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Peripheral blood (PB) T cells from 56 patients with B-cell chronic lymphocytic leukemia (B-CLL) were analyzed by two- and three-color immunofluorescence (IF) to determine the expansion of distinct T-cell subsets and their relationship with the clinical and biological features of the disease. We detected the expansion of an unusual T-cell subpopulation expressing lower CD4 or CD8 levels (CD4lo, CD8lo) than classic T cells (CD4hi, CD8hi). This subpopulation also expressed low levels of the CD3/TCR alpha/beta complex and was CD19-CD13-CD14-. A phenotypic analysis probing the activation level of CD4lo, CD8lo, CD4hi, and CD8hi cells showed that they comprised increased counts of HLA-DR+, CD11b+, CD45R0+, and CD45RA+ cells. Subset expansion ranged from 2.1- to 13.6-fold. Statistical analysis showed that the size of some of these subsets was correlated to intrinsic features of the tumor. First, CD4loHLA-DR+ cell counts were higher in patients with stage A than those with stages B and C disease. Second, CD8loHLA-DR+ cell counts were higher in patients in stable remission than in those at diagnosis. Third, CD4loHLA-DR+, CD4loCD45R0+, CD4loCD45RA+, and CD4hiCD11b+ cell counts were higher in patients whose tumor cells expressed high levels of surface immunoglobulin (sIg) than in those expressing low levels. The involvement of CD4lo and CD8lo cells in most of these correlations suggests that they may be tumor-reactive cells. Similar cells described in human and murine autoimmune disease have been shown to be autoreactive anergic cells, which may derive from nonclassic pathways of T-cell development.

Summary Argyrophilic nucleolar organizer region (Ag-NOR) analysis was performed on bone marrow biopsies from 90 patients with multiple myeloma (MM) at presentation. The pattern of AgNOR expression and its relationship to histological features were evaluated. The mean AgNOR number per plasma cell was directly correlated with the degree of MM differentiation (3.18 for G1, 4.36 for G2, 6.13 for G3;P<0.0001), with the per-centage of bone marrow plasma cells (BMPC%) (3.06 for BMPC%=20, 4.28 for BMPC% 21–50, 5.14 for BMPC%>50;P<0.0001), with the pattern of medullary involvement (3.63 for interstitial, 4.44 for nodular, 5.17 for diffuse involvement;P<0.001) and with medullary fibrosis (5.23 for cases with fibrosis, 4.29 for cases without fibrosis;P<0.05). The plasma cells of G1 MM showed 2–3 large AgNORs, tightly grouped in a central nuclear cluster; those of G2 MM showed a central nuclear cluster composed of 4–5 medium-size dots and/or two clusters of 2–3 dots; the G3 MM plasma cells showed many small dots scattered in the nucleolus or dispersed in the nucleus. Our results indicate the diagnostic value of AgNOR analysis in MM and suggest the use of this method for identifying clones of atypical plasma cells with different proliferative activity in bone marrow biopsies. It allows simultaneous evaluation of the morphology and kinetics of MM cells in routinely fixed, decalcified, paraffin-embedded material.

Background. Recent studies suggest that Flow Cytometry (FCM) might provide information useful in managing Myelodysplastic Syndromes (MDS) patients. Aim of the work. The purpose of this work is to describe the prognostic relevance of FCM analysis on a cohort of 303 myelodysplastic patients (64 RA; 15 RARS; 59 RCMD; 12 5q-MDS; 20 U-MDS, 72 RAEB-I, 44 RAEB-II, 17 LMMC), registered in the Multicenter Piedmont MDS Registry. Patients, materials, and methods. Marrow of 303 patients was studied by seven local FCM laboratories, using monoclonal antibodies against: CD3, CD8, CD4, CD16, CD19, CD34, CD117, CD33, CD14, CD11b, CD66b, HLADR, GPA and CD45. Myeloid maturation was evaluated according to the expression of both CD11b and CD66b antigens. Immature compartment of myeloblasts was analysed with CD117 and CD11b−CD66b−, and myeloid maturation with CD11b−CD66b+ (promyelocytes), CD11b+CD66b++ (myelocytes and metamyelocytes), CD11b+CD66b+ (neutrophils and bands). This definition of myeloid subpopulations was confirmed by sorting and by citospin. The overall survival (OS) and Leukemia-Free Survival (LFS) was calculated from the time of diagnosis until death or until leukaemia transformation. Survival curves were obtained by the Kaplan-Meier method and compared by the log-rank test. To evaluate the impact of FCM parameters on OS and LFS, we determined quantitative expression of the following antigen: CD117 and CD11b−CD66b− (≤5% vs >5%); CD11b−CD66b+ (≤12% vs >12%); CD11b+CD66b++ (≤15% vs >15%); CD11b+CD66b+ (≤25% vs >25%). Results. In our patients, quantitative expression of CD117 correlated with a shorter OS and LFS: the 40 months-OS was 66,5% versus 26,4% (p<0.00001) while LFS was 91.6% versus 27.6% (p<0.00001) (Fig 1). Similarly OS and LFS decreased when CD11b−CD66b− (myeloblasts) increased. Conversely, we observed a higher OS and LFS in patients with a high percentage of subpopulations CD11b+CD66++ and CD11b+CD66+ (Table 1). Discussion. Cytometric recognitions of dysplasia is difficult to perform, as it requires subjective interpretation by a skilled pathologist. This study emphasizes that FCM analysis of myeloid maturation, expressed as percentage of positive cells, may provide meaningful prognostic information. Both blast percentage expressed as CD117+ or CD11b−CD66b− and the quantitative assessment of myeloid lineage maturation showed, in our experience, a high prognostic value. These FCM parameters should be proposed as a prognostic tool easy to reproduce and perform. Results of quantitative FCM markers FCM variable Cut off 40 months-OS p 40 months-LFS p CD117+ ≤5% vs >5% 66.5% vs 26.4% 0.00001 91.6% vs 27.6% 0.00001 CD11b−CD66b− ≤5% vs >5% 71.5% vs 45.9% 0.0002 90.8% vs 51.3% 0.00001 CD11b−CD66b+ ≤12% vs >12% 49.8% vs 58.1% NS 80.7% vs 76.6% NS CD11b+CD66++ ≤15% vs >15% 33.0% vs 63.3% 0.001 58.9% vs 87.5% 0.002 CD11b+CD66b+ ≤25% vs >25% 30.3% vs 64.3% 0.00003 53.3% vs 76.6% 0.0008 Leukaemia transformation free Survival (Kaplan-Meier) ○ Complete + Censored Leukaemia transformation free Survival (Kaplan-Meier) ○ Complete + Censored

Background. Recent studies suggest that Flow Cytometry (FCM) might provide information useful in managing Myelodysplastic Syndromes (MDS) patients.

BACKGROUND. TheWHO classification of myelodysplastic syndromes (MDS) is based on the evaluation of bone marrow morphology. The two categories of REAB-I and RAEB-II are apparently easy to differentiate on the basis of bone marrow blast percent. However there are no so far data about the differences among cytology, histology and immunophenotypic evaluation of blasts in order to discrimante non-RAEB from RAEB-I and RAEB-II categories. PATIENTS AND METHODS. The Piemonte MDS Registry was born in 1999 thanks to the cooperation of both Haematology and Internal Medicine departments of our region, with the following aims: a) to follow homogeneous guidelines in diagnosis and treatment of MDS; b) to collect epidemiological and clinical information on a large group of patients; c) to cryopreserve bone marrow cells for molecular biology studies. When obtaining an informed consent, data of patients were prospectively centrally recorded through our web site. A retrospective analysis on differences in diagnosing RAEB, comparing conventional cytology on bone marrow smears (CBM), histochemical evaluation of CD34+ cells on bone marrow trephine biopsy (HBM), and cytofluorimetric count of CD34+ and CD117+ cells (IBM) has been done. RESULTS. From June 1999 to December 2003, 633 MDS patients were registered from 37 different institutions: 364 (57%) from haematology and/or academic institutions and 269 (43%) from internal medicine departments of community hospitals. Mean age was 72 (range 23–69). The actual diagnostic distribution of cases according to the WHO criteria based on only morphology evaluation of bone marrow smears was: non-RAEB 429 (68%), RAEB-I 134 (21%), and RAEB-II 70 (11%). Information about the quantification of blasts with both CBM and HBM techniques was avilable in 243 cases. An IBM evaluation was also available in 89 out of this 243 cases. A disagreement between CBM and HBM was evident in 65/243 cases (27%), with HBM over-evaluating and under-evaluating WHO class on the basis of blasts count in 54/243 (22%) and 11/243 cases respectively. When comparing CBM and IBM the disagreement was even higher in 29/89 cases (33%), with IBM over-evaluating blast percent in 9 (10%) and under-evaluating it in 20 cases (23%). The disagreement betwen HBM and IBM was maximum with a value of 39%. The role of CBM in predicting a different prognosis of non-RAEB, RAEB-I and RAEB-II was confirmed. However, when comparing the prognostic value of the three different methods of computing bone marrow blasts, IBM was the best in order to define the good prognostic non-RAEB group. CONCLUSIONS. The distinction among non-RAEB, RAEB-I and RAEB-II is far from beeing highly accurate and reproducible. Important differences are present among CBM, HBM and IBM. While CBM remain the conventional standard system, IBM could offer a tool better and more reproducible than CBM in order to define MDS categories on the basis of blast percentage. A large multicenter study could be useful in order to clarify this point.

BACKGROUND. TheWHO classification of myelodysplastic syndromes (MDS) is based on the evaluation of bone marrow morphology. The two categories of REAB-I and RAEB-II are apparently easy to differentiate on the basis of bone marrow blast percent. However there are no so far data about the differences among cytology, histology and immunophenotypic evaluation of blasts in order to discrimante non-RAEB from RAEB-I and RAEB-II categories.