Michael Bousamra

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

BackgroundMetabolic 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.ResultsMany 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.ConclusionPC 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.

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.

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.

Early and late morbidity in patients undergoing pulmonary resection with low diffusion capacity

BACKGROUND We sought to determine whether low diffusion capacity of the lung to carbon monoxide (DLCO) is a predictor of high postoperative mortality and morbidity after major pulmonary resection and whether major pulmonary resection in patients with low DLCO results in substantial long-term morbidity. METHODS Sixty-two major pulmonary resections were performed in 61 patients with low DLCO (DLCO < or = 60% predicted for pneumonectomy or bilobectomy; < or = 50% predicted for lobectomy). Contemporaneously, 262 other patients underwent 263 major pulmonary resections (group II). Long-term morbidity was assessed in subsets of patients with low (n = 24) and high (n = 22; DLCO > 60% predicted) DLCO. RESULTS The hospital mortality rates were equivalent (4.8% low DLCO versus 4.9% group II), whereas respiratory complications were more frequent in patients with low DLCO (18% versus 9.5%; p = 0.05). In the subgroup analyses, patients with low DLCO had more hospitalizations for respiratory compromise and worse median dyspnea scores. Analysis of patients with substantial dyspnea revealed an association with extended pulmonary resection and postoperative radiation therapy in patients with low DLCO. CONCLUSIONS Patients with low DLCO underwent major pulmonary resection with a low mortality rate and an acceptable, but increased, respiratory complication rate. Long-term respiratory morbidity was increased in patients with low DLCO; however, the extent of pulmonary resection and the use of postoperative radiation therapy may have contributed to the development of dyspnea in these patients.

Stable Isotope-Resolved Metabolomics (SIRM) in Cancer Research with Clinical Application to NonSmall Cell Lung Cancer

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. Clinical metabolomics using stable isotope resolved metabolomics (SIRM) for pathway tracing represents an important new approach to obtaining metabolic parameters in human cancer subjects in situ. Here we provide an overview of the technology development of labeling from cells in culture and mouse models. The high throughput analytical methods NMR and mass spectrometry, especially Fourier transform ion cyclotron resonance, for analyzing the resulting metabolite isotopomers and isotopologues are described with examples of applications in cancer biology. Special technical considerations for clinical applications of metabolomics using stable isotope tracers are described. The whole process from concept to analysis will be exemplified by our on-going study of nonsmall cell lung cancer (NSCLC) metabolomics. This powerful new approach has already provided important new insights into metabolic adaptations in lung cancer cells, including the upregulation of anaplerosis via pyruvate carboxylation in NSCLC.

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.

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.

Mortality and neurologic morbidity after repair of traumatic aortic disruption

BACKGROUND Traumatic disruption of the thoracic aorta frequently results in death before operative repair. The determinants of mortality after repair, however, are uncertain. In addition, intraoperative strategies for reducing the incidence of spinal cord injury remain controversial. METHODS The records of 45 consecutive patients undergoing repair of traumatic disruption of the thoracic aorta at a single institution during a 9-year period were reviewed in a retrospective fashion. Patient age ranged from 15 to 81 years (mean age, 33.9 years). Twenty-two patients (49%) had multiple associated injuries, and 8 (18%) had isolated aortic injuries. Nine patients (20%) experienced preoperative hypotension (systolic blood pressure of less than 90 mm Hg). Repair was performed with partial bypass in 22 patients, a heparinized shunt in 2, and no distal perfusion (clamp and sew technique) in 21. RESULTS Nine patient (20%) died after operation. Multivariate logistic regression analysis of preoperative and intraoperative variables identified advancing age and preoperative hypotension as independent predictors of operative death. The presence of associated injuries was not an independent predictor of operative death. All 4 patients with injuries proximal to the aortic isthmus died. Ten patients were excluded from analysis of spinal cord injury either because of preoperative neurologic deficit or because of death before postoperative evaluation. Six (17%) of the remaining 35 patients had development of paraplegia: 5 of the 15 patients having the clamp and sew technique, 1 of the 2 with a shunt, and 0 of the 18 patients with bypass (p < 0.05, clamp and sew versus bypass). In the clamp and sew group, patients in whom paraplegia developed had significantly longer aortic clamp times than those without neurologic injury (40.6 +/- 4.4 minutes versus 28.7 +/- 2.9 minutes, respectively; p < 0.05). CONCLUSIONS Advancing age, preoperative hypotension, and perhaps injury location are important determinants of death after repair of traumatic disruption of the thoracic aorta. Adjunctive perfusion with partial bypass should be used during repair to reduce the incidence of spinal cord injury.

Dectin-1 Activation by a Natural Product β-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 β-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 β-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 β-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 β-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 β-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 β-glucan treatment in cancer.

Pharyngeal dysphagia in postesophagectomy patients: correlation with deglutitive biomechanics

BACKGROUND Because of the transient nature of pharyngeal phase dysphagia, posttranshiatal esophagectomy patients provide a model for studying the correlation of dysphagic symptoms and aspiration with deglutitive biomechanics. METHODS We studied 8 transhiatal esophagectomy patients (age range, 51 to 78 years) and 8 normal age-matched controls in upright position using lateral and anteroposterior (AP) projection videofluoroscopy during three 5 mL barium swallows. RESULTS The maximum upper esophageal sphincter (UES) AP diameter and maximum anterior excursion of the hyoid bone in patients with transhiatal esophagectomy who experienced aspiration (6.2+/-0.6 and 9.0+/-2.0 mm, respectively) were significantly smaller than those of age-matched normal controls (9.4+/-0.7 and 17.0+/-1.0 mm, respectively). Resolution of aspiration was associated with a significant increase in AP diameter of the UES as well as anterior and superior excursion of the hyoid bone (p<0.05). CONCLUSIONS Dysphagic symptoms and aspiration in posttranshiatal esophagectomy patients are associated with significant abnormalities of deglutitive biomechanics. Improvement in deglutitive biomechanics is associated with resolution of dysphagic symptoms as well as postdeglutitive aspiration in these patients.