Early-stage Human Lung Cancer Research Articles

Magnetic Resonance Spectroscopy-based Metabolomic Biomarkers for Typing, Staging, and Survival Estimation of Early-Stage Human Lung Cancer

Low-dose CT has shown promise in detecting early stage lung cancer. However, concerns about the adverse health effects of radiation and high cost prevent its use as a population-wide screening tool. Effective and feasible screening methods to triage suspicious patients to CT are needed. We investigated human lung cancer metabolomics from 93 paired tissue-serum samples with magnetic resonance spectroscopy and identified tissue and serum metabolomic markers that can differentiate cancer types and stages. Most interestingly, we identified serum metabolomic profiles that can predict patient overall survival for all cases (p = 0.0076), and more importantly for Stage I cases alone (n = 58, p = 0.0100), a prediction which is significant for treatment strategies but currently cannot be achieved by any clinical method. Prolonged survival is associated with relative overexpression of glutamine, valine, and glycine, and relative suppression of glutamate and lipids in serum.

Abstract 3790: Human tumor-infiltrating monocytes/macrophages do not predominantly inhibit tumor-specific effector T cell responses in early-stage lung cancer: The role of macrophage versus tumor PD-L1

Abstract Our current understanding of tumor-associated monocyte/macrophage lineage cells (MMLC) as being immunosuppressive is based largely on murine tumor models that generated a widespread interest in targeting these cells therapeutically. Unfortunately, this approach has not yet been successful in many clinical trials. One potential explanation is that there are fundamental differences between mice and humans in tumor evolution and immunity that might impact the function of human MMLC, limiting the success of myeloid cell-related therapies. Moreover, despite recent successes with checkpoint blockade therapy, the contribution of PD-L1 expressed on MMLC to T cell suppression in humans remains unclear. Thus, the overall purpose of this study was to determine the phenotypic composition of monocyte-macrophage cell lineage in a large cohort of early stage (resected) human lung tumors and delineate how different cell populations of this lineage regulate the effector phase of tumor-specific T cell responses. In addition we also explored the role of MMLC-expressed PD-L1 by interrogating the tripartite functional interactions between tumor-specific effector T cells, PD-L1+ or PD-L1- tumor cells, and TAM that possess a varied surface PD-L1 expression. Phenotypically, we find that (i) MMLC are not the predominate cell population of infiltrating leukocytes within the early-stage lung tumors, (ii) tumor MMLC consist primarily of tumor-associated macrophages (TAM), (iii) the TAM phenotype is complex and does not fit the conventional M1 and M2 phenotype denominations, (iv) TAM are able to co-express T cell co-inhibitory and co-stimulatory receptors. Functionally, MMLC are not primarily suppressive, but have diverse effects, including stimulation of the effector phase of tumor-specific T cell responses. Mechanistically, TAM-expressed PD-L1 (in contrast to tumor-expressed PD-L1), does not inhibit the interaction between tumor-specific effector T cells and tumor cells, but does protect TAMs expressing a tumor antigenic peptide/MHC class I complex from being killed by effector T cells recognizing the cognate peptide on the tumor. These data provide new insights into the phenotype of tumor MMLC and functional cross-talk between TAM, tumor-specific T cells, and tumor cells. Our results show that the prevailing premise that TAMs are predominantly immunosuppressive does not apply to early-stage human lung cancer and thus the current MMLC-targeted approaches would not be helpful in majority of early-stage lung cancer patients. Citation Format: Jason Stadanlick, Abhishek Rao, Sunil Singhal, Michael Annunziata, Pratick Bhojnagarwala, Shaun Obrien, Edmund Moon, Edward Cantu, Gwenn Danet-Desnoyers, Hyun-Jeong Ra, Leslie Litzky, Wayne W. Hancock, Steven M. Albelda, Evgeniy Eruslanov. Human tumor-infiltrating monocytes/macrophages do not predominantly inhibit tumor-specific effector T cell responses in early-stage lung cancer: The role of macrophage versus tumor PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3790.

Abstract 3707: Tumor-associated neutrophils with antigen-presenting features in early-stage human lung cancer

Abstract To date there has been an increasing focus on the interactions between inflammatory myeloid cells and T cells in the tumor microenvironment because cytotoxic anti-tumoral T cells represent the chief effector mechanism of anti-tumoral immunity. Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in lung tumors; however, whether specialized neutrophil subpopulations capable of regulating T cell responses exist in human cancers is unknown. Our goal was to identify subsets of TANs and determine their specific roles in the regulation of T cell responses in patients with early stage lung cancer. An extensive phenotypic analysis of 55 early-stage human lung tumors revealed that TANs, defined as CD11b+Arg1+MPO+CD66b+CD15+ cells, contained two major sub-populations. One subset of “canonical” TANs expressed classic neutrophil markers. A second subset of TANs displayed a combination of neutrophil markers plus markers (CD14+HLA-DR+CCR7+CD86+) normally expressed on antigen-presenting cells (APC). We termed this unique neutrophil population “APC-like hybrid TANs”. The frequency of these hybrid TANs varied widely within lung cancers and ranged from 0.5-25% of all TANs. Interestingly, the frequency of this hybrid population declined as tumors enlarged, and they were almost completely absent in tumors greater than 5 cm in diameter. Mechanistically, we determined that low doses of IFN-γ and GM-CSF in the tumors were required for the development of APC-like hybrid neutrophils. The high proportion of hybrid TANs (>10% of all TANs) directly correlated with the presence of IFN-γ and GM-CSF in the autologous tumor tissue. Using bone marrow-derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC-like hybrid neutrophils originate from CD11b+CD15+CD10-CD16-/low/int neutrophil progenitors in the presence of IFN-γ and GM-CSF or in tumor-conditioned media. Functionally, the APC-like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) stimulate antigen non-specific autologous T cell responses 2) directly stimulate antigen-specific autologous memory T cell responses, 4) augment NY-ESO-1 specific effector T cell responses by providing a co-stimulatory signals through the OX40L, 4-1BBL CD86, CD54 molecules, and 5) cross present tumor-associated antigen as IgG-immune complex. In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early-stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC-like hybrid neutrophils. These hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction Citation Format: Jason Stadanlick, Abhishek Rao, Michael Annunziata, Edmund Moon, Shaun O'brien, Pratik Bhojnagarwala, Michael Feldman, Wayne Hancock, Jose Conejo-Garcia, Sunil Singhal, Stephen Albelda, Evgeniy Eruslanov. Tumor-associated neutrophils with antigen-presenting features in early-stage human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3707. doi:10.1158/1538-7445.AM2017-3707

Phenotype and function of tumor-associated neutrophils and their subsets in early-stage human lung cancer.

Neutrophils accumulate in many types of human and murine tumors and represent a significant portion of tumor-infiltrating myeloid cells. Our current understanding of the role of neutrophils in tumor development has depended primarily on murine models of cancer. However, there are crucial species differences in the evolution of tumors, genetic diversity, immune and inflammatory responses, and intrinsic biology of neutrophils that might have a profound impact on the tumor development and function of neutrophils in mouse versus human tumors. To date, the majority of experimental approaches to study neutrophils in cancer patients have been limited to the examination of circulating blood neutrophils. The phenotype and function of tumor-associated neutrophils (TANs) in humans, particularly in the early stages of tumor development, have not been extensively investigated. Thus, the long-term goal of our work has been to characterize human TANs and determine their specific role in tumor development. Here, we summarize our findings on human TANs obtained from human early stage lung cancer patients. We will describe the phenotypes of different TAN subsets identified in early stage lung tumors, as well as their functional dialog with T cells.

Abstract A02: The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer

Abstract To date there has been an increasing focus on the interactions between inflammatory myeloid cells and T cells in the tumor microenvironment because cytotoxic anti-tumoral T cells represent the chief effector mechanism of anti-tumoral immunity. Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in lung tumors; however, whether specialized neutrophil subpopulations capable of regulating T cell responses exist in human cancers is unknown. Our goal was to identify subsets of TANs and determine their specific roles in the regulation of T cell responses in patients with early stage lung cancer. To address this question, freshly isolated tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. An extensive phenotypic analysis of 55 early-stage human lung tumors revealed that TANs, defined as CD11b+Arg1+MPO+CD66b+CD15+ cells, contained two major sub-populations. One subset of canonical TANs expressed classic neutrophil markers. A second subset of TANs displayed a combination of neutrophil markers plus markers (CD14+HLA-DR+CCR7+CD86+) normally expressed on antigen-presenting cells (APC). This subset of TANs was found in lung tumor tissue but not in adjacent uninvolved lung. We termed this unique neutrophil population “APC-like hybrid TANs”. The frequency of these hybrid TANs varied widely within lung cancers and ranged from 0.5-25% of all TANs. Interestingly, the frequency of this hybrid population declined as tumors enlarged, and they were almost completely absent in tumors greater than 5 cm in diameter. Mechanistically, we determined that low doses of IFN-γ and GM-CSF in the tumors were required for the development of APC-like hybrid neutrophils. The high proportion of hybrid TANs (>10% of all TANs) directly correlated with the presence of IFN-γ and GM-CSF in the autologous tumor tissue. Using bone marrow-derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC-like hybrid neutrophils originate from CD11b+CD15+CD10-CD16-/low/int neutrophil progenitors in the presence of IFN-γ and GM-CSF or in tumor-conditioned media. By contrast, mature CD11b+CD15+CD10+CD16hi neutrophils did not acquire the phenotype of hybrid TANs, when cultured under these conditions. In addition, we determined that IFN-γ and GM-CSF synergistically exerted APC promoting effects on immature neutrophils via the downregulation of the transcription factor, Ikaros. However, the development of APC-like hybrid neutrophils was profoundly inhibited under hypoxic conditions. Functionally, the APC-like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) produce APC cytokines such as TNF and IL-12 after stimulation, 2) stimulate antigen non-specific autologous T cell responses induced by plate-bound anti-CD3 antibodies 3) directly stimulate antigen-specific autologous memory T cell responses to virus-derived antigens, 4) augment NY-ESO-1 specific effector T cell responses by providing a co-stimulatory signals through the OX40L, 4-1BBL CD86, CD54 molecules, and 5) cross present tumor-associated antigen as IgG-immune complex. In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early-stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC-like hybrid neutrophils. These hybrid TANs had enhanced ability to trigger and support T cell responses in direct cell-cell interactions. This property of hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Shaun O'Brien, Edmund Moon, Tom Stephen, Abhishek Rao, Alfred Garfall, Wayne Hancock, Jose Conejo-Garcia, Charuhas Deshpande, Michael Feldman, Sunil Singhal, Steven Albelda. The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A02.

Origin and Role of a Subset of Tumor-Associated Neutrophils with Antigen-Presenting Cell Features in Early-Stage Human Lung Cancer

Based on studies in mouse tumor models, granulocytes appear to play a tumor-promoting role. However, there are limited data about the phenotype and function of tumor-associated neutrophils (TANs) in humans. Here, we identify a subset of TANs that exhibited characteristics of both neutrophils and antigen-presenting cells (APCs) in early-stage human lung cancer. These APC-like "hybrid neutrophils," which originate from CD11b(+)CD15(hi)CD10(-)CD16(low) immature progenitors, are able to cross-present antigens, as well as trigger and augment anti-tumor Tcell responses. Interferon-γ and granulocyte-macrophage colony-stimulating factor are requisite factors in the tumor that, working through the Ikaros transcription factor, synergistically exert their APC-promoting effects on the progenitors. Overall, these data demonstrate the existence of a specialized TAN subset with anti-tumor capabilities in human cancer.

Abstract A66: Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation

Abstract Tumor-recruited myeloid cells represent a significant portion of inflammatory cells within the tumor microenvironment and influence nearly all steps of tumor progression. Although tumor-associated neutrophils (TANs) are present in large numbers, the role of TANs in cancer progression remains unclear and has only been recently investigated in murine models. The goal of this study was to provide a phenotypic and functional characterization of TANs in freshly harvested surgically resected lung cancer samples. Tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. Importantly, fresh lung tumors from patients were processed within 20 minutes of removal from the patient. We developed an approach to prepare human lung tumors for immunological studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This approach retrieved all major tumor-associated cell populations with high cell yield and maintained the expression of cell surface markers and the functions of isolated cells. In multicolor tracings, TANs were defined as CD15hi/CD66b+/CD11b+/MPO+/Arg1+/IL-5Ra-/ cells. We found that TANs comprised 5—25% of cells isolated from all digested human lung tumors (n=42). Compared to blood neutrophils, TANs displayed an activated phenotype (CD62Llo/CD54hi) and a novel repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. They also produced substantial quantities of the pro-inflammatory factors MCP-1, IL-8, MIP-1a, and IL-6, as well as the anti-inflammatory IL-1R antagonist. TANs were not hypofunctional cells, since they were able to phagocytize bacteria and produce reactive oxygen species. Understanding the role of TANs in regulating T cell responses in cancer patients is particularly important because cytotoxic T lymphocytes are the chief effector cells mediating antigen-driven anti-tumor immunity. We found that TANs were able to stimulate antigen non-specific T cell proliferation and IFN-γ release. Crosstalk between TANs and activated T cells led to substantial up-regulation of CD54, CD86, OX40L, and 4-1BBL co-stimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive feedback loop. This stimulatory activity of TANs appeared to be related to tumor size, as TANs from the majority of the smaller tumors (< 3cm) were more stimulatory than those from larger tumors. We also identified a novel subset of TANs with a hybrid “composite” phenotype of neutrophils and antigen-presenting cells (MPO+CD66b+CD14+HLA-DR+CCR7+CD86+). We found that this hybrid subset had the ability to: 1) dramatically stimulate antigen non-specific T cell proliferation, 2) induce the proliferation of allogeneic T cells in mixed lymphocyte reaction and 3) trigger antigen-specific memory T cell response to a mixture of T cell epitopes from human CMV, Epstein-Barr, flu viruses, as well as tetanus toxoid from all the common HLA types. Thus, our data suggest that in patients with early-stage lung cancer, TANs do not significantly contribute to inhibition of T cell responses. Rather, the majority of neutrophils recruited into the tumor microenvironment undergo phenotypic and functional changes that result in the formation of cells that resemble the murine anti-tumor “N1 TANs” and could potentially augment and support T cells responses. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Tom Stephen, Anjana Ranganathan, Charuhas Deshpande, Tatiana Akimova, Anil Vachani, Leslie Litzky, Wayne Hancock, Jose Conejo-Garcia, Michael Feldman, Sunil Singhal, Steven Albelda. Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A66.

Abstract 1291: Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells

Abstract Aim: Advances in immunotherapy require a detailed understanding of host immunity. Data from murine models indicate solid tumors of various types can recruit Tregs and/or promote iTreg development, and thereby curtail antitumor responses. However, clinical data have been restricted to either evaluation of Treg numbers in tumors, or studies of PBMC Treg in patients with cancers. Surprisingly, no studies of Treg function have ever been reported using Tregs isolated from tumors themselves. Methods: Using our recently reported technique, we isolated Tregs within 20 mins of resection from 13 lung cancer patients, stages Ia-IIIa, and 2 control non-cancer patients (diaphragmatic hernia and lung granuloma). The mean age of the cancer patients (10 males, 3 females) was 65.4±2.2 years; 7 patients had adenocarcinoma, 3 had squamous cell carcinoma, and the remainder had large cell neuroendocrine carcinoma, colon adenocarcinoma and high-grade carcinoma. The mean size of their tumors was 4.0±0.8 cm, and 1 of the 13 patients developed metastases during the follow-up period. We compared the suppressive function of Tregs isolated from each patient's freshly resected tumor, LN and blood, using cryopreserved healthy donor PBMC aliquots as responders, and area-under-curve (AUC) analysis. We controlled for comparable CD4+ and FOXP3+ purity in all isolated Treg samples, and cell viability, excluding from further analysis all data from non-comparable FOXP3+ subsets. Thus, we were able to avoid artifacts arising from different cell conditions (e.g. use of autologous responder cells) or from unequal FOXP3+ content in Treg isolates. Results: Tumor-infiltrating Tregs had significantly enhanced suppressive function when compared with Tregs isolated from other locations in the same patient. Thus, tumor Tregs were significantly more suppressive than lung LN Tregs (p = 0.0003), and significantly more suppressive than blood Tregs (p = 0.03). Tregs isolated from LN in close proximity to tumors had comparable suppressive function to Tregs isolated from distant LN (p>0.05), and blood Tregs had comparable suppressive function to that of LN Tregs (p>0.05). Additional analyses showed that the enhanced suppressive function of tumor Treg was not due to Treg-induced killing/apoptosis of responder cells, cytokine and/or growth factor deprivation, or soluble factors produced by tumor Treg cells. Conclusions: We demonstrate for the first time that tumor-infiltrating human lung cancer Treg have significantly increased suppressive function. This finding is important for the development of effective anti-tumor therapy. While we are continuing to study the basis for such differences, our data suggest that studies of peripheral blood Tregs in cancer patients may be inadequate and inefficient approaches for understanding the fundamental mechanisms of limited host antitumor immune responses. Citation Format: Tatiana Akimova, Evgeniy B. Eruslanov, Pratik S. Bhojnagarwala, Jon G. Quatromoni, Jacqueline Morgen, Sunil Singhal, Steven M. Albelda, Wayne W. Hancock. Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1291. doi:10.1158/1538-7445.AM2015-1291

In the Hunt for Therapeutic Targets: Mimicking the Growth, Metastasis, and Stromal Associations of Early-Stage Lung Cancer Using a Novel Orthotopic Animal Model

The existing shortage of animal models that properly mimic the progression of early-stage human lung cancer from a solitary confined tumor to an invasive metastatic disease hinders accurate characterization of key interactions between lung cancer cells and their stroma. We herein describe a novel orthotopic animal model that addresses these concerns and consequently serves as an attractive platform to study tumor-stromal cell interactions under conditions that reflect early-stage lung cancer. Unlike previous methodologies, we directly injected small numbers of human or murine lung cancer cells into murine's left lung and longitudinally monitored disease progression. Next, we used green fluorescent protein-tagged tumor cells and immuno-fluorescent staining to determine the tumor's microanatomic distribution and to look for tumor-infiltrating immune cells and stromal cells. Finally, we compared chemokine gene expression patterns in the tumor and lung microenvironment. We successfully generated a solitary pulmonary nodule surrounded by normal lung parenchyma that grew locally and spread distally over time. Notably, we found that both fibroblasts and leukocytes are recruited to the tumor's margins and that distinct myeloid cell attracting and CCR2-binding chemokines are specifically induced in the tumor microenvironment. Our orthotopic lung cancer model closely mimics the pathologic sequence of events that characterizes early-stage human lung cancer propagation. It further introduces new means to monitor tumor-stromal cell interactions and offers unique opportunities to test therapeutic targets under conditions that reflect early-stage lung cancer. We argue that for such purposes our model is superior to lung cancer models that are based either on genetic induction of epithelial transformation or on ectopic transplantation of malignant cells.

Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

Diagnosis of lung cancer based on direct-infusion electrospray mass spectrometry of blood plasma metabolites

Lung cancer is one of the most prevalent types of cancer in men and women, and is a leading cause of cancer-related death. Early detection of lung cancer may profoundly reduce cancer death rates. It is therefore extremely important to develop laboratory tests to detect human lung cancer, including at clinically asymptomatic stages. To this end, mass spectrometric metabolite analysis was performed on blood samples collected from patients with lung cancer ( N = 100) and age-matched controls ( N = 100). Proteins were extracted from blood plasma with methanol, and the remaining metabolite fractions were directly analyzed using electrospray ionization (ESI) mass spectrometry. Mass spectra obtained were converted into binary format, aligned, reduced to several variables by principal component analysis (PCA), and finally, classified as cancer cases versus controls by a support vector machine (SVM) algorithm. Repeated random sub-sampling validation revealed an accuracy of classification as high as 93.3% (sensitivity 94.1%, selectivity 92.4%), strongly indicating that direct-infusion ESI mass spectrometry of blood plasma metabolites offers great clinical potential in the diagnosis of early-stage human lung cancer.