This was a Phase 1, single-arm, open-label, pilot study of NY-ESO-1 protein vaccination with imiquimod as an adjuvant in patients with resected Stage IIB, IIC, and III malignant melanoma. The primary study objective was to determine the safety of NY-ESO-1 protein/imiquimod treatment, and the secondary objective was to evaluate the immunogenicity of treatment.

Patients applied imiquimod (250 mg) topically to a designated area of healthy skin on the upper inner arm or inner thigh (the cream remained on the skin overnight for 6-10 hours) every day for 5 consecutive days (i.e., for the first 5 days of Cycles 1-3 and for the first 4 days of Cycle 4). The NY-ESO-1 protein (100 μg) was injected intradermally into the imiquimod-pretreated area on Day 3 of each cycle for 4 consecutive 21-day cycles.

Safety was monitored continuously. Immunization was assessed by the generation of NY-ESO-1-specific cluster of differentiation (CD)4+ and CD8+ T cell responses in enzyme-linked immunosorbent spot (ELISPOT) assays and by the development or augmentation of NY-ESO-1-specific antibody titers, assessed by enzyme-linked immunosorbent assay (ELISA).

Blood samples were obtained for the assessment of clinical biochemistry and hematology, and physical examinations were performed at baseline, on Day 1 of each cycle, and at a follow-up visit at Week 13.

Skin biopsies of the vaccinated area were obtained 48 hours after the last injection (Day 5 of Cycle 4). To avoid irritation, imiquimod was not applied after the biopsies.

Inclusion Criteria:

• Had histologically confirmed, resected American Joint Committee on Cancer Stage IIB, IIC or III malignant melanoma

• Fully recovered from surgery

• Age ≥ 18 years; children were excluded from this study, as the safety of imiquimod had not been established in patients below the age of 18

• Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2

• Adequate organ and marrow function as defined below:

  • absolute neutrophil count: ≥ 1500/μL
  • hemoglobin: ≥ 9 g/dL
  • platelets: ≥ 100,000/μL
  • total bilirubin: ≤ 1.5 × institutional upper limit of normal (ULN)
  • aspartate aminotransferase/alanine aminotransferase (AST/ALT): ≤ 2.5 × institutional ULN
  • creatinine: ≤ 1.5 × institutional ULN

• Ability to understand and the willingness to sign a written informed consent document

Exclusion Criteria:

• Received chemotherapy, immunotherapy (including interferon), or radiotherapy within 4 weeks prior to first dosing of study agent
• Prior treatment with NY-ESO-1 vaccines
• Known human immunodeficiency virus infection or autoimmune disease (rheumatoid arthritis, systemic lupus erythematosus), as these conditions could have interfered with the evaluation of the induced immune response; patients with vitiligo or melanoma-associated hypopigmentation were not excluded
• History of allergic reactions attributed to compounds of similar chemical or biologic composition to imiquimod or other agents used in the study
• Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection,symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would have limited compliance with study requirements
• Pregnancy or lactation
• Women of childbearing potential not using a medically acceptable means of contraception
• Known history of inflammatory skin disorders, as imiquimod might have exacerbated these conditions
• Chronic corticosteroid or immunosuppressive therapies, as these might have interfered with the evaluation of the induced immune response
• Lack of availability for immunological and clinical follow-up assessments

The population comprises all enrolled patients.

All adverse events (AEs), regardless of causality to study drug, were documented from informed consent through the final follow-up visit, which was up to 4 months for each patient.

AE documentation included onset/resolution dates, severity using the NCI CTCAE (version 3.0), frequency, seriousness, relationship to study drug, study drug action taken, treatment, and outcome. In summaries, preferred terms were counted only once per patient at the maximum reported grade.