Paul B. Chapman

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently educating bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45−C-KITlow/+TIE2+ bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.

Cross-presentation of Disialoganglioside GD3 to Natural Killer T Cells

GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2− CD1−) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of ∼1:2,000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-γ followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1− tumors.

Prognostic factors in patients with metastatic malignant melanoma: A multivariate analysis

Background. Current methods to predict survival in patients with advanced, metastatic melanoma are limited. To determine clinical prognostic factors that accurately predict survival in patients with metastatic melanoma, a retrospective analysis was performed.

Effect of Selumetinib vs Chemotherapy on Progression-Free Survival in Uveal Melanoma A Randomized Clinical Trial

IMPORTANCEnUveal melanoma is characterized by mutations in GNAQ and GNA11, resulting in mitogen-activated protein kinase pathway activation.nnnOBJECTIVEnTo assess the efficacy of selumetinib, a selective, non-adenosine triphosphate competitive inhibitor of MEK1 and MEK2, in uveal melanoma.nnnDESIGN, SETTING, AND PARTICIPANTSnRandomized, open-label, phase 2 clinical trial comparing selumetinib vs chemotherapy conducted from August 2010 through December 2013 among 120 patients with metastatic uveal melanoma at 15 academic oncology centers in the United States and Canada.nnnINTERVENTIONSnOne hundred one patients were randomized in a 1:1 ratio to receive selumetinib, 75 mg orally twice daily on a continual basis (nu2009=u200950), or chemotherapy (temozolomide, 150 mg/m2 orally daily for 5 of every 28 days, or dacarbazine, 1000 mg/m2 intravenously every 21 days [investigator choice]; nu2009=u200951) until disease progression, death, intolerable adverse effects, or withdrawal of consent. After primary outcome analysis, 19 patients were registered and 18 treated with selumetinib without randomization to complete the planned 120-patient enrollment. Patients in the chemotherapy group could receive selumetinib at the time of radiographic progression.nnnMAIN OUTCOMES AND MEASURESnProgression-free survival, the primary end point, was assessed as of April 22, 2013. Additional end points, including overall survival, response rate, and safety/toxicity, were assessed as of December 31, 2013.nnnRESULTSnMedian progression-free survival among patients randomized to chemotherapy was 7 weeks (95% CI, 4.3-8.4 weeks; median treatment duration, 8 weeks; interquartile range [IQR], 4.3-16 weeks) and among those randomized to selumetinib was 15.9 weeks (95% CI, 8.4-21.1 weeks; median treatment duration, 16.1 weeks; IQR, 8.1-25.3 weeks) (hazard ratio, 0.46; 95% CI, 0.30-0.71; Pu2009<u2009.001). Median overall survival time was 9.1 months (95% CI, 6.1-11.1 months) with chemotherapy and 11.8 months (95% CI, 9.8-15.7 months) with selumetinib (hazard ratio, 0.66; 95% CI, 0.41-1.06; Pu2009=u2009.09). No objective responses were observed with chemotherapy. Forty-nine percent of patients treated with selumetinib achieved tumor regression, with 14% achieving an objective radiographic response to therapy. Treatment-related adverse events were observed in 97% of patients treated with selumetinib, with 37% requiring at least 1 dose reduction.nnnCONCLUSIONS AND RELEVANCEnIn this hypothesis-generating study of patients with advanced uveal melanoma, selumetinib compared with chemotherapy resulted in a modestly improved progression-free survival and response rate; however, no improvement in overall survival was observed. Improvement in clinical outcomes was accompanied by a high rate of adverse events.nnnTRIAL REGISTRATIONnclinicaltrials.gov Identifier: NCT01143402.

The History and Future of Chemotherapy for Melanoma

Melanoma is considered a chemotherapy-resistant cancer, but in reality there are several chemotherapy drugs with significant single-agent activity. Response rates to combination regimens are reproducibly higher than with standard dacarbazine, but of the randomized trials comparing combination regimens with dacarbazine, none were of sufficient size to detect a realistic effect on survival. Similarly, adjuvant chemotherapy has not had a realistic test in melanoma. Response to chemotherapy is associated reproducibly with better survival rates suggesting that regimens with higher response rates are needed. Recent observations suggest that combining antiangiogenic agents with either dacarbazine or temozolomide can double response rates. These combinations are worthy of further investigation and might serve as a foundation on which to build a combination regimen that improves overall survival in metastatic melanoma patients.

Combination of RAF and MEK Inhibition for the Treatment of BRAF-Mutated Melanoma: Feedback Is Not Encouraged

In BRAF V600E melanoma patients, RAF inhibitor treatment causes a MEK-inhibitor-sensitive, RAF-inhibitor-resistant adaptive reactivation of ERK signaling. In clinical trials combining MEK and RAF inhibitors, therapeutic efficacy was modestly enhanced, suggesting the utility of inhibiting feedback-reactivated pathways. Strategies for optimally inhibiting ERK signaling should be explored.

Measuring Toxic Effects and Time to Treatment Failure for Nivolumab Plus Ipilimumab in Melanoma

Importance Nivolumab plus ipilimumab (nivou2009+u2009ipi) is a standard treatment of advanced melanoma. Two randomized trials describe high objective response rates by Response Evaluation Criteria in Solid Tumors. The trials assessed toxic effects using the Common Terminology Criteria for Adverse Events (CTCAE), which may underestimate incidence of clinically significant immune-related adverse events (AEs). Objective To describe detailed toxic effects and time to treatment failure of patients with melanoma treated with nivou2009+u2009ipi in a prospective cohort. Design, Setting, and Participants A cohort of 64 adults with advanced or unresectable melanoma were examined at a single tertiary cancer and enrolled in an expanded access program of nivou2009+u2009ipi conducted from December 2014 to January 2016. Interventions Intravenous nivolumab (1 mg/kg) and ipilimumab (3 mg/kg) administered every 3 weeks for up to 4 doses, followed by nivolumab (3 mg/kg) every 2 weeks or pembrolizumab (2 mg/kg) every 3 weeks until unacceptable toxic effects, disease progression, or complete response. Main Outcomes and Measures Clinically significant immune-related AEs were defined as CTCAE grade 2 or higher or any immune-related AEs requiring systemic steroids. Time to treatment failure was defined as the interval between initiating therapy and the earliest of clinical progression, new locally directed or systemic treatment other than anti–programmed cell death 1 protein (anti–PD-1) monotherapy, or death. Results Overall 64 adults with advanced or unresectable melanoma were enrolled (male to female ratio, 1:1; median [range] age, 56 [22-82] years); 25 patients (39%) received all 4 doses of nivou2009+u2009ipi, and 31 patients (48%) received no maintenance anti–PD-1 therapy. Most who discontinued treatment (nu2009=u200931 [80%]) stopped because of toxic effects. Among those patients who were progression free at 12 weeks, time to treatment failure was similar between those who did or did not modify therapy for toxic effects. Fifty-eight patients (91%) had a clinically significant immune-related AE (median, 2/patient), and 46 patients (72%) required systemic steroids. Infliximab or mycophenolate was required in 16 patients (25%) for steroid-refractory immune-related AEs. Seven patients (11%) developed hyperglycemia, 32 patients (50%) had an emergency department visit, and 23 patients (36%) required a hospital admission related to an immune-related AE. Four of 31 patients (13%) who stopped combination therapy early for toxic effects developed a new, clinically significant immune-related AE more than 16 weeks after the last treatment. Conclusions and Relevance We observed a 91% incidence of clinically significant immune-related AEs leading to frequent emergency department visits, hospitalizations, and systemic immunosuppression. Immuno-oncology trials should routinely report these metrics. Most patients do not tolerate 4 doses of nivou2009+u2009ipi; however, 4 doses may not be required for clinical benefit.

Peripheral blood clinical laboratory variables associated with outcomes following combination nivolumab and ipilimumab immunotherapy in melanoma

Both the combination of nivolumab + ipilimumab and single‐agent anti‐PD‐1 immunotherapy have demonstrated survival benefit for patients with advanced melanoma. As the combination has a high rate of serious side effects, further analyses in randomized trials of combination versus anti‐PD‐1 immunotherapy are needed to understand who benefits most from the combination. Clinical laboratory values that were routinely collected in randomized studies may provide information on the relative benefit of combination immunotherapy. To prioritize which clinical laboratory factors to ultimately explore in these randomized studies, we performed a single‐center, retrospective analysis of patients with advanced melanoma who received nivolumab + ipilimumab either as part of a clinical trial (n = 122) or commercial use (n = 87). Baseline routine laboratory values were correlated with overall survival (OS) and overall response rate (ORR). Kaplan–Meier estimation and Cox regression were performed. Median OS was 44.4 months, 95% CI (32.9, Not Reached). A total of 110 patients (53%) responded (CR/PR). Significant independent variables for favorable OS included the following: high relative eosinophils, high relative basophils, low absolute monocytes, low LDH, and a low neutrophil‐to‐lymphocyte ratio. These newly identified factors, along with those previously reported to be associated with anti‐PD‐1 monotherapy outcomes, should be studied in the randomized trials of nivolumab + ipilimumab versus anti‐PD‐1 monotherapies to determine whether they help define the patients who benefit most from the combination versus anti‐PD‐1 alone.

Clinical and Morphologic Characteristics of MEK Inhibitor–Associated Retinopathy:Differences from Central Serous Chorioretinopathy

PURPOSEnTo investigate the clinical and morphologic characteristics of serous retinal disturbances in patients taking mitogen-activated protein kinase kinase (MEK) inhibitors.nnnPARTICIPANTSnA total of 313 fluid foci in 50 eyes of 25 patients receiving MEK inhibitors for treatment of their metastatic cancer, who had evidence of serous retinal detachments confirmed by optical coherence tomography (OCT).nnnDESIGNnSingle-center, retrospective cohort study.nnnMETHODSnClinical examination and OCT were used to evaluate MEK inhibitor-associated subretinal fluid. The morphology, distribution, and location of fluid foci were serially evaluated for each eye. Choroidal thickness was measured at each time point (baseline, fluid accumulation, and fluid resolution). Two independent observers performed all measurements. Statistical analysis was used to correlate interobserver findings and compare choroidal thickness and visual acuity at each time point.nnnMAIN OUTCOME MEASURESnComparison of OCT characteristics of retinal abnormalities at baseline to fluid accumulation.nnnRESULTSnThe majority of patients had fluid foci that were bilateral (92%) and multifocal (77%) and at least 1 focus involving the fovea (83.3%). All fluid foci occurred between the interdigitation zone and an intact retinal pigment epithelium. The 313 fluid foci were classified into 4 morphologies, as follows: 231 (73.8%) dome, 36 (11.5%) caterpillar, 31 (9.9%) wavy, and 15 (4.8%) splitting. Best-corrected visual acuity at fluid resolution was not statistically different from baseline; and no eye lost more than 2 Snellen lines from baseline at the time of fluid accumulation. There was no statistical difference in the choroidal thickness between the different time points (baseline, fluid accumulation, and fluid resolution). A strong positive interobserver correlation was obtained for choroidal thickness measurements (rxa0= 0.97, P < 0.0001) and grading of foci morphology (rxa0= 0.97, P < 0.0001).nnnCONCLUSIONnThe subretinal fluid foci associated with MEK inhibitors have unique clinical and morphologic characteristics, which can be distinguished from the findings of central serous chorioretinopathy. In this series,xa0MEK inhibitors did not cause irreversible loss of vision or serious eye damage.

PTEN Loss-of-Function Alterations Are Associated With Intrinsic Resistance to BRAF Inhibitors in Metastatic Melanoma

PurposenThe clinical use of BRAF inhibitors in patients with melanoma is limited by intrinsic and acquired resistance. We asked whether next-generation sequencing of pretreatment tumors could identify coaltered genes that predict for intrinsic resistance to BRAF inhibitor therapy in patients with melanoma as a prelude to rational combination strategies.nnnPatients and MethodsnWe analyzed 66 tumors from patients with metastatic BRAF-mutant melanoma collected before treatment with BRAF inhibitors. Tumors were analyzed for > 250 cancer-associated genes using a capture-based next-generation sequencing platform. Antitumor responses were correlated with clinical features and genomic profiles with the goal of identifying a molecular signature predictive of intrinsic resistance to RAF pathway inhibition.nnnResultsnAmong the 66 patients analyzed, 11 received a combination of BRAF and MEK inhibitors for the treatment of melanoma. Among the 55 patients treated with BRAF inhibitor monotherapy, objective responses, as assessed by Response Evaluation Criteria in Solid Tumors (RECIST), were observed in 30 patients (55%), with five (9%) achieving a complete response. We identified a significant association between alterations in PTEN that would be predicted to result in loss of function and reduced progression-free survival, overall survival, and response grade, a metric that combines tumor regression and duration of treatment response. Patients with melanoma who achieved an excellent response grade were more likely to have an elevated BRAF-mutant allele fraction.nnnConclusionnThese results provide a rationale for cotargeting BRAF and the PI3K/AKT pathway in patients with BRAF-mutant melanoma when tumors have concurrent loss-of-function mutations in PTEN. Future studies should explore whether gain of the mutant BRAF allele and/or loss of the wild-type allele is a predictive marker of BRAFi sensitivity.