- Email: [email protected]
Evidence That Inflammation Encourages Pulmonary Adenocarcinoma Formation in Mice
New Targets for Detection and Treatment of Small Cell Lung Cancer* Nina Pedersen, MSc, PhD; Mikkel W. Pedersen, MSc, PhD; Thomas T. Poulsen, MSc(c); Shila Mortensen, MSc; Susanne B. Sørensen, MSc; Klaus Rieneck, MD; Lone F. Bovin, MSc, PhD; and Hans Skovgaard Poulsen, MD, DrMedSci
(CHEST 2004; 125:154S) Abbreviation: SCLC ⫽ small cell lung cancer
the purpose of identifying novel targets for the W ithdiagnosis, staging, and treatment of small cell lung
cancer (SCLC), a global gene expression analysis using oligonucleotide microarrays (Affymetrix; Santa Clara, CA) was performed on a large number of human SCLC cell lines and their xenografts. The expression was compared to the expression profiles of 18 normal human tissues. The analysis confirmed the high expression of a number of genes that previously had been identified as highly being expressed in SCLC, including neuroendocrine markers, oncogenes, and genes involved in cell proliferation and division. However, the analysis also identified a number of molecules that had not previously been associated with SCLC. Several of these are expressed in low or undetectable amounts in the majority of normal tissues and, therefore, are potential targets for new therapeutic approaches. By comparison with the published array profiles of five resected SCLC tumors,1 we found that most of these potential targets also were expressed in tumors and, therefore, were bona fide targets. Several of the novel identified targets are surface molecules, and therefore are potential targets for diagnostic and therapeutic approaches, such as positron emission tomography imaging, and for radiotherapy. Some of these surface molecules are internalizing receptors, and therefore are potential targets for radiotherapy, toxin therapy, and gene therapy. The expression of several of these surface molecules on a protein level has been verified by Western blotting, and the receptors are currently under investigation for functionality in ligand binding. Many other genes have been found with high and cancer-specific expression. Some of these may be directly involved in a malignant phenotype, such as oncogenes, and are therefore potential targets in themselves. Others with *From the Department of Radiation Biology (Drs. N. Pedersen, M.W. Pedersen, and H.S. Poulsen, Mr. T.T. Poulsen, and Mss. Mortensen and Sørensen), Institute for Inflammation Research (Drs. Rieneck and Bovin), National University Hospital, Copenhagen, Denmark. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Hans Skovgaard Poulsen, MD, DrMedSci, Chief Physician, Department of Radiation Biology, Finsen Center, Section 6321, National University Hospital, DK-Copenhagen 2100, Denmark; e-mail: skovgaard @rh.dk 154S
unknown functions may be able to confer high and cancer-specific gene expression, and are therefore candidates for use in gene therapy for the cancer-specific expression of a therapeutic gene. Gene expression analysis also was used for a hierarchical cluster analysis, which revealed that the cell lines grouped into four subclusters, two of which contained the classic SCLC cell line types and one of which contained the variant types. The distinction between these types has previously been made in only a few gene products, and the analysis therefore has identified many additional genes with differential expression between the two types of SCLC.
Reference 1 Bhattacharjee A, Richards WG, Staunton J, et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci U S A 2001; 98:13790 –13795
Evidence That Inflammation Encourages Pulmonary Adenocarcinoma Formation in Mice* Clinical Implications Alvin M. Malkinson, PhD
(CHEST 2004; 125:154S–155S) associations between inflammation and E pidemiologic lung cancer include an increased cancer risk in COPD
and asthma patients, familial clustering of pulmonary inflammatory diseases and lung cancer, and a decreased cancer incidence among long-term aspirin users. Experimental evidence that inflammation enhances the development of lung tumors in mice includes the following: chromosomal sites that determine susceptibilities to lung injury/ inflammation in response to environmental toxins also regulate lung tumor susceptibility; reduced or raised tumorigenesis in mice that are null for, or that overexpress, enzymes that synthesize inflammatory mediators and their receptors; decreased tumor formation following the administration of antiinflammatory drugs such as budesonide and indomethacin; biochemical markers of inflammation such as the elevated expression of enzymes catalyzing eicosanoid
*From the University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, CO. This research was supported by USPHS grants CA33497 and CA96133. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Alvin M. Malkinson, PhD, The University of Colorado Cancer Center, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Denver, CO 80262; e-mail: [email protected]
Thomas L. Petty 46th Annual Aspen Lung Conference; Lung Cancer: Early Events, Early Interventions
biosynthesis and nitric oxide formation within and adjacent to tumor sites; and the incursion of activated inflammatory cells into and peripheral to the tumor mass. This association between inflammation and neoplasia opens up new avenues for investigating the mechanisms of neoplastic progression, such as the shifting intercellular communication between mutated epithelial cells and inflammatory cells (ie, macrophages, lymphocytes, and mast cells), and determining which inflammatory mediators act at different stages of progression. Practically, this association affords the potential for exploring new biomarkers of early disease (eg, increased serum surfactant protein-D,1 increased the exhalation of nitric oxide by lung cancer patients2), using anti-inflammatory drugs to prevent the growth of early lesions and even to cause their regression, and to consider including such drugs in combination therapy with cytotoxic agents during more advanced disease stages. Even negative results in the mouse model (eg, our finding that celecoxib not only does not diminish the number of chemically induced tumors but actually enhances their rate of growth3) will be informative in the safe and effective design of prevention trials in high-risk groups and in understanding the interplay of different mediators. The eventual identification of the genes that influence inflammation, and hence tumorigenesis, the delineation of the proinflammatory and antiinflammatory mediators that participate in neoplastic conversion, and the detection of additional inflammatory markers in body fluids that may indicate incipient neoplasia will have practical consequences.
References 1 Zhang R, Pao W, Umphress SJ, et al. Serum levels of surfactant protein D are increased in mice with lung tumors. Cancer Res 2003; 63:5889 –5894 2 Liu CY, Wand CH, Chen TAC, et al. Increased level of exhaled nitric oxide and up-regulation of inducible nitric oxide synthase in patients with primary lung cancer. Br J Cancer 1998; 78:534 –541 3 Kisley L, Barrett BS, Dwyer-Nield LD, et al. Celecoxib reduces pulmonary inflammation but not lung tumorigenesis in mice. Carcinogenesis 2002; 23:1653–1660
Use of Standardized Reverse Transcription-Polymerase Chain Reaction and the Standardized Expression Measurement Center in Multiinstitutional Trials To Develop Meaningful Lung Cancer Classification Based on Molecular Genetic Criteria* James C. Willey, MD; Charles R. Knight, MS; Erin L. Crawford, MS; Dan E. Olson, MD, PhD, FCCP; Jeffrey Hammersly, MD; Youngsook Yoon, MD, FCCP; and Imran Sharief, MD
(CHEST 2004; 125:155S–156S) Abbreviations: PCR ⫽ polymerase chain reaction; SEM ⫽ Standardized Expression Measurement; StaRT ⫽ standardized reverse transcription
the Director’s Challenge Program of the NaT hrough tional Cancer Institute, we have joined with others in
an effort to establish a more meaningful classification of lung cancer based on molecular genetic criteria. Specifically, this program has funded our laboratory in developing and implementing the standardized reverse transcription (StaRT) polymerase chain reaction (PCR) method. StaRT-PCR allows numerical, standardized measurement of hundreds of genes to be made simultaneously. Using this method, data from multiple experiments and laboratories may be entered into the same databank, thereby facilitating multi-institutional trials. We have developed software that allows the rapid sorting of data by samples according to annotated criteria and the averaging of the gene expression values among the sorted group of samples. This will enable the establishment of gene expression values for important classes, and this, in turn, will allow the identification of diagnostic criteria. We have optimized the methods for the measurement of gene expression by StaRT-PCR in cytologic specimens obtained by transthoracic fine-needle aspiration or bronchoscopy. As an example, using these methods we have identified an index comprising the product of c-myc and E2F1 gene expression values divided by the p21 expression value that is 100% accurate for diagnosing lung cancer in cytologic specimens. This is an improvement over the
*From the Department of Medicine, Medical College of Ohio, Toledo, OH. Dr. Willey and Ms. Crawford have a significant interest in Gene Express, Inc, which produces and markets StaRT-PCR reagents. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: James C. Willey, MD, George Isaac Professor for Cancer Research, Rm 0012, Ruppert Health Bldg, Medical College of Ohio, 3000 Arlington Ave, Toledo, OH 43614; e-mail: [email protected] www.chestjournal.org
CHEST / 125 / 5 / MAY, 2004 SUPPLEMENT
155S
(CHEST 2004; 125:154S) Abbreviation: SCLC ⫽ small cell lung cancer
the purpose of identifying novel targets for the W ithdiagnosis, staging, and treatment of small cell lung
cancer (SCLC), a global gene expression analysis using oligonucleotide microarrays (Affymetrix; Santa Clara, CA) was performed on a large number of human SCLC cell lines and their xenografts. The expression was compared to the expression profiles of 18 normal human tissues. The analysis confirmed the high expression of a number of genes that previously had been identified as highly being expressed in SCLC, including neuroendocrine markers, oncogenes, and genes involved in cell proliferation and division. However, the analysis also identified a number of molecules that had not previously been associated with SCLC. Several of these are expressed in low or undetectable amounts in the majority of normal tissues and, therefore, are potential targets for new therapeutic approaches. By comparison with the published array profiles of five resected SCLC tumors,1 we found that most of these potential targets also were expressed in tumors and, therefore, were bona fide targets. Several of the novel identified targets are surface molecules, and therefore are potential targets for diagnostic and therapeutic approaches, such as positron emission tomography imaging, and for radiotherapy. Some of these surface molecules are internalizing receptors, and therefore are potential targets for radiotherapy, toxin therapy, and gene therapy. The expression of several of these surface molecules on a protein level has been verified by Western blotting, and the receptors are currently under investigation for functionality in ligand binding. Many other genes have been found with high and cancer-specific expression. Some of these may be directly involved in a malignant phenotype, such as oncogenes, and are therefore potential targets in themselves. Others with *From the Department of Radiation Biology (Drs. N. Pedersen, M.W. Pedersen, and H.S. Poulsen, Mr. T.T. Poulsen, and Mss. Mortensen and Sørensen), Institute for Inflammation Research (Drs. Rieneck and Bovin), National University Hospital, Copenhagen, Denmark. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Hans Skovgaard Poulsen, MD, DrMedSci, Chief Physician, Department of Radiation Biology, Finsen Center, Section 6321, National University Hospital, DK-Copenhagen 2100, Denmark; e-mail: skovgaard @rh.dk 154S
unknown functions may be able to confer high and cancer-specific gene expression, and are therefore candidates for use in gene therapy for the cancer-specific expression of a therapeutic gene. Gene expression analysis also was used for a hierarchical cluster analysis, which revealed that the cell lines grouped into four subclusters, two of which contained the classic SCLC cell line types and one of which contained the variant types. The distinction between these types has previously been made in only a few gene products, and the analysis therefore has identified many additional genes with differential expression between the two types of SCLC.
Reference 1 Bhattacharjee A, Richards WG, Staunton J, et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci U S A 2001; 98:13790 –13795
Evidence That Inflammation Encourages Pulmonary Adenocarcinoma Formation in Mice* Clinical Implications Alvin M. Malkinson, PhD
(CHEST 2004; 125:154S–155S) associations between inflammation and E pidemiologic lung cancer include an increased cancer risk in COPD
and asthma patients, familial clustering of pulmonary inflammatory diseases and lung cancer, and a decreased cancer incidence among long-term aspirin users. Experimental evidence that inflammation enhances the development of lung tumors in mice includes the following: chromosomal sites that determine susceptibilities to lung injury/ inflammation in response to environmental toxins also regulate lung tumor susceptibility; reduced or raised tumorigenesis in mice that are null for, or that overexpress, enzymes that synthesize inflammatory mediators and their receptors; decreased tumor formation following the administration of antiinflammatory drugs such as budesonide and indomethacin; biochemical markers of inflammation such as the elevated expression of enzymes catalyzing eicosanoid
*From the University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, CO. This research was supported by USPHS grants CA33497 and CA96133. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Alvin M. Malkinson, PhD, The University of Colorado Cancer Center, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Denver, CO 80262; e-mail: [email protected]
Thomas L. Petty 46th Annual Aspen Lung Conference; Lung Cancer: Early Events, Early Interventions
biosynthesis and nitric oxide formation within and adjacent to tumor sites; and the incursion of activated inflammatory cells into and peripheral to the tumor mass. This association between inflammation and neoplasia opens up new avenues for investigating the mechanisms of neoplastic progression, such as the shifting intercellular communication between mutated epithelial cells and inflammatory cells (ie, macrophages, lymphocytes, and mast cells), and determining which inflammatory mediators act at different stages of progression. Practically, this association affords the potential for exploring new biomarkers of early disease (eg, increased serum surfactant protein-D,1 increased the exhalation of nitric oxide by lung cancer patients2), using anti-inflammatory drugs to prevent the growth of early lesions and even to cause their regression, and to consider including such drugs in combination therapy with cytotoxic agents during more advanced disease stages. Even negative results in the mouse model (eg, our finding that celecoxib not only does not diminish the number of chemically induced tumors but actually enhances their rate of growth3) will be informative in the safe and effective design of prevention trials in high-risk groups and in understanding the interplay of different mediators. The eventual identification of the genes that influence inflammation, and hence tumorigenesis, the delineation of the proinflammatory and antiinflammatory mediators that participate in neoplastic conversion, and the detection of additional inflammatory markers in body fluids that may indicate incipient neoplasia will have practical consequences.
References 1 Zhang R, Pao W, Umphress SJ, et al. Serum levels of surfactant protein D are increased in mice with lung tumors. Cancer Res 2003; 63:5889 –5894 2 Liu CY, Wand CH, Chen TAC, et al. Increased level of exhaled nitric oxide and up-regulation of inducible nitric oxide synthase in patients with primary lung cancer. Br J Cancer 1998; 78:534 –541 3 Kisley L, Barrett BS, Dwyer-Nield LD, et al. Celecoxib reduces pulmonary inflammation but not lung tumorigenesis in mice. Carcinogenesis 2002; 23:1653–1660
Use of Standardized Reverse Transcription-Polymerase Chain Reaction and the Standardized Expression Measurement Center in Multiinstitutional Trials To Develop Meaningful Lung Cancer Classification Based on Molecular Genetic Criteria* James C. Willey, MD; Charles R. Knight, MS; Erin L. Crawford, MS; Dan E. Olson, MD, PhD, FCCP; Jeffrey Hammersly, MD; Youngsook Yoon, MD, FCCP; and Imran Sharief, MD
(CHEST 2004; 125:155S–156S) Abbreviations: PCR ⫽ polymerase chain reaction; SEM ⫽ Standardized Expression Measurement; StaRT ⫽ standardized reverse transcription
the Director’s Challenge Program of the NaT hrough tional Cancer Institute, we have joined with others in
an effort to establish a more meaningful classification of lung cancer based on molecular genetic criteria. Specifically, this program has funded our laboratory in developing and implementing the standardized reverse transcription (StaRT) polymerase chain reaction (PCR) method. StaRT-PCR allows numerical, standardized measurement of hundreds of genes to be made simultaneously. Using this method, data from multiple experiments and laboratories may be entered into the same databank, thereby facilitating multi-institutional trials. We have developed software that allows the rapid sorting of data by samples according to annotated criteria and the averaging of the gene expression values among the sorted group of samples. This will enable the establishment of gene expression values for important classes, and this, in turn, will allow the identification of diagnostic criteria. We have optimized the methods for the measurement of gene expression by StaRT-PCR in cytologic specimens obtained by transthoracic fine-needle aspiration or bronchoscopy. As an example, using these methods we have identified an index comprising the product of c-myc and E2F1 gene expression values divided by the p21 expression value that is 100% accurate for diagnosing lung cancer in cytologic specimens. This is an improvement over the
*From the Department of Medicine, Medical College of Ohio, Toledo, OH. Dr. Willey and Ms. Crawford have a significant interest in Gene Express, Inc, which produces and markets StaRT-PCR reagents. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: James C. Willey, MD, George Isaac Professor for Cancer Research, Rm 0012, Ruppert Health Bldg, Medical College of Ohio, 3000 Arlington Ave, Toledo, OH 43614; e-mail: [email protected] www.chestjournal.org
CHEST / 125 / 5 / MAY, 2004 SUPPLEMENT
155S