Odnos faktora rasta i diferencijacije-15 i hepcidina sa biomarkerima metabolizma gvožđa u limfoproliferativnim bolestima

Abstract

UVOD: Limfoproliferativne neoplazme nastaju klonskom proliferacijom ćelija limfoidne loze različitog stepena zrelosti. Agresivne limfoproliferativne neoplazme se leče odmah po postavljanju dijagnoze i cilj je postizanje kompletne remisije. Lečenje indolentnih limfoproliferativnih neoplazmi, ako je oboleli bez simptoma i velike tumorske mase treba odložiti uz redovno kliničko praćenje, režim poznat kao „watch and wait“. Ako je anemija u limfoproliferativnoj bolesti umerena ili teška i posledica limfoproliferacije onda je i indikacija za započinjanje terapije. Serumske koncentracije biomarkera metabolizma gvožđa se redovno određuju u sklopu dijagnostičkog algoritma anemije. Glavni regulator metabolizma gvožđa je hepcidin na čiju regulaciju utiče eritropoetska aktivnost, putem eritroferona, ali i faktora rasta i diferencijacije-15 (GDF-15). GDF-15 ima ulogu u različitim metaboličkim i imunološkim procesima. Ispitivan je u brojnim malignitetima i povezan sa godinama i mortalitetom bilo kog uzroka. Sem u multiplom mijelomu nema rezultata ispitivanja uloge GDF-15 u drugim limfoproliferativnim neoplazmama. CILJ: Ispitati cirkulišući nivo biomarkera metabolizma gvožđa, parametre kompletne krvne slike i utvrditi njihovu vezu s hepcidinom i GDF-15 kod obolelih od zrelih B limfoproliferativnih neoplazmi, kao i ispitati povezanost biomarkera metabolizma gvožđa, hepcidina i GDF-15 sa standardnim laboratorijskim pokazateljima inflamatorne i aktivnosti osnovne bolesti. Pored toga, utvrditi postoji li razlika u koncentraciji parametara metabolizma gvožđa, hepcidina i GDF-15 između obolelih koji imaju indikaciju za lečenje i obolelih na „watch and wait“ režimu, kao i ispitati korelaciju koncentracija biomarkera metabolizma gvožđa, hepcidina i GDF-15 s postignutim terapijskim odgovorom u grupi lečenih. MATERIJAL I METODE: Prospektivno istraživanje obuhvatilo je 71 ispitanika: 40 u grupi lečenih od agresivnih i indolentnih limfoproliferativnih neoplazmi s indikacijom za terapiju i 31 u „watch and wait“ grupi. U grupi lečenih analize su rađene u tri navrata: pre terapije, tokom terapije, na prvoj reviziji i posle terapije kod procene terapijskog odgovora. U „watch and wait“ grupi materijal za analize je uzorkovan dva puta u razmaku od 3 do 4 meseca. Sve analize su rađene po standardnoj laboratorijskoj proceduri, a hepcidin i GDF- 15 su određivani nakon odmrzavanja iz prethodno zamrznutih seruma čuvanih na -700C, ELISA metodom. Terapijski odgovor je procenjivan u skladu s Lugano klasifikacijom. Analiza podataka je izvršena upotrebom statističkog paketa IBM SPSS Statistics 22, a obrada metodama deskriptivne i inferencijalne statistike. Svi testovi su dvostrani s nivoom značajnosti p˂0,05. REZULTATI: U grupi lečenih posle u odnosu na vreme pre terapije značajno je niži broj leukocita, limfocita i neutrofilnih granulocita (p˂0,001), niža je serumska koncentracija beta 2 mikroglobulina (p˂0,001) i hepcidina (p=0,01), a viša GDF-15 (p=0,027). U podgrupi lečenih s agresivnim limfoproliferativnim neoplazmama, nakon lečenja su značajno više koncentracije gvožđa (p=0,025), transferina (p=0,007), albumina (p=0,013), a niža je brzima sedimentacije (p=0,033). U podgrupi lečenih s indolentnim limfoproliferativnim neoplazmama po završetku lečenja značajno su više koncentracije feritina (p=0,009), fibrinogena (p=0,01), GDF-15 (p=0,028), a niže beta 2 mikroglobulina (p˂0,001) i hepcidina (p=0,002). Hepcidin značajno pozitivno korelira s feritinom pri svim merenjima u obe grupe (p˂0,001) i negativno s koncentracijom transferina u grupi lečenih inicijalno (p˂0,05) i pri sva tri merenja s indeksom feritina (p˂0,05). U grupi lečenih pre započinjanja terapije postoji značajna pozitivna korelacija koncentracije gvožđa, transferina i solubilnog transferinskog receptora (sTfR) sa albuminom (p˂0,05), a negativna s brzinom sedimentacije (p=0,001) i plazmatskom koncentracijom fibrinogena (p˂0,001). Posle terapije postoji značajna pozitivna povezanost koncentracije transferina i albumina (p=0,005) i značajna negativna korelacija koncentracije transferina s brzinom sedimentacije (p=0,026) i plazmatskom koncentracijom fibrinogena (p=0,011). Nakon terapije feritin je značajno povezan s koncentracijom LDH (p=0,022) i fibrinogenom (p=0,043), a sTfR i GDF-15 s beta 2 mikroglobulinom(p=0,03). Statistički su značajno niže serumske koncentracije gvožđa (p˂0,001), kao i inicijalne koncentracije transferina (p˂0,001), a posle terapije značajno više vrednosti feritina (p=0,034) i sTfR (p=0,001) u grupi lečenih u odnosu na „watch and wait“ grupu . Postoji statistički značajna razlika (p˂0,05) u brzini sedimentacije i plazmatskoj koncentraciji fibrinogena u odnosu na terapijski odgovor. Najniže vrednosti su kod ispitanika s kompletnom remisijom, a najviše kod onih sa progresijom bolesti. ZAKLJUČAK: Nedostatak korelacije hepcidina s parametrima krvne slike u našem istraživanju posledica je većeg broja ispitanika bez anemije i s koncentracijom biomarkera metabolizma gvožđa u referentnom opsegu u obe grupe, na šta su uticali strogi kriterijumi isključivanja s ciljem da se dobijeni rezultati povežu isključivo s limfoproliferativnim neoplazmama. Serumske koncentracije gvožđa su značajno niže kod obolelih od agresivnih u odnosu na obolele od indolentnih limfoproliferativnih neoplazmi, takođe i značajno niže kod obolelih od indolentnih limfoproliferacija koji imaju indikaciju za terapiju u odnosu na one koji je nemaju. Pozitivna korelacija feritina sa laktat dehidrogenazom i fibrinogenom i negativna korelacija serumske koncentracije transferina s koncentracijom CRP, fibrinogenom i brzinom sedimentacije u grupi ispitanika koji imaju indikaciju za lečenje mogla bi biti osnova za dalje istraživanje prognoznog značaja ovih biomarkera kod obolelih od LPN kod kojih je indikovana terapija. Statistički signifikantna korelacija GDF-15 s beta 2 mikroglobulinom, pre i posle terapije i fibrinogenom pre terapije ukazuje na moguće mesto ovog faktora u proceni rizika kod obolelih od indolentnih limfoproliferativnih neoplazmi kod kojih je indikovano lečenje. Ne postoji značajna povezanost koncentracije feritina, hepcidina i GDF-15 sa standardnim markerima aktivnosti LPN u grupi ispitanika kod kojih nije indikovano lečenje.

INTRODUCTION: Lymphoproliferative neoplasms arise from the clonal proliferation of malignant lymphoid cells at different maturation stages. Treatment of aggressive lymphoma starts upon diagnosis and staging with the aim to achieve complete remission. Treatment of indolent lymphoproliferative neoplasms in the absence of symptoms and bulky mass should be postponed with careful follow up, an approach known as „watch and wait “. Moderate or severe anemia due to indolent lymphoproliferative disease is an indication for lymphoma/leukemia treatment initiation. Concentrations of iron metabolism biomarkers in serum are an essential part in the diagnostic algorithm of anemia. Hepcidin is the main systemic regulator of iron metabolism which depends on the erythropoietic activity, via erythroferrone and the growth and differentiation factor-15 (GDF-15). GDF-15 has a role in different metabolic and immune states. It has been examened in numerous malignancies and has been linked to age and mortality of any cause Except in multiple myeloma there are no published data about the role of GDF-15 in other lymphoproliferative neoplasms. AIM: To investigate the relation between the circulating levels of iron metabolism biomarkers, complete blood count values in regards to hepcidin and GDF-15 levels in patients with mature B cell neoplasms as well as the relation of iron metabolism biomarkers, hepcidin and GDF-15 with standard laboratory markers of lymphoproliferative disease activity. To assess the differences between the circulating levels of those aforementioned parameters among patients who need lymphoma/leukemia treatment and those on “watch and wait” regimen and to find out if there is a correlation between iron metabolism biomarkers, hepcidin and GDF-15 levels and therapeutic response. MATERIAL AND METHODS: A prospective research included 71 patients, 40 in a group with aggressive or indolent lymphoproliferative neoplasms who needed and received treatment and 31 in “watch and wait” group. In treated patients laboratory analyses were performed before therapy, during treatment at 1st revision, and after, at treatment response evaluation. In the “watch and wait” group blood samples were taken on two separate occasions, 3 to 4 months apart. All analyses were done according to standard procedures, while ELISA technique from stored sera at -700C was used for hepcidin and GDF-15 level measurement. Treatment response evaluations were done according to Lugano criteria. Statistical analyses were performed by IBM SPSS Statistics 22 using methods of descriptive and inferential statistics. All tests are twotailed with significance level p˂0, 05. RESULTS: In the group of treated patients after receiving therapy, significantly lower white blood cells count, lymphocyte and neutrophil granulocyte count (p˂0,001), lower concentrations of β2microglobulin (p˂0,001) and hepcidin (p=0,01), and higher concentration of GDF-15 (p=0,027) were noticed when compared to prior treatment values. In the subgroup of patients treated for aggressive lymphoma after therapy, concentrations of iron (p=0,025), transferrin (p=0,007) and albumin (p=0,013) were significantly higher, while erythrocyte sedimentation rate (ESR) was lower (p=0,033). In the subgroup of patients treated for indolent lymphoproliferative neoplasms, significantly higher levels of ferritin (p=0,009) fibrinogen (p=0, 01) and GDF-15 (p=0,028) as well as lower concentrations of β2microglobulin (p˂0,001) and hepcidin (p=0,002) were observed after treatment completion. There is a positive correlation between hepcidin and ferritin in both groups (p˂0,001) in treated patients. A negative correlation exists between the levels of hepcidin and transferrin before treatment (p˂0, 05) as well as between hepcidin level and ferritin index (p˂0, 05) in treated patients. Also, in the group of treated patients before treatment there are positive correlation between iron, transferrin and soluble transferrin receptor (sTfR) levels and albumin (p˂0,05) while the level of the same iron metabolism biomarkers negatively correlate with erythrocyte sedimentation rate (p=0,001) and fibrinogen concentration (p˂0,001). After treatment completion transferrin positively correlates with albumin (p=0,005) and negatively with ESR (p=0,026) and fibrinogen concentration (p=0,011); ferritin positively correlates with lactate dehydrogenase level (LDH) (p=0,022) and fibrinogen (p=0,043), while sTfR and GDF-15 positively correlate with β2microglobulin (p=0, 03). Iron and transferrin concentrations before treatment are significantly lower in treated vs. „watch and wait “group (p˂0,001), while ferritin (p=0,034) and sTfR (p=0,001) concentrations are significantly higher in treated patients after therapy in comparison with those on “watch and wait“ regimen. ESR and fibrinogen level significantly correlate with treatment response; the lowest levels are in patients with complete response and highest in those with progressive disease. CONCLUSION: The vast majority of patients in both groups were non anemic with iron metabolism biomarkers within referent range, due to the strict exclusion criteria, as the aim was to link the findings exclusively to lymphoproliferative neoplasms, which in part led to the absence of correlation between hepcidin level and blood cell count. Serum iron concentration is lower in treated patients for aggressive than indolent lymphoma and also in patients with indolent lymphoproliferative diseases who need treatment than in those on „watch and wait” regimen. The positive correlation between ferritin levels, LDH and plasmatic fibrinogen concentration and the negative correlation of transferrin with ESR and fibrinogen which were found in treated patients might be the base for further investigation of the prognostic impact of those biomarkers in patients with lymphoproliferative neoplasms who need to be treated. Statistically significant correlation of GDF-15 with β2microglobulin and fibrinogen before and β2microglobulin after therapy suggests its possible role in the risk evaluation of patients with indolent lymphoproliferative neoplasms who need to be treated. There are no significant correlations between ferritin, hepcidin and GDF-15 levels with standard markers of disease activity in patients with lymphoproliferative neoplasms without indication for therapy.