Branko Vitale

Total Tumour Mass Score (TTM): a New Parameter in Chronic Lymphocytic Leukaemia

Summary. Total tumour mass score (TTM) is introduced as a new parameter in chronic lymphocytic leukaemia (CLL) in order to assess the tumour mass within all major body compartments. TTM is the sum of: (1) the square root of the number of peripheral blood lymphocytes per nl, (2) the diameter of the largest palpable lymph node in centimetres, and (3) the enlargement of the spleen below left costal margin in centimetres. The validity of the proposed scoring system was evaluated in a prognostic study on 256 CLL patients. Patients with high TTM (>9.0) at presentation had the expected median survival (EMS) of 39 months whereas patients with low TTM (<8.9) had EMS of 101 months (P< 0.0005). TTM was a significant prognostic factor even when adjustment was performed for age, sex, lymphocyte count, response to therapy, TTM‐distribution pattern and bone marrow failure. In contrast, the lymphocyte count was not prognostically significant when adjustment was performed for TTM. This suggests that TTM is a better indicator of tumour cell burden than the lymphocyte count. TTM measurement enables the analysis of the tumour mass size independently of other factors. TTM is a simple, objective parameter that can be measured as a continuous quantitative variable allowing derivation of new factors. TTM‐doubling time and TTM‐response to therapy were significant for prognosis (P< 0.0005). The proposed scoring system can also be used for the study of the tumour mass distribution pattern.

Phenotypic analysis of receptor-Ligand pairs on B-Cells in B-Chronic lymphocytic leukemia

Whole-blood three-color immunofluorescence analysis was used to investigate the role of CD5/CD72 and CD21/CD23 receptor-ligand pair formation on B-chronic lymphocytic leukemia (B-CLL) cells as well as sCD23 and bcl-2 oncoprotein expression in disease progression and activity and total tumor mass in B-cell chronic leukemia (B-CLL) patients. Thirty-four patients with B-CLL and 19 controls were included in the study. The majority of B-cells in B-CLL patients coexpressed CD5 and CD72 as well as the CD23 antigen. Unlike B-cells in B-CLL patients, B-cells in all healthy controls tested had high expression of CD21 antigen. We identified two groups of B-CLL patients according to high (n = 20) or low levels (n = 14) of CD21 expression on CD19+CD23+ B-cells. Only in the patients with high CD21 expression, were sCD23 levels positively correlated with factors known to have prognostic significance in B-CLL (Rai stage and TTM) and could, therefore, be used as a prognostic parameter for these B-CLL patients. Bcl-2 oncoprotein expression did not differ between these patient groups. We presumed that in patients with a lower expression of CD21 antigen, the contribution of the CD21 molecule to homotypic adhesion was lacking. Further studies are necessary to determine the possible association of higher expression of the CD21 antigen with disease progression and the aggressive character of the B-CLL.

Decision‐tree approach to the immunophenotype‐based prognosis of the B‐cell chronic lymphocytic leukemia

Use of a nonlinear prediction method, such as machine learning, is a valuable choice in predicting progression rate of disease when applied to the highly variable and correlated biological data such as those in patients with chronic lymphocytic leukemia (CLL). In this work, decision‐tree approach to cell phenotype‐based prognosis of CLL was adopted. The panel of 33 (32 different phenotypic features and serum concentration of sCD23) parameters was simultaneously presented to the C4.5 decision tree which extracted the most informative of them and subsequently performed classification of CLL patients against the modified Rai staging system. It has been shown that substantial correlation between the percentage of expression of the CD23 molecule on CD19+ B‐cells, the level of sCD23, the percentage of CD45RA+, and the absolute number of CD4CD45RA+RO+ T‐cells and the clinical stages, exists. The prediction vector, composed of their concatenated values, was able to correctly associate 83% of the cases in the low‐risk group (Rai stage 0), 100% of the cases in the intermediate‐risk group (Rai stage I and II), and 89% of the cases in the high‐risk group (Rai stage III and IV) of CLL patients. Predictivity of this vector was 100%, 95%, and 89%, respectively. In conclusion, from the described analysis, it may be inferred that two processes play important roles in the progression rate of CLL: 1. deregulated function of the CD23 gene in B‐cells accompanied by the appearance of its cleaved product sCD23 in the sera; and 2. functionally impaired and imbalanced CD4 T‐cell subpopulations found in the peripheral blood of CLL patients. Am. J. Hematol. 59:143–148, 1998.

Functional Differences of T cells in B-Chronic Lymphocytic Leukemia

B cell chronic lymphocytic leukemia (B-CLL) is a disease characterized by an accumulation of monoclonal lymphocytes of B cell origin. Although the neoplastic process involves the B lymphocyte compartment, phenotypic and functional defects within the T lymphocyte population implicate their possible role in the pathogenesis of the disease. We analyzed the functional and morphological integrity of T lymphocytes from the peripheral blood of 64 patients with B-CLL. The activation of B-CLL T cells after PHA stimulation was determined by measuring [3H]-thymidine incorporation, assessing cell numbers in parallel cultures, and by monitoring the lymphocyte subsets during 9 days of cultivation. Our results indicate the presence of three functionally different populations of T cells in the peripheral blood of B-CLL patients. We present evidence for an increased proliferative potential of T lymphocytes from a group of patients with B-CLL.

Prolegomenon for Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is a unique lymphoproliferative disorder that scarcely occurs under the age of 40; thereafter the incidence of CLL increases exponentially with age. CLL is characterized by progressive expansion of malignant CD5+ME+ B‐cell clone accompanied by a myriad of cellular and humoral immune defects. Each of them might be linked to different clinically manifested complications such as increasing rate of infections, autoimmune disorders and disturbed immune surveillance against tumour cells. We assume that CLL occurs as a consequence of age‐dependent, genetically related functional restrictions of the thymic microenvironment in supporting common lymphoid progenitor cells (CD5+ME+CD4–CD8–) to differentiate into mature T‐cell and B‐cell descendants. In conjunction with genetic abnormalities developing in B‐cell progenitors, presumably expressing P glycoprotein (Pgp+), we postulate that developmentally altered T‐cell descendants, along with quantitative imbalance among CD4+, their subsets and CD8+ lymphocytes in the peripheral blood, play an important additional role in facilitating the malignant B‐cell clone emergence and in modulating the CLL clinical evolution. Namely, imbalance of any of T‐cell‐mediated cell interactive homeostatic mechanisms accompanied by imbalance in the production of various cytokines might in CLL influence leukaemic B‐cell growth by deregulating inducer (c‐myc and p53) and/or suppressor (bcl‐2 and mutant p53) oncogenes responsible for the promotion or suppression of B‐cell mitogenesis that may in turn further contribute to their impaired differentiation and/or differentiation arrest. In conclusion, CLL might be interpreted as a primary immunodeficiency syndrome developing in elderly population due to gradually evolving restriction of genetically controlled programs in the thymic microenvironment responsible for irregular maturation of common lymphoid progenitor cells that constitutively express CD5 antigen and ME receptor into T‐cell and B‐cell descendants.

A NEW AVENUE FOR ASSESSING CARDIOVASCULAR RISK - HOMOCYSTEINE AS A RISK FACTOR

Background and aims: Mild hyperhomocysteinemia (>12 mM) is considered as a risk factor for many age-associated phenotypes, including: atherosclerosis, thrombosis, depression, neurodegenerative diseases and cancer. In parallel, it may be due many underlying causes, all impairments with increasing age, such as: B-vitamins deficiency, chronic gastritis, chronic renal failure, other chronic conditions and medical treatment, as well. The aim was to shed more light upon these complex relationships, involving hyperhomocysteinemia, and to suggest a new avenue for assessing cardiovascular risk. Methods: Total of 93 volunters, general medicine patients, 50-89 years old, were enrolled in the study. They were divided in two subgroups, depending on whether their homocysteine levels were above or below the median value (>11.8 mM). Data, such as: age, sex, physician`s diagnoses of the main diseases, antropometric measures, medical treatment, hemathological and biochemical tests, 40 parameters in total, were used to describe their health status. Data were analysed by using Data-Mining methodology, applicable to find patterns (models) in a Data-Base. Results: Six best descriptors (a cluster) of the health status of subjects with mild hyperhomocysteinemia were extracted. By additional models, derived from this basic one, cluster was further branched out. In such way, the pattern of disorders, linking: folic acid and vitamin B12 deficiency, low creatinine clearance, neuropsychiatric and gastroduodenal disorders, hypothyreoidism and hyperhomocysteinemia, could be constructed. Conclusion: This approach indicates that wider background of common aging disorders and clustering of data are to be considered when assess cardiovascular risk.

Transient early metabolic and functional hyperreactivity and late areactivity of lymphocytes in preleukemic AKR mice

The dynamics of changes in the metabolic and functional activities of thymus, lymph node and spleen lymphocytes and spleen macrophages of AKR mice was examined during the preleukemic period. The MTT colorimetric assay was used to determine the mitochondrial enzyme activity of viable cells, the local xenogeneic GVH reaction and the IL-2 assay for measuring T cell responses, and the IL-1 assay as an indicator of macrophage activity. In the early preleukemic period (at 1.5 months of age), lymphocyte dehydrogenase enzyme hyperactivity was accompanied by a highly increased production of IL-2, positive local xenogeneic GVH reaction and increased IL-1 production. Later on, at the age of 4–5 months, AKR mice demonstrated a progressive decrease in the metabolic activity of lymphocytes, negative local GVH reaction and reduction or lack in IL-2 and IL-1 production. This early hyperreactivity and late, gradually evolving, areactivity of lymphocytes and macrophages was not found in other, non-leukemic strains of mice (REM, CBA, C57BL).

An old and simple solution for a new problem--more on clinical staging and evaluation of response in B-cell chronic lymphocytic leukaemia in the era of new therapies.

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PO15-409 SOLUBLE ADHESION MOLECULES IN HYPERTENSION ARE REFLECTIVE OF PROFOUND PATHOGENESIS DISTURBANCE

Mediated by upregulated adhesion molecules on endothelial cells, mononuclear leukocytes participate in the formation of atherosclerotic plaques. Hypertensio is a major cardiovascular risk faktor. In this study, levels of soluble adhesion molecules E-selection and ICAM-1 Werw determined in a group of 79 subject with uncomplicated hypertensio and free of diabetes mellitus, aged 50-65 years, and 35 nonhypertensive subjects as the controlos. The relationships werw also assessed with parameters indicating: 1. risk factor (BMI, fasting glucose, total cholesterol and triglycerides), 2. markers of inflammation (total leukocyte count, C - reactive protein and red blood cell sedimentation rate) and 3. markers of renal dysfunction (24 h urine albumin, total protein in urine and creatinine clearance). Results showed that soluble adhesion molecules are not raised in hypertensive patients without manifest cardiovascular diseases, as comparedto the nonhypertensive controlos. Parameters selected in multiple regression analyses, performed for soluble E-selectin, were: BMI, triglicerides, total leukocytes count and 24 urine albumin – representing features of the insulinresistance syndrome. Parameters selected to independently correlate withthe plasma levels of soluble P-selection: red blood cell sedimentation rate, total leukocyte count and total cholesterol – indicate disturbed hemortheology. Soluble ICAM-1 showed correlations with C-reactive protein and markers of renal dysfunction (total protein in urine and reverse correlation with creatinine clearance), but only in univariate correlation with creatinine clearance), but only in univariate correlations. All this leads to the conclusion that the conclusion that the raised levels of soluble adhesion molecules may in hypertensive patients not be merely the consequence of high blood pressure, but rather reflect common underlying pathogenetic disturbances.