Description: Purpose: A CTEP-sponsored phase II trial was conducted to evaluate safety and clinical activity of combination therapy with CCI-779 (temsirolimus) and bevacizumab in patients with advanced melanoma. Experimental Design: Patients with unresectable stage III to IV melanoma were treated intravenously with temsirolimus 25 mg weekly and bevacizumab 10 mg every 2 weeks. Adverse events were recorded using CTCAE v3.0. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors and overall survival was recorded. Correlative studies measured protein kinases and histology of tumor biopsies and immune function in peripheral blood. Results: Seventeen patients were treated. Most patients tolerated treatment well, but 2 had grade 4 lymphopenia and 1 developed reversible grade 2 leukoencephalopathy. Best clinical response was partial response (PR) in 3 patients [17.7%, 90% confidence interval (CI) 5, 0–39.6], stable disease at 8 weeks (SD) in 9 patients, progressive disease (PD) in 4 patients, and not evaluable in 1 patient. Maximal response duration for PR was 35 months. Ten evaluable patients had BRAF WT tumors, among whom 3 had PRs, 5 had SD, and 2 had PD. Correlative studies of tumor biopsies revealed decreased phospho-S6K (d2 and d23 vs. d1, P 〈 0.001), and decreased mitotic rate (Ki67 + ) among melanoma cells by d23 ( P = 0.007). Effects on immune functions were mixed, with decreased alloreactive T-cell responses and decreased circulating CD4 + FoxP3 + cells. Conclusion: These data provide preliminary evidence for clinical activity of combination therapy with temsirolimus and bevacizumab, which may be greater in patients with BRAF wt melanoma. Mixed effects on immunologic function also support combination with immune therapies. Clin Cancer Res; 19(13); 3611–20. ©2013 AACR .

Description: Toll-like receptors (TLR) are overexpressed on many types of cancer cells, including colorectal cancer cells, but little is known about the functional relevance of these immune regulatory molecules in malignant settings. Here, we report frequent single-nucleotide polymorphisms (SNP) in the flagellin receptor TLR5 and the TLR downstream effector molecules MyD88 and TIRAP that are associated with altered survival in a large cohort of Caucasian patients with colorectal cancer (n = 613). MYD88 rs4988453, a SNP that maps to a promoter region shared with the acetyl coenzyme-A acyl-transferase-1 (ACAA1), was associated with decreased survival of patients with colorectal cancer and altered transcriptional activity of the proximal genes. In the TLR5 gene, rs5744174/F616L was associated with increased survival, whereas rs2072493/N592S was associated with decreased survival. Both rs2072493/N592S and rs5744174/F616L modulated TLR5 signaling in response to flagellin or to different commensal and pathogenic intestinal bacteria. Notably, we observed a reduction in flagellin-induced p38 phosphorylation, CD62L shedding, and elevated expression of interleukin (IL)-6 and IL-1β mRNA in human primary immune cells from TLR5 616LL homozygote carriers, as compared with 616FF carriers. This finding suggested that the well-documented effect of cytokines like IL-6 on colorectal cancer progression might be mediated by TLR5 genotype-dependent flagellin sensing. Our results establish an important link between TLR signaling and human colorectal cancer with relevance for biomarker and therapy development. Cancer Res; 73(24); 7232–42. ©2013 AACR.

Description: Purpose: To investigate the mechanism(s) of resistance to the RAF-inhibitor vemurafenib, we conducted a comprehensive analysis of the genetic alterations occurring in metastatic lesions from a patient with a BRAF V600E -mutant cutaneous melanoma who, after a first response, underwent subsequent rechallenge with this drug. Experimental Design: We obtained blood and tissue samples from a patient diagnosed with a BRAF V600E -mutant cutaneous melanoma that was treated with vemurafenib and achieved a near-complete response. At progression, he received additional lines of chemo/immunotherapy and was successfully rechallenged with vemurafenib. Exome and RNA sequencing were conducted on a pretreatment tumor and two subcutaneous resistant metastases, one that was present at baseline and previously responded to vemurafenib (PV1) and one that occurred de novo after reintroduction of the drug (PV2). A culture established from PV1 was also analyzed. Results: We identified two NRAS-activating somatic mutations, Q61R and Q61K, affecting two main subpopulations in the metastasis PV1 and a BRAF alternative splicing, involving exons 4–10, in the metastasis PV2. These alterations, known to confer resistance to RAF inhibitors, were tumor-specific, mutually exclusive, and were not detected in pretreatment tumor samples. In addition, the oncogenic PIK3CA H1047R mutation was detected in a subpopulation of PV1, but this mutation did not seem to play a major role in vemurafenib resistance in this metastasis. Conclusions: This work describes the coexistence within the same patient of different molecular mechanisms of resistance to vemurafenib affecting different metastatic sites. These findings have direct implications for the clinical management of BRAF-mutant melanoma. Clin Cancer Res; 19(20); 5749–57. ©2013 AACR .

Description: Autophagy attenuates the efficacy of conventional chemotherapy but its effects on immunotherapy have been little studied. Here, we report that chemotherapy renders tumor cells more susceptible to lysis by CTL in vivo. Moreover, bystander tumor cells that did not express antigen were killed by CTL. This effect was mediated by transient but dramatic upregulation of the mannose-6-phosphate receptor (MPR) on the tumor cell surface. Antitumor effects of combined treatment related to the kinetics of MPR upregulation and abrogation of this event abolished the combined effect of immunotherapy and chemotherapy. MPR accumulation on the tumor cell surface during chemotherapy was observed in different mouse tumor models and in patients with multiple myeloma. Notably, this effect was the result of redistribution of the receptor caused by chemotherapy-inducible autophagy. Together, our findings reveal one molecular mechanism through which the antitumor effects of conventional cancer chemotherapy and immunotherapy are realized. Cancer Res; 72(21); 5483–93. ©2012 AACR.

Description: Purpose: This retrospective analysis was conducted to characterize ipilimumab exposure–response relationships for measures of efficacy and safety in patients with advanced melanoma. Experimental Design: Data were pooled from 498 patients who received ipilimumab monotherapy at 0.3, 3, or 10 mg/kg in 1 of 4 completed phase II clinical trials. The relationships between steady–state ipilimumab trough concentration (Cminss), complete or partial tumor response (CR or PR), and safety [immune-related adverse events (irAEs)] were described by logistic regression models. The relationship between exposure and overall survival was characterized using a Cox proportional–hazards model. Results: The steady-state trough concentration of ipilimumab was found to be a significant predictor of a CR or PR ( P 〈 0.001). Model-based estimates indicate that the probabilities of a CR or PR at median Cminss for the 0.3, 3, and 10 mg/kg groups were 0.6%, 4.9%, and 11.6%, respectively. Overall survival at the median Cminss for ipilimumab at 0.3 mg/kg was estimated to be 0.85- and 0.58-fold lower relative to that at the median Cminss for 3 and 10 mg/kg, respectively. Model-based estimates indicate that the probabilities of a grade 3 or more irAE at the median Cminss for the 0.3, 3, and 10 mg/kg doses were 3%, 13%, and 24%, respectively. Conclusions: Higher doses of ipilimumab produce greater Cminss that may be associated with increased tumor responses, longer survival, and higher rates of irAEs. The efficacy and safety of ipilimumab at 3 versus 10 mg/kg in patients with advanced melanoma is being evaluated in an ongoing phase III trial. Clin Cancer Res; 19(14); 3977–86. ©2013 AACR .

Description: Purpose: Although modern cure rates for childhood acute lymphoblastic leukemia (ALL) exceed 80%, the outlook remains poor in patients with high-risk disease and those who relapse, especially when allogeneic hematopoietic stem cell transplantation is not feasible. Strategies to improve outcome and prevent relapse are therefore required. Immunotherapy with antigen-specific T cells can have antileukemic activity without the toxicities seen with intensive chemotherapy, and therefore represents an attractive strategy to improve the outcome of high-risk patients with ALL. We explored the feasibility of generating tumor antigen-specific T cells ex vivo from the peripheral blood of 50 patients with ALL [26 National Cancer Institute (NCI) high-risk and 24 standard-risk] receiving maintenance therapy. Experimental Design: Peripheral blood mononuclear cells were stimulated with autologous dendritic cells pulsed with complete peptide libraries of WT1, Survivin, MAGE-A3, and PRAME, antigens frequently expressed on ALL blasts. Results: T-cell lines were successfully expanded from all patients, despite low lymphocyte counts and irrespective of NCI risk group. Antigen-specificity was observed in more than 50% of patients after the initial stimulation and increased to more than 90% after three stimulations as assessed in IFN--enzyme-linked immunospot (ELISpot) and 51 Cr-release assays. Moreover, tumor-specific responses were observed by reduction of autologous leukemia blasts in short- and long-term coculture experiments. Conclusion: This study supports the use of immunotherapy with adoptively transferred autologous tumor antigen-specific T cells to prevent relapse and improve the prognosis of patients with high-risk ALL. Clin Cancer Res; 19(18); 5079–91. ©2013 AACR .

Description: Mutations in TP53 are common in non–small cell lung cancer. Apart from the loss of tumor-suppressor functions, TP53 mutations may result in gain of function favoring cellular proliferation, inhibition of apoptosis, and genomic instability. Some TP53 mutations are more likely to affect the course of the disease than others. Clin Cancer Res; 20(17); 4419–21. ©2014 AACR .

Description: The von Hippel–Lindau (VHL) tumor suppressor protein pVHL is commonly mutated in clear cell renal cell carcinoma (ccRCC) and has been implicated in the control of multiple cellular processes that might be linked to tumor suppression, including promoting proper spindle orientation and chromosomal stability. However, it is unclear whether pVHL exerts these mitotic regulatory functions in vivo as well. Here, we applied ischemic kidney injury to stimulate cell division in otherwise quiescent mouse adult kidneys. We show that in the short term (5.5 days after surgery), Vhl-deficient kidney cells demonstrate both spindle misorientation and aneuploidy. The spindle misorientation phenotype encompassed changes in directed cell division, which may manifest in the development of cystic lesions, whereas the aneuploidy phenotype involved the occurrence of lagging chromosomes but not chromosome bridges, indicative of mitotic checkpoint impairment. Intriguingly, in the long term (4 months after the ischemic insult), Vhl-deficient kidneys displayed a heterogeneous pattern of ccRCC precursor lesions, including cysts, clear cell–type cells, and dysplasia. Together, these data provide direct evidence for a key role of pVHL in mediating oriented cell division and faithful mitotic checkpoint function in the renal epithelium, emphasizing the importance of pVHL as a controller of mitotic fidelity in vivo. Cancer Res; 74(9); 2422–31. ©2013 AACR.