With the successful development of immune checkpoint blockade, there remains the continued need to improve efficacy and decrease toxicities. The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to ipilimumab has previously demonstrated both an improvement in efficacy and decrease in the incidence of high-grade adverse events. ICOS+CD4+ or ICOS+CD8+ peripheral blood T cells are significantly greater in the patients treated with ipilimumab plus GM-CSF than in the patients treated with ipilimumab alone. To better understand the effects of GM-CSF on inducible T-cell costimulator (ICOS) and clinical outcomes, the relative roles of identified soluble ICOS and membrane-bound ICOS were evaluated. The ICOS splice variant was secreted and found to have immunologic suppressive effects. Changes in soluble ICOS splice variant levels in treated patients correlated with clinical outcomes. GM-CSF enhanced membrane-bound ICOS in an IL12-dependent manner but did not increase soluble ICOS levels. Whereas soluble ICOS plays a role in immune suppression, GM-CSF efficacy involves increasing membrane-bound ICOS and induction of dendritic cell development. Thus, soluble ICOS splice variants may be used as a biomarker for GM-CSF and immune checkpoint blockade-based therapies.

Although progression-based endpoints, such as progression-free survival, are often key clinical trial endpoints for anticancer agents, the clinical meaning of "objective progression" is much less certain. As scrutiny of progression-based endpoints in clinical trials increases, it should be remembered that the Response Evaluation Criteria In Solid Tumors (RECIST) progression criteria were not developed as a surrogate for survival. Now that progression-free survival has come to be an increasingly important trial endpoint, the criteria that define progression deserve critical evaluation to determine whether alternate definitions of progression might facilitate the development of stronger surrogate endpoints and more meaningful trial results. In this commentary, we review the genesis of the criteria for progression, highlight recent data that question their value as a marker of treatment failure, and advocate for several research strategies that could lay the groundwork for a clinically validated definition of disease progression in solid tumor oncology.

Background

Melanoma of unknown primary (MUP) represents a poorly understood group of patients both clinically and immunologically. We investigated differences in prognosis and candidate immune biomarkers in patients with unknown compared with those with known primary melanoma enrolled in the E1609 adjuvant trial that tested ipilimumab at 3 and 10 mg/kg vs high-dose interferon-alfa (HDI).

Patients and methods

MUP status was defined as initial presentation with cutaneous, nodal or distant metastasis without a known primary. Relapse-free survival (RFS) and overall survival (OS) rates were estimated by the Kaplan-Meier method. Stratified (by stage) log-rank test was used to compare RFS and OS by primary tumor status. Gene expression profiling (GEP) was performed on the tumor biopsies of a subset of patients. Similarly, peripheral blood samples were tested for candidate soluble and cellular immune biomarkers.

Results

MUP cases represented 12.8% of the total population (N=1699) including 11.7% on the ipilimumab arms and 14.7% on the HDI arm. Stratifying by stage, RFS (p=0.001) and overall survival (OS) (p=0.009) showed outcomes significantly better for patients with unknown primary. The primary tumor status remained prognostically significant after adjusting for treatment and stage in multivariate Cox proportional hazards models. Including only ipilimumab-treated patients, RFS (p=0.005) and OS (p=0.023) were significantly better in favor of those with unknown primary. Among patients with GEP data (n=718; 102 MUP, 616 known), GEP identified pathways and genes related to autoimmunity, inflammation, immune cell infiltration and immune activation that were significantly enriched in the MUP tumors compared with known primaries. Further investigation into infiltrating immune cell types estimated significant enrichment with CD8 +and CD4+T cells, B cells and NK cells as well as significantly higher major histocompatibility complex (MHC)-I and MHC-II scores in MUP compared with known primary. Among patients tested for circulating biomarkers (n=321; 66 unknown and 255 known), patients with MUP had significantly higher circulating levels of IL-2R (p=0.04).

Conclusion

Patients with MUP and high-risk melanoma had significantly better prognosis and evidence of significantly enhanced immune activation within the TME and the circulation, supporting the designation of MUP as a distinct prognostic marker in patients with high-risk melanoma.

Background

Mucosal melanoma is an aggressive melanoma with poor prognosis. We assessed efficacy of pembrolizumab in patients with advanced mucosal melanoma in KEYNOTE-001 (NCT01295827), -002 (NCT01704287), and -006 (NCT01866319).

Methods

Patients received pembrolizumab 2 mg/kg every 3 weeks (Q3W) or 10 mg/kg Q2W or Q3W. Response was assessed by independent central review per RECIST v1.1.

Results

1567 patients were treated and 84 (5%) had mucosal melanoma. Fifty-one of 84 were ipilimumab-naive. In patients with mucosal melanoma, the objective response rate (ORR) was 19% (95% CI 11-29%), with median duration of response (DOR) of 27.6 months (range 1.1 + to 27.6). Median progression-free survival (PFS) was 2.8 months (95% CI 2.7-2.8), with median overall survival (OS) of 11.3 months (7.7-16.6). ORR was 22% (95% CI 11-35%) and 15% (95% CI 5-32%) in ipilimumab-naive and ipilimumab-treated patients.

Conclusion

Pembrolizumab provides durable antitumour activity in patients with advanced mucosal melanoma regardless of prior ipilimumab.

KEYNOTE-030 (ClinicalTrials.gov ID, NCT02083484) was a global expanded access program that allowed access to pembrolizumab, an antiprogrammed death 1 antibody, for patients with advanced melanoma before its regulatory approval. Patients with unresectable stage III/IV melanoma that progressed after standard-of-care therapy, including ipilimumab and, if BRAF mutant, a BRAF inhibitor, were eligible to receive pembrolizumab 2 mg/kg every 3 weeks. Response was assessed by immune-related response criteria by investigator review. Adverse events (AEs) were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. In the United States, 979 patients enrolled between April and September 2014. Of the 947 evaluable patients, 621 (65.6%) remained on treatment and transitioned to receive commercial pembrolizumab following approval by the Food and Drug Administration, whereas 326 (34.4%) discontinued, most commonly for disease progression (39.6%) or death (26.4%). Objective response rate was 14.5% (95% confidence interval, 12.2%-16.8%) in the treated population (n=947) and 22.1% (95% confidence interval, 18.8%-25.5%) in patients who had ≥1 response assessment reported (n=619). Twelve patients achieved complete response. One hundred eighty-one (19.1%) patients experienced ≥1 treatment-related AE, most commonly general disorders (8.0%), skin/subcutaneous tissue disorders (7.3%), and gastrointestinal disorders (6.4%); 29 (3.1%) patients experienced ≥1 grade 3/4 treatment-related AE. Immune-mediated AEs were also reported. There were no treatment-related deaths. The safety and efficacy observed in this expanded access program were consistent with those previously reported for similar populations and support the use of pembrolizumab for patients with advanced melanoma.

Oral leukoplakia is common and may, in some cases, progress to carcinoma. Proliferative leukoplakia is a progressive, often multifocal subtype with a high rate of malignant transformation compared with the more common localized leukoplakia. We hypothesized that the immune microenvironment and gene expression patterns would be distinct for proliferative leukoplakia compared with localized leukoplakia. We summarize key clinicopathologic features among proliferative leukoplakia and localized leukoplakia and compare cancer-free survival (CFS) between subgroups. We analyze immunologic gene expression profiling in proliferative leukoplakia and localized leukoplakia tissue samples (NanoString PanCancer Immune Oncology Profiling). We integrate immune cell activation and spatial distribution patterns in tissue samples using multiplexed immunofluorescence and digital image capture to further define proliferative leukoplakia and localized leukoplakia. Among N = 58 patients (proliferative leukoplakia, n = 29; localized leukoplakia, n = 29), only the clinical diagnosis of proliferative leukoplakia was associated with significantly decreased CFS (HR, 11.25; P < 0.01; 5-year CFS 46.8% and 83.6% among patients with proliferative leukoplakia and localized leukoplakia, respectively). CD8⁺ T cells and T regulatory (Treg) were more abundant among proliferative leukoplakia samples (P < 0.01) regardless of degree of epithelial dysplasia, and often colocalized to the dysplasia-stromal interface. Gene set analysis identified granzyme M as the most differentially expressed gene favoring the proliferative leukoplakia subgroup (log₂ fold change, 1.93; P _adj < 0.001). Programmed death ligand 1 (PD-L1) was comparatively overexpressed among proliferative leukoplakia samples, with higher (>5) PD-L1 scores predicting worse CFS (P _adj < 0.01). Proliferative leukoplakia predicts a high rate of malignant transformation within 5 years of diagnosis. A prominent CD8⁺ T-cell and Treg signature along with relative PD-L1 overexpression compared with localized leukoplakia provides strong rationale for PD-1/PD-L1 axis blockade using preventative immunotherapy.

Significance

This is the first in-depth profiling effort to immunologically characterize high-risk proliferative leukoplakia as compared with the more common localized leukoplakia. We observed a notable cytotoxic T-cell and Treg signature with relative overexpression of PD-L1 in high-risk proliferative leukoplakia providing a strong preclinical rationale for investigating PD-1/PD-L1 axis blockade in this disease as preventative immunotherapy.

Oncogene-targeted therapy with B-Raf proto-oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors induces a high initial response rate in patients with BRAF^V600-mutated melanoma, with a median duration of response of approximately 1 year^1-3. Immunotherapy with antibodies to programmed death 1 (PD-1) produces lower response rates but with long response duration. Preclinical models suggest that combining BRAF and MEK inhibitors with PD-1 blockade therapy improves antitumor activity^4-6, which may provide additional treatment options for patients unlikely to have long-lasting responses to either mode of therapy alone. We enrolled 15 patients with BRAF^V600-mutated metastatic melanoma in a first-in-human clinical trial of dabrafenib, trametinib and pembrolizumab ( NCT02130466 ). Eleven patients (73%) experienced grade 3/4 treatment-related adverse events, the most common being elevation of liver function tests and pyrexia, most of which resolved with drug interruption or discontinuation of either the anti-PD-1 antibody or the targeted therapy combination. Eleven patients (73%; 95% confidence interval = 45-92%) had an objective response, and six (40%; 95% confidence interval = 16-68%) continued with a response at a median follow-up of 27 months (range = 10.3-38.4+ months) for all patients. This study suggests that this triple-combined therapy may benefit a subset of patients with BRAF^V600-mutated metastatic melanoma by increasing the frequency of long-lasting antitumor responses.

[This corrects the article DOI: 10.1158/2767-9764.CRC-21-0060.][This corrects the article DOI: 10.1158/2767-9764.CRC-21-0060.].

Background

We hypothesized that a gender difference in clinical response may exist to adjuvant CTLA4 blockade with ipilimumab versus high-dose IFNα (HDI). We investigated differences in candidate immune biomarkers in the circulation and tumor microenvironment (TME).

Patients and methods

This gender-based analysis was nested within the E1609 trial that tested adjuvant therapy with ipilimumab 3 mg/kg (ipi3) and 10 mg/kg (ipi10) versus HDI in high risk resected melanoma. We investigated gender differences in treatment efficacy with ipi3 and ipi10 versus HDI while adjusting for age, stage, ECOG performance (PS), ulceration, primary tumor status and lymph node number. Forest plots were created to compare overall survival (OS) and relapse free survival (RFS) between ipi and HDI. Gene expression profiling (GEP) was performed on tumors of 718 (454 male, 264 female) patients. Similarly, serum and peripheral blood mononuclear cells (PBMC) samples were tested for soluble and cellular biomarkers (N = 321 patients; 109 female and 212 male).

Results

The subgroups of female, stage IIIC, PS = 1, ulcerated primary, in-transit metastasis demonstrated significant improvement in RFS and/or OS with ipi3 versus HDI. Female gender was significant for both OS and RFS and was further explored. In the RFS comparison, a multivariate Cox regression model including significant variables indicated a significant interaction between gender and treatment (P = 0.024). In peripheral blood, percentages of CD3+ T cells (P = 0.024) and CD3+ CD4+ helper T cells (P = 0.0001) were higher in females compared to males. Trends toward higher circulating levels of IL1β (P = 0.07) and IL6 (P = 0.06) were also found in females. Males had higher percentages of monocytes (P = 0.03) with trends toward higher percentages of regulatory T cells (T-reg). Tumor GEP analysis supported enhanced infiltration with immune cells including gammadelta T cells (P = 0.005), NK cells (P = 0.01), dendritic cells (P = 0.01), CD4+ T cells (P = 0.03), CD8+ T cells (P = 0.03) and T-reg (P = 0.008) in the tumors of females compared to males and a higher T-effector and IFNγ gene signature score (P = 0.0244).

Conclusion

Female gender was associated with adjuvant CTLA4 blockade clinical benefits and female patients were more likely to have evidence of type1 immune activation within the TME and the circulation. Trial registration ClinicalTrials.gov NCT01274338. Registered 11 January 2011, https://www.

Clinicaltrials

gov/ct2/show/NCT01274338.

Purpose Expression of programmed death-ligand 1 (PD-L1) is a potential predictive marker for response and outcome after treatment with anti-programmed death 1 (PD-1). This study explored the relationship between anti-PD-1 activity and PD-L1 expression in patients with advanced melanoma who were treated with pembrolizumab in the phase Ib KEYNOTE-001 study (clinical trial information: NCT01295827). Patients and Methods Six hundred fifty-five patients received pembrolizumab10 mg/kg once every 2 weeks or once every 3 weeks, or 2 mg/kg once every 3 weeks. Tumor response was assessed every 12 weeks per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 by independent central review. Primary outcome was objective response rate. Secondary outcomes included progression-free survival (PFS) and overall survival (OS). Membranous PD-L1 expression in tumor and tumor-associated immune cells was assessed by a clinical trial immunohistochemistry assay (22C3 antibody) and scored on a unique melanoma (MEL) scale of 0 to 5 by one of three pathologists who were blinded to clinical outcome; a score ≥ 2 (membranous staining in ≥ 1% of cells) was considered positive. Results Of 451 patients with evaluable PD-L1 expression, 344 (76%) had PD-L1-positive tumors. Demographic and staging variables were equally distributed among PD-L1-positive and -negative patients. An association between higher MEL score and higher response rate and longer PFS (hazard ratio, 0.76; 95% CI, 0.71 to 0.82) and OS (hazard ratio, 0.76; 95% CI, 0.69 to 0.83) was observed ( P < .001 for each). Objective response rate was 8%, 12%, 22%, 43%, 57%, and 53% for MEL 0, 1, 2, 3, 4, and 5, respectively. Conclusion PD-L1 expression in pretreatment tumor biopsy samples was correlated with response rate, PFS, and OS; however, patients with PD-L1-negative tumors may also achieve durable responses.