Genetska variabilnost in serumski nivoji kalretinina kot diagnostični in prognostični označevalec pri azbestnih boleznih

Zupanc, Cita (author), Goričar, Katja (mentor)

POVZETEK Ozadje. Azbestne bolezni, kot so plevralni plaki, azbestoza in maligni mezoteliom (MM), so velik javno zdravstveni problem v Sloveniji in po svetu. Zgodnja diagnostika bi lahko izboljšala zdravljenje in prognozo bolnikov. Kalretinin je histološki označevalec, ki ga uporabljajo pri diagnostiki MM. V nekaterih raziskavah so že pokazali, da bi tudi serumski kalretinin lahko služil kot biološki označevalec pri azbestnih boleznih, predvsem pri MM. Namen dela. V naši raziskavi smo želeli ovrednotiti serumski nivo kalretinina in genetske polimorfizme v genih, ki lahko sodelujejo pri uravnavanju izražanja kalretinina, kot diagnostični in prognostični označevalec pri azbestnih boleznih. Opis metod. V raziskavo smo vključili 83 preiskovancev, ki so bili izpostavljeni azbestu, a niso zboleli za nobeno azbestno boleznijo, 380 preiskovancev s plevralnimi plaki, 153 preiskovancev z azbestozo in 288 bolnikov z MM. V serumu preiskovancev smo s komercialno dostopnim encimskim imunskim testom (ELISA) določili nivo kalretinina. Z bioinformatskimi pristopi smo poiskali pogoste polimorfizme v regulatornih regijah gena CALB2 ter polimorfizme v genih za miRNA in transkripcijske dejavnike, udeležene v uravnavanju izražanja kalretinina. DNA smo izolirali iz vzorcev periferne venske krvi, za genotipizacijo pa smo uporabili alelno-specifično verižno reakcijo s polimerazo. Za statistično analizo rezultatov smo uporabili standardne statistične teste (logistična regresija, neparametrični testi, analiza preživetja). Rezultati. Bolniki z MM so imeli znatno višji serumski nivo kalretinina kot osebe brez bolezni, osebe s plevralnimi plaki ali osebe z azbestozo (P<0,001). Histološki tip je bil pomembno povezan s serumskim kalretininom: bolniki s sarkomatoidnim MM so imeli nižji kalretinin kot bolniki z epiteloidnim tipom. Nivo kalretinina je bil dober diagnostični označevalec MM (površina pod krivuljo 0,826 (0,782-0,869); P<0,001). Pri mejni vrednosti 0,32 ng/ml je bila občutljivost 0,683, specifičnost pa 0,886. Genetski dejavniki so vplivali na nivo kalretinina in tveganje za pojav MM. Nosilci vsaj enega polimorfnega alela CALB2 rs889704 so imeli nižji nivo kalretinina (P=0,036). Pri osebah brez MM so imeli nosilci dveh polimorfnih alelov MIR335 rs3807348 višji nivo kalretinina (P=0,027), nosilci vsaj enega polimorfnega alela NRF1 rs13241028 pa nižji nivo kalretinina (P=0,034). Nosilci dveh polimorfnih alelov E2F2 rs2075995 so imeli manjšo verjetnost, da bodo razvili MM (OR=0,64, 95 % IZ=0,43-0,96, P=0,032), vendar povezava ni bila več statistično značilna po prilagoditvi za starost (P=0,093). Mejne vrednosti kalretinina v serumu, ki lahko razlikujejo bolnike z MM od drugih oseb, izpostavljenih azbestu, so se razlikovale glede na genotipe CALB2, NRF1, E2F2 in MIR335. Višji nivo kalretinina v serumu je bil povezan s krajšim preživetjem brez napredovanja bolezni (PFS) in krajšim celokupnim preživetjem (OS) od diagnoze (P=0,023), vendar povezava ni bila statistično značilna po prilagoditvi za klinične dejavnike. SEPTIN7 rs3801339 je bil povezan s krajšim PFS, tudi po prilagoditvi za klinične dejavnike (P=0,007). MIR335 rs3807348 pa je bil povezan z daljšim OS, tudi po prilagoditvi za klinične dejavnike (P=0,028). Nivo kalretinina je bil višji pri bolnikih, pri katerih je prišlo po zdravljenju s kemoterapijo na osnovi cisplatina do napredovanja bolezni (P=0,001). Nivo kalretinina nad 0,89 ng/ml je bil povezan tudi s krajšim PFS in OS od začetka kemoterapije (P=0,001 oziroma P=0,004), tudi po prilagoditvi za klinične dejavnike (P=0,006 oziroma P=0,049). MIR335 rs3807348 je bil povezan z boljšim odzivom na kemoterapijo (P=0,015) , tudi po prilagoditvi za klinične dejavnike (P=0,020). Zaključki. V raziskavi smo ovrednotili serumski nivo kalretinina kot diagnostični in prognostični označevalec pri različnih azbestnih boleznih. Potrdili smo, da je nivo kalretinina v serumu pri MM povišan. Prav tako so naši rezultati pokazali, da bi lahko genetska variabilnost vplivala na nivo kalretinina v serumu. Pokazali smo, da lahko serumski kalretinin vpliva na preživetje in izid kemoterapije pri MM in bi lahko služil kot napovedni biološki označevalec. Te ugotovitve bi lahko prispevale k boljšemu razumevanju regulacije kalretinina in potencialno k diagnostiki in napovedi odgovora na zdravljenje pri MM.

• Language: Slovenian
• Keywords: maligni mezoteliom, kalretinin, azbestne bolezni, biološki označevalec, polimorfizem
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2024
• PID: 20.500.12556/RUL-159270
• Publication date in RUL: 05.07.2024

Secondary language

• Language: English
• Title: Genetic variability and serum levels of calretinin as a diagnostic and prognostic biomarker in asbestos diseases

Abstract

SHORT SUMMARY Background. Asbestos-related diseases such as pleural plaques, asbestosis, and malignant mesothelioma (MM) are a major public health problem in Slovenia and around the world. Early diagnosis could improve the treatment and prognosis of patients. Calretinin is a histological marker used in the diagnosis of MM. Some studies have already shown that serum calretinin could also serve as a biological marker in asbestos diseases, especially in MM. Hypothesis. In our study we wanted to evaluate serum calretinin levels and genetic polymorphisms in genes that may regulate calretinin expression as a diagnostic and prognostic marker in asbestos-related diseases. Methods. We included in the study 83 subjects who were exposed to asbestos but did not develop any asbestos-related disease, 380 subjects with pleural plaques, 153 subjects with asbestosis and 288 patients with MM. Serum calretinin levels were determined using a commercially available enzyme immunoassay (ELISA). Using bioinformatic approaches, we identified common polymorphisms in the regulatory regions of the CALB2 gene, as well as polymorphisms in genes for miRNAs and transcription factors involved in the regulation of calretinin expression. DNA was isolated from peripheral venous blood samples, and allele-specific polymerase chain reaction was used for genotyping. Standard statistical tests (logistic regression, non-parametric tests, survival analysis) were used for statistical analysis. Results. Patients with MM had significantly higher calretinin concentrations than subjects without asbestos-related diseases, subjects with pleural plaques, or subjects with asbestosis (P<0.001). Histological type was significantly associated with serum calretinin: patients with sarcomatoid MM had lower calretinin than patients with epithelioid type. Calretinin level was a good diagnostic marker of MM (area under the curve 0.826 (0.782-0.869); P<0.001). At a cutoff of 0.32 ng/ml, the sensitivity was 0.683 and the specificity was 0.886. Genetic factors were associated with serum calretinin levels and MM risk. Carriers of at least one CALB2 rs889704 polymorphic allele had lower calretinin levels (P=0.036). In subjects without MM, carriers of two MIR335 rs3807348 polymorphic alleles had higher calretinin (P=0.027), while carriers of at least one NRF1 rs13241028 polymorphic allele had lower calretinin levels (P=0.034). Carriers of two E2F2 rs2075995 polymorphic alleles were less likely to develop MM (OR=0.64, 95% CI=0.43-0.96, P=0.032), but the association was no longer statistically significant after adjustment for age (P=0.093). Serum calretinin cut-off values differentiating MM patients from other asbestos-exposed subjects differed according to CALB2, NRF1, E2F2, and MIR335 genotypes. Higher serum calretinin concentration was associated with shorter progression-free (PFS) and overall survival (OS) from diagnosis (P=0.023), but the association was not significant after adjusting for clinical factors. SEPTIN7 rs3801339 was associated with shorter PFS, even after adjustment for clinical factors (P=0.007). MIR335 rs3807348 was associated with longer OS, even after adjustment for clinical factors (P=0.028). Calretinin concentration was higher in patients who progressed after treatment with cisplatin-based chemotherapy (P=0.001). Calretinin concentration above 0.89 ng/mL was associated with shorter PFS and OS from the start of chemotherapy (P=0.001 and P=0.004, respectively), even after adjustment for clinical factors (P=0,006 and P=0,049, respectively). MIR335 rs3807348 was associated with a better response to chemotherapy (P=0.015), even after adjustment for clinical parameters (P=0.020). Conclusions. In our study, we evaluated serum calretinin levels as a diagnostic and prognostic marker in various asbestos diseases. We confirmed that serum calretinin levels are increased in MM. Our results also indicated that genetic variability could affect serum calretinin levels. We showed that serum calretinin can influence survival and chemotherapy outcome in MM and could serve as a predictive biomarker. These findings could contribute to a better understanding of calretinin regulation and potentially to diagnosis and treatment response prediction in MM.

• Keywords: malignant mesothelioma, calretinin, asbestos-related disease, biomarker, polymorphism