Description: Purpose: Cultured tumor fragments from melanoma metastases have been used for years as a source of tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy (ACT). The expansion of tumor-reactive CD8 + T cells with interleukin-2 (IL2) in these early cultures is critical in generating clinically active TIL infusion products, with a population of activated 4-1BB CD8 + T cells recently found to constitute the majority of tumor-specific T cells. Experimental Design: We used an agonistic anti–4-1BB antibody added during the initial tumor fragment cultures to provide in situ 4-1BB costimulation. Results: We found that addition of an agonistic anti–4-1BB antibody could activate 4-1BB signaling within early cultured tumor fragments and accelerated the rate of memory CD8 + TIL outgrowth that were highly enriched for melanoma antigen specificity. This was associated with NFB activation and the induction of T-cell survival and memory genes, as well as enhanced IL2 responsiveness, in the CD8 + T cells in the fragments and emerging from the fragments. Early provision of 4-1BB costimulation also affected the dendritic cells (DC) by activating NFB in DC and promoting their maturation inside the tumor fragments. Blocking HLA class I prevented the enhanced outgrowth of CD8 + T cells with anti–4-1BB, suggesting that an ongoing HLA class I–mediated antigen presentation in early tumor fragment cultures plays a role in mediating tumor-specific CD8 + TIL outgrowth. Conclusions: Our results highlight a previously unrecognized concept in TIL ACT that the tumor microenvironment can be dynamically regulated in the initial tumor fragment cultures to regulate the types of T cells expanded and their functional characteristics. Clin Cancer Res; 21(3); 611–21. ©2014 AACR.

Description: Purpose: The anti-programmed death-1 (PD-1) antibody nivolumab (BMS-936558) has clinical activity in patients with metastatic melanoma. Nivolumab plus vaccine was investigated as adjuvant therapy in resected stage IIIC and IV melanoma patients. Experimental Design: HLA-A*0201 positive patients with HMB-45, NY-ESO-1, and/or MART-1 positive resected tumors received nivolumab (1 mg/kg, 3 mg/kg, or 10 mg/kg i.v.) with a multi-peptide vaccine (gp100, MART-1, and NY-ESO-1 with Montanide ISA 51 VG) every 2 weeks for 12 doses followed by nivolumab maintenance every 12 weeks for 8 doses. Primary objective was safety and determination of a maximum tolerated dose (MTD). Secondary objectives included relapse-free survival (RFS), overall survival (OS), and immunologic correlative studies. Results: Thirty-three patients were enrolled. Median age was 47 years; 55% were male. Two patients had stage IIIC disease; 31 patients had stage IV disease. Median follow-up was 32.1 months. MTD was not reached. Most common related adverse events (〉40%) were vaccine injection site reaction, fatigue, rash, pruritus, nausea, and arthralgias. Five related grade 3 adverse events [hypokalemia (1), rash (1), enteritis (1), and colitis (2)] were observed. Ten of 33 patients relapsed. Estimated median RFS was 47.1 months; median OS was not reached. Increases in CTLA-4 + /CD4 + , CD25 + Treg/CD4 + , and tetramer specific CD8 + T-cell populations were observed with treatment ( P 〈 0.05). Trends for lower baseline myeloid-derived suppressor cell and CD25 + Treg/CD4 + populations were seen in nonrelapsing patients; PD-L1 tumor status was not significantly associated with RFS. Conclusions: Nivolumab with vaccine is well tolerated as adjuvant therapy and demonstrates immunologic activity with promising survival in high-risk resected melanoma, justifying further study. Clin Cancer Res; 21(4); 712–20. ©2014 AACR .

Description: Purpose: We decided to construct a novel oncolytic adenovirus whose replication was driven by the CDC25B promoter for its use in preclinical models of pancreatic cancer. Experimental Design: We placed the essential E1A gene under control of the CDC25B promoter. Based on preliminary data, we pseudotyped the adenovirus with a chimeric fiber of serotypes 5/3. We investigated the in vitro lytic effect and the in vivo therapeutic efficacy in combination with gemcitabine on human pancreatic tumor xenografts orthotopically growing in nude mice and in tumors growing in Syrian hamsters. We also assessed biochemical markers of hepatic toxicity and CA19.9 levels. Results: AV25CDC exhibited a strong in vitro lytic effect on pancreatic cancer cells. In vivo administration of AV25CDC combined with gemcitabine in mice harboring subcutaneously growing SW1990 pancreatic tumors almost abrogated tumor growth. Nude mice harboring 15-day-old orthotopic tumors, treated intratumorally or systemically with AV25CDC combined with gemcitabine, exhibited 70% to 80% reduction in tumor size compared with control mice that lasted for at least 60 days. Chemovirotherapy treatment induced a return to normal levels of biochemical parameters of hepatic toxicity; these mice exhibited more than 90% reduction in CA19.9 serum levels compared with control. Chemovirotherapy efficacy was confirmed in mice harboring Mia PaCa-2 tumors and in Syrian hamster harboring HaP-T1 tumors. We observed that viral treatment disrupted tumor architecture and induced an increase in MMP-9 activity that might facilitate gemcitabine penetrability. Conclusion: These data demonstrate that AV25CDC is an effective oncolytic agent candidate for pancreatic cancer chemovirotherapy combination. Clin Cancer Res; 21(7); 1665–74. ©2015 AACR .

Description: Purpose: Although durable responses can be achieved with tyrosine kinase inhibitors such as imatinib in melanomas harboring KIT mutations, the efficacy of alternative inhibitors after progression to imatinib and the activity of these agents on brain metastases are unknown. Experimental Design: We conducted a phase II study of nilotinib 400 mg twice a day in two cohorts of patients with melanomas harboring KIT mutations or amplification: (A) those refractory or intolerant to a prior KIT inhibitor; and (B) those with brain metastases. The primary endpoint was 4-month disease control rate. Secondary endpoints included response rate, time-to-progression (TTP), and overall survival (OS). A Simon two-stage and a single-stage design was planned to assess for the primary endpoint in cohorts A and B, respectively. Results: Twenty patients were enrolled and 19 treated (11 in cohort A; 8 in cohort B). Three patients on cohort A [27%; 95% confidence interval (CI), 8%–56%] and 1 on cohort B (12.5%; 90% CI, 0.6%–47%) achieved the primary endpoint. Two partial responses were observed in cohort A (18.2%; 90% CI, 3%–47%); none were observed in cohort B. The median TTP and OS was 3.3 (90% CI, 2.1–3.9 months) and 9.1 months (90% CI, 4.3–14.2 months), respectively, in all treated patients. Conclusions: Nilotinib may achieve disease control in patients with melanoma harboring KIT alterations and whose disease progressed after imatinib therapy. The efficacy of this agent in KIT-altered melanoma with brain metastasis is limited. Clin Cancer Res; 21(10); 2289–96. ©2015 AACR .

Description: Purpose: Adoptive transfer of Epstein–Barr virus (EBV)–specific and cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) genetically modified to express a chimeric antigen receptor (CAR) induces objective tumor responses in clinical trials. In vivo expansion and persistence of these cells are crucial to achieve sustained clinical responses. We aimed to develop an off-the-shelf whole-cell vaccine to boost CAR-redirected virus-specific CTLs in vivo after adoptive transfer. As proof of principle, we validated our vaccine approach by boosting CMV-specific CTLs (CMV-CTLs) engineered with a CAR that targets the GD2 antigen. Experimental Design: We generated the whole-cell vaccine by engineering the K562 cell line to express the CMV-pp65 protein and the immune stimulatory molecules CD40L and OX40L. Single-cell–derived clones were used to stimulate CMV-CTLs in vitro and in vivo in a xenograft model. We also assessed whether the in vivo boosting of CAR-redirected CMV-CTLs with the whole-cell vaccine enhances the antitumor responses. Finally, we addressed potential safety concerns by including the inducible safety switch caspase9 ( iC9 ) gene in the whole-cell vaccine. Results: We found that K562-expressing CMV-pp65, CD40L, and OX40L effectively stimulate CMV-specific responses in vitro by promoting antigen cross-presentation to professional antigen-presenting cells (APCs). Vaccination also enhances antitumor effects of CAR-redirected CMV-CTLs in xenograft tumor models. Activation of the iC9 gene successfully induces growth arrest of engineered K562 implanted in mice. Conclusions: Vaccination with a whole-cell vaccine obtained from K562 engineered to express CMV-pp65, CD40L, OX40L and iC9 can safely enhance the antitumor effects of CAR-redirected CMV-CTLs. Clin Cancer Res; 21(13); 2952–62. ©2015 AACR .

Description: Because of its heterogeneity, lack of prognostic markers, tumor-escape mechanisms, and frequent relapse upon surgical intervention, treatment of hepatocellular carcinoma (HCC) remains challenging. In this issue of Clinical Cancer Research , Groß and colleagues characterize a rodent model that might help identify novel drugs for combinatorial sorafenib-based therapies for HCC. Clin Cancer Res; 21(19); 4254–6. ©2015 AACR . See related article by Groß et al., p. 4440

Description: Purpose: Adenoid cystic carcinoma (ACC) is an indolent salivary gland malignancy, characterized by t(6;9) translocations and MYB – NFIB gene fusions in approximately 50% of the tumors. The genetic alterations underlying t(6;9)-negative and t(6;9)-positive/ MYB–NFIB fusion–negative ACC remain unknown. To uncover the genetic alterations in ACC lacking the canonical translocation and fusion transcript and identify new abnormalities in translocation positive tumors. Experimental Design: We performed whole-genome sequencing in 21 salivary ACCs and conducted targeted molecular analyses in a validation set (81 patients). Microarray gene-expression data were also analyzed to explore the biologic differences between fusion positive and negative tumors. Results: We identified a novel MYBL1–NFIB gene fusion as a result of t(8;9) translocation and multiple rearrangements in the MYBL1 gene in 35% of the t(6;9)-negative ACCs. All MYBL1 alterations involved deletion of the C-terminal negative regulatory domain and were associated with high MYBL1 expression. Reciprocal MYB and MYBL1 expression was consistently found in ACCs. In addition, 5'- NFIB fusions that did not involve MYB / MYBL1 genes were identified in a subset of t(6;9)-positive/fusion-negative tumors. We also delineated distinct gene-expression profiles in ACCs associated with the length of the MYB or MYBL1 fusions, suggesting a biologic importance of the C-terminal part of these fusions. Conclusions: Our study defines new molecular subclasses of ACC characterized by MYBL1 rearrangements and 5'- NFIB gene fusions. Clin Cancer Res; 22(3); 725–33. ©2015 AACR .

Description: Purpose: Lenalidomide, thalidomide, and pomalidomide (LTP) are immunomodulatory agents approved for use in multiple myeloma, but in some settings, especially with alkylating agents, an increase in Hodgkin lymphoma and other secondary primary malignancies (SPM) has been noted. Some of these malignancies have been linked to Epstein–Barr virus (EBV), raising the possibility that immunomodulatory drugs disrupt latent EBV infection. Experimental Design: We studied the ability of LTP to reactivate latently infected EBV-positive cell lines in vitro and in vivo , and evaluated the EBV viral load in archived serum samples from patients who received a lenalidomide, thalidomide, and dexamethasone (LTD) combination. Results: Treatment of EBV-infected B-cell lines with LTP at physiologically relevant concentrations induced the immediate early gene BZLF1 , the early gene BMRF1 , and the late proteins VCA and BCFR1. This occurred in the potency order pomalidomide 〉 lenalidomide 〉 thalidomide, and the nucleoside analogue ganciclovir enhanced the cytotoxic effects of lenalidomide and pomalidomide in Burkitt lymphoma cells in vitro and in vivo . EBV reactivation was related to PI3K stimulation and Ikaros suppression, and blocked by the PI3K inhibitor idelalisib. Combinations of lenalidomide with dexamethasone or rituximab increased EBV reactivation compared with lenalidomide alone and, importantly, lenalidomide with melphalan produced even greater reactivation. Conclusions: We conclude LTP may reactivate EBV-positive resting memory B cells thereby enhancing EBV lytic cycle and host immune suppression. Clin Cancer Res; 22(19); 4901–12. ©2016 AACR .

Description: Purpose: BRAF inhibitors are clinically active in patients with advanced BRAF V600 -mutant melanoma, although acquired resistance remains common. Preclinical studies demonstrated that resistance could be overcome using concurrent treatment with the HSP90 inhibitor XL888. Patients and Methods: Vemurafenib (960 mg p.o. b.i.d.) combined with escalating doses of XL888 (30, 45, 90, or 135 mg p.o. twice weekly) was investigated in 21 patients with advanced BRAF V600 -mutant melanoma. Primary endpoints were safety and determination of a maximum tolerated dose. Correlative proteomic studies were performed to confirm HSP inhibitor activity. Results: Objective responses were observed in 15 of 20 evaluable patients [75%; 95% confidence interval (CI), 51%–91%], with 3 complete and 12 partial responses. Median progression-free survival and overall survival were 9.2 months (95% CI, 3.8–not reached) and 34.6 months (6.2–not reached), respectively. The most common grade 3/4 toxicities were skin toxicities, such as rash ( n = 4, 19%) and cutaneous squamous cell carcinomas ( n = 3, 14%), along with diarrhea ( n = 3, 14%). Pharmacodynamic analysis of patients' peripheral blood mononuclear cells (PBMC) showed increased day 8 HSP70 expression compared with baseline in the three cohorts with XL888 doses ≥45 mg. Diverse effects of vemurafenib-XL888 upon intratumoral HSP client protein expression were noted, with the expression of multiple proteins (including ERBB3 and BAD) modulated on therapy. Conclusions: XL888 in combination with vemurafenib has clinical activity in patients with advanced BRAF V600 -mutant melanoma, with a tolerable side-effect profile. HSP90 inhibitors warrant further evaluation in combination with current standard-of-care BRAF plus MEK inhibitors in BRAF V600 -mutant melanoma. Clin Cancer Res; 24(22); 5516–24. ©2018 AACR . See related commentary by Sullivan, p. 5496

Description: Purpose: The purpose of this study was to assess the association of baseline tumor size (BTS) with other baseline clinical factors and outcomes in pembrolizumab-treated patients with advanced melanoma in KEYNOTE-001 (NCT01295827). Experimental Design: BTS was quantified by adding the sum of the longest dimensions of all measurable baseline target lesions. BTS as a dichotomous and continuous variable was evaluated with other baseline factors using logistic regression for objective response rate (ORR) and Cox regression for overall survival (OS). Nominal P values with no multiplicity adjustment describe the strength of observed associations. Results: Per central review by RECIST v1.1, 583 of 655 patients had baseline measurable disease and were included in this post hoc analysis. Median BTS was 10.2 cm (range, 1–89.5). Larger median BTS was associated with Eastern Cooperative Oncology Group performance status 1, elevated lactate dehydrogenase (LDH), stage M1c disease, and liver metastases (with or without any other sites; all P ≤ 0.001). In univariate analyses, BTS below the median was associated with higher ORR (44% vs. 23%; P 〈 0.001) and improved OS (HR, 0.38; P 〈 0.001). In multivariate analyses, BTS below the median remained an independent prognostic marker of OS ( P 〈 0.001) but not ORR. In 459 patients with available tumor programmed death ligand 1 (PD-L1) expression, BTS below the median and PD-L1–positive tumors were independently associated with higher ORR and longer OS. Conclusions: BTS is associated with many other baseline clinical factors but is also independently prognostic of survival in pembrolizumab-treated patients with advanced melanoma. Clin Cancer Res; 24(20); 4960–7. ©2018 AACR . See related commentary by Warner and Postow, p. 4915