Melanoma Lymph Node Metastases Research Articles

Lymph node metastasis in melanoma: a debate on the significance of nodal metastases, conditional survival analysis and clinical trials.

While there is no doubt that regional lymph node metastases are an enormously important factor in melanoma staging and treatment, the biology behind this significance and its precise implications for treatment planning have been a leading controversy in melanoma and other solid tumors for over a century. Recent clinical data, including data from prospective randomized clinical trials have refined our understanding of the process of nodal metastases and the advantages and disadvantages of different clinical management strategies. This review presents two points of view in this debate and discusses the results of new data analyses as well as pivotal clinical trials informing the discussion.

Unique Genes in Tumor-Positive Sentinel Lymph Nodes Associated with Nonsentinel Lymph Node Metastases in Melanoma.

Current risk assessment tools to estimate the risk of nonsentinel lymph node metastases after completion lymphadenectomy for a positive sentinel lymph node (SLN) biopsy in cutaneous melanoma are based on clinical and pathologic factors. We identified a novel genetic signature that can predict non-SLN metastases in patients with cutaneous melanoma staged with a SLN biopsy. RNA was collected for tumor-positive SLNs in patients staged by SLN biopsy for cutaneous melanoma. All patients with a tumor-positive SLN biopsy underwent completion lymphadenectomy. A 1:10 case:control series of positive and negative non-SLN patients was analyzed by microarray and quantitative RT-PCR. Candidate differentially expressed genes were validated in a 1:3 case:control separate cohort of positive and negative non-SLN patients. The 1:10 case:control discovery set consisted of 7 positive non-SLN cases matched to 70 negative non-SLN controls. The cases and controls were similar with regards to important clinicopathologic factors, such as gender, primary tumor site, age, ulceration, and thickness. Microarray and RT-PCR identified six potential differentially expressed genes for validation. In the 40-patient separate validation set, 10 positive non-SLN patients were matched to 30 negative non-SLN controls based on gender, ulceration, age, and thickness. Five of the six genes were differentially expressed. The five gene panel identified patients at low (7.1%) and high risk (66.7%) for non-SLN metastases. A novel, non-SLN gene score based on differential expressed genes in a tumor-positive SLN can identify patients at high and low risk for non-SLN metastases.

Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration in the Diagnosis of Hilar and Mediastinal Lymph Node Metastases of Melanoma.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique for investigating hilar and mediastinal lymphadenopathy. This study reports eleven cases in which EBUS-TBNA was used to assess mediastinal and hilar lymph nodes for the presence of metastatic melanoma. A retrospective study was performed of all patients who had a history of melanoma and underwent EBUS-TBNA to assess hilar or mediastinal lymphadenopathy for the presence of metastatic melanoma. In seven cases, molecular analysis to detect mutations in the BRAF gene was also used. Eight patients had been diagnosed with malignant melanoma in the past (mean 54.4 months, range 18 to 115 months) while in the other three this tumor was primarily diagnosed in the staging phase. The male-female ratio was 6:5, and the mean age was 60.3 years (range 42 to 88 years). The mean hilar or mediastinal lymph node size detected with computed tomography was 3.0 cm (range 1.1 to 8.1 cm). Eight (72.7%) cases had metastases to the lung associated with metastases in the mediastinal lymph nodes. In four (50%) of these cases, the lung metastasis was solitary. Three (27.3%) cases had metastases in the mediastinal lymph nodes in absence of lung metastases. Metastatic melanoma was diagnosed by cytology and confirmed by cell block study with immunohistochemistry in all cases. BRAF mutations were detected in two (28.6%) of seven cases studied. Cytology and tissue samples obtained from EBUS-TBNA are adequate to detect metastatic melanoma and permit in some cases the determination of biomarkers and identify the presence or absence of mutations in the BRAF gene. The procedure is safe, fast, and precise for the staging of melanoma.

Association Between Patient Age and Lymph Node Positivity in Thin Melanoma

More than half of all new melanoma diagnoses present as clinically localized T1 melanoma, yet sentinel lymph node biopsy (SLNB) is controversial in this population given the overall low yield. Guidelines for SLNB have focused on pathologic factors, but patient factors, such as age, are not routinely considered. To identify indicators of lymph node (LN) metastasis in thin melanoma in a large, generalizable data set and to evaluate the association between patient age and LN positivity. A retrospective cohort study using the National Cancer Database, an oncology database representing patients from more than 1500 hospitals throughout the United States, was performed (2010-2013). Data analysis was conducted from October 1, 2016, to January 15, 2017. A total of 8772 patients with clinical stage I 0.50 to 1.0 mm thin melanoma undergoing wide excision and surgical evaluation of regional LNs were included for study. The primary outcome of interest was presence of melanoma in a biopsied regional LN. Clinicopathologic factors associated with LN positivity were characterized, using logistic regression. Age was categorized as younger than 40 years, 40 to 64 years, and 65 years or older for multivariable analysis. Classification tree analysis was performed to identify high-risk groups for LN positivity. Among the study cohort (n = 8772), 333 patients had nodal metastases, for an overall positivity rate of 3.8% (95% CI, 3.4%-4.2%). A total of 4087 (54.0%) patients were women. Median age was 56 years (interquartile range [IQR], 46-67) in patients with negative LNs and 52 years (IQR, 41-61) in those with positive LNs (P < .001). In multivariable analysis, younger age, female sex, thickness of 0.76 mm or larger, increasing Clark level, mitoses, ulceration, and lymphovascular invasion were independently associated with LN positivity. In decision tree analysis, patient age was identified as an important risk stratifier for LN metastases, after mitoses and thickness. Patients younger than 40 years with category T1b tumors 0.50 to 0.75 mm, who would generally not be recommended for SLNB, had an LN positivity rate of 5.6% (95% CI, 3.3%-8.6%); conversely, patients 65 years or older with T1b tumors 0.76 mm or larger, who would generally be recommended for SLNB, had an LN positivity rate of only 3.9% (95% CI, 2.7%-5.3%). Patient age is an important factor in estimating lymph node positivity in thin melanoma independent of traditional pathologic factors. Age therefore should be taken into consideration when selecting patients for nodal biopsy.

Abstract 2842: Dissecting the transcriptional profiles of metastatic and primary disease across cancer types

Abstract Cancer metastasis is the principal cause of death in individuals with cancer; nevertheless, the molecular basis of metastases is poorly understood. Genetic and epigenetic changes in malignant cells and their interaction with the tumor microenvironment are presumably key events in the establishment of metastases. In this study, we pursue to provide insights into potential importance of various fundamental cancer-related pathways in the metastatic process. We have performed a pathway - based analysis on mRNA expression data (Agilent 8X60K array) from colorectal liver metastases (CLM; N = 38 patients, n = 44 metastases), breast lymph-node metastases (BCM; N = n = 43) and melanoma lymph-node metastases (MLM; N = n= 44). For each of the 186 pathways in the KEGG pathway database, each sample is represented as points in a ‘n’ dimensional space according to their gene expression values, where ‘n’ is the number of genes in the pathway. We first calculated centroids for each of the three metastases types (CLM, BCM and MLM) and then calculated the Euclidean distance between centroids to get a measure of how similar or different each cancer type is for that particular pathway. The findings were compared to the corresponding primary cancer types and normal tissue available from TCGA to uncover biological processes specific to metastases. The pathways were functionally classified into genetic information processing, cellular processes, environmental information processing and metabolic pathways as per KEGG database. Across metastases from different cancer types, pathways involved in genetic information processing (e.g. translation, transcription, protein folding and degradation, DNA replication and repair) and metabolic pathways were more similar than the pathways involved in cellular processes (e.g. cell-cycle, apoptosis, adherence junction), and signal transduction pathways (e.g. Notch, MTOR, JAK STAT) responsible for environmental information processing. The genetic information processing pathways were more similar across cancer types also in the primary setting than the pathways involved in cellular and environmental information processing, whereas the metabolic pathways were more similar across cancer types in the metastatic setting compared to the primary setting. Preliminary findings suggest that the basic molecular architecture of the primary tumors is maintained, except for the metabolic pathways, which become more similar in the metastatic setting; and hence may be a common denominator across cancer types during metastatic process. Citation Format: Laxmi Silwal-Pandit, Vigdis Nygaard, Hege Russnes, Vegar Johansen Dagenborg, Veronica Skarpeteig, Olga Østrup, Silje Nord, Vivi Ann Flørnes, Anne Hansen Ree, Kjersti Flatmark, Anne-Lise Børresen-Dale, Ole Christian Lingjærde, Gunhild Mari Mælandsmo. Dissecting the transcriptional profiles of metastatic and primary disease across cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2842. doi:10.1158/1538-7445.AM2017-2842

Abstract NG02: Lymphatic vessels: Balancing immune priming and immune evasion in melanoma

Abstract NG02: Lymphatic vessels: Balancing immune priming and immune evasion in melanoma

Lymphatic invasion predicts sentinel lymph node metastasis and adverse outcome in primary cutaneous melanoma.

Sentinel lymph node (SLN) metastasis is a powerful predictor of survival in primary cutaneous melanoma. Lymphatic invasion (LI) may correlate with increased risk of SLN metastasis. Intralymphatic metastases, often difficult to detect on hematoxylin and eosin (H&E) stained sections, are readily identified with dual immunohistochemistry for melanocytic and lymphatic markers. We used dual S100/D240 immunohistochemistry to detect LI in 125 melanomas from patients who underwent SLN biopsy and correlated LI with melanoma staging parameters and disease status. Dual immunohistochemistry allowed for the identification of LI in 33 cases (26%), compared to only 2% on H&E stained sections. Melanomas with LI showed greater thickness, higher mitotic rate and more frequent ulceration. Eleven of 33 cases with LI (33%) and 10 of 92 cases without LI (11%) were associated with a positive SLN (P = .006). More patients without LI were disease-free at last follow-up (80%) than patients with LI (50%; P = .002); LI was significantly associated with decreased progression-free survival. The detection of LI is improved by dual immunohistochemistry and predicts SLN metastasis. The presence of LI may impact therapeutic planning in melanoma, such as the decision to perform a SLN biopsy.

Role of lymphadenectomy in vulvar melanoma

Objective: Malignant melanoma of the vulva is rare, representing less than 1% of melanomas and 10% of vulvar malignancies. Stage is based on the cutaneous melanoma AJCC system. Lymphadenectomy is not performed in thin cutaneous melanomas without risk factors (presence of ulceration, mitotic rate). Clinicopathologic factors of vulvar melanoma and management are based on small series. The objective of this study was to identify pathologic factors associated with lymph node metastasis and survival in vulvar melanoma.

Paraneoplastic acral vascular syndrome in a patient with metastatic melanoma under immune checkpoint blockade

BackgroundParaneoplastic acral vascular syndrome (PAVS) is a rare phenomenon which is observed in patients with adenocarcinomas and other malignancies. Various potential pathogenic mechanisms such as tumour invasion of sympathetic nerves, hyperviscosity, hypercoagulability, vasoactive tumour-secreted substances, and immunological mechanisms have been suggested.Case presentationWe report a 60-year-old Caucasian male attended our hospital with a bulky lymph node mass in the right axilla. Extirpation of a lymph node conglomerate revealed 5 melanoma lymph node metastases. Computed tomography showed a liver metastasis (diameter: 3.8 cm), several retroperitoneal metastases, bilateral metastases in the lung hilus, and prepectoral subcutaneous metastases (Stage IV; pTx, N3, M1c). Lactate dehydrogenase and S100B were slightly elevated. Combination therapy of nivolumab (1 mg/kg BW) and ipilimumab (3 mg/kg BW) was started. Three weeks after the first combination therapy he developed progressive erythema, paraesthesia and pain on the fingertips of both hands. Both cold and warmth was not well tolerated by the patient. Complete work-up excluded associated conditions or factors such as haematological disorders, rheumatologic disorders, hypertension, diabetes or smoking. Treatment was initiated with prostacyclin 20 μg twice daily and oral prednisolone 50 mg in tapering dosage. However, prostacyclin was stopped after the first applications because the pain increased during infusion. The second course of nivolumab and ipilimumab was administered. About 2 weeks later, the patient presented with increased pain and small subungual necrosis. We treated the patient with oral analgetics and intravenous prednisolone 500 mg in tapering dosage. On digital substraction angiography occlusion of all arteries of the fingers was demonstrated. Further rheologic and anti-melanoma treatments were refused by the patient. About 2 months after the second course of nivolumab and ipilimumab combination therapy several fingers showed severe gangrene which finally led to amputations of end phalanges of several fingers. Histopathology did not reveal evidence for vasculitis or other primary vascular pathologies. During the following 2 months the patient experienced dramatic progress of his metastatic disease and finally died at multi-organ failure.ConclusionPresence of rapidly progressive digital ischemia in an elderly patient with cancer should always raise clinical suspicion of a paraneoplastic phenomenon when other possible causes have been excluded. In patients treated with immune checkpoint inhibitors such as CTLA-4 and PD-L1 blockers PVAS-like events have not been reported so far. However, it is debatable whether immune checkpoint blockade may play a pathogenetic role in the development of PAVS in patients with malignancies.

Melanoma growth and lymph node metastasis is independent of host CD169 expression

Metastasis to the lymph node is a frequent and early event in tumour dissemination. Tumour soluble factors, including extracellular vesicles, condition host organs for metastatic tumour spread, thereby facilitating tumour cell migration and survival. In the peripheral lymphatics, extracellular vesicles are captured via their sialic acids by lymph node macrophages expressing the CD169 (sialoadhesin) molecule, thereby suppressing the immune response. We hypothesised that the CD169 molecule could modulate primary tumour growth and invasion into the regional lymph node by altering the immune response to tumour extracellular vesicles, or by directly interacting with invading tumour cells. No significant difference was noted in primary tumour growth between wild-type and CD169−/− mice, and protection against tumour challenge with tumour extracellular vesicle immunisation was similar between the strains. Subcutaneous implantation of B16 (F1 or F10) into the ventral-carpal aspect of forelimb resulted in melanoma infiltration into the axillary and brachial lymph nodes. CD169−/− mice displayed a lower level of metastatic lymph node lesions, however this failed to reach statistical significance. Although CD169 participates in the immune response to tumour antigen and appears to be a positive prognostic marker for human cancers, its role in modulating melanoma growth and metastasis is less clear.

Abstract A71: Tumor HLA Class I expression influences immune cells infiltration in metastatic melanoma tumor microenvironment

Abstract The variable clinical success with immune checkpoint inhibitors in the treatment of solid tumors challenges us to further understand the regulatory mechanisms of immune cells infiltration – a critical step of tumor elimination – in a highly heterogenous tumor microenvironment (TME). Systemic analyses of the tumor-immune cell interface and interactions are of particular significance with direct clinical impact. We employed a multiplexed immunofluorescence (MxIF) method, a novel technology that allows in situ quantitative single cell characterization with high order protein multiplexing on whole tumor sections, to elucidate the key components in TME governing the immune cells' composition and function in metastatic melanoma lymph node metastases. Here, we show that the expression of tumor cell Human Leukocyte Antigen 1 (HLA-1) is highly heterogeneous both within the TME and between patients. The level of tumor HLA-1 expression directly correlates with the magnitude of cytotoxic T lymphocytes (CTLs) infiltration in the TME. In addition, decreased tumor HLA-1 expression is associated with accumulation of CD20+ cells at the edge of the tumor as well as an increased population of CD4+FOXP3+ cells. The association between tumor HLA-1 expression and immune cell distribution is highly consistent yet heterogenous across the entire tumor mass. Moreover, using quantitative single cell data, we developed algorithms for computational and spatial modeling of tumor-immune cell interfaces, allowing statistical comparison of cellular interactions in the context of tumor heterogeneity. Spatial analysis demonstrates that tumor HLA-1 expression favors CTL invasion, while lack of tumor HLA-1 expression results in CTL evasion. Furthermore, in a cohort of 166 Stage III melanoma patients, we confirm that CTLs infiltration is only present in regions with high tumor HLA-1 expressions, and that patients with tumors high in both tumor HLA-1 and CTLs have significantly improved progression free survival (PFS) compared with those with tumors low in HLA-1 and/or CTLs. Our study demonstrates that the heterogeneous tumor HLA-1 expression results in various immune cell distribution patterns in the TME, directly contributing to the varied antitumor immunities and ultimately influencing tumor outcomes. As part of the antigen presentation machinery, HLA-1 expression on tumors is necessary for CTLs infiltration and is indispensable for T cell mediated tumor elimination with direct clinical benefits. We also establish a novel platform for visualization and spatial representation of the cellular heterogeneity within the TME, providing data processing and modeling algorithms necessary for understanding the cellular interplay mediating tumor surveillance, which will ultimately improve therapeutic efficacy and clinical outcomes. Citation Format: Yiyi Yan, Alexey Leontovich, Thomas Flotte, Michael Gerdes, Fiona Ginty, Alberto Santamaria-Pang, Anup Sood, Keyur Desai, Chrystal Chadwick, Rong Zhang, Svetomir N. Markovic. Tumor HLA Class I expression influences immune cells infiltration in metastatic melanoma tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A71.

Heterogeneity of Metastatic Melanoma: Correlation of MITF With Its Transcriptional Targets MLSN1, PEDF, HMB-45, and MART-1.

Histologic and molecular heterogeneity is well recognized in malignant melanoma; however, the diversity of expression of new and classic melanoma markers has not been correlated in serial sections of metastases. We examined and correlated the expression of microphthalmia transcription factor (MITF) with its transcriptional targets, including melastatin (MLSN1/TRPM1), pigment epithelium-derived factor (SERPINF1/PEDF), SILV/PMEL17/GP100 (human melanoma black 45 [HMB-45]), and melanoma antigen recognized by T cells 1 (MART-1)/MLANA, in 13 melanoma metastases in lymph nodes of 13 patients. The expression levels and patterns of marker expression were recorded by a semiquantitative, 4-point ordinal reactivity method. Our results showed a consistently robust and diffuse expression of MITF protein in 12 (92%) of 13 metastatic tumors compared with variable expression of MLSN1 (46%) messenger RNA or PEDF (75%), HMB-45 (54%), and MART-1 (46%) proteins. Overall, in melanoma lymph node metastases, MITF protein expression was not tightly correlated with its gene targets. Moreover, the immunoreactivity for MITF, compared with MART-1 and HMB-45, was retained, supporting immunohistochemical detection of MITF as a more sensitive method of detecting metastatic melanoma.

PD-L1 Negative Status is Associated with Lower Mutation Burden, Differential Expression of Immune-Related Genes, and Worse Survival in Stage III Melanoma.

Understanding why some melanomas test negative for PD-L1 by IHC may have implications for the application of anti-PD-1 therapies in melanoma management. This study sought to determine somatic mutation and gene expression patterns associated with tumor cell PD-L1 expression, or lack thereof, in stage III metastatic melanoma to better define therapeutically relevant patient subgroups. IHC for PD-L1 was assessed in 52 American Joint Committee on Cancer stage III melanoma lymph node specimens and compared with specimen-matched comprehensive clinicopathologic, genomic, and transcriptomic data. PD-L1-negative status was associated with lower nonsynonymous mutation (NSM) burden (P = 0.017) and worse melanoma-specific survival [HR = 0.28 (0.12-0.66), P = 0.002] in stage III melanoma. Gene set enrichment analysis identified an immune-related gene expression signature in PD-L1-positive tumors. There was a marked increase in cytotoxic T-cell and macrophage-specific genes in PD-L1-positive melanomas. CD8A(high) gene expression was associated with better melanoma-specific survival [HR = 0.2 (0.05-0.87), P = 0.017] and restricted to PD-L1-positive stage III specimens. NF1 mutations were restricted to PD-L1-positive tumors (P = 0.041). Tumor negative PD-L1 status in stage III melanoma lymph node metastasis is a marker of worse patient survival and is associated with a poor immune response gene signature. Lower NSM levels were associated with PD-L1-negative status suggesting differences in somatic mutation profiles are a determinant of PD-L1-associated antitumor immunity in stage III melanoma. Clin Cancer Res; 22(15); 3915-23. ©2016 AACR.

Elevated Serum Leptin Levels are Associated With an Increased Risk of Sentinel Lymph Node Metastasis in Cutaneous Melanoma.

The metabolic hormone leptin has been implicated in the pathogenesis of various malignancies and may contribute to the high rate of cancer in obese individuals. We reported that leptin and its receptor are expressed by melanoma tumors and cell lines, and that leptin stimulates proliferation of cultured melanoma cells. Here, we tested the hypothesis that leptin contributes to early melanoma progression by assessing its association with sentinel node positivity in cutaneous melanoma patients. The study enrolled 72 patients who were scheduled to undergo lymphatic mapping and sentinel node biopsy. Fasting blood was obtained before surgery, and serum leptin levels were measured by enzyme-linked immunosorbent assay (ELISA) with a "raw" (assay value) and an "adjusted" value (raw value divided by body mass index). Leptin levels and other clinicopathologic parameters were compared between sentinel node positive and negative groups. Logistic regression models were used to predict sentinel node status using leptin and other relevant clinical parameters. The raw and adjusted leptin levels were significantly higher in the 15 patients with positive sentinel nodes. These findings could not be attributed to differences in body mass indices. Univariate models revealed raw leptin, adjusted leptin, Breslow thickness, and mitotic rate as significant predictors of sentinel node status. Leptin levels and Breslow thickness remained significant in multivariate models. Survival and follow-up analysis revealed more aggressive disease in diabetic patients. Elevated serum leptin levels predict sentinel node metastasis in melanoma. Validation of this finding in larger cohorts should enable better stratification of early stage melanoma patients.

Ultrasound-guided core needle biopsy of superficial lymph nodes: an alternative to fine-needle aspiration cytology for the diagnosis of lymph node metastasis in cutaneous melanoma.

To investigate the diagnostic value of ultrasound-guided core needle biopsy (US-CNB) in suspected cases of lymph node metastasis from cutaneous melanoma. All patients with cutaneous melanoma followed in Saint-Louis Hospital between 2006 and 2010 who underwent US-CNB for suspicion of melanoma lymph node metastasis were reviewed retrospectively. Histopathological results of US-CNB samples were classified as melanoma, other malignancy, suspicious, inadequate, or benign. The diagnostic accuracy of US-CNB was assessed by comparison with two reference standards: histopathological examination of the radical lymph node dissection or, when this was not available, clinical and radiological follow-up. The data from 72 US-CNB were analyzed. Forty-four melanomas, 22 benign, three other malignancies, three inadequate samples, and no inconclusive specimens were diagnosed. Seventy-one US-CNB results were confirmed (98.6%). US-CNB achieved high sensitivity, specificity, and positive predictive value (respectively, 97.9, 100, and 100%). No adverse events were reported after the procedure. US-CNB provided a mean tissue volume of 16.7 mm per lymphadenopathy. US-CNB has diagnostic value similar to that of fine-needle aspiration cytology. It represents a reliable alternative method in melanoma lymph node metastasis to avoid surgery in patients who will not benefit from it. US-CNB provides relatively large samples of tissue suitable for comprehensive genomic analyses currently needed for research and personalized care of melanoma patients.

SNPs at miR-155 binding sites of TYRP1 explain discrepancy between mRNA and protein and refine TYRP1 prognostic value in melanoma.

Background:We previously demonstrated an inverse correlation between tyrosinase-related protein 1 (TYRP1) mRNA expression in melanoma metastases and patient survival. However, TYRP1 protein was not detected in half of tissues expressing mRNA and did not correlate with survival. Based on a study reporting that 3′ untranslated region (UTR) of TYRP1 mRNA contains two miR-155-5p (named miR-155) binding sites exhibiting single-nucleotide polymorphisms (SNPs) that promote (matched miRNA–mRNA interaction) mRNA decay or not (mismatched), we aimed to investigate the role of miR-155 in the regulation of TYRP1 mRNA expression and protein translation accounting for these SNPs.Methods:The effect of miR-155 on TYRP1 mRNA/protein expression was evaluated in two melanoma cell lines harbouring matched or mismatched miR-155–TYRP1 mRNA interaction after transfection with pre-miR-155. In parallel, 192 skin and lymph node melanoma metastases were examined for TYRP1 mRNA/protein, miR-155 and SNPs and correlated with patient survival. TYRP1 mRNA, SNPs at its 3′UTR and miR-155 were analysed by RT–qPCR, whereas TYRP1 protein was evaluated by western blot in cell lines and by immunohistochemistry in metastatic tissues.Results:The miR-155 induced a dose-dependent TYRP1 mRNA decay and hampered its translation into protein in the line with the ‘match' genotype. In melanoma metastases, TYRP1 mRNA inversely correlated with miR-155 expression but not with TYRP1 protein in the ‘match' group, whereas it positively correlated with protein but not with miR-155 in the ‘mismatch' group. Consequently, in the latter group, TYRP1 protein inversely correlated with survival.Conclusion:Polymorphisms in 3′UTR of TYRP1 mRNA can affect TYRP1 mRNA regulation by miR-155 and its subsequent translation into protein. These SNPs can render TYRP1 mRNA and protein expression nonsusceptible to miR-155 activity and disclose a prognostic value for TYRP1 protein in a subgroup of melanoma patients. These data support the interest in the prognostic value of melanogenic markers and propose TYRP1 to refine prognosis in patients with advanced disease.

Canine cutaneous melanocytic tumours: significance of β-catenin and survivin immunohistochemical expression.

Recent investigations have highlighted the controversial role of Wnt/β-catenin pathway activation in human cutaneous melanoma. Survivin has been proposed as a valid prognostic marker for invasive and metastatic melanomas and lymph node melanoma metastasis in human cutaneous melanoma and is a promising therapeutic target. Our aim was to investigate the immunohistochemical expression of survivin and β-catenin in canine cutaneous melanocytic tumours, in order to understand their prognostic significance. Twenty-one melanocytic tumours (10 melanocytomas and 11 melanomas) were investigated by immunohistochemistry using specific anti-survivin and anti-β-catenin antibodies. A semi-quantitative method was used to analyse the results; β-catenin immunolabelling in neoplastic cells was evaluated as cytoplasmic, membranous or nuclear. The number of survivin-positive cells was counted within ~1000 neoplastic cells. Results were related to histopathological features, evaluated in haematoxylin- and eosin-stained slides, and to the clinical data obtained through a telephone survey with referring veterinarians. Despite a low level of expression in the majority of cases, β-catenin was found to be correlated strongly with malignant behaviour (P < 0.01). An overexpression of nuclear survivin was statistically related to histological features of malignancy, presence of metastasis and death related to melanoma spread (P < 0.01). The low nuclear β-catenin expression, mainly found in metastatic cases, would indicate that β-catenin activation may have only limited importance in the development or progression of canine cutaneous melanoma. The correlation of nuclear survivin expression with malignancy would indicate that survivin is possibly a useful prognostic marker and therapeutic target in canine melanoma patients.

Predictors of Sentinel Lymph Node Status in Cutaneous Melanoma: A Classification and Regression Tree Analysis

ObjectiveThe main aim of this study was to identify predictors of sentinel lymph node (SN) metastasis in cutaneous melanoma. Patients and methodsThis was a retrospective cohort study of 818 patients in 2 tertiary-level hospitals. The primary outcome variable was SN involvement. Independent predictors were identified using multiple logistic regression and a classification and regression tree (CART) analysis. ResultsUlceration, tumor thickness, and a high mitotic rate (≥6 mitoses/mm2) were independently associated with SN metastasis in the multiple regression analysis. The most important predictor in the CART analysis was Breslow thickness. Absence of an inflammatory infiltrate, patient age, and tumor location were predictive of SN metastasis in patients with tumors thicker than 2mm. In the case of thinner melanomas, the predictors were mitotic rate (>6 mitoses/mm2), presence of ulceration, and tumor thickness. Patient age, mitotic rate, and tumor thickness and location were predictive of survival. ConclusionsA high mitotic rate predicts a higher risk of SN involvement and worse survival. CART analysis improves the prediction of regional metastasis, resulting in better clinical management of melanoma patients. It may also help select suitable candidates for inclusion in clinical trials.

Factores predictivos del estado del ganglio centinela en el melanoma cutáneo: análisis mediante un árbol de clasificación y regresión

ObjectiveThe main aim of this study was to identify predictors of sentinel lymph node (SN) metastasis in cutaneous melanoma. Patients and methodsThis was a retrospective cohort study of 818 patients in 2 tertiary-level hospitals. The primary outcome variable was SN involvement. Independent predictors were identified using multiple logistic regression and a classification and regression tree (CART) analysis. ResultsUlceration, tumor thickness, and a high mitotic rate (≥6 mitoses/mm2) were independently associated with SN metastasis in the multiple regression analysis. The most important predictor in the CART analysis was Breslow thickness. Absence of an inflammatory infiltrate, patient age, and tumor location were predictive of SN metastasis in patients with tumors thicker than 2mm. In the case of thinner melanomas, the predictors were mitotic rate (>6 mitoses/mm2), presence of ulceration, and tumor thickness. Patient age, mitotic rate, and tumor thickness and location were predictive of survival. ConclusionsA high mitotic rate predicts a higher risk of SN involvement and worse survival. CART analysis improves the prediction of regional metastasis, resulting in better clinical management of melanoma patients. It may also help select suitable candidates for inclusion in clinical trials.

Prospective immunohistochemical analysis of BRAF V600E mutation in melanoma.

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation is the most common activating genetic alteration of this oncogene and a predictive marker for the therapeutic use of BRAF inhibitors in melanoma. Our aim was to evaluate the performance of BRAF V600E mutation-specific monoclonal antibody (VE1) in a prospective diagnostic setting of melanoma patients (n = 102). All 41 cases (40.2%) that showed a V600E mutation in the cyclic minisequencing analysis of the DNA were also initially scored immunopositive. Two cases that were scored as BRAF V600E mutation positive by immunohistochemistry were negative in the DNA-based mutation analysis and determined to be immunonegative in a repeated staining with more representative specimens. Thus, BRAF V600E mutation detection using immunohistochemistry was 100% sensitive and 96.8% specific, when compared with the analysis of the DNA. None of the BRAF V600K mutations was detected by the VE1 antibody (n = 7). However, the VE1 antibody detected a rare V600E2 mutation. We also studied the role of BRAF V600E mutation in a set of melanoma patients who had been investigated for sentinel node metastasis. Melanoma lymph node metastases were diagnosed in 21.8% (12/55) of the sentinel nodes, and BRAF V600E immunopositivity was detected in 34.5% (19/55) of the cases. BRAF V600E mutation status did not correlate with any clinicopathological parameters. In conclusion, analysis of BRAF V600E mutation in melanoma by immunohistochemistry is a sensitive and specific method, which can be used to identify BRAF inhibitor-sensitive melanoma patients as a first-line method due to its rapid and affordable nature.