Effective Treatment of Advanced Human Melanoma Metastasis In

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Published OnlineFirst July 21, 2009; DOI: 10.1158/1078-0432.CCR-08-3275 Published Online First on July 21, 2009 as 10.1158/1078-0432.CCR-08-3275 Cancer Therapy: Preclinical

Effective Treatment of Advanced Human Melanoma Metastasis in Immunodeficient Mice Using Combination Metronomic Chemotherapy Regimens William Cruz-Munoz, Shan Man, and Robert S. Kerbel

Abstract Purpose: The development of effective therapeutic approaches for treatment of metastatic melanoma remains an immense challenge. Present therapies offer minimal benefit. Although dacarbazine chemotherapy remains the standard therapy, it mediates only low response rates, usually of short duration, even when combined with other chemotherapeutic agents. Thus, new therapeutic strategies are urgently needed. Experimental Design: Using a newly developed preclinical model, we evaluated the efficacy of various doublet metronomic combination chemotherapy against established advanced melanoma metastasis and compared these with the standard maximum tolerated dose dacarbazine (alone or in combination with chemotherapeutic agents or vascular endothelial growth factor receptor–blocking antibody). Results: Whereas maximum tolerated dose dacarbazine therapy did not cause significant improvement in median survival, a doublet combination of low-dose metronomic vinblastine and low-dose metronomic cyclophosphamide induced a significant increase in survival with only minimal toxicity. Furthermore, we show that the incorporation of the low-dose metronomic vinblastine/low-dose metronomic cyclophosphamide combination with a low-dose metronomic dacarbazine regimen also results in a significant increase in survival, but not when combined with maximum tolerated dose dacarbazine therapy. We also show that a combination of metronomic vinblastine therapy and a vascular endothelial growth factor receptor 2–blocking antibody (DC101) results in significant control of metastatic disease and that the combination of low-dose metronomic vinblastine/DC101 and low-dose metronomic dacarbazine induced a significant improvement in median survival. Conclusions: The effective control of advanced metastatic melanoma achieved by these metronomic-based chemotherapeutic approaches warrants clinical consideration of this treatment concept, given the recent results of a number of metronomic-based chemotherapy clinical trials.

Metastatic melanoma is generally associated with poor prog- agent chemotherapy with dacarbazine has long been the stan- nosis. The aggressiveness of the disease is compounded by the dard systemic treatment for metastatic melanoma, it is associated lack of effective treatment, with only modest progress being with low response rates of short duration (1, 2). Various combi- made over the last few decades in the discovery of chemother- nations of chemotherapeutic agents, some of which have inclu‐ apeutic drugs or other therapies, showing a meaningful clinical ded dacarbazine, have also shown only very modest effects on survival benefit in randomized clinical trials. Although single- increasing response rates and with little or no effect on improving median survival times (3–6). All of these approaches have involved conventional maximal tolerated dose regimens in

Authors' Affiliation: Sunnybrook Health Sciences Centre, Molecular and which prolonged breaks are used between treatments to allow Cellular Biology Research, Toronto, Ontario, Canada recovery from induced toxic side effects. Significant efforts have Received 12/18/08; revised 3/9/09; accepted 4/8/09; published OnlineFirst been made to enhance the efficacy of dacarbazine therapy by 7/21/09. using it in combination with other chemotherapeutic agents. Grant support: NIH (CA-41233), the National Cancer Institute of Canada, In the face of the disappointing results achieved thus far with and the Canadian Institutes for Health Research (R.S. Kerbel). The costs of publication of this article were defrayed in part by the payment of such maximum tolerated dose approaches, it is obvious that page charges. This article must therefore be hereby marked advertisement new drugs and/or treatment strategies are needed. In this regard, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the effectiveness of metronomic chemotherapy, in which drugs Requests for reprints: Robert S. Kerbel, Sunnybrook Health Sciences Cen- are given in low, minimally toxic doses at frequent regular inter- tre, Molecular and Cellular Biology Research, S-217, 2075 Bayview Avenue, vals with no prolonged breaks, usually over long periods of time Toronto, Ontario M4N 3M5, Canada. Phone: 416-480-5711; Fax: 416-480- 5884; E-mail: [email protected]. (7, 8), has been assessed in only a few situations in patients with F 2009 American Association for Cancer Research. metastatic or primary melanoma, thus far (9–12). Metronomic doi:10.1158/1078-0432.CCR-08-3275 chemotherapy regimens have several potential advantages,

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Cancer Therapy: Preclinical

may potentiate mechanisms leading to increased angiogenesis Translational Relevance and enhanced tumor growth, as well as metastasis. As such, therapeutic inhibition of VEGF inhibition might be an impor- Despite significant efforts over the last two dec- tant option to consider when designing effective dacarbazine- ades aimed at improving the efficacy of standard based approaches for treating melanoma metastasis. Previous treatment (maximum tolerated dose of dacarbazine), results from our laboratory indicate that treatment with a met- there has been no significant increase in the median ronomic combination of low-dose metronomic vinblastine survival of patients suffering from metastatic mela- and optimal biological doses of a VEGF receptor 2-inhibiting noma. Given the lack of success achieved, a rethink- antibody (DC101) results in sustained regression of estab- ing of alternative treatment strategies is needed. lished s.c. tumor (neuroblastoma) without inducing overt tox- Using preclinical models of advanced melanoma icity (18). Thus, we also examined whether the addition of the metastasis, we show that metronomic chemothera- antiangiogenic drug DC101 can enhance the effectiveness of peutic combinations results in improved survival, dacarbazine-based therapies for the treatment of advanced me- which is achieved with minimal toxicity. These re- tastatic melanoma. sults compare favorably with minimal effectiveness achieved by maximum tolerated dose dacarbazine therapy (alone or in combination with other chemo- Materials and Methods therapeutic agents or a vascular endothelial growth Preclinical models of spontaneous melanoma metastasis. factor receptor–blocking antibody), often accompa- The 113/6- 4L model of spontaneous melanoma metastasis has been previously nied by higher toxicity. Successes in preclinical set- described. The line was derived from the WM239 human melanoma cell ting of metastatic breast cancer have led to a clinical line by serial selection of lung metastases arising in mice after orthotopic trial to examine the efficacy of metronomic therapy. implantation of WM239 cells followed by surgical resection of the prima- Asimilar extension of the metronomic chemothera- ry tumor (16). Briefly, one million 113/6-4L melanoma cells were in- peutic combinations presented here into the clinical jected subdermally into CB-17 SCID female mice, and primary tumors setting of melanoma metastasis may be warranted. were allowed to develop to a size of 400 to 500 mm3, at which time they were resected. Mice in the control and treatment groups were monitored regularly in accordance with the institutional guidelines stipulated by the Sunnybrook Health Sciences Centre Animal Care Committee and sacri- including reduced acute toxic side effects, convenience when us- ficed when they showed signs of stress (e.g., breathing difficulty and ing oral drugs, and activity in some cases against refractory drug- severe weight loss). A similar approach was taken in the case of the resistant tumors; in addition, they are easily combined for pro- MDA-MB-435 model of spontaneous metastasis. This cell line was ini- longed periods with targeted biological therapies such as antian- tially isolated and studied as a breast cancer model but has now been giogenic drugs, with such combinations sometimes causing recognized as representative of a human melanoma based on genomic surprisingly robust and prolonged tumor responses (7, 8, 13–15). and protein profiling (19–23). Extensive visceral metastases were Evaluation of metronomic chemotherapy for the treatment observed in these models within 1 mo of removal of the primary tumor. Impact of metronomic, maximum tolerated dose, and combination of advanced metastatic disease was initially done in a model chemotherapy on survival. In the 113/6-4L model, chemotherapy using breast cancer xenografts (13). The underlying rationale was initiated 56 d after primary tumor resection, at which time exten- was that such models might have superior predictive value sive visceral metastatic tumor burden was present, especially evident in for showing a clinical benefit in patients having the respective lungs and liver (16). Mice were treated for long periods, between 100 to cancer with advanced metastatic disease. Our breast cancer 133 d in the various experiments done. The study consisted of three results showed that a highly effective treatment could be experiments: experiment 1 examined the efficacy of low-dose metro- achieved with the doublet combination of concurrent low- nomic vinblastine/low-dose metronomic cyclophosphamide versus dose metronomic oral cyclophosphamide and uracil-tegafur, maximum tolerated dose dacarbazine; experiment 2examined the effi- a fluorouracil oral prodrug (13). More recent work from our cacy of maximum tolerated dose dacarbazine and low-dose metronom- lab using the 113/6-4L preclinical model of advanced melano- ic dacarbazine, as well as whether the addition of low-dose metronomic ma metastasis, wherein visceral metastases in sites such as the vinblastine or low-dose metronomic vinblastine/low-dose metronomic cyclophosphamide could improve the efficacy of these dacarbazine- liver, lung, and lymph nodes arise from orthotopically trans- based regimens; whereas experiment 3 examined whether the incorpo- planted and then surgically resected tumors, has also shown ration of the low-dose metronomic vinblastine + DC101 combination that the use of low-dose metronomic chemotherapy using could enhance the efficacy of maximum tolerated dose or low-dose the combination of low-dose metronomic vinblastine (admin- metronomic dacarbazine regimens. Descriptions of all treatment regi- istered i.p. thrice per week) and low-dose metronomic oral cy- mens (including doses, routes of delivery, and number of mice) are clophosphamide results in a beneficial survival effect with outlined in Table 1. minimal toxicity (16). Here, we expand on this initial preclin- In the case of the MDA-MB-435 metastasis model, treatment was ini- ical therapy trial and examined how this doublet metronomic tiated 8 d post–primary tumor removal and continued for 84 d. Mice chemotherapy regimen compares with a standard maximum were assigned to specific treatment groups: group 1, control (normal n tolerated dose dacarbazine therapeutic regimen, as well as saline i.p. thrice weekly; = 11); group 2, low-dose metronomic vinblastine (0.33 mg/kg; i.p. thrice weekly) plus DC101 (800 mg/kg; i.p. whether the efficacy of maximum tolerated dose dacarbazine twice weekly; n = 10); and group 3, DC101 (800 mg/kg; i.p. twice can be enhanced by its combination with metronomic vin- weekly; n = 10). blastine/cyclophosphamide. Dacarbazine treatment is associat- The efficacy of these regimens on primary tumor growth was also ex- ed with the transcriptional up-regulation of genes encoding amined. In this case, 1 × 106 cells were implanted subdermally and pri- tumor growth promoting factors such as vascular endothelial mary tumors allowed to develop. Once tumors reached an average size of growth factor (VEGF) in melanoma cells (17). This up-regulation 200 mm3, treatment was initiated and given in the regimens described

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Treatment of Melanoma Metastasis by Metronomic Therapy above for control, low-dose metronomic vinblastine + DC101, and therapy patients (24) and highlights both the poor efficacy DC101. Additional treatment regimens included low-dose metronomic of this standard therapy, as well as the importance of devel- cyclophosphamide (20 mg/kg p.o.) + normal saline, low-dose metro- oping alternative and more effective therapies. nomic vinblastine (0.33 mg/kg; i.p. thrice weekly) + normal saline, Superior survival achieved by vinblastine/cyclophosphamide and low-dose metronomic cyclophosphamide (20 mg/kg p.o.) + combination metronomic therapy. DC101 (800 mg/kg; i.p. twice weekly). For this experiment, five mice Preliminary examination per group were used. Mice were monitored regularly, and the experiment of effectiveness of metronomic combination of low-dose met- was terminated when tumors reached an average size of 1,500 mm3. ronomic vinblastine/low-dose metronomic cyclophosphamide Statistical significance difference in survival outcomes between con- suggested that this therapy can also induce an increase in me- trol and each treatment group was done using log rank test (P < 0.05 dian survival times in our preclinical model of advanced mel- indicated statistical significance). Hazard ratio was equally done by anoma metastasis (16). Further assessment of the efficacy of comparing control versus each treatment group. Statistical analysis this combination showed that it mediates a significant in- was done with the aid of GraphPad Prism4 software. crease in median survival when compared with control (saline alone) with an improved hazard ratio (P = 0.026; hazard ra- Results tio, 0.29; Fig. 1A; Table 2). Importantly, long-term treatment with the low-dose metronomic vinblastine/low-dose metro- Maximum tolerated dose dacarbazine chemotherapy does not nomic cyclophosphamide combination was associated with improve survival of mice with advanced metastatic melanoma. minimal toxicity in metastasis-bearing mice, as denoted by a Using the 113/6-4L model, we examined the efficacy of stable body weight in the treatment group (Fig. 1B). Mice in standard maximum tolerated dose dacarbazine therapy and maximum tolerated dose dacarbazine groups maintained sta- compared the results to those achieved by observation ble body weight but showed signs of distress (scruffiness and alone. Our results show that maximum tolerated dose dacar- lethargy), which became more evident after each additional bazine therapy is associated with minimal increases in me- cycle of treatment. No such signs of distress were noted in dian survival, which are not significantly different form the low-dose metronomic vinblastine/low-dose metronomic control and associated with a high hazard ratio (P = 0.42; cyclophosphamide treatment group. Survival and hazard ratio hazard ratio, 0.71; Fig. 1A; Table 2). This result is similar to mediated by this metronomic chemotherapy regimen were su- those obtained in meta-analysis of patient outcomes com- perior to values achieved by maximum tolerated dose dacar- paring median survival of untreated and dacarbazine mono- bazine therapy alone.

Table 1. Description of regimens examined using the 113/6-4L model of advanced metastatic disease

Experiment 1 Treatment No. of mice

Saline Normal saline daily in drinking water and i.p. thrice weekly 17 LDM Vbl + LDM CTX Vbl 0.33 mg/kg i.p. thrice weekly; 20 mg/kg CTX p.o. 14 MTD DTIC DTIC 50 mg/kg i.p. in days 1-5 of a 21-d cycle 17

Experiment 2 Treatment No. of mice

Saline Normal saline daily in drinking water and i.p. thrice weekly 9 MTD DTIC DTIC 50 mg/kg i.p. in days 1-5 of a 21-d cycle 9 MTD DTIC + LDM Vbl DTIC 50 mg/kg i.p. in days 1-5 of a 21-d cycle 11 Vbl 0.33 mg/kg i.p. thrice weekly MTD DTIC + LDM Vbl + LDM CTX DTIC 50 mg/kg i.p. in days 1-5 of a 21-d cycle 6 Vbl 0.33 mg/kg i.p. thrice weekly; 20 mg/kg CTX p.o. LDM DTIC DTIC 5mg/kg i.p. thrice weekly 9 LDM DTIC + LDM Vbl DTIC 5mg/kg i.p. thrice weekly 11 Vbl 0.33 mg/kg i.p. thrice weekly LDM DTIC + LDM Vbl + LDM CTX DTIC 5 mg/kg i.p. thrice weekly 11 Vbl 0.33 mg/kg i.p. thrice weekly; 20 mg/kg CTX p.o.

Experiment 3 Treatment No. of mice

Saline Normal saline daily in drinking water and i.p. thrice weekly 8 LDM Vbl + DC101 Vbl 0.33 mg/kg i.p. thrice weekly; DC101 800 mg/kg i.p. twice weekly 7 LDM Vbl + LDM CTX + DC101 Vbl 0.33 mg/kg i.p. thrice weekly; 20 mg/kg CTX p.o. 8 DC101 800 mg/kg i.p. twice weekly MTD DTIC + LDM Vbl + DC101 DTIC 50 mg/kg i.p. in days 1-5 of a 21-d cycle 8 Vbl 0.33 mg/kg i.p. thrice weekly; DC101 800 mg/kg i.p. twice weekly LDM DTIC + LDM Vbl + DC101 DTIC 5mg/kg i.p. thrice weekly; Vbl 0.33 mg/kg i.p. thrice weekly 7 DC101 800 mg/kg i.p. twice weekly

NOTE: Vinblastine/cyclophosphamide and vinblastine/DC101 combinations were examined alone or in combination with maximum tolerated dose dacarbazine and low-dose metronomic DTIC. Abbreviations: DTIC, dacarbazine; MTD, maximum tolerated dose; LDM, low-dose metronomic; Vbl, vinblastine; CTX, cyclophosphamide.

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Cancer Therapy: Preclinical

Given the efficacy of low-dose metronomic vinblastine/low- dose metronomic cyclophosphamide combination therapy, we next examined whether addition of this combination to standard maximum tolerated dose dacarbazine or low-dose metronomic dacarbazine therapy could improve survival. In the case of maximum tolerated dose treatment, the addition of low-dose metronomic vinblastine alone or low-dose metronomic vinblastine/ low-dose metronomic cyclophosphamide combination resulted in an increase in median survival from 147 days for maximum tolerated dose dacarbazine to 178 and 168 days, respectively. However, this increase did not reach statistical significance when compared with control (P = 0.063; Fig. 2A; Table 2). Mice in these groups, especially the maximum tolerated dose dacarbazine/ low-dose metronomic vinblastine/low-dose metronomic cyclophosphamide group, showed signs of toxicity associated with weight loss (Fig. 2B). This toxicity highlights one of the limita- tions associated with maximum tolerated dose regimens, especially when it is used in polychemotherapeutic combinations. On the other hand, the addition of low-dose metronomic vinblastine alone or low-dose metronomic vinblastine/low-dose metronomic cyclophosphamide to low-dose metronomic dacarbazine resulted in significant increase in survival relative to control (Fig. 2C; Table 2) and moreover was associated with minimal toxicity (Fig. 2D). Interestingly, low-dose metronomic dacarbazine therapy was just as effective as the standard maximum tolerated dose dacarbazine regimen; however, the former had the added advantage of lower toxicity (Fig. 2E). Fig. 1. Efficacy of standard maximum tolerated dose dacarbazine in The combination of low-dose metronomic vinblastine/DC101 combination with metronomic vinblastine/cyclophosphamide in the results in improved survival in the MDA-MB-435 model of 113/6-4L model of advanced metastatic melanoma. A, examination of the efficacy of standard maximum tolerated dose dacarbazine and vinblastine/ spontaneous metastasis. We had previously shown in the cyclophosphamide metronomic chemotherapeutic regimens against MDA-MB-435 melanoma model of advanced metastatic dis- advanced melanoma metastatic disease. B, examination of body weights of mice treated with standard maximum tolerated dose dacarbazine, ease that DC101 alone exerts minimal effect on survival, where- vinblastine/cyclophosphamide, and saline (control). Error bars, ±SEM. as metronomic vinblastine alone could significantly prolong

Table 2. Summary of median survival times

Experiment 1 Median survival (d) P (vs saline) Hazard ratio

Control 156 MTD DTIC 172 0.420 0.71 LDM Vbl + LDM CTX Undefined 0.026 0.29

Experiment 2 Median survival (d) P (vs saline) Hazard ratio

Control 110 MTD DTIC 147 0.120 0.47 MTD DTIC + LDM Vbl 178 0.063 0.37 MTD DTIC + DLM Vbl + LDM CTX 168 0.063 0.35 LDM DTIC 154 0.115 0.47 LDM DTIC + LDM Vbl 192 0.026 0.35 LDM DTIC + LDM Vbl + LDM CTX 195.5 0.024 0.33

Experiment 3 Median survival (d) P (vs control) Hazard ratio

Control 156 MTD DTIC 191 0.66 0.76 MTD DTIC + LDM Vbl + DC101 189 0.33 0.59 LDM DTIC + LDM Vbl + DC101 Undefined 0.01 0.11 LDM Vbl + DC101 Undefined 0.11 0.3 LDM Vbl + LDM CTX + DC101 183 0.30 0.49

NOTE: Metronomic combinations showed improved survival compared with control. None of the therapies that incorporated maximum tolerated dose dacarbazine resulted in significant increase in survival.

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Treatment of Melanoma Metastasis by Metronomic Therapy survival (25). Here, our results show that the combination of metastatic melanoma. We examined whether incorporation of low-dose metronomic vinblastine and DC101 resulted in a the doublet low-dose metronomic vinblastine + DC101 combi- dramatic suppression of metastatic disease and resulted in a nation could enhance the efficacy of maximum tolerated dose or significant increase in survival (P < 0.0001; hazard ratio, low-dose metronomic dacarbazine chemotherapy. At the same 0.04; Fig. 3A). In addition, this combination was found to be time, we examined whether combination of low-dose metro- associated with significant inhibition of primary tumor growth nomic vinblastine + DC101 with low-dose metronomic cyclo- (Fig. 3B). No such inhibition was noted by treatment with phosphamide could result in increased antitumor effectiveness DC101 alone or in the case of low-dose metronomic vinblas- and survival. Using the 113/6-4L model, we found that low-dose tine + normal saline, low-dose metronomic cyclophosphamide metronomic vinblastine + DC101 did result in an improved out- + normal saline, or low-dose metronomic cyclophosphamide + come compared with that in control mice (P = 0.08; hazard ratio, DC101. The last two regimens had also shown no significant 0.2; Fig. 4A; Table 2). The beneficial effect of the low-dose met- effect on survival when used in the treatment of advanced ronomic vinblastine + DC101 therapy did not reach statistical metastatic disease (25). significance because a number of mice succumbed to toxicity me- The incorporation of metronomic vinblastine + DC101 to diated by long-term DC101 treatment (characterized by loss of low-dose metronomic dacarbazine results in effective control of weight; Fig. 4B). In these mice, no metastases were observed in

Fig. 2. Vinblastine and vinblastine/cyclophosphamide enhance survival when combined with low-dose metronomic dacarbazine therapy in the 113/6-4L preclinical model of advanced metastatic melanoma. A, survival curves associated with maximum tolerated dose dacarbazine as well as those in which this standard treatment was combined with vinblastine and vinblastine/cyclophosphamide in the preclinical treatment of advanced metastasis. B, body weights during the course of treatment with various regimens incorporating maximum tolerated dose DITC. C, survival curves associated with low-dose metronomic dacarbazine as well as those in which this treatment was combined with vinblastine and vinblastine/cyclophosphamide. D, body weights during the course of treatment with various chemotherapeutic combinations incorporating low-dose metronomic dacarbazine. E, survival curves associated with low-dose metronomic dacarbazine and maximum tolerated dose dacarbazine regimens. Error bars, ±SEM.

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Cancer Therapy: Preclinical

to improve efficacy; indeed, a large number of clinical trials remain underway evaluating such combinations.1 The testing of these combinations has not been generally evaluated precli- nically using primary tumor models, let alone those involving advanced metastatic disease. However, taking such empirical approaches into the clinic may lead to the unnecessary and premature exposure of patients to drugs for which adequate proof of activity has not been shown and for which possible toxicities have not been fully evaluated (26). Thus, preclinical evaluation of antimetastatic activity could be an essential step in the development of more effective therapeutic approaches for malignant melanoma. However, to increase relevance, such evaluation should be conducted in models that reflect the typ- ical presentation of metastatic disease frequently seen in patients involved in clinical trials, namely, advanced, high volume metastatic disease (25, 27). In this regard, we have observed several instances wherein the outcomes of antitumor activity using primary xenograft tumor models did not corre- late with antimetastatic activity (13, 16). Such a disconnect may be an important factor in the frequent disparity observed between promising preclinical therapeutic results using such primary tumor models and the subsequent, usually less im- pressive results observed in clinical trials. Because they more closely reflect the clinical presentation of disease, preclinical models of advanced metastatic disease likely represent a more useful tool to examine therapies to be used for the treatment of late-stage metastases (25, 28). In support of Fig. 3. Vinblastine/DC101 therapy suppresses metastatic disease and this argument, attention is drawn to the results of a recently re- primary tumor growth in the MDA-MB-435 model of advanced metastatic ported nonrandomized phase II clinical trial in metastatic disease. A, survival curves associated with vinblastine/DC101 and DC101 metronomic regimens in the treatment of advanced metastatic disease. breast cancer patients using a doublet metronomic chemother- B, examination of efficacy of vinblastine/DC101 and DC101 against growth apy combination, namely, daily cyclophosphamide and capeci- of primary MDA-MB-435 tumors. Error bars, ±SEM. tabine, an oral 5-FU prodrug, used in combination with bevacizumab, a monoclonal anti-VEGF antibody (29). This trial was undertaken, in part, based on our previous preclinical re- either the chest cavity or in the central nervous system. The sults showing the potent activity of long-term daily low-dose addition of cyclophosphamide or maximum tolerated dose cyclophosphamide with daily low-dose UFT (another 5-FU oral dacarbazine to the low-dose metronomic vinblastine + DC101 prodrug), which was used to treat established advanced visceral combination did not result in enhanced survival but was associ- metastatic human breast cancer using a single newly developed – ated with an exacerbation in toxicity (Fig. 4A D; Table 2). Inter- preclinical model of advanced spontaneous metastasis (13). estingly, a high proportion of mice (3 of 4) that succumbed to The 113/6-4L model of spontaneous melanoma metastasis metastatic disease in the maximum tolerated dose dacarba- used in the studies described herein recapitulates all the events zine/low-dose metronomic vinblastine/DC101 group showed and steps involved in the multistep process of metastasis seen extensive signs of central nervous system melanoma metastases; in clinic, including brain metastases (16). Using this model, we such a high frequency of central nervous system metastasis was have shown that metronomic chemotherapy combination regi- not observed in any other treatment group. Contrary to the lack mens are effective in controlling one of the most intractable of – of additional activity achieved by maximum tolerated dose metastatic diseases, late-stage melanoma metastasis and more- based therapies, the addition of low-dose metronomic vinblas- over do so with surprisingly little associated toxicity. Further- tine + DC101 to low-dose metronomic dacarbazine resulted in a more, our results show that the standard maximum tolerated dramatic suppression of metastatic disease and improved medi- dose dacarbazine therapy for treatment is not as effective, and P an survival ( = 0.01; hazard ratio, 0.11; Fig. 4E; Table 2). This other results suggest that, even when combined with other triplet combination also showed minimal toxicity in the treated agents (either chemotherapeutics or antiangiogenics), dacarba- mice (Fig. 4F) even after 13 weeks of continuous treatment. zine therapy provides minimal improvement in survival. The results we have obtained should be considered in the Discussion context of the numerous clinical trials that are underway exam- ining polychemotherapeutic approaches based on the use of Dacarbazine has minimal impact on survival of melanoma maximum tolerated dose dacarbazine. The antitumor effects patients. Indeed, its effectiveness has not been compared di- achieved by the low-dose metronomic vinblastine/low-dose rectly with observation alone, and as such, there is no clear metronomic cyclophosphamide or low-dose metronomic indication about whether it improves survival (24). Given its limited therapeutic activity, dacarbazine has been combined with a number of other chemotherapeutic drugs in an effort 1 http://clinicaltrials.gov

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Treatment of Melanoma Metastasis by Metronomic Therapy dacarbazine/low-dose metronomic vinblastine/DC101 treat- clophosphamide/UFT in previous breast cancer studies) against ments suggest that improved survival in melanoma patients advanced metastatic disease is often contrasted by their relative might be achieved by such combination metronomic therapy ineffectiveness against primary tumors (13, 16). This disparity approaches and, moreover, with lesser toxicity. Our data sug- may be ascribed to differential effects mediated by metronomic gest that consideration might be given to combining antiangio- therapy against endothelial cells in specific sites (e.g., lung pa- genic agents and dacarbazine using a metronomic regimen, renchyma metastases versus subdermal primary tumor). In perhaps immediately following upfront conventional dacarba- turn, this divergence in sensitivity may be attributed to the het- zine chemotherapy alone or in combination with an antiangio- erogeneity of endothelial cells present in different organ sites, as genic agent. The feasibility of such metronomic chemotherapy well as to differences in the microenvironment itself (e.g., pres- might be improved by substituting dacarbazine (an agent ad- ence of distinct growth factors, cytokines, etc.). Given that the ministered i.v.) with orally bioavailable agents such as temozo- primary target of metronomic chemotherapy schedules is pre- lomide, an agent that is equally effective as dacarbazine in the sumed to be the endothelial cell population of the neovascula- treatment of malignant melanoma but also shows superior ture of the tumor, it reasons that this antiangiogenic effect can penetration into the brain parenchyma (2, 30). be enhanced by the incorporation of "dedicated" antiangio- It is interesting to note that the effectiveness achieved by the genic agents. In this regard, the effective suppression of both metronomic chemotherapeutic regimens that we have exam- metastatic disease and primary tumor growth that was mediat- ined (low-dose metronomic vinblastine/low-dose metronomic ed by the combination of metronomic vinblastine and the cyclophosphamide in this study and low-dose metronomic cy- VEGF receptor –targeting antibody (DC101) suggests that this

Fig. 4. Examination of the effect of vinblastine/DC101 in the 113/6-4L model of spontaneous melanoma metastasis. A, survival curves associated with vinblastine/DC101 and vinblastine/DC101/cyclophosphamide regimens in the treatment of advanced metastatic disease in the 113/6-4L model. B, body weights during the course of treatment with vinblastine/DC101 or vinblastine/DC101/cyclophosphamide. C, survival curves associated with maximum tolerated dose dacarbazine or maximum tolerated dose dacarbazine + vinblastine + DC101 regimens in the treatment of metastatic disease. D, body weights during treatment with maximum tolerated dose dacarbazine or maximum tolerated dose dacarbazine/vinblastine/DC101 regimens. E, survival curves associated with metronomic regimen incorporating low-dose metronomic dacarbazine + vinblastine + DC101 in the treatment of advanced melanoma metastasis. F, body weights during the course of treatment with low-dose metronomic dacarbazine + vinblastine + DC101 therapy. Error bars, ±SEM.

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Cancer Therapy: Preclinical combination therapy approach is more effective against tumors noted herein, consideration should be given to evaluating the growing in different sites. efficacy of the metronomic chemotherapy approach in the In addition to the current lack of effective therapies for ad- adjuvant setting. vanced melanoma metastasis, there are also few alternatives for adjuvant therapy. Presently, interferon-α-2b is the only Disclosure of Potential Conflicts of Interest Food and Drug Administration–approved agent for the treat- R.S. Kerbel holds a Tier I Canada Research Chair in Tumor Biology, ment of melanoma patients who are free of disease but at Angiogenesis, and Antiangiogenic Therapy. high risk for recurrence (31). However, despite the initial promise, this agent has been shown to induce only a small Acknowledgments improvement on overall survival; moreover, this minimal beneficial effect is achieved with high levels of toxicity (31, We thank ImClone Systems for their gift of the VEGF receptor targeting 32). Given the efficacy and minimal toxicity of the regimens antibody, DC101.

References 1. Comis RL. DTIC (NSC-45388) in malignant melano- murine melanoma model. Int J Cancer 2007; 21. Christgen M, Lehmann U. MDA-MB-435: the ma: a perspective. Cancer Treat Rep 1976;60:165–76. 121:666–74. questionable use of a melanoma cell line as a 2. Middleton MR, Grob JJ, Aaronson N, et al. Ran- 12. Reichle A, Bross K, Vogt T, et al. Pioglitazone model for human breast cancer is ongoing. domized phase III study of temozolomide versus and rofecoxib combined with angiostatically Cancer Biol Ther 2007;6:1355–7. dacarbazine in the treatment of patients scheduled trofosfamide in the treatment of far- 22. Rae JM, Ramus SJ, Waltham M, et al. Common with advanced metastatic malignant melanoma. advanced melanoma and soft tissue sarcoma. origins of MDA-MB-435 cells from various sources J Clin Oncol 2000;18:158–66. Cancer 2004;101:2247–56. with those shown to have melanoma properties. 3. Ringborg U, Jungnelius U, Hansson J, Strander 13. Munoz R, Man S, Shaked Y, et al. Highly effica- Clin Exp Metastasis 2004;21:543–52. H. Dacarbazine-vindesine-cisplatin in dissemi- cious nontoxic preclinical treatment for advanced 23. Ellison G, Klinowska T, Westwood RF, et al. nated malignant melanoma. Aphase I-II trial. metastatic breast cancer using combination oral Further evidence to support the melanocytic Am J Clin Oncol 1990;13:214–7. UFT-cyclophosphamide metronomic chemother- origin of MDA-MB-435. Mol Pathol 2002;55: 4. Buzaid AC, Murren J. Chemotherapy for ad- apy. Cancer Res 2006;66:3386–91. 294–9. vanced malignant melanoma. Int J Clin Lab 14. Emmenegger U, Morton GC, Francia G, et al. 24. Khan MA, Andrews S, Ismail-Khan R, et al. Res 1992;21:205–9. Low-dose metronomic daily cyclophosphamide Overall and progression-free survival in meta- 5. Chapman PB, Einhorn LH, Meyers ML, et al. and weekly tirapazamine: a well-tolerated combi- static melanoma: analysis of a single-institution Phase III multicenter randomized trial of the nation regimen with enhanced efficacy that ex- database. Cancer Control 2006;13:211–7. Dartmouth regimen versus dacarbazine in pa- ploits tumor hypoxia. Cancer Res 2006;66:1664–74. 25. Kerbel RS. Human tumor xenografts as predic- tients with metastatic melanoma. J Clin Oncol 15. Pietras K, Hanahan D. Amultitargeted, metro- tive preclinical models for anticancer drug activ- 1999;17:2745–51. nomic, and maximum-tolerated dose "chemo- ity in humans: better than commonly perceived 6. Eigentler TK, Caroli UM, Radny P, Garbe C. Pal- switch" regimen is antiangiogenic, producing —but they can be improved. Cancer Biol Ther liative therapy of disseminated malignant mela- objective responses and survival benefit in a mouse 2003;2:S134–9. noma: a systematic review of 41 randomised model of cancer. J Clin Oncol 2005;23:939–52. 26. Gogas HJ, Kirkwood JM, Sondak VK. Chemo- clinical trials. Lancet Oncol 2003;4:748–59. 16. Cruz-Munoz W, Man S, Xu P, Kerbel RS. Devel- therapy for metastatic melanoma: time for a 7. Kerbel RS, Kamen BA. The anti-angiogenic ba- opment of a preclinical model of spontaneous change?. Cancer 2007;109:455–64. sis of metronomic chemotherapy. Nat Rev Can- human melanoma central nervous system me- 27. Bibby MC. Orthotopic models of cancer cer 2004;4:423–36. tastasis. Cancer Res 2008;68:4500–5. for preclinical drug evaluation: advantages 8. Kerbel RS, Klement G, Pritchard KI, Kamen B. Con- 17. Lev DC, Ruiz M, Mills L, et al. Dacarbazine causes and disadvantages. Eur J Cancer 2004;40: tinuous low-dose anti-angiogenic/metronomic transcriptional up-regulation of interleukin 8 and 852–57. chemotherapy: from the research laboratory into vascular endothelial growth factor in mela‐ 28. Man S, Munoz R, Kerbel RS. On the develop- the oncology clinic. Ann Oncol 2002;13:12–5. noma cells: a possible escape mechanism from ment of models in mice of advanced visceral 9. Hermans IF, Chong TW, Palmowski MJ, et al. chemotherapy. Mol Cancer Ther 2003;2:753–63. metastatic disease for anti-cancer drug testing. Synergistic effect of metronomic dosing of cy- 18. Klement G, Baruchel S, Rak J, et al. Continu- Cancer Metastasis Rev 2007;26:737–47. clophosphamide combined with specific antitu- ous low-dose therapy with vinblastine and VEGF 29. Dellapasqua S, Bertolini F, Bagnardi V, et al. mor immunotherapy in a murine melanoma receptor-2 antibody induces sustained tumor re- Metronomic cyclophosphamide and capecita- model. Cancer Res 2003;63:8408–13. gression without overt toxicity. J Clin Invest bine combined with bevacizumab in advanced 10. Spieth K, Kaufmann R, Gille J. Metronomic 2000;105:R15–24. breast cancer. J Clin Oncol 2008;26:4899–905. oral low-dose treosulfan chemotherapy com- 19. Lacroix M. MDA-MB-435 cells are from mela- 30. Quirbt I, Verma S, Petrella T, et al. Temozolo- bined with cyclooxygenase-2 inhibitor in pre- noma, not from breast cancer. Cancer Che- mide for the treatment of metastatic melanoma. treated advanced melanoma: a pilot study. mother Pharmacol 2008;63:567. Curr Oncol 2007;14:27–33. Cancer Chemother Pharmacol 2003;52:377–82. 20. Rae JM, Creighton CJ, Meck JM, et al. MDA- 31. Lawson DH. Choices in adjuvant therapy of 11. Jia LJ, Wei DP, Sun QM, et al. Tumor-targeting MB-435 cells are derived from M14 melanoma melanoma. Cancer Control 2005;12:236–41. Salmonella typhimurium improves cyclophos- cells—a loss for breast cancer, but a boon for 32. Ascierto PA, Kirkwood JM. Adjuvant therapy phamide chemotherapy at maximum tolerated melanoma research. Breast Cancer Res Treat of melanoma with interferon: lessons of the past dose and low-dose metronomic regimens in a 2007;104:13–9. decade. J Transl Med 2008;6:62.

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Effective Treatment of Advanced Human Melanoma Metastasis in Immunodeficient Mice Using Combination Metronomic Chemotherapy Regimens

William Cruz-Munoz, Shan Man and Robert S. Kerbel

Clin Cancer Res Published OnlineFirst July 21, 2009.

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