| Abstract | 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. |
| Abstract (english) | 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. |
