A Multidimensional Bioinformatic Platform for the Study of Human Response to Surgery.

OBJECTIVE: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing. BACKGROUND: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes. METHODS: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury. RESULTS: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints. CONCLUSIONS: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.

Immune response profiling in patients with traumatic injuries associated with alcohol ingestion.

Traumatic injuries afflict more than 5 million people globally every year. Current and past animal research has demonstrated association among alcohol, trauma, and impaired immune function, whereas human registries have shown association between alcohol and morbidity as well as mortality. The purpose of this study is to elucidate the immune interactions with alcohol in traumatically injured patients. We prospectively enrolled 379 patients after trauma at three medical centers in the Surgical Critical Care Initiative. Plasma was analyzed using Luminex for up to 35 different cytokines. Collected samples were grouped by patients with detectable plasma alcohol levels versus those without. Univariate testing determined differences in analytes between groups. We built Bayesian belief networks with multiple minimum descriptive lengths to compare the two groups. All 379 patient samples were analyzed. Two hundred eighty-two (74.4%) patients were men, and 143 (37.7%) were White. Patients had a median intensive care unit length of stay (LOS) of 5.8 days and hospital LOS of 12 days. Using single variate analyses, eight different cytokines were differentially associated with alcohol. Cytokines IL-12 and IL-6 were important nodes in both models and IL-10 was a prominent node in the nonalcohol model. This study found select immune function differed between traumatically injured patients with measurable serum alcohol levels as compared with those without. Traumatically injured patients with positive blood alcohol content appear less able to inhibit inflammatory stress. Alcohol appears to suppress pro-inflammatory IL-12 and IL-6, whereas patients without alcohol have greater levels of anti-inflammatory IL-10 expressed at injury and may better regulate anti-inflammatory pathways. Future studies should determine the relationship with these markers with clinically oriented outcomes.

Multi-Institutional Prospective Validation of Prognostic mRNA Signatures in Early Stage Squamous Lung Cancer (Alliance).

INTRODUCTION: Surgical resection is curative for some patients with early lung squamous cell carcinoma. Staging and clinical factors do not adequately predict recurrence risk. We sought to validate the discriminative performance of proposed prognostic gene expression signatures at a level of rigor sufficient to support clinical use. METHODS: The two-stage validation used independent core laboratories, objective quality control standards, locked test parameters, and large multi-institutional specimen and data sets. The first stage validation confirmed a signature's ability to stratify patient survival. The second-stage validation determined which signature(s) optimally improved risk discrimination when added to baseline clinical predictors. Participants were prospectively enrolled in institutional (cohort I) or cooperative group (cohort II) biospecimen and data collection protocols. All cases underwent a central review of clinical, pathologic, and biospecimen parameters using objective criteria to determine final inclusion (cohort I: n = 249; cohort II: n = 234). Primary selection required that a signature significantly predict a 3-year survival after surgical resection in cohort I. Signatures meeting this criterion were further tested in cohort II, comparing risk prediction using baseline risk factors alone versus in combination with the signature. RESULTS: Male sex, advanced age, and higher stage were associated with shorter survival in cohort I and established a baseline clinical model. Of the three signatures validated in cohort I, one signature was validated in cohort II and statistically significantly enhanced the prognosis relative to the baseline model (C-index difference 0.122; p < 0.05). CONCLUSIONS: These results represent the first rigorous validation of a test appropriate to direct adjuvant treatment or clinical trials for patients with lung squamous cell carcinoma.

Correlation of PD-L1 Expression with Tumor Mutation Burden and Gene Signatures for Prognosis in Early-Stage Squamous Cell Lung Carcinoma.

OBJECTIVES: Anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy has demonstrated success in the treatment of advanced NSCLC. Recently, PD-1/PD-L1 blockade also has demonstrated interesting results in small trials of neoadjuvant treatment in stage IB to IIIA NSCLC. In addition, several clinical trials using anti-PD-1/PD-L1 immunotherapy as an adjuvant or neoadjuvant treatment in patients with resectable stage NSCLC are ongoing. However, few analyses of anti-PD-1/PD-L1 immunotherapy-related biomarkers in early-stage squamous cell lung carcinoma (SqCLC) have been reported. In this study, we evaluated PD-L1 protein expression, tumor mutation burden, and expression of an immune gene signature in early-stage SqCLC, providing data for identifying the potential role for patients with anti-PD-1/PD-L1 treatment in early-stage SqCLC. METHODS: A total of 255 specimens from patients with early-stage SqCLC were identified within participating centers of the Strategic Partnering to Evaluate Cancer Signatures program. PD-L1 protein expression by immunohistochemistry was evaluated by using the Dako PD-L1 22C3 pharmDx kit on the Dako Link 48 auto-stainer (Dako, Carpinteria, CA). Tumor mutation burden (TMB) was calculated on the basis of data from targeted genome sequencing. The T-effector and interferon gamma (IFN-γ) gene signature was determined from Affymetrix gene chip data (Affymetrix, Santa Clara, CA) from frozen specimens. RESULTS: The prevalence of PD-L1 expression was 9.8% at a tumor proportion score cutoff of at least 50%. PD-L1 mRNA and programmed cell death 1 ligand 2 mRNA positively correlated with PD-L1 protein expression on tumor cells (TCs) and tumor-infiltrating immune cells. PD-L1 protein expression on tumor-infiltrating immune cells was correlated with the T-effector and IFN-γ gene signature (p < 0.001), but not with TMB. For TCs, all of these biomarkers were independent of each other and neither PD-L1 protein expression, TMB, or T-effector and IFN-γ gene signatures were independently prognostic for patient outcomes. CONCLUSIONS: Evaluation of PD-L1 expression, TMB, and T-effector and IFN-γ gene signatures in the cohort with early-stage SqCLC found them to be independent of each other, and none was associated with overall survival. Our results also support the hypothesis that PD-L1 expression is regulated by an intrinsic mechanism on TCs and an adaptive mechanism on immune cells.

Consensus-Driven Development of a Terminology for Biobanking, the Duke Experience.

Biobanking at Duke University has existed for decades and has grown over time in silos and based on specialized needs, as is true with most biomedical research centers. These silos developed informatics systems to support their own individual requirements, with no regard for semantic or syntactic interoperability. Duke undertook an initiative to implement an enterprise-wide biobanking information system to serve its many diverse biobanking entities. A significant part of this initiative was the development of a common terminology for use in the commercial software platform. Common terminology provides the foundation for interoperability across biobanks for data and information sharing. We engaged experts in research, informatics, and biobanking through a consensus-driven process to agree on 361 terms and their definitions that encompass the lifecycle of a biospecimen. Existing standards, common terms, and data elements from published articles provided a foundation on which to build the biobanking terminology; a broader set of stakeholders then provided additional input and feedback in a secondary vetting process. The resulting standardized biobanking terminology is now available for sharing with the biobanking community to serve as a foundation for other institutions who are considering a similar initiative.

Neighborhood-level socioeconomic determinants impact outcomes in nonsmall cell lung cancer patients in the Southeastern United States.

BACKGROUND: Studies examining the impact of lower socioeconomic status (SES) on the outcomes of patients with nonsmall cell lung cancer (NSCLC) are inconsistent. The objective of this study was to clearly elucidate the association between SES, education, and clinical outcomes among patients with NSCLC. METHODS: The study population was derived from a consecutive, retrospective cohort of patients with NSCLC who received treatment within the Duke Health System between 1995 and 2007. SES determinants were based on the individual's census tract and corresponding 2000 Census data. Determinants included the percentage of the population living below poverty, the median household income, and the percentages of residents with at least a high school diploma and at least a bachelor's degree. The SES and educational variables were divided into quartiles. Statistical comparisons were performed using the 25th and 75th percentiles. RESULTS: Individuals who resided in areas with a low median household income or in which a high percentage of residents were living below the poverty line had a shorter cancer-specific 6-year survival than individuals who resided in converse areas (P = .0167 and P = .0067, respectively). Those living in areas in which a higher percentage of residents achieved a high school diploma had improved disease outcomes compared with those living in areas in which a lower percentage attained a high school diploma (P = .0033). A survival advantage also was observed for inhabitants of areas in which a higher percentage of residents attained a bachelor's degree (P = .0455). CONCLUSIONS: Low SES was identified as an independent prognostic factor for poor survival in patients with both early and advanced stage NSCLC. Patients who lived in areas with high poverty levels, low median incomes, and low education levels had worse mortality.

Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients.

BACKGROUND: Early stage lung cancer has a variable prognosis, and there are currently no markers that predict which patients will recur. This study examined the relation between tumor-regulatory T (Treg) cells and total tumor-infiltrating T-cell lymphocytes (TIL) to determine whether they correlated with recurrence. METHODS: The authors reviewed all patients in our tissue databank from 1996 to 2001 and identified 64 consecutive pathologic stage I non-small cell lung cancer (NSCLC) patients who had surgical resection and at least a 2.5 years disease-free follow-up or documented recurrence within 2 years. Immunohistochemical analyses were performed on paraffin-embedded lung cancer tissue and the relation between Treg cells, TIL, and disease-specific survival was determined. A risk index was devised deductively for various possible combinations of Treg cells and TIL. RESULTS: Treg cells and TIL were detected in 33 of 64 (51%) and 53 of 64 (83%) patients, respectively. When data were analyzed by using a Treg/TIL Combination Risk Index, patients with high-risk and intermediate-risk indices had hazard ratios of 8.2 (P = .007) and 3.3 (P = .109), respectively. CONCLUSIONS: Patients with stage I NSCLC who have a higher proportion of tumor Treg cells relative to TIL had a significantly higher risk of recurrence. These data may be useful, particularly if combined with a panel of tumor markers, to suggest at the time of diagnosis which patients with seemingly early-stage NSCLC will relapse.

Serum protein expression predicts recurrence in patients with early-stage lung cancer after resection.

BACKGROUND: Patients with early stage nonsmall-cell lung cancer who have undergone complete resection have a recurrence rate of approximately 50%, predominately due to the development of systemic metastases. This study is a prospective analysis of the expression of seven serum protein markers of invasion and metastasis, collected preoperatively (baseline) and serially after resection, to determine the relationship between marker expression and recurrence. METHODS: Serum was collected from 196 patients with clinical stage I nonsmall-cell lung cancer who underwent resection over a 5-year period (1996 to 2000). Samples were drawn before resection and 1, 4, 6, 12, 18, and 24 months postoperatively. All patients were followed for at least 24 months or until death. Serum protein levels of vascular endothelial growth factor, hepatocyte growth factor), E-selectin, CD44, basic fibroblast growth factor, urokinase plasminogen activator, and urokinase plasminogen activator receptor were determined using enzyme-linked immunosorbent assay. RESULTS: To date, 73 patients (37%) have demonstrated recurrence. Baseline levels of only 1 marker (CD44) correlated with pathologic stage (p = 0.02). Analysis of the serial samples demonstrated that recurrence was predicted (before clinical or radiographic determination) by decreasing levels of E-selectin (p = 0.002), increasing levels of CD44 (p = 0.001), and increasing levels of urokinase plasminogen activator receptor (p = 0.03). CONCLUSIONS: This study demonstrates the potential to predict recurrence after resection in patients with early-stage nonsmall-cell lung cancer using a panel of serum protein markers. Early identification of patients with recurrence may improve the efficacy of systemic therapy.

Oncogenic pathway signatures in human cancers as a guide to targeted therapies.

The development of an oncogenic state is a complex process involving the accumulation of multiple independent mutations that lead to deregulation of cell signalling pathways central to the control of cell growth and cell fate. The ability to define cancer subtypes, recurrence of disease and response to specific therapies using DNA microarray-based gene expression signatures has been demonstrated in multiple studies. Various studies have also demonstrated the potential for using gene expression profiles for the analysis of oncogenic pathways. Here we show that gene expression signatures can be identified that reflect the activation status of several oncogenic pathways. When evaluated in several large collections of human cancers, these gene expression signatures identify patterns of pathway deregulation in tumours and clinically relevant associations with disease outcomes. Combining signature-based predictions across several pathways identifies coordinated patterns of pathway deregulation that distinguish between specific cancers and tumour subtypes. Clustering tumours based on pathway signatures further defines prognosis in respective patient subsets, demonstrating that patterns of oncogenic pathway deregulation underlie the development of the oncogenic phenotype and reflect the biology and outcome of specific cancers. Predictions of pathway deregulation in cancer cell lines are also shown to predict the sensitivity to therapeutic agents that target components of the pathway. Linking pathway deregulation with sensitivity to therapeutics that target components of the pathway provides an opportunity to make use of these oncogenic pathway signatures to guide the use of targeted therapeutics.

High gene expression of TS1, GSTP1, and ERCC1 are risk factors for survival in patients treated with trimodality therapy for esophageal cancer.

PURPOSE: To assess the relationship between molecular markers associated with chemotherapy resistance and survival in esophageal cancer patients treated with trimodality therapy. EXPERIMENTAL DESIGN: The original pretreatment formalin-fixed, paraffin-embedded endoscopic esophageal tumor biopsy material was obtained from 99 patients treated with concurrent cisplatin plus 5-fluorouracil plus 45 Gy radiation followed by resection at Duke University Medical Center (Durham, NC) from 1986 to 1997. cDNA was derived from the biopsy and analyzed to determine mRNA expression relative to an internal reference gene (beta-actin) using fluorescence-based, real-time reverse transcription-PCR. Possible markers of platinum chemotherapy association [glutathione S-transferase pi (GSTP1) and excision cross-complementing gene 1 (ERCC1)] and 5-fluorouracil association [thymidylate synthase 1 (TS1)] were measured. RESULTS: Cox proportional hazards model revealed a significant inverse, linear effect for TS1 with respect to survival (P = 0.007). An inverse relationship between TS1 expression and treatment response was also detected (P < or = 0.001). Univariate analysis identified an association with decreased survival for GSTP1 > or = 3.0 (P = 0.05). In multivariate analyses, TS1 >6.0, ERCC1 >3, and GSTP1 >3 were statistically significant predictors of decreased survival (P = 0.007). Additionally, the presence of ERCC1 >3.0 or TS1 >6.0 was associated with an approximately 2-fold increase in the risk of cancer recurrence (P = 0.086 and 0.003, respectively). CONCLUSION: The measurement of relative gene expression of molecular markers associated with chemoresistance in endoscopic esophageal tumor biopsies may be a useful tool in assessing outcome in patients with trimodality-treated esophageal cancer. These data should be validated further in larger prospective studies.

Measurement of chemoresistance markers in patients with stage III non-small cell lung cancer: a novel approach for patient selection.

BACKGROUND: The long-term survival of patients with stage III non-small cell lung cancer treated with a combination of chemotherapy and radiation is 10% to 20%. Survival could potentially be increased and toxicity limited if one could identify patients most likely to respond to a particular treatment regimen. This project prospectively evaluated a panel of potential immunohistochemical markers of chemoresistance in a population of patients with pathology-confirmed stage III non-small cell lung cancer in order to determine the prognostic value of each marker in relation to response to chemotherapy or survival. METHODS: Immunohistochemical staining was performed on histologically positive mediastinal nodal specimens obtained from 59 patients (mean age, 62 years; range, 41 to 79 years) without evidence of distant metastatic disease treated with navelbine-based chemotherapy and external beam radiation therapy between 1996 and 2001. Included were markers for apoptosis (p53, bcl-2), drug efflux/degradation (MDR, GST-pi), growth factors (EGFr, Her2-neu), and mismatch repair (hMLH1, hMSH2). After chemotherapy, patients underwent radiologic evaluation for response measured by standard criteria. RESULTS: After a median 41 months of follow-up (range, 17 to 55 months), 43 patients had recurrent disease and 38 of these patients were dead of cancer (median cancer-free survival of 10 months and overall survival of 18 months). Patients who demonstrated a complete or partial response (n = 38) had a significantly improved survival (p = 0.002) compared with those with stable or progressive cancer (n = 21). Multivariable Cox step-wise regression analysis of marker expression associated overexpression of p53 and low expression of hMSH2 with poor treatment response and cancer death. CONCLUSIONS: These preliminary data suggest that marker expression may allow the separation of patients into low- and high-risk groups with respect to survival after combined navelbine-based chemotherapy and XRT. This could represent a novel method of selecting patients for a particular treatment regimen if these data are reproduced in a larger prospective trial.