Sažetak | Klinička i morfološka slika leukemijskih oblika kroničnih limfoproliferativnih bolesti (KLLPB) pokazuje značajnu heterogenost. Zbog nepredvidivog tijeka, dosadašnji klinički stadiji i drugi pokazatelji nisu mogli izdvojiti bolesnike visokog rizika za progresiju bolesti. Zbog toga je cilj ovog rada bio naći nove morfometrijske, proliferacijske i/ili kinetičke pokazatelje unutar različitih odjeljaka tumorske mase: koštane srži (KS), periferne krvi (PK) i limfnog čvora (LČ) koji će predvidjeti preživljenje i progresiju bolesti sa svrhom definiranja proliferacijsko kinetičkog indeksa (PKI). Uz to, ocijeniti dužinom preživljenja dijagnostičku i prognostičku vrijednost pokazatelja koje karakteriziraju podtip bolesti, kliničke stadije, tumorsku masu, vrijeme udvostručenja tumorske mase i broja limfocita u ukupnoj populaciji KLLPB i njenim podskupinama. Na osnovu proliferacijskih i kinetičkih pokazatelja ocijeniti pokušaj obrazloženja podrijetla i distribucije malignih limfatičnih stanica u različitim kliničkim i morfološkim entitetima. Ispitanici, materijal i metode. Istraživanje je obuhvatilo 155 ispitanika dijagnosticiranih kao KLLPB. Ukupno je analizirano 657 razmaza punktata KS (236), LČ (146) i PK (275). Analiza je obuhvatila morfometrijsku analizu, analizu osobina regije nukleolarne organizacije (AgNOR) te statičku DNA citometriju (ICM), ukupno 71895 stanica, na osobnom računalu “SFORM” tvrtke VAMSTEC, Zagreb. Za statističku obradu su korištene univarijatne i bivarijatne analize te Kaplan-Meierova metoda za analizu preživljenja (Statistika 7.1). Rezultati. Prognostički su se pokazali brojni klinički, hematološki, biokemijski i imunofenotipski pokazatelji, morfološke karakteristike diferencijalne krvne slike i koštane srži, faktori progresije bolesti te morfometrijski, proliferacijski i kinetički pokazatelji. Postavljena je teorija „začaranog kruga“ te hipoteza «single» i multiplih” «programiranih stopova» u nastanku tipičnih oblika leukemija i limfoma te subakutnih i subkroničnih leukemija. Poremećaj diferencijacije može nastati na bilo kojem stupnju, a različito mjesto “stopa” rezultira različitom morfologijom, različitim afinitetom prema akumulaciji u koštanoj srži, perifernoj krvi i limfnom čvoru. Novi pokazatelji modificirane analize diploidnog tipa histograma pokazali su se adekvatni za kinetičku analizu metodom statičke DNA citometrije. Isto kao i novoopisani tipovi AgNOR-a: homogeni, inhomogeni i prstenasti pokazali su statistički značajnu korelaciju s osobinama DNA histograma i morfometrijskim karakteristikama stanice i jezgre, kao i ulogu u preživljenju, tipu distribucije tumorske mase, biološkom ponašanju tumorske bolesti te morfološkim karakteristikama limfatičnih stanica u koštanoj srži i perifernoj krvi. Međusobnom analizom morfometrijskih, proliferacijskih i kinetičkih pokazatelja vidjelo se kako nisko proliferativne stanice imaju male homogene AgNOR-e te većinu stanica u vršku DNA histograma. Visoko proliferativne stanice su s inhomogenim AgNOR-ima, od kojih većina sadrži količinu DNA veću od stanica od u vršku ili patološke mitoze (DNA>4N) ili ih je veći broj stanica u S-fazi staničnog ciklusa. Negdje u sredini su srednje proliferativne stanice s prstenastim AgNOR-ima. Analizom u različitim odjeljcima tumorske mase vidljivo je kako limfatične stanice koje imaju afinitet prema akumulaciji u koštanoj srži („oštećeni“ naivni B-limfociti) u pravilu imaju malu proliferativnu aktivnost (najveći postotak stanica u vršku Go/G1 faze). Stanice koje su „programirane“ i imaju afinitet prema akumulaciji u limfne čvorove, migriraju u limfne čvorove gdje se transformiraju do stupnja „programiranog stopa“ (veći broj ukupnih AgNOR-a, veća površina proliferativnijih, inhomogenih AgNOR-a i najmanji postotak stanica u Go/G1 fazi). Sama migracija stanica iz koštane srži prema limfnim čvorovima te između limfnih čvorova se događa u perifernoj krvi (mješavina stanica s nižom i višom proliferativnom aktivnosti: viši postotak stanica u S-fazi i istodobno u Go/G1 fazi staničnog ciklusa kao i prisustvo većih stanica, ali manjih jezgara, s prstenastim AgNOR-ima intermedijalnog stupnja proliferativnosti). Izgleda kako periferna krv ima samo transportnu ulogu. Analizirajući veličinu stanica i njihovu proliferativnu aktivnost u različitim odjeljcima tumorske mase uočena je pravilnost unutar ukupne populacije KLLPB te podskupina BKLL+ V i tipičnih B-KLL-a. Dok se stanice u KS i PK ne razlikuju bitno po veličini i proliferativnoj aktivnosti, obrnuta je situacija ako se gleda periferna krv i limfni čvor. Nasuprot očekivanju, kako su male stanice mirnije, a veće proliferativnije, analiza je pokazala da su u PK stanice najveće i najmirnije, za razliku od limfnog čvora gdje su najmanje i najaktivnije. Na osnovu najreprezentativnijih pokazatelja AgNOR-a i DNA (vezanim za preživljenje) u različitim odjeljcima tumorske mase izračunat je „score“ za PKI, posebno za cjelokupnu populaciju KLLPB, a posebno za B-KLL+V. Statistički značajno bolju prognozu imali su bolesnici s KLLPB (p=0,00118) i u grupi B-KLL+V (p=0,03589) kad je PKI bio manji od 4. Zaključak. Uzorak samo iz periferne krvi nije reprezentativan i dostatan za istraživanja progresivnosti ili stabilnosti bolesti. Morfometrijska, proliferacijska i kinetička obilježja tumorskih stanica, po prvi put analizirana paralelno u različitim odjeljcima tumorske mase, jasnije obilježavaju procese širenja i progresivnosti bolesti. Morfometrijske karakteristike homogenih, inhomogenih i prstenastih AgNOR-a te karakteristike diploidnog tipa histograma potvrdili su hipotezu kako se i unutar relativno nisko malignih neoplazmi kao što je KLLPB i njene podskupine mogu izdvojiti prognostički lošije/bolje podskupine te predvidjeti tijek bolesti. |
Sažetak (engleski) | The clinical and morphological picture of the leukemic types of chronic lymphoproliferative disorders (CLLPD) shows considerable heterogeneity. Due to the unpredictable course of the disease, the clinical stages and other parameters used to date have been unable to identify patients at a high risk of disease progression. Therefore, the aim of the study was to find new morphometric, proliferative and/or kinetic parameters in different tumor mass compartments, i.e. bone marrow, peripheral blood and lymph node, which would be able to predict survival and disease progression, in order to define the proliferative kinetic index (PKI). In addition, the aim was to evaluate, in terms of survival, the diagnostic and prognostic value of the variables characterizing disease subtypes, clinical stages, tumor mass, time to tumor mass doubling, and lymphocyte count in total CLLPD population and its subgroups. Then, the proliferative and kinetic parameters were used in an attempt to assess the origin and distribution of malignant lymphatic cells in various clinical and morphological entities. Patients, materials and methods. The study included 155 patients diagnosed with CLLPD. A total of 657 puncture smears of bone marrow (n=236), lymph nodes (n=146) and peripheral blood (n=275) were analyzed. The analysis consisted of morphometric studies, assessment of the nucleolar organization region (AgNOR) characteristics, and image cytometry (ICM), performed in 71895 cells on a SFORM PC (VAMSTEC, Zagreb). The univariate and bivariate analyses were used on statistical data processing, and Kaplan-Meier method for the analysis of survival (Statistica 7.1). Results. Prognostic potential was demonstrated for many clinical, hematological, biochemical, immunophenotypic parameters, morphological characteristics of differential blood count and bone marrow, factors of disease progression, and morphometric, proliferative and kinetic parameters. The "single" and "multiple” programmed stops in the development of typical forms of leukemias and lymphomas, subacute and subchronic leukemias were hypothesized. Differentiation impairment may occur at any stage, and different "stop" locations result in different morphology and affinity to accumulation in bone marrow, peripheral blood and lymph nodes. The new parameters of modified analysis of diploid histogram were found to be appropriate for kinetic analysis by the method of image DNA cytometry. Also, the newly characterized types of AgNOR points, homogeneous, inhomogeneous, and annular, yielded a statistically significant correlation with DNA histogram properties and morphometric characteristics of the cell and nucleus, and were additionally found to play a role in the survival, type of tumor mass distribution, biological behavior of tumor disease, and morphological characteristics of lymphatic cells in bone marrow and peripheral blood. Correlation analysis of the morphometric, proliferative and kinetic characteristics revealed the low proliferative cells to possess small, homogeneous AgNOR, with the majority of cells in the peak of DNA histogram. The high proliferative cells had inhomogeneous AgNOR, mostly containing greater DNA amount than peak cells, or pathologic mitoses (DNA >4N), or the majority of cells being in the S-phase of the cell cycle. Cells with medium proliferative activity and annular AgNOR were in-between. Analysis of different tumor mass compartments showed the lymphatic cells with affinity to accumulation in bone marrow ("damaged" naïve B-lymphocytes) to regularly exhibit low proliferative activity (a lower percentage of cells in SFC and highest percentage of cells in the peak of the G0/G1 phase). The "programmed" cells with affinity to accumulation in lymph nodes migrated to lymph nodes, where they transformed to the stage of "programmed stop", exhibiting the characteristics of proliferative cells (an increased number of AgNOR, larger more proliferative inhomogeous AgNOR and lowest percentage of cells in the G0/G1 phase). The migration of cells from bone marrow to lymph nodes and between lymph nodes occurs in peripheral blood (a mixture of cells with low and high proliferative activity: a higher proportion of cells in SFC and at the same time in the G0/G1 phase of the cell cycle). Analysis of cell size and proliferative activity in different compartments of tumor mass revealed a regular pattern within total CLLPD population, and in the subgroups of B-chronic lymphocytic leukemia with variants (B-CLL+V) and typical B-chronic lymphocytic leukemia (B-CLL). Whereas the cells in bone marrow and peripheral blood did not differ substantially according to size and proliferative activity, an inverse pattern was observed between peripheral blood and lymph node. As small cells are inactive and larger cells more proliferative, the analysis quite unexpectedly showed the peripheral blood cells to be largest and most inactive, in contrast to lymph node where the cells were smallest and most active. Based on the most representative AgNOR and DNA characteristics (related to survival) in various tumor mass compartments, the PKI score was calculated for the CLLPD population as a whole and for B-CLL+V in separate. The CLLPD (p=0,00118) and B-CLL+V (p=0,03589) patients had a statistically significantly better prognosis when score of PKI was less than 4. Conclusion. Peripheral blood sample is inadequate and not representative for the study of disease progression or stability. The morphometric, proliferative and kinetic properties of tumor cells, now for the first time analyzed simultaneously in different tumor mass compartments, provide better assessment of the disease dissemination and progression. The morphometric characteristics of homogeneous, inhomogeneous and annular AgNOR, and characteristics of diploid histogram have confirmed the hypothesis that prognostically unfavorable/favorable subgroups could be identified and the course of the disease predicted even within the groups of neoplasms of relatively low malignancy such as CLLPD with subgroups. |