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Malignant melanoma (MM) is known to avoid the host's immune response. Studies on in vitro melanoma cell lines link the microphthalmia-associated transcription factor (MITF) with the regulation of the PD-L1 expression. It seems that MITF affects the activation of the gene responsible for PD-L1 protein expression. Several proteins, including Bcl-2 and Cyclin D1, play major roles in malignant melanoma cell cycle regulation and survival. Our study aims to assess the relationship between MITF, Bcl-2, and cyclin D1 protein expression and the expression of the PD-L1 molecule. Additionally, we examined the association of BRAF mutation, MITF, and CCND1 gene amplification with PD-L1 protein expression. We performed immunohistochemical staining on fifty-two tumour samples from patients diagnosed with nodular melanoma (NM). BRAF V600 mutation, MITF, and CCND1 gene amplification analyses were analyzed by the Sanger sequencing and QRT-PCR methods, respectively. Statistical analyses confirmed the significant inverse correlation between cyclin D1 and PD-L1 expression (p = 0.001) and correlation between PD-L1 and MITF protein expression (p = 0.023). We found a statistically significant inverse correlation between the present MITF gene amplification and PD-L1 (p = 0.007) and MITF protein expression (p = 3.4 ×10-6), respectively. Our study, performed on clinical NM materials, supports the in vitro study findings providing a rationale for the potential MITF-dependent regulation of PD-L1 expression in malignant melanoma., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: The spread and invasion of malignant melanoma cells involve degradation and reorganization of the extracellular matrix by the activation of several matrix metalloproteinases (MMPs). This study analyzed the expression of MMP-1, MMP-2, and MMP-13 proteins in primary nodular melanoma (NM) and dysplastic nevi (DN) as a significant risk factor for melanoma development. The secondary goal was to analyze the correlation of MMPs protein expression in NM with tumor invasion, BRAF V600 mutation status, and overall survival., Methods: Immunohistochemistry for MMP-1, MMP-2, and MMP-13 was performed on nodular melanoma (n = 52) and dysplastic nevi (n = 28) on tissue microarray (TMA). BRAF V600 mutation analysis on NM samples was performed by the Sanger sequencing method., Results: A high level of MMPs expression in NM samples (>30%) compared with DN (<8%) was statistically significant (P < 0.001). BRAF V600 mutations were detected in 15 of 39 (38.5%) NM samples. This study revealed an interesting finding that MMP-1 and MMP-13 protein expression in the BRAF V600 mutated melanomas were significantly lower than in the BRAF V600 wild type (P < 0.05)., Conclusion: Cox analysis revealed that Clark categories, Breslow thickness, and MMP-1 high protein expression are predictive factors for shorter overall survival (P < 0.05)., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Since there are no standardized protocols regarding the detection of microscopic melanoma deposits in sentinel lymph nodes (SLN), the aim of this study was to present our experience with intraoperative cytological evaluation of SLN in patients with melanoma. The study included 475 SLN biopsies (SLNB) from 201 patients with primary cutaneous melanoma of intermediate thickness. Each lymph node was cut in half; touch imprint cytology (TIC) preparations of all cut surfaces were performed and stained according to a modified May-Grünwald-Giemsa method. The results were compared to definitive postoperative histology. Twenty of 25 SLNB positive on TIC proved to be metastatic when compared to definitive histology. Most of 32 SLN that were suspicious but not diagnostic on TIC were proven negative (23/32, 71.8%), while 7 nodes had metastases (one micrometastasis and one with isolated tumor cells only). The majority (94%) of SLNBs negative on TIC remained negative on final histology, while 6% or 25 nodes were positive, mostly with micrometastases or isolated tumor cells (17/25). In frozen sections performed in cases of suspicious or positive SLN cytology, metastasis was confirmed in 80% of positive and in 21.9% of suspicious TIC. Altogether, 49% (27/55) of positive SLNB were identified intraoperatively in 57% (24/42) of patients, and in those cases a complete regional lymph node dissection was performed in the first step. TIC assessment of SLNB with 99% specificity and 57% sensitivity for intraoperative identification of metastasis is useful and beneficial for avoiding a second operative procedure.

Melanoma ulceration is the third most powerful survival predictor in the analysis for the currently valid 2010 AJCC melanoma staging system. However, there is still no detailed explanation for melanoma ulceration mechanism. Although thicker tumors are more commonly ulcerated, ulceration is a factor that predicts the disease outcome independently of melanoma thickness. It is our hypothesis that EGFR activates melanoma cell mobility. Melanoma cells with increased mobility move through tissue in diverse direction. Cells moving toward the epithelium weaken the intercellular bonds between the cells causing the loss of epithelium and subsequently ulceration., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Considering that nodular melanoma (NM) has the potential to show an early distant metastasis, there is an urgent need for the discovery and evaluation of new diagnostic and prognostic biomarkers. We aimed to investigate the protein expression of membrane and nuclear epidermal growth factor receptor (EGFR), cyclin D1, and the corresponding gene status in NM samples and correlate the results obtained with clinicopathological parameters and overall survival of patients. Immunohistochemical and fluorescence in-situ hybridization analyses were carried out on tissue microarrays constructed from 110 NM samples, 30 compound nevi, and 38 dysplastic nevi. NM samples showed 24% strong cyclin D1 and 37% strong Ki67 protein expression compared with 3 and 0% strong cyclin D1 and Ki67 expression in the control group. Membrane EGFR expression was detected in 50% of NM cases, whereas EGFR gene amplification was detected in only 4% of NM cases. Multiple NM samples presented simultaneous membrane and nuclear EGFR expression. We found a negative correlation between tumor thickness and membrane EGFR expression. It was also observed that membrane EGFR 3+ NM samples presented ulceration significantly more often than membrane EGFR-negative (0) NM samples. In univariate analysis, carried out on 44 patients with follow-up data, both nuclear and membrane EGFR overexpression showed a correlation with a shorter overall survival. Nuclear EGFR (++, +++) showed 3.06 and membrane EGFR (2+, 3+) showed 2.76 higher risk of mortality compared with patients with low and negative nuclear and membrane EGFR expression (P<0.05).

There is a global rising incidence of melanoma. For different reasons, the patterns of the incidence, appearance, gender, anatomical distribution and outcome vary among different geographic areas. Screening programs have led to better early detection of melanoma in Australia and some world areas. National Cancer Registry and practice data show the incidence in Croatia to be constantly rising. Despite public education programs about early detection, at clinical departments there are still many new advanced stage melanoma patients. We analyzed data on 157 patients treated and followed up for 10 years for T1b-T4aN0 skin melanoma. There was a difference in anatomical distribution of melanoma lesions in correlation with patient age (ANOVA test, F=3.51, p=0.009). A higher prevalence of shoulder melanoma was found in young people and of head/neck melanoma in the elderly (post-hoc Sheffe test, p=0.038). T4 lesions were more commonly found in men and T1 mainly in women (Pearson χ(2)-test, χ(2)=12.08, p=0.016). There was no difference in Clark level, but a significantly higher Breslow stage was found in men (t=-2.52, p=0.013). Men were much more prone to have head and neck, body and shoulder melanoma, whereas women had more melanoma on their legs and arms. Clark and Breslow levels were strongly correlated in leg melanoma; head localization showed no correlation at all. In conclusion, more attention should be devoted to improve the results in melanoma detection in men, especially considering the prevalence of body (back) and head/neck localizations, sometimes not readily accessible for visual detection. The pattern of distribution also pointed to the need for more attention to pay to shoulder melanoma in younger people.

Skin melanoma is by far the most lethal skin cancer, it is unpredictable by nature and presents a severe diagnostic problem. One of the major issues in melanoma diagnostics is to differentiate it with confidence from a dysplastic nevus. Thus, the aim of this study was to evaluate hTERT expression on a spectrum of dermal lesions (from normal skin toprimary melanoma) in order to examine its possible role as a diagnostic marker in melanoma diagnostics. In this study we analyzed the expression of hTERT by real-time PCR on 58 freshly obtained biopsy samples (4 samples of normal skin, 12 dermal nevi, 23 dysplastic nevi, 19 primary melanomas). Our results showed slightly greater hTERT expression in dysplastic nevi than melanomas with major data overlap. Considering the given results, hTERT does not seem to be a reliable diagnostic marker for melanoma.

Global heating and increased solar ultraviolet irradiance have caused an increase in number of many diseases, particularly skin malignant diseases. Aim of this study was to investigate the influence of climate changes on the health of the population of the Primorsko-goranska and Istria Counties. We gathered and analyzed data about the frequency of skin malignant melanoma in the period of eight years (1998-2005). The data were collected from the Croatian cancer registry. The incidence of malignant skin cancer was estimated overall, by age group and gender. We found that the incidence of the skin melanoma was approximately the same in both counties during the period 1998-2005. However, significant increase has been noted when compared to the situation in the period 1977-1996 (p = 4.95 E-13) The incidence of malignant skin melanoma has risen during the last ten years. It is differently distributed between gender and age groups in Primorsko-goranska and Istria County. It can be related to climate changes, but also to different ways way of life between these two counties.

A case of rare tumor, Merkel cell carcinoma, located in the ear canal of a 25-year-old woman is presented. A polypoid tumor mass was extirpated, and tympanoplasty was done at the first operation, whereas at the second operation, all the bones of the ear canal were removed. Epitympanum and cavum were filled with tumor, and the tumor mass was removed in toto. The histopathology and immunohistochemical staining characteristics of tumor confirmed the presence of Merkel cell tumor. Postoperatively, radiation therapy to the tumor bed was completed. There was no clinical or radiographic evidence of recurrence or metastasis of Merkel cell tumor for 3 years.

Involution displayed by keratoacanthoma (KA) represents an important difference between KA and squamous cell carcinoma (SCC). It has been suggested that apoptosis plays a part in process of involution of KA. Altogether 150 specimens were included in this study, 30 cases of each; normal skin (NS), proliferative (pKA) and regressing keratoacanthoma (rKA), well differentiated (wdSCC) and poorly differentiated (pdSCC) squamous cell carcinoma. All samples were examined immunohistochemically for expression of M30 protein. A significantly lower number of M30 positive cells has been detected in NS as compared to skin tumors examined (p<0.001), except for rKA (p=0.057). The highest percentage of M30 positive cells was detected in pdSCC (p<0.001) as compared with all other examined groups. Keratinocytes of normal and changed epidermis expressing higher levels of M30 protein were predominately found in sun-exposed areas (chi2=14.93; p=0.060). There was an increasing trend of M30 protein expression with increasing age of the patient in NS and skin tumors examined. Majority of skin tumors with higher percentage of M30 positive cells tended to display higher Ki-67 expression. M30 expression was highly correlated with bak (r=0.811; p=0.048) and granzyme B expression in rKA (r=0.733; p=0.015). Cell apoptosis as assessed by M30 expression is, generally, increased in examined skin tumors and related to cell proliferation. Cell apoptosis mediated by bak and granzyme B expression could contribute to KA regression.

UV radiation is present in sunlight and can be emitted from numerous artificial sources. Outdoor workers are exposed to sunlight in a wide variety of occupations like sailors, fishers, construction workers, farmers, and other. Presented are the skin diseases caused by sunlight exposure. They may be of little medical importance such as stigmata or create problems like photoaging, skin carcinoma, melanoma, phototoxic and photoallergic reactions. Shown are briefly data on skin cancer in the Rijeka region in outdoor occupations, the legislation and necessity for prevention. Psoriatic patients need particular caution because they are exposed to UVR, tars, and immunosuppressive drugs during the treatment.

The carcinogenicity (photocarcinogenicity) of sunlight to human skin has been recognized more than a century ago. Last decades numerous experimental studies show that UV rays damage DNA, cause gene mutations leading to the development of malignant tumors such basal cell carcinomas, squamous cell carcinomas and melanomas. The tumors occur most frequently in fair skinned people, and the mutations typically are found at dipyrimidine sites with C-T or / and CC-TT tandem double mutations. The authors briefly summarize their investigation of the p53 suppressor gene, and expose their hypothesis of hTERT involvement in cancerogenesis. Also their underline the importance of UV induced immunosuppression in photocarcinogenesis. Psoriatic patients are exposed to numerous cancerogens in their treatment. A better understanding of the mechanisms of photocarcinogenesis could provide new ways in the treatment of skin tumors.

Non-melanoma skin cancers (NMSC) are the most common malignant tumors in white population and their incidence has been increasing worldwide. Molecular events regulating cell survival, apoptosis, growth arrest as well as cell differentiation, are important contributors to the overall kinetics of benign and malignant cell growth and play a role in their development, progression and regression. Failure of these pathways can result in the loss of control over proliferation and lead to tumor development through the inactivation of tumor suppressor genes or the activation of oncogenes. Also, immunological mechanisms have been implicated in a phenomenon of tumor progression as well as spontaneous tumor regression. We have tried to summarize the main events in etiopatogenesis, development, progression and in some cases skin cancer regression. Further studies are needed to elucidate completely the details of apoptotic control in normal skin and determine factors resulting in apoptotic disbalance and disease.

Telomerase is a ribonucleoprotein reverse transcriptase which RNA component (TERC) and reverse transcriptase (TERT) function together to elongate telomeres. If cells are to survive and proliferate indefinitely, telomere preservation is essential for the immortalization process. Somatic cells rarely possess TA, but over 90% of tumor cells express active telomerase. Increased cell proliferation and deregulation of cell cycle occur in human cancers, including cutaneous melanoma. The exact nature of links between TA, cell proliferation and apoptosis has not been extensively elucidated in cutaneous melanoma. We hypothesize a relationship between TA and cutaneous melanoma cell proliferation in a way that TA in telomere elongation is only an early event in cell immortalization. The telomere elongation makes their proliferation possible and being, at the same time, one of its limiting factors. But the TA other than telomere elongation (TERC independent) is crucial to initiate or restore melanoma cell proliferation. On the other hand, TA in telomere elongation, together with other factors (for example TNF), has an active anti-apoptotic role. This way melanoma cells overwhelm the apoptotic defense mechanisms, finally resulting in their indefinite proliferation. In evaluation of our hypothesis, we suggest thorough studies of both telomerase activity and proliferation in cutaneous melanoma on multiple checkpoints and targets. We also suggest combined analyses of TA and telomere length. This approach seems inevitable since it is obvious that telomerase is no longer just for the elongation of telomeres and, to our knowledge, most of the studies conducted so far evaluated TA as an expression of a single subunit or associated molecule.

To gain insight into the role and association of cell cycle and apoptosis regulatory proteins and telomerase activity in the course of progression of melanocitic lesions we have examined immunohistochemicaly, expression and the distribution of p53, bcl-2, Ki-67 and telomerase in 25 samples of common and dysplastic nevi, and 45 samples of primary invasive melanomas. Protein p53 expression was significantly increased in dysplastic as compared with common nevi and melanomas (p < 0.001). Bcl-2 protein expression was significantly increased in melanomas as compared with common aquired and dysplastic nevi (p = 0.001). Nevi and melanomas exhibited clear-cut differences in terms of Ki-67 expression. Telomerase expression was significantly increased in melanomas as compared with common acquired (p = 0.014) and dysplastic nevi (p < 0.001). Enhanced telomerase activity in association with increased bcl-2 expression in the course of melanoma progression could contribute to development and progression of melanoma.

Background: Recently reverse transcriptase-polymerase chain reaction (RT-PCR) has been proposed as a new sensitive method for the detection of submicroscopic melanoma nodal metastases. Sentinel lymph node (SLN) status is considered the most important prognostic factor for melanoma patients. Thus, in recent years, melanoma research has been focused on identifying new molecular markers of micrometastases., Methods: In this study, 129 SLNs were collected and analyzed by RT-PCR for tyrosinase and melanoma inhibitory activity (MIA) messenger RNA (mRNA) expression. RESULTS from PCR analysis were then compared with those obtained by hematoxylin and eosin and immunohistochemistry and related to progression of disease., Results: MIA gene expression was positive by RT-PCR in 27% of the tyrosinase-positive SLNs. When the correlation between tyrosinase and/or MIA mRNA expression and disease-free survival was evaluated by the Kaplan-Meier exact test, there was a statistically significant correlation between simultaneous tyrosinase and MIA gene expression in SLNs and progression of disease., Conclusions: RT-PCR analysis for both MIA and tyrosinase mRNA may identify a subset of melanoma patients with a worse prognosis whom the routine methods, such as histology and immunohistochemistry, fail to identify because of the poor sensitivity of these methods.

Aims and Background: The p53 protein is essential for the regulation of cell proliferation and its aberrant accumulation is usually seen in malignant tumors but also occurs in squamous epithelium of inflammatory skin diseases characterized by hyperproliferation. The aim of this study is to elucidate the role of the p53 tumor suppressor protein in the pathogenesis of different hyperproliferative, non-malignant and malignant skin diseases, and to determine the association between p53 overexpression and cell proliferation. We also investigated the influence of aging on p53 and Ki-67 protein expression., Methods: One hundred and fifty skin specimens divided into 30 samples each of normal skin (NS), psoriatic skin (PS), keratoacanthomas (KA), basal cell carcinomas (BCC), and squamous cell carcinomas (SCC) were examined immunohistochemically to assess p53 and Ki-67 protein expression., Results: p53 immunostaining of NS, PS, KA, BCC and SCC was detected in 39.0%, 46.7%, 66.7%, 80% and 86.7% of cases, respectively. Median values and ranges of p53 protein expression were as follows: 0.0% (range, 0.0-1.8%) in NS, 0.0% (range, 0.0-6.5%) in PS, 9.2% (range, 0.0-24.0%) in KA, 19.3% (range, 0.0-48.1%) in BCC and 30.1% (range, 0.0-68.1%) in SCC. p53- and Ki-67-positive cells were present in basal (NS) and suprabasal layers (PS), and not only in cancer nests of KA, BCC and SCC but also in dysplastic and even morphologically normal epidermis adjoining cancers. The positivity of p53 and Ki-67 proteins differed significantly among the groups, with no differences in p53 expression between NS and PS and in Ki-67 expression between PS and KA. Within all groups there was a significant correlation between p53 and Ki-67 expression. Lesion location and patient age, with the exception of location in PS and age in BCC, were significantly related to p53 and Ki-67 expression in all groups., Conclusions: Our findings suggest that p53 overexpression occurs mainly in neoplastic skin lesions, although it may also occur in squamous epithelium of inflammatory skin diseases such as PS, as well as in normal skin epithelium. It is associated with cell proliferation in normal as well as altered epithelium. p53 protein overexpression is an age-related process and significantly associated with sun exposure, especially in NS and PS but also in KA and SCC. Our findings suggest that Ki-67 rate and p53 protein expression reflect the degree of malignancy in the examined cutaneous neoplasms.

Depth of invasion and stage of the disease are well known prognostic indicators in cutaneous melanoma (CM). However, the role of other parameters, such as the variations in cytomorphology of melanocyte tumours, mitotic activity and angiogenesis is still open to question. The aim of this study was to analyse proliferation by mitotic activity index (MAI) and immunostaining of proliferating cell nuclear antigen (PCNA), and the intensity of neovascularization (microvessel density; MVD) in CM clinical stage I in relation to epithelioid, spindle and nevoid cell type, histological type (superficial spreading melanoma and nodular melanoma), Clark's level and Breslow thickness. Finally, the role of all parameters in the prognosis of CM was evaluated. Statistical analysis demonstrated that cytological characteristics of CM correlate only with Clark's level, while histological types correlate with MAI, PCNA and MVD. MAI and PCNA also showed correlation between groups according to Clark's level and Breslow thickness. Finally, tumour cell PCNA was found to correlate with MVD. Survival of patients with CM correlated significantly with MAI. These results suggest that cytological variation, histological type, PCNA and MVD alone are not independent prognostic parameters, whereas MAI is a potentially important prognostic marker in CM.

Eighty-eight cases of cutaneous melanoma (CM) were analyzed in order to elucidate the relationship between thickness, angiogenesis, and prognosis. The thickness of the tumor was measured according to the Breslow method, and the microvessels were identified by an immunohistochemical study using anti-factor VIII monoclonal antibody on specimens from 40 patients with superficial spreading melanoma (SSM), and 48 with nodular type (NM). Microvessels were counted in the area of highest density. The overall survival and disease-free period were analyzed retrospectively. The proportion of patients with thicker CMs (> 1.5 mm) increased with age in both sexes. Mean vascular count was statistically significant different only between thinner and thicker tumors in the SSM group (P < 0.05). Prognosis was correlated with the thickness of CM (P = 0.0002), mean vascular count alone (P = 0.004), mean vascular count in association with CM thickness less than 1.5 mm (P = 0.0005), and with mean vascular count in NM (P = 0.02). These findings suggest that increasing microvessel density indicates a worsening prognosis.

A retrospective study was made of the incidence of cutaneous melanoma among the population of the district of Rijeka (Croatia) during the period 1977-96. A total of 397 patients with cutaneous melanoma was documented during this period. Over the 20-year period the incidence of the tumour increased, the mean annual rate being 4. 8 (4.0 in males and 5.4 in females) in the first 10-year period and 7.16 in the second (7.1 in males and 7.3 in females). The number of registered cases in males and females was almost identical (1 : 1.05). The number of melanoma cases increased with age in both sexes, whereas it was rare in children. The most affected anatomical location was the trunk in males and the lower limbs in females. The results indicate the need for active prevention and educational programmes in this population.

Anaplastic large cell lymphoma (ALCL) is a high-grade non-Hodgkin's lymphoma recognized by the expression of the CD30 marker and by its morphology. We report an unusual case of ALCL in a 42-year-old woman. For ten years the patient only had pruritic erythematous skin lesions, then a plaque with nodules and ulcers on the right thigh and leg developed, followed by ichthyosis acquisita. The development of ALCL in women and the association of ichthyosis acquisita with this type of lymphoma are uncommon.