20.500.12556/RUL-177985 Pomen histopatoloških, imunohistokemičnih in molekularnogenetskih dejavnikov pri opredeljevanju malignega potenciala Spitz melanocitnih proliferacij The role of histologic, immunohistochemical and genetic factors in determining the malignant potential of Spitz proliferations Namen: Spitz melanocitne proliferacije so podskupina melanocitnih tumorjev, genetsko opredeljenih s fuzijami kinaznih receptorjev ali mutacijo/amplifikacijo gena HRAS, ki obsegajo spekter od Spitz nevusov do Spitz melanomov. Zaradi morfoloških značilnosti so pogosto napačno diagnosticirani kot konvencionalni melanom. Ker so kriteriji opredelitve Spitz proliferacij v različne biološke skupine številni, subjektivni in slabo definirani, je bil cilj naše raziskave v veliki kohorti identificirati tiste histopatološke, imunohistokemične in molekularne parametre, ki so za pravilno razvrščanje Spitz proliferacij najbolj pomembni. Dodatna cilja sta bila ustvariti po meri narejena testa tarčnega sekvenciranja na nivoju DNA in RNA ter s sekvenciranjem celotnega transkriptoma odkriti možne nove biološke označevalce. Metode: V raziskavo smo vključili 276 primerov Spitz proliferacij. Pri vseh smo opravili enak nabor imunohistokemičnih barvanj in opredelili morfološko diagnozo na podlagi treh kriterijev: prisotnost hudih citoloških atipij, ? 2 mitozi/mm2 in prisotnost difuzne rasti. V podskupini smo izvedli analizo mutacij BRAF/NRAS/HRAS, tarčno sekvenciranje DNA/RNA in/ali sekvenciranje celotnega transkriptoma (WTS). V podskupini s sekvenciranjem DNA smo postavili integrirano končno diagnozo. Za diagnozo Spitz melanoma je bila potrebna morfološka diagnoza atipičnega Spitz tumorja visokega gradusa ter molekularni znak malignosti (mutacija promotorja TERT in/ali ? 4 spremembe števila kopij (CNV)). Rezultati: Morfološko smo 164 primerov diagnosticirali kot Spitz nevus, 78 kot atipični Spitz tumor nizkega gradusa in 34 kot atipični Spitz tumor visokega gradusa. Upoštevajoč rezultate molekularnih analiz, smo identificirali 9 Spitz melanomov. Pri nobenem bolniku v času sledenja (mediana 74 mesecev, razpon 6 - 172 mesecev) ni prišlo do napredovanja bolezni. Kot najpomembnejši parametri so se za opredelitev Spitz proliferacij izkazali starost, debelina po Breslowu, globoke mitoze, izrazita pagetoidna rast, proliferacijski indeks Ki-67 ? 10 %, izguba izražanja p16, difuzno pozitivna reakcija na PRAME ter prisotnost številnih CNV. Z WTS smo prepoznali več možnih bioloških označevalcev, ki se različno izražajo med biološkimi podskupinami Spitz proliferacij. Zaključki: Z integracijo morfoloških in molekularnogenetskih podatkov smo zanesljivo opredelili najbolj atipične Spitz proliferacije in potrdili, da predstavljajo Spitz melanomi le približno 3 % vseh Spitz proliferacij ter imajo praviloma indolenten klinični potek. Z uporabo molekularnogenetskih analiz smo pomembno zmanjšali delež tumorjev z nejasnim malignim potencialom in pokazali, da je za ustrezno nadaljnjo klinično obravnavo bolnikov ključna opredelitev gonilnih in sekundarnih genetskih sprememb. Naše ugotovitve tako podpirajo uporabo integriranega morfološko-molekularnega pristopa pri rutinski diagnostiki, zlasti pri morfološko atipičnih primerih. Goals: Spitz proliferations are a subset of melanocytic tumours, genetically defined by kinase-receptor fusions or HRAS mutations/amplifications, that span the spectrum from benign naevi to melanomas. They are often misdiagnosed as conventional melanoma due to their morphologic resemblance. Classification into biologic subgroups is often unreliable due to numerous and poorly defined criteria. Based on an analysis of a large cohort of Spitz proliferations, our goal was to identify the histomorphologic, immunohistochemical, and molecular parameters that are most significant for the proper classification of these lesions. Additional goals were to develop custom-made panels for DNA and RNA sequencing, and to identify potential novel biomarkers based on results from whole-transcriptome sequencing (WTS). Methods: Our study included 276 Spitz proliferations. The same immunohistochemical panel was performed for all cases. A morphologic diagnosis was rendered for all cases using three distinct morphologic criteria: severe cytologic atypia, ≥ 2 mitotic figures/mm2, and a diffuse growth pattern. In a subset of cases, additional molecular analyses were performed: BRAF/NRAS/HRAS hotspot mutations, targeted DNA/RNA sequencing, and/or WTS. In the subset with targeted DNA sequencing, a final (integrated) diagnosis was also made based on two criteria: (1) a morphologic diagnosis of high-grade atypical Spitz tumour, and (2) the presence of molecular markers of malignancy (pTERT mutation and/or ≥ 4 copy-number variations [CNVs]). Results: Morphologically, we classified 164 Spitz naevi, 78 low-grade atypical Spitz tumours, and 34 high-grade atypical Spitz tumours. Considering molecular findings, 9 of 49 cases met criteria for Spitz melanoma. All tumours followed an indolent clinical course during the follow-up period (median, 74 months; range, 6-172 months). The most significant parameters for classifying Spitz proliferations were age, Breslow thickness, deep dermal mitoses, prominent pagetoid spread, Ki-67 proliferation index of ≥ 10 %, loss of p16 expression, diffuse PRAME positivity, and numerous CNVs. WTS revealed several candidate biomarkers differentially expressed across Spitz subgroups. Conclusions: Integrating morphologic and molecular-genetic data enabled reliable identification of the most atypical Spitz proliferations. We confirmed that Spitz melanomas account for only about 3% of all Spitz proliferations and have an indolent clinical course. Molecular-genetic analyses substantially reduced the proportion of tumours with uncertain malignant potential and demonstrated that identifying driver and secondary genetic alterations is essential for appropriate clinical management. Our findings support adopting an integrated morphologic-molecular approach in routine diagnostics, especially in morphologically atypical cases. Spitz nevus atipični Spitz tumor Spitz melanom tarčno sekvenciranje PRAME p16 sekvenciranje celotnega genoma Spitz naevus atypical Spitz tumour Spitz melanoma targeted sequencing PRAME p16 whole-transcriptome sequencing true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-01-15 07:15:47 2026-01-15 07:15:59 2026-01-16 04:22:23 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 38451 8505.pdf 8505.pdf 1 19E2A0CA558E40213AA2DBDB99FC3EB9 1b2a60e7d2f72fc5e305dc83a20bb8216bc857dd7d34a42c0479db9492bdbc0f a5ea76d1-f1d9-11f0-bf92-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=225460 Medicinska fakulteta 0 0 0