Faculty Bibliography

Background: The high mortality of cutaneous melanoma (CM) is partly due to unpredictable patterns of disease progression in patients with early-stage lesions. The reliable prediction of advanced disease risk from early-stage CM, is an urgent clinical need, especially given the recent expansion of immune checkpoint inhibitor therapy to the adjuvant setting. In our study, we comprehensively investigated the role of germline variants as CM prognostic markers. Methods: We performed a genome-wide association analysis in two independent cohorts of N=551 (discovery), and N=550 (validation) early-stage immunotherapy-naïve melanoma patients. A multivariable Cox proportional hazard regression model was used to identify associations with overall survival in the discovery group, followed by a validation analysis. Transcriptomic profiling and survival analysis were used to elucidate the biological relevance of candidate genes associated with CM progression. Results: We found two independent associations of germline variants with melanoma prognosis. The alternate alleles of these two SNPs were both associated with an increased risk of death [rs60970102 in MELK: HR=3.14 (2.05"“4.81), p=1.48×10^-7; and rs77480547 in SH3BP4: HR=3.02 (2.02"“4.52), p=7.58×10^-8, both in the pooled cohort]. The addition of the combined risk alleles (CRA) of the identified variants into the prognostic model improved the predictive power, as opposed to a model of clinical covariates alone. Conclusions: Our study provides suggestive evidence of novel melanoma germline prognostic markers, implicating two candidate genes: an oncogene MELK and a tumor suppressor SH3BP4, both previously suggested to affect CM progression. Pending further validation, these findings suggest that the genetic factors may improve the prognostic stratification of high-risk early-stage CM patients, and propose putative biological insights for potential therapeutic investigation of these targets to prevent aggressive outcome from early-stage melanoma.

BACKGROUND:Recurrence-free survival (RFS) and overall survival (OS) data for adjuvant nivolumab versus placebo (proxy for routine surveillance) in patients with high-risk, resected melanoma are lacking. This post hoc, indirect treatment comparison (ITC) used pooled data from the phase 3 EORTC 18,071 (ipilimumab vs. placebo) and CheckMate 238 (nivolumab vs. ipilimumab) trials to assess RFS and OS with nivolumab versus placebo and the numbers needed to treat (NNT) over 4 years. METHODS:Patients with resected stage IIIB-C cutaneous melanoma (American Joint Committee on Cancer seventh edition) were included. Inverse probability treatment weighting (IPTW) was used to balance baseline characteristics. RFS NNTs were calculated for nivolumab versus ipilimumab and placebo. OS NNTs were calculated for nivolumab versus placebo. To adjust for different post-recurrence treatments, the difference in post-recurrence survival between the two ipilimumab arms was added to OS of the placebo arm. RESULTS:This ITC included 278, 643, and 365 patients treated with nivolumab, ipilimumab, and placebo, respectively. Following IPTW, nivolumab was associated with improved RFS versus placebo (hazard ratio [HR]: 0.49; 95% confidence interval [CI] 0.39-0.61) and ipilimumab (HR: 0.69; 95% CI 0.56-0.85). RFS NNT was 4.2 for nivolumab versus placebo and 8.9 for nivolumab versus ipilimumab. After post-recurrence survival adjustment, weighted 4-year OS rates were 75.8% for nivolumab and 64.1% for placebo; OS NNT for nivolumab versus placebo was 8.5. CONCLUSIONS:In patients with resected stage IIIB-C cutaneous melanoma in this ITC, nivolumab improved RFS versus placebo and ipilimumab, and OS versus placebo after post-recurrence survival adjustment.

Background/Purpose: Treatment with a combination of immune checkpoint inhibitors (ICI) has promising outcomes in many tumor types but carries higher adverse event risk than ICI monotherapy. Also, patients with pre-existing autoimmune disease (AID) have largely been excluded from ICI clinical trials due to concern for pre-existing AID flare or immune-related adverse events (irAEs). This is the first study to analyze safety and effectiveness of ICI combination versus monotherapy for this at-risk population.
Method(s): We conducted a multi-center retrospective study in patients with pre-existing AIDs receiving ICIs (i.e., antiprogrammed cell death protein 1 (PD-1) single-agent (monotherapy) or ICI combination). Primary endpoints included the time to occurrence of any-type ICI AE (irAE or AID flare), time to irAEs and time to AID flares in the presence of the competing risk of death with progression free survival (PFS, time to progression or death) and overall survival (OS) as secondary endpoints. We used Fine-Gray models and Cox regression models to investigate the factors associated with these endpoints.
Result(s): 133 patients with pre-existing AID who received ICIs were identified: 69 (52%) monotherapy and 64 (48%) combination (Table 1). About half the patients had melanoma (44%) and 25% had lung cancer. Rheumatic (34%) or dermatologic (22%) pre-existing AIDs were the most common. Most patients (95%) had controlled autoimmune disease at ICI start. Six of 7 patients with active AID at baseline experienced some AE. Patients receiving baseline DMARD(s) were more likely to experience an AE (95%CI 1.079-2.996, p=0.024). The cumulative incidence of irAEs was higher for ICI combination compared to monotherapy (subdistribution hazard ratio (sHR) 2.28, 95%CI 1.36-3.84), adjusting for age at malignancy, but there was no significant difference between rate of high-grade toxicity for patients treated with ICI combination versus monotherapy (See Figure 1). On subgroup analysis for patients with melanoma or lung cancer, the cumulative incidence of irAEs or AID flares were not statistically different between treatment groups. PFS was longer (but not statistically significant) for combination therapy for any tumor type compared to single agent (median 12.3mo, 95%CI 5.0-23.2 versus 7.3mo, 95%CI 5.2-11.3, p=0.116). Similar trend was noted for PFS for melanoma (median 23.2mo combination vs. 14.0mo monotherapy, p=0.4237), while the opposite relation was noted for lung cancer subgroup (4.4mo combination vs. 7.1mo monotherapy, p=0.2933).
Conclusion(s): Efficacy of ICI combination versus monotherapy was not statistically significant and so still remains unclear in this patient population, but there was no significant difference in rates of high-grade toxicity between the two cohorts. Our data supports the notion that patients with pre-existing AIDs should not be indiscriminately precluded from getting ICI combination. Our results provide guidance for future prospective clinical trials studying combination therapy for subgroups of this at-risk population. No statistically significant difference appreciated in high grade adverse events between patients with pre-existing autoimmune disease treated with ICI combination versus monotherapy. Grading determined by the Common Terminology Criteria for Adverse Events rubric with grade 3 or higher considered "high grade."

BACKGROUND:Patients with advanced melanoma have limited treatment options after progression on immune checkpoint inhibitors (ICI). Lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, demonstrated an investigator-assessed objective response rate (ORR) of 36% in 66 patients who progressed after ICI and targeted therapy. Herein, we report independent review committee (IRC)-assessed outcomes of 153 patients treated with lifileucel in a large multicenter Phase 2 cell therapy trial in melanoma. METHODS:Eligible patients had advanced melanoma that progressed after ICI and targeted therapy, where appropriate. Melanoma lesions were resected (resected tumor diameter ≥1.5 cm) and shipped to a central good manufacturing practice facility for 22-day lifileucel manufacturing. Patients received a non-myeloablative lymphodepletion regimen, a single lifileucel infusion, and up to six doses of high-dose interleukin-2. The primary endpoint was IRC-assessed ORR (Response Evaluation Criteria in Solid Tumors V.1.1). RESULTS:The Full Analysis Set consisted of 153 patients treated with lifileucel, including longer-term follow-up on the 66 patients previously reported. Patients had received a median of 3.0 lines of prior therapy (81.7% received both anti-programmed cell death protein 1 and anti-cytotoxic lymphocyte-associated protein 4) and had high disease burden at baseline (median target lesion sum of diameters (SOD): 97.8 mm; lactate dehydrogenase (LDH) >upper limit of normal: 54.2%). ORR was 31.4% (95% CI: 24.1% to 39.4%), with 8 complete responses and 40 partial responses. Median duration of response was not reached at a median study follow-up of 27.6 months, with 41.7% of the responses maintained for ≥18 months. Median overall survival and progression-free survival were 13.9 and 4.1 months, respectively. Multivariable analyses adjusted for Eastern Cooperative Oncology Group performance status demonstrated that elevated LDH and target lesion SOD >median were independently correlated with ORR (p=0.008); patients with normal LDH and SOD <median had greater likelihood of response than those with either (OR=2.08) or both (OR=4.42) risk factors. The most common grade 3/4 treatment-emergent adverse events (≥30%) were thrombocytopenia (76.9%), anemia (50.0%), and febrile neutropenia (41.7%). CONCLUSIONS:Investigational lifileucel demonstrated clinically meaningful activity in heavily pretreated patients with advanced melanoma and high tumor burden. Durable responses and a favorable safety profile support the potential benefit of one-time lifileucel TIL cell therapy in patients with limited treatment options in ICI-refractory disease.

Background Therapies targeting T-cell checkpoints have resulted in anti-tumor responses leading to FDA approval of multiple immunotherapies for metastatic melanoma and an expanding list of other malignancies. Despite having unprecedented efficacy, PD1 and CTLA4 antagonist antibodies still fail to benefit many patients. Critically, an understanding of mechanisms of resistance to immunotherapies is lacking. We recently identified and validated a novel population of peripheral blood T-cells predictive of resistance in nivolumab (alphaPD1) treated metastatic melanoma patients. This population is defined by the marker set CD3+CD4+CD127-GARP-CD38 +CD39+. Based on the co-expression of CD38 and CD39, we have termed the population ectoenzyme expressing T-cells (Teee). Methods We utilized high-dimension flow cytometry to assess Teee frequencies in adjuvant immunotherapy treated metastatic melanoma patient peripheral blood samples. To characterize the phenotype of Teee, we utilized flow cytometry and CITESeq on melanoma patient tumor specimens. We also evaluated the presence and phenotype of Teee in several l syngeneic murine tumor models. Results We found that Teee were of low frequency in demographic matched healthy donors and increased in both resected (p=0.018) and active disease metastatic melanoma patients (p=0.003). Circulating Teee frequencies positively correlated with frequencies of Tregs (R²=0.4367, p<0.0001), MDSCs (R²=0.2706, p=0.0004) and M2-like monocytes (R²=0.1514, p=0.0109). Increases in circulating Teee were associated with relapse in resected melanoma patients treated with adjuvant combination ipilimumab (alphaCTLA4) and nivolumab (p=0.0213). Human tumors showed high frequencies of Teee in tumor infiltrate. We also demonstrated the existence of this population in MC38 and YUMM mouse tumor models. Intratumor frequencies of Teee positively correlated with tumor volume (R²=0.96, p=0.0006) and inversely correlated with overall immune infiltrate (R²=0.3198, p=0.0280). Our characterization of this population showed an enhanced adenosine generating phenotype (i.e. CD73^high, CD26^low), a terminal exhaustion phenotype (i.e. TOX^high, TCF1^low), expression of inhibitory receptors (e.g. CTLA4, TIM3) and ligands (e.g. PDL1, B7-H4), and expression of immunosuppressive cytokines (e.g. IL-8, TGFbeta). Supporting a mechanistic relationship to resistance, Teee suppressed autologous T-cell proliferation (p=0.0278) and inflammatory function. Conclusions We have identified a novel population of ectoenzyme expressing T-cells associated with immunotherapy resistance in metastatic melanoma patients. This population of cells had phenotypes associated with immune suppression and suppressed autologous T-cells in vitro. Collectively, our data support evaluation of targeting Teee to augment the efficacy of immunotherapy

Background Despite significant improvements in treatment outcomes with ICIs, deeper understanding is needed to improve outcomes for patients with melanoma who experience no clinical benefit from ICI therapy and exhibit early disease progression (primary resistance).1 We analyzed clinical and translational factors using data from 9 BMS-sponsored clinical trials of nivolumab (NIVO), ipilimumab (IPI) and their combination in patients with advanced cutaneous melanoma (Check- Mate 003, 037, 038, 064, 066, 067, 069, 511, 742) to predict resistance to ICIs and explore underlying mechanisms. Methods Analyses of pre-treatment clinical covariates and biomarkers (determined by tumor immunohistochemistry, whole exome DNA sequencing, RNA sequencing, and serum cytokine analysis) were performed in patients with melanoma who received NIVO, IPI or NIVO+IPI and were evaluable for resistance. Several methods were used to develop and test models predicting ICI primary resistance, defined as death due to disease or best overall response of progressive disease (excluding pseudoprogression) before the first radiographic scan (<=13 weeks of treatment). Model performance was assessed using cross-validated area under the receiver operating characteristic curve (AUROC) and bootstrap bias-adjusted calibration metrics. Additionally, we evaluated individual biomarker associations with primary resistance. Results Across 1803 patients from 9 clinical trials, 1638 were evaluable for cutaneous melanoma primary resistance and 585 (36%) met the criteria. A multivariable logistic regression model for predicting primary resistance yielded an AUROC of 0.78 (R2=0.29) and included 17 commonly available clinical factors and PD-L1 (table 1). External validation using data from an independent study yielded an AUROC of 0.70 (R2=0.17). Higher levels of acute phase cytokines and expression of genes associated with suppressive tumor microenvironment or stromal factors, including epithelial-mesenchymal transition, cancer-associated fibroblasts, transforming growth factor beta, and angiogenesis, were associated with increased risk of primary resistance. In contrast, higher levels of tumor mutational burden, PD-L1, CD8, mutations in MAPK-pathway genes, and immune gene signature scores were associated with decreased risk (figure 1, table 2). Conclusions This exploratory analysis of clinical and translational factors in patients with advanced cutaneous melanoma identified associations with primary resistance to NIVO, IPI, or NIVO+IPI, with the best model yielding an AUROC of 0.78. Factors associated with primary resistance included higher levels of prognostic cytokines, gene expression indicating an immune suppressive tumor microenvironment, lower levels of anti-tumor immunity, and lack of MAPK-pathway mutations. These findings are supportive of future clinical trial stratification of patients and mechanistic studies targeting the biology of primary resistance to ICI therapy

Background Tumor Infiltrating Lymphocyte (TIL) Adoptive Cell Transfer (ACT) is effective in treating malignant melanoma and other solid tumors. The success of TIL ACT relies on the adequate expansion of TIL, with previous studies showing a positive association between number of TIL infused and patient response. To identify characteristics important for TIL expansion, we performed a multiomic analysis of patients' TIL product. Methods Expanded TIL products from metastatic melanoma patients enrolled in clinical trials at Moffitt Cancer Center were collected before infusion. CD4+ and CD8+ were isolated and analyzed by RNA-seq (n=13) and pan-acetyl histone 3 ChIP-seq (n=20). The number of TIL, percentage of CD4/ CD8 infused, progression free survival (PFS), and overall survival (OS) were recorded. To more evenly divide both RNASeq and ChIP-seq samples between groups, patients were categorized into TIL high vs TIL low based on division at the geometric mean of the number of TIL infused (geometric mean= 4.6e10, range= 9.05e+09 - 1.13e+11). Log2 fold changes of +/-0.5 and q-values of <0.1 were considered significant. Results Individuals in the TIL high group had longer (PFS) (median=92 vs 4, p< 0.0001) and OS (OS median=92 vs 10 months , p<0.0001) than those in the TIL low group, and the percentage of CD4+ infused was negatively correlated with the number of TIL infused (R2=-0.72, p=7.7e-05). RNA-seq revealed 30 differentially expressed genes (DEGs) in CD4+ between groups. The upregulated genes in the TIL low group included TNFRSF4, PTGDR2, IL5, and CCL4, which are associated with Th2 and Treg phenotypes. Pathway analysis of the RNA-seq data further suggested increased Th2 and Th17 polarization in the TIL low group. ChIP-seq showed 18 differentially acetylated genes (DAGs) between TIL high and TIL low CD4+, including increased acetylation at known inducers of Tregs CD200R1, IL12RB2. RNA-seq revealed 3 DEGs between CD8+ in the TIL high and TIL low groups. ChIP-seq revealed 18 DAGs between TIL high and TIL low CD8+, with increased acetylation at genes known to be upregulated during CD8+ activation (e.g., SLC4A10, TIGIT and P2RY1). Conclusions Our results and those of other groups have shown that increased number of TIL infused are associated with positive patient outcomes. Our results further indicate that CD4+ Th2, Th17 and Treg phenotypes in the TIL product are associated with decreased numbers of TIL achieved during expansion. Consequently, we hypothesize that approaches to directing T-cell polarization during TIL expansion may increase patient response rates

Background mRNA-2752 is a first-in-class mRNA-based therapeutic agent encoding T cell co-stimulator OX40L, and proinflammatory cytokines IL-23 and IL-36g. Preclinical data demonstrated activity in a range of tumor microenvironments (TMEs), including immune checkpoint inhibitor (CPI)- refractory cancer models, and synergy when combined with alpha-PDL1 antibodies. Data from the dose escalation phase of the study was previously reported and the recommended mRNA- 2752 dose for expansion (RDE) was up to 8mg ITu. Methods We evaluated the safety and efficacy of ITu mRNA- 2752 administered as monotherapy (Arm A; previously reported ASCO 2020) and in combination with the PD-L1 inhibitor durvalumab (Arm B) in patients (pts) with tumors that were palpable or accessible with image guidance. Here we report preliminary data for the expansion cohorts in TNBC, CPI-refractory melanoma, and HNSCC. Biomarker analyses included IHC/F-IHC of immune status markers and whole transcriptome assessments of paired tumor biopsies. Protein quantification of IL-23, IL-36g and other pro-inflammatory cytokines were performed in plasma and tumors. Results As of 01JUL 2022, 88 pts were treated, 69 in Arm B with mRNA-2752 dosed by tumor size ranging from 0.25-8 mg. The most common treatment related adverse events occurring in >= 10% of pts in Arm B included grade 1/2 injection site erythema/pain/swelling, fever, chills, fatigue, nausea, and flushing. Grade 3 events included injection site pain/erythema, and fever. There were no grade 4/5 related events. Objective responses were observed in immune refractory tumors by RECIST and iRECIST, including a confirmed PR in a PD-L1 low TNBC and confirmed iCR in an immune checkpoint- refractory melanoma pt, respectively. Biomarker analyses of plasma and tumor show mRNA-2752 treatment was associated with elevated pro-inflammatory cytokines, including IL- 23, IL-36g, IFNgamma, and TNFalpha. F-IHC of paired tumor biopsies showed increase in proliferating CD8+ T cells. Transcriptional profiling of the TME demonstrates a pronounced immune response including dendritic cell recruitment and T cell activation, which remained elevated in longitudinal samples. The greatest increases in immune response and markers of cytolytic activity were observed in pts deriving clinical benefit. Conclusions ITu mRNA-2752 + IV durvalumab is safe, tolerable, and shows preliminary efficacy in immune refractory tumors, including TNBC and melanoma. Biomarker analyses indicate mRNA-2752 drives cytokine responses. Consistent with the expected mechanism of action, a productive and sustained inflammatory response is observed in the TME in response to treatment, including signatures of increased innate and adaptive immune cell abundance and effector response

Background Though FDA-approved for use after surgery for stage III/IV melanoma in the adjuvant setting, ipilimumab (anti-CTLA-4) and interferon-a are not typically used because of the improved toxicity profile and relapse-free survival (RFS) benefits seen with anti-PD-1-based regimens in this patient population. However, it is important to study samples derived from anti-CTLA-4 treated patients from the mechanistic perspective to understand its contribution to ipilimumab/ nivolumab therapy and to study single agent CTLA4 therapy in the adjuvant cases of BRAF wild-type patients who were re-resected and previously received anti-PD-1 therapy. Recently published data from our group indicated that nitric oxide (NO) increased in immune effector cells among patients treated with longer RFS, whereas NO also increased in immune suppressor cells among patients with shorter RFS. Given this unexpected dichotomy, we measured the posttranslational modification in the immune cells that is a direct result of increased NO levels on the interferon response protein STAT1 important for response to anti-CTLA-4 therapy/interferon responsiveness. Nitration is a stable post-translational modification of NO metabolism and STAT1 is nitrated at the same position (Y701) as it is phosphorylated. Methods Peripheral blood mononuclear cells (PBMCs) collected from 35 patients at day 0 and day 155 of ipilimumab therapy were analyzed for phosphorylated STAT1 (pSTAT1; using flow cytometry) and nitrated STAT1 at position 701 (nSTAT1; using liquid chromatography with tandem mass spectrometry). Patients were divided into groups based on RFS post ipilimumab therapy. Results Pre- and post-therapy PBMCs were available from 35 patients with stage IIIC/IV melanoma. The median patient age was 58 years (range, 21-78 years), and 63% of patients were male. pSTAT1 levels had the least variability in the tercile of patients with the longest RFS (P=0.002). Higher pSTAT1 levels in PBMCs were associated with worse RFS before (P = 0.007) and after therapy (P = 0.036) with ipilimumab. Expectedly, pSTAT1 levels in PBMCs increased following ipilimumab therapy. Patients whose STAT1 remained nitrated had decreased RFS, but intriguingly, increases in nitration of STAT1 following ipilimumab therapy was associated with favorable outcomes (P=0.01). Conclusions These data suggest that interferon responsiveness is regulated by changes in nitration of STAT1. Nitration of Y701 on STAT1 may regulate excessive interferon responses by limiting pSTAT1 phosphorylation for successful ipilimumabbased therapy in melanoma and deserves further study in the adjuvant and metastatic settings.