Chuong D. Hoang

Robust enumeration of cell subsets from tissue expression profiles

We introduce CIBERSORT, a method for characterizing cell composition of complex tissues from their gene expression profiles. When applied to enumeration of hematopoietic subsets in RNA mixtures from fresh, frozen and fixed tissues, including solid tumors, CIBERSORT outperformed other methods with respect to noise, unknown mixture content and closely related cell types. CIBERSORT should enable large-scale analysis of RNA mixtures for cellular biomarkers and therapeutic targets (http://cibersort.stanford.edu/).

The prognostic landscape of genes and infiltrating immune cells across human cancers

Molecular profiles of tumors and tumor-associated cells hold great promise as biomarkers of clinical outcomes. However, existing data sets are fragmented and difficult to analyze systematically. Here we present a pan-cancer resource and meta-analysis of expression signatures from ∼18,000 human tumors with overall survival outcomes across 39 malignancies. By using this resource, we identified a forkhead box MI (FOXM1) regulatory network as a major predictor of adverse outcomes, and we found that expression of favorably prognostic genes, including KLRB1 (encoding CD161), largely reflect tumor-associated leukocytes. By applying CIBERSORT, a computational approach for inferring leukocyte representation in bulk tumor transcriptomes, we identified complex associations between 22 distinct leukocyte subsets and cancer survival. For example, tumor-associated neutrophil and plasma cell signatures emerged as significant but opposite predictors of survival for diverse solid tumors, including breast and lung adenocarcinomas. This resource and associated analytical tools (http://precog.stanford.edu) may help delineate prognostic genes and leukocyte subsets within and across cancers, shed light on the impact of tumor heterogeneity on cancer outcomes, and facilitate the discovery of biomarkers and therapeutic targets.

Non–Small Cell Lung Cancer: Identifying Prognostic Imaging Biomarkers by Leveraging Public Gene Expression Microarray Data—Methods and Preliminary Results

PURPOSE To identify prognostic imaging biomarkers in non-small cell lung cancer (NSCLC) by means of a radiogenomics strategy that integrates gene expression and medical images in patients for whom survival outcomes are not available by leveraging survival data in public gene expression data sets. MATERIALS AND METHODS A radiogenomics strategy for associating image features with clusters of coexpressed genes (metagenes) was defined. First, a radiogenomics correlation map is created for a pairwise association between image features and metagenes. Next, predictive models of metagenes are built in terms of image features by using sparse linear regression. Similarly, predictive models of image features are built in terms of metagenes. Finally, the prognostic significance of the predicted image features are evaluated in a public gene expression data set with survival outcomes. This radiogenomics strategy was applied to a cohort of 26 patients with NSCLC for whom gene expression and 180 image features from computed tomography (CT) and positron emission tomography (PET)/CT were available. RESULTS There were 243 statistically significant pairwise correlations between image features and metagenes of NSCLC. Metagenes were predicted in terms of image features with an accuracy of 59%-83%. One hundred fourteen of 180 CT image features and the PET standardized uptake value were predicted in terms of metagenes with an accuracy of 65%-86%. When the predicted image features were mapped to a public gene expression data set with survival outcomes, tumor size, edge shape, and sharpness ranked highest for prognostic significance. CONCLUSION This radiogenomics strategy for identifying imaging biomarkers may enable a more rapid evaluation of novel imaging modalities, thereby accelerating their translation to personalized medicine.

A Rare Population of CD24+ITGB4+Notchhi Cells Drives Tumor Propagation in NSCLC and Requires Notch3 for Self-Renewal

Sustained tumor progression has been attributed to a distinct population of tumor-propagating cells (TPCs). To identify TPCs relevant to lung cancer pathogenesis, we investigated functional heterogeneity in tumor cells isolated from Kras-driven mouse models of non-small-cell lung cancer (NSCLC). CD24(+)ITGB4(+)Notch(hi) cells are capable of propagating tumor growth in both a clonogenic and an orthotopic serial transplantation assay. While all four Notch receptors mark TPCs, Notch3 plays a nonredundant role in tumor cell propagation in two mouse models and in human NSCLC. The TPC population is enriched after chemotherapy, and the gene signature of mouse TPCs correlates with poor prognosis in human NSCLC. The role of Notch3 in tumor propagation may provide a therapeutic target for NSCLC.

Prognostic PET 18F-FDG Uptake Imaging Features Are Associated with Major Oncogenomic Alterations in Patients with Resected Non–Small Cell Lung Cancer

Although 2[18F]fluoro-2-deoxy-d-glucose (FDG) uptake during positron emission tomography (PET) predicts post-surgical outcome in patients with non-small cell lung cancer (NSCLC), the biologic basis for this observation is not fully understood. Here, we analyzed 25 tumors from patients with NSCLCs to identify tumor PET-FDG uptake features associated with gene expression signatures and survival. Fourteen quantitative PET imaging features describing FDG uptake were correlated with gene expression for single genes and coexpressed gene clusters (metagenes). For each FDG uptake feature, an associated metagene signature was derived, and a prognostic model was identified in an external cohort and then tested in a validation cohort of patients with NSCLC. Four of eight single genes associated with FDG uptake (LY6E, RNF149, MCM6, and FAP) were also associated with survival. The most prognostic metagene signature was associated with a multivariate FDG uptake feature [maximum standard uptake value (SUV(max)), SUV(variance), and SUV(PCA2)], each highly associated with survival in the external [HR, 5.87; confidence interval (CI), 2.49-13.8] and validation (HR, 6.12; CI, 1.08-34.8) cohorts, respectively. Cell-cycle, proliferation, death, and self-recognition pathways were altered in this radiogenomic profile. Together, our findings suggest that leveraging tumor genomics with an expanded collection of PET-FDG imaging features may enhance our understanding of FDG uptake as an imaging biomarker beyond its association with glycolysis.

Selective Activation of Insulin Receptor Substrate-1 and -2 in Pleural Mesothelioma Cells Association with Distinct Malignant Phenotypes

Molecular mechanisms active in transforming human pleural cells remain incompletely understood. Our previous microarray analysis of malignant pleural mesothelioma revealed alterations in components of the insulin-like growth factor (IGF) system, implicating this signaling axis in tumorigenesis. Therefore, in this current study, we characterized the molecular phenotype and investigated the key signaling pathways of the IGF system in malignant pleural mesothelioma specimens. For the major IGF components, we assessed mRNA abundance and total protein levels. We measured IGF-I ligand-dependent activation of signaling pathways downstream of the type I IGF receptor in a subset of malignant pleural mesothelioma cell lines and determined the corresponding biological consequences. At the transcriptional level, we observed consistent changes in IGF components that may contribute to a malignant phenotype. IGF-I stimulation of cells resulted in enhanced activation of type I IGF receptor and IRS adaptor proteins. Differential activation of IRS-1 signaling was associated with cell growth, whereas IRS-2 signaling was associated with cell motility. Thus, these data suggest that multiple mechanisms likely contribute to malignant pleural mesothelioma tumorigenesis. Therefore, IGF system components represent novel malignant pleural mesothelioma therapeutic targets for investigation.

Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems

Glycolysis is the initial step of glucose catabolism and is up‐regulated in cancer cells (the Warburg Effect). Such shifts toward a glycolytic phenotype have not been explored widely in other biological systems, and the molecular mechanisms underlying the shifts remain unknown. With proteomics, we observed increased glycolysis in disused human diaphragm muscle. In disused muscle, lung cancer, and H2O2‐treated myotubes, we show up‐regulation of the rate‐limiting glycolytic enzyme muscle‐type phosphofructokinase (PFKm, >2 fold, P<0.05) and accumulation of lactate (>150%, P< 0.05). Using microRNA profiling, we identify miR‐320a as a regulator of PFKm expression. Reduced miR‐320a levels (to ~50% of control, P<0.05) are associated with the increased PFKm in each of these diverse systems. Manipulation of miR‐320a levels both in vitro and in vivo alters PFKm and lactate levels in the expected directions. Further, miR‐320a appears to regulate oxidative stress‐induced PFKm expression, and reduced miR‐320a allows greater induction of glycolysis in response to H2O2 treatment. We show that this microRNA‐mediated regulation occurs through PFKms 3′ untranslated region and that Ets proteins are involved in the regulation of PFKm via miR‐320a. These findings suggest that oxidative stress‐responsive microRNA‐320a may regulate glycolysis broadly within nature.—Tang, H., Lee, M., Sharpe, O., Salamone, L., Noonan, E. J., Hoang, C. D., Levine, S., Robinson, W. H., Shrager, J. B. Oxidative stress‐responsive microRNA‐320 regulates glycolysis in diverse biological systems. FASEB J. 26, 4710–4721 (2012). www.fasebj.org

A meta-analysis of lung cancer gene expression identifies PTK7 as a survival gene in lung adenocarcinoma

Lung cancer remains the most common cause of cancer-related death worldwide and it continues to lack effective treatment. The increasingly large and diverse public databases of lung cancer gene expression constitute a rich source of candidate oncogenic drivers and therapeutic targets. To define novel targets for lung adenocarcinoma, we conducted a large-scale meta-analysis of genes specifically overexpressed in adenocarcinoma. We identified an 11-gene signature that was overexpressed consistently in adenocarcinoma specimens relative to normal lung tissue. Six genes in this signature were specifically overexpressed in adenocarcinoma relative to other subtypes of non-small cell lung cancer (NSCLC). Among these genes was the little studied protein tyrosine kinase PTK7. Immunohistochemical analysis confirmed that PTK7 is highly expressed in primary adenocarcinoma patient samples. RNA interference-mediated attenuation of PTK7 decreased cell viability and increased apoptosis in a subset of adenocarcinoma cell lines. Further, loss of PTK7 activated the MKK7-JNK stress response pathway and impaired tumor growth in xenotransplantation assays. Our work defines PTK7 as a highly and specifically expressed gene in adenocarcinoma and a potential therapeutic target in this subset of NSCLC.

Morbidity and Mortality After Esophagectomy Following Neoadjuvant Chemoradiation

BACKGROUND Neoadjuvant chemoradiation (CRT) is an accepted treatment for locally advanced esophageal carcinoma. A survival benefit has not been definitively established, and there is concern that chemoradiation may increase postoperative morbidity and mortality. METHODS A retrospective review was made of 138 patients treated for esophageal carcinoma between January 1999 and December 2009. Fifty-four patients who underwent CRT followed by esophagectomy were compared with 84 patients who underwent esophagectomy alone. RESULTS The chemoradiation and esophagectomy alone cohorts were well matched on all preoperative variables. There was a higher percentage of Ivor Lewis procedures in the esophagectomy alone cohort (82.0%) compared with the CRT cohort (59.3%; p = 0.006). Thirty-five percent of the CRT group underwent transhiatal esophagectomy. Thirty-day mortality was 6.0% (5 of 84) in the esophagectomy alone cohort compared with 1.9% (1 of 54) in the CRT cohort (p = 0.5). Similarly, mean intensive care unit stay (4.7 versus 6.5 days; p = 0.5), ventilator time (2.4 versus 4.2 days; p = 0.5), and length of stay (13.5 versus 17 days; p = 0.2) did not differ significantly between the groups. The overall major complication rates were similar in the CRT and esophagectomy alone cohorts: 57.4% versus 56% (p = 0.98). Multivariate analysis determined that coronary artery disease (p = 0.01; odds ratio 3.5) and transthoracic esophagectomy (p = 0.05; odds ratio 1.4) were predictive of development of postoperative complications. Only cervical anastomotic location (p = 0.04; odds ratio 3.0) was predictive of anastomotic leak on multivariate analysis. CONCLUSIONS Neoadjuvant chemoradiation does not appear to increase postoperative morbidity or mortality after esophagectomy. Major postoperative complications are associated with the transthoracic approach and preoperative coronary artery disease.

Intrinsic apoptosis in mechanically ventilated human diaphragm: linkage to a novel Fos/FoxO1/Stat3-Bim axis

Mechanical ventilation (MV) is a life‐saving measure in many critically ill patients. However, prolonged MV results in diaphragm dysfunction that contributes to the frequent difficulty in weaning patients from the ventilator. The molecular mechanisms underlying ventilator‐induced diaphragm dysfunction (VIDD) remain poorly understood. We report here that MV induces myonuclear DNA fragmentation (3‐fold increase; P<0.01) and selective activation of caspase 9 (P<0.05) and Bcl2‐interacting mediator of cell death (Bim; 2‐ to 7‐fold increase; P<0.05) in human diaphragm. MV also statistically significantly down‐regulates mitochondrial gene expression and induces oxidative stress. In cultured muscle cells, we show that oxidative stress activates each of the catabolic pathways thought to underlie VIDD: apoptotic (P<0.05), proteasomal (P<0.05), and autophagic (P<0.01). Further, silencing Bim expression blocks (P<0.05) oxidative stress‐induced apoptosis. Overlapping the gene expression profiles of MV human diaphragm and H2O2‐treated muscle cells, we identify Fos, FoxO1, and Stat3 as regulators of Bim expression as well as of expression of the catabolic markers atrogin and LC3. We thus identify a novel Fos/FoxO1/Stat3‐Bim intrinsic apoptotic pathway and establish the centrality of oxidative stress in the development of VIDD. This information may help in the design of specific drugs to prevent this condition.—Tang, H., Lee, M., Budak, M. T., Pietras, N., Hittinger, S., Vu, M., Khuong, A., Hoang, C. D., Hussain, S. N. A., Levine, S., Shrager, J. B. Intrinsic apoptosis in mechanically ventilated human diaphragm: linkage to a novel Fos/FoxO1/Stat3‐Bim axis. FASEB J. 25, 2921–2936 (2011). www.fasebj.org