- Email: [email protected]
German Testicular Cancer Study Group cooperative study
original article
Annals of Oncology 22: 2654–2660, 2011 doi:10.1093/annonc/mdr026 Published online 17 March 2011
Preclinical and clinical activity of sunitinib in patients with cisplatin-refractory or multiply relapsed germ cell tumors: a Canadian Urologic Oncology Group/German Testicular Cancer Study Group cooperative study K. Oechsle1 , F. Honecker1 , T. Cheng2, F. Mayer3, P. Czaykowski4, E. Winquist5, L. Wood6, M. Fenner7, S. Glaesener1, J. T. Hartmann3, K. Chi8, C. Bokemeyer1 & C. Kollmannsberger8*
Received 29 November 2010; revised 31 December 2010 & revised 3 January 2011; accepted 17 January 2011
Background: The objective of the study was to investigate the activity of sunitinib in a cell line model and subsequently in patients with cisplatin-refractory or multiply relapsed germ cell tumors (GCT).
original article
Methods: The effect of sunitinib on cell proliferation in cisplatin-sensitive and cisplatin-refractory GCT cell lines was evaluated after 48-h sunitinib exposure by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and IC50 (concentration that causes 50% inhibition of growth) doses were determined. Sunitinib was subsequently administered at a dose of 50 mg/day for 4 weeks followed by a 2-week break to 33 patients using a Simon two-stage design. Results: Sunitinib demonstrated comparable dose-dependent growth inhibition in cisplatin-sensitive and cisplatinresistant cell lines, with IC50 between 3.0 and 3.8 lM. Patient characteristics were as follows: median of 2 (1–6) cisplatin-containing regimens; high-dose chemotherapy 67%; late relapse 33%; and cisplatin refractory or absolute cisplatin refractory 54%. Toxic effects included fatigue (39%), anorexia (21%), diarrhea (27%), mucositis (45%), nausea (33%), hand–foot syndrome (12%), dyspepsia (27%), and skin rash (18%). No unexpected side-effects were observed. Thirty -two of 33 patients were assessable for response. Three confirmed partial responses (PRs) and one unconfirmed PR were seen for a total response rate of 13%. Median progression-free survival (PFS) was 2 months, with a 6-month PFS rate of 11%. Conclusions: Sunitinib shows in vitro activity in cisplatin-resistant GCT cell lines. Modest clinical activity in heavily pretreated GCT patients was observed. Key words: angiogenesis, cisplatin resistance, germ cell tumor, relapse, sunitinib
introduction Although the vast majority of patients with metastatic germ cell tumors (GCT) are cured, those with cisplatin-refractory relapse or relapse after high-dose chemotherapy (HD-CT) still exhibit a very poor prognosis, and <5% of these patients will achieve long-term survival [1]. For these patients, the evaluation of new active drugs and treatment combinations remains a priority. *Correspondence to: Dr C. Kollmannsberger, Division of Medical Oncology, British Columbia Cancer Agency, University of British Columbia–Vancouver Cancer Centre, 600 West 10th Avenue, Vancouver, Canada BC V5Z 4E6. Tel: +1-604-877-6000 ext. 2674; Fax: +1-604-708-2144; E-mail: [email protected]
Both authors contributed equally as senior authors.
Various agents have been evaluated in intensively pretreated or cisplatin-refractory patients. As single agents, only paclitaxel, gemcitabine, oxaliplatin, and orally administered etoposide have been shown to be active, with selected patients achieving long-term survival [2–6]. Different combinations of these agents demonstrated response rates up to 51%, including 10%– 15% long-term survivors, although duration of remission is usually moderate [7–10]. The molecular mechanisms involved in cisplatin resistance are still incompletely understood. Mismatch repair deficiency and microsatellite instability are thought to be associated with cisplatin resistance in human GCT [11]. Preliminary studies suggest that vascular endothelial growth factor (VEGF) may play an important role in development and metastasizing of
ª The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected]
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
1 Division of Oncology, Hematology, Bone Marrow Transplantation and Section Pneumology, University Medical Centre Eppendorf, Hamburg University, Hamburg, Germany; 2Department of Oncology, Tom Baker Cancer Centre, Calgary, Canada; 3Department of Medical Oncology, Hematology, Rheumatology, Immunology and Pulmonology, University of Tuebingen, Tuebingen, Germany; 4Department Medical Oncology/Haematology, CancerCare Manitoba, University of Manitoba, Winnipeg; 5 Division of Medical Oncology, London Health Science Centre, London; 6Division of Medical Oncology, Queen Elizabeth II Health Sciences Centre, Halifax, Canada; 7 Department of Hematology/Hemostaseology/Oncology/Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; 8Division of Medical Oncology, British Columbia Cancer Agency, University of British Columbia–Vancouver Cancer Centre, Vancouver, Canada
original article
Annals of Oncology
patients and methods preclinical study Sunitinib was kindly provided by Pfizer Inc., Berlin, Germany, and was dissolved in dimethyl sulfoxide (DMSO). Cisplatin was obtained from Teva, Radebeul, Germany, and was dissolved in 0.9% saline. NTERA2 cells, first described by Andrews [18], were obtained from the ‘Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH’, Braunschweig, Germany. NCCIT cells, first described by Teshima et al. [19], were obtained from American Type Culture Collection, Manassas, VA. 2102Ep cells were kindly provided by L. Looijenga, Rotterdam, The Netherlands. NCCIT and 2102Ep cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and F12 medium (1 : 1) (Gibco-BRL; Invitrogen, Paisley, UK) containing 10% fetal calf serum (FCS) (Gibco-BRL; Invitrogen), and NTERA2 cells with DMEM supplemented with GlutamaxI (Gibco-BRL; Invitrogen) containing 10% FCS (Gibco-BRL; Invitrogen). Cells were incubated at 37C in a humidified atmosphere with 5% CO2. From these well-established GCT cell lines, all derived from human embryonal carcinomas, cisplatin-resistant sublines exhibiting 5.2–12.2-fold increase in cisplatin resistance were generated in our laboratory over a time period of 18 months by intermittent exposure to increasing doses of cisplatin, starting with the IC50 (concentration that causes 10% inhibition of growth) dose of the parental cell lines. At reaching 50% lethality, the addition of cisplatin was paused and cells were allowed to recover over three passages until the next dose escalation step. The MTT assay was carried out as previously described [20]. The MTT assay is based on the cleavage of the tetrazolium salt MTT to purple formazan crystal by metabolically active cells. In brief, the cell lines were rinsed with phosphate-buffered saline, trypsinized, and resuspended in 1 ml of the appropriate culture medium to count the cells in a hemaocytometer chamber. In total, 4 · 103 cells per well were seeded in 96-well plates to ensure logarithmic growth. Cells were allowed to adhere over night, and serial dilutions of the chemotherapeutic agents were added to octuplicate
Volume 22 | No. 12 | December 2011
wells at concentrations from 0.2 to 25.6 lM of both sunitinib and cisplatin. The cells were exposed to the drug for 48 h. Thereafter, the drug-containing medium was removed and 0.2 ml MTT solution (final concentration: 0.5 mg/ml MTT; Sigma, Hamburg, Germany) was added. The plates were incubated for 2 h before the medium was removed and 0.1 ml DMSO was added. The plates were agitated for 15 min before reading absorbance using a photometer (Victor2 Wallac 1420; Perkin Elmer Life Sciences, Wellesley, MA) at 570 nm. All experiments were carried out in triplicates and were repeated at least twice. The results are expressed as IC50 (inhibitory concentration).
clinical study Eligibility criteria for this open-label, multicenter phase II trial included histologically confirmed seminomatous or nonseminomatous GCT, and relapse within 2 months after cisplatin-based chemotherapy, tumor progression during or relapse after salvage HD-CT, tumor progression during salvage cisplatin-based chemotherapy, or ineligibility for cisplatinbased chemotherapy or HD-CT due to severe comorbidities. Patients had to have measurable disease and documented tumor progression on imaging. A sole elevation of alpha fetoprotein (AFP) or beta-human chorionic gonadotropin (b-HCG) without disease on imaging was accepted as measurable disease if an increase of at least 25% within 4 weeks before study inclusion had been observed. Further inclusion criteria consisted of a Karnofsky performance status ‡60% and a life expectancy ‡3 months. Adequate bone marrow function (neutrophils >1500/ll, platelet count >100 000/ll), liver function (bilirubin <1.5-fold upper normal limit), and renal function (serum creatinine £twofold upper normal limit) were mandatory. All patients gave written informed consent. The study (clinical trial identifier: NCT00371553) was approved by the ethics committees of the British Columbia Cancer Agency and the University of Hamburg-Eppendorf, Germany, as well as by local ethics committees of all participating centers. Sunitinib was given at a dose of 50 mg daily for 4 weeks followed by a 2week break to form 6-week cycles. Dose reductions to 37.5 or 25 mg daily for 4 weeks on/2 weeks off were recommended for significant toxicity. No more than two dose reductions were permitted in any patient. If clinically deemed appropriate or necessary by the investigator, a continuous administration schedule utilizing 37.5 or 25 mg daily without breaks was allowed. Intrapatient reescalation back to the previous dose level was permitted in the absence of grade ‡3 hematologic or grade ‡2 non-hematologic toxicity in the previous cycle. Treatment was continued until disease progression, occurrence of unacceptable toxicity, intercurrent illness that prevented further administration of therapy, or patient’s decision to withdraw from the study. Pretreatment evaluation consisted of medical history, assessment of the Karnofsky performance status, physical examination, electrocardiogram, Multi Gated Acquisition Scan or echocardiogram, routine laboratory, thyroid function tests, urinnalysis, tumor markers (AFP and b-HCG), as well as radiological tumor assessments.
definitions Disease was considered cisplatin refractory when at least tumor stabilization or a remission had been achieved during cisplatin-based chemotherapy, but tumor progression occurred again within 4 weeks of the last cisplatin-based chemotherapy. Disease was considered absolutely cisplatin refractory when tumor progression had occurred while patients were receiving cisplatinbased therapy [21, 22].
response/toxicity assessment Response assessments were carried out on day 28 of each 6-week cycle. Response was evaluated according to RECIST version 1.0 criteria [23]. In addition, patients with reduction of the size of a tumor lesion and normalization of previously elevated tumor markers were considered partial remission with tumor marker normalization (PR negative). Patients with
doi:10.1093/annonc/mdr026 | 2655
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
GCT [12–14]. A substantially higher VEGF and platelet-derived growth factor (PDGF) expression has been found in patients with GCT compared with normal testis tissue in several studies, indicating that VEGF and PDGF expression could play an important role in tumor angiogenesis, tumor progression, and metastases [12–15]. A significantly higher progression-free survival (PFS) rate was seen in a murine testicular cancer xenograft model when an angiostatic agent was combined with carboplatin or cisplatin as compared with either carboplatin– cisplatin or the angiostatic agent alone [16, 17]. Sunitinib is an orally administered small molecule and a potent multityrosine kinase inhibitor for VEGF receptor, PDGF receptor, as well as the stem cell factor receptor c-KIT. Sunitinib has demonstrated clinical activity in several tumors including thymic carcinoma and pancreatic neuroendocrine tumors, and is currently approved for the treatment of gastrointestinal stromal tumors and renal cell carcinoma. Based on this rationale, we used a cell line model to investigate the preclinical activity of sunitinib in different GCT cell lines with defined levels of cisplatin resistance by MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Having observed in vitro activity of sunitinib in cisplatinresistant GCT cell lines at the same level as in their cisplatinsensitive counterparts, we carried out a phase II study in patients with cisplatin-refractory or multiply relapsed GCT.
original article a partial remission of tumor lesions without complete tumor marker normalization were considered a marker-positive partial remission (PR positive). If elevated markers were the only evidence of progressive disease, a decrease of at least 90% was required for a PR. Levels of serum tumor markers were measured every 3 weeks. All responses as well as the diagnosis of a stable disease had to be confirmed after a 4-week interval. Toxicity was assessed after each cycle and graded by the National Cancer Institute—Common Terminology Criteria for Adverse Events version 3.0.
statistical considerations
results proliferation and cytotoxicity assays MTT assay. After long-term exposure to cisplatin, resistance levels substantially increased in all three cell lines: NTERA2, 2102Ep, and NCCIT (Figure 1). Whereas the IC50 values of the sensitive parental cell lines were 0.45 lM [60.1 standard deviation (SD)], 0.75 lM (60.1 SD), and 1.75 lM (60.1 SD), respectively, the three cisplatin-resistant sublines exhibited IC50 values of 5.1 lM (60.1 SD) in NTERA-2R, 4.9 lM (60.1 SD) in 2102Ep-R, and 4.7 lM (60.2 SD) in NCCIT-R (P < 0.001 for all). Sunitinib substantially decreased proliferation of all
Figure 1. IC50 (concentration that causes 50% inhibition of growth) values of cisplatin and sunitinib in three embryonal carcinoma cell lines (NTERA2, 2102Ep, and NCCIT) and their derivatives, relatively cisplatinresistant sublines NTERA2-R, 2102Ep-R, and NCCIT-R. Cells were treated with either cisplatin or sunitinib for 48 h. Whereas the differences between the IC50 values for cisplatin-sensitive parental and their cisplatin-resistant sublines were all statistically significant (àP < 0.001), differences in sensitivity to sunitinib were only significant for NTERA2-R versus NTERA, with the cisplatin-resistant line showing better sensitivity than the cisplatin-sensitive cell line ( P = 0.02).
2656 | Oechsle et al.
GCT cell lines in comparison with control cells treated with the solvent DMSO. By MTT assay, IC50 values for sunitinib were within a narrow range in all three cell lines: NTERA2 3.8 (60.1 SD), NTERA2-R 3.5 (60.1 SD), 2102Ep 3.0 (60.1 SD), 2102Ep-R 3.3 (60.4 SD), NCCIT 3.7 (60.2), and NCCIT-R 3.4 (60.6 SD) (Figure 1). Apart from NTERA2-R, which were slightly more sensitive to sunitinib than NTERA2 (P = 0.02), there was no difference in sensitivity to sunitinib between cisplatin-sensitive parental cell lines and their cisplatin-resistant sublines, demonstrating that there is no cross-resistance of cisplatin and sunitinib in GCT cell lines in vitro.
clinical study Thirty-three patients with heavily pretreated or cisplatinrefractory GCT were entered into the study between February 2007 and January 2010. Patient characteristics are listed in Table 1. Fifteen percent of patients had a primary mediastinal GCT. All patients were heavily pretreated with a median number of 2 (range, 1–6) platinum-based regimens. Sixty-six percent of patients had relapsed after HD-CT, and 33% were considered late relapses (‡2 years after initial therapy). Fifty-four percent of patients were considered cisplatin refractory or absolutely refractory. A total of 66 cycles of sunitinib were administered with a median number of 1 cycle per patient (range, 1–9). No complete remission was observed. Three patients achieved a partial remission (9%) (Tables 2 and 3). Time to progression for these three patients was 5.0, 6.4, and 12.2 months. One additional seminoma patient achieved a response but subsequently went off study before a confirmatory scan was carried out (Table 3). Stable disease was recorded for 41% of patients with a median duration of 2.3 months (range 1.6–6.5 months). After a median follow-up of 14.4 months (range, 6–18 months), all patients are off study. In 30 patients, treatment termination was due to progression, 2 patients refused further treatment, and 1 patient was stopped due to hyperbilirubinemia. Median PFS for all patients was 2.0 months [95% confidence interval (CI) 1.4–2.60], with 11% and 3.7% of patients being progression free at 6 and 12 months, respectively (Figure 2). Median OS was 3.8 months (95% CI 3.0–6.6), with 36.4% and 9.9% of patients being alive at 6 and 12 months, respectively. Sunitinib treatment was feasible and toxicity in these heavily pretreated patients was generally acceptable. However, treatment duration was short in the majority of patients and thus toxicity assessment, and in particular evaluation of longterm toxicity, is limited (Table 4). The most common sunitinib-related toxic effects included fatigue (39%), anorexia (21%), diarrhea (27%), mucositis (45%), nausea/vomiting (24%), and dyspepsia (27%). Grade 3/4 toxic effects were rare and no unexpected side-effects were observed.
discussion Despite the overall success of cisplatin-based chemotherapy in patients with metastatic GCT, the prognosis for cisplatin-
Volume 22 | No. 12 | December 2011
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
The primary end point of this study was response rate. Tolerability, PFS, and overall survival (OS) time represented secondary end points. An optimal Simon two-stage design was used to determine the number of patients required [24]. Assuming a response rate of clinical interest of >20%, a minimal response rate of 5%, a probability of 5% for rejecting an active drug combination (type II error), and a probability of 20% to further evaluate an ineffective drug combination (type I error), 16 patients had to be enrolled into the first cohort. If no response to study therapy was observed among the first 16 patients, the study was to be terminated. If at least one objective remission occurred, the study was to be continued with a second cohort of 16 patients.
Annals of Oncology
original article
Annals of Oncology
Table 1. Patients’ characteristics (N = 33)
Table 2. Treatment efficacy (N = 33) n 32 (22–54)
14 17 2
42 52 6
26 0 5 2
79 0 15 6
4 29
12 88
20 11 8 15 18 25
61 33 24 35 55 76
9 kU/l (1–490 000) 111 U/l (0.3–39 000) 278 (110–2107) 15 18 18 6 (1–18)
48 62 60
22
66
9 9 15 21
27 27 45 64
5
15
11
33
a
n = 31. n = 29. c n = 30. AFP, alpha fetoprotein; HCG, human chorionic gonadotropin; LDH, lactate dehydrogenase; TUM, tumor markers. b
refractory patients remains very poor. Gemcitabine–oxaliplatin and paclitaxel–gemcitabine or gemcitabine–oxaliplatin– paclitaxel are the most commonly used regimens for these patients resulting in response rates of 25%–40%, with selected patients achieving prolonged survival [7–9, 25]. Limited clinical data exist regarding targeted therapies in GCT [26, 27]. Based on previous reports on the potential role of VEGF and VEGFR and the very high expression of tyrosine kinases in GCT, we
Volume 22 | No. 12 | December 2011
Total cycles Median cycles per patient (range) Response (n = 32), n (%) Confirmed PR Unconfirmed PR Stable disease Progression Time to progression Median, months (95% CI) 3 months PFS, % (95% CI) 6 months PFS, % (95% CI) OS Median, months (95% CI) 3 months OS, % (95% CI) 6 months OS, % (95% CI) 9 months OS, % (95% CI) 12 months OS, % (95% CI)
66 1 (1–9) 3 1 13 15
(9) (3) (41) (47%)
2 (1.4–2.6) 26 (13.8% to 47.5%) 11 (3.8% to 31.6%) 3.8 66 36 17 10
(3.0–6.6) (52.4% to 84.9%) (23.2% to 57.1%) (6.4% to 35.9%) (0.8% to 31.0%)
PR, partial response; CI, confidence interval; PFS, progression-free survival; OS, overall survival.
investigated the multityrosine kinase inhibitor sunitinib preclinically and subsequently clinically in cisplatin-resistant GCT [28]. Analysis of the efficacy of sunitinib in GCT cell lines in vitro demonstrated inhibition of proliferation (MTT assay) at an IC50 in the range of 3.0–3.8 lM after 48 h of drug exposure. There was no difference between cisplatin-sensitive and cisplatin-resistant cell lines, indicating that there is no cross-resistance between sunitinib and cisplatin. Furthermore, this dose range for in vitro activity is at the lower end of previously determined doses in different other tumor cell lines including renal cell carcinoma, nasopharyngeal carcinoma, or lung cancer, where IC50 doses of 3–10 lM have been reported [29–31]. An IC50 of 6 lM has previously been reported for the human yolk sac tumor cell line 1411H after 24-h treatment [17]. As was the case in our three cell lines and their cisplatinresistant sublines, no cross-resistance between cisplatin and sunitinib was observed by Castillo-Avila et al. [17] who, in addition to direct action on receptor tyrosine kinases, also did observe a strong antiangiogenic effect of sunitinib in GCT xenografts. This suggests that in GCT cells, resistance to cisplatin does not confer resistance to sunitinib. Despite this promising preclinical activity of sunitinib in cisplatin-resistant GCT cells, shown both in vitro in our analysis and in vivo by Castillo-Avila et al. [17], sunitinib exhibited only modest activity with three confirmed temporary partial responses (PRs) in patients with cisplatin -refractory or multiply relapsed GCT and thus our study failed to meet its primary end point of a 20% response rate. This study was conducted in a prognostically unfavorable group of patients, comparable with those treated within our previous trials evaluating the role of paclitaxel, gemcitabine, and oxaliplatin, or the combination of gemcitabine–paclitaxel in relapsed GCT [7–9]. Thirty-three percent of the patients presented with a late relapse, 15% initially had presented with a primary mediastinal GCT, 66% were pretreated with HD-CT, and 54% of our patients were classified as cisplatin refractory
doi:10.1093/annonc/mdr026 | 2657
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
Median age, years (range) Eastern Cooperative Oncology Group performance status 0 1 2 Location of primary tumor Gonadal Retroperitoneal Mediastinal Extragonadal not further specified Histology Pure seminoma Mixed nonseminomatous Metastases at study entry Lungs Liver Bones Lymph nodes Other Visceral (liver, pancreas, colon) Tumor markers at study entry Median AFP valuea Median beta-HCG valueb Median LDH valuec Elevated TUM at baseline AFP HCG LDH Median no. of previous platin-based cycles (range) Previous high-dose chemotherapy Refractoriness after previous platin-based chemotherapy Absolute cisplatin refractory Cisplatin refractory Not refractory Previous gemcitabine–oxaliplatin-based regimen Previous gemcitabine–paclitaxel-based regimen Late relapse (>2 years after initial chemotherapy)
%
original article
Annals of Oncology
Table 3. Characteristics of patients achieving a confirmed or an unconfirmed PR to sunitinib Patient no.
Primary histology
Prior therapy
Late relapse
Refractory status
Site of metastases at study entry
Best response to sunitinib
Time to progression (months)
007-007
Seminoma
No
Refractory
Liver, bone metastases
PR
5.3
007-013
Nonseminoma
BEP, TIP, HD-CT, Gem–Oxali BEP, VIP, Gem–Oxali
No
Lung, bone metastases
PR-
6.4
007-014
Nonseminoma
Yes
Absolute refractory Sensitive
Marker relapse
PR
12.2
04-001
Seminoma
No
Refractory
Lymph nodes
PR unconfirmed
2.3
PVB, VIP, actinomycin– cyclophosphamide BEP, VIP, HD-CT
BEP, Bleomycin, Etoposide, Cisplatin; HD-CT, high-dose chemotherapy; PR, partial response; PVB, Cisplatin, Vinblastine, Bleomycin; TIP, Paclitaxel, Etoposide, Cisplatin; VIP, Etoposide, Ifosfamide, Cisplatin.
Table 4. Toxic effects attributable to sunitinib in ‡7% of patients (N = 33)
Anorexia Diarrhea Fatigue Heartburn Mucositis Vomiting
Grade I/II, n (%)
Grade III/IV, n (%)
7 9 12 9 13 8
0 0 1 (3) 0 2 (6) 0
(21) (27) (36) (27) (39) (24)
(Table 1). In addition, the majority of patients had also been pretreated with gemcitabine, paclitaxel, and/or oxaliplatin or combinations of these drugs. The PR rate of 13% and disease stabilization rate of 41% with 11% of patients being progression free at 6 months suggest activity in this patient population but is lower than the response rates of 25%–45% recently reported for conventional combination chemotherapy such as oxaliplatin–gemcitabine
2658 | Oechsle et al.
Volume 22 | No. 12 | December 2011
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
Figure 2. Progression-free survival (PFS). CI, confidence interval.
(Table 2). Achieving a high response rate in refractory patients, as seen in studies with combination regimens, e.g. gemcitabine– oxaliplatin, is important because the induction of a response may subsequently allow the resection of residual masses and may thus be a chance to still achieve long-term survival. This is underlined by a proportion of 10%–15% of long-term survivors in the trials with conventional chemotherapy [10]. No complete remission was observed in the current trial with sunitinib. Interestingly, however, three of the responding patients were classified as cisplatin refractory before sunitinib therapy. Two patients had refractory seminoma, which can harbor c-KIT mutations as a potential target for sunitinib [32, 33]. Further molecular analysis of these cases is warranted to potentially identify predictive factors for response to sunitinib. Forty-one percent of patients in our study achieved disease stabilization, although the duration of stabilization was generally brief and nonresponding patients progressed rapidly, similar to the duration of stable disease seen in previous chemotherapy studies. It remains unclear whether off-target activity of sunitinib may have contributed to the activity seen in our study. However, any activity in refractory GCT patients is uncommon, and the overall control rate seen in this study is noteworthy and suggests that further investigation of antiangiogenic agents, in particular in combination with chemotherapy, is warranted. Sunitinib was associated with acceptable toxicity in this heavily pretreated patient population. No unexpected toxic effects were observed, and myelotoxicity was not treatment limiting in these extensively pretreated patients. However, the majority of patients received sunitinib only for a short period of time, which limits our assessment of toxicity. Our results are slightly better than results previously reported in another small exploratory phase II trial [34]. In this trial, some marker decline was seen; however, no objective response was observed, and the trial was stopped prematurely after enrollment of only 10 patients. In summary, sunitinib appears to have good preclinical activity and antitumor responses were observed in refractory GCT suggesting a role of the VEGF pathway in GCT. Exploring the molecular characteristics of responding patients may allow the identification of a molecular profile suitable for sunitinib or treatment with other targeted therapies. However, taking into account the results by Feldman et al. [34] as well as our results, it is unlikely that sunitinib as a single agent will clinically play
Annals of Oncology
acknowledgements We thank Ozmosis Inc., Toronto, for excellent data management and logistical support. Presented in part at the 45th Annual Meeting of the American Society of Clinical Oncology, Chicago, IL, June 2010.
funding Pfizer Inc., New York (investigator-initiated research grant).
disclosure The authors declare no conflict of interest.
references 1. Porcu P, Bhatia S, Einhorn LH. Results of treatment after relapse from high-dose chemotherapy in germ cell tumors. J Clin Oncol 2000; 18: 1181–1186. 2. Miller JC, Einhorn LH. Phase II study of daily oral etoposide in refractory germ cell tumors. Semin Oncol 1990; 17: 36–39. 3. Motzer RJ, Bajorin D, Schwartz LH et al. Phase II trial of paclitaxel shows antitumor activity in patients with previously treated germ cell tumors. J Clin Oncol 1994; 12: 2277–2283. 4. Bokemeyer C, Gerl A, Scho¨ffski P et al. Gemcitabine in patients with relapsed or cisplatin-refractory testicular cancer. J Clin Oncol 1999; 17: 512–516. 5. Einhorn LH, Stender MJ, Williams SD. Phase II trial of gemcitabine in refractory germ cell tumors. J Clin Oncol 1999; 17: 509–511. 6. Kollmannsberger C, Rick O, Derigs H-G et al. Activity of oxaliplatin in patients with relapsed or cisplatin-refractory germ cell cancer: a study of the German Testicular Cancer Study Group. J Clin Oncol 2002; 20: 2031–2037. 7. Hinton S, Catalano PJ, Einhorn L et al. Phase II trial of paclitaxel and gemcitabine in refractory germ cell tumors. J Clin Oncol 2001; 20: 1859–1863. 8. Bokemeyer C, Oechsle K, Honecker F et al. Combination chemotherapy with gemcitabine, oxaliplatin, and paclitaxel in patients with cisplatin-refractory or multiply relapsed germ-cell tumors: a study of the German Testicular Cancer Study Group. Ann Oncol 2008; 19: 448–453. 9. Kollmannsberger C, Beyer J, Liersch R et al. Combination chemotherapy with gemcitabine plus oxaliplatin in patients with intensively pretreated or refractory germ cell cancer: a study of the German Testicular Cancer Study Group. J Clin Oncol 2004; 22: 108–114.
Volume 22 | No. 12 | December 2011
10. Oechsle K, Kollmannsberger C, Honecker F et al. Long term outcome in patients with cisplatin-refractory or multiply relapsed germ cell tumors following treatment with gemcitabine and oxaliplatin 6 paclitaxel. J Clin Oncol 2010; 28: 15s (Abstr 4585). 11. Honecker F, Wermann H, Mayer F et al. Microsatellite instability, mismatch repair deficiency, and BRAF mutation in treatment-resistant germ cell tumors. J Clin Oncol 2009; 27: 2129–2136. 12. Viglietto G, Romano A, Maglione D et al. Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor. Oncogene 1996; 13: 577–587. 13. Bentas W, Beecken W, Glienke W et al. Serum levels of basic fibroblast growth factor reflect disseminated disease in patients with testicular germ cell tumors. Urol Res 2003; 30: 390–393. 14. Fukuda S, Shirahama T, Imazono Y et al. Expression of vascular endothelial growth factor in patients with testicular germ cell tumors as an indicator of metastatic disease. Cancer 1999; 85: 1323–1330. 15. Adam M, Schmidt D, Wardelmann E et al. Angiogenetic protooncogene ets-1 induced neovascularization is involved in the metastatic process of testicular germ cell tumors. Eur Urol 2003; 44: 329–336. 16. Abraham D, Abri S, Hofmann M et al. Low dose carboplatin combined with angiostatic agents prevents metastasis in human testicular germ cell tumor xenografts. J Urol 2003; 170: 1388–1393. 17. Castillo-Avila W, Piulats JM, Garcia Del Muro X et al. Sunitinib inhibits tumor growth and synergizes with cisplatin in orthotopic models of cisplatin-sensitive and cisplatin-resistant human testicular germ cell tumors. Clin Cancer Res 2009; 15: 3384–3395. 18. Andrews PW. Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev Biol 1984; 103: 285–293. 19. Teshima S, Shimosato Y, Hirohashi S et al. Four new human germ cell tumor cell lines. Lab Invest 1988; 59: 328–336. 20. Glaesener S, Honecker F, Veltman IM et al. Comparative proteome, transcriptome, and genome analysis of a gonadal and an extragonadal germ cell tumor cell line. J Proteome Res 2008; 7: 3890–3899. 21. Nichols CR, Tricot G, Williams SD et al. Dose-intensive chemotherapy in refractory germ cell cancer—a phase I/II trial of high-dose carboplatin and etoposide with autologous bone marrow transplantation. J Clin Oncol 1989; 7: 932–939. 22. Beyer J, Kramar A, Mandanas R et al. High-dose chemotherapy as salvage treatment in germ cell tumors: a multivariate analysis of prognostic variables. J Clin Oncol 1996; 14: 2638–2645. 23. Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000; 92: 205–216. 24. Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials 1989; 10: 1–10. 25. Pectasides D, Pectasides M, Farmakis D et al. Gemcitabine and oxaliplatin (GEMOX) in patients with cisplatin-refractory germ cell tumors: a phase II study. Ann Oncol 2004; 15: 493–497. 26. Fenner MH, Beutel G, Grunwald V. Targeted therapies for patients with germ cell tumors. Expert Opin Investig Drugs 2008; 17: 511–522. 27. Kollmannsberger C, Honecker F, Bokemeyer C. Pharmacotherapy of relapsed metastatic testicular cancer. Expert Opin Pharmacother 2008; 9: 2259–2272. 28. Muller-Tidow C, Schwable J, Steffen B et al. High-throughput analysis of genome-wide receptor tyrosine kinase expression in human cancers identifies potential novel drug targets. Clin Cancer Res 2004; 10: 1241–1249. 29. Abrams TJ, Lee LB, Murray LJ et al. SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer. Mol Cancer Ther 2003; 2: 471–478. 30. Potapova O, Laird AD, Nannini MA et al. Contribution of individual targets to the antitumor efficacy of the multitargeted receptor tyrosine kinase inhibitor SU11248. Mol Cancer Ther 2006; 5: 1280–1289. 31. Hui EP, Lui VW, Wong CS et al. Preclinical evaluation of sunitinib as single agent or in combination with chemotherapy in nasopharyngeal carcinoma. Invest New Drugs 2010. [Epub ahead of print].
doi:10.1093/annonc/mdr026 | 2659
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
a major role in cisplatin-refractory patients at standard doses and standard schedules unless the underlying mechanism of response to sunitinib is identified. GCT as a highly chemotherapy-sensitive malignancy may provide an opportunity to combine sunitinib with classic cytotoxic cisplatin-based chemotherapy [17, 35]. Ramasubbaiah et al. [36] recently published preliminary data on the combination of oxaliplatin and bevacizumab. Five of eight patients with late relapse responded, suggesting some activity of a combination approach in this particular patient population. A major concern, however, with regard to combination treatment using a multityrosine kinase inhibitor like sunitinib and a cytotoxic drug is overlapping and often additive toxicity, e.g. myelosuppression [37, 38]. Further research to unravel the molecular biology of testicular GCT and in particular the mechanisms of chemotherapy resistance is urgently needed, but the identification of new targets will hopefully allow more tailored therapy approaches in GCT.
original article
original article 32. Kemmer K, Corless CL, Fletcher JA et al. KIT mutations are common in testicular seminomas. Am J Pathol 2004; 164: 305–313. 33. Coffey J, Linger R, Pugh J et al. Somatic KIT mutations occur predominantly in seminoma germ cell tumors and are not predictive of bilateral disease: report of 220 tumors and review of literature. Genes Chromosomes Cancer 2008; 47: 34–42. 34. Feldman DR, Turkula S, Ginsberg MS et al. Phase II trial of sunitinib in patients with relapsed or refractory germ cell tumors. Invest New Drugs 2009; 28: 523–528. 35. Suddek GM. Sunitinib improves chemotherapeutic efficacy and ameliorates cisplatin-induced nephrotoxicity in experimental animals. Cancer Chemother Pharmacol 2010. [Epub ahead of print].
Annals of Oncology
36. Ramasubbaiah R, Brames J, Johnston EL et al. Phase II study of oxaliplatin (O) and bevacizumab (B) chemotherapy in refractory germ cell tumors (GCT). J Clin Oncol 2010; 28: (Abstr e15054). 37. Galsky MD, Sonpavde G, Hellerstedt BA et al. Phase II study of gemcitabine, cisplatin, and sunitinib in patients with advanced urothelial carcinoma (UC). J Clin Oncol 2010; 28: 15s (Abstr 4573). 38. Reck M, Frickhofen N, Cedres S et al. Sunitinib in combination with gemcitabine plus cisplatin for advanced non-small cell lung cancer: a phase I dose escalation study. Lung Cancer 2010; 70: 180–187.
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
2660 | Oechsle et al.
Volume 22 | No. 12 | December 2011
Annals of Oncology 22: 2654–2660, 2011 doi:10.1093/annonc/mdr026 Published online 17 March 2011
Preclinical and clinical activity of sunitinib in patients with cisplatin-refractory or multiply relapsed germ cell tumors: a Canadian Urologic Oncology Group/German Testicular Cancer Study Group cooperative study K. Oechsle1 , F. Honecker1 , T. Cheng2, F. Mayer3, P. Czaykowski4, E. Winquist5, L. Wood6, M. Fenner7, S. Glaesener1, J. T. Hartmann3, K. Chi8, C. Bokemeyer1 & C. Kollmannsberger8*
Received 29 November 2010; revised 31 December 2010 & revised 3 January 2011; accepted 17 January 2011
Background: The objective of the study was to investigate the activity of sunitinib in a cell line model and subsequently in patients with cisplatin-refractory or multiply relapsed germ cell tumors (GCT).
original article
Methods: The effect of sunitinib on cell proliferation in cisplatin-sensitive and cisplatin-refractory GCT cell lines was evaluated after 48-h sunitinib exposure by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and IC50 (concentration that causes 50% inhibition of growth) doses were determined. Sunitinib was subsequently administered at a dose of 50 mg/day for 4 weeks followed by a 2-week break to 33 patients using a Simon two-stage design. Results: Sunitinib demonstrated comparable dose-dependent growth inhibition in cisplatin-sensitive and cisplatinresistant cell lines, with IC50 between 3.0 and 3.8 lM. Patient characteristics were as follows: median of 2 (1–6) cisplatin-containing regimens; high-dose chemotherapy 67%; late relapse 33%; and cisplatin refractory or absolute cisplatin refractory 54%. Toxic effects included fatigue (39%), anorexia (21%), diarrhea (27%), mucositis (45%), nausea (33%), hand–foot syndrome (12%), dyspepsia (27%), and skin rash (18%). No unexpected side-effects were observed. Thirty -two of 33 patients were assessable for response. Three confirmed partial responses (PRs) and one unconfirmed PR were seen for a total response rate of 13%. Median progression-free survival (PFS) was 2 months, with a 6-month PFS rate of 11%. Conclusions: Sunitinib shows in vitro activity in cisplatin-resistant GCT cell lines. Modest clinical activity in heavily pretreated GCT patients was observed. Key words: angiogenesis, cisplatin resistance, germ cell tumor, relapse, sunitinib
introduction Although the vast majority of patients with metastatic germ cell tumors (GCT) are cured, those with cisplatin-refractory relapse or relapse after high-dose chemotherapy (HD-CT) still exhibit a very poor prognosis, and <5% of these patients will achieve long-term survival [1]. For these patients, the evaluation of new active drugs and treatment combinations remains a priority. *Correspondence to: Dr C. Kollmannsberger, Division of Medical Oncology, British Columbia Cancer Agency, University of British Columbia–Vancouver Cancer Centre, 600 West 10th Avenue, Vancouver, Canada BC V5Z 4E6. Tel: +1-604-877-6000 ext. 2674; Fax: +1-604-708-2144; E-mail: [email protected]
Both authors contributed equally as senior authors.
Various agents have been evaluated in intensively pretreated or cisplatin-refractory patients. As single agents, only paclitaxel, gemcitabine, oxaliplatin, and orally administered etoposide have been shown to be active, with selected patients achieving long-term survival [2–6]. Different combinations of these agents demonstrated response rates up to 51%, including 10%– 15% long-term survivors, although duration of remission is usually moderate [7–10]. The molecular mechanisms involved in cisplatin resistance are still incompletely understood. Mismatch repair deficiency and microsatellite instability are thought to be associated with cisplatin resistance in human GCT [11]. Preliminary studies suggest that vascular endothelial growth factor (VEGF) may play an important role in development and metastasizing of
ª The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected]
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
1 Division of Oncology, Hematology, Bone Marrow Transplantation and Section Pneumology, University Medical Centre Eppendorf, Hamburg University, Hamburg, Germany; 2Department of Oncology, Tom Baker Cancer Centre, Calgary, Canada; 3Department of Medical Oncology, Hematology, Rheumatology, Immunology and Pulmonology, University of Tuebingen, Tuebingen, Germany; 4Department Medical Oncology/Haematology, CancerCare Manitoba, University of Manitoba, Winnipeg; 5 Division of Medical Oncology, London Health Science Centre, London; 6Division of Medical Oncology, Queen Elizabeth II Health Sciences Centre, Halifax, Canada; 7 Department of Hematology/Hemostaseology/Oncology/Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; 8Division of Medical Oncology, British Columbia Cancer Agency, University of British Columbia–Vancouver Cancer Centre, Vancouver, Canada
original article
Annals of Oncology
patients and methods preclinical study Sunitinib was kindly provided by Pfizer Inc., Berlin, Germany, and was dissolved in dimethyl sulfoxide (DMSO). Cisplatin was obtained from Teva, Radebeul, Germany, and was dissolved in 0.9% saline. NTERA2 cells, first described by Andrews [18], were obtained from the ‘Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH’, Braunschweig, Germany. NCCIT cells, first described by Teshima et al. [19], were obtained from American Type Culture Collection, Manassas, VA. 2102Ep cells were kindly provided by L. Looijenga, Rotterdam, The Netherlands. NCCIT and 2102Ep cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and F12 medium (1 : 1) (Gibco-BRL; Invitrogen, Paisley, UK) containing 10% fetal calf serum (FCS) (Gibco-BRL; Invitrogen), and NTERA2 cells with DMEM supplemented with GlutamaxI (Gibco-BRL; Invitrogen) containing 10% FCS (Gibco-BRL; Invitrogen). Cells were incubated at 37C in a humidified atmosphere with 5% CO2. From these well-established GCT cell lines, all derived from human embryonal carcinomas, cisplatin-resistant sublines exhibiting 5.2–12.2-fold increase in cisplatin resistance were generated in our laboratory over a time period of 18 months by intermittent exposure to increasing doses of cisplatin, starting with the IC50 (concentration that causes 10% inhibition of growth) dose of the parental cell lines. At reaching 50% lethality, the addition of cisplatin was paused and cells were allowed to recover over three passages until the next dose escalation step. The MTT assay was carried out as previously described [20]. The MTT assay is based on the cleavage of the tetrazolium salt MTT to purple formazan crystal by metabolically active cells. In brief, the cell lines were rinsed with phosphate-buffered saline, trypsinized, and resuspended in 1 ml of the appropriate culture medium to count the cells in a hemaocytometer chamber. In total, 4 · 103 cells per well were seeded in 96-well plates to ensure logarithmic growth. Cells were allowed to adhere over night, and serial dilutions of the chemotherapeutic agents were added to octuplicate
Volume 22 | No. 12 | December 2011
wells at concentrations from 0.2 to 25.6 lM of both sunitinib and cisplatin. The cells were exposed to the drug for 48 h. Thereafter, the drug-containing medium was removed and 0.2 ml MTT solution (final concentration: 0.5 mg/ml MTT; Sigma, Hamburg, Germany) was added. The plates were incubated for 2 h before the medium was removed and 0.1 ml DMSO was added. The plates were agitated for 15 min before reading absorbance using a photometer (Victor2 Wallac 1420; Perkin Elmer Life Sciences, Wellesley, MA) at 570 nm. All experiments were carried out in triplicates and were repeated at least twice. The results are expressed as IC50 (inhibitory concentration).
clinical study Eligibility criteria for this open-label, multicenter phase II trial included histologically confirmed seminomatous or nonseminomatous GCT, and relapse within 2 months after cisplatin-based chemotherapy, tumor progression during or relapse after salvage HD-CT, tumor progression during salvage cisplatin-based chemotherapy, or ineligibility for cisplatinbased chemotherapy or HD-CT due to severe comorbidities. Patients had to have measurable disease and documented tumor progression on imaging. A sole elevation of alpha fetoprotein (AFP) or beta-human chorionic gonadotropin (b-HCG) without disease on imaging was accepted as measurable disease if an increase of at least 25% within 4 weeks before study inclusion had been observed. Further inclusion criteria consisted of a Karnofsky performance status ‡60% and a life expectancy ‡3 months. Adequate bone marrow function (neutrophils >1500/ll, platelet count >100 000/ll), liver function (bilirubin <1.5-fold upper normal limit), and renal function (serum creatinine £twofold upper normal limit) were mandatory. All patients gave written informed consent. The study (clinical trial identifier: NCT00371553) was approved by the ethics committees of the British Columbia Cancer Agency and the University of Hamburg-Eppendorf, Germany, as well as by local ethics committees of all participating centers. Sunitinib was given at a dose of 50 mg daily for 4 weeks followed by a 2week break to form 6-week cycles. Dose reductions to 37.5 or 25 mg daily for 4 weeks on/2 weeks off were recommended for significant toxicity. No more than two dose reductions were permitted in any patient. If clinically deemed appropriate or necessary by the investigator, a continuous administration schedule utilizing 37.5 or 25 mg daily without breaks was allowed. Intrapatient reescalation back to the previous dose level was permitted in the absence of grade ‡3 hematologic or grade ‡2 non-hematologic toxicity in the previous cycle. Treatment was continued until disease progression, occurrence of unacceptable toxicity, intercurrent illness that prevented further administration of therapy, or patient’s decision to withdraw from the study. Pretreatment evaluation consisted of medical history, assessment of the Karnofsky performance status, physical examination, electrocardiogram, Multi Gated Acquisition Scan or echocardiogram, routine laboratory, thyroid function tests, urinnalysis, tumor markers (AFP and b-HCG), as well as radiological tumor assessments.
definitions Disease was considered cisplatin refractory when at least tumor stabilization or a remission had been achieved during cisplatin-based chemotherapy, but tumor progression occurred again within 4 weeks of the last cisplatin-based chemotherapy. Disease was considered absolutely cisplatin refractory when tumor progression had occurred while patients were receiving cisplatinbased therapy [21, 22].
response/toxicity assessment Response assessments were carried out on day 28 of each 6-week cycle. Response was evaluated according to RECIST version 1.0 criteria [23]. In addition, patients with reduction of the size of a tumor lesion and normalization of previously elevated tumor markers were considered partial remission with tumor marker normalization (PR negative). Patients with
doi:10.1093/annonc/mdr026 | 2655
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
GCT [12–14]. A substantially higher VEGF and platelet-derived growth factor (PDGF) expression has been found in patients with GCT compared with normal testis tissue in several studies, indicating that VEGF and PDGF expression could play an important role in tumor angiogenesis, tumor progression, and metastases [12–15]. A significantly higher progression-free survival (PFS) rate was seen in a murine testicular cancer xenograft model when an angiostatic agent was combined with carboplatin or cisplatin as compared with either carboplatin– cisplatin or the angiostatic agent alone [16, 17]. Sunitinib is an orally administered small molecule and a potent multityrosine kinase inhibitor for VEGF receptor, PDGF receptor, as well as the stem cell factor receptor c-KIT. Sunitinib has demonstrated clinical activity in several tumors including thymic carcinoma and pancreatic neuroendocrine tumors, and is currently approved for the treatment of gastrointestinal stromal tumors and renal cell carcinoma. Based on this rationale, we used a cell line model to investigate the preclinical activity of sunitinib in different GCT cell lines with defined levels of cisplatin resistance by MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Having observed in vitro activity of sunitinib in cisplatinresistant GCT cell lines at the same level as in their cisplatinsensitive counterparts, we carried out a phase II study in patients with cisplatin-refractory or multiply relapsed GCT.
original article a partial remission of tumor lesions without complete tumor marker normalization were considered a marker-positive partial remission (PR positive). If elevated markers were the only evidence of progressive disease, a decrease of at least 90% was required for a PR. Levels of serum tumor markers were measured every 3 weeks. All responses as well as the diagnosis of a stable disease had to be confirmed after a 4-week interval. Toxicity was assessed after each cycle and graded by the National Cancer Institute—Common Terminology Criteria for Adverse Events version 3.0.
statistical considerations
results proliferation and cytotoxicity assays MTT assay. After long-term exposure to cisplatin, resistance levels substantially increased in all three cell lines: NTERA2, 2102Ep, and NCCIT (Figure 1). Whereas the IC50 values of the sensitive parental cell lines were 0.45 lM [60.1 standard deviation (SD)], 0.75 lM (60.1 SD), and 1.75 lM (60.1 SD), respectively, the three cisplatin-resistant sublines exhibited IC50 values of 5.1 lM (60.1 SD) in NTERA-2R, 4.9 lM (60.1 SD) in 2102Ep-R, and 4.7 lM (60.2 SD) in NCCIT-R (P < 0.001 for all). Sunitinib substantially decreased proliferation of all
Figure 1. IC50 (concentration that causes 50% inhibition of growth) values of cisplatin and sunitinib in three embryonal carcinoma cell lines (NTERA2, 2102Ep, and NCCIT) and their derivatives, relatively cisplatinresistant sublines NTERA2-R, 2102Ep-R, and NCCIT-R. Cells were treated with either cisplatin or sunitinib for 48 h. Whereas the differences between the IC50 values for cisplatin-sensitive parental and their cisplatin-resistant sublines were all statistically significant (àP < 0.001), differences in sensitivity to sunitinib were only significant for NTERA2-R versus NTERA, with the cisplatin-resistant line showing better sensitivity than the cisplatin-sensitive cell line ( P = 0.02).
2656 | Oechsle et al.
GCT cell lines in comparison with control cells treated with the solvent DMSO. By MTT assay, IC50 values for sunitinib were within a narrow range in all three cell lines: NTERA2 3.8 (60.1 SD), NTERA2-R 3.5 (60.1 SD), 2102Ep 3.0 (60.1 SD), 2102Ep-R 3.3 (60.4 SD), NCCIT 3.7 (60.2), and NCCIT-R 3.4 (60.6 SD) (Figure 1). Apart from NTERA2-R, which were slightly more sensitive to sunitinib than NTERA2 (P = 0.02), there was no difference in sensitivity to sunitinib between cisplatin-sensitive parental cell lines and their cisplatin-resistant sublines, demonstrating that there is no cross-resistance of cisplatin and sunitinib in GCT cell lines in vitro.
clinical study Thirty-three patients with heavily pretreated or cisplatinrefractory GCT were entered into the study between February 2007 and January 2010. Patient characteristics are listed in Table 1. Fifteen percent of patients had a primary mediastinal GCT. All patients were heavily pretreated with a median number of 2 (range, 1–6) platinum-based regimens. Sixty-six percent of patients had relapsed after HD-CT, and 33% were considered late relapses (‡2 years after initial therapy). Fifty-four percent of patients were considered cisplatin refractory or absolutely refractory. A total of 66 cycles of sunitinib were administered with a median number of 1 cycle per patient (range, 1–9). No complete remission was observed. Three patients achieved a partial remission (9%) (Tables 2 and 3). Time to progression for these three patients was 5.0, 6.4, and 12.2 months. One additional seminoma patient achieved a response but subsequently went off study before a confirmatory scan was carried out (Table 3). Stable disease was recorded for 41% of patients with a median duration of 2.3 months (range 1.6–6.5 months). After a median follow-up of 14.4 months (range, 6–18 months), all patients are off study. In 30 patients, treatment termination was due to progression, 2 patients refused further treatment, and 1 patient was stopped due to hyperbilirubinemia. Median PFS for all patients was 2.0 months [95% confidence interval (CI) 1.4–2.60], with 11% and 3.7% of patients being progression free at 6 and 12 months, respectively (Figure 2). Median OS was 3.8 months (95% CI 3.0–6.6), with 36.4% and 9.9% of patients being alive at 6 and 12 months, respectively. Sunitinib treatment was feasible and toxicity in these heavily pretreated patients was generally acceptable. However, treatment duration was short in the majority of patients and thus toxicity assessment, and in particular evaluation of longterm toxicity, is limited (Table 4). The most common sunitinib-related toxic effects included fatigue (39%), anorexia (21%), diarrhea (27%), mucositis (45%), nausea/vomiting (24%), and dyspepsia (27%). Grade 3/4 toxic effects were rare and no unexpected side-effects were observed.
discussion Despite the overall success of cisplatin-based chemotherapy in patients with metastatic GCT, the prognosis for cisplatin-
Volume 22 | No. 12 | December 2011
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
The primary end point of this study was response rate. Tolerability, PFS, and overall survival (OS) time represented secondary end points. An optimal Simon two-stage design was used to determine the number of patients required [24]. Assuming a response rate of clinical interest of >20%, a minimal response rate of 5%, a probability of 5% for rejecting an active drug combination (type II error), and a probability of 20% to further evaluate an ineffective drug combination (type I error), 16 patients had to be enrolled into the first cohort. If no response to study therapy was observed among the first 16 patients, the study was to be terminated. If at least one objective remission occurred, the study was to be continued with a second cohort of 16 patients.
Annals of Oncology
original article
Annals of Oncology
Table 1. Patients’ characteristics (N = 33)
Table 2. Treatment efficacy (N = 33) n 32 (22–54)
14 17 2
42 52 6
26 0 5 2
79 0 15 6
4 29
12 88
20 11 8 15 18 25
61 33 24 35 55 76
9 kU/l (1–490 000) 111 U/l (0.3–39 000) 278 (110–2107) 15 18 18 6 (1–18)
48 62 60
22
66
9 9 15 21
27 27 45 64
5
15
11
33
a
n = 31. n = 29. c n = 30. AFP, alpha fetoprotein; HCG, human chorionic gonadotropin; LDH, lactate dehydrogenase; TUM, tumor markers. b
refractory patients remains very poor. Gemcitabine–oxaliplatin and paclitaxel–gemcitabine or gemcitabine–oxaliplatin– paclitaxel are the most commonly used regimens for these patients resulting in response rates of 25%–40%, with selected patients achieving prolonged survival [7–9, 25]. Limited clinical data exist regarding targeted therapies in GCT [26, 27]. Based on previous reports on the potential role of VEGF and VEGFR and the very high expression of tyrosine kinases in GCT, we
Volume 22 | No. 12 | December 2011
Total cycles Median cycles per patient (range) Response (n = 32), n (%) Confirmed PR Unconfirmed PR Stable disease Progression Time to progression Median, months (95% CI) 3 months PFS, % (95% CI) 6 months PFS, % (95% CI) OS Median, months (95% CI) 3 months OS, % (95% CI) 6 months OS, % (95% CI) 9 months OS, % (95% CI) 12 months OS, % (95% CI)
66 1 (1–9) 3 1 13 15
(9) (3) (41) (47%)
2 (1.4–2.6) 26 (13.8% to 47.5%) 11 (3.8% to 31.6%) 3.8 66 36 17 10
(3.0–6.6) (52.4% to 84.9%) (23.2% to 57.1%) (6.4% to 35.9%) (0.8% to 31.0%)
PR, partial response; CI, confidence interval; PFS, progression-free survival; OS, overall survival.
investigated the multityrosine kinase inhibitor sunitinib preclinically and subsequently clinically in cisplatin-resistant GCT [28]. Analysis of the efficacy of sunitinib in GCT cell lines in vitro demonstrated inhibition of proliferation (MTT assay) at an IC50 in the range of 3.0–3.8 lM after 48 h of drug exposure. There was no difference between cisplatin-sensitive and cisplatin-resistant cell lines, indicating that there is no cross-resistance between sunitinib and cisplatin. Furthermore, this dose range for in vitro activity is at the lower end of previously determined doses in different other tumor cell lines including renal cell carcinoma, nasopharyngeal carcinoma, or lung cancer, where IC50 doses of 3–10 lM have been reported [29–31]. An IC50 of 6 lM has previously been reported for the human yolk sac tumor cell line 1411H after 24-h treatment [17]. As was the case in our three cell lines and their cisplatinresistant sublines, no cross-resistance between cisplatin and sunitinib was observed by Castillo-Avila et al. [17] who, in addition to direct action on receptor tyrosine kinases, also did observe a strong antiangiogenic effect of sunitinib in GCT xenografts. This suggests that in GCT cells, resistance to cisplatin does not confer resistance to sunitinib. Despite this promising preclinical activity of sunitinib in cisplatin-resistant GCT cells, shown both in vitro in our analysis and in vivo by Castillo-Avila et al. [17], sunitinib exhibited only modest activity with three confirmed temporary partial responses (PRs) in patients with cisplatin -refractory or multiply relapsed GCT and thus our study failed to meet its primary end point of a 20% response rate. This study was conducted in a prognostically unfavorable group of patients, comparable with those treated within our previous trials evaluating the role of paclitaxel, gemcitabine, and oxaliplatin, or the combination of gemcitabine–paclitaxel in relapsed GCT [7–9]. Thirty-three percent of the patients presented with a late relapse, 15% initially had presented with a primary mediastinal GCT, 66% were pretreated with HD-CT, and 54% of our patients were classified as cisplatin refractory
doi:10.1093/annonc/mdr026 | 2657
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
Median age, years (range) Eastern Cooperative Oncology Group performance status 0 1 2 Location of primary tumor Gonadal Retroperitoneal Mediastinal Extragonadal not further specified Histology Pure seminoma Mixed nonseminomatous Metastases at study entry Lungs Liver Bones Lymph nodes Other Visceral (liver, pancreas, colon) Tumor markers at study entry Median AFP valuea Median beta-HCG valueb Median LDH valuec Elevated TUM at baseline AFP HCG LDH Median no. of previous platin-based cycles (range) Previous high-dose chemotherapy Refractoriness after previous platin-based chemotherapy Absolute cisplatin refractory Cisplatin refractory Not refractory Previous gemcitabine–oxaliplatin-based regimen Previous gemcitabine–paclitaxel-based regimen Late relapse (>2 years after initial chemotherapy)
%
original article
Annals of Oncology
Table 3. Characteristics of patients achieving a confirmed or an unconfirmed PR to sunitinib Patient no.
Primary histology
Prior therapy
Late relapse
Refractory status
Site of metastases at study entry
Best response to sunitinib
Time to progression (months)
007-007
Seminoma
No
Refractory
Liver, bone metastases
PR
5.3
007-013
Nonseminoma
BEP, TIP, HD-CT, Gem–Oxali BEP, VIP, Gem–Oxali
No
Lung, bone metastases
PR-
6.4
007-014
Nonseminoma
Yes
Absolute refractory Sensitive
Marker relapse
PR
12.2
04-001
Seminoma
No
Refractory
Lymph nodes
PR unconfirmed
2.3
PVB, VIP, actinomycin– cyclophosphamide BEP, VIP, HD-CT
BEP, Bleomycin, Etoposide, Cisplatin; HD-CT, high-dose chemotherapy; PR, partial response; PVB, Cisplatin, Vinblastine, Bleomycin; TIP, Paclitaxel, Etoposide, Cisplatin; VIP, Etoposide, Ifosfamide, Cisplatin.
Table 4. Toxic effects attributable to sunitinib in ‡7% of patients (N = 33)
Anorexia Diarrhea Fatigue Heartburn Mucositis Vomiting
Grade I/II, n (%)
Grade III/IV, n (%)
7 9 12 9 13 8
0 0 1 (3) 0 2 (6) 0
(21) (27) (36) (27) (39) (24)
(Table 1). In addition, the majority of patients had also been pretreated with gemcitabine, paclitaxel, and/or oxaliplatin or combinations of these drugs. The PR rate of 13% and disease stabilization rate of 41% with 11% of patients being progression free at 6 months suggest activity in this patient population but is lower than the response rates of 25%–45% recently reported for conventional combination chemotherapy such as oxaliplatin–gemcitabine
2658 | Oechsle et al.
Volume 22 | No. 12 | December 2011
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
Figure 2. Progression-free survival (PFS). CI, confidence interval.
(Table 2). Achieving a high response rate in refractory patients, as seen in studies with combination regimens, e.g. gemcitabine– oxaliplatin, is important because the induction of a response may subsequently allow the resection of residual masses and may thus be a chance to still achieve long-term survival. This is underlined by a proportion of 10%–15% of long-term survivors in the trials with conventional chemotherapy [10]. No complete remission was observed in the current trial with sunitinib. Interestingly, however, three of the responding patients were classified as cisplatin refractory before sunitinib therapy. Two patients had refractory seminoma, which can harbor c-KIT mutations as a potential target for sunitinib [32, 33]. Further molecular analysis of these cases is warranted to potentially identify predictive factors for response to sunitinib. Forty-one percent of patients in our study achieved disease stabilization, although the duration of stabilization was generally brief and nonresponding patients progressed rapidly, similar to the duration of stable disease seen in previous chemotherapy studies. It remains unclear whether off-target activity of sunitinib may have contributed to the activity seen in our study. However, any activity in refractory GCT patients is uncommon, and the overall control rate seen in this study is noteworthy and suggests that further investigation of antiangiogenic agents, in particular in combination with chemotherapy, is warranted. Sunitinib was associated with acceptable toxicity in this heavily pretreated patient population. No unexpected toxic effects were observed, and myelotoxicity was not treatment limiting in these extensively pretreated patients. However, the majority of patients received sunitinib only for a short period of time, which limits our assessment of toxicity. Our results are slightly better than results previously reported in another small exploratory phase II trial [34]. In this trial, some marker decline was seen; however, no objective response was observed, and the trial was stopped prematurely after enrollment of only 10 patients. In summary, sunitinib appears to have good preclinical activity and antitumor responses were observed in refractory GCT suggesting a role of the VEGF pathway in GCT. Exploring the molecular characteristics of responding patients may allow the identification of a molecular profile suitable for sunitinib or treatment with other targeted therapies. However, taking into account the results by Feldman et al. [34] as well as our results, it is unlikely that sunitinib as a single agent will clinically play
Annals of Oncology
acknowledgements We thank Ozmosis Inc., Toronto, for excellent data management and logistical support. Presented in part at the 45th Annual Meeting of the American Society of Clinical Oncology, Chicago, IL, June 2010.
funding Pfizer Inc., New York (investigator-initiated research grant).
disclosure The authors declare no conflict of interest.
references 1. Porcu P, Bhatia S, Einhorn LH. Results of treatment after relapse from high-dose chemotherapy in germ cell tumors. J Clin Oncol 2000; 18: 1181–1186. 2. Miller JC, Einhorn LH. Phase II study of daily oral etoposide in refractory germ cell tumors. Semin Oncol 1990; 17: 36–39. 3. Motzer RJ, Bajorin D, Schwartz LH et al. Phase II trial of paclitaxel shows antitumor activity in patients with previously treated germ cell tumors. J Clin Oncol 1994; 12: 2277–2283. 4. Bokemeyer C, Gerl A, Scho¨ffski P et al. Gemcitabine in patients with relapsed or cisplatin-refractory testicular cancer. J Clin Oncol 1999; 17: 512–516. 5. Einhorn LH, Stender MJ, Williams SD. Phase II trial of gemcitabine in refractory germ cell tumors. J Clin Oncol 1999; 17: 509–511. 6. Kollmannsberger C, Rick O, Derigs H-G et al. Activity of oxaliplatin in patients with relapsed or cisplatin-refractory germ cell cancer: a study of the German Testicular Cancer Study Group. J Clin Oncol 2002; 20: 2031–2037. 7. Hinton S, Catalano PJ, Einhorn L et al. Phase II trial of paclitaxel and gemcitabine in refractory germ cell tumors. J Clin Oncol 2001; 20: 1859–1863. 8. Bokemeyer C, Oechsle K, Honecker F et al. Combination chemotherapy with gemcitabine, oxaliplatin, and paclitaxel in patients with cisplatin-refractory or multiply relapsed germ-cell tumors: a study of the German Testicular Cancer Study Group. Ann Oncol 2008; 19: 448–453. 9. Kollmannsberger C, Beyer J, Liersch R et al. Combination chemotherapy with gemcitabine plus oxaliplatin in patients with intensively pretreated or refractory germ cell cancer: a study of the German Testicular Cancer Study Group. J Clin Oncol 2004; 22: 108–114.
Volume 22 | No. 12 | December 2011
10. Oechsle K, Kollmannsberger C, Honecker F et al. Long term outcome in patients with cisplatin-refractory or multiply relapsed germ cell tumors following treatment with gemcitabine and oxaliplatin 6 paclitaxel. J Clin Oncol 2010; 28: 15s (Abstr 4585). 11. Honecker F, Wermann H, Mayer F et al. Microsatellite instability, mismatch repair deficiency, and BRAF mutation in treatment-resistant germ cell tumors. J Clin Oncol 2009; 27: 2129–2136. 12. Viglietto G, Romano A, Maglione D et al. Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor. Oncogene 1996; 13: 577–587. 13. Bentas W, Beecken W, Glienke W et al. Serum levels of basic fibroblast growth factor reflect disseminated disease in patients with testicular germ cell tumors. Urol Res 2003; 30: 390–393. 14. Fukuda S, Shirahama T, Imazono Y et al. Expression of vascular endothelial growth factor in patients with testicular germ cell tumors as an indicator of metastatic disease. Cancer 1999; 85: 1323–1330. 15. Adam M, Schmidt D, Wardelmann E et al. Angiogenetic protooncogene ets-1 induced neovascularization is involved in the metastatic process of testicular germ cell tumors. Eur Urol 2003; 44: 329–336. 16. Abraham D, Abri S, Hofmann M et al. Low dose carboplatin combined with angiostatic agents prevents metastasis in human testicular germ cell tumor xenografts. J Urol 2003; 170: 1388–1393. 17. Castillo-Avila W, Piulats JM, Garcia Del Muro X et al. Sunitinib inhibits tumor growth and synergizes with cisplatin in orthotopic models of cisplatin-sensitive and cisplatin-resistant human testicular germ cell tumors. Clin Cancer Res 2009; 15: 3384–3395. 18. Andrews PW. Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev Biol 1984; 103: 285–293. 19. Teshima S, Shimosato Y, Hirohashi S et al. Four new human germ cell tumor cell lines. Lab Invest 1988; 59: 328–336. 20. Glaesener S, Honecker F, Veltman IM et al. Comparative proteome, transcriptome, and genome analysis of a gonadal and an extragonadal germ cell tumor cell line. J Proteome Res 2008; 7: 3890–3899. 21. Nichols CR, Tricot G, Williams SD et al. Dose-intensive chemotherapy in refractory germ cell cancer—a phase I/II trial of high-dose carboplatin and etoposide with autologous bone marrow transplantation. J Clin Oncol 1989; 7: 932–939. 22. Beyer J, Kramar A, Mandanas R et al. High-dose chemotherapy as salvage treatment in germ cell tumors: a multivariate analysis of prognostic variables. J Clin Oncol 1996; 14: 2638–2645. 23. Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000; 92: 205–216. 24. Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials 1989; 10: 1–10. 25. Pectasides D, Pectasides M, Farmakis D et al. Gemcitabine and oxaliplatin (GEMOX) in patients with cisplatin-refractory germ cell tumors: a phase II study. Ann Oncol 2004; 15: 493–497. 26. Fenner MH, Beutel G, Grunwald V. Targeted therapies for patients with germ cell tumors. Expert Opin Investig Drugs 2008; 17: 511–522. 27. Kollmannsberger C, Honecker F, Bokemeyer C. Pharmacotherapy of relapsed metastatic testicular cancer. Expert Opin Pharmacother 2008; 9: 2259–2272. 28. Muller-Tidow C, Schwable J, Steffen B et al. High-throughput analysis of genome-wide receptor tyrosine kinase expression in human cancers identifies potential novel drug targets. Clin Cancer Res 2004; 10: 1241–1249. 29. Abrams TJ, Lee LB, Murray LJ et al. SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer. Mol Cancer Ther 2003; 2: 471–478. 30. Potapova O, Laird AD, Nannini MA et al. Contribution of individual targets to the antitumor efficacy of the multitargeted receptor tyrosine kinase inhibitor SU11248. Mol Cancer Ther 2006; 5: 1280–1289. 31. Hui EP, Lui VW, Wong CS et al. Preclinical evaluation of sunitinib as single agent or in combination with chemotherapy in nasopharyngeal carcinoma. Invest New Drugs 2010. [Epub ahead of print].
doi:10.1093/annonc/mdr026 | 2659
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
a major role in cisplatin-refractory patients at standard doses and standard schedules unless the underlying mechanism of response to sunitinib is identified. GCT as a highly chemotherapy-sensitive malignancy may provide an opportunity to combine sunitinib with classic cytotoxic cisplatin-based chemotherapy [17, 35]. Ramasubbaiah et al. [36] recently published preliminary data on the combination of oxaliplatin and bevacizumab. Five of eight patients with late relapse responded, suggesting some activity of a combination approach in this particular patient population. A major concern, however, with regard to combination treatment using a multityrosine kinase inhibitor like sunitinib and a cytotoxic drug is overlapping and often additive toxicity, e.g. myelosuppression [37, 38]. Further research to unravel the molecular biology of testicular GCT and in particular the mechanisms of chemotherapy resistance is urgently needed, but the identification of new targets will hopefully allow more tailored therapy approaches in GCT.
original article
original article 32. Kemmer K, Corless CL, Fletcher JA et al. KIT mutations are common in testicular seminomas. Am J Pathol 2004; 164: 305–313. 33. Coffey J, Linger R, Pugh J et al. Somatic KIT mutations occur predominantly in seminoma germ cell tumors and are not predictive of bilateral disease: report of 220 tumors and review of literature. Genes Chromosomes Cancer 2008; 47: 34–42. 34. Feldman DR, Turkula S, Ginsberg MS et al. Phase II trial of sunitinib in patients with relapsed or refractory germ cell tumors. Invest New Drugs 2009; 28: 523–528. 35. Suddek GM. Sunitinib improves chemotherapeutic efficacy and ameliorates cisplatin-induced nephrotoxicity in experimental animals. Cancer Chemother Pharmacol 2010. [Epub ahead of print].
Annals of Oncology
36. Ramasubbaiah R, Brames J, Johnston EL et al. Phase II study of oxaliplatin (O) and bevacizumab (B) chemotherapy in refractory germ cell tumors (GCT). J Clin Oncol 2010; 28: (Abstr e15054). 37. Galsky MD, Sonpavde G, Hellerstedt BA et al. Phase II study of gemcitabine, cisplatin, and sunitinib in patients with advanced urothelial carcinoma (UC). J Clin Oncol 2010; 28: 15s (Abstr 4573). 38. Reck M, Frickhofen N, Cedres S et al. Sunitinib in combination with gemcitabine plus cisplatin for advanced non-small cell lung cancer: a phase I dose escalation study. Lung Cancer 2010; 70: 180–187.
Downloaded from http://annonc.oxfordjournals.org/ at Chapman University on October 28, 2012
2660 | Oechsle et al.
Volume 22 | No. 12 | December 2011