Phase II Study of Adjuvant Immunotherapy with the CSF-470 Vaccine Plus Bacillus Calmette-Guerin Plus Recombinant Human Granulocyte Macrophage-Colony Stimulating Factor vs Medium-Dose Interferon Alpha 2B in Stages IIB, IIC, and III Cutaneous Melanoma Patients: A Single Institution, Randomized Study

Abstract

The irradiated, allogeneic, cellular CSF-470 vaccine plus Bacillus Calmette-Guerin (BCG) and recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) is being tested against medium-dose IFN-α2b in stages IIB-III cutaneous melanoma (CM) patients (pts) after surgery in an open, randomized, Phase II/III study. We present the results of the Phase II part of the ongoing CASVAC-0401 study (ClinicalTrials.gov: NCT01729663). Thirty-one pts were randomized to the CSF-470 vaccine (n = 20) or to the IFN-α2b arm (n = 11). During the 2-year treatment, immunized pts should receive 13 vaccinations. On day 1 of each visit, 1.6 × 107 irradiated CSF-470 cells plus 106 colony-forming units BCG plus 100 µg rhGM-CSF were administered intradermally, followed on days 2-4 by 100 µg rhGM-CSF. IFN-α2b pts should receive 10 million units (MU)/day/5 days a week for 4 weeks; then 5 MU thrice weekly for 23 months. Toxicity and quality of life (QOL) were evaluated at each visit. With a mean and a maximum follow-up of 39.4 and 83 months, respectively, a significant benefit in the distant metastasis-free survival (DMFS) for CSF-470 was observed (p = 0.022). Immune monitoring showed an increase in antitumoral cellular and humoral response in vaccinated pts. CSF-470 was well tolerated; 20/20 pts presented grades 1-2 dermic reactions at the vaccination site; 3/20 pts presented grade 3 allergic reactions. Other adverse events (AEs) were grade 1. Pts in the IFN-α2b arm presented grades 2-3 hematological (7/11), hepatic (2/11), and cardiac (1/11) toxicity; AEs in 9/11 pts forced treatment interruptions. QOL was significantly superior in the vaccine arm (p < 0.0001). Our results suggest that CSF-470 vaccine plus BCG plus GM-CSF can significantly prolong, with lower toxicity, the DMFS of high-risk CM pts with respect to medium-dose IFN-α2b. The continuation of a Phase III part of the CASVAC-0401 study is encouraged.

Keywords: Bacillus Calmette–Guerin; CSF-470 allogeneic cell vaccine; IFN-α2b; Phase II clinical study; cutaneous melanoma; rhGM-CSF.

Figures

Figure 1

Antigenic profile of cutaneous melanoma (CM) cell lines that compose the CSF-470 vaccine as determined by RNAseq (A). Expression of GD2 and GD3 gangliosides in CSF-470 vaccine as determined by flow cytometry (B); isotype-matched immunoglobulins were used as controls to set unspecific labeling.

Figure 2

Randomized Phase II CASVAC-0401 study diagram.

Figure 3

CASVAC-0401 consort flow diagram.

Figure 4

Distant metastasis-free survival (DMFS) of eligible patients (pts) participating in the CASVAC-0401 study (*p = 0.022, Gehan–Breslow–Wilcoxon test) (A). Quality of life (QOL) in all eligible pts participating in CASVAC-0401, evaluated by the European Organization for Research and Treatment of Cancer—Quality of Life Questionnaire (“EORTC QLQ-C30, v 3.0”). Mean and SD are shown (score 1 ***p < 0.0001; score 2 ***p < 0.0001). Score 1 = a higher value implies a poorer QOL; score 2 = a lower value implies a poorer QOL (B).

Figure 5

Mean and SD of delayed-type hypersensitivity (DTH) scores from vaccinated patients (pts) (A). Vaccinated pts were grouped according to progressed (PRO) or with no evidence of disease (NED). DTH scores are shown from PRO and NED pts after seven vaccinations (visit 8, p = 0.025, Mann–Whitney test) (B). Peripheral blood mononuclear cell immune populations gating strategy and results comparing PRE vs POST1 samples from CSF-470 and IFN-α2b arms. CSF-470: NK PRE vs POST1, **p = 0.008; and Tregs PRE vs POST1, *p = 0.021. IFN-α2b: CD3 PRE vs POST1, **p = 0.0036; CD4 PRE vs POST1, *p = 0.035; and CD8 PRE vs POST1, **p = 0.0075. For all comparisons, paired t-test was used. Ns, not significant (C).

Figure 6

IFN-γ enzyme-linked immunospot assay (ELISPOT) analysis in PRE and POST1 peripheral blood mononuclear cells (PBMC) samples of patients (pts) participating in CASVAC-0401 study. (A) Each bar corresponds to the mean number of IFN-γ spots quantified for PRE and POST1 PBMC stimulated with vaccine lysate as described in Section “Materials and Methods.” PBMC from a healthy donor (HD) were equally stimulated to set unspecific baseline. Spots could be properly quantified from 1 to 350 spots/well, due to saturation of the signal. CSF-470: PRE vs POST1, ***p = 0.0001, paired t-test (B) Selected ELISPOT wells are shown to illustrate IFN-γ secreting cells detected after stimulation with CSF-470 lysate from two vaccinated pts and one IFN-α2b-treated pt (duplicates). Positive controls correspond to PBMCs stimulated with anti-CD3 mAb plus PHA as described in Section “Materials and Methods.”

Figure 7

Serum reactivity to MelC of PRE and POST1 samples of patients (pts) participating in CASVAC-0401 study, detected by ELISA (unpaired t-test, ***p < 0.0001). Optical density (O.D.450 nm) from each sample is shown as O.D.450nm = O.D. pt serum sample − O.D. healthy donor serum sample. A total of 1/10 serum dilution is shown (A). Serum reactivity to autologous tumor cells of patient #006 was detected by ELISA. A total of 1/10, 1/100, and 1/1,000 serum dilutions are shown (B). Immunofluorescence staining of autologous patient #006 tumor cells with PRE and POST 1 sera showing cell surface reactivity (original magnification: 400×). Antibodies were detected by FITC-rabbit anti IgA, G, and M (DAKO). PRE, POST1, POST2, and POST3 correspond to samples obtained at 0, 6, 12, and 24 months from protocol start. F-UP (follow-up) sample was obtained 24 months after ending protocol participation (C).