Adjuvant interferon alfa in malignant melanoma: An interdisciplinary and multinational expert review

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1 Critical Reviews in Oncology/Hematology 85 (2013) Adjuvant interferon alfa in malignant melanoma: An interdisciplinary and multinational expert review Paolo A. Ascierto a,, Helen J. Gogas b, Jean Jacques Grob c, Salvador Martín Algarra d, Peter Mohr e, Johan Hansson f, Axel Hauschild g a Unit of Melanoma, Cancer Immunotherapy and Innovative Therapy, Istituto Nazionale per lo Studio e la Cura dei Tumori Fondazione G. Pascale, Naples, Italy b First Department of Internal Medicine, University of Athens, Athens, Greece c Aix-Marseille Université, Service de Dermatologie, Hopital de la Timone, Marseille, France d Department of Oncology, University of Navarra, Pamplona, Spain e Department of Dermatology, Elbeklinikum Buxtehude, Buxtehude, Germany f Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden g Department of Dermatology, Venereology and Allergology, University Hospital Schleswig-Holstein, Kiel, Germany Accepted 10 July 2012 Contents 1. Introduction Mechanism of action of IFN in melanoma Review of recent clinical studies High-dose IFN 2b Intermediate-dose IFN Low-dose IFN PegIFN 2b Predictive biomarkers of IFN response Tumor stage and disease burden Ulceration Autoantibodies Other potential predictive markers Recommendations regarding the use of adjuvant IFN Conclusions Conflict of interest statement Reviewers Acknowledgements References Biographies Abstract Interferon alfa (IFN ) and pegylated IFN 2b (PegIFN 2b) are the only agents currently approved for the adjuvant treatment of resected melanoma at high risk of recurrence. Meta-analyses showed statistically significant disease-free survival (DFS) and overall survival (OS) benefits versus controls but did not clarify optimal dose/duration. We review data from all recent clinical trials to provide the latest information Corresponding author at: Unit of Melanoma, Cancer Immunotherapy and Innovative Therapy, Istituto Nazionale per lo Studio e la Cura dei Tumori Fondazione G. Pascale, Via Mariano Semmola, Naples, Italy. Tel.: ; fax: address: paolo.ascierto@gmail.com (P.A. Ascierto) /$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.

2 150 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) on dose, duration, and potential predictive factors of treatment success. Recent data largely confirm DFS and OS benefits but optimal dose/duration is not clarified. The data suggest greater responses in patients with stage III micro-metastatic versus macro-metastatic disease, and ulceration may also predict greater sensitivity to therapy, although further investigation is needed. Presently, IFN and PegIFN 2b remain valid adjuvant therapies following resection of high-risk melanoma; the most appropriate treatment regimen should be determined on an individual patient basis according to patient lifestyle and approach, potential for toxicity, and the available clinical evidence Elsevier Ireland Ltd. All rights reserved. Keywords: Adjuvant; Disease-free survival; Immunomodulation; Interferon alfa-2a; Interferon alfa-2b; Malignant melanoma; Peginterferon alfa-2b; Survival 1. Introduction Melanoma incidence and mortality have been increasing steadily in Europe over the past decades, although rates vary by geographical region [1,2]. The age-standardized estimated incidence of melanoma in the European Union in 2008 was 11.3 per 100,000 and the mortality rate was 2.1 per 100,000 [3]. Survival rates are strongly correlated with disease stage at diagnosis and are generally favorable for localized earlystage tumors; however, for more advanced disease, survival rates are low [4]. Currently, the only approved agents for the adjuvant treatment of resected melanoma that is at high risk of recurrence are interferon alfa-2b (IFN 2b) in Europe and the United States, the pegylated form of IFN 2b (PegIFN 2b) in the United States and Switzerland, and IFN alfa-2a (IFN 2a) in Europe. High-dose IFN 2b (HDI) is the approved dosing regimen in the United States for American Joint Committee on Cancer (AJCC) stage IIB III melanoma, comprising an induction phase of 20 MIU/m 2 intravenously (IV) 5 times/week for 4 weeks followed by a maintenance phase of 10 MIU/m 2 subcutaneously (SC) 3 times/week for 48 weeks. Three Eastern Cooperative Oncology Group (ECOG) trials of HDI showed significant improvements in relapse-free survival (RFS) versus the control arm [5 7]; in the first of these trials a significant improvement in overall survival (OS) was also seen versus alone [5]. In Europe, intermediate (e.g. 10 MIU for 5 days/week for 4 weeks then 3 times/week for 1 2 years) and low (e.g. 3 MIU 3 times/week for 6 18 months) IFN dosing regimens are used; most clinical studies indicate RFS benefits with these regimens compared with. PegIFN 2b was recently approved in the United States for adjuvant treatment of resected melanoma with microscopic or gross nodal involvement at a dose of 6 g/kg/week SC for 8 weeks followed by 3 g/kg/week SC for up to 5 years. The large, randomized, phase III European Organisation for Research and Treatment of Cancer (EORTC) trial demonstrated significant improvement in RFS with this regimen compared with (hazard ratio [HR] = 0.82; 95% confidence interval [CI] ; p = 0.01) [8]. The significance of this effect was maintained at the 7.6-year update (HR = 0.87; 95% CI ; p = 0.05) [9]. The first meta-analysis of adjuvant IFN in high-risk melanoma, which included 12 trials of high-, intermediate-, and low-dose IFN 2a and IFN 2b, indicated a clear improvement in RFS versus control (HR = 0.83; 95% CI ; p = ) and some OS benefit (HR = 0.93; 95% CI ; p = 0.1) [10]. There was evidence for a significant trend in increasing RFS benefit with increasing IFN dose (p = 0.02) although the data were insufficient to provide clear conclusions regarding optimal dose and duration. A followup individual patient data meta-analysis of 13 trials indicated statistically significant increases in event-free survival (odds ratio [OR] = 0.87; 95% CI ; p = ) and OS (OR = 0.9; 95% CI ; p = 0.008) versus control but no evidence of differences according to dose or duration [11]. A more recent meta-analysis, which also included the EORTC PegIFN 2b trial, indicated statistically significant improvements in both disease-free survival (DFS) (HR for recurrence = 0.82; 95% CI ; p < 0.001) and OS (HR for death = 0.89; 95% CI ; p = 0.002), but again no optimal dose and/or treatment duration could be identified [12]. Overall, the available evidence clearly indicates significant RFS benefits and modest OS benefits with adjuvant IFN, but the optimal dose and treatment duration have not yet been determined. Thus, guidance is needed for physicians regarding the adjuvant treatment protocols that they should recommend to their patients with high-risk melanoma. Emerging evidence suggests that only certain patient subgroups respond to adjuvant therapy. The lack of clarity from the meta-analyses regarding dose and duration of therapy may be expected if the key factor for success is individual patient responsiveness to adjuvant therapy rather than total dose achieved. This was suggested by a small phase II study published in 2000, which found that, in patients experiencing melanoma recurrence during adjuvant low-dose IFN 2b therapy, increasing the dose resulted in negative clinical outcomes. These findings suggest that these patients were unresponsive to IFN 2b regardless of dose or treatment duration [13]. As it is not without side effects, there is a significant medical need for markers that will predict response to adjuvant IFN, thereby helping physicians to identify those patients who will benefit most from treatment and to improve the therapeutic index of adjuvant therapy. The purpose of this report is to review the recent evidence and make recommendations to European physicians regarding the adjuvant treatment of patients with high-risk melanoma.

3 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) Mechanism of action of IFN in melanoma The mechanism of action of IFN in melanoma appears to be mainly immunomodulatory although it does also have antiproliferative activity [14]. IFN binds to cell surface receptors causing activation of a number of signaling pathways, primarily the JAK STAT pathway [15 17]. Evidence for the involvement of several immunomodulatory pathways has been derived from a number of studies [17]. A gene expression profiling study found evidence for immune dysfunction related to a defect in IFN signaling in the T and B cells of patients with metastatic melanoma compared with healthy controls [18]. This dysfunction could be partially corrected in lymphocytes from some patients by prolonged exposure to high concentrations of IFN in vitro. A number of analyses have been carried out on samples from a study enrolling 20 patients with stage IIIB C melanoma who received neoadjuvant HDI induction therapy for 4 weeks [19]. Assessment of tumor specimens from 17 patients indicated significantly greater increases in CD11c+ antigen-presenting dendritic cells and CD3+ T-lymphocytes, and significantly greater decreases in CD83+ dendritic cells, at the 4-week time point in tumors of patients who responded to HDI versus nonresponders [19]. In a further analysis of samples from this study, HDI upregulated STAT1 and downregulated STAT3 in tumor cells, thereby significantly increasing the pstat1/pstat3 ratio [16]. This was relevant because higher baseline pstat1/pstat3 ratios were associated with longer OS. STAT1 is a tumor suppressor that restricts cell growth through the effector immune response, and STAT3 is implicated in tumor progression and downregulates response to IFNs. The antigen peptide transporter TAP2 was also upregulated by HDI, which may enhance antigen presentation [16]. Furthermore, analysis of peripheral blood lymphocytes from 14 patients in this study indicated that those having a clinical response had lower peripheral blood lymphocyte IFN signaling capacity at baseline (before treatment) compared with non responders, based on assessment of STAT1 phosphorylation. Also, clinical responders and those with good long-term outcomes had a significant increase in STAT1 activation in peripheral T cells with HDI therapy versus nonresponders and patients who developed metastases [20]. IFN has been shown to modulate T cells in other clinical studies. In a study of 21 patients with stage II III melanoma who received adjuvant low-dose IFN (LDI), decreases were observed in both CD3+CD4+ (helper) and CD3+CD8+ (cytotoxic) T-cell subsets, with greater reductions in memory CD4+ subsets as compared with CD4+ naïve lymphocytes [21]. A substantial increase in IFN -induced protein 10 (IP- 10/CXCL10), which inversely correlated with a significant decrease in CD8+ T lymphocytes expressing the chemokine receptor CXCR3, was also seen with LDI treatment. Based on these findings, the authors suggest that LDI therapy might aid in the induction of an orchestrated Th1 response that relies upon chemokines like IP-10/CXCL10, which could represent potential surrogate markers of adjuvant IFN activity and may contribute to the overall clinical benefit. Levels of T-regulatory cells, which suppress immune system activation, appear to be significantly higher in patients with melanoma compared with healthy controls [22 24], and may therefore contribute to disease-related immunosuppression and treatment resistance. In a pilot study of patients with melanoma, there was a trend toward a decrease in serum T-regulatory cell levels with HDI treatment [22]. This effect may help to potentiate an antitumor immune response, although further research is warranted. A multiplex analysis of samples from the ECOG 1694 study indicated that 3 months of HDI therapy was associated with decreases in levels of angiogenic and growth factors, and significant increases in anti-angiogenic protein IP-10, monocyte chemoattractant protein (MIP)-1, interleukins (IL)-12p40 and -2R, tumor necrosis factor (TNF)-RI, TNF-RII, and IFN. While no correlation was found in this study between the IFN-induced reduction in angiogenic factor concentrations and RFS of analyzed melanoma patients, RFS was longer in patients with the highest pretreatment levels of the proinflammatory cytokines IL-1, IL-1, IL-6, TNF, and the chemokines MIP-1 and MIP- 1 [25]. An earlier model of melanoma progression, based on a Cox regression analysis of prognostic factors for OS in 546 patients with melanoma, also suggested that tumor vascularity could be a surrogate marker of both vascular involvement and ulceration [26]; however, these data have only been presented in an abstract form. Additional evidence supporting the hypothesis that antitumor effects of interferon are mediated through angiogenic pathways is needed. Analysis of a subgroup of samples from the ECOG 1690 study showed dose-dependent modulation of tumor cell major histocompatibility complex class II molecule HLA- DR, adhesion molecule expression (ICAM-1), natural killer cell function, and T-cell function by HDI, suggesting modulation of tumor cell-host immune responses [27]. Further clinical data suggest an association between autoimmune responses and antitumor responses [28]; these data will be discussed in Section Review of recent clinical studies In this section, we summarize the efficacy results from all the recently published clinical studies of adjuvant IFN 2a, IFN 2b, and PegIFN 2b that were not included in the most recent meta-analysis, to determine whether new evidence provides clarity on dose and duration. Relevant clinical trials of adjuvant therapy in melanoma were identified by searching PubMed using appropriate search terms; relevant abstracts presented at the American Society of Clinical Oncology (ASCO) 2011 annual meeting were identified by searching the ASCO abstract database online.

4 152 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) Table 1 Summary of recent clinical studies of IFN and PegIFN 2b. Trial Patients Treatment arms N DFS (95% CI) OS (95% CI) Italian Melanoma Intergroup [29] E1697 [30] AJCC stage III Breslow thickness > 1.5 mm or any thickness N1a N2a (microscopic nodal disease) AJCC stage IIIA B (T2 4, N1a) AJCC stage IB IIC (T2 4, N0) but positive for melanoma-specific mrna AJCC stage IIB III (T3b excluded) IHDI (4 courses of 20 MIU/m 2 IV on 5 days/week for 4 weeks every other month) versus standard HDI HDI induction only versus Sunbelt [31] Protocol A: standard HDI versus Protocol B: CLND plus HDI induction only versus Hellenic [32] 15 MIU/m 2 IV 5 days/week for 4 weeks versus 15 MIU/m 2 IV 5 days/week for 4 weeks then 10 MIU SC TIW for 48 weeks Mao [33] AJCC stage IIB IIIC (acral) Arm A: 15 MIU/m 2 days 1 5 per week for 4 weeks versus arm B: same induction then 9 MIU TIW for 48 weeks Nordic [37] DeCOG [38] DeCOG [39] EORTC follow-up [9] EADO [40] AJCC stage IIB IIC (T4N0M0) or stage III (TxN1 3M0) >1.5 mm thickness, node negative (clinically or by SLNB) 1.5 mm thickness, node negative (clinically or by SLNB) AJCC stage III (TxN1 2M0) 1.5 mm without clinically detectable regional node or distant metastases (AJCC stage IB IIIB) 10 MIU 5 days/week for 4 weeks then 10 MIU TIW for 1 year versus 10 MIU 5 days/week for 4 weeks then 10 MIU TIW for 2 years versus 10 MIU/m 2 5/week IV for 2 weeks then SC for 2 weeks then 3 MIU SC TIW for 23 months versus 3 MIU SC TIW for 24 months 3 MIU IFN 2a TIW SC for either 18 months (arm A) or 60 months (arm B) PegIFN 2b 6 g/kg/week for 8 weeks then 3 g/kg/week for up to 5 years versus PegIFN 2b flat dose 100 g/week SC for 36 months versus IFN 2b 3 MIU SC TIW for 18 months 172/158 Median: 47.9 versus 35.6 months 5-year: 45.8% ( ) versus 44.3% ( ) 581/ years ( ) versus 7.3 years ( ) 112/106 HR = 0.82 ( ), p = /180 HR = 0.90 ( ), p = /176 Median (months) 24.1 ( ) versus 27.9 ( ) p = 0.9 HR = 0.94 ( ) a, p = /79 Median (months) 17.9 ( ) versus 22.5 ( ), p = /284 HR = 0.77 ( ), p = /284 HR = 0.83 ( ), p = /329 5-year: 68.0% versus 67.1%, p = /419 HR = 1.05 ( ), p = /629 HR = 0.87 ( ), p = 0.05 Median: 88.7 (61.2 to nr) versus 82.6 (45.8 to nr) 5-year: 60.1% ( ) versus 52.7% ( ) 5-year: 0.82 ( ) versus 0.85 ( ) HR = 1.07 ( ), p = 0.79 HR = 0.96 ( ), p = 0.88 Median (months) 64.4 ( ) versus 65.3 ( ), p = 0.49 HR = 0.91 ( ), p = HR = 0.91 ( ), p = year: 80.2% versus 82.9%, p = 0.66 HR = 1.03 ( ), p = 0.86 HR = 0.96 ( ), p = /453 HR = 0.91 ( ) HR = 1.09 ( ) AJCC: American Joint Committee on Cancer; CLND: complete lymph-node dissection; DFS: disease-free survival; HDI: high-dose interferon- 2b; HR: hazard ratio; IHDI: intensified HDI; IV: intravenous; OS: overall survival; nr: not reached; SC: subcutaneous; SLNB: sentinel lymph node biopsy; TIW: three times weekly. a Multivariate analysis after adjusting for lymph node positivity High-dose IFNα2b The Italian Melanoma Intergroup carried out a randomized phase III trial of intensified HDI (IHDI) [29]. Eligible patients had stage III histologically confirmed melanoma (staged according to AJCC 2002 criteria), but those with in-transit metastases and satellitosis were excluded. Patients were randomly assigned to either IHDI (4 courses of IFN 2b 20 MIU/m 2 IV for 5 days/week for 4 weeks every other month) or standard HDI (ECOG 1684 regimen), with randomization performed within 12 weeks of complete lymphadenectomy. Baseline characteristics were well balanced between treatment arms. For the IHDI and standard HDI arms, ulceration was present in 42% versus 47% of patients, respectively, and macroscopic disease was found in 41% versus 45%. Nodal status in this study was as follows: N1 (64% versus 61%), N2 (22% versus 28%), and N3 (13% versus 9%). There were no statistically significant differences between the treatment arms for RFS or OS (median or 5-year survival rate) (Table 1). Subgroup analysis indicated that the treatment effect was similar across all subgroups for both RFS and OS. However, IHDI was not associated

5 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) with greater toxicity compared with the standard HDI regimen. Patients on the IHDI arm did have higher completion rates (66% IHDI versus 49% HDI) and lower discontinuation rates due to toxicity (20% IHDI versus 28% HDI), suggesting that this is a more feasible and less toxic regimen compared with HDI. However, this study provided no information about the role of intensification in melanoma adjuvant therapy. The Intergroup E1697 randomized, phase III trial compared the 4-week HDI induction phase with in intermediate- and high-risk melanoma [30]. At a third interim analysis, there was no significant difference between the treatment and arms in median RFS or 5-year OS (Table 1) and accrual was terminated. In the prospective, randomized Sunbelt Melanoma Trial, patients with a single positive sentinel lymph node (SLN) (N1a micrometastases) were randomized to standard HDI versus (protocol A). Patients with a negative SLN biopsy underwent molecular staging to detect melanoma-specific mrna and those who were positive were randomized to complete lymph-node dissection only, complete lymph-node dissection plus IFN 2b (4-week HDI induction phase only), or (protocol B). At a median follow-up of 64 months, there were no significant differences in DFS or OS between the treatment and arms in either protocol (Table 1) [31]. The results of the E1697 and Sunbelt trials suggest that the 4-week HDI induction phase alone is not sufficient for benefit. However, in both trials the patient population was heterogeneous (e.g. up to 70% of patients enrolled in E1697 had stage IB IIB disease). The majority of patients therefore appear to be at a lower risk for disease recurrence compared with the high-risk population (stage IIB III) in which the HDI regimen has been validated. The Hellenic trial was a prospective, randomized study comparing the efficacy of 4 weeks of an alternative high-dose regimen of IFN 2b at 15 MIU/m 2 IV on 5 days/week (arm A) with the same regimen followed by 10 MIU flat-dose SC 3 times/week for 48 weeks (arm B) [32]. This dose is lower than the standard (approved) HDI regimen, and RFS and OS were very similar between the 2 treatment arms (Table 1). This trial was not able to validate the benefit of a longer intermediate regimen. However, it was designed as a non-inferiority trial to detect a 15% difference between the 2 treatment arms, and this difference is unlikely to be achievable with 2 active treatment arms in this patient population given that the observed differences for HDI versus no treatment in other trials was lower than 15%. In a randomized phase II trial of acral melanoma that was based on the Hellenic trial design [33], 158 patients were randomized to receive IFN 2b 15 MIU/m 2 on days 1 5/week for 4 weeks (arm A) or the same induction phase followed by 9 MIU 3 times/week for 48 weeks (arm B). At the median follow-up of 36.1 months, there was no significant difference between the 2 arms, which was similar to the Hellenic trial (Table 1) [33]. However, subgroup analysis showed that median RFS was significantly longer with the 1-year versus the 4-week regimen in patients with IIIB C disease (7.6 versus 12.0 months; p = 0.02) and in those with 3 nodal metastases (3.3 versus 11.9 months; p = 0.004). These findings are in line with other data suggesting differences between stage IIB IIIA and IIIB C disease in terms of response to adjuvant treatment with IFN. Other recent studies have explored the potential of intermittent HDI and whether HDI is superior to biochemotherapy as adjuvant therapy for high-risk melanoma. The phase III Dermatologic Cooperative Oncology Group (DeCOG) MM- ADJ-5 trial randomized high-risk patients with stage III resected or in-transit or lymph node metastasis to receive either standard HDI or an intermittent (pulsed) regimen (3 courses of IFN 2b 20 MU/m 2 IV on 5 days a week for 4 weeks repeated every 4 months). There were no statistically significant differences in DMFS or OS, however, although the pulsed regimen was associated with less cumulative toxicity and a better QoL profile compared with standard HDI [34,35]. The S-0008 Intergroup trial compared HDI to a shortcourse biochemotherapy (BCT) regimen (BCT consisting of dacarbazine 800 mg/m 2 on day 1, cisplatin 20 mg/m 2 on days 1 4, vinblastine 1.2 mg/m 2 on days 1 4, IL-2 9 MIU/m 2 /day continuous IV on days 1 4, IFN 5 MU/m 2 /day SC on days 1 4, 8, 10, and 12, and G-CSF 5 g/kg/day SC on days 7 16; BCT cycles were given every 21 days 3 cycles) as adjuvant therapy for high-risk stage III melanoma. While biochemotherapy improved RFS, there was no improvement in OS, and grade 4 toxicity was greater on the chemotherapy arm [36] Intermediate-dose IFNα A randomized, open-label, phase III, parallel-group trial by the Nordic Melanoma Cooperative Group investigated whether a much longer duration of intermediate-dose adjuvant IFN would improve outcomes [37]. Patients were randomized to 1 of 3 arms: only; or IFN 2b 10 MIU flat-dose SC 5 days/week for 4 weeks (induction) followed by 10 MIU flat-dose SC 3 days/week for 12 months (maintenance); or the same regimen as in the other treatment arm but with the maintenance period extended to 24 months. At a median follow-up of 72.4 months, there were no differences for either of the treatment arms versus arm in OS (primary endpoint); according to the authors this was likely to be due to insufficient power. There was a significant improvement in RFS in patients receiving 1-year IFN 2b therapy versus but not in those receiving 2-year IFN 2b therapy versus (Table 1). Prolonging maintenance treatment to 2 years did not appear to improve the outcome as there was no statistically significant difference in RFS or OS between the 2 IFN 2b arms. Overall, these data support RFS improvements versus with intermediate IFN doses, consistent with the pivotal HDI studies and meta-analyses. Longer follow-up and

6 154 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) further analysis may be required to determine the true effects on OS Low-dose IFNα DeCOG carried out a prospective, randomized, multicenter trial investigating the effect of adding a 4-week, intermediate-dose induction phase to an LDI regimen (IFN 2b) in node-negative patients with resected primary melanoma of >1.5 mm thickness (Table 1). RFS and OS rates were similar in both treatment arms, suggesting that the addition of an intermediate-dose induction phase to a long-term LDI regimen did not provide additional benefit [38]. In a separate randomized, multicenter DeCOG trial, node-negative patients with resected primary melanoma of >1.5 mm thickness received low-dose IFN 2a at 3 MIU 3 times/week SC for either 18 months or 60 months [39]. There were no differences between the treatment arms in RFS or OS (Table 1), suggesting that prolonging the duration of LDI therapy had no clinical benefit in this population PegIFNα2b The pivotal EORTC study investigated the PegIFN 2b dosing regimen that is now approved for adjuvant therapy in the United States and Switzerland (3 6 g/kg/week). This dosing regimen had a significant effect on RFS in patients with stage III melanoma, with a 6.7% (95% CI ) absolute difference in estimated 4-year recurrence rate versus. Although distant metastasis-free survival (DMFS) was longer with PegIFN 2b versus, the difference did not reach statistical significance and there was no difference in OS between the groups. Long-term follow-up of the study was presented at ASCO 2011 [9], and at a median follow-up of 7.6 years a significant impact on RFS was maintained for PegIFN 2b versus (Table 1). These findings suggest durability of the effect of PegIFN 2b throughout the course of treatment. The open-label, randomized, phase III European Association of Dermato-Oncology (EADO) study compared a lower and flat dose of PegIFN 2b (100 g SC once/week for 36 months) with the active comparator of LDI (IFN 2b 3 MIU SC 3 times/week for 18 months) in patients at intermediate risk in which LDI has been validated [40]. At median followup of 56.9 months and 55.0 months in the PegIFN 2b and IFN 2b arms, respectively, there was no significant difference regarding DFS and OS (Table 1). Surprisingly, adverse events were more frequent in the PegIFN 2b arm compared with the IFN 2b arm in this study. It is uncertain whether a higher dose of PegIFN 2b would show superiority over LDI. It is noteworthy that, despite an intended duration of treatment with PegIFN 2b of 36 months, median treatment duration was 19.2 months in the PegIFN 2b arm. Together with EORTC 18991, these data suggest that it can be difficult to maintain patients on PegIFN 2b treatment for several years. Overall, these 2 studies show that PegIFN 2b is efficacious in the adjuvant treatment of melanoma, although the optimal regimen is still unknown, and superiority over regular IFN has not been demonstrated to date. Overall, the clinical studies of adjuvant therapy reported since the Mocellin meta-analysis that compared IFN or PegIFN 2b with largely support the existing evidence that adjuvant therapy has clear DFS benefits and modest OS benefits. These studies were not able to provide clarity on the optimal dose or duration of adjuvant therapy, although the data suggest that HDI induction only is not sufficient in stage IB IIB disease, and that increasing the duration of treatment with intermediate doses does not significantly increase efficacy in the overall high-risk population. 4. Predictive biomarkers of IFN response Although clinical trials and meta-analyses support the efficacy of IFN and PegIFN in terms of RFS, with modest OS benefits, emerging data indicate that within the broad high-risk patient population, only a proportion of patients experience long-term extensions of DFS and OS. Therefore, the current focus of clinical research for adjuvant therapy in melanoma is to determine which subgroups will benefit most and how these patients can be readily identified in practice so that the most appropriate treatment course can be initiated. A number of markers that may be predictive of adjuvant therapy outcomes have been identified and now need verification in larger trials designed specifically to confirm their predictive value Tumor stage and disease burden Two of the most important markers are tumor stage and disease burden. Data from trials carried out before the development of the new AJCC staging categories had already suggested that LDI may be less effective in patients with macro-metastases [41], but appeared to provide benefit in patients without macro-metastases [42,43]. However, these studies had not stratified patients by stage and were therefore unable to compare efficacy by disease stage. Subgroup analyses of efficacy by disease stage were inconsistent in the ECOG HDI studies [5 7]. Subanalysis of data from a number of recent studies strengthens the hypothesis that efficacy differs according to whether microscopic or macroscopic disease is present. In a retrospective review of patients with stage III melanoma who received adjuvant IFN, a covariate-adjusted analysis indicated a significant improvement in RFS with IFN versus at a median follow-up time of 5.2 years in patients with stage IIIA disease (microscopic nodal involvement N1) (Table 2), but not in stage IIIB and IIIC patients [44]. No OS differences were observed in any subgroup.

7 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) Table 2 Summary of efficacy outcomes by disease stage and ulceration. Trial Treatment arm Stage N DFS (95% CI) d OS (95% CI) Anaya retrospective analysis [44] EORTC [45] EORTC [8,9] IFN (dose not specified) versus no treatment 13 months intermediate IFN 2b versus 25 months intermediate IFN 2b versus 6 g/kg/week for 8 weeks then 3 g/kg/week for up to 5 years versus Nordic [37] Observation (arm A) versus 10 MIU 5 days/week for 4 weeks then 10 MIU TIW for 1 year (arm B) versus 10 MIU 5 days/week for 4 weeks then 10 MIU TIW for 2 years (arm C) Sunbelt [46] Protocol A: HDI versus Protocol B: CLND plus IFN induction only versus IIIA ( ), p = 0.02 IFN not a predictor of OS IIIB ( ), p = 0.4 in any sub-stage IIIC ( ), p = 0.9 IIB a ( ), p = ( ), p = 0.14 IIIN1 b ( ), p = ( ), p = 1.00 IIIN2 b ( ), p = ( ), p = 0.57 IIB a ( ), 0.54 ( ), p = p = IIIN1 b ( ), p = ( ), p = 0.17 IIIN2 b ( ), p = ( ), p = 0.83 Microscopic node c 271/ ( ), p = ( ), p = 0.22 Clinically palpable node c 356/ ( ), p = ( ), p = 0.70 Microscopic node and 96/ ( ), p = ( ), p = 0.03 ulceration Only 1 positive LN c 339/ ( ), 0.82 ( ), p = 0.15 p = positive LNs 204/ ( ), p = ( ), p = positive LNs 76/ ( ), p = ( ), p = 0.93 Microscopic (follow-up) ( ), p = ( ), p = 0.26 Clinically palpable ( ), p = ( ), p = 0.97 (follow-up) Microscopic and ( ), p = ( ), ulcerated (follow-up) p = IIB IIC 166 Greater benefit in arm B versus arms A and C (p = 0.082) Stage III with clinically palpable LN 554 Improved in arms B plus C versus arm A (p = 0.015) Stage III with 1 LN 373 No significant differences Stage III with 2 3 LNs 200 No significant differences Stage III with 4 LNs 124 Improved in arms B plus C versus arm A (p = 0.038) Ulcerated 238 Arm B plus C: HR = 1.04 ( ), p = Arm B plus C: HR = 1.05 ( ), p = Non-ulcerated 441 Arm B plus C: HR = 0.69 ( ), p = Arm B plus C: HR = 0.75 ( ), p = Ulcerated 75 p = for IFN versus Non-ulcerated 142 p = for IFN versus Ulcerated 127 p = for IFN versus Non-ulcerated 425 p = for IFN versus p = for IFN versus p = for IFN versus p = for IFN versus p = for IFN versus CI: confidence interval; CLND: complete lymph-node dissection; DFS: disease-free survival; HDI: high-dose interferon- 2b; HR: hazard ratio; LN: lymph node; OS: overall survival; TIW: three times weekly. a Adjusted for sex, tumor site, and ulceration. b Adjusted for sex, tumor site, ulceration, and stage/number of positive LNs (0 = IIB, 1 = microscopic and 1 4 positive LNs, 2 = macroscopic and 1 positive LN, 3 = macroscopic and 2 4 positive LNs, 4 = 5 positive LNs); 11 patients with in-transit metastases at randomization excluded from these analyses. c Multivariate analysis. d In distant metastasis-free interval is reported instead of DFS. The EORTC study randomized patients with stage IIB III resected melanoma to or intermediate IFN 2b (4 weeks of 10 MIU 5 days/week followed by either 10 MIU 3 times/week for 1 year or 5 MIU 3 times/week for 2 years) [45]. At a median follow-up of 4.5 years, there were no significant differences between the treatment arms and in distant metastasis-free interval or OS in the overall population. However, subgroup analysis indicated that tumor stage was a predictive factor (Table 2), with a significant difference between the 25-month IFN group and in

8 156 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) distant metastasis-free interval and OS (i.e. greater difference with lower disease burden) [45]. Similarly, in the study, the benefits of PegIFN 2b were greater in patients with earlier stage III disease (micrometastases) than in those with later-stage disease (macrometastases), and greater in those with only 1 lymph node involved than in those with >1 node involved (Table 2) [8]. Kaplan Meier curves for the micrometastatic patient subgroup indicated that the benefit in this group was apparent early on in the study and was maintained throughout followup; the absolute difference in 4-year RFS was approximately 12% in this subgroup. In the long-term follow-up of the EORTC PegIFN 2b study (median 7.6 years), subgroup analyses indicated that the significant improvement in RFS versus in micrometastatic patients had been maintained for a long-term, with marginal benefits on DMFS and OS in this subgroup. In the original analysis, the benefit of PegIFN 2b appeared to be greatest in the subgroup of patients with micrometastatic melanoma that was ulcerated (N = 186). Significant improvements were observed in these patients for RFS, DMFS, and OS, and were maintained in this long-term follow-up (Table 2). These data support the hypothesis of different sensitivity to IFN according to disease stage. Contrary to the studies summarized previously, exploratory subgroup analyses of the Nordic study data suggested that the greatest RFS benefit is in patients with the highest tumor burden prior to lymph-node dissection, i.e. stage III disease with clinically palpable lymph-node metastases and 4 involved lymph nodes (Table 2) [37]. According to the authors, these findings may be due to the fact that the majority of patients in this trial were in the higher disease burden category and that in these patients more relapses and deaths occurred, thereby allowing detection of a modest adjuvant therapy effect. No difference in outcomes was observed by tumor stage in the Hellenic trial [32]. Taken together, these data suggest that there may be differences in tumor biology according to the phase of the disease, before or after emergence of macro-metastases in the lymph node(s), which could account for differences in response according to the dose/regimen of adjuvant therapy given Ulceration Exploratory analyses of the data from the EORTC trial suggested that ulcerated primary melanomas are more sensitive to PegIFN 2b compared with non-ulcerated melanomas [8]. Therefore, response to therapy according to this tumor characteristic has been analyzed in other studies, although the molecular mechanism behind this potential effect is not known to date. In the Sunbelt Melanoma Trial, subanalysis of data from protocol A (N1a micrometastases) by ulceration status indicated that HDI had no significant effect on DFS or OS in patients with non-ulcerated melanoma (N = 142), but there was a significant improvement in DFS (p = ) in patients with ulceration (N = 75) (Table 2) [46]. No significant differences related to treatment were observed according to ulceration in protocol B (negative SLN biopsy). In contrast, exploratory analysis of the Nordic study data indicated that the significant benefits of IFN 2b on OS and RFS versus were observed only in patients with non-ulcerated primary tumors (Table 2). However, the authors noted that these were unplanned retrospective analyses with insufficient power to detect a difference and that most patients in this study had stage III disease with clinically detectable lymph node metastases [36], a patient subgroup for which a previous analysis showed no significant association between ulceration and IFN 2b response [47]. These findings are supported by a retrospective analysis of the EORTC and EORTC trials that further explored the effects of ulceration and stage on response to IFN in high-risk patients [47]. Treatment effect was greater for patients with ulceration than those without ulcerated tumors for RFS (p = 0.02), DMFS (p < 0.001), and OS (p < 0.001). Notably, risk reductions were greatest in patients with both ulceration and IIb/III-N1; estimated HRs for RFS, DMFS, and OS were 0.69 (p = 0.003), 0.59 (p < ), and 0.58 (p < ), respectively. Patients with stage III-N2 disease with ulceration had a lower risk reduction, while there was no effect in patients without ulceration. If confirmed by prospective studies, these data suggest that the benefits of adjuvant IFN and Peg-IFN therapy may be limited to a subpopulation of high-risk patients with melanoma Autoantibodies In a subgroup analysis of stage IIB III patients enrolled in the Hellenic trial, OS was longer in the 26% of patients with autoantibodies or clinical manifestations of autoimmunity compared with those without autoimmunity (median not reached [range, months] versus 37.6 months [ months]) [28]. Univariate and multivariate regression analyses indicated that autoimmunity, as a timedependent variable, was an independent prognostic marker for improved RFS and OS (p < 0.001). These findings were not confirmed by similar analyses from the intermediatedose EORTC or Nordic studies. In these studies, antibodies appeared in 36% and 39% of patients receiving IFN 2b, respectively (rates in the arms were 26% and 14%). In both trials, in patients receiving IFN 2b, autoantibody-positive patients had statistically significantly better RFS versus antibody negative-patients using time-independent Cox models; however, in contrast to the Gogas study, when time-dependent Cox models were used the significance was lost [48]. In the EORTC PegIFN 2b study, antibodies occurred in 35% of patients analyzed, but Cox models that considered autoantibody appearance as a time-independent variable indicated no association between autoimmunity and improved RFS in patients receiving PegIFN 2b [49]. In summary, development of

9 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) Table 3 Summary of other potential predictive markers of IFN 2b and PegIFN 2b efficacy. Marker Study type Dose Stage Outcomes STAT1 MTAP STAT1 Analysis of peripheral blood lymphocytes [20] Tissue microarray analysis [50] Neoadjuvant HDI induction IIIB C Median ratio for fold change in STAT1 phosphorylation post- and pre-treatment: <1.15 (negative/minimal) and >1.35 ( hyper response) = poor outcome (modest increase) = favorable outcome Low-dose IFN 3 MIU SC TIW for 24 months versus Breslow thickness mm (IB IV) Significantly longer RFS on IFN for MTAP+ tumors versus MTAP tumors (p < 0.05) Longer OS in MTAP+ versus MTAP tumors (median 80 versus 35 months; p = 0.119) No RFS benefit in STAT1+ tumors Significant OS benefit in STAT1+ tumors (p < 0.05) S100B E1694 analysis [51] Standard HDI IIB III Univariate analysis: patients with a high baseline S100B level ( 0.15 g/l) had a longer RFS (p = 0.018) and longer OS (p = 0.028) when treated with HDI S100B analysis [52] Intermediate IFN 2b for 13 months or 25 months versus HLA class I and II type B2M and sil-2r pstat1/pstat3 ratio WBC WBC Hellenic [53] Prospective randomized trial [54] 15 MIU/m 2 IV 5 days/week for 4 weeks versus 15 MIU/m 2 IV 5 days/week for 4 weeks then 10 MIU SC TIW for 48 weeks 10 MIU SC 5 days/week then either 5 or 10 MIU SC TIW for 2 years IIB III IIB III (T3b excluded) II III Higher S100B levels at baseline or during follow-up associated with worse DMFS (S100B 0.2 versus S100B < 0.2): HR = 5.57 (95% CI ); p < Prognostic impact of S100B 0.2 g/l more pronounced in stage III versus IIB disease HLA-Cw*06 positive versus negative patients: Median RFS versus 37.3 months (p = 0.013) Median OS not reached versus 78.9 months (p = 0.025) Unable to clarify if HLA-Cw*06 positivity is a prognostic or predictive marker B2M and sil-2r increased throughout therapy Baseline TNF /B2M/sIL-2R had positive predictive value for relapse of 82.9% (multivariate analysis) Neoadjuvant trial [16] Standard HDI induction IIIB C May have prognostic relevance and may assist in predicting treatment outcome, although no significant difference in this ratio was identified between clinical responders and nonresponders in this small study French cooperative group [57] Corollary study of randomized E1690 trial [27] IFN 2a 3 MIU TIW for 18 months Ferritin CRP EORTC [55] Intermediate IFN for 1 or 2 years Breslow thickness 1.5 mm without clinically detectable node metastases In patients with high baseline WBC ( /L) DFI was highly significantly prolonged for IFN versus control HR = 0.50 (95% CI ), p = No treatment effect in patients with low baseline WBC (< /L) HR = 1.27 (95% CI ), p = 0.26 Standard HDI IIB III No significant quantitative interaction between WBC and treatment outcomes p = 0.53 RR = 1.17 (95% CI ), p = 0.32 IIB III The higher ferritin ratios observed at end of induction and at 6 months in IFN-treated versus non-treated patients were not associated with improved DMFS No differences between treatment groups or association with DMFS for CRP ratios B2 M: beta-2 microglobulin; CI: confidence interval; CRP: C-reactive protein; DFI: disease-free interval; DMFS: distant metastasis-free survival; HDI: highdose interferon- 2b; HR: hazard ratio; IV: intravenous; MTAP: methylthioadenosine phosphorylase; OS: overall survival; RFS: relapse-free survival; RR: risk ratio; SC: subcutaneous; TIW: three times weekly; WBC: white blood cell count.

10 158 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) autoantibodies should not be considered as a prognostic marker Other potential predictive markers A number of other potential predictive markers for IFN efficacy have been assessed in clinical trials and are summarized in Table 3. Those that have shown potential as predictors of treatment outcomes include methylthioadenosine phosphorylase expression [50], STAT1 expression [20,50], S100B levels [51,52], HLA class I and II type [53], -2 microglobulin [54], and serum IL-2R [54]. These potential markers need further study to confirm their predictive value. Other markers that did not show predictive value include pstat1/pstat3 ratio [16], ferritin and C-reactive protein [55], the presence of tumor-infiltrating lymphocytes [56], and white blood cell count [27,57]. Analysis of peripheral blood lymphocytes from 14 patients receiving neoadjuvant HDI induction indicated that differences in STAT1 activation pre- and post-hdi distinguished between patients who were likely to have a favorable versus unfavorable outcome (Table 3), and may therefore be useful as a predictive indicator of response to therapy, although further verification is required [20]. 5. Recommendations regarding the use of adjuvant IFN At present, native IFN and PegIFN remain valid as adjuvant therapy following resection of high-risk melanoma. No other adjuvant therapies with proven benefits are currently available or approved for use in the adjuvant setting. Ongoing phase III trials are investigating the efficacy of ipilimumab versus placebo and MAGE-A3 antigen-specific cancer immunotherapeutic versus placebo as adjuvant therapy [58] but no data are yet available. The primary endpoint in these studies is RFS, with OS being investigated as a secondary endpoint. Additionally, the ongoing ECOG 1609 phase III randomized trial is comparing adjuvant ipilimumab versus high-dose IFN -2b in patients with resected high-risk stage III or stage IV melanoma. In metastatic melanoma, OS has been improved in trials of ipilimumab and vemurafenib (in patients with the BRAF V600E mutation) [59,60], but their impact on long-term survival and potential role in the adjuvant setting is yet to be determined. On the basis of the recent Mocellin meta-analysis and reported clinical studies, while waiting for data to emerge from the ongoing studies of potential new adjuvant therapies, PegIFN 2b can be offered to patients based on its proven benefits in both DFS and OS, even though the OS impact was not statistically significant except in patients who were sentinel node-positive and had ulcerated melanoma. The benefits seem to be largely independent of dosage and duration. In the Wheatley and Mocellin meta-analyses, there appeared to be a numerically greater reduction in risk of disease recurrence with HDI (25% and 26% versus control, respectively) compared with low/intermediate doses (15% and 17%, respectively), although no differences were seen for reduction in risk of death [10,12]. However, currently, there is no compelling evidence to support an optimal dose (low, intermediate, or high-dose PegIFN ), or the use of any particular dosing schedule or duration. Physicians should discuss the potential options with individual patients in order to determine which regimen, if any, may be best suited to that patient based on factors such as patient lifestyle and attitude, potential for toxicity, and contraindications. Based on clinical trial data, it seems that the stage of disease/disease burden may be considered a key factor when discussing the adjuvant therapy options with individual patients. Other potential predictive markers need further investigation before they can be used in the decision-making process. 6. Conclusions Adjuvant treatment with IFN or PegIFN 2b provided a substantial benefit in terms of RFS in almost all the clinical trials conducted, with an HR for disease recurrence of 0.82 (p < 0.001) in the most recent meta-analysis by Mocellin et al. [12]. It also provided an OS benefit, albeit a much smaller one, with an HR for death of 0.89 (p = 0.002) in this meta-analysis [12]. There is currently no definitive evidence regarding the optimal dose and duration of therapy for this agent, and recent studies have not answered the question. In this context, low and intermediate doses have the advantage of better tolerability. The overall clinical evidence should be discussed with all patients with melanoma who have a high risk of relapse following resection, in order to help determine the right course of treatment. A well-balanced discussion with each patient regarding the advantages and disadvantages of the different regimens, including potential side effects and contraindications, may assist in deciding whether or not adjuvant therapy is prescribed and, if so, the selection of the dose and regimen. It should be mandatory to provide patients with an opportunity to review this information in an accessible manner before an informed choice can be made by the patient and physician together. This information should be updated by the physician as soon as new evidence becomes available. Conflict of interest statement PAA: Advisory boards for Bristol-Myers Squibb, Merck Sharp & Dohme, Roche-Genentech, GlaxoSmithKline, Amgen, Celgene, Medimmune, Novartis; honoraria from Bristol-Myers Squibb, Merck Sharp & Dohme, Roche- Genentech. HJG: Consultant to Bristol-Myers Squibb, Merck & Co. Inc. (paid to institution). JJG: Advisory boards, honoraria, and travel/ accommodation expenses from Merck & Co. Inc. (previously Schering-Plough Corp.).

11 P.A. Ascierto et al. / Critical Reviews in Oncology/Hematology 85 (2013) SMA: Consultant to Bristol-Myers Squibb. PM: Consultant to Merck & Co. Inc., Roche, Bristol- Myers Squibb; expert testimony for Merck & Co. Inc., Roche, Bristol-Myers Squibb; payment for development of educational presentations from Merck & Co. Inc., Bristol-Myers Squibb. JH: Expert testimony for Merck & Co. Inc., Roche, Glaxo- SmithKline, Bristol-Myers Squibb; grants paid to institution from Merck & Co. Inc. (previously Schering-Plough Corp.), Roche; travel/accommodation expenses paid to institution from Merck & Co. Inc., Roche, GlaxoSmithKline, Bristol- Myers Squibb. AH: Consultant to Merck Sharp & Dohme, Roche; honoraria from Merck Sharp & Dohme, Roche; payment for the development of educational presentations from Merck Sharp & Dohme, Roche; travel/accommodation expenses from Merck Sharp & Dohme, Roche. 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