Amod A. Sarnaik

Safety, Efficacy, and Biomarkers of Nivolumab With Vaccine in Ipilimumab-Refractory or -Naive Melanoma

PURPOSE Nivolumab, a human immunoglobulin G4-blocking antibody against the T-cell programmed death-1 checkpoint protein, has activity against metastatic melanoma. Its safety, clinical efficacy, and correlative biomarkers were assessed with or without a peptide vaccine in ipilimumab-refractory and -naive melanoma. PATIENTS AND METHODS In this phase I study, 90 patients with unresectable stage III or IV melanoma who were ipilimumab naive and had experienced progression after at least one prior therapy (cohorts 1 to 3, 34 patients) or experienced progression after prior ipilimumab (cohorts 4 to 6, 56 patients) received nivolumab at 1, 3, or 10 mg/kg every 2 weeks for 24 weeks, then every 12 weeks for up to 2 years, with or without a multipeptide vaccine. RESULTS Nivolumab with vaccine was well tolerated and safe at all doses. The RECIST 1.1 response rate for both ipilimumab-refractory and -naive patients was 25%. Median duration of response was not reached at a median of 8.1 months of follow-up. High pretreatment NY-ESO-1 and MART-1-specific CD8(+) T cells were associated with progression of disease. At week 12, increased peripheral-blood T regulatory cells and decreased antigen-specific T cells were associated with progression. PD-L1 tumor staining was associated with responses to nivolumab, but negative staining did not rule out a response. Patients who experienced progression after nivolumab could respond to ipilimumab. CONCLUSION In patients with ipilimumab-refractory or -naive melanoma, nivolumab at 3 mg/kg with or without peptide vaccine was well tolerated and induced responses lasting up to 140 weeks. Responses to nivolumab in ipilimumab-refractory patients or to ipilimumab in nivolumab-refractory patients support combination or sequencing of nivolumab and ipilimumab.

The HSP90 Inhibitor XL888 Overcomes BRAF Inhibitor Resistance Mediated through Diverse Mechanisms

Purpose: The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance. Experimental Design: The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry–based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis. Results: XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein–extracellular signal–regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition. Conclusions: HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy. Clin Cancer Res; 18(9); 2502–14. ©2012 AACR.

Extended Dose Ipilimumab with a Peptide Vaccine: Immune Correlates Associated with Clinical Benefit in Patients with Resected High-Risk Stage IIIc/IV Melanoma

Purpose: To determine safety and feasibility of adjuvant ipilimumab following resection of high-risk melanoma and to identify surrogate markers for benefit. Experimental Design: In this phase II trial, 75 patients with resected stage IIIc/IV melanoma received the CTLA-4 antibody ipilimumab every 6 to 8 weeks for 1 year. Eligible patients received further maintenance treatments. The first 25 patients received 3 mg/kg of ipilimumab, and an additional 50 patients received 10 mg/kg. HLA-A*0201+ patients received multipeptide immunizations in combination with ipilimumab. Leukapheresis was performed prior to and 6 months after initiation of treatment. Results: Median overall and relapse-free survivals were not reached after a median follow-up of 29.5 months. Significant immune-related adverse events were observed in 28 of 75 patients and were positively associated with longer relapse-free survival. Antigen-specific T cell responses to vaccine were variable, and vaccine combination was not associated with additional benefit. No effects on T regulatory cells were observed. Higher changes in Th-17 inducible frequency were a surrogate marker of freedom from relapse (P = 0.047), and higher baseline C-reactive protein (CRP) levels were associated with freedom from relapse (P = 0.035). Conclusions: Adjuvant ipilimumab following resection of melanoma at high risk for relapse appeared to be associated with improved outcome compared to historical reports. Significant immune-related adverse events were generally reversible and appeared to be associated with improved relapse-free survival. Although vaccination failed to induce a consistent in vitro measurable response, a higher change in Th-17 inducible cells and higher baseline CRP levels were positively associated with freedom from relapse. Clin Cancer Res; 17(4); 896–906. ©2010 AACR.

Routine Omission of Sentinel Lymph Node Biopsy for Merkel Cell Carcinoma ≤ 1 cm Is Not Justified

TO THE EDITOR: Stokes et al recently reported a retrospective review of resected primary Merkel cell carcinoma (MCC) utilizing the Department of Veterans’ Affairs national health care database. The authors report 213 patients who had a resection of their primary MCC as well as an assessment of the regional lymph nodes by either sentinel lymph node biopsy (SNB) or complete lymphadenectomy (CLD). The patients were stratified into three groups based on the diameter of the primary tumor. The authors report that in the subgroup of 54 patients whose tumor diameter was 1 cm, only two patients had evidence of gross regional metastasis at presentation. None of the remaining 52 patients had nodal disease diagnosed by either SNB or elective CLD, although no data are presented to indicate who may have failed in the draining regional nodal basin at risk during the follow-up period. The authors conclude that regional lymph node assessment can be “reasonably and safely omitted” in patients who present with MCC 1 cm in diameter and without palpable disease. However, the routine omission of SNB in patients with MCC 1 cm diameter may not be justified based on the evidence presented in the report. Caution must be taken when utilizing national databases from multiple institutions, especially when attempting to characterize rare and diagnostically challenging malignancies such as MCC. In the methods section, the authors fail to report the precise manner in which the pathologic diagnosis of MCC was rendered in the primary tumor and whether this was standardized among all the various Veterans’ Affairs medical centers that contributed data toward this retrospective review. Without the use of appropriate immunostains, it is possible non-MCC cases were erroneously included in the study, which could potentially account for the relatively low recurrence rate of this study when compared with other reports. In addition, there is inherent difficulty and subjectivity in assessing tumor diameter, and precisely how this critical parameter was established was not mentioned by the authors (probably because it was impossible to determine in a retrospective database review). The authors also report the use of either SNB or elective CLD when staging clinically negative regional lymph nodes, but they failed to state the frequency of each modality. In addition, they do not elucidate the details of the pathologic examination of the nodal specimens. Specifically, elective CLD may not have been subjected to the thorough analysis to which SLN are typically subjected, with serial sectioning and immunohistochemistry. This could potentially lead to a higher false negative rate. The authors do report a 17% overall recurrence rate in the cohort of MCC with diameters 1 cm. However, the specific situations that led to recurrence are not enumerated: how many of these patients failed in the regional nodal basin as a false negative SNB? How many demonstrated regional nodal disease as the first site of recurrence in those patients previously treated without any nodal staging? How many failed in the regional nodes after CLD? These statistics would be important to note before concluding that small diameter MMCs routinely do not metastasize to lymph nodes. In addition, the 17% recurrence rate could omit cases that were missed due to the difficulty in establishing a diagnosis of microscopic metastasis in a complete lymph node dissection specimen. It should also be noted that it is difficult to interpret the significance of the recurrence rate, because the length of follow-up for any of the individual subgroups was not reported. In an attempt to validate the results reported in this study, we recently reviewed our single institution’s experience regarding the use of SNB to diagnosis microscopic regional lymph node metastasis in MCC with diameters 1 cm. In contrast to the findings of this study, we found evidence of clinically occult regional lymph node metastasis in five of 12 cases (42%). This marked difference may be due to the exclusive use of SNB rather than elective CLD as well as our routine application of appropriate immunostaining of the sentinel lymph node. Our findings from a large referral center with a relatively high volume of patients with MCC refute the premise that it is safe to omit SNB in 1 cm diameter MCC based on an anticipated low yield of positive sentinel nodes. Changes in “standard” surgical practice in a rare disease such as MCC should require strong evidence, preferably from prospective trials, before they are adopted.

Biomarkers on melanoma patient T Cells associated with ipilimumab treatment

BackgroundIpilimumab induces long-lasting clinical responses in a minority of patients with metastatic melanoma. To better understand the mechanism(s) of action and to identify novel biomarkers associated with the clinical benefit and toxicity of ipilimumab, baseline characteristics and changes in CD4+ and CD8+ T cells from melanoma patients receiving ipilimumab were characterized by gene profiling and flow cytometry.MethodsMicroarray analysis of flow-cytometry purified CD4+ and CD8+ T cells was employed to assess gene profiling changes induced by ipilimumab. Selected molecules were further investigated by flow cytometry on pre, 3-month and 6-month post-treatment specimens.ResultsIpilimumab up-regulated Ki67 and ICOS on CD4+ and CD8+ cells at both 3- and 6-month post ipilimumab (p ≤ 0.001), decreased CCR7 and CD25 on CD8+ at 3-month post ipilimumab (p ≤ 0.02), and increased Gata3 in CD4+ and CD8+ cells at 6-month post ipilimumab (p ≤ 0.001). Increased EOMES+CD8+, GranzymeB+EOMES+CD8+ and decreased Ki67+EOMES+CD4+ T cells at 6 months were significantly associated with relapse (all p ≤ 0.03). Decreased Ki67+CD8+ T cells were significantly associated with the development of irAE (p = 0.02). At baseline, low Ki67+EOMES+CD8+ T cells were associated with relapse (p ≤ 0.001), and low Ki67+EOMES+CD4+ T cells were associated with irAE (p ≤ 0.008).ConclusionsUp-regulation of proliferation and activation signals in CD4+ and CD8+ T cells were pharmacodynamic markers for ipilimumab. Ki67+EOMES+CD8+ and Ki67+EOMES+CD4+T cells at baseline merit further testing as biomarkers associated with outcome and irAEs, respectively.

HDAC Inhibition Upregulates PD-1 Ligands in Melanoma and Augments Immunotherapy with PD-1 Blockade.

Combining other agents with immune-based approaches can enhance treatment for melanoma. PDL-1 gene expression was increased after inhibition of histone deacetylases. Combining PD-1–blockade immunotherapy with histone deacetylase inhibition increased responses in a mouse model of melanoma. Expression of PD-1 ligands by tumors and interaction with PD-1–expressing T cells in the tumor microenvironment can result in tolerance. Therapies targeting this coinhibitory axis have proven clinically successful in the treatment of metastatic melanoma, non–small cell lung cancer, and other malignancies. Therapeutic agents targeting the epigenetic regulatory family of histone deacetylases (HDAC) have shown clinical success in the treatment of some hematologic malignancies. Beyond direct tumor cell cytotoxicity, HDAC inhibitors have also been shown to alter the immunogenicity and enhance antitumor immune responses. Here, we show that class I HDAC inhibitors upregulated the expression of PD-L1 and, to a lesser degree, PD-L2 in melanomas. Evaluation of human and murine cell lines and patient tumors treated with a variety of HDAC inhibitors in vitro displayed upregulation of these ligands. This upregulation was robust and durable, with enhanced expression lasting past 96 hours. These results were validated in vivo in a B16F10 syngeneic murine model. Mechanistically, HDAC inhibitor treatment resulted in rapid upregulation of histone acetylation of the PD-L1 gene leading to enhanced and durable gene expression. The efficacy of combining HDAC inhibition with PD-1 blockade for treatment of melanoma was also explored in a murine B16F10 model. Mice receiving combination therapy had a slower tumor progression and increased survival compared with control and single-agent treatments. These results highlight the ability of epigenetic modifiers to augment immunotherapies, providing a rationale for combining HDAC inhibitors with PD-1 blockade. Cancer Immunol Res; 3(12); 1375–85. ©2015 AACR.

Efficacy of adoptive cell transfer of tumor-infiltrating lymphocytes after lymphopenia induction for metastatic melanoma.

A single-institution pilot clinical trial was performed combining nonmyeloablative chemotherapy and the adoptive transfer of tumor-infiltrating lymphocytes with interleukin-2 in patients with metastatic melanoma. Nineteen patients were enrolled with 13 patients (68%) successfully completing treatment. An overall response rate (partial and complete responses) of 26% by intention to treat was achieved with a median follow-up time of 10 months. Of the 13 treated patients, there were 2 complete responses and 3 partial responses (38% response rate among treated patients), along with 4 patients with stable disease ranging from 2+ to 24+months. Three of the 4 patients with stable disease have had disease control without additional therapy, including one at 24+ months. Adoptive therapy with infiltrating lymphocytes is labor intensive but feasible and has a high response rate in treated patients.

Blockade of myeloid-derived suppressor cells after induction of lymphopenia improves adoptive T cell therapy in a murine model of melanoma.

Administration of nonmyeloablative chemotherapeutic agents or total body irradiation (TBI) prior to adoptive transfer of tumor-specific T cells may reduce or eliminate immunosuppressive populations such as T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC). Little is known about these populations during immune reconstitution. This study was designed to understand the reconstitution rate and function of these populations post TBI in melanoma tumor‑bearing mice. Reconstitution rate and suppressive activity of CD4+CD25+Foxp3+ Tregs and CD11b+Gr1+ MDSC following TBI-induced lymphopenia was measured in B16 melanoma tumor‑bearing mice. To ablate the rapid reconstitution of suppressive populations, we treated mice with docetaxel, a known chemotherapeutic agent that targets MDSC, in combination with adoptive T cell transfer and dendritic cell immunotherapy. Both Treg and MDSC populations exhibited rapid reconstitution after TBI-induced lymphopenia. Although reconstituted Tregs were just as suppressive as Tregs from untreated mice, MDSC demonstrated enhanced suppressive activity of CD8+ T cell proliferation compared with endogenous MDSC from tumor-bearing mice. TBI-induced lymphopenia followed by docetaxel treatment improved the efficacy of adoptive T cell transfer and dendritic cell immunotherapy in melanoma-bearing mice, inducing a significant reduction in tumor growth and enhancing survival. Tumor regression correlated with increased CTL activity and persistence of adoptively transferred T cells. Overall, these findings suggest that TBI-induced MDSC are highly immunosuppressive and blocking their rapid reconstitution may improve the efficacy of vaccination strategies and adoptive immunotherapy.

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8+ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8+ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8+ T cells, on the yield, phenotype, and functional activity of expanded CD8+ T cells during the REP. We found that CD8+ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG4 (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8+ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8+ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.

Recent advances using anti-CTLA-4 for the treatment of melanoma.

Metastatic melanoma is a disease associated with poor prognosis, with a median survival reported to range from 6 to 9 months. Patients who are not candidates for surgical resection have an even worse expected survival. This is largely due to the lack of effective chemotherapeutic regimens and has led to the investigation of alternative treatment strategies including immunotherapy. Although melanoma is felt to be an immunogenic tumor and has been associated with the development of spontaneous tumor-specific immune responses in patients, the implementation of vaccine-based treatment has had limited success. Because the administration of a melanoma-specific vaccine alone has not been sufficient to generate robust and reproducible clinical responses, investigators are currently pursuing additional methods to augment antimelanoma immune responses by optimizing T-cell activation. T-cell activation requires both antigen presentation to the T-cell receptor and a second signal mediated by CD80 and CD86 on antigen-presenting cells and CD28 on the T cell. Ligand binding to CD28 on the T-cell surface leads to T-cell proliferation and expression of activating cytokines such as interleukin-2. Cytotoxic T-lymphocyte antigen-4 (CTLA-4), an inhibitory protein expressed on T cells, competes for the same ligands as CD28 and modulates T-cell activation. Because CTLA-4 has a significantly higher binding efficiency than CD28, CTLA-4 is critical in maintaining immune tolerance to self-antigens and may also limit responses to tumor antigens and vaccine therapy. CTLA-4 blockade either alone or in combination with melanoma-specific vaccines has been explored as a potential means to treat advanced stage melanoma. In this article, we review the spectrum of clinical trials involving CTLA-4 blockade and also review recent correlative studies attempting to elucidate the potential mechanisms by which CTLA-4 blockade achieves its therapeutic effects.