Patients and inclusion/exclusion criteria

Informed consent was obtained before entry into the study, which was approved by the Royal Adelaide Hospital Human Research Ethics Committee and was registered with the Australian Clinical Trials Registry [ACTRN 12605000425695]. 54 stage IV/IIIc advanced melanoma patients were enrolled in these studies for the primary aim. Most patients had failed other therapies. Confidentiality of patient data was preserved. Overall quality of life was measured by assessing functional status using the standard Eastern Cooperative Oncology Group (ECOG) and Union for International Cancer Control (UICC) scores. CT scans were performed each 3 months and ophthalmology examinations (for possible melanoma associated retinopathy/iritis) each year, or as clinically indicated.

Inclusion criteria

Patients ≥ 18 years of age; ECOG 0-2; evaluable metastases from primary cutaneous melanoma; advanced non-surgically resectable AJCC Stage IV or Stage IIIb/c disease; tumour volume < 20 cm diameter or < 70% of organ replacement; +/- post-surgical treatment of brain metastases; voluntary informed consent.

Exclusion criteria

Second primary invasive cancer (not BCC, SCC of skin or resected in-situ malignancy); untreated brain metastases, extremely extensive metastatic disease, bone metastases only; high-dose oral steroid therapy; pregnant or lactating; severely atopic individuals; severe cachexia; immunodeficiency; HIV, Hepatitis B or C positive. Three patients with inoperable brain metastases were excluded. No patient was both eligible and refused to participate.

VMCL vaccine

Vaccinia Melanoma Cell Lysate (VMCL) vaccine was manufactured using successive aliquots of a single stable culture seed lot of the allogeneic melanoma cell line MM200, which were thawed as required, briefly cultured then infected with vaccinia virus (CSL laboratories, Melbourne) to cause cell lysis. The thawed MM200 aliquots were determined to be stable over time using karyotype, western blot and antigenic analysis. Lysed cells were ultrasonicated and centrifuged to create the allogeneic cell lysate vaccine product for use as described previously [15, 17–20]. Each vaccine had a protein content of 100mcg per 0.3 ml dose, equivalent to 5 × 106 cells per dose. Vaccine doses were frozen to preserve protein content at -20°C and thawed to room temperature before use. The process had been previously used successfully in a previous Australian randomised clinical trial for earlier-stage, completely resected high-risk melanoma [20].

VMCL vaccinations

All patients received regular 2-weekly single-dose intradermal vaccinations for 6 months; then monthly for 6 months; then if stabilisation or a CR was obtained, 3-monthly thereafter. Injection sites were rotated between upper outer aspects of all 4 limbs, but avoiding any limb where lymph node dissection was performed. Previous VMCL studies using 0.3 ml of re-suspended sonicated lysate had determined safety and efficacy of this dose and schedule [15, 17–20, 24]. Occasional minimal skin reactions were noted in the pilot or previous studies, and precautionary resuscitation facilities were available (but never required) with patients being observed for 30 minutes after each vaccination.

Chemotherapy

Melanoma disease progression during vaccine therapy indicated addition of concurrent standard chemotherapy (either dacarbazine (DTIC) 1000 mg/m2 at 3-weekly intervals intravenously), or fotemustine (100 mg/m2 weekly intravenously for 3 weeks, then 4 weekly thereafter) [25, 26]. Vaccinations were maintained at 2-weekly intervals throughout the chemotherapy period, including between doses and during breaks in chemotherapy. Occasional vaccine schedule adjustment was required to suit the chemotherapy schedule.

Skin Delayed Type Hypersensitivity (DTH)

The 1st and the 4th VMCL vaccination doses (0.3 ml) were investigated to examine DTH responses. Each read-out was 48 hours after vaccine administration. Erythema and the induration were recorded and independently recorded in two directions perpendicular to each other. Responses at the vaccination sites > 10 mm were considered positive.

Clinical end-points

Primary end-point

Overall survival was assessed by survival in months from the time of commencement of vaccination to the date of analysis or death of the patient.

Secondary end-points

(i) Toxicity and tolerability: local or systemic reactions were recorded.

(ii) Tumour response rates: Rates of Complete Response (CR), Partial Response (PR), Stable Disease (SD) and Progressive Disease (PD) were recorded using the WHO criteria [27]. Observable subcutaneous lesions were assessed using direct size measurement using calipers or a ruler, and internal metastases were assessed using CT scans at 3-monthly intervals or as clinically otherwise determined, and where appropriate using ultrasound, MRI or Positron Emission Tomographic (PET) scans. Measurements were in two directions perpendicular to each other.

Statistical analysis

This was performed using mean and median calculations, Kaplan-Meier analysis and time series analysis with the assistance of statisticians and a mathematician (TS; NB; AC). A significance level of p < 0.05 was set for all analyses.