Long-Term Survival after Complete Surgical Resection and Adjuvant Immunotherapy for Distant Melanoma Metastases.

BACKGROUND: This phase III study was undertaken to evaluate the efficacy of an allogeneic whole-cell vaccine (Canvaxin™) plus bacillus Calmette-Guerin (BCG) after complete resection of stage IV melanoma. METHODS: After complete resection of ≤5 distant metastases, patients were randomly assigned to BCG+Canvaxin (BCG/Cv) or BCG+placebo (BCG/Pl). The primary endpoint was overall survival (OS); secondary endpoints were disease-free survival (DFS), and immune response measured by skin test (ClinicalTrials.gov identifier: NCT00052156). RESULTS: Beginning in May 1998, 496 patients were randomized. In April 2005, the Data Safety Monitoring Board recommended stopping enrollment due to a low probability of efficacy. At that time, median OS and 5-year OS rate were 38.6 months and 44.9%, respectively, for BCG/Pl versus 31.4 months and 39.6% in the BCG/Cv group (hazard ratio (HR), 1.18; p = 0.250). Follow-up was extended at several trial sites through March 2010. Median OS and 5-year and 10-year survival was 39.1 months, 43.3 and 33.3%, respectively, for BCG/Pl versus 34.9 months, 42.5 and 36.4%, in the BCG/Cv group (HR 1.053; p = 0.696). Median DFS, 5- and 10-year DFS were 7.6 months, 23.8 and 21.7%, respectively, for BCG/Pl versus 8.5 months, 30.0%, and 30.0%, respectively, for the BCG/Cv group (HR 0.882; p = 0.260). Positive DTH skin testing correlated with increased survival. DISCUSSION: In this, the largest study of postsurgical adjuvant therapy for stage IV melanoma reported to date, BCG/Cv did not improve outcomes over BCG/placebo. Favorable long-term survival among study patients suggests that metastasectomy should be considered for selected patients with stage IV melanoma.

Inflammatory Marker Testing Identifies CD74 Expression in Melanoma Tumor Cells, and Its Expression Associates with Favorable Survival for Stage III Melanoma.

PURPOSE: Inflammatory marker expression in stage III melanoma tumors was evaluated for association with outcome, using two independent cohorts of stage III melanoma patients' tumor tissues. EXPERIMENTAL DESIGN: Fifteen markers of interest were selected for analysis, and their expression in melanoma tissues was determined by immunohistochemistry. Proteins associating with either overall survival (OS) or recurrence-free survival (RFS) in the retrospective discovery tissue microarray (TMA; n = 158) were subsequently evaluated in an independent validation TMA (n = 114). Cox proportional hazards regression models were used to assess the association between survival parameters and covariates, the Kaplan-Meier method to estimate the distribution of survival, and the log-rank test to compare distributions. RESULTS: Expression of CD74 on melanoma cells was unique, and in the discovery TMA, it associated with favorable patient outcome (OS: HR, 0.53; P = 0.01 and RFS: HR, 0.56; P = 0.01). The validation data set confirmed the CD74 prognostic significance and revealed that the absence of macrophage migration inhibitory factor (MIF) and inducible nitric oxide synthase (iNOS) was also associated with poor survival parameters. Consistent with the protein observation, tumor CD74 mRNA expression also correlated positively (P = 0.003) with OS in the melanoma TCGA data set. CONCLUSIONS: Our data validate CD74 as a useful prognostic tumor cell protein marker associated with favorable RFS and OS in stage III melanoma. Low or negative expression of MIF in both TMAs and of iNOS in the validation set also provided useful prognostic data. A disease-specific investigation of CD74's functional significance is warranted, and other markers appear intriguing to pursue. Clin Cancer Res; 22(12); 3016-24. ©2016 AACR.

Brain metastasis is predetermined in early stages of cutaneous melanoma by CD44v6 expression through epigenetic regulation of the spliceosome.

Melanoma brain metastasis (MBM) is frequent and has a very poor prognosis with no current predictive factors or therapeutic molecular targets. Our study unravels the molecular alterations of cell-surface glycoprotein CD44 variants during melanoma progression to MBM. High expression of CD44 splicing variant 6 (CD44v6) in primary melanoma (PRM) and regional lymph node metastases from AJCC Stage IIIC patients significantly predicts MBM development. The expression of CD44v6 also enhances the migration of MBM cells by hyaluronic acid and hepatocyte growth factor exposure. Additionally, CD44v6-positive MBM migration is reduced by blocking with a CD44v6-specific monoclonal antibody or knocking down CD44v6 by siRNA. ESRP1 and ESRP2 splicing factors correlate with CD44v6 expression in PRM, and ESRP1 knockdown significantly decreases CD44v6 expression. However, an epigenetic silencing of ESRP1 is observed in metastatic melanoma, specifically in MBM. In advanced melanomas, CD44v6 expression correlates with PTBP1 and U2AF2 splicing factors, and PTBP1 knockdown significantly decreases CD44v6 expression. Overall, these findings open a new avenue for understanding the high affinity of melanoma to progress to MBM, suggesting CD44v6 as a potential MBM-specific factor with theranostic utility for stratifying patients.

Clinical Benefit from Ipilimumab Therapy in Melanoma Patients may be Associated with Serum CTLA4 Levels.

Stage IV metastatic melanoma patients historically have a poor prognosis with 5-10% 5-year survival. Ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte antigen 4 (CTLA4), is one of the first treatments to provide beneficial durable responses in advanced melanoma. However, less than 25% of those treated benefit, treatment is expensive, and side effects can be fatal. Since soluble (s) CTLA4 may mediate inhibitory effects previously ascribed to the membrane-bound isoform (mCTLA4), we hypothesized patients benefiting from ipilimumab have higher serum levels of sCTLA4. We found that higher sCTLA4 levels correlated both with response and improved survival in patients treated with ipilimumab in a small patient cohort [patients with (n = 9) and without (n = 5) clinical benefit]. sCTLA4 levels were statistically higher in ipilimumab-treated patients with response to ipilimumab. In contrast, sCTLA4 levels did not correlate with survival in patients who did not receive ipilimumab (n = 11). These preliminary observations provide a previously unrecognized link between serum sCTLA4 levels and response to ipilimumab as well as to improved survival in ipilimumab-treated melanoma patients and a potential mechanism by which ipilimumab functions.

DNA methylation and gene deletion analysis of brain metastases in melanoma patients identifies mutually exclusive molecular alterations.

BACKGROUND: The brain is a common target of metastases for melanoma patients. Little is known about the genetic and epigenetic alterations in melanoma brain metastases (MBMs). Unraveling these molecular alterations is a key step in understanding their aggressive nature and identifying novel therapeutic targets. METHODS: Genome-wide DNA methylation analyses of MBMs (n = 15) and normal brain tissues (n = 91) and simultaneous multigene DNA methylation and gene deletion analyses of metastatic melanoma tissues (99 MBMs and 43 extracranial metastases) were performed. BRAF and NRAS mutations were evaluated in MBMs by targeted sequencing. RESULTS: MBMs showed significant epigenetic heterogeneity. RARB, RASSF1, ESR1, APC, PTEN, and CDH13 genes were frequently hypermethylated. Deletions were frequently detected in the CDKN2A/B locus. Of MBMs, 46.1% and 28.8% had BRAF and NRAS missense mutations, respectively. Compared with lung and liver metastases, MBMs exhibited higher frequency of CDH13 hypermethylation and CDKN2A/B locus deletion. Mutual exclusivity between hypermethylated genes and CDKN2A/B locus deletion identified 2 clinically relevant molecular subtypes of MBMs. CDKN2A/B deletions were associated with multiple MBMs and frequently hypermethylated genes with shorter time to brain metastasis. CONCLUSIONS: Melanoma cells that colonize the brain harbor numerous genetically and epigenetically altered genes. This study presents an integrated genomic and epigenomic analysis that reveals MBM-specific molecular alterations and mutually exclusive molecular subtypes.

A 20-year experience of hepatic resection for melanoma: is there an expanding role?

BACKGROUND: Melanoma liver metastasis is most often fatal, with a 4- to 6-month median overall survival (OS). Over the past 20 years, surgical techniques have improved in parallel with more effective systemic therapies. We reviewed our institutional experience of hepatic melanoma metastases. STUDY DESIGN: Overall and disease-specific survivals were calculated from hepatic metastasis diagnosis. Potential prognostic factors including primary tumor type, depth, medical treatment response, location, and surgical approach were evaluated. RESULTS: Among 1,078 patients with melanoma liver metastases treated at our institution since 1991, 58 (5.4%) received surgical therapy (resection with or without ablation). Median and 5-year OS were 8 months and 6.6 %, respectively, for 1,016 nonsurgical patients vs 24.8 months and 30%, respectively, for surgical patients (p < 0.001). Median OS was similar among patients undergoing ablation (with or without resection) relative to those undergoing surgery alone. On multivariate analysis of surgical patients, completeness of surgical therapy (hazard ratio [HR] 3.4, 95% CI 1.4 to 8.1, p = 0.007) and stabilization of melanoma on therapy before surgery (HR 0.38, 95% CI 0.19 to 0.78, p = 0.008) predicted OS. CONCLUSIONS: In this largest single-institution experience, patients selected for surgical therapy experienced markedly improved survival relative to those receiving only medical therapy. Patients whose disease stabilized on medical therapy enjoyed particularly favorable results, regardless of the number or size of their metastases. The advent of more effective systemic therapy in melanoma may substantially increase the fraction of patients who are eligible for surgical intervention, and this combination of treatment modalities should be considered whenever it is feasible in the context of a multidisciplinary team.

Genome-wide characterization of circulating tumor cells identifies novel prognostic genomic alterations in systemic melanoma metastasis.

BACKGROUND: Circulating tumor cells (CTC) have been found in patients with metastatic melanoma and are associated with advanced melanoma stage and poor patient outcome. We hypothesize that CTC harbor genomic changes critical in the development of distant systemic metastasis. Here, we present the first genome-wide copy-number aberration (CNA) and loss of heterozygosity (LOH)-based characterization of melanoma CTC. METHODS: CTC were isolated from peripheral blood monocytes of 13 melanoma patients with regional metastasis stage IIIB/C using antibodies against melanoma-associated cell surface gangliosides. RESULTS: We characterized 251 CNA in CTC. Comparative analysis demonstrated >90% concordance in single-nucleotide polymorphism profiles between paired CTC and tumor metastases. In particular, there were notable recurring CNA across patients. In exploratory studies, the presence of several top CTC-associated CNA was verified in distant metastasis (stage IV) from 27 patients, suggesting that certain genomic changes are propagated from regional metastasis to CTC and to distant systemic metastases. Lastly, an exploratory biomarker panel derived from 5 CTC-associated CNA [CSMD2 (CUB and Sushi multiple domains 2), 1p35.1; CNTNAP5 (contactin associated protein-like 5), 2q14.3; NRDE2 (NRDE-2, necessary for RNA interference, domain containing), 14q32.11; ADAM6 (ADAM metallopeptidase domain 6, pseudogene), 14q32.33; and TRPM2 (transient receptor potential cation channel, subfamily m, member 2), 21q22.3] conferred prognostic utility for melanoma recurrence [hazard ratio (HR), 1.14; CI, 1.00-1.44; P = 0.0471] and death (HR, 2.86; CI, 1.23-14.42; P = 0.0014) in 35 patients with stage IIIB/C melanoma, with a 5-year disease-free survival of 13% vs 69% (P = 0.0006) and overall survival of 28% vs 94% between high-risk and low-risk groups defined by the biomarker panel, respectively. CONCLUSIONS: This study provides the first detailed CNA-based profile of melanoma CTC and illustrates how CTC may be used as a novel approach for identification of systemic metastasis.

Age as a predictor of sentinel node metastasis among patients with localized melanoma: an inverse correlation of melanoma mortality and incidence of sentinel node metastasis among young and old patients.

PURPOSE: We have previously reported that older patients with clinical stage I and II primary cutaneous. Melanoma had lower survival rates compared to younger patients. We postulated that the incidence of nodal metastasis would therefore be higher among older melanoma patients. METHODS: The expanded American Joint Committee on Cancer melanoma staging database contains a cohort of 7,756 melanoma patients who presented without clinical evidence of regional lymph node or distant metastasis and who underwent a sentinel node biopsy procedure as a component of their staging assessment. RESULTS: Although older patients had primary melanoma features associated with more aggressive biology, we paradoxically observed a significant decrease in the incidence of sentinel node metastasis as patient age increased. Overall, the highest incidence of sentinel node metastasis was 25.8 % in patients under 20 years of age, compared to 15.5 % in patients 80 years and older (p < 0.001). In contrast, 5-year mortality rates for clinical stage II patients ranged from a low of 20 % for those 20-40 years of age up to 38 % for those over 70 years of age. Patient age was an independent predictor of sentinel node metastasis in a multifactorial analysis (p < 0.001). CONCLUSIONS: Patients with clinical stage I and II melanoma under 20 years of age had a higher incidence of sentinel lymph node metastasis but, paradoxically, a more favorable survival outcome compared to all other age groups. In contrast, patients >70 years had the most aggressive primary melanoma features and a higher mortality rate compared to all other age groups but a lower incidence of sentinel lymph node metastasis.

Final trial report of sentinel-node biopsy versus nodal observation in melanoma.

BACKGROUND: Sentinel-node biopsy, a minimally invasive procedure for regional melanoma staging, was evaluated in a phase 3 trial. METHODS: We evaluated outcomes in 2001 patients with primary cutaneous melanomas randomly assigned to undergo wide excision and nodal observation, with lymphadenectomy for nodal relapse (observation group), or wide excision and sentinel-node biopsy, with immediate lymphadenectomy for nodal metastases detected on biopsy (biopsy group). Results No significant treatment-related difference in the 10-year melanoma-specific survival rate was seen in the overall study population (20.8% with and 79.2% without nodal metastases). Mean (± SE) 10-year disease-free survival rates were significantly improved in the biopsy group, as compared with the observation group, among patients with intermediate-thickness melanomas, defined as 1.20 to 3.50 mm (71.3 ± 1.8% vs. 64.7 ± 2.3%; hazard ratio for recurrence or metastasis, 0.76; P=0.01), and those with thick melanomas, defined as >3.50 mm (50.7 ± 4.0% vs. 40.5 ± 4.7%; hazard ratio, 0.70; P=0.03). Among patients with intermediate-thickness melanomas, the 10-year melanoma-specific survival rate was 62.1 ± 4.8% among those with metastasis versus 85.1 ± 1.5% for those without metastasis (hazard ratio for death from melanoma, 3.09; P<0.001); among patients with thick melanomas, the respective rates were 48.0 ± 7.0% and 64.6 ± 4.9% (hazard ratio, 1.75; P=0.03). Biopsy-based management improved the 10-year rate of distant disease-free survival (hazard ratio for distant metastasis, 0.62; P=0.02) and the 10-year rate of melanoma-specific survival (hazard ratio for death from melanoma, 0.56; P=0.006) for patients with intermediate-thickness melanomas and nodal metastases. Accelerated-failure-time latent-subgroup analysis was performed to account for the fact that nodal status was initially known only in the biopsy group, and a significant treatment benefit persisted. CONCLUSIONS: Biopsy-based staging of intermediate-thickness or thick primary melanomas provides important prognostic information and identifies patients with nodal metastases who may benefit from immediate complete lymphadenectomy. Biopsy-based management prolongs disease-free survival for all patients and prolongs distant disease-free survival and melanoma-specific survival for patients with nodal metastases from intermediate-thickness melanomas. (Funded by the National Cancer Institute, National Institutes of Health, and the Australia and New Zealand Melanoma Trials Group; ClinicalTrials.gov number, NCT00275496.).

Epigenome-wide DNA methylation landscape of melanoma progression to brain metastasis reveals aberrations on homeobox D cluster associated with prognosis.

Melanoma brain metastasis (MBM) represents a frequent complication of cutaneous melanoma. Despite aggressive multi-modality therapy, patients with MBM often have a survival rate of <1 year. Alteration in DNA methylation is a major hallmark of tumor progression and metastasis; however, it remains largely unexplored in MBM. In this study, we generated a comprehensive DNA methylation landscape through the use of genome-wide copy number, DNA methylation and gene expression data integrative analysis of melanoma progression to MBM. A progressive genome-wide demethylation in low CpG density and an increase in methylation level of CpG islands according to melanoma progression were observed. MBM-specific partially methylated domains (PMDs) affecting key brain developmental processes were identified. Differentially methylated CpG sites between MBM and lymph node metastasis (LNM) from patients with good prognosis were identified. Among the most significantly affected genes were the HOX family members. DNA methylation of HOXD9 gene promoter affected transcript and protein expression and was significantly higher in MBM than that in early stages. A MBM-specific PMD was identified in this region. Low methylation level of this region was associated with active HOXD9 expression, open chromatin and histone modifications associated with active transcription. Demethylating agent induced HOXD9 expression in melanoma cell lines. The clinical relevance of this finding was verified in an independent large cohort of melanomas (n = 145). Patients with HOXD9 hypermethylation in LNM had poorer disease-free and overall survival. This epigenome-wide study identified novel methylated genes with functional and clinical implications for MBM patients.

Staging of regional lymph nodes in melanoma: a case for including nonsentinel lymph node positivity in the American Joint Committee on Cancer staging system.

IMPORTANCE: Survival varies widely in patients with stage III melanoma. The existence of clinical significance for positive nonsentinel lymph node (NSLN) status would warrant consideration for incorporation into the American Joint Committee on Cancer staging system and better prediction of survival. OBJECTIVE: To evaluate whether disease limited to sentinel lymph nodes (SLNs) represents different clinical significance than disease spread into NSLNs. DESIGN, SETTING, AND PARTICIPANTS: The database of the John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, California, was queried for all patients with SLNs positive for cutaneous melanoma who subsequently underwent completion lymph node dissection. MAIN OUTCOMES AND MEASURES: Disease-free survival, melanoma-specific survival (MSS), and overall survival. RESULTS: A total of 4223 patients underwent SLN biopsy from 1986 to 2012. Of these patients, 329 had a tumor-positive SLN. Of the 329, 250 patients (76.0%) had no additional positive nodes and 79 (24.0%) had a tumor-positive NSLN. Factors predictive of NSLN positivity included older age (P = .04), greater Breslow thickness (P < .001), and ulceration (P < .02). Median overall survival was 178 months for the SLN-only positive group and 42.2 months for the NSLN positive group (5-year overall survival, 72.3% and 46.4%, respectively). Median MSS was not reached for the SLN-only positive group and was 60 months for the NSLN positive group (5-year MSS, 77.8% and 49.5%, respectively). On multivariate analysis, NSLN positivity had a strong association with recurrence (hazard ratio [HR], 1.75; 95% CI, 1.23-2.50; P = .002), shorter overall survival (HR, 2.24; 95% CI, 1.48-3.40; P < .001), and shorter MSS (HR, 2.23; 95% CI, 1.46-3.07; P < .001). To further control for the effects of total positive lymph nodes, comparison was done for patients with only N2 disease (2-3 total positive lymph nodes); the results of this comparison confirmed the independent effect of NSLN status (MSS; P = .04). CONCLUSIONS AND RELEVANCE: Nonsentinel lymph node positivity is one of the most significant prognostic factors in patients with stage III melanoma. Subclassification of melanoma by NSLN tumor status should be considered for the American Joint Committee on Cancer staging system.

Age as a prognostic factor in patients with localized melanoma and regional metastases.

BACKGROUND: We postulated that the worse prognosis of melanoma with advancing age reflected more aggressive tumor biology and that in younger patients the prognosis would be more favorable. MATERIALS AND METHODS: The expanded AJCC melanoma staging database contained 11,088 patients with complete data for analysis, including mitotic rate. RESULTS: With increasing age by decade, primary melanomas were thicker, exhibited higher mitotic rates, and were more likely to be ulcerated. In a multivariate analysis of patients with localized melanoma, thickness and ulceration were highly significant predictors of outcome at all decades of life (except for patients younger than 20 years). Mitotic rate was significantly predictive in all age groups except patients <20 and >80 years. For patients with stage III melanoma, there were four independent variables associated with patient survival: number of nodal metastases, patient age, ulceration, and mitotic rate. Patients younger than 20 years of age had primary tumors with slightly more aggressive features, a higher incidence of sentinel lymph node metastasis, but, paradoxically, more favorable survival than all other age groups. In contrast, patients >70 years old had primary melanomas with the most aggressive prognostic features, were more likely to be head and neck primaries, and were associated with a higher mortality rate than the other age groups. Surprisingly, however, these patients had a lower rate of sentinel lymph node metastasis per T stage. Among patients between the two age extremes, clinicopathologic features and survival tended to be more homogeneous. CONCLUSIONS: Melanomas in patients at the extremes of age have a distinct natural history.

Late recurrence in melanoma: clinical implications of lost dormancy.

BACKGROUND: For patients with melanoma, if there has been no recurrence of disease 10 years after initial treatment, additional disease is believed to be very unlikely. However, such late recurrences are known to occur. The frequency of this phenomenon and its clinical significance are not well characterized due to the difficulty in obtaining relevant data. We examined a large, mature, institutional database to evaluate late recurrence. STUDY DESIGN: The late recurrence cohort was defined as having a disease-free interval of 10 or more years after potentially curative treatment and was compared with an early recurrence cohort recurring within 3 years. Actuarial late recurrence frequency and factors associated with late recurrence were examined. Post-recurrence overall and melanoma-specific survival and prognostic variables were analyzed. RESULTS: Among all patients, 408 exhibited late recurrence (mean disease-free interval 15.7 years). For patients who received primary treatment at our institution with 10 or more years follow-up, 327 of 4,731 (6.9%) showed late recurrence. On an actuarial basis, late recurrence rates were 6.8% and 11.3% at 15 and 20 years, respectively, for those with no recurrence at 10 years. Late recurrence was associated with both tumor (thin, non-ulcerated, non-head/neck, node negative) and patient (younger age, less male predominant) characteristics. Multivariate analysis confirmed younger age, thinner and node negative tumors in the late recurrence group. Late recurrences were more likely to be distant, but were associated with better post-recurrence survival on univariate and multivariate analyses. CONCLUSIONS: Late melanoma recurrence is not rare. It occurs more frequently in certain clinical groups and is associated with improved post-recurrence survival.

BCL2A1 is a lineage-specific antiapoptotic melanoma oncogene that confers resistance to BRAF inhibition.

Although targeting oncogenic mutations in the BRAF serine/threonine kinase with small molecule inhibitors can lead to significant clinical responses in melanoma, it fails to eradicate tumors in nearly all patients. Successful therapy will be aided by identification of intrinsic mechanisms that protect tumor cells from death. Here, we used a bioinformatics approach to identify drug-able, "driver" oncogenes restricted to tumor versus normal tissues. Applying this method to 88 short-term melanoma cell cultures, we show that the antiapoptotic BCL2 family member BCL2A1 is recurrently amplified in ∼30% of melanomas and is necessary for melanoma growth. BCL2A1 overexpression also promotes melanomagenesis of BRAF-immortalized melanocytes. We find that high-level expression of BCL2A1 is restricted to melanoma due to direct transcriptional control by the melanoma oncogene MITF. Although BRAF inhibitors lead to cell cycle arrest and modest apoptosis, we find that apoptosis is significantly enhanced by suppression of BCL2A1 in melanomas with BCL2A1 or MITF amplification. Moreover, we find that BCL2A1 expression is associated with poorer clinical responses to BRAF pathway inhibitors in melanoma patients. Cotreatment of melanomas with BRAF inhibitors and obatoclax, an inhibitor of BCL2A1 and other BCL2 family members, overcomes intrinsic resistance to BRAF inhibitors in BCL2A1-amplified cells in vitro and in vivo. These studies identify MITF-BCL2A1 as a lineage-specific oncogenic pathway in melanoma and underscore its role for improved response to BRAF-directed therapy.

B7-H3 associated with tumor progression and epigenetic regulatory activity in cutaneous melanoma.

B7-H3, a cell surface transmembrane glycoprotein, was assessed for its functional and prognostic role in cutaneous melanoma progression. B7-H3 expression in melanoma cells was shown to be related to specific downstream signal transduction events as well as associated with functional epigenetic activity. B7-H3 expression and prognostic utility were shown by reverse transcription and real-time PCR and immunohistochemistry analysis on individual melanoma specimens and then verified in clinically annotated melanoma stage III and stage IV metastasis tissue microarrays in a double-blind study. B7-H3 messenger RNA expression was shown to be significantly increased with stage of melanoma (P<0.0001) and significantly associated with melanoma-specific survival in both stage III (P<0.0001) and stage IV (P<0.012) melanoma patients. B7-H3 expression was related to migration and invasion; overexpression of B7-H3 increased migration and invasion, whereas knockdown of B7-H3 reduced cell migration and invasion. MiR-29c expression was shown to inversely regulate B7-H3 expression. Furthermore, we demonstrated that melanoma B7-H3 expression was correlated to phosphorylated signal transducer and activator of transcription-3 activity level in melanoma tissues and cell lines. These studies demonstrate that B7-H3 is a significant factor in melanoma progression and events of metastasis.

Combined intralesional Bacille Calmette-Guérin (BCG) and topical imiquimod for in-transit melanoma.

The introduction of numerous immunotherapeutic agents into the clinical arena has allowed the long-time promise of immunotherapy to begin to become reality. Intralesional immunotherapy has demonstrated activity in multiple tumor types, and as the number of locally applicable agents has increased, so has the opportunity for therapeutic combinations. Both intralesional Bacille Calmette-Guérin (ILBCG) and topical 5% imiquimod cream have been used as single agents for the treatment of dermal/subcutaneous lymphatic metastases or in-transit melanoma, but the combination has not previously been reported. We used this combination regimen in 9 patients during the period from 2004 to 2011 and report their outcomes here. All patients were initially treated with ILBCG, followed by topical imiquimod after development of an inflammatory response to BCG. In this retrospective study, we examined their demographics, tumor characteristics, clinical and pathologic response to treatment, associated morbidities, local and distant recurrence, and overall survival. The 9 patients (8 male) had a mean age of 72 years (range, 56-95 y). Mild, primarily local toxicities were noted. Five patients (56%) had complete regression of their in-transit disease and 1 had a partial response. The 3 others had "surgical" complete responses with resection of solitary resistant lesions. The mean interval between the first treatment and complete resolution of in-transit disease was of 6.5 months (range, 2-12 mo). With a mean follow-up of 35 months (range 12-58 mo), 7 patients (78%) had not developed recurrent in-transit disease. Two patients (22%) have died of nonmelanoma causes, and none have died due to melanoma.

Preoperative imaging of pulmonary metastases in patients with melanoma: implications for minimally invasive techniques.

HYPOTHESIS: Preoperative imaging underestimates the number of pulmonary melanoma metastases. Although thoracoscopic resection is less invasive than resection via thoracotomy, it does not allow manual palpation of the lung to identify any metastases not visible on the preoperative scan or at the time of resection. DESIGN: Retrospective review of a prospectively maintained database. SETTING: Tertiary referral center. PATIENTS: A total of 170 patients who underwent preoperative computed tomography of the chest, followed within 30 days by thoracotomy for resection of pulmonary metastatic melanoma. MAIN OUTCOME MEASURES: Number of pathology-confirmed pulmonary metastases detected by preoperative chest computed tomography vs intraoperative manual palpation. RESULTS: The mean age of the patients was 49.5 years at initial diagnosis of melanoma and 57.1 years at diagnosis of pulmonary metastases; 69% of patients were male. A total of 334 pulmonary metastases were resected; the mean lesion size was 2.0 cm (range, 0.1-14.0 cm). In 49 of 190 pulmonary resections (26%), manual palpation of the subpleural parenchyma revealed lesions not identified during preoperative imaging. The rate of 5-year overall survival was 33%. CONCLUSIONS: Preoperative imaging underestimates the number of pulmonary lesions in patients with metastatic melanoma. Because incomplete resection of metastatic disease is associated with worse outcomes, we recommend caution when considering a minimally invasive approach for the resection of pulmonary metastatic melanoma.

Association between circulating tumor cells and prognosis in patients with stage III melanoma with sentinel lymph node metastasis in a phase III international multicenter trial.

PURPOSE: The outcomes of patients with melanoma who have sentinel lymph node (SLN) metastases can be highly variable, which has precluded establishment of consensus regarding treatment of the group. The detection of high-risk patients from this clinical setting may be helpful for determination of both prognosis and management. We report the utility of multimarker reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) detection of circulating tumor cells (CTCs) in patients with melanoma diagnosed with SLN metastases in a phase III, international, multicenter clinical trial. PATIENTS AND METHODS: Blood specimens were collected from patients with melanoma (n = 331) who were clinically disease-free after complete lymphadenectomy (CLND) before entering onto a randomized adjuvant melanoma vaccine plus bacillus Calmette-Guérin (BCG) versus BCG placebo trial from 30 melanoma centers (United States and international). Blood was assessed using a verified multimarker RT-qPCR assay (MART-1, MAGE-A3, and GalNAc-T) of melanoma-associated proteins. Cox regression analyses were used to evaluate the prognostic significance of CTC status for disease recurrence and melanoma-specific survival (MSS). RESULTS: Individual CTC biomarker detection ranged from 13.4% to 17.5%. There was no association of CTC status (zero to one positive biomarkers v two or more positive biomarkers) with known clinical or pathologic prognostic variables. However, two or more positive biomarkers was significantly associated with worse distant metastasis disease-free survival (hazard ratio [HR] = 2.13, P = .009) and reduced recurrence-free survival (HR = 1.70, P = .046) and MSS (HR = 1.88, P = .043) in a multivariable analysis. CONCLUSION: CTC biomarker status is a prognostic factor for recurrence-free survival, distant metastasis disease-free survival, and MSS after CLND in patients with SLN metastasis. This multimarker RT-qPCR analysis may therefore be useful in discriminating patients who may benefit from aggressive adjuvant therapy or stratifying patients for adjuvant clinical trials.

Lymphatics, lymph nodes and the immune system: barriers and gateways for cancer spread.

Metastasis to the regional lymph node is the most important prognostic indicator for the outcomes of patients with sold cancer. In general, it is well recognized that cancer development is genetically determined with progression from the microenvironment of the primary tumor site, oftentimes via the SLN gateway, to the distant sites. In about 20 % of the time, the cancer cells may spread directly through the blood vascular system to the distant sites. Thus, in general, cancer progression is consistent with Hellman's spectrum theory in that development of nodal and systemic metastasis from a localized cancer growth is a progressive process. Cancer proliferation within the tumor microenvironment may give rise to increased tumor heterogeneity, which is further complicated by its continuous change through its evolution within the host in a Darwinian sense. It is crucial to understand the molecular process of lymphangiogenesis and hemangiogenesis in the tumor microenvironment with respect to the initial steps of cancer cells entering into the lymphatic and vascular systems so that rational therapy can be developed to curb the process of specific routes of metastasis. This chapter elucidates the role of lymphatics, nodal metastasis and antitumor immunity. We present novel immune targets in nodal metastases, the importance of the lymph node as a pre-metastatic niche, and immune-related proteins as biomarkers of metastasis.

A landscape of driver mutations in melanoma.

Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which-RAC1, PPP6C, and STK19-harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.