Peter Shepard

A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling

The use of gene expression signatures to classify compounds, identify efficacy or toxicity, and differentiate close analogs relies on the sensitivity of the method to identify modulated genes. We used a novel ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq®, to determine whether previously unreported compound-responsive genes could be identified and incorporated into a broad but specific compound signature. TempO-Seq exhibits 99.6% specificity, single cell sensitivity, and excellent correlation with fold differences measured by RNA-Seq (R2 = 0.9) for 20,629 targets. Unlike many expression assays, TempO-Seq does not require RNA purification, cDNA synthesis, or capture of targeted RNA, and lacks a 3′ end bias. To investigate the sensitivity of the TempO-Seq assay to identify significantly modulated compound-responsive genes, we derived whole transcriptome profiles from MCF-7 cells treated with the histone deacetylase inhibitor Trichostatin A (TSA) and identified more than 9,000 differentially expressed genes. The TSA profile for MCF-7 cells overlapped those for HL-60 and PC-3 cells in the Connectivity Map (cMAP) database, suggesting a common TSA-specific expression profile independent of baseline gene expression. A 43-gene cell-independent TSA signature was extracted from cMAP and confirmed in TempO-Seq MCF-7 data. Additional genes that were not previously reported to be TSA responsive in the cMAP database were also identified. TSA treatment of 5 cell types revealed 1,136 differentially expressed genes in common, including 785 genes not previously reported to be TSA responsive. We conclude that TSA induces a specific expression signature that is consistent across widely different cell types, that this signature contains genes not previously associated with TSA responses, and that TempO-Seq provides the sensitive differential expression detection needed to define such compound-specific, cell-independent, changes in expression.

Extraction-Free Whole Transcriptome Gene Expression Analysis of FFPE Sections and Histology-Directed Subareas of Tissue

We describe the use of a ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq™, to profile formalin-fixed paraffin-embedded (FFPE) tissue, including H&E stained FFPE tissue, by directly lysing tissue scraped from slides without extracting RNA or converting the RNA to cDNA. The correlation of measured gene expression changes in unfixed and fixed samples using blocks prepared from a pellet of a single cell type was R2 = 0.97, demonstrating that no significant artifacts were introduced by fixation. Fixed and fresh samples prepared in an equivalent manner produced comparable sequencing depth results (+/-20%), with similar %CV (11.5 and 12.7%, respectively), indicating no significant loss of measurable RNA due to fixation. The sensitivity of the TempO-Seq assay was the same whether the tissue section was fixed or not. The assay performance was equivalent for human, mouse, or rat whole transcriptome. The results from 10 mm2 and 2 mm2 areas of tissue obtained from 5 μm thick sections were equivalent, thus demonstrating high sensitivity and ability to profile focal areas of histology within a section. Replicate reproducibility of separate areas of tissue ranged from R2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R2 = 0.90 across nine tissue types. The average %CVs were 16.8% for genes expressed at greater than 200 counts, and 20.3% for genes greater than 50 counts. Tissue specific differences in gene expression were identified and agreed with the literature. There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years. Similarly, there was negligible impact of H&E staining, facilitating accurate visualization for scraping and assay of small focal areas of specific histology within a section.

Abstract 1992: Changes in gene expression of stromal and epithelial cells during prostate cancer progression

Prostatic intraepithelial neoplasia (PIN) is a histologic abnormality which arises within the secretory epithelium of prostate glands, without disrupting the layer of basal cells which separates the epithelium from the surrounding stroma. PINs are generally considered to be a step towards development of prostatic adenocarcinoma (PCA). Molecular steps in the transition from normal glands to PIN to PCA are poorly understood, in part due to the difficulty of accessing and characterizing individual PINs without contamination from surrounding tissue. Furthermore, stromal cells surrounding pre-cancerous glands may play an active role in cancer development and progression, but are difficult to profile. We utilized the ultra-sensitive and quantitative TempO-Seq gene expression assay of the whole transcriptome to profile microdisections of stromal vs. epithelial cells in archival prostatectomy FFPE tissue. TempO-Seq is highly insensitive to RNA degradation (measurements of intact RNA RIN=9.1 to degraded RNA RIN=3.0 correlate with R2=0.97) and does not require RNA extraction, allowing measurement of both soluble and insoluble cross-linked RNA. This allowed deparaffinization and HE 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1992. doi:10.1158/1538-7445.AM2017-1992

Abstract 1729: Detection of gene expression biomarkers from enriched CTC preparations

Circulating tumor cells (CTCs) offer an opportunity for non-invasive characterization of metastatic cancer, including the ability to follow subtype evolution, development of resistance, tissue homing biomarkers, and changes in cell signaling mechanisms required for metastasis and continued growth and survival of the tumor. Analysis of CTCs and translation of a CTC assay into a clinical test has been difficult due to issues of obtaining high purity, high yield preparations and/or isolating individual CTCs. This is further compounded by the heterogeneity of CTCs, hurdles in profiling their molecular signatures, and difficulty correlating CTC number/subtype to actionable therapy. We pursued a detailed analysis of CTC gene expression using whole transcriptome TempO-Seq targeted sequencing, a highly sensitive, direct lysis, addition only assay. As this assay does not require purified CTC isolation or RNA extraction, there is no risk of CTC nor RNA loss. We exploited the single cell sensitivity of TempO-Seq to profile enriched CTC preparations prepared by RosetteSep gradient centrifugation, allowing us to characterize the phenotypes of CTCs within a high background of normal blood cells. To handle this large and potentially overwhelming background we developed and exploited a proprietary method of sample normalization that increased the sensitivity and consistency of CTC gene signature measurements. We demonstrated that the TempO-Seq assay could detect a single MCF7 (EpCAM+) Luminal-like cell in a background of 1,000 MDA MB 231 (EpCAM-) Basal-like cells (positive for an Endothelial Mesenchymal Transition, EMT), and vice versa. Next, we prepared mixtures of different proportions of these cultured cells spiked into normal blood, enriched the model “CTCs” using RosetteSep, and profiled the bulk preparation. Proportional discrimination of cell-specific signatures were obtained with as few as 40 spike-in cells. Finally, we profiled enriched CTC preparations from the blood of patients with metastatic breast cancer. The results demonstrated that the expression of HER2, ER, and EpCAM could be measured as well as biomarkers of drug resistance, bone/brain homing, EMT, and all 50 Hallmark cell signaling pathways. TempO-Seq EpCAM gene level correlated with the percent EpCAM+ cells measured by flow analysis while EMT signature levels correlated inversely. Patients with HER2- primary tumor and HER2+ CTCs could be identified, as could patients with elevated chemotherapy resistance pathways. These data demonstrate that it is possible to profile CTC phenotypes that are important in metastases and useful for selecting therapy using a bulk CTC preparation combined with the sensitivity of the TempO-Seq whole transcriptome assay. This suggests the opportunity for the development of a predictive test using currently available purification platforms without need for expensive specialized equipment or separation into single cells. Citation Format: Bruce Seligmann, Marilyn Marron, Milos Babic, Elliot Imler, Peter Shepard, Pavani Chalasani, Alison Stopeck, Joanne Yeakley. Detection of gene expression biomarkers from enriched CTC preparations [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 1729. doi:10.1158/1538-7445.AM2017-1729

Abstract 1840: Differential expression and mechanistic pathways of prostate cancer identified from FFPE tissue using surrogate or whole transcriptome TempO-Seq targeted gene expression assays

Highly sensitive and quantitative whole transcriptome and surrogate transcriptome targeted sequencing TempO-Seq assays were used to profile gene expression from 1 mm sq focal areas of FFPE sections (5 μm thick) of normal and cancerous tissue within the same prostate. The whole transcriptome assay directly measures changes within the whole transcriptome. The surrogate assay targets fewer genes yet permits in silico extrapolation to identify changes across the whole transcriptome. Both approaches identified differentiating biomarkers and mechanistic pathways in prostate cancer, both corroborating what has been reported and adding new mechanistic insights which will be discussed. These new observations likely result from the sensitivity of the TempO-Seq assays and their robust performance measuring gene expression from focal areas of FFPE, which in turn permit improved isolation of histology. Benefits derived from the TempO-Seq platform which will be discussed are: 1) Insensitivity to RNA degradation (measurements from intact RNA RIN = 9.1 correlate to measurements from degraded RNA RIN = 3.0, R2 = 0.97). 2) Does not require RNA extraction. Measures total RNA from FFPE samples, both soluble RNA in the lysate and crosslinked insoluble RNA. 3) Simple capture-free ligation-based assay protocol, without 3’ or 5’ bias to the probes used to measure each gene. No requirement for poly-adenylation or capture of the RNA (typically required in ligation-based assays). 4) Single base specificity derived from the specificity of probe ligation. 5) Misligation rates 6) High signal-to-noise provides single cell sensitivity and ability to measure differential gene expression from small focal areas of tissue. 7) Excellent reproducibility (average ∼5% CV) between biological replicates across all expressed genes results in high significance of even small fold change differences. 8) Sequencing only the ligated probes complementary to a 50-base sequence of each gene eliminates the need for bioinformatics to analyze the sequencing data. Pooling of samples into a single sequencing run permits 42 or 865 surrogate samples and 8 or 153 whole transcriptome samples to be sequenced at the same time on the MiSeq or NextSeq, respectively, at an average of 250 counts/expressed gene. In combination, this results in low sequencing cost/sample without sacrifice of information or quality. 9) Simple, robust protocol requires only a PCR or qPCR instrument, and access to a sequencer. Citation Format: Milos Babic, Peter Shepard, Joanne Yeakley, Ruchir Shah, Deepak Mav, Raymond B. Nagle, Bruce Seligmann. Differential expression and mechanistic pathways of prostate cancer identified from FFPE tissue using surrogate or whole transcriptome TempO-Seq targeted gene expression assays. [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 1840.