Sera Of Melanoma Patients Research Articles

Melanoma Clinical Decision Support System: An Artificial Intelligence-Based Tool to Diagnose and Predict Disease Outcome in Early-Stage Melanoma Patients.

This study set out to assess the performance of an artificial intelligence (AI) algorithm based on clinical data and dermatoscopic imaging for the early diagnosis of melanoma, and its capacity to define the metastatic progression of melanoma through serological and histopathological biomarkers, enabling dermatologists to make more informed decisions about patient management. Integrated analysis of demographic data, images of the skin lesions, and serum and histopathological markers were analyzed in a group of 196 patients with melanoma. The interleukins (ILs) IL-4, IL-6, IL-10, and IL-17A as well as IFNγ (interferon), GM-CSF (granulocyte and macrophage colony-stimulating factor), TGFβ (transforming growth factor), and the protein DCD (dermcidin) were quantified in the serum of melanoma patients at the time of diagnosis, and the expression of the RKIP, PIRIN, BCL2, BCL3, MITF, and ANXA5 proteins was detected by immunohistochemistry (IHC) in melanoma biopsies. An AI algorithm was used to improve the early diagnosis of melanoma and to predict the risk of metastasis and of disease-free survival. Two models were obtained to predict metastasis (including "all patients" or only patients "at early stages of melanoma"), and a series of attributes were seen to predict the progression of metastasis: Breslow thickness, infiltrating BCL-2 expressing lymphocytes, and IL-4 and IL-6 serum levels. Importantly, a decrease in serum GM-CSF seems to be a marker of poor prognosis in patients with early-stage melanomas.

Comprehensive Analyses of miRNAs Revealed miR-92b-3p, miR-182-5p and miR-183-5p as Potential Novel Biomarkers in Melanoma-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are important mediators in the intercellular communication, influencing the function and phenotype of different cell types within the tumor micro-milieu and thus promote tumor progression. Since EVs safely transport packages of proteins, lipids and also nucleic acids such as miRNAs, EVs and their cargo can serve as diagnostic and prognostic markers. Therefore, the aim of this study was to investigate EV embedded miRNAs specific for melanoma, which could serve as potential biomarkers. In contrast to previous studies, we not only analysed miRNAs from EVs, but also included the miRNA profiles from the EV-secreting cells to identify candidates as suitable biomarkers. While the characterization of EVs derived from normal melanocytes and melanoma cells showed largely comparable properties with regard to size distribution and expression of protein markers, the NGS analyses yielded marked differences for several miRNAs. While miRNA load of EVs derived from normal human epidermal melanocytes (NHEMs) and melanoma cells were very similar, they were highly different from their secreting cells. By comprehensive analyses, six miRNAs were identified to be enriched in both melanoma cells and melanoma cell-derived EVs. Of those, the accumulation of miR-92b-3p, miR-182-5p and miR-183-5p in EVs could be validated in vitro. By functional network generation and pathway enrichment analysis we revealed an association with different tumor entities and signaling pathways contributing melanoma progression. Furthermore, we found that miR-92b-3p, miR-182-5p and miR-183-5p were also enriched in EVs derived from serum of melanoma patients. Our results support the hypothesis that miRNAs derived from EVs can serve as prognostic or diagnostic liquid biopsy markers in melanoma. We identified EV-derived miRNAs and showed that those miRNAs, which were enriched in melanoma cells and EVs, are also found elevated in serum-derived EVs of patients with metastatic melanoma, but not in healthy subjects.

The soluble form of CD160 acts as a tumor mediator of immune escape in melanoma

Melanoma is responsible for 90% of skin cancer-related deaths. Major therapeutic advances have led to a considerable improvement in the prognosis of patients, with the development of targeted therapies (BRAF or MEK inhibitors) and immunotherapy (anti-CTLA-4 or -PD-1 antibodies). However, the tumor constitutes an immunosuppressive microenvironment that prevents the therapeutic efficacy and/or promotes the development of secondary resistances. CD160 is an activating NK-cell receptor initially described as delineating the NK and CD8+T-cell cytotoxic populations. Three forms of CD160 have been described: (1) the GPI isoform, constitutively expressed and involved in the initiation of NK-cells' cytotoxic activity, (2) the transmembrane isoform, neo-synthesized upon cell activation, allowing the amplification of NK cells' cytotoxic functions and (3) the soluble form, generated after cleavage of the GPI isoform, which presents an immuno-suppressive activity. By performing immunohistochemistry analyses, we observed a strong expression of CD160 at the primary cutaneous tumor site of melanoma patients. We further demonstrated that melanoma cells express CD160-GPI isoform and constitutively release the soluble form (sCD160) into the tumor environment. sCD160 was shown to inhibit the cytotoxic activity of NK-cells towards their target cells. In addition, it was found in the serum of melanoma patients and associated with increased tumor dissemination. Altogether these results support a role for sCD160 in the mechanisms leading to the inhibition of anti-tumor response and immune surveillance in melanoma.

Using autoantibody signatures to predict immunotherapy discontinuation in melanoma patients.

3069 Background: Immune checkpoint inhibitors (ICIs), e.g., ipilimumab (IPI) and/or nivolumab (NIVO), produce durable survival benefit in a substantial proportion of melanoma patients but can also induce severe immune-related adverse events (irAEs) requiring treatment discontinuation. There is no biomarker to predict irAEs in ICI-treated melanoma patients. Given the similar clinical manifestation between irAEs and autoimmune disorders, we hypothesized that a subset of patients possess a subclinical baseline predisposition to developing irAEs that is characterized by specific autoantibodies (autoAbs). Methods: Pre-treatment melanoma patient sera from the CheckMate-238 Phase III trial of adjuvant IPI vs. NIVO were used for autoAb profiling with HuProt proteomic arrays (CDI Labs). The outcome of interest is to predict toxicity events that caused treatment discontinuation. For each treatment arm, we allocated patients to training and testing datasets in a 3:1 ratio. We calculated the area under the curve (AUC) of the receiver operating characteristic curve to select a probability threshold, which was applied to the testing dataset to assess accuracy, sensitivity, and specificity. Functional enrichment among autoAb protein targets was assessed using Metascape. Results: There were 707 irAEs among 597 patients (IPI = 423, NIVO = 174), of which 355 required treatment discontinuation (IPI = 287, NIVO = 68). In the training sets, we identified a 170 autoAbs signature consisting of 102 autoAbs for IPI treatment and 68 autoAbs for NIVO treatment. In the independent testing set, the signatures showed AUC of 0.85 (0.78, 0.92), 82% sensitivity, 78% specificity, and overall accuracy of 81% to predict IPI discontinuation, and AUC of 0.87 (0.74, 0.99), 75% sensitivity, 97% specificity, and overall accuracy of 88% to predict NIVO discontinuation. Enrichment of nuclear lumen-associated protein targets was identified among autoAb signatures that predict IPI or NIVO discontinuation. Conclusions: The identified signature within a large Phase III trial cohort highlights the potential utility of pre-treatment autoAbs for prediction of patients at high risk of developing irAEs in the adjuvant setting necessitating treatment termination. We are currently validating and refining toxicity-associated autoAb signatures with the goal of developing a Clinical Laboratory Improvement Amendments (CLIA)-certified assay to enable clinicians to optimize immunotherapy delivery and patient selection.

Soluble NKG2D ligands are biomarkers associated with the clinical outcome to immune checkpoint blockade therapy of metastatic melanoma patients

ABSTRACTThe introduction of immune checkpoint blockade into the clinical practice resulted in improvement of survival of a significant portion of melanoma patients. Consequently, predictive biomarkers of response are needed to optimize patient's stratification and the development of combination therapies. The aim of this study was to determine whether levels of soluble NKG2D ligands (MICA, MICB, ULBP1, 2 and 3; sNKG2DLs) in the serum of melanoma patients can serve as useful predictors of response to the treatment with immune checkpoint blockade. sNKG2DLs were measured by ELISA in baseline and post-treatment serum and these results were correlated with the clinical outcome of melanoma patients (N = 194). The same determinations were performed also in a cohort of patients (N = 65) treated with either chemotherapy, radiotherapy, or mutated BRAF inhibitors (BRAFi). Absence of soluble MICB and ULBP-1 in baseline serum correlated with improved survival (OS = 21.6 and 25.3 mo and p = 0.02 and 0.01, respectively) of patients treated with immunological therapies while detectable levels of these molecules were found in poor survivors (OS = 8.8 and 12.1 mo, respectively). Multivariate analysis showed that LDH (p <0.0001), sULBP-1 (p = 0.02), and sULBP-2 (p = 0.02) were independent predictors of clinical outcome for the cohort of melanoma patients treated with immune checkpoint blockade. Only LDH but not sNKG2DLs was significantly associated with the clinical outcome of patients treated with standard or BRAFi regimens. These findings highlight the relevance of sNKG2DLs in the serum of melanoma patients as biomarkers for patients' stratification and optimization of immune checkpoint inhibition regimens.

High-sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients' sera.

The characterization of peptides bound to human leukocyte antigen (HLA) class I is of fundamental importance for understanding CD8+ T cell-driven immunological processes and for the development of immunomodulatory therapeutic strategies. However, until now, the mass spectrometric analysis of HLA-bound peptides has typically required billions of cells, still resulting in relatively few high-confidence peptide identifications. Capitalizing on the recent developments in mass spectrometry and bioinformatics, we have implemented a methodology for the efficient recovery of acid-eluted HLA peptides after purification with the pan-reactive antibody W6/32 and have identified a total of 27 862 unique peptides with high confidence (1% false discovery rate) from five human cancer cell lines. More than 93% of the identified peptides were eight to 11 amino acids in length and contained signatures that were in excellent agreement with published HLA binding motifs. Furthermore, by purifying soluble HLA class I complexes (sHLA) from sera of melanoma patients, up to 972 high-confidence peptides could be identified, including melanoma-associated antigens already described in the literature. Knowledge of the HLA class I peptidome should facilitate multiplex tetramer technology-based characterization of T cells, and allow the development of patient selection, stratification and immunomodulatory therapeutic strategies.

Serum markers in early-stage and locally advanced melanoma.

The identification of prognostic factors in cutaneous melanoma allows choosing the most effective treatment, especially in group of patients with locoregional disease. Markers related to carcinogenesis and angiogenesis in particular have effect on the course of the disease. The aim of this study was to evaluate clinical utility of vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), MMP-9, tissue inhibitors of metalloproteinase 1 (TIMP-1), and YKL-40 in serum of melanoma patients at pathological stages I–III. We included 148 adult patients with melanoma. The median follow-up was 40 months. Disease recurrence was observed in 43 patients; 3-year disease-free survival (DFS) rate was 71.7 %; 35 patients died; and the 3-year overall survival (OS) rate was 85 %. Concentrations of VEGF, MMP-2, MMP-9, TIMP-1, and YKL-40 were measured by ELISA kits. VEGF, MMP-9, TIMP-1, and YKL-40 were significantly higher in group of patients than in controls. Increased concentrations of TIMP-1 were related to patient survival, which in the group of lower and increased TIMP-1, disease-free survival amounted to 81 vs. 61 % (p = 0.014) and overall survival −88 vs. 82 % (p = 0.050), respectively. An increased concentration of YKL-40 was observed in 59 % of patients with ulceration and in 26 % of patients without ulceration (p = 0.012). We have found a clinically significant correlation between YKL-40 and MMP-9 (rho = 0.363; p = 0.004) as well as YKL-40 and VEGF (rho = 0.306; p = 0.018). In melanoma patients at stages I–III, the high concentrations of TIMP-1 in serum predicted adverse prognosis. YKL-40 was associated with ulceration of primary tumor, which is a very important prognostic factor.

Circulating prognosis markers in melanoma: proteomic profiling and clinical studies

Cutaneous melanoma is the most dangerous skin cancer because of its ability to metastasize. Several clinical factors (number of invaded lymph nodes and/or presence of distant metastases) and histopathology (depth of tumor and presence of ulceration) are available to the clinician for determining prognosis and suggesting appropriate therapeutic management. However, these factors are often insufficient, especially in early forms of melanoma. Much research has focused on the identification of effective prognostic markers in serum. The only serum marker, which has been incorporated into the current AJCC classification for clinical use is lactate dehydrogenase dosage, a historical marker, restricted to the prognosis of metastatic disease. The recent development of technologies for proteome analysis offers new perspectives in this field. This review summarizes the specific considerations for each of the proteomic techniques used to date and presents the results of recent clinical investigations conducted to identify prognostic biomarkers in the serum of melanoma patients.

Serum Anti-BPAG1 Auto-Antibody Is a Novel Marker for Human Melanoma

Malignant melanoma is one of the most aggressive types of tumor. Because malignant melanoma is difficult to treat once it has metastasized, early detection and treatment are essential. The search for reliable biomarkers of early-stage melanoma, therefore, has received much attention. By using a novel method of screening tumor antigens and their auto-antibodies, we identified bullous pemphigoid antigen 1 (BPAG1) as a melanoma antigen recognized by its auto-antibody. BPAG1 is an auto-antigen in the skin disease bullous pemphigoid (BP) and anti-BPAG1 auto-antibodies are detectable in sera from BP patients and are used for BP diagnosis. However, BPAG1 has been viewed as predominantly a keratinocyte-associated protein and a relationship between BPAG1 expression and melanoma has not been previously reported. In the present study, we show that bpag1 is expressed in the mouse F10 melanoma cell line in vitro and F10 melanoma tumors in vivo and that BPAG1 is expressed in human melanoma cell lines (A375 and G361) and normal human melanocytes. Moreover, the levels of anti-BPAG1 auto-antibodies in the sera of melanoma patients were significantly higher than in the sera of healthy volunteers (p<0.01). Furthermore, anti-BPAG1 auto-antibodies were detected in melanoma patients at both early and advanced stages of disease. Here, we report anti-BPAG1 auto-antibodies as a promising marker for the diagnosis of melanoma, and we discuss the significance of the detection of such auto-antibodies in cancer biology and patients.

Increased shedding of HU177 correlates with worse prognosis in primary melanoma

BackgroundIncreased levels of cryptic collagen epitope HU177 in the sera of melanoma patients have been shown to be associated with thicker primary melanomas and with the nodular histologic subtype. In this study, we investigate the association between HU177 shedding in the sera and clinical outcome in terms of disease-free survival (DFS) and overall survival (OS).MethodsSerum samples from 209 patients with primary melanoma prospectively enrolled in the Interdisciplinary Melanoma Cooperative Group at the New York University Langone Medical Center (mean age = 58, mean thickness = 2.09 mm, stage I = 136, stage II = 41, stage III = 32, median follow-up = 54.9 months) were analyzed for HU177 concentration using a validated ELISA assay. HU177 serum levels at the time of diagnosis were used to divide the study cohort into two groups: low and high HU177. DFS and OS were estimated by Kaplan-Meier survival analysis, and the log-rank test was used to compare DFS and OS between the two HU177 groups. Multivariate Cox proportional hazards regression models were employed to examine the independent effect of HU177 category on DFS and OS.ResultsHU177 sera concentrations ranged from 0-139.8 ng/ml (mean and median of 6.2 ng/ml and 3.7 ng/ml, respectively). Thirty-eight of the 209 (18%) patients developed recurrences, and 34 of the 209 (16%) patients died during follow-up. Higher HU177 serum level was associated with an increased rate of melanoma recurrence (p = 0.04) and with increasing mortality (p = 0.01). The association with overall survival remained statistically significant after controlling for thickness and histologic subtype in a multivariate model (p = 0.035).ConclusionsIncreased shedding of HU177 in the serum of primary melanoma patients is associated with poor prognosis. Further studies are warranted to determine the clinical utility of HU177 in risk stratification compared to the current standard of care.

Differential Clinical Significance of Individual NKG2D Ligands in Melanoma: Soluble ULBP2 as an Indicator of Poor Prognosis Superior to S100B

Cytotoxic lymphocytes interact with human tumor cells via the activating immunoreceptor NKG2D, recognizing a variety of stress-associated MIC and ULBP surface molecules. However, tumors can escape from this immunosurveillance by shedding NKG2D ligands (NKG2DL), rendering the soluble products detectable in patients' sera. To elucidate the clinical significance of NKG2DL diversity, we studied their expression on melanoma tissues and their presence as soluble molecules in sera from >200 melanoma patients and compared the latter with the well-established serum marker S100B. Immunohistochemistry revealed a heterogeneous expression of MIC and ULBP2 molecules between and within melanoma metastases. Compared with MIC, ULBP2 was less frequently expressed. Accordingly, elevated levels of soluble ULBP2 (sULBP2) were detected in sera of melanoma patients less frequently than elevated levels of soluble MICA (sMICA), although both soluble NKG2DL (sNKG2DL) were significantly increased compared with sera of healthy controls (P < 0.0001). Strikingly, elevated concentrations of sULBP2, but not of sMICA, were strongly associated with disease progression (P < 0.0001) and tumor load (P = 0.0003). Elevated serum levels of either sNKG2DL correlated with reduced overall survival, albeit considerably stronger for sULBP2 (P < 0.0001) than for sMICA (P = 0.011). In early-stage (I-III) melanoma patients, only sULBP2 (P < 0.0001) but neither sMICA nor S100B revealed prognostic significance. Multivariate analysis identified sULBP2 (P = 0.0015) and S100B (P = 0.013) but not sMICA as independent predictors of prognosis. Our data reveal marked differences in the clinical significance of individual sNKG2DL. Only sULBP2 is an independent predictor of prognosis, the significance of which is superior to the well-established and widely used melanoma serum marker S100B.

Association between HU177 serum level and prognosis in patients with primary melanoma

9044 Background: Cleavage of type IV collagen during extracellular matrix (ECM) remodeling leads to exposure of cryptic regulatory sites within the ECM shown to be involved in tumor angiogenesis. Increased levels of a soluble form of the cryptic epitope HU177 in sera of melanoma patients have been shown to be associated with greater tumor thickness and nodular histological subtype. In this study, we investigate the association between HU177 serum levels and melanoma patients' clinical outcomes. Methods: Sera from 209 patients with primary melanoma prospectively enrolled in the Interdisciplinary Melanoma Cooperative Group at the New York University Langone Medical Center (85 females, 124 males, mean age=58, mean thickness=2.09 mm, Stage I n=140, Stage II n=40, Stage III n=29) were analyzed for HU177 level. HU177 serum levels at the time of diagnosis were then correlated with disease-free survival (DFS) and overall survival (OS). Results: Median follow-up time for survivors was 54.9 months (range 2–81 months). Thirty-eight of the 209 (18%) patients developed recurrences, and 34 of the 209 (16%) patients died during follow-up. HU177 sera levels ranged from 0–139.9 ng/ml (mean=6.2 ng/ml; median=3.7 ng/ml). Because the distribution of HU177 levels was positively skewed, we analyzed the data using the median in addition to the mean. HU177 level &gt; 3.7 ng/ml (the median) was associated with a higher rate of melanoma recurrence (p=0.04) and increasing mortality (p=0.01) in a Kaplan Meier analysis. HU177 remained an independent prognostic factor for DFS and OS when controlling for tumor thickness and histological subtype in multivariate Cox proportional hazards regression models. In the DFS hazard model controlling for tumor thickness and histology, the hazard ratio for HU177 &gt;3.7 ng/ml (the median) was 2.01 (95% CI= 1.002, 4.04; p=0.049). In the OS hazard model controlling for tumor thickness and histology, the hazard ratio for HU177 &gt;3.7 ng/ml (the median) was 2.23 (95% CI=1.06, 4.70; p=0.03). Conclusions: Increased serum level of HU177 identifies a subset of primary melanoma patients with worse prognosis and suggests that anti-angiogenic therapy in the adjuvant setting may be a rational approach. [Table: see text]

Assessing the clinical utility of measuring Insulin-like Growth Factor Binding Proteins in tissues and sera of melanoma patients

BackgroundDifferent 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.MethodsThe 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.ResultsMedian 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 μg/ml vs. 3.4 μg/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.ConclusionDecreased 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.

Shedding of Distinct Cryptic Collagen Epitope (HU177) in Sera of Melanoma Patients

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. 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. 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. 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.

Correlation of shedding cryptic epitope (HU177) levels and treatment response in sera of melanoma patients

20018 Background: We have previously shown that shedding of the recently identified cryptic epitope HU177 can be measured in melanoma patients’ sera and correlates with invasiveness of primary melanomas. Here we prospectively measured HU177 collagen epitope (HCE) titers in sera of stage IV melanoma patients (pts) enrolled in two clinical trials conducted at our institution. Methods: Serum samples from 37 pts (11-M1a, 10-M1bM, 16-M1c) who received Sorafenib on an NCI phase II trial (6617) (study A) and from 18 stage IV pts who were treated 2nd line with Carboplatin, Paclitaxel, and PABA on a phase I trial (study B), were analyzed for HCE by a dual sandwich ELISA during each of up to 12 cycles. Serum was aliquoted into 200 μl volumes and stored at - 20°C until assayed. Correlation of HCE levels with treatment response as measured by PET/CT or CT imaging was examined. Results: In 2 of 3 radiographically responding pts in study A correlation with HCE was seen. In one pt, HCE levels dropped from 74.4 ng/ml to undetectable by cycle 2. Another pt who had a response that lasted >200 days, HCE titers progressively decreased from 55.1 to 15.8 ng/ml over the first 3 cycles (71.5% drop) and subsequently remained in the range from 0 - 9.7 ng/ml from cycle 4 to cycle 7 (mean decrease (± SD) from baseline of 89.6±6.1% below pre-treatment levels). During this time, the mean standard uptake values (SUV) measured in PET/CT scans decreased from 11.3 (pre-treatment) to 4.3 and 2.3, respectively. In study B, 4 of 6 responders demonstrated a drop in HCE titers that correlated with treatment response. For example, one patient had HCE levels decrease from 54.0 ng/ml to 30.2 ng/ml by cycle 1 and remained in the range from 20.6 ng/ml to 41.1 ng/ml from cycle 2 to cycle 9 (mean 38.6±12.0 % below pre-treatment levels) while serial CT imaging demonstrated continued treatment response lasting > 200 days. Conclusions: Our data demonstrate, in two separate trials, the utility of serial HCE measurements for the monitoring of therapeutic responses in metastatic melanoma pts. Since the assay is not tumor-type specific, it may not only prove useful in future melanoma studies, but other cancer trials as well. No significant financial relationships to disclose.

Circulating levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase-3 (MMP-3), and BCL-2 in malignant melanoma

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and metastasis of melanoma. In literature, all studies concerning influences of matrix metalloproteinases (MMPs) and antiapoptotic proteins on VEGF-induced angiogenesis in melanoma patients have been performed in tissue scale in melanoma. The objective of this study was to determine the value of circulating serum VEGF and its possible mechanisms of angiogenesis by circulating VEGF, MMP-3, and Bcl-2 in patients with melanoma. Fifty-one patients with cutaneous melanoma pathologically verified at different stages, and eighteen healthy controls were investigated. Serum VEGF, MMP-3, and Bcl-2 levels were quantitatively analyzed by ELISA. The serum VEGF (P = 0.034) and Bcl-2 (P = 0.005) levels were significantly higher in patients with melanoma than in the control group. However, there was no significant difference in the serum MMP-3 level between melanoma patients and controls (P = 0.51). The serum levels of VEGF were significantly influenced only by Breslow thickness (P = 0.045) and mitosis (0.039) and were not positively correlated with the stage of the disease. Among serum parameters, a significant relationship was found only between serum levels of VEGF and MMP-3 (r = 0.32, P = 0.023). In conclusion, our study demonstrates increased concentrations of VEGF and Bcl-2, but not MMP-3, in serum of melanoma patients regardless of the stage of the disease. VEGF may be a potential endothelial cell growth and survival factor. The mechanism of VEGF regulation of angiogenesis may be in part due to enhanced proliferation and survival of endothelial cells by differential expression of antiapoptotic genes and in part by activation of MMPs.

Shedding of distinct cryptic epitope (HU177) in sera of melanoma patients

8550 Background: Our preclinical data suggest that a newly identified cryptic epitope HU177 within collagen regulates endothelial and tumor cell adhesion in-vitro and angiogenesis and tumor growth in-vivo. We investigated whether 1) HU177 shedding can be measured in melanoma patients’ sera, and 2) if HU177 concentration correlates with clinicopathological features, recurrence and survival. Methods: Sera from 291 melanoma patients (primary Stage I, n=140; II n=41; III n=29; recurrent/metastatic n=81) and 30 normal volunteers prospectively enrolled at the NYU School of Medicine were analyzed for HU177 epitope concentration (ng/ml) by ELISA. Microtiter plates were incubated overnight with monoclonal antibody (anti-HU177 epitope). Patients’ sera and standards (denatured type-IV collagen) were added in duplicate followed by dilutions of biotinylated anti-collagen type IV polyclonal and HRP-labeled anti-biotin antibodies with multiple washes after each incubation. Absorbance was monitored at 400 nm. Results: The mean concentration of anti-HU177 epitope was 5.8 ng/ml (range 0.1 to 139.8). Normal volunteers were 1.6±0.3 (mean ± SEM). A significant correlation was observed between HU177 concentration and tumor thickness in patients who presented with primary melanoma (=1.00 mm, n=113, 3.8±0.4; 1.01–3.99 mm, n=72, 8.9 ±2.1; =4.00 mm, n=22, 10.3±2.2; P=0.003 by ANOVA test) and nodular melanoma histological subtype (nodular, n=47, 10.5±3.1; superficial spreading, n=95, 4.4±0.5; others, n=68, 6.0±2.1; P=0.02, ANOVA). Multivariate analysis confirmed the independent correlation between higher HU177 concentration and nodular subtype after controlling for tumor thickness (P=0.04). Primary patients with ulcerated melanomas and those who developed recurrences both showed higher HU177 epitope levels, however, the difference was not significant (P&gt;0.05). Conclusions: This is the first study on HU177 epitope shedding in melanoma patients. Data demonstrate the clinical feasibility of HU177 testing in melanoma patients’ sera and suggest that HU177 epitope shedding correlates with thicker primary melanomas. Longer follow-up is required to better define the prognostic value of HU177 epitope shedding in melanoma patients. [Table: see text]

Recombinant interferon α-2b and coenzyme Q10 as a postsurgical adjuvant therapy for melanoma: a 3-year trial with recombinant interferon-α and 5-year follow-up

Early surgical intervention remains the most successful therapy for melanoma. Despite better outcomes observed in soft tissue and lymph node metastases, the results of pharmacological therapies are still disappointing. Currently, there is no standard adjuvant therapy for melanoma. Low concentrations of coenzyme Q10 have been demonstrated in melanoma cell lines and in sera of melanoma patients. These data and the results of clinical trials of patients with other advanced cancers prompted this study of the long-term administration of an optimized dose of recombinant interferon alpha-2b and coenzyme Q10 to patients with stage I and II melanoma. A 3-year trial envisaging uninterrupted treatment with low-dose recombinant interferon alpha-2b (9 000 000 000 IU weekly) administered twice daily and coenzyme Q10 (400 mg/day) was conducted in patients with stage I and II melanoma (American Joint Committee on Cancer criteria 2002) and surgically removed lesions. Treatment efficacy was evaluated as incidence of recurrences at 5 years. All patients completed the treatment and the follow-up. Significantly different rates of disease progression were observed in the interferon+coenzyme Q10 and the interferon group for both stages. No patient withdrew from the study owing to side effects. Long-term administration of an optimized dose of recombinant interferon alpha-2b in combination with coenzyme Q10 seemed to induce significantly decreased rates of recurrence and had negligible adverse effects. A survival study could not be undertaken owing to the small patient sample and the short duration of follow-up.

Prognostic utility of combined multiple methylation and microsatellite biomarkers as circulating DNA in serum of melanoma patients

Tanemura, A.1; Mori, T.1; O'Day, S.2; Martinez, S.1; Kitago, M.1; Koyanagi, K.1; Sunami, E.1; Hoon, D.1Author Information

Methylated estrogen receptor alpha in tumors and sera of melanoma patients

8026 Background: The mechanisms regulating the variable expression of estrogen receptor alpha (ER-α) in melanoma are unknown. Silencing of ER-α expression in melanoma may be regulated by the hypermethylation of promoter region CpG islands. To determine the significance of ER-α hypermethylation-induced transcription silencing in melanoma progression, we assessed hypermethylation of ER-α DNA in primary tumors, metastatic tumors, and sera of AJCC stage I-IV melanoma patients. Methods: The methylation status of theER-α DNA promoter region in tumor (n = 107) and sera (n = 119) from AJCC stage I-IV melanoma patients was examined by methylation-specific PCR (MSP) and capillary array electrophoresis (CAE). To determine the significance of free circulating hypermethylated ER-α DNA, we assessed the therapeutic response, progression-free and overall survival among AJCC stage IV melanoma patients receiving biochemotherapy. Results: The frequency of ER-α DNA methylation in AJCC stage I, II, and III primary melanomas was 36% (4 of 11), 26% (5 of 19), and 35% (8 of 23), respectively. The frequency of methylated ER-α DNA detected in stage III and IV metastatic melanomas was 42% (8 of 19) and 86% (30 of 35), respectively. ER-α DNA was methylated in 38 of 54 (70%) metastatic tumors, a more than two-fold increase compared to primaries (p = 0.0003). In the analysis of 109 melanoma patients’ sera, the frequency of methylated ER-α DNA in AJCC stage I, II, III, and IV was 10% (2 of 20), 15% (3 of 20), and 26% (5 of 19), and 32% (16 of 50), respectively. The frequency of methylated ER-α DNA detected in stage III/IV was significantly higher than in stage I/II (p=0.034). In a multivariate analysis, circulating hypermethylated ER-α DNA was the only independent factor predicting progression-free (risk ratio 2.64, 95% CI 1.36–5.13, p = 0.004) and overall survival (risk ratio 2.31, 95% confidence interval 1.41–5.58, p = 0.003) among stage IV patients receiving biochemotherapy. Conclusion: Hypermethylation of ER-α DNA in primary and metastatic melanoma tumors and serum is a significant factor in melanoma progression. The detection of methylated serum ER-α DNA predicts poor response to systemic therapy and is an unfavorable prognostic factor in advanced melanoma. [Table: see text]