Predictive Value of Intraoperative Sentinel Lymph Node Imprint Cytology Analysis for Metastasis in Patients with Melanoma.

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.

A polymorphic GGC repeat in the NPAS2 gene and its association with melanoma.

Circadian clock regulation in mammals is controlled by feedback loops of a set of circadian genes. One of these circadian genes, NPAS2, encodes for a member of the bHLH-PAS class of transcription factors and is expressed in the forebrain and in some peripheral organs such as liver and skin. Other biological processes are also regulated by circadian genes. For example, NPAS2 is involved in cell proliferation, DNA damage repair and malignant transformation. Aberrant expression of clock genes has been previously observed in melanoma which led to our effort to sequence the NPAS2 promoter region in this cancer type. The NPAS2 putative promoter and 5' untranslated region of ninety-three melanoma patients and ninety-six control subjects were sequenced and several variants were identified. Among these is a novel microsatellite comprising a GGC repeat with different alleles ranging from 7 to 13 repeats located in the 5' untranslated exon. Homozygosity of an allele with nine repeats (9/9) was more prevalent in melanoma than in control subjects (22.6% and 13.5%, respectively, P: 0.0206) suggesting that some NPAS2 variants might contribute to melanoma susceptibility. Impact statement This report describes a variable microsatellite repeat sequence located in the 5' untranslated exon of NSPAS2, a gene encoding a clock transcription factor. Significantly, this study is the first to show that a variant copy number GGC repeat sequence in the NPAS2 clock gene associates with melanoma risk and which may be useful in the assessment of melanoma predisposition.

Inflammatory Gene Expression Upon TGF-ß1-Induced p38 Activation in Primary Dupuytren's Disease Fibroblasts.

OBJECTIVES: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytren's disease (DD) patients. METHODS: Primary non-Dupuytren's disease cells (ND) were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-ß1 and/or inhibitor of p38 phosphorylation. RESULTS: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11, and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro. CONCLUSIONS: TGF-ß1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed toward inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence.

EGFR and cyclin D1 in nodular melanoma: correlation with pathohistological parameters and overall survival.

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).

Running for time: circadian rhythms and melanoma.

Circadian timing system includes an input pathway transmitting environmental signals to a core oscillator that generates circadian signals responsible for the peripheral physiological or behavioural events. Circadian 24-h rhythms regulate diverse physiologic processes. Deregulation of these rhythms is associated with a number of pathogenic conditions including depression, diabetes, metabolic syndrome and cancer. Melanoma is a less common type of skin cancer yet more aggressive often with a lethal ending. However, little is known about circadian control in melanoma and exact functional associations between core clock genes and development of melanoma skin cancer. This paper, therefore, comprehensively analyses current literature data on the involvement of circadian clock components in melanoma development. In particular, the role of circadian rhythm deregulation is discussed in the context of DNA repair mechanisms and influence of UV radiation and artificial light exposure on cancer development. The role of arylalkylamine N-acetyltransferase (AANAT) enzyme and impact of melatonin, as a major output factor of circadian rhythm, and its protective role in melanoma are discussed in details. We hypothesise that further understanding of clock genes' involvement and circadian regulation might foster discoveries in the field of melanoma diagnostics and treatment.

Microarray analysis of Dupuytren's disease cells: the profibrogenic role of the TGF-ß inducible p38 MAPK pathway.

BACKGROUND: Dupuytren's disease (DD) is a nodular palmar fibromatosis that causes irreversible permanent contracture of fingers and results in the loss of hand function. Surgery still remains the only available solution for DD patients but cannot permanently cure the disease nor reduce high recurrence rates. With this rationale, we designed a study aimed at an improved understanding of the molecular mechanisms underlying DD. Our major focus was an analysis of the global gene expression profile and signalling pathways in DD cells with the aim of identifying novel biomarkers and/or therapeutic targets. METHODS: Primary cells were cultured from surgically removed diseased and healthy tissue. Microarray expression analysis (HG-U133A array, Affymetrix) and qPCR was performed with total RNA isolated from primary DD cells. Mechanistic studies involving inhibition of p38 phosphorylation were performed on normal human fibroblasts' and primary DD cells' in vitro models. Expression of stem cell markers in primary fibroblasts/myofibroblasts was assessed as well. RESULTS: We identified 3 p38MAPK signalling pathway regulatory genes, THBS1, GADD45α and NUAK1, all involved in cellular proliferation and production of the extracellular matrix proteins. Inhibition of the p38MAPK signalling pathway induced down-regulation of myofibroblast markers, α-smooth muscle actin and palladin. A stem-cell like subpopulation positive for CD90 marker was identified among primary DD cells. CONCLUSION: The study reveals involvement of the p38 MAPK pathway as a possible signalling cascade in the pathogenesis of Dupuytren's disease. Moreover, a particular stem cell-like CD90(+) subpopulation was identified that might contribute to DD development.

Chondromyxoid fibroma of the second metacarpal bone--a case report.

This report describes a chondromyxoid fibroma of the second metacarpal bone in a 32-year-old female patient. Chondromyxoid fibroma is a rare, benign, slow-growing bone tumor of cartilaginous origin. Tumor has a high recurrance rate. Our aim was to show successful treatment of a metacarpal chondromyxoid fibroma with wide resection and implantation of finger join endoprosthesis.

Advantages of intramedullar fixation in treatment of congenital tibial pseudoarthrosis--a case report.

This report describes a case of a 29-year old patient with congenital pseudoarthrosis of the distal tibia previously treated unsuccessfully by a conventional surgical method. Tibial congenital pseudoarthrosis is a rare disease characterized by segmental osseous weakness resulting in deformation of the bone and spontaneous fractures which progresses to a tibial nonunion. In our case we used intramedullary stabilization with bone grafting and six month after operation congenital pseudarthrosis of the tibia healed.

The advantages of humeral anteromedial plate osteosynthesis in the middle third shaft fractures.

BACKGROUND: Humeral shaft fractures account for 1.2% of all fractures and occur in a slightly younger population. Their causes include a fall from standing or from height, motor vehicle accident, but can be also pathological. In order to clarify which of both surgeries we performed in our Department for treating humeral shaft fractures had more advantages (anterolateral or anteromedial plating through anterolateral approach) we analyzed incidence of postoperative iatrogenic radial palsies and mean operation time required to complete each surgery. METHODS: During January 1992 to December 2009 on Department of Surgery, Division for Traumatology of Clinical Hospital Center Rijeka, 420 patients (340 males and 80 females with mean age of 38.11 ± 9.29 years) were treated for middle third humeral shaft fracture by anterolateral approach and internal fixation using AO/DCP or LCP plates that was positioned on anteromedial humeral surface in 141 patients (33.57%) and on anterolateral humeral surface in 279 patients (66.43%). RESULTS: None of the patients who had osteosynthesis by using plate on anteromedial humeral sufrace had lesions of the radial nerve. Therefore, χ(2) test revealed significantly higher frequency of postsurgical radial nerve injuries in patients who were treated by anterolateral plating than in patients where anteromedial plating was performed (χ(2) = 17.51; p< 0.05). Anterolateral plating required longer mean operation time than anteromedial plating and the difference in its duration determined by t-test for independent samples showed statistically significant difference (t= 14.57; p< 0.05). CONCLUSION: An anteromedial plating of humeral shaft fractures through anterolateral approach was determinated to be a simple, safe, effective and also fast surgical treatment and we highly recommend it as operative technique for treating humeral shaft fractures.

Primary large-cell neuroendocrine carcinoma of the scrotum.

Neuroendocrine tumors (NETs) mostly develop from the neural crest cells but a few arise from neuroectoderm. They are common in the lungs and gastrointestinal tract but rare in the genitourinary tract. A 78-year-old man with no family history of malignant or hereditary diseases presented with a 3-month history of a rapidly growing asymptomatic scrotal nodule and swelling in the groin. He had a negative history of sexually transmitted disease and of trauma, fungal infection or chronic irritation in the scrotal area; there was no history of radiotherapy or exposure to chemicals or arsenic. Both the scrotal and groin lesions were excised with a minimum of 1.2 cm of normal skin. Examination of the specimen revealed a confined poorly differentiated large-cell neuroendocrine carcinoma with a metastasis to the inguinal lymph nodes. Three months after the excision we found a local recurrence. The recurrent tumor revealed tumor tissue concurrent with the primary lesion. To the best of our knowledge, there have been no previously published case reports on neuroendocrine tumor of the scrotum.

Emerging risk for viral hepatitis A in Croatian adults.

An objective of the study was to determine the changes in the risk of developing hepatitis A in the 30-years period and discuss the need for vaccination against HAV infection in Croatia and the city of Rijeka comparing incidence of hepatitis A between 1970-1974 and 2000-2004 periods. Hepatitis A declined in both populations and affected more prominently older population groups. Improvement of hygiene and sanitary conditions appears to have decreased hepatitis A incidence among children and adults, but only a seroepidemiological study can give more accurate data as a basis for discussion on the necessity of vaccination as a further measure in reducing hepatitis A incidence.

Real-time expression of hTERT in primary melanoma biopsies.

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.

Functional genomics in identification of drug targets in Dupuytren's contracture.

Although functional genomics methods offer new viewpoint on molecular processes involved in particular pathological state, these methods, in particular proteomics, are still under-represented in Dupuytren's contracture research. However, several recent papers based on functional genomics technologies represent a breakthrough in studying Dupuytren's contracture as they revealed new molecular players that had been impossible to detect by traditional molecular biology methods. Using computational tools to provide biological context for such broad arrays of data accelerates the process of homing in on the potential molecular markers and drug targets. Interactomes, maps of protein-protein interactions characteristic for the disease and as such putative models of its molecular pathology, are especially useful for this purpose, facilitating the transition from global screening methods to specific experiments aimed at therapy development. The combination of these approaches in Dupuytren's contracture research might therefore facilitate the discovery of novel therapeutic targets and diagnostic markers indicative of disease progression.

Malignant skin melanoma in Croatia.

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.

Screening of potential prodrugs on cells derived from Dupuytren's disease patients.

Dupuytren's disease (DD) is a fibroproliferative disorder, the cure for which is still limited to surgical excision of the affected fascia, often leading to high recurrence rates. Due to this fact, non-surgical treatments are being investigated, among them those targeting molecular processes of proliferation and differentiation in Dupuytren's cell cultures. Drugs with antiproliferative action may be valuable in DD treatment. Through characterization of changes on DD-specific cells, we, therefore, decided to test the therapeutic potential of new cytostatic drugs for DD treatment and/or for reduction of post-operative recurrence rates. The N-sulfonylpyrimidine derivative, amidino-substituted benzimidazo[1,2-a]quinoline, and amidino dihydrothienothienyl[2,3-c]quinolone hydrochloride, known to affect proliferation processes, were tested for their antiproliferative activity on primary fibroblasts/myofibroblasts cell cultures derived from the palmar fascia of patients with DD. Only amidino dihydrothienothienyl[2,3-c]quinolone hydrochloride acted in a highly specific manner on cells derived from diseased fascia of DD patients and exhibited a low cytotoxic effect. This result might be a consequence of its specific activity on cytoskeleton changes occurring in differentiating cells. A similar short-term differential antiproliferative effect was observed by the N-sulfonylpyrimidine derivative that was, however, completely lost after 6- and 14-day treatments. The amidino-substituted benzimidazo[1,2-a]quinoline exerted a strong non-specific, dose-related antiproliferative activity on cell types.

An integrated proteomics approach for studying the molecular pathogenesis of Dupuytren's disease.

Dupuytren's disease (DD) is a fibromatosis characterized by non-malignant transformation of palmar fascia leading to permanent contraction of one or more fingers. Despite the extensive knowledge of its clinical pathogenesis, the aetiology of this disease remains obscure. In the present paper, we report for the first time on the proteomic profiling of diseased versus unaffected patient-matched palmar fasciae tissues from DD patients using two-dimensional gel electrophoresis coupled with mass spectrometry analysis. The herein identified proteins were then used to create the protein-protein interaction network (interactome). Such an integrated approach revealed the involvement of several different molecular processes related to DD progression, including extra- and intra-cellular signalling, oxidative stress, cytoskeletal changes, and alterations in cellular metabolism. In particular, autocrine regulation through ERBB-2 and IGF-1R receptors and the Akt signalling pathway have emerged as novel components of pro-survival signalling in Dupuytren's fibroblasts and thus might provide a basis for a new therapeutic strategy in Dupuytren's disease.

Dupuytren's disease characteristics in Primorsko-goranska County, Croatia.

Dupuytren's disease predominantly occurs among Europeans, with higher reported prevalence in Northern than in Mediterranean European countries. We evaluated the recurrence and extension rates among Caucasian patients from Primorsko-goranska County in Croatia who underwent partial fasciectomy as treatment for Dupuytren's disease. Furthermore, we investigated the influence of diathesis factors on disease progression. Recurrent disease was observed in 68 out of 93 patients (73%). There where 48 (52%) patients with extension of the disease. Differences were found between patients with recurrent disease and those without recurrence regarding age at onset, age at operation and duration of disease. We compared patients younger and older than 50 years at disease onset, and found that older patients had a significantly higher recurrence rate. Characteristics of our patients fit into the picture of typical Dupuytren's disease except for the influence of early age at onset. Among our patients late age at onset proved to be a diathesis factor.

The role of telomerase activity in psoriatic skin lesions.

Psoriasis is a benign, chronic skin disease characterized by keratinocyte hyperproliferation and abnormal differentiation. Telomerase is an enzyme-reverse transcriptase that protects chromosomes from degradation by stabilizing telomere length. Recent studies suggest that telomerase activity (TA) may be responsible in part for some of nonmalignant proliferative skin diseases. There is evidence that telomerase has an active anti-apoptotic role. TA in general is associated with cellular proliferation. We hypothesize a relationship between TA, keratinocyte proliferation and apoptosis in psoriatic skin lesions. The TA in telomere elongation makes keratinocyte hyperproliferation possible and is at the same time, one of its limiting factors. This hyperproliferation in psoriasis occurs as a result of significant keratinocyte damage caused by self-reactive T-cells through induction of various apoptotic pathways. On the other hand, TA in telomere elongation, together with other factors, has an active anti-apoptotic role, preserving the necessary amount of equilibrium between these two processes (apoptosis and proliferation) therefore being the main reason why conversion of a psoriatic plaque to squamous cell carcinoma is rare. As there is little data on TA in psoriatic lesions, in evaluation of our hypothesis we suggest thorough parallel studies of TA, telomere length, apoptosis and proliferation in psoriatic lesional skin on multiple checkpoints and targets, using more samples so the reliability of the results would be higher. This is important since a better understanding of these factors might provide new possible therapeutic targets.