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Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that mediates multiple cellular functions such as survival, invasion, and migration. FAK has been found to be over-expressed in various human cancers, including melanoma. FAK molecule has several tyrosine, serine, and threonine phosphorylation sites which have an important regulatory role. Tyrosine phosphorylation of FAK has been extensively studied, however little is known about the role of serine phosphorylation. We sought to examine the frequency and extent of serine-910 phosphorylated FAK (P-FAKSer910) expression in a spectrum of melanocytic proliferations as well as it's correlation with other histopathologic predictors and its effect on patient's survival. Clinicopathologic features and immunohistochemical expression of P-FAKSer910 were evaluated in 147 melanocytic proliferations: 73 primary melanoma (PM), 19 metastatic melanoma (MetM), 2 melanoma in situ, and 53 melanocytic nevi (MN). Higher cytoplasmic intensity predicted better overall survival (OS) in PM (χ=5.69; P=0.034) and was associated with 48% decrease in death risk (HR, 0.52; 95% CI, 0.28-0.95; P=0.036). In contrast, increased nuclear intensity was significantly associated with better disease-free survival (DFS) when stratified by tumor stage Log-rank test, trend of survival (χ=5.83, P=0.015) and independently on multivariate analysis when subcategorized into 3-tier categories (HR, 0.43; 95% CI, 0.18-0.98; P=0.045). Also, Clark's level and tumor-infiltrating lymphocytes (TILS) were independent predictors of DFS. Cytoplasmic intensity correlated inversely with American Joint Commission on Cancer stage in primary melanoma cases as well with vascularity in both primary and metastatic melanoma. Nuclear intensity independently correlated negatively with angioinvasion and TILS when subcategorized to 3 tier system. We found American Joint Commission on Cancer tumor stage, Clark's level, depth, ulceration, TILS, mitosis, angioinvasion, and tumor vascularity predictors of both DFS and OS. There was no significant difference in cytoplasmic or nuclear expression among the major categories of melanocytic proliferation. In this pilot immunohistochemistry-based study, we found P-FAKSer910 expression in melanoma by cytoplasmic intensity to correlate with better OS while nuclear intensity correlated with better DFS.

Background: The expression of TRPM1 (melastatin) mRNA is an independent marker, as measured by radioactive in situ hybridization (RISH), of disease-free survival in primary cutaneous melanoma (PM). The aim of the study was to determine if chromogenic in situ hybridization (CISH) can reproduce results examining diagnostic and prognostic utility of TRPM1 mRNA expression in melanocytic proliferations as measured by RISH., Methods: The expression of TRPM1 mRNA was detected by CISH in melanocytic nevi (MN, n = 61), PM (n = 145) and metastatic melanomas (MMs, n = 15)., Results: A progressive loss of TRPM1 was found moving from MN to PM to MM. The histologic stepwise model of melanoma progression revealed that loss of TRPM1 occurred at the transition of RGP PM to VGP PM. As a diagnostic marker, TRPM1 gradient loss showed 93.8% sensitivity and 52.4% specificity for PM. Loss of TRPM1 mRNA correlated with melanoma aggressiveness markers and was independent predictor of disease-free and overall survival. The corresponding survival curves for degree of melanoma pigmentation matched those for degree of loss of TPRM1 mRNA., Conclusion: Loss of TRPM1 mRNA expression appears to be a crucial event in the progression of melanoma to a more malignant, metastatic phenotype., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Melastatin (TRPM1), a.k.a. transient receptor potential cation channel, subfamily M, member 1 (TRPM-1) regulates melanocyte differentiation and proliferation. TRPM1 is transcriptionally regulated by the essential melanocyte transcription factor MITF (microphthalmia-associated transcription factor). For the most part, MITF expression is preserved during melanoma progression, while TRPM1 mRNA expression decreases or is completely lost. The loss of TRPM1 is associated with melanomas that are more aggressive., Objective: To assess the relationship between TRPM1 mRNA expression and the expression of MITF and nine other markers of melanocytes and melanin-related proteins by immunohistochemistry in normal skin, scars, hair follicles and ordinary melanocytic nevi., Methods: Samples of normal skin (n = 102; from tumor excisions and plastic procedures), scars (n = 5; from re-excision specimens) and compound melanocytic nevi (n = 4) were evaluated for the presence of TRPM1 mRNA transcripts as detected by chromogenic in situ hybridization (CISH). Immunohistochemical techniques were used to detect melanin-related proteins including: MITF, S100 protein, Mart-1, tyrosinase, Mel5, HMB45, tyrosinase-related protein-1 (TRP1), TRP2 and alpha-melanocyte stimulating hormone (alphaMSH). The labeling index (LI) was defined as the number of intraepidermal cells expressing mRNA or protein per one hundred basal keratinocytes., Results: A wide range of LI was found for all markers (0-33 positive cells/100 keratinocytes). When these LI were compared, no significant differences in the expression of MITF, S100, Mart1, tyrosinase proteins and TRPM1 mRNA were identified. The LI for TRPM1 mRNA expression ranged from 74% of that for MITF to 86% for tyrosinase. The LI for TRP-1, TRP-2 and Mel5 was similar to that of TRPM1, while HMB-45 had a significantly lower LI than all other markers. TRPM1 mRNA correlated most tightly with MITF and tyrosinase expression (r = 0.81 and 0.68, respectively, both p = 0.0001). Likewise, the strongest correlation among all the melanin-related proteins existed between tyrosinase and MITF (r = 0.79, p = 0.0001). There was variable expression of melanin-related proteins when LI were analyzed by anatomic site, patient age, extent of sun-damage and proximity to a melanocytic tumor. Anogenital skin showed the highest and acral skin the lowest LI for TRPM1, MITF, S100 protein, Tyrosinase, Mel5 and HMB45. Advanced age (> 60 years) was associated with decreased TRPM1 expression. Sun-damaged skin exhibited significantly increased LI as measured by MITF, S100 protein, Mart1, tyrosinase and HMB-45, but no differences for TRPM1. However, the MITF-TRPM1 differential (i.e. MITF LI-TRPM1 LI = MITF+TRPM1--melanocytes) was significantly increased in site-matched skin (4.6 +/- 4.4 vs. 1.5 +/- 2.5, p = 0.01). There was a suggestion of reduced LI in normal skin in the proximity of melanoma (from melanoma re-excision specimens) for S100, HMB45 and TRPM1 mRNA. TRPM1 LI was significantly decreased in scars compared to normal skin (5.6 +/- 1.4 vs. 9.7 +/- 4.3, p = 0.02), this was reflected in an increase in the MITF-TRPM1 differential (9.6 +/- 7.5 vs. 3.2 +/- 3.1, p = 0.0001). MITF LI were consistently higher than MSLN LI at all levels of the hair follicle; notably, MITF was expressed by isthmic-bulge cells. In ordinary melanocytic nevi, MITF and TRPM1 expression decreased with melanocyte descent: there was more signal for both markers in superficial epithelioid type A melanocytes than deeper type C melanocytes., Conclusions: By CISH, TRPM1 mRNA expression is specific for melanocytes and strongly associated with MITF and tyrosinase expression, the latter implicating a mature melanocyte phenotype. However, in normal skin, TRPM1 mRNA expression appears to be dynamic, labeling most but not all melanocytes, with variable expression ostensibly related to local environmental factors.

Objective: To determine whether loss of melastatin (MLSN) is a universal phenomenon in American Joint Committee on Cancer (AJCC) stage I and II melanoma patients who experienced recurrence., Material and Methods: Paraffin blocks of primary melanomas (PMs) were retrieved from 30 patients who had a negative sentinel lymph node biopsy and developed recurrent melanoma (AJCC stage I and II). Chromogenic in situ hybridization (CISH) methods were utilized to evaluate the expression of MLSN mRNA. These results were correlated with clinicopathologic data., Results: Variable, heterogeneous expression of MLSN mRNA was identified in normal, in situ and invasive melanocytes within and between cases. For the invasive PM component, 24 (80%) had focal, regional or complete loss of MLSN mRNA. The remaining 20% had either regional or total partial downregulation of MLSN mRNA. Intact MLSN mRNA expression was present regionally in 14/30 (47%), with mean relative tumor area of 38%, range 5-85%. Increasing loss of MLSN mRNA significantly correlated with increasing tumor depth and microsatellites (r = 0.1/0.4, p = 0.04). However, thin, AJCC T stage 1a PM had higher relative mean loss than intermediate AJCC T stage 2a/2b/3a thickness PM (65% vs. 34%/48%/25%). Increasing loss of MLSN mRNA significantly impacted on disease free survival (DFS) by multivariate analysis (58 vs. 0% 2 years DFS, < or = 75 vs. > 75% mRNA loss, p = 0.02). Decreased overall survival significantly correlated with increasing age and vascular invasion on multivariate analysis., Conclusion: Extensive loss of MLSN in PM correlated with aggressive metastatic melanoma. Ancillary testing for MLSN mRNA expression by CISH could offer a means to more accurately identify AJCC stage I and II patients at risk for metastatic disease, who could benefit from adjuvant therapy.

Background: S-phase kinase associated protein-2 (Skp2) ubiquitin ligase p45(SKP2) is important in the degradation of p27kip1 (a cyclin dependent kinase inhibitor) and progression through the G1-S cell-cycle checkpoint. Low levels of p27 and high levels of Skp2 are related to poor prognosis in some cancers., Methods: Clinicopathologic features and immunohistochemical expression of Skp2 and p27kip1 were investigated in 198 melanocytic proliferations: 21 melanocytic nevi, 23 melanoma in situ, 119 primary melanoma, and 35 metastatic melanoma samples. Comparative and survival analyses were performed., Results: Progressive and significant increases and decreases in the nuclear expression of Skp2 and p27kip1, respectively, was identified moving from melanocytic nevi (0.05 +/- 0.2/85 +/- 15) to melanoma in situ (3 +/- 2/45 +/- 20) to primary cutaneous melanoma (12 +/- 9/30 +/- 25) to metastatic melanoma (25 +/- 15/15 +/- 20) (p < or = 0.006). Expression of these proteins also significantly correlated with increasing American Joint Committee on Cancer (AJCC) T (tumor) classification and AJCC stage (p < or = 0.01). Moreover, the level of these two proteins exhibited a significant inverse relationship (r = -0.4, p = 0.0001). Skp2 cytoplasmic labeling index of >20% predicted worse 10-year overall survival (38% vs. 86%, p = 0.04) in primary melanoma. Neither p27 nor Skp2 nuclear expression impacted significantly on prognosis., Conclusions: Gain of Skp2 and loss of p27kip1 protein expression are implicated in melanoma progression where the level of p27kip1 may be regulated by targeted proteolysis via Skp2. Cytoplasmic expression of Skp2 defines a subset of aggressive melanomas and could represent another pathway of deregulation of the cell cycle.

Background: Although various studies have shown mutations of the tumor suppressor gene, PTEN/MMAC1, in primary, metastatic, and cultured cutaneous melanoma specimens, little is known about the pattern of PTEN protein expression in early melanocytic tumor progression., Objective: To further investigate the role of PTEN in melanocytic tumor development., Methods: We assessed the level and distribution of PTEN in normal skin, 39 acquired melanocytic nevi, and 30 primary cutaneous melanomas, including lentigo malignas, by immunostaining., Results: We found high levels of PTEN expression in cutaneous muscles, nerves, and muscular arteries, and moderate-to-high amounts of PTEN in the epidermis, follicular epithelium, and sebaceous and eccrine glands. PTEN staining in cutaneous lymphatics, dermal and periadnexal adventitial fibroblasts, and chondrocytes were variably absent. Junctional melanocytes and chondrocytes frequently exhibited preferential nuclear staining. We found uniformly strong PTEN expression in the cytoplasm of almost all benign and dysplastic nevi. However, there was some evidence of nuclear PTEN loss even in the benign melanocytic proliferations. In addition, out of 30 primary cutaneous melanomas and lentigo malignas, we detected diffuse expression of PTEN in 11 (37%) tumors, widespread loss of PTEN in 11 (37%) tumors and mixed PTEN expression in 8 (27%) lesions. In the primary cutaneous melanomas, PTEN was largely localized to the cytoplasm., Conclusions: The presence of PTEN in benign melanocytic tumors and the absence of PTEN in a significant proportion of primary cutaneous melanomas support a role for PTEN loss in the pathogenesis of melanoma.

Oncostatin M (OSM) is a 28-kDa glycoprotein, produced by stimulated macrophages and T lymphocytes, that inhibits the proliferation and induces differentiation of a number of different cell lines derived from solid tumors. To determine whether keratoacanthoma (KA) is unique or a variant of squamous cell carcinoma (SCC), we compared the immunohistochemical expression of OSM in the tumor cells and peri- and intratumoral macrophages of 21 mature KAs, 7 regressing KAs, and 27 SCCs. An inverse correlation was identified between OSM tumor labeling and the density of OSM-labeled tumor-associated macrophages for KAs (r = -.4; P = .09). OSM tumor expression was significantly more frequent and more intense in KAs than in SCCs (95% versus 63%; P < .01). In contrast, the density of OSM-labeled macrophages was significantly higher in SCCs compared with mature KAs (7/3 high power fields versus 4/3 high power fields; P = .02). These OSM-positive macrophages were predominantly located at the advancing, infiltrative margins of both neoplasms. Regressing KAs demonstrated a decreased level of OSM tumor expression compared with mature KAs (53% versus 95%; P = .001), but there was no difference in density of OSM-labeled macrophages. Both the above differences and the overlapping patterns of OSM expression suggest that KAs are a variant of SCC where OSM, possibly as an autocrine factor, may mediate KA's overwhelming but not absolute tendency to involute.