Maligní melanom (MM) představuje nejagresivnější typ kožních nádorů s vysokým metastatickým potenciálem a nepříznivou prognózou. Rozvoj poznatků týkajících se molekulárních mechanismů podmiňujících rozvoj MM a nalezení nových biomarkerů je potřebné pro přesnou diagnostiku a volbu léčebných postupů. V předkládaném projektu plánujeme provést komplexní studii založenou na sekvenování nové generace, in situ hybridizaci a imunohistochemické analýze se zaměřením na defekty v onkogenech a tumor supresorových genech zapojených do rozvoje MM, expresi proteinů a charakteristiku stromálního mikroprostředí. Funkční význam vybraných mutací bude dále testován na kulturách melanocytů, především se zaměřením na jejich roli v odpovědi na poškození DNA a regulaci buněčného cyklu. Nalezené změny na genetické úrovni budou korelovány s histopatologickým obrazem, imunohistochemickým profilem a s klinickými daty. Očekáváme prohloubení znalostí o známých a identifikaci nových biomarkerů umožňujících lepší predikci, diagnostiku a léčbu MM.; Malignant melanoma represents the most aggressive type of skin cancer with high metastatic potential and poor prognosis. Better understanding of molecular mechanisms involved in pathogenesis of melanoma and identification of novel biomarkers is needed for precise diagnostic classification and prediction of therapeutical outcomes. Here we aim to perform a comprehensive study employing next-generation sequencing, in situ hybridisation and immunohistochemical analysis to identify new defects in oncogenes and tumour suppressor genes involved in melanoma, protein expression and characterization of stromal microenvironment. Selected mutations will be functionally tested in culture of melanocytes, focusing mainly on their role in DNA damage response and cell cycle checkpoints. Identified genetic changes will be correlated with histopathological findings, immunohistochemical profile and clinical data. We expect deepening of known and identification of new markers allowing better prediction, diagnosis and treatment of melanoma.

• MeSH
• geny p53 genetika   MeSH
• hybridizace in situ MeSH
• imunohistochemie metody   MeSH
• lidé MeSH
• melanom genetika   mikrobiologie   MeSH
• mutace MeSH
• nádorové biomarkery MeSH
• onkogeny genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• genetika, lékařská genetika
• onkologie
• mikrobiologie, lékařská mikrobiologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Cutaneous melanoma is the deadliest skin malignity with a rising prevalence worldwide. Patients carrying germline mutations in melanoma-susceptibility genes face an increased risk of melanoma and other cancers. To assess the spectrum of germline variants, we analyzed 264 Czech melanoma patients indicated for testing due to early melanoma (at <25 years) or the presence of multiple primary melanoma/melanoma and other cancer in their personal and/or family history. All patients were analyzed by panel next-generation sequencing targeting 217 genes in four groups: high-to-moderate melanoma risk genes, low melanoma risk genes, cancer syndrome genes, and other genes with an uncertain melanoma risk. Population frequencies were assessed in 1479 population-matched controls. Selected POT1 and CHEK2 variants were characterized by functional assays. Mutations in clinically relevant genes were significantly more frequent in melanoma patients than in controls (31/264; 11.7% vs. 58/1479; 3.9%; p = 2.0 × 10-6). A total of 9 patients (3.4%) carried mutations in high-to-moderate melanoma risk genes (CDKN2A, POT1, ACD) and 22 (8.3%) patients in other cancer syndrome genes (NBN, BRCA1/2, CHEK2, ATM, WRN, RB1). Mutations in high-to-moderate melanoma risk genes (OR = 52.2; 95%CI 6.6-413.1; p = 3.2 × 10-7) and in other cancer syndrome genes (OR = 2.3; 95%CI 1.4-3.8; p = 0.003) were significantly associated with melanoma risk. We found an increased potential to carry these mutations (OR = 2.9; 95%CI 1.2-6.8) in patients with double primary melanoma, melanoma and other primary cancer, but not in patients with early age at onset. The analysis revealed affected genes in Czech melanoma patients and identified individuals who may benefit from genetic testing and future surveillance management of mutation carriers.

• Publikační typ
• časopisecké články MeSH

Phosphatase and tensin homologue (PTEN) is a tumour suppressor gene implicated in tumorigenesis of melanoma, with distinct cytoplasmic and nuclear functions. Cytoplasmic PTEN negatively regulates the PI3K/AKT/mTOR signalling pathway, while nuclear PTEN works as a tumour suppressor. Clinical data suggest that the loss of PTEN function in melanoma is associated with aggressive tumour behaviour. We performed a comprehensive analysis of PTEN in 112 primary cutaneous melanomas including immunohistochemical (IHC), fluorescent in situ hybridization (FISH), next-generation sequencing (NGS), and epigenetic analysis. The goal of our study was to: (a) correlate PTEN expression with selected clinico-pathological variables, and assess its prognostic significance; (b) correlate molecular aberrations with PTEN expression to consider the utility of immunohistochemical analysis of PTEN protein expression for screening PTEN genetic alterations; (c) review the literature and evaluate the PTEN expression level in melanoma with respect to possible therapeutic targeting. Our results showed that PTEN molecular alterations were present in 4/20 (20 %) cases with a loss of expression, 3/11 (27 %) cases with clonal-like expression, and 1/81 (1 %) cases with positive PTEN expression. No PTEN promoter methylation was found in any of the cases. Even though the value of our observation is limited by the low number of cases fully evaluated by IHC (112 cases), FISH (19 cases) and NGS (30 cases), our data suggest that IHC is not an appropriate method for the screening of PTEN genetic alterations. Our survival analysis suggests that patients with positive cytoplasmic PTEN expression show better disease-free survival (P < 0.05).

• MeSH
• fosfohydroláza PTEN genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• melanom * genetika   MeSH
• nádory kůže * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The objective of our study was to compare the five different scoring methods of tumor-infiltrating lymphocytes (TILs) assessment in a group of 213 cases of superficial spreading and nodular melanoma. The scoring methods include (a) Clark scoring; (b) Melanoma Institute Australia system; (c) scoring system used in the study of Saldanha et al.; (d) scoring system used in the TCGA study and modified by Park et al.; and (e) the system recently proposed by the "International Immuno-Oncology Biomarker Working Group" for TILs scoring in all solid tumors. Prediction of survival with three main outcomes-disease-specific-free survival, local recurrence-free survival, and distant metastasis-free survival-was evaluated. The prognostic value of TILs showed statistical significance in univariate analysis regarding all three of the outcomes only for three of the five evaluated methods; the Clark scoring, the Melanoma Institute Australia system, and the system proposed by the "International Immuno-Oncology Biomarker Working Group". However, in multivariate analysis with covariants including Breslow thickness, type of melanoma, location, sex, and age, we did not find TILs to be an independent prognostic factor.

• MeSH
• dospělí MeSH
• dvojitá slepá metoda MeSH
• lidé středního věku MeSH
• lidé MeSH
• melanom klasifikace   imunologie   patologie   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádory kůže imunologie   patologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tumor infiltrující lymfocyty imunologie   MeSH
• výzkumný projekt MeSH
• zánět imunologie   patologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The most common histological subtypes of cutaneous melanoma include superficial spreading and nodular melanoma. However, the spectrum of somatic mutations developed in those lesions and all potential druggable targets have not yet been fully elucidated. We present the results of a sequence capture NGS analysis of 114 primary nodular and superficial spreading melanomas identifying driver mutations using biostatistical, immunohistochemical and/or functional approach. The spectrum and frequency of pathogenic or likely pathogenic variants were identified across 54 evaluated genes, including 59 novel mutations, and the newly identified TP53 loss-of-function mutations p.(L194P) and p.(R280K). Frequently mutated genes most commonly affected the MAPK pathway, followed by chromatin remodeling, and cell cycle regulation. Frequent aberrations were also detected in the genes coding for proteins involved in DNA repair and the regulation and modification of cellular tight junctions. Furthermore, relatively frequent mutations were described in KDR and MET, which represent potential clinically important targets. Those results suggest that with the development of new therapeutic possibilities, not only BRAF testing, but complex molecular testing of cutaneous melanoma may become an integral part of the decision process concerning the treatment of patients with melanoma.

• MeSH
• buněčný cyklus genetika   MeSH
• dospělí MeSH
• frekvence genu genetika   MeSH
• genetická predispozice k nemoci genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• MAP kinasový signální systém genetika   MeSH
• melanom genetika   patologie   MeSH
• mladý dospělý MeSH
• mutace ztráty funkce genetika   MeSH
• nádorové biomarkery genetika   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• nádory kůže genetika   patologie   MeSH
• oprava DNA genetika   MeSH
• protoonkogenní proteiny B-raf genetika   MeSH
• restrukturace chromatinu genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• těsný spoj genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The glucose transporter-1 (Glut-1) is a cell membrane glycoprotein involved in glucose uptake. An increased expression of Glut-1 is an important cell adaptation mechanism against hypoxia. An upregulation of Glut-1 can be found in several types of malignant tumors, which are able to reprogram their metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect). However, the data regarding melanocytic lesions is equivocal. We performed comprehensive immunohistochemical analysis of the Glut-1 expression in 225 malignant melanomas (MM) and 175 benign nevi. Only the membranous expression of Glut-1 was regarded as positive. The expression of Glut-1 (the cut-off for positivity was determined as H-score 15) was found in 69/225 malignant melanomas. The number of positive cases and the H-score of Glut-1 increased where there was a higher Breslow thickness (p < 0.00001) when comparing pT1- pT4 MM groups. All benign nevi were classified as negative. In conclusion, the membranous expression of Glut-1 is a common feature of a malignant melanoma but this type of expression is very rare in benign melanocytic nevi. Our results suggest that the membranous expression of Glut-1 can be used as a surrogate marker in the assessing of the biological nature of benign and malignant melanocytic lesions. However, despite its high specificity, the sensitivity of this marker is relatively low. Moreover, due to the fact that the increased expression of Glut-1 correlates with a shorter survival period (10-year disease free survival, recurrence free survival and metastasis free survival and MFS), it can be used as a prognostically adverse factor.

• MeSH
• dospělí MeSH
• imunohistochemie metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• melanom metabolismus   patologie   MeSH
• míra přežití MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádorové biomarkery metabolismus   MeSH
• následné studie MeSH
• pigmentový névus metabolismus   patologie   MeSH
• přenašeč glukosy typ 1 metabolismus   MeSH
• prognóza MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The steadily increasing incidence of malignant melanoma (MM) and its aggressive behaviour makes this tumour an attractive cancer research topic. The tumour microenvironment is being increasingly recognised as a key factor in cancer biology, with an impact on proliferation, invasion, angiogenesis and metastatic spread, as well as acquired therapy resistance. Multiple bioactive molecules playing cooperative roles promote the chronic inflammatory milieu in tumours, making inflammation a hallmark of cancer. This specific inflammatory setting is evident in the affected tissue. However, certain mediators can leak into the systemic circulation and affect the whole organism. The present study analysed the complex inflammatory response in the sera of patients with MM of various stages. Multiplexed proteomic analysis (Luminex Corporation) of 31 serum proteins was employed. These targets were observed in immunohistochemical profiles of primary tumours from the same patients. Furthermore, these proteins were analysed in MM cell lines and the principal cell population of the melanoma microenvironment, cancer‑associated fibroblasts. Growth factors such as hepatocyte growth factor, granulocyte‑colony stimulating factor and vascular endothelial growth factor, chemokines RANTES and interleukin (IL)‑8, and cytokines IL‑6, interferon‑α and IL‑1 receptor antagonist significantly differed in these patients compared with the healthy controls. Taken together, the results presented here depict the inflammatory landscape that is altered in melanoma patients, and highlight potentially relevant targets for therapy improvement.

• MeSH
• chemokiny krev   MeSH
• dospělí MeSH
• fibroblasty asociované s nádorem metabolismus   MeSH
• krevní proteiny analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• melanom krev   metabolismus   MeSH
• nádorové biomarkery krev   MeSH
• nádorové buněčné linie MeSH
• pilotní projekty MeSH
• prognóza MeSH
• proteomika metody   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

In several solid tumors, an increased stathmin expression is associated with both poor prognosis and resistance to certain chemotherapy types. However, the data regarding melanocytic lesions are very limited. The goals of our study are as follows: the assessment of stathmin expression in benign and malignant melanocytic lesions, and the significance of its expression for the differential diagnostics between benign and malignant lesions; the analysis of the prognostic significance of stathmin expression in melanoma; and the evaluation of stathmin expression in melanoma and melanoma metastases with respect to possible therapeutic targeting. Immunohistochemical analysis of stathmin expression was done in 323 melanocytic lesions, including 205 primary cutaneous melanomas, 60 melanoma metastases, and 58 melanocytic nevi. Stathmin expression was found in all analyzed groups of melanocytic lesions. Using the H-scoring system, the observed intensity of expression was as follows: melanocytic nevi: 146.1 (mean) and 150 (median); melanomas: 116.7 (mean) and 110 (median); and melanoma metastases: 136.8 (mean) and 140 (median). The stathmin expression was significantly lower in the cohort of primary melanomas when compared with metastases and nevi (P=0.001). The stathmin expression showed no prognostic significance. The high stathmin expression in melanoma suggests that stathmin might be a promising marker for therapeutic targeting in ongoing clinical trials. Compared with several other solid tumors, stathmin expression in melanoma showed no prognostic significance. The potential use of stathmin expression in differential diagnostics is limited by its common expression, and despite the statistically significant differences between nevi and melanoma, it may not be used in this setting.

• MeSH
• imunohistochemie MeSH
• lidé středního věku MeSH
• lidé MeSH
• melanocyty metabolismus   patologie   MeSH
• melanom krev   patologie   MeSH
• nádorové biomarkery metabolismus   MeSH
• nádory kůže krev   patologie   MeSH
• prognóza MeSH
• stathmin metabolismus   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Melanom patří mezi výrazně imunogenní nádory, což umožňuje imunoonkologické přístupy. Samotná imunoterapie u melanomu se uplatňuje jak v adjuvantním podání, tak i u metastatického postižení. Pro adjuvantní podání byl řadu let dostupný pouze interferon alfa, který v dlouhodobém sledování neprokázal příliš významný efekt, nověji jsou v klinických studiích používány checkpoint inhibitory (anti-CTLA-4, PD-1/PD-L1), které jsou již standardem při metastatickém postižení. Práce diskutuje nejvýznamnější klinické studie poslední doby a poukazuje i na možnosti kombinované terapie s cílenou léčbou. Jsou diskutovány i nové přístupy a budoucí směr imunookologické terapie melanomu.

Melanoma belongs to significantly immunogenic tumors, allowing for immunological approaches. Melanoma immunotherapy is possibly used in adjuvant administration as well as in metastatic disease. Interferon alpha is used for adjuvant treatment without a significant effect in long-term follow-up. More recently, checkpoint inhibitors (anti-CTLA4, PD1/PD-L1) are used in clinical trials also for adjuvant treatment, which are already standard in metastatic disease. The work discusses the most recent clinical trials and points to the possibilities of combined therapy with targeted therapy. New approaches and future directions for immunotherapy of melanoma are also discussed.

• MeSH
• adjuvantní chemoterapie metody   trendy   MeSH
• antigen CTLA-4 antagonisté a inhibitory   genetika   imunologie   MeSH
• antigeny CD279 antagonisté a inhibitory   terapeutické užití   MeSH
• imunoterapie * dějiny   metody   trendy   MeSH
• interferon alfa aplikace a dávkování   škodlivé účinky   terapeutické užití   MeSH
• kombinovaná terapie * metody   trendy   MeSH
• kontrolní body buněčného cyklu imunologie   MeSH
• lidé MeSH
• melanom * dějiny   imunologie   terapie   MeSH
• metaanalýza jako téma MeSH
• metastázy nádorů imunologie   prevence a kontrola   terapie   MeSH
• onkolytické viry imunologie   účinky léků   MeSH
• statistika jako téma MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

We performed comprehensive molecular analysis of five cases of metastasizing uveal malignant melanoma (UM) (fresh-frozen samples) with an NGS panel of 73 genes. A likely pathogenic germline TP53 mutation c.760A > G (p.I254V) was found in two tumor samples and matched nontumor tissue. In three cases, pathogenic BAP1 mutation was detected together with germline missense variants of uncertain significance in ATM. All cases carried recurrent activating GNAQ or GNA11 mutation. Moreover, we analyzed samples from another 16 patients with primary UM by direct Sanger sequencing focusing only on TP53 coding region. No other germline TP53 mutation was detected in these samples. Germline TP53 mutation, usually associated with Li-Fraumeni syndrome, is a rare event in UM. To the best of our knowledge, only one family with germline TP53 mutation has previously been described. In our study, we detected TP53 mutation in two patients without known family relationship. The identification of germline aberrations in TP53 or BAP1 is important to identify patients with Li-Fraumeni syndrome or BAP1 cancer syndrome, which is also crucial for proper genetic counseling.

• MeSH
• ATM protein genetika   MeSH
• dospělí MeSH
• genetická predispozice k nemoci MeSH
• lidé středního věku MeSH
• lidé MeSH
• melanom genetika   patologie   MeSH
• míra přežití MeSH
• mladý dospělý MeSH
• mutační analýza DNA MeSH
• nádorové supresorové proteiny genetika   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• nádory jater genetika   sekundární   MeSH
• nádory uvey genetika   patologie   MeSH
• prognóza MeSH
• senioři MeSH
• thiolesterasa ubikvitinu genetika   MeSH
• zárodečné mutace * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH