Search

Figure S5 shows that complement factor B (CFB), component 7 (C7) and alpha-1B-glycoportein (A1BG) expression are increased upon melanoma progression and recurrence.

Table S3 shows the patient characteristics PRADO

Figure S2 shows a flowchart of patients PRADO study

Figure S6 shows that neutrophil count and neutrophil-to-lymphocyte ratio (NLR) are not associated with recurrence in non-responding patients

Background In the multicenter investigator-initiated OpACIN-neo trial, patients (pts) with macroscopic stage III melanoma were randomized (stratified by center) to three different dosing schemes of neoadjuvant ipilimumab (IPI) + nivolumab (NIVO). Two cycles IPI 1 mg/kg + NIVO 3 mg/kg was identified as the most favorable regimen with 20% grade 3-4 adverse events and a pathologic response rate (pRR) of 77%. After a median follow-up of 17.7 months, relapses were observed in 1/64 (2%) of the pts with a pathologic response, and in 13/21 (62%) of the non-responders. Post-hoc analyses according to continent of study inclusion were conducted to investigate potential differences between pts treated in Europe (EU) and in Australia (AUS). Methods We evaluated baseline patient characteristics, safety and efficacy in terms of pathologic response in pts treated in EU (n=48) and AUS (n=38). Mutational (mut) load of baseline biopsies was assessed using whole exome sequencing. Multivariate analyses were performed using the logistic regression method. Median follow-up was 18.2 months for EU pts and 16.6 months for AUS pts. Results Baseline characteristics (AUS vs EU) differed in age (median 60 vs 53 year [yr], p=0.017). There were numerically more male pts in the Australian cohort (65.8 vs 50.0%, p=0.142) and more pts with unknown primary melanoma (36.8 vs 20.8%, p=0.100). A numerical higher pRR was observed in AUS pts vs EU pts (84.2% vs 64.7%, OR 2.50, p=0.092). The pRR was significantly higher for older pts (OR per yr 1.059, p=0.003), males (83.7% vs 63.9%, OR 2.90, p=0.041), and pts with higher mut load (OR per mutation 1.002, p=0.014). Mut load was higher in pts with pathologic response (p=0.0013) and in AUS pts (p=0.0003). There was a positive correlation between age and mut load (R=0.26, p=0.043). Multivariate analysis including continent, age, gender and mut load revealed that only mut load was significantly associated with response (OR 1.002, p=0.037).The frequency of grade 3-5 toxicities was the same in pts 60 yr (42.3% vs 32.4%, p=0.353). Conclusion The numerical higher pRR in AUS vs EU melanoma pts upon neoadjuvant IPI + NIVO appears mostly driven by a higher mut load found in the melanomas of AUS pts. AUS pts were older and there was a positive correlation between age and mut load, indicating that the higher mut load in AUS pts might be explained by higher age. It remains to be elucidated if continental variance in sun exposure also contributed to the difference in mut load. The fact that older pts achieve a higher response rate in absence of increased toxicity rates indicates that older pts should not be withheld neoadjuvant IPI + NIVO. Citation Format: Irene L. Reijers, Elisa A. Rozeman, Alexander M. Menzies, Judith M. Versluis, Bart A. van de Wiel, Karolina Sikorska, Hanna Eriksson, Kerwin Shannon, Carolien Bierman, Harm van Tinteren, Maria Gonzalez, Andrew J. Spillane, Robyn P. Saw, Alexander C. van Akkooi, Richard A. Scolyer, Johan Hansson, Georgina V. Long, Christian U. Blank. Different pathologic response rates between Australia and Europe in macroscopic stage III melanoma patients upon neoadjuvant ipilimumab plus nivolumab in the phase II OpACIN-neo trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3271.

Introduction The outcome of high-risk stage III melanoma patients was poor with a 5-year overall survival (OS) rate of Methods The phase 1b OpACIN trial randomized 20 stage IIIB/IIIC melanoma patients to receive either 4 cycles of adjuvant IPI 3 mg/kg plus NIVO 1 mg/kg or 2 cycles of neoadjuvant IPI plus NIVO at the same dose followed by 2 cycles adjuvant IPI plus NIVO. In the OpACIN-neo trial, 86 patients were randomized to 2 cycles neoadjuvant in arm A: 2x IPI 3 mg/kg plus NIVO 1 mg/kg q3w (n=30), arm B: 2x IPI 1 mg/kg plus NIVO 3 mg/kg q3w (n=30), and arm C: 2x IPI 3 mg/kg q3w followed immediately by 2x NIVO 3 mg/kg q3w (n=26). Pathologic response was defined as Results After a median follow-up of 36 months for the OpACIN and 18 months for the OpACIN-neo trial, only 1 of 71 patients (1.4%) with a pathologic response on neoadjuvant therapy had relapsed, versus 15 of 23 patients (65.2%) without a pathologic response. The estimated 3-year RFS rate for the neoadjuvant arm was 80% (95% CI: 59%-100%) versus 60% (95% CI: 36%-100%) for the adjuvant arm in the OpACIN trial. The median RFS was not reached in any of the arms within the OpACIN-neo trial. Estimated 18-months RFS rate was 85% (95% CI: 78%-93%) for all patients; for arm A 90% (95% CI: 80%-100%), for arm B 82% (95% CI: 70%-98%) and for arm C 83% (95% CI: 70%-100%). Translational analyses showed that tumor mutational burden and interferon-γ gene expression score at baseline, both separate and combined, can function as predictors of response. Conclusions OpACIN showed for the first time a potential benefit of neoadjuvant versus adjuvant immunotherapy, while OpACIN-neo confirmed the high pathologic response rates which can be achieved by neoadjuvant IPI plus NIVO. Both trials argue for pathologic response as a surrogate markers for RFS. Clinical trial information: NCT02437279, NCT02977052 Citation Format: Christian U. Blank, Judith M. Versluis, Elisa A. Rozeman, Alexander M. Menzies, Irene L. Reijers, Oscar Krijgsman, Esmée P. Hoefsmit, Bart A. van de Wiel, Karolina Sikorska, Carolien Bierman, Petros Dimitriadis, Maria Gonzalez, Annegien Broeks, Ron M. Kerkhoven, Andrew J. Spillane, John B. Haanen, Winan J. van Houdt, Robyn P. Saw, Hanna Eriksson, Alexander C. van Akkooi, Richard A. Scolyer, Ton N. Schumacher, Georgina V. Long. 36-months and 18-months relapse-free survival after (neo)adjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma patients - update of the OpACIN and OpACIN-neo trials [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3412.

The immune cell composition within tumors plays a major role in response to immunotherapy. Therefore, comprehensive characterization of the diverse functional states exhibited by immune cell infiltrates, and their association with treatment outcome, is critical for the development of more effective immunotherapies. We combined single-cell RNA- and TCR-sequencing with an autologous tumor reactivity assay, tracing immune cell heterogeneity and dynamics in melanoma patients. Analysis of 70,000 cells revealed the presence of two effector T-cell subsets, of which only one transitions into a dysfunctional T-cell population, as based both on TCR sharing as well as a gradient of expression of dysfunction-associated genes present in virtually all samples. Notably, we denote dysfunctional T cells— and in particular early dysfunctional cells—as the major proliferating immune cell compartment. Furthermore, TCR-seq of autologous tumor reactive T cells illustrated that presence of reactive potential is associated with the magnitude of dysfunctional signature and is in concordance with response to immunotherapy. Data from 25 melanoma patients confirm the universality of these observations and distinguish shared and divergent regulatory modules between dysfunctional CD8 and regulatory T cells. Our analysis also puts forward novel candidate genes, which are highly correlated with known checkpoint targets within specific cellular subsets and may prove to be effective targets for checkpoint blockade. In conclusion, our findings proclaim that CD8 T cells previously associated with a dysfunctional or exhausted state are in fact a highly proliferating and dynamically interacting cell population within the human tumor microenvironment. This abstract is also being presented as Poster B58. Citation Format: Ido Yofe, Hanjie Li, Anne van der Leun, Yaniv Lubling, Dikla Gelbard Solodkin, Alexander van Akkooi, Hugo M. Horlings, Eyal David, Yael Baran, Akihad Berkovitz, Aviezer Lifshitz, Ton N. Schumacher, Amos Tanay, Ido Amit. Single-cell analysis illuminates dysfunctional CD8+ T cells as a proliferative, dynamically regulated compartment within human melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR04.

Checkpoint blockade therapies that aim to reactivate antitumor immune responses have revolutionized cancer treatment, resulting in durable responses in a significant proportion of patients with advanced tumor progression. Nevertheless, many patients fail to reach long-term clinical benefit due to lack of response or acquired resistance. Inconsistency in therapy outcomes may be explained in part by recent findings suggesting that immune cell infiltrates in tumors are highly heterogeneous among patients. Therefore, comprehensive characterization of the diverse functional states exhibited by immune cell infiltrates is critical for the development of more effective immunotherapies. Here, we characterized immune cell infiltrates within tumors derived from 28 metastatic melanoma patients by single-cell RNA-seq of ~100,000 immune cells, and parallel T-cell receptor (TCR) sequencing, thereby generating an unbiased map of the expression signatures of immune cells, as well as clonality of T-cells within and between metastases. We identified within the tumor infiltrates naïve, semi- and fully-activated effector, dysfunctional, and regulatory T-cells, as well as NK cells, and various myeloid subsets. While various immune cell types and cellular states are shared among patients, their frequency in each is highly heterogeneous even among similar tumor progression stages and treatment background. We noticed that clonally expanded T-cells predominantly adapted similar expression profiles. The frequencies of certain sub-populations were found to be correlated; notably, dysfunctional CD8 T-cell states were associated with prevalence of regulatory T-cells and follicular helper cells. The high-resolution map demonstrated gradient transitions between activation and dysfunctional states of CD8 T-cells, as well as variability within regulatory T-cell states. We used computational modeling to find the key transcription factors driving these gradients of expression states. Our analysis also puts forward novel candidate genes, which are highly correlate with known checkpoint targets within specific single-cell clusters, and may prove to be effective targets for checkpoint blockade. In order to validate our results, we performed in vitro reactivity assays (for available samples), which allowed us to determine autologous-tumor reactive and nonreactive TCRs, adding the reactive potential of T-cells as another layer of information to the single-cell expression signatures, facilitating inference of the functionality of clonally expanded T-cell populations in the tumor. Linking autologous tumor reactivity with single-cell expression profiles, we found that T-cells presenting reactive capability in vitro show a highly dysfunctional signature in the original tumor. The lack or presence of such reactive CD8 T-cells was also associated with patient response to immunotherapy (n=10). In conclusion, we present an atlas of immune cell infiltrates of human melanoma, revealing intra- and inter-patient heterogeneity of tumor immune infiltrates, highlighting regulatory circuits underlying different immune populations and their interactions. Our findings demonstrate how single-cell analysis may serve in the future as a tool for predicting response to therapy, provide rapid and effective tumor-immune state characterization, and ultimately lead to optimization of personalized immunotherapy. Citation Format: do Yofe, Hanjie Li, Anne van der Leun, Yaniv Lubling, Dikla Gelbard, Alexander C. J. van Akkooi, Amos Tanay, Ton N.M. Schumacher, Ido Amit. Single-cell analysis illuminates the gradients of immune cell functional states within human melanoma tumors, and facilitates characterization of tumor-reactive T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B054.