- Email: [email protected]
Prevalence of in vitro chemotherapeutic drug resistance in primary malignant pleural mesothelioma: Result in a cohort of 203 resection specimens
GENERAL THORACIC SURGERY
Prevalence of in vitro chemotherapeutic drug resistance in primary malignant pleural mesothelioma: Result in a cohort of 203 resection specimens Aneil A. Mujoomdar, MD, Tamara R. Tilleman, MD, PhD, William G. Richards, PhD, Raphael Bueno, MD, and David J. Sugarbaker, MD Objective: Optimizing the multimodality treatment of malignant pleural mesothelioma depends on many factors including an adequate chemotherapeutic response. Currently, chemotherapy regimens for patients with mesothelioma are empirically selected. In vitro chemotherapy resistance in human mesothelioma has not been reported. Our goal was to determine the prevalence of drug resistance in a large sample of malignant pleural mesothelioma using a commercially available assay.
GTS
Methods: Tumors specimens (n ¼ 203) were cultured for analysis of chemoresistance using the extreme drug resistance assay. Evaluable results were obtained in 168 (168/203 ¼ 83%) specimens. Each specimen was tested with 3 drugs: cisplatin, gemcitabine, and vinorelbine. Drug resistance was characterized as low, intermediate, or extreme. Median age was 64 years (30–85 years). Forty-four (26%) patients received neoadjuvant chemotherapy before sampling and testing. The distribution of histopathologic cell types was epithelial (103; 61%), mixed (57; 34%), and sarcomatoid (8; 5%). Results: A significant proportion of tumors had extreme/intermediate drug resistance to cisplatin (27%), gemcitabine (31%), or vinorelbine (59%). Nineteen tumors (11%) had extreme/intermediate resistance to all 3 drugs. Resistance (extreme/intermediate) to cisplatin was more prevalent in epithelial tumors than in nonepithelial (33% vs 18%; P ¼ .0394). No significant differences in chemoresistance were found in tumors of patients who had received neoadjuvant chemotherapy compared with those who had not. Conclusions: The feasibility of performing off-site in vitro drug resistance assays on resected malignant mesothelioma specimens is reported. A significant proportion of mesothelioma tumors exhibited extreme/intermediate resistance to cisplatin, gemcitabine, or vinorelbine. (J Thorac Cardiovasc Surg 2010;140:352-5) Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer that is associated with a poor prognosis. Despite improvements in overall median survival with the multimodality approach,1,2 survival in MPM still lags behind other thoracic malignancies, such as non–small cell lung cancer. Response rates of mesothelioma tumors to various single chemotherapy agents consistently have been low,3 suggesting the need for improved chemotherapy efficacy and/or selection to optimize multimodality treatment. Although combination chemotherapy has been associated with higher response rates in patients with MPM, these response rates do not necessarily translate into longer median survival.4 With current methods, the response of an individual tumor to a particular drug combination can only be tested empiriFrom the Division of Thoracic Surgery, Department of Surgery, Brigham and Women’s Hospital, Boston, Mass. Disclosures: None. Received for publication May 29, 2009; revisions received Oct 19, 2009; accepted for publication Nov 14, 2009; available ahead of print May 31, 2010. Address for reprints: Tamara Raveh Tilleman, MD, PhD, Medical Director Clinical Research, Division of Thoracic Surgery, Brigham and Women’s Hospital, 15 Francis St, Boston, MA 02115 (E-mail: [email protected]). 0022-5223/$36.00 Copyright Ó 2010 by The American Association for Thoracic Surgery doi:10.1016/j.jtcvs.2009.11.072
352
cally, risking significant toxicity in patients who may not respond to the treatment and losing precious time in a disease associated with a short survival.5 Therefore, investigation of new methods to assay individual tumor characteristics is warranted. Drug resistance profiles have been reported for various other malignant diseases, including non&small cell lung cancer, ovarian, breast, colon, esophageal cancer, and carcinoid.6-11 Some patients who have undergone chemoresistance assay– directed treatment have had significantly longer disease-free and overall survival duration.12 We report the results of a single-center experience with an in vitro chemoresistance assay in a pathologically diverse cohort of MPM tumors. We used a currently available commercial assay to test for drug resistance to cisplatin, gemcitabine, and vinorelbine, 3 agents commonly used to treat mesothelioma. To our knowledge, this is the first study to characterize in vitro chemoresistance in MPM. METHODS MPM Specimens Fresh MPM specimens were obtained from patients undergoing surgery at the Brigham and Women’s Hospital, placed in transport medium, and express shipped to Oncotech (now known as Exiqon Diagnostics) in Tustin, California, for analysis by extreme drug resistance assay (EDR Assay;
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General Thoracic Surgery
TABLE I. Patient characteristics
Abbreviations and Acronyms EDR ¼ extreme drug resistance IDR ¼ intermediate drug resistance LDR ¼ low drug resistance MPM ¼ malignant pleural mesothelioma OR ¼ odds ratio
Gender Laterality Neoadjuvant treatment Resectable* Histopathologic cell type
N (%)
N (%)
139 male (83) 91 right (54) 44 yes (26) 146 yes (94) 103 epithelial (61)
29 female (17) 77 left (46) 124 no (74) 10 no (6) 57 mixed (34) 8 sarcomatoid (5)
Oncotech/Exiqon, Vedbaek, Denmark).13 Specimens were assayed over a 2-year period (2006–2008). We excluded from the study cohort cases that were not primary MPM and specimens that demonstrated only partial growth. We also excluded repeat assays from individuals with recurrent tumors because it has been reported that recurrent lesions may exhibit greater heterogeneity and more frequent EDR. In total, 203 MPM tumor samples were obtained. Tissue culture was successful in 168 (83%) of 203 resection specimens. This evaluability rate was similar to previously reported rates for other diseases.7
Patient Data EDR assay results were entered into the clinical record for each patient and then were obtained retrospectively along with demographic, pathologic, and prior treatment data from the International Mesothelioma Program Patient Data Registry under an institutional review board–approved protocol.
Specimen Handling and in Vitro EDR Assay The protocol for specimen handling has been previously published.13,14 In brief, viable tumor cells are cultured in soft-agarose medium at approximately 30,000 cells per well in 24-well plates, each with a single chemotherapeutic agent (cisplatin, gemcitabine, or vinorelbine). After 72 hours of incubation, tritiated thymidine is added to each well and left for an additional 48 hours. Tumor cell DNA is harvested and tritium incorporation is measured on a scintillation counter as an indicator of cellular proliferation. Positive controls (supralethal exposure to cisplatin, gemcitabine, and vinorelbine) and negative controls (exposure to media only) are performed with each assay. The results are reported as in vitro cell proliferation, relative to the median of a historical database of over 100,000 human tumors of varied histologic types submitted for EDR assay. This measure is corrected using the negative and positive controls. Specimens are considered to have EDR when the in vitro cellular proliferation rate is greater than 1 SD above the median. This indicates that tumor cell growth in the specimen has been virtually unaffected by exposure to the cytotoxic agent. Intermediate drug resistance (IDR) indicates moderate tumor growth (in vitro cell proliferation is greater than the median but less than 1 SD above the median). Low drug resistance (LDR) indicates that tumor cell proliferation was inhibited by the tested agent (in vitro cell proliferation is less than the median).
or vinorelbine (59%) (Figure 1). Fifteen (88%) of 17 tumors with EDR to cisplatin also had EDR to either gemcitabine (n ¼ 10) or vinorelbine (n ¼ 9). Four (24%) of the 17 tumors with EDR to cisplatin had EDR to all 3 drugs. Nineteen (11%) tumors had EDR or IDR to all 3 chemotherapy drugs (Figure 2). EDR or IDR to cisplatin was more prevalent in epithelial (33%) than nonepithelial tumors (18%) (P ¼ .0394). Trends in the same direction were observed with gemcitabine (35% vs 25%; P ¼ .1581) and vinorelbine (62% vs 54%; P ¼ .2875) (Figures 3 and 4). All 4 patients with triple EDR had epithelial type tumors. Forty-four (25%; 44/168) patients had chemotherapy before the specimens were sent for EDR assay. No significant differences were detected in EDR/IDR versus LDR between those who received chemotherapy treatment and those who did not. Those who had prior chemotherapy tended to have more EDR to cisplatin (odds ratio [OR] ¼ 1.6; P ¼ .2453), but there were no differences with respect to gemcitabine (OR ¼ 0.91; P ¼ .8142) and vinorelbine (OR ¼ 1.0; P ¼ .9797). No significant differences were detected in EDR/IDR versus LDR between those who were ultimately resectable and those who were not. DISCUSSION Drug resistance profiles for MPM have not been described in the literature, although the lack of efficacy of most chemotherapy is widely acknowledged. Such profiles may contribute to the individualized treatment of MPM. The 83% evaluability rate of the MPM cultures in this study is similar to previous reports for other cancers6,9 and confirms the feasibility of shipping viable samples of MPM tumors to Drug Resistance Assay
Statistical analysis and data collection were performed pursuant to an institutional review board–approved protocol. Patient tumor cultures were tested against a panel of 3 individual drugs. Statistical analysis was done using a 2-way c2 test. We are reporting the exact P values.
RESULTS The characteristics of the study population are summarized in Table 1. A significant proportion of MPM tumors had IDR or EDR to cisplatin (27%), gemcitabine (31%),
Number of specimens
Statistical Analysis 140 120 100 60
Low Intermediate
40
Extreme
80
20 0
Cisplatin
Gemcitabine
Vinorelbine
FIGURE 1. Drug resistance assay results among malignant pleural mesothelioma tumors.
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*This category applies only to the 156 patients who underwent definitive cytoreductive surgery (extrapleural pneumonectomy or pleurectomy/decortication).
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Mujoomdar et al
Drug Resistance - Non-epithelial Histology 100 Low Intermediate Extreme
Percentage
80 60 40 20 0
Cisplatin
Gemcitabine
Vinorelbine
FIGURE 4. Drug resistance in tumors with nonepithelial histology. Note: Y-axis is percentage.
GTS FIGURE 2. Venn diagram: EDR and IDR for mesothlioma specimens. EDR, Extreme drug resistance; IDR, intermediate drug resistance.
a commercial reference laboratory for in vitro analysis. We were particularly interested in examining specimen growth, in consideration of the desmoplastic response associated with MPM, which may interfere with cell growth. A significant proportion of tumors demonstrated EDR/IDR (ranging from 27% to 59%) to at least one of the drugs in the assay panel. Other cancers also have been shown to exhibit significant levels of drug resistance using this assay.6,9,10 Approximately 27% of all tumors had EDR or IDR to cisplatin. Interestingly, tumors with epithelial histology were more resistant to cisplatin than tumors of nonepithelial subtype. The finding is counterintuitive, inasmuch as nonepithelial tumors have worse outcome and, in addition, any mixed tumor also would contain some epithelial cells. In view of the limited chemotherapeutic options available to treat MPM, new tools for measuring tumor response, including in vitro testing, may extend the therapeutic possibilDrug Resistance - Epithelial Histology 100
Percentage
80 Low
60
Intermediate Extreme
40 20 0 Cisplatin
Gemcitabine
Vinorelbine
FIGURE 3. Drug resistance in tumors with epithelial histology. Note: Y-axis is percentage.
354
ities by permitting optimal combination treatments on an individualized basis. Especially in MPM tumors, where both the effectiveness of chemotherapy and survival are low,5,14 tailoring the treatment regimen to optimize efficacy is of utmost importance. Clearly, this is possible only if more than one therapy is being contemplated, in which case a resistance assay might be used as a guide to select among regimens. Currently, the therapeutic options for MPM are few in number, and essentially all commonly used regimens include a platinum compound. However, as additional agents and combinations are considered, the ability to simulate a range of combination therapies in vitro by using the target tumor material may better inform the therapeutic decision.15 In 2004, the American Society of Clinical Oncology failed to find a firm basis for integrating chemosensitivity and resistance assay into the clinical setting.16 Yet recent publications have shown a positive correlation between tumor response and a treatment tailored according to chemoresistance assays.12,17 Chemosensitivity assays analyze the sensitivity of a tumor to chemotherapy by measuring the number of tumor cells that are killed by a particular cancer drug. The test assumes that a short exposure to the drug, in concentrations at or below clinical peak plasma levels, is representative of tumor exposure in vivo. This assumption fails to account for the individual’s biologic reaction to the cytotoxic agent (eg, metabolism or vascular supply) and therefore cannot be extrapolated to the in vivo setting. Chemoresistance assays, on the other hand, expose tumors to chemotherapy agents for longer periods of time at higher concentrations. Tumor growth under such conditions in vitro might reasonably be expected to correlate with in vivo drug resistance.18,19 Indeed, Cortazar and associates14 demonstrated that individualized treatment was associated with prolonged survival for patients with small-cell lung cancer who were randomized to receive assay-directed therapy. A significant association between in vitro EDR to platinum and clinical outcomes was also observed in patients with ovarian cancer. Patients with tumors demonstrating in vitro EDR to platinum were at significantly increased risk for progression and death when treated with standard platinum-based regimens.8
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Overall and progression-free survival was significantly better in the EDR assay–guided treatment groups.8,9,12 The limitations of this study are twofold. The EDR assay used for this study only tested single drugs, whereas platinum-based combination treatment now is standard for MPM. Second, we were unable to test pemetrexed (Alimta), another drug commonly used to treat MPM, because the EDR assay relies on tritiated thymidine uptake as the end point for determining tumor resistance, and pemetrexed is an antifolate that increases thymidine uptake.20 Hence, an alternative assay that does not rely on tritiated thymidine incorporation would have to be developed. Despite these limitations, this study not only establishes the prevalence of chemotherapy resistance in cultured mesothelioma tumors, but also suggests differences in drug resistance based on histopathologic cell type. Our findings indicate that a significant proportion of MPM tumors have IDR or EDR to 3 common chemotherapeutic agents. Clinical implementation of this strategy awaits a proliferation test applicable to pemetrexed, the availability of additional chemotherapeutic options, and the integration of biology and genomic diagnostic tests to accompany the chemoresistance assay.21 The effectiveness of assay-directed therapy will need to be addressed in prospective trials. We acknowledge Dr John Mannick, Ann S. Adams, and William Francis Powell, Jr, for their valuable contributions to this article.
5.
6.
7.
8.
9.
10.
11.
12.
13. 14.
15. 16.
References 1. Sugarbaker DJ, Flores RM, Jaklitsch MT, Richards WG, Strauss GM, Corson JM, et al. Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: results in 183 patients. J Thorac Cardiovasc Surg. 1999;117:54-63; discussion 63-5. 2. Krug LM, Pass HI, Rusch VW, Kindler HL, Sugarbaker DJ, Rosenzweig KE, et al. Multicenter phase II trial of neoadjuvant pemetrexed plus cisplatin followed by extrapleural pneumonectomy and radiation for malignant pleural mesothelioma. J Clin Oncol. 2009;27:3007-13. 3. Ellis P, Davies AM, Evans WK, Haynes AE, Lloyd NS. Lung Cancer Disease Site Group of Cancer Care Ontario’s Program in Evidence-based Care. The use of chemotherapy in patients with advanced malignant pleural mesothelioma: a systematic review and practice guideline. J Thorac Oncol. 2006;1:591-601. Review. 4. Kalmadi SR, Rankin C, Kraut MJ, Jacobs AD, Petrylak DP, Adelstein DJ, et al. Gemcitabine and cisplatin in unresectable malignant mesothelioma of the pleura:
17.
18.
19. 20.
21.
a phase II study of the Southwest Oncology Group (SWOG 9810). Lung Cancer. 2008;60:259-63. Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E, Ruffie P, et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol. 2003;21: 2636-44. d’Amato TA, Landreneau RJ, Ricketts W, Huang W, Parker R, Mechetner E, et al. Chemotherapy resistance and oncogene expression in non–small cell lung cancer. J Thorac Cardiovasc Surg. 2007;133:352-63. d’Amato TA, Landreneau RJ, McKenna RJ, Santos RS, Parker RJ. Prevalence of in vitro extreme chemotherapy resistance in resected nonsmall-cell lung cancer. Ann Thorac Surg. 2006;81:440-6; discussion 446-7. Holloway RW, Mehta RS, Finkler NJ, Li KT, McLaren CE, Parker RJ, et al. Association between in vitro platinum resistance in the EDR assay and clinical outcomes for ovarian cancer patients. Gynecol Oncol. 2002;87:8-16. Mehta RS, Bornstein R, Yu IR, Parker RJ, McLaren CE, Nguyen KP, et al. Breast cancer survival and in vitro tumor response in the extreme drug resistance assay. Breast Cancer Res Treat. 2001;66:225-37. Haroun RI, Clatterbuck RE, Gibbons MC, Burger PC, Parker R, Fruehauf JP, et al. Extreme drug resistance in primary brain tumors: in vitro analysis of 64 resection specimens. J Neurooncol. 2002;58:115-23. Lyons JM 3rd, Abergel J, Thomson JL, Anthony CT, Wang YZ, Anthony LB, et al. In vitro chemoresistance testing in well-differentiated carcinoid tumors. Ann Surg Oncol. 2009;16:649-55. Epub 2009 Jan 8. Loizzi V, Chan JK, Osann K, Cappuccini F, DiSaia PJ, Berman ML. Survival outcomes in patients with recurrent ovarian cancer who were treated with chemoresistance assay–guided chemotherapy. Am J Obstet Gynecol. 2003;189:1301-7. Exiquon diagnostics. http://www.exiqon.com/dxps/Documents/edr_4_pager. pdf_Last entered 2009 Oct 17. van Meerbeeck JP, Gaafar R, Manegold C, Van Klaveren RJ, Van Marck EA, Vincent M, et al. Randomized phase III study of cisplatin with or without raltitrexed in patients with malignant pleural mesothelioma: an intergroup study of the European Organisation for Research and Treatment of Cancer Lung Cancer Group and the National Cancer Institute of Canada. J Clin Oncol. 2005;23: 6881-9. Schinko¨the T, Haeger S, Gabri MR. Practical guidelines for diagnostic use of in vitro chemosensitivity tests. Anticancer Res. 2007;27:1365-7. Schrag D, Garewal HS, Burstein HJ, Samson DJ, Von Hoff DD, Somerfield MR. American Society of Clinical Oncology Technology Assessment: chemotherapy sensitivity and resistance assays. J Clin Oncol. 2004;22:3631-8. Cortazar P, Gazdar AF, Woods E, Russell E, Steinberg SM, Williams J, et al. Survival of patients with limited-stage small cell lung cancer treated with individualized chemotherapy selected by in vitro drug sensitivity testing. Clin Cancer Res. 1997;3:741-7. Kern DH, Weisenthal LM. Highly specific prediction of antineoplastic drug resistance with an in vitro assay using suprapharmacologic drug exposures. J Nat Cancer Inst. 1990;82:582-8. Fruehauf JP, Alberts DS. In vitro drug resistance versus chemosensitivity: two sides of different coins. J Clin Oncol. 2005;23:3641-2. Chattopadhyay S, Zhao R, Krupenko SA, Krupenko N, Goldman ID. The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line. Mol Cancer Ther. 2006;5:438-49. McAlpine JN, Eisenkop SM, Spirtos NM. Tumor heterogeneity in ovarian cancer as demonstrated by in vitro chemoresistance assays. Gynecol Oncol. 2008;110: 360-4.
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Prevalence of in vitro chemotherapeutic drug resistance in primary malignant pleural mesothelioma: Result in a cohort of 203 resection specimens Aneil A. Mujoomdar, MD, Tamara R. Tilleman, MD, PhD, William G. Richards, PhD, Raphael Bueno, MD, and David J. Sugarbaker, MD Objective: Optimizing the multimodality treatment of malignant pleural mesothelioma depends on many factors including an adequate chemotherapeutic response. Currently, chemotherapy regimens for patients with mesothelioma are empirically selected. In vitro chemotherapy resistance in human mesothelioma has not been reported. Our goal was to determine the prevalence of drug resistance in a large sample of malignant pleural mesothelioma using a commercially available assay.
GTS
Methods: Tumors specimens (n ¼ 203) were cultured for analysis of chemoresistance using the extreme drug resistance assay. Evaluable results were obtained in 168 (168/203 ¼ 83%) specimens. Each specimen was tested with 3 drugs: cisplatin, gemcitabine, and vinorelbine. Drug resistance was characterized as low, intermediate, or extreme. Median age was 64 years (30–85 years). Forty-four (26%) patients received neoadjuvant chemotherapy before sampling and testing. The distribution of histopathologic cell types was epithelial (103; 61%), mixed (57; 34%), and sarcomatoid (8; 5%). Results: A significant proportion of tumors had extreme/intermediate drug resistance to cisplatin (27%), gemcitabine (31%), or vinorelbine (59%). Nineteen tumors (11%) had extreme/intermediate resistance to all 3 drugs. Resistance (extreme/intermediate) to cisplatin was more prevalent in epithelial tumors than in nonepithelial (33% vs 18%; P ¼ .0394). No significant differences in chemoresistance were found in tumors of patients who had received neoadjuvant chemotherapy compared with those who had not. Conclusions: The feasibility of performing off-site in vitro drug resistance assays on resected malignant mesothelioma specimens is reported. A significant proportion of mesothelioma tumors exhibited extreme/intermediate resistance to cisplatin, gemcitabine, or vinorelbine. (J Thorac Cardiovasc Surg 2010;140:352-5) Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer that is associated with a poor prognosis. Despite improvements in overall median survival with the multimodality approach,1,2 survival in MPM still lags behind other thoracic malignancies, such as non–small cell lung cancer. Response rates of mesothelioma tumors to various single chemotherapy agents consistently have been low,3 suggesting the need for improved chemotherapy efficacy and/or selection to optimize multimodality treatment. Although combination chemotherapy has been associated with higher response rates in patients with MPM, these response rates do not necessarily translate into longer median survival.4 With current methods, the response of an individual tumor to a particular drug combination can only be tested empiriFrom the Division of Thoracic Surgery, Department of Surgery, Brigham and Women’s Hospital, Boston, Mass. Disclosures: None. Received for publication May 29, 2009; revisions received Oct 19, 2009; accepted for publication Nov 14, 2009; available ahead of print May 31, 2010. Address for reprints: Tamara Raveh Tilleman, MD, PhD, Medical Director Clinical Research, Division of Thoracic Surgery, Brigham and Women’s Hospital, 15 Francis St, Boston, MA 02115 (E-mail: [email protected]). 0022-5223/$36.00 Copyright Ó 2010 by The American Association for Thoracic Surgery doi:10.1016/j.jtcvs.2009.11.072
352
cally, risking significant toxicity in patients who may not respond to the treatment and losing precious time in a disease associated with a short survival.5 Therefore, investigation of new methods to assay individual tumor characteristics is warranted. Drug resistance profiles have been reported for various other malignant diseases, including non&small cell lung cancer, ovarian, breast, colon, esophageal cancer, and carcinoid.6-11 Some patients who have undergone chemoresistance assay– directed treatment have had significantly longer disease-free and overall survival duration.12 We report the results of a single-center experience with an in vitro chemoresistance assay in a pathologically diverse cohort of MPM tumors. We used a currently available commercial assay to test for drug resistance to cisplatin, gemcitabine, and vinorelbine, 3 agents commonly used to treat mesothelioma. To our knowledge, this is the first study to characterize in vitro chemoresistance in MPM. METHODS MPM Specimens Fresh MPM specimens were obtained from patients undergoing surgery at the Brigham and Women’s Hospital, placed in transport medium, and express shipped to Oncotech (now known as Exiqon Diagnostics) in Tustin, California, for analysis by extreme drug resistance assay (EDR Assay;
The Journal of Thoracic and Cardiovascular Surgery c August 2010
Mujoomdar et al
General Thoracic Surgery
TABLE I. Patient characteristics
Abbreviations and Acronyms EDR ¼ extreme drug resistance IDR ¼ intermediate drug resistance LDR ¼ low drug resistance MPM ¼ malignant pleural mesothelioma OR ¼ odds ratio
Gender Laterality Neoadjuvant treatment Resectable* Histopathologic cell type
N (%)
N (%)
139 male (83) 91 right (54) 44 yes (26) 146 yes (94) 103 epithelial (61)
29 female (17) 77 left (46) 124 no (74) 10 no (6) 57 mixed (34) 8 sarcomatoid (5)
Oncotech/Exiqon, Vedbaek, Denmark).13 Specimens were assayed over a 2-year period (2006–2008). We excluded from the study cohort cases that were not primary MPM and specimens that demonstrated only partial growth. We also excluded repeat assays from individuals with recurrent tumors because it has been reported that recurrent lesions may exhibit greater heterogeneity and more frequent EDR. In total, 203 MPM tumor samples were obtained. Tissue culture was successful in 168 (83%) of 203 resection specimens. This evaluability rate was similar to previously reported rates for other diseases.7
Patient Data EDR assay results were entered into the clinical record for each patient and then were obtained retrospectively along with demographic, pathologic, and prior treatment data from the International Mesothelioma Program Patient Data Registry under an institutional review board–approved protocol.
Specimen Handling and in Vitro EDR Assay The protocol for specimen handling has been previously published.13,14 In brief, viable tumor cells are cultured in soft-agarose medium at approximately 30,000 cells per well in 24-well plates, each with a single chemotherapeutic agent (cisplatin, gemcitabine, or vinorelbine). After 72 hours of incubation, tritiated thymidine is added to each well and left for an additional 48 hours. Tumor cell DNA is harvested and tritium incorporation is measured on a scintillation counter as an indicator of cellular proliferation. Positive controls (supralethal exposure to cisplatin, gemcitabine, and vinorelbine) and negative controls (exposure to media only) are performed with each assay. The results are reported as in vitro cell proliferation, relative to the median of a historical database of over 100,000 human tumors of varied histologic types submitted for EDR assay. This measure is corrected using the negative and positive controls. Specimens are considered to have EDR when the in vitro cellular proliferation rate is greater than 1 SD above the median. This indicates that tumor cell growth in the specimen has been virtually unaffected by exposure to the cytotoxic agent. Intermediate drug resistance (IDR) indicates moderate tumor growth (in vitro cell proliferation is greater than the median but less than 1 SD above the median). Low drug resistance (LDR) indicates that tumor cell proliferation was inhibited by the tested agent (in vitro cell proliferation is less than the median).
or vinorelbine (59%) (Figure 1). Fifteen (88%) of 17 tumors with EDR to cisplatin also had EDR to either gemcitabine (n ¼ 10) or vinorelbine (n ¼ 9). Four (24%) of the 17 tumors with EDR to cisplatin had EDR to all 3 drugs. Nineteen (11%) tumors had EDR or IDR to all 3 chemotherapy drugs (Figure 2). EDR or IDR to cisplatin was more prevalent in epithelial (33%) than nonepithelial tumors (18%) (P ¼ .0394). Trends in the same direction were observed with gemcitabine (35% vs 25%; P ¼ .1581) and vinorelbine (62% vs 54%; P ¼ .2875) (Figures 3 and 4). All 4 patients with triple EDR had epithelial type tumors. Forty-four (25%; 44/168) patients had chemotherapy before the specimens were sent for EDR assay. No significant differences were detected in EDR/IDR versus LDR between those who received chemotherapy treatment and those who did not. Those who had prior chemotherapy tended to have more EDR to cisplatin (odds ratio [OR] ¼ 1.6; P ¼ .2453), but there were no differences with respect to gemcitabine (OR ¼ 0.91; P ¼ .8142) and vinorelbine (OR ¼ 1.0; P ¼ .9797). No significant differences were detected in EDR/IDR versus LDR between those who were ultimately resectable and those who were not. DISCUSSION Drug resistance profiles for MPM have not been described in the literature, although the lack of efficacy of most chemotherapy is widely acknowledged. Such profiles may contribute to the individualized treatment of MPM. The 83% evaluability rate of the MPM cultures in this study is similar to previous reports for other cancers6,9 and confirms the feasibility of shipping viable samples of MPM tumors to Drug Resistance Assay
Statistical analysis and data collection were performed pursuant to an institutional review board–approved protocol. Patient tumor cultures were tested against a panel of 3 individual drugs. Statistical analysis was done using a 2-way c2 test. We are reporting the exact P values.
RESULTS The characteristics of the study population are summarized in Table 1. A significant proportion of MPM tumors had IDR or EDR to cisplatin (27%), gemcitabine (31%),
Number of specimens
Statistical Analysis 140 120 100 60
Low Intermediate
40
Extreme
80
20 0
Cisplatin
Gemcitabine
Vinorelbine
FIGURE 1. Drug resistance assay results among malignant pleural mesothelioma tumors.
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*This category applies only to the 156 patients who underwent definitive cytoreductive surgery (extrapleural pneumonectomy or pleurectomy/decortication).
General Thoracic Surgery
Mujoomdar et al
Drug Resistance - Non-epithelial Histology 100 Low Intermediate Extreme
Percentage
80 60 40 20 0
Cisplatin
Gemcitabine
Vinorelbine
FIGURE 4. Drug resistance in tumors with nonepithelial histology. Note: Y-axis is percentage.
GTS FIGURE 2. Venn diagram: EDR and IDR for mesothlioma specimens. EDR, Extreme drug resistance; IDR, intermediate drug resistance.
a commercial reference laboratory for in vitro analysis. We were particularly interested in examining specimen growth, in consideration of the desmoplastic response associated with MPM, which may interfere with cell growth. A significant proportion of tumors demonstrated EDR/IDR (ranging from 27% to 59%) to at least one of the drugs in the assay panel. Other cancers also have been shown to exhibit significant levels of drug resistance using this assay.6,9,10 Approximately 27% of all tumors had EDR or IDR to cisplatin. Interestingly, tumors with epithelial histology were more resistant to cisplatin than tumors of nonepithelial subtype. The finding is counterintuitive, inasmuch as nonepithelial tumors have worse outcome and, in addition, any mixed tumor also would contain some epithelial cells. In view of the limited chemotherapeutic options available to treat MPM, new tools for measuring tumor response, including in vitro testing, may extend the therapeutic possibilDrug Resistance - Epithelial Histology 100
Percentage
80 Low
60
Intermediate Extreme
40 20 0 Cisplatin
Gemcitabine
Vinorelbine
FIGURE 3. Drug resistance in tumors with epithelial histology. Note: Y-axis is percentage.
354
ities by permitting optimal combination treatments on an individualized basis. Especially in MPM tumors, where both the effectiveness of chemotherapy and survival are low,5,14 tailoring the treatment regimen to optimize efficacy is of utmost importance. Clearly, this is possible only if more than one therapy is being contemplated, in which case a resistance assay might be used as a guide to select among regimens. Currently, the therapeutic options for MPM are few in number, and essentially all commonly used regimens include a platinum compound. However, as additional agents and combinations are considered, the ability to simulate a range of combination therapies in vitro by using the target tumor material may better inform the therapeutic decision.15 In 2004, the American Society of Clinical Oncology failed to find a firm basis for integrating chemosensitivity and resistance assay into the clinical setting.16 Yet recent publications have shown a positive correlation between tumor response and a treatment tailored according to chemoresistance assays.12,17 Chemosensitivity assays analyze the sensitivity of a tumor to chemotherapy by measuring the number of tumor cells that are killed by a particular cancer drug. The test assumes that a short exposure to the drug, in concentrations at or below clinical peak plasma levels, is representative of tumor exposure in vivo. This assumption fails to account for the individual’s biologic reaction to the cytotoxic agent (eg, metabolism or vascular supply) and therefore cannot be extrapolated to the in vivo setting. Chemoresistance assays, on the other hand, expose tumors to chemotherapy agents for longer periods of time at higher concentrations. Tumor growth under such conditions in vitro might reasonably be expected to correlate with in vivo drug resistance.18,19 Indeed, Cortazar and associates14 demonstrated that individualized treatment was associated with prolonged survival for patients with small-cell lung cancer who were randomized to receive assay-directed therapy. A significant association between in vitro EDR to platinum and clinical outcomes was also observed in patients with ovarian cancer. Patients with tumors demonstrating in vitro EDR to platinum were at significantly increased risk for progression and death when treated with standard platinum-based regimens.8
The Journal of Thoracic and Cardiovascular Surgery c August 2010
General Thoracic Surgery
Overall and progression-free survival was significantly better in the EDR assay–guided treatment groups.8,9,12 The limitations of this study are twofold. The EDR assay used for this study only tested single drugs, whereas platinum-based combination treatment now is standard for MPM. Second, we were unable to test pemetrexed (Alimta), another drug commonly used to treat MPM, because the EDR assay relies on tritiated thymidine uptake as the end point for determining tumor resistance, and pemetrexed is an antifolate that increases thymidine uptake.20 Hence, an alternative assay that does not rely on tritiated thymidine incorporation would have to be developed. Despite these limitations, this study not only establishes the prevalence of chemotherapy resistance in cultured mesothelioma tumors, but also suggests differences in drug resistance based on histopathologic cell type. Our findings indicate that a significant proportion of MPM tumors have IDR or EDR to 3 common chemotherapeutic agents. Clinical implementation of this strategy awaits a proliferation test applicable to pemetrexed, the availability of additional chemotherapeutic options, and the integration of biology and genomic diagnostic tests to accompany the chemoresistance assay.21 The effectiveness of assay-directed therapy will need to be addressed in prospective trials. We acknowledge Dr John Mannick, Ann S. Adams, and William Francis Powell, Jr, for their valuable contributions to this article.
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