Search

Summary Background Monoclonal T-cell receptor (TCR) rearrangement is detected in 57–75% of early-stage mycosis fungoides (MF) at diagnosis. A retrospective study showed molecular residual disease (MRD) in 31% of patients in complete clinical remission (CR) after 1 year of treatment. Objectives To confirm the frequency of MRD at 1 year and to determine its prognostic value for further relapse. Methods Patients with T1-, T2- or T4-stage MF were prospectively included in this multicentre study. At diagnosis, clinical lesions and healthy skin were biopsied. After 1 year of topical treatment, previously involved skin of patients in CR was biopsied for histology and analysis of TCR-γ gene rearrangement. The results were compared with the clinical status each year for 4 years. Results We included 214 patients, 133 at T1, 78 at T2 and three at T4 stage. At diagnosis, 126 of 204 cases (61·8%) showed TCR clonality in lesional skin. After 1 year, 83 of 178 patients (46·6%) still being followed up were in CR and 13 of 63 (21%) showed MRD. At 4 years, 55 of 109 patients (50·5%) still being followed up were in CR and 44 of 109 (40·4%) were in T1 stage. MRD did not affect clinical status at 4 years (CR vs. T1/T2, P = 1·0; positive predictive value 36·4%; negative predictive value 67·6%). Conclusions T-cell clonality at diagnosis and MRD at 1 year are not prognostic factors of clinical status at 4 years.

The less differentiated stage (CD10-) of B-lineage acute lymphoblastic leukaemia (ALL) described as preB1-ALL in the GEIL nomenclature, accounts for less than 10% of ALL. It is classically considered to be associated with translocation (4;11)(q21;q23), and to have a poor prognosis. We report an extensive immunophenotypic, genomic and clinical study of a series of 64 preB-1 ALL patients, representing 6.3% of a cohort of consecutive ALLs. The engagement of preB1-ALL cells in the B-lineage was confirmed by their B-lineage score, equal to or higher than 2. In addition, more than 90% of the cases tested showed rearranged IGH genes. Translocation (4;11) was the most frequent karyotypic anomaly seen, but only accounted for 24% of the preB1-ALL cases tested. Expression of the myeloid associated antigen CD15 was also found with high incidence in this subset. Clinical and biological features at presentation showed more significant differences between preB1- and T-ALL than between preB1- and preB2-ALL (CD10+). However, outcome characteristics of the 22 children with preB1-ALL confirmed the worse prognosis of this entity.

Resume Introduction Le syndrome d’hypersensibilite medicamenteuse ou DRESS (drug reaction with eosinophilia and systemic symptoms) est une toxidermie grave car elle s’accompagne d’une atteinte viscerale letale dans 6 a 10 p. 100 des cas. Sa physiopathologie reste mal connue. Dans le but de preciser les caracteristiques immunologiques de cette toxidermie, nous avons analyse, de facon prospective, le rearrangement des genes du recepteur des lymphocytes T (TCR) sanguins et cutanes des malades ayant un syndrome d’hypersensibilite medicamenteuse entre avril 1998 et avril 2000. Malades et methodes Les criteres d’inclusion etaient : un âge superieur a 18 ans, la survenue d’une eruption generalisee declenchee par un medicament, l’existence d’une atteinte systemique associee (ganglionnaire ou viscerale), la presence d’une hypereosinophilie superieure a 0,5 G/l et/ou de lymphocytes atypiques circulants. Six malades (3 hommes, 3 femmes), d’âge moyen, 54 ans, ont ete inclus. Le medicament imputable etait un anticonvulsivant dans 3 cas, l’allopurinol dans 2 cas, l’oxazepam dans 1 cas. La guerison est survenue dans un delai de 10 a 30 jours apres la phase aigue. Deux malades ont eu plusieurs poussees. Resultats Aucun rearrangement clonal des genes du TCR n’a ete detecte dans les prelevements cutanes. Un rearrangement clonal des genes du TCR a ete detecte initialement dans les lymphocytes sanguins de 3 des 6 malades (allopurinol : n = 2, oxazepam : n = 1). Ce rearrangement clonal restait detectable pendant l’evolution, lors d’une 2e ou une 3e poussee du syndrome d’hypersensibilite medicamenteuse chez 2 malades (allopurinol : n = 1, oxazepam : n = 1). Discussion La presence de clones T circulant detectables pendant plusieurs mois apres la survenue d’un syndrome d’hypersensibilite medicamenteuse traduit une expansion, mono ou oligoclonale de lymphocytes T actives, declenchee par le medicament en cause. Leur persistance pendant plusieurs mois correspond a une activation remanente du systeme immunitaire pouvant expliquer l’evolution prolongee et/ou recidivante du SHM chez quelques malades.

Resume Introduction Le syndrome de Melkersson Rosenthal est une maladie rare qui associe classiquement : un œdeme orofacial, une paralysie faciale peripherique et une langue plicaturee. La macrocheilite de Miescher en represente la forme monosymptomatique. Son etiopathogenie reste inconnue. Nous rapportons 2 cas de macrocheilite de Miescher au cours desquels ont ete mises en evidence des expansions lymphocytaires monoclonales posant la question d’un lien eventuel entre ces 2 anomalies. Observations Cas 1. Un homme de 30 ans, sans antecedents medicaux, etait suivi depuis 3 ans pour une macrocheilite de Miescher. Les examens complementaires permettaient d’eliminer une cause infectieuse, une maladie de Crohn, une sarcoidose ou une allergie de contact. Une proteine monoclonale IgG kappa serique etait mise en evidence fortuitement. Les radiographies du squelette et le myelogramme etaient normaux. Il n’existait pas de rearrangement monoclonal detectable des genes du recepteur des lymphocytes T ou B sanguins et medullaires. En l’absence d’evolution vers une hemopathie maligne apres un recul de 3 ans, nous avons conclu a une gammapathie monoclonale benigne. Cas 2. Un homme de 36 ans d’origine algerienne, sans antecedents medicaux, etait suivi depuis 8 ans pour une macrocheilite granulomateuse. La recherche d’une maladie de Crohn, d’une sarcoidose ou d’une allergie de contact etait negative et le diagnostic de syndrome de Melkersson Rosenthal incomplet etait retenu. La numeration formule sanguine montrait une hyperlymphocytose sanguine persistante. La recherche etiologique de l’hyperlymphocytose, mettait en evidence un rearrangement monoclonal des genes du recepteur des lymphocytes T dans les lymphocytes sanguins. Le myelogramme etait normal. Commentaires Le syndrome de Melkersson Rosenthal est une maladie granulomateuse rare de la muqueuse buccale. L’etiopathogenie de cette affection est inconnue et discutee mais plusieurs observations suggerent qu’il s’agirait d’une affection d’origine immunologique. Une population lymphocytaire T clonale a ete mise en evidence dans les lesions labiales d’un malade âge de 12 ans atteint de syndrome de Melkersson Rosenthal au cours d’une observation ponctuelle sans que le role de cette population lymphocytaire n’ait pu etre determine. Nous rapportons 2 autres cas associant une proliferation lymphocytaire sanguine a un syndrome de Melkersson Rosenthal. Cette association n’est peut-etre pas fortuite, compte tenu de la rarete du syndrome de Melkersson Rosenthal d’une part et du caractere inhabituel de la detection de clones lymphocytaires chez des sujets jeunes d’autre part. La presence d’une population clonale peut etre interpretee de 2 facons. Elle peut traduire une stimulation antigenique chronique, qui par un effet superantigenique entraine l’expansion d’une population lymphocytaire devenant detectable. L’autre hypothese serait une secretion accrue de cytokines par le clone lymphocytaire provoquant une organisation granulomateuse, comme au cours des lymphomes granulomateux.

The analytical performance and the abnormality messages on differential (flags) of the new analyzer Beckman Coulter DxH 800 were compared with those of the LH 755.First, we evaluated the accuracy of the results of the DxH 800, in comparison with the LH 755, in 125 samples without alarm using unflagged sample results on both analyzers. Second, flagged samples on the LH 755 but not flagged by the DxH 800 were evaluated by flow cytometry for accuracy of the DxH 800 results. Finally, we evaluated the sensitivity and specificity of abnormality messages on differential given by the analyzers, in comparison with manual blood smears.The correlation coefficients (R) for complete blood count parameters and differential demonstrated that the DxH 800 results were similar to that of LH 755. Excellent correlation coefficients between DxH 800 and flow cytometry results were found for white blood cell count (R = 0.985, n = 31), platelet count (R = 0.976, n = 51) and nucleated red blood cells (R = 0.966, n = 37). The overall performance showed an increased sensitivity (0.892) and specificity (0.864) of the flags on DxH 800 when compared to the LH 755 (0.846 and 0.733, respectively).The DxH 800 provides reliable results and increases laboratory efficiency by reducing working time and costs associated with the optical validation of the results.

Some patients suffering from malignancies may benefit of myeloablative chemotherapy followed by hematological reconstitution with autologous peripheral blood reinfusion. A quick evaluation of the number of hematopoietic progenitors present in leukapheresis blood samples is necessary to ensure the collection of a sufficient number of these cells. A study was performed on a series of 25 leukapheresis following initial chemotherapy. The number of granulomonocytic colony-forming unit (CFU-GM) and the number of CD34+ cells were evaluated simultaneously, in each sample. Results have shown a relatively strong linear correlation between both methods of evaluation of hematopoietic progenitors, suggesting that immunophenotyping could be a useful method to estimate the number of progenitors.

Peripheral human blood contains granulo-monocyte (CFU-GM) and eosinophil (CFU-Eo) progenitors. I n vitro , the number of colony forming units is thought to range from 0.1–14 per 2 × 10 5 plated cells. We show that the number of CFU-GM, Eo depends on culture methods. By modifying the usual assay method (using human umbilical cord plasma and the association of 2 stimulating conditioned media: activated lymphocyte conditioned medium and bone marrow fibroblast conditioned medium), we found different circulating CFU-GM, Eo numbers. The mean number of circulating CFU-GM, Eo in 107 healthy adults was 22.4 per 2 × 10 5 plated cells (range: 1–84). There was a slight difference between males (mean number: 23.6) and females (mean number: 20.4). The mean number of CFU-GM, Eo harvested on Percoll gradient was 123/ml of peripheral blood (range: 7–513). These results are far over those commonly reported in literature. This suggests that these latter results were probably underestimated. The use of recombinant human interteukin 3 and recombinant human GM (granulocyte-monocyte) colony-stimulating factors shows that CFU-GM, Eo numbers are found to be comparatively increased compared to that obtained with our modified method (using rhIL-3 alone), or that the size of those colonies is notably increased (using rhIL-3 + rhGM-CSF).

Introduction Les facteurs pronostiques de l’evolution du mycosis fongoide (MF) sont mal connus et les etudes de long terme peu nombreuses. L’objectif de notre etude etait de determiner la frequence et la valeur pronostique d’une maladie residuelle moleculaire (MRD) apres un an de traitement. Patients et methodes Cette etude prospective multicentrique menee de 2003 a 2007 a inclus les adultes avec un MF en plaques (T1 10 %) ou erythrodermiques (T4). A l’inclusion puis tous les ans pendant 4 ans, des biopsies cutanees etaient realisees, en peau atteinte (PA) et en peau saine au diagnostic, puis en peau saine anterieurement atteinte (PSAA) au cours du suivi, pour histologie et etude de rearrangement du TCR (PCRγ-DGGE). Les patients etaient traites localement (chlormethine ou puvatherapie). La MRD a 1 an etait definie par la persistance d’un clone T dans la PSAA des patients en remission clinique complete (T0). Resultats Deux cent quinze patients etaient inclus, d’âge moyen 56 ans (extremes 27–87). 1/Au diagnostic : 134 patients etaient T1 (62 %), 78 T2 (37 %), 3 T4 (1 %). Quatre-vingt-sept pour cent des patients etaient traites par chlormethine, 7 % par PUVA, 6 % par un autre traitement (dermocorticoides, methotrexate). Un clone T etait detecte : – dans la peau chez 65 % des T1 et 68 % des T2 ( p = 0,7) ; – dans le sang chez 41 % des T1 et 36 % des T2 ( p = 0,58), ce dernier etant identique au clone cutane dans 6 % des cas. 2/Au cours du suivi : a 1 an, parmi 175 patients encore suivis (82 %), 82 (46 %) etaient T0, 71 T1 (41 %) et 14 T2 (8,5 %). On notait une tendance a une moindre frequence d’un clone positif en PSAA chez les patients T0 (25 %) par rapport aux patients evolutifs (T1/T2, 38 %, p = 0,17). A 4 ans, parmi 83 patients encore suivis (39 %), 43 etaient T0 (52 %), 30 T1 (34 %), 4 T2 (5 %) et la moitie n’avait plus de traitement. La detection initiale du clone cutane n’avait pas d’impact sur le stade T (T0 vs T1/T2) a 1 an ( p = 0,9) ou a 4 ans ( p = 0,25). 3/Maladie residuelle : a 1 an, parmi 52 patients T0 ayant eu une etude de clonalite, 13 (25 %), avaient une MRD. Au diagnostic, ces patients etaient T1 dans 10 cas et T2 dans 3 cas, avec un clone detecte dans 12 cas. A 3 ou 4 ans, 7/13 etaient T0 et 3/13 etaient T1 (3 perdus de vue), alors que parmi les 39 sans MRD, 19 etaient T0 et 8 etaient T1 ou T2 (6 T1, 2 T2, 12 perdus de vue, p = 1). Discussion Nous confirmons l’evolution indolente de la maladie avec plus de 80 % des patients aux stades T0 ou T1 a 4 ans. Au diagnostic, 1/3 des infiltrats sont polyclonaux et la positivite initiale du clone ne prejuge pas de l’evolution ulterieure. Dans notre etude, la MRD moleculaire a 1 an ne semble pas etre un facteur pronostique de l’evolution clinique, mais le nombre de patients reste faible pour conclure. Conclusion L’existence d’une MRD moleculaire a 1 an ne semble pas influencer l’evolution ulterieure du MF.

Cases of secondary acute myeloid leukemia (AML) occurring after treatment for an Ewing's sarcoma are uncommon. Therapy-related AML with t(8;21) translocation is an entity which has been well characterized. A case of AML-2 with t(8;21) and t(3;15) occurring 4 years after treatment for an Ewing's sarcoma with cyclophosphamide, doxorubicin, vincristine, dactinomycin, and radiotherapy, is reported. Autologous bone marrow transplantation was performed during second remission, 23 months after diagnosis. Reverse transcriptase polymerase chain reaction of the AML1/ETO fusion gene product was performed in order to monitor the quality of the remission. The patient currently remains in remission 24 months after the bone marrow transplantation.

Enumeration of CD34 + cells is the standard assay procedure for optimization of peripheral blood stem cell harvesting. High fluorescence reticulocytes (HFR) have been shown to signal a rebound in haematopoiesis after chemotherapy. The aim of this study was to evaluate HFR determination, as compared to CD34 + cell counts and CFU-GM, as a potential predictor of PBSC counts after recovery from chemotherapy and/or bone marrow transplantation. Twenty-five paediatric patients undergoing intensive courses of chemotherapy and 9 undergoing bone marrow auto or allografts were investigated. In most of our cases, HFR recovered at the same time or earlier than CD34 + cells. Similarly, the rise in HFR preceeded the CFU-GM peak in most of these cases. In no case did we observe a CFU-GM peak without a rise in HFR%. In our experience, the daily relative HFR increase may be used to predict the optimal time for mobilization of stem cells and was therefore of value clinically to confirm the timing of apheresis.

To determine whether cutaneous involvement in patients with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is related to a clonal T-cell proliferation.Retrospective study.University hospitals.Ten patients with AILD and cutaneous involvement.The T-cell receptor-gamma (TCRG)gene rearrangement was studied with the use of polymerase chain reaction and denaturing gradient gel electrophoresis in blood, nodal, and skin samples. Skin and nodal samples were investigated also for the presence of Epstein-Barr virus (EBV) RNA by in situ hybridization.A transient morbilliform eruption of the trunk was seen most often. Other cutaneous features were infiltrated plaques and purpuric or urticarial lesions. A clonal TCRG gene rearrangement was detected in 7 skin samples, corresponding to a maculopapular eruption with a histological pattern of nonspecific mild lymphoid dermal infiltrate in 6 patients, and to erythematous plaques with histological findings of typical cutaneous lymphoma in 1 patient. In the 5 patients in whom a TCRG gene rearrangement was evidenced in skin and lymph node samples, identical clones were detected in both. Five patients died by the end of the study, with a mean survival of 33.2 months. Four of these 5 patients had a clonal infiltrate in skin and lymph nodes. The EBV RNA was detected in only 1 of 10 skin biopsy specimens and in 5 of 8 lymph nodes tested.Cutaneous involvement is often related to a clonal T-cell proliferation in AILD, even when clinical and histological features are nonspecific. Cutaneous infiltrate seems to be clonally related to the nodal T-cell proliferation. The role of EBV infection in skin lesions was not evidenced.

The differential diagnosis of cutaneous lymphoid hyperplasia and B-cell lymphoma may be difficult. Whether the detection of clonal immunoglobulin gene rearrangement in the cutaneous lesion is predictive of a malignant outcome remains controversial. We therefore studied cases of cutaneous lymphoid hyperplasia by polymerase chain reaction analysis.Retrospective study of patients seen between 1988 and 1996.Two dermatology university departments.Twenty-four patients with cutaneous lymphoid hyperplasias were included according to clinical, histopathological, and immunophenotypic criteria.Clinical, histopathological, and laboratory findings.There were 13 men and 11 women (mean age, 49 years) who presented with erythematous or violaceous papules or nodules. The lesions were unique in 13 cases and multiple in 11 cases. All patients had immunochemical evidence of a mixed T- and B-cell infiltrate with polytypic B cells. Polyclonality was demonstrated in 23 patients, whereas a dominant B-cell clone was detected in 1 patient. No lymphoma developed during the follow-up (median, 4 years). In the same period, we studied 53 cases of B-cell lymphomas. Thirty-five (66%) of the 53 cases had a detectable clonal immunoglobulin gene rearrangement.In the majority of our cases, polyclonality demonstrated by polymerase chain reaction analysis was in accordance with the diagnosis of cutaneous lymphoid hyperplasia. In 1 of the 24 patients, the presence of a B-cell clone could be evidenced. This fact did not modify the treatment as there were no histological or immunophenotypic signs suggestive of a lymphoma.

We report seven cases of particular cutaneous tumors selected from the register of the French Study Group on Cutaneous Lymphomas. The patients (three men, four women) were aged 37-86 years. They initially presented with cutaneous nodules or papules. Three cases presented with regional lymph nodes. Stagings were negative, except for one patient with bone marrow involvement. Histological features were relevant with pleomorphic medium T-cell lymphoma, but these cells exhibited a distinguishing phenotype. They were positive for CD4, CD56, and also CD45, CD43, and HLA-DR. All other T-cell and B-cell markers were negative. The myelomonocytic markers (CD13, CD14, CD15, CD33, CD117, myeloperoxidase, and lysozyme) were negative excepted CD68, which was clearly positive in four cases and weakly in two cases. Others natural killer cell markers (CD16, CD57, TiA1, granzyme B), TdT, and CD34 were negative. Polymerase chain reaction studies did not detect any B or T clonal rearrangement. The cytogenetic studies, performed in five cases, showed a del(5q) in two cases. All patients were treated successfully by polychemotherapy, but relapsed quickly in the skin, between 4 and 28 months. Five patients developed bone marrow involvement, with leukemia in three cases, and they died in 5-27 months. One patient died at 17 months with skin progression. The seventh patient is alive at 33 months, with cutaneous progression. The origin of these cells is unclear. Despite expression of CD4 or CD56, we failed to demonstrate a T-cell, natural killer cell origin. However, CD4 and CD56 are not specific for T or natural killer lineages. Although these two markers are also known to be expressed by monocytic cells, classic myeloid antigens were negative. These seven cases, together with other rare similar cases already reported, seem to represent a distinct entity likely developed from hematological precursor cells.

The less differentiated stage (CD10-) of B-lineage acute lymphoblastic leukaemia (ALL) described as preB1-ALL in the GEIL nomenclature, accounts for less than 10% of ALL. It is classically considered to be associated with translocation (4;11)(q21;q23), and to have a poor prognosis. We report an extensive immunophenotypic, genomic and clinical study of a series of 64 preB-1 ALL patients, representing 6.3% of a cohort of consecutive ALLs. The engagement of preB1-ALL cells in the B-lineage was confirmed by their B-lineage score, equal to or higher than 2. In addition, more than 90% of the cases tested showed rearranged IGH genes. Translocation (4;11) was the most frequent karyotypic anomaly seen, but only accounted for 24% of the preB1-ALL cases tested. Expression of the myeloid associated antigen CD15 was also found with high incidence in this subset. Clinical and biological features at presentation showed more significant differences between preB1- and T-ALL than between preB1- and preB2-ALL (CD10+). However, outcome characteristics of the 22 children with preB1-ALL confirmed the worse prognosis of this entity.

Cases of secondary acute myeloid leukemia (AML) occurring after treatment for an Ewing's sarcoma are uncommon. Therapy-related AML with t(8;21) translocation is an entity which has been well characterized. A case of AML-2 with t(8;21) and t(3;15) occurring 4 years after treatment for an Ewing's sarcoma with cyclophosphamide, doxorubicin, vincristine, dactinomycin, and radiotherapy, is reported. Autologous bone marrow transplantation was performed during second remission, 23 months after diagnosis. Reverse transcriptase polymerase chain reaction of the AML1/ETO fusion gene product was performed in order to monitor the quality of the remission. The patient currently remains in remission 24 months after the bone marrow transplantation.

This retrospective study aimed to evaluate the long-term prognostic impact of phenotypic remission in B-cell chronic lymphocytic leukaemia (CLL) patients who have achieved clinical, haematological and bone-marrow complete remission (CR) after conventional chemotherapy. The clinical and phenotypic data of 77 CLL patients in CR with a median follow-up from CR achievement of 54 months (range 5-127) were analysed. 32 patients (42%) displayed a normalized phenotype as evaluated by k:lambda ratio or by CD5+/CD19+ cell numbers. Patients with normalized phenotype demonstrated a significantly higher incidence of female sex, a lower relapse rate, a trend for higher prevalence of stage A and a lower occurrence of CLL-related deaths. The relapse-free survival of patients with normalized phenotype was significantly longer (P = 0.02), whereas no difference in overall survival was found between the two groups. Interestingly, Binet's stage at diagnosis was highly predictive of the overall survival following CR achievement. From the results of the present study we conclude that a phenotype normalization at CR obtained with conventional chemotherapy indicates a higher probability of a longer CR but it does not translate into prolonged survival. Clinical features at diagnosis, such as stage distribution, are apparently stronger predictors of the final outcome. These results emphasize, however, the need for a routine assessment of the quality of response since this information could be crucial in designing therapeutic strategies for young patients suffering from advanced CLL.

Discrepancies in the literature on acute leukemia blast cell immunophenotypes are sometimes related to differences between the epitopes recognized by various monoclonal antibodies (MoAb) in the same cluster of differentiation. CD15 is one example of such a variation. CD15 expression has been reported in 1.6% to 39% of acute lymphoblastic leukemias (ALL). We studied the expression of CD15 using 10 different commercially available anti-CD15 MoAbs and we observed three different expression patterns using anti-CD15 MoAbs by flow cytometry in 158 cases of ALL: Smy15c was found in 70% of B lineage ALLs, Smy15a and FMC-13 in 30 to 40% of cases and all others in less than 9% of B-ALL cases (p < 0.0001). In T lineage ALLs, Smy15c, Smy15a and FMC-10 identified CD15 in 30% of the cases and all others in less than 8% of the cases. Logistic regression revealed that Smy15a, CD34 and CD14 correlated significantly with Smy15c expression. We conclude that CD15 MoAbs have to be chosen carefully when ALL immunophenotype and subsequent studies of prognostic significance are performed particularly in assessing multiphenotypic ALLs.

The recently cloned cytotoxic lymphocyte maturation factor [CLMF] also called NK cell stimulatory factor [NKSF] or interleukin-12 [IL-12] has been described as a growth factor for mature lymphoid cells. The present study investigated whether purified recombinant human IL-12 could stimulate CFU colony growth. Source of progenitor cells were peripheral blood cells depleted of adherent, CD2- and CD56-positive cells. RhIL-12 was investigated either alone or in combination with rhIL-3, rhIL-6 and rhGM-CSF. RhIL-12 alone did not support colony formation of myeloid or erythroid progenitors. RhIL-12 in combination with rhIL-3 increased the numbers of BFU-E and CFU-GM. No synergism or additive effect was seen with the combination of rhIL-12 and rhGM-CSF or rhIL-12 and rhIL-6. An additive increase in the number of granulocytic colonies was observed when rhIL-3, rhIL-6 and rhGM-CSF were used together with rhIL-12. Our result therefore suggest that, in addition to being a potent lymphopoietic stimulator, IL-12 acts synergistically with IL-3 in enhancing the sensitivity of hemopoietic progenitors to IL-3.

To gain additional informations on the role played by Natural Killer cells (NK) in the differentiation of human hematopoietic precursors, we have studied the effect of NK-cell depletion on the in vitro proliferation of hematopoietic cells. NK cells were depleted from blood mononuclear cells by FACS using anti CD3 and anti CD56 monoclonal antibodies. Depletion of NK cells suppressed CFU-GM up to 69% (P < 0,01), while no significant effect on either BFU-E and CFU-Mix growth was observed. To define the threshold of CD56+ cells required to support CFU-GM formation, NK cells were added to NK-depleted cells in a titrated fashion. Enhancement of CFU-GM colony growth was observed at NK/NK - depleted cells ratio of 0.15/1. A dose dependent suppression of CFU growth was observed at ratios ranging from 0.25/1 to 0.5/1. Addition of neutralizing antibodies against IL3 and GM-CSF abrogate the stimulating effect of NK cells. Our results suggest that cells with LGL morphology and NK markers play an important role in differentiation of myeloid precursors and exert a moderate influence on erythroid progenitors. The modulatory effect on hematopietic progenitors depend on the number of NK cells present in the mixed culture. © 1994 Wiley-Liss, Inc.

Twenty-two B-cell chronic lymphocytic leukemia (CLL) patients were investigated to evaluate residual disease in clinico-hematological remission. Residual disease was determined by monotypy of surface light-chain expression and by dual-color staining with CD5 and CD19 markers. Samples were analyzed on flow cytometer. Total CD19+ cells above 25%, the CD5+CD19+/total CD19+ cells ratio above 0.25, clonal excess above 0.4 were considered positive for residual disease. According to these immunological criteria, only four cases achieved phenotypic remission. Our data confirm that dual marker analysis is more sensitive than clonal excess and may predict an early relapse. Ig gene rearrangements were studied by Southern blot analysis using IGHJ and IGKC probes in fifteen cases. All 12 cases that retained a detectable rearrangement displayed a phenotypic residual disease. Conversely, in two cases, DNA analysis failed to detect the residual disease characterized by flow cytometry. In conclusion, this study suggests that in B-CLL, dual marker analysis is sensitive in predicting an early relapse in sequential evaluations of residual disease, whereas rearranged bands are undetectable when the proportion of malignant cells is low.

Hyaluronectin (HN), a hyaluronan (hyaluronic acid, HA)-binding glycoprotein is normally expressed in the nervous system, found in the desmoplasia of tumours, and is also produced in vitro by peripheral blood mononuclear cells. We have therefore investigated the expression and the production of HN by leukemic cells, with the hypothesis that HN would be expressed in leukemias of the myeloid lineage. Fresh and frozen leukemic cells were studied from 70 patients of whom 53 had acute myeloblastic leukemia (AML). HN was strongly expressed (80% blood cells) in two out of 13 M4 AMLs and four out of four M5B AMLs. One further M4 AML displayed 25% positive cells and two 20% cell positivity cases were seen, in one case of M4 AML and in one case of chronic myelomonocytic leukemia (CMML). The rest of the cases of AML as well as all cases of acute lymphoblastic leukemia (ALL) showed almost no positivity (1%). The residual positive cells appeared to be normal blood promonocytes. Taken togetheror = 20% positive cells was seen in eight out of 56 (14%) examined myeloid leukemias. The HN production was significantly higher (p0.0001) in cell culture media of M4 and M5 AML cells than in other AML or ALL cell culture media. A significant correlation was found (p0.0001) between the number of HN-positive leukemic cells and the number of cells with a monocytic morphology, suggesting that HN is a marker for the promonocyte.

We report a series of 20 non-Hodgkin's lymphomas (NHL) in which cytogenetic analysis showed a translocation involving band 3q27 and the site of one of the three Ig genes (14q32, 2p12, 22q11) in the neoplastic cells. These cases were found in a series of 319 patients with clonal chromosomal abnormalities studied over a 7-year period. Fourteen patients had diffuse lymphoma, mainly of large cell type and the remaining six were follicular lymphomas. All cases studied were of B-cell phenotype. A t(3;14)(q27;q32) was commonest, found in 15 patients (4.7%), with the two variant translocations, t(3;22)(q27;q11) and t(2;3)(p12;q27), being found in three and two patients, respectively. Additional chromosomal defects were present in most patients, but two patients had this type of translocation as the sole abnormality. These results indicate that translocations involving band 3q27 and Ig genes are not uncommon, and suggest that a novel oncogene, located at band 3q27, may be implicated in B-cell NHL.

Twenty-five patients with B-cell chronic lymphocytic leukemia (B-CLL) were investigated to correlate the immunological phenotype with the description of the Ig gene rearrangements of the B-cell clone. All patients were positive for the CD19 antigen and one pan B-antigen, markers of late cells (CD20, CD37 or Y2955). Twenty-four of the 25 patients tested expressed monoclonal cell surface immunoglobulin (SIg). The CD5 antigen was present in 21 of the 25 tested patients. Immunoglobulin gene rearrangements were detected by hybridization of the BamHI, EcoRI, BgIII, and HindIII digested genomic DNAs to the IGHJ, IGKC, IGLC, and IGLJ2 probes. Twenty-four of 25 patients had two rearranged IGH loci. The IGKC rearrangements were observed in 20 patients. In four patients, the IGK loci were deleted on both chromosomes. One patient without SIg displayed a germline pattern. All six patients with lambda producing B-CLL showed a lambda gene rearranged band, although the use of IGL polymorphism to investigate IGL rearrangements must be noted. These clonal rearrangements of IGL genes, together with the detection of either the kappa or lambda light chain of SIg, confirm that patients with B-CLL meet the developmental scheme of ordered light chain gene rearrangements.