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Supplemental figure 2. Phenotype of NFAT.IL12 gene modified TIL compared to matched untransduced TIL.Shown are the percentage of cells with a phenotype consistent with effector memory (CD62L-CD45RA-),central memory (CD62L+CD45RA-), Naive (CD62+, CD45RA+) and EMRA (CD62L-CD45RA+).UT, untransduced.

Purpose: Uveal melanoma is a rare melanoma variant with no effective therapies once metastases develop. Although durable cancer regression can be achieved in metastatic cutaneous melanoma with immunotherapies that augment naturally existing antitumor T-cell responses, the role of these treatments for metastatic uveal melanoma remains unclear. We sought to define the relative immunogenicity of these two melanoma variants and determine whether endogenous antitumor immune responses exist against uveal melanoma.Experimental Design: We surgically procured liver metastases from uveal melanoma (n = 16) and cutaneous melanoma (n = 35) patients and compared the attributes of their respective tumor cell populations and their infiltrating T cells (TIL) using clinical radiology, histopathology, immune assays, and whole-exomic sequencing.Results: Despite having common melanocytic lineage, uveal melanoma and cutaneous melanoma metastases differed in their melanin content, tumor differentiation antigen expression, and somatic mutational profile. Immunologic analysis of TIL cultures expanded from these divergent forms of melanoma revealed cutaneous melanoma TIL were predominantly composed of CD8+ T cells, whereas uveal melanoma TIL were CD4+ dominant. Reactivity against autologous tumor was significantly greater in cutaneous melanoma TIL compared with uveal melanoma TIL. However, we identified TIL from a subset of uveal melanoma patients which had robust antitumor reactivity comparable in magnitude with cutaneous melanoma TIL. Interestingly, the absence of melanin pigmentation in the parental tumor strongly correlated with the generation of highly reactive uveal melanoma TIL.Conclusions: The discovery of this immunogenic group of uveal melanoma metastases should prompt clinical efforts to determine whether patients who harbor these unique tumors can benefit from immunotherapies that exploit endogenous antitumor T-cell populations. Clin Cancer Res; 22(9); 2237–49. ©2015 AACR.

Purpose: Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies.Experimental Design: From a cohort of 1,514 patients with metastatic melanoma, biopsies were procured over a 17-year period from 3,086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we used established immunohistochemical methods to perform semiquantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDA), including gp100, MART-1, and tyrosinase. To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4+ and CD8+ T-cell infiltrate.Results: Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Furthermore, tyrosinase expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8+ and CD4+ T-cell infiltrates.Conclusion: Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen-specific immunotherapies. Clin Cancer Res; 20(10); 2607–16. ©2014 AACR.

Purpose: Although adoptive cell therapy can be highly effective for the treatment of patients with melanoma, the application of this approach to the treatment of other solid tumors has been limited. The observation that the cancer germline (CG) antigen NY-ESO-1 is expressed in 70% to 80% and in approximately 25% of patients with synovial cell sarcoma and melanoma, respectively, prompted us to perform this first-in-man clinical trial using the adoptive transfer of autologous peripheral blood mononuclear cells that were retrovirally transduced with an NY-ESO-1–reactive T-cell receptor (TCR) to heavily pretreated patients bearing these metastatic cancers.Experimental Design: HLA-*0201 patients with metastatic synovial cell sarcoma or melanoma refractory to standard treatments and whose cancers expressed NY-ESO-1 received autologous TCR-transduced T cells following a lymphodepleting preparative chemotherapy. Response rates using Response Evaluation Criteria in Solid Tumors (RECIST), as well as immunologic correlates of response, are presented in this report.Results: Eleven of 18 patients with NY-ESO-1+ synovial cell sarcomas (61%) and 11 of 20 patients with NY-ESO-1+ melanomas (55%) who received autologous T cells transduced with an NY-ESO-1–reactive TCR demonstrated objective clinical responses. The estimated overall 3- and 5-year survival rates for patients with synovial cell sarcoma were 38% and 14%, respectively, whereas the corresponding estimated survival rates for patients with melanoma were both 33%.Conclusions: The adoptive transfer of autologous T cells transduced with a retrovirus encoding a TCR against an HLA-A*0201 restricted NY-ESO-1 epitope can be an effective therapy for some patients bearing synovial cell sarcomas and melanomas that are refractory to other treatments. Clin Cancer Res; 21(5); 1019–27. ©2014 AACR.

PDF file - 51K, Pairwise comparison of antigen expression by individual anatomic site cluster. Shown are p value results of Cochran-Armitage test for trend. p

Legends for Supplementary Figures 1 and 2

Supplemental figure 4. IL6 and IL10 levels. Shown are data from five patients with elevated IL12 and IFNg levels that were subject to cytokine multiplex analysis. Only cytokine IL6 and IL10 were shown to be consistently elevated post-infusion.

Purpose:Cellular therapy is an emerging cancer treatment modality, but its application to epithelial cancers has been limited. This clinical trial evaluated tumor-infiltrating lymphocyte (TIL) therapy for the treatment of patients with metastatic human papillomavirus (HPV)-associated carcinomas.Patients and Methods:The trial was a phase II design with two cohorts, cervical cancers and noncervical cancers. Cell infusion was preceded by a lymphocyte-depleting conditioning regimen and followed by systemic high-dose aldesleukin.Results:Objective tumor responses occurred in 5 of 18 (28%) patients in the cervical cancer cohort and 2 of 11 (18%) patients in the noncervical cancer cohort. Two of the responses in cervical cancer were complete and are ongoing 67 and 53 months after treatment. Responses in the noncervical cancer cohort were in anal cancer and oropharyngeal cancer. The HPV reactivity of the infused T cells correlated with clinical response. Peripheral blood repopulation with HPV-reactive T cells also correlated with clinical response.Conclusions:These findings support the concept that cellular therapy can mediate the regression of epithelial cancers, and they suggest the importance of predictive biomarkers and novel treatment platforms for more effective therapies.

Supplementary Figure Legend. Weak and insufficient tumor responses after MDA specific CD8+ T cell clone infusion

PDF file - 47K, Individual dendrogram distance matrices for the melanoma differentiation antigens. Dendrogram distant matrices calculated from DendroUPGMA.

PDF file - 57K, Pairwise comparison of antigen expression by individual anatomic site. Shown are p value results of Cochran-Armitage test for trend. p

Purpose: Treatment with ipilimumab can cause objective tumor responses in patients with metastatic melanoma. We have treated 177 evaluable patients in three clinical trials and have long-term follow-up to evaluate the durability of responses.Experimental Design: Patients with metastatic melanoma were treated in three trials from 2002 to 2005. In protocol 1, 56 patients received ipilimumab with gp100 peptides. In protocol 2, 36 patients received ipilimumab with interleukin-2. In protocol 3, 85 patients received ipilimumab with intrapatient dose-escalation and were randomized to receive gp100 peptides. We have analyzed their long-term follow-up and survival data.Results: With median follow-up for protocols 1, 2, and 3 being 92, 84, and 71 months, median survival was 14, 16, and 13 months with 5-year survival rates being 13%, 25%, and 23%, respectively. Patients in protocol 2 had a 17% complete response (CR) rate, compared with 7% in protocol 1 and 6% in protocol 3. These CR rates are higher than previously reported for the same trials because some patients who eventually became complete responders had continual tumor regression months to years after therapy. All but one of the 15 complete responders are ongoing at 54+ to 99+ months.Conclusions: This report provides the longest follow-up of patients with melanoma treated with ipilimumab and shows that ipilimumab can induce durable, potentially curative tumor regression in a small percentage of patients with metastatic melanoma. The combination of ipilimumab and interleukin-2 seems to have an increased CR rate, but this needs to be tested in a randomized trial. Clin Cancer Res; 18(7); 2039–47. ©2012 AACR.

PDF file - 73K, MDA and MHC II expression demonstrates a site cluster-specific pattern. (A) The distribution of antigen expression for each anatomic site cluster. The p-values indicate the probability that no variability exists across the site clusters as determined by the Kruskal-Wallis test.

Supplementary Figure 1. PET-CT scan images

supplemental figure legend

PDF file - 65K, TYR expression varies with the degree of CD8+ and CD4+ T cell infiltrate. (A) The distribution of antigen expression for given CD8 (blue) infiltrate score (0-3+, column). (B) The distribution of antigen expression for given CD4 (green) infiltrate score (0-3+, column).

PDF file - 50K, Dendrogram distance matrix for all melanoma differentiation antigens. Dendrogram distant matrix calculated from DendroUPGMA.

Supplementary Figure 1. In situ MRI assessment of tumor melanin content. Supplementary Figure 2. UM and CM liver metastases demonstrate similar immunophenotype profile.

Supplementary Figure S1; Supplementary Table S1.

CCR Translation for This Article from Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell Transfer Immunotherapy

Figures for data supplement

CCR Translation for This Article from CTLA-4 Blockade with Ipilimumab: Long-term Follow-up of 177 Patients with Metastatic Melanoma

Supplementary Figure 1 and 2. Supplementary Figure 1. Evaluation of persistence and function of TCR-transduced T cells following adoptive transfer in responding and non-responding patients. Supplementary Figure 2. Evaluation of function of TCR-transduced T cells in vaccinated and unvaccinated patients.

Supplementary Figures S1-S2; Supplementary Tables S1-S2.

Supplementary Table 1. Melanoma patients: Prior treatments

Supplemental figure 5. Treg reconstitution. Shown are the percentages of circulating T cells with a phenotype consistent with a Treg cell (CD4+/CD25+/FoxP3+). Data from parallel TIL trial using TIL administration but without exogenous IL2 support (TIL No IL2) and TIL engineered with the NFAT.IL2 vector (TIL IL12).

Purpose: Adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) can mediate durable cancer regression in selected patients with metastatic melanoma. However, the tumor antigens associated with these favorable responses remain unclear. We hypothesized that a clinical strategy involving the iterative adoptive transfer of selected autologous antigen-specific T-cell clones could help systematically define immunologic targets associated with successful cancer therapy, without the interpretative ambiguity of transferring polyclonal populations. Here, we evaluated the clinical efficacy of CD8+ T-cell clones specific for the melanocyte differentiation antigens (MDA), gp100 and MART-1, respectively.Experimental Design: We conducted two consecutive phase II clinical trials involving the adoptive transfer of highly selected autologous antigen-specific CD8+ T-cell clones against gp100 and MART-1, respectively. Fifteen patients with HLA-A2+ treatment-refractory metastatic melanoma received highly avid MDA-specific CD8+ T-cell clones specific for either gp100 (n = 10) or MART-1 (n = 5) with or without intravenous interleukin-2 (IL2) after a lymphodepleting myeloablative preparative regimen.Results: Of the 15 treated patients, we observed immune-mediated targeting of skin melanocytes in 11 patients (73%) and clonal engraftment in eight patients (53%) after cell transfer. There were only transient minor tumor regressions observed, but no objective tumor responses based on Response Evaluation Criteria in Solid Tumor (RECIST) criteria.Conclusions: Despite successful clonal repopulation and evidence of in vivo antigen targeting, the poor therapeutic efficacy after the adoptive transfer of autologous MDA-specific T cells raises significant concerns regarding future immunotherapy efforts targeting this class of tumor antigens. Clin Cancer Res; 21(3); 534–43. ©2014 AACR.

Purpose: Tumor‐infiltrating lymphocytes (TIL) and interleukin (IL)-2 administered following lymphodepletion can cause the durable complete regression of bulky metastatic melanoma in patients refractory to approved treatments. However, the generation of a unique tumor-reactive TIL culture for each patient may be prohibitively difficult. We therefore investigated the clinical and immunologic impact of unscreened, CD8+ enriched “young” TIL.Experimental Design: Methods were developed for generating TIL that minimized the time in culture and eliminated the individualized tumor-reactivity screening step. Thirty-three patients were treated with these CD8+ enriched young TIL and IL-2 following nonmyeloablative lymphodepletion (NMA). Twenty-three additional patients were treated with CD8+ enriched young TIL and IL-2 after lymphodepletion with NMA and 6 Gy of total body irradiation.Results: Young TIL cultures for therapy were successfully established from 83% of 122 consecutive melanoma patients. Nineteen of 33 patients (58%) treated with CD8+ enriched young TIL and NMA had an objective response (Response Evaluation Criteria in Solid Tumors) including 3 complete responders. Eleven of 23 patients (48%) treated with TIL and 6 Gy total body irradiation had an objective response including 2 complete responders. At 1 month after TIL infusion the absolute CD8+ cell numbers in the periphery were highly correlated with response.Conclusions: This study shows that a rapid and simplified method can be used to reliably generate CD8+ enriched young TIL for administration as an individualized therapy for advanced melanoma, and may allow this potentially effective treatment to be applied at other institutions and to reach additional patients. Clin Cancer Res; 16(24); 6122–31. ©2010 AACR.

Supplemental figure 1. Gene transfer efficiency. Shown are 6 representative determinations of NFAT.IL12 transduction efficiency. NFAT.IL2 engineered TIL cultures were stimulated by PMA/ionomycin and then subject to surface staining for CD3 and intracellular staining for IL-12 with resultant scatter plots shown. The patient numbers and response were as labeled with the percent IL-12+ cells shown. UT, untransduced.

Purpose: Most treatments for patients with metastatic melanoma have a low rate of complete regression and thus overall survival in these patients is poor. We investigated the ability of adoptive cell transfer utilizing autologous tumor-infiltrating lymphocytes (TIL) to mediate durable complete regressions in heavily pretreated patients with metastatic melanoma.Experimental Design: Ninety-three patients with measurable metastatic melanoma were treated with the adoptive transfer of autologous TILs administered in conjunction with interleukin-2 following a lymphodepleting preparative regimen on three sequential clinical trials. Ninety-five percent of these patients had progressive disease following a prior systemic treatment. Median potential follow-up was 62 months.Results: Objective response rates by Response Evaluation Criteria in Solid Tumors (RECIST) in the 3 trials using lymphodepleting preparative regimens (chemotherapy alone or with 2 or 12 Gy irradiation) were 49%, 52%, and 72%, respectively. Twenty of the 93 patients (22%) achieved a complete tumor regression, and 19 have ongoing complete regressions beyond 3 years. The actuarial 3- and 5-year survival rates for the entire group were 36% and 29%, respectively, but for the 20 complete responders were 100% and 93%. The likelihood of achieving a complete response was similar regardless of prior therapy. Factors associated with objective response included longer telomeres of the infused cells, the number of CD8+CD27+ cells infused, and the persistence of the infused cells in the circulation at 1 month (all P2 < 0.001).Conclusions: Cell transfer therapy with autologous TILs can mediate durable complete responses in patients with metastatic melanoma and has similar efficacy irrespective of prior treatment. Clin Cancer Res; 17(13); 4550–7. ©2011 AACR.

Purpose: Infusion of interleukin-12 (IL12) can mediate antitumor immunity in animal models, yet its systemic administration to patients with cancer results in minimal efficacy and severe toxicity. Here, we evaluated the antitumor activity of adoptively transferred human tumor-infiltrating lymphocytes (TILs) genetically engineered to secrete single-chain IL12 selectively at the tumor site.Experimental Design: Thirty-three patients with metastatic melanoma were treated in a cell dose–escalation trial of autologous TILs transduced with a gene encoding a single-chain IL12 driven by a nuclear factor of the activated T cells promoter (NFAT.IL12). No IL2 was administered.Results: The administration of 0.001 to 0.1 × 109 NFAT.IL12–transduced TILs to 17 patients resulted in a single, objective response (5.9%). However, at doses between 0.3 and 3 × 109 cells, 10 of 16 patients (63%) exhibited objective clinical responses. The responses tended to be short, and the administered IL12-producing cells rarely persisted at 1 month. Increasing cell doses were associated with high serum levels of IL12 and IFNγ as well as clinical toxicities, including liver dysfunction, high fevers, and sporadic life-threatening hemodynamic instability.Conclusions: In this first-in-man trial, administration of TILs transduced with an inducible IL12 gene mediated tumor responses in the absence of IL2 administration using cell doses 10- to 100-fold lower than conventional TILs. However, due to toxicities, likely attributable to the secreted IL12, further refinement will be necessary before this approach can be safely used in the treatment of cancer patients. Clin Cancer Res; 21(10); 2278–88. ©2015 AACR.

The optimal T-cell attributes for adoptive cancer immunotherapy are unclear. Recent clinical trials of ex vivo–expanded tumor-infiltrating lymphocytes indicated that differentiated T effector cells can elicit durable antitumor responses in some patients with cancer, with their antitumor activity tightly correlated with their persistence in the host. Thus, there is great interest in the definition of intrinsic biomarkers that can predict the conversion of short-lived tumor antigen–specific T effector cells into long-lived T memory cells. Long-term persistence of ex vivo–expanded tumor-specific CD8+ T effector clones has been reported in refractory metastatic melanoma patients after adoptive T-cell transfer. By using highly homogeneous clone populations from these preparations, we performed a comparative transcriptional profiling to define preinfusion molecular attributes that can be ascribed to an effector-to-memory transition. Through this route, we discovered that preinfusion T-cell clones that expressed the IL7 receptor (IL7R) and c-myc were more likely to persist longer after adoptive transfer to patients. The predictive value of these two biomarkers was strengthened by using IL7R protein, IL7-induced pSTAT5, and c-myc mRNA expression to prospectively identify human tumor-specific T effector clones capable of engraftment into immunodeficient mice. Overall, our findings reveal IL7R and c-myc expression as intrinsic biomarkers that can predict the fate of CD8+ T effector cells after adoptive transfer. Cancer Res; 75(16); 3216–26. ©2015 AACR.

Supplementary Table and Figures Supplementary Table 1: Differentially expressed genes between persisting and non-persisting clones. Supplementary Figure 1: % IL-7R expressing cells in the infusion bag correlates with persistence of CD8+ effector clones. Supplementary Figure 2: Representative phenotypic profiling of IL7R+ and IL7R- subsets found within infused effector clones. Supplementary Figure 3: IL-7R expression correlates with c-myc mRNA expression in CD8+ effector clones. Supplementary Figure 4: IL-7R expression correlates with induced pSTAT5 expression in CD8+ effector clones.

Supplementary Methods: Detailed information regarding additional materials and methods.

The majority of lncRNAs’ roles in tumor immunology remain elusive. This project performed a CRISPR activation screening of 9744 lncRNAs in melanoma cells cocultured with human CD8 + T cells. We identified 16 lncRNAs potentially regulating tumor immune response. Further integrative analysis using tumor immunogenomics data revealed that IL10RB-DT and LINC01198 are significantly correlated with tumor immune response and survival in melanoma and breast cancer. Specifically, IL10RB-DT suppresses CD8 + T cells activation via inhibiting IFN-γ–JAK–STAT1 signaling and antigen presentation in melanoma and breast cancer cells. On the other hand, LINC01198 ’s up-regulation sensitizes the killing of tumor cells by CD8 + T cells. Mechanistically, LINC01198 interacts and activates NF-κB component p65 to trigger the type I and type II interferon responses in melanoma and breast cancer cells. Our study systematically characterized novel lncRNAs involved in tumor immune response.

Adoptive cell therapy (ACT) using tumor-specific tumor-infiltrating lymphocytes (TILs) has demonstrated success in patients where tumor-antigen specific TILs can be harvested from the tumor, expanded, and re-infused in combination with a preparatory regimen and IL2. One major issue for non-immunogenic tumors has been that the isolated TILs lack tumor specificity and thus possess limited in vivo therapeutic function. An oncolytic virus (OV) mediates an immunogenic cell death for cancer cells, leading to elicitation and dramatic enhancement of tumor-specific TILs. We hypothesized that the tumor-specific TILs elicited and promoted by an OV would be a great source for ACT for solid cancer. In this study, we show that a local injection of oncolytic poxvirus in MC38 tumor with low immunogenicity in C57BL/6 mice, led to elicitation and accumulation of tumor-specific TILs in the tumor tissue. Our analyses indicated that IL2-armed OV-elicited TILs contain lower quantities of exhausted PD-1hiTim-3+ CD8+ T cells and regulatory T cells. The isolated TILs from IL2-expressing OV-treated tumor tissue retained high tumor specificity after expansion ex vivo. These TILs resulted in significant tumor regression and improved survival after adoptive transfer in mice with established MC38 tumor. Our study showcases the feasibility of using an OV to induce tumor-reactive TILs that can be expanded for ACT.

ObjectivesNovel immunotherapeutic options for refractory metastatic cancer patients include adoptive cell therapies such as tumor infiltrating lymphocytes (TILs). This study characterizes the clinicopathologic findings in a cohort of TIL specimens.MethodsPatients with metastatic malignancy who were eligible had TILs from their metastases grown and expanded and then sent to pathology.ResultsA total of 11 TIL specimens (10 melanoma, 1 adenocarcinoma) from patients enrolled in an experimental clinical trial were reviewed. All specimens showed more than 200 lymphoid cells, stained positive for lymphoid markers confirming an activated cytotoxic T-cell immunophenotype, and morphologically showed an intermediate-sized population with immature chromatin and frequent mitoses. Six cases (55%) showed large cells with nucleomegaly and prominent nucleoli.ConclusionsThis report is the first describing cytopathologic findings of autologous TIL therapy including adequacy guidelines and expected cytomorphologic and immunophenotypic findings. To meet this novel clinical demand, a predefined cytology protocol to rapidly process and interpret these specimens needs to be established.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) has been tested in advanced melanoma patients at various centers. We conducted a systematic review and meta-analysis to assess its efficacy on previously treated advanced metastatic cutaneous melanoma. The PubMed electronic database was searched from inception to 17 December 2018 to identify studies administering TIL-ACT and recombinant interleukin-2 (IL-2) following non-myeloablative chemotherapy in previously treated metastatic melanoma patients. Objective response rate (ORR) was the primary end point. Secondary end points were complete response rate (CRR), overall survival (OS), duration of response (DOR) and toxicity. Pooled estimates were derived from fixed or random effect models, depending on the amount of heterogeneity detected. Analysis was carried out separately for high dose (HD) and low dose (LD) IL-2. Sensitivity analyses were carried out. Among 1211 records screened, 13 studies (published 1988 − 2016) were eligible for meta-analysis. Among 410 heavily pretreated patients (some with brain metastasis), 332 received HD-IL-2 and 78 LD-IL-2. The pooled overall ORR estimate was 41% [95% confidence interval (CI) 35% to 48%], and the overall CRR was 12% (95% CI 7% to 16%). For the HD-IL-2 group, the ORR was 43% (95% CI 36% to 50%), while for the LD-IL-2 it was 35% (95% CI 25% to 45%). Corresponding pooled estimates for CRR were 14% (95% CI 7% to 20%) and 7% (95% CI 1% to 12%). The majority of HD-IL-2 complete responders (27/28) remained in remission during the extent of follow-up after CR (median 40 months). Sensitivity analyses yielded similar results. Higher number of infused cells was associated with a favorable response. The ORR for HD-IL-2 compared favorably with the nivolumab/ipilimumab combination following anti-PD-1 failure. TIL-ACT therapy, especially when combined with HD-IL-2, achieves durable clinical benefit and warrants further investigation. We discuss the current position of TIL-ACT in the therapy of advanced melanoma, particularly in the era of immune checkpoint blockade therapy, and review future opportunities for improvement of this approach.

While T cell-based cancer immunotherapies have shown great promise, there remains a need to understand how individual metastatic tumor environments impart local T cell dysfunction. At advanced stages, cancers that metastasize to the pleural space can result in a malignant pleural effusion (MPE) that harbors abundant tumor and immune cells, often exceeding 108 leukocytes per liter. Unlike other metastatic sites, MPEs are readily and repeatedly accessible via indwelling catheters, providing an opportunity to study the interface between tumor dynamics and immunity. In the current study, we examined CD8+ T cells within MPEs collected from patients with heterogeneous primary tumors and at various stages in treatment to determine (1) if these cells possess anti-tumor activity following removal from the MPE, (2) factors in the MPE that may contribute to their dysfunction, and (3) the phenotypic changes in T cell populations that occur following ex vivo expansion. Co-cultures of CD8+ T cells with autologous CD45― tumor containing cells demonstrated cytotoxicity (p = 0.030) and IFN&gamma, production (p = 0.003) that inversely correlated with percent of myeloid derived suppressor cells, lactate, and lactate dehydrogenase (LDH) within the MPE. Ex vivo expansion of CD8+ T cells resulted in progressive differentiation marked by distinct populations expressing decreased CD45RA, CCR7, CD127, and increased inhibitory receptors. These findings suggest that MPEs may be a source of tumor-reactive T cells and that the cellular and acellular components suppress optimal function.

EPIC1-EZH2 axis activation emerges as a targetable mechanism for tumor immune evasion and immunotherapy resistance., Through an integrative analysis of the lincRNA expression and tumor immune response in 9,626 tumor samples across 32 cancer types, we developed a lincRNA-based immune response (LIMER) score that can predict the immune cells infiltration and patient prognosis in multiple cancer types. Our analysis also identified tumor-specific lincRNAs, including EPIC1, that potentially regulate tumor immune response in multiple cancer types. Immunocompetent mouse models and in vitro co-culture assays demonstrated that EPIC1 induces tumor immune evasion and resistance to immunotherapy by suppressing tumor cell antigen presentation. Mechanistically, lincRNA EPIC1 interacts with the histone methyltransferase EZH2, leading to the epigenetic silencing of IFNGR1, TAP1/2, ERAP1/2, and MHC-I genes. Genetic and pharmacological inhibition of EZH2 abolish EPIC1’s immune-related oncogenic effect and its suppression of interferon-γ signaling. The EPIC1-EZH2 axis emerges as a potential mechanism for tumor immune evasion that can serve as therapeutic targets for immunotherapy.

Brain metastases cause significant morbidity and mortality in patients with metastatic melanoma. Although adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) can achieve complete and durable remission of advanced cutaneous melanoma, the efficacy of this therapy for brain metastases is unclear. Records of patients with M1c melanoma treated with ACT using TIL, including patients with treated and untreated brain metastases, were analyzed. Treatment consisted of preparative chemotherapy, autologous TIL infusion, and high-dose interleukin-2. Treatment outcomes, sites of initial tumor progression, and overall survival were analyzed. Among 144 total patients, 15 patients with treated and 18 patients with untreated brain metastases were identified. Intracranial objective responses (OR) occurred in 28% patients with untreated brain metastases. The systemic OR rates for patients with M1c disease without identified brain disease, treated brain disease, and untreated brain disease, and were 49%, 33% and 33%, respectively, of which 59%, 20% and 16% were durable at last follow-up. The site of untreated brain disease was the most likely site of initial tumor progression (61%) in patients with untreated brain metastases. Overall, we found that ACT with TIL can eliminate small melanoma brain metastases. However, following TIL therapy these patients frequently progress in the brain at a site of untreated brain disease. Patients with treated or untreated brain disease are less likely to achieve durable systemic ORs following TIL therapy compared with M1c disease and no history of brain disease. Melanoma brain metastases likely require local therapy despite the systemic effect of ACT.

Summary Background Uveal melanoma is a rare tumour with no established treatments once metastases develop. Although a variety of immune-based therapies have shown efficacy in metastatic cutaneous melanoma, their use in ocular variants has been disappointing. Recently, adoptive T-cell therapy has shown salvage responses in multiple refractory solid tumours. Thus, we sought to determine if adoptive transfer of autologous tumour-infiltrating lymphocytes (TILs) could mediate regression of metastatic uveal melanoma. Methods In this ongoing single-centre, two-stage, phase 2, single-arm trial, patients (aged ≥16 years) with histologically confirmed metastatic ocular melanoma were enrolled. Key eligibility criteria were an Eastern Cooperative Oncology Group performance status of 0 or 1, progressive metastatic disease, and adequate haematological, renal, and hepatic function. Metastasectomies were done to procure tumour tissue to generate autologous TIL cultures, which then underwent large scale ex-vivo expansion. Patients were treated with lymphodepleting conditioning chemotherapy (intravenous cyclophosphamide [60 mg/kg] daily for 2 days followed by fludarabine [25 mg/m 2 ] daily for 5 days, followed by a single intravenous infusion of autologous TILs and high-dose interleukin-2 [720 000 IU/kg] every 8 h). The primary endpoint was objective tumour response in evaluable patients per protocol using Response to Evaluation Criteria in Solid Tumors, version 1.0. An interim analysis of this trial is reported here. The trial is registered at ClinicalTrials.gov, number NCT01814046. Findings From the completed first stage and ongoing expansion stage of this trial, a total of 21 consecutive patients with metastatic uveal melanoma were enrolled between June 7, 2013, and Sept 9, 2016, and received TIL therapy. Seven (35%, 95% CI 16–59) of 20 evaluable patients had objective tumour regression. Among the responders, six patients achieved a partial response, two of which are ongoing and have not reached maximum response. One patient achieved complete response of numerous hepatic metastases, currently ongoing at 21 months post therapy. Three of the responders were refractory to previous immune checkpoint blockade. Common grade 3 or worse toxic effects were related to the lymphodepleting chemotherapy regimen and included lymphopenia, neutropenia, and thrombocytopenia (21 [100%] patients for each toxicity); anaemia (14 [67%] patients); and infection (six [29%] patients). There was one treatment-related death secondary to sepsis-induced multiorgan failure. Interpretation To our knowledge, this is the first report describing adoptive transfer of autologous TILs to mediate objective tumour regression in patients with metastatic uveal melanoma. These initial results challenge the belief that metastatic uveal melanoma is immunotherapy resistant and support the further investigation of immune-based therapies for this cancer. Refinement of this T-cell therapy is crucial to improve the frequency of clinical responses and the general applicability of this treatment modality. Funding Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Background The use of routine CT imaging for surveillance in asymptomatic patients with cutaneous melanoma is controversial. We report our experience using a surveillance strategy that included CT imaging for a cohort of patients with high-risk melanoma. Methods A total of 466 patients with high-risk cutaneous melanoma enrolled in adjuvant immunotherapy trials were followed for tumor progression by physical examination, labs, and CT imaging as defined by protocol. Evaluations were obtained at least every 6 months for year 1, every 6 months for year 2, and then annually for the remainder of the 5-year study. Time to tumor progression, sites of recurrence, and the method of relapse detection were identified. Results The patient cohort consisted of 115 stage II patients, 328 stage III patients, and 23 patients with resected stage IV melanoma. The medium time to progression for the 225 patients who developed tumor progression was 7 months. Tumor progression was detected by patients, physician examination or routine labs, or by CT imaging alone in 27, 14, and 59% of cases respectively. Melanoma recurrences were noted to be locoregional in 36% of cases and systemic in 64% of cases. Thirty percent of patients with locoregional relapse and 75% of patients with systemic relapse were detected solely by CT imaging. Conclusions CT imaging alone detected the majority of sites of disease progression in our patients with high-risk cutaneous melanoma. This disease was not heralded by symptoms, physical examination, or blood work. Although the benefit of the early detection of advanced melanoma is unknown, this experience is relevant because of the rapid development and availability of potentially curative immunotherapies.

Background Immunotherapeutic treatment strategies including adoptive cell transfer (ACT) for metastatic melanoma are capable of mediating complete and durable responses, as well as partial responses and prolonged disease stabilization. Unfortunately, many patients ultimately develop progressive disease. The role of salvage metastasectomy in managing these patients has not been evaluated. Methods Records of patients with metastatic melanoma treated with ACT at a single institution between 2000 and 2014 were reviewed. Patients with an objective response by RECIST criteria or disease stabilization of at least 6 months and who subsequently developed progressive melanoma and were managed with metastasectomy as the next therapeutic strategy were studied for progression-free survival (PFS) and overall survival (OS). Five additional clinical parameters were also reviewed for association with outcomes. Results Of 115 patients treated with ACT who met our response criteria and then developed progressive disease, 26 (23%) had surgery. There were no mortalities related to surgical intervention. Median follow-up after surgery was 62 months. Median PFS after surgery was 11 months and five-year OS was 57%. The development of a new site of metastasis after ACT was associated with poor PFS and OS. Conclusions Surgery after immunotherapy is safe. Long PFS and OS can be achieved by metastasectomy in selected patients with progressive melanoma following treatment with ACT. Clinical variables important for patient selection for metastasectomy after immunotherapy remain largely undefined. Improvements in immunotherapeutic treatment strategies may increase the role of surgery for patients with advanced disease.

Purpose: Uveal melanoma is a rare melanoma variant with no effective therapies once metastases develop. Although durable cancer regression can be achieved in metastatic cutaneous melanoma with immunotherapies that augment naturally existing antitumor T-cell responses, the role of these treatments for metastatic uveal melanoma remains unclear. We sought to define the relative immunogenicity of these two melanoma variants and determine whether endogenous antitumor immune responses exist against uveal melanoma. Experimental Design: We surgically procured liver metastases from uveal melanoma (n = 16) and cutaneous melanoma (n = 35) patients and compared the attributes of their respective tumor cell populations and their infiltrating T cells (TIL) using clinical radiology, histopathology, immune assays, and whole-exomic sequencing. Results: Despite having common melanocytic lineage, uveal melanoma and cutaneous melanoma metastases differed in their melanin content, tumor differentiation antigen expression, and somatic mutational profile. Immunologic analysis of TIL cultures expanded from these divergent forms of melanoma revealed cutaneous melanoma TIL were predominantly composed of CD8+ T cells, whereas uveal melanoma TIL were CD4+ dominant. Reactivity against autologous tumor was significantly greater in cutaneous melanoma TIL compared with uveal melanoma TIL. However, we identified TIL from a subset of uveal melanoma patients which had robust antitumor reactivity comparable in magnitude with cutaneous melanoma TIL. Interestingly, the absence of melanin pigmentation in the parental tumor strongly correlated with the generation of highly reactive uveal melanoma TIL. Conclusions: The discovery of this immunogenic group of uveal melanoma metastases should prompt clinical efforts to determine whether patients who harbor these unique tumors can benefit from immunotherapies that exploit endogenous antitumor T-cell populations. Clin Cancer Res; 22(9); 2237–49. ©2015 AACR.

Purpose: Cellular therapy is an emerging cancer treatment modality, but its application to epithelial cancers has been limited. This clinical trial evaluated tumor-infiltrating lymphocyte (TIL) therapy for the treatment of patients with metastatic human papillomavirus (HPV)-associated carcinomas. Patients and Methods: The trial was a phase II design with two cohorts, cervical cancers and noncervical cancers. Cell infusion was preceded by a lymphocyte-depleting conditioning regimen and followed by systemic high-dose aldesleukin. Results: Objective tumor responses occurred in 5 of 18 (28%) patients in the cervical cancer cohort and 2 of 11 (18%) patients in the noncervical cancer cohort. Two of the responses in cervical cancer were complete and are ongoing 67 and 53 months after treatment. Responses in the noncervical cancer cohort were in anal cancer and oropharyngeal cancer. The HPV reactivity of the infused T cells correlated with clinical response. Peripheral blood repopulation with HPV-reactive T cells also correlated with clinical response. Conclusions: These findings support the concept that cellular therapy can mediate the regression of epithelial cancers, and they suggest the importance of predictive biomarkers and novel treatment platforms for more effective therapies.