Block, Matthew S.

Abstract Genome-wide identification and characterization of long noncoding RNAs (lncRNA) in individual immune cell lineages helps us better understand the driving mechanisms behind melanoma and advance personalized patient treatment. To elucidate the transcriptional landscape in diverse immune cell types of peripheral blood cells (PBC) in stage IV melanoma, we used whole transcriptome RNA sequencing to profile lncRNAs in CD4+, CD8+, and CD14+ PBC from 132 patient samples. Our integrative computational approach identified 27,625 expressed lncRNAs, 2,744 of which were novel. Both T cells (i.e., CD4+ and CD8+ PBC) and monocytes (i.e., CD14+ PBC) exhibited differential transcriptional expression profiles between patients with melanoma and healthy subjects. Cis- and trans-level coexpression analysis suggested that lncRNAs are potentially involved in many important immune-related pathways and the programmed cell death receptor 1 checkpoint pathways. We also identified nine gene coexpression modules significantly associated with melanoma status, all of which were significantly enriched for three mRNA translation processes. Age and melanoma traits closely correlated with each other, implying that melanoma contains age-associated immune changes. Our computational prediction analysis suggests that many cis- and trans-regulatory lncRNAs could interact with multiple transcriptional and posttranscriptional regulatory elements in CD4+, CD8+, and CD14+ PBC, respectively. These results provide novel insights into the regulatory mechanisms involving lncRNAs in individual immune cell types in melanoma and can help expedite cell type-specific immunotherapy treatments for such diseases. Significance: These findings elucidate melanoma-associated changes to the noncoding transcriptional landscape of distinct immune cell classes, thus providing cell type-specific guidance to targeted immunotherapy regimens. Cancer Res; 78(15); 4411–23. ©2018 AACR.

Abstract Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients. Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later. Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months. Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014–25. ©2018 AACR.

Abstract Ligation of programmed cell death-1 (PD-1) in the tumor microenvironment is known to inhibit effective adaptive antitumor immunity. Blockade of PD-1 in humans has resulted in impressive, durable regression responses in select tumor types. However, durable responses have been elusive in ovarian cancer patients. PD-1 was recently shown to be expressed on and thereby impair the functions of tumor-infiltrating murine and human myeloid dendritic cells (TIDC) in ovarian cancer. In the present work, we characterize the regulation of PD-1 expression and the effects of PD-1 blockade on TIDC. Treatment of TIDC and bone marrow–derived dendritic cells (DC) with IL10 led to increased PD-1 expression. Both groups of DCs also responded to PD-1 blockade by increasing production of IL10. Similarly, treatment of ovarian tumor–bearing mice with PD-1 blocking antibody resulted in an increase in IL10 levels in both serum and ascites. While PD-1 blockade or IL10 neutralization as monotherapies were inefficient, combination of these two led to improved survival and delayed tumor growth; this was accompanied by augmented antitumor T- and B-cell responses and decreased infiltration of immunosuppressive MDSC. Taken together, our findings implicate compensatory release of IL10 as one of the adaptive resistance mechanisms that undermine the efficacy of anti–PD-1 (or anti–PD-L1) monotherapies and prompt further studies aimed at identifying such resistance mechanisms. Cancer Res; 77(23); 6667–78. ©2017 AACR.

The optimal surveillance for patients with resected high-risk melanoma is controversial. Select locoregional or oligometastatic recurrences can be cured with salvage resection. Data on the ability of PET/CT to detect such recurrences are sparse. We evaluated whether surveillance PET/CT in patients with resected stage III–IV melanoma led to detection of clinically occult recurrences amenable to curative-intent salvage treatment. We retrospectively identified 1429 melanoma patients who underwent PET/CT between January 2008 and October 2012 at Mayo Clinic (Rochester, Minnesota). A total of 1130 were excluded because of stage I–II, ocular or mucosal melanoma, incomplete resection, PET/CT not performed for surveillance or performed at a different institution, and records not available. A total of 299 patients were eligible. Overall, 162 (52%) patients developed recurrence [locoregional: 77 (48%), distant: 85 (52%)]. The first recurrence was clinically occult in 98 (60%) and clinically evident in 64 (40%). Clinically evident recurrences were more often superficial (skin, subcutaneous, or nodal) or in the brain, whereas clinically occult recurrences more often visceral. Overall, 90% of all recurrences were detected by 2.8 years. In all, 70% of patients with recurrence underwent curative-intent salvage treatment (locoregional: 94%, distant: 48%), with similar rates for clinically occult versus clinically evident recurrences (66 vs. 75%, P=0.240). Overall survival was superior among those who underwent curative-intent salvage treatment [5.9 vs. 1.2 years; hazard ratio=4.27, 95% confidence interval (CI)=2.68–6.80; P<0.001], despite 79% developing recurrence again. PET/CT had high sensitivity (88%, 95% CI=79.94–93.31%), specificity (90%, 95% CI=88.56–91.56%), and negative predictive value (99%, 95% CI=98.46–99.52%). However, the positive predictive value was only 37% (95% CI=31.32–43.68%). In patients with resected stage III–IV melanoma, surveillance PET/CT detected a large proportion of clinically occult recurrences amenable to curative-intent salvage treatment. Despite a high rate of second relapse, curative-intent salvage treatment was associated with superior overall survival. Even though PET/CT had high sensitivity, specificity, and negative predictive value, positive predictive value was poor, highlighting the need for histologic confirmation of PET/CT-detected abnormalities.

Abstract PD-1 and PD-L1 form a major inhibitory axis that acts to suppress tumor-rejecting effector responses. In addition to its expression and inhibitory functions in T and B cells, PD-1 has also been shown to be expressed on and hence impact the functions of cells of innate arm, such as dendritic cells (DCs), primarily at the tumor site but not in the periphery. Our aim in this study was to identify tumor-associated factor(s) responsible for PD-1 regulation on DCs and investigate how DCs in the tumor microenvironment respond to anti-PD-1-based immunotherapy. Both bone marrow-derived dendritic cells (BMDCs) as well as murine ovarian tumor ascites-derived DCs were used in the studies. Using in vitro cultures, we show that the cytokine IL-10; which is expressed in abundance in many malignancies including ovarian cancer, is potent regulator of PD-1 expression on DCs. Based on inhibition and siRNA knockdown studies; we show that IL-10 mediated PD-1 expression depends on STAT3 activation. Treatment of DCs with IL-10 also led to an increase in expression of PD-L1 on surface of DCs as well as an increase in release of soluble PD-L1. Antibody-mediated blockade of PD-1 on DCs led to greatly increased production of IL-10 (&gt; 4 fold) in cultures in vitro. Furthermore, treatment of tumor bearing mice with anti-PD-1 antibody led to a significant increase of IL-10 in ascites (&gt; 6 fold) and periphery (&gt; 4 fold). This compensatory release of IL-10 correlated with a further increase in PD-1 expression on DCs both in vitro and in vivo. In in vivo studies, while the blockade of PD-1 or IL-10 neutralization as monotherapy were ineffective, blockade of PD-1 and IL-10 neutralization as combination therapy augmented the anti-tumor response in ovarian tumor bearing mice; leading to a decrease in tumor growth and a significant increase in survival. These results show that the IL-10 and PD-1 pathways intersect with DCs in the tumor microenvironment. PD-1 blockade on DCs leads to compensatory release of IL-10 resulting in the maintenance of immunosuppression, and a combination regimen of PD-1 blockade and IL-10 neutralization has therapeutic benefit in ovarian tumors. These results serve as catalyst to explore the feasibility/efficacy of exploiting these targets in clinical settings for treatment of human ovarian cancer. Citation Format: Purushottam Lamichhane, Lavakumar Karyampudi, Barath Shreeder, James Krempski, Deborah Bahr, Matthew Block, Keith Knutson. Blockade of PD-1 signaling in tumor-associated dendritic cells results in compensatory IL-10 release maintaining immune suppression in ovarian cancer microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B66.

Immune checkpoint therapy with PD-1 blockade has emerged as an effective therapy for many advanced cancers; however, only a small fraction of patients achieve durable responses. To date, there is no validated blood-based means of predicting the response to PD-1 blockade. We report that Bim is a downstream signaling molecule of the PD-1 pathway, and its detection in T cells is significantly associated with expression of PD-1 and effector T cell markers. High levels of Bim in circulating tumor-reactive (PD-1 + CD11a hi CD8 + ) T cells were prognostic of poor survival in patients with metastatic melanoma who did not receive anti–PD-1 therapy and were also predictive of clinical benefit in patients with metastatic melanoma who were treated with anti–PD-1 therapy. Moreover, this circulating tumor-reactive T cell population significantly decreased after successful anti–PD-1 therapy. Our study supports a crucial role of Bim in both T cell activation and apoptosis as regulated by PD-1 and PD-L1 interactions in effector CD8 + T cells. Measurement of Bim levels in circulating T cells of patients with cancer may provide a less invasive strategy to predict and monitor responses to anti–PD-1 therapy, although future prospective analyses are needed to validate its utility.

Uveal melanoma is a rare type of melanoma, with only five to seven cases per one million persons diagnosed each year. Patients with metastatic melanoma of uveal origin tend to have lower response rates on traditional therapies. Herein we report our experience with 10 patients with metastatic uveal melanoma (MUM) who received pembrolizumab. Eligible patients were more than or equal to 18 years old, had unresectable MUM, progressed on prior ipilimumab therapy, had good performance status (Eastern Cooperative Oncology Group of 0 or 1), and adequate organ and marrow function. Patients could have central nervous system disease, but needed to be clinically stable. Patients were treated with 2 mg/kg pembrolizumab intravenously over 30 min every 3 weeks until disease progression, unacceptable toxicity, or for up to 2 years. Between April 2014 and October 2014, we treated a total of 10 patients with MUM with pembrolizumab. Median age was 65 years, with 70% being female. As of the data cutoff date of 14 May 2015, median progression-free survival was 18 weeks (range 3.14–49.3 weeks), with four patients still currently receiving therapy. Of eight evaluable patients, there was one complete response, two partial responses, and one patient with stable disease. Four patients had rapidly progressive disease. Toxicities were as expected and were usually grade 1/2 in nature. Although this cohort of patients was small, to our knowledge this is the first such report of outcomes in uveal melanoma patients being treated with anti-PD1 therapy. In the absence of a clinical trial, treatment with pembrolizumab appears to be a viable option for patients with MUM.