cancer intrinsic subtype. Of 302 genes in our discovery analysis, 22 were further evaluated in relation to OS among 134 TNBC cases from the Nashville Breast Health Study and the Southern Community Cohort Study; 16 genes were further evaluated in relation to DFS in 335 TNBC cases from four gene expression omnibus datasets. Fixed-effect meta-analysis was used to combine results across data sources. Twofold higher expression of EOMES (HR 0.90, 95% CI 0.83-0.97), RASGRP1 (HR 0.89, 95% CI 0.82-0.97), and SOD2 (HR 0.80, 95% CI 0.66-0.96) was associated with better OS. Twofold higher expression of EOMES (HR 0.89, 95% CI 0.81-0.97) and RASGRP1 (HR 0.87, 95% CI 0.81-0.95) was also associated with better DFS. On the contrary, a doubling of FA2H (HR 1.14, 95% CI 1.06-1.22) and GSPT1 (HR 1.33, 95% CI 1.14-1.55) expression was associated with shorter DFS. We identified five genes (EOMES, FA2H, GSPT1, RASGRP1, and SOD2) that may serve as potential prognostic biomarkers and/or therapeutic targets for TNBC.

Abstract P5-14-01: Telapristone acetate abrogates PR-dependent paracrine-mediated mammary cell proliferation

Abstract Background: Blockade of the progesterone (P)-progesterone receptor (PR) axis is a novel but untested strategy for breast cancer prevention. We report preclinical data evaluating telapristone acetate (TPA) compared with mifepristone (MFP), the prototype PR-antagonist. We hypothesize that the progesterone-PR blockade by TPA will inhibit PR-dependent paracrine expression (RANKL, WNT4, ID4, and calcitonin) attenuating cell proliferation and abrogating side branching and alveoli formation of mammary glands induced by hormones similar in extent or superior to MFP. Methods:Adult virgin FVB mice at 12 weeks of age were randomized to four treatment groups: no treatment control, EP (0.3 mg E + 30 mg P), EP + TPA (30mg), and EP + MFP (30mg). Hormone and drug pellets were subcutaneously implanted in flank area between the neck and shoulder. After 28-day treatments, the mice were euthanized to collect mammary glands, and processed as mammary whole mounts and as formalin-fixed paraffin embedding (FFPE) specimens. We evaluated cell proliferation (Ki67) by immunohistochemistry. Total RNA was extracted from FFPE specimens and the Nanostring nCounter assay was utilized to assess paracrine gene expression. The Mann Whitney test was used to calculate statistical significance (p&amp;lt;0.05). Results: We observed a considerable increase in side branches and alveoli in EP treatment group compared to the controls. The growth of the mammary gland stimulated by EP treatment was corroborated by a significant increase in Ki67 compared to control mice (median 41% vs 24%, respectively, p &amp;lt; 0.0001). Both TPA and MFP treatment abrogated side branching and alveoli formation and significantly reduced median Ki67 to 1/3 of that of control mice having endogenous estrus cycle hormone levels (24%, 8%, and 9% for control, EP+TPA and EP+MFP groups, respectively).TPA induced greater Ki67 reduction than MFP (7.5 ± 1.9% vs. 9.4 ± 1.9%, p= 0.04). As expected, EP treatment upregulated the RANKL expression compared to the control group while the administration of TPA and MFP significantly inhibited the RANKL expression stimulated by EP treatment. The mRNA expression measurement revealed that Rankl expression was 18 fold increased by EP treatment but completely inhibited by TPA and MFP treatment (p&amp;lt;0.001 for both drugs). Similarly the Rank expression was increased two-fold by EP treatment (p&amp;lt; 0.05) but down-regulated by TPA and MFP treatment (p&amp;lt;0.01 for both drugs). We observed significant upregulation of Wnt4, and Calca expression by EP treatment compared to controls (p&amp;lt;0.01 for both). Id4 expression showed the same trend but the change was non-significant. The addition of TPA completely opposed the hormone-stimulated increase of three paracrine molecules (Wnt4, Calca, and Id4) to a level below control group (p&amp;lt; 0.001 for all). Conclusions: We have demonstrated that TPA abrogates cell proliferation induced by exogenous EP hormones in an ovary intact mouse model. The blockade of PR-P binding was evident by complete inhibition of paracrine expression (not only RANKL/RANK expression but also WNT4, Calcitonin, and ID4 expression). TPA was efficacious as MFP in opposing paracrine-induced mammary cell proliferation, and warrants further testing in a breast cancer prevention trial. Citation Format: Lee O, Sun L, Karavites LC, Clare SE, Khan SA. Telapristone acetate abrogates PR-dependent paracrine-mediated mammary cell proliferation [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-14-01.

Development and Evaluation of a Pan-Sarcoma Fusion Gene Detection Assay Using the NanoString nCounter Platform

The NanoString nCounter assay is a high-throughput hybridization technique using target-specific probes that can be customized to test for numerous fusion transcripts in a single assay using RNA from formalin-fixed, paraffin-embedded material. We designed a NanoString assay targeting 174 unique fusion junctions in 25 sarcoma types. The study cohort comprised 212 cases, 96 of which showed fusion gene expression by the NanoString assay, including all 20 Ewing sarcomas, 11 synovial sarcomas, and 5 myxoid liposarcomas tested. Among these 96 cases, 15 showed fusion expression not identified by standard clinical assay, including EWSR1-FLI1, EWSR1-ERG, BCOR-CCNB3, ZC3H7B-BCOR, HEY1-NCOA2, CIC-DUX4, COL1A1-PDGFB, MYH9-USP6, YAP1-TFE3, and IRF2BP2-CDX1 fusions. There were no false-positive results; however, four cases were false negative when compared with clinically available fluorescence in situ hybridization or RT-PCR testing. When batched as six cases, the per-sample reagent cost was less than conventional techniques, such as fluorescence in situ hybridization, with technologist hands-on time of 1.2 hours per case and assay time of 36 hours. In summary, the NanoString nCounter Sarcoma Fusion CodeSet reliably and cost-effectively identifies fusion genes in sarcomas using formalin-fixed, paraffin-embedded material, including many fusions missed by standard clinical assays, and can serve as a first-line clinical diagnostic test for sarcoma fusion gene identification, replacing multiple individual clinical assays.

HDAC6 regulates microRNA-27b that suppresses proliferation, promotes apoptosis and target MET in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. Histone deacetylase 6 (HDAC6) is frequently altered in DLBCL and inhibition of HDAC6 has potent anti-tumor effects in vitro and in vivo. We profiled miRNAs that altered in the HDAC6 knockdown DLBCL cells with NanoString nCounter assay and identified microRNA-27b (miR-27b) as the most significantly increased miRNA. We validated decreased expression of miR-27b in DLBCL tissues, and we found that low expression of miR-27b was associated with poor overall survival of patients with DLBCL. In addition, forced expression of miR-27b suppressed DLBCL cell viability and proliferation in vitro, and inhibited tumor growth in vivo. Mechanistically, Rel A/p65 is found to negatively regulate miR-27b expression, and its acetylation and block of nuclear translocalization caused by HDAC6 inhibition significantly elevates miR-27b expression. Furthermore, miR-27b targets MET and thus represses the MET/PI3K/AKT pathway. These findings highlight an important role of miR-27b in the development of DLBCL and uncover a HDAC6-Rel A/p65-miR-27b-MET signaling pathway. Elevating miR-27b through HDAC6 inhibition would be a promising strategy for DLBCL treatment.

Abstract 811: Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma

Abstract Introduction: The receptor tyrosine kinase (RTK) inhibitor sunitinib is the first line treatment for advanced clear cell renal cell carcinoma (ccRCC). Sunitinib inhibits angiogenesis via blocking signaling through VEGFR. About 80% of patients develop resistance after a drug-sensitive period. Molecular changes early in treatment may impact drug resistance, but are poorly understood. Experimental Procedures: ACHN, 786-O and Renca cell lines were treated with 1 µM sunitinib. NSG mice were s.c. xenografted with the model cell lines and were treated with sunitinib at 40 mg/kg/day dose. mRNA expression was screened using Illumina HT-12 bead chip array and miRNA expression was assessed by Nanostring nCounter assay. R statistical packages were used for data processing. Reactome and miRPath softwares were used for downstream analysis. Results: Sunitinib treatment of ccRCC xenografts led to several early changes in tumor histology, such as the emergence of live tumor areas within the necrotic spaces. These areas showed membranous staining for E-cadherin, and β-catenin, while the rest of the tumor and vehicle-treated tumors were negative. In vitro model cell lines developed cancer spheroids when treated with sunitinib. Cancer spheroids were highly tumorigenic and metastatic, and expressed several established cancer stem cell markers. ccRCC cancer spheres, but not the 2D adherent cells, showed membranous staining for E-cadherin and β-catenin; similarly to the live tumor areas observed in in vivo sunitinib treatment. In vitro inhibition of E-cadherin by EGTA or by siRNA, interfered with viability of sunitinib treated ccRCC cell lines. Conclusions: Sunitinib treatment causes early phenotypic changes of the tumor in vivo and in vitro. The formation of highly metastatic and tumorigenic cancer spheres in model cell lines is the most prominent effect in vitro. We provide preliminary evidence that sunitinib induced in vitro cancer spheres and the live tumor areas that survive within necrotic patches of the sunitinib-treated xenografts, are related. Finally, membranous expression of E-cadherin enhances the survival of ccRCC cell lines under sunitinib treatment. Citation Format: Zsuzsanna Lichner, Rola Saleeb, Henriett Butz, Roy Nofech-Mozes, Sara Riad, Mina Farag, Andras Kapus, George Yousef. Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2017-811

Abstract 2251: Tumor tissue gene expression in association with survival of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive type of cancer with limited treatment options. Previous studies have shown that gene expression profiles were associated with TNBC prognosis. Information on specific genes that are predictive of TNBC outcome is limited. Using data and samples from a cohort of 469 TNBC cases from Shanghai Breast Cancer Survival Study (SBCSS), we systematically evaluated expressions of 302 genes in tumor tissue with survival of TNBC. Genes included in the study are PAM50 genes, genes encoding drug metabolizing enzymes and, genes implicated in TNBC biology and progression based on literature reviews. Gene expression levels were measured in total RNA isolated from formalin-fixed paraffin-embedded breast cancer tissues using the NanoString nCounter assay. Cox regression was applied to evaluate disease-free survival (DFS) and overall survival (OS) in association with gene expression. Analysis was adjusted for known predictors of TNBC outcome, including age, disease stage, basal like subtype and DUSP4 gene expression. During a median follow-up of 5.3 years (range: 0.7-8.9 years), 100 deaths and 92 recurrences/breast cancer deaths were documented. Expression levels of 17 genes were significantly associated with OS, and 15 genes with DFS (P&amp;lt;0.05). The top 5 most significant genes are EOMES (Hazard Ratio, HR = 0.88; 95% Confidence Interval, 95% CI: 0.82-0.96), GZMK (HR = 0.91, 95% CI: 0.85-0.97), TUBB3 (HR = 1.12, 95% CI: 1.03-1.21), SIT1 (HR = 0.90, 95% CI: 0.83-0.98) and ZNF224 (HR = 0.89, 95% CI: 0.81-0.97) for OS, and TUBB3 (HR = 1.14, 95% CI: 1.05-1.24), KRT14 (HR = 1.08, 95% CI: 1.02-1.14), BAG1 (HR = 1.19, 95% CI: 1.05-1.35), CD44 (HR = 0.73, 95% CI: 0.58-0.92) and EOMES (HR = 0.89, 95% CI: 0.82-0.97) for DFS. Study is on-going to replicate the findings of this study. Citation Format: Shuyang Wang, Qiuyin Cai, Hui Cai, Pingping Bao, Jie Wu, Fei Ye, Wei Zheng, Ying Zheng, Xiao-Ou Shu. Tumor tissue gene expression in association with survival of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2251. doi:10.1158/1538-7445.AM2017-2251

An investigation of the role of gene copy number variations in sorafenib sensitivity in metastatic hepatocellular carcinoma patients.

Background: Metastatic hepatocellular carcinoma (HCC) is a highly aggressive tumor with limited treatment options. While sorafenib has recently been shown to provide a survival advantage in patients with advanced HCC, the overall outcomes such as time to progression (TTP) and overall survival (OS) ought to be further improved. To that end, several targeted agents aimed at amplified oncogenes such as HER2 and FGFR2 have recently been developed. In this study, we aimed to identify genetic markers in the form of copy number variations (CNVs) that influence clinical outcomes post-sorafenib treatment in advanced HCC patients.Methods: We surveyed 38 metastatic HCC patients who were treated with sorafenib for the presence of CNVs using the NanoString nCounter assay.Results: The median TTP and OS for all patients were 2.7 months (95% confidence interval [CI]: 2.0-3.3 months) and 13.4 months (95% CI: 8.4-18.4 months), respectively. Several genes previously implicated in liver cancer were amplified, including CCND1 (n = 4), CDKN1A (n = 2), KRAS (n = 2), MDM2 (n = 1), and ERBB2 (n = 1). However, we found no correlations between CNVs and survival in our sorafenib-treated patients.Conclusions: The clinical features and biomarkers that account for sensitivity to sorafenib in HCC are complicated and remain unclear. Further investigation to identify predictive biomarkers and therapeutic strategies, including combining sorafenib with other target agents, are warranted.

An mRNA Gene Expression–Based Signature to Identify FGFR1-Amplified Estrogen Receptor–Positive Breast Tumors

Fibroblast growth factor receptor 1 (FGFR1) amplification drives poor prognosis and is an emerging therapeutic target. We sought to construct a multigene mRNA expression signature to efficiently identify FGFR1-amplified estrogen receptor-positive (ER+) breast tumors. Five independent breast tumor series were analyzed. Genes discriminative for FGFR1 amplification were screened transcriptome-wide by receiver operating characteristic analyses. The METABRIC series was leveraged to construct/evaluate four approaches to signature composition. A locked-down signature was validated with 651 ER+formalin-fixed, paraffin-embedded tissues (the University of British Columbia-tamoxifen cohort). A NanoString nCounter assay was designed to profile selected genes. For a gold standard, FGFR1 amplification was determined by fluorescent in situ hybridization (FISH). Prognostic effects of FGFR1 amplification were assessed by survival analyses. Eight 8p11-12 genes (ASH2L, BAG4, BRF2, DDHD2, LSM1, PROSC, RAB11FIP1, and WHSC1L1) together with the a priori selected FGFR1 gene, highly discriminated FGFR1 amplification (area under the receiver operating characteristic curve ≥0.82, all genes and all cohorts). The nine-gene signature Call-FGFR1-amp accurately identified FGFR1 FISH-amplified ER+tumors in the University of British Columbia-tamoxifen cohort (specificity,0.94; sensitivity,0.96) and exhibited prognostic effects (disease-specific survival hazard ratio, 1.57; 95% CI, 1.14-2.16; P=0.005). Call-FGFR1-amp includes several understudied 8p11-12 amplicon-driven oncogenes and accurately identifies FGFR1-amplified ER+breast tumors. Our study demonstrates an efficient approach to diagnosing rare amplified therapeutic targets with FISH as a confirmatory assay.

Long-term soy consumption and tumor tissue MicroRNA and gene expression in triple-negative breast cancer.

Soy food intake may have protective effects against the risk for breast cancer, including estrogen receptor (ER)-negative breast cancer. However, the underlying molecular mechanisms remain unclear. To evaluate the association of soy intake with the expression of microRNAs (miRNAs) and genes in the tumor tissue of patients with triple-negative breast cancer (TNBC; ie, breast cancer lacking expression of ER, progesterone receptor, and human epidermal growth factor receptor 2), the expression of 800 miRNAs and 302 genes were measured with NanoString nCounter assays in formalin-fixed, paraffin-embedded tumor tissue from 272 TNBC patients. Soy intake during the 1-year period before the cancer diagnosis was assessed with a validated food-frequency questionnaire. The association of soy intake with the expression of miRNAs and genes was evaluated via linear regression analysis with adjustments for patient age and TNM stage. A total of 14 miRNAs and 24 genes were significantly associated with soy food intake (P < .05): Thirteen of the 14 miRNAs (92.9%) and 9 of the 24 genes (37.5%), including tumor suppressors miR-29a-3p and IGF1R, showed overexpression for those women with high soy intake, whereas the remaining miRNAs and genes, including oncogenes KRAS and FGFR4, showed underexpression. Furthermore, cell growth-related genes showed a predominantly underexpression pattern according to a comparison of tumor samples from women with high soy food intake and samples from women with lower soy food intake. This study suggests that long-term prediagnosis soy intake may lead to increased expression of tumor suppressors and decreased expression of oncogenes, especially cell growth-related genes, in breast tumor tissues. Cancer 2016;122:2544-51. © 2016 American Cancer Society.

Ibrutinib Responsive Micro-RNAs and Upregulation of Tumor Suppressor Targets in Chronic Lymphocytic Leukemia

Background: MicroRNAs (miRNAs, miRs) are small (~22nt) noncoding RNAs that are involved in post-transcriptional regulation of specific mRNA targets. Aberrant miRNA expression has been shown to play a role in chronic lymphocytic leukemia (CLL) pathogenesis and to have prognostic implications. The lymph node (LN) has been demonstrated to be the site of CLL cell proliferation and activation. In particular, CLL cells in the LN display significantly increased BCR and NF-kB signaling in comparison to CLL cells in the peripheral blood (PB). Ibrutinib is a clinically approved BTK inhibitor that disrupts BCR and NF-kB signaling; and has been shown to be effective in relapsed or refractory CLL. We hypothesized that 1) miRNAs would be differentially expressed between activated CLL cells in the LN and CLL cells in the PB; and 2) ibrutinib would alter miRNA expression in CLL, which may affect therapeutic response by regulating the expression of miR target genes.Methods: RNA was isolated from CD19 positive selected cells from two separate cohorts of CLL patients. The first group included 17 treatment naïve patients donating paired LN and PB CD19 positive cells (10 IGHV un-mutated [59%], 4 IGHV mutated [23.5%] and 3 IGHV unknown [17.5%]); the second group contained 38 pairs of PB CD19 positive cells (22 IGHV un-mutated [58%] and 16 IGHV mutated [42%]) before and after ibrutinib treatment on day 28. A panel of 22 miRs previously reported to play a role in CLL pathogenesis, prognosis, or BCR signaling was profiled by qPCR. The expression level of a custom panel of genes encoding putative miR targets was quantified using the nanoString nCounter assay and verified by qPCR. Functional studies were performed in MEC-1 and SP-53 cell lines, which were treated separately with 1 μM ibrutinib or 1 μM fludarabine for 48 hours. Live cells were identified by trypan blue staining; miR and mRNA expression was quantified by qPCR.Results: Nineteen of 22 (86.4%) miRs were significantly increased (p < 0.05) in CLL cells from the LN in comparison to paired CLL cells from the PB, correlating with an activated B cell phenotype. Examination of mRNA transcripts of putative miR targets in 13 paired LN and PB samples showed that multiple tumor suppressor transcripts including CYLD, ARID1B and FOXP1 were significantly decreased in LN-derived CLL cells. Assay of 22 miRs in PB CLL cells from 38 patients before and after ibrutinib treatment indicated that a subset of miRs (miR-22, miR-34a, miR-146b and miR-181b) were significantly decreased (p < 0.05) in response to ibrutinib. Quantification of mRNA transcripts of putative targets of the ibrutinib responsive miRs by the nanoString nCounter assay showed that several tumor suppressor transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) were significantly increased. Functional studies in MEC-1 and SP-53 cell lines confirmed that miR-22, miR-34a, miR-146b and miR-181b were down-regulated and the tumor suppressor targets of these miRs (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, PTEN and SMAD4) were significantly increased after ibrutinib treatment. The alteration of miR and gene expression was specific to the effects of ibrutinib as similar changes were not observed post treatment with the chemotherapeutic agent fludarabine.Conclusions: miRNA expression patterns are significantly altered between CLL cells in the LN and those in the PB. An overall increase in the expression of miRs and down-regulation of mRNAs of tumor suppressor genes (ARID1B, CYLD and FOXP1) was observed in CLL cells in the LN, correlating with an activated B-cell phenotype. Ibrutinib treatment and inhibition of BCR signaling resulted in down-regulation of miR-22, miR-34a, miR-146b and miR-181b and concomitant up-regulation of multiple tumor suppressor targets including ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4 in PB CLL cells. Similar findings were also observed in ibrutinib treated MEC-1 and SP-53 cell lines. Additional studies are needed to address how much the down-regulation of miRs and consequent up-regulation of tumor suppressors in response to ibrutinib contributes to the overall disease response and whether this effect could sensitize CLL cells to treatment with cytotoxic agents. DisclosuresWiestner:Pharmacyclics: Research Funding.

Abstract 2773: Association of ALDH1A1 gene expression with survival of triple-negative breast cancer

Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that catalyze aldehyde into carboxylic acids via the NAD(P)+-dependent oxidation. Several ALDH family members have been identified in humans, including ALDH1A1, ALDH1A3, ALDH2, ALDH3A1, and ALDH4A1. ALDH1A1 is a crucial element in the retinoic acid signaling pathway regulating the self-renewal and differentiation of normal stem cells. It ALDH1A1 has been suggested as one of breast cancer stem cell markers and may play an important role in breast cancer prognosis. However, previous research on ALDH1A1 and breast cancer prognosis has yielded conflicting results. We analyzed the association between mRNA expression of ALDH1A1 in tumor tissues and survival of triple-negative breast cancer (TNBC: ER-/PR-/HER2-) using data and samples from three cohorts of breast cancer patients, including 469 cases from the Shanghai Breast Cancer Survival Study (SBCSS), 86 cases from the Nashville Breast Health Study (NBHS), and 47 cases from the Southern Community Cohort Study (SCCS). Gene expression levels were measured in total RNA isolated from microdissected archival formalin-fixed paraffin-embedded breast cancer tissues using Nanostring nCounter assays. The associations between ALDH1A1 mRNA expression levels and recurrence/breast cancer mortality and overall mortality were evaluated by multivariate survival analysis using the Cox regression model. Data from the SCCS and NBHS were combined in the analysis because both studies were conducted among US women and had a small sample size. The levels of ALDH1A1 mRNA expression were consistently and positively associated with disease free and overall survival independent of age at diagnosis and TNM stage in both Chinese and US women with TNBC. Compared to those with ALDH1A1 mRNA expression below the median, TNBC patients with ALDH1A1 mRNA expression above the median had a reduced risk of recurrence/breast cancer mortality (HR = 0.60, 95% CI: 0.39-0.92) and overall mortality (HR = 0.70, 95% CI: 0.47-1.05) in the SBCSS and reduced overall mortality in the SCCS/NBHS (HR = 0.27,95% CI: 0.10-0.74). Additional analyses of the SBCSS cohort stratified by basal-like breast cancer subtype showed that ALDH1A1 mRNA expression was similarly associated with reduced risk of recurrence/breast cancer mortality and total mortality in both basal-like and non-basal like TNBC patients, although the point estimates were not significant due to the smaller sample size. In addition, we found that patients with a higher tumor grade had a lower level of ALDH1A1 mRNA expression but did not observe an association with TNM stage. Our study suggests that the mRNA expression of ALDH1A1 in tumor tissue may be associated with favorable clinical outcomes in patients with TNBC. Further investigations are needed to understand the biological mechanism(s) underlying the observed association. Citation Format: Yan Liu, Qiuyin Cai, Ying Zheng, Michelle L. Baglia, Yinghao Su, Sarah Nechuta, Ping-Ping Bao, William Blot, Wei Zheng, Xiao-Ou Shu. Association of ALDH1A1 gene expression with survival of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2773. doi:10.1158/1538-7445.AM2015-2773