Advancements in prognostication have improved the subdivision of chronic lymphocytic leukaemia (CLL) into diverse prognostic subgroups. In CLL, IGHV unmutated and IGHV3-21 genes are associated with a poor-prognosis, conversely, IGHV mutated genes with a favourable outcome. The finding of multiple CLL subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has suggested a role for antigen(s) in leukemogenesis. Patients belonging to certain stereotyped subsets share clinical and biological characteristics, yet limited knowledge exists regarding the genetic and epigenetic events that may influence their clinical behaviour. This thesis aimed to, further investigate Swedish IGHV3-21-utilising patients, screen for genetic and DNA-methylation events in CLL subgroups/subsets and study DNA methylation over time and within different CLL compartments. In paper I, IGHV gene sequencing of 337 CLL patients from a Swedish population-based cohort revealed a lower (6.5%) IGHV3-21 frequency relative to previous Swedish hospital-based studies (10.1-12.7%). Interestingly, this frequency remained higher compared to other Western CLL (2.6-4.1%) hospital-based cohorts. Furthermore, we confirmed the poor-outcome for IGHV3-21 patients to be independent of mutational and stereotypy status. In paper II, genomic events in stereotyped IGHV3-21-subset #2, IGHV4-34-subset #4 and subset #16 and their non-stereotyped counterparts were investigated via SNP arrays (n=101). Subset #2 and non-subset #2 carried a higher frequency of events compared to subset #4. A high frequency of del(11q) was evident in IGHV3-21 patients particularly subset #2 cases, which may partially explain their poor-prognosis. In contrast, the lower prevalence of aberrations and absence of poor-prognostic alterations may reflect the inherent low-proliferative disease seen in subset #4 cases. In papers III and IV, differential methylation profiles in IGHV mutated and IGHV unmutated patients were identified using DNA-methylation microarrays. CLL prognostic genes (CLLU1, LPL), tumor-suppressor genes (TSGs) (ABI3, WISP3) and genes belonging to TGF-ß and NF-kB/TNFR1 pathways were differentially methylated between the subgroups. Additionally, the re-expression of methylated TSGs by use of methyl and deacetyl inhibitors was demonstrated. Interestingly, analysis of patient-paired diagnostic/follow-up samples and patient-matched lymph node (LN) and peripheral blood (PB) cases revealed global DNA methylation to be relatively stable over time and remarkably similar within the different compartments. Altogether, this thesis provides insight into the aberrant genomic and DNA methylation events in divergent CLL subgroups. Moreover this thesis helps distinguish the extent to which DNA methylation changes with respect to time and microenvironment in CLL.

Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) VH genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated VH genes display superior survival compared to unmutated cases. Biased VH gene usage has also been reported in CLL which may reflect antigen selection. We performed VH gene analysis in 265 CLL cases and confirmed the prognostic impact of the VH mutation status. Preferential VH gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular VH gene, VH3-21, displayed poor outcome despite that two-thirds showed mutated VH genes. Many of the VH3-21 cases expressed λ light chains, rearranged a Vλ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the VH3-21 subset comprises an additional CLL entity. To further explore the B-cell receptors in CLL, we analyzed the VH gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted VL gene usage as in the VH3-21-using group. Our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of CLL. Different cutoffs have been suggested to distinguish mutated CLL in addition to the 2% cutoff. Using three levels of somatic mutations, i.e. <2%, 2-5% and >5%, we divided 323 CLLs into subsets with divergent survival. This division revealed a low-mutated subgroup (2-5%) with inferior outcome that would have been masked using the traditional 2% cutoff. A 1513A/C polymorphism in the P2X7 receptor gene was reported to be more frequent in CLL, but no difference in genotype frequencies was revealed in our 170 CLL cases and 200 controls. However, CLL cases with the 1513AC genotype showed superior survival than 1513AA cases and this was in particular confined to CLL with mutated VH genes. In summary, we could define new prognostic subgroups in CLL using Ig gene rearrangement analysis. This also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of CLL.

Η αναιμία σχετιζόμενη με τη νόσο στη χρόνια λεμφογενή λευχαιμία (ΧΛΛ) είναι διάγνωση εξ΄ αποκλεισμού και διαπιστώνεται στις περιπτώσεις που δεν ανευρίσκεται άλλο εμφανές αίτιο αναιμίας. Δεδομένου ότι η παθογένεια της αναιμίας αυτής δεν είναι διευκρινισμένη, μελετήσαμε διάφορες παραμέτρους της ερυθροποίησης σε πρωτοδιαγνωσθέντες ασθενείς με ΧΛΛ και αναιμία σχετιζόμενη με τη νόσο και τις συγκρίναμε με τα αντίστοιχα ευρήματα ασθενών με ΧΛΛ χωρίς αναιμία και χωρίς να έχουν λάβει θεραπεία και με αυτά των υγιών μαρτύρων. Η διήθηση του μυελού των οστών από τα κακοήθη Β-λεμφοκύτταρα δεν ήταν αποκλειστικά υπεύθυνη για την πρόκληση αναιμίας. Τα CD34+ κύτταρα του μυελού των οστών στη ΧΛΛ δεν εμφάνισαν ενδογενή διαταραχή στη στροφή και περαιτέρω διαφοροποίησή τους προς την ερυθρά σειρά. Επιπλέον, δε διαπιστώθηκαν ανεπαρκή επίπεδα ερυθροποιητίνης ορού ούτε ελαττωματική απάντηση των ερυθροποιητικών προδρομικών κυττάρων στην ΕΡΟ στον άξονα ΕΡΟ-ΕΡΟ υποδοχέα κατά τη διέγερσή τους με ΕΡΟ ± TNF-α. Από την άλλη μεριά, τα επίπεδα του TNF-α βρέθηκαν αυξημένα στους ασθενείς με ΧΛΛ και αναιμία σχετιζόμενη με τη νόσο και φάνηκε ο TNF-α να ασκεί άμεση κατασταλτική δράση στη διαφοροποίηση των CD34+ κυττάρων προς κύτταρα της ερυθράς σειράς in vitro. Η μελέτη μας έδειξε ότι η αναιμία σχετιζόμενη με τη νόσο στη ΧΛΛ δεν οφείλεται σε ενδογενείς διαταραχές των ερυθροποιητικών προδρομικών κυττάρων, αλλά πιθανότατα να αποδίδεται στην απευθείας κατασταλτική επίδραση του TNF-α . / Disease-related anemia in chronic lymphocytic leukemia (CLL) occurs when the obvious causes are excluded while its pathogenesis is still obscure. To investigate its underlying mechanisms we studied parameters of erythropoiesis at cellular and molecular level in newly diagnosed CLL patients with disease-related anemia in comparison with those of non-anemic CLL patients and normal controls. Bone marrow (BM) infiltration by leukemic B cells was not exclusively responsible for the presence and the severity of disease-related anemia and BM CD34+ cells were intrinsically capable of generating erythroid precursors. No deficiency of serum erythropoietin (EPO) or defective intracellular response of erythroid precursors to EPO ± Tumor Necrosis Factor-α (TNF-α) stimulation was also observed. On the other hand, serum TNF-α levels were found increased in patients with CLL and disease-related anemia and TNF-α appeared to exert a direct inhibitory effect on the differentiation of CD34+ cells towards the erythroid lineage in vitro. Our study showed that disease-related anemia in CLL was not due to intrinsic defects of erythroid precursors, but might result from the direct suppressive effect of TNF-α on the erythroid production.

Monoclonal B lymphocytosis (MBL) has been recognized as a novel diagnostic condition. This study aims at the identification of clonal lymphocytosis in the patients with asymptomatic lymphocytosis. A total of 203 patients were evaluated for clonal B and T lymphocytosis by using flow cytometry and multiplex-PCR. Among them clonal B- or T-cells were detected in 54.2% of the cases, of which 38.4% were clonal B-cells and 15.8% were clonal T-cells cases. By immunophenotype, MBL was classified into the chronic lymphocytic leukemia (CLL) type (21.7%) and non-CLL-type (7.4%). Flow cytometry analysis and cell counts were used to determine the size of clonal population, and the data indicate that MBL and CLL are present in a continuous spectrum of clonal expansion. The findings may contribute to the current understanding of MBL and evaluation of incidental lymphocytosis. Further studies are required to evaluate clonal progression as a precursor stage of lymphoid malignancy.

To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.

Monoclonal B lymphocytosis (MBL) has been recognized as a novel diagnostic condition. This study aims at the identification of clonal lymphocytosis in the patients with asymptomatic lymphocytosis. A total of 203 patients were evaluated for clonal B and T lymphocytosis by using flow cytometry and multiplex-PCR. Among them clonal B- or T-cells were detected in 54.2% of the cases, of which 38.4% were clonal B-cells and 15.8% were clonal T-cells cases. By immunophenotype, MBL was classified into the chronic lymphocytic leukemia (CLL) type (21.7%) and non-CLL-type (7.4%). Flow cytometry analysis and cell counts were used to determine the size of clonal population, and the data indicate that MBL and CLL are present in a continuous spectrum of clonal expansion. The findings may contribute to the current understanding of MBL and evaluation of incidental lymphocytosis. Further studies are required to evaluate clonal progression as a precursor stage of lymphoid malignancy.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

Resumo: Leucemia Linfóide Crônica (LLC) é uma neoplasia maligna de linfócitos B maduros com aspecto monomórfico, que se acumulam em tecidos linfóides secundários, medula óssea e sangue periférico. O diagnóstico é realizado com base em achados clínico-morfológicos e imunofenotípicos. Os pacientes com LLC são estratificados conforme o estadiamento clínico para definição de terapêutica; atualmente a descoberta de marcadores prognósticos, como ZAP-70, trouxe novas perspectivas ao tratamento, principalmente no estádio precoce. Os objetivos deste estudo foram: avaliar a eficácia da pesquisa da ZAP-70 em biópsia de medula óssea (BMO) e em inclusão do coágulo, utilizando-se a técnica de imuno-histoquímica; avaliar possíveis correlações deste marcador na BMO com evolução clínica e risco de progressão; correlacionar a imuno-expressão da ZAP- 70 com o arcabouço reticulinico das BMO; avaliar se este arcabouço possui correlação com o prognostico da doença e, por fim, traçar um perfil epidemiológico dos pacientes com LLC atendidos no Hospital das Clinicas da Faculdade de Medicina de Botucatu (FMB). Foram selecionados 153 pacientes com LLC, atendidos no ambulatório do Serviço de Hematologia da FMB-UNESP, de 1980 a 2008, e 9 pacientes assistidos no Serviço de Onco-Hematologia do Hospital Amaral Carvalho-Jaú, no período de 2000 a 2008, com o mesmo diagnóstico, perfazendo 162 casos. Destes, 79 possuíam prontuários que foram revisados, bem como dados morfológicos da BMO e AMO. Foi realizada a pesquisa da ZAP- 70 pela técnica de imuno-histoquímica nas amostras parafinadas de BMO e inclusão do coágulo. Observou-se que 55,7% dos pacientes eram do sexo masculino; 86,1% de etnia branca; a mediana de idade foi 65 anos; relação homem:mulher de 1,2:1. 40,5% dos pacientes tiveram diagnóstico por achado incidental e 73,4%, já com adenopatia secundária à admissão. 17,2% ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chronic lymphocytic leukemia (CLL) is a malignant neoplasm consisting of mature monomorphic B lymphocytes that accumulate in secondary lymphoid tissues, bone marrow and peripheral blood. Diagnosis is based on clinical, morphological and immunophenotypic findings. CLL patients are categorized according to clinical aspects for treatment management and currently, certain tools, such as prognostic marker ZAP- 70, have determined new treatment perspectives, especially for patients in early stages. The study aimed to evaluate the efficiency of ZAP-70 investigation in bone marrow biopsies (BMBs) and inclusion coagulate using immunohistochemistry; identify possible correlations of this marker in clinical evolution and risk of progression; correlate the immunoexpression of ZAP-70 with BMB reticulin network; evaluate whether this network correlates with disease prognosis and; outline an epidemiological profile of patients with CLL attended in the Clinics Hospital of Botucatu Faculty of Medicine (FMB). The sample consisted of 153 CLL patients attended at hematology outpatient clinic of the FMB-UNESP, from 1980 to 2008, and 9 CLL patients attended at the oncohematology service of the Amaral Carvalho Hospital, Jaú, from 2000 to 2008, totaling 162 cases. In 79 cases, medical records including BMB and bone marrow aspirate (BMA) morphological data were reviewed. Immunohistochemistry for ZAP-70 was conducted on paraffinated samples of BMB and inclusion coagulate. Observation revealed that: 55.7% were male, 86.1% white, with a median age of 65 years-old; 40.5% of cases were diagnosed by incidental findings in routine blood smears; 73.4% were diagnosed with peripheral adenomegaly on admission. 17.2% were diagnosed in the early stages and 15% of cases showed prolonged remission. BMB and BMA morphology revealed that 94.8% of patients presented increased cellularity... (Complete abstract click electronic access below) / Orientador: Lígia Niéro-Melo / Coorientador: Maria Aparecida Custódio Domingues / Banca: Lucilene Silva Ruiz e Resende / Banca: Lisandro Ferreria Lopes / Mestre

The estrogen receptor alpha (ERa) and estrogen receptor beta (ER~) have been demonstrated to be important for immune system regulation and studies have suggested an antiproliferative effect of ER~ in lymphoid malignancies. We have studied the expression of ERa and ER~ in peripheral blood mononuclear cells from patients with chronic lymphocytic leukemia (CLL). Expression of ERa was low, while ER~ was highly expressed in CLL cells. In order to investigate the possible inhibitory effect of ligand-activated ER~ , we treated CLL cells and Mecl cell lines with the selective ER~ agonist diarypropionitrile (DPN) in culture. Treating Mecl cell lines with DPN showed an antiproliferative effect of ER~ agonist by significantly inhibit the growth of Mec 1 cell lines. This suggests that ER~ agonist may be useful in the treatment of CLL.

Chronic lymphocytic leukemia (CLL) cells are considered to be derived from antigen-exposed B cells. To further explore the antigen-driven selection behind the leukemogenesis of CLL, we performed immunoglobulin (Ig) specificity screening of 7 CLL cell lines and 23 primary CLL clones from patient peripheral blood. We also included a recombinant monovalent monoclonal antibody (mAb) belonging to a subset of CLL cases with identical or semiidentical heavy chain complementarity determining region 3 (HCDR3) of the IGHV3-21 gene rearrangement. We found CLL mAb specificities against vimentin, filamin B, cofilin-1, proline-rich acidic protein 1, cardiolipin, oxidized low density lipoprotein and Streptococcus pneumoniae polysaccarides. These molecules are functionally associated with microbial infection and/or apoptotic cell removal. An antigen-driven selection would therefore imply that CLL B cell precursors are involved in the elimination and scavenging of pathogens and apoptotic cells, which could trigger the development of the disease. The limited in vitro survival of CLL cells makes Epstein-Barr virus (EBV) immortalization of CLL cells a useful experimental model for studies on antibody-specificity screening. Considering the intricate procedure of EBV transformation of CLL cells and the many false cell lines used worldwide, we also wanted to characterize and evaluate the authentic origin of several previously established CLL cell lines and their normal lymphoblastoid counterparts. Three of the CLL cell lines tested were truly authentic (I83-E95, CLL-HG3 and CII), two had features of a biclonal Ig expression (232B4 and WaC3CD5+), one was only tentatively verified (PGA-1), whereas one cell line could not be verified (EHEB) due to lack of original patient cells for comparison. Two of the presumed normal lymphoblastoid cell lines tested were shown to be a neoplastic CLL clone. This study emphasizes the importance of proper cell line authentication and we will continue to verify additional cell lines not yet proven authentic. In conclusion, we provide evidence for natural Ab production by CLL cells and suggest that these cells might be derived from B cell precursors involved in the innate immunity and, thus, providing a first-line-defence against pathogens and in elimination of apoptotic cells.