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Genetski označevalci raka Huerthlejevih celic ščitnice : doktorsko delo
ID Krhin, Blaž (Author), ID Bešić, Nikola (Mentor) More about this mentor... This link opens in a new window, ID Dolžan, Vita (Comentor)

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Abstract
Tumorje Huerthlejevih celic ščitnice uvrščamo v skupino folikularnih tumorjev ščitnice in so relativno redka bolezen. Kljub velikemu napredku v onkologiji in diagnostiki rakavih bolezni je razlikovanje med benignim tumorjem (gomolj Huerthlejevih celic ščitnice - HCTN in adenom Huerthlejevih celic ščitnice - HCTA) ter rakom Huerthlejevih celic ščitnice (HCTC) možno le s histološko preiskavo ščitničnega tkiva, kjer se ugotavlja morebitna prisotnost intravaskularne in/ali transkapsularne invazije, ki je značilna za HCTC. Ker je operacija ščitnice zahtevna, se kirurg običajno najprej odloči za lobektomijo (delno odstranitev ščitnice). Po operaciji patolog opravi histološko analizo odstranjenega ščitničnega tkiva. Če je odstranjeno tkivo rakavo, je potreben ponovni kirurški poseg, s katerim kirurg odstrani še preostanek ščitnice. Poznavanje ustreznih genetskih označevalcev HCTC bi v klinični praksi pripomoglo k možnosti zgodnjega razlikovanja med benignim tumorjem in rakom, oziroma bi omogočilo lažje načrtovanje zdravljenja HCTC, vključno z optimalnim operativnim posegom (delna oziroma popolna odstranitev ščitnice). Hipoteze V naši raziskavi smo preverili naslednji hipotezi: a) specifične mutacije onkogenov in tumor zaviralnih genov so genetski označevalci HCTC, oziroma tveganja za njegovo ponovitev ali zasevanje; b) specifični polimorfizmi genov antioksidativnih encimov in/ali genov popravljalnih mehanizmov DNA so genetski označevalci HCTC, oziroma tveganja za njegovo ponovitev ali zasevanje. Preiskovanci in metode Z retrospektivno raziskavo smo zajeli 139 preiskovancev, pri katerih je bila zaradi suma na neoplazmo Huerthlejevih celic ščitnice, opravljena operacija ščitnice. Genomsko DNA smo izolirali iz tumorskega in zdravega tkiva arhiviranih vzorcev tumorja, fiksiranih s formalinom in vklopljenih v parafin. S postopki, ki temeljijo na principu sekveniranja druge generacije, smo na desetih reprezentativnih vzorcih tumorske DNA opravili presejalno iskanje 739 mutacij v 46 onkogenih in tumor zaviralnih genih. Prisotnost nekaterih odkritih mutacij, ki so se nakazovale kot možni označevalci HCTC, smo preverili z visoko občutljivim testom verižne reakcije s polimerazo v realnem času, tudi v ostalih vzorcih tumorske DNA. Pri bolnikih z mutacijo v tumorski DNA smo nato z analizo DNA iz zdravega tkiva preverili tudi ali gre za somatsko ali zarodno mutacijo. V vzorcih DNA iz zdravega tkiva, smo preverili tudi prisotnost nekaterih polimorfizmov genov antioksidativnih encimov (CAT, SOD2, GSTM1, GSTT1, GSTP1, GPX1) in genov popravljalnih mehanizmov DNA (OGG1, XRCC3, NBN, RAD51, XRCC3). Rezultati Skupno 53 preiskovancev je imelo HCTC, 37 HCTA in 21 HCTN. Ostalih 18 preiskovancev je imelo multinodozno golšo (MNG) ali folikularni adenom (FTA) ali limfocitni tiroiditis (LT). Pri 20 preiskovancih s HCTC so bili prisotni zasevki, pri 16 pa ponovitev bolezni. Preiskovanci s HCTC so bili značilno starejši od preiskovancev s HCTA in HCTN (p = 0,004), imeli so značilno večji začetni premer tumorja (p < 0,001), kot tudi drugačno razmerje spolov (p = 0,043). Pri presejalnem iskanju mutacij smo našli mutacije v 26 od skupno 46 onkogenov in tumor zaviralnih genov in sicer so bile mutacije pogostejše v skupini preiskovancev s HCTC. Prisotnost mutacij v izbranih genih (BRAF, KRAS, NRAS) smo preverili tudi v ostalih vzorcih tumorske DNA, vendar v primerjavi HCTC s HCTA in HCTN, nismo ugotovili statistično značilnih povezav. Potrdili pa smo, da so bile vse odkrite mutacije somatske. Frekvenčna razporeditev genotipov antioksidativnih genov ter DNA popravljalnih genov se med preiskovanci s HCTC ter s HCTA in HCTN ni razlikovala (p > 0,050). Preiskovani polimorfizmi tudi niso bili povezani s pojavom oddaljenih zasekov (p > 0,050). Od proučevanih polimorfizmov pa je bil polimorfizem GPX1 povezan s ponovitvijo HCTC (p = 0,040). Zaključki Z našo raziskavo smo pokazali, da izbrane mutacije BRAF, KRAS, NRAS niso statistično značilno povezane s HCTC ali pojavom zasevkov. Vse odkrite mutacije so bile somatske. Z našo raziskavo smo tudi pokazali, da polimorfizmi genov za antioksidativne encime in genov popravljalnih mehanizmov DNA niso povezani s tveganjem za nastanek HCTC, oziroma tveganjem za pojav oddaljenih zasevkov. Polimorfizem GPX1 pa je povezan s tveganjem za ponovitev HCTC.

Language:Slovenian
Keywords:genetika medicinska, ščitnične novotvorbe, oksifilne celice, genetski označevalci, genetske tehnike, genetski polimorfizem, oksidativni stres
Work type:Dissertation
Typology:2.08 - Doctoral Dissertation
Organization:MF - Faculty of Medicine
Place of publishing:Ljubljana
Publisher:[B. Krhin]
Year:2016
Number of pages:156 str.
PID:20.500.12556/RUL-88440 This link opens in a new window
UDC:616.4(043.3)
COBISS.SI-ID:2507899 This link opens in a new window
Publication date in RUL:05.01.2017
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Downloads:559
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Secondary language

Language:English
Title:Genetic markers of Huerthle cell carcinoma
Abstract:
Hurthle cell tumours of thyroid are regarded as a subtype of follicular thyroid tumours and are a rare type of disease. Despite of recent large improvement in diagnostics of cancer patients, a definitive way to differentiate a benign tumour (Hurthle cell thyroid adenoma - HCTA and Hurthle cell thyroid nodule - HCTN) from Hurthle cell thyroid carcinoma (HCTC) is based on histological examination of thyroid tissue, where vascular and/or transcapsular invasion is characteristic for HCTC. For HCTA or HCTN, lobectomy is a sufficient surgical procedure. However, if HCTC is diagnosed by histopathology after lobectomy, then complete thyroidectomy is performed as a second surgical procedure. Use of appropriate genetic markers of HCTC, could help in early differentiation between benign tumour and cancer, as well as enable better treatment planning, including optimal surgical procedure (lobectomy or total thyroidectomy) in patients with HCTN. Hypothesis The following hypotheses were verified: a) Specific mutation of oncogenes and tumour suppressor genes are genetic markers of HCTC, disease recurrence, or distant metastasis; b) Specific polymorphisms of antioxidative genes or DNA repair genes are genetic markers of HCTC, disease recurrence, or distant metastasis. Patients and methods A retrospective study included 139 patients treated by thyroid surgery for Hurthle cell neoplasm. We isolated genomic DNA from tumour and normal thyroid tissue of formalin-fixed and paraffin-embedded samples. We have screened 10 representative tumour DNA samples for 739 mutations of 46 oncogenes and tumour suppresion genes, using second generation DNA sequencing. Selected mutations, that were detected as possible markers of HCTC, were verified on all 139 tumour DNA samples, using high sensitivity real time polymerase chain reaction. In patients harbouring specific mutations in their tumour DNA, DNA analysis was repeated in their normal thyroid tissue, to verify whether the mutation is somatic or germinal. DNA from normal thyroid tissue was also analyzed for common functional polymorphism of antioxidant genes (CAT, SOD2, GSTM1, GSTT1, GSTP1, GPX1) and DNA repair genes (OGG1, XRCC1, NBN, RAD51, XRCC3). Results Patients were diagnosed as follows: 53 had HCTC, 37 HCTA, 21 HCTN and 18 had multinodular goiter, or follicular thyroid adenoma, or lymphocytic thyroiditis. Altogether 20 of 53 patients with HCTC had metastatic disease. Recurrent disease was observed in 16 patients with HCTC. The patients from the HCTC group had different gender (F/M) ratio (p = 0.043), were older (p = 0.004) and had larger initial tumour diameter (p < 0.001) in comparison with the patients from the HCTA or HCTN group. By second generation DNA sequencing, 26 (of totally 46) mutated oncogenes and tumour suppression genes were found and were more often in HCTC groups. Mutations in selected genes (BRAF, KRAS, NRAS) were verified in all the remaining samples, but no associations between gene mutations and presence of HCTC and HCTA or HCTN groups were found. All the detected mutations in these genes were somatic. Under the dominant genetic model, no significant differences in the genotype frequency distribution of antioxidative genes and DNA repair genes were observed, when HCTC group was compared to HCTA and HCTN groups. These polymorphisms were also not associated with presence of metastatic disease. However, GPX1 polymorphism was associated with the presence of recurrent disease (p = 0.040). Conclusions In our study we did not find any association between selected mutations of BRAF, KRAS and NRAS and presence of HCTC, or metastatic, or recurrent disease. All detected mutations of these genes were somatic. In our study we also did not find any association between common functional polymorphism of antioxidant genes or DNA repair genes and development of HCTC, or metastatic disease. However, GPX1 polymorphism is associated with the risk of HCTC recurrence.

Keywords:genetics medical, thyroid neoplasms, oxyphil cells, genetic markers, genetic techniques, polymorphism genetic, oxidative stress

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