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Originally published as JCO Early Release 10.1200/JCO.2004.07.060 on January 15 2004 © 2004 American Society of Clinical Oncology. Selective CD4+ Lymphopenia in Melanoma Patients Treated With Temozolomide: A Toxicity With Therapeutic ImplicationsFrom the Clinical Immunology and Infectious Disease Services, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY Address reprint requests to Paul B. Chapman, MD, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021; e-mail: chapmanp{at}mskcc.org
PURPOSE: Standard schedule temozolomide (TMZ; daily for 5 days every 4 weeks) is often used in melanoma patients, but phase III data show that it is no more effective than standard dacarbazine. Extended TMZ dosing regimens may be superior by delivering the drug continuously at a higher dose over time. Using an extended dosing schedule, we noted a high incidence of lymphopenia and occasional opportunistic infections (OIs). Here we report our retrospective experience in the first 97 patients. MATERIALS AND METHODS: TMZ was administered at 75 mg/m2/d orally for 6 weeks every 8 weeks, although nine patients were treated continuously without a break. Seventeen patients were treated with TMZ alone; 73 patients received TMZ with thalidomide; seven patients received TMZ with low-dose interferon alfa.
RESULTS: Median duration of TMZ treatment was 113 days; 29% received CONCLUSION: TMZ at this dose and schedule results in CD4+ lymphopenia in a majority of patients that can result in OIs. Pneumocystis pneumonia prophylaxis should be considered for patients who develop sustained lymphopenia on TMZ.
Standard therapy for metastatic melanoma usually includes dacarbazine (DTIC) alone or in combination with other chemotherapeutic and/or immunomodulatory drugs. Response rates to DTIC alone generally range from 5% to 20%, and few responses are complete. Many DTIC-based combination treatment regimens have been described and although response rates as high as 40% to 50% have been reported, no treatment has been shown in randomized trials to be superior to DTIC. Temozolomide (TMZ) is an orally available drug that is converted spontaneously to 5-(3-methyltriazen-1-yl)imidazole-4-carboximide, the active metabolite of DTIC. TMZ has been approved by the US Food and Drug Administration for treatment of anaplastic astrocytoma at a dose of 150 to 200 mg/m2 orally (PO) daily for 5 days every 4 weeks. This dosing schedule has also been used extensively in melanoma, and although tumor responses have been seen, the overall response rate was only 13.5% in a phase III randomized trial, not significantly different from the response rate to DTIC [1]. In addition, there was no significant improvement in median survival. An extended dosing regimen was explored by Brock et al [2] who found that TMZ could be administered at a dose of 75 mg/m2/d for 6 to 7 weeks followed by a 1 week rest period. We were attracted to this extended dosing schedule for melanoma because it results in continuous exposure of tumor cells to the drug and delivers a substantially higher total drug dose over a 2-month cycle compared to the standard regimen. Given this, and the disappointing results from the phase III trial administering TMZ for 5 days every 4 weeks, we incorporated an extended dosing schedule of TMZ into our clinical trials and began using the extended dosing schedule in 1999 in melanoma patients not eligible for protocols at Memorial Sloan-Kettering Cancer Center (MSKCC; New York, NY). An unexpected toxicity of this treatment was lymphopenia and opportunistic infections indicative of T-cell dysfunction. In this study, we report our retrospective experience with TMZ-induced lymphopenia in the first 97 assessable patients with metastatic melanoma. Although this is a retrospective analysis, it points out an important and novel toxicity of TMZ using an extended dosing schedule.
Patients and TMZ Treatment Between November 1999 and February 2002, 105 patients with American Joint Commission on Cancer stage II, III, or IV melanoma were treated at MSKCC either with TMZ alone, TMZ plus thalidomide, or TMZ plus interferon alfa using an extended dosing schedule. Patients who received other chemotherapy or radiation therapy with TMZ were excluded from the analysis. All patients were treated at MSKCC. Adequate follow-up was not available for eight patients and they were excluded from the study population. Of the 97 remaining patients, one had stage II, 15 had stage III, and 81 had stage IV disease (Table 1). In 88 of the patients, TMZ was administered PO at a dose of 75 mg/m2/d in cycles of 6 weeks followed by a 2-week break; in nine patients, TMZ was administered continually for more than 6 weeks without a break. Seventeen patients were treated with TMZ alone and seven patients received TMZ along with interferon alfa at doses of  2 million units/d. Seventy-three patients were treated with TMZ and thalidomide at a thalidomide dose of 200 mg/d with dose escalation to a maximum dose of 400 mg/d. In patients older than 70 years, thalidomide was started at a dose of 100 mg and escalated to a maximum dose of 250 mg/d. Forty-nine of the patients treated with thalidomide were participants in a phase I/II clinical trial ongoing at MSKCC, and for these patients, written informed consent was obtained. The other patients received TMZ with thalidomide off protocol.
This retrospective analysis was performed with institutional review board approval which determined that this was exempt research under 45 CFR 46.101.b.
Identification and Definition of Cases Opportunistic infections (OIs) were defined a priori to be Pneumocystis pneumonia (PCP) or systemic fungal infections. To be counted as TMZ-associated, the OI had to be diagnosed either while the patient was taking TMZ or after TMZ had been stopped but before any other cytotoxic chemotherapy or radiotherapy had been started. Cases of Varicella zoster, Herpes simplex, and mucocutaneous Candidiasis were recorded as infections indicative of possible immune suppression and T-cell dysfunction but were not strictly defined as OIs. Infections that occurred while patients were taking steroids (two patients with mucocutaneous Candidiasis) were not considered as TMZ-related. Research records, clinical charts, laboratory, and microbiologic records were reviewed. All patients who developed lymphopenia or OIs were identified.
Statistical Analysis
The median duration of TMZ treatment in all 97 patients was 113 days (range, 25 to 745). Twenty-nine percent of the patients received treatment for more than 24 weeks.
Development of Lymphopenia
We observed little evidence of generalized bone marrow suppression among the patients treated with TMZ. Only seven patients (7%) developed neutropenia and eight patients (8%) developed thrombocytopenia. As a result, we cannot attribute the lymphopenia to a generalized suppressive effect on bone marrow function. We did note that 18.5% (n = 18) of patients developed eosinophilia on TMZ treatment, which was not associated with respiratory or vascular symptoms. We tested whether the development of lymphopenia was associated with other factors such as age, sex, stage of melanoma, concomitant treatment with thalidomide, interferon alfa, or steroids. The proportion of patients developing lymphopenia was not associated with any of these variables. There was a trend for lymphopenia to be more frequent in the nine patients treated with TMZ without a break during each 8-week cycle compared to the 79 patients treated with a 2-week break every 8-week cycle (88.9% v 57%; P = .08). Although the small number of patients in this group makes it difficult to come to a definite conclusion about the association of the 2-week treatment break and lymphopenia, patients treated without a break received 25% more TMZ over an 8-week cycle and so it may not be surprising that this group experienced a more profound effect on lymphocyte counts. In 17 of the patients who developed lymphopenia, TMZ was discontinued for various reasons and complete blood counts were available during the period after TMZ treatment before additional therapy was administered. In these patients, we were able to follow the duration of lymphopenia after TMZ was discontinued. Lymphopenia persisted over a range of 9 to 245+ days. Using Kaplan-Meier analysis, the estimated proportion of patients who were still lymphopenic 2 months after discontinuing TMZ was 61% (Fig 2). Because of the small number of patients in this analysis, the 95% CI is fairly wide (37% to 86%), and at later time points, the confidence intervals were too wide to be meaningful. However, this analysis indicates that the lymphopenia induced by TMZ is long-lasting and implies that TMZ affects an early lymphocyte progenitor.
Analysis of Lymphopenia In 11 patients who developed lymphopenia (median CD3+ T-cell nadir, 291/µL; range, 76 to 438), T-cell subsets were studied by flow cytometry at various time points during the lymphopenia. The characteristics of the 11 patients are shown in Table 2. In all 11 patients, the absolute CD4+ T-cell counts were depressed (median CD4+ lymphocyte nadir, 150/µL; range, 44 to 346); the absolute CD8+ T-cell counts were also decreased in five of the patients (Fig 3). In three of the lymphopenic patients who had received TMZ alone (one without a break, two with a break every 6 weeks), we measured CD4+CD25+ T-cell subsets, B cells (CD3-CD19+), and natural killer cells (CD3-, CD56+, and CD16+) at various time points (Fig 4). In all three patients tested, the CD4+CD25+ T-cell subset was markedly depressed. B-cell numbers were generally within the lower limits of normal or slightly decreased; the natural killer cell number was normal. These data indicate that the lymphopenic effect of TMZ is relatively specific for the CD4+ subset although CD8+ T cells and B cells can be affected as well.
Opportunistic Infections Two patients developed documented OIs while on TMZ treatment; there was one case of PCP and one case of Aspergillus pneumonia. Both of these cases were in lymphopenic patients who were being treated on TMZ without a break and both recovered after appropriate antimicrobial therapy. Two additional lymphopenic patients developed presumptive PCP based on symptoms, radiographic imaging, gallium scans, and recovery after treatment with trimethoprim/sulfamethoxazole and steroids, although these were not biopsy-proven. One of these patients had been treated with a 2-week break, the other on a continuous schedule without a break. We noted a fourth patient who developed lymphopenia on TMZ and who developed biopsy-proven PCP 1 month after finishing 15 months of TMZ treatment (using the interrupted schedule) and 7 months after lomustine had been added to the patient's TMZ regimen. In addition to cases of PCP, we observed a variety of infections indicative of T-cell dysfunction, but not formally considered an OI, in 21 patients. There were four cases of Herpes simplex (one proven, three probable based on clinical assessment), four cases of Herpes zoster (one proven, three probable based on clinical assessment), 11 patients with mucocutaneous Candidiasis, and one proven case of rapidly progressive classic Kaposi's sarcoma (KS). In this melanoma patient who also had classic KS confined to the foot that did not require therapy, two cycles of TMZ treatment induced lymphopenia accompanied by the development of numerous KS lesions and lymphedema involving the entire leg, which required treatment with liposomal doxorubicin. This observation is of interest due to the viral etiology of KS and its strong association with immunodeficiency in patients with AIDS and solid organ transplants [3]. All of the patients who developed infections were lymphopenic except for two patients who developed Herpes zoster and two patients who developed mucocutaneous Candidiasis.
As we and others have demonstrated, TMZ can be administered at low daily doses using an extended treatment schedule [2,4-6]. The theoretical advantage of this approach over the standard dose and schedule (150 to 200 mg/m2/d for 5 days every 4 weeks) is that extended dosing results in prolonged exposure of tumor cells to the drug. In addition, the extended dosing schedule delivers a total dose of TMZ over 8 weeks that is 57% higher than the 5-day course at a dose of 200 mg/m2/d. Given that the standard dose and schedule of TMZ is not more effective than standard DTIC [1], we have used an extended dosing schedule routinely in treating melanoma patients. In our article we report that this treatment regimen results in an observed lymphopenia rate of 60% (and an estimated rate of 77% for patients treated at least 200 days) without inducing generalized suppression of bone marrow function. The estimated median time to lymphopenia was 101 days. TMZ-induced lymphopenia appears to be long-lived, as an estimated 61% of lymphopenic patients were still lymphopenic 2 months after drug discontinuation. This was a retrospective analysis; one of the potentially confounding variables is that patients were treated with TMZ alone, TMZ with thalidomide, or TMZ with interferon alfa, and nine patients were treated using an uninterrupted TMZ dosing schedule. However, concomitant treatment with thalidomide was not associated with an increased risk of lymphopenia. There were too few patients treated with TMZ and interferon alfa to allow the incidence of lymphopenia in this group to be compared meaningfully with the incidence of lymphopenia in patients treated with TMZ alone. In patients treated with TMZ using the continuous dosing schedule, there was a trend towards an increased risk of lymphopenia. Another potentially confounding variable is that since most patients were not part of a clinical trial, the timing of blood counts was not uniform across our study population. This could affect the precision of our estimates of time to lymphopenia and time to recovery, but does not affect our overall conclusions. In patients receiving TMZ according to the standard dose and schedule, lymphopenia seems to be uncommon. For example, in the randomized phase III trial in which 156 patients were treated with TMZ, lymphopenia was not reported [1]. However, lymphopenia is not always noted as a toxicity in clinical trials and it is possible that the true incidence of lymphopenia using standard doses of TMZ may be underreported. For the same reason, there is little information about lymphopenia after DTIC therapy; this needs to be assessed. Lymphopenia has been seen in patients with brain cancer treated with TMZ using an extended dosing schedule [6,7], although no detailed analysis of the lymphopenia was reported. In one report, two of 64 patients reportedly developed PCP, but these patients were also pancytopenic and receiving steroids and radiation [7]. Other cytotoxic drugs are known to cause lymphopenia, especially purine analogues fludarabine and 2-chlorodeoxyadenosine, which also can predispose patients to OIs. However, these drugs usually result in generalized marrow suppression and isolated lymphopenia is relatively rare. After marrow recovery, T lymphocyte recovery can be significantly delayed [8,9], but this is quite different from our TMZ-treated patients in whom we rarely observed neutropenia or thrombocytopenia. There is evidence that fludarabine can also cause immunosuppression by interfering with STAT1 signaling by depleting STAT1 protein [10], raising the possibility that chemotherapy drugs can interfere with lymphocyte function as well as lymphocyte number. We found that TMZ primarily affected the CD4+ T-cell compartment, although in some patients CD8+ T cells and B cells were also decreased. The CD4+CD25+ subset was among the CD4+ T cells that were depleted by TMZ. This phenotype identifies regulatory T cells that function to suppress T-cell responses [11]. Depletion of regulatory T cells can markedly enhance CD8+ T-cell immunity in animal models [11-13]. The TMZ-induced CD4+ lymphopenia we observed was long-lived. For example, in one patient, a specific CD4+ lymphopenia has persisted for more than 5 months off TMZ, despite normal numbers of CD8+ lymphocytes, neutrophils, and platelets. The lymphocyte-specific effects of TMZ might be as a result of effects either on the thymus or at the level of a common lymphoid precursor. We cannot rule out the possibility that TMZ is preferentially transported into CD4+ lymphocytes and causes cytotoxic effects specifically within the CD4+ compartment, although this mechanism would not be expected to result in the long-term lymphopenia we observed. Lymphopenia observed in our patients treated with TMZ was clinically significant in that patients developed OIs, some of which were potentially life-threatening. A striking feature was the occurrence of PCP. One lymphopenic patient developed PCP during TMZ treatment and a second lymphopenic patient developed PCP just after finishing TMZ and after another cytotoxic drug had been added to the patient's TMZ regimen. In addition, we saw two other lymphopenic patients with presumptive PCP. In a review of all PCP cases over the past 25 years at Memorial Sloan-Kettering Cancer Center, there had not been a single case of PCP in a melanoma patient [14]. The occurrence of four cases of PCP in this 3-year period in patients who developed lymphopenia on the TMZ extended dosing schedule highlights the uniqueness of this adverse effect and indicates that the extended dosing schedule of TMZ is significantly different than treatment strategies used in the past (eg, DTIC, biochemotherapy) which had not increased the risk of PCP. Physicians treating patients with TMZ using this dose and schedule should consider administering PCP prophylaxis to patients who develop lymphopenia. Two of the patients who developed Herpes zoster infections were not lymphopenic. It is possible that these represented sporadic cases and were not related to TMZ treatment. Alternatively, it is possible that TMZ induces a functional defect in lymphocytes not necessarily reflected by lymphopenia. Although little is known about the effects of TMZ on lymphocyte function, TMZ can suppress lymphocyte proliferation in vitro in response to interleukin-2 or allogeneic cells [15]. Recently, Wick et al [16] have shown that TMZ can activate caspase 3 in glioma cells leading to focal adhesion kinase degradation and the loss of the invasive phenotype. These observations suggest the hypothesis that TMZ might interfere with lymphocyte function through disruption of intracellular signaling pathways. Therefore, it would be of interest to study lymphocyte function in patients treated with TMZ. Our limited T-cell analysis indicated that CD4+ lymphocytes were uniformly depleted with TMZ. This lymphopenia was long-lived and we speculate that TMZ at this dose and schedule could be useful in treating malignancies of CD4+ lymphocytes such as cutaneous T-cell lymphomas and other T-cell malignancies. In this regard, it is interesting to note that in a phase I trial using TMZ on a 5-day schedule, the only patient with mycosis fungoides experienced a complete response [17]. Based on our experience, it would be of interest to test the hypothesis that the 6-week dosing schedule would be even more effective. The CD4+ lymphopenia induced by TMZ may have complex implications for experimental vaccine therapy. CD4+ T-cell help is very important for immunity against melanoma in mouse models. Vaccines meant to induce T-cell responses might be compromised by CD4+ lymphopenia induced by TMZ and patients who have been treated with TMZ may be poor vaccine candidates. Alternatively, our observation that TMZ depleted CD4+CD25+ regulatory T cells suggests that TMZ might offer a pharmacologic strategy that could actually lead to an augmented immune responses. For instance, selective depletion of CD4+CD25+ T cells enhances immunity induced by vaccine in mouse models [12]. This is an area that deserves further investigation.
The authors indicated no potential conflicts of interest.
We are grateful to Drs Alan N. Houghton and Wen-Jen Hwu for critically reading the manuscript and making many valuable suggestions, to Dr Marcel R.M. van den Brink for helpful discussions, and to Katherine Smith of the Clinical Cellular Immunology Laboratory for the flow cytometry analyses.
Supported in part by NIH grants CA81293 and AI52239-01. Presented in part in abstract form at the 38th Annual Meeting of the American Society of Clinical Oncology, May 18-21, 2002, Orlando, FL and the 40th Annual Meeting of the Infectious Diseases Society of America, October 24-27, 2002, Chicago IL. Authors' disclosures of potential conflicts of interest 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. 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 3. Biggar RJ, Rabkin CS: The epidemiology of AIDS–related neoplasms. Hematol Oncol Clin North Am 10:997-1010, 1996[CrossRef][Medline] 4. Margolin K, Atkins B, Thompson A, et al: Temozolomide and whole brain irradiation in melanoma metastatic to the brain: A phase II trial of the Cytokine Working Group. J Cancer Res Clin Oncol 128:214-218, 2002[CrossRef][Medline]
5. 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
6. Khan RB, Raizer JJ, Malkin MG, et al: A phase II study of extended low-dose temozolomide in recurrent malignant gliomas. Neuro-oncol 4:39-43, 2002
7. Stupp R, Dietrich PY, Ostermann Kraljevic S, 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
8. Mackall CL, Fleisher TA, Brown MR, et al: Distinctions between CD8+ and CD4+ T-cell regenerative pathways result in prolonged T-cell subset imbalance after intensive chemotherapy. Blood 89:3700-3707, 1997 9. Azuma E, Nagai M, Qi J, et al: CD4+ T-lymphocytopenia in long-term survivors following intensive chemotherapy in childhood cancers. Med Pediatr Oncol 30:40-45, 1998[CrossRef][Medline] 10. Frank DA, Mahajan S, Ritz J: Fludarabine-induced immunosuppression is associated with inhibition of STAT1 signaling. Nat Med 5:444-447, 1999[CrossRef][Medline] 11. Sakaguchi S, Sakaguchi N, Shimizu J, et al: Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: Their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 182:18-32, 2001[CrossRef][Medline]
12. Sutmuller RP, van Duivenvoorde LM, van Elsas A, et al: Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 194:823-832, 2001
13. Kursar M, Bonhagen K, Fensterle J, et al: Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses. J Exp Med 196:1585-1592, 2002
14. Sepkowitz KA, Brown AE, Telzak EE, et al: Pneumocystis carinii pneumonia among patients without AIDS at a cancer hospital. JAMA 267:832-837, 1992
15. Alvino E, Pepponi R, Pagani E, et al: O(6)-benzylguanine enhances the in vitro immunotoxic activity of temozolomide on natural or antigen-dependent immunity. J Pharmacol Exp Ther 291:1292-1300, 1999
16. Wick W, Wick A, Schulz JB, et al: Prevention of irradiation-induced glioma cell invasion by temozolomide involves caspase 3 activity and cleavage of focal adhesion kinase. Cancer Res 62:1915-1919, 2002 17. Newlands ES, Blackledge GR, Slack JA, et al: Phase I trial of temozolomide (CCRG 81045: M&B 39831: NSC 362856). Br J Cancer 65:287-291, 1992[Medline] Submitted July 9, 2003; accepted October 29, 2003.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
24 weeks of therapy. Lymphopenia was seen in 60% of patients (absolute lymphocyte count < 800/µL) with a median of 101 days to lymphopenia. TMZ did not cause significant neutropenia or thrombocytopenia. Lymphopenia was not more common in patients treated concomitantly with thalidomide. In all patients analyzed for lymphocyte subsets, lymphopenia induced by TMZ affected the CD4+ compartment preferentially. There were two documented OIs (Pneumocystis and Aspergillus pneumonia) as well as other infections indicative of T-cell dysfunction in another 21 patients. 
2 or Fisher's exact test where appropriate. Differences in continuous variables, such as age at start of TMZ treatment and median duration of lymphopenia, were analyzed using nonparametric methods. The proportion of patients developing lymphopenia over time was estimated using the Kaplan-Meier method and plotted using Prism (Version 3.03, GraphPad Software Inc, San Diego, CA). Univariate and multivariate analyses were performed using Cox regression models. All reported P values are two-sided with P 
