Faculty Bibliography

Several challenges face the development and operation of a biospecimen bank linked to clinical information, a critical component of any effective translational research program. Melanoma adds particular complexity and difficulty to such an endeavor considering the unique characteristics of this malignancy. We describe here a review of biospecimen bank and our experience in establishing a multi-disciplinary, prospective, integrated clinicopathological-biospecimen database in melanoma. The Interdisciplinary Melanoma Cooperative Group (IMCG), a prospective clinicopathological and biospecimen database, was established at the New York University (NYU) Langone Medical Center. With patients' informed consent, biospecimens from within and outside NYU, clinicopathological data, and follow-up information are collected using developed protocols. Information pertaining to biospecimens is recorded in 35 fields, and clinicopathological information is recorded in 371 fields within 5 modules in a virtual network system. Investigators conducting research utilizing the IMCG biospecimen resource are blind to clinicopathological information, and molecular data generated using biospecimens are linked independently with clinicopathological data by biostatistics investigators. This translational research enterprise acts as a valuable resource to efficiently translate laboratory discoveries to the clinic

BACKGROUND: Nodular melanoma (NM) may be biologically aggressive compared with the more common superficial spreading melanoma (SSM), with recent data suggesting underlying genetic differences between these 2 subtypes. To better define the clinical behavior of NMs, the authors compared their clinical and histopathologic features to those of SSMs at their institution, a tertiary referral center, over 3 decades. METHODS: A total of 1,684 patients diagnosed with 1,734 melanomas were prospectively enrolled. Of these, 1,143 patients (69% SSM, 11% NM, 20% other) were diagnosed between 1972 and 1982; 541 patients (54% SSM, 23% NM, 23% other) were diagnosed between 2002 and the present. Differences between the features of NM and SSM within each time period as well as changes over time were analyzed. RESULTS: The authors found that SSMs are now diagnosed as thinner lesions (P < .0001) with a low incidence of histologic ulceration (P < .0001), whereas there was no significant change in the median tumor thickness or ulceration status of NMs over time (P = .10, P = .30, respectively). The median age at diagnosis of NM, however, did significantly increase over time (51 years to 63 years, P < .01). The median duration of NMs was reported to be only 5 months compared with 9 months in SSM patients. CONCLUSIONS: The authors' data suggest that improvements have been made in the early detection of SSM but not NM. Modifications of current screening practices, including increased surveillance of high-risk patients with an emphasis on the 'E' for 'evolution' criterion of the ABCDE acronym used for early detection of melanoma, are thus warranted

BACKGROUND: Different Insulin-like Growth Factor Binding Proteins (IGFBPs) have been investigated as potential biomarkers in several types of tumors. In this study, we examined both IGFBP-3 and -4 levels in tissues and sera of melanoma patients representing different stages of melanoma progression. METHODS: The study cohort consisted of 132 melanoma patients (primary, n = 72; metastatic, n = 60; 64 Male, 68 Female; Median Age = 56) prospectively enrolled in the New York University School of Medicine Interdisciplinary Melanoma Cooperative Group (NYU IMCG) between August 2002 and December 2006. We assessed tumor-expression and circulating sera levels of IGFBP-3 and -4 using immunohistochemistry and ELISA assays. Correlations with clinicopathologic parameters were examined using Wilcoxon rank-sum tests and Spearman-rank correlation coefficients. RESULTS: Median IGFBP-4 tumor expression was significantly greater in primary versus metastatic patients (70% versus 10%, p = 0.01) A trend for greater median IGFBP-3 sera concentration was observed in metastatic versus primary patients (4.9 microg/ml vs. 3.4 microg/ml, respectively, p = 0.09). However, sera levels fell within a normal range for IGFBP-3. Neither IGFBP-3 nor -4 correlated with survival in this subset of patients. CONCLUSION: Decreased IGFBP-4 tumor expression might be a step in the progression from primary to metastatic melanoma. Our data lend support to a recently-described novel tumor suppressor role of secreting IGFBPs in melanoma. However, data do not support the clinical utility of measuring levels of IGFBP-3 and -4 in sera of melanoma patients

Melanoma patients may exhibit a T(H)2-skewed cytokine profile within blood and tumor-infiltrating lymphocytes. Therapies that induce beneficial T(H)1-type tumor-specific immune responses, therefore, are highly desirable. Dendritic cells (DC) are widely used as immune adjuvants for cancer. Before their administration, DC are generally induced to mature with a cocktail of recombinant cytokines [interleukin (IL)-1beta, tumor necrosis factor alpha, and IL-6] and prostaglandin E(2) (PGE(2)), which is added to preserve the ability of DC to migrate to draining lymph nodes. However, PGE(2) suppresses the production of IL-12p70, a cytokine essential for differentiation of T(H)1 responses. In this study, human DC were transfected with IL-12p70 mRNA and tested for their ability to alter the T(H)2 type bias manifested by blood T cells of patients with melanoma. Transfected DC secreted high levels of bioactive IL-12p70, as indicated by their capacity to enhance natural killer cell activity, skew T(H)1 responses in allogeneic mixed lymphocyte reactions through reduction of IL-4 and IL-5, and prime CD8(+) T cells to the melanoma-associated antigen Melan A/MART-1. Furthermore, T-cell lines primed in vitro from the blood of melanoma patients showed strong type 2 skewing that was dramatically reversed by IL-12p70 transfection of autologous DC. Thus, IL-12p70 transfection of clinical DC preparations may enhance type 1 antitumor responses and may thereby contribute to effective immune-based therapy

PURPOSE: Extracellular matrix remodeling during tumor growth plays an important role in angiogenesis. Our preclinical data suggest that a newly identified cryptic epitope (HU177) within collagen type IV regulates endothelial and melanoma cell adhesion in vitro and angiogenesis in vivo. In this study, we investigated the clinical relevance of HUI77 shedding in melanoma patient sera. EXPERIMENTAL DESIGN: Serum samples from 291 melanoma patients prospectively enrolled at the New York University Medical Center and 106 control subjects were analyzed for HU177 epitope concentration by a newly developed sandwich ELISA assay. HU177 serum levels were then correlated with clinical and pathologic parameters. RESULTS: Mean HU177 epitope concentration was 5.8 ng/mL (range, 0-139.8 ng/mL). A significant correlation was observed between HU177 concentration and nodular melanoma histologic subtype [nodular, 10.3 +/- 1.6 ng/mL (mean +/- SE); superficial spreading melanoma, 4.5 +/- 1.1 ng/mL; all others, 6.1 +/- 2.1 ng/mL; P = 0.01 by ANOVA test]. Increased HU177 shedding also correlated with tumor thickness (< or =1.00 mm, 3.8 +/- 1.1 ng/mL; 1.01-3.99 mm, 8.7 +/- 1.3 ng/mL; > or =4.00 mm, 10.3 +/- 2.4 ng/mL; P = 0.003 by ANOVA). After multivariate analysis controlling for thickness, the correlation between higher HU177 concentration and nodular subtype remained significant (P = 0.03). The mean HU177 epitope concentration in control subjects was 2.4 ng/mL. CONCLUSIONS: We report that primary melanoma can induce detectable changes in systemic levels of cryptic epitope shedding. Our data also support that nodular melanoma might be biologically distinct compared with superficial spreading type melanoma. As targeted interventions against cryptic collagen epitopes are currently undergoing phase I clinical trial testing, these findings indicate that patients with nodular melanoma may be more susceptible to such targeted therapies

T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod's adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity

Background: DCs are widely used in experimental immune-based therapies for melanoma. Prior to injection, DCs are matured with a cocktail of recombinant cytokines and PGE-2, which preserves the ability of DCs to migrate to draining lymph nodes. However, PGE-2 also suppresses the ability of DCs to express IL-12p70. To overcome this paradox, we transfected mature DCs with synthetic mRNA encoding IL-12p70 and evaluated both their innate and adaptive immune functions in vitro. Methods: DCs from healthy donors and melanoma patients were cultured in vitro and transfected with IL-12p70 mRNA. The ability of these DCs to secrete bioactive IL-12p70 was tested by ELISA. Bioactivity of the secreted IL-12 was tested using NK cell assays and allogeneic MLRs. T cell assays were also performed to assess TH1 skewing towards melanoma antigens: T cells from healthy donors and melanoma patients were isolated and stimulated with autologous DCs previously transfected with MART-1 and IL-12p70 mRNA. The quantity and quality of the primed MART-1 specific T cells was determined. For comparison, melanoma tumor-infiltrating T cells (TILs) isolated from a resected tumor were used to examine TH phenotype. Results: IL-12p70-transfected DCs produced bioactive IL-12p70, and enhanced the response of NK cells to MHC class I-negative tumor targets. IL-12p70 transfected DC skewed TH responses away from TH2 in allogeneic MLRs. Cotransfection of DCs derived from the blood of healthy donors with IL-12p70 and MART-1 enhanced in vitro priming of melanoma- specific CD8 T cells, enhanced their capacity to secrete IFN? and inhibited the production of the TH2 cytokines IL-4 and IL-5, compared to MART-1 alone. In contrast, TILs from resected tumor exhibited a strong TH2 phenotype. T cell lines primed in vitro from the blood of melanoma patients by autologous DCs also showed strong TH2 skewing that was reversed by IL-12p70 transfection. Conclusions: Clinical DC preparations may be improved by IL-12p70 transfection, which enhances both innate and antigen-specific anti-tumor immune responses in vitro. This approach can be applied in vivo, thereby contributing to effective immune-based therapy against melanoma

Background: Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. Here we use gene expression profiling of metastatic melanoma samples to search for a molecular basis for this observation. Methods: We analyzed gene expression profiles of 44 metastatic melanoma specimens collected from 38 patients who were followed clinically for a median of 20 months after surgery. RNA was processed using standard methods and hybridized to Affymetrix Human Genome HU133 Plus 2.0 arrays. Data were analyzed using GeneSpring and PathwayAssist software, normalizing using a PLIER algorithm and utilizing ANOVA statistical tests. Results: Unsupervised clustering yielded 4 distinct groups of subjects, 2 of which had large differences in overall survival. We then used supervised clustering to compare patients whose post-surgery survival was less then 1.5 years (11 subjects, 9.5 month median survival) to those who survived greater than 1.5 years (23 subjects, 26 month median survival, 17 alive at last follow-up). Only genes with changes in expression of > 2 with a p value of < 0.05 were accepted, resulting in a group of approximately 200 genes. Genes associated with immune response (TNFa, IRF1, Granzyme B, CD8a, CXCL11, IL27AR, GBP1, CXCL10, CCBP2, GBP2, CCR9 and IFIT4) or with cell proliferation (FGFR1, MET, MDM2, CDC25A, RFC2, SOS1, HOXA3, MCM4, MCM7, ORC5L, KIF23 and VAV3) were highly represented. Prolonged survival was associated with elevated expression of immune response genes and decreased expression of genes associated with cell proliferation. Conclusions: Gene expression profiling of metastatic melanoma samples identified a set of genes associated with patient survival, and suggests that immune surveillance is a mechanism for prolonged survival in these patients

BACKGROUND: Tumorigenic breast cancer cells that express high levels of CD44 and low or undetectable levels of CD24 (CD44(>)/CD24(>/low)) may be resistant to chemotherapy and therefore responsible for cancer relapse. These tumorigenic cancer cells can be isolated from breast cancer biopsies and propagated as mammospheres in vitro. In this study, we aimed to test directly in human breast cancers the effect of conventional chemotherapy or lapatinib (an epidermal growth factor receptor [EGFR]/HER2 pathway inhibitor) on this tumorigenic CD44(>) and CD24(>/low) cell population. METHODS: Paired breast cancer core biopsies were obtained from patients with primary breast cancer before and after 12 weeks of treatment with neoadjuvant chemotherapy (n = 31) or, for patients with HER2-positive tumors, before and after 6 weeks of treatment with the EGFR/HER2 inhibitor lapatinib (n = 21). Single-cell suspensions established from these biopsies were stained with antibodies against CD24, CD44, and lineage markers and analyzed by flow cytometry. The potential of cells from biopsy samples taken before and after treatment to form mammospheres in culture was compared. All statistical tests were two-sided. RESULTS: Chemotherapy treatment increased the percentage of CD44(>)/CD24(>/low) cells (mean at baseline vs 12 weeks, 4.7%, 95% confidence interval [CI] = 3.5% to 5.9%, vs 13.6%, 95% CI = 10.9% to 16.3%; P < .001) and increased mammosphere formation efficiency (MSFE) (mean at baseline vs 12 weeks, 13.3%, 95% CI = 6.0% to 20.6%, vs 53.2%, 95% CI = 42.4% to 64.0%; P < .001). Conversely, lapatinib treatment of patients with HER2-positive tumors led to a non-statistically significant decrease in the percentage of CD44(>)/CD24(>/low) cells (mean at baseline vs 6 weeks, 10.0%, 95% CI = 7.2% to 12.8%, vs 7.5%, 95% CI = 4.1% to 10.9%) and a statistically non-significant decrease in MSFE (mean at baseline vs 6 weeks, 16.1%, 95% CI = 8.7% to 23.5%, vs 10.8%, 95% CI = 4.0% to 17.6%). CONCLUSION: These studies provide clinical evidence for a subpopulation of chemotherapy-resistant breast cancer-initiating cells. Lapatinib did not lead to an increase in these tumorigenic cells, and, in combination with conventional therapy, specific pathway inhibitors may provide a therapeutic strategy for eliminating these cells to decrease recurrence and improve long-term survival.