Lung cancer basics and current areas of research

1. Lung cancer basics and current areas of research Doug Arenberg Associate Professor of Medicine

2. In what context are we discussing this?

3. NIH research spending per life lost

4. Morpholgical classification of lung cancer Small cell lung cancer (15%) Non-small cell lung cancer (85%) Squamous cell carcinoma Adenocarcinoma Bronchoalveolar carcinoma Large cell carcinoma Rarer entities

5. Lung cancer is staged according to three parameters: tumour (T), lymph nodes (N) and metastatic involvement (M).1 For each of these, x indicates that the value cannot be assessed and 0 indicates that no evidence of the parameter is found. Additional stages are outlined below. Tumour stages: TIS: carcinoma in situ (confined to airway lining) T1-T4: ranging from tumour <3 cm in greatest dimension through to tumour of any size that invades the mediastinum, heart, great vessels, trachea, oesophagus, vertebral body or carina. Lymph node stages: N1: metastasis to ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodes involved by direct extension of primary tumour N2: metastasis to ipsilateral mediastinal and/or subcarinal lymph node(s) N3: metastasis to contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph node(s). Distant metastasis stages: M1: distant metastasis present. The schematic diagram shows examples of some tumour stages: Early stage 0 (carcinoma in situ): tumour is confined to the airway lining stage IA (T1 N0 M0): tumour has spread to nearby lung tissue but has not reached the main bronchus Localised stage IIB (T2 N1 M0/T3 N0 M0): tumour has reached main bronchus and local lymph nodes or direct local invasion into chest wall, diaphragm, mediastinal pleura or parietal pericardium Advanced stage IIIB (T4 Any N M0/Any T N3 M0): tumour has invaded chest wall, trachea and the contralateral lymph nodes stage IV (Any T Any N M1): distant metastasis present in the brain. Fewer than 30% of patients present with stage I or II disease.2 References Mountain CF. Chest 1997; 111: 1710-1717. Ihde DC. N Engl J Med 1992; 327: 1434-1441.Lung cancer is staged according to three parameters: tumour (T), lymph nodes (N) and metastatic involvement (M).1 For each of these, x indicates that the value cannot be assessed and 0 indicates that no evidence of the parameter is found. Additional stages are outlined below. Tumour stages: TIS: carcinoma in situ (confined to airway lining) T1-T4: ranging from tumour <3 cm in greatest dimension through to tumour of any size that invades the mediastinum, heart, great vessels, trachea, oesophagus, vertebral body or carina. Lymph node stages: N1: metastasis to ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodes involved by direct extension of primary tumour N2: metastasis to ipsilateral mediastinal and/or subcarinal lymph node(s) N3: metastasis to contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph node(s). Distant metastasis stages: M1: distant metastasis present. The schematic diagram shows examples of some tumour stages: Early stage 0 (carcinoma in situ): tumour is confined to the airway lining stage IA (T1 N0 M0): tumour has spread to nearby lung tissue but has not reached the main bronchus Localised stage IIB (T2 N1 M0/T3 N0 M0): tumour has reached main bronchus and local lymph nodes or direct local invasion into chest wall, diaphragm, mediastinal pleura or parietal pericardium Advanced stage IIIB (T4 Any N M0/Any T N3 M0): tumour has invaded chest wall, trachea and the contralateral lymph nodes stage IV (Any T Any N M1): distant metastasis present in the brain. Fewer than 30% of patients present with stage I or II disease.2 References Mountain CF. Chest 1997; 111: 1710-1717. Ihde DC. N Engl J Med 1992; 327: 1434-1441.

10. Oncogenes Usually proteins involved in growth factor signaling, promotion of cell growth Many are growth factor receptors or factors involved in their signalling Ras Activates cell proliferation in response to growth factors c-Kit/c-Kit ligand in SCLC May inactivate tumor suppressor proteins MDM2 binds to and antagonizes p53

11. Mechanism of activation of proto-oncogenes

12. Tumor suppressor genes Tumor suppressor genes (Understanding these as they relate to the cellular properties of the tumor is helpful) Genes that negatively regulate cell cycle progression Genes that activate apoptosis or programmed cell death

13. Tumor suppressor genes p53 What does it mean to “detect” p53 in a tumor? Defective, non-functional protein with prolonged half-life Controls both cell cycle and apoptotic machinery Induced by cellular “stress” such as DNA damage or hypoxia Via Rb, arrests cell cycle transition from G1 to S Promotes apoptosis via Bax/Bcl-2 balance leading to activation of caspases

14. Tumor suppressor genes Rb The “lynch-pin” of the cell cycle Binds to and blocks the activity of the E2F transcription factor Recruits chromatin remodeling machinery E2F promotes the transcription of genes that are required for DNA synthesis (S phase) RB “releases” E2F when it gets phosphorylated by a complex called CyclinD1-cdk4

21. Rb protein Also acts to recruit chromatin remodeling complexes Histone de-acetylases (De-acetylation returns histones to their native/active conformation) ATP-dependent chromatin remodeling enzymes (swi/snf in yeast, BRG-1/BRM in human cells) Many viral oncoproteins target and inactivate Rb SV40 large T antigen Adenoviral E1B HPV

24. Tumor cell recipe: Add lots of active Cyclin-CDK complexes.

25. Oncogene types:

26. Mechanisms of activation of cellular proto-oncogenes Point mutation Deletion Chromosomal translocation Gene amplification Overexpression of protein

27. Methods of oncogene activation Point mutation Ususlly generate an overactive protein Loss of regulatory sites Ligand independence Gene Amplification Many copies of a given gene Fusion proteins (translocation) Promoter mutations Chromosomal translocations Bcr-abl in leukemia, EML-Alk4 in lung cancer

28. Ras point mutation

29. Methods of gene “silencing” Homozygous deletion Absence of the gene Promoter methylation Inability to activate transcription of the gene Point mutation Non-functional protein

30. “Loss of heterozygosity” Loss of an allele at a chromosomal locus One hit of the “two-hit” theory Implies that the 2nd allele is mutated or epigenetically modified in a way that allows homozygous loss of a gene Usually prompts search for a tumor suppressor gene at that locus In lung cancer 3p12-23 LOH is a nearly universal event Several candidate TSGs have been reported in this region

31. EGFR: downstream signaling The epidermal growth factor receptor is overexpressed in 40-80% of NSCLC patients. It regulates cell proliferation, survival, angiogenesis, and tumor metastasisThe epidermal growth factor receptor is overexpressed in 40-80% of NSCLC patients. It regulates cell proliferation, survival, angiogenesis, and tumor metastasis

32. EGFR: downstream signaling The epidermal growth factor receptor is overexpressed in 40-80% of NSCLC patients. It regulates cell proliferation, survival, angiogenesis, and tumor metastasisThe epidermal growth factor receptor is overexpressed in 40-80% of NSCLC patients. It regulates cell proliferation, survival, angiogenesis, and tumor metastasis

33. Two mutually exclusive pathways to lung adenocarcinoma

34. Tumors as wounds:Is a cancer cell enough?

35. “Translational” “… to translate the basic science discoveries into clinical applications” “…use clinical observations to generate research foci for basic sciences”

36. Lung cancer research: Areas of opportunity Screening Will screening be effective? Will screening be cost effective? Inversely proportional to the prevalence of the disease Focus on the population at highest risk Studies which elucidate those individuals at greatest risk are of enormous value in determining future screening recommendations When we consider the role we as pulmonary specialists can occupy in the study, of and care for patients with, lung cancer we have to recognize that very few of us are actually oncologists, but as investigators and clinicians in a multidisciplinary context we have a very important role in advocating for screening (who, if anybody, should be screened, and how do we approach the patient with a positive finding on a screening test?) With all the focus on low-dose helical CT screening, we have to consider the potential cost effectiveness of such a strategy if it were ever put in place. At least decision analysis approaches (1-2) have been published top address this question, and while the conclusions differed between the two as to the cost effectiveness of screening (assuming it was effective at reducing lung cancer mortality) in both of them cost effectiveness was highly effected by the actual prevalence of caner in the screened population. Biomarkers of risk for, and the presence of, cancer will of necessity be a part of the future efforts at a) restricting screening to those at highest risk, and b) reducing the number of invasive procedures done on patients with benign disease. Mahadevia, P. J., Fleisher, L. A., Frick, K. D., Eng, J., Goodman, S. N., and Powe, N. R. Lung Cancer Screening With Helical Computed Tomography in Older Adult Smokers: A Decision and Cost-effectiveness Analysis. JAMA, 289: 313-322, 2003. Wisnivesky, J. P., Mushlin, A. I., Sicherman, N., and Henschke, C. The Cost-Effectiveness of Low-Dose CT Screening for Lung Cancer: Preliminary Results of Baseline Screening. Chest, 124: 614-621, 2003. When we consider the role we as pulmonary specialists can occupy in the study, of and care for patients with, lung cancer we have to recognize that very few of us are actually oncologists, but as investigators and clinicians in a multidisciplinary context we have a very important role in advocating for screening (who, if anybody, should be screened, and how do we approach the patient with a positive finding on a screening test?) With all the focus on low-dose helical CT screening, we have to consider the potential cost effectiveness of such a strategy if it were ever put in place. At least decision analysis approaches (1-2) have been published top address this question, and while the conclusions differed between the two as to the cost effectiveness of screening (assuming it was effective at reducing lung cancer mortality) in both of them cost effectiveness was highly effected by the actual prevalence of caner in the screened population. Biomarkers of risk for, and the presence of, cancer will of necessity be a part of the future efforts at a) restricting screening to those at highest risk, and b) reducing the number of invasive procedures done on patients with benign disease. Mahadevia, P. J., Fleisher, L. A., Frick, K. D., Eng, J., Goodman, S. N., and Powe, N. R. Lung Cancer Screening With Helical Computed Tomography in Older Adult Smokers: A Decision and Cost-effectiveness Analysis. JAMA, 289: 313-322, 2003. Wisnivesky, J. P., Mushlin, A. I., Sicherman, N., and Henschke, C. The Cost-Effectiveness of Low-Dose CT Screening for Lung Cancer: Preliminary Results of Baseline Screening. Chest, 124: 614-621, 2003.

37. Lung cancer Screening CXR & sputum too insensitive Changing spectrum of histology Low dose helical CT screening Likely will result in stage shift compared to an unscreened population Almost certainly will result in increased costs related to evaluation of benign lesions Unless additional biomarkers can be developed to focus testing on highest risk individuals When we consider the role we as pulmonary specialists can occupy in the study, of and care for patients with, lung cancer we have to recognize that (unless your last name is Jett) very few of us are actually oncologists, but as investigators and clinicians in a multidisciplinary context we have a very important role in advocating for screening (who, if anybody, should be screened, and how do we approach the patient with a positive finding on a screening test?) With all the focus on low-dose helical CT screening, we have to consider the potential cost effectiveness of such a strategy if it were ever put in place. At least 2 decision analysis approaches (1-2) have been published to address this question, and while the conclusions differed between the two as to the cost effectiveness of screening (assuming it was effective at reducing lung cancer mortality), in both of them cost effectiveness was highly effected by the actual prevalence of caner in the screened population. Biomarkers of risk for, and the presence of, cancer will of necessity be a part of the future efforts at a) restricting screening to those at highest risk, and b) reducing the number of invasive procedures done on patients with benign disease. Mahadevia, P. J., Fleisher, L. A., Frick, K. D., Eng, J., Goodman, S. N., and Powe, N. R. Lung Cancer Screening With Helical Computed Tomography in Older Adult Smokers: A Decision and Cost-effectiveness Analysis. JAMA, 289: 313-322, 2003. Wisnivesky, J. P., Mushlin, A. I., Sicherman, N., and Henschke, C. The Cost-Effectiveness of Low-Dose CT Screening for Lung Cancer: Preliminary Results of Baseline Screening. Chest, 124: 614-621, 2003. When we consider the role we as pulmonary specialists can occupy in the study, of and care for patients with, lung cancer we have to recognize that (unless your last name is Jett) very few of us are actually oncologists, but as investigators and clinicians in a multidisciplinary context we have a very important role in advocating for screening (who, if anybody, should be screened, and how do we approach the patient with a positive finding on a screening test?) With all the focus on low-dose helical CT screening, we have to consider the potential cost effectiveness of such a strategy if it were ever put in place. At least 2 decision analysis approaches (1-2) have been published to address this question, and while the conclusions differed between the two as to the cost effectiveness of screening (assuming it was effective at reducing lung cancer mortality), in both of them cost effectiveness was highly effected by the actual prevalence of caner in the screened population. Biomarkers of risk for, and the presence of, cancer will of necessity be a part of the future efforts at a) restricting screening to those at highest risk, and b) reducing the number of invasive procedures done on patients with benign disease. Mahadevia, P. J., Fleisher, L. A., Frick, K. D., Eng, J., Goodman, S. N., and Powe, N. R. Lung Cancer Screening With Helical Computed Tomography in Older Adult Smokers: A Decision and Cost-effectiveness Analysis. JAMA, 289: 313-322, 2003. Wisnivesky, J. P., Mushlin, A. I., Sicherman, N., and Henschke, C. The Cost-Effectiveness of Low-Dose CT Screening for Lung Cancer: Preliminary Results of Baseline Screening. Chest, 124: 614-621, 2003.

38. Lung cancer Chemoprevention? Primary prevention (Smoking cessation) Secondary prevention block the effects of carcinogen(s) Tertiary prevention Chemoprevention trials Must have surrogate endpoints (biomarkers) What markers and from what tissue? The complexity of the transformed cell means that there are many potential pathways to exploit that might or moght not be effective in reducing the incidence of lung cancer. Trials designed to assess chemoprevention are by necessity exremely effective, requiring large numbers if patients and long periods of follow up. Intermediate endpoints are needed to allow multiple trials to proceed with smaller numbers of patients. These endpoints ARE among the biomarkers we seek, and determing the ones which accurately reflect the The complexity of the transformed cell means that there are many potential pathways to exploit that might or moght not be effective in reducing the incidence of lung cancer. Trials designed to assess chemoprevention are by necessity exremely effective, requiring large numbers if patients and long periods of follow up. Intermediate endpoints are needed to allow multiple trials to proceed with smaller numbers of patients. These endpoints ARE among the biomarkers we seek, and determing the ones which accurately reflect the

39. Biomarkers:In a perfect world Surrogate markers for the presence of, or risk for, a disease In isolation as a diagnostic indicator (blood glucose, cholesterol) As an adjunctive test, should enhance the sensitivity and specificity of other modalities of detecting a disease Ideally come from known molecular mechanisms of pathogenesis Levels should be (?) proportional to risk Characteristics of biomarkersCharacteristics of biomarkers

40. The search for useful cancer biomarkers Mutated genes, polymorphisms, or epigenetic changes LOH Histologic dysplasia or proliferative markers DNA adducts Blood Oral mucosal epithelial cells Sputum Bronchial bushings or biopsies The range of possible biomarkers and sources of tissue are broad and each type may be complementary. It is highly likely that any effective biomarker will actually be used as a panel of markers to include history, blood tests, oral mucosal or sputum samples, with the more invasive teating reserved for individuals whose non-invasive testing indicated very high riskThe range of possible biomarkers and sources of tissue are broad and each type may be complementary. It is highly likely that any effective biomarker will actually be used as a panel of markers to include history, blood tests, oral mucosal or sputum samples, with the more invasive teating reserved for individuals whose non-invasive testing indicated very high risk

41. The search for useful cancer biomarkers Mutated genes or epigenetic changes Hypermethylation of the tumor suppressor p16 (Belinsky, PNAS, 95(20), 1998) LOH at 9p or 3p RAR expression Telomerase FHIT protein Histologic dysplasia, “angiogenic squamous dysplasia” Proliferative markers (Ki-67) Here is a list of pitential biomarkers that have been tested with varying degrees of predictive value Keith RL, et al, Links Angiogenic squamous dysplasia in bronchi of individuals at high risk for lung cancer.Clin Cancer Res. 2000 May;6(5):1616-25. Clin Cancer Res. 2000 May;6(5):1616-25. Kurie JM, Lotan R, Lee JJ, Lee JS, Morice RC, Liu DD, Xu XC, Khuri FR, Ro JY, Hittelman WN, Walsh GL, Roth JA, Minna JD, Hong WK.Related Articles, Links Treatment of former smokers with 9-cis-retinoic acid reverses loss of retinoic acid receptor-beta expression in the bronchial epithelium: results from a randomized placebo-controlled trial. J Natl Cancer Inst. 2003 Feb 5;95(3):206-14. Here is a list of pitential biomarkers that have been tested with varying degrees of predictive value Keith RL, et al, Links Angiogenic squamous dysplasia in bronchi of individuals at high risk for lung cancer.Clin Cancer Res. 2000 May;6(5):1616-25. Clin Cancer Res. 2000 May;6(5):1616-25. Kurie JM, Lotan R, Lee JJ, Lee JS, Morice RC, Liu DD, Xu XC, Khuri FR, Ro JY, Hittelman WN, Walsh GL, Roth JA, Minna JD, Hong WK.Related Articles, Links Treatment of former smokers with 9-cis-retinoic acid reverses loss of retinoic acid receptor-beta expression in the bronchial epithelium: results from a randomized placebo-controlled trial. J Natl Cancer Inst. 2003 Feb 5;95(3):206-14.

42. Figure 1. Odds Ratios for Recurrence among Case Patients as Compared with Controls, According to Methylated Gene and Site. Multivariate logistic-regression analysis was performed with the use of data for the four genes that had the largest univariate differences in distribution with regard to methylation: the adenomatous polyposis coli gene APC, the Ras association domain family 1 gene RASSF1A, the cyclin-dependent kinase inhibitor 2A gene p16, and the H-cadherin gene CDH13. The prognostic value of each gene was adjusted for stage (IA or IB), age, sex, race (white or black), histologic feature of the tumor (adenocarcinoma, squamous-cell, or other), and smoking status (current or former smoker or nonsmoker) and then graphed as a forest plot. Among single genes, the methylation of either p16 or CDH13 was associated with the most significant odds ratios of recurrence, regardless of the type of tissue. Methylation of either RASSF1A or APC in the primary tumor or in mediastinal lymph nodes was associated with an elevation in the odds of recurrence that was not significant. Patients with methylation of both p16 and CDH13 in both tumor and mediastinal lymph nodes had a significantly higher odds of lung-cancer recurrence than those without methylation of this pair of genes (P=0.03).Figure 1. Odds Ratios for Recurrence among Case Patients as Compared with Controls, According to Methylated Gene and Site. Multivariate logistic-regression analysis was performed with the use of data for the four genes that had the largest univariate differences in distribution with regard to methylation: the adenomatous polyposis coli gene APC, the Ras association domain family 1 gene RASSF1A, the cyclin-dependent kinase inhibitor 2A gene p16, and the H-cadherin gene CDH13. The prognostic value of each gene was adjusted for stage (IA or IB), age, sex, race (white or black), histologic feature of the tumor (adenocarcinoma, squamous-cell, or other), and smoking status (current or former smoker or nonsmoker) and then graphed as a forest plot. Among single genes, the methylation of either p16 or CDH13 was associated with the most significant odds ratios of recurrence, regardless of the type of tissue. Methylation of either RASSF1A or APC in the primary tumor or in mediastinal lymph nodes was associated with an elevation in the odds of recurrence that was not significant. Patients with methylation of both p16 and CDH13 in both tumor and mediastinal lymph nodes had a significantly higher odds of lung-cancer recurrence than those without methylation of this pair of genes (P=0.03).

43. Table 3. Multivariate Odds Ratios and 95% Confidence Intervals for the Estimated Risk of Recurrence among Case Patients as Compared with Controls, According to the Methylation Status of Four Genes.Table 3. Multivariate Odds Ratios and 95% Confidence Intervals for the Estimated Risk of Recurrence among Case Patients as Compared with Controls, According to the Methylation Status of Four Genes.

45. Approximate Cancer Stage at Diagnosis This graph shows the likely effect of widespread screening on various cancers, showing distribution of cancers at the time of diagnosis. Breast and prostate cancer (which are more widely screened for) are found predominantly in local stages. Colon cancer has more than 50% of cases with regional or distant spread at time of diagnosis, more like lung cancer (for which no proven screening exists)This graph shows the likely effect of widespread screening on various cancers, showing distribution of cancers at the time of diagnosis. Breast and prostate cancer (which are more widely screened for) are found predominantly in local stages. Colon cancer has more than 50% of cases with regional or distant spread at time of diagnosis, more like lung cancer (for which no proven screening exists)

46. Biomarkers for lung cancer

47. Markers of risk:Gene environment interactions

48. Stem cells Stem cell traits Self renewing Tissue specific stem cells are able to differentiate and re-constitute a damaged organ Relatively quiescent Drug resistant ATP-binding cassette (ABC) family of proteins

49. Stem cells and the tumor microenvironment Embryonic stem cells all have the same genetic material Destiny is determined by the cells’ interaction with their environment Microenvironment determines the destiny of a stem cell and which genes to activate & silence Microenvironment is an epigenetic control switch Mina Bissel showed in 1990 that Rous sarcoma transformed tumors were associated with high expression of the angiogenic chemokine KC (CXCL1), but when tumor cells were taken out and cultured in vitro the expression of CXCL1 mRNA was not detected.Mina Bissel showed in 1990 that Rous sarcoma transformed tumors were associated with high expression of the angiogenic chemokine KC (CXCL1), but when tumor cells were taken out and cultured in vitro the expression of CXCL1 mRNA was not detected.

50. Tumor stem cells and microenvironment Mintz and Illmenesse (1975) Teratocarcinoma cells, tumorigenic when injected into mice When injected into blastocysts, they developed normally into chimeric mice Immortalized mammary cells (Barcellos-Hoff 2000) or myoblasts (Morgan 2002) Form normal cells when injected into tissue Form tumors when injected into pre-irradiated tissue Mary Helen Barcellos-Hoff and Shraddha A. Ravani Irradiated Mammary Gland Stroma Promotes the Expression of Tumorigenic Potential by Unirradiated Epithelial Cells [CANCER RESEARCH 60, 1254–1260, March 1, 2000] Morgan JE, Gross JG, Pagel CN, Beauchamp JR, Fassati A, Thrasher AJ, Di Santo JP, Fisher IB, Shiwen X, Abraham DJ, Partridge TA.Related Articles, Links Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site. J Cell Biol. 2002 May 13;157(4):693-702. Epub 2002 May 13. Mary Helen Barcellos-Hoff and Shraddha A. Ravani Irradiated Mammary Gland Stroma Promotes the Expression of Tumorigenic Potential by Unirradiated Epithelial Cells [CANCER RESEARCH 60, 1254–1260, March 1, 2000] Morgan JE, Gross JG, Pagel CN, Beauchamp JR, Fassati A, Thrasher AJ, Di Santo JP, Fisher IB, Shiwen X, Abraham DJ, Partridge TA.Related Articles, Links Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site. J Cell Biol. 2002 May 13;157(4):693-702. Epub 2002 May 13.

51. Mouse models Allow us to study many aspects of tumor biology Carcinogenesis Molecular “road map” to malignant transformation Tumor stem cells Tumorigenesis Inflammation in tumor growth/metastasis Tumor microenvironment Testing novel therapies

52. Lung cancer & the pulmonologist Therapy We MUST understand the nature of the problem to be part of the solution Gene therapy can be used to target all apsect of tumor biology Transformation Angiogenesis, metastatic “cascade” Immunotherapy

53. Transformation vs tumorigenesis Transformation is a cellular phenomenon Genetic and biochemical changes that result in changes in the cells behavior Unrestricted growth Soft agar colony formation (anchorage independence) Invasiveness/motility

54. Transformation vs tumorigenesis Tumorigenesis is a tissue phenomenon The end result of the cellular changes of transformation Also requires cooperative interactions of other tissue constituents Tumor stroma formation Recruitment of host cells Immune modulation Angiogenesis