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In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large ( 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

OBJECTIVES Morphology and cytogenetics are currently used to define prognosis in myelodysplastic syndromes (MDS). However, these parameters have some limits. Flow cytometry has been recently included in the diagnostic panel for MDS, and its prognostic significance is under evaluation. METHODS Marrow aspirates from 424 MDS patients were analyzed by flow cytometry to evaluate the impact of bone marrow cell immunophenotype on overall survival (OS) and leukemia-free survival (LFS). The immature compartment of myeloblasts was analyzed by the quantitative expression of CD34 (

T-cell antigen expression can be observed in B-cell non-Hodgkin lymphoma (B-NHL). Although CD5 is expressed in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, the presence of other T-cell antigens is less common. This article reports a retrospective multicenter analysis in which flow cytometry was used to evaluate aberrant CD8 expression on the pathologic B cells of 951 bone marrow samples from patients with various types of B-NHL. In a total of 18 patients, CD8 was coexpressed: 10 had B-CLL; 1, small lymphocytic lymphoma (SLL); 1, marginal zone lymphoma; 1, lymphoplasmacytic lymphoma; 2, diffuse large B-cell lymphoma; and 3, follicular lymphoma. There was a 1.89% overall frequency of CD8 coexpression in which B-CLL/SLL had a higher frequency (3.03%) than did the other B-cell neoplasms (1.18%). Most cases were characterized by a favorable outcome.

Abstract 4028 BACKGROUND. The corner stone of the WHO classification and prognostic scores of myelodysplastic syndromes (MDS) is the blast count in bone marrow. The standard cytology evaluation of at least 500 bone marrow cells is easy to perform, but some concerns arise about reproducibility of this method. Nowadays bone marrow trephine biopsy and flow cytometry are frequently considered for the diagnosis of MDS. However there is so far paucity of data comparing cytology, histology and flow cytometry in quantifying bone marrow blasts in order to differentiate non RAEB from BAEB-I and RAEB-II cases. AIM OF THE WORK. The Aim of the work was to analyse the differences and the prognostic impact of cytology, histology and flow cytometry in differentiating non RAEB from BAEB-I and RAEB-II. PATIENTS AND METHODS. Since 1999, clinical and laboratory data from 1256 new cases of MDS were prospectively recorded into the Piemonte MDS Registry. Blast count could be performed with the three different methods: BMC (bone marrow cytology) has been performed in 844 cases, BMH (bone marrow histology) in 874 cases, and BMF (bone marrow flow cytometry) in 636. In order to quantify blasts, immune-histochemistry evaluation of CD34+ cells was used in BMH, while both CD34+ and CD117+ cells were considered in BMF. Out of the total of the 636 patients analysed by BMF only 420 had an accurate and complete registration of CD34 and CD117 positivity and were considered for the present analysis. In two hundred and thirty six cases all three evaluations were contemporary available. The concordance of each diagnostic method with the others and their prognostic value were evaluated in both univariate and multivariate analyses. A comparison between BMC and BMH was available in 571 cases, between BMC and BMF in 228 cases, and between BMH and BMF in 279 cases. RESULTS. The disagreement in classifying patients as non-RAEB or RAEB-I or RAEB-II between BMC and BMH was 156/571 (27%), with BMH over-evaluating blasts in 114/571 cases (20%) and under-evaluating blasts in 42/571 cases (7%). The disagreement between BMC and BMF was 80/228 (35%), with BMF over-evaluating and under-evaluating blast percentage in comparison to BMC in 53/228 (23%) and in 27/228 (12%) cases respectively. The disagreement between BMH and BMF was present in 113/279 (41%), with BMF over-evaluating and under-evaluating blast percentage in comparison to BMH in 44/279 (16%) and in 69/279 (25%) cases respectively. In univariate analysis all three methods of quantifing blasts and differentiating non-RAEB from RAEB-I and RAEB-II retained an important prognostic value for both leukemic evolution and survival. However when the three models were tested in multivariate analysis in order to define the best predictor of leukemic evolution, BMC retained the best predictive value. CONCLUSIONS. When BMH or BMF are used instead of BMC in order differentiate non-RAEB from RAEB-I and RAEB-II, the shift to a different WHO category is evident in at least 30% of patients and BMH and BMF do not play the same role as BMC. BMC still remain the standard method to quantify blasts for classification and prognostic evaluation of MDS. Disclosures: Off Label Use: Lenalidomide in Mantle Cell Lymphoma. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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 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