HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Bryan A. Whitson Jonathan D'Cunha Rafael S. Andrade Rosemary F. Kelly Jeffrey S. Miller Michael A. Maddaus Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Whitson, B. A. Articles by Maddaus, M. A. Search for Related Content PubMed PubMed Citation Articles by Whitson, B. A. Articles by Maddaus, M. A. Related Collections Lung - cancer Molecular biology Related Article

---

Original Articles: General Thoracic

Thoracoscopic Versus Thoracotomy Approaches to Lobectomy: Differential Impairment of Cellular Immunity

^a Department of Surgery, University of Minnesota, Minneapolis, Minnesota
^b Department of Medicine, University of Minnesota, Minneapolis, Minnesota
^c Department of Surgery, Minneapolis Veterans Affairs Medical Center, Minneapolis, Minnesota
^d Division of Biostatistics, University of Minnesota School of Public Health, Minneapolis, Minnesota

Accepted for publication July 2, 2008.

^_* Address correspondence to Dr Maddaus, University of Minnesota, Department of Surgery, MMC 207, 420 Delaware St SE, Minneapolis, MN 55455 (Email: madda001{at}umn.edu).

	Abstract

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background: Video-assisted thoracoscopic surgery (VATS) for patients with early-stage non–small-cell lung cancer is associated with lower stress responses and potentially improved outcomes, as compared with thoracotomy. The goal of our study was to examine the cellular components of the postoperative immune response. Specifically, we assessed the cytotoxic capacity of peripheral blood mononuclear cells (PBMCs) of patients undergoing lobectomy for non–small-cell lung cancer by either VATS or thoracotomy.

Methods: We performed a prospective cohort study of lobectomy patients undergoing either VATS or thoracotomy. We isolated PBMCs from perioperative blood samples, and performed cytokine analysis on plasma fractions. Using flow cytometry, we analyzed PBMC phenotype (CD3, CD16/56, CD4, CD8) and T-cell activation markers (CD25, CD69, HLA-DR). Using a chromium release assay, we quantified cellular cytotoxicity. To assess gene expression differences, we used Affymetrix messenger ribonucleic acid microarray and polymerase chain reaction analysis.

Results: A total of 13 patients enrolled in our study: 6, VATS; 7, thoracotomy. On postoperative day 1, interleukin-6 and matrix metalloproteinase-9 were significantly different between the two groups. On day 2, cellular cytotoxicity (0.34) was significantly greater (p < 0.05) after VATS, as compared with thoracotomy (0.18). In both groups, cytotoxicity returned to baseline and was equivalent at first follow-up (VATS, 29.4 days versus thoracotomy, 29.3 days; p > 0.05). We noted minimal yet significant differences in PBMC phenotype, but no differences in T-cell activation makers. A 9-gene polymerase chain reaction–validated subset clustered the two groups with complete concordance.

Conclusions: Video-assisted thoracoscopic surgery lobectomy for non–small-cell lung cancer is associated with less impairment of cellular cytotoxicity, as compared with thoracotomy. We found that this difference was not accounted for by PBMC phenotypic changes. Instead, PBMC gene expression changes likely represent the molecular basis of this differential immune response.

	Introduction

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Cancer has immunosuppressive effects on patients' immune systems [1]. Surgery itself perturbs baseline immune status and cytokine milieus [2, 3]. Research clarifying the effect of surgical approach on postoperative stress has used models focused on nononcologic patients or on small animal models [4–6].

For patients with early-stage non–small-cell lung cancer (NSCLC) who undergo resection with curative intent, typically a thoracotomy lobectomy, the survival rate is poor and tumor recurrence rates vary [7, 8]. Recent reports have suggested that the video-assisted thoracoscopic surgery (VATS) approach to lobectomy for NSCLC patients may have potential survival advantages, as compared with the traditional thoracotomy [9, 10]. These two approaches to lobectomy have been touted as being oncologically equivalent; however, the underlying mechanism(s) for the potential differences in survival have not been elucidated. Studies on these two approaches to lobectomy have focused on the postoperative cytokine stress response [3, 11, 12].

The perioperative cytokine response is relatively well characterized, but the effect on the peripheral blood mononuclear cells (PBMCs), or immune effector cells, is not. The cytokine response may be a reflection of changes in PBMCs, some of which may be functional and at the level of gene expression. Investigations into mechanisms of immunologic benefits associated with thoracoscopic surgery must begin with PBMCs. If PBMCs are differently affected by surgical approach, elucidating these functional changes may begin to explain the observed differences in morbidity and survival. We theorize that, in patients undergoing lobectomy for NSCLC, surgical stress may inhibit their baseline immune function, leaving them susceptible to infectious complications or to lessened tumor surveillance, thus potentially diminishing survival. In this study, we focus on the PBMC leukocyte fraction, which consists of natural killer cells and T-cell lymphocytes. The hypothesis of our study is that the VATS approach to lobectomy for early-stage NSCLC has a less detrimental effect on PBMC function, as compared with thoracotomy, and this difference is driven by differential gene expression.

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Institutional review boards of the University of Minnesota and the Minneapolis Veteran Affairs Medical Center, as well as the University of Minnesota Cancer Center Cancer Protocol Review Committee, approved this study. Individual patients gave informed consent. We had freedom in study design and publication, with no financial conflicts of interest.

We performed a prospective, observational study in consecutive patients who underwent lobectomy for early-stage NSCLC. The choice of the surgical approach to lobectomy was not influenced by study design. We collected blood samples from patients at four predetermined time intervals: the morning of surgery (postoperative day [POD] 0), POD 1, POD 2, and at the first follow-up clinic visit.

The VATS and thoracotomy were performed as previously described. In brief, the VATS is a non–rib-spreading approach using three or four 1-cm incisions for trocars and a 4- to 6-cm access incision. The thoracotomy approach used a traditional posterolateral thoracotomy incision at the fifth intercostal space [13].

Sample Collection
Patients underwent a peripheral venipuncture collection of a 30-mL blood sample in acid citrate dextrose vacuum containers. Within 2 hours of collection the blood was centrifuged; the plasma fraction was isolated and frozen (–80°C) for batch cytokine analysis processing. Peripheral blood mononuclear cells were isolated by Ficoll density gradient separation [6]. Viability was determined by standard 0.4% trypan dye exclusion with a hemacytometer [6]. Subsequently, PBMCs were divided into aliquots for phenotypic analysis, cellular cytotoxicity analysis, and gene expression analysis.

Cytokine Analysis
To quantify cytokine levels, we used a Bio-Plex suspension array system (Bio-Rad Laboratories, Inc, Hercules, CA) incorporating Luminex microsphere technology (Luminex Corp, Austin, TX) [6]. Interleukin (IL) 6, IL-8, matrix metalloproteinase-9 (MMP9), and insulin-like growth factor binding protein-3 (IGFBP3) levels were measured for each sample.

Phenotypic Analysis
The PBMC aliquot slated for phenotypic analysis was stained with antibody or the appropriate isotype control (Becton, Dickinson and Company, San Jose, CA, or eBioscience, Inc, San Diego, CA) and washed, as previously described [6]. Next, PBMCs underwent four-color immunophenotypic fluorescent-activated cell sorting analysis on a FACSCaliber flow cytometer with CellQuest (Becton, Dickinson and Company) [6]. Cell surface markers CD3⁺/CD4⁺ denoted T-helper cells, CD3⁺/CD8⁺ denoted cytotoxic T cells, and CD3^–/CD16⁺CD56 denoted natural killer cells. The CD3⁺/CD4⁺ fraction was analyzed for T-cell activation markers (CD25, CD69, and HLA-DR).

Cellular Cytotoxicity Analysis
We used a standard chromium 51 (⁵¹Cr) release assay [6] to analyze cellular cytotoxicity. K562 target cells, a lymphoblastic cell line derived from a patient with chronic myelogenous leukemia, which predominantly evaluates unstimulated natural killer cell cytotoxicity, were incubated with ⁵¹Cr for 1 hour and rinsed. Then 100,000 effector cells were pipetted into 96-well microtiter plates and mixed with K562 target cells at a 20:1 concentration. Each sample was run in triplicate. With each analysis, we obtained target cell background and total gamma radiation release values in sextuplets. Cells were incubated at 37°C and 5% CO₂ for 4 hours. The supernatant was collected and percent specific lysis (PSL) was calculated using:

We calculated normalized PSL by dividing the values by the POD 0 PSL values. Postoperative day 0 was, by definition, 100%.

Gene Expression Analysis
The aliquot for gene expression analysis underwent total messenger ribonucleic acid (mRNA) extraction using a combined Trizol and silica gel column method (RNEasy, Qiagen, Valencia, CA) [6]. Isolated total mRNA was frozen (–80°C) until either biotin labeling for microarray analysis or gene expression analysis. For complementary ribonucleic acid (cRNA) microarray analysis, we used mRNA samples from POD 2 for the first 5 patients in both groups (n = 10 arrays total). The POD 2 time was chosen because of significant observed differences in cytotoxic function. Biotin-labeled cRNA was made, purified, quantified, fragmented, and hybridized to Affymetrix human U133 plus 2.0 microarray chips (Affymetrix, Santa Clara, CA) [6].

Gene Signature Validation
To validate the reliability of our Affymetrix microarray data, we selected nine genes (all underexpressed in VATS patients relative to thoracotomy patients) to determine the relative expression differences in all of the patients' POD 2 mRNA samples through quantitative real-time reverse-transcriptase polymerase chain reaction analysis (QRT-PCR) [14]. Two-step QRT-PCR was performed with complementary deoxyribonucleic acid and the QuantiTect SYBR Green PCR Kit (Qiagen, Inc) on the ABI Prism 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) following the manufacturers' specifications. Each gene and sample was run in triplicate.

To determine the relative quantitation of gene transcripts, we used the comparative threshold cycle method (C_t) [15, 16] with the following formulas:

and

The cycle number, C_t, was determined in the exponential phase of the amplification plot.

Statistical Analysis
Within both groups, we performed statistical analysis to determine any postoperative variance from baseline; between groups at each time to determine differences between the two surgical approaches ( = 0.05). We used the Student's t test for analysis between groups; within groups, a one-way analysis of variance was used in addition to the Student's t test. For nominal and ordinal variables, we used the Chi-square test. Results are reported as mean ± standard errors of the mean. We obtained the normalized microarray gene expression data using the robust multiarray average expression measure [17]. To detect differentially expressed genes between groups, we performed significance analysis of microarray [18]. To control the proportion of false positives, we calculated the false discovery rate [19].

	Results

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Surgical Groups
Thirteen patients enrolled in our study; 7 thoracotomy, 6 VATS. The two groups were well matched in terms of their patient characteristics (Table 1). We noted no statistically significant difference in the mean ages. The pulmonary function test results of the two groups were significantly worse for the VATS group. Both groups were well matched in terms of their comorbidities.

Phenotypic Changes
The perioperative PBMC phenotype was very similar between the VATS and thoracotomy groups. We saw no significant differences in concentrations of CD3^–CD16⁺CD56 natural killer cells or of CD3⁺ T cells postoperatively. Similarly, we saw no differences postoperatively in the CD3⁺/CD4⁺ lymphocyte subset. However, in the CD3⁺/CD8⁺ subset, the differences were statistically significant between groups, but only preoperatively (POD 0; Fig 1A). In terms of the CD4/CD8 ratio, the differences between groups were significantly different on POD 0 and at first follow-up (Fig 1B). We found no differences between groups' T-lymphocyte activation markers for CD25, CD69, or HLA-DR. Within both groups, we saw postoperative differences only in HLA-DR.

View larger version (7K): [in this window] [in a new window]   Fig 1. Flow cytometry analysis of the phenotype of the peripheral blood mononuclear cells perioperatively. (A) Cytotoxic (CD3+/CD8+) and helper (CD3+/CD4+) T-cell subsets. (B) CD4/CD8 ratio. (OT = open thoracotomy; POD = postoperative day; PREOP = preoperatively; VATS = video-assisted thoracoscopic surgery.)

View larger version (36K): [in this window] [in a new window]   Fig 2. Cellular cytotoxicity. (A) Percent specific lysis. (B) Normalized (to postoperative day [POD] 0) percent specific lysis. (OT = open thoracotomy; PREOP = preoperatively; VATS = video-assisted thoracoscopic surgery.)

We cross-referenced this 99-gene set with the Affymetrix probe set database (www.affymetrix.com), the National Institutes of Health's National Center for Biotechnology Information Entrez Gene (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene), and the Weizmann Institute of Science's GeneAnnot (http://bioinfo2.weizmann.ac.il/geneannot/) and GeneCards (http://www.genecards.org). Pathway associations were evaluated though Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA). We identified a highly selective nine-gene subset (Table 3) that were all downregulated in the mRNA of our VATS patients. We used this nine-gene subset for QRT-PCR validation of our microarray analysis in all POD 2 mRNA samples.

This highly selective subset of genes showed a high degree of concordance between the relative QRT-PCR fold expression changes and the microarray fold expression changes, with a regression coefficient of 0.84 (Fig 3). In this analysis we did not collect cytosolic samples for protein quantification associated with the gene expression. Collection for this type of analysis in future studies would be of value. We measured the levels of plasma IGFBP3 and MMP9 in our samples (Fig 4). Both IGFBP3 and MMP9 have been associated with the differential postoperative immune response [12] and found in our gene expression analysis. For IGFBP3 levels, we found no significant differences within or between groups. We observed elevated IGFBP3 levels in the VATS group on POD 1 and POD 2. On POD 1, the MMP9 level in the VATS group was significantly greater, as compared with the thoracotomy group. Within the VATS group, MMP9 levels significantly increased postoperatively and then returned to baseline levels at first follow-up.

View larger version (7K): [in this window] [in a new window]   Fig 3. Gene signature validation. (RT-PCR = reverse-transcriptase polymerase chain reaction; VATS = video-assisted thoracoscopic surgery.)

View larger version (7K): [in this window] [in a new window]   Fig 4. Protein validation of gene expression. (A) Matrix metalloproteinase 9 concentrations. (B) Insulin-like growth factor binding protein 3 concentrations. (OT = open thoracotomy; POD = postoperative day; PREOP = preoperatively; VATS = video-assisted thoracoscopic surgery.)

	Comment

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

In patients undergoing resection for gastric and colon cancers, Ogawa and coworkers [22] demonstrated through cytokine and cytometric analysis that surgery caused a decrease in PBMC function for up to 2 weeks postoperatively. Until now, human data on the immunosuppressive effect of surgery for NSCLC have been limited to cytokine and cytometric analysis [3, 11, 12]. In a rat model of surgical stress that used laparotomy and thoracotomy to modulate the extent and duration of the trauma, Hirai and associates [23] correlated increased surgical stress with an increased number of lung cancer metastases and with a decrease in survival. They termed this surgical facilitation of metastasis "surgical oncotaxis."

Cytokines and Surgical Stress Model
The observed postoperative cytokine response experienced by our patients was similar to that described in the literature, in terms of its character and duration [3, 11]. Immediately postoperatively, inflammation surges, then subsides with time. Evidence in the literature increasingly shows that minimally invasive surgical approaches are less immunosuppressive than their traditional open counterparts [2, 3, 11], and this observation has been ascribed to the cytokine response [2, 3]. The response they have observed is similar to that which we observed in our study: a quick postoperative rise, muted in the minimally invasive approaches, then a relatively rapid return to baseline.

Peripheral Blood Mononuclear Cell Response
Characterizing the cytokine response postoperatively is important, but the deep-seated question we intended to answer was whether or not there are functional differences in PBMCs. By measuring the cytotoxic function of our PBMC fraction with the ⁵¹Cr release assay, we demonstrated that surgical stress (lobectomy for NSCLC) caused a decrease in the function of the innate arm of the immune system. Furthermore, we observed that thoracotomy has a more deleterious effect than VATS. The differential effect is greatest on POD 2. The time of the effect lags the cytokine response by about a day. Video-assisted thoracoscopic surgery patients rebounded toward baseline more quickly than thoracotomy patients (whose nadir did not occur until at least POD 2). In both groups, changes in innate immune function were not accounted for by PBMC phenotype differences. We saw preoperative differences in T lymphocytes, but no postoperative differences. Similarly, between groups, T-lymphocyte activation markers did not differ.

Three potential explanations for the lack of demonstrable differences in T-lymphocyte activation markers relate to the time course of the samples, the nonpurified nature of the samples, and the possible absence of activated T-lymphocyte fractions in peripheral blood at the time of phlebotomy. Expression of CD69, CD25, and HLA-DR has been associated with the very early, early, and late activation of T lymphocytes [24]. We assumed that differential changes in activation would be apparent at the times we chose; all seemed clinically relevant and convenient, yet our assumption may be incorrect. Activated T lymphocytes might not circulate in peripheral blood postoperatively. They might be sequestered at the site of injury (eg, incision, dissection, resection), or at a nidus of inflammation or chemotaxis.

Many investigators have used enriched or purified, sorted, or stimulated samples to measure responses. Given the volume of blood needed for such measurements, we were not able to do so in our study. Future endeavors to further elucidate the lymphocyte response could consist of proliferation or stimulation assays and measurement of antibody production.

Differential Gene Expression
Using the high-density Affymetrix chip for our differential gene expression analysis, we were able to identify a 99-gene signature that helped us stratify data relatively well. Our QRT-PCR validation of the nine-gene subset on the entire POD 2 sample set allowed us to demonstrate that our microarray data were accurate. Our study combines phenotype analysis with functional data and differential gene expression in a human model of cancer.

The genes in our subset are associated with immune activation and cellular regulation. The BMX nonreceptor tyrosine kinase regulates eukaryotic initiation factor 4E, a requisite component for cap-dependent translation. Overexpression of eukaryotic initiation factor 4E has been implicated in oncogenesis [25]. Additionally, BMX nonreceptor tyrosine kinase is part of IL-6 induced differentiation. Leukocyte immunoglobulin-like receptor, subfamily B, member 3 modulates cellular immunoreceptor responses. Carcinoembryonic antigen-related cell adhesion molecule 1 is relevant to carcinogenesis and cellular signaling. Histone cluster 1, H3h expression increases during the S-phase and enhances access to deoxyribonucleic acid through histones that facilitate deoxyribonucleic acid wrapping and regulate transcription. Complement component 3a receptor 1 is a chemotactic factor for the complement component C3a. Signal-regulatory protein alpha is associated with an immunoglobulin-like cell surface receptor as well as intracellular signaling and cell cycle control.

Matrix metalloproteinase 9 is an inflammatory mediator. Insulin-like growth factor binding protein 7 is elevated in senescent and nonactivated cells. Matrix metalloproteinase 9, along with IGFBP3, has been associated with differential expression in thoracotomy and VATS patients. Ng and colleagues [12] reported that thoracotomy was associated with higher levels of MMP9 and IGFBP3. In our study, we observed similar findings with our plasma concentrations of IGFBP3. However, we observed that VATS patients had a higher level of MMP9, as compared with thoracotomy patients. In contrast, Ng and coworkers [12] used serum (instead of plasma) to measure MMP9 and IGFBP3. The type of container used was not reported in their study, and the tumor sizes differed. Why we observed the plasma values that we did for MMP9 is unclear, but collection and preservation differences might have played a role. However, in the mRNA of the PBMCs, we observed, by microarray analysis and then QRT-PCR validation, downregulation in MMP9 and insulin-like growth factor binding protein 7 expression. Our gene expression findings could be consistent with the findings reported by Ng and colleagues [12]. The recurrence of insulin-like growth factor pathway components (insulin receptor substrate 2, IGFBP3 and insulin-like growth factor binding protein 7, eukaryotic initiation factor 4E) underscores the importance of its contribution to cellular signaling and translational control.

Potential Limitations
Two potential limitations to our study were its nonrandomized nature and the small sample size. Given the nature of the surgical practices at our institutions and of our patients' disease presentations, we were not able to randomly assign patients. However, as a prospective study, consecutive patients were enrolled in both arms, without preselection. Both groups were well matched in terms of disease and comorbidities. The small sample size may lead to bias and enhance the potential for type II error. Nevertheless, we were able to observe functional differences on POD 2.

Conclusion
Our study showed that lobectomy for early-stage NSCLC was associated with transient inflammation, as demonstrated by cytokine changes consistent with the literature. The PBMC phenotype of thoracotomy and VATS patients was not significantly altered postoperatively. However, even though the PBMC phenotype did not change, thoracotomy had a greater decrease in innate immunity, as compared with VATS. Gene expression changes may represent the molecular basis of this differential immune response. Our study combined phenotype analysis with functional data and differential gene expression in a human model of cancer. Our encouraging and preliminary data set lays the groundwork to correct previous limitations. A larger, prospective, randomized, multicenter study is needed to continue our focus on the immunologic effects of surgical approach used to treat patients with early-stage NSCLC.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The authors would like to acknowledge Mary Knatterud, PhD, for her editorial assistance with this manuscript, the Engeland and Saluja/Vickers Laboratories at the University of Minnesota Department of Surgery for the use of their equipment, and the University of Minnesota Supercomputing Institute for the use of their hardware and software resources.

	References

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

1. Kim R, Emi M, Tanabe K, Arihiro K. Tumor-driven evolution of immunosuppressive networks during malignant progression Cancer Res 2006;66:5527-5536.[Abstract/Free Full Text]
2. Nguyen NT, Goldman CD, Ho HS, et al. Systemic stress response after laparoscopic and open gastric bypass J Am Coll Surg 2002;194:557-566.[Medline]
3. Yim AP, Wan S, Lee TW, Arifi AA. VATS lobectomy reduces cytokine responses compared with conventional surgery Ann Thorac Surg 2000;70:243-247.[Abstract/Free Full Text]
4. Allendorf JD, Bessler M, Whelan RL, et al. Postoperative immune function varies inversely with the degree of surgical trauma in a murine model Surg Endosc 1997;11:427-430.[Medline]
5. Bouvy ND, Marquet RL, Jeekel J, Bonjer HJ. Laparoscopic surgery is associated with less tumour growth stimulation than conventional surgery: an experimental study Br J Surg 1997;84:358-361.[Medline]
6. Whitson BA, d'Cunha J, Hoang CD, et al. Minimally invasive versus open roux-en-Y gastric bypass: impact on immune effector cells. Surg Obes Relat Dis (in press).
7. American College of Surgeons National Cancer Data Base Detailed Guide: Lung Cancer - Non-small Cell, in AJCC Manual, American Cancer Society.
8. Mountain CF. Revisions in the International System for Staging Lung Cancer Chest 1997;111:1710-1717.[Medline]
9. Roviaro G, Varoli F, Vergani C, et al. Long-term survival after videothoracoscopic lobectomy for stage I lung cancer Chest 2004;126:725-732.[Medline]
10. Walker WS, Codispoti M, Soon SY, et al. Long-term outcomes following VATS lobectomy for non-small cell bronchogenic carcinoma Eur J Cardiothorac Surg 2003;23:397-402.[Abstract/Free Full Text]
11. Sugi K, Kaneda Y, Esato K. Video-assisted thoracoscopic lobectomy reduces cytokine production more than conventional open lobectomy Jpn J Thorac Cardiovasc Surg 2000;48:161-165.[Medline]
12. Ng CS, Lee TW, Wan S, et al. Thoracotomy is associated with significantly more profound suppression in lymphocytes and natural killer cells than video-assisted thoracic surgery following major lung resections for cancer J Invest Surg 2005;18:81-88.[Medline]
13. Whitson BA, Andrade RS, Boettcher A, et al. Video-assisted thoracoscopic surgery is more favorable than thoracotomy for resection of clinical stage I non-small cell lung cancer Ann Thorac Surg 2007;83:1965-1970.[Abstract/Free Full Text]
14. D'Cunha J, Corfits AL, Herndon 2nd JE, et al. Molecular staging of lung cancer: real-time polymerase chain reaction estimation of lymph node micrometastatic tumor cell burden in stage I non-small cell lung cancer—preliminary results of Cancer and Leukemia Group B Trial 9761 J Thorac Cardiovasc Surg 2002;123:484-491.[Abstract/Free Full Text]
15. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Methods 2001;25:402-408.[Medline]
16. Baker RWR, Nissim JA. Expressions for combining standard errors of two groups and for sequential standard error Nature 1963;198:1020.
17. Irizarry RA, Hobbs B, Collin F, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data Biostatistics 2003;4:249-264.[Abstract]
18. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response Proc Natl Acad Sci USA 2001;98:5116-5121.[Abstract/Free Full Text]
19. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing J R Stat Soc Ser B Methodol 1995;57:289-300.
20. Imai K, Matsuyama S, Miyake S, Suga K, Nakachi K. Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population Lancet 2000;356:1795-1799.[Medline]
21. Shakhar G, Ben-Eliyahu S. Potential prophylactic measures against postoperative immunosuppression: could they reduce recurrence rates in oncological patients? Ann Surg Oncol 2003;10:972-992.[Medline]
22. Ogawa K, Hirai M, Katsube T, et al. Suppression of cellular immunity by surgical stress Surgery 2000;127:329-336.[Medline]
23. Hirai T, Matsumoto H, Yamashita K, et al. Surgical oncotaxis—excessive surgical stress and postoperative complications contribute to enhancing tumor metastasis, resulting in a poor prognosis for cancer patients Ann Thorac Cardiovasc Surg 2005;11:4-6.[Medline]
24. Rea IM, McNerlan SE, Alexander HD. CD69, CD25, and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNF-alpha, IFN-gamma, and sIL-2R levels in aging Exp Gerontol 1999;34:79-93.[Medline]
25. Richter JD, Sonenberg N. Regulation of cap-dependent translation by eIF4E inhibitory proteins Nature 2005;433:477-480.[Medline]

This article has been cited by other articles:

				S. Gilbert, A. Kilic, K. Yaeger, Y. Toyoda, C. Bermudez, M. P. Siegenthaler, and R. L. Kormos Minimally invasive approach to thoracic effusions in patients with ventricular assist devices Interact CardioVasc Thorac Surg, January 1, 2012; 14(1): 44 - 47. [Abstract] [Full Text] [PDF]

				M. Nosotti, L. Rosso, P. Mendogni, A. Palleschi, D. Tosi, P. Bonara, and L. Santambrogio Leukocyte subsets dynamics following open pulmonary lobectomy for lung cancer: a prospective, observational study Interact CardioVasc Thorac Surg, September 1, 2011; 13(3): 262 - 266. [Abstract] [Full Text] [PDF]

				M. Kamiyoshihara, T. Nagashima, H. Igai, J. Atsumi, T. Ibe, S. Kakegawa, and K. Shimizu Video-assisted thoracic lobectomy with bronchoplasty for lung cancer, with special reference to methodology Interact CardioVasc Thorac Surg, April 1, 2011; 12(4): 534 - 539. [Abstract] [Full Text] [PDF]

				M. Kamiyoshihara, T. Nagashima, T. Ibe, and I. Takeyoshi A tip for controlling the main pulmonary artery during video-assisted thoracic major pulmonary resection: the outside-field vascular clamping technique Interact CardioVasc Thorac Surg, November 1, 2010; 11(5): 693 - 695. [Abstract] [Full Text] [PDF]

				G. Vanni, F. Tacconi, F. Sellitri, V. Ambrogi, T. C. Mineo, and E. Pompeo Impact of Awake Videothoracoscopic Surgery on Postoperative Lymphocyte Responses Ann. Thorac. Surg., September 1, 2010; 90(3): 973 - 978. [Abstract] [Full Text] [PDF]

				K. E. Rusthoven and T. J. Pugh Stereotactic Body Radiation Therapy for Inoperable Lung Cancer JAMA, June 16, 2010; 303(23): 2354 - 2355. [Full Text] [PDF]

				C. E. Nwogu, S. Yendamuri, and T. L. Demmy Does Thoracoscopic Pneumonectomy for Lung Cancer Affect Survival? Ann. Thorac. Surg., June 1, 2010; 89(6): S2102 - S2106. [Abstract] [Full Text] [PDF]

				N. M. Rueth and R. S. Andrade Is VATS Lobectomy Better: Perioperatively, Biologically and Oncologically? Ann. Thorac. Surg., June 1, 2010; 89(6): S2107 - S2111. [Abstract] [Full Text] [PDF]

				S. Paul, N. K. Altorki, S. Sheng, P. C. Lee, D. H. Harpole, M. W. Onaitis, B. M. Stiles, J. L. Port, and T. A. D'Amico Thoracoscopic lobectomy is associated with lower morbidity than open lobectomy: a propensity-matched analysis from the STS database. J. Thorac. Cardiovasc. Surg., February 1, 2010; 139(2): 366 - 378. [Abstract] [Full Text] [PDF]

				H. Pass Invited Commentary Ann. Thorac. Surg., December 1, 2008; 86(6): 1744 - 1744. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Bryan A. Whitson Jonathan D'Cunha Rafael S. Andrade Rosemary F. Kelly Jeffrey S. Miller Michael A. Maddaus Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Whitson, B. A. Articles by Maddaus, M. A. Search for Related Content PubMed PubMed Citation Articles by Whitson, B. A. Articles by Maddaus, M. A. Related Collections Lung - cancer Molecular biology Related Article