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Survival and biomarker analyses from the OpACIN-neo and OpACIN neoadjuvant immunotherapy trials in stage III melanoma


Neoadjuvant ipilimumab plus nivolumab showed high pathologic response rates (pRRs) in patients with macroscopic stage III melanoma in the phase 1b OpACIN (NCT02437279) and phase 2 OpACIN-neo (NCT02977052) studies1,2. While the results are promising, data on the durability of these pathologic responses and baseline biomarkers for response and survival were lacking. After a median follow-up of 4 years, none of the patients with a pathologic response (n = 7/9 patients) in the OpACIN study had relapsed. In OpACIN-neo (n = 86), the 2-year estimated relapse-free survival was 84% for all patients, 97% for patients achieving a pathologic response and 36% for nonresponders (P < 0.001). High tumor mutational burden (TMB) and high interferon-gamma-related gene expression signature score (IFN-γ score) were associated with pathologic response and low risk of relapse; pRR was 100% in patients with high IFN-γ score/high TMB; patients with high IFN-γ score/low TMB or low IFN-γ score/high TMB had pRRs of 91% and 88%; while patients with low IFN-γ score/low TMB had a pRR of only 39%. These data demonstrate long-term benefit in patients with a pathologic response and show the predictive potential of TMB and IFN-γ score. Our findings provide a strong rationale for a randomized phase 3 study comparing neoadjuvant ipilimumab plus nivolumab versus standard adjuvant therapy with antibodies against the programmed cell death protein-1 (anti-PD-1) in macroscopic stage III melanoma.

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Fig. 1: Relapse-free survival and ongoing toxicities.
Fig. 2: Baseline IFN-γ signature and tumor mutational burden associated with response and relapse.
Fig. 3: Plasma analysis using Olink proteomic assay.
Fig. 4: Continental differences between European and Australian patients.

Data availability

DNA-sequencing and RNA-sequencing data generated during the study will be deposited in the European Genome-phenome Archive (EGA) under the accession codes EGAS00001004832 (DNA) and EGAS00001004833 (RNA), and will be made available on reasonable request. Data requests will be reviewed by the institutional review board of the Netherlands Cancer Institute and applying researchers will need to sign a data access agreement after approval.


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We thank the patients and their families for participating in the study. We thank all investigators and members of the clinical trial teams in Melanoma Institute Australia, the Netherlands Cancer Institute and the Karolinska Institutet; the Netherlands Cancer Institute-Antoni van Leeuwenhoek Core Facility Molecular Pathology & Biobanking for supplying biobank material and/or laboratory support; the Genomics Core Facility for their support regarding sequencing; and S. Vanhoutvin for financial management. We acknowledge A. Evans and B. Stegenga from Bristol Myers Squibb for scientific input and support. G.V.L. is supported by an Australian National Health and Medical Research Council (NHMRC) Practitioner Fellowship and the Medical Foundation at the University of Sydney. A.M.M. is supported by Cancer Institute New South Wales fellowship and Melanoma Institute Australia. R.A.S. is supported by NHMRC Practitioner Fellowship. Support from an Australian NHMRC program grant (to G.V.L. and R.A.S.), the Ainsworth Foundation, the Fairfax Foundation and the Cameron family is also gratefully acknowledged. The authors also acknowledge assistance from colleagues at their various institutions.

Author information

Authors and Affiliations



C.U.B. designed the study and wrote the study protocol. A.C.J.v.A. and T.N.M.S. provided additional input to the study design. E.A.R. coordinated the trial, analyzed and interpreted clinical and translational data and wrote the first draft of the manuscript with E.P.H., I.L.M.R. and J.M.V. C.U.B. co-wrote the manuscript. E.A.R., R.P.M.S., H.E., K. Shannon, J.B.A.G.H., J.S., S. Ch’ng, O.E.N., H.A.M., S.A., W.M.C.K., C.L.Z., W.J.v.H., A.J.S., A.C.J.v.A., A.M.M., G.V.L. and C.U.B. recruited and treated patients and collected data. B.A.v.d.W. and R.A.S. reviewed and scored the pathology of all cases, including grading pathologic responses. P.D. and O.K. (under the supervision of D.S.P.) performed the bioinformatics analysis. K. Sikorska performed the statistical analysis. A.T.A. and L.G.G.-O. were responsible for central and local data management. M.G. was a clinical project manager involved in the trial. E.P.H. performed the analysis of the plasma proteomics data. R.M.K. was responsible for the sequencing. S. Cornelissen performed DNA and RNA isolations. A.B. coordinated and contributed to translational laboratory logistics and immunohistochemistry and molecular laboratory work. Every author contributed to the initial draft of the manuscript and agreed on submission for publication. All authors interpreted the data, reviewed the manuscript and approved the final version.

Corresponding author

Correspondence to C. U. Blank.

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Competing interests

E.P.H., I.L.M.R., J.M.V., O.K., P.D., K. Sikorska, H.E., M.G., A.T.A., L.G.G.-O., K. Shannon, J.S., S. Ch’ng, O.E.N., H.A.M., S.A., R.M.K., S. Cornelissen, A.B., W.M.C.K., C.L.Z., W.J.v.H. and A.J.S. declared no competing interests. E.A.R. reports travel support from NanoString Technologies and MSD. R.P.M.S. has served on advisory boards for MSD Novartis Qbiotics and received honoraria from BMS. B.A.v.d.W. has served on advisory boards for BMS. J.B.A.G.H. has served on advisory boards for AIMM, Achilles, AZ/MEDimmune, Amgen, Bayer, BMS, GSK, Ipsen, Immunocore, MSD, Merck Sorono, Neon Therapeutics, Neogene Therapeutics, Novartis, Pfizer, Roche/Genentech, Sanofi, Seattle Genetics, Third Rock Ventures and Vaximm, and reports research fees paid to the institute from BMS, MSD, Novartis and Neon Therapeutics. D.S.P. is cofounder, shareholder and advisor of Immagene BV. A.C.J.v.A. has served on advisory boards for Amgen, BMS, Novartis, MSD-Merck, Merck-Pfizer, Sanofi and 4SC, and reports research fees paid to the institute from Amgen, BMS and Merck-Pfizer. R.A.S. reports financial support from Qbiotics, Novartis, NeraCare, AMGEN, BMS, Myriad Genetics, GlaxoSmithKline and Merck Sharp & Dohme. T.N.M.S. has served on advisory boards for Adaptive Biotechnologies, AIMM Therapeutics, Allogene Therapeutics, Merus, BioNTech, Scenic Biotech; reports financial support from Merck KGaA; and is stockholder in AIMM Therapeutics, Allogene Therapeutics, Merus, Neogene Therapeutics, BioNTech and Scenic Biotech. A.M.M. has served on advisory boards for BMS, MSD, Novartis, Roche, Pierre Fabre and QBiotics. G.V.L. has served on advisory boards for Aduro, Amgen, BMS, Highlight Therapeutics S.L., Mass-Array, Merck, MSD, Novartis, OncoSec Medical, Pierre Fabre, Roche, Qbiotics and Sandoz. C.U.B. has served on advisory boards for BMS, MSD, Roche, Novartis, GSK, AZ, Pfizer, Lilly, GenMab, Pierre Fabre and Third Rock Ventures, for which the institute received funding; received research funding from BMS, Novartis and NanoString, all paid to the institute; stock ownership: Uniti Cars; and is cofounder of Immagene BV.

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Extended data

Extended Data Fig. 1 Study design OpACIN, event-free survival and overall survival of OpACIN.

a, Study design of the OpACIN study. Patients were randomized to receive 4 cycles of ipilimumab 3 mg/kg + nivolumab 1 mg/kg every 3 weeks after surgery (adjuvant arm, n = 10) or 2 cycles of ipilimumab 3 mg/kg + nivolumab 1 mg/kg every 3 weeks followed by surgery and thereafter again 2 cycles of ipilimumab 3 mg/kg + nivolumab 1 mg/kg (neoadjuvant arm, n = 10). A biopsy was taken at screening and blood samples were taken at screening, baseline, week 6, week 12 and week 18. IPI; ipilimumab, NIVO; nivolumab, PBMC; peripheral blood mononuclear cells. b, Event-free survival by treatment arm and c, Overall survival by treatment arm of the OpACIN study. Kaplan-Meier curves were generated including all patients from the adjuvant arm (red, n = 10) and neoadjuvant arm (blue, n = 10).

Extended Data Fig. 2 Study design and flowchart OpACIN-neo.

a, Study design of the OpACIN-neo study. Patients were randomized to receive 2 cycles of ipilimumab 3 mg/kg + nivolumab 1 mg/kg every 3 weeks (arm A, n = 30), 2 cycles of ipilimumab 1 mg/kg + nivolumab 3 mg/kg every 3 weeks (arm B, n = 30) or 2 cycles of ipilimumab 3 mg/kg every 3 weeks directly followed by 2 cycles nivolumab 3 mg/kg every 2 weeks (arm C, n = 26). Surgery was planned after 6 weeks. A biopsy was taken at screening and blood samples were taken at screening, baseline, week 6 and week 12. IPI; ipilimumab, NIVO; nivolumab, PBMC; peripheral blood mononuclear cells. b, Flowchart of the OpACIN-neo study showing the number of patients screened, allocated to a treatment arm, starting immunotherapy and undergoing surgery per treatment arm.

Extended Data Fig. 3 Event-free survival and overall survival of OpACIN-neo.

a, EFS for the total population of the OpACIN-neo study. A Kaplan-Meier curve for EFS of all patients (n = 86) was generated. The corresponding 95% CI is displayed and was computed using log transformation. b, EFS of the OpACIN-neo study by treatment arm including all patients from arm A (blue, n = 30), arm B (orange, n = 30) and arm C (purple, n = 26). P values were calculated using the log-rank test (two-sided). c, OS for the total population of the OpACIN-neo study. A Kaplan-Meier curve for OS of all patients (n = 86) was generated. d, OS of the OpACIN-neo study by treatment arm including all patients from arm A (blue, n = 30), arm B (orange, n = 30) and arm C (purple, n = 26). a-b, The asterisk denotes the patient who died due to irAEs.

Extended Data Fig. 4 Ongoing surgery-related adverse events of OpACIN-neo.

Frequency of maximum grade and ongoing surgery-related adverse events (AEs) of the OpACIN-neo study. Frequencies of maximum grade AEs are displayed in light blue (grade 1–2) and dark blue (grade 3–5), and frequencies of ongoing AEs in orange (grade 1–2) and red (grade 3–5). AEs that were reported at a frequency of >5% and all grade 3–5 AEs were included. All patients (n = 86) were included in the analysis of maximum grade AEs; for ongoing AEs only patients alive at time of data cutoff (n = 81) were included.

Extended Data Fig. 5 Pathologic response rates according to subgroups.

Forest plot of data for all patients who underwent surgery (n = 85). pRRs according to demographic, clinical and tumor characteristics are displayed. The 95% CIs were calculated using the Clopper-Pearson method. PD-L1 expression on pretreatment tumor biopsies was assessed centrally with an automated lab-validated immunohistochemistry assay, using the 22C3 antibody on a Ventana platform. PD-L1 expression was determined by the Tumor Proportion Score (TPS; the percentage of tumor cells with complete or partial membranous staining at any intensity).

Extended Data Fig. 6 Whole-exome sequencing and RNA sequencing analysis of pretreatment tumor biopsies.

a, Mutational load and mutational patterns of recurrent mutated cutaneous melanoma genes found by whole-exome sequencing. The frequency, mutation type and base changes are indicated. Each column represents one patient (n = 60 patients). b, Correlation between the IFN-γ score (values displayed as the average z-score of all the genes within the IFN-γ signature14) and TMB (displayed in log scale) for patients with pathologic response (n = 42, green) and no pathologic response (n = 17, red). The correlation coefficient and P value were calculated using the Pearson’s correlation method. c, TMB and baseline average expression of IFN-γ score of patients with a response (green dots) and patients without a response (red dots). The quadrants are determined by the optimal cutoff for each of the biomarkers as defined by the sROC curves. Each quadrant indicates the number of responding patients and total number of patients in the corresponding quadrant. Data were available for 59 patients. d, sROC curve showing the AUC for the combination of the IFN-γ score and TMB (0.83) (n = 59). e, Heatmap of the MCP counter15 RNA gene signature ordered according to average signature expression per response category of baseline tumor biopsies (n = 65). The MCP counter signature expresses the abundance of eight immune and two stromal cell populations. Each cell type is represented by the averaged z-score of the genes that it is consisted of, which were previously normalized by DESeq2. The score was computed from the average expression of all the ten cell types that form the MCP counter signature. Columns represent patients (green: pathological response/no relapse; red: no pathological response/relapse; grey: not evaluable (NE); blue: treatment arm A; orange: treatment arm B; purple: treatment arm C) and rows represent genes. Positive values (red) indicate higher expression and negative (blue) indicate lower expression. f, Gene set enrichment analysis displaying hallmark gene sets that are significantly enriched in responders (green) or nonresponders (red). Pathways are ordered according to the FDR. FDRs were computed as previously described37.

Extended Data Fig. 7 Extended plasma analysis using Olink proteomic assay.

a, PDCD1, CXCL9, CXCL10 normalized protein expression (NPX) in plasma of patients (n = 85) before treatment (pretreatment; round dots) and after treatment (post-treatment; triangle dots) measured with Olink immunoassay (green: patients with pathologic response, n = 64; red: patients without pathologic response, n = 21). Data for PDCD1 are missing for pretreatment samples of 9 patients (all responders) because values were below detection limit. The mean and SD are shown. P values were calculated using the paired Student’s t-test (two-sided). b, Heatmap of VEGFR2, CX3CL1 and PD-L2 NPX in plasma of patients before start of treatment. The heatmap depicts the ordered mean expression of these three genes of the 86 patients included in the OpACIN-neo cohort. The score of each patient expresses the baseline averaged z-score of the three mentioned genes mentioned beforehand which was previously normalized by DESeq2. Each column represents a different patient (green: response/no relapse; red: no response/relapse; blue: arm A; orange: arm B; purple: arm C) and rows indicate protein expression. Positive values (red) indicate higher expression and negative values (blue) indicate lower expression.

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Supplementary Tables 1–9 and study protocols.

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Rozeman, E.A., Hoefsmit, E.P., Reijers, I.L.M. et al. Survival and biomarker analyses from the OpACIN-neo and OpACIN neoadjuvant immunotherapy trials in stage III melanoma. Nat Med 27, 256–263 (2021).

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