Bevacizumab Plus Fotemustine As First-Line Treatment in Metastatic Melanoma Patients: Clinical Activity and Modulation of Angiogenesis and Lymphangiogenesis Factors

Published OnlineFirst October 28, 2010; DOI: 10.1158/1078-0432.CCR-10-2363

Clinical Cancer Cancer Therapy: Clinical Research

Bevacizumab plus Fotemustine as First-line Treatment in Metastatic Melanoma Patients: Clinical Activity and Modulation of Angiogenesis and Lymphangiogenesis Factors

Michele Del Vecchio1, Roberta Mortarini2, Stefania Canova1, Lorenza Di Guardo1, Nicola Pimpinelli3, Mario R. Sertoli4, Davide Bedognetti4, Paola Queirolo5, Paola Morosini6, Tania Perrone7, Emilio Bajetta1, and Andrea Anichini2

Abstract Purpose: To assess the clinical and biological activity of the association of bevacizumab and fotemustine as first-line treatment in advanced melanoma patients. Experimental Design: Previously untreated, metastatic melanoma patients (n ¼ 20) received bevacizumab (at 15 mg/kg every 3 weeks) and fotemustine (100 mg/m2 by intravenous administration on days 1, 8, and 15, repeated after 4 weeks) in a multicenter, single-arm, open-label, phase II study. Primary endpoint was the best overall response rate; other endpoints were toxicity, time to progression (TTP), and overall survival (OS). Serum cytokines, angiogenesis, and lymphangiogenesis factors were monitored by multiplex arrays and by in vitro angiogenesis assays. Effects of fotemustine on melanoma cells, in vitro, on vascular endothelial growth factor (VEGF)-C release and apoptosis were assessed by ELISA and flow cytometry, respectively. Results: One complete response, 2 partial responses (PR), and 10 patients with stable disease were observed. TTP and OS were 8.3 and 20.5 months, respectively. Fourteen patients experienced adverse events of toxicity grade 3–4. Serum VEGF-A levels in evaluated patients (n ¼ 15) and overall serum proangiogenic activity were significantly inhibited. A significant reduction in VEGF-C levels was found in several post-versus pretherapy serum samples. In vitro, fotemustine inhibited VEGF-C release by melanoma cells without inducing significant cell death. Serum levels of interleukin (IL)-10 and IL-12p70 showed the highest levels in sera of PR patients, compared with patients with stable or progressive disease whereas IL- 23 showed the opposite pattern. Conclusions: The combination of bevacizumab plus fotemustine has clinical activity in advanced melanoma and promotes systemic modulation of angiogenesis and lymphangiogenesis factors. Clin Cancer Res; 16(23); 5862–72. 2010 AACR.

Authors' Affiliations: 1Unit of Medical Oncology 2, Department of Medical Oncology, 2Human Tumors Immunobiology Unit, Department of Experi- Treatmentofadvancedmelanomaremainsunsatisfac- mental Oncology and Molecular Medicine, Fondazione IRCCS Istituto tory, because no therapy has demonstrated to affect over- 3 Nazionale dei Tumori, Milan; Department of Dermatological Sciences all survival (OS), with the exception of a recent and Department of Human Pathology and Oncology, University of Flor- ence, Florence; 4Department of Oncology, Biology, and Genetics, Uni- immunotherapy study based on administration of the versity of Genova; 5Department of Medical Oncology A, Istituto Nazionale anti-CTLA-4 monoclonal antibody (mAb) ipilimumab 6 per la Ricerca sul Cancro, Genova; Medical Affairs, Roche S.p.A., Monza; with/without a gp100 vaccine (1). Moreover, there is and 7Scientific Direction, Italfarmaco, Cinisello Balsamo, Italy no evidence that polychemotherapy is better than mono- Note: Supplementary data for this article are available at Cancer therapy, and no regimen can be considered of choice in Research Online (http://cancerres.aacrjournals.org/). metastatic disease. Nowadays, the CVD combination Current address for S. Canova: Unit of Medical Oncology, S. Gerardo (cisplatin, vinblastine or vindesine, and dacarbazine) is Hospital, 20052 Monza, Italy. widely used (2), as it can yield high response rates with Current address for E. Bajetta: Institute of Oncology, Policlinico di Monza, 20052 Monza, Italy. manageable toxicity. However, all phase III trials have M. Del Vecchio and R. Mortarini contributed equally to this work. failed to show an effective advantage of the combined therapy over the single approaches (immunotherapy or Corresponding Author: Andrea Anichini, Department of Experi- mental Oncology and Molecular Medicine, Fondazione IRCCS Istituto chemotherapy; ref. 3, 4), with the exception of a phase III Nazionale dei Tumori, 20133 Milan, Italy. Phone: 39-0223902817; Fax trial by Eton et al. (2). In such study, comparing CVD 39-0223903237; E-mail: [email protected] or Emilio Bajetta, Institute of Oncology, Policlinico di Monza, 20052 Monza, Italy. versus CVD plus interferon (IFN)-a and interleukin (IL)- Phone: 39-0392810661; Fax: 39-0392810331; E-mail: emilio.bajetta@ 2, in 190 melanoma patients, the response rate was 48% policlinicodimonza.it. for the combination versus 25% for CVD (P ¼ 0.001), doi: 10.1158/1078-0432.CCR-10-2363 time to progression (TTP) was 4.9 versus 2.4 months (P ¼ 2010 American Association for Cancer Research. 0.008), and the median survival was 11.9 versus 9.2

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Translational Relevance On the basis of these clinical data, we carried out a phase II study to investigate the clinical and biological There is an urgent need for improved therapies in activity of the bevacizumab–fotemustine combination in advanced cutaneous melanoma. So far only ipilimumab untreated metastatic melanoma patients. On the basis of has shown significant impact on overall survival, recent evidence (14, 15) of the relevance of serum biomar- although durable complete responses have been kers (detected by multiplex assays) as prognostic factors of observed in some patients treated with high-dose inter- response to therapy with biological agents such as IFN-a2b leukin 2. However, in randomized trials, biochemother- or IL-2, we designed multiplex ELISA arrays to detect 16 apy regimens have been shown to improve response different angiogenesis, lymphangiogenesis, and immuno- rates in advanced disease, thus suggesting that this is a modulatory factors. The results of the study indicated that promising approach that could be further developed by the combination of bevacizumab plus fotemustine has new combinations of cytotoxic drugs and biological clinical activity and modulates both angiogenesis and agents. In this study, we show that the combination lymphangiogenesis factors. of the antiangiogenesis monoclonal antibody bevacizumab with the nitrosourea fotemustine has clinical Materials and Methods activity as first-line treatment in metastatic disease. Moreover, this biochemotherapy regimen significantly Patients reduced systemic levels not only of vascular endothelial Eligible patients were at least 18 years of age, with growth factor (VEGF)-A, but also of VEGF-C, suggesting histologically or cytologically confirmed measurable that this therapeutic approach may contribute to the diagnosis of metastatic, inoperable nonchoroidal mela- inhibition of both angiogenesis and lymphangiogen- noma; an Eastern Cooperative Oncology Group (ECOG) esis, 2 relevant processes in melanoma development performance status (PS) of 0 to 1, no previous treatment and progression. of metastatic disease (exception for a vaccination with a wash-out period of at least 4 weeks), normal hematologic, hepatic, and renal functions; absence of brain metastases; and life expectancy of at least 3 months. Exclusion criteria months (P ¼ 0.006). Among the new chemotherapeutic were history of bleeding, diathesis, or coagulopathy, treatments of advanced melanoma, fotemustine is a pro- clinically significant cardiovascular disease, arterial mising drug, and there is a growing interest for bevaci- thromboembolism less than 6 months before the first zumab, a recombinant humanized mAb that binds to and study treatment, uncontrolled hypertension, and a his- inhibits the biological activity of human vascular tory of other malignancy within the past 5 years (except endothelial growth factor (VEGF)-A (see ref. 5 for review). curatively treated nonmelanoma skin cancer or carci- Bevacizumab is clinically active in different solid tumors noma in situ of the cervix). The trial was approved by (6–8): in colorectal cancer the addition of bevacizumab the Institutional Review Board or ethics committee of to irinotecan/fluorouracil/leucovorin has been shown to each participating center and was conducted in accor- improve survival of metastatic patients (9). Bevacizumab- dance with the principles of the Declaration of Helsinki based approaches in advanced melanoma were reported and with the Good Clinical Practice. All patients provided in a randomized phase II trial comparing bevacizumab written, informed consent. versus bevacizumab plus low-dose IFN-a2b (10). In that study,8of32patients(5inthebevacizumabarmand3in Study design and treatment the bevacizumab plus IFN-a2b arm) showed prolonged This was a multicenter, single-arm, open-label, phase II stabilization of disease (10). Additional evidence has study. The primary endpoint was the best tumor response been reported more recently, in a phase II biochemother- at any time during therapy. Secondary objectives were apy study based on paclitaxel, carboplatin, and bevaci- toxicity assessment, duration of response, TTP, and OS. zumab (11). The authors reported that 17% of the Eligible patients were treated with a combination of bev- patients achieved partial remission and 57% obtained acizumab and fotemustine. Fotemustine (Muphoran, stable disease (SD) for at least 8 weeks (11). Servier; Thissen Laboratories) dose was 100 mg/m2 (intra- Fotemustine is a cytotoxic alkylating agent of the nitro- venous infusion over 1 hour) and was given on days 1, 8, sourea family showing activity in melanoma (12). In a and 15 (induction phase), to be repeated after 4 weeks randomized trial (13) comparing fotemustine with dacar- every 21 days (maintenance phase). Bevacizumab (Avastin; bazine, as first-line chemotherapy in 229 patients, with or Roche Ltd.) was intravenously administered at a dose of 15 without brain metastases, overall response rate in the mg/kg every 3 weeks, after chemotherapy (i.e., at day 1, 21, intent-to-treat population was respectively 15.5% versus 42, and so on), initially over 90 minutes, and then over 60 6.8% (P ¼ 0.043). Interestingly, among patients without to 30 minutes according to degree of tolerance. Before brain metastases at inclusion, median time to development starting each cycle, patients underwent physical examina- of central nervous system relapses was 22.7 months in the tion, toxicity assessment, and complete biochemistry and fotemustine arm versus 7.2 months in the group receiving hematologic tests. If the absolute neutrophil count (ANC) dacarbazine (P ¼ 0.059). was less than 2,000/mm3, or if the platelets were less than

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100,000/mm3, treatment was delayed for 1 week. If these Imaging System). Analysis of SearchLight images was car- low counts persisted, the dose of fotemustine was reduced ried out with Array Analyst software (Pierce). Nonlinear by 25%. This dose reduction was maintained for the regression analysis by Prism software (GraphPad) was then successive cycles. If the ANC was less than 1,500/mm3, used to fit the following variable slope, 4-parameter equa- Y ¼ þð the dose of fotemustine was reduced by 50%. The same tion ofÀÁ the standard curve: Bottom Top dose was maintained for the successive cycles. No dose BottomÞ= 1 þ 10½ðlog EC50XÞHill Slope . The arrays were modifications of bevacizumab were done unless the sub- designed to detect and quantitate the following molecules: ject’s weight changed by more than 10%, in which case the VEGF-A, VEGF-C, VEGF-R1, VEGF-R2, sE-selectin, sP-selec- dose was recalculated. Further mAb administration was tin, soluble intercellular adhesion molecule (sICAM)-1, halted if the patient developed any of the following toxi- C-reactive protein (CRP), IL-8, IL-10, CXCL10, IL-12p70, cities attributable to bevacizumab: grade 4 toxicity, grade IL-17, IL-23, IFN-g, and tumor necrosis factor (TNF)-a.In 3 toxicity that did not resolve to grade 1 or less within addition to patients’ sera, a pool of sera from 5 healthy 4 weeks, and arterial thromboembolic events, gastrointest- donors was also evaluated for each of the 16 molecules. inal perforation. If fotemustine was held or delayed for toxicity reasons, bevacizumab was delayed as well and In vitro angiogenesis assay was resumed with the chemotherapy. A treatment delay Overall proangiogenic activity present in the serum of was requested until ANC and platelet count recovery enrolled patients was evaluated by an in vitro angiogenic 3 (1,500 and 100,000/mm , respectively). In the case assay (AngioKit; CellWorks). AngioKit wells, containing of a dose delay of 1 of the 2 study drugs for more than human endothelial cells at the earliest stage of tubule 3 weeks, the patient withdrew from the study for toxicity. formation, on a layer of human fibroblasts, were seeded Adverse events were graded according to the National at day 1 with dilutions of pre- and posttherapy serum Cancer Institute Common Toxicity Criteria (Version 3). samples from enrolled patients. Three replicate wells were All patients who received 1 dose of chemotherapy were set up for each serum sample. Negative and positive con- assessable for toxicity. trols included wells fed with medium only, serum from healthy donors, VEGF-A (2 ng/mL), or suramin (20 mmol/ Evaluation L). Medium from each well was replaced at days 4, 7, and 9 Tumor response was assessed according to the Response with aliquots of each test sample. Tubule formation was Evaluation Criteria in Solid Tumors (RECIST). The disease then assessed at day 11 after staining wells with CD31. evaluation during treatment was performed every 9 weeks. Briefly, wells were stained with anti-CD31 antibody at In the case of objective response or SD, bevacizumab was 37C for 60 minutes followed by the addition of alkaline continued until disease progression, unacceptable toxicity, phosphatase-conjugated secondary goat anti-mouse Ig. patient refusal, or physician decision, and fotemustine was Colorimetric reaction was then developed by adding p- continued up to 6 cycles of maintenance. TTP was defined NPP buffered substrate. Digital images of each well were as the time from the beginning of the first cycle to the acquired (magnification 5) on an Axiovert (Zeiss) micro- evidence of progressive disease (PD), or death, or the last scope. Images were then processed with Angioquant soft- date the patient was known to be progression free or alive. ware to quantitate the extent of tubule formation (lengths, OS was calculated from the beginning of the first cycle to sizes, and number of junctions) in each replicate well. death from any cause, or the last date the patient was known to be alive. Biomarker analysis was carried out Modulation of VEGF-C release by melanoma cells on serum samples obtained from the 15 patients enrolled Melanoma cell lines established from surgical specimens in one of the participating centers (Fondazione IRCCS of neoplastic lesions of patients admitted to Fondazione Istituto Nazionale dei Tumori, Milan). IRCCS Istituto Nazionale dei Tumori, Milan, were used. Cell lines Me4405P and Me26635P were isolated from Serum biomarker analysis primary melanomas of the right cheek and of the scalp Serum samples were obtained immediately before and of 2 patients in 1989 and 1995, respectively. Clone 1 hour after administration of therapy (days 1, 21, 42, 63, 2/21 was isolated by soft agar cloning from a subcutaneous and so on, as long as therapy continued). Blood samples metastatic melanoma cell line (Me 665/2) obtained in were centrifuged at room temperature and serum sam- 1986. All the lesions were histologically confirmed to be ples were frozen and stored at 80C until use. Serum cutaneous malignant melanomas. Molecular and biologi- samples were evaluated by custom-made, antibody-based cal characterization of these cell lines has been reported chemiluminescent SearchLight multiplex arrays (Pierce previously (16–18). Release of VEGF-C in melanoma SearchLight Proteome Arrays). Briefly, after plate incuba- supernatants after fotemustine treatment for 72 hours tion with biotinylated detecting antibodies, streptavidin– was evaluated by ELISA (Bender). horseradish peroxidase was added, which then reacted with a chemiluminescent substrate (SuperSignal ELISA Femto Apoptosis assay Chemiluminescent Substrate) and the reaction produced a Extent of apoptosis in melanoma cells treated with chemiluminescent signal that was detected by a charge fotemustine or temozolomide (Temodal; Schering-Plough) coupled device (CCD) camera (SearchLight Plus CCD was evaluated at 24 to 72 hours by staining tumor cells with

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Annexin V-FITC and propidium iodide (PI; BD Bios- a PS of 0. Eight patients had M1c disease; in particular, 5 of ciences) as described (19), followed by flow cytometric them had liver metastases. analysis with FACScalibur flow cytometer (BD Biosciences Pharmingen). Clinical activity All the patients completed the induction phase and Statistical analysis received a median of 5 (range, 2–8) administrations of Data are reported as percentage of complete response bevacizumab. There was 1 CR in a patient (#13) with groin (CR), partial response (PR), SD and PD. Data from all and pelvic lymph nodes, but such response developed after patients, who received at least 1 drug delivery and for patient withdrawal from trial due to toxicity (Table 2). Two whom at least 1 tumor evaluation was performed, were patients (#11 and #12), with M1a disease, showed a PR included in the response analysis. According to a typical 2- (Table 2). Ten patients obtained SD, yielding an overall stage optimal design, if less than 10% is considered an disease control rate (clinical benefit) of 65% (13/20). The unacceptable response rate and more than 30% acceptable, clinical benefit increased to 68.4% after excluding patient for 5% significance level and 90% power, then 18 patients 16 from the analysis. Two patients (#8 and #17), bearing are needed in the first stage. If patients (n 2) have an M1c stage disease for liver metastases, had SD but pro- objective response, then the trial must be closed because of gressed after 7.1 and 25.8 months, respectively, from the poor response. If 3 or more patients respond, the trial goes beginning of therapy. All the 20 patients were assessable for on to the second stage by recruiting 18 additional patients TTP and OS. Median TTP was 8.3 months (range, 4.1–24.2) to reach a total of 36 patients. Exact binomial methods and median OS was 20.5 months (range, 9.9–26.5). These were used to estimate the response rates and their 95% efficacy parameters did not change after excluding patient confidence intervals. TTP or death was estimated by the 16 from analysis. Nine patients are still alive, 2 of whom product-limit method of Kaplan and Meier. All results of have liver disease. the serum marker analyses were evaluated by ANOVA, followed by the SNK multiple comparison test. Toxicity Of the 20 enrolled patients, 18 experienced 1 or more Results adverse events: 14 of them developed at least 1 adverse event of grade 3–4 toxicity. The most frequently observed grade 3– Patient characteristics 4 hematologic toxicity was neutropenia, which occurred in Twenty patients entered the study and were included in 10 patients (Supplementary Table S1). Two patients (10%) the safety and efficacy analysis. The study recruited patients developed febrile neutropenia. Nonhematologic toxicities between December 2006 and November 2007 and was were generally mild; 4 patients (20%) developed grade 3–4 closed in March 2010. One patient (#16), initially with a hypertension. Treatment was discontinued because of toxi- diagnosis of M1b melanoma, was then confirmed to have city in 5 of the 20 patients. In particular, therapy was histiocytosis. The patient characteristics are summarized interrupted for thrombocytopenia in 3 patients and for in Table 1. Median age was 54 years, and all patients had pulmonary thromboembolism in 1 patient. Serious adverse events included 1 case of metrorrhagia in a patient with thrombocytopenia, 1 case of pulmonary thromboembolism, and 1 case of dyspnea in a patient with pleural Table 1. Patient characteristics effusion. No treatment-related deaths were observed.

n % Serum VEGF-A analysis To assess systemic levels and modulation of VEGF-A – Median age (range), y 54 (22 75) (bevacizumab target), serum samples obtained from 15 Sex patients at several time points during therapy were evaluated Male 12 60 by the SearchLight multiplex array approach. Among the 15 Female 8 40 patients, 2 (#11, #12) showed a PR, 7 (#2, #4, #7, #8, #10, PS (ECOG): 0 20 100 #17, #19) SD, and 6 (#3, #5, #6, #9, #15, #20) PD. In all Prior therapy assessed patients, pretherapy VEGF-A levels (average 419 Adjuvant IFN-a 525 252 pg/mL, n ¼ 15) were about 23-fold higher than those Surgical resection of primary tumor 15 75 a in serum from a pool of healthy donors (18 7 pg/mL). Sites of metastases Within 1 hour after the first administration of therapy M1a 5 25 (Fig. 1, black symbols), VEGF-A serum levels were signifi- M1b 7 35 cantly reduced compared with pretherapy values. In addi- M1c 8 40 tion, by looking at all patients, a significant reduction in aM1a, subcutaneous, skin, and/or lymph nodes only; M1b, VEGF-A levels was also observed by comparing serum sam- M1a, and/or lung only; M1c, other visceral organs involved ples taken immediately before and 1 hour after administra- P ¼ and/or elevated lactate dehydrogenase. tion of bevacizumab alone at day 21 ( 0.0005). In each of the patients, with only 1 exception (pt #2, third serum

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Table 2. Characteristics of patients who achieved clinical benefit

Patient no. Best Age/Sex Duration Disease sites Adjuvant therapy TTPb,mo response of study, moa

1 SD 55/F 6.11 Skin IFN-a2a 16.8 2 SD 33/F 3.65 Skin, lung No 4.0 4 SD 59/M 4.73 Lymph nodes, No 8.2 abdominal lymph nodes 7 SD 35/F 2.73 Left iliac crest, lung No 28.2 8 SD 66/M 5.32 Left axillary lymph HD IFN-a 7.1 nodes, liver, and pancreas 10 SD 69/M 5.19 Left axillary lymph nodes, No 26.1 left inguinal iliac lymph nodes, lungs 11 PR 67/M 4.57 Laterocervical lymph nodes, No 14.8 left supraclavicular lymph nodes, left mastoid region, left clavicular, and laterocervical region 12 PR 52/M 5.75 Right mammary region, No 7.1 right axillary lymph nodes 13 CR 75/F 10.84 Groin lymph nodes, No 28.1 pelvic lymph nodes 14 SD 68/M 0.76 Right axillary lymph nodes, No 2.4 mediastinum lymph nodes, right pleura 17 SD 38/F 4.17 Axillary lymph nodes, liver IFN-a 25.8 18 SD 56/M 8.31 Laterocervical lymph node (right) IFN-a 25.1 19 SD 49/F 5.49 Left inguinal lymph nodes, No 23.9 bilateral ovaries, lungs, and perineal region

Abbreviations: F, female; M, male. aDuration of study was calculated as time elapsed between the date of baseline visit and the date of study discontinuation. bTTP was defined as the time from the first treatment administration until objective tumor progression or death. If a patient had no events, TTP was censored at the date of the last adequate tumor assessment.

sample), all VEGF-A serum values observed during therapy (AngioKit) that allowed to test whether posttherapy serum (66/67 posttherapy serum samples) remained significantly samples had less growth-promoting activity for human lower than pretherapy levels (see Fig. 1). Moreover, with the endothelial cells than pretherapy samples. Using this 11- exception of some samples of patients 2, 7, and 11, VEGF-A day in vitro assay, we could score the extent of development levels dropped upon therapy administration to values simi- of a network of capillary-like tubules by human endothelial lar to those found in serum from a pool of healthy donors cells seeded on an underlying matrix of fibroblasts. Com- (horizontal dotted line in Fig. 1). Interestingly, in one of the pared with the extent of tubule formation induced by a patients (patient 19), 2 cycles of therapy were skipped (due pool of pretherapy serum samples from 6 patients, post- to intervening toxicity) between day 21 and day 77, but therapy sera from the same patients showed a dramatic VEGF-A levels remained low for as long as 56 days after reduction in their proangiogenic activity as documented by administration of therapy at day 21 (Fig. 1). These results the marked reduction in tubule size, length, and number of indicate that administration of bevacizumab plus fotemus- junctions (see Supplementary Fig. S1). Assessment of tine was associated with profound and durable inhibition of tubule lengths, sizes, and junctions indicated that post- systemic levels of VEGF-A in all assessed patients. therapy serum samples from treated patients had an overall proangiogenic activity reduced to that seen in control Modulation of proangiogenic activity at the systemic serum from healthy donors (Fig. 2). In addition, in agree- level in enrolled patients ment with the known half-life of bevacizumab (20), at the To corroborate the evidence for VEGF-A inhibition after end of therapy, serum samples could still suppress the therapy, we carried out an in vitro angiogenesis assay proangiogenic activity of recombinant VEGF-A. In fact,

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Fig. 1. Modulation of VEGF-A serum levels in 15 patients upon treatment with fotemustine plus bevacizumab. Patients were identified by their randomization number and grouped according to response to therapy (PR, SD, or PD). VEGF-A levels were evaluated in serum samples taken immediately before (empty symbols) and 1 hour after (black symbols) therapy administration, according to the schedule described in the Materials and Methods section. Timing of therapy administration is indicated by vertical dotted lines. VEGF-A levels were also evaluated in serum from a pool of healthy donors (horizontal dotted line). In all patients, all VEGF-A values, with exception of the third serum sample from patient 2, were significantly lower than the pretherapy (day 1) values (SNK test, P < 0.01). end-of-therapy serum sample from one of the patients bevacizumab plus fotemustine leads not only to suppres- (patient 4) could significantly inhibit tubule formation sion of VEGF-A serum levels but also to profound inhibi- induced by recombinant VEGF-A (Fig. 2). Taken together, tion of the overall proangiogenic activity detected in these results indicated that the treatment of patients with periphery of enrolled patients.

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A B C

Fig. 2. Modulation of serum proangiogenic activity upon therapy with fotemustine plus bevacizumab. Human endothelial cell growth was assessed in an 11-day assay by measuring total tubule lengths (A), sizes (B), and junctions (C) upon culture with medium alone (Ct) or in the presence of 1:8 dilution of serum from healthy donors or from patients. Serum from patients was a pool of pretherapy (Pre, day 1) and end-of-therapy (Post, last day of therapy) samples from 6 patients (#3, #4, #10, #12, #17, and #19). Endothelial cell growth was also scored after growth in the presence of recombinant VEGF-A (2 ng/mL) supplemented or not with serum from donors (þDo) or from end of therapy sample from patient 4 (þ Pt.P.). Statistical analysis, by ANOVA followed by SNK multiple comparison test, was annotated as follows: ***P < 0.001; **P < 0.01.

Modulation of the lymphangiogenesis factor VEGF-C fotemustine on VEGF-C release by these tumors was upon treatment with bevacizumab plus fotemustine not due to induction of cell death. In fact, apoptosis The multiplex ELISA array was designed to include VEGF- assays showed that fotemustine did not induce significant C. Pretherapy serum samples from all patients contained cell death up to 72 hours in Me26635P and Me4405P high levels of the lymphangiogenesis factor VEGF-C (aver- tumors. However, the same tumors were susceptible to age pretherapy value: 2,458 938 pg/mL, n ¼ 15). In cell death induced by a different alkylating agent (temo- contrast, no VEGF-C could be measured in serum from a zolomide; Fig. 4B), and fotemustine could induce apop- pool of healthy donors. Analysis of the VEGF-C serum tosis of a different tumor (Me2/21; Fig. 4B). Gene- levels indicated significant reductions (marked by asterisks, profiling experiments, by whole genome arrays under Fig. 3) compared with pretherapy levels (day 0) in 47 of 68 the Illumina platform, also showed that VEGF-C was posttherapy samples. Only 6 posttherapy serum samples among the genes significantly downmodulated in mela- (patients 11, 4, and 6, samples marked by arrows, Fig. 3) noma cells upon 6-hour treatment with fotemustine (data showed significantly higher VEGF-C levels than pretherapy not shown). Taken together, these results indicate that values. Collectively, comparison of end-of-therapy VEGF-C fotemustine can impact on VEGF-C release by human levels versus day 0 values provided evidence for significant melanoma cells. reduction in 2 of 2 PR, 5 of 7 SD, and 5 of 6 PD patients (Supplementary Fig. S2). Taken together, these results Association of distinct cytokines with clinical indicate that treatment with bevacizumab plus fotemustine response groups markedly inhibits serum levels of the lymphangiogenesis We tested the hypothesis that the treatment with bev- factor VEGF-C. acizumab plus fotemustine could affect serum levels of additional molecules involved in the regulation of angio- Fotemustine inhibits VEGF-C release by melanoma genesis and of the immune response. To this end, the cells in vitro without inducing cell death multiplex ELISA arrays were designed to allow detection No available data indicate that bevacizumab can of levels of VEGF-R2 and VEGF-R1, of endothelial adhe- induce suppression of VEGF-C serum levels. Therefore, sion molecules (E-selectin, P-selectin, and sICAM-1), of we hypothesized a role of fotemustine in the modulation cytokines IL-8, IL-10, IL-12p70, IL-17, IL-23, IFN-g,and of this factor. To address this possibility, culture super- TNF-a, and of the chemokine CXCL10. CRP, a known natants from several melanoma cell lines established biomarker of tumor load in melanoma (21), was also from patients were evaluated by ELISA for VEGF-C secre- included in the analysis. Levels of CRP showed significant tion (data not shown). Two cell lines producing VEGF-C increases at the end of therapy compared with pretherapy (Me26635P and Me4405P) were selected for further ana- values in 4 of 7 SD patients and in 5 of 6 PD patients but lysis. Fotemustine-treated melanoma cells from one of not in the 2 PR patients (Supplementary Fig. S2). A these cell lines (Me26635P) showed a dose-dependent similar comparison of end-of-therapy versus pretherapy and significant decrease in VEGF-C secretion in a 72-hour values of all factors showed changes in some of the assay (Fig. 4A). Similar results were obtained with patients but were not associated with the response groups Me4405P (data not shown). The inhibitory effect of (Supplementary Fig. S2). We then assessed whether an

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Fig. 3. Modulation of serum VEGF-C levels in 15 patients upon treatment with fotemustine plus bevacizumab. Patients were identified by their randomization number and grouped according to response to therapy (PR, SD, or PD). VEGF-C levels were evaluated in serum samples taken immediately before (empty symbols) and 1 hour after (black symbols) therapy administration. Timing of therapy administration is indicated by vertical dotted lines. VEGF-C was also evaluated in serum from a pool of healthy donors (horizontal dotted line) but was undetectable. All VEGF-C values identified by asterisks (**) were significantly lower than day 1, pretherapy values (SNK test, P < 0.01). VEGF-C levels marked by arrows were significantly higher than pretherapy values (SNK test, P < 0.01). association of any of the investigated factors with the 3 cantassociationwiththe3responsegroups(PR,SD,and main response groups (PR, SD, and PD) could be found PD; Supplementary Fig. S3). IL-10 and IL-12p70 showed by analysis of all available serum samples from all the highest levels in sera of PR patients compared with SD patients rather than looking only at pre- versus end-of- and PD patients. IL-23 showed the opposite pattern, with therapy samples. By this approach, serum levels of 3 the highest levels found in sera of PD patients compared cytokines (IL-10, IL-12p70, and IL-23) showed a signifi- with SD and PR patients (Supplementary Fig. S3).

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Fig. 4. Fotemustine inhibits VEGF- C release by melanoma cells without inducing cell death. A, melanoma cells (7.5 105/mL) from a melanoma cell line (Me26635P) were cultured for 72 hours with the indicated concentration levels of fotemustine or in medium alone (). VEGF-C levels were then evaluated in supernatants by ELISA. Statistical analysis by SNK test was annotated as follows: ***P < 0.001; **P < 0.01. B, melanoma B cells from Me26635P and from 2 additional melanoma lines (Me4405P and Me2/21) were treated for 72 hours with the indicated concentrations of fotemustine (FTM) or of temozolomide (TMZ), or cultured in medium without drugs (Ctrl). Extent of early apoptosis (lower right quadrant in each dot plot) or of late apoptosis (upper right quadrant in each dot plot) was then assessed by flow cytometry after staining cells with Annexin V and PI. Numbers in each dot plot represent percentage of cells in each quadrant.

Discussion patients in whom it showed good safety and efficacy (22). Patients received intravenous administration of In this study, the choice of fotemustine, as chemother- bevacizumab 10 mg/kg on day 1 and day 15 and fote- apy agent, was based on 2 main reasons. The first one mustine 75 mg/m2 on day 1 and day 8 as an induction was that fotemustine, when compared in a randomized phase; after 3 weeks, patients received bevacizumab 10 trial to dacarbazine (DTIC), a conventional chemother- mg/kg and fotemustine 75 mg/m2 every 3 weeks (main- apy drug for advanced melanoma, was shown to yield a tenance phase). The rationale for using bevacizumab higher overall response rate (13). The second one was even in melanoma has been strengthened by preclinical that fotemustine, in contrast with DTIC, has the ability studies (23) and early-phase clinical trials (10, 11). to cross the blood–brain barrier, thus potentially pre- Moreover, the only clinical case reported so far of meta- venting the development of brain metastases (12). The static melanoma treatment with this biochemotherapy choice of bevacizumab was initially based on the evi- regimenshowedanearlytumorresponseintermsof dence for its clinical activity in different solid tumors (6– extensive necrosis (24). 8) and on the emerging impact on OS, progression-free In this study, we found that a biochemotherapy regimen survival, and response rate found in colorectal cancer based on the combination of bevacizumab plus fotemus- when adding this mAb to chemotherapy (9). The com- tine has clinical activity as first-line treatment in metastatic bination of bevacizumab and fotemustine has been melanoma and promotes suppression, at the systemic recently investigated in recurrent malignant glioma level, of soluble factors involved in angiogenesis and

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Biochemotherapy for Melanoma

lymphangiogenesis. Moreover, given the relevance of fotemustine may impact on both angiogenesis and lym- angiogenesis and lymphangiogenesis in melanoma pro- phangiogenesis, 2 highly relevant processes in melanoma gression and in the development of metastases (25), the progression. In fact, strong evidence (see ref. 33 for review) association of bevacizumab and fotemustine might be indicates that VEGF-C expression in the tumor significantly considered for testing in the adjuvant setting in high-risk correlates with lymph node metastasis. Moreover, experi- stage III patients, in which, so far, only IFN-a2b has shown mental models have indicated that neoplastic cells can impact on OS (26). Of course, randomized trials compar- promote lymphangiogenesis even within draining lymph ing the biochemotherapy approach versus chemotherapy nodes and that this process promotes further metastases to alone in a large series of stage IV melanoma patients should distant organs (34). be carried out before moving to the adjuvant setting. Finally, investigation on the expression of several cyto- In agreement with previous bevacizumab-based studies kines in enrolled patients showed a significant associa- in melanoma (11, 27, 28), our results confirmed the tion of IL-10, IL-12p70, and IL-23 serum levels with the 3 clinical benefit of this biochemotherapy approach. On response groups. The converse levels of IL-12p70 and IL- the other hand, of possible concern for future trials invol- 23 in the PR and SD versus PD patients seem to fit the ving the combination of bevacizumab plus fotemustine recent model describing the contrasting immunoregula- was the number of patients (n ¼ 5) who discontinued tory functions of these 2 cytokines under the control of treatment because of toxicity. In spite of the response rate STAT3 in the tumor microenvironment (35). According (1 CR and 2 PR), clinical benefit (65% of patients), median to this model, as well as to a wealth of previous evidence OS (20.5 months), and good compliance with both drugs, (see ref. 36 for review), IL-12p70 can promote adaptive based on the results of the safety analysis, we did not feel T cell–mediated immunity and suppress angiogenesis comfortable to proceed to the second stage of the study. whereas IL-23 can have a protumoral activity by stimulat- Fourteen patients (70%) experienced adverse events of ing regulatory T cells (35). The results obtained for IL-10 toxicity grade 3–4, although no treatment-related death are unexpected, as this cytokine has been considered an was detected during the study. Therefore, the bevacizu- anti-inflammatory immunosuppressive factor (37) and a mab–fotemustine schedule used in brain tumors could marker of poor prognosis in advanced melanoma (38). be helpful, in terms of safety profile, in future trials in However, IL-10 may also exert antitumor activity, thanks melanoma. to its ability to inhibit MHC class I expression, thus The serum biomarker analysis showed that this bio- favoring NK-mediated antitumor responses (37), and chemotherapy was associated with a quick and highly to suppress angiogenesis (39). In spite of the limited significant drop in VEGF-A levels, which remained low, in sample size, these results suggest that these immunomo- all patients, throughout the whole duration of therapy. dulatory molecules may be evaluated in larger trials The reduction in VEGF-A levels was observed in all involving this drug combination as predictive biomarkers evaluated patients and therefore it did not correlate with of response to therapy. the clinical response. In addition, an in vitro angiogenesis In conclusion, the good clinical impact of bevacizumab– assay indicated that the overall proangiogenic activity fotemustine biochemotherapy, in terms of both disease detectable in peripheral blood was completely inhibited control rate and median OS, suggests that this therapeutic by the therapy. We also found that posttherapy serum approach should be investigated in future studies, possibly from treated patients could completely inhibit the angio- by modulating drug schedules (22) to improve the safety genic activity of recombinant VEGF-A. Taken together, profile. even though melanoma patients can have measurable levels of additional angiogenic molecules, such as IL-8 Disclosure of Potential Conflicts of Interest and bFGF (29–31), these results suggest that the thera- No potential conflicts of interest were disclosed. peutic approach described in this study can durably revert the systemic "proangiogenic phenotype" of advanced Acknowledgments melanoma patients to a condition similar to peripheral blood of healthy donors. The authors thank A. Molla, C. Vegetti, and P. Baldassari (Fondazione Unexpectedly, we found that bevacizumab plus fotemus- IRCCS Istituto Nazionale dei Tumori, Milan) for their excellent technical work. tine combination therapy also affected serum levels of VEGF-C, a molecule that together with VEGF-D plays a key role in the lymphangiogenesis process (32). Although Grant Support we cannot rule out that both bevacizumab and fotemustine concur to VEGF-C inhibition in vivo, nevertheless, in vitro Supported in part by grants from the Italian Association for Cancer Research (A.I.R.C., Milan, Italy) to A.A. and R.M., from Ministry of Health, assays indicated that fotemustine, used alone, could inhibit Rome to A.A., from "Alleanza Contro il Cancro", Rome to A.A. and from VEGF-C gene expression (data not shown) in melanoma Italfarmaco to A.A. The patient trial was sponsored by Roche Ltd. Clin- cells and significantly suppressed VEGF-C release from icalTrials.Gov Identifier:NCT01069627. melanoma cells, a finding not due to induction of apoptosis. Therefore, these results suggest that a biochemother- Received 09/02/2010; revised 10/14/2010; accepted 10/15/2010; apy based on the combination of bevacizumab and published OnlineFirst 10/28/2010.

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Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Clinical Cancer Correction Research Correction: Bevacizumab plus Fotemustine as First-line Treatment in Metastatic Melanoma Patients: Clinical Activity and Modulation of Angiogenesis and Lymphangiogenesis Factors

In this article (Clin Cancer Res 2010;16:5862–72), which was published in the December 1, 2010 issue of Clinical Cancer Research (1), an affiliation for Prof. Mario R. Sertoli was missing. The correct affiliations for Prof. Sertoli are Department of Medical Oncology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy and Department of Oncology, Biology, and Genetics, University of Genova, Genova, Italy.

Reference 1. Del Vecchio M, Mortarini R, Canova S, Di Guardo L, Pimpinelli N, Sertoli MR, et al. Bevacizumab plus fotemustine as first-line treatment in metastatic melanoma patients: clinical activity and modulation of angiogenesis and lymphangiogenesis factors. Clin Cancer Res 2010;16:5862–72.

Published Online First January 25, 2011. Ó2011 American Association for Cancer Research. doi: 10.1158/1078-0432.CCR-10-3193

630 Clin Cancer Res; 17(3) February 1, 2011 Published OnlineFirst October 28, 2010; DOI: 10.1158/1078-0432.CCR-10-2363

Bevacizumab plus Fotemustine as First-line Treatment in Metastatic Melanoma Patients: Clinical Activity and Modulation of Angiogenesis and Lymphangiogenesis Factors

Michele Del Vecchio, Roberta Mortarini, Stefania Canova, et al.

Clin Cancer Res 2010;16:5862-5872. Published OnlineFirst October 28, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-10-2363

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