Uloga citokina i čimbenika rasta u nastanku i napredovanju gastroenteropankreatičnih neuroendokrinih tumora (GEP-NET) [The role of cytokines and growth factors on development and progression of neuroendocrine tumors of gastrointestinal tract and pancreas (GEP-NETs)]

Cigrovski Berković, Maja (2009) Uloga citokina i čimbenika rasta u nastanku i napredovanju gastroenteropankreatičnih neuroendokrinih tumora (GEP-NET) [The role of cytokines and growth factors on development and progression of neuroendocrine tumors of gastrointestinal tract and pancreas (GEP-NETs)]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) represent a rare group of tumors arising from cells of diffuse endocrine system. Patients with inherited GEP-NETs, occurring as a part of multiple endocrine neoplasia type 1 syndrome usually show a mutation in MEN1 gene, while genetic basis of more frequent sporadic tumors remains unclear. There is growing evidence of involvement of chronic inflammation in the pathogenesis of cancer. Inflammation, through network of proinflammatory cytokines and growth factors predisposes protumorigenic environment, supports neoangiogenesis and tumor spread. Current investigations of growth pathways underlining GEP-NETs suggest the role of cytokines as tumor promoters via autocrine and paracrine manner and predict success of new therapeutic options targeting proinflammatory cytokines involved in tumorigenesis. Expression of proinflammatory cytokines and growth factors is regulated on transcriptional level. Many SNPs (Single Nucleotide Polymoprhisms) in the promoter regions of proinflammatory genes not only affect the serum and/or tumor tissue cytokine levels, but also, through specific haplotypes, predispose to development of inflammation-mediated cancer. We hypothesized that proinflammatory cytokine gene polymorphisms occur more frequently among patients with GEP-NETs than in healthy population, and that they contribute to different tumor origin and functional status. Moreover, we hypothesized polymorphisms of proinflammatory cytokine genes and growth factors to alter cytokine expression, leading to their elevated levels in sera and tumor tissue, and influencing clinical presentation of patients. The aim of present study was to investigate the role of proinflammatory cytokine genes in GEP-NET susceptibility through comparison of frequencies of polymorphisms IL-1β -511 T/C, IL-2 -330 G/T, IL-6 -174 G/C, TNF-α -238 A/G, TNF-α -308 A/G, TNF-α -857 T/C, TNF-α -1031 C/T and VEGF -1154 G/A between 150 healthy volunteers and 101 patients diagnosed with GEPNETs. Moreover, we tested the correlation of specific alleles/genotypes of cytokine polymorphisms with levels of proinflammatory cytokines and growth factor VEGF in serum and tumor tissue of patients. Through comparison with standard neuroendocrine markers such as chromogranin A (CgA) and 5-hydroxyindolacetic acid (5-HIAA) and Ki67 index significance of cytokines and growth factor VEGF was tested. Patients were divided into two groups according to the tumor origin, and further divided according to the tumor functionality. First group, comprised of 55 patients with gastrointestinal neuroendocrine tumors had 13 patients with functioning and 42 with nonfunctioning tumors, and in second group there were 46 patients with pancreatic neuroendocrine tumors, of which 20 with functioning and 27 with nonfunctioning tumors. RFLP method was used for detection of IL-2 -330 T/G and IL-6 -174 C/G, while for detection of TNF-α -238 A/G, -308 A/G, -857 C/T, -1031 C/T, IL-1β -511 C/T and VEGF -1154 A/G polymorphisms we used «real» time PCR with allelic discrimination. According to obtained results IL-2 -330 G/T and VEGF -1154 G/A polymorphisms were statistically more frequent among patients with GEP-NETs than in healthy controls (p=0.0033 and p=0.0368, respectively), while polymorphisms IL-2 -330 G/T, IL-6 -174 G/C, TNF-α -1031 C/T and VEGF -1154 G/A were statistically more frequent in patients with gastrointestinal neuroendocrine tumors (p=0.0491, p=0.0445, p=0.0462 and p=0.0492 respectively). Patients with pancreatic neuroendocrine tumors were statistically more frequent carriers of IL-2 -330 G/T (p=0.0031) and TNF-α -308 A/G (p=0.0476) polymorphisms, while polymorphism IL-6 -174 G/C appeared to be of special importance for the development of nonfunctioning pancreatic neuroendocrine tumors (p=0.0216). Alleles IL-2 -330 G, IL-6 -174 G, TNF-α -238 A and -857 C, and VEGF -1154 G correlated with elevated serum concentrations of cytokines (p=0.03, p=0.00003, p=0.02, p=0.01 and p=0.003 respectively). In comparison to 5-HIAA, IL-2, IL-6, TNF-α and VEGF were more sensitive markers of GEP-NETs (p<0.0001, p<0.0001, p=0.0002 and p=0.0006, respectively), while IL-2 was also superior to CgA (p= p<0.0001), a standard neuroendocrine marker regardless of tumor functional status. Serum levels of IL-6 appeared to be as sensitive as CgA (p=0.13). Moreover expression of TNF-α and VEGF in GEP-NET tissue correlated with Ki67 index, and proliferative activity of tumors (p=0.03 and p=0.09, respectively). In conclusion, this study suggests the role of chronic inflammation in GEP-NET development. Specific haplotypes of proinflammatory genes seem to increase susceptibility to GEP-NETs and amplify serum and tumor tissue levels of cytokines and growth factors, contributing to prognostic value of standard neuroendocrine markers. Also, proinflammatory cytokines and growth factors, expressed in tumor tissue might add to histological diagnosis and/or prognosis of GEP-NETs.

Abstract in Croatian

Neuroendokrini tumori probavnog sustava i gušterače (GEP-NET) rijetki su tumori stanica difuznog endokrinog sustava. Javljaju li se u sklopu sindroma multiple endokrine neoplazije za njihov nastanak često su odgovorne mutacije tumor-supresorskog gena MEN1, dok je etiopatogeneza sporadičnih oblika tumora još uvijek nedovoljno istražena. Sve je više dokaza da kronična upala, aktivacijom proupalnih citokina i čimbenika rasta, pogoduje neoangiogenezi i stvaranju povoljnog mikrookoliša za razvoj tumora. Novijim istraživanjima dokazana je ekspresija citokina i čimbenika rasta u tkivu GEP-NET koji, djelujući autokrino i parakrino, aktiviraju signalne putove odgovorne za rast tumorskih stanica te time predstavljaju i buduće mete ciljane anti-tumorske terapije. Ekspresija citokina i čimbenika rasta regulirana je na nivou gena u čijim promotorskim regijama postoje genetski određene varijacije (SNP, od engl. Single Nucleotide Polymorphisms) s posljedičnom izmijenjenom ekspresijom citokina, koje dodatno mijenjaju i sklonost obolijevanju od različitih sporadičnih tumora. Hipoteza ovog istraživanja bila je da su polimorfizmi gena za proupalne citokine i čimbenike rasta češći među oboljelima od GEP-NET nego u zdravoj populaciji te da se prema učestalosti alela/genotipova povezanih s izmijenjenom ekspresijom međusobno dodatno razlikuju GEP-NET obzirom na podrijetlo i funkcionalni status tumora. Također je pretpostavljeno da genotipovi povezani s izmijenjenom ekspresijom u GEP-NET koreliraju s povećanim razinama citokina i čimbenika rasta u serumu i tumorskom tkivu te tako utječu i na težinu kliničke slike oboljelih. U ovoj disertaciji, uspoređena je učestalost polimorfizama IL-1β -511 T/C, IL-2 -330 G/T, IL-6 -174 G/C, TNF-α -238 A/G, TNF-α -308 A/G, TNF-α -857 T/C, TNF-α -1031 C/T i VEGF - 1154 G/A u 150 zdravih nesrodnika te 101 oboljelog od GEP-NET s ciljem utvrđivanja uloge gena za proupalne citokine i čimbenik rasta VEGF u obolijevanju od GEP-NET. Prisutnost alela/genotipova polimorfizama gena za citokine i čimbenik rasta VEGF korelirana je sa razinama citokina u serumu i dostupnom tkivu oboljelih od GEP-NET. Serumske razine proupalnih citokina i čimbenika rasta VEGF također su uspoređene s uvriježenim biljezima GEP-NET poput kromogranina A (CgA) i 5-hidroksiindoloctene kiseline (5-HIAA), a izraženost citokina i čimbenika rasta u tumorskom tkivu s proliferativnim biljegom Ki67 kako bi se utvrdila njihova prognostička valjanost. Oboljeli su prema sijelu i funkcionalnom statusu tumora podijeljeni u skupine. Prvu su skupinu (55 oboljelih) sačinjavali bolesnici s neuroendokrinim tumorima crijeva; od čega je njih 13 imalo funkcionalne, a 42 nefunkcionalne tumore. Skupinu oboljelih od neuroendokrinih tumora gušterače činilo je 46 bolesnika, od čega ih je 20 imalo funkcionalno aktivne, a 27 neaktivne tumore. Polimorfizmi gena za citokine IL-2 -330 T/G te IL-6 -174 C/G određivani su RFLP metodom, dok su polimorfizmi TNF-α -238 A/G, -308 A/G, -857 C/T, -1031 C/T, IL-1β -511 C/T i VEGF -1154 A/G određivani metodom „real time“ PCR te alelnom diskriminacijom. Istraživanje je pokazalo kako su polimorfizmi IL-2 -330 G/T i VEGF -1154 G/A statistički značajno češći u oboljelih od GEP-NET (p=0.0033 i p=0.0368), dok su polimorfizmi IL-2 -330 G/T, IL-6 -174 G/C, TNF-α -1031 C/T i VEGF -1154 G/A statistički značajno češći u oboljelih od neuroendokrinih tumora podrijetla crijeva (p=0.0491, p=0.0445, p=0.0462 i p=0.0492). U oboljelih pak od neuroendokrinih tumora gušterače statistički značajno češći bili su polimorfizmi IL-2 -330 G/T (p=0.0031) i TNF-α -308 A/G (p=0.0476), dok se za nastanak nefunkcionalnih neuroendokrinih tumora gušterače kao posebno važan izdvojio IL-6 -174 G/C (p=0.0216). Aleli IL-2 -330 G, IL-6 -174 G, TNF-α -238 A i -857 C te VEGF -1154 G u oboljelih od GEP-NET korelirali su s povišenim serumskim razinama citokina i čimbenika rasta (p=0.03, p=0.00003, p=0.02, p=0.01 i p=0.003). Usporedbom serumskih razina citokina s CgA i 5-HIAA proizašla je njihova vrijednost u serološkoj dijagnostici GEP-NET obzirom da su IL-2, IL-6, TNF-α i VEGF imali veću osjetljivost od 5-HIAA (p<0.0001, p<0.0001, p=0.0002 i p=0.0006), a u usporedbi s CgA su bili ili podjednako osjetljivi (IL-6, p=0.13), odnosno čak i više osjetljivi (IL-2, p= p<0.0001). Dodatno je utvrđeno kako ekspresija TNF-α i VEGF u tkivu GEP-NET korelira s Ki67 te time utječe na proliferativnu aktivnost tumora (p=0.03 i p=0.09). Na temelju rezultata ovog istraživanja zaključujemo da specifični haplotipovi gena za proupalne citokine i čimbenike rasta potencijalno mijenjaju sklonost obolijevanju od GEP-NET, odnosno utječu na ekspresiju proupalnih citokina i čimbenika rasta u serumu i tumorskom tkivu oboljelih. Određivanje proupalnih citokina i čimbenika rasta u serumu i tumorskom tkivu oboljelih moglo bi upotpuniti dijagnostiku GEP-NET te pomoći probiru posebno ugroženih bolesnika.

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