NanoString nCounter System Research Articles

Understanding of altered N-glycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting.

To understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10 mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes. Among these queried genes, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5 times in the culture with ammonium addition after 5 days of culture; however, none of the 54 genes were significantly different after 3 days of culture. In particular, the expression level of neu1 (sialidase-1) and neu3 (sialidase-3), which play a role in reduction of sialylation, increased over 2 times. Likewise, the protein expression levels of sialidase-1 and sialidase-3 determined by Western blot analysis were also increased significantly in the culture with ammonium addition. Transient transfection of neu-1 or neu3-targeted siRNAs significantly improved the sialic acid content of the Fc-fusion protein in the culture with ammonium addition, indicating that the decreased sialic acid content was in part due to the increased expression level of sialidase. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of ammonium on N-glycosylation, especially sialylation, in rCHO cells.

GE-16 * JAPANESE PEDIATRIC MOLECULAR NEURO-ONCOLOGY GROUP (JPMNG): ESTABLISHMENT OF A NATIONWIDE MOLECULAR DIAGNOSTIC NETWORK FOR PEDIATRIC MALIGNANT BRAIN TUMORS IN JAPAN

Recent intensive molecular biological analyses revealed that pediatric brain tumors can be subclassified into multiple distinct molecular subgroups. The current consensus is that medulloblastomas consist of four core subgroups, and the sub-grouping of ependymomas is being proposed. This molecular sub-grouping system will not only improve our understanding of the biology of these tumors, but may also contribute for the development of novel therapeutic targets and strategies in the future. We have formed the Japanese Pediatric Molecular Neuro-oncology Group (JPMNG) and initiated a new clinical research project to establish a nationwide network of a molecular diagnosis system for pediatric brain tumors in Japan with the aim to provide a standardized molecular diagnosis according to the international consensus. We initially focus on medulloblastomas and ependymomas. Fresh, fresh-frozen and/or formalin-fixed paraffin-embedded archived tissue specimens are collected. Optimal diagnostic methods are being set up to reliably and reproducibly classify them into molecular subgroups according to the consensus criteria. These include gene expression analysis using the NanoString nCounter system, immunohistochemistry, RT-PCR, DNA sequencing, fluorescence in situ hybridization, and DNA methylation analysis. We have so far collected a total of 85 tissue samples including 30 medulloblastomas and 55 ependymomas. Preliminary study using 17 meduloblastomas indicated that proportions of four core subgroups were WNT (12%), SHH (35%), Group 3 (0%) and Group 4 (53%), respectively. Driver gene analysis showed that two WNT medulloblastomas had CTNNB1 mutations and TP53 mutations were found in one WNT and one SHH tumors. Mutation of TERT promoter was also found in one adult SHH tumors. We expect that this research will provide a highly accurate molecular diagnosis of medulloblastoma and ependymoma comparable to the international standard, leading to form a basis for better clinical management and outcomes and to develop new therapeutic strategies of the pediatric brain tumors in Japan.

Prognostic gene signature profiles of hepatitis C-related early-stage liver cirrhosis.

The rate of hepatitis C virus (HCV) related liver cirrhosis and subsequent cancer development is increasing and raising the risk of related mortality and morbidity. To address this issue, we aimed to develop a prognostic index that can be used to stratify patients for risk of disease progression. This index was developed in part by using a gene signature test implemented in a clinically applicable digital transcript counting platform (NanoString nCounter system). A cohort of 145 U.S. patients with HCV-related early-stage cirrhosis was analyzed by using the assay. This dataset (GEO accession number GPL17230) provides information of expression levels of the prognostic genes in the cohort.

Abstract 5100: Development of a direct-measurement molecular assay to determine telomere length in human samples

Abstract Telomere length (TL) is a recognized biomarker in cancer. Epidemiology studies suggest that blood and/or buccal cell TL is associated with certain cancers; however, TL measurement is still an under-utilized biomarker in clinical trials. The molecular testing platforms currently used for TL determination are not optimal due to inherent technical limitations. While qPCR based assays have high throughput and are inexpensive, they are difficult to develop and are very sensitive to subtle permutations in reaction components. We evaluated a strategy that relies on direct hybridization and detection of the telomere target utilizing the NanoString nCounter System (NS). The NS is a semi-quantitative assay that compares the number of reporter probe hybridization events of telomere-specific versus chromosomal invariant regions in DNA samples. Performed without nucleic acid amplification, it has potential to more accurately quantify telomeric sequences than any other assay currently in use. NS uses moderate DNA quantities per test (600ng), and has a throughput that can be expanded in multiples of 12. In each sample, the Ratio of Telomere Length (RTL) is obtained by normalizing the signal produced by the telomere specific code set probes to the signal produced by multiple control probes as compared to commercial normal human DNA. NS was validated in a three stage process using commercial DNA (n=3), cell lines (n=11) and human clinical samples (n=43) that had previously been tested by other TL tests. A blinded panel of six research samples previously tested in-house by multiplex monochrome quantitative PCR (qPCR) was sent to NanoString for in-house testing. NS exactly reproduced the panel's previous RTL ranking. The telomere code sets were subsequently tested at NCI on a panel of 11 established cell lines. Good correlation was observed compared with our in-house qPCR assay (R2 = 0.89). For the third stage of the validation, we evaluated the assay's utility on human clinical samples for which 43 whole blood leukocyte DNA samples were tested (18 dyskeratosis congenita (DC) patients with very short TL, 25 mutation-free (normal) DC relatives) and compared against leukocyte flow cytometry with in situ hybridization (flow-FISH, FF) TL measurement; FF is the gold standard in clinical testing for DC. The correlation between NS and FF lymphocyte TL was high, R2 = 0.72. Notably, the two sample t test showed that the NS assay could discriminate RTL between normals and DC patients (p = 4.6x10-8); normal relatives have longer telomeres. These data suggest that the NS RTL assay has the ability to be specific and sensitive to differences in TL between individuals with DC and healthy individuals. To determine the assay's exact clinical parameters a greater number of samples will need to be tested. The NS assay is less costly than the FF test by at least one half and if shown to be reproducible would be a viable alternative to FF and qPCR, especially in a diagnostic setting. Citation Format: Daniel C. Edelman, David Petersen, Allison Gomez, Shahinaz Gadalla, Philippa Webster, Lucas Dennis, Mike Krouse, Sharon Savage, Paul S. Meltzer. Development of a direct-measurement molecular assay to determine telomere length in human samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5100. doi:10.1158/1538-7445.AM2014-5100

Abstract 1595: Human mammary tumor virus (HMTV) detection in surgical specimens and metastases of human breast cancer by the Nanostring nCounter system and FISH analysis

Abstract The purpose of this investigation was to confirm previous PCR studies that showed the presence of Human Mammary Tumor Virus (HMTV) in human breast cancer. HMTV a retrovirus 90-98% homologous to Mouse Mammary tumor Virus (MMTV), the etiological agent of murine mammary cancer, has been detected by PCR in 40% of American women's breast cancers, but not in healthy tissues of the same breast (Cancer Research 1995, 55:5173-79). A complete 9.9kb HMTV proviral sequence has been detected from breast cancer genomic DNA and found to be inserted at different chromosomes (Cancer Res 2001,61:1754-9). HMTV viral particles with betaretrovirus characteristics and RNA sequence 85-95% homologous to MMTV have been isolated from primary cultures of metastatic breast cancer established from pleural and ascitic effusions of patients (MSSM cells). Expression of HMTV proteins has been detected in MSSM cells but not in normal mammary epithelial cells by western blot, fluorescence-activated cell sorting analysis, and immunofluorescence assays (J Virol Methods 2010, 163(1): 157-61). PCR is a sensitive molecular technique susceptible of contamination, which due to amplification, can result in false positive detection. Although our samples were tested for cox2 sequences indicative of murine mitochondrial DNA and Intracisternal A type Particle (IAP) sequences characteristic of mouse genomic DNA, the possibility still exists that laboratory contamination accounted for mistaken HMTV detection. This possibility of contamination has led to doubt that murine-like viruses exist as human pathogens. We now report two new methods for HMTV detection to reinforce previous findings. Using fluorescence in situ hybridization (FISH) assays with a HMTV probe, HMTV proviral DNA has been visualized in the nuclei of surgical samples of breast cancer as well as MSSM cells. The Nanostring nCounter system, a new technology using specific 100mer HMTV oligonucleotides as probes bound to reporter molecules, detected HMTV gag, env and LTR sequences in the absence of cox2 and IAP in a single reaction without amplification. Results from FISH analysis and Nanostring nCounter reactions show the presence of HMTV DNA in surgical cancer specimens and in MSSM metastatic cells. The data exclude contamination and confirm the authenticity of HMTV in human breast cancer. Studies are being pursued to determine if HMTV, like MMTV, plays a role in causation. Citation Format: Stella M. Melana, Polly Etkind, Teiko Nartey, Yetunde Agbaje, James F. Holland, Beatriz G-T Pogo. Human mammary tumor virus (HMTV) detection in surgical specimens and metastases of human breast cancer by the Nanostring nCounter system and FISH analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1595. doi:10.1158/1538-7445.AM2014-1595

Robust global microRNA expression profiling using next-generation sequencing technologies

miRNAs are a class of regulatory molecules involved in a wide range of cellular functions, including growth, development and apoptosis. Given their widespread roles in biological processes, understanding their patterns of expression in normal and diseased states will provide insights into the consequences of aberrant expression. As such, global miRNA expression profiling of human malignancies is gaining popularity in both basic and clinically driven research. However, to date, the majority of such analyses have used microarrays and quantitative real-time PCR. With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal many more options. To make effective use of these different platforms, the strengths and pitfalls of several miRNA profiling technologies were assessed, including a microarray platform, NGS technologies and the NanoString nCounter System. Overall, NGS had the greatest detection sensitivity, largest dynamic range of detection and highest accuracy in differential expression analysis when compared with gold-standard quantitative real-time PCR. Its technical reproducibility was high, with intrasample correlations of at least 0.95 in all cases. Furthermore, miRNA analysis of formalin-fixed, paraffin-embedded (FFPE) tissue was also evaluated. Expression profiles between paired frozen and FFPE samples were similar, with Spearman's ρ>0.93. These results show the superior sensitivity, accuracy and robustness of NGS for the comprehensive profiling of miRNAs in both frozen and FFPE tissues.

Application of NanoString nCounter System in biomedical fields

NanoString nCounter Analysis System is a newly developed gene expression detection platform that directly measures multiplexed mRNA levels through digital counting of individual mRNA transcripts.This technology uses as little as 100 ng of RNA and can obtain accurate gene quantitative data from up to 800 genes in one reaction.It requires no reverse transcription,enzymes and amplifications,and its sensitivity and accuracy are comparable to real time quantitative PCR.NanoString technology has been more and more extensively used in frontiers of biomedical research and clinics such as in validation of data from high-throughput platforms,gene expression profiling,gene regulatory network,molecular subtyping,diagnosis and prognosis of diseases. Key words: NanoString; Digital gene analysis; Multiplexed gene quantitation; Biomedical fields

Digital mRNA profiling of N-glycosylation gene expression in recombinant Chinese hamster ovary cells treated with sodium butyrate

To understand the effects of sodium butyrate (NaBu) on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing Fc-fusion glycoprotein were subjected to 3mM NaBu. The addition of NaBu to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of the glycoprotein. Fifty-two N-glycosylation-related gene expressions were also assessed by the NanoString nCounter system, which can provide a direct digital readout using custom-designed color-coded probes. Among them, ten genes (ugp, slc35a2, ganc, man1a, man1c, mgat5a, st3gal5, glb1, neu1, and neu3) were up-regulated and three genes (b4galt2, st3gal3, and neu2) were down-regulated significantly. Altered expression patterns in st3gal3, neu1, and neu3, which have roles in the sialic acid biosynthesis pathway, correlated with reduced sialic acid content of the glycoprotein by NaBu. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of NaBu on N-glycosylation in rCHO cells.

Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation

About 50% of all malignant peripheral nerve sheath tumors (MPNSTs) arise as neurofibromatosis type 1 associated lesions. In those patients malignant peripheral nerve sheath tumors are thought to arise through malignant transformation of a preexisting plexiform neurofibroma. The molecular changes associated with this transformation are still poorly understood. We sought to test the hypothesis that dysregulation of expression of kinases contributes to this malignant transformation. We analyzed expression of all 519 kinase genes in the human genome using the nanostring nCounter system. Twelve cases of malignant peripheral nerve sheath tumor arising in a background of preexisting plexiform neurofibroma were included. Both components were separately sampled. Statistical analysis compared global changes in expression levels as well as changes observed in the pairwise comparison of samples taken from the same surgical specimen. Immunohistochemical studies were performed on tissue array slides to confirm expression of selected proteins. The expression pattern of kinase genes can separate malignant peripheral nerve sheath tumors and preexisting plexiform neurofibromas. The majority of kinase genes is downregulated rather than overexpressed with malignant transformation. The patterns of expression changes are complex without simple recurring alteration. Pathway analysis demonstrates that differentially expressed kinases are enriched for kinases involved in the direct regulation of mitosis, and several of these show increased expression in malignant peripheral nerve sheath tumors. Immunohistochemical studies for the mitotic regulators BUB1B, PBK and NEK2 confirm higher expression levels at the protein level. These results suggest that the malignant transformation of plexiform neurofibroma is associated with distinct changes in the expression of kinase genes. The patterns of these changes are complex and heterogeneous. There is no single unifying alteration. Kinases involved in mitotic regulation are particularly enriched in the pool of differentially expressed kinases. Some of these are overexpressed and are therefore possible targets for kinase inhibitors.

MAGIC trial gene expression profiling study.

4020 Background: The MRC MAGIC trial established perioperative epirubicin, cisplatin and 5FU (ECF) chemotherapy as a standard of care for patients (pts) with operable oesophagogastric (OG) cancer (Cunningham NEJM 2006). We performed transcriptomic profiling of archival MAGIC tissue to evaluate an existing intrinsic gastric cancer (GC) gene signature, to describe expression patterns of biologically informative genes and to identify prognostic or predictive markers. Methods: RNA was extracted from formalin fixed paraffin embedded (FFPE) resections and analysed with the NanoString nCounter system. Our panel comprised 151 genes including intrinsic GC (G-INT and G-DIFF) signature genes (Tan Gastroenterology 2011), and genes amplified/deleted in GC including the therapeutic targets FGFR2, EGFR, ERBB2 and c-MET (Deng Gut 2012). Data was preprocessed using nsolver analysis software with 2-step normalisation and log2 transformed. G-INT and G-DIFF subtype classification used the nearest template prediction algorithm; these were then correlated with pt characteristics and survival. The overexpression threshold was defined as deviation above the normal quantile-quantile plot. Results: Sufficient RNA for analysis was available for 209 resected pts. A pilot study (n=23) confirmed RNA quality and correlation between NanoString and Affymetrix results. RNA was more often available for pts with GC (p<0.001) and pts in the surgery alone arm (p=0.049). 70% of pts were classified into G-INT or G-DIFF intrinsic signature subtype (false discovery rate (FDR) <0.05); 61 (30%) were ambiguous. Predominantly mutually exclusive overexpression of receptor tyrosine kinase (RTK) targets EGFR, ERBB2, FGFR, and c-MET was seen in 5%, 12%, 9% and 6% of pts respectively. Conclusions: Although bias may exist in tissue availability for chemosensitive pts, transcriptomic profiling of archival FFPE from this mature phase 3 study provides powerful insight into the molecular heterogeneity of OG cancer. 70% of evaluable MAGIC pts were classified unambiguously into intrinsic GC subtypes. Mutually exclusive overexpression of targetable RTKs supports a personalized approach in OG cancer evaluating targeted drugs in relevant biological subgroups. Survival analyses are ongoing.

Micro-RNA signature differences in lung adenocarcinoma with specific driver alterations.

11066 Background: Oncogenic driving alterations define types of lung adenocarcinoma with different prognosis and sensitivity to targeted agents. MicroRNAs (miRNAs) are a new class of non-coding RNAs involved in gene expression regulation. How miRNAs are dysregulated in lung cancer with ALK translocation, EGFR or KRAS mutation is unknown. In this study we aimed to identify miRNA signatures according to the presence of specific driver and to correlate miRNAs deregulation with patient outcome. Methods: The study was conducted in a cohort of 70 lung cancer patients (pts) including 18 ALK+ tumors, 11 ALK-/EGFR mutation+, 15 ALK-/KRAS mutation+, 26 ALK-/EGFR and KRAS wild-type and defined as triple negative. Matched normal lung tissue from 18 cases representative of the entire cohort were also included onto the analysis. RNA was isolated from formalin-fixed paraffin-embedded tissue (FFPE), using the Recover ALL kit (Ambion). NanoString nCounter system platform was used to generate the miRNA profile. We used Limma to test for differential expression analysis of data. The miR-515 family expression between tissues was validated by RT-qPCRs, analyzed using the parametric t-test (unpaired, 2-tailed for validation). Results: miRNA expression profile clusters distinctly ALK+ pts from ALK- and normal lung tissue. Within the ALK- group we found specific miRNAs subsets able to sub-stratify KRAS versus EGFR careers clustering sharply triple negative versus EGFR mutation+ and triple negative versus KRAS mutation+. miRNAs belonging to the miR-515 family seems to be the most deregulated in the ALK+ versus ALK-. Although their expression is stably high in normal tissues and ALK+ class, they are highly downregulated in KRAS mutated versus EGFR mutated and versus triple negative (p-value <0.001 for all comparisons). Conclusions: miRNAs profile significantly differs in pts with ALK translocation, EGFR mutations and KRAS mutations. Analysis of miR-515 family members is ongoing in order to correlate their expression levels with pts’ outcome. In vitro modulation of miR-515 family expression levels, together with drugs treatment are ongoing in order to find possible chemo-resistance/chemo-sensitivity miRNA dependent, in ALK+ and ALK- model.

Forensic Body Fluid Identification by Analysis of Multiple RNA Markers Using NanoString Technology

RNA analysis has become a reliable method of body fluid identification for forensic use. Previously, we developed a combination of four multiplex quantitative PCR (qRT-PCR) probes to discriminate four different body fluids (blood, semen, saliva, and vaginal secretion). While those makers successfully identified most body fluid samples, there were some cases of false positive and negative identification. To improve the accuracy of the identification further, we tried to use multiple markers per body fluid and adopted the NanoString nCounter system instead of a multiplex qRT-PCR system. After measuring tens of RNA markers, we evaluated the accuracy of each marker for body fluid identification. For body fluids, such as blood and semen, each body fluid-specific marker was accurate enough for perfect identification. However, for saliva and vaginal secretion, no single marker was perfect. Thus, we designed a logistic regression model with multiple markers for saliva and vaginal secretion and achieved almost perfect identification. In conclusion, the NanoString nCounter is an efficient platform for measuring multiple RNA markers per body fluid and will be useful for forensic RNA analysis.

Gene expression analysis of novel genes in the prefrontal cortex of major depressive disorder subjects

Dysregulation of the glutamatergic system has been implicated not only in the treatment of major depressive disorder (MDD), but also in the excitotoxic effects of stress and anxiety on the prefrontal cortex, which may precede the onset of a depressive episode. Our previous studies demonstrate marked deficits in prominent postsynaptic proteins involved in glutamate neurotransmission in the prefrontal cortex (PFC), Brodmann's area 10 (BA 10) from subjects diagnosed with major depressive disorder (MDD). In the same group of subjects we have identified deficits in expression and phosphorylation level of key components of the mammalian target of rapamycin (mTOR) signaling pathway, known to regulate translation initiation. Based on our previous findings, we have postulated that glutamate-dependent dysregulation of mTOR-initiated protein synthesis in the PFC may underlie the pathology of MDD. The aim of this study was to use the NanoString nCounter System to perform analysis of genes coding for glutamate transporters, glutamate metabolizing enzymes, neurotrophic factors and other intracellular signaling markers involved in glutamate signaling that were not previously investigated by our group in the PFC BA 10 from subjects with MDD. We have analyzed a total of 200 genes from 16 subjects with MDD and 16 healthy controls. These are part of the same cohort used in our previous studies. Setting our cutoff p-value≤0.01, marked upregulation of genes coding for mitochondrial glutamate carrier (GC1; p=0.0015), neuropilin 1 (NRP-1; p=0.0019), glutamate receptor ionotropic N-methyl-d-aspartate-associated protein 1 (GRINA; p=0.0060), and fibroblast growth factor receptor 1 (FGFR-1; p=0.010) was identified. No significant differences in expression of the remaining 196 genes were observed between MDD subjects and controls. While upregulation of FGFR-1 has been previously shown in MDD; abnormalities in GC-1, GRINA, and NRP-1 have not been reported. Therefore, this postmortem study identifies GC1, GRINA, and NRP-1 as novel factors associated with MDD; however, future studies will be needed to address the significance of these genes in the pathophysiology of depression and antidepressant activity.

Abstract P1-07-07: Inflammatory gene expression variations in the interval between core needle biopsy and excisional biopsy in early breast cancer

Abstract Introduction: Advancements in molecular biology have unveiled multiple breast cancer promoting pathways and potential therapeutic targets. Large randomized clinical trials remain the ultimate means of validating therapeutic efficacy, but they require large cohorts of patients and are lengthy and costly. An alternative approach is to conduct a window of opportunity study in which patients are exposed to a drug pre-surgically during the interval between the core needle biopsy (CNB) and the definitive surgery (excisional biopsy (EB)). These are non-therapeutic studies and the end point is not clinical or pathological response but rather evaluation of molecular changes in the tumor specimens that can predict response. However, since the end points of the non-therapeutic studies are biologic, it is critical to first define any biologic changes that occur in the absence of treatment. In this study, we compared the molecular profiles of breast cancer tumors at the time of the diagnostic biopsy versus the definitive surgery in the absence of any intervention. Methods: The study was conducted with DFCI/HCC IRB approval and patient consent. Post-menopausal women with a breast lesion suspected to be cancerous were eligible for this study. We obtained a tissue specimen at the time of a CNB and if determined to be consistent with invasive carcinoma a second specimen was obtained at the time of the EB. We used the Nanostring Ncounter system to study the expression level of 148 transcripts. Since we expected that most of the tumors will be hormone receptor positive (HR+), the library included; genes that have been shown to be prognostic in HR+ tumors (Oncotype DX®, PAM50), estrogen receptor (ER) modulators, ER responsive genes and inflammatory genes. The Wilcoxon's signed rank test was used to evaluate for changes in gene expression levels between the paired samples. Results: 25 patients were enrolled in this study and paired tumor tissue samples were obtained from all patients. 21 of the paired samples were successfully analyzed by the nanostring system. 86% of the patients are HR+/Her2−. We found that the gene expression levels of 14 out of the 148 genes (9%) did change between the CNB and EB without any intervention (p < 0.05). 8 of these 14 genes can be classified as inflammatory genes that also have known functions in tumor progression. The expression of these 8 genes was upregulated between the biopsies and include; CD68, ADM, CD14, IL6, VEGFA, CD52, CD44 and SNAI1. These changes may be due to an inflammatory response to the CNB. Ki67 expression did not change significantly between biopsies. Conclusions: In this study we found significant gene expression variations between CNBs and EBs in 9% of the genes tested, without any therapeutic intervention. Our findings suggest that when conducting a “Window of Opportunity” clinical study to evaluate for biomarkers of response or resistance, changes in expression of inflammatory genes cannot be attributed to treatment and a control arm should be considered. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-07.

Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome

Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesis-related mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis-related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓placental growth factor), adhesion molecules (↓angiopoietin-like-3; ↓collagen-18A1; ↓endoglin), proteases/matrix proteins/inhibitors (↓alanyl aminopeptidase; ↓collagen-4A3; ↓heparanase; ↓plasminogen, ↑plasminogen activator urokinase; ↓platelet factor-4; ↓plexin domain containing-1; ↓tissue inhibitor of metalloproteinases-3), transcription/signaling molecules (↓heart and neural crest derivatives-2; ↓DNA-binding protein inhibitor; ↓NOTCH-4; ↓ribosomal protein-L13a1; ↓ribosomal protein large-P1), cytokines/chemokines (↓interleukin-1B), and miscellaneous growth factors (↓leptin; ↓platelet-derived growth factor-α); ↓transforming growth factor (TGF-α; ↑TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional questions about FASD from both mechanistic as well as ameliorative perspectives.

Transcriptional Profiling by Sequencing of Oropharyngeal Cancer

ObjectiveTo compare full transcriptome expression levels of matched tumor and normal samples from patients with oropharyngeal carcinoma stratified by known tumor etiologic factors. Patients and MethodsFull transcriptome sequencing was analyzed for 10 matched tumor and normal tissue samples from patients with previously untreated oropharyngeal carcinoma. Transcriptomes were analyzed using massively parallel messenger RNA sequencing and validated using the NanoString nCounter system. Global gene expression levels were compared in samples grouped by smoking status and human papillomavirus status. This study was completed between June 10, 2010, and June 30, 2011. ResultsGlobal gene expression analysis indicated tumor tissue from former smokers grouped more closely to the never smokers than the current smokers. Pathway analysis revealed alterations in the expression of genes involved in the p53 DNA damage-repair pathway, including CHEK2 and ATR, which display patterns of increased expression that is associated with human papillomavirus–negative current smokers rather than former or never smokers. ConclusionThese findings support the application of messenger RNA sequencing technology as an important clinical tool for more accurately stratifying patients based on individual tumor biology with the goal of improving our understanding of tumor prognosis and treatment response, ultimately leading to individualized patient care strategies.

Linking expression of FOXM1 , CEP55 and HELLS to tumorigenesis in oropharyngeal squamous cell carcinoma

To investigate the relationship between the expression of FOXM1, CEP55, and HELLS in oropharyngeal squamous cell carcinoma to human papillomavirus (HPV), smoking, and tumor stage. Retrospective cohort study. Transcriptome data were analyzed from matched tumor-normal samples taken from patients with oropharyngeal squamous cell carcinoma. Data were previously generated using deep-sequencing techniques (mRNA-Seq). Transcript levels of all three genes were validated using the NanoString nCounter system in a larger group of patients. Analyses were conducted to assess possible associations between expression levels and HPV infection status, smoking status, or tumor staging. FOXM1, CEP55, and HELLS were all overexpressed in oropharyngeal squamous cell carcinoma tissue when compared to normal tissue. Significant trends were found between expression levels of FOXM1, CEP55, and HELLS and tumor staging. Tumors staged 3 or greater showed significantly higher levels of expression compared with those staged 1. No significant association or trend was found between expression of any genes of interest and etiologic subgroupings (i.e., HPV, smoking). FOXM1, CEP55, and HELLS were all overexpressed in oropharyngeal squamous cell carcinoma. Gene expression is related to tumor stage. The significant association between the expression of these genes and advanced tumor stage suggest that they may play a role in tumorigenesis.

Abstract LB-327: Analytical performance of the nCounter analysis system for gene expression cancer signatures

Abstract Background: The nCounter Analysis System is a platform for performing highly multiplexed, digital quantification of hundreds of different nucleic acid species in a single reaction. The system is being developed for use as a platform for in vitro diagnostic applications. The current study aimed to evaluate the analytical performance of the system by implementing a 50-gene signature used for determining the intrinsic subtype and prognostic risk of a breast cancer tumor from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The gene expression profiles can be used to divide breast cancer into four intrinsic subtypes: Basal-like, Luminal A, Luminal B, and HER2 enriched. A normal-like subtype identifies tumor specimens contaminated with a high percentage of normal tissue. A prognostic Risk-Of-Relapse (ROR) score is also calculated. Methods: The nCounter assay is performed by direct, multiplexed hybridization of molecular barcodes to target mRNAs. For this study, a CodeSet containing probes for 50 classification genes and 8 normalizing genes was developed. The intrinsic subtyping algorithm was trained by supervised hierarchical clustering of data from 538 samples. Prototypical centroids for tumor subtypes were chosen as statistically significant clusters, while the normal-like centroid was trained from reduction mammoplasty samples. All samples were correlated to the five prototypical centroids and assigned the subtype with the largest positive correlation. The analytical precision of the assay was measured by testing the same sample across replicates. The precision of the subtyping test was determined by assaying 40 different samples across 9 different combinations of reagent lots. The performance of the assay was also evaluated with varying input levels of RNA. Results: The precision of the nCounter assay was found to be driven by Poisson noise in the digital measurements at low expression levels. When samples were subtyped across multiple reagent lots, the correlations to each of the five subtypes were narrowly distributed. One sample varied in the categorical subtype call between Luminal B and HER2-enriched across all lots, however, this sample was similarly correlated between both Luminal B and HER2-enriched across all lot combinations. Not surprisingly, the variability for the ROR was minimal, even for the case where the categorical subtype call changed. Finally, the subtype call and ROR score were stable across a 10x range of RNA input. Conclusions: The NanoString nCounter system has the sensitivity and precision to be implemented as a distributed platform for multiplexed gene expression profiling of tumors. The sensitivity and direct digital detection without the need for enzymatic amplification make the technology compatible with FFPE tissue samples. The precision should make the technology robust under the many varied conditions seen in testing labs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-327. doi:10.1158/1538-7445.AM2011-LB-327