| Sažetak | Ciljevi istraživanja: Multipli mijelom (MM) je B-stanična neoplazma obilježena
multifokalnom infiltracijom koštane srži s malignim plazma stanicama. Molekularna je
osnova bolesti nejasna. Angiogeneza, kao biološki fenomen stvaranja novih krvnih
žila, prisutna je u koštanoj srži kod raznih plazma staničnih poremećaja i posebno je
značajna karakteristika aktivnog MM. Zbog toga se inhibicija tumorske angiogeneze
pokazala kao obećavajući pristup liječenju mijeloma. Ciljevi istraživanja su bili ispitati
angiogenezu i ekspresiju vaskularnog endotelnog faktora rasta (VEGF), monocitnog
kemotaktičnog proteina (MCP-1) u odnosu prema nuklearnom faktoru – kappa B
(NF-êB) na molekularnoj razini, razini proteina u stanici i u plazmi bolesnika s MM.
Ispitanici i metode: U istraživanje je ukljuceno 79 bolesnika s dijagnozom MM i s
kliničkim podatcima uključujuci klinički stadij bolesti, odgovor na terapiju i preživljenje.
Bioptički uzorci koštane srži korišteni su za dvostruku imunohistokemijsku analizu u
cilju utvrđivanja gustoće krvnih žila (MVD), Ki67 proliferacijskog indeksa te izražaja
VEGF, MCP-1 i subcelularne lokalizacije NF-êB. Krv 19 bolesnika korištena je za
određivanje plazmatske koncentracije VEGF i MCP-1 ELISA metodom, dok su
stanice koštane srži korištene za izolaciju plazma stanica metodom magnetske
separacije ili protočnom citometrijom u cilju utvrđivanja mRNA VEGF i MCP-1 RTPCR
metodom.
Rezultati: Rezultati su pokazali pozitivnu udruženost između MVD, % tumorskog
infiltrata (p<0,001), pojave atipičnih plazma stanica (p=0,001) i kliničkog stadija
bolesti (p=0,001). Nadalje, uočena je značajna povezanost većeg MVD sa slabijim
odgovorom na terapiju (p=0,029) i kraćim preživljenjem (p=0,04). Izražaj VEGF u
tumorskim stanicama je bio značajno niži kod većeg MVD (p=0,002). Slabija
ekspresija oba citokina, VEGF (p =0,006) i MCP-1 (p = 0,03) bila je udružena s većim izražajem NF-êB. U bolesnika je utvrđena veća plazmatska koncentracija VEGF u
odnosu na kontrolnu skupinu (p=0,008). Koncentracije oba citokina, VEGF (p=0,05) i
MCP-1 (p=0,05) bile su udružene s većim % tumorskih infiltrata, a samo VEGF s
većim kliničkim stadijem bolesti (p=0,001). Vrijednosti mRNA VEGF i MCP-1 nisu
pokazale udruženost s ispitivanim parametrima.
Zakljucci: P otvrđena je pozitivna korelacija između angiogeneze i nepovoljnih
prognostičkih čimbenika te kraćeg preživljenja u MM. Koncentracije secerniranih
citokina u krvi, VEGF i MCP-1, proporcionalne su tumorskoj masi i dugorocno mogu
biti važan klinički pokazatelj bolesti što se daljnjim studijama mora potvrditi. Opažena
obrnuta ekspresija između NF-êB i citokina ne isključuje mogućnost njihove
međusobne povezanosti u regulaciji sinteze i sekrecije ovih proteina. |
| Sažetak (engleski) | The aim: Multiple myeloma (MM) is B-cell neoplasm characterized with multifocal
malignant plasma cell bone infiltration, while the molecular basis of disease remains
unclear. Angiogenesis, biological phenomenon of new blood vessel formation is
present in different plasma cellular disorders and is of importance in active MM.
Because of that the inhibition of tumour angiogenesis has shown to be promising
target in myeloma therapy. The aim of this study was to determine angiogenesis and
expression of vascular endothelial growth factor (VEGF), monocyte chemotactic
protein (MCP-1) in relation to nuclear factor kappa B (NF-kB) on molecular level,
cellular level as well as in plasma of MM patients.
Patients and Methods: The study was conducted on 79 patients with diagnosed MM
including clinical parameters of stage, therapy response and overall survival. The
bone marrow biopsy specimens were used for double immunostaining method for
detection of mean vascular density (MVD), Ki67 proliferation index (PI), VEGF, MCP-
1 and subcellular localization of NF-kB. Plasma samples obtained from 19 patients
were used for detection of VEGF and MCP-1 using ELISA method, while the bone
aspirates were used for magnetic cell labeling or flow cytometry separation in order
to separate myeloma cells and determine mRNA VEGF and MCP-1 by RT-PCR.
Results: The results have shown positive correlation of MVD, % of bone marrow
tumour infiltrate (p<0,001), atypical plasma cell morphology (p=0,001) and clinical
stage (p=0,001) the patients were in. Further, there was significant correlation of MVD and poor therapy response (p=0,029) as well as shorter survival (p=0, 04) when
high MVD values were observed. The VEGF expression was significantly lower when
MVD was higher (p=0,002). The lower expression of both cytokines VEGF (p =0,006)
and MCP-1 (p =0, 03) was related to higher NF-êB expression. The higher plasma
concentrations of VEGF compared to the control group were observed (p=0.008).
Concentration of both cytokines VEGF (p=0.05) and MCP-1(p=0.05) correlated with
% of tumour cell infiltrate (p=0.05), while VEGF correlated with the clinical stage
(p=0,001), respectively. Values of mRNA VEGF and MCP-1 showed lack of
significance when compared with any of parameters in the study.
Conclusions: The results of this study confirmed correlation of angiogenesis and poor
prognostic parameters in myeloma as well as shorter survival observed. The
concentrations of cytokines in blood, VEGF and MCP-1 are in proportion with tumour
mass and could be important clinical parameters which remain to be confirmed in
further studies. Observed inverse correlation of NF-kB in myeloma cells and
angiogenic cytokines does not rule out the possibility of their mutual connection in
regulation of synthesis and secretion of these proteins. |
