Prolongation of survival of dogs with oral malignant melanoma treated by en bloc surgical resection and adjuvant CSPG4-antigen electrovaccination

Abstract

Reported post-surgery 1-year survival rate for oral canine malignant melanoma (cMM) is around 30%; novel treatments are needed as the role of adjuvant chemotherapy is unclear. This prospective study regards adjuvant electrovaccination with human chondroitin sulfate proteoglycan-4 (hCSPG4)-encoded plasmid in 23 dogs with resected II/III-staged CSPG4-positive oral cMM compared with 19 dogs with resected only II/III-staged CSPG4-positive oral cMM. Vaccination resulted in 6-, 12-, 18- and 24-month survival rate of 95.6, 73.9, 47.8 and 30.4%, respectively [median survival time (MST) 684 days, range 78-1694, 8 of 23 dogs alive] and 6-, 12-, 18- and 24-month disease-free interval (DFI) rate of 82.6, 47.8, 26.1 and 17.4%, respectively (DFI 477 days, range 50-1694). Non-vaccinated dogs showed 6-, 12-, 18- and 24-month survival rate of 63.2, 26.3, 15.8 and 5.3%, respectively (MST 200 days, range 75-1507, 1 of 19 dogs alive) and 6-, 12-, 18- and 24-month DFI rate of 52.6, 26.3, 10.5 and 5.3%, respectively (DFI 180 days, range 38-1250). Overall survival and DFI of vaccinated dogs was longer in those <20 kg. In vaccinated and non-vaccinated dogs local recurrence rate was 34.8 and 42%, respectively while lung metastatic rate was 39 and 79%, respectively.

Keywords: CSPG4; DNA electroporation; adjuvant immunotherapy; canine oral malignant melanoma; comparative oncology.

Figure 1.

Vaccination-induced anti-hCSPG4 humoral response in sera of cMM dogs. (A) Detection of hCSPG4 antibodies in sera collected before (white bar) and after the fourth DNA vaccination (black bar) by ELISA. Results are expressed as the mean OD at 450 nm ± SD values of all vaccinated dogs. ***P < 0.0001, Student’s t-test. (B) Evaluation of hCSPG4 antibody response in sera collected from vaccinated dogs in relation to BW. Results are shown as fold change values expressing the ratio between post-vaccination OD/pre-vaccination OD values measured by ELISA. (C) Assessment of the effect of vaccine-induced anti-hCSPG4 antibodies on hCSPG4 expression. hCSPG4-positive SK-MEL28 cells were incubated with medium alone, CSPG4-specific mAb or canine sera before and after the fourth immunization at 37°C for 48h. Representative immunoblot analysis of CSPG4 modulation induced by the sera of 2 vaccinated dogs (#5 and #3) and the corresponding percentage of CSPG4 reduction compared with the medium is shown. Actin was used as loading control.

Figure 2.

Kaplan–Meier curves comparing survival and DFI in the two groups. (A) Survival (in days) of CSPG4-positive MM, vaccinated dogs (group A, black line) and of CSPG4-positive MM, non-vaccinated dogs (group B, grey dotted line; *** log-rank test P = 0.0005). (B) DFI (in days) of CSPG4-positive MM, vaccinated dogs (group A, black line) and of CSPG4-positive MM, non-vaccinated dogs (group B, grey dotted line; * log-rank test P = 0.0174).

Figure 3.

Kaplan–Meier curves comparing survival (in days) in relation to BW of dogs. (A) Survival of CSPG4-positive cMM, vaccinated dogs (group A) with BW <20 kg (black line) and with BW >20 kg (black dotted line). (B, C) Survival of CSPG4-positive cMM, vaccinated dogs (group A) with BW <20 kg (black line) in comparison with survival of (B) the entire population of non-vaccinated dogs (group B, grey dotted line; ** log-rank test P = 0.0015) or of (C) non-vaccinated dogs with BW <20 kg (group B, grey line; *** log-rank test P = 0.0002). (D, E) Survival of CSPG4-positive cMM vaccinated dogs (group A) with BW > 20 kg (black dotted line) in comparison with survival of (D) the entire population of non-vaccinated dogs (group B, grey dotted line) or of (E) non-vaccinated dogs with BW >20 kg (group B, grey line).

Figure 4.

Kaplan–Meier curves comparing DFI (in days) in relation to BW of dogs. (A) DFI of CSPG4-positive cMM vaccinated dogs (group A) with BW <20 kg (black line) and with BW >20 kg (black dotted line). (B, C) DFI of CSPG4-positive cMM vaccinated dogs (group A) with BW <20 kg (black line) in comparison with DFI of (B) the entire population of non-vaccinated dogs (group B, grey dotted line; * log-rank test P = 0.0252) or of (C) non-vaccinated dogs with BW <20 kg (group B, grey line; ** log-rank test P = 0.0070). (D, E) DFI of CSPG4-positive cMM vaccinated dogs (group A) with BW >20 kg (black dotted line) in comparison with DFI of (D) the entire population of non-vaccinated dogs (group B, grey dotted line) or of (E) non-vaccinated dogs with BW >20 kg (group B, grey line).

Figure 5.

Kaplan–Meier curves comparing survival (in days) of dogs in relation to CSPG4-positivity score of cMM. (A) Survival of vaccinated dogs (group A) bearing a cMM with CSPG4-positivity score ≥5 (black line) or <5 (black dotted line). (B, C) Survival of vaccinated dogs (group A) bearing a cMM with CSPG4-positivity score ≥5 (black line) in comparison with the survival of (B) the entire population of non-vaccinated dogs (group B, grey dotted line; *** log-rank test P = 0.0004) or of (C) non-vaccinated dogs bearing a cMM with CSPG4-positivity score ≥5 (group B, grey line; *** log-rank test P = 0.0006). (D, E) Survival of vaccinated dogs (group A) bearing a cMM with CSPG4-positivity score <5 (black dotted line) in comparison with the survival of (D) the entire population of non-vaccinated dogs (group B, grey dotted line) or of (E) non-vaccinated dogs bearing a cMM with CSPG4-positivity score <5 (group B, grey line).

Figure 6.

Kaplan–Meier curves comparing DFI (in days) of dogs in relation to cMM CSPG4-positivity score. (A) DFI of vaccinated dogs (group A) bearing a cMM with CSPG4-positivity score ≥5 (black line) or <5 (black dotted line). (B, C) DFI of vaccinated dogs (group A) bearing a cMM with CSPG4-positivity score ≥5 (black line) in comparison with DFI of (B) the entire population of non-vaccinated dogs (group B, grey dotted line; * log-rank test P = 0.0167) or of (C) non-vaccinated dogs bearing a cMM with CSPG4-positivity score ≥5 (group B, grey line; * log-rank test P = 0.0249). (D, E) DFI of vaccinated dogs (group A) bearing a cMM with CSPG4-positivity score <5 (black dotted line) in comparison with DFI of (D) the entire population of non-vaccinated dogs (group B, grey dotted line) or of (E) non-vaccinated dogs bearing a cMM with CSPG4-positivity score <5 (group B, grey line).