Quantitative Nuclease Protection Assay Research Articles

Usefulness of an RNA extraction-free test for the multiplexed detection of ALK, ROS1, and RET Gene Fusions in Real Life FFPE Specimens of Non-Small Cell Lung Cancers

ABSTRACT Background ALK, ROS1 and RET rearrangements occur, respectively, in 5%, 2%, and 1% non-small cell lung cancers (NSCLC). ALK and ROS1 fusion proteins detection by immunohistochemistry (IHC) has been validated for rapid patient screening, but ROS1 fusions need to be confirmed by another technique and no RET IHC test is available for clinical use. Research design and methods We report herein the usefulness of the HTG EdgeSeq Assay, an RNA extraction-free test combining a quantitative nuclease protection assay with NGS, for the detection of ALK, ROS1 and RET fusions from ‘real-life’ small NSCLC samples. A total of 203 FFPE samples were collected from 11 centers. They included 143 rearranged NSCLC (87 ALK, 39 ROS1, 17 RET) and 60 ALK-ROS1-RET negative controls. Results The assay had a specificity of 98% and a sensitivity for ALK, ROS1 and RET fusions of 80%, 94% and 100% respectively. Among the 19 HTG-assay false negative samples, the preanalytical conditions were identified as the major factors impacting the assay efficiency. Conclusions Overall, the HTG EdgeSeq assay offers comparable sensitivities and specificity than other RNA sequencing techniques, with the advantage that it can be used on very small and old samples collected multicentrically.

Platform comparison of HTG EdgeSeq and RNA-Seq for gene expression profiling of tumor tissue specimens.

3566 Background: Clinical biomarker studies are often hindered by the availability of tissue specimens of sufficient quality and quantity. While RNA-Seq is often considered the gold standard for measuring mRNA expression levels in cancer tissue, it typically requires multiple formalin-fixed paraffin-embedded (FFPE) tissue sections to extract a sufficient amount of quality RNA for subsequent gene expression profiling analysis. The HTG EdgeSeq technology is a gene expression profiling platform that combines quantitative nuclease protection assay technology with next-generation sequencing detection. Unlike RNA-Seq, the HTG EdgeSeq technology does not require RNA extraction, and can use small amounts of tissue material, typically several mm2, to generate reproducible gene expression profiles. Methods: This study compares the performance of RNA-Seq and HTG's profiling panel, the HTG EdgeSeq Precision Immuno-Oncology Panel (PIP), which is designed to measure expression levels of 1,392 genes focused on tumor/immune interaction. Approximately 1,200 samples from three tumor indications (gastric cancer, colorectal cancer and ovarian cancer) were tested using both technologies. Results: Up to four FFPE slides were used for RNA extraction to support RNA-Seq testing; out of the 1,202 samples processed, 1,099 generated extracted RNA of sufficient quality and quantity (as measured by RNA concentration, RIN score and %DV200) to proceed to sequencing, which resulted in a pass rate of 91.4% for RNA-Seq. The HTG EdgeSeq PIP panel resulted in a pass rate of 97.3% (samples passing QC metrics) when the same 1,200 samples were tested, and required only a single FFPE section owing to the small sample requirement. The t-SNE (a non-linear dimensionality reduction method) analysis of the common 1,358 genes revealed similar clustering of the three cancer indications between the two methods. Correlations across individual genes by sample resulted in the mean Spearman correlation coefficient of 0.73 (95% confidence interval of 0.61 - 0.80). Additionally, gene-wise comparisons across all samples were also evaluated. Conclusions: These data demonstrate that HTG EdgeSeq gene expression panels can be used as a competitive alternative to RNA-Seq, generating equivalent gene expression results, while offering the added benefits of a small sample size requirement, lack of RNA extraction bias, and fully automated data analysis pipeline.

Reliable Gene Expression Profiling from Small and Hematoxylin and Eosin–Stained Clinical Formalin-Fixed, Paraffin-Embedded Specimens Using the HTG EdgeSeq Platform

Clinical biomarker studies are often hindered by the scarcity or suboptimal quality of biological specimens. EdgeSeq, a transcriptomics analysis platform, combines quantitative nuclease protection assay technology with next-generation sequencing, using small amounts of starting material and delivering reproducible gene expression profiles from challenging material, such as formalin-fixed, paraffin-embedded (FFPE) tissue. To evaluate EdgeSeq for analysis of archives of stained FFPE tissue, EdgeSeq was performed on unstained, hematoxylin and eosin (H&E)-stained, and immunohistochemistry-stained slides from patients with small-cell and non-small-cell lung cancer. Pairwise comparisons of gene expression profiles from stained and unstained slides showed higher Pearson correlation coefficients with H&E staining (0.86 to 0.97) than with immunohistochemistry staining (0.21 to 0.56). A 25-gene interferon-γ signature score from unstained slides showed a Pearson correlation coefficient of 0.92 with H&E-stained slides and a significant Spearman correlation (P=0.0025) with immune scores. To test gene expression profiling in small samples, FFPE sample equivalents were examined from 5.0 to 0.08 mm2 of a section (5 μm thick); sample equivalents ≥0.31 mm2 showed alignment rates >69% and pairwise Pearson correlation coefficients ≥0.87. EdgeSeq can, thus, be used to profile small and H&E-stained FFPE tumor specimens to obtain biomarker data from limited tissue in oncology clinical trials and enable research into tumor microenvironment and immune cell engagement with tumors at the locoregional level.

An assessment of candidate genes to assist prognosis in gastric cancer.

Gastric cancer (GC) is the fourth commonest cancer worldwide, with the second highest mortality rate. Its poor mortality is linked to delayed presentation. There is a drive towards non-invasive biomarker screening and monitoring of many different types of cancer, although with limited success so far. We aimed to determine if any genes from a 32-gene panel could be used to determine GC prognosis. We carried out a retrospective study on the expression of 32 genes, selected for their proven or potential links to GC, on historic formalin fixed paraffin-embedded (FFPE) GC specimens from our unit. Gene expression was measured using quantitative nuclease protection assays (qNPA) technology. Following statistical analysis of the results, immunohistochemical staining for eight genes, both discriminating and non-discriminating, was conducted in seven age and sex matched non-metastatic: metastatic GC pairings. The stained samples were reviewed by two blinded consultant histopathologists. Multivariate Cox analysis of the gene expression data revealed metastatic status, age, sex and five genes appeared to influence GC survival. Genes negatively influencing survival included BCAS1, P53 and HSP90AA1 (relative risks 2.20, 3.73 and 7.53 respectively). Genes conveying survival benefit included CASP3 and TERT (relative risks 0.10 and 0.24 respectively). Immunohistochemical staining of seven age and sex matched non-metastatic: metastatic pairs revealed no association between gene expression and protein expression. Our study found several genes whose expression may affect GC prognosis. However, immunohistochemical analysis revealed no association between gene expression and protein expression. It remains to be determined whether gene expression or protein expression are reliable means of assessing GC prognosis.

1505P - Analysis of expression of immunomodulation factors in alveolar soft-part sarcoma: a retrospective study from the Spanish Group for Research on Sarcoma (GEIS)

Background: Alveolar soft-part sarcoma (ASPS) is an exceedingly rare entity. We previously reported on a series of 12 ASPS the expression of PD-1/PD-L1 and lymphocytic infiltrates. PD-L1 staining was positive in 50% of the cases, suggesting a role for immunomodulatory interventions. We now enlarge the series and explore the expression of immune-related genes with the HTG EdgeSeq System and analyze their possible prognostic role. Methods: Patients (pts) diagnosed with ASPS from Dec 1994 to Jul 2016 were reviewed. Clinical characteristics and outcome were collected. Immunohistochemical expression was tested on archived Formalin-Fixed Paraffin-Embedded (FFPE) blocks using PD-L1 (ab205921; Abcam), CD8 (ab4055; Abcam) antibodies. An ImmunOncology (IO) panel of 549 mRNAs was evaluated with the HTG EdgeSeq System from one 5µm FFPE section. This novel technology consists in a tissue digestion followed by a pre-hybridization with specific probes using quantitative nuclease protection assay (qNPA) and quantified by a standard RNA-seq protocol in a NGS sequencer. Results: We identified 16 ASPS pts: median age 23y (6-62), M/F=5/11. Stage localized/metastatic in 11/5. With a median FU of 91 mos (4-151), 6/11 pts (54%) relapsed, with a median RFS of 19 mos (95% CI 1-75) and 2 pts died. PD-L1 was pos in tumor in 10/16 (62%) pts. HTG showed differential expression of immune-related genes according to stage (localized vs metastatic) in MNDA (log2 fold change: -3.01, p < 0.00001) and PRAME (log2 fold change: 1.9, p < 0.0002). Pts relapsing differentially expressed ABCC2 (log2 fold change -1,64, p < 0.04) when compared with those not relapsing. PD-L1 pos vs neg tumors (pos>5% membrane staining for PD-L1) differentially expressed TNFSF11 (fold change: -1,77, p = 0.05). Tumors infiltrated/not for CD8 lymphs (pos>10% of CD8) had a differential expression of CCL18 (log2 fold change: 4,89, p < 0.0001) and SLAMF7 (log2 fold change 2,37, p < 0.0089). Conclusions: PD-L1 is expressed in more than a half of ASPS of our series. Differential expression in immune-associated genes suggest an immune related gene profile on this entity with clinical and pathological implications. Further exploration of immune-modulation in ASPS is ongoing. Legal entity responsible for the study: Spanish Group for Research on Sarcoma Funding: Spanish Group for Research on Sarcoma (GEIS). Disclosure: All authors have declared no conflicts of interest.

Abstract 5348: Reliable gene expression profiling from smaller and H&amp;E stained clinical FFPE specimens using EdgeSeq platform

Abstract Background: The scarcity of biological specimens and suboptimal quality of collected material, make clinical biomarker studies challenging. The EdgeSeq system, a novel genomics analysis platform which combines quantitative nuclease protection assay (qNPA) technology with next-generation sequencing (NGS), has the potential to mitigate the aforementioned challenges. The technology allows for a limited sample input and delivers high quality gene expression profiling data from challenging material such as FFPE tissues. However, there is further unmet need toward even smaller tissue input or in taking advantage of archival stained FFPE slides to fill the data gap, specifically for studying tumor microenvironment in the oncology clinical trials Methods: We performed EdgeSeq analysis on six triplets of unstained, H&amp;E stained and IHC stained slides from non-small cell lung cancer (NSCLC) patients tumor tissue using Oncology biomarker panel (OBP). Next, we extended the EdgeSeq OBP analysis to 44 pairs of unstained and H&amp;E stained slides from NSCLC to confirm the good correlation. To assess feasibility of limited tissue input, we first optimized bead-to-DNA volume ratio during NGS library cleanup by testing 4 ratios of bead-to-DNA from 2.5 to 1.0. Next, crude lysate prepared from small cell lung cancer (SCLC) FFPE tissue was 2-fold diluted from equivalent tissue area of 5mm2 to 0.08 mm2, and was subjected to EdgeSeq OBP profiling using the optimal bead-to-DNA ratio. Finally, correlation of gene expression profiles between stained and unstained pairs were calculated. Results: Within the six triplets of unstained, H&amp;E stained and IHC stained slides, the gene expression profiles from H&amp;E slides showed good correlation with the profiles from matched unstained FFPE. An extended NSCLC sample set of 44 pairs of unstained and H&amp;E stained FFPE further confirmed overall good correlation with 84% (37 out of 44 ) pairs with Pearson correlation coefficients of 0.9 or higher. In assessing the limited tissue input, crude lysate was diluted to as little as equivalent tissue area of 0.08mm2. The tissue area as small as 0.31 mm2 resulted in Pearson correlation coefficients of 0.9 with 5mm2 surface area input. Conclusion: We demonstrated feasibility of using EdgeSeq to generate reliable gene expression data from H&amp;E stained FFPE tissues. In addition, we showed that EdgeSeq platform can be used to generate reliable expression data from crude FFPE tissue lysate equivalent to surface area as low as 0.31 mm2 (size of larger laser microdissection tile or half of a tissue microarray core). The data show that EdgeSeq can be considered as a platform of choice to analyze specimens of suboptimal quality or of limited quantity and thus may help to fill the biomarker data gap in the oncology clinical trials, including enabling research into patterns of interactions and mechanisms of response and resistance of immune cell engagement with tumor at the loco-regional level. Citation Format: Zhenhao Qi, Lisu Wang, Keyur Desai, John Cogswell, Mark Stern, Byron Lawson, Bonnie LaFleur, Patrik Vitazka. Reliable gene expression profiling from smaller and H&amp;E stained clinical FFPE specimens using EdgeSeq platform [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 5348. doi:10.1158/1538-7445.AM2017-5348

Validation of a Multiplexed Gene Signature Assay for Diagnosis of Canine Cancers from Formalin‐Fixed Paraffin‐Embedded Tissues

BackgroundUse of molecular‐based diagnostics for companion animals is impeded by availability of technology platforms, tissue acquisition requirements, and species‐specific reagents.Hypothesis/ObjectivesTo validate a quantitative nuclease protection assay (qNPA) to simultaneously measure RNA expression of multiple genes in archived formalin‐fixed paraffin‐embedded (FFPE) tumors from dogs.AnimalsAll tumor biopsy samples were collected retrospectively from surgical biopsies and in the care of veterinarians.MethodsRetrospective case series. A qNPA 96‐well ArrayPlate was built using 30 canine‐specific genes, 5 housekeeping genes, positive and negative controls with qualified gene‐specific oligonucleotides. Pearson's correlation, coefficient of variation (CV), and multivariate analysis were used to determine analytical performance using 40 FFPE dog tumors. Once validated, 70 FFPE dog tumors were analyzed for differences in gene expression using hierarchical clustering and analysis of variance of log transformed data. Immunohistochemistry (IHC) was performed to correlate gene expression and protein expression in a subset of tumors.ResultsThe assay was linear with decreasing sample input (R 2 = 0.978), reproducible within and between 96‐well plates (r = 0.988 and 0.95, respectively) and between different laboratories (CV = 0.96). Hierarchical cluster analysis showed grouping of tumors by histogenesis and oncogenes. Significant differences were found between BCl2, E2F transcription factor 1, MDM2, COX‐2, MET proto‐oncogene receptor kinase, and other biologically relevant gene expression in tumor subtypes. Immunohistochemistry confirmed protein expression.Conclusions and Clinical ImplicationsBecause this technology works reliably on FFPE specimens, it can help expedite the broad introduction of multiplexed genomic information for improved diagnostics and discovery of new targets for therapies in veterinary oncology.

Evaluation and selection of a non-PCR based technology for improved gene expression profiling from clinical formalin-fixed, paraffin-embedded samples.

Formalin-fixed, paraffin-embedded (FFPE) clinical tissue samples have the potential to provide valuable gene-expression data for the development of cancer biomarkers. However, FFPE RNA is extensively fragmented, presenting a significant challenge for reliably detecting gene expression using traditional qPCR methods. We evaluated three novel methodologies along with the traditional qPCR method for their ability to detect Notch pathway gene expression in colorectal cancer FFPE tissue RNAs. We found that quantitative nuclease protection assay-detected gene expression in high-quality RNAs as sensitively as qPCR, and consistently detected mRNAs in highly fragmented FFPE tissue RNAs. Quantitative nuclease protection assay represents an improved methodology for detecting gene expression in FFPE tissue and has the potential to advance the development of cancer biomarkers.

Association of tumor and plasma microRNA expression with tumor monosomy-3 in patients with uveal melanoma.

BackgroundEpigenetic events mediated by methylation and histone modifications have been associated with the development of metastasis in patients with uveal melanoma. The role of epigenetic events mediated by microRNA (miR) is less clear. Tumor and plasma miR expression was examined in patients with primary uveal melanoma with tumor monosomy-3, a predictor of metastasis.ResultsmiR profiling of tumors by microarray found six miRs over-expressed and 19 under-expressed in 33 tumors with monosomy-3 compared to 22 without. None of the miRs differentially expressed in tumors with and without monosomy-3 was differentially expressed in tumors with and without tumor infiltrating lymphocytes. Tumors manifesting monosomy-3 were also characterized by higher levels of TARBP2 and DDX17 and by lower levels of XPO5 and HIWI, miR biogenesis factors. miR profiling of plasma by a quantitative nuclease protection assay found elevated levels of 11 miRs and reduction in four in patients with tumor monosomy-3. Only three miRs differentially expressed in the tumor arrays were detectable in plasma. miRs implicated in uveal melanoma development were not differentially expressed. Elevated plasma levels in patients with tumor monosomy-3 of miR-92b, identified in the tumor array, and of miR-199-5p and miR-223, identified in the plasma array, were confirmed by quantitative real-time polymerase chain reaction. Levels were also higher in patients compared to normal controls.ConclusionsThese results support a role for epigenetic mechanisms in the development of metastasis in patients with uveal melanoma and the analysis of miRs as biomarkers of metastatic risk. They also suggest that potentially useful blood miRs may be derived from the host response as well as the tumor.

Abstract 2237: Gene expression measurement of immuno-oncology targets in a single FFPE section using a novel targeted sequencing assay

Abstract Background and Purpose: The field of immuno-oncology (IO) covers a broad set of research disciplines and presents a highly diverse set of experimental requirements. The challenges include sample types of varying quality and quantity of material (including both small fixed samples and blood products) as well as an expanding multiplicity of targets to assay for immunological response. The HTG EdgeSeq system combines HTG's proprietary quantitative nuclease protection assay chemistry with a Next Generation Sequencing (NGS) platform to enable semiquantitative analysis of hundreds of targeted genes in a single assay. The novel HTG EdgeSeq Immuno-Oncology Assay measures 549 IO-related genes and can be used with most clinically relevant samples. Methods: An internal verification study was performed to evaluate a five-point, two-fold titration series (6 concentrations) in a variety of sample types with as few as a 250 cells, 32 μl of PAXgene, or 1.56 mm2 of a 5 μm FFPE section. FFPE sample types included: melanoma; DLBCL; and tissue from lung, breast, colon and renal carcinomas. Additionally, examples of the biological relevance of this data are provided by examination of expression from several pairs of samples, each profiled using a single 5 μm FFPE section. Results: Equivalent expression across the dynamic range was obtained within each of the sample types (Pearson correlations ranging between 0.84 to 0.98). For DLBCL, while most canonical markers of immune cells showed similar patterns of expression, different patterns of expression of T cell and NK cell genes were obtained, likely indicating a different composition of the immune cell infiltrates within these tumors. Specific to the two different melanoma tumors the lymphocyte infiltrates appear to be similar in these tumors. Interestingly, a markedly different immune response appears to be occurring in the melanoma tumors. One tumor appears to be mounting a significant type I interferon response, which is not as apparent in the second tumor. Differential expression of other well-known metastasis-associated genes are also observed between the two tumors. These observations suggest that patterns of differential expression could be used to assist in directing patients to targeted therapies. Conclusion: The HTG EdgeSeq Immuno-Oncology Assay provides a valuable tool for researchers exploring the immune response to tumors across a wide variety of tissues. Combining highly reproducible results with very small sample input amounts allows the assay to be utilized for the very small and precious samples available to researchers in this field. Citation Format: Monica M. Reinholz, Debrah Thompson, James Cooley, Xiao-Bo Chen, Iris Howlett, John Luecke, Patrick Roche, Bonnie LaFleur. Gene expression measurement of immuno-oncology targets in a single FFPE section using a novel targeted sequencing assay. [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 2237.

Hsa-miR-24-3p increases nasopharyngeal carcinoma radiosensitivity by targeting both the 3'UTR and 5'UTR of Jab1/CSN5.

Radiotherapy is the standard therapy for nasopharyngeal carcinoma (NPC); however, radioresistance can hinder successful treatment. Here, we report that miR-24 acts as a tumor suppressor and radiosensitizer in NPC cells and xenografts by targeting Jab1/CSN5. Although accumulating evidence has shown that Jab1/CSN5 functions as an oncoprotein in human cancers, its regulation through miRs has not been described. In this study, we found that Jab1/CSN5 functioned in a manner opposite that of miR-24 in NPC tumorigenesis and radioresistance. We demonstrated that miR-24 inhibits Jab1/CSN5 translation via direct binding to its 3’UTR and 5’UTR, leading to tumor growth inhibition, and sensitizes NPC tumors to radiation in vivo. Furthermore, silencing Jab1/CSN5 phenocopied the function of miR-24 in NPC cells after ionizing radiation treatment, resulting in increased apoptosis. Finally, we analyzed 50 paired samples of primary and matched recurrent NPC tissues from 25 NPC patients and subjected them to high-throughput genomic quantitative nuclease protection assay for quantifying simultaneously miR and mRNA levels. Our results showed that miR-24 levels were significantly decreased in recurrent NPC and that levels of Jab1/CSN5, as its target, were higher than those in primary NPC. Together, our findings indicate that miR-24 inhibits NPC tumor growth and increases NPC radiosensitivity by directly regulating Jab1/CSN5 and that both miR-24 and Jab1/CSN5 can serve as prognostic markers for NPC recurrence; this, in turn, may provide a promising therapeutic strategy for reversing NPC radioresistance.

Evaluation of a hypoxia regulated gene panel in ovarian cancer.

A panel of nine hypoxia regulated genes, selected from a previously published fifty gene panel, was investigated for its ability to predict hypoxic ovarian cancer phenotypes. All nine genes including vascular endothelial growth factor A, glucose transporter 1, phosphoglycerate mutase 1, lactate dehydrogenase A, prolyl 4-hydroxylase, alpha-polypeptide 1, adrenomedullin, N-myc downstream regulated 1, aldolase A, and carbonic anhydrase 9 were upregulated in the HEY and OVCAR-3 human ovarian cell lines cultured in vitro under hypoxic compared to normoxic conditions as measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The gene panel was also elevated in HEY xenograft tumor tissue compared to HEY cells cultured in normoxia. The HEY xenograft tissue demonstrated heterogeneous positive immunohistochemical staining for the exogenous hypoxia biomarker pimonidazole, and the hypoxia regulated protein carbonic anhydrase IX. A quantitative nuclease protection assay (qNPA) was developed which included the nine hypoxia regulated genes. The qNPA assay provided similar results to those obtained using qRT-PCR for cultured cell lines. The qNPA assay was also evaluated using paraffin embedded fixed tissues including a set of five patient matched primary and metastatic serous cancers and four normal ovaries. In this small sample set the average gene expression was higher in primary and metastatic cancer tissue compared to normal ovaries for the majority of genes investigated. This study supports further evaluation by qNPA of this gene panel as an alternative or complimentary method to existing protein biomarkers to identify ovarian cancers with a hypoxic phenotype.

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

Abstract 1904: RNA expression based screening of ALK gene fusion from formalin fixed non-small cell lung cancer samples

Abstract The ALK gene fusion results in the constitutive activation of the oncogenic ALK kinase activity through the expression of 3`ALK gene downstream of fusion partners like EML4 and KIF5B. Due to this rearrangement, fusion mRNA show high expression of 3` regions compared to 5` region of ALK gene and can be used as an indicator for presence of gene fusion. However, due to the low intrinsic expression of normal ALK mRNA, detection of the 5′ region of the ALK mRNA requires a highly sensitive assay system. To achieve this high sensitivity, we adapted the quantitative nuclease protection assay to combine biotinylated tiled probes for 3′ and 5′ ALK mRNA with the use of streptavidin conjugated polymeric horseradish peroxidase for detection. This assay can robustly detect baseline levels of ALK mRNA from normal and ALK-negative NSCLC specimen. We then tested the ability of the assay to measure the over expression of 3′ ALK gene by spiking in 3′ and 5′ in vitro transcribed (IVT) ALK RNAs into normal lung samples. Signal over baseline was detected for ALK5` and ALK3` IVT at attomolar concentrations even in the presence of a ratio of 95:5 in the background of normal lung tissue. Known ALK fusion cell lines also showed exquisite sensitivity for detection of fusion by comparing 5′ and 3′ expression ratios with sample input as few as 65 cells per reaction. We screened 293 (222 Adenocarcinoma, 47 Squamous cell carcinoma and 24 other NSCLC subtypes) surgically resected NSCLC FFPE clinical samples from multiple sources for presence of EML4-ALK fusions. Endogenous ALK mRNA expression was detectable in all samples with 12 (4.09 %) samples showing ALK fusion based on aberrant overexpression of 3′ region compared with 5′ region. All the 12 samples were of the adenocarcinoma or large cell subtype consistent with reported ALK fusion epidemiology (table). Confirmation studies with positives show greater sensitivity of detection of ALK fusion than is possible currently by FISH. ALK fusion positive samples epidemiologyALK FUSION POSITIVE SAMPLE IDNSCLC SUBTYPEP63 IHCTTF-1 IHCRACESEXAGEHISTOLOGY041535T2(1)ADENO-+CaucasianFemale69NA042210T2ADENO++CaucasianMale61Poorly differentiatedB610155ADENO++AsianFemale35Undifferentiated carcinomaCU2010/22 15825ADENO++CaucasianNA68Adenosquamous carcinomaCU2010/22 16787ADENO++CaucasianMale71Poorly differentiatedCU2010/22 20035ADENO-+CaucasianMale64Adenosquamous carcinoma with clear cell- and signet cell-like structuresCU2010/22 8119ADENO++CaucasianFemale53NACU2010/24 259313ADENO++CaucasianFemale50NARln020161ADENONANAAsianMale27Mucinous branchioalveolar carcinomaRln020207ADENONANAAsianMale59Mucinous branchioalveolar carcinomaRln060392Large CellNANAAsianFemale45Undifferentiated large cellRln060453Large Cell--AsianMale66Poorly differentiated Citation Format: Krishna Maddula, Mary-Beth Joshi, Vijay Modur, David H. Harpole. RNA expression based screening of ALK gene fusion from formalin fixed non-small cell lung cancer 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 1904. doi:10.1158/1538-7445.AM2014-1904

Abstract 4132: Expression of pattern recognition receptor genes and survival in colorectal adenocarcinoma

Abstract Chronic inflammation is one of the hallmarks of colorectal cancer. Pattern recognition receptors (PRRs) recognize dietary nutrients, bacterial components, and damage signals, and hence play an important role in innate immunity and chronic inflammation. Toll-like receptors (TLRs) and receptors for advanced glycation end products (RAGE) are PRRs that trigger NF-kB activation by binding to ligands, such as high mobility group 1 (HMGB1) and S100 protein family. However, the potential role of PRRs in colorectal cancer prognosis is unclear. We investigated the association between epithelial gene expression of TLRs, RAGE, their ligands, and inflammatory effectors and survival of patients with colorectal adenocarcinoma (CRAC). From a clinical surgical registry (2002-2012) at the Michael E. DeBakey VA Medical Center in Houston, we randomly selected 32 male patients and obtained their formalin-fixed, paraffin-embedded specimens of primary CRAC and paired adjacent normal mucosa. The mucosal epithelia were macrodissected. Multiplex quantitative nuclease protection assay was used to simultaneously quantify the expression levels for TLRs 2, 3, 4, and 5, RAGE, HMGB1, S100P, MyD88, IL-1β, and IL-8. Levels of expression were calculated as average pixel intensity for the spot image on the array plate and normalized to RPL6 and RPS13. Demographic and clinical information was abstracted from the electronic medical records. Wilcoxon test was used to compare expression levels of each gene between the tumor and the paired normal adjacent epithelia. Log-rank test was used to compare the survival function between those with high versus low expression of genes (using the median as the cutoff) in the tumor epithelia, and multivariate Cox regression model was used to estimate hazard ratio of dying. Survival time was calculated from the date of diagnosis to death or May 31, 2013. Of 32 patients with mean age 64, 18 had stage 0-IIA tumor and 24 did not receive neoadjuvant therapy. During a mean follow-up of 54 months, 15 (46.9%) patients died and 13 died within 36 months after diagnosis. Expression levels of TLR3, TLR5, MyD88, and RAGE were significantly lower, and TLR4 and IL-8 were significantly higher in the tumor epithelia compared with the paired normal mucosa (P &amp;lt; 0.05). Three-year survival rate was 82% versus 42% for those with higher versus lower expression of TLR5 (P = 0.01); and 76% versus 47% for those with higher versus lower expression of RAGE (P =0.05), in tumor epithelia respectively. Cox regression analysis adjusting for age and stage showed that higher expression levels of TLR5 in tumor epithelia were associated with reduced hazard of dying (HR = 0.23, 95% CI: 0.07-0.80). This inverse association was observed for both early and late stage of CRAC. We did not observe any consistent or significant association for other genes. Our study supports the notion that TLR5, a PRR that detects bacterial flagellins, may play a role in anti-tumor response in CRAC. Citation Format: Liang Chen, David Berger, Michael Ittmann, Hashem B. El-Serag, Courtney J. Balentine, David Y. Graham, Peter A. Richardson, Margaret R. Spitz, Susan G. Hilsenbeck, Rolando Rumbaut, Zhigang Duan, Donna L. White, Li Jiao. Expression of pattern recognition receptor genes and survival in colorectal adenocarcinoma. [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 4132. doi:10.1158/1538-7445.AM2014-4132

Effects of acute exposure to the non-steroidal anti-inflammatory drug ibuprofen on the developing North American Bullfrog (Rana catesbeiana) tadpole.

A variety of pharmaceutical chemicals can represent constituents of municipal effluent outflows that are dispersed into aquatic receiving environments worldwide. Increasingly, there is concern as to the potential of such bioactive substances to interact with wildlife species at sensitive life stages and affect their biology. Using a combination of DNA microarray, quantitative real-time polymerase chain reaction, and quantitative nuclease protection assays, we assessed the ability of sub-lethal and environmentally relevant concentrations of ibuprofen (IBF), a non-steroidal anti-inflammatory agent and prevalent environmental contaminant, to function as a disruptor of endocrine-mediated post-embryonic development of the frog. While the LC50 of IBF for pre-metamorphic Rana catesbeiana tadpoles is 41.5 mg/L (95% confidence interval: 32.3-53.5 mg/L), exposure to concentrations in the ppb range elicited molecular responses both in vivo and in organ culture. A nominal concentration of 15 μg/L IBF (actual = 13.7 μg/L) altered the abundance of 26 mRNA transcripts within the liver of exposed pre-metamorphic R. catesbeiana tadpoles within 6 d. IBF-treated animals demonstrated subsequent disruption of thyroid hormone-mediated reprogramming in the liver transcriptome affecting constituents of several metabolic, developmental, and signaling pathways. Cultured tadpole tail fin treated with IBF for 48 h also demonstrated altered mRNA levels at drug concentrations as low as 1.5 μg/L. These observations raise the possibility that IBF may alter the post-embryonic development of anuran species in freshwater environs, where IBF is a persistent or seasonal pollutant.

In Vivo Molecular Markers for Pro-inflammatory Cytokine M1 Stage and Resident Microglia in Trimethyltin-Induced Hippocampal Injury

Microglia polarization to the classical M1 activation state is characterized by elevated pro-inflammatory cytokines; however, a full profile has not been generated in the early stages of a sterile inflammatory response recruiting only resident microglia. We characterized the initial M1 state in a hippocampal injury model dependent upon tumor necrosis factor (TNF) receptor signaling for dentate granule cell death. Twenty-one-day-old CD1 male mice were injected with trimethyltin (TMT 2.3mg/kg, i.p.) and the hippocampus was examined at an early stage (24-h post-dosing) of neuronal death. Glia activation was assessed using a custom quantitative nuclease protection assay. We report elevated mRNA levels for glia response such as ionizing calcium-binding adapter molecule-1 and glial fibrillary acidic protein (Gfap); Fas, hypoxia inducible factor alpha, complement component 1qb, TNF-related genes (Tnf, Tnfaip3, Tnfrsfla); interleukin-1 alpha, Cd44, chemokine (C-C motif) ligand (Ccl)2, Cc14, integrin alpha M, lipocalin (Lcn2), and secreted phosphoprotein 1 (Spp1). These changes occurred in the absence of changes in matrix metalloproteinase 9 and 12, neural cell adhesion molecule, metabotropic glutamate receptor (Grm)3, and Ly6/neurotoxin 1 (Lynx1), as well as, a decrease in neurotrophin 3, glutamate receptor subunit epsilon (Grin)-2b, and neurotrophic tyrosine kinase receptor, type 3. The M2 anti-inflammatory marker, transforming growth factor beta-1 (Tgfb1) was elevated. mRNAs associated with early stage of injury-induced neurogenesis including fibroblast growth factor 21 and Mki67 were elevated. In the "non-injured" temporal cortex receiving projections from the hippocampus, Lynx1, Grm3, and Grin2b were decreased and Gfap increased. Formalin fixed-paraffin-embedded tissue did not generate a comparable profile.

Developing a Predictive Gene Classifier for Autism Spectrum Disorders Based upon Differential Gene Expression Profiles of Phenotypic Subgroups.

Autism spectrum disorders (ASD) are neurodevelopmental disorders which are currently diagnosed solely on the basis of abnormal stereotyped behavior as well as observable deficits in communication and social functioning. Although a variety of candidate genes have been identified on the basis of genetic analyses and up to 20% of ASD cases can be collectively associated with a genetic abnormality, no single gene or genetic variant is applicable to more than 1-2 percent of the general ASD population. In this report, we apply class prediction algorithms to gene expression profiles of lymphoblastoid cell lines (LCL) from several phenotypic subgroups of idiopathic autism defined by cluster analyses of behavioral severity scores on the Autism Diagnostic Interview-Revised diagnostic instrument for ASD. We further demonstrate that individuals from these ASD subgroups can be distinguished from nonautistic controls on the basis of limited sets of differentially expressed genes with a predicted classification accuracy of up to 94% and sensitivities and specificities of ~90% or better, based on support vector machine analyses with leave-one-out validation. Validation of a subset of the "classifier" genes by high-throughput quantitative nuclease protection assays with a new set of LCL samples derived from individuals in one of the phenotypic subgroups and from a new set of controls resulted in an overall class prediction accuracy of ~82%, with ~90% sensitivity and 75% specificity. Although additional validation with a larger cohort is needed, and effective clinical translation must include confirmation of the differentially expressed genes in primary cells from cases earlier in development, we suggest that such panels of genes, based on expression analyses of phenotypically more homogeneous subgroups of individuals with ASD, may be useful biomarkers for diagnosis of subtypes of idiopathic autism.

Development of an automated RNA-based nuclease protection assay for the detection of expressed EML-ALK fusions, EGFR, and KRAS mutations in NSCLC.

e22185 Background: The management of patients for NSCLC requires appropriate molecular pathology assessment to determine treatment. However current methods require the use of more than one technology including sequencing for EGFR and KRAS mutation and in situ hybridization for EML-ALK fusion, resulting in slow turnaround, and technical as well as logistical complexities. Here, we report the development of an automated microarray based quantitative nuclease protection assay that determines gene fusions, deletions and point mutations in target genes such as EML4/ALK, EGFR and KRAS from expressed mRNA directly from FFPE tissue without prior RNA extraction. Methods: We designed mutation and fusion specific probes for nuclease protection and measured the sensitivity, specificity, and reproducibility of gene mutations and fusions using in vitro transcribed RNA and cell lysates known to contain the target mutations. In addition to synthetic samples, we also screened 88 lung cancer specimen for EML-ALK fusions. Results: Mutations in KRAS (G12D, G12C, G12V); EGFR (D761Y, T790M, L858R); EML-ALK fusions (v1 v2, v3a, v3b-3, v4, v5a, v5b-3 and v6) were detected at concentrations of 3 copies/cell in 15,000 cells even when present as low as 5% of wild type IVT RNA. The sensitivity corresponds to an amount of FFPE sample ranging from 0.3 to 0.6 square cm of a 5mm FFPE section per replicate assay making it suitable for needle biopsy specimen. Six samples with probable EML4-ALK fusions were identified in the 88 adenomatous NSCLC samples and will be subjected to further confirmation. Additional clinical validation of all the assays will be presented in our poster. Conclusions: An automated, multiplexed, EGFR and KRAS mutation and EML-ALK gene fusion from expressed mRNA is feasible. The assay can be run directly on FFPE sections, does not require RNA extraction or amplification, and is completed within 24 hours.

Abstract 1202: Expression signature for classification of non-small cell lung cancer.

Abstract Background: Approximately 85-90% of lung cancers are non-small cell lung cancers (NSCLCs) consisting of adenocarcinoma (ADC) and squamous cell carcinoma (SCC) and several rare types including undifferentiated large cell carcinomas. Most NSCLCs are diagnosed from small biopsies or cytology materials and, even with the use of immunostains, some cannot be classified further and are referred to as NSCLC - not otherwise specified (NSCLC-NOS). For several clinically relevant reasons and for selection of therapy the correct identification of NSCLC typing is important. Specific Aim: To develop a highly specific and sensitive RNA expression signature as an adjunct test for routine pathological classification. Methods: A microarray dataset of 263 resected NSCLC cases (183 ADC, 80 SCC) obtained from MD Anderson Cancer Center and profiled on the Illumina WG-6 V3 platform was used as the learning set. The Cancer Genome Atlas (TCGA; http://cancergenome.nih.gov/) microarray dataset (105 ADC, 205 SCC), which were profiled on an RNAseq platform, was used as the validation set. Histologic typing on the TCGA specimens was performed by light microscopy according to the WHO classification. Materials for immunostaining were not always available. Results: We developed a 44-gene signature which contained many of the genes encoding proteins routinely used in immunostains for NSCLC typing, including TP63, TTF1, SOX2, and several high molecular weight keratins. For testing, a nearest neighbor approach with Pearson metric was used, which yielded two scores (Pearson correlations, ranging from -1 to +1) for ADC and SCC histologies. Scores above 0.4 were considered positive for the respective tumor type, while values below 0.4 were considered to be null (equivalent to poorly/undifferentiated histology). The learning set had 7% discrepancies for the major tumor type and 1% was scored as null. Initial validation of the TCGA data indicated a relatively high percentage of discrepancies (10% of ADC, 12% of SCC). After discussions with TCGA reference pathologists Drs. Travis and Rekhtman and further evaluation by them a revised pathological classification was generated, which included 25 NSCLC-NOS diagnoses. The discrepancies were reduced to 9 % and 4 % for ADC and SCC respectively. The NSCLC-NOS cases were classified as ADC, SCC or null types. Future plans: We are utilizing the expression signature to develop and validate a quantitative nuclease protection assay (qNPA by High Throughput Genomics) that can be applied to small biopsies and 5-micron sections of archival paraffin embedded formalin fixed material in a 96 well format. Summary and significance: The development and application of a sensitive and specific molecular signature for the classification of NSCLC provides an important adjunct test for routine pathological diagnosis, especially for application to small specimens (which constitute about 70% of current diagnostic lung cancer samples). Citation Format: Luc Girard, Ignacio Wistuba, William D. Travis, Natasha Rekhtman, Milind Suraokar, Carmen Behrens, John D. Minna, Adi F. Gazdar. Expression signature for classification of non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1202. doi:10.1158/1538-7445.AM2013-1202