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Targeting lethal minimal residual disease in small cell lung cancer
Targeting Lethal Minimal Residual Disease in Small Cell Lung Cancer Jyoti D. Patel, Lee M. Krug, Christopher G. Azzoli, Jorge Gomez, Mark G. Kris, and Vincent A. Miller In the last three decades, treatment for small cell lung cancer has improved with advances in chemotherapy and radiotherapy. Almost all patients respond initially to standard chemotherapy, and some patients with limited stage disease are cured with the combination of chemotherapy and thoracic irradiation. Nonetheless, the majority of patients will experience lethal relapse from chemotherapy-resistant micrometastatic disease, and this has resulted in poor long-term survival for most patients. Addressing the problem of relapse requires unique approaches to eliminating minimal residual disease. This review will focus on the detection of minimal residual disease as well as strategies with which to treat it, including matrix metalloproteinase inhibitors, tyrosine kinase inhibitors, and vaccine therapy. Semin Oncol 30:79-85. Copyright 2003, Elsevier Science (USA). All rights reserved.
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ESPITE HIGH initial response rates, most patients with small cell lung cancer (SCLC) relapse soon after discontinuation of chemotherapy. Even though combination chemoradiotherapy can cure some patients with limited-stage disease, the majority of patients will experience lethal relapse from chemotherapy-resistant micrometastatic disease. Five-year survival for patients with extensive-stage disease remains only between 2% and 8%.1 The most studied approach to eradicating residual disease after initial therapy has focused on treatment with more chemotherapy. Although the initial response of SCLC to chemotherapy suggests that maintenance chemotherapy may be effective in prolonging remission, multiple trials have tested this concept using different regimens without success. Only two studies showed any survival benefit, but at the expense of increased toxicity.2 Investigators have also tested high-dose chemotherapy with stem cell support, but treatment-related mortality is excessive, and the advanced age and other smoking-related comorbidities of most patients with SCLC preclude high-dose therapy. The lack of data supporting these approaches has left us with our current standard of care, which is expectant observation after completion of initial chemotherapy or chemoradiation.3 Although maintaining response is an area of ongoing research, we are substantially limited by Seminars in Oncology, Vol 30, No 1 (February), 2003: pp 79-85
the lack of reproducible assays measuring residual disease. Immunohistochemical methods have been used to identify SCLC in the bone marrow. Persistent disease identified by this technique correlates with poorer survival.4 Over the past decade, new approaches, such as reverse transcriptasepolymerase chain reaction assays, have been studied as a method for detecting and quantitating minimal residual disease (MRD). The polymerase chain reaction method has been applied successfully for the detection of MRD in hematologic malignancies, largely because specific chromosomal translocations exist in certain leukemias and lymphomas, such as the t(9;22) in chronic myeloid leukemia, t(15;17) in acute promyelocytic leukemia, and t(14;18) in non-Hodgkin’s lymphoma.5 Thus, mRNA for the characteristic fusion proteins can be sought. However, solid tumors tend to have more heterogeneity and often multiple genomic abnormalities. The detection of circulating cancer cells has lagged behind the work in hematologic malignancies. The detection of MRD can be accomplished by reverse transcriptase-polymerase chain reaction amplification of tumor-specific or tissue-specific mRNA. Reverse transcriptase-polymerase chain reaction has the sensitivity to detect one tumor cell per 1-10 million cells. Multiple putative markers including cytokeratin 19, prepro-gastrin releasing peptide, and neuromedin B receptor have been correlated with extent of disease and recurrent disease in SCLC,6-8 but this technique has yet to prove its clinical applicability. Another method of detecting MRD being studied is aimed at detecting alterations in circulating DNA. Increased quantities of DNA have been found in the plasma of patients with different
From the Thoracic Oncology Service, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, Weill Medical College of Cornell University, New York, NY. Address reprint requests to Lee M. Krug, MD, Memorial SloanKettering Cancer Center, 1275 York Ave, New York, NY 10021. Copyright 2003, Elsevier Science (USA). All rights reserved. 0093-7754/03/3001-0008$30.00/0 doi:10.1053/sonc.2003.50018 79
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Table 1. Characteristics of Minimal Residual Disease Detection
Sensitivity Specificity Rapidity
Immunohistochemistry
RT-PCR for Tumor-Specific RNA
Detection of DNA Microsatellite Alterations
1 in 104-105 ⫹⫹ ⫹⫹⫹
1 in 105-106 ⫹ ⫹
1 in 104-105 ⫹ ⫹
cancers.9-11 The circulating tumor-related DNA shows decreased strand stability, hypermethylation of promoter regions, and microsatellite instability. Microsatellite DNA is composed of simple repeats of unknown function and is unstable in cancer cells.12 These changes in DNA may be used prognostically. Gonzalez et al13 studied the correlation of abnormal DNA with survival in 35 patients with SCLC. Three polymorphic markers (ACTBP2, UT762, and AR) as well as mutations of TP53 were analyzed in the serum and primary tumor. In 71% of patients, at least one molecular change precisely matched that of the primary tumor. In 60% of patients, treatment response correlated with the persistence or disappearance of abnormal plasma DNA. In one patient, the reappearance of plasma DNA alterations preceded a clinical recurrence by 12 weeks. The value of these molecular markers as possible indicators of MRD and relapse needs further study. Table 1 provides a comparison of these techniques. Until these molecular techniques become validated prognostic tools, all patients who achieve complete or partial response to initial therapy are considered to be at significant risk of relapse. The challenge we face is to maintain a durable remission. Since maintenance and high-dose chemotherapy have not afforded survival benefits, new approaches are needed. Targeted therapies such as matrix metalloproteinase inhibitors, tyrosine kinase inhibitors, and immunologic strategies each have the potential to fill this therapeutic void. MATRIX METALLOPROTEINASE INHIBITORS
Inhibitors of matrix metalloproteinases (MMPIs) have been extensively tested in SCLC after patients have achieved maximal response to cytotoxic chemotherapy. Matrix metalloproteinases (MMPs) are extracellular enzymes that degrade connective tissue and play an important role in
bone development, wound healing, and vascular remodeling. They have been shown to be important to cancer cell invasion and metastasis and angiogenesis. The more than 20 MMPs can be subdivided into three classes based on enzymatic activity. The collagenases degrade collagen, the stromelysins degrade proteoglycans, laminin, and fibronectin, and the gelatinases degrade denatured collagen (gelatin).14,15 Secretion of these enzymes is upregulated in cancer cells, allowing them to penetrate basement membranes, invade blood vessels, and colonize distant sites, all necessary steps in the development of metastases. These enzymes are ordinarily kept in check through natural tissue inhibitors of MMPs, also called TIMPs. It is felt that elevation of tumor and stromal MMPs is associated with increased incidence of nodal and distant metastases,16 as well as shorter survival.17,18 Given that SCLC has such a high initial relapse rate, this tumor type provides a good clinical model for MMP inhibition. Synthetic MMP inhibitors have been developed. These compounds are not cytotoxic agents but rather inhibit the events initiating metastases and angiogenesis. Marimastat was the first MMPI to be tested in lung cancer. Marimastat inhibits a number of different MMPs. The primary toxicities of marimastat are joint stiffness and arthralgias.14,15 Two phase III studies of marimastat in SCLC have been completed. In both, patients received at least four cycles of chemotherapy, with or without thoracic radiotherapy. Within 28 days of completion of initial therapy, patients who had achieved a complete or partial response were randomized to receive marimastat or placebo. The results of the National Cancer Institute of Canada-European Organization for the Research and Treatment of Cancer trial were reported at the American Society of Clinical Oncology meeting in 2001.19 Five hundred fifty-five patients were enrolled on this trial between January 1997 and April 2000. Myal-
MINIMAL RESIDUAL DISEASE IN SCLC
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Table 2. Matrix Metalloproteinase Inhibitors in Lung Cancer
Compound
Disease
Chemotherapy
Phase
Study or Sponsor
No. of Patients
Outcome
Marimastat Marimastat BA 12-9566 Prinomastat Prinomastat
SCLC SCLC SCLC NSCLC NSCLC
⬎ 4 cycles ⫹/⫺ RT ⬎ 4 cycles ⫹/⫺ RT ⬎ 4 cycles ⫹/⫺ RT Carboplatin ⫹ paclitaxel Cisplatin ⫹ gemcitabine
III III III III III
Shepherd et al19 British Biotech Bayer Smylie et al20 Bissett et al21
555 — — 686 362
No improvement in survival Not reported Closed after interim analysis No improvement in survival No improvement in survival
gias and arthralgias necessitated dose reduction or drug discontinuation in 20% and 23%, respectively, of the patients receiving marimastat. Median survival was 9.5 months with 20% 2-year survival in both groups. There was no difference between the marimastat and placebo groups. In addition, the presenters concluded that marimastat adversely affected the quality of life in these patients. A similar trial in hundreds of patients was completed in the United States, but results have not yet been published or presented. Another compound, BAY 12-9566, was being tested in a similar trial design, but this trial was closed early based on the results of an interim analysis that showed a shorter survival in patients receiving BAY 12-9566. The MMPIs have also shown a lack of efficacy in non–small cell lung cancer patients. A phase III trial of the MMPI prinomastat (AG3340) in combination with paclitaxel and carboplatin in 686 patients with non–small cell lung cancer was presented at the American Society of Clinical Oncology meeting in 2001.20 Significant numbers of patients experienced myalgias and arthralgias. More importantly, there were no differences in overall survival, 1-year survival, response rate, or progression-free survival between the two groups.20 A similar trial with the same drug, prinomastat, in combination with gemcitabine and cisplatin in 362 patients with non–small cell lung cancer was presented at the American Society of Clinical Oncology meeting in 2002.21 Forty percent of patients in the prinomastat arm experienced grade 2 or greater musculoskeletal effects. Median survival was 11.5 months in the prinomastat group and 10.8 months in the placebo group (P ⫽ .82). The presenters concluded that the addition of prinomastat did not enhance the effi-
cacy of this chemotherapy regimen. Trials of the various MMPIs are listed in Table 2. With this record of toxicity, degraded quality of life, reproducible ineffectiveness, and shortened survival, further trials of any MMPIs must be approached with extreme caution. Only investigations with the strongest preclinical rationale, safety, and excellent tolerability in well-conducted phase I studies and strict early stopping rules should be considered. TYROSINE KINASE INHIBITORS
Protein tyrosine kinases are key enzymes controlling cellular growth, invasion, proliferation, and apoptosis. Imatinib (STI571, Gleevec, Novartis Pharmaceuticals, Hanover, NJ) is a rationally designed inhibitor of tyrosine kinases in the platelet-derived growth factor family, including c-kit. Subsequent work has shown that in addition to its designed inhibition of platelet-derived growth factor signal transduction pathway, imatinib blocks the activity of bcr-abl tyrosine kinase created by the fusion protein formed from the translocation of chromosomes 9 and 22 (t(9;22)) that has impressive efficacy in the treatment of chronic myelogenous leukemia. Constitutively activated c-kit has been shown to play a role in the pathogenesis of gastrointestinal stromal tumors,22 another disease for which imatinib has proven efficacy. In gastrointestinal stromal tumors, it appears that the growth inhibition by imatinib correlates with the presence of an activating mutation of c-kit.23 Preclinical data supports the notion that c-kit activation plays a central role in SCLC. Some investigators have reported c-kit expression in 80% to 90% of SCLC samples,24 although others have found a much lower expression rate.25 c-kit
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activation can lead to enhanced proliferation and inhibition of apoptosis in SCLC cell lines.26 An experiment using a cell line that expresses the kit ligand SCF showed that transfection of c-kit expression vector increased cell growth.26 In addition, the growth of an SCLC cell line that expresses SCF and c-kit was markedly decreased when defective c-kit was transfected. These data support the notion that SCF and c-kit are important factors in autocrine and paracrine growth stimulation in SCLC. Imatinib has shown growth inhibition of multiple SCLC cell lines in vitro.27,28 Growth inhibition correlates with c-kit expression and blockade of c-kit phosphorylation. A phase II trial of imatinib in chemotherapynaive patients or in patients with chemotherapysensitive relapse with SCLC was reported at the American Society of Clinical Oncology meeting in 2002.25 Nineteen patients were treated on this study, and treatment was generally well tolerated. There were no responses. One caveat to interpreting these results is that, of the 14 tumor blocks that were available for CD117 (c-kit) immunohistochemical staining, only four stained positive for the presence of c-kit. These phase II results suggest that other growth factors and survival signals drive tumor cell growth in SCLC. Imatinib also could have this effect when combined with cytotoxic chemotherapy or irradiation in SCLC. Because the single-agent toxicity of imatinib is mild and not obviously overlapping with chemotherapy, it could theoretically increase overall cell kill by inhibiting tumor cell regrowth between chemotherapy cycles. More importantly, one could continue imatinib after the completion of chemotherapy after maximal response to slow the regrowth of any MRD and thus, improve overall survival. Further testing in SCLC trials should require confirmation of c-kit expression and should explore c-kit– expressing tumors for the presence of an activating mutation. At Memorial Sloan-Kettering Cancer Center, we are currently testing the combination of cisplatin, irinotecan, and imatinib in a phase I trial in patients with extensive-stage SCLC. Patients will continue imatinib monotherapy for 1 year or until disease progression. Another trial, led by the Medical College of Virginia, combines imatinib with etoposide and cisplatin.
PATEL ET AL
Table 3. Cancer Cell Surface Targets for Small Cell Lung Cancer Vaccine Construction Antigens GD2 GD3 GM2 Fucosyl GM1
Polysialic Acid Globo H Sialyl Lea KSA Bombesin
VACCINES
Another approach to eradication of MRD in SCLC capitalizes on advances in our knowledge of tumor biology and immunology. Vaccination therapy has been studied in a variety of tumors as a way to eliminate persistent disease after maximal response has been achieved from standard therapy. For SCLC, the ideal time for immune intervention is after completion of definitive chemoradiotherapy, when tumor burden has been reduced to the maximum extent possible with current therapies. If immune effectors can be induced against tumor antigens, we would at last have a therapy targeted to micrometastatic disease. Tumor cells overexpress a variety of antigens that are not found on normal cells. Some of these represent altered molecules that have undergone subtle biochemical changes that distinguish them from the native antigens. Others are proteins that are involved in, or are markers of, cellular differentiation. Small cell lung cancer offers a unique opportunity to study vaccine therapy because the differentiation antigens expressed on these cells are relatively tumor-specific and have restricted expression. Tumor-associated antigens that are thought to play a role in SCLC, and which are potential targets for vaccine therapy, are listed in Table 3. These targets include the gangliosides GM2, GD2, GD3, bombesin, and Fucosyl-GM1, as well as the polysialic acid epitope characteristic of the embryonic neural-cell adhesion molecule, the natural glycolipid Globo H, and the glycoprotein KSA.29-36 GD3 is a ganglioside that is present in both melanoma and SCLC cells. It is poorly immunogenic by itself. The development of an immune strategy to effectively induce an immune response against self-antigens represents one of the great
MINIMAL RESIDUAL DISEASE IN SCLC
challenges in the immunotherapy of cancer. One method to overcome self-tolerance for this antigen is through an anti-idiotype approach. Here, the critical antigen is presented in a different way to enhance its antigenic potential. The mouse monoclonal antibody, R24, is specific for GD3 and when used as an immunogen, R24 generates a series of antibodies that bind to R24 antigen combining sites. These anti-idiotypic antibodies effectively mimic the three-dimensional structures and functions of GD3 and can be used as surrogate antigens for active specific immunotherapy.37 The monoclonal antibody BEC2 is an antibody directed against the antigen binding site of R24. McCaffrey et al showed that vaccination with BEC2, when given with the immunologic adjuvant BCG (Bacillus Calmette-Gue´ rin), was found to induce anti-GD3 antibodies in three of 14 highrisk melanoma patients.38 Because GD3 is expressed on SCLC cells, a pilot study was conducted in which BEC2 plus BCG was used to immunize 15 patients with SCLC who had achieved a major response to chemotherapy or chemoradiotherapy.39 Patients received a series of five intradermal immunizations of BEC2 plus BCG over a 10-week period. Median overall survival was 21 months. Median time to relapse was 11 months in patients with extensive-stage disease and was not reached at 47 months for patients with limited-stage disease. These results were significantly better than historical controls. The only significant toxicity was local skin reaction at immunization sites. Although GD3 is also expressed on some normal tissues, there was no evidence of any toxicity caused by auto-immunity. Because of these results, the European Organization for Research and Treatment of Cancer has initiated a phase III, prospective, randomized trial, SILVA (Survival in an International Phase III Prospective Randomized LD Small Cell Lung Cancer Vaccination Study with Adjuvant BEC2 and BCG). Patients with limited-stage SCLC are treated in standard fashion with chemotherapy and chest radiation. Prophylactic cranial irradiation is also recommended. Patients with a partial or complete responses are eligible, and are randomized to receive BEC2 plus BCG vaccination versus observation. Thus far, over 400 of the planned 500 patients have been accrued. This trial is scheduled to complete accrual in the Fall of 2002.
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Other gangliosides are also being investigated. Fucosyl-GM1 was initially isolated from the bovine thyroid gland40 With the use of a highly specific mouse monoclonal antibody, F12, the ganglioside Fuc-GM1 was identified in the majority of SCLC tissue samples and in the serum of some patients.30,31,41,42 Fuc-GM1 has a more restricted distribution on normal tissues than some other gangliosides, including GD3. Serum antibodies against Fuc-GM1 have also been described in the serum of a few patients with sensory neuropathies but not in other settings, suggesting that the molecule itself is poorly immunogenic.43 However, conjugation with KLH (keyhole limpet hemocyanin), a shellfish-derived protein, followed by a mixture with the immunoadjuvant QS-21, augments its immunogenicity greatly. A phase I/II study using Fucosyl-GM1–KLH conjugate vaccine was conducted in 13 patients with SCLC who had a documented major response to chemotherapy.44 Ten patients completed five vaccinations and were evaluable for immunologic response. All 10 patients mounted both IgM and IgG titers of ⱖ 1:40 against Fucosyl-GM1 as measured by enzyme-linked immunosorbent assay. Mild transient erythema and induration at injection sites were the only consistent toxicities. Three patients with SCLC were free of relapse at 18, 24, and 30 months at the time of manuscript submission. A synthetic version of Fucosyl-GM1 is now available, and a study has completed enrollment with this vaccine.45 This construct allows easier vaccine production and reduces the theoretical risk of prion transmission from bovine tissues. Because tumor cells express multiple antigens and there is heterogeneity in human immune response, a “polyvalent” vaccine containing multiple antigens (GM2, Globo H, polysialic acid, and Fucosyl GM1) may provide an even better approach to attack MRD. A polyvalent vaccine eliminates escape by tumor cells that fail to express any one antigen, and also increases the number of antibodies reacting against each tumor cell. We plan to test this concept with a vaccine combining GM2, Globo H, polysialic acid, and synthetic fucosyl GM1 and QS21 adjuvant in SCLC patients who have completed chemotherapy and chemoradiotherapy.
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SUMMARY
Because the majority of patients with SCLC relapse from persistent but clinically undetectable micrometastatic disease, we continue to evaluate therapies and strategies to attack these resistant tumor cells. Improvements in our ability to quantify residual disease will help us better monitor our efforts and identify patients most in need of additional therapy. Targeted therapies may help us halt regrowth of micrometastatic disease without substantially increasing toxicity. The incorporation of these new approaches and improvements in our knowledge of the molecular mechanism that underlie the growth of SCLC will ultimately improve survival in our patients with this disease. REFERENCES 1. Ihde DC: Chemotherapy of lung cancer. N Engl J Med 327:1434-1441, 1992 2. Sandler AB: Current management of small cell lung cancer. Semin Oncol 24:463-476, 1997 3. Armitage JO: Bone marrow transplantation. N Engl J Med 330:827-838, 1994 4. Pelosi G, Pasini F, Pavenel F, et al: Effects of different immunolabeling techniques on the detection of small-cell lung cancer cells in bone marrow. J Histochem Cytochem 47:10751087, 1999 5. Zippelius A, Pantel K: RT-PCR-cased detection of occult disseminated tumor cells in peripheral blood and bone marrow of patients with solid tumors. An overview. Ann N Y Acad Sci 906:110-123, 2000 6. Peck K, Sher Y, Shih J, et al: Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients. Cancer Res 58:2761-2765, 1998 7. Lacroix J, Becker HD, Woerner SM, et al: Sensitive detection of rare cancer cells in sputum and peripheral blood samples of patients with lung cancer by preprogrp-specific RT PCR. Int J Cancer 92:1-8, 2001 8. Bessho A, Tabata M, Kiura K, et al: Detection of occult tumor cells in peripheral blood from patients with small cell lung cancer by reverse transcriptase polymerase chain reaction. Anticancer Res 20:1149-1154, 2000 9. Leon S, Shapiro B, Sklaroff D, et al: Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 37:646-650, 1977 10. Stroun M, Anker P, Lyautey J, et al: Isolation and characterization of DNA from the plasma of cancer patients. Eur J Cancer Clin Oncol 23:707-712, 1989 11. Anker P, Mulcahy H, Chen X, et al: Detection of circulating tumour DNA in blood (plasma/serum) of cancer patients. Cancer Metastasis Rev 18:65-73, 1999 12. Anker P, Stroun M: Tumor-related alterations in circulation DNA, potential for diagnosis, prognosis and detection of minimal residual disease. Leukemia 15:289-291, 2001 13. Gonzalez R, Silva J, Sanchez A, et al: Microsatellite alterations and TP53 mutations in plasma DNA of small cell
lung cancer patients: Follow up study and prognostic significance. Ann Oncol 11:1097-1104, 2000 14. Hunt JD, Robert EG, Zieske AW, et al: Orthotopic human lung carcinoma xenografts in BALB/c mice immunosuppressed with anti-CD4 monoclonal antibodies and chronic alcohol consumption. Cancer 88:468-479, 2000 15. Hidalgo M, Siu L, Nemaunaitis J, et al: Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 19:3267-3279, 2001 16. Nawrocki B, Polette M, Marchand V, et al: Expression of matrix metalloproteinases and their inhibitors in human bronchopulmonary carcinomas: Quantificative and morphologic analyses. Int J Cancer 72:556-564, 1997 17. Michael M, Babic B, Khokha R, et al: Expression and prognostic significance of metalloproteinases and their tissue inhibitors in patients with small-cell lung cancer. J Clin Oncol 17:1802-1808, 1999 18. Ylisirnio S, Hoyhtya M, Turpeenniemi-Hujanen T: Serum matrix metalloproteinases -2, -9 and tissue inhibitors of metalloproteinases -1, -2 in lung cancer–TIMP-1 as a prognostic marker. Anticancer Res 20:1311-1316, 2000 19. Shepherd F, Giaccone G, Debruyne C: Randomized double-blind placebo-controlled trial of marimastat in patients with small cell lung cancer (SCLC) following response to first-line chemotherapy: An NCIC and EORTC study. Proceedings of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001 (abstr 1) 20. Smylie M, Mercier R, Aboulafia D, et al: Phase III study of the matrix metalloproteinase inhibitor prinomastat in patients having advanced non–small cell lung cancer. Proceedings of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001 (abstr 1226) 21. Bissett D, Pawel JV, Mercier R, et al: Phase III study of the matrix metalloproteinase inhibitor prinomastat in combination with gemcitabine and cisplatin in non–small cell lung cancer. Proceedings of the American Society of Clinical Oncology. Orlando, FL, May 17-21, 2002 (abstr 1183) 22. Hirota S, Isozaki K, Moriyama Y, et al: Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279:577-580, 1998 23. Heinrich M, Corless C, Blanke C, et al: KIT mutational status predicts clinical response to STI571 in patients with metastatic gastrointestinal stromal tumors (GISTs). Proceedings of the American Society of Clinical Oncology, Orlando, FL, May 17-21, 2002 (abstr 6) 24. Sekido Y, Obata Y, Ueda R, et al: Preferential expression of c-kit protooncogene transcripts in small cell lung cancer. Cancer Res 51:2416-2419, 1999 25. Johnson B, Fisher B, Fisher T, et al: A phase II study of STI571 (Gleevec) for patients with small cell lung cancer. Proceedings of the American Society of Clinical Oncology, Orlando, FL, May 17-21, 2002 (abstr 1171) 26. Krystal GW, Hines SJ, Organ CP: Autocrine growth of small cell lung cancer mediated by coexpression of c-kit and stem cell factor. Cancer Res 56:370-376, 1996 27. Wang W, Healy M, Sattler M, et al: Growth inhibition and modulation of kinase pathways of small cell lung cancer cell lines by the novel tyrosine kinase inhibitor STI571. Oncogene 19:3521-3528, 2000 28. Krystal GW, Honsawek S, Litz J, et al: The selective
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tyrosine kinase inhibitor STI571 inhibits small cell lung cancer growth. Clin Cancer Res 6:3319-3326, 2000 29. Hamilton WB, Helling F, Livingston PO: Ganglioside expression on sarcoma and small cell lung carcinoma compared to tumors of neuroectodermal origin. Proc Am Assoc Cancer Res 34:491, 1993 (abstr) 30. Zhang S, Cordon-Cardo C, Zhang H, et al: Selection of tumor antigens as targets for immune attack using immunuhistochemistry: I. Focus on gangliosides. Int J Cancer 73:42-49, 1997 31. Brezicka F-T, Olling S, Nilsson O, et al: Immunohistological detection of Fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies. Cancer Res 49:1300-1305, 1989 32. Fuentes R, Allman R, Mason MD: Ganglioside expression in lung cancer cell lines. Lung Cancer 18:21-33, 1997 33. Brezicka FT, Olling S, Bergman B, et al: Coexpression of ganglioside antigen Fuc-GM1, neural-cell adhesion molecule, carcinoembryonic antigen, and carbohydrate tumor-associated antigen CA 50 in lung cancer. Tumor Biol 13:308-315, 1992 34. Cheresh DA, Pierschbacher MD, Herzig MA, et al: Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins. J Cell Biol 102:688-696, 1986 35. Grant SC, Kostakoglu L, Kris MG, et al: Targeting of small-cell lung cancer using the anti-GD2 ganglioside monoclonal antibody 3f8: A pilot trial. Eur J Nucl Med 23:145-149, 1996 36. Zhang S, Zhang HS, Cordon-Cardo C, et al: Selection of tumor antigens for immune attack using immunohistochemistry: III. Protein antigens. Clin Cancer Res 4:2669-2676, 1998 37. Bhattacharya-Chatterjee M, Chatterjee S, Foon K: The
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anti-idiotypic vaccines for immunotherapy. Curr Opin Molec Ther 3:63-69, 2001 38. McCaffery M, Yao T-J, Williams L, et al: Immunization of melanoma patients with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: Enhanced immunogenicity when combined with adjuvant. Clin Cancer Res 2:679686, 1996 39. Grant SC, Kris MG, Houghton AN, et al: Long survival of patients with small cell lung cancer after adjuvant treatment with the anti-idiotypic antibody BEC2 plus Bacillus CalmetteGuerin. Clin Cancer Res 5:1319-1323, 1999 40. Macher BA, Pacuzska T, Mullin BR, et al: Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland. Biochim Biophys Acta 588:35-43, 1979 41. Fredman P, Brezicka T, Holmgren J, et al: Binding specificity of monoclonal antibodies to ganglioside, Fuc-GM1. Biochim Biophys Acta 875:316-323, 1986 42. Vangsted AJ, Clausen H, Kjeldsen TB, et al: Immunochemical detection of a small cell lung cancer-associated ganglioside (FucGM1) antigen in serum. Cancer Res 51:28792884, 1991 43. Yoshino H, Ariga T, Latov N, et al: Fucosyl-GM1 in human sensory nervous tissue is a target antigen in patients with autoimmune neuropathies. J Neurochem 61:658-663, 1993 44. Dickler MN, Ragupathi G, Liu NX, et al: Immunogenicity of a Fucosyl-GM1-keyhole limpet hemocyanin conjugate vaccine in patients with small cell lung cancer. Clin Cancer Res 5:2773-2779, 1999 45. Krug LM, Ragupathi G, Livingston P, et al: Pilot trial of a synthetic fucosyl-GM1 ganglioside vaccine in patients with small cell lung cancer. Proceedings of the American Society of Clinical Oncology, Orlando, FL, May 17-21, 2002 (abstr 1824)
D
ESPITE HIGH initial response rates, most patients with small cell lung cancer (SCLC) relapse soon after discontinuation of chemotherapy. Even though combination chemoradiotherapy can cure some patients with limited-stage disease, the majority of patients will experience lethal relapse from chemotherapy-resistant micrometastatic disease. Five-year survival for patients with extensive-stage disease remains only between 2% and 8%.1 The most studied approach to eradicating residual disease after initial therapy has focused on treatment with more chemotherapy. Although the initial response of SCLC to chemotherapy suggests that maintenance chemotherapy may be effective in prolonging remission, multiple trials have tested this concept using different regimens without success. Only two studies showed any survival benefit, but at the expense of increased toxicity.2 Investigators have also tested high-dose chemotherapy with stem cell support, but treatment-related mortality is excessive, and the advanced age and other smoking-related comorbidities of most patients with SCLC preclude high-dose therapy. The lack of data supporting these approaches has left us with our current standard of care, which is expectant observation after completion of initial chemotherapy or chemoradiation.3 Although maintaining response is an area of ongoing research, we are substantially limited by Seminars in Oncology, Vol 30, No 1 (February), 2003: pp 79-85
the lack of reproducible assays measuring residual disease. Immunohistochemical methods have been used to identify SCLC in the bone marrow. Persistent disease identified by this technique correlates with poorer survival.4 Over the past decade, new approaches, such as reverse transcriptasepolymerase chain reaction assays, have been studied as a method for detecting and quantitating minimal residual disease (MRD). The polymerase chain reaction method has been applied successfully for the detection of MRD in hematologic malignancies, largely because specific chromosomal translocations exist in certain leukemias and lymphomas, such as the t(9;22) in chronic myeloid leukemia, t(15;17) in acute promyelocytic leukemia, and t(14;18) in non-Hodgkin’s lymphoma.5 Thus, mRNA for the characteristic fusion proteins can be sought. However, solid tumors tend to have more heterogeneity and often multiple genomic abnormalities. The detection of circulating cancer cells has lagged behind the work in hematologic malignancies. The detection of MRD can be accomplished by reverse transcriptase-polymerase chain reaction amplification of tumor-specific or tissue-specific mRNA. Reverse transcriptase-polymerase chain reaction has the sensitivity to detect one tumor cell per 1-10 million cells. Multiple putative markers including cytokeratin 19, prepro-gastrin releasing peptide, and neuromedin B receptor have been correlated with extent of disease and recurrent disease in SCLC,6-8 but this technique has yet to prove its clinical applicability. Another method of detecting MRD being studied is aimed at detecting alterations in circulating DNA. Increased quantities of DNA have been found in the plasma of patients with different
From the Thoracic Oncology Service, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, Weill Medical College of Cornell University, New York, NY. Address reprint requests to Lee M. Krug, MD, Memorial SloanKettering Cancer Center, 1275 York Ave, New York, NY 10021. Copyright 2003, Elsevier Science (USA). All rights reserved. 0093-7754/03/3001-0008$30.00/0 doi:10.1053/sonc.2003.50018 79
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Table 1. Characteristics of Minimal Residual Disease Detection
Sensitivity Specificity Rapidity
Immunohistochemistry
RT-PCR for Tumor-Specific RNA
Detection of DNA Microsatellite Alterations
1 in 104-105 ⫹⫹ ⫹⫹⫹
1 in 105-106 ⫹ ⫹
1 in 104-105 ⫹ ⫹
cancers.9-11 The circulating tumor-related DNA shows decreased strand stability, hypermethylation of promoter regions, and microsatellite instability. Microsatellite DNA is composed of simple repeats of unknown function and is unstable in cancer cells.12 These changes in DNA may be used prognostically. Gonzalez et al13 studied the correlation of abnormal DNA with survival in 35 patients with SCLC. Three polymorphic markers (ACTBP2, UT762, and AR) as well as mutations of TP53 were analyzed in the serum and primary tumor. In 71% of patients, at least one molecular change precisely matched that of the primary tumor. In 60% of patients, treatment response correlated with the persistence or disappearance of abnormal plasma DNA. In one patient, the reappearance of plasma DNA alterations preceded a clinical recurrence by 12 weeks. The value of these molecular markers as possible indicators of MRD and relapse needs further study. Table 1 provides a comparison of these techniques. Until these molecular techniques become validated prognostic tools, all patients who achieve complete or partial response to initial therapy are considered to be at significant risk of relapse. The challenge we face is to maintain a durable remission. Since maintenance and high-dose chemotherapy have not afforded survival benefits, new approaches are needed. Targeted therapies such as matrix metalloproteinase inhibitors, tyrosine kinase inhibitors, and immunologic strategies each have the potential to fill this therapeutic void. MATRIX METALLOPROTEINASE INHIBITORS
Inhibitors of matrix metalloproteinases (MMPIs) have been extensively tested in SCLC after patients have achieved maximal response to cytotoxic chemotherapy. Matrix metalloproteinases (MMPs) are extracellular enzymes that degrade connective tissue and play an important role in
bone development, wound healing, and vascular remodeling. They have been shown to be important to cancer cell invasion and metastasis and angiogenesis. The more than 20 MMPs can be subdivided into three classes based on enzymatic activity. The collagenases degrade collagen, the stromelysins degrade proteoglycans, laminin, and fibronectin, and the gelatinases degrade denatured collagen (gelatin).14,15 Secretion of these enzymes is upregulated in cancer cells, allowing them to penetrate basement membranes, invade blood vessels, and colonize distant sites, all necessary steps in the development of metastases. These enzymes are ordinarily kept in check through natural tissue inhibitors of MMPs, also called TIMPs. It is felt that elevation of tumor and stromal MMPs is associated with increased incidence of nodal and distant metastases,16 as well as shorter survival.17,18 Given that SCLC has such a high initial relapse rate, this tumor type provides a good clinical model for MMP inhibition. Synthetic MMP inhibitors have been developed. These compounds are not cytotoxic agents but rather inhibit the events initiating metastases and angiogenesis. Marimastat was the first MMPI to be tested in lung cancer. Marimastat inhibits a number of different MMPs. The primary toxicities of marimastat are joint stiffness and arthralgias.14,15 Two phase III studies of marimastat in SCLC have been completed. In both, patients received at least four cycles of chemotherapy, with or without thoracic radiotherapy. Within 28 days of completion of initial therapy, patients who had achieved a complete or partial response were randomized to receive marimastat or placebo. The results of the National Cancer Institute of Canada-European Organization for the Research and Treatment of Cancer trial were reported at the American Society of Clinical Oncology meeting in 2001.19 Five hundred fifty-five patients were enrolled on this trial between January 1997 and April 2000. Myal-
MINIMAL RESIDUAL DISEASE IN SCLC
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Table 2. Matrix Metalloproteinase Inhibitors in Lung Cancer
Compound
Disease
Chemotherapy
Phase
Study or Sponsor
No. of Patients
Outcome
Marimastat Marimastat BA 12-9566 Prinomastat Prinomastat
SCLC SCLC SCLC NSCLC NSCLC
⬎ 4 cycles ⫹/⫺ RT ⬎ 4 cycles ⫹/⫺ RT ⬎ 4 cycles ⫹/⫺ RT Carboplatin ⫹ paclitaxel Cisplatin ⫹ gemcitabine
III III III III III
Shepherd et al19 British Biotech Bayer Smylie et al20 Bissett et al21
555 — — 686 362
No improvement in survival Not reported Closed after interim analysis No improvement in survival No improvement in survival
gias and arthralgias necessitated dose reduction or drug discontinuation in 20% and 23%, respectively, of the patients receiving marimastat. Median survival was 9.5 months with 20% 2-year survival in both groups. There was no difference between the marimastat and placebo groups. In addition, the presenters concluded that marimastat adversely affected the quality of life in these patients. A similar trial in hundreds of patients was completed in the United States, but results have not yet been published or presented. Another compound, BAY 12-9566, was being tested in a similar trial design, but this trial was closed early based on the results of an interim analysis that showed a shorter survival in patients receiving BAY 12-9566. The MMPIs have also shown a lack of efficacy in non–small cell lung cancer patients. A phase III trial of the MMPI prinomastat (AG3340) in combination with paclitaxel and carboplatin in 686 patients with non–small cell lung cancer was presented at the American Society of Clinical Oncology meeting in 2001.20 Significant numbers of patients experienced myalgias and arthralgias. More importantly, there were no differences in overall survival, 1-year survival, response rate, or progression-free survival between the two groups.20 A similar trial with the same drug, prinomastat, in combination with gemcitabine and cisplatin in 362 patients with non–small cell lung cancer was presented at the American Society of Clinical Oncology meeting in 2002.21 Forty percent of patients in the prinomastat arm experienced grade 2 or greater musculoskeletal effects. Median survival was 11.5 months in the prinomastat group and 10.8 months in the placebo group (P ⫽ .82). The presenters concluded that the addition of prinomastat did not enhance the effi-
cacy of this chemotherapy regimen. Trials of the various MMPIs are listed in Table 2. With this record of toxicity, degraded quality of life, reproducible ineffectiveness, and shortened survival, further trials of any MMPIs must be approached with extreme caution. Only investigations with the strongest preclinical rationale, safety, and excellent tolerability in well-conducted phase I studies and strict early stopping rules should be considered. TYROSINE KINASE INHIBITORS
Protein tyrosine kinases are key enzymes controlling cellular growth, invasion, proliferation, and apoptosis. Imatinib (STI571, Gleevec, Novartis Pharmaceuticals, Hanover, NJ) is a rationally designed inhibitor of tyrosine kinases in the platelet-derived growth factor family, including c-kit. Subsequent work has shown that in addition to its designed inhibition of platelet-derived growth factor signal transduction pathway, imatinib blocks the activity of bcr-abl tyrosine kinase created by the fusion protein formed from the translocation of chromosomes 9 and 22 (t(9;22)) that has impressive efficacy in the treatment of chronic myelogenous leukemia. Constitutively activated c-kit has been shown to play a role in the pathogenesis of gastrointestinal stromal tumors,22 another disease for which imatinib has proven efficacy. In gastrointestinal stromal tumors, it appears that the growth inhibition by imatinib correlates with the presence of an activating mutation of c-kit.23 Preclinical data supports the notion that c-kit activation plays a central role in SCLC. Some investigators have reported c-kit expression in 80% to 90% of SCLC samples,24 although others have found a much lower expression rate.25 c-kit
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activation can lead to enhanced proliferation and inhibition of apoptosis in SCLC cell lines.26 An experiment using a cell line that expresses the kit ligand SCF showed that transfection of c-kit expression vector increased cell growth.26 In addition, the growth of an SCLC cell line that expresses SCF and c-kit was markedly decreased when defective c-kit was transfected. These data support the notion that SCF and c-kit are important factors in autocrine and paracrine growth stimulation in SCLC. Imatinib has shown growth inhibition of multiple SCLC cell lines in vitro.27,28 Growth inhibition correlates with c-kit expression and blockade of c-kit phosphorylation. A phase II trial of imatinib in chemotherapynaive patients or in patients with chemotherapysensitive relapse with SCLC was reported at the American Society of Clinical Oncology meeting in 2002.25 Nineteen patients were treated on this study, and treatment was generally well tolerated. There were no responses. One caveat to interpreting these results is that, of the 14 tumor blocks that were available for CD117 (c-kit) immunohistochemical staining, only four stained positive for the presence of c-kit. These phase II results suggest that other growth factors and survival signals drive tumor cell growth in SCLC. Imatinib also could have this effect when combined with cytotoxic chemotherapy or irradiation in SCLC. Because the single-agent toxicity of imatinib is mild and not obviously overlapping with chemotherapy, it could theoretically increase overall cell kill by inhibiting tumor cell regrowth between chemotherapy cycles. More importantly, one could continue imatinib after the completion of chemotherapy after maximal response to slow the regrowth of any MRD and thus, improve overall survival. Further testing in SCLC trials should require confirmation of c-kit expression and should explore c-kit– expressing tumors for the presence of an activating mutation. At Memorial Sloan-Kettering Cancer Center, we are currently testing the combination of cisplatin, irinotecan, and imatinib in a phase I trial in patients with extensive-stage SCLC. Patients will continue imatinib monotherapy for 1 year or until disease progression. Another trial, led by the Medical College of Virginia, combines imatinib with etoposide and cisplatin.
PATEL ET AL
Table 3. Cancer Cell Surface Targets for Small Cell Lung Cancer Vaccine Construction Antigens GD2 GD3 GM2 Fucosyl GM1
Polysialic Acid Globo H Sialyl Lea KSA Bombesin
VACCINES
Another approach to eradication of MRD in SCLC capitalizes on advances in our knowledge of tumor biology and immunology. Vaccination therapy has been studied in a variety of tumors as a way to eliminate persistent disease after maximal response has been achieved from standard therapy. For SCLC, the ideal time for immune intervention is after completion of definitive chemoradiotherapy, when tumor burden has been reduced to the maximum extent possible with current therapies. If immune effectors can be induced against tumor antigens, we would at last have a therapy targeted to micrometastatic disease. Tumor cells overexpress a variety of antigens that are not found on normal cells. Some of these represent altered molecules that have undergone subtle biochemical changes that distinguish them from the native antigens. Others are proteins that are involved in, or are markers of, cellular differentiation. Small cell lung cancer offers a unique opportunity to study vaccine therapy because the differentiation antigens expressed on these cells are relatively tumor-specific and have restricted expression. Tumor-associated antigens that are thought to play a role in SCLC, and which are potential targets for vaccine therapy, are listed in Table 3. These targets include the gangliosides GM2, GD2, GD3, bombesin, and Fucosyl-GM1, as well as the polysialic acid epitope characteristic of the embryonic neural-cell adhesion molecule, the natural glycolipid Globo H, and the glycoprotein KSA.29-36 GD3 is a ganglioside that is present in both melanoma and SCLC cells. It is poorly immunogenic by itself. The development of an immune strategy to effectively induce an immune response against self-antigens represents one of the great
MINIMAL RESIDUAL DISEASE IN SCLC
challenges in the immunotherapy of cancer. One method to overcome self-tolerance for this antigen is through an anti-idiotype approach. Here, the critical antigen is presented in a different way to enhance its antigenic potential. The mouse monoclonal antibody, R24, is specific for GD3 and when used as an immunogen, R24 generates a series of antibodies that bind to R24 antigen combining sites. These anti-idiotypic antibodies effectively mimic the three-dimensional structures and functions of GD3 and can be used as surrogate antigens for active specific immunotherapy.37 The monoclonal antibody BEC2 is an antibody directed against the antigen binding site of R24. McCaffrey et al showed that vaccination with BEC2, when given with the immunologic adjuvant BCG (Bacillus Calmette-Gue´ rin), was found to induce anti-GD3 antibodies in three of 14 highrisk melanoma patients.38 Because GD3 is expressed on SCLC cells, a pilot study was conducted in which BEC2 plus BCG was used to immunize 15 patients with SCLC who had achieved a major response to chemotherapy or chemoradiotherapy.39 Patients received a series of five intradermal immunizations of BEC2 plus BCG over a 10-week period. Median overall survival was 21 months. Median time to relapse was 11 months in patients with extensive-stage disease and was not reached at 47 months for patients with limited-stage disease. These results were significantly better than historical controls. The only significant toxicity was local skin reaction at immunization sites. Although GD3 is also expressed on some normal tissues, there was no evidence of any toxicity caused by auto-immunity. Because of these results, the European Organization for Research and Treatment of Cancer has initiated a phase III, prospective, randomized trial, SILVA (Survival in an International Phase III Prospective Randomized LD Small Cell Lung Cancer Vaccination Study with Adjuvant BEC2 and BCG). Patients with limited-stage SCLC are treated in standard fashion with chemotherapy and chest radiation. Prophylactic cranial irradiation is also recommended. Patients with a partial or complete responses are eligible, and are randomized to receive BEC2 plus BCG vaccination versus observation. Thus far, over 400 of the planned 500 patients have been accrued. This trial is scheduled to complete accrual in the Fall of 2002.
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Other gangliosides are also being investigated. Fucosyl-GM1 was initially isolated from the bovine thyroid gland40 With the use of a highly specific mouse monoclonal antibody, F12, the ganglioside Fuc-GM1 was identified in the majority of SCLC tissue samples and in the serum of some patients.30,31,41,42 Fuc-GM1 has a more restricted distribution on normal tissues than some other gangliosides, including GD3. Serum antibodies against Fuc-GM1 have also been described in the serum of a few patients with sensory neuropathies but not in other settings, suggesting that the molecule itself is poorly immunogenic.43 However, conjugation with KLH (keyhole limpet hemocyanin), a shellfish-derived protein, followed by a mixture with the immunoadjuvant QS-21, augments its immunogenicity greatly. A phase I/II study using Fucosyl-GM1–KLH conjugate vaccine was conducted in 13 patients with SCLC who had a documented major response to chemotherapy.44 Ten patients completed five vaccinations and were evaluable for immunologic response. All 10 patients mounted both IgM and IgG titers of ⱖ 1:40 against Fucosyl-GM1 as measured by enzyme-linked immunosorbent assay. Mild transient erythema and induration at injection sites were the only consistent toxicities. Three patients with SCLC were free of relapse at 18, 24, and 30 months at the time of manuscript submission. A synthetic version of Fucosyl-GM1 is now available, and a study has completed enrollment with this vaccine.45 This construct allows easier vaccine production and reduces the theoretical risk of prion transmission from bovine tissues. Because tumor cells express multiple antigens and there is heterogeneity in human immune response, a “polyvalent” vaccine containing multiple antigens (GM2, Globo H, polysialic acid, and Fucosyl GM1) may provide an even better approach to attack MRD. A polyvalent vaccine eliminates escape by tumor cells that fail to express any one antigen, and also increases the number of antibodies reacting against each tumor cell. We plan to test this concept with a vaccine combining GM2, Globo H, polysialic acid, and synthetic fucosyl GM1 and QS21 adjuvant in SCLC patients who have completed chemotherapy and chemoradiotherapy.
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PATEL ET AL
SUMMARY
Because the majority of patients with SCLC relapse from persistent but clinically undetectable micrometastatic disease, we continue to evaluate therapies and strategies to attack these resistant tumor cells. Improvements in our ability to quantify residual disease will help us better monitor our efforts and identify patients most in need of additional therapy. Targeted therapies may help us halt regrowth of micrometastatic disease without substantially increasing toxicity. The incorporation of these new approaches and improvements in our knowledge of the molecular mechanism that underlie the growth of SCLC will ultimately improve survival in our patients with this disease. REFERENCES 1. Ihde DC: Chemotherapy of lung cancer. N Engl J Med 327:1434-1441, 1992 2. Sandler AB: Current management of small cell lung cancer. Semin Oncol 24:463-476, 1997 3. Armitage JO: Bone marrow transplantation. N Engl J Med 330:827-838, 1994 4. Pelosi G, Pasini F, Pavenel F, et al: Effects of different immunolabeling techniques on the detection of small-cell lung cancer cells in bone marrow. J Histochem Cytochem 47:10751087, 1999 5. Zippelius A, Pantel K: RT-PCR-cased detection of occult disseminated tumor cells in peripheral blood and bone marrow of patients with solid tumors. An overview. Ann N Y Acad Sci 906:110-123, 2000 6. Peck K, Sher Y, Shih J, et al: Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients. Cancer Res 58:2761-2765, 1998 7. Lacroix J, Becker HD, Woerner SM, et al: Sensitive detection of rare cancer cells in sputum and peripheral blood samples of patients with lung cancer by preprogrp-specific RT PCR. Int J Cancer 92:1-8, 2001 8. Bessho A, Tabata M, Kiura K, et al: Detection of occult tumor cells in peripheral blood from patients with small cell lung cancer by reverse transcriptase polymerase chain reaction. Anticancer Res 20:1149-1154, 2000 9. Leon S, Shapiro B, Sklaroff D, et al: Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 37:646-650, 1977 10. Stroun M, Anker P, Lyautey J, et al: Isolation and characterization of DNA from the plasma of cancer patients. Eur J Cancer Clin Oncol 23:707-712, 1989 11. Anker P, Mulcahy H, Chen X, et al: Detection of circulating tumour DNA in blood (plasma/serum) of cancer patients. Cancer Metastasis Rev 18:65-73, 1999 12. Anker P, Stroun M: Tumor-related alterations in circulation DNA, potential for diagnosis, prognosis and detection of minimal residual disease. Leukemia 15:289-291, 2001 13. Gonzalez R, Silva J, Sanchez A, et al: Microsatellite alterations and TP53 mutations in plasma DNA of small cell
lung cancer patients: Follow up study and prognostic significance. Ann Oncol 11:1097-1104, 2000 14. Hunt JD, Robert EG, Zieske AW, et al: Orthotopic human lung carcinoma xenografts in BALB/c mice immunosuppressed with anti-CD4 monoclonal antibodies and chronic alcohol consumption. Cancer 88:468-479, 2000 15. Hidalgo M, Siu L, Nemaunaitis J, et al: Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 19:3267-3279, 2001 16. Nawrocki B, Polette M, Marchand V, et al: Expression of matrix metalloproteinases and their inhibitors in human bronchopulmonary carcinomas: Quantificative and morphologic analyses. Int J Cancer 72:556-564, 1997 17. Michael M, Babic B, Khokha R, et al: Expression and prognostic significance of metalloproteinases and their tissue inhibitors in patients with small-cell lung cancer. J Clin Oncol 17:1802-1808, 1999 18. Ylisirnio S, Hoyhtya M, Turpeenniemi-Hujanen T: Serum matrix metalloproteinases -2, -9 and tissue inhibitors of metalloproteinases -1, -2 in lung cancer–TIMP-1 as a prognostic marker. Anticancer Res 20:1311-1316, 2000 19. Shepherd F, Giaccone G, Debruyne C: Randomized double-blind placebo-controlled trial of marimastat in patients with small cell lung cancer (SCLC) following response to first-line chemotherapy: An NCIC and EORTC study. Proceedings of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001 (abstr 1) 20. Smylie M, Mercier R, Aboulafia D, et al: Phase III study of the matrix metalloproteinase inhibitor prinomastat in patients having advanced non–small cell lung cancer. Proceedings of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001 (abstr 1226) 21. Bissett D, Pawel JV, Mercier R, et al: Phase III study of the matrix metalloproteinase inhibitor prinomastat in combination with gemcitabine and cisplatin in non–small cell lung cancer. Proceedings of the American Society of Clinical Oncology. Orlando, FL, May 17-21, 2002 (abstr 1183) 22. Hirota S, Isozaki K, Moriyama Y, et al: Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279:577-580, 1998 23. Heinrich M, Corless C, Blanke C, et al: KIT mutational status predicts clinical response to STI571 in patients with metastatic gastrointestinal stromal tumors (GISTs). Proceedings of the American Society of Clinical Oncology, Orlando, FL, May 17-21, 2002 (abstr 6) 24. Sekido Y, Obata Y, Ueda R, et al: Preferential expression of c-kit protooncogene transcripts in small cell lung cancer. Cancer Res 51:2416-2419, 1999 25. Johnson B, Fisher B, Fisher T, et al: A phase II study of STI571 (Gleevec) for patients with small cell lung cancer. Proceedings of the American Society of Clinical Oncology, Orlando, FL, May 17-21, 2002 (abstr 1171) 26. Krystal GW, Hines SJ, Organ CP: Autocrine growth of small cell lung cancer mediated by coexpression of c-kit and stem cell factor. Cancer Res 56:370-376, 1996 27. Wang W, Healy M, Sattler M, et al: Growth inhibition and modulation of kinase pathways of small cell lung cancer cell lines by the novel tyrosine kinase inhibitor STI571. Oncogene 19:3521-3528, 2000 28. Krystal GW, Honsawek S, Litz J, et al: The selective
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tyrosine kinase inhibitor STI571 inhibits small cell lung cancer growth. Clin Cancer Res 6:3319-3326, 2000 29. Hamilton WB, Helling F, Livingston PO: Ganglioside expression on sarcoma and small cell lung carcinoma compared to tumors of neuroectodermal origin. Proc Am Assoc Cancer Res 34:491, 1993 (abstr) 30. Zhang S, Cordon-Cardo C, Zhang H, et al: Selection of tumor antigens as targets for immune attack using immunuhistochemistry: I. Focus on gangliosides. Int J Cancer 73:42-49, 1997 31. Brezicka F-T, Olling S, Nilsson O, et al: Immunohistological detection of Fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies. Cancer Res 49:1300-1305, 1989 32. Fuentes R, Allman R, Mason MD: Ganglioside expression in lung cancer cell lines. Lung Cancer 18:21-33, 1997 33. Brezicka FT, Olling S, Bergman B, et al: Coexpression of ganglioside antigen Fuc-GM1, neural-cell adhesion molecule, carcinoembryonic antigen, and carbohydrate tumor-associated antigen CA 50 in lung cancer. Tumor Biol 13:308-315, 1992 34. Cheresh DA, Pierschbacher MD, Herzig MA, et al: Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins. J Cell Biol 102:688-696, 1986 35. Grant SC, Kostakoglu L, Kris MG, et al: Targeting of small-cell lung cancer using the anti-GD2 ganglioside monoclonal antibody 3f8: A pilot trial. Eur J Nucl Med 23:145-149, 1996 36. Zhang S, Zhang HS, Cordon-Cardo C, et al: Selection of tumor antigens for immune attack using immunohistochemistry: III. Protein antigens. Clin Cancer Res 4:2669-2676, 1998 37. Bhattacharya-Chatterjee M, Chatterjee S, Foon K: The
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anti-idiotypic vaccines for immunotherapy. Curr Opin Molec Ther 3:63-69, 2001 38. McCaffery M, Yao T-J, Williams L, et al: Immunization of melanoma patients with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: Enhanced immunogenicity when combined with adjuvant. Clin Cancer Res 2:679686, 1996 39. Grant SC, Kris MG, Houghton AN, et al: Long survival of patients with small cell lung cancer after adjuvant treatment with the anti-idiotypic antibody BEC2 plus Bacillus CalmetteGuerin. Clin Cancer Res 5:1319-1323, 1999 40. Macher BA, Pacuzska T, Mullin BR, et al: Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland. Biochim Biophys Acta 588:35-43, 1979 41. Fredman P, Brezicka T, Holmgren J, et al: Binding specificity of monoclonal antibodies to ganglioside, Fuc-GM1. Biochim Biophys Acta 875:316-323, 1986 42. Vangsted AJ, Clausen H, Kjeldsen TB, et al: Immunochemical detection of a small cell lung cancer-associated ganglioside (FucGM1) antigen in serum. Cancer Res 51:28792884, 1991 43. Yoshino H, Ariga T, Latov N, et al: Fucosyl-GM1 in human sensory nervous tissue is a target antigen in patients with autoimmune neuropathies. J Neurochem 61:658-663, 1993 44. Dickler MN, Ragupathi G, Liu NX, et al: Immunogenicity of a Fucosyl-GM1-keyhole limpet hemocyanin conjugate vaccine in patients with small cell lung cancer. Clin Cancer Res 5:2773-2779, 1999 45. Krug LM, Ragupathi G, Livingston P, et al: Pilot trial of a synthetic fucosyl-GM1 ganglioside vaccine in patients with small cell lung cancer. Proceedings of the American Society of Clinical Oncology, Orlando, FL, May 17-21, 2002 (abstr 1824)