nCounter Assay Research Articles

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.

Urinary miRNAs as Biomarkers for Noninvasive Evaluation of Radiation-Induced Renal Tubular Injury.

Radiation nephropathy is one of the common late effects in cancer survivors who received radiotherapy as well as in victims of radiation accidents. The clinical manifestations of radiation nephropathy occur months after exposure. To date, there are no known early biomarkers to predict the future development of radiation nephropathy. This study focuses on the development of urinary biomarkers providing readout of acute responses in renal tubular epithelial cells. An amplification-free hybridization-based nCounter assay was used to detect changes in mouse urinary miRNAs after irradiation. After a single LD50 of total-body irradiation (TBI) or clinically relevant fractionated doses (2 Gy twice daily for 3 days), changes in urinary levels of microRNAs followed either an early pattern, peaking at 6-8 h postirradiation and gradually declining, or later pattern, peaking from 24 h to 7 days. Of 600 miRNAs compared, 12 urinary miRNAs showed the acute response and seven showed the late response, common to both irradiation protocols. miR-1224 and miR-21 were of particular interest, since they were the most robust acute and late responders, respectively. The early responding miR-1224 also exhibited good dose response after 2, 4, 6 and 8 Gy TBI, indicating its potential use as a biomarker for radiation exposure. In situ hybridization of irradiated mouse kidney sections and cultured mouse primary renal tubular cells confirmed the tubular origin of miR-1224. A significant upregulation in hsa-miR-1224-3p expression was also observed in human proximal renal tubular cells after irradiation. Consistent with mouse urine data, a similar expression pattern of hsa-miR-1224-3p and hsa-miR-21 were observed in urine samples collected from human leukemia patients preconditioned with TBI. This proof-of-concept study shows the potential translational utility of urinary miRNA biomarkers for radiation damage in renal tubules with possible prediction of late effects.

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 5563: 3D Biology™ view of cancer: Simultaneous detection of somatic DNA mutations and expression profiling of genes and signaling proteins from melanoma tumor FFPE samples

Abstract Prognosis is favorable in patients with primary localized melanoma but poor in patients with metastatic disease. With more than 76,000 cases expected to be diagnosed in 2016, more precise prognostic technologies and new therapies are needed. Although targeted treatment regimens have been approved in recent years, resistance has emerged in large part due to adaptive response mechanisms and intratumoral heterogeneity. Analysis of tumor samples across multiple molecular platforms will help elucidate the complexities within and across tumors which may underlie response to therapy as well as assist in identifying predictive biomarkers; however, these approaches require significant amounts of sample, time, and resources. In order to integrate the strengths of analyzing different molecular analytes, we have modularized Nanostring Technologies' molecular barcoding technology to permit simultaneous digital measurement of cancer-associated DNA mutation variants, mRNA expression, and protein expression in one assay from the same sample (3D Biology). Novel nucleotide variant probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection of ≤ 5% from 5 ng of FFPE-extracted genomic DNA. Gene expression is measured via unique digital barcoding technology to measure mRNA transcripts, and protein expression and activity (via phosphorylation) is measured by DNA-labeled antibodies. The multi-omic workflow requires only two 5-10 micron sections of FFPE tissue, whereby DNA and RNA are extracted from one section and multiplex digital protein profiling is conducted on the second. As proof of concept demonstrating the utility of this 3D Biology platform, we have simultaneously analyzed DNA variants, RNA expression, and protein expression using NanoString’s nCounter Vantage 3D™ Solid Tumor Panel on 12 FFPE melanoma tumor samples and one normal tissue from six patients. This sample set included two metastatic tumors from each patient, and in one instance multiple regions from each metastatic site (7 and 2 regions) in order to assess intratumoral heterogeneity. Importantly, this sample set has associated mRNA expression measured using the nCounter® PanCancer Pathways for Human panel as well as whole exome sequencing (WES) data. Somatic variants seen in this latter dataset were compared with the results from the DNA SNV Solid Tumor Panel. Samples with variants that were detected in the nCounter assay but not WES were subjected to deep sequencing for validation. Overall, we show that this multiplex and multi-omic platform has the potential for rapid and sensitive assessment of patient samples that will impact clinical care. Citation Format: Jinho Lee, Christopher P. Vellano, Gavin Meredith, Jill Mckay-Fleisch, P. Martin Ross, Michael Tetzlaff, Alexandre Reuben, Courtney Hudgens, Jennifer Wargo, Jessica Garber, Andrew White, Joseph Pan, Mike Krouse, Mekala Pansalawatta, Lucas Dennis, Anisha Kharkia, Erin Piazza, Afshin Mashadi-Hossein, Rich Boykin, Nathan Elliott, Brian Filanoski, Gokhan Demirkan, Sarah Warren, Gary Geiss, Dae Kim, Joseph Beechem, Gordon B. Mills. 3D Biology™ view of cancer: Simultaneous detection of somatic DNA mutations and expression profiling of genes and signaling proteins from melanoma tumor FFPE samples [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 5563. doi:10.1158/1538-7445.AM2017-5563

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<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

Molecular classification with NanoString nCounter system in triple-negative breast cancer.

e23198 Background: Previous studies have shown that several distinct subtypes identified by gene expression profiling (GEP) consisted of triple-negative breast cancer (TNBC). Compared with the subtypes defined by GEP, we developed molecular classification with NanoString nCounter system in TNBC. Methods: GEP was conducted on 188 FFPE containing chemotherapy-naïve TNBC tumors collected at Gangnam Severance Hospital. To select core genes for classification, other 120 samples from public GEP database were used. Correlation between nCounter system and GEP using identical RNA was done. In a part of tumors, BRCA1 methylation, homologous recombination deficiency (HRD) assay, and drug response assay with ATP was comprehensively assessed. Results: To classify TNBC into 4 major subtypes (Basal-like: BL, Luminal androgen receptor: LAR, Mesenchymal: M, and Immune-modulatory: IM) according to the Vanderbilt classification, we selected 110 genes in 220 samples with GEP (100 from Gangnam Severance Hospital and 120 from public database). In other 88 samples, the classification with the 110 genes were validated. In 149 tumors excluding UNS subtype by the Vanderbilt, a correlation between 110 genes-classification and the Vanderbilt system was 74.4% (111 of 149, Pearson’s R = 0.726). In 180 tumors with GEP and nCounter assay, a correlation between them was 85.0% (153 of 180, Pearson’s R = 0.827). Compared with tumors of the IM type by 110-genes, the recurrence-free survival was significantly reduced in tumors of the non-IM type by 110-genes ( P= 0.049). In cases with BRCA1 methylation test (n = 147), a significant higher rate of BRCA1 promoter methylation was found in BL type by nCounter system (BL: 41.0% vs. non-BL: 19.4%). In cases with HRD test, a significant lower rate of HRD was found in nCounter-identified LAR type (LAR: 0% vs. non-LAR: 79.3%). In patients with in vitro drug response assay with cisplatin (n = 36), tumors with nCounter-BL had a significant higher responsiveness than tumors with others ( P= 0.028). Conclusions: Our work shows the feasibility of molecular classification with nCounter system in TNBC. Future study warrants the clinical utility of this classification to guide the subtype-specific therapy in patients with TNBC.

Allergic environment enhances airway epithelial pro-inflammatory responses to rhinovirus infection.

Airway epithelial cells (AEC) exhibit a pro-inflammatory phenotype in patients with allergic asthma. We examined the effect of an allergic cytokine environment on the response of AEC to rhinovirus (RV), the most common trigger of acute exacerbations of asthma. Calu-3 cells, a well-differentiated human AEC line, were cultured with or without the T-helper type2 cytokines interleukin (IL)-4 and IL-13, then stimulated with a toll-like receptor (TLR) 3 agonist (poly I:C, dsRNA) or a TLR7 agonist (imiquimod), or infected with RV 16. Expression of pro-inflammatory and antiviral mediators, and of viral pattern-recognition molecules, was assessed using nCounter assays, quantitative real-time PCR (qRT-PCR) and protein immunoassays. Both dsRNA and imiquimod stimulated expression of mRNA for IL6 and IL8 whereas expression of several chemokines and antiviral response genes was induced only by dsRNA. Conversely, expression of other cytokines and growth factors was induced only by imiquimod. RV infection not only stimulated expression of the inflammation-related genes induced by dsRNA, but also of complement factor B and the novel pro-inflammatory cytokine IL-32. In the T helper type2 (Th2) cytokine environment, several mediators exhibited significantly enhanced expression, whereas expression of interferons was either unchanged or enhanced. The allergic environment also increased expression of pattern-recognition receptors and of intercellular adhesion molecule 1, the cell surface receptor for RV. We conclude that Th2 cytokines promote increased production of pro-inflammatory mediators by AEC following infection with RV. Increased viral entry or enhanced signalling via pattern-recognition receptors could also contribute to the exaggerated inflammatory response to RV observed in allergic asthmatics.

Molecular characterization of urothelial carcinoma of bladder and upper urinary tract.

361 Background: It is a considerable unmet need to increase understanding of the molecular basis of urothelial carcinoma (UC) to refine the clinical decision-making process. We also aim to assess that whether we can apply similar principles in the management of upper tract UC (UTUC) based on the behavior of urinary bladder UC (UBUC). Methods: To study the molecular nature of UC, we performed next-generation sequencing to investigate the mutational and transcriptional profiles of commonly mutated genes using the Ampliseq v2. Copy number variations (CNVs) were detected with nCounter assay. Genetic alterations between UTUC and UBUC were compared. Results: Tumor samples from 34 UTUC and 63 UBUC patients were eligible for analyses. Two groups showed similar clinicopathologic features including tumor grade and stage. Median survival was, although statistically insignificant, longer in UTUC than UBUC patients (59 vs. 41 months, P = 0.63). In total, we found 920 genetic alterations from 97 samples: most common type of somatic mutation was single nucleotide variants (SNVs; 494/525, 94.1%). After analyzing total genomic alteration frequency, we found that the frequency of CNVs was not significantly different between UTUC and UBUC group: most commonly observed mutation in the UBUC patients was TP53 (18.9%), followed by KDR (7.2%), FGFR3 (4.1%) and PIK3CA (4.1%). In the UTUC patients, TP53 (22.9%), KDR (6.5%), NOTCH1 (5.3%) and FGFR3 (4.1%) are most frequentl somatic mutations. We also identified five translocations in total UC cohort including one case with FGFR3–TACC3 (Chr4) fusion. Conclusions: Within the limitation of small sample size, we found no relevant differences in somatic mutation frequencies between UTUC and UBUC, indicating a basis for similar management strategies.

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.

694P - Prognostic value of the immune-related transcriptome in biliary tract cancers

Biliary Tract Cancers (BTC) are inflammatory cancers with derangement of cytokines and recruitment of immune cells. Here we investigate if a transcriptomic immune profile (IP) is activated in BTC and represents a feature of biological behaviour. RNA was extracted by tumour tissues (TT) and matched adjacent tissues (AT). IP of 700+ immune-related transcripts was performed in TT and AT of 24 BTCs by nCounter assay. CD80 expression was assessed by immunohistochemistry (IHC). Cox regression analysis and Kaplan Meier methods were used to correlate with Relapse Free Survival (RFS) and derive a prognostic gene signature. 132 transcripts were aberrantly expressed in TT vs AT. Ingenuity Pathway Analysis showed that leucocyte migration was the top function annotation. De-regulation of PDZ Binding Kinase (PBK) in TT appeared to differentiate cases with worse prognosis (nCounter p:0.08; Taqman p:0.07). We derived a list of genes whose expression was associated with RFS. We observed that risk of recurrence was associated with a greater number of genes deregulated in AT than in TT. We shortlisted genes that maintained statistical significance at multivariate analysis (gene expression, tumour site, adjuvant chemotherapy (AC) and R0/1 resection). We observed correlation between high expression of cytotoxic T-lymphocyte antigen-4 (CTLA-4) in the AT and RFS (p:0.0004). Cases with low CTLA4 had reduced expression of CD80, while cases with high CTLA4 had increased CD80 expression. IHC expression of CD80 varied in TT and AT. No association was seen between AT CD80 expression and RFS. However, CD80 expression seemed to differentiate prognosis in patients who did not receive AC (p:0.01), suggesting that activation of this pathway may promote relapse and may be affected from adjuvant treatment. Number of CD8+ and CD4+ cells did not correlate with RFS. We also derived a immuno-gene signature that could significantly differentiate relapsed cases and was an independent prognostic factor (HR 29.43, p:0.001). We showed that IP is deregulated in the AT of resected BTC and correlates with relapse suggesting that deregulation of immune infiltrate in the normal tissue creates a favourable soil for cancer cell growth.

Abstract 1666: Biomarkers for early detection of radiation nephropathy

Abstract Purpose/Background: Chronic kidney disease is among the common late effects detected in patients receiving chemotherapy and/or radiotherapy. Currently, there are no biomarkers that allow detection of normal tissue toxicity in late responding organs such as kidney. Developing predictive biomarkers could help identify individuals that are at high risk for close follow-up and prophylactic treatments. Our studies show that miRNAs detectable in cell-free body fluids can be developed as biomarkers of acute radiation syndromes. In the present study, we sought to investigate the potential of serum and urine miRNAs as indicators of radiation induced kidney damage in rodent models. Methods and Results: An amplification-free hybridization based nCounter assay was optimized for evaluating the changes of miRNAs in body fluids. Comparison of cell-free miRNAs in urine as well as serum, collected from control versus irradiated mice, enabled us to identify candidate biomarkers that are potential indicators of radiation response. Over 40 miRNAs were detectable in urine samples collected from mice and over 80 miRNAs were detectable in serum. Molecules such as miR-1224, miR-804, miR-714 and miR-709 showed significant increase in their urine level after radiation, which peaked 6-8 hours after exposure to acute doses. On the other hand, markers such as miR-378 that are also abundant in urine did not show significant change after radiation, hence serving as an internal control. Comparison of urinary response in mice exposed to 2, 4, 6 and 8 Gy TBI revealed good dose response. Furthermore, the same markers exhibited a dose and time dependent changes following exposure to fractionated myeloablative regimen (6 × 2 Gy, bid) that is commonly used in leukaemia patients, prior to stem cell transplantation. Specifically, two of these biomarkers, miR-714 and miR-1224 exhibited a dose dependent increase in their serum level after fractionated or acute single dose exposure. In-situ hybridization displayed an overall increase in signals of miR-1224 and miR-714 at outer medulla after radiation. Increased signals detected in tubular epithelial cells indicate the possible tubular origin of the urinary miRNAs that are responsive to radiation. Conclusion: We have developed a panel of miRNAs for non-/minimally invasive detection of radiation response in kidney. Several of the urinary miRNA biomarkers exhibited a dose and time dependent changes, suggesting their potential as biomarkers for non-invasive evaluation of radiation response in organs such as kidney for which early detection of damage is a major challenge. Citation Format: Feifei Song, Jidhin Jacob, Arnab Chakravarti, Naduparambil K. Jacob. Biomarkers for early detection of radiation nephropathy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1666.

Abstract 1371: Spatially-resolved, multiplexed digital characterization of protein distribution and abundance in FFPE tissue sections

Abstract Intratumoral heterogeneity has emerged as a critical challenge to the implementation of targeted therapeutics. Historically, immunohistochemistry (IHC) has been used to assess spatial heterogeneity of proteins; however, it has been difficult to quantify protein abundance at high multiplex and wide dynamic range. Here, we report the development and validation of a spatially-resolved, antibody-based proteomic approach with a “barcoding-potential” to quantify up to 800 targets with 5.5 logs (base 10) of dynamic range in a single formalin-fixed paraffin-embedded (FFPE) slide. By labeling antibodies with photocleavable oligos which are recognized by NanoString® nCounter® fluorescent barcodes and subsequently exposing them to focused UV light, we have developed an nCounter assay capable of quantifying protein abundance in a predefined spatial region of a tissue section. Methods: A slide-mounted FFPE tissue section is bound with a multiplexed cocktail of primary antibody-oligo conjugates, and a microfluidic flow cell is attached to the slide. Using a simple modification of a standard microscope, regions of interest are identified by light or fluorescence microscopy and are sequentially illuminated with UV light to release the oligos. Following each illumination cycle, an eluent is collected and analyzed, resulting in digital counts that correspond to the abundance of each targeted protein in sequentially illuminated areas. We demonstrate a high degree of linearity (0.97 < R2 < 0.99) for the number of observed counts versus area of UV illumination, with current detection spatial resolution down to 100 μm x 100 μm, or approximately 100 cells. Application: FFPE slides from resected breast cancers are bound with an antibody cocktail (10+ plex, including HER2, EGFR, PR and others) and visualized by light microscopy. Regions of interest are identified, and oligo barcodes from those regions are released by UV illumination and digitally quantified by nCounter analysis. This enables multiplexed detection and comparison of proteins of interest from discrete regions within the tumor and adjacent normal tissue, enabling systematic interrogation of the heterogeneous tumor microenvironment. Conclusion: Application of this NanoString barcoded antibody platform to ongoing clinical studies is intended to elucidate novel responses to immunotherapy and other targeted therapies. Further development of this technology will enable the multiplexed analysis of up to 800 protein targets from a single FFPE section and facilitate detailed interrogation of spatial interactions within a tissue. The ability to measure DNA, RNA, and protein from FFPE tissue may enable the discovery of immune biomarkers in tumors and the development of companion diagnostics. Citation Format: Alessandra Cesano, Joseph Beechem, Philippa Webster, Chris Merritt, Jaemyeong Jung, Dwayne Dunaway, Gary Geiss, Sarah Warren, Gordon Mills. Spatially-resolved, multiplexed digital characterization of protein distribution and abundance in FFPE tissue sections. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1371.

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.

A nCounter CNV Assay to Detect HER2 Amplification: A Correlation Study with Immunohistochemistry and In Situ Hybridization in Advanced Gastric Cancer.

Screening amplified genes for targeted therapy with high-throughput technology is very important. The NanoString nCounter system allows multiplexed digital quantification of target molecules through the use of color-coded barcodes with the great advantage that formalin-fixed, paraffin-embedded (FFPE) tissue can be utilized. We tested nCounter custom copy number variation (CNV) panels in 220 gastric cancer samples and evaluated the utility of this method as a screening tool for the detection of CNV using HER2. For the validation of results, we compared the nCounter results with immunohistochemistry (IHC), and we further performed in situ hybridization (ISH) in discrepant cases. The average HER2 gene copy numbers (CNs) by nCounter were 17.25, 2.0 and 2.61 for the HER2 IHC positive (3+), equivocal (2+), and negative cases, respectively. Out of the 16 IHC 3+ cases, 13 (81.3%) were reported as HER2 CN gain (≥4). Gastric cancers with homogeneous HER2 overexpression or high tumor purity showed HER2 CN ≥10. Among the 192 cases with HER2 IHC negative and without HER2 gene amplification, 29 showed a HER2 CN ≥4 with the nCounter assay. The nCounter assay had a concordance rate of 83.4% (kappa value, 0.35), a sensitivity of 66.7%, a specificity of 85.2%, a negative predictive value of 96%, and a positive predictive value of 32.6% compared with HER2 IHC/ISH results. Fresh frozen (FF) samples revealed a higher concordance rate (91.5%, kappa value, 0.59) than FFPE samples (78.5%, kappa value 0.27) and showed a high specificity (97.2%). The nCounter CNV assay is a reliable and practical method to detect high CN variations. Given the intra-tumoral HER2 heterogeneity and normal cell contamination, additional IHC and/or FISH is necessary and needs caution in interpretation, especially in FFPE tissue samples.

To Excavate Biomarkers Predictive of the Response for Capecitabine plus RAD001 through Nanostring-Based Multigene Assay in Advanced Gastric Cancer Patients.

Comprehensive characterization of individual patients' tumour is important to realize personalized medicine. Here, we investigate to identify subsets that benefit from capecitabine plus RAD001 in advanced gastric cancer (GC) patients by comprehensive high-throughput genomic analysis (nCounter assay). Archival tumour tissue blocks, if possible, were collected at phase II trial of capecitabine plus RAD001 in 47 refractory GC patients (at clinicaltrials.gov NCT#01099527). A total of 42 formalin-fixed, paraffin-embedded (FFPE) tumour samples were available for nanostring based-multigene Assay. An nCounter assay of 519 kinase panels has been used. We performed correlation analyses between expression levels of kinase genes and response for capecitabine plus RAD001. Among 42 patients with An nCounter assay of 519 kinase panels, 4 patients achieved confirmed partial response and 15 patients revealed stable disease, resulting in an overall response rate (ORR) of 9.5%. No difference in ORR was observed in terms of gender, performance status, primary tumour site, gastric resection, histologic subtype, Lauren classification, No. of metastatic site and No. of chemotherapy. In subgroups with response for capecitabine plus RAD001, there is significant overexpression of 6 genes among 519 kinase gene such as EPHA2 (P = 0.0025), PIM1 (P = 0.0031), KSR1 (P = 0.0033), and EIF2AK4 (P = 0.0046) that are related to the activation of mTOR signalling. This study is first report that investigated to identify biomarkers predictive of the response for RAD001 containing treatment in refractory GC patients, by comprehensive high-throughput genomic analysis (nCounter assay).

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.

Clinical Application of Prognostic Gene Expression Signature in Fusion Gene-Negative Rhabdomyosarcoma: A Report from the Children's Oncology Group.

Pediatric rhabdomyosarcoma (RMS) has two common histologic subtypes: embryonal (ERMS) and alveolar (ARMS). PAX-FOXO1 fusion gene status is a more reliable prognostic marker than alveolar histology, whereas fusion gene-negative (FN) ARMS patients are clinically similar to ERMS patients. A five-gene expression signature (MG5) previously identified two diverse risk groups within the fusion gene-negative RMS (FN-RMS) patients, but this has not been independently validated. The goal of this study was to test whether expression of the MG5 metagene, measured using a technical platform that can be applied to routine pathology material, would correlate with outcome in a new cohort of patients with FN-RMS. Cases were taken from the Children's Oncology Group (COG) D9803 study of children with intermediate-risk RMS, and gene expression profiling for the MG5 genes was performed using the nCounter assay. The MG5 score was correlated with clinical and pathologic characteristics as well as overall and event-free survival. MG5 standardized score showed no significant association with any of the available clinicopathologic variables. The MG5 signature score showed a significant correlation with overall (N = 57; HR, 7.3; 95% CI, 1.9-27.0; P = 0.003) and failure-free survival (N = 57; HR, 6.1; 95% CI, 1.9-19.7; P = 0.002). This represents the first, validated molecular prognostic signature for children with FN-RMS who otherwise have intermediate-risk disease. The capacity to measure the expression of a small number of genes in routine pathology material and apply a simple mathematical formula to calculate the MG5 metagene score provides a clear path toward better risk stratification in future prospective clinical trials.

Gastrointestinal malignancies harbor actionable MET exon 14 deletions.

Recently, MET exon 14 deletion (METex14del) has been postulated to be one potential mechanism for MET protein overexpression. We screened for the presence of METex14del transcript by multiplexed fusion transcript analysis using nCounter assay followed by confirmation with quantitative reverse transcription PCR with correlation to MET protein expression by immunohistochemistry (IHC) and MET amplification by fluorescence in situ hybridization (FISH). We extracted RNAs from 230 patients enrolled onto the prospective molecular profiling clinical trial (NEXT-1) (NCT02141152) between November 2013 and August 2014. Thirteen METex14del cases were identified including 3 gastric cancer, 4 colon cancer, 5 non-small cell lung cancer, and one adenocarcinoma of unknown primary. Of these 13 METex14del cases, 11 were MET IHC 3+ and 2 were 2+. Only one out of the 13 METex14del cases was MET amplified (MET/CEP ratio > 2.0). Growths of two (gastric, colon) METex14del+ patient tumor derived cell lines were profoundly inhibited by both MET tyrosine kinase inhibitors and a monoclonal antibody targeting MET. In conclusion, METex14del is a unique molecular aberration present in gastrointestinal (GI) malignancies corresponding with overexpression of MET protein but rarely with MET amplification. Substantial growth inhibition of METex14del+ patient tumor derived cell lines by several MET targeting drugs strongly suggests METex14del is a potential actionable driver mutation in GI malignancies.