TPM, FPKM, or Normalized Counts? A Comparative Study of Quantification Measures for the Analysis of RNA-seq Data from the NCI Patient-Derived Models Repository.

BACKGROUND: In order to correctly decode phenotypic information from RNA-sequencing (RNA-seq) data, careful selection of the RNA-seq quantification measure is critical for inter-sample comparisons and for downstream analyses, such as differential gene expression between two or more conditions. Several methods have been proposed and continue to be used. However, a consensus has not been reached regarding the best gene expression quantification method for RNA-seq data analysis. METHODS: In the present study, we used replicate samples from each of 20 patient-derived xenograft (PDX) models spanning 15 tumor types, for a total of 61 human tumor xenograft samples available through the NCI patient-derived model repository (PDMR). We compared the reproducibility across replicate samples based on TPM (transcripts per million), FPKM (fragments per kilobase of transcript per million fragments mapped), and normalized counts using coefficient of variation, intraclass correlation coefficient, and cluster analysis. RESULTS: Our results revealed that hierarchical clustering on normalized count data tended to group replicate samples from the same PDX model together more accurately than TPM and FPKM data. Furthermore, normalized count data were observed to have the lowest median coefficient of variation (CV), and highest intraclass correlation (ICC) values across all replicate samples from the same model and for the same gene across all PDX models compared to TPM and FPKM data. CONCLUSION: We provided compelling evidence for a preferred quantification measure to conduct downstream analyses of PDX RNA-seq data. To our knowledge, this is the first comparative study of RNA-seq data quantification measures conducted on PDX models, which are known to be inherently more variable than cell line models. Our findings are consistent with what others have shown for human tumors and cell lines and add further support to the thesis that normalized counts are the best choice for the analysis of RNA-seq data across samples.

IL-4 and IL-17A Cooperatively Promote Hydrogen Peroxide Production, Oxidative DNA Damage, and Upregulation of Dual Oxidase 2 in Human Colon and Pancreatic Cancer Cells.

Dual oxidase 2 (DUOX2) generates H2O2 that plays a critical role in both host defense and chronic inflammation. Previously, we demonstrated that the proinflammatory mediators IFN-γ and LPS enhance expression of DUOX2 and its maturation factor DUOXA2 through STAT1- and NF-κB‒mediated signaling in human pancreatic cancer cells. Using a panel of colon and pancreatic cancer cell lines, we now report the induction of DUOX2/DUOXA2 mRNA and protein expression by the TH2 cytokine IL-4. IL-4 activated STAT6 signaling that, when silenced, significantly decreased induction of DUOX2. Furthermore, the TH17 cytokine IL-17A combined synergistically with IL-4 to increase DUOX2 expression in both colon and pancreatic cancer cells mediated, at least in part, by signaling through NF-κB. The upregulation of DUOX2 was associated with a significant increase in the production of extracellular H2O2 and DNA damage-as indicated by the accumulation of 8-oxo-dG and γH2AX-which was suppressed by the NADPH oxidase inhibitor diphenylene iodonium and a DUOX2-specific small interfering RNA. The clinical relevance of these experiments is suggested by immunohistochemical, microarray, and quantitative RT-PCR studies of human colon and pancreatic tumors demonstrating significantly higher DUOX2, IL-4R, and IL-17RA expression in tumors than in adjacent normal tissues; in pancreatic adenocarcinoma, increased DUOX2 expression is adversely associated with overall patient survival. These data suggest a functional association between DUOX2-mediated H2O2 production and induced DNA damage in gastrointestinal malignancies.

Hydrogen peroxide-producing NADPH oxidases and the promotion of migratory phenotypes in cancer.

The cellular microenvironment plays a critical role in cancer initiation and progression. Exposure to oxidative stress, specifically hydrogen peroxide (H2O2), has been linked to aberrant cellular signaling through which the development of cancer may be promoted. Three members of the NADPH oxidase family (NOX4, DUOX1 and DUOX2) explicitly generate this non-radical oxidant in a wide range of tissues, often in support of the inflammatory response. This review summarizes the contributions of each H2O2-producing NOX to the invasive behaviors of tumors and/or the epithelial-mesenchymal transition (EMT) in cancer that plays an essential role in metastasis. Tissue localization in tumorigenesis is also highlighted, with patient-derived TCGA microarray data profiled across 31 cancer cohorts to provide a comprehensive guide to the relevance of NOX4/DUOX1/DUOX2 in cancer studies.

Discovery of pathway-independent protein signatures associated with clinical outcome in human cancer cohorts.

Proteomic data provide a direct readout of protein function, thus constituting an information-rich resource for prognostic and predictive modeling. However, protein array data may not fully capture pathway activity due to the limited number of molecules and incomplete pathway coverage compared to other high-throughput technologies. For the present study, our aim was to improve clinical outcome prediction compared to published pathway-dependent prognostic signatures for The Cancer Genome Atlas (TCGA) cohorts using the least absolute shrinkage and selection operator (LASSO). RPPA data is particularly well-suited to the LASSO due to the relatively low number of predictors compared to larger genomic data matrices. Our approach selected predictors regardless of their pathway membership and optimally combined their RPPA measurements into a weighted risk score. Performance was assessed and compared to that of the published signatures using two unbiased approaches: 1) 10 iterations of threefold cross-validation for unbiased estimation of hazard ratio and difference in 5-year survival (by Kaplan-Meier method) between predictor-defined high and low risk groups; and 2) a permutation test to evaluate the statistical significance of the cross-validated log-rank statistic. Here, we demonstrate strong stratification of 445 renal clear cell carcinoma tumors from The Cancer Genome Atlas (TCGA) into high and low risk groups using LASSO regression on RPPA data. Median cross-validated difference in 5-year overall survival was 32.8%, compared to 25.2% using a published receptor tyrosine kinase (RTK) prognostic signature (median hazard ratios of 3.3 and 2.4, respectively). Applicability and performance of our approach was demonstrated in three additional TCGA cohorts: ovarian serous cystadenocarcinoma (OVCA), sarcoma (SARC), and cutaneous melanoma (SKCM). The data-driven LASSO-based approach is versatile and well-suited for discovery of new protein/disease associations.

Molecular Profiling-Based Assignment of Cancer Therapy (NCI-MPACT): A Randomized Multicenter Phase II Trial.

This trial assessed the utility of applying tumor DNA sequencing to treatment selection for patients with advanced, refractory cancer and somatic mutations in one of four signaling pathways by comparing the efficacy of four study regimens that were either matched to the patient's aberrant pathway (experimental arm) or not matched to that pathway (control arm). MATERIALS AND METHODS: Adult patients with an actionable mutation of interest were randomly assigned 2:1 to receive either (1) a study regimen identified to target the aberrant pathway found in their tumor (veliparib with temozolomide or adavosertib with carboplatin [DNA repair pathway], everolimus [PI3K pathway], or trametinib [RAS/RAF/MEK pathway]), or (2) one of the same four regimens, but chosen from among those not targeting that pathway. RESULTS: Among 49 patients treated in the experimental arm, the objective response rate was 2% (95% CI, 0% to 10.9%). One of 20 patients (5%) in the experimental trametinib cohort had a partial response. There were no responses in the other cohorts. Although patients and physicians were blinded to the sequencing and random assignment results, a higher pretreatment dropout rate was observed in the control arm (22%) compared with the experimental arm (6%; P = .038), suggesting that some patients may have had prior tumor mutation profiling performed that led to a lack of participation in the control arm. CONCLUSION: Further investigation, better annotation of predictive biomarkers, and the development of more effective agents are necessary to inform treatment decisions in an era of precision cancer medicine. Increasing prevalence of tumor mutation profiling and preference for targeted therapy make it difficult to use a randomized phase II design to evaluate targeted therapy efficacy in an advanced disease setting.

Inhibiting the Activity of NADPH Oxidase in Cancer.

Significance: The primary function of NADPH oxidases (NOX1-5 and dual oxidases DUOX1/2) is to produce reactive oxygen species (ROS). If inadequately regulated, NOX-associated ROS can promote oxidative stress, aberrant signaling, and genomic instability. Correspondingly, NOX isoforms are known to be overexpressed in multiple malignancies, thus constituting potential therapeutic targets in cancer. Recent Advances: Multiple genetic studies aimed at suppressing the expression of NOX proteins in cellular and animal models of cancer have provided support for the notion that NOXs play a pro-tumorigenic role. Further, large drug screens and rational design efforts have yielded inhibitor compounds, such as the diphenylene iodonium (DPI) analog series developed by our group, with increased selectivity and potency over "first generation" NOX inhibitors such as apocynin and DPI. Critical Issues: The precise role of NOX enzymes in tumor biology remains poorly defined. The tumorigenic properties of NOXs vary with cancer type, and precise tools, such as selective inhibitors, are needed to deconvolute NOX contribution to cancer development. Most NOX inhibitors developed to date are unspecific, and/or their mechanistic and pharmacological characteristics are not well defined. A lack of high-resolution crystal structures for NOX functional domains has hindered the development of potent and selective inhibitors. Future Directions: In-depth studies of NOX interactions with the tumor microenvironment (e.g., cytokines, cell-surface antigens) will help identify new approaches for NOX inhibition in cancer.

NADPH oxidase 1 is highly expressed in human large and small bowel cancers.

To facilitate functional investigation of the role of NADPH oxidase 1 (NOX1) and associated reactive oxygen species in cancer cell signaling, we report herein the development and characterization of a novel mouse monoclonal antibody that specifically recognizes the C-terminal region of the NOX1 protein. The antibody was validated in stable NOX1 overexpression and knockout systems, and demonstrates wide applicability for Western blot analysis, confocal microscopy, flow cytometry, and immunohistochemistry. We employed our NOX1 antibody to characterize NOX1 expression in a panel of 30 human colorectal cancer cell lines, and correlated protein expression with NOX1 mRNA expression and superoxide production in a subset of these cells. Although a significant correlation between oncogenic RAS status and NOX1 mRNA levels could not be demonstrated in colon cancer cell lines, RAS mutational status did correlate with NOX1 expression in human colon cancer surgical specimens. Immunohistochemical analysis of a comprehensive set of tissue microarrays comprising over 1,200 formalin-fixed, paraffin-embedded tissue cores from human epithelial tumors and inflammatory disease confirmed that NOX1 is overexpressed in human colon and small intestinal adenocarcinomas, as well as adenomatous polyps, compared to adjacent, uninvolved intestinal mucosae. In contradistinction to prior studies, we did not find evidence of NOX1 overexpression at the protein level in tumors versus histologically normal tissues in prostate, lung, ovarian, or breast carcinomas. This study constitutes the most comprehensive histopathological characterization of NOX1 to date in cellular models of colon cancer and in normal and malignant human tissues using a thoroughly evaluated monoclonal antibody. It also further establishes NOX1 as a clinically relevant therapeutic target in colorectal and small intestinal cancer.

Clinical Evolution of Epithelial-Mesenchymal Transition in Human Carcinomas.

The significance of the phenotypic plasticity afforded by epithelial-mesenchymal transition (EMT) for cancer progression and drug resistance remains to be fully elucidated in the clinic. We evaluated epithelial-mesenchymal phenotypic characteristics across a range of tumor histologies using a validated, high-resolution digital microscopic immunofluorescence assay (IFA) that incorporates ß-catenin detection and cellular morphology to delineate carcinoma cells from stromal fibroblasts and that quantitates the individual and colocalized expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (V) at subcellular resolution ("EMT-IFA"). We report the discovery of ß-catenin+ cancer cells that coexpress E-cadherin and vimentin in core-needle biopsies from patients with various advanced metastatic carcinomas, wherein these cells are transitioning between strongly epithelial and strongly mesenchymal-like phenotypes. Treatment of carcinoma models with anticancer drugs that differ in their mechanism of action (the tyrosine kinase inhibitor pazopanib in MKN45 gastric carcinoma xenografts and the combination of tubulin-targeting agent paclitaxel with the BCR-ABL inhibitor nilotinib in MDA-MB-468 breast cancer xenografts) caused changes in the tumor epithelial-mesenchymal character. Moreover, the appearance of partial EMT or mesenchymal-like carcinoma cells in MDA-MB-468 tumors treated with the paclitaxel-nilotinib combination resulted in upregulation of cancer stem cell (CSC) markers and susceptibility to FAK inhibitor. A metastatic prostate cancer patient treated with the PARP inhibitor talazoparib exhibited similar CSC marker upregulation. Therefore, the phenotypic plasticity conferred on carcinoma cells by EMT allows for rapid adaptation to cytotoxic or molecularly targeted therapy and could create a form of acquired drug resistance that is transient in nature. SIGNIFICANCE: Despite the role of EMT in metastasis and drug resistance, no standardized assessment of EMT phenotypic heterogeneity in human carcinomas exists; the EMT-IFA allows for clinical monitoring of tumor adaptation to therapy.

Molecular function limits divergent protein evolution on planetary timescales.

Functional conservation is known to constrain protein evolution. Nevertheless, the long-term divergence patterns of proteins maintaining the same molecular function and the possible limits of this divergence have not been explored in detail. We investigate these fundamental questions by characterizing the divergence between ancient protein orthologs with conserved molecular function. Our results demonstrate that the decline of sequence and structural similarities between such orthologs significantly slows down after ~1-2 billion years of independent evolution. As a result, the sequence and structural similarities between ancient orthologs have not substantially decreased for the past billion years. The effective divergence limit (>25% sequence identity) is not primarily due to protein sites universally conserved in all linages. Instead, less than four amino acid types are accepted, on average, per site across orthologous protein sequences. Our analysis also reveals different divergence patterns for protein sites with experimentally determined small and large fitness effects of mutations. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

The NCI Transcriptional Pharmacodynamics Workbench: A Tool to Examine Dynamic Expression Profiling of Therapeutic Response in the NCI-60 Cell Line Panel.

: The intracellular effects and overall efficacies of anticancer therapies can vary significantly by tumor type. To identify patterns of drug-induced gene modulation that occur in different cancer cell types, we measured gene-expression changes across the NCI-60 cell line panel after exposure to 15 anticancer agents. The results were integrated into a combined database and set of interactive analysis tools, designated the NCI Transcriptional Pharmacodynamics Workbench (NCI TPW), that allows exploration of gene-expression modulation by molecular pathway, drug target, and association with drug sensitivity. We identified common transcriptional responses across agents and cell types and uncovered gene-expression changes associated with drug sensitivity. We also demonstrated the value of this tool for investigating clinically relevant molecular hypotheses and identifying candidate biomarkers of drug activity. The NCI TPW, publicly available at https://tpwb.nci.nih.gov, provides a comprehensive resource to facilitate understanding of tumor cell characteristics that define sensitivity to commonly used anticancer drugs. SIGNIFICANCE: The NCI Transcriptional Pharmacodynamics Workbench represents the most extensive compilation to date of directly measured longitudinal transcriptional responses to anticancer agents across a thoroughly characterized ensemble of cancer cell lines.