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We here describe 13 patients with non-Hodgkin's lymphoma (NHL) and a translocation t(11:14)(q13:q32). They were part of a series of 163 patients with NHL and an abnormal karyotype, serially referred to our institution between January 1984 and 1990. Patients with t(11:14) seem to present several common and interesting features. Males are more frequently affected than females, and old people more than young. They present at diagnosis with advanced disease and usually show involvement of epithelium and bone marrow. With respect to histologic diagnoses, these patients are usually considered to be of low-grade malignancies. However, most of them do very poorly, have short complete remission and frequent relapses whatever the treatment. As a whole, the median survival rate is rather low. The cytologic, histologic as well as the immunologic patterns tend to be uniform: tumours are composed of small cells and display features of mantle zone/intermediate lymphocytic lymphoma. They express high IgM and low IgD levels and more commonly bear Ig lambda light chains. They also express all pan-B antigens (except CD23) as well as the CD5 antigen, but usually lack the CD10. According to these characteristics, these tumours could be placed in between lymphocytic lymphomas (which usually express CD23) and follicular lymphomas (which commonly lack IgD and CD5 and bear CD10 as well as a t(14:18).

The possible role of T lymphocytes in the formation of granulomatous reactions seen in certain malignant lymphoid tumours was investigated by measuring the granulopoietic colony-stimulating activity (CSA) and granulopoietic-inhibiting activity (IA) produced by stimulated T-lymphocytes isolated from peripheral blood, spleen and lymph nodes of patients and normal subjects. Lymph-node T-cells from patients with benign lymphoid hyperplasia, B-cell non-Hodgkin's lymphoma (B-NHL), and non-granulomatous Hodgkin's disease (HD) showed no CSA, but the cells produced IA of 40 +/- 23%, 40 +/- 24% and 50.5 +/- 22.5% respectively. The corresponding cells from patients with HD accompanied by granulomatous reactions produced CSA of 6.85 +/- 6.5 u/microliters and IA of 23.5 +/- 21%. The presence of a granulomatous reaction in malignant lymphoma was correlated with the stimulation of granulopoiesis in vitro by T lymphocytes associated with malignant cells. A correlation was demonstrated between neutrophilic and eosinophilic colonies obtained in vitro under the influence of CSA-producing T cells isolated from malignant lymphomas and the neutrophils and eosinophils present in the granuloma. These results showed that tumour-infiltrating T cells play a role in the presence of granulomatous reactions seen in lymphomas. Peripheral-blood T cells from healthy subjects, and from patients with B-NHL, or with HD unaccompanied by granulocytic reactions produced CSAs of, respectively, 5 +/- 0.5 u/microliter, 4.8 +/- 2.2 u/microliters and 5.3 +/- 0.4 u/microliters, and IAs of 45 +/- 18%. 50 +/- 5.5% and 50.5 +/- 7% respectively. The corresponding values for HD patients with granulocytic reactions were CSA. 17 +/- 15.5 u/microliters, and IA, 9.5 +/- 9%. No correlation was demonstrated between neutrophilic colonies obtained in vitro under the influence of HD blood T cells and neutrophils present in blood. Only one correlation was found: between the percentage of eosinophilic colonies and the number of blood eosinophils. HD blood T cells did not seem to explain completely granulocytic reactions seen in blood.

A new entity of acute leukemia co-expressing CD4 and CD56 markers without any other lineage-specific markers has recently been identified as arising from plasmacytoid dendritic cells (pDC). We proposed to call it plasmacytoid dendritic cell leukemia (pDCL) (Chaperot et al., 2001; Feuillard et al., 2002). We previously reported that pDCL may express the CD33 myeloid associated marker (Garnache-Ottou et al., 2005). This expression of CD33 on CD4+CD56+ lineage negative cells should not exclude the diagnosis of pDCL and underlines that pDCL specific markers should be identified. In order to better characterize pDCL, we organized a French pDCL network to collect cells and data on this rare leukemia. The aim of this study was to identify specific markers for the routine diagnosis. We focused on previously described pDC specific markers: CD123, BDCA-2, BDCA-4 (neuropilin-1). Fourteen pDCL and 69 other acute leukemias (10 B-ALL, 3 T-ALL, 3 BAL, 2 unclassified AML, 4 AML M0, 12 AML M1, 11 AML M2, 1 AML M3, 11 AML M4, 9 AML M5 according to FAB classification and 3 blastic transformations of myeloproliferative or myelodysplastic syndromes) were analyzed by flow cytometry. Leukemic cells were identified on their low expression of CD45 and using lineage specific markers. pDCL have been functionally characterized by their capacities to activate naïve cord blood CD4+ T cells, to induce a Th2 polarization and to produce IFN-α in response to viral supernatant. BDCA-4 was not specific for pDCL since 10% of acute leukemias (7/69) expressed this marker at the same levels as pDCL. This was not surprising since BDCA-4 has been reported to be expressed by myeloid cells as well as by some B and T cell subsets. Unlike BDCA-4, BDCA-2 was never expressed in the 69 tested acute leukemias. However, BDCA-2 expression was not systemically detected in all the pDCL tested (4 negative cases out of the 14 tested). CD123, also know as IL-3 receptor α-chain, was expressed on nearly all the acute leukemias tested. However, the levels of expression discriminated pDCL from other acute leukemias. These latter acute leukemias expressed lower levels of CD123 (fluorescence intensity ratio: 22 [mean] +/- 8 [SEM]; range: 2–71) when compared to pDCL (fluorescence intensity ratio: 171 [mean] +/− 36 [SEM]; range: 71–353). Overall, these results show that BDCA-4 is not a useful marker to identify pDCL. When BDCA-2 is expressed on leukemic cells, this is a strong argument in favor of pDCL. Finally, the level of CD123 expression is an interesting tool to characterize pDCL. We plan to analyze other acute leukemias, and in particular, acute leukemias aberrantly expressing CD4 and/or CD56 markers. This will allow us to confirm that pDCL can be identified on the basis of BDCA-2 and CD123 expression. Since the prognosis of pDCL was poor, an early diagnosis is needed to treat patients with specific therapeutic options (e.g., allogeneic hematopoietic cell transplantation). This can be achieved in the future by using anti-BDCA-2 and CD123 antibodies in addition to classical lineage specific markers. supported by the Goelam and the GEIL

Cytogenetic studies were performed at the time of diagnosis on two patients with diffuse small cell lymphocytic lymphoma. Both patients had a similar simple karyotype with a t(13;17)(q12–14;p12–13). These observations confirm the nonrandom involvement of band 13q13 in chronic lymphoproliferative diseases.

Total T lymphocytes separated from twelve lymph nodes involved by B-NHL were studied in limiting dilution experiments for their ability to proliferate in the presence of both R-IL2 used at a final concentration of 40 U/ml and irradiated autologous malignant B cells as feeders. The number of proliferating T-lymphocyte precursors (PTL-P) thus estimated was low in each case (mean: 1/4503; range, 1/200 to 1/11013). Once expanded, proliferation of the IL2 responsive T cells in the presence of autologous malignant B cells remained strictly dependent on the addition of exogenous IL2. Control cases consisted of T lymphocytes separated from peripheral blood of six healthy subjects and cultured in the presence of both R-IL2 (40 U/ml) and irradiated autologous total mononuclear cells as feeders; the mean frequency of PTL-P thus obtained (1/173; range, 1/49 to 1/457) was significantly higher than in malignant lymph nodes (p less than 0.01). These findings do not support the hypothesis that, in this series of patients, expansion of malignant B cells may lead to the activation and growth of T cells sensitized against the tumour.

Mémoire de DES (Diplôme d'Etudes Spécialisées) tenant lieu de thèse d'exercice.; Bone marrow immunophenotyping by flow cytometry is an essential tool to screen, classify and monitor hematological malignancies. Our aim in the present work is to determine reference values for myeloid and lymphoid bone marrow subpopulations. A major issue for bone marrow cell subsets quantification is contamination by peripheral blood-derived cells which modify cell counts. Thus, in a first step, we validated a criterion for assessing cytometric bone marrow purity. It’s the ratio of immature granulocytes (IGRA) on neutrophils. In a cohort of 106 patients, we have shown that this parameter is highly correlated to the standard Holdrinet method. This first validated cytometric test is unique, easy to calculate, applicable for children and adults, for normal or pathological samples. Secondly we used this cytometric criterion to select 97 cytomorphologically normal, undiluted bone marrow samples, in an oligocentric study with Saint-Etienne and Lyon hospitals. We determined the reference values of 16 subpopulations in 5 age groups, from infants to over 70 years old adults. They are the only values available by flow cytometry for as many populations and for as many age classes. Finally, our study is the last step before testing the capacity of this 8-color tube to screen hematological disorders in bone marrow samples.; L'immunophénotypage de la moelle osseuse par cytométrie en flux est un outil indispensable au screening, classement et suivi des hémopathies malignes. Notre objectif dans ce travail est de déterminer des valeurs de référence pour les sous populations lymphoïdes et myéloïdes de la moelle. Un problème important est la fréquente hémodilution des moelles osseuses qui modifie les proportions des populations. Dans un premier temps, nous avons donc validé un critère cytométrique d'appréciation de la pureté médullaire : le rapport des granuleux immatures (GRIM) sur les PNN. Sur une cohorte de 106 patients, nous avons montré que ce paramètre était fortement corrélé à la méthode d'Holdrinet. Il s'agit ainsi du premier critère cytométrique validé. Il est unique, facile à calculer, utilisable chez l'enfant comme chez l'adulte, que la moelle soit normale ou pathologique. Nous avons dans un second temps utilisé ce critère cytométrique pour sélectionner 97 moelles osseuses cytologiquement normales, non diluées, dans une étude oligocentrique avec les CHU de Saint-Etienne et de Lyon et nous avons déterminé les valeurs de référence de 16 sous-populations pour 5 classes d'âge, du bébé à l'adulte de plus de 70 ans. A ce jour, il s'agit des seules valeurs disponibles en cytométrie pour autant de populations et pour tous les âges de la vie. Finalement, notre étude est l’étape ultime avant l’évaluation des capacités de screening des hémopathies de ce tube en 8 couleurs dans la moelle osseuse.

Mémoire de DES (Diplôme d'Etudes Spécialisées) tenant lieu de thèse d'exercice.; Bone marrow analysis by flow cytometry is part of the routine diagnosis of haematological disorders in medical laboratories. Leukocyte differential and search for abnormal subsets is yet usually performed previously through morphological examination on glass smears by skilled microscopists. In the present work we propose a single 8-color tube i.e. CD3-FITC/ CD10-PE/ CD38-PerCP-Cy5.5/ CD19-PECy7/ CD36-APC/ CD16-APC-H7/ CD34-BV421/ CD45-V500, aiming at equivalent information using flow cytometry. Ninety nine bone marrow samples have been included and classified in 2 groups, i) 51 normal samples from healthy donors or patients suffering from different diseases at diagnosis or during remission, and ii) 48 pathological samples involving quantitative and/or qualitative abnormalities. Analysis has been performed using a FACSCanto-II cytometer. We first proposed a standardized strategy of sequential regions leading to identification of 14-part normal leukocyte differential within lymphoid and myeloid lineages. We validated this approach by flow cytometry against microscopic morphological examination, in case of normal as well as tumour cells. Very interestingly, we showed that cell identification and numeration by flow cytometry was very easily performed and highly reproducible. Finally, we proposed criteria indicative of pathological conditions and orienting further analysis for the identification of abnormal cells. In conclusion, the present flow cytometric approach using a combination of 8 antibodies proved to be very simple, rapid, and reproducible for the determination of the bone marrow cell composition with high precision.; L’analyse de la moelle osseuse par cytométrie en flux (CMF) fait partie du diagnostic de routine des hémopathies dans un laboratoire d’analyses médicales. La formule leucocytaire et la recherche de populations anormales sont habituellement réalisées au préalable grâce à l’analyse morphologique microscopique sur frottis par un cytologiste averti. Nous proposons ici une combinaison de 8 couleurs en un seul tube, CD3-FITC/ CD10-PE/ CD38-PerCP-Cy5.5/ CD19-PECy7/ CD36-APC/ CD16-APC-H7/ CD34-BV421/ CD45-V500, visant à obtenir ces données grâce à la CMF. Nous avons inclus 99 moelle osseuses, classés en 2 groupes, i) 51 échantillons normaux, provenant de donneurs sains ou de patients avec diverses pathologies et ii) 48 échantillons pathologiques avec des anomalies quantitatives ou qualitatives. L’analyse a été réalisée sur un FACSCanto-II. Dans ce travail, nous proposons une stratégie standardisée d’identification de 14 sous-populations normales, myéloïdes et lymphoïdes. Nous avons validé cette approche de CMF en corrélant les résultats avec ceux de l’analyse morphologique, pour des cellules normales comme pathologiques. Nous montrons que cette approche d’identification et de numération cellulaire est facilement réalisable et très reproductible. Nous proposons aussi une série de critères permettant de détecter les moelles pathologiques et d’orienter vers un panel complémentaire approprié pour l’identification des cellules anormales. En conclusion, cette approche déterminant la composition cellulaire de la moelle osseuse par CMF avec une combinaison 8 couleurs est particulièrement intéressante en raison de sa précision, simplicité, rapidité, et reproductibilité.

Mémoire de DES (Diplôme d'Etudes Spécialisées) tenant lieu de thèse d'exercice.; Le concentré plaquettaire d'aphérèse est préparé par l'extraction sélective de plaquettes, ex vivo, grâce à un séparateur de cellules qui restitue au donneur ses autres composants sanguins puis il est conservé à température ambiante pendant 5 jours maximum. Le séparateur COBE Spectra 5.1 a été amélioré afin de permettre une meilleure extraction des plaquettes ; la nouvelle version est appelée Turbo. Le but de cette étude était de comparer selon des critères analytiques in vitro la qualité des deux types de CP A. Trente donneurs volontaires en thrombaphérèse ont été prélevés deux fois à 8 semaines d'intervalle : une fois sur la version 5 .1 et une autre fois sur la version Turbo. Les paramètres suivants ont été mesurés à JO, J3 et J5 sur la suspension plaquettaire indice de tournoiement, pH, leucocytes résiduels, VPM, CD62P membranaire, LDH, glucose, PTG et GMP140. Nous avons étudié 60 CPA qui sont tous conformes à la norme AFS (pH, volume, nombre de plaquettes, nombre de leucocytes résiduels). L'ensemble des paramètres mesurés est similaire pour les deux versions à l'exception du VPM (supérieur pour les plaquettes issues de la version Turbo). Nous avons observé sur la totalité des poches, conformément aux résultats de la littérature, une légère acidification du milieu, une consommation de glucose et une lyse plaquettaire très modérée. La modification majeure à l'origine de la "storage lesion" est l'activation plaquettaire progressive de JO à J5 responsable d'une part de la dégranulation alpha (29 % à 57 % de plaquettes CD62P+ en moyenne et relargage de deux facteurs solubles PTG et GMP140) et d'autre part du flip-flop membranaire (exposition de la phosphatidylsérine à l'extérieur de la membrane). L'intérêt de ce travail est de servir de base pour définir ultérieurement les critères majeurs d'altération des fonctions plaquettaires et leurs valeurs seuil dans le cadre du contrôle qualité prétransfusionnel.