The role of immunofluorescence, immunohistochemistry and nonlinear scanning microscopy in diagnosis of renal cell tumors according to new classification

[G. Z. Nikolić]

73 str.

Medicina - Epidemiologija, Patologija /Medicine - Epidemiology, Pathology

Uvod: Karcinomi porekla bubrežnog parenhima (RCC) čine 2,4% svih malignih neoplaѕmi odraslih. Češće se javlja kod muškaraca između 60 i 70 godina starosti. Zahvaljujući ranom otkrivanju, petogodišnje preživljavanje je preko 93%, dok je kod pacijenata sa uznapredovalim karcinomom i metastazama petogodišnje preživljavanje svega 12% i sa terapijom. Patohistološka analiza predstavlja zlatni standard u dijagnostici tumora bubrega (RCT). Imunohistohemijska bojenja (IHH) kao dodatne analize koriste se za dijagnostiku RCT. Decenijama se istražuju i druge metode kao što su spektroskopske za dijagnostiku RCT. Poslednje dve decenije značajan napredak u mikroskopskom oslikavanju različitih vrsta ćelija i tkiva donela je upotreba femtosekundnih lasera koji su temelji nelinearne mikroskopije (NLM). Ciljevi: Cilj studije je poređenje rezultata optičke, konfokalne (KM), NLM u različitim RCT sa akcentom na detekciji mitohondrija koje predstavljaju ključne organele kod pojedinih RCT-ova. Materijal i metoda: Istraživanjem je obuhvaćeno 100 nefrektomisanih pacijenata u periodu 2019-2021. godine od kojih su četiri izuzeta zbog dijagnoze urotelnog karcinoma. Od 96 uzoraka, 43 su dijagnostikovana kao svetloćelijski RCC (ccRCC), 15 kao papilarni RCC (pRCC), 20 kao hromofobni RCC (chRCC) i 18 kao onkocitom (RO). Od IHH koristili smo Mitohondria i Prohibitin antitelo za detekciju mitohondrije. Iste uzorke smo analizirali i na KM, dok smo na NLM analizirali nebojene uzorke. Statistička analiza je izvršena korišćenjem statističkog paketa za društvene nauke, verzija 17 (SPSS). Rezultati: Kliničke i patohistološke karakteristike 96 pacijenata su analizirane u tri različite grupe, na osnovu IHH bojenja antimitohondrijalnog antitela. Pacijenti su bili slične starosne dobi, bez statističke značajnosti (p=0.960). Poređenjem lokalizacije tumora sa distribucijom IHH bojenja nismo dobili statističku značajnost (p=0.478). Prosečne vrednosti dimenzija tumora se nisu statistički razlikovale (p=0.865). Između RCT uočeni su statistički značajno različiti obrasci ekspresije. ccRCC je dijagnostikovan sa grubim oskudnim IHH bojenjem, sa izuzetkom dva slučaja sa višim nuklearnim gradusom sa difuznim granularnim obrascima bojenja umerenog intenziteta (p = 0,005). Svi pRCC i RO su imali difuzno granularno citoplazmatsko bojenje, pri čemu je razlika u distribuciji bojenja, dok je umereno granuliran obrazac uočen kod chRCC. Ove razlike među pokazale su statističku značajnost (p<0,001). Poređenjem histopatološkog stadijuma bolesti i pola, uočeno je da se pT1 stadijum se češće dijagnostikovao kod muškaraca, bez statističke značajnosti (p=0.804). Analizom slika dobijenih NLM, uočeni su jasni obrasci rasta tumora, i karakteristike signala u različitim RCT. Harmonikom druge generacije detektovali smo kolagensku mrežu, kako u bubrežnom parenhimu tako i u, i pored tumora. Zaključak: Upotreba antimitohondrijalnog antitela je pokazala visoku specifičnost u određivanju podtipa RCT, sa jedinstvenim intenzitetom bojenja i distribucijom. IF analiza KM potvrđuje ovu specifičnost, omogućavajući jasnu vizualizaciju mitohondrija u RCT-ovima. NLM analiza pokazuje efikasnost za vizualizaciju uzoraka bez pripreme tkiva. Svaki analizirani podtip RCT-a ima specifične karakteristike koje se mogu prepoznati na osnovu signala, što olakšava preciznu dijagnozu.

neoplasms in adults. It occurs more frequently in men between the ages of 60 and 70. Due to early detection, the five-year survival rate is over 93%, while in patients with advanced carcinoma and metastases, the five-year survival rate is only 12% with therapy. Pathohistological analysis represents the gold standard in the diagnosis of renal cell tumors (RCT). Immunohistochemical staining (IHC) as additional analysis is used for the diagnosis of RCT. For decades, other methods have been researched, such as spectroscopy for the diagnosis of RCT. The last two decades have brought significant progress in microscopic imaging of different types of cells and tissues with the use of femtosecond lasers, which are the basis of nonlinear microscopy (NLM). Objectives: The aim of the study is to compare the results of optical, confocal (CM), NLM in different RCTs with an emphasis on detecting mitochondria, which are key organelles in certain RCTs. Material and methods: The study included 100 nephrectomized patients from 2019- 2021, with four excluded due to a diagnosis of urothelial carcinoma. Of the 96 samples, 43 were diagnosed as clear cell RCC (ccRCC), 15 as papillary RCC (pRCC), 20 as chromophobe RCC (chRCC), and 18 as renal oncocytoma (RO). For IHC Mitochondria and Prohibitin antibodies are used for mitochondria detection. The same samples were analyzed on CM, while on NLM, unstained samples were used. Statistical analysis was performed using the Statistical Package for the Social Sciences, version 17 (SPSS). Results: Clinical and pathohistological characteristics of 96 patients were analyzed in three different groups, based on IHC staining of antimitochondrial antibody. Patients were similar in age, with no statistical significance (p=0.960). Comparing tumor localization with the distribution of IHC staining did not yield statistical significance (p=0.478). The average tumor dimension did not differ statistically (p=0.865). Statistically significant different expression patterns were observed between RCTs. ccRCC was diagnosed with coarse sparse IHC staining, except for two cases with higher nuclear grade showing diffuse granular staining patterns of moderate intensity (p=0.005). All pRCC and RO showed diffuse granular cytoplasmic staining, with differences in staining distribution, while moderate granular patterns were observed in chRCC. These differences were statistically significant (p<0.001). Comparing the histopathological stage of the disease and gender, pT1 stage was more frequently diagnosed in males, without statistical significance (p=0.804). NLM image analysis revealed distinct tumor growth patterns and signal characteristics in different RCTs. Second-generation harmonic detected the collagen network, both in renal parenchyma and in, and around the tumor. Conclusion: The use of antimitochondrial antibodies showed high specificity in determining the subtype of RCT, with a unique staining intensity and distribution. CM analysis confirms this specificity, allowing clear visualization of mitochondria in RCTs. NLM analysis shows efficacy in visualizing tissue-prepared samples. Each analyzed subtype of RCT has specific characteristics that can be recognized based on signals, facilitating precise diagnosis.

RCC, RCT, konfokalna mikroskopija, nelinearna skenirajuća mikroskopija

RCC, RCT, confocal microscopy, nonlinear scanning microscopy

616.61-006.6-07(043.3)

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