MOLECULAR CHANGES AND PHARMACOGENOMIC BIOMARKERS FOR CROHN DISEASE TREATMENT USING MONOCLONAL ANTIBODIES AGAINST TNFα

Kronična vnetna črevesna bolezen (KVČB) je kompleksna bolezen in se pojavlja v dveh podtipih, kot Crohnova bolezen (CB) in ulcerozni kolitis (UK). CB lahko prizadene celoten prebavni trakt, medtem ko UC prizadene skoraj izključno danko in debelo črevo. Na potek in razvoj bolezni pomembno vplivajo tako okoljski dejavniki kot tudi genetska zasnova posameznika. Asociacijske študije na celotnem genomu (GWA študije) so do danes identificirale 163 lokusov na človeškem genomu, ki so povezani z nastankom KVČB. Zdravljenje KVČB poteka v več stopnjah. Navadno začnemo z aminosalicilati, nadaljujemo s kortikosteroidi in imunosupresivi. Če zdravljenje s standardno terapijo ni uspešno, pa si v zadnjem času pomagamo z biološkimi zdravili in sicer zaviralci provnetnega citokina TNF-α, katerih prva predstavnika sta infliksimab (IFX) in adalimumab (ADA). Terapija z zaviralci TNF-α je naredila izjemen napredek v zdravljenju ne samo CB, ampak tudi ostalih oblik KVČB. V naši študiji smo se osredotočili na zdravljenje bolnikov s CB z adalimumabom (ADA). ADA je humano monoklonsko protitelo. Z odmerjanjem vsaki drugi teden dosegamo zelo dober odziv, v indukcijski kot tudi vzdrževalni terapiji, z zelo solidnim procentom bolnikov v remisiji, vendar se kljub temu približno tretjina bolnikov ne odziva na terapijo z zaviralci TNF-α. Cilj naloge je bil izvesti prospektivno farmakogenetsko raziskavo, pri čemer bi odkrili genetske biooznačevalce, s katerimi bi lahko vnaprej napovedali, kakšen bo izid zdravljenja z ADA pri posameznem bolniku. Dodatno smo želeli ugotoviti ali obstaja povezava med odzivom na terapijo z ADA in izražanjem genov pri bolnikih s CB, pri čemer smo ugotovili, da prav tako izstopa gen ATG16L1, pri katerem se je izražanje statistično značilno spreminjalo skozi celotno obdobje zdravljenja. Prav tako smo v nalogi analizirali potencialne povezave med imunološkimi, biokemijskimi in hematološkimi parametri. V študijo smo zajeli 102 bolnika s CB. Bolnike smo spremljali tako, da smo jim pred terapijo ter po 4., 12., 20., in 30. tednu odvzeli kri, izmerili vrednost C-reaktivnega proteina (CRP) kot pokazatelja vnetja ter ovrednotili uspešnost zdravljenja z IBDQ vprašalnikom, ki poleg vprašanj kliničnega značaja zelo dobro odraža psihološko stanje in počutje bolnika. Glede na število točk IBDQ vprašalnika smo ovrednotili uspešnost terapije, tako da je število nad 170 ali relativni dvig IBDQ za 22 točk pomenilo remisijo. Uspešnost terapije smo ovrednotili tudi z vrednostjo CRP in sicer smo menili, da je terapija uspešna, v kolikor se je CRP zmanjšal za 25% glede na začetno stanje pred zdravljenjem oz. je CRP dosegel normalne vrednosti. DNA polimorfizme posameznega nukleotida (SNP) in kandidatne gene smo izbrali po sledečih kriterijih: SNP-ji, močno povezani s KVČB v posameznih kandidatnih študijah, SNP-ji, ki so jih potrdile GWA študije za KVČB, ter SNP-ji, ki so bili v predhodnih študijah povezani z odzivom na IFX. Skupno smo genotipizirali 33 izbranih SNP-jev in izmerili izražanje 5 genov, predhodno povezanih s KVČB ter primerjali genotipe izbranih SNP-jev in nivo izražanja genov z odzivom na zdravljenje z ADA. Gensko tipizacijo polimorfizmov SNP v izbranih genih/lokusih smo izvajali z metodo analize talilnih krivulj visoke ločljivosti (HRM, ang. high resolution melting) in tehniko polimorfizmov dolžin restrikcijskih fragmentov (RFLP, ang. restriction fragment length polymorphism). Ugotovili smo, da je bil pozitivni odziv na zdravljenje z ADA merjen z IBDQ po 4 tednih pri 62.9% bolnikov, po 12 tednih 68%, po 20 tednih 66% in po 30 tednih 71%. V primeru merjenja odziva z CRP pa je imelo po 4 tednih pozitivni odziv 82,6% bolnikov, po 12 tednih 71,9% , po 20 tednih 72,7% in po 30 tednih 72,5% bolnikov. Najmočnejšo povezavo smo našli med polimorfizmom SNP rs10210302 v genu ATG16L1 in biološkim odgovorom na terapijo z ADA, merjenim s CRP, pri čemer so imeli bolniki z genotipom TT boljši odgovor po 12, 20 in 30 tednih zdravljenja v primerjavi z bolniki z genotipom CC (p=8.11E-04, p (po korekciji za multiplo testiranje = 0,027)). Nadalje smo močno povezavo odkrili med polimorfizmom SNP rs10512734, ki je lociran v genski puščavi na kromosomu 5p13.1 in za katerega je bilo dokazano, da vpliva na izražanje gena PTGER4 ter odgovorom na ADA, pri čemer so imeli bolniki z genotipom GG boljši odgovor v primerjavi z bolniki z genotipom AA po 12 tednih zdravljenja (p=4.62E-03). Ostale statistično najbolj značilne povezave smo ugotovili med SNP-ji v genih CASP9, IL27, CCNY, C11orf30, NR1I2 in IL13 ter odzivom na zdravljenje z ADA. V skladu s predhodnimi kliničnimi študijami smo tudi v naši študiji potrdili povezavo med nekaterimi kliničnimi značilnostmi bolnikov in odzivom na zdravljenje. Bolniki, ki predhodno še niso bili zdravljeni z IFX, so se signifikantno boljše odzvali na terapijo. Glede vzporedne terapije s standardnimi zdravili nismo našli značilnih povezav z odzivom na zdravljenje. Boljši odziv na zdravljenje smo opazili pri bolnikih, ki so bili predhodno kirurško zdravljeni. In boljši odziv na terapijo je bil opažen tudi pri nekadilcih. Bolniki, ki so bili ob postavitvi diagnoze mlajši, so se na terapijo boljše odzivali. Rezultati naše študije kažejo, da je odziv na zdravljenje z ADA deloma genetsko pogojen s polimorfizmi SNP, povezanimi z nastankom CB, pri čemer se je kot najbolj obetaven kandidatni gen za odgovor na zdravljenje z ADA izkazal gen ATG16L1. Glede na to, da smo našli številne povezave z drugimi polimorfizmi SNP ter odgovorom na ADA, bodo v prihodnje potrebne nadaljnje farmakogenetske študije, ki bi te povezave potrdile na večjem številu bolnikov s CB s ciljem razviti molekularne biološke označevalce in napovedne modele, s katerimi bi lahko načrtovali individualizirano zdravljenje bolnikov.The inflammatory bowel disease (IBD) is a complex disease which occurs in two subtypes - Crohn\u27s disease (CD) and ulcerative colitis (UC). CD may affect any part of the gastrointestinal tract and UC, in contrast, is almost exclusively restricted to the colon and the rectum. The disease course and development are caused by a combination of environmental and genetic factors. Until now genome wide association studies (GWAS) discovered 163 IBD risk loci. The approach to treatment of IBD is stepwise. Initially we introduce aminosalicylates, followed by corticosteroids and immunosuppressants in monotherapy or in combination. If the treatment is not successful, biologics are the next option, e.g. inhibitors of proinflammatory cytokine tumor necrosis factor alpha (TNF-α). The first two representatives of this group of drugs are infliximab (IFX) and adalumumab (ADA). The therapy with TNF-α inhibitors has made an exceptional progress in treatment of not only CD but also other forms of IBD. In our study we focused on ADA therapy in CD patients. ADA is a fully human monoclonal antibody against TNF-α. Induction and maintenance therapy with ADA every other week achieves a very good response and a solid percentage of patients in remission. But still approximately a third of the patients do not respond to therapy with THF-α inhibitors. The aim of this research project was to carry out a prospective pharmacogenetic study, with the attempt to discover genetic biomarkers that could be able to predict treatment outcomes with TNF-α in an individual patient. In the study we enrolled 102 patients with CD. Before and after week 4., 12., 20., and 30. of the treatment we collected blood samples, measured CRP as a marker of inflammation, and evaluated treatment success with the IBDQ questionnaire, which in addition to the clinical status also includes quality of life aspects. Cutoff IBDQ values of >170 points or an increase for 22 points indicated remission. We also considered treatment as successful if the CRP dropped by 25% as compared to baseline value or if it was lowered bellow 3 mg/l. Single nucleotide polymorphisms (SNPs) were chosen based on the following criteria: SNPs strongly correlated with IBD in candidate gene studiesSNPs identified by GWAS in IBDand SNPs associated with response to IFX. In total, we genotyped 33 SNPs and measured expression of 5 genes, which were correlated with IBD in previous studies, we also compared genotypes of these SNPs with gene expression regarding response to ADA therapy. After 30 weeks more than 70% of patients responded to therapy with respect to both criteria (71.1% based on IBDQ and 72.5% based on biologic response - CRP). Regarding CRP 82.6% of patients had a positive response after 4 weeks, and after 30 weeks of therapy the response was positive in 72.5% of patients. The strongest correlation was found between SNP rs10210302 (ATG16L1 gene) and biologic response measured with CRP: patients with TT genotype had a better response after weeks 12., 20., and 30. compared to patients with CC genotype (p=8,11E-04). Another strong correlation found was between SNP rs10512734, that is located in a gene dessert on chromosome 5p13.1 and has an impact on PTGER4 expression, and response to therapy with ADA - patients with GG genotype had a better response compared to patients with AA genotype after 12 weeks of treatment (p=4,62E-03). Other statistically the most significant correlations were found between response to therapy with ADA and SNPs in genes CASP9, IL27, CCNY, C11orf30, NR1I2 and IL13. Additionally, we wanted to determine whether there is a correlation between response to treatment with ADA and gene expression in patients with CD, where we found that ATG16L1 gene stands out again. The expression of ATG16L1 significantly changed during the whole treatment time. In the study we also analyzed potential links between immunological, biochemical and hematological parameters, but we did not find statistically significant correlations. The results of our study suggest that the response to treatment with ADA is partly genetically determined by SNPs associated with CD susceptibility and as the most promising candidate gene ATG16L1 was demonstrated. Given that we have found a number of correlations between SNPs and treatment response, further pharmacogenetic studies on a larger number of patients with CD are needed to develop molecular biomarkers and predictive models in order to plan individualized therapy regimes