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Journal of Clinical Oncology, Vol 26, No 14 (May 10), 2008: pp. 2299-2304 © 2008 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.14.5292 Phase II Study of Extended-Dose Temozolomide in Patients With Melanoma
From the Departments of Medicine, Radiology, Pathology, Clinical Laboratories, and Biostatistics and Epidemiology, Memorial Sloan-Kettering Cancer Center, New York, NY Corresponding author: Paul B. Chapman, MD; Memorial Sloan-Kettering Cancer Center; 1275 York Ave, Room Z1402; New York, NY 10065; e-mail: chapmanp{at}mskcc.org
Purpose We conducted a phase II trial of extended-dose temozolomide (TMZ) in patients with melanoma to test the hypothesis that the approximately 30% response rate observed in patients treated with extended-dose TMZ with antiangiogenic agents was caused by TMZ alone. We hypothesized that expression of methylguanine methyltransferase (MGMT) in the tumor would correlate with drug resistance to TMZ. Patients and Methods Patients with stage IV or unresectable stage III melanoma were treated with TMZ 75 mg/m2/d for 6 weeks followed by a 2-week rest period. Cycles were repeated until progression. Patients were stratified by M1c disease or not. The primary end point was objective response proportion. MGMT expression was assessed by methylation-specific pyrosequencing of the promoter and by immunohistochemistry. Results Forty-nine patients (25 with M1c disease) were assessable. Three patients (12.5%) in each cohort experienced partial responses; there were no complete responses. Ten patients (21%) had stable disease lasting more than 24 weeks. Median time to progression was 3.3 months. Median survival was 10.1 months; survival was similar in the two cohorts. The estimated 18-month survival was 27%. There was no correlation between response and either immunohistochemistry staining for MGMT or for MGMT promoter methylation. Seventy-five percent of patients developed CD4+ lymphopenia after three cycles. Conclusion Extended-dose TMZ therapy did not result in a 30% responses rate, which has been observed using extended-dose TMZ with antiangiogenic agents. Response did not correlate with MGMT expression or promoter methylation as a continuous variable, suggesting that other resistance mechanisms are important.
Patients with advanced melanoma have a poor prognosis; the reported median survival ranges from 3 to 11 months, depending on the subgroup analyzed. Dacarbazine (DTIC) induces objective tumor responses in only 5% to 20% of patients. Most responses are partial, and there is no evidence that DTIC or combinations of chemotherapy drugs prolong median survival, although complete responses do occur and are occasionally durable. Temozolomide (TMZ) is an orally bioavailable prodrug that, like DTIC, converts to the active compound 5(3-methyltriazen-1-yl)imidazole-4-carboxamide. TMZ administered at 150 to 200 mg/m2 x 5 days every 4 weeks showed equivalent response rates to DTIC in patients with metastatic melanoma.1
We2-4 and others5-7 have reported the use of TMZ using an extended dosing schedule of 75 mg/m2/d x 6 weeks every 8 weeks. This schedule results in chronic drug exposure over 6 weeks and delivers 50% more drug over 8 weeks than does the standard 5-day TMZ regimen. Because TMZ depletes levels of methylguanine methyltransferase (MGMT), a DNA repair enzyme thought to play a role in TMZ resistance7,8 by repairing alkylated guanine nucleotides, extended dosing of TMZ should allow prolonged TMZ exposure after MGMT has been depleted. Further, extended dosing may take advantage of the metronomic low-dose chemotherapy strategy9-11 thought to shrink tumors through antiendothelial effects. In fact, there is evidence that TMZ at low concentrations can have antiangiogenic effects.12 Two phase II trials that combined extended-dose TMZ with thalidomide13 or with pegylated interferon- Prior trials using TMZ in patients with glioblastoma reported an association between antitumor response and silencing of the MGMT gene as measured by increased MGMT promoter methylation.15,16 Earlier melanoma studies in which MGMT expression was measured retrospectively by immunohistochemistry (IHC) or bioassay could not identify an association between MGMT expression and sensitivity of melanoma to DTIC17 or TMZ.7 We wished to study prospectively the association between intratumoral MGMT and response to TMZ in patients with melanoma. We previously observed that TMZ induces lymphopenia in a large proportion of patients and affects CD4+ T cells predominantly.3 In this phase II trial, we planned to test whether TMZ inhibits CD4+ T-cell function before causing a decrease in CD4+ T-cell numbers.
Patients This single-institution phase II trial was open to patients with stage IV or unresectable stage III melanoma. Patients could not have had prior chemotherapy, although prior interleukin (IL)-2 or adjuvant immunotherapy was allowed. Patients with mucosal or uveal melanomas were excluded. Patients had to have measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST) criteria, Karnofsky performance status (KPS) of at least 60%, and adequate organ function: absolute neutrophil count more than 1,500/µL, platelets more than 150,000/µL, creatinine less than 2.0 mg/dL, and liver function tests less than 1.5x upper limits of normal. Patients who had brain metastases (unless treated and free from recurrence for at least 6 months), history of HIV, or were taking immunosuppressive drugs were excluded. Neither concurrent anticancer therapy nor high-dose vitamin/herb intake were allowed. The trial was approved by the Memorial Sloan-Kettering (MSKCC; New York, NY) institutional review board.
Treatment Before each treatment cycle, patients underwent CBC, comprehensive chemistry panel, lactate dehydrogenase, and T-cell subset analysis (CD3, CD4, CD8). CD4+ T-cell counts lower than 200/µL were considered severe lymphopenia, and these patients received pneumocystis pneumonia (PCP) prophylaxis. Tumors were evaluated by physical examination and/or radiographically before treatment and at the end of each treatment cycle. All radiographic tumor measurements were confirmed by a single reference radiologist (S.G.). Tumor responses were evaluated according to RECIST criteria.
Biostatistics
T-Cell Functional Studies
For validation purposes, 27 healthy donors were tested in parallel with patient samples. Some of these donors were assayed at several time points and some samples were assayed several times to determine ranges of normal variation. The lower limit of detection was 1 CD4+CD69+IFN
Detection of MGMT
Using a hematoxylin and eosin–stained slide as a guide, paraffin-embedded tumor tissue was manually microdissected. DNA was extracted and 1 µg of sample DNA was bisulfate-treated using the Zymo DNA Methylation Kit (Zymo Research, Orange, CA). Bisulfite-treated DNA was eluted in 10 µL volume. PCR fragments were amplified in a PCR reaction mix containing 1 µL of bisulfite-converted DNA (test samples) or internal controls, 10x PCR buffer, 3.0 mmol/L MgCl2, 200 µmol/L of each dNTP, 0.2 µmol/L each of forward and reverse primers (Table A1, online only), and 1.25 U of HotStart DNA polymerase (Qiagen Inc, Valencia, CA). Products were immobilized and denatured at 80°C for 2 minutes. For the pyrosequencing reaction, the corresponding sequencing primer (Table A1) was added to the single stranded DNA and nucleotides dispensed automatically by the PSQ HS 96A instrument and software (Biotage, Uppsala, Sweden). For each reaction, peripheral-blood mononuclear cells served as unmethylated controls, the human colon cancer cell line SW48 served as the methylated control. Pyrograms were read at least twice. For IHC analysis, 5-µm sections were deparaffinized and rehydrated in xylene and a series of graded alcohols. Sections were blocked with 3% bovine-specific antigen/phosphate-buffered saline (PBS) solution. For detection of MGMT, monoclonal antibody MT3.1 (Kamiya Biomedical Co, Seattle, WA) was used. Antigen retrieval was performed by immersing slides in preheated (95°C) EDTA buffer solution (1 mmol/L, pH 8.0) for 30 minutes. The primary antibody (0.5 µg/mL) was incubated overnight at 4°C and then detected by biotinylated horse-antimouse secondary antibody (Vector Labs, Burlingame, CA) followed by an avidin-biotin system (ABC-Elite, Vector Labs). Diaminobenzidine (liquid DAB, Biogenex, San Ramon, CA) served as a chromogen. Finally, slides were counterstained with Gill's hematoxylin. Slides were evaluated in a blinded manner by a single pathologist (A.A.J.) and scored 0 to 4+ as previously described.20
Patient Demographics Fifty patients (25 in each cohort) were accrued between January 2005 and June 2007. All patients were assessable for response except for one patient in the M1c cohort who died within 2 weeks of start of treatment. The characterization of the 50 patients is presented in Table 1. There were no differences in sex proportion or age between the patients in the two cohorts. All patients had a KPS of 90% except for two patients in the stage III/M1a/M1b cohort who had a KPS of 70%. The median number of treatment cycles administered was two (range, one to six). Thirty-two patients have died; 17 are still alive. The median follow-up of the patients still alive is 12.1 months (range, 1.8 to 29.4 months) as of September 2007.
Clinical Responses Overall, six patients showed partial responses (12.5%), three in each cohort; the median duration of response was 7.7 months. There were no complete responses. The median time to progression was 3.3 months, corresponding to two cycles of treatment. Twenty-two patients (45%) had stable disease after the first 8-week cycle, and 10 patients (21%) had stable disease for at least three cycles. Thus, there were 16 patients (33%) who had either an objective antitumor response or stable disease for at least 24 weeks. The median survival of all patients was 10.1 months (Fig 2). The overall survival in each of the cohorts was not significantly different from each other (not shown). The estimated survival at 18 months was 27%.
Toxicities Extended-dose TMZ was generally well-tolerated. The most common grade 3/4 toxicities were lymphopenia and anemia (Table 2). Nausea, when seen, was generally limited to the first 3 days of each cycle. Typically, antiemetics were discontinued by day 3 of each cycle.
Effects on Circulating T Lymphocytes We3 and others7 have observed that TMZ induces lymphopenia. In this study, T-cell subsets were measured in all patients at the start of each cycle. In some patients, T-cell subsets were also measured at week 4. Figure 3 shows the cumulative effect of 1, 2, or 3 cycles of TMZ on mean numbers of total CD3+, CD4+, and CD8+ T cells. TMZ induced a steady decline in CD3+ T cells, which was largely a result of CD4+ T-cell depletion (Table 3). After two cycles of TMZ, 64% of patients had severe CD4+ lymphopenia (defined as CD4+ < 200/µL); after three cycles, 75% of patients had severe CD4+ lymphopenia. TMZ had less of an effect on CD8+ T cells. After two cycles of TMZ, only seven of 22 (32%) of patients had CD8+ lymphopenia.
All patients who developed severe CD4+ lymphopenia received PCP prophylaxis. No opportunistic infections were observed in patients on this trial, although three patients developed dermatomal Herpes zoster.
CD4+ T-Cell Function Studies At the pretreatment time point, we found that only 12 (44%) of 27 patients had detectable CD4+ T-cell responses against CMV. There was no difference between the cohorts, and this was not significantly different from the proportion of normal healthy donors with detectable CD4+ T-cell responses against CMV. In patients, the median number of the anti-CMV T cells was four per 1,000 CD4+ T cells; for healthy controls, we observed a median of seven per 1,000 CD4+ T cells, which was not a significant difference. Among the 12 patients with detectable CD4+ T cell responses against CMV at baseline, five showed a decrease in anti-CMV CD4+ T-cell reactivity during the first cycle of TMZ, five patients showed no change, and two showed an increase (Fig A1 , online only).
MGMT Analysis For 12 patients, we had sufficient tumor material to stain for MGMT expression by IHC. There was no detectable MGMT expression in seven patients including one patient who had a partial response. Three patients had 1+ staining, including one patient who had a partial response. Two patients exhibited strong staining, neither of whom had a clinical response. Thus, in this small subset of patients, there was no correlation between MGMT staining and clinical response. Eight of the patients with tumor available for IHC staining also had tumor analyzed for MGMT promoter methylation. We found that promoter methylation more than 12% correlated with negative staining for MGMT (Fisher's exact test P = .0179). This suggests that partial promoter methylation is sufficient to turn off expression of MGMT below the level detectable by IHC. This needs to be confirmed with a larger data set.
Because our prior studies of extended-dose TMZ combined with either thalidomide or interferon-  in patients with metastatic melanoma resulted in response rates of approximately 30%, this trial was powered to test whether extended-dose TMZ alone induced a response rate of 30% or more. We observed a response rate of 12.5% in each cohort, and so were not able to reject the null hypothesis. We observed no difference between the two cohorts stratified on the basis of M1c disease. Previous response rates using the standard 5-day regimen have been similar to our results and ranged from 13.5% to 17.3%.1,7,21,22 Although these trials cannot be compared in a formal manner, our results are consistent with the hypothesis that the extended-dose schedule is not more effective than the standard 5-day schedule in inducing responses.
Contrary to our expectations, we were not able to reproduce the response rates observed when patients were treated with extended-dose TMZ combined with an antiangiogenic agent. In phase II trials, response rates were 32% when thalidomide was added13 and 31% when interferon- In retrospective analyses, we2-4 and others5-7 have shown that TMZ can induce lymphopenia. The current study confirms this in a prospective manner. We had also previously shown that the lymphopenia is largely a result of loss of CD4+ T cells. In this trial, we showed that severe CD4+ lymphopenia was very uncommon after a single cycle of extended-dose TMZ but the incidence of lymphopenia increased with additional treatment cycles. Among patients who completed three cycles, 75% had severe CD4+ lymphopenia. We measured CD4+ T-cell function (reactivity to CMV) during cycle 1 in 12 patients and found decreased function in five patients. This suggests that TMZ may have effects on T-cell function as well as T-cell number. One of the methylation targets of TMZ is the O6 position of guanine, which is repaired by MGMT. Loss of MGMT expression, as measured by MGMT promoter methylation, has been correlated with improved response rate to TMZ in glioblastoma15 and glioma,16 and with progression-free survival in glioblastoma.15 In melanoma, studies to date have not shown a correlation with response to DTIC17 or TMZ,7 although different methods of assessing MGMT were used. In one case, IHC was used to distinguish between tumors less than 50% positive and more than 50% positive; in the other case, melanoma lysates were assayed for MGMT activity. Efforts to inhibit MGMT have not been successful to date.21
In our study, we measured the degree of MGMT promoter methylation and found a low level of methylation ( Our initial hypothesis was that extended-dose TMZ would induce objective responses in at least 30% of patients; our results do not support this hypothesis. Response rates of 30% (twice the response rate observed in the current trial) were observed in two previous phase II trials combining TMZ with antiangiogenic agents. Although these results cannot be compared in a formal manner, it seems reasonable to investigate further the addition of antiangiogenic agents to extended-dose TMZ in future trials. It would be highly desirable to be able to predict which tumors were sensitive to TMZ. Although quantitative MGMT promoter methylation as a continuous variable did not correlate with sensitivity to TMZ, our data suggest that there may be a threshold of methylation that correlates with response. This should be tested in future studies.
Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: Paul B. Chapman, Schering-Plough (C) Stock Ownership: None Honoraria: None Research Funding: Paul B. Chapman, Schering-Plough Expert Testimony: None Other Remuneration: None
Conception and design: Jedd D. Wolchok, Paul B. Chapman Provision of study materials or patients: Petra Rietschel, Jedd D. Wolchok, Susan Krown, Paul B. Chapman Collection and assembly of data: Scott Gerst, Achim A. Jungbluth, Klaus Busam, Katherine Smith, Paul B. Chapman Data analysis and interpretation: Petra Rietschel, Achim A. Jungbluth, Katherine Smith, Irene Orlow, Katherine Panageas, Paul B. Chapman Manuscript writing: Petra Rietschel, Susan Krown, Achim A. Jungbluth, Katherine Smith, Irene Orlow, Katherine Panageas, Paul B. Chapman Final approval of manuscript: Petra Rietschel, Jedd D. Wolchok, Susan Krown, Scott Gerst, Achim A. Jungbluth, Klaus Busam, Katherine Smith, Irene Orlow, Katherine Panageas, Paul B. Chapman
We acknowledge Sharon Lu and Liying Yan (EpigenDx, Inc) for technical expertise, Christy Giordano and Jacqueline Simpronio for data management, and Virginia Murphy, Carolyn Quinn, and Ruth Roman for nursing assistance critical for the successful treatment of the patients.
Supported by a grant from Schering-Plough Corp. Presented in part at the 43rd Annual Meeting of the American Society of Clinical Oncology, June 1-5, 2007, Chicago, IL. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Middleton MR, Grob JJ, Aaronson N, et al: Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma. J Clin Oncol 18:158-166, 2000 2. Hwu WJ, Krown SE, Panageas KS, et al: Temozolomide plus thalidomide in patients with advanced melanoma: Results of a dose-finding trial. J Clin Oncol 20:2610-2615, 2002 3. Su YB, Sohn S, Krown SE, et al: Selective CD4 lymphopenia in melanoma patients treated with temozolomide: A toxicity with therapeutic implications. J Clin Oncol 22:610-616, 2004 [Erratum: J Clin Oncol 22:2038, 2004] 4. Krown SE, Hwu WJ, Mennell JH, et al: A phase II study of temozolomide and pegylated interferon alpha2b in the treatment of advanced melanoma. J Clin Oncol 22:715s, 2004 (suppl; abstr 7533) 5. Brock CS, Newlands ES, Wedge SR, et al: Phase I trial of temozolomide using an extended continuous oral schedule. Cancer Res 58:4363-4367, 1998 6. Stupp R, Dietrich PY, Kraljevic SO, et al: Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide. J Clin Oncol 20:1375-1382, 2002 7. Middleton MR, Lunn JM, Morris C, et al: O6-methylguanine-DNA methyltransferase in pretreatment tumour biopsies as a predictor of response to temozolomide in melanoma. Br J Cancer 78:1199-1202, 1998[Medline] 8. Middleton MR, Thatcher N, McMurry TB, et al: Effect of O6-(4-bromothenyl)guanine on different temozolomide schedules in a human melanoma xenograft model. Int J Cancer 100:615-617, 2002[CrossRef][Medline] 9. Hermans IF, Chong TW, Palmowski MJ, et al: Synergistic effect of metronomic dosing of cyclophosphamide combined with specific antitumor immunotherapy in a murine melanoma model. Cancer Res 63:8408-8413, 2003 10. Glode LM, Barqawi A, Crighton F, et al: Metronomic therapy with cyclophosphamide and dexamethasone for prostate carcinoma. Cancer 98:1643-1648, 2003[CrossRef][Medline] 11. Bertolini F, Paul S, Mancuso P, et al: Maximum tolerable dose and low-dose metronomic chemotherapy have opposite effects on the mobilization and viability of circulating endothelial progenitor cells. Cancer Res 63:4342-4346, 2003 12. Kurzen H, Schmitt S, Naher H, et al: Inhibition of angiogenesis by non-toxic doses of temozolomide. Anticancer Drugs 14:515-522, 2003[CrossRef][Medline] 13. Hwu WJ, Krown SE, Menell JH, et al: Phase II study of temozolomide plus thalidomide for the treatment of metastatic melanoma. J Clin Oncol 21:3351-3356, 2003 14. Hwu WJ, Panageas KS, Menell JH, et al: Phase II study of temozolomide plus pegylated interferon-alpha-2b for metastatic melanoma. Cancer 106:2445-2451, 2006[Medline] 15. Hegi ME, Diserens A-C, Gorlia T, et al: MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352:997-1003, 2005 16. Paz MF, Yaya-Tur R, Rojas-Marcos I, et al: CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas. Clin Cancer Res 10:4933-4938, 2004 17. Ma S, Egyhazi S, Ueno T, et al: O6-methylguanine-DNA-methyltransferase expression and gene polymorphisms in relation to chemotherapeutic response in metastatic melanoma. Br J Cancer 89:1517-1523, 2003[CrossRef][Medline] 18. Colella S, Shen L, Baggerly KA, et al: Sensitive and quantitative universal pyrosequencing methylation analysis of CpG sites. Biotechniques 35:146-150, 2003[Medline] 19. Ronaghi M: Pyrosequencing for SNP genotyping. Methods Mol Biol 212:189-195, 2003[Medline] 20. Jungbluth AA, Busam KJ, Gerald WL, et al: A103: An anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues. Am J Surg Pathol 22:595-602, 1998[CrossRef][Medline] 21. Ranson M, Hersey P, Thompson D, et al: Randomized trial of the combination of lomeguatrib and temozolomide compared with temozolomide alone in chemotherapy naive patients with metastatic cutaneous melanoma. J Clin Oncol 25:2540-2545, 2007 22. Kaufmann R, Spieth K, Leiter U, et al: Temozolomide in combination with interferon-alfa versus temozolomide alone in patients with advanced metastatic melanoma: A randomized, phase III, multicenter study from the Dermatologic Cooperative Oncology Group. J Clin Oncol 23:9001-9007, 2005 23. Amaravadi RK, Schuchter LM, Kramer A, et al: Updated results of a randomized phase II study comparing two schedules of temozolomide in combination with sorafenib in patients with advanced melanoma. J Clin Oncol 25:478s, 2007 (suppl; abstr 8527) 24. Trivedi RN, Almeida KH, Fornsaglio JL, et al: The role of base excision repair in the sensitivity and resistance to temozolomide-mediated cell death. Cancer Res 65:6394-6400, 2005 Submitted October 9, 2007; accepted January 25, 2008.
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Copyright © 2008 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
in response to cytomegalovirus (CMV) were detected in whole blood by intracellular cytokine staining (FastImmune CD4 Intracellular Cytokine Detection Kit, BD Biosciences, San Diego, CA) following manufacturer's instructions. Cells were analyzed on a BD FACSCalibur flow cytometer. Patients were scored as having had an increase (or decrease) in CMV-reactive T-cell function if there was more than a two-fold increase (or decrease) in the number of CMV-reactive CD4+ T cells/106 CD4+ T cells. 
2 test). However, we found that there was a correlation of partial response with MGMT promoter methylation level more than 25% (
10%) in half of the tumors tested. There was no statistical correlation with clinical response, although we noticed that levels of promoter methylation more than 25% correlated with partial response. This observation is worthy of further study in larger numbers of patients. In a small number of patients, we measured tumor MGMT expression by IHC and similarly found no correlation with clinical response. Thus, our results are consistent with prior studies in melanoma. Mechanisms of resistance other than MGMT have been proposed in melanoma, such as loss of mismatch repair or activation of base-excision repair mechanisms.
