Chapter 4 Advances in Adjuvant Therapy: Potential for Prognostic and Predictive Biomarkers Diwakar Davar, Ahmad A. Tarhini, Helen Gogas, and John M. Kirkwood Abstract Melanoma is the third most common skin cancer but accounts for the majority of skin cancer-related mortality. The rapidly rising incidence and younger age at diagnosis has made melanoma a leading cause of lost productive years of life and has increased the urgency of finding improved adjuvant therapy for melanoma. Interferon-α was approved for the adjuvant treatment of resected high-risk melanoma following studies that demonstrated improvements in relapse-free survival and overall survival that were commenced nearly 30 years ago. The clinical benefits associated with this agent have been consistently observed across multiple studies and meta-analyses in terms of relapse rate, and to a smaller and less-consistent degree, mortality. However, significant toxicity and lack of prognostic and/or predictive biomarkers that would allow greater risk–benefit ratio have limited the more widespread adoption of this modality. Recent success with targeted agents directed against components of the MAP-kinase pathway and checkpoint inhibitors have transformed the treatment landscape in metastatic disease. Current research efforts are centered around discovering predictive/prognostic biomarkers and exploring the options for more effective regimens, either singly or in combination. Key words Melanoma, Adjuvant, Interferon, IFN-α, CTLA-4, Ipilimumab, BRAF, Vemurafenib, Debrafenib, MEK, Trametinib

1

Introduction In 2012 alone, invasive melanoma accounted for approximately 76,180 new cases and 9,250 deaths in the USA alone according to US Surveillance, Epidemiology, and End Results (SEER) registry estimates [1]. The increasing incidence of this disease, tripling the rate observed in the Caucasian population over the last two decades, adds national and international importance to this most lethal of all cancers of the skin, the largest organ of the human body. When discovered early, definitive surgical resection is often curative for melanoma. However, patients with advanced disease (>stage IIB) are at increased risk of recurrence and death. Adjuvant interferon-α (IFN-α), originally licensed by the US Food and Drug

Magdalena Thurin and Francesco M. Marincola (eds.), Molecular Diagnostics for Melanoma: Methods and Protocols, Methods in Molecular Biology, vol. 1102, DOI 10.1007/978-1-62703-727-3_4, © Springer Science+Business Media New York 2014

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Administration (US FDA) on the basis of a low but reproducible reduction in risk of relapse, has been the mainstay of adjuvant therapy in high-risk disease for the preceding two decades. Following early experiments that suggested broad anti-tumor effects in various laboratory models through a variety of potential mechanisms, multiple international and US Intergroup trials were conducted that tested various doses and schedules to derive maximal therapeutic benefit. High-dose interferon (HDI) comprising an induction phase (20 MU/m2 daily for 5 days per week for 4 weeks) followed by a subcutaneous maintenance phase (10 MU/ m2 for 48 weeks) has reliably and reproducibly demonstrated improvement in relapse-free survival (RFS) and, to a lesser extent, overall survival (OS) in patients with high-risk cutaneous melanoma [2]. European trials studying the pegylated form of interferon (peg IFN-α) have shown improvement in RFS although, unlike the experience with HDI, no impact upon overall survival (OS) has been observed. The advances of the past decade have elucidated some of the molecular underpinnings and elements of host immunological response to melanoma and have resulted in transformational new therapies targeting the MAP kinase signaling pathway (RAS/RAF/ MEK/ERK) and immune checkpoints (CTLA-4, PD-1). The BRAF inhibitor vemurafenib and the CTLA-4 blocking antibody ipilimumab have independently demonstrated survival benefits in phase III trials leading to regulatory approval for these new treatment options in the setting of advanced disease. However, their potential role in the adjuvant and neo-adjuvant arenas remains a topic of ongoing investigation. This chapter will focus on the indications for adjuvant therapy, current management of high-risk resected melanoma, ongoing adjuvant and neo-adjuvant trials and the development of prognostic and predictive biomarkers in this arena. 1.1 Indications for Adjuvant Therapy

Adjuvant therapy is indicated in patients whose risk of recurrence following definitive surgery remains high, and is pursued with the intent of treating micro-metastatic disease and thereby reducing the risk of local and distant relapse. As not all patients have the same risk, and among those at risk, not all may benefit from adjuvant systemic therapy, the decision to pursue adjuvant therapy requires assessment of an individual patient’s risk for recurrence and fractional likelihood of reducing risks of recurrence or mortality, weighed against the risks and the costs of treatment. A number of independent prognostic factors have been identified based on analysis of data obtained from patients compiled in the American Joint Committee on Cancer (AJCC) Melanoma Staging Database and these form the basis of the revised 2009 AJCC staging manual for melanoma [3]. These include:

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●

Primary tumor thickness—single greatest predictor of recurrence and survival which are directly commensurate to thickness measured in millimeters.

●

Ulceration—adversely affects survival at every T sub-stage such that ulceration in any given T level is associated with a similar risk of death and relapse as the next higher non-ulcerated T level (i.e. T2b–T3a).

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Mitotic rate—defined as the number of mitoses per square millimeter in the primary tumor with the threshold of significance being 1/mm2. High mitotic rate connotes a more aggressive phenotype and being the second most powerful predictor of survival after tumor thickness is strongly associated with adverse outcomes. In sub-mm lesions (T1), mitotic rate separates T1a (non-ulcerated T1 melanomas with a mitotic rate of less than 1/mm2) from T1b lesions (ulcerated T1 melanomas with a mitotic rate of greater than 1/mm2).

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Lymph node burden—risk of lymph node involvement increases with tumor thickness by approximately 2–5 % for Breslow’s depth ≤1.00 mm and reaches 34 % for T4 lesions [3]. Survival decreases commensurate with increasing lymph node tumor burden—5 year survival rates for stage IIIA, IIIB, and IIIC disease ranging from 78 % to 59 % and 40 % respectively. The importance of accurate assessment of lymph node involvement was underscored by two changes in AJCC 7th edition staging system: (a) use of immunohistochemistry to define nodal disease rather than hematoxylin and eosin (H&E) staining alone, and (b) inclusion of micro-metastases (0.1–1.0 mm) in addition to macro-metastases (>1.0 mm) in defining lymph node involvement. However, patients with sub-micrometastases (3

3.4

6.0

Median follow-up at time of reporting (years) OS

HR: 0.74 (LDI vs. obs) (S)

Rate of relapse: (24.0 % LDI vs. 36.3 % obs)

RFS/DMFS not reported

IFN 2 year versus obs (NS)

HR: 0.70 (LDI vs. obs) (S)

Not available

23.2 months (A) versus 56.1 months (A) versus 37.8 months (B) 72.1 months versus 28.6 months (B) versus (C) 64.3 months IFN versus obs & IFN (C) (NS) 1 year versus obs (S);

DFS/RFS

0

0

81

% Node positive

N/A

N/A

ANA, anti-TG, ACL development (HR = 0.85 for improved RFS; NS after adjusting for guarantee-time bias) [58]

ANA, anti-TG, ACL development (HR = 0.51 for improved RFS; S before adjusting for guarantee-time bias) [58]

Biomarkers evaluated

II–III (T3-4N0M0/ TanyN+M0)

II–III (T3-4N0M0/ TanyN+M0)

II–III (T3-4N0M0/ TanyN+M0)

III (T3anyN+M0)

96 Scottish Melanoma Cooperative Group [26]

728 EORTC 18871/DKG 80-1 [27]

UKCCCR/ 674 AIM HIGH [28]

840

444

DeCOG [29]

DeCOG [30]

S.C. 3 MU 3 days a week for 6 months

S.C. 3MU 3 days a week for 36 months

IFNα-2a

IFNα-2a

IFNα-2a versus observation 4.3

3.1

8.2

>6

7.3

3.9 S.C. 3MU 3 days a week for 24 months (A) versus S.C. 3MU 3 days a week for 24 months + DTIC 850 mg/m2 every 4–8 weeks for 24 months (B) versus observation (C)

S.C. 3 MU 3 days a week for 18 months (A) versus 5 years (B)

S.C. 3 MU 3 days a week for 24 months

IFNα-2b versus IFN-α2b: S.C. 1MU every IFN-γ versus other day for 12 months ISCADOR M® IFN-γ: S.C. 0.2 mg every versus observation other day for 12 months

IFNα-2a versus observation

IFNα-2a versus observation

HR: 0.69 (A) versus 1.01 (B) versus 1.0 (C)

5 years DMFS 81.9 % (A) versus 79.7 % (B) (NS)

NS

NS

NS

NS

N/A

N/A

N/A

Not N/A available

Not N/A available

58

Not N/A available

100

HR: 0.62 (A) 100 % versus 0.96 (B) versus 1.0 (C)

5 years OS 85.9 % (A) versus 84.9 % (B) (NS)

NS

NS

NS

NS

NS not significant, S significant, HR hazard ratio, DFS disease-free survival, DMFS distant metastases-free survival, OS overall survival, ANA anti-nuclear antibody, anti-TPO anti-thyroid peroxidase antibody, anti-TG anti-thyroglobulin antibody, AMA anti-mitochondrial antibody, ACL anti-cardiolipin autoantibodies

III (TanyN+M0)

III (TanyN+M0)

WHO 444 Melanoma Program Trial 16 [25]

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The Italian Melanoma Group evaluated the same hypothesis using an intensified regimen—4 cycles of induction dosing for a month with HDI administered every other month (intensified HDI, IHDI) compared to the standard E1684 full-year regimen— in a randomized phase III study [19]. Although RFS and OS were improved in the IHDI arm compared to the standard HDI arm, the differences were not statistically significant. However, given that the overall toxicity profiles were relatively similar in both groups, an argument can be made for this shorter (and more feasible) regimen. The pegylated form of IFN-α allows once-weekly administration without sacrificing potency. Several EORTC trials assessed various pegylated IFN-α schedules to determine if similar benefits could be obtained. EORTC 18991 was an adjuvant study in which 1,256 patients with resected AJCC stage III melanoma were randomized in a nonblinded fashion to either pegylated IFN-α for 5 years versus observation. The choice of an extended duration of therapy (5 years) was designed to assess whether the benefit of IFN eroded after cessation of therapy—as suggested by earlier European trials of low-dose IFN [20, 23]. Study investigators presented initial results in 2007—noting a significant improvement in RFS (HR = 0.82, p = 0.011) with no improvement in either of the original primary endpoints of the study, DMFS or OS at 3.8 years median follow-up [33]. Patients with microscopic node-positive disease appeared to benefit predominantly in comparison to the other groups with no evidence of any benefit in the macroscopic nodal disease (N2) subset. Although the study results were published early, the study was criticized due to the lack of maturity of the results, given their lesser magnitude compared to E1684 (which was first reported at 6.9 years median follow-up). Nonetheless, the results served as the basis for a successful application for FDA approval of pegylated IFN-α (Sylatron™, Merck Corporation) for adjuvant therapy of stage III melanoma with either microscopic or macroscopic nodal involvement following definitive surgical resection including complete lymphadenectomy. Recently, the mature results of this trial were published [34]. At mature follow-up, the overall RFS benefit has diminished from the prior HR of 0.82 to a current HR of 0.87 (95 % CI, 0.76– 1.00) and the prior significance has diminished to a marginal (p = 0.055) without significant benefits in the originally specified endpoints of DMFS/OS. Subgroup analysis suggests that patients with microscopic nodal disease and ulcerated primaries derive relatively greater RFS benefit while patients with N2 disease lack benefit in any parameter. In light of these results, practitioners should consider several factors when deciding between HDI and pegylated IFN-α: first, the overall hazard of benefit in PFS terms is greater with conventional high-dose IFN—HR 0.69 at 6.9 years

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and 0.72 at 12.6 years (E1684) versus HR 0.82 at 3.8 years and 0.87 at more mature 7.6 years with pegylated IFN-α as tested in EORTC 18991. Second, the collective US intergroup experience over time demonstrates RFS and OS in patients with macroscopic as well as microscopic nodal disease while pegylated IFN-α has no benefit in this sub-group of patients whose risk of recurrence/ death is the highest in all of the trials of various dosages and formulations of IFN over the years. The quest for biomarkers that would allow the more selective application of IFN in general for adjuvant therapy of melanoma is thus even more compelling for pegylated IFN-α; for the present and apart from biomarkers that may allow the improvement of therapeutic index for IFNs, pegylated IFN-α may be considered for patients with N1 disease who are either unwilling or unable to tolerate the HDI regimen. The benefit of adjuvant IFN-α has been comprehensively evaluated in several meta-analyses [35, 36], a systematic review [37] and a pooled data analysis [38]. The 2003 meta-analysis by Wheatley and colleagues pooled data from 12 randomized controlled trials (RCT) that compared varying doses of IFN-α to observation and demonstrated RFS (significant) and OS (nonsignificant) benefits with a trend towards improved outcomes in patients receiving higher doses of IFN-α. The second meta-analysis by Wheatley and colleagues in 2007 utilized individual patient level data from 13 RCTs that included vaccine and observation comparators and concluded that IFN-α significantly improved RFS and OS as did a more recent 2010 meta-analysis of 14 RCTs. The accumulated evidence reliably indicates that adjuvant HDI reduces RFS by 30 % with a lesser impact on OS. 1.3 Ongoing Adjuvant Trials

The current agenda of cooperative group trials for adjuvant therapy involve the introduction of new targeted molecular signaling inhibitor therapies (BRAF, MEK), and inhibitors of immune checkpoints (anti-CTLA-4, anti-PD1, and anti-PDL1) and nextgeneration immunotherapeutic options that are summarized in Table 2. Building upon the earlier observations of Ives et al., in the meta-analysis which demonstrated a potential selective advantage of patients with ulcerated primary melanomas receiving adjuvant IFN, and trials that have shown this in the EORTC including 18952 and 18991 the proposed EORTC 18081 is designed to prospectively test the benefit of 2 years of pegylated IFN-α versus observation in patients with node-negative melanoma and ulcerated primaries greater than 1 mm (T2-4bN0M0, n = 1,200). MAGE-A3 is a tumor-associated protein expressed in >60 % of metastatic melanoma that elicits humoral and cell-mediated immune responses in the autologous host. Previously, a phase II trial of MAGE-A3 vaccine (GSK Biologicals, Rixensart, Belgium) showed objective responses in 5/72 treated patients and long lasting

OS, DMFS, FFR, safety, toxicity

OS, safety, toxicity

RFS

Phase III, randomized, double-blinded study in high-risk (IIIA-C) resected BRAF V600E/K mutant melanoma

Open label phase RFS II study in high-risk (IIIC) resected BRAF V600E/K mutant melanoma

COMBI-AD 850 (NCT01682083)

Adjuvant dabrafenib 23 (NCT01682213)

OS, DMFS, quality of life

Primary Secondary endpoint endpoint(s)

RFS Phase III, randomized, open-label study in T2b4bN0M0 melanoma

Estimated enrollment Study design

EORTC 18081 1,200 (NCT01502696)

Study reference

Table 2 Ongoing adjuvant studies in high-risk resected melanoma

Dabrafenib 150 mg B.I.D. per cycle (28 days) for 4 cycles

September 2012

September Unknown 2014

Unknown

July 2015

November Dabrafenib 150 mg 2012 B.I.D. AND Trametinib 2 mg once daily versus placebos for 12 months

Estimated Biomarker completion evaluation April 2020 Unknown

Start date

Pegylated IFN-α2b 3 μg/ April 2012 kg weekly injections for 2 years

Dose and schedule— treatment arm

RFS, OS Toxicity, global Phase III, quality of life randomized, open-label study in high-risk (IIIB-C or resected IVA) resected melanoma

June 2016

May 2018

September 2012

Ipilimumab in 2 dose May 2011 levels vs. HDI Ipilimumab: Induction—3/10 mg/kg q3weeks for 4 doses Maintenance—3/10 mg/ kg q6weeks until week 48 then q12weeks afterwards HDI: Induction—I.V. 20 MU/ m2 5 days a week for 4 weeks Maintenance—S.C. 10 MU/m2 3 days a week for 48 weeks

OS, DMFS, quality Vemurafenib 960 mg B.I.D. versus placebo of life, for 12 months pharmacokinetics, safety, toxicity

E1609 1,500 (NCT01274338)

DFS

Phase III, randomized, double-blinded study in high-risk (IIC or IIIA-C) resected BRAF V600E mutant melanoma

Adjuvant 725 vemurafenib (NCT01667419)

(continued)

Ipilimumab— MDSC, Treg, IL-17 HDI— S100B

Unknown

OS, DMFS, anti-MAGE-A3 and anti-protein D seropositivity status, quality of life

DFS Phase III, randomized, open-label study in T2b4bN0M0 melanoma

MAGE-A3 vaccine 1,349 (NCT00796445)

Expression of MAGE-A3 AntigenSpecific Cancer Immunotherapeutic (ASCI) gene signature

October 2016

MAGE-A3 vaccine with 2 December 2008 adjuvants including QS-21 (Stimulon™, Agenus), and a proprietary Toll-like receptor 9 agonist (VaxImmune™, Coley Pharmaceuticals)

Estimated Biomarker completion evaluation September Unknown 2014

Start date

Ipilimumab versus June 2008 placebo Ipilimumab: Induction—10 mg/kg q3weeks for 4 doses Maintenance—10 mg/kg q6weeks until week 48 then q12weeks afterwards

Dose and schedule— treatment arm

OS overall survival, RFS relapse-free survival, DFS disease-free survival, DMFS distant metastasis-free survival, FFR freedom from relapse, DLT dose-limiting toxicities

OS, DMFS, quality of life, quality-oflife-adjusted survival

Primary Secondary endpoint endpoint(s)

RFS Phase III, randomized, open-label study in T2b4bNxM0 melanoma following resection

Estimated enrollment Study design

EORTC 18071 950 (NCT00636168)

Study reference

Table 2 (continued)

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stable disease in 11/72 patients [39]. A phase III trial of the vaccine combined with two potent new immunopotentiators including a toll-like receptor 9 agonist designed to induce potent antibody and CD8+ T-cell responses completed accrual in 2011 and results of this trial are awaited in 2013. Ipilimumab (Yervoy™, Medarex Inc/Bristol-Myers Squibb) is a fully humanized immunoglobulin G1κ monoclonal antibody that competitively inhibits CTLA-4. CTLA-4 and PD-1 are T-cell surface receptors that down modulate T-cell responses following binding of cognate ligands present on antigen presenting cells— B7-1/2 and PD-L1, respectively. By blocking negative regulators known as immune checkpoints, CTLA-4 (and PD-1) inhibitors appear to enhance T-cell activation and proliferation in early nodal phases of the immune response to melanoma, and later tissuebased immune responses to melanoma. Ipilimumab has demonstrated improved survival in two large RCTs involving different dosages (10 mg/kg and 3 mg/kg), directed at different settings (untreated first line versus relapsed prior treated patients) and compared against different controls (dacarbazine versus gp 100 peptide vaccine) [40, 41]. EORTC 18071 is prospectively comparing ipilimumab at 10 mg/kg to placebo in stage III melanoma following resection in 950 patients. Accrual has completed and results are expected in 2013. ECOG’s companion study (E1609) is an open-label RCT that seeks to compare ipilimumab at 2 dose levels (3 mg/kg and 10 mg/kg) to HDI in highest-risk melanoma (stage IIIB-C and resected stage IV). The mitogen-activated protein (MAP) kinase pathway comprises several (RAS/RAF/MEK/ERK) serine/threonine-specific protein kinases that direct cellular responses to a diverse array of stimuli including growth signals. Activating mutations in BRAF are present in approximately 40–60 % of cutaneous melanomas most of which are V600E mutations and result in unregulated cellular proliferation. MEK lies directly downstream of RAF and is an attractive target as MEK inhibition would effectively target BRAFand RAS-mutant melanoma. When compared against dacarbazine in previously untreated patients with metastatic melanoma, the BRAF V600E inhibitor vemurafenib (Zelboraf™, Roche/Plexxikon) demonstrated improved overall survival and decreased risk of tumor progression with an overall response rate (ORR) of 48 %. Dabrafenib (GSK2118436, Glaxo-SmithKline) is another BRAF inhibitor with activity in V600E and V600K mutants that appears similar in efficacy, and different in its profile of toxicities that are in general, well tolerated for both agents. In a phase III trial that compared dabrafenib to dacarbazine in previously untreated patients with advanced V600E mutant melanoma, dabrafenib demonstrated significant improvement in PFS over dacarbazine with a confirmed response rate of 53 % [42]. Trametinib (GSK1120212, GlaxoSmithKline) is a MEK inhibitor that has also demonstrated improved

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PFS and OS in V600E/K mutant melanoma when tested against dacarbazine or paclitaxel [43]. Success in the metastatic setting has prompted the development of investigations of these agents in the adjuvant setting. Currently there are three proposed or initiated studies evaluating RAF/MEK inhibitors either singly or in combination for adjuvant treatment of melanoma. COMBI-AD (NCT01682083) and NCT01667419 are randomized, double-blind phase III studies enrolling high-risk stage III patients to placebo versus combined RAF/MEK inhibition with dabrafenib and trametinib (COMBI-AD) or RAF inhibition alone with vemurafenib (NCT01667419). Primary endpoints are RFS (COMBI-AD) and disease-free survival (NCT01667419) with the proposed duration of treatment in both studies being 12 months. Investigators from Memorial Sloan-Kettering Cancer Center are performing a phase II adjuvant study of 4 cycles of monthly dabrafenib in resected stage IIIC patients with RFS as a primary endpoint (NCT01682213). These trials are slated to open in 2013 with estimated completion between 2014 and 2016. RAF/MEK inhibition is associated with the acquisition of resistance at a median PFS of 4.8–5.3 months. The treatment of patients who have progressed on RAF/MEK inhibitors has been problematic—and an important consideration for adjuvant therapy has to be the longer term (years in the future) impact of the drug to be tested. The rationale and mechanism of action for the agents under evaluation are important to consider for adjuvant therapy, given the lack of durability of direct antitumor effects with BRAF inhibitors alone, and BRAF-MEK inhibitor combinations to date. There is emerging evidence of immunological effects with BRAF inhibitors alone and these lend support to the notion that BRAF inhibitors might add to the impact of immunotherapy in the adjuvant setting [44, 45]. We have proposed the sequenced combination of BRAF inihibitors prior to IFNα, for which the clinical experience with BRAF inhibitors provides partial support, and additional evidence preclinical evidence is also promising (Ferrone, Zarour, Davar & Kirkwood unpublished). The consideration of BRAF inhibitors and MEK inhibitors together has been advanced in view of the reduced toxicity and potential to prolong the interval to progression with the combination. However, the impact of MEK inhibitors upon the immune response needs to be evaluated, as the potential inhibition of immune responses in the adjuvant setting may be a larger issue. The use of tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer suggests that timing of recurrence is associated with resistance mediated by EGFR kinase domain mutation (T790M) observed on treatment, but not after treatment. Thus, it may be that recurrence after finite intervals of BRAF inhibitor therapy may still allow successful retreatment with BRAF inhibitors at later intervals [46].

Advances in Adjuvant Therapy: Potential for Prognostic and Predictive Biomarkers

1.4 Prognostic and Predictive Biomarkers

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The identification of predictive and prognostic biomarkers of therapy for melanoma is the highest priority for translational research in melanoma both in the advanced and in the adjuvant settings of disease, to allow the individualization of therapy to optimize efficacy and minimize toxicity and cost. While the effort to individualize therapy will ultimately refine the population to be treated, this narrowing of the therapeutic window will be more than compensated by the improvement in the therapeutic index. One potential avenue for tailoring adjuvant IFN therapy lies in the pathologic variable of primary tumor ulceration already discussed. The aggregate data from EORTC 18952 and 18991 in 2,644 patients report that ulceration is strongly correlated with benefit from intermediate dosage or pegylated IFN-α [20, 33]. The more limited Sunbelt melanoma trial also identified ulceration as a predictive factor, but neither the Sunbelt trial nor the EORTC trials have adopted rigorous pathology review for subjects enrolled into these trials, so they rely upon institutional pathology reporting that has not been verified [47, 48]. Unfortunately, the US Intergroup trials conducted to date and the E1684 pivotal study have not identified ulceration as a predictive factor and these studies have uniformly required central pathology review. The possibility that a gene signature will predict the benefit of immunotherapy has already been noted, and the MAGE A3 DERMA vaccine trial for Stage IIIB melanoma is accompanied by a prospective formal gene signature analysis that may further refine the application of this vaccine, and enhance the potential therapeutic index of this vaccine [49]. Gene signatures of the tumor may guide future therapies apart from vaccines such as MAGE A3, and current questions in regard to the pathological variable of tumor ulceration may ultimately be understood in terms of the genetic profile of the tumor. The development of autoimmunity has been advanced as a surrogate of IFN response following on observations of autoimmune thyroid and cutaneous depigmentary response with IL-2 [50]. The prospective clinical and serological analysis in the Hellenic Oncology Group trial 13A/98 provided rigorous evidence that the development of autoimmunity was associated with statistically significant improvements in RFS and OS in patients with melanoma treated with adjuvant IFN [51]. Analyses of US Intergroup trials E2696 and E1694 have shown a correlation between improved survival and HDI-induced autoimmunity, but did not have the advantage of assessing clinical features of autoimmunity [52]. US Intergroup trial E1697 evaluated 1 month induction only course of adjuvant IFN-α2b versus observation in patients with intermediate-risk resected melanoma. Following interim analysis that suggested futility, the trial was closed early. However biomarker analysis in 268 patients suggested that month IL2Rα, IL-12p40, and IFN levels at 1 month in the treatment group significantly predicted 1 year RFS (Kirkwood et al., presented at

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ASCO 2013). Other analyses using different methodologies in relation to intermediate-dosage IFN regimens and focusing only on the serological development of autoimmunity have confirmed this correlation, but lacked prospective clinical evaluations [53]. These analyses are delineated in the “Biomarkers Evaluated” section of Table 1. Other candidate biomarkers include methylthioadenosine phosphorylase (MTAP) protein expression, YKL-40 (a mammalian chitinase-like protein) levels, S100B, melanoma-inhibiting activity (MIA), and tumor-associated antigen 90 immune complex (TA90IC)—reviewed in detail elsewhere [54–60]. While these candidate biomarkers have, to varying degrees, been linked with outcomes on retrospective analyses they have not been evaluated as markers for IFN adjuvant therapy for melanoma.

2

Conclusions Multiple prior US and European intergroup studies (E1684, E1690, E1694, EORTC 18952, and EORTC 18991) have demonstrated in aggregate that adjuvant therapy with IFN-α reliably consistently improves RFS and to a lesser magnitude, OS in patients with deep primary melanomas, and in those with nodal disease— without differential effects in resected nodal disease of macroscopic and microscopic extent. The pivotal study and subsequent evaluations of HDI have revealed benefit across the operable high-risk disease spectrum, and no differential benefit according to disease bulk, or the presence of primary tumor ulceration. The benefits of high-dose recombinant IFN upon OS appears to be real, but less in magnitude than upon RFS, and diminish with long-term followup past 10 years. Trials of pegylated IFN-α show improvement in RFS without any evidence of an impact upon OS, and the magnitude of the benefit upon RFS is less than that of HDI, and restricted to patient populations with microscopic nodal disease, where the benefit, like that of other lower-dose regimes, has been linked with primary tumor ulceration. Although certain clinical features have been advanced that may allow the improvement in the therapeutic index of IFN (such as ulceration of the primary and laboratory indices of immune reactivity) (such as the development of autoimmunity, MTAP expression, and serum levels of YKL-40, S100B, MIA, and TA90IC), predictive biomarkers used to predict therapeutic benefit to IFN adjuvant therapy, or any of the newer candidate immunomodulators are still lacking. These are the compelling need of the field, as we develop new immune checkpoint inhibitors, signaling pathway inhibitors, and combinations for adjuvant therapy. E1609, the current US Intergroup trial, will evaluate candidate blood biomarkers of immunotherapeutic benefit

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with anti-CTLA4 blocking antibody ipilimumab, as well as IFNα and EORTC 18081 will evaluate the role of Peg-IFNα in patients with ulcerated primaries (T2b-4b) to shed light on this issue in the future. The past several years have witnessed an exponential increase in our understanding of the molecular events underpinning melanoma tumorigenesis and the subtleties of the host-tumor immune interactions, culminating in regulatory approval of the BRAF inhibitor vemurafenib and the CTLA-4 inhibitor ipilimumab in advanced melanoma. These agents have transformed the therapeutic landscape in advanced melanoma—where molecular profiling of BRAF and NRAS is now routine in patients with metastatic disease. The role of these genetic mutations in guiding adjuvant therapy is likely in the future, but for the large population without identified BRAF or NRAS mutations, immunotherapeutic options with either IL-2 or ipilimumab are considerations in metastatic disease, and IFN-α has an established role in the adjuvant setting. Despite the impressive and frequent remission of advanced metastatic BRAFV600E melanoma with vemurafenib (ORR 48.4 %), and the recently identified immunological effects of this agent, the relatively short duration of response secondary to the rapid acquisition of resistance at a median PFS of 5.3 months is an issue for adjuvant application of this agent as a single intervention. Ipilimumab has demonstrated durable responses in a significant but small proportion of patients in two phase III trials involving different dosages against different comparators in the treatment-naïve and previously untreated populations. The low ORR of 10.9–15.2 % may be improved in the adjuvant setting for this agent, as has been observed with IFNα, where a response rate of 16 % in advanced disease translates to a hazard ratio of 0.67 or relapse reduction of 33 %. This is the basis for current efforts to evaluate ipilimumab in randomized trials for high-risk resected melanoma compared against HDI in ECOG 1609 (ipilimumab at 10 mg/kg and 3 mg/kg vs. HDI) and against placebo in EORTC 18071 (ipilimumab 10 mg/kg vs. placebo). Vemurafenib and the dabrafenib/tremetinib combination are each being considered for evaluation randomized trials in similar highrisk groups. Our understanding of the mechanism of action for IFN, and now for ipilimumab, has arisen from neoadjuvant studies conducted over the years and argues for the prospective application of this approach to accelerate the development of combinations of immunotherapy and molecularly-targeted therapy in the neoadjuvant setting. This approach provides us the needed access to essential biomarker information, and a rapid read-out of correlations between the antitumor effects at the level of tumor tissue that

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is otherwise lost in the adjuvant postoperative treatment setting. The effort to optimize our new agents and combinations as well as sequences of these agents will be best served by the pursuit of neoadjuvant studies in parallel with the definitive clinical evaluation of these approaches. Clinical trials currently underway may answer these questions and for now, patients with high-risk melanoma who are unable to participate in clinical trials are reasonably offered high-dose IFN-α following surgery.

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