Metabolic reprogramming and Notch activity distinguish between non-small cell lung cancer subtypes.

BACKGROUND: Previous studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking. METHODS: Publicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices. RESULTS: Analysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture. CONCLUSIONS: Our data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.

Yeast-Derived Particulate ß-Glucan Treatment Subverts the Suppression of Myeloid-Derived Suppressor Cells (MDSC) by Inducing Polymorphonuclear MDSC Apoptosis and Monocytic MDSC Differentiation to APC in Cancer.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. In this study, we demonstrated that activation of a C-type lectin receptor, dectin-1, in MDSC differentially modulates the function of different MDSC subsets. Yeast-derived whole ß-glucan particles (WGP; a ligand to engage and activate dectin-1, oral treatment in vivo) significantly decreased tumor weight and splenomegaly in tumor-bearing mice with reduced accumulation of polymorphonuclear MDSC but not monocytic MDSC (M-MDSC), and decreased polymorphonuclear MDSC suppression in vitro through the induction of respiratory burst and apoptosis. On a different axis, WGP-treated M-MDSC differentiated into F4/80(+)CD11c(+) cells in vitro that served as potent APC to induce Ag-specific CD4(+) and CD8(+) T cell responses in a dectin-1-dependent manner. Additionally, Erk1/2 phosphorylation was required for the acquisition of APC properties in M-MDSC. Moreover, WGP-treated M-MDSC differentiated into CD11c(+) cells in vivo with high MHC class II expression and induced decreased tumor burden when inoculated s.c. with Lewis lung carcinoma cells. This effect was dependent on the dectin-1 receptor. Strikingly, patients with non-small cell lung carcinoma that had received WGP treatment for 10-14 d prior to any other treatment had a decreased frequency of CD14(-)HLA-DR(-)CD11b(+)CD33(+) MDSC in the peripheral blood. Overall, these data indicate that WGP may be a potent immune modulator of MDSC suppressive function and differentiation in cancer.

Dectin-1 Activation by a Natural Product ß-Glucan Converts Immunosuppressive Macrophages into an M1-like Phenotype.

Tumor-associated macrophages (TAM) with an alternatively activated phenotype have been linked to tumor-elicited inflammation, immunosuppression, and resistance to chemotherapies in cancer, thus representing an attractive target for an effective cancer immunotherapy. In this study, we demonstrate that particulate yeast-derived ß-glucan, a natural polysaccharide compound, converts polarized alternatively activated macrophages or immunosuppressive TAM into a classically activated phenotype with potent immunostimulating activity. This process is associated with macrophage metabolic reprograming with enhanced glycolysis, Krebs cycle, and glutamine utilization. In addition, particulate ß-glucan converts immunosuppressive TAM via the C-type lectin receptor dectin-1-induced spleen tyrosine kinase-Card9-Erk pathway. Further in vivo studies show that oral particulate ß-glucan treatment significantly delays tumor growth, which is associated with in vivo TAM phenotype conversion and enhanced effector T cell activation. Mice injected with particulate ß-glucan-treated TAM mixed with tumor cells have significantly reduced tumor burden with less blood vascular vessels compared with those with TAM plus tumor cell injection. In addition, macrophage depletion significantly reduced the therapeutic efficacy of particulate ß-glucan in tumor-bearing mice. These findings have established a new paradigm for macrophage polarization and immunosuppressive TAM conversion and shed light on the action mode of ß-glucan treatment in cancer.

Transcription factor c-Maf is a checkpoint that programs macrophages in lung cancer.

Macrophages have been linked to tumor initiation, progression, metastasis, and treatment resistance. However, the transcriptional regulation of macrophages driving the protumor function remains elusive. Here, we demonstrate that the transcription factor c-Maf is a critical controller for immunosuppressive macrophage polarization and function in cancer. c-Maf controls many M2-related genes and has direct binding sites within a conserved noncoding sequence of the Csf-1r gene and promotes M2-like macrophage-mediated T cell suppression and tumor progression. c-Maf also serves as a metabolic checkpoint regulating the TCA cycle and UDP-GlcNAc biosynthesis, thus promoting M2-like macrophage polarization and activation. Additionally, c-Maf is highly expressed in tumor-associated macrophages (TAMs) and regulates TAM immunosuppressive function. Deletion of c-Maf specifically in myeloid cells results in reduced tumor burden with enhanced antitumor T cell immunity. Inhibition of c-Maf partly overcomes resistance to anti-PD-1 therapy in a subcutaneous LLC tumor model. Similarly, c-Maf is expressed in human M2 and tumor-infiltrating macrophages/monocytes as well as circulating monocytes of human non-small cell lung carcinoma (NSCLC) patients and critically regulates their immunosuppressive activity. The natural compound ß-glucan downregulates c-Maf expression on macrophages, leading to enhanced antitumor immunity in mice. These findings establish a paradigm for immunosuppressive macrophage polarization and transcriptional regulation by c-Maf and suggest that c-Maf is a potential target for effective tumor immunotherapy.

Altered regulation of metabolic pathways in human lung cancer discerned by (13)C stable isotope-resolved metabolomics (SIRM).

BACKGROUND: Metabolic perturbations arising from malignant transformation have not been systematically characterized in human lung cancers in situ. Stable isotope resolved metabolomic analysis (SIRM) enables functional analysis of gene dysregulations in lung cancer. To this purpose, metabolic changes were investigated by infusing uniformly labeled 13C-glucose into human lung cancer patients, followed by resection and processing of paired non-cancerous lung and non small cell carcinoma tissues. NMR and GC-MS were used for 13C-isotopomer-based metabolomic analysis of the extracts of tissues and blood plasma. RESULTS: Many primary metabolites were consistently found at higher levels in lung cancer tissues than their surrounding non-cancerous tissues. 13C-enrichment in lactate, Ala, succinate, Glu, Asp, and citrate was also higher in the tumors, suggesting more active glycolysis and Krebs cycle in the tumor tissues. Particularly notable were the enhanced production of the Asp isotopomer with three 13C-labeled carbons and the buildup of 13C-2,3-Glu isotopomer in lung tumor tissues. This is consistent with the transformations of glucose into Asp or Glu via glycolysis, anaplerotic pyruvate carboxylation (PC), and the Krebs cycle. PC activation in tumor tissues was also shown by an increased level of pyruvate carboxylase mRNA and protein. CONCLUSION: PC activation - revealed here for the first time in human subjects - may be important for replenishing the Krebs cycle intermediates which can be diverted to lipid, protein, and nucleic acid biosynthesis to fulfill the high anabolic demands for growth in lung tumor tissues. We hypothesize that this is an important event in non-small cell lung cancer and possibly in other tumor development.

Prospects for clinical cancer metabolomics using stable isotope tracers.

Metabolomics provides a readout of the state of metabolism in cells or tissue and their responses to external perturbations. For this reason, the approach has great potential in clinical diagnostics. For more than two decades, we have been using stable isotope tracer approaches to probe cellular metabolism in greater detail. The ability to enrich common compounds with rare isotopes such as carbon ((13)C) and nitrogen ((15)N) is the only practical means by which metabolic pathways can be traced, which entails following the fate of individual atoms from the source molecule to products via metabolic transformation. Changes in regulation of pathways are therefore captured by this approach, which leads to deeper understanding of the fundamental biochemistry of cells. Using lessons learned from pathways tracing in cells and organs, we have been applying this methodology to human cancer patients in a clinical setting. Here we review the methodologies and approaches to stable isotope tracing in cells, animal models and in humans subjects.

Metabolic profiling identifies lung tumor responsiveness to erlotinib.

A subtype of non-small cell lung cancer, bronchioalveolar adenocarcinoma (BAC), is more prevalent in Asian female non-smokers, and is more likely to respond to treatment with tyrosine kinase inhibitors such as erlotinib and gefitinib. Nuclear magnetic resonance and mass spectrometry-based metabolomic analysis of extracts from two different lung lesions and surrounding non-cancerous tissues of a BAC patient showed novel protein and phospholipid-associated metabolic differences that correlated with tumor development as well as PET and erlotinib sensitivity.

Single Versus Double Lung Retransplantation Does Not Affect Survival Based on Previous Transplant Type.

BACKGROUND: Survival following retransplantation with a single lung is worse than after double lung transplant. We sought to characterize survival of patients who underwent lung retransplantation based on the type of their initial transplant, single or double. METHODS: The United Network for Organ Sharing database was queried for adult patients who underwent lung retransplantation from 2005 onward. Patients were excluded if they underwent more than one retransplantation. The patient population was divided into 4 groups based on first followed by second transplant type, respectively: single then single, double then single, double then double, and single then double. Descriptive analysis and Kaplan-Meier survival analysis were performed. A p value less than 0.05 was considered significant. RESULTS: A total of 410 patients underwent retransplantation in the study time period. Overall mean survival for all patients who underwent retransplantation was 1,213 days. Kaplan-Meier survival analysis demonstrated no difference in graft survival between the 4 study groups (p = 0.146). CONCLUSIONS: There was no significant difference in graft survival between recipients of retransplant with single or double lungs when stratified by previous transplant type. These results suggest that when retransplantation is performed, single lung retransplantation should be considered, regardless of previous transplant type, in an effort to maximize organ resources.

Normalization of Exhaled Carbonyl Compounds After Lung Cancer Resection.

BACKGROUND: Quantitative analysis of specific exhaled carbonyl compounds (ECCs) has shown promise for the detection of lung cancer. The purpose of this study is to demonstrate the normalization of ECCs in patients after lung cancer resection. METHODS: Patients from a single center gave consent and were enrolled in the study from 2011 onward. Breath analysis was performed on lung cancer patients before and after surgical resection of their tumors. One liter of breath from a single exhalation was collected and evacuated over a silicon microchip. Carbonyls were captured by oximation reaction and analyzed by mass spectrometry. Concentrations of four cancer-specific ECCs were measured and compared by using the Wilcoxon test. A given cancer marker was considered elevated at 1.5 or more standard deviations greater than the mean of the control population. RESULTS: There were 34 cancer patients with paired samples and 187 control subjects. The median values after resection were significantly lower for all four ECCs and were equivalent to the control patient values for three of the four ECCs. CONCLUSIONS: The analysis of ECCs demonstrates reduction to the level of control patients after surgical resection for lung cancer. This technology has the potential to be a useful tool to detect disease after lung cancer resection. Continued follow-up will determine whether subsequent elevation of ECCs is indicative of recurrent disease.

High sensitivity for lung cancer detection using analysis of exhaled carbonyl compounds.

OBJECTIVE: Several volatile carbonyl compounds in exhaled breath have been identified as cancer-specific markers. The potential for these markers to serve as a screening test for lung cancer is reported. METHODS: Patients with computed tomography-detected intrathoracic lesions and healthy control participants were enrolled from 2011 onward. One liter of breath was collected from a single exhalation from each participant. The contents were evacuated over a silicon microchip, captured by oximation reaction, and analyzed by mass spectrometry. Concentrations of 2-butanone, 3-hydroxy-2-butanone, 2-hydroxyacetaldehyde, and 4-hydroxyhexanal were measured. The overall population was divided into 3 groups: those with lung cancer, benign disease, and healthy controls. An elevated cancer marker was defined as ≥1.5 SDs above the mean concentration of the control population. One or more elevated cancer markers constituted a positive breath test. RESULTS: In all, 156 subjects had lung cancer, 65 had benign disease, and 194 were healthy controls. A total of 103 (66.0%) lung cancer patients were early stage (stage 0, I, and II). For ≥1 elevated cancer marker, breath analysis showed a sensitivity of 93.6%, and a specificity of 85.6% for lung cancer patients. Additionally, 83.7% of stage I tumors ≤2 cm were detected; whereas only 14% of the control population tested positive. In a comparison of cancer to benign disease, specificity was proportional to the number of elevated cancer markers present. CONCLUSIONS: Screening using a low-dose CT scan is associated with high cost, repeated radiation exposure, and low accrual. The high sensitivity, convenience, and low cost of breath analysis for carbonyl cancer markers suggests that it has the potential to become a primary screening modality for lung cancer.

Breath carbonyl compounds as biomarkers of lung cancer.

OBJECTIVE: Lung cancer dysregulations impart oxidative stress which results in important metabolic products in the form of volatile organic compounds (VOCs) in exhaled breath. The objective of this work is to use statistical classification models to determine specific carbonyl VOCs in exhaled breath as biomarkers for detection of lung cancer. MATERIALS AND METHODS: Exhaled breath samples from 85 patients with untreated lung cancer, 34 patients with benign pulmonary nodules and 85 healthy controls were collected. Carbonyl compounds in exhaled breath were captured by silicon microreactors and analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The concentrations of carbonyl compounds were analyzed using a variety of statistical classification models to determine which compounds best differentiated between the patient sub-populations. Predictive accuracy of each of the models was assessed on a separate test data set. RESULTS: Six carbonyl compounds (C(4)H(8)O, C(5)H(10)O, C(2)H(4)O(2), C(4)H(8)O(2), C(6)H(10)O(2), C(9)H(16)O(2)) had significantly elevated concentrations in lung cancer patients vs. CONTROLS: A model based on counting the number of elevated compounds out of these six achieved an overall classification accuracy on the test data of 97% (95% CI 92%-100%), 95% (95% CI 88%-100%), and 89% (95% CI 79%-99%) for classifying lung cancer patients vs. non-smokers, current smokers, and patients with benign nodules, respectively. These results were comparable to benchmarking based on established statistical and machine-learning methods. The sensitivity in each case was 96% or higher, with specificity ranging from 64% for benign nodule patients to 86% for smokers and 100% for non-smokers. CONCLUSION: A model based on elevated levels of the six carbonyl VOCs effectively discriminates lung cancer patients from healthy controls as well as patients with benign pulmonary nodules.

Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation.

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non-small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.

Comorbidity and KPS are independent prognostic factors in stage I non-small-cell lung cancer.

PURPOSE: To determine the prognostic role of comorbidity in Stage I non-small-cell lung cancer (NSCLC) treated with surgery or radiotherapy (RT). MATERIALS AND METHODS: One hundred sixty-three patients with clinical Stage I NSCLC were analyzed for overall survival (OS) and comorbidity. One hundred thirteen patients underwent surgery (surgical group) and 50 patients received definitive radiotherapy (RT group). Ninety-six percent of the surgical group had lobectomy or pneumonectomy, and negative margins were achieved in 96% of the patients. The median dose to the tumor for the RT group was 61.2 Gy (range 30.8-77.4). The Cumulative Illness Rating Scale for Geriatrics (CIRS-G) and the Charlson scale were used to rate comorbidity. Karnofsky performance scores (KPS) were available in 42 patients; the rest of the scores were determined retrospectively by two physicians independently, with 97% agreement. RESULTS: The OS was 44% for the surgical group and 5% for the RT group at 5 years. Noncancer-related mortality was observed in 31% and 62% of the surgical and RT patients, respectively. On univariate analysis, performed on all patients (n = 163), squamous cell histologic type (p <0.001), clinical Stage T2 (p = 0.062), tumor size >4 cm (p = 0.065), >40 pack-year tobacco use (p <0.001), presence of a CIRS-G score of 4 (extremely severe, CIRS-G4: [+]) (p <0.001), severity index of >2 (p <0.001), Charlson score >2 (p = 0.004), KPS <70 (p <0.001), and treatment with RT (p <0.001) were associated with a statistically significant inferior OS. Multivariate analysis with histologic features, clinical T stage, age, tobacco use, KPS, comorbidity [CIRS-G(4)] and treatment group on all patients showed that squamous cell histology, >40 pack-year tobacco use, KPS <70, and presence of CIRS-G(4) were independently associated with an inferior OS. Treatment modality, T stage, and age did not have any statistically significant effect on OS. Statistically significant differences were found between the surgical and RT groups in Charlson score (p = 0.001), CIRS-G total score (p = 0.004), severity index (p = 0.006), CIRS-G4(+) (p <0.001), KPS (p <0.001), amount of tobacco use (p = 0.002), clinical tumor size (p <0.001), clinical T stage (p = 0.01), forced expiratory volume in 1 s (p = 0.001), and age (p = 0.008), in favor of the surgical group. CONCLUSION: The presence of significant comorbidity and KPS of <70 are both important prognostic factors, but were found to be independent of each other in Stage I NSCLC. Therefore, comorbidity and KPS assessment are recommended when analyzing the prognostic effects of tumor or treatment-related factors on OS.

Hemoptysis resulting from unilateral pulmonary artery agenesis.

Unilateral pulmonary artery agenesis is a rare congenital anomaly often associated with other cardiovascular abnormalities. It is usually diagnosed and surgically treated in childhood. Subjects without associated cardiac anomalies (isolated unilateral pulmonary artery agenesis) may be asymptomatic or have recurrent respiratory infections. We report a case of left pulmonary artery agenesis in a 35-year-old man complicated by hemoptysis and treated by pneumonectomy. Physicians should be aware of unilateral pulmonary artery agenesis presenting later in life as a source of chronic respiratory symptomatology or hemoptysis.

A decade of experience with transthoracic and transhiatal esophagectomy.

BACKGROUND: Morbidity and mortality remain significant for transthoracic (TT) and transhiatal (TH) esophagectomy. We report a case-specific approach employing either resection to minimize perioperative morbidity and mortality. METHODS: All primary esophageal resections performed for benign and malignant esophageal disease were reviewed over a 10-year period. The operative approach was tailored to the location and extent of disease and the physiologic reserve of the patient. RESULTS: In all, 115 patients underwent esophagectomy for benign (25) and malignant (90) disease. Fifty-six TT and 59 TH resections were performed. Four emergent TT cases did not have reconstruction. There was 1 hospital mortality. Perioperative transfusion was avoided in 65 patients. Respiratory complications occurred in 15. Three patients had a cervical anastomotic leak requiring open wound drainage. No association between resection type and complication was evident. CONCLUSIONS: The judicious use of both TT and TH esophagectomy resulted in an operative mortality of less than 1%, reduced operative blood loss, and a relatively low rate of perioperative complications.

Late superior vena cava perforation and aortic laceration after stenting to treat superior vena cava syndrome secondary to fibrosing mediastinitis.

We describe a case of late perforation of the superior vena cava and laceration of the ascending aorta after stent implantation for superior vena cava syndrome. The etiology of the late perforation is unclear, and could be secondary to either flaring of the trailing edge of the stent or chest trauma.

Double lung transplants have significantly improved survival compared with single lung transplants in high lung allocation score patients.

BACKGROUND: Historically, double lung transplantation survival rates are higher than those of single lung transplantation, but in critically ill patients a single lung transplant, with less associated operative morbidity, could afford a better outcome. This article evaluates how survival is affected in patients who have a high lung allocation score (LAS) and receive a single versus a double lung transplant. METHODS: The UNOS Thoracic Transplant Database for lung transplants from January 2005 to June 2012 was used for analysis. Propensity matching was used to minimize differences between the high and low LAS groups and between single and double lung transplants in the high LAS group. RESULTS: Within this database, there were 8,778 patients, of whom 8,050 had an LAS less than 75 and 728 had an LAS greater than or equal to 75. Kaplan-Meier survival curves stratified by high and low LAS, and by single versus double lung transplants, showed a marked decrease in survival (p<0.001) in those with a high LAS who received a single lung transplant when compared with those with a high LAS who received a double lung transplant. This was a much greater difference in survival than was present in the low LAS patient population. CONCLUSIONS: Despite a higher operative morbidity, patients who had a high LAS did substantially better in terms of survival if two lungs were transplanted rather than only one, with a larger difference in survival than for patients with a lower LAS.

Noninvasive detection of lung cancer using exhaled breath.

Early detection of lung cancer is a key factor for increasing the survival rates of lung cancer patients. The analysis of exhaled breath is promising as a noninvasive diagnostic tool for diagnosis of lung cancer. We demonstrate the quantitative analysis of carbonyl volatile organic compounds (VOCs) and identification of lung cancer VOC markers in exhaled breath using unique silicon microreactor technology. The microreactor consists of thousands of micropillars coated with an ammonium aminooxy salt for capture of carbonyl VOCs in exhaled breath by means of oximation reactions. Captured aminooxy-VOC adducts are analyzed by nanoelectrospray Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry (MS). The concentrations of 2-butanone, 2-hydroxyacetaldehyde, 3-hydroxy-2-butanone, and 4-hydroxyhexenal (4-HHE) in the exhaled breath of lung cancer patients (n = 97) were significantly higher than in the exhaled breath of healthy smoker and nonsmoker controls (n = 88) and patients with benign pulmonary nodules (n = 32). The concentration of 2-butanone in exhaled breath of patients (n = 51) with stages II though IV non-small cell lung cancer (NSCLC) was significantly higher than in exhaled breath of patients with stage I (n = 34). The carbonyl VOC profile in exhaled breath determined using this new silicon microreactor technology provides for the noninvasive detection of lung cancer.