Faculty Bibliography

A potential strategy for diagnosing lung cancer, the leading cause of cancer-related death, is to identify metabolic signatures (biomarkers) of the disease. Although data supports the hypothesis that volatile compounds can be detected in the breath of lung cancer patients by the sense of smell or through bioanalytical techniques, analysis of breath samples is cumbersome and technically challenging, thus limiting its applicability. The hypothesis explored here is that variations in small molecular weight volatile organic compounds ("odorants") in urine could be used as biomarkers for lung cancer. To demonstrate the presence and chemical structures of volatile biomarkers, we studied mouse olfactory-guided behavior and metabolomics of volatile constituents of urine. Sensor mice could be trained to discriminate between odors of mice with and without experimental tumors demonstrating that volatile odorants are sufficient to identify tumor-bearing mice. Consistent with this result, chemical analyses of urinary volatiles demonstrated that the amounts of several compounds were dramatically different between tumor and control mice. Using principal component analysis and supervised machine-learning, we accurately discriminated between tumor and control groups, a result that was cross validated with novel test groups. Although there were shared differences between experimental and control animals in the two tumor models, we also found chemical differences between these models, demonstrating tumor-based specificity. The success of these studies provides a novel proof-of-principle demonstration of lung tumor diagnosis through urinary volatile odorants. This work should provide an impetus for similar searches for volatile diagnostic biomarkers in the urine of human lung cancer patients.

The Cub and Sushi Multiple Domains-1 (CSMD1) is a tumor suppressor gene on 8p23.2, where allelic loss is both frequent and associated with poor prognosis in head and neck squamous cell carcinoma (HNSCC). To understand the extent of CSMD1 aberrations in vivo, we characterized 184 primary tumors from the head and neck, lung, breast and skin for gene copy number and analyzed expression in our HNSCCs and lung squamous cell carcinomas (SCCs). We detected loss of CSMD1 in a large proportion of HNSCCs (50%), lung (46%) and breast cancers (55%), and to a lesser extent in cutaneous SCCs (29%) and basal cell carcinomas (BCCs, 17%) using array-based comparative genomic hybridization (aCGH). Studying the region more closely with quantitative real-time PCR (qPCR), the loss of CSMD1 increased to 80% in HNSCCs and 93% in lung SCCs. CSMD1 expression was decreased in tumors compared to adjacent benign tissue (65%, 13/20) and was likely due to gene loss in 45% of cases (9/20). We also identified truncated transcripts lacking exons due to DNA copy number loss (30%, 5/17) or aberrant splicing (24%, 4/17). We show loss of CSMD1 in primary HNSCC tissues, and document for the first time that CSMD1 is lost in breast, lung and cutaneous SCCs. We also show that deletions of CSMD1 and aberrant splicing contribute to altered CSMD1 function in vivo.

Flaxseed (FS) has high contents of omega-3 fatty acids and lignans with antioxidant properties. Its use in preventing thoracic X-ray radiation therapy (XRT)-induced pneumonopathy has never been evaluated. We evaluated FS supplementation given to mice given before and post-XRT. FS-derived lignans, known for their direct antioxidant properties, were evaluated in abrogating ROS generation in cultured endothelial cells following gamma radiation exposure. Mice were fed 10% FS or isocaloric control diet for three weeks and given 13.5 Gy thoracic XRT. Lungs were evaluated at 24 hours for markers of radiation-induced injury, three weeks for acute lung damage (lipid peroxidation, lung edema and inflammation), and at four months for late lung damage (inflammation and fibrosis). FS-Lignans blunted ROS generation in vitro, resulting from radiation in a dose-dependent manner. FS-fed mice had reduced expression of lung injury biomarkers (Bax, p21 and TGF-beta1) at 24 hours following XRT and reduced oxidative lung damage as measured by malondialdehyde (MDA) levels at 3 weeks following XRT. In addition, FS-fed mice had decreased lung fibrosis as determined by hydroxyproline content and decreased inflammatory cell influx into lungs at 4 months post XRT. Importantly, when Lewis lung carcinoma cells were injected systemically in mice, FS dietary supplementation did not appear to protect lung tumors from responding to thoracic XRT. Dietary FS is protective against pulmonary fibrosis, inflammation and oxidative lung damage in a murine model. Moreover, in this model, tumor radioprotection was not observed. FS lignans exhibited potent radiation-induced ROS scavenging action. Taken together, these data suggest that dietary flaxseed may be clinically useful as an agent to increase the therapeutic index of thoracic XRT by increasing the radiation tolerance of lung tissues.

OBJECTIVE:The electronic nose is a sensor of volatile molecules that is useful in the analysis of expired gases. Our hypothesis is that the electronic nose can distinguish between different types of upper aerodigestive tract tumor cells in vitro. STUDY DESIGN/METHODS:Cells from both tumor and normal cell lines were suspended in saline, and a polymer composite electronic nose was used to evaluate the headspace gases. The data were subjected to principal components analysis, and Mahalanobis distances were calculated to demonstrate the ability of the electronic nose to distinguish among samples. RESULTS:The tumor cell lines, including adenocarcinoma, squamous cell carcinoma, and mesothelioma, were distinct from each other, and from the normal fibroblast and smooth muscle cells as seen on canonical discrimination plots. CONCLUSION/CONCLUSIONS:The electronic nose can distinguish between tumor cell lines in vitro and has the potential to be a useful screening test for cancer.

PURPOSE/OBJECTIVE:The risk of developing metastatic squamous cell carcinoma for patients with head and neck squamous cell carcinoma (HNSCC) is very high. Because these patients are often heavy tobacco users, they are also at risk for developing a second primary cancer, with squamous cell carcinoma of the lung (LSCC) being the most common. The distinction between a lung metastasis and a primary LSCC is currently based on certain clinical and histologic criteria, although the accuracy of this approach remains in question. EXPERIMENTAL DESIGN/METHODS:Gene expression patterns derived from 28 patients with HNSCC or LSCC from a single center were analyzed using penalized discriminant analysis. Validation was done on previously published data for 134 total subjects from four independent Affymetrix data sets. RESULTS:We identified a panel of 10 genes (CXCL13, COL6A2, SFTPB, KRT14, TSPYL5, TMP3, KLK10, MMP1, GAS1, and MYH2) that accurately distinguished these two tumor types. This 10-gene classifier was validated on 122 subjects derived from four independent data sets and an average accuracy of 96% was shown. Gene expression values were validated by quantitative reverse transcription-PCR derived on 12 independent samples (seven HNSCC and five LSCC). The 10-gene classifier was also used to determine the site of origin of 12 lung lesions from patients with prior HNSCC. CONCLUSIONS:The results suggest that penalized discriminant analysis using these 10 genes will be highly accurate in determining the origin of squamous cell carcinomas in the lungs of patients with previous head and neck malignancies.

Flaxseed (FS) is a nutritional supplement with high concentrations of (n-3) fatty acids and lignans that have anti-inflammatory and antioxidant properties. The use of FS in the prevention or treatment of acute lung disease is unknown. In this study, we evaluated diets with high FS content in experimental murine models of acute lung injury and inflammation. The kinetics of lignan accumulation in blood, following 10% FS supplementation, was determined using liquid chromatography tandem mass spectrometry. Mice were fed isocaloric control and 10% FS-supplemented diets for at least 3 wk and challenged by hyperoxia (80% oxygen), intratracheal instillation of lipopolysaccharide, or acid aspiration. Bronchoalveolar lavage was evaluated for white blood cells, neutrophils, and proteins after a 24 h postintratracheal challenge of hydrochloric acid or lipopolysaccharide, or after 6 d of hyperoxia. Lung lipid peroxidation was assessed by tissue malondialdehyde concentrations. The plasma concentrations of the FS lignans, enterodiol and enterolactone, were stable after mice had eaten the diets for 2 wk. Following hyperoxia and acid aspiration, bronchoalveolar lavage neutrophils decreased in FS-supplemented mice (P = 0.012 and P = 0.027, respectively), whereas overall alveolar white blood cell influx tended to be lower (P = 0.11). In contrast, neither lung injury nor inflammation was ameliorated by FS following lipopolysaccharide instillation. Lung malondialdehyde levels were lower in hyperoxic mice than in unchallenged mice (P = 0.0001), and decreased with FS treatment following acid aspiration (P = 0.011). Dietary FS decreased lung inflammation and lipid peroxidation, suggesting a protective role against pro-oxidant-induced tissue damage in vivo.

Tumor-derived cyclooxygenase-2 (COX-2) and its product, prostaglandin E2, exert strong immunoinhibitory effects that block dendritic cell function and CD4+ and CD8+ T-cell proliferation and function. We have shown previously that the addition of an oral COX-2 inhibitor to immunogene therapy using IFN-beta markedly augmented therapeutic efficacy in murine tumor models. In this study, we hypothesized that COX-2 inhibition might also augment an antitumor vaccination strategy. Mice bearing tumors derived from TC1 cells, a tumor line that expresses the human papillomavirus (HPV) E7 protein, were thus vaccinated with an adenoviral vector expressing HPV E7 protein (Ad.E7). This vaccine approach effectively generated E7-specific CD8+ cells and slowed the growth of small tumors but had little effect on large tumors. However, feeding mice with the COX-2 inhibitor, rofecoxib, restored the effectiveness of the vaccine against large tumors and prolonged survival. This effect was accompanied by a larger percentage of E7-specific CD8+ cells in the regional draining lymph nodes and a markedly increased number of tumor-infiltrating E7-specific CD8+ cells (as determined by flow cytometry) and total CD8+ T cells (as determined by immunohistochemical staining). Increased immunocyte trafficking was likely mediated by the generation of a Th1-type tumor microenvironment because COX-2 inhibition increased expression levels of mRNA for IFN-gamma, interleukin-12, IP-10, and MIG while lowering the expression of vascular endothelial growth factor within tumors. This study shows that the effectiveness of a cancer vaccine can be significantly improved by adding COX-2 inhibition.

BACKGROUND:Lung carcinoma remains the major cause of cancer death in North America and is even more common among military veterans. The objective of this study was to determine whether there were differences in the characteristics and survival of Pennsylvania patients with lung carcinoma in the Veterans Administration (VA) hospital system compared with patients in the rest of the state. METHODS:The Pennsylvania Cancer Registry was used to identify all patients who were diagnosed with lung carcinoma in the State of Pennsylvania from 1995 to 1999. Patients who were treated within the Veterans Administration Health Care Network were identified by hospital code. Survival from the date of diagnosis of lung carcinoma was determined by using the Pennsylvania state mortality files from 1995 to 2001. RESULTS:From 1995 to 1999, 48,994 patients were newly diagnosed with lung carcinoma in Pennsylvania (41.2% women), including 856 patients in the VA system (6 women). The current analysis was restricted to male patients (n = 28,798 men). There was no major difference in age of VA patients compared with non-VA patients, and the proportions of patients who had localized or regional stage disease were similar (49% of VA patients vs. 48% of non-VA patients). The proportion of black patients was much higher in the VA population (23%) compared with the non-VA population (9%). The median survival was 6.3 months for VA patients compared with 7.9 months for patients in the rest of the state, and the 5-year overall survival rate was 12% for VA patients compared with 15% for patients in the rest of the state. When survival was analyzed according to race, there was a significant difference in the age-adjusted survival of white patients in the VA system compared with patients in the rest of the state (P = 0.0007), but no significant difference was observed among black patients (P = 0.92). CONCLUSIONS:The overall survival of VA patients with lung carcinoma in Pennsylvania was inferior to that of patients in the remainder of the state and this was due primarily to differences in survival among the white patients. Further investigation will be necessary to determine whether this disparity was caused by differences in socioeconomic status or comorbidities or whether there are systematic differences in the diagnosis, staging, or treatment of lung carcinoma between VA patients and civilian patients.

Lung cancer is the leading cause of cancer death in the U.S. with survival restricted to a subset of those patients able to undergo surgical resection. However, even with surgery, recurrence rates range from 30% to 60%, depending on the pathologic stage. With the advent of partially effective, but potentially toxic adjuvant chemotherapy, it has become increasingly important to discover biomarkers that will identify those patients who have the highest likelihood of recurrence and who thus might benefit most from adjuvant chemotherapy. Hundreds of papers have appeared over the past several decades proposing a variety of molecular markers or proteins that may have prognostic significance in non-small cell lung cancer. This review analyzes the largest and most rigorous of these studies with the aim of compiling the most important prognostic markers in early stage non-small cell lung cancer. In this review, we focused on biomarkers primarily involved in one of three major pathways: cell cycle regulation, apoptosis, and angiogenesis. Although no single marker has yet been shown to be perfect in predicting patient outcome, a profile based on the best of these markers may prove useful in directing patient therapy. The markers with the strongest evidence as independent predictors of patient outcome include cyclin E, cyclin B1, p21, p27, p16, survivin, collagen XVIII, and vascular endothelial cell growth factor.

Thrombolytic therapy unquestionably leads to more rapid and complete clot lysis with a significantly higher risk of bleeding when compared with anticoagulation. The most definite indication for thrombolytic therapy in patients with VTE is massive PE associated with hemodynamic instability. Other potential indications, although not widely accepted or proven, include PE-related respiratory failure with severe hypoxemia and massive iliofemoral thrombosis with the risk of phlegmasia cerulea dolens. Routine use of thrombolytic therapy in all other cases of PE and DVT cannot be justified. Future research using randomized controlled studies should focus on the following key questions: Do hemodynamically stable patients with PE and right ventricular dysfunction benefit from thrombolysis, and, if so, is there a subset of patients within this group who are most likely to benefit? Does thrombolytic therapy improve long-term outcomes of DVT with a favorable risk-to-benefit ratio, and, if so, which patients are most likely to benefit long-term? What is the precise role of catheter-directed thrombolysis in the treatment of VTE, particularly the use of a low-dose thrombolytic agent in conjunction with mechanical clot disruption to minimize bleeding in patients at high risk? Until these questions are answered, clinicians must approach decision-making regarding the use of thrombolytic therapy in PE and DVT with careful consideration of the potential risks and benefits for the patient within the framework of currently available data.