Faculty Bibliography

Phenotyping of immune cell subsets in clinical trials is limited to well-defined phenotypes, due to technological limitations of reporting flow cytometry multi-dimensional phenotyping data. We developed a multi-dimensional phenotyping analysis tool and applied it to detect nitric oxide (NO) levels in peripheral blood immune cells before and after adjuvant ipilimumab co-administration with a peptide vaccine in melanoma patients. We analyzed inhibitory and stimulatory markers for immune cell phenotypes that were felt to be important in the NO analysis. The pipeline allows visualization of immune cell phenotypes without knowledge of clustering techniques and to categorize cells by association with relapse-free survival (RFS). Using this analysis, we uncovered the potential for a dichotomous role of NO as a pro- and anti-melanoma factor. NO was found in subsets of immune-suppressor cells associated with shorter-term (≤ 1 year) RFS, whereas NO was also present in immune-stimulatory effector cells obtained from patients with significant longer-term (> 1 year) RFS. These studies provide insights into the cell-specific immunomodulatory role of NO. The methods presented herein can be applied to monitor the pro- and anti-tumor effects of a variety of immune-based therapeutics in cancer patients. Clinical Trial Registration Number: NCT00084656 (https://clinicaltrials.gov/ct2/show/NCT00084656).

The impact on survival of steroids and TNF-alpha blockade to treat immune-related toxicity from checkpoint blockade with ipilimumab, nivolumab/pembrolizumab or combined ipilimumab and nivolumab was assessed using data from a large national database. Using steroids was associated with better survival than the use of TNF-alpha blocking antibodies such as infliximab.

BACKGROUND:The American Joint Committee on Cancer (AJCC) maintains that the eighth edition of its Staging Manual (AJCC8) has improved accuracy compared to the seventh (AJCC7). However, there are concerns that implementation may disrupt analysis of active clinical trials for stage III patients. We used an independent cohort of melanoma patients to test the extent to which AJCC8 has improved prognostic accuracy compared to AJCC7. METHODS:We analyzed a cohort of 1,315 prospectively enrolled patients. We assessed primary tumor and nodal classification of stage I-III patients using AJCC7 and AJCC8 to assign disease stages at diagnosis. We compared recurrence-free (RFS) and overall survival (OS) using Kaplan-Meier curves and log-rank tests. We then compared concordance indices of discriminatory prognostic ability and area under the curve (AUC) of 5-year survival to predict RFS/OS. All statistical tests were two-sided. RESULTS:Stage IIC continued to have worse outcomes than those for stage IIIA patients, with 5-year RFS of 26.5% (95%CI=12.8-55.1%) vs. 56.0% (95%CI=37.0-84.7%) by AJCC8 (P = 0.002). For stage I, removing mitotic index as T classification factor decreased its prognostic value, although not statistically significantly (RFS C-index=0.63 [95%CI=0.56-0.69] to 0.56 [95%CI=0.49-0.63], P = 0.07; OS C-index=0.48 [95%CI=0.38-0.58] to 0.48 [95%CI=0.41-0.56], P = 0.90). For stage II, prognostication remained constant (RFS C-index=0.65 [95%CI=0.57-0.72]; OS C-index=0.61 [95%CI=0.50-0.72]), and. For stage III, AJCC8 yielded statistically significantly enhanced prognostication for RFS (C-index=0.65 [95%CI=0.60-0.70] to 0.70 [95%CI=0.66-0.75], P = 0.01). CONCLUSIONS:Compared with AJCC7, we demonstrate that AJCC8 enables more accurate prognosis for patients with stage III melanoma. Restaging a large cohort of patients can enhance the analysis of active clinical trials.

BACKGROUND:Pembrolizumab demonstrated robust antitumor activity and safety in the phase Ib KEYNOTE-001 study (NCT01295827) of advanced melanoma. Five-year outcomes in all patients and treatment-naive patients are reported herein. Patients whose disease progressed following initial response and who received a second course of pembrolizumab were also analyzed. PATIENTS AND METHODS:Patients aged ≥18 years with previously treated or treatment-naive advanced/metastatic melanoma received pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks until disease progression, intolerable toxicity, or patient/investigator decision to withdraw. Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS) were calculated. Objective response rate and PFS were based on immune-related response criteria by investigator assessment (data cut-off, September 1, 2017). RESULTS:KEYNOTE-001 enrolled 655 patients with melanoma; median follow-up was 55 months. Estimated 5-year OS was 34% in all patients and 41% in treatment-naive patients; median OS was 23.8 months (95% CI, 20.2-30.4) and 38.6 months (95% CI, 27.2-not reached), respectively. Estimated 5-year PFS rates were 21% in all patients and 29% in treatment-naive patients; median PFS was 8.3 months (95% CI, 5.8-11.1) and 16.9 months (95% CI, 9.3-35.5), respectively. Median response duration was not reached; 73% of all responses and 82% of treatment-naive responses were ongoing at data cut-off; the longest response was ongoing at 66 months. Four patients [all with prior response of complete response (CR)] whose disease progressed during observation subsequently received second-course pembrolizumab. One patient each achieved CR and partial response (after data cut-off). Treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8%; 17% experienced grade 3/4 TRAE. CONCLUSIONS:This 5-year analysis of KEYNOTE-001 represents the longest follow-up for pembrolizumab to date and confirms the durable antitumor activity and tolerability of pembrolizumab in advanced melanoma. CLINICAL TRIAL REGISTRY:ClinicalTrials.gov, NCT01295827.

Background: The phase III study CheckMate 238 demonstrated improved relapse-free survival (RFS) with NIVO 3 mg/kg vs IPI 10 mg/kg in patients (pts) with resected stage III/IV melanoma. Sustained efficacy benefit at 24 mo of follow-up with NIVO vs IPI was previously reported. Here we present a 36mo analysis of efficacy and biomarker data from this study.
Method(s): Pts aged >= 15 y with completely resected stage IIIB/C or IV melanoma were randomized 1:1 to receive NIVO (3 mg/kg Q2W; N=453) or IPI (10 mg/kg Q3W for 4 doses; Q12W thereafter; N=453) for <= 1 y or until disease recurrence/unacceptable toxicity. RFS was the primary endpoint; exploratory endpoints included distant metastases-free survival (DMFS) in pts with stage III disease and potential biomarkers of efficacy.
Result(s): With 36mo of follow-up, NIVO continued to demonstrate superior RFS vs IPI (HR, 0.68; P<0.0001; 3-y RFS rates, 58%vs 45%and 188/453 vs 239/453 pts with events, respectively). Prespecified subgroup analyses demonstrated a consistent pattern similar to that of the 24-mo analysis, with HRs favoring NIVO (Table). DMFS analysis also continued to favor NIVO (Table). High levels of all biomarkers analyzed (interferon-gamma gene expression signature, tumor mutational burden, and CD8+ T-cell infiltration by immunohistochemistry) showed an association with improved RFS for both NIVO and IPI.Median RFS (mo; 24-mo follow-up) by high vs low values for each biomarker for NIVO was 30.8 vs 24.1, not reached (NR) vs 30.8, and 30.8 vs 24.9, respectively; and for IPI was NR vs 15.9, NR vs 18.3, and NR vs 13.8, respectively.
Conclusion(s): With 36 mo of follow-up, NIVO continued to demonstrate superior efficacy over IPI in pts with stage III/IV melanoma at high risk of recurrence across pt subgroups. Additional analyses using composite scoring of biomarker combinations will be presented

Background: Tumor-specific indicators, such as tumor mutation burden (TMB) have been shown to affect overall survival (OS) in melanoma. Recently, pan-cancer analyses from The Cancer Genome Atlas (TCGA) discovered specific tumor immune signatures predictive of overall survival (OS), yet it is unclear how these interact with other prognostic markers, independently of immunotherapy (IT). We aimed to combine the immune landscape signatures with TMB and other prognostic markers to improve melanoma OS prediction in patients, independent of IT.
Method(s): We examined the whole-exome data in conjunction with the molecular, clinical and immune features from 278 metastatic melanomas from TCGA, not treated by IT, to develop an improved prognostic model of melanoma OS. Using the discovery (N=139) and validation (N=139) design we performed multivariate Cox proportional hazards (Cox HR) models, adjusted for age and tumor stage at primary diagnosis, to identify interaction between TMB and melanoma immune features (n=59), refining the prediction of melanoma OS.
Result(s): We identified 4 immune features that were significantly associated with OS in both the discovery and validation cohorts. The multivariate Cox HR models revealed that IFN-c response (IFN-c R) and macrophage regulation (MR) signatures in combination with TMB were the most significantly associated with OS (p=8.80E-14). After further refinement, we observed that patients with high TMB, high IFN-c R and high MRhad significantly better OS compared to high TMB, low IFN-y R and low MR (HR=2.8, p=3.55E-08). This association was not observed in low TMB patients.
Conclusion(s): We show, for the first time, that TMB and tumor immune features are significantly associated with improved OS, independent of IT. Further analysis of patients revealed that high TMB associates with improved OS in patients with high IFN- c R and MR but not in low IFN- c R and MR. Hence, this data provides first evidence that patients with high TMB have distinct OS outcome depending on other tumor immune features. Beside biological link between TMB and IFN-y and MR, our data suggest that these associations may significantly improve the current melanoma prognostic models

Background: Peripheral blood T cell and myeloid-derived suppressor cells (MDSC), as well as myeloid and macrophage subsets, have been associated with a poor clinical outcome in a variety of cancers. We analyzed circulating cells from patients (pts) that received either nivolumab (NIVO) then ipilimumab (IPI) (cohort A, 16 pts) or IPI then NIVO (cohort B, 17 pts) in a randomized clinical trial to determine if peripheral blood phenotypes were associated either at baseline or on treatment with outcome. Method(s): Frozen peripheral blood mononuclear cells (PBMC) from the ChekMate 064 study were assessed at baseline and on treatment at week 13 for circulating cell subsets by 28-color, high-dimensional flow cytometry with CytoBrute, a rapid computational platform that performs high-parameter Boolean analysis. Correlations with response and survival as well as toxicity were evaluated using the machine learning algorithm ElasticNet. Result(s): In cohort B pts the frequency of resting Ki67-, long-lived memory CD45+/ CD45RO+/CD127+ T cells was reduced (p=0.005), and dividing Ki-67+ CD4+/ CD45RO+/CD95+ T cells susceptible to apoptosis were increased after IPI (p=0.007), but were associated at baseline with a poor outcome with cohort A (p=0.0002). Subsets of myeloid cells that were CD66b+/CD33+/41-BB+/CD86+ at baseline were associated with survival in cohort B (p=0.0006). A macrophage subset that was PD-L2+/CD163+/41-BBL+/CD40+ was associated with survival for cohort A (p=0.0001). Additional phenotypes were associated with grade 1 compared with grades 2-4 toxicity that differentiated side effects from either IPI or NIVO, and other phenotypes distinguished normals and pts (AUC=0.96). Conclusion(s): A circulating CD4+/CD45RO+/CD95+ proliferating memory T cell phenotype signature is augmented after IPI and is associated at baseline with poor survival with NIVO in CheckMate 064. We discriminated pts and healthy controls with great specificity and sensitivity at baseline, and demonstrated new phenotypes associated with immune-related toxicity. Peripheral blood immune monitoring may be of value in selecting melanoma pts to be treated with immunotherapy

Background: Melanoma that metastasizes to the brain has a poor prognosis, and accounts for up to 54% of melanoma deaths. Clinical data show that treatment with BRAF-targeted agents induces responses in RAFV600-mutant MBM. Duration of response in the brain is shorter than that observed with extracranial disease. The BRAF/MEK-targeted combination encorafenib +binimetinib demonstrated favorable efficacy and safety for patients with BRAF^V600- mutant melanoma in the COLUMBUS study, but excluded patients with active MBM. The aim of this study is to evaluate encorafenib + binimetinib in a population of patients with BRAF^V600-mutated active MBM. A higher dose of combination therapy will be studied versus a standard dose to evaluate whether greater efficacy may be achieved with acceptable safety for patients with BRAF^V600 MBM. Trial design: This multicenter, randomized, open-label phase II study will evaluate two dosing regimens of encorafenib + binimetinib combination in adults with BRAF^V600- mutant MBM. Eligible patients will have at least 1 measurable MBM, no prior local MBMtherapy, no corticosteroids for MBM, and no prior BRAF or MEK inhibitors in the metastatic setting. One prior line of checkpoint inhibitor or prior adjuvant BRAF or MEK inhibitors is permitted. Patients will be randomized (1:1) to either the standard dose (450 mg orally QD and binimetinib 45 mg orally BID) or high-dose (encorafenib 300 mg BID and binimetinib 45 mg BID) stratified by baseline tumor burden (1 to 2 brain lesions vs. >= 3 brain lesions at baseline) and by prior checkpoint inhibitor (yes vs. no). The first 9 evaluable patients in the high-dose arm will constitute the safety lead-in cohort. If the high-dose is not tolerated, subsequent patients will receive standard-dose therapy. Assessments include intracranial response (assessed as per modified RECIST using gadolinium enhanced MRI), extracranial response, global response rate, DCR, DOR, PFS, OS, PK, and safety. The study will enroll approximately 100 patients

Background: A goal of adjuvant therapy is to prolong disease-free survival, with the risk of mortality approaching that of the general population. From the literature, recurrences in stage III melanoma are most likely to occur within 3 years after surgery. Mixture-cure models (MCMs) capture survival heterogeneity of cancer patients by assuming they are either "cured" or "uncured", where cured patients are defined as having a low risk of relapse. Method(s): We applied MCMs to 3-year recurrence-free survival (RFS) outcomes in patients with resected melanoma treated with adjuvant nivolumab (NIVO) or ipilimumab (IPI) in the double-blind, phase III CheckMate 238 trial (NCT02388906) and assumed that the cured fraction has mortality risks similar to that of the general population. Patient age, sex, and geographic region information from CheckMate 238 and mortality rates from the World Health Organization were used to derive a cohort-level background survival distribution representative of "cured" or "disease-free" patients. Parametric functions were used to model uncured population outcomes. Parameters of theMCMwere estimated by means of maximum likelihood methods. Statistical goodness- of-fit metrics and visual inspection showed that generalized gamma and log-logistic distributions were best suited for survival analyses of cured and uncured patients, respectively. Result(s): The 3-year RFS rates were explained by the fraction of patients with a low risk of relapse in the NIVO arm at 55.1% (95% CI, 49.2-60.1) and IPI arm at 39.9% (95% CI, 33.6-46.1). Sensitivity analyses indicated that the estimated fraction of patients with a low risk of relapse was > 50% in the NIVO arm. Estimated RFS distributions of the uncured patients exhibited similar patterns for both arms, implying the differences in RFS rates between treatment arms can be explained with the elicited differences in proportions of patients at high vs low risk of relapse. Conclusion(s):MCManalyses from CheckMate 238 suggested that adjuvant treatment with NIVO leads to a higher proportion of patients with low risk of relapse compared with IPI. Validation of the results from MCMs will include analyses of longitudinal RFS and overall survival data from the EORTC 18071 trial