Overall Survival In Melanoma Patients Research Articles

Polyfunctional Melan-A-specific tumor-reactive CD8+ T cells elicited by dacarbazine treatment before peptide-vaccination depends on AKT activation sustained by ICOS

ABSTRACTThe identification of activation pathways linked to antitumor T-cell polyfunctionality in long surviving patients is of great relevance in the new era of immunotherapy. We have recently reported that dacarbazine (DTIC) injected one day before peptide-vaccination plus IFN-α improves the antitumor lytic activity and enlarges the repertoire of Melan-A-specific T-cell clones, as compared with vaccination alone, impacting the overall survival of melanoma patients. To identify the mechanisms responsible for this improvement of the immune response, we have analyzed the endogenous and treatment-induced antigen (Ag)-specific response in a panel of Melan-A-specific CD8+ T-cell clones in terms of differentiation phenotype, inhibitory receptor profile, polyfunctionality and AKT activation. Here, we show that Melan-A-specific CD8+ T cells isolated from patients treated with chemoimmunotherapy possess a late differentiated phenotype as defined by the absence of CD28 and CD27 co-stimulatory molecules and high levels of LAG-3, TIM-3 and PD-1 inhibitory receptors. Nevertheless, they show higher proliferative potential and an improved antitumor polyfunctional effector profile in terms of co-production of TNF-α, IFNγ and Granzyme-B (GrB) compared with cells derived from patients treated with vaccination alone. Polyfunctionality is dependent on an active AKT signaling related to the engagement of the co-stimulatory molecule ICOS. We suggest that this phenotypic and functional signature is dictated by a fine-tuned balance between TCR triggering, AKT activation, co-stimulatory and inhibitory signals induced by chemoimmunotherapy and may be associated with antitumor T cells able to protect patients from tumor recurrence.

Microphthalmia transcription factor in malignant melanoma predicts occult sentinel lymph node metastases and survival.

Microphthalmia transcription factor (Mitf) is involved in melanocyte development and differentiation. We previously reported that Mitf expression, as detected by immunohistochemical analysis, is an independent prognostic marker in patients with intermediate-thickness melanoma. However, the clinical significance of Mitf expression in melanoma is not well delineated. In this prospective study, we attempted to demonstrate the correlation between Mitf expression in primary melanoma and the sentinel lymph node status and prognosis. We prospectively examined primary cutaneous melanomas from 94 patients undergoing nodal staging by sentinel lymph node biopsy. We quantified the percentage of tumor cells whose nuclei stained with the Mitf antibody visually. Survival curves were generated using the Kaplan-Meier method. The correlation between Mitf expression and nodal status was evaluated using the Mann-Whitney U-test. Here we demonstrate that Mitf expression is directly correlated with both disease-free survival (DFS) and overall survival (OS) over a median follow-up of 28.5 months. The mean DFS and OS in the eight patients whose melanomas did not stain positive for Mitf were 15.75±3.36 months (median, 12 months) and 38.17±5.18 months (median, 29 months), respectively. These results are significantly lower than those for patients who showed evidence of Mitf expression, in whom the mean DFS and OS were 66.1±4.03 months (median, not reached, P=0.0001) and 66.75±38.17 months (median, not reached, P=0.0001), respectively. The mean DFS and OS with greater than 25% (67 patients) of the melanoma cells staining positive for Mitf expression were 78.37±2.78 and 82.38±1.6 months, respectively, compared with 26.37±3.2 months (P=0.0001) and 44.53±4.5 months (P=0.0001), respectively, with up to 25% (27 patients) of cells stained positive for Mitf expression. In addition, there was a significant relationship between Mitf expression and nodal status, as evaluated by sentinel node biopsy. For example, none of the melanomas with greater than 50% Mitf expression had a positive sentinel node biopsy. Our study shows that expression of the molecular marker Mitf in primary cutaneous melanomas is a useful tool in assessing lymph node status. Mitf immunostaining in the primary tumor serves as a reliable predictor of occult lymph node metastases, as well as a favorable prognosticator of DFS and OS in melanoma patients.

Abstract A2-25: Identification of RASA1 as a novel melanoma tumor suppressor gene

Abstract Hyperactivation of the Ras-Raf-Mitogen-activated protein kinase (MAPK) pathway has been commonly observed in melanoma via frequent activating mutations in NRAS and BRAF. Novel mutations in other components of this pathway such as MEK1, MEK2, MAP3K5, and MAP3K9 have been reported recently by high-throughput sequencing efforts. In addition, NF1 (neurofibromatosis type 1), one of the Ras GTPase activating proteins (RasGAPs) that negatively regulate Ras, has been shown to be mutated or suppressed in melanoma. However, importance of other RasGAPs has not been addressed in melanoma. To obtain a comprehensive view of melanoma genomes, we conducted whole genome sequencing (WGS) of 15 metastatic melanomas and matched normal PBMC genomes from 13 melanoma patients. All melanoma genomes from these 13 patients contained at least one mutation in genes of Ras-Raf-MAPK pathway (MAPK1, MAP3K1, MAP4K2, MAP3K14, NRAS, and BRAF). In addition, we identified two novel, clustered somatic missense mutations (p.Tyr472His and p.Leu481Phe) in RASA1 (RAS p21 protein activator 1, p120RasGAP). In this study, we addressed functional roles of RASA1 in melanoma tumorigenesis. The RNAi-mediated down-regulation of RASA1 promoted, while ectopic expression of wild type RASA1 decreased, anchorage-independent colony formation, tumor growth, and RAS activation. Interestingly, RASA1 Y472H mutant enhanced soft agar colony formation and tumor growth, while RASA1 L481F mutant lost its tumor suppressive activity. Mechanistically, RASA1 required RasGAP activity to suppress colony formation and showed higher activity toward R-Ras (related RAS viral (r-ras) oncogene homolog) isoform among the Ras superfamily of small GTPases. In addition, RASA1 suppressed Ral-A among Ras downstream effectors. Interestingly, RASA1 expression was frequently down-regulated in metastatic melanoma samples (11.4% (4/35) of lymph node metastasis and 3.4% (1/29) of distal metastases) compared to primary melanomas (33.3% (21/63)) and dysplastic nevi (44.1% (15/34)). We also observed significantly shorter overall survival of melanoma patients with BRAF mutations when RASA1 mRNA expression is low. Thus, these data support that RASA1 is a novel melanoma tumor suppressor that is inactivated by suppressed expression or by mutation. Citation Format: Hyeran Sung, Krishna L. Kanchi, Jane Messina, Ji-Hyun Lee, Youngchul Kim, Li Ding, Richard K. Wilson, Jeffrey S. Weber, Minjung Kim. Identification of RASA1 as a novel melanoma tumor suppressor gene. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-25.

Abstract A10: Soluble CD25 and C-reactive protein predict overall survival in melanoma patients receiving anti-CD40 monoclonal antibody CP-870,893 (αCD40) and anti-CTLA4 monoclonal antibody tremelimumab

Abstract Purpose: Identifying baseline patient biomarkers that predict clinical outcomes to cancer immunotherapy is a major goal for the field. In a phase I dose escalation study of αCD40 CP-870,893 (0.1-0.2 mg/kg) and tremelimumab (6-15 mg/kg) for patients (pts) with metastatic melanoma, we determined associations between pre-treatment clinical (stage, LDH) and immune variables and tumor response, progression-free survival (PFS) and overall survival (OS). Methods: Twenty-two metastatic melanoma pts (6 stage 4a, 6 stage 4b and 10 stage 4c) enrolled and contributed serum specimens for immunologic evaluation, which included: soluble CD25 (sCD25), C-reactive protein (CRP), EGF, HGF, VEGF, IFNα, IFNγ, TNFα, G-/GM-CSF, MIP1A/1B, MIG, MCP1, IL1B, IL1RA, IL2, IL4, IL5, IL6, IL8, IL10, IL12 and IL13. There were 6 responders (2 CR, 4 PR) and 16 nonresponders (3 SD, 13 PD). With a median follow-up of 26 months (mos), 13 pts have died. Median PFS and OS were 2.5 and 26.1 mos, respectively. Associations with continuous and binary immune variables were tested, cut points for the latter were determined by median or classification and regression tree (CART) analysis. Univariate logistic or Cox regression analyses determined predictors for response or PFS and OS, respectively. Variables with univariate significance <0.05 were then tested in multivariable models using stepwise regression. Results: Tumor Response. Response was associated with stage (50% in 12 stage 4a/b pts vs 0% in 10 stage 4c pts, p=0.02). Baseline LDH was not associated with response or PFS. No immune variables predicted response. Overall Survival. sCD25 (p=0.01), CRP (p=0.005), LDH (p=0.006) and stage (p=0.01) were highly significant predictors of OS. Natural log transformed sCD25 was highly correlated with CRP (r=0.56, p=0.006) and moderately correlated with LDH (r=0.41, p=0.06) and stage (rho=0.32, p=0.09). CART analysis identified sCD25 >110 and CRP >7 as the optimal cut points for OS. Accordingly, binary sCD25 (>110 vs <110, HR=13.4, p<0.001) and binary CRP (>7 vs <7, HR=7.2, p=0.003) were highly significant predictors. These cut points defined very similar risk groups. Of 6 pts with sCD25 >110, 5 had CRP >7 and of 16 pts with sCD25 <110, 14 had CRP <7. Thus, these CART-defined binary variables were strongly associated (p=0.004). In multivariate modeling, only binary sCD25 remained statistically significant since it was slightly superior to binary CRP, although both variables produced identical estimates of median OS. For pts with sCD25 <110 (or CRP <7), median OS was 27.1 mos while for pts with sCD25 >110 (or CRP >7), median OS was 4.6 mos. Moreover, binary sCD25 remained highly significant (HR=11.2, p=0.002) after adjusting for LDH (p=0.06). Progression-free Survival. Stage (p=0.01) and sCD25 (p=0.02), but not CRP (p=0.10), were significant predictors of PFS. Binary sCD25 defined by median (>60 vs <60, HR=3.1, p=0.02) and binary CRP (>7 vs <7, HR=3.1, p=0.02) were also significant predictors. Binary CRP (p=0.001), but not binary sCD25 (p=0.20), was associated with stage. In multivariable modeling, only stage remained significant. For stage 4a/b pts, median PFS was 4.8 mos while for stage 4c pts, median PFS was 2.3 mos. sCD25, CRP and IL6. IL6 was not correlated with sCD25 (p=0.99) or CRP (p=0.83) nor was it associated with response (p=0.26), PFS (p=0.48) or OS (p=0.74). Conclusion: sCD25 and CRP are both highly significant predictors for OS, although they are not independent predictors due to their strong co-linearity. sCD25 remains a significant predictor for OS after adjusting for baseline LDH. To confirm these findings, sCD25 and CRP should be investigated as possible predictive biomarkers in future studies of immune checkpoint inhibitors. Citation Format: Rosemarie Mick, David Bajor, Lee Richman, Robert Vonderheide. Soluble CD25 and C-reactive protein predict overall survival in melanoma patients receiving anti-CD40 monoclonal antibody CP-870,893 (αCD40) and anti-CTLA4 monoclonal antibody tremelimumab. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A10.

Resistance to BRAF inhibitors induces glutamine dependency in melanoma cells

BRAF inhibitors can extend progression-free and overall survival in melanoma patients whose tumors harbor mutations in BRAF. However, the majority of patients eventually develop resistance to these drugs. Here we show that BRAF mutant melanoma cells that have developed acquired resistance to BRAF inhibitors display increased oxidative metabolism and increased dependency on mitochondria for survival. Intriguingly, the increased oxidative metabolism is associated with a switch from glucose to glutamine metabolism and an increased dependence on glutamine over glucose for proliferation. We show that the resistant cells are more sensitive to mitochondrial poisons and to inhibitors of glutaminolysis, suggesting that targeting specific metabolic pathways may offer exciting therapeutic opportunities to treat resistant tumors, or to delay emergence of resistance in the first-line setting.

Abstract 3704: Novel panRAF inhibitors active in melanomas that are resistant to BRAF-selective, or BRAF-selective/MEK inhibitor combinations

Abstract The protein kinase BRAF is mutated ∼40% of human melanomas. BRAF is a component of the RAS/RAF/MEK/ERK pathway and BRAF or MEK inhibitors increase progression-free and overall survival in melanoma patients with BRAF mutations. However, most patients relapse with acquired resistance and ∼20% of patients present intrinsic resistance and do not respond to these drugs. We describe here two novel compounds that target mutant BRAF and wild-type CRAF. Our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors. ERK pathway reactivation is responsible for resistance to BRAF targeted therapies in ∼60% of the patients and in ∼25% of patients resistance is driven by acquisition of mutations in NRAS. We show that our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors due to pathway reactivation mediated by different mechanisms. We show that the drugs were active against patient derived xenografts (PDXs) from patients with acquired or intrinsic resistance to BRAF-selective inhibitors and in whose tumors resistance was associated with ERK pathway reactivation. Further, our compounds are active in a PDX from a patient whose tumor developed acquired resistance to a combination of a BRAF-selective plus a MEK inhibitor and associated with acquisition of an NRAS mutation. Thus, our panRAF inhibitors can inhibit melanomas with different mechanisms of acquired or intrinsic resistance to BRAF-selective and BRAF-selective/MEK inhibitor combinations, potentially providing first-line treatment for naïve patients and second-line treatments for a range of relapsed patients. Citation Format: Maria R. Girotti, Filipa Lopes, Natasha Preece, Dan Niculescu-Duvaz, Alfonso Zambon, Lawrence Davies, Steven Whittaker, Grazia Saturno, Amaya Viros, Malin Pedersen, Bart MJM Suijkerbuijk, Delphine Menard, Robert Mcleary, Louise Johnson, Laura Fish, Sarah Ejiama, Berta Sanchez-Laorden, Neil Carragher, Kenneth Macleod, Garry Ashton, Anna Marusiak, Alberto Fusi, John Brognard, Margaret Frame, Paul Lorigan, Caroline J. Springer, Richard Marais. Novel panRAF inhibitors active in melanomas that are resistant to BRAF-selective, or BRAF-selective/MEK inhibitor combinations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3704. doi:10.1158/1538-7445.AM2014-3704

β-Blocker use and all-cause mortality of melanoma patients: Results from a population-based Dutch cohort study

BackgroundResults from preclinical and observational studies suggest that β-adrenoreceptor inhibition might influence disease progression of melanoma. Patients and methodsPatients ⩾18years with cutaneous melanoma (Breslow thickness >1mm) registered in the Eindhoven Cancer Registry between January 1, 1998 and December 31, 2010, who were also registered with PHARMO record linkage system (RLS), were eligible. Randomly selected patients using β-blockers from PHARMO record linkage system (RLS) matched on age and gender served as a control cohort. Adjusted time-dependent and time-fixed Cox proportional hazard models were employed to estimate the hazard ratio of all-cause mortality. Five-year relative survival rates for all-cause mortality were calculated to estimate disease specific survival. Results203 of 709 eligible patients used β-blockers after melanoma diagnosis. The use of β-blockers was not associated with the risk of dying (adjusted hazard ratio (HR) 0.82, 95% confidence interval (CI) 0.55–1.24). Neither duration of exposure nor β-blocker dosage showed significant influence on survival. Five-year relative survival for β-blocker users was lower than in non-users amongst melanoma patients (80.9% and 83.7%, respectively) but higher among the β-blocker control group compared to the general population (101.4%). ConclusionOur results do not show a statistically significant impact of β-blocker exposure on overall survival of melanoma patients, regardless of the timing, duration or dosage of β-blocker use.

Photosensitivity and Other Cutaneous Side Effects of Vemurafenib Therapy

Vemurafenib has recently been approved for treating advanced melanoma. Targeting the V600 mutation in BRAF , this agent improves progression-free and overall survival of melanoma patients with tumors carrying that mutation. Keratoacanthomas and squamous cell carcinomas (SCCs) are well-known adverse effects of vemurafenib. Two study groups recently characterized other adverse cutaneous reactions in vemurafenib recipients. Gelot and colleagues describe vemurafenib-associated photosensitivity in 10 of 18 previously untreated …

Recent clinical trials of tumor immunotherapy

Many recent clinical trials of immunotherapy for advanced tumors have reported remarkable results. For example, blockade of cytotoxic T-lymphocyte antigen 4 (CTLA-4) significantly improved median overall survival of melanoma patients. Blockade of programmed cell death 1 (PD-1) or programmed cell death 1 ligand 1 (PD-L1) induced durable tumor regression and prolonged disease stabilization in patients with advanced cancers, including melanoma, non-small-cell lung cancer, and renal cell cancer. In addition, adoptive cell transfer of tumor infiltrating lymphocytes (TILs) and T cells transduced with high avidity T cell receptor (TCR) genes have been reported to elicit marked durable tumor regression in melanoma patients. Further, clinical trials of cancer vaccines targeting various tumors have been conducted to prolong overall survival. In this review, some remarkable results achieved in recent clinical trials are summarized.

Dabrafenib in the treatment of advanced melanoma

Advanced melanoma has long been a challenging malignancy to treat due to a relative paucity of efficacious therapeutic options. However, the identification of activating BRAF mutations in approximately 50% of patients with cutaneous melanoma has ushered in the era of targeted therapy for melanoma patients. Similar to the first-in-class selective serine/threonine-protein kinase B-raf inhibitor vemurafenib, dabrafenib is highly efficacious in melanoma patients with BRAF V600E mutations, with response rates of approximately 50% and progression-free survival of 6 months. There is data to suggest that dabrafenib not only shows activity in V600E-mutated melanoma, but also in non-V600E BRAF-mutated disease such as V600K. There is also early data to suggest that dabrafenib is effective in controlling metastases in the brain. Combining dabrafenib with the selective mitogen-activated protein kinase kinase (MEK) inhibitor trametinib has been effective in improving both the progression-free survival and overall survival of melanoma patients over those patients treated with dabrafenib alone. Dabrafenib is still being evaluated in several clinical trials in melanoma as well as a variety of other solid tumors with BRAF mutations. The U.S. Food and Drug Administration has recently approved dabrafenib as a single agent for the treatment of unresectable or metastatic melanoma in adult patients with BRAF V600E mutation.

Pitfalls in retrospective analyses of biomarkers: A case study with metastatic melanoma patients

BackgroundReliable prognostic biomarkers of survival and response to treatment are clearly important in oncology, and many studies have been carried out with the objective of identifying new prognostic biomarkers. Retrospective analysis of blood banked from patients is a frequently used paradigm for these studies. We describe a new study of the association of serum biomarker level with overall survival in melanoma patients, and the problems encountered in carrying it out. MethodsBlood samples from 56 patients with stage IV metastatic melanoma were drawn prior to initiation of any treatment for their disease. Sera from the samples were stored for up to 94months at −80°C, and were subsequently thawed at the same time and tested by multiplex Luminex assay for 30 analytes (cytokines, chemokines and growth factors). Cox regression analysis was used to assess the association between these analytes and time-to-death. ResultsOf the 30 analytes, 17 were associated with survival, most strongly so, and in all cases, a higher analyte level was associated with increased survival. In addition, the correlations of the levels of all possible pairs of analytes were all positive and in almost all cases highly significant. However, these results are artifacts that arise from the combination of two peculiarities of the data: the apparent decrease in analyte level with storage time, and the uniformly shorter storage times of the samples from censored patients than the storage times of the samples from patients who died. ConclusionsAll retrospective studies can have hidden biases, and thus investigators should not claim new findings before examining the data in detail with the goal of determining whether the findings could be spurious. There were several suspicious findings in our initial analyses: too many analytes found significant, too many very small p-values, a uniformly positive association of analyte level with survival, and a uniformly positive correlation between analyte levels. We were convinced that these findings must be artifacts, and further analyses showed that the findings could be explained by an apparent decrease of analyte level storage time.

The Impact of Primary Melanoma Thickness and Microscopic Tumor Burden in Sentinel Lymph Nodes on Melanoma Patient Survival

The primary objectives of this work are to (1) quantitate tumor burden in sentinel lymph nodes (SLNs), and (2) assess the independent contributions of SLN tumor burden and primary melanoma thickness (PMT) with respect to progression-free survival (PFS) and overall survival (OS). Sixty-three patients (41 male and 22 female) with one or more positive SLNs were available for review in this study, with median follow-up of 6.8 years. PMT was measured and SLN metastases were assessed for size, as maximum metastasis size (MMS) in mm, by hematoxylin and eosin (H&E) and immunohistochemistry (S100 and HMB45). PFS and OS were calculated from time of SLN resection until melanoma recurrence or death. Univariate and multivariate analyses and trend test were performed. Kaplan-Meier estimates of PFS and OS differed significantly by MMS (log-rank P = 0.031 for PFS and P = 0.016 for OS) and PMT (log-rank P = 0.036 for PFS and P < 0.001 for OS). After adjusting for age and gender, the hazard ratio (HR) associated with MMS was 1.09 per mm increase (P = 0.05) for PFS, and 6.30 (P = 0.014) and 5.41 (P = 0.048) for OS in patients, respectively, with MMS of 0.6-5.5 mm and MMS ≥5.5 mm compared with those with MMS <0.6 mm. When patients were stratified by their tumor characteristics of PMT, the risk for disease progression and worse OS was substantially higher for the group with PMT ≥ 4.5 mm (HR = 13.10 and P = 0.022 for PFS; HR = 17.26 and P < 0.001 for OS) relative to the baseline group with PMT <1.6 mm. All patients had completion lymph node dissection (CLND) except for four patients. Patients with positive CLND (14, 22.2%) showed significant worse PFS (P = 0.002) and OS (P = 0.0003) than the negative CLND group (45, 71.4%). PMT and MMS were independently prognostic of PFS and OS in melanoma patients. Patients with negative CLND had significantly better PFS and OS than those with positive CLND.

Lower tumour burden and better overall survival in melanoma patients with regional lymph node metastases and negative preoperative ultrasound

BackgroundThe purpose of the study was to evaluate the ability of ultrasound (US) and fine needle aspiration biopsy (FNAB) in reducing the number of melanoma patients requiring a sentinel node biopsy (SNB); to compare the amount of metastatic disease in regional lymph nodes in SNB candidates with clinically uninvolved lymph nodes and of those with US uninvolved lymph nodes; and to compare the overall survival (OS) of both groups.MethodsBetween 2000 and 2007, a SNB was successfully performed in 707 patients with melanoma. The preoperative US of the regional lymph node basins was performed in 405 SNB candidates. In 14 of these patients, the US-guided FNAB was positive and they proceeded directly to lymph node dissection. In 391 patients, the preoperative US was either negative (343 patients) or suspicious (48 patients) (US group). In the remaining 316 patients the preoperative US was not performed (non-US group).ResultsThe proportion of macrometastatic sentinel lymph nodes (SN), number of metastatic lymph nodes per patient and proportion of nonsentinel lymph node metastases were found to be lower in the US group compared to the non-US group. The smaller tumour burden of the US group was reflected in a significantly better OS of patients with SN metastases.ConclusionsThe preoperative US of regional lymph nodes spares some patients with melanoma from undergoing a SNB. Patients with regional metastases and a negative preoperative US have a significantly lower tumour burden in comparison to those with clinically negative lymph nodes, which is also reflected in a better OS.

Combination of targeted therapy and immunotherapy in melanoma.

The treatment of human melanoma has progressed markedly in recent years. Building on the observation that immune recognition is a frequent event in melanoma, a series of immunotherapeutic approaches have been evaluated in clinical trials, culminating in the first phase III study improving overall survival of melanoma patients since 20 years. However, the response rates seen upon immunotherapeutic interventions such as anti-CTLA4 treatment are often low. Furthermore, clinical responses can take several weeks to develop, during which time stage IV melanoma patients often deteriorate. Recent advances in our understanding of the genetic lesions in human melanoma now also allow the specific targeting of the signaling pathway alterations in this disease. Such targeted therapies can lead to high response rates, although the duration of these responses is thus far relatively short. We suggest that the combination of immuno and targeted therapy offers potential for synergy for both conceptual and practical reasons. In this review, we will discuss the potential and possible limitations for such combination therapy, and we describe the most promising combinations of targeted therapy and immunotherapy that can be tested in the clinic in the coming years. The concept of induction therapy by small molecule administration and consolidation by immunotherapeutics also has potential for the treatment of other human cancers.

Large-Scale Analysis of KIT Aberrations in Chinese Patients with Melanoma

KIT aberrations were described in acral and mucosal melanomas in largely Caucasian populations. Asian populations are more prone to develop acral and mucosal than cutaneous melanomas, and may harbor a high frequency of KIT aberrations. Melanoma subtypes (n = 502) were analyzed histologically to determine melanoma subtype. Tissue samples were analyzed for mutations in exons 9, 11, 13, 17, and 18 of KIT gene in genomic DNA by PCR amplification and Sanger sequencing. The copy numbers of the KIT gene were analyzed by quantitative PCR, and protein expression levels of KIT (CD117) were determined by immunohistochemistry. The most common melanoma subtypes were acral (38.4%) and mucosal (33.3%) melanomas in this population. The overall incidence of somatic mutations within the KIT gene was 10.8% (54/502), and all subtypes of melanoma contained KIT mutations. Increases in KIT gene copy numbers were correlated to CD117 overexpression. The genetic mutations of KIT were unrelated to the age, gender, stage, thickness, and ulceration of primary melanomas. Importantly, the overall survival of melanoma patients with KIT mutations (P = 0.001) or with KIT aberrations (mutation plus amplification, P = 0.0002) was significantly shorter than that of patients without such alterations. In China, the prevalent melanomas are acral and mucosal melanomas. KIT mutations are detected in all melanoma subtypes. Our study suggests that increases in KIT gene copy numbers, but not KIT mutations, may be correlated to CD117 overexpression. For the first time, our study suggests that genetic KIT aberration is an adverse prognostic factor for melanoma.

Upregulated ankyrin repeat-rich membrane spanning protein contributes to tumour progression in cutaneous melanoma

Background:We have previously demonstrated that overexpression of ankyrin repeat-rich membrane spanning (ARMS) protein facilitates melanoma formation via conferring apoptotic resistance. This study aims to investigate whether ARMS contributes to melanoma progression.Method:Using immunohistochemistry, we graded the expression level of ARMS in 54 cases of primary melanoma and 46 cases of metastatic melanoma. The immunointensity of ARMS was statistically correlated with individual clinicopathological characteristics. By RNA interference, stable melanoma cell clones with ARMS-knockdown were constructed, and were used for in vitro scratch wound, transwell invasion assays, and in vivo lung metastasis experiment.Results:Stronger immunointensity of ARMS was observed mostly in melanomas with Breslow tumour thickness >1.0 mm (Fisher's exact test, P=0.002) or with nodal metastasis (Fisher's exact test, P=0.026), and was correlated with a worse overall survival in melanoma patients (log-rank test, P=0.04). Depletion of ARMS inhibited migration, invasion, and metastatic potential of melanoma cells in vitro and in vivo. Moreover, ARMS mediated melanoma cell migration and invasion through activation of the extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signalling pathway.Conclusion:Ankyrin repeat-rich membrane spanning expression, conjunctly with tumour thickness or ulceration, may serve as a prognostic factor in patients with cutaneous melanoma.

FC19 Index risk on sentinel lymph nodes features that could predict non-sentinel lymph node involvement, disease-free survival and overall survival in melanoma patients

FC19 Index risk on sentinel lymph nodes features that could predict non-sentinel lymph node involvement, disease-free survival and overall survival in melanoma patients

Abstract 4652: Identification and validation of a novel nine-gene signature predicting clinical outcome in malignant melanoma

Abstract Incidence of malignant melanoma is rapidly increasing - with a doubling rate of 10-20 years. Conventional histopathological and clinical staging (based on Breslow tumor thickness, lymph node status, and ulceration) is largely inadequate for predicting clinical outcome of malignant melanoma. On the other hand, molecular prognostic markers are not yet available. Here, we identified a nine-gene signature which is closely associated with overall survival of melanoma patients. To identify prognostic genes we correlated gene expression profiles of 136 primary melanomas with patient overall survival using Cox regression analysis: Initially, a comparative analysis of 20 high-risk vs. 20 low-risk primary melanomas with a clinical follow-up of more than 20 years (training cohort) was performed using whole-genome DNA microarray analysis and yielded 92 prognostically relevant candidate genes. Technical and statistical validation using TaqMan Array real-time RT-PCR and correlation of gene expression with patient overall survival reduced candidate gene number to 11. Expression of these 11 genes was further analyzed in an extended group of 91 primary melanomas (study cohort), yielding a nine-gene signature with prognostic significance. This novel prognostic melanoma gene signature was successfully validated using an independent set of 45 primary melanomas (validation cohort). A risk score, based on the expression of the nine genes of the signature (KRT9, SPINK7/ECG2, KBTBD10, DCD, HES6, COL6A6, PIP, SCGB1D2, SCGB2A2), or any subgroup thereof, predicted patient overall survival in the study cohort (p = 0.0004, hazard ratio 3.83), independently of conventional AJCC 2002 staging. When combining gene expression score and AJCC staging, approximately two thirds (29/45, 64%) of patients with AJCC intermediate prognosis (i.e. stages IIA, IIB, and IIIA) were reclassified into good prognosis, exhibiting a long-term overall survival probability of 95%. Misclassification rate of all patients classified into good prognosis (low-risk gene score combined with AJCC stages I IIA/B, or IIIA) was very low at 4.6% and 6.25% in the training and validation cohorts, respectively. The prognostic value of this novel signature-based risk score is its ability to identify patients at low risk, not identified by AJCC staging. This re-classification may allow these patients to stay treatment-free while experiencing excellent long-term survival. The remaining patients are risk patients and are in need of adjuvant therapies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4652.

XPG D1104 single-nucleotide polymorphisms and the prognosis of melanoma

11096 Background: Sunlight is a major risk factor for melanoma. Since UV radiation causes DNA damage, it is not surprisingly, that genetic variants in DNA repair enzymes contribute to the susceptibility to cutaneous melanoma. Methods: Presence of common non-synonymous single-nucleotide polymorphism in different DNA repair enzymes were established and correlated with overall survival of melanoma patients. To this end, the SNPs of 6 different DNA repair enzymes were evaluated in a cohort of 742 melanoma patients. The impact of these polymorphisms on overall survival was subsequently calculated by the cox hazard model. Results: This analysis demonstrated that after adjustment to gender and primary tumor T classification XPG 1104 His/His as well as XPD 751 Lys/Lys genotypes were significantly associated with improved survival. Cox hazard coefficients were 0.744 for XPG 1104 His/His (p = 0.0059) and 0.651 for XPD 751 Lys/Lys (p = 0.017). Importantly, bootstrapping confirmed theses results for subpopulations. Furthermore, multivariate analysis demonstrated that XPG 1104 His/His is an independent factor affecting overall survival (cox coefficient 0.95788; p = 0.0011). Conclusions: XPG codon 1104 polymorphism may be predictive of survival outcome in patients with cutaneous melanoma. No significant financial relationships to disclose.

Molecular and biochemical testing in stage III melanoma: multimarker reverse transcriptase-polymerase chain reaction assay of lymph fluid after lymph node dissection and preoperative serum lactate dehydrogenase level

There is a need for biomarkers to identify patients at risk for disease progression after resection of melanoma regional lymph node metastasis. This study assessed the prognostic value of multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) assay in lymphatic drainage (LY) after lymph node dissection (LND) and of preoperative serum lactate dehydrogenase (LDH) levels in American Joint Committee on Cancer (AJCC) stage III melanoma patients. We collected 24-h LY from 255 stage III melanoma patients after radical LND [114, completion LND after positive sentinel node biopsy (CLND); 141, therapeutic LND for clinically/cytologically detected regional nodal metastases (TLND)]. For detection of melanoma cells, RT-PCR assays with primers specific for tyrosinase, MART1 (MelanA) and uMAGE mRNA were conducted. The LY sample was deemed positive if at least one marker was detected. In 244 patients, the preoperative serum LDH level was available. Median follow-up time was 25 months (range 5-60). The LY multimarker RT-PCR assay results were positive in 82 of 255 patients (32%). A significantly higher rate of melanoma recurrence was observed in patients with positive LY multimarker RT-PCR results (P = 0.01). Significant relationships were observed between positive LY multimarker RT-PCR results and shorter 3-year overall survival (OS) and disease-free survival (DFS), both in univariate and multivariate analyses (P = 0.007). Preoperative serum LDH level was increased in 79 of 244 patients (32%) [40.5% in TLND group and 23.0% in CLND group (P = 0.003)]. There were significant differences in OS between patients with normal and high preoperative LDH levels (P = 0.007), and these differences were seen mainly in patients in the TLND group. The multimarker RT-PCR assay detected melanoma cells in approximately 32% of LY after LND, which correlated significantly with early melanoma recurrence and shorter survival. Increased pre-LND serum LDH level had an additional negative impact on OS of melanoma patients with palpable nodal metastases after TLND.