Faculty Bibliography

Advances in immune checkpoint and combination therapy have led to improvement in overall survival for patients with advanced melanoma. Improved understanding of the tumor, tumor microenvironment and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. Combination modalities with other immunotherapy agents, chemotherapy, radiotherapy, electrochemotherapy are also being explored to overcome resistance and to potentiate the immune response. In addition, novel approaches such as adoptive cell therapy, oncogenic viruses, vaccines and different strategies of drug administration including sequential, or combination treatment are being tested. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic theràapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers, but they have yet to be fully characterized and implemented clinically. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. Overall, the future research efforts in melanoma therapeutics and translational research should focus on several aspects including: (a) developing robust biomarkers to predict efficacy of therapeutic modalities to guide clinical decision-making and optimize treatment regimens, (b) identifying mechanisms of therapeutic resistance to immune checkpoint inhibitors that are potentially actionable, (c) identifying biomarkers to predict therapy-induced adverse events, and (d) studying mechanism of actions of therapeutic agents and developing algorithms to optimize combination treatments. During the Melanoma Bridge meeting (December 2nd-4th, 2021, Naples, Italy) discussions focused on the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine as well as the impact of COVID-19 pandemic on management of melanoma patients.

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."

OBJECTIVE:The objective of this study was to determine the impact of multiple sclerosis (MS) disease-modifying therapies (DMTs) on the development of cellular and humoral immunity to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. METHODS:Patients with MS aged 18 to 60 years were evaluated for anti-nucleocapsid and anti-Spike receptor-binding domain (RBD) antibody with electro-chemiluminescence immunoassay; antibody responses to Spike protein, RBD, N-terminal domain with multiepitope bead-based immunoassays (MBI); live virus immunofluorescence-based microneutralization assay; T-cell responses to SARS-CoV-2 Spike using TruCulture enzyme-linked immunosorbent assay (ELISA); and IL-2 and IFNγ ELISpot assays. Assay results were compared by DMT class. Spearman correlation and multivariate analyses were performed to examine associations between immunologic responses and infection severity. RESULTS:Between January 6, 2021, and July 21, 2021, 389 patients with MS were recruited (mean age 40.3 years; 74% women; 62% non-White). Most common DMTs were ocrelizumab (OCR)-40%; natalizumab -17%, Sphingosine 1-phosphate receptor (S1P) modulators -12%; and 15% untreated. One hundred seventy-seven patients (46%) had laboratory evidence of SARS-CoV-2 infection; 130 had symptomatic infection, and 47 were asymptomatic. Antibody responses were markedly attenuated in OCR compared with other groups (p ≤0.0001). T-cell responses (IFNγ) were decreased in S1P (p = 0.03), increased in natalizumab (p <0.001), and similar in other DMTs, including OCR. Cellular and humoral responses were moderately correlated in both OCR (r = 0.45, p = 0.0002) and non-OCR (r = 0.64, p <0.0001). Immune responses did not differ by race/ethnicity. Coronavirus disease 2019 (COVID-19) clinical course was mostly non-severe and similar across DMTs; 7% (9/130) were hospitalized. INTERPRETATION/CONCLUSIONS:DMTs had differential effects on humoral and cellular immune responses to SARS-CoV-2 infection. Immune responses did not correlate with COVID-19 clinical severity in this relatively young and nondisabled group of patients with MS. ANN NEUROL 2022.

BACKGROUND:Immune-related adverse events (irAEs) are common, clinically significant autoinflammatory toxicities observed with immune checkpoint inhibitors (ICI). Preexisting immune-mediated inflammatory disease (pre-IMID) is considered a relative contraindication to ICI due to the risk of inciting flares. Improved understanding of the risks and benefits of treating pre-IMID patients with ICI is needed. METHODS:We studied melanoma patients treated with ICI and enrolled in a prospective clinicopathological database. We compiled a list of 23 immune-mediated inflammatory diseases and evaluated their presence prior to ICI. We tested the associations between pre-IMID and progression-free survival (PFS), overall survival (OS), and irAEs. RESULTS:In total, 483 melanoma patients were included in the study; 74 had pre-IMID and 409 did not. In patients receiving ICI as a standard of care (SoC), pre-IMID was significantly associated with irAEs (p = 0.04) as well as improved PFS (p = 0.024) and OS (p = 0.007). There was no significant association between pre-IMID and irAEs (p = 0.54), PFS (p = 0.197), or OS (p = 0.746) in patients treated through a clinical trial. Pre-IMID was significantly associated with improved OS in females (p = 0.012), but not in males (p = 0.35). CONCLUSIONS:The dichotomy of the impact of pre-IMID on survival and irAEs in SoC versus clinical trial patients may reflect the inherit selection bias in patients accrued in clinical trials. Future mechanistic work is required to better understand the differences in outcomes between female and male pre-IMID patients. Our data challenge the notion that clinicians should avoid ICI in pre-IMID patients, although close monitoring and prospective clinical trials evaluating ICI in this population are warranted.

Objective: To examine antibody and T-cell responses to mRNAplatform COVID-19 vaccines in Ocrelizumab-treated MS patients over a 12-month period. Introduction: B-cell depletion with Ocrelizumab attenuates humoral responses to vaccines. The kinetics of humoral and cellular immune responses to COVID-19 vaccines in B-cell depleted MS patients has not been reported.
Method(s): VIOLA (NCT04843774) is an open-label, observational study enrolling 60 MS patients on Ocrelizumab from NYU and Rocky Mountain at the University of Colorado MS Centers. First vaccine dose occurred >=2 weeks after ocrelizumab infusion; second-dose >=8 weeks before the next infusion. Antibody responses to SARS-COV-2 spike proteins were assessed with Elecsys Anti-SARS-CoV-2 (Roche Diagnostics) and multiplex bead-based immunoassays. T-cell responses to SARS-CoV-2 Spike protein were assessed with IFNgamma ELISpot (Invitrogen) and TruCulture (Myriad RBM) and high-dimensional immunophenotyping. Samples are collected pre-vaccination and at 4, 12, 24, and 48-weeks post-vaccination.
Result(s): As of 7/15/2021, 52 subjects have been enrolled (39.7+/-10.0 years; 73% female; 47% non-white), of whom 47 were fully vaccinated (85% Pfizer, 15% Moderna). Anti-spike RBD antibody (Elecsys Anti-SARS-CoV-2) were available for pre- and post-vaccine timepoints for 15 patients. Pre-vaccine, 1/15 (7%) patients had detectable titers, while at 4-weeks postvaccine, 10/15 (66%) patients had detectible titers (mean for positives: 1189 U/ml; 5 patients had positive titers <25 U/ml). T-cell activation based on induced IFNgamma secretion (TruCulture) at baseline and 4-week post-vaccine timepoints were available for 13 patients, of whom 12 (92%) were increased (mean pre-vaccine: 24 pg/ml; mean post-vaccine: 366 pg/ml, two-tailed t-test, p=0.0032).
Conclusion(s): This prospective study of humoral and cellular immune responses to COVID-19 vaccines in Ocrelizumab-treated patients will generate data to help guide management of MS patients on anti-CD20 therapies. Early results suggest that 4-weeks post-vaccination nearly all Ocrelizumab-treated MS patients develop T-cell immunity and two-thirds showed evidence of humoral response. Additional 4-week and 12-week post-vaccination data will be presented

Objective: To compare humoral and T-cell responses to COVID- 19 vaccines in 400 MS patients who were on Ocrelizumab ('OCR') v. other disease-modifying therapies ('non-OCR') at the time of vaccination. Introduction: Peripheral B-cell depletion with anti-CD20 therapies attenuates humoral responses to vaccines. Whether immune responses to COVID-19 vaccines differ between B-cell depleted and non-B cell depleted MS patients is not known.
Method(s): Consecutive MS patients from NYU MS Care Center were invited to participate if they completed COVID-19 vaccination >=6 weeks previously. Immune testing included anti-spike RBD antibody (Elecsys Anti-SARS-CoV-2) (Roche Diagnostics); multiplex bead-based immunoassays of antibody-responses to SARS-COV-2 spike proteins; T-cell responses to SARS-CoV-2 Spike protein using IFNgamma enzyme-linked immune-absorbent spot (Invitrogen) and TruCulture (Myriad RBM) assays; high dimensional immunophenotyping; and live virus immunofluorescencebased microneutralization assay.
Result(s): As of 7/15/2021, 105 MS subjects were enrolled (mean age: 40.5 years; 76% female; 41% non-white; 38% on OCR; 12% with prior COVID-19 infection). 95% were fully vaccinated with mRNA vaccines (Pfizer/Moderna); 5% - with adenovirus-based vaccine (Johnson&Johnson). Median time from sample collection to last vaccine was 79 days. Positive Elecsys Anti-SARS-CoV-2 Ab titers post-vaccine were detected in 11/37 (30%) in OCR (mean level: 702 U/mL among seropositives) and 54/54 (100%) patients in non-OCR (mean level: 2310 U/mL; p<0.0001). Positive response by multiplex assay (threshold of 'positive' defined as 2 SD below the mean for the non-OCR) were detected in 10/27 (37%) OCR and 29/31 (94%) non-OCR (p<0.00001). T-cell activation based on induced IFNgamma secretion (TruCulture) was detected in 20/25 (80%) OCR and 16/19 (84%) non-OCR patients (p=0.71).
Conclusion(s): Preliminary results suggest robust T-cell immune response to SARS-CoV2 vaccines in approximately 80% of both OCR and non-OCR MS patients. Antibody responses were markedly attenuated in OCR compared to non-OCR group. Updated results will be presented

Advances in immune checkpoint therapy and targeted therapy have led to improvement in overall survival for patients with advanced melanoma. Single agent checkpoint PD-1 blockade and combination with BRAF/MEK targeted therapy demonstrated benefit in overall survival (OS). Superior response rates have been demonstrated with combined PD-1/CTLA-4 blockade, with a significant OS benefit compared with single-agent PD-1 blockade. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic therapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers but they have yet to be fully characterized and implemented clinically. Overall, the progress in melanoma therapeutics and translational research will help to optimize treatment regimens to overcome resistance and develop robust biomarkers to guide clinical decision-making. During the Melanoma Bridge meeting (December 3rd-5th, 2020, Italy) we reviewed the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine.

PURPOSE/OBJECTIVE:Immunotherapy has recently been shown to improve outcomes for advanced PD-L1-positive triple-negative breast cancer (TNBC) in the Impassion130 trial, leading to FDA approval of the first immune checkpoint inhibitor in combination with taxane chemotherapy. To further develop predictive biomarkers and improve therapeutic efficacy of the combination, interrogation of the tumor immune microenvironment before therapy as well as during each component of treatment is crucial. Here we use single-cell RNA sequencing (scRNA-seq) on tumor biopsies to assess immune cell changes from two patients with advanced TNBC treated in a prospective trial at predefined serial time points, before treatment, on taxane chemotherapy and on chemo-immunotherapy. METHODS:Both patients (one responder and one progressor) received the trial therapy, in cycle 1 nab-paclitaxel given as single agent, in cycle 2 nab-paclitaxel in combination with pembrolizumab. Tumor core biopsies were obtained at baseline, 3 weeks (after cycle 1, chemotherapy alone) and 6 weeks (after cycle 2, chemo-immunotherapy). Single-cell RNA sequencing (scRNA-seq) of both cancer cells and infiltrating immune cells isolated were performed from fresh tumor core biopsy specimens by 10 × chromium sequencing. RESULTS:). In contrast, tumors from the patient with rapid disease progression showed a prevalent and persistent myeloid compartment. CONCLUSIONS:Our study provides a deep cellular analysis of on-treatment changes during chemo-immunotherapy for advanced TNBC, demonstrating not only feasibility of single-cell analyses on serial tumor biopsies but also the heterogeneity of TNBC and differences in on-treatment changes in responder versus progressor.

T cell recognition of antigen and resulting proximal signaling are key steps in the initiation of the adaptive immune response. Previous studies targeting antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity. Recently, we used nuclear magnetic resonance (NMR) spectroscopy, mutational analysis and computational docking to derive a 3D structure of the extracellular TCRCD3 assembly. Further, biomolecular force probe (BFP) measurements allowed us to determine how 2D affinity and force-modulated TCR-pMHC kinetics depend on TCR-CD3 interaction sites and affect transduction of extracellular pMHC-TCR ligation into T cell function. Based on our TCR-CD3 structural model and binding data, we generated TCR libraries for a melanoma-specific TCR (DMF5) using site-specific mutagenesis in the Cbhelix 3 and helix 4-F strand regions of the TCR to optimize the TCR-CD3 interaction and to select for mutants with enhanced T-cell effector function. One Cb helix 4-F strand mutant, NP202203AA showed increased T cell response to antigen and showed enhanced TCR-pMHC bond lifetime (catch-bonds) in BFP assays leading to prolonged T cell signaling. In the future, DMF5 TCR with reengineered CD3 binding regions will be used in tumor rejection in pre-clinical mouse melanoma models for eficacy and toxicity to develop more effective T cell therapies for human targets

BACKGROUND:High C reactive protein (CRP) levels have been reported to be associated with a poor clinical outcome in a number of malignancies and with programmed cell death protein 1 immune checkpoint blockade in patients with advanced cancer. Little is known about the direct effects of CRP on adaptive immunity in cancer. Therefore, we investigated how CRP impacted the function of T cells and dendritic cells (DCs) from patients with melanoma. METHODS:The effects of CRP on proliferation, function, gene expression and phenotype of patient T cells and DCs, and expansion of MART-1 antigen-specific T cells were analyzed by multicolor flow cytometry and RNA-seq. Additionally, serum CRP levels at baseline from patients with metastatic melanoma treated on the Checkmate-064 clinical trial were assessed by a Luminex assay. RESULTS:In vitro, CRP inhibited proliferation, activation-associated phenotypes and the effector function of activated CD4+ and CD8+ T cells from patients with melanoma. CRP-treated T cells expressed high levels of interleukin-1β, which is known to enhance CRP production from the liver. CRP also suppressed formation of the immune synapse and inhibited early events in T-cell receptor engagement. In addition, CRP downregulated the expression of costimulatory molecules on mature DCs and suppressed expansion of MART-1-specific CD8+ T cells in a dose-dependent manner by impacting on both T cells and antigen-presenting cells. High-serum CRP levels at baseline were significantly associated with a shorter survival in both nivolumab-treated and ipilimumab-treated patients. CONCLUSIONS:in melanoma and support the blockade of CRP as a therapeutic strategy to enhance immune checkpoint therapies in cancer. TRIAL REGISTRATION NUMBER/BACKGROUND:NCT01783938 and NCT02983006.