Faculty Bibliography

Blockade of the co-inhibitory receptor PD-1 has had unprecedented success in the treatment of metastatic melanoma, with response rates of approximately 40% in previously untreated patients. However, since the majority of patients fail to respond, a better understanding of the mechanisms of action involved in response/resistance and novel approaches to overcome resistance are needed. We evaluated T-cells present in peripheral blood samples of resected stage III/IV melanoma patients treated with adjuvant nivolumab. Data from intra-patient, paired pre-treatment/post-treatment samples showed a significant (p<0.05) increase in phosphorylated STAT3 (pSTAT3) in patients without relapse, but not in patients relapsing. Increases in regulatory T-cells (Tregs) (p<0.05) in relapsers, but not in non-relapsers, and OX40 expression on Tregs (p<0.05) in both relapsers and non-relapsers were also observed. Based on these data we evaluated the ability of PD-1 blocking antibodies to induce pSTAT3 signaling in vitro. Culturing with anti-PD-1 significantly increased levels of pSTAT3 in T-cells, including Tregs, both at the basal state (p<0.05) and post-CD3/CD28 activation (p<0.01). Additionally, culturing Tcells with PD-1 blocking antibodies increased the production of the STAT3 regulated cytokine IL-10 (p<0.05), the number of Tregs (p<0.01) and Treg expression of OX40 (p<0.05). Increases in all these markers/populations were abrogated with the addition of a STAT3 inhibitor (p<0.01). Similar increases in the number of Tregs and the expression of OX40 were also found when T-cells were treated in vitro with recombinant IL-10. Addition of IL-10 neutralizing antibodies ameliorated upregulation of Treg numbers and OX40 expression resulting from PD-1 blockade. Additionally, serum levels of IL-10 were found to be significantly elevated in non-relapsing patients relative to relapsers (p<0.05). RNA-seq analysis comparing Tregs from relapsers to non-relapsers further supported the observed increase in Tregs resulting from nivolumab treatment, with pathway analyses demonstrating significantly increased cell cycle, DNA replication, mitosis and other proliferation related pathways in Tregs from non-relapsed patients. Next we evaluated changes in the function of patient Tregs in an allogenic mixed lymphocyte suppression assay. Tregs from non-relapsers had a significant reduction in suppressive capacity post-nivolumab treatment in paired analyses (p<0.05), while Tregs from relapsers had no significant change. Post-treatment samples from relapses were also found to be significantly more suppressive (p<0.01) than those of non-relapsers. Additionally, decreased percentages of CCR4+CD45RA+ Tregs, previously described as having enhanced suppressive capacity, were found in non-relapsers post-treatment, but not in relapsers. A similar decrease in CCR4+CD45RA+ Tregs was achieved in vitro using OX40 agonist antibodies. Therefore, we propose that PD-1 blockade triggers STAT3 signaling, a hereunto unknown effect. These data support a model in which STAT3 induction by PD-1 blockade results in IL-10 production, which induces proliferation of Tregs with reduced suppressive capacity and triggers OX40 expression. Based on these results and previous findings of others demonstrating that ligation of OX40 expressed on Tregs hinders suppressive capacity, combining OX40 agonist antibodies may enhance the effectiveness of PD-1 blockade by reducing Treg suppression

Immunotherapy strategies for the treatment of melanoma have achieved impressive clinical outcomes over the past decade. Response rates to checkpoint blockade by PD-1 and CTLA-4 antibodies range from 15-40%, while in adoptive cell therapy using tumor infiltrating lymphocytes (TILs), anti-tumor response is observed in approximately 50%. However, the need to improve immunotherapies is evident as the majority of patients are unresponsive to treatment. Dysfunctional T-cells are thought to contribute to failed responses to checkpoint inhibition. As such we sought to investigate the ability of drugs targeting the epigenetic regulatory machinery as a means to alter T-cell function(s) and improve the anti-melanoma response. Here we demonstrate that the HDAC6 selective inhibitor ACY1215 disrupts mTORC signaling pathways in T-cells obtained from melanoma patients. Phosphorylation of mTOR, RAPTOR and the downstream molecules AKT, SGK1, PKCa and S6K were reduced on CD4 and CD8⁺ T-cells after ACY1215 in vitro treatment (p<0.05). The levels of the Th2 cytokines IL-4, IL-6, IL-10 generated by ACY1215-treated T-cells (p<0.05) were also decreased. Similar results were achieved with an SGK1 inhibitor, in agreement with published data demonstrating SGK1 as a regulator of Th2 polarization. Since the mTOR/RAPTOR complex is known to be involved in determining T regulatory (Treg) function, the effects of ACY1215 on Tregs were evaluated. Treatment in vitro with ACY1215 decreased phosphorylated mTOR and RAPTOR in Tregs, and reduced the levels of FOXP3. In a functional suppression assay, ACY1215-treated Tregs displayed a reduced ability to impair proliferation of effector T-cells (Teff) compared to control (DMSO: 10% vs ACY1215: 25% Teff proliferation, p<0.05). To explore whether HDAC inhibition during expansion of tumor infiltrating lymphocytes (TIL) for adoptive transfer would improve their quality and anti-tumor reactivity, TIL isolated from melanoma surgical biopsies were cultured in vitro with IL-2 and ACY1215. Treatment with ACY1215 led to an accumulation of central memory CD4 and CD8⁺ TILs (p<0.01 and p<0.05, respectively), which was maintained even after rapid expansion with anti-CD3 and anti-CD28 stimulation in vitro. Similarly, ACY1215 treatment of T-cells derived from peripheral blood of melanoma patients and healthy donors also displayed an increased central memory phenotype, characterized by expression of CD45RO, CD62L and CCR7 (p<0.05). Inhibition of AKT has been shown to increase T-cells with memory characteristics, and the use of an AKT inhibitor also resulted in accumulation of central memory T-cells. Confirming the observed phenotypic changes, microarray analysis of ACY1215-treated TILs revealed up-regulation of genes associated with a T-cell central memory and inflammatory response (e.g. SELL, LEF1, TNFRSF9) and downregulation of genes associated with Treg function (e.g. LGMN, CXCL8). Collectively these data suggest that reprogramming T-cells with epigenetic modulators may improve melanoma immunotherapy by reducing Treg suppression and production of immunosuppressive cytokines, while favoring generation of central memory T-cells

: Immune checkpoint inhibitors have emerged as a mainstay of melanoma therapy and are playing an increasingly important role in the treatment of other tumor types. The clinical benefit afforded by these treatments can be accompanied by a unique spectrum of adverse events, called immune-related adverse events (irAEs), which reflect the drug's immune-based mechanism of action. IrAEs typically originate in the skin, gastrointestinal tract, liver, and endocrine system, although other organ systems may also be affected. This article provides an overview of irAEs associated with anti-programmed death-1 (anti-PD-1) antibodies (nivolumab and pembrolizumab) as monotherapy or in combination with anti-cytotoxic T-lymphocyte antigen-4 inhibition (ipilimumab), followed by a discussion of irAEs of special clinical interest based on the potential for morbidity, frequent steroid use, and inpatient admission. We review clinical trial data and provide recommendations on how to manage irAEs associated with anti-PD-1 agents based on clinical experience and established management guidelines. We further illustrate the practical considerations of managing irAEs by presenting three cases of immune-related toxicity in melanoma patients treated with nivolumab or pembrolizumab. A better understanding of the identification and management of irAEs will help inform health care providers about the risks associated with anti-PD-1 treatment, to ensure the safe and appropriate use of these important new treatments. IMPLICATIONS FOR PRACTICE: Immune checkpoint inhibitors have demonstrated significant clinical benefit in advanced melanoma and other tumor types. These treatments are associated with immune-related adverse events (irAEs), which most commonly affect the skin and gastrointestinal tract, and, to a lesser extent, the liver, endocrine system, and other organs. This review focuses on the management of irAEs after treatment with anti-programmed death-1 (anti-PD-1) antibodies (nivolumab or pembrolizumab) as monotherapy or in combination with anti-cytotoxic T-lymphocyte antigen-4 inhibition (ipilimumab) in patients with advanced melanoma. A better understanding of the management of irAEs will help ensure the safe and appropriate use of anti-PD-1 agents in melanoma and other tumor types.

BACKGROUND: Concurrent administration of the immune checkpoint inhibitors nivolumab and ipilimumab has shown greater efficacy than either agent alone in patients with advanced melanoma, albeit with more high-grade adverse events. We assessed whether sequential administration of nivolumab followed by ipilimumab, or the reverse sequence, could improve safety without compromising efficacy. METHODS: We did this randomised, open-label, phase 2 study at nine academic medical centres in the USA. Eligible patients (aged >/=18 years) with unresectable stage III or IV melanoma (treatment-naive or who had progressed after no more than one previous systemic therapy, with an Eastern Cooperative Oncology Group performance status of 0 or 1) were randomly assigned (1:1) to induction with intravenous nivolumab 3 mg/kg every 2 weeks for six doses followed by a planned switch to intravenous ipilimumab 3 mg/kg every 3 weeks for four doses, or the reverse sequence. Randomisation was done by an independent interactive voice response system with a permuted block schedule (block size four) without stratification factors. After induction, both groups received intravenous nivolumab 3 mg/kg every 2 weeks until progression or unacceptable toxicity. The primary endpoint was treatment-related grade 3-5 adverse events until the end of the induction period (week 25), analysed in the as-treated population. Secondary endpoints were the proportion of patients who achieved a response at week 25 and disease progression at weeks 13 and 25. Overall survival was a prespecified exploratory endpoint. This study is registered with ClinicalTrials.gov, number NCT01783938, and is ongoing but no longer enrolling patients. FINDINGS: Between April 30, 2013, and July 21, 2014, 140 patients were enrolled and randomly assigned to nivolumab followed by ipilimumab (n=70) or to the reverse sequence of ipilimumab followed by nivolumab (n=70), of whom 68 and 70 patients, respectively, received at least one dose of study drug and were included in the analyses. The frequencies of treatment-related grade 3-5 adverse events up to week 25 were similar in the nivolumab followed by ipilimumab group (34 [50%; 95% CI 37.6-62.4] of 68 patients) and in the ipilimumab followed by nivolumab group (30 [43%; 31.1-55.3] of 70 patients). The most common treatment-related grade 3-4 adverse events during the whole study period were colitis (ten [15%]) in the nivolumab followed by ipilimumab group vs 14 [20%] in the reverse sequence group), increased lipase (ten [15%] vs 12 [17%]), and diarrhoea (eight [12%] vs five [7%]). No treatment-related deaths occurred. The proportion of patients with a response at week 25 was higher with nivolumab followed by ipilimumab than with the reverse sequence (28 [41%; 95% CI 29.4-53.8] vs 14 [20%; 11.4-31.3]). Progression was reported in 26 (38%; 95% CI 26.7-50.8) patients in the nivolumab followed by ipilimumab group and 43 (61%; 49.0-72.8) patients in the reverse sequence group at week 13 and in 26 (38%; 26.7-50.8) and 42 (60%; 47.6-71.5) patients at week 25, respectively. After a median follow-up of 19.8 months (IQR 12.8-25.7), median overall survival was not reached in the nivolumab followed by ipilimumab group (95% CI 23.7-not reached), whereas over a median follow-up of 14.7 months (IQR 5.6-23.9) in the ipilimumab followed by nivolumab group, median overall survival was 16.9 months (95% CI 9.2-26.5; HR 0.48 [95% CI 0.29-0.80]). A higher proportion of patients in the nivolumab followed by ipilimumab group achieved 12-month overall survival than in the ipilimumab followed by nivolumab group (76%; 95% CI 64-85 vs 54%; 42-65). INTERPRETATION: Nivolumab followed by ipilimumab appears to be a more clinically beneficial option compared with the reverse sequence, albeit with a higher frequency of adverse events. FUNDING: Bristol-Myers Squibb.

PURPOSE: We evaluated atypical response patterns and the relationship between overall survival and best overall response measured per immune-related response criteria (irRC) and Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1) in patients with advanced melanoma treated with pembrolizumab in the phase Ib KEYNOTE-001 study (clinical trial information: NCT01295827). PATIENTS AND METHODS: Patients received pembrolizumab 2 or 10 mg/kg every 2 weeks or every 3 weeks. Atypical responses were identified by using centrally assessed irRC data in patients with >/= 28 weeks of imaging. Pseudoprogression was defined as >/= 25% increase in tumor burden at week 12 (early) or any assessment after week 12 (delayed) that was not confirmed as progressive disease at next assessment. Response was assessed centrally per irRC and RECIST v1.1. RESULTS: Of the 655 patients with melanoma enrolled, 327 had >/= 28 weeks of imaging follow-up. Twenty-four (7%) of these 327 patients had atypical responses (15 [5%] with early pseudoprogression and nine [3%] with delayed pseudoprogression). Of the 592 patients who survived >/= 12 weeks, 84 (14%) experienced progressive disease per RECIST v1.1 but nonprogressive disease per irRC. Two-year overall survival rates were 77.6% in patients with nonprogressive disease per both criteria (n = 331), 37.5% in patients with progressive disease per RECIST v1.1 but nonprogressive disease per irRC (n = 84), and 17.3% in patients with progressive disease per both criteria (n = 177). CONCLUSION: Atypical responses were observed in patients with melanoma treated with pembrolizumab. Based on survival analysis, conventional RECIST might underestimate the benefit of pembrolizumab in approximately 15% of patients; modified criteria that permit treatment beyond initial progression per RECIST v1.1 might prevent premature cessation of treatment.

Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to target a variety of other proteins unrelated to the chromatin environment. In this context, our present work demonstrates that the pharmacological or genetic abrogation of HDAC6 in primary melanoma samples and cell lines, down-regulates the expression of PD-L1, an important co-stimulatory molecule expressed in cancer cells, which activates the inhibitory regulatory pathway PD-1 in T-cells. Our data suggests that this novel mechanism of PD-L1 regulation is mainly mediated by the influence of HDAC6 over the recruitment and activation of STAT3. Additionally, we observed that selective HDAC6 inhibitors impairs tumor growth and reduce the in vivo expression of several inhibitory check-point molecules and other regulatory pathways involved in immunosurveillance. Most importantly, these results provide a key pre-clinical rationale and justification to further study isotype selective HDAC6 inhibitors as potential immuno-modulatory agents in cancer.

Inactivation of Ras GTPase activating proteins (RasGAPs) can activate Ras, increasing the risk for tumor development. Utilizing a melanoma whole genome sequencing (WGS) data from 13 patients, we identified two novel, clustered somatic missense mutations (Y472H and L481F) in RASA1 (RAS p21 protein activator 1, also called p120RasGAP). We have shown that wild type RASA1, but not identified mutants, suppresses soft agar colony formation and tumor growth of BRAF mutated melanoma cell lines via its RasGAP activity toward R-Ras (related RAS viral (r-ras) oncogene homolog) isoform. Moreover, R-Ras increased and RASA1 suppressed Ral-A activation among Ras downstream effectors. In addition to mutations, loss of RASA1 expression was frequently observed in metastatic melanoma samples on melanoma tissue microarray (TMA) and a low level of RASA1 mRNA expression was associated with decreased overall survival in melanoma patients with BRAF mutations. Thus, these data support that RASA1 is inactivated by mutation or by suppressed expression in melanoma and that RASA1 plays a tumor suppressive role by inhibiting R-Ras, a previously less appreciated member of the Ras small GTPases.

IMPORTANCE: The programmed death 1 (PD-1) pathway limits immune responses to melanoma and can be blocked with the humanized anti-PD-1 monoclonal antibody pembrolizumab. OBJECTIVE: To characterize the association of pembrolizumab with tumor response and overall survival among patients with advanced melanoma. DESIGN, SETTINGS, AND PARTICIPANTS: Open-label, multicohort, phase 1b clinical trials (enrollment, December 2011-September 2013). Median duration of follow-up was 21 months. The study was performed in academic medical centers in Australia, Canada, France, and the United States. Eligible patients were aged 18 years and older and had advanced or metastatic melanoma. Data were pooled from 655 enrolled patients (135 from a nonrandomized cohort [n = 87 ipilimumab naive; n = 48 ipilimumab treated] and 520 from randomized cohorts [n = 226 ipilimumab naive; n = 294 ipilimumab treated]). Cutoff dates were April 18, 2014, for safety analyses and October 18, 2014, for efficacy analyses. EXPOSURES: Pembrolizumab 10 mg/kg every 2 weeks, 10 mg/kg every 3 weeks, or 2 mg/kg every 3 weeks continued until disease progression, intolerable toxicity, or investigator decision. MAIN OUTCOMES AND MEASURES: The primary end point was confirmed objective response rate (best overall response of complete response or partial response) in patients with measurable disease at baseline per independent central review. Secondary end points included toxicity, duration of response, progression-free survival, and overall survival. RESULTS: Among the 655 patients (median [range] age, 61 [18-94] years; 405 [62%] men), 581 had measurable disease at baseline. An objective response was reported in 194 of 581 patients (33% [95% CI, 30%-37%]) and in 60 of 133 treatment-naive patients (45% [95% CI, 36% to 54%]). Overall, 74% (152/205) of responses were ongoing at the time of data cutoff; 44% (90/205) of patients had response duration for at least 1 year and 79% (162/205) had response duration for at least 6 months. Twelve-month progression-free survival rates were 35% (95% CI, 31%-39%) in the total population and 52% (95% CI, 43%-60%) among treatment-naive patients. Median overall survival in the total population was 23 months (95% CI, 20-29) with a 12-month survival rate of 66% (95% CI, 62%-69%) and a 24-month survival rate of 49% (95% CI, 44%-53%). In treatment-naive patients, median overall survival was 31 months (95% CI, 24 to not reached) with a 12-month survival rate of 73% (95% CI, 65%-79%) and a 24-month survival rate of 60% (95% CI, 51%-68%). Ninety-two of 655 patients (14%) experienced at least 1 treatment-related grade 3 or 4 adverse event (AE) and 27 of 655 (4%) patients discontinued treatment because of a treatment-related AE. Treatment-related serious AEs were reported in 59 patients (9%). There were no drug-related deaths. CONCLUSIONS AND RELEVANCE: Among patients with advanced melanoma, pembrolizumab administration was associated with an overall objective response rate of 33%, 12-month progression-free survival rate of 35%, and median overall survival of 23 months; grade 3 or 4 treatment-related AEs occurred in 14%. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01295827.