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We have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4+CD8dim+ and CD4+CD8− phenotype. Both clones mediated a specific MHC class I–restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vβ gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vβ7/Jβ2.3, Vβ13/Jβ2.5, and Vβ22/Jβ2.5 rearrangements. Phenotypic analysis using specific anti-Vβ monoclonal antibodies indicated that only Vβ13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vβ gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vβ transcript, corresponding, respectively, to Vβ5/Jβ2.3 and Vβ17/Jβ2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vβ TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.

The rate of detection of chromosome abnormalities in T-cell proliferations is lower than that observed in B-cell malignancies. The former frequently involve the TCRα/δ locus at chromosome band 14q11. We have identified a YAC encompassing 70% of the TCRα/δ locus, which has been used as a fluorescence in situ hybridization probe to detect chromosome rearrangements involving 14q11, both at metaphase and within interphase nuclei, in patients with a variety of T-lymphoproliferative disorders. Its use allowed detection of previously unsuspected TCRα/δ rearrangements in 4/13 (30%) immature T-lineage acute leukemias, including two t(10; 14) and 2 minor inversion 14s. It also clarified interpretation of complex chromosome 14 abnormalities in mature T-cell proliferations (T-prolymphocytic leukemia and ataxia telangiectasia). Use of this probe will aid the detection and characterization of abnormalities involving the TCRα/δ locus, particularly in cases with normal or complex karyotypes and in those proliferations for which mitoses are difficult to obtain.

Fifty-six cases of anaplastic large cell lymphoma (ALCL), 23 cases of Hodgkin's disease, and 16 cases of diffuse large cell lymphoma were investigated for the t(2;5)(p23;q35) translocation. The translocation was detected by using cytogenetic analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry with P80 antibody directed against the kinase domain of anaplastic lymphoma kinase (ALK) of the chimeric NPM/ALK protein. In all but three cases of ALCL, we found an agreement between cytogenetic analysis, RT-PCR, and P80 staining. However, in one case, the t(2;5) translocation was detected with cytogenetic analysis, but RT-PCR and P80 staining were found to be negative. Conversely, in another case the karyotype was normal, but the hybrid mRNA and P80 staining were found to be positive. In one case, malignant cells showed a translocation involving chromosomes 1q25 and 2p23 and were strongly positive for P80 staining. Such a result could be expected because P80 antibody detects the kinase domaine of the ALK protein encoded by chromosome 2p23. Overall 73.2% (41 of 56) of cases were found to be positive. However, the highest percentage (23 of 26 cases; 88.5%) of P80 positive cases was found in children compared with 60% (18 of 30 cases) in adult ALCL (P < .05). In Hodgkin's disease, Reed-Sternberg cells were found to be clearly negative by RT-PCR and with P80 antibody. The latter results suggest that Hodgkin's disease and t(2;5)-positive ALCL are distinct biological entities and that the demonstration of the t(2;5) translocation is of diagnostic importance in differentiating these two entities. The results of the present study indicate that immunohistochemistry with P80 antibody is a reliable method for detecting NPM/ALK chimeric protein.

To reveal the relationship between hypodiploidy with 30 to 39 chromosomes and near-triploidy in acute lymphoblastic leukemia (ALL), we studied 24 patients presenting with one of these aneuploidies among 623 adults with ALL registered in the Leucemie Algue Lymphoblastique de l'Adulte (LALA) protocols. The 2 ploidy groups presented a striking similarity of their cytogenetic profiles: chromosomes 2, 3, 4, 7, 13, 15, 16, and 17, significantly monosomic in hypodiploidy 30 to 39, were also frequently disomic in near-triploidy, whereas those retained in pairs in hypo diploidy 30 to 39 were frequently tetrasomic in near-triploidy. DNA content data revealed the simultaneous presence of 2 aneuploid peaks in most tested cases (DNA indexes: 0.72-0.87/1.39-1.89) and a multiple correspondence analysis applied on cytogenetic profiles ascertained their strong relationship. We thus assumed that near-triploidy derives from the duplication of hypodiploidy with 30 to 39 chromosomes and that both aneuploid groups are 2 expressions of the same disease. These 24 patients presented with B-cell phenotype, low leukocytoses (median white blood cell count, 4.2 x 10(9)/L), and poor prognosis (complete remission, 57%; median disease-free-survival, 8 months; median survival, 10.4 months) comparable to that of Ph+ patients treated according to the same protocol. We suggest that hypodiploidy with 30 to 39 chromosomes or near-triploidy should be regarded as a new high-risk factor in the risk stratification of adult ALL protocols. (C) 2004 by The American Society of Hematology.

During differentiation, megakaryocytes increase ploidy through a process called endomitosis, whose mechanisms remain unknown. As it corresponds to abortive mitosis at anaphase and is associated with a multipolar spindle, investigation of chromosome segregation may help to better understand this cell-cycle abnormality. To examine this variation, a new method was developed to combine primed in situ labeling to label centromeres of one chromosome category and immunostaining of tubulin. Human megakaryocytes were obtained from normal bone marrow culture. By confocal microscopy, this study demonstrates an asymmetrical distribution of chromosomes (1 or 7) either between the spindle poles at anaphase stage of endomitosis and between the different lobes of interphase megakaryocyte nuclei. The metaphase/anaphase checkpoint appears normal on the evidence that under nocodazole treatment megakaryocytes progressively accumulate in pseudo-metaphase, without spontaneous escape from this blockage. Immunostaining of p55CDC/hCDC20 with similar kinetochore localization and dynamics as during normal mitosis confirms this result. HCdh1 was also expressed in megakaryocytes, and its main target, cyclin B1, was normally degraded at anaphase, suggesting that the hCdh1-anaphase–promoting complex checkpoint was also functional. This study found the explanation for these unexpected results of an asymmetrical segregation coupled to normal checkpoints by careful analysis of multipolar endomitotic spindles: whereas each aster is connected to more than one other aster, one chromosome may segregate symmetrically between 2 spindle poles and still show asymmetrical segregation when the entire complex spindle is considered.

We have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4(+)CD8dim+ and CD4(+)CD8(-) phenotype. Both clones mediated a specific MHC class I-restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vbeta gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vbeta7/Jbeta2.3, Vbeta13/Jbeta2.5, and Vbeta22/Jbeta2.5 rearrangements. Phenotypic analysis using specific anti-Vbeta monoclonal antibodies indicated that only Vbeta13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vbeta gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vbeta transcript, corresponding, respectively, to Vbeta5/Jbeta2.3 and Vbeta17/Jbeta2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vbeta TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.

The rate of detection of chromosome abnormalities in T-cell proliferations is lower than that observed in B-cell malignancies. The former frequently involve the TCR alpha/delta locus at chromosome band 14q11. We have identified a YAC encompassing 70% of the TCR alpha/delta locus, which has been used as a fluorescence in situ hybridization probe to detect chromosome rearrangements involving 14q11, both at metaphase and within interphase nuclei, in patients with a variety of T-lymphoproliferative disorders. Its use allowed detection of previously unsuspected TCR alpha/delta rearrangements in 4/13 (30%) immature T-lineage acute leukemias, including two t(10;14) and 2 minor inversion 14s. It also clarified interpretation of complex chromosome 14 abnormalities in mature T-cell proliferations (T-prolymphocytic leukemia and ataxia telangiectasia). Use of this probe will aid the detection and characterization of abnormalities involving the TCR alpha/delta locus, particularly in cases with normal or complex karyotypes and in those proliferations for which mitoses are difficult to obtain.

We have recently reported the superiority of prolonged ambulatory versus short intensive post-remission therapy in 416 patients with AML aged 65 years or more (median age, 72 years) selected to be treated intensively in the ALFA-9803 trial (C. Gardin et al. Blood 2007). We report here on the prognostic value of cytogenetic abnormalities in 339 of these patients with available karyotype (27 not done, 50 failures). When classical endpoints (CR rate, DFS, OS) were used, we were only able to identify one very unfavorable subgroup of 14% patients (N=49 with −7, abnormalities of both chromosomes 5 and 7, 3q abnormality, or complex karyotype with 5 abnormalities or more; CR rate, 30%). No significant differences in outcome were found between patients with any other abnormality and the 171 patients with a normal karyotype/−Y (overall CR rate, 62%). When cumulative incidences of AML-related events (primary refractory AML, relapses) were used as endpoints with induction deaths and deaths in first CR as competing events, three distinct subsets were more easily identified: 14% very unfavorable-risk patients, as detailed above; 59% standard-risk patients (N=200 with t(8;21), inv(16), normal karyotype/−Y, or isolated +8; CR rate, 65%); and 27% unfavorable-risk patients (N=90 with other abnormalities, including notably 5q− and 7q−; CR rate, 58%). Median failure-free interval (Fig 1; P 1. Failure-free interval 1. Failure-free interval 2. Relapse-free interval 2. Relapse-free interval

Introduction: BL is the main childhood B-cell NHL, characterized by the 8q24 translocation [t(8q24)] leading to a c-myc deregulation. Additional chromosomal alterations have been described in BL as well as in DLBCL, but their prognostic value is not clearly evaluated, especially in children. We have already shown the prognostic value of complex karyotype in childhood B-cell NHL in general (Lugano 2005). We now study the role of c-myc rearrangement and of other chromosomal alterations in each sub entity, BL and DLBCL. Methods: Karyotypes from 237 children enrolled in the FAB/LMB96 study were centrally reviewed by each participating cooperative group and obtained an international morphological consensus diagnosis: BL 76.8%, BL-like (BLL) 7.6%, DLBCL 12.2%, unclassifiable 3.4%. Prognosis analyses (event free survival -EFS- and overall survival -OS-) were performed after adjustment on the national cooperative group, the therapeutic stratification group (C vs A & B), the morphological consensus classification (DLBCL vs BL & BLL), the LDH level (>2 vs Results: The BL/BLL were usually diploid with a t(8q24) in 92% cases and associated with other chromosomal aberrations in 69%, mainly +1q, del(13q), +7q. The DLBCL were more heterogeneous and more complex than BL/BLL. t(8q24) incidence (34%) was higher than in adult DLBCL and the pattern of chromosomal alterations varied according to the presence of t(8q24) [diploidy, +1q, del(6q), +7q] or not [hyperdiploidy, der(11q) and +12q]. Multivariate analysis identified two different associations of independent cytogenetic factors with worse prognosis on both OS and EFS. Model (A) for EFS: complex karyotype (more than 3 chromosomal alterations) [HR=3, p=0.0008] and t(8q24) [HR=5.1, p=0.04] or model (B) for EFS: del(13q) [HR=2.6, p=0.0008], +7q [HR=2.6, p=0.01] and t(8q24) [HR=5.7, p=0.03]. (+1q had no significant effect). In the BL entity (N=182), only the complexity [HR=2.5, p=0.02] persists in the model A and del(13q) [HR=4.2, p=0.002] in the model B. In the group of 29 DLBCL, the only significant variable (univariate analysis) is the t(8q24): the 3-yr EFS in DLBCL without t(8q24) is 100% while 50% in DLBCL with t(8q24) [p=0.0005]. Conclusion: In BL/BLL, the strong effect of complex karyotype is partly explained by the role of del(13q) and to a lesser extent by +7q. The strong prognosis effect of t(8q24) in B-cell mature childhood NHL is mainly due to its role in DLBCL. This study emphasizes the impact of cytogenetics in both diagnostic characterization and prognostic stratification of childhood mature B-cell NHL.

A cytologic and cytogenetic study of 10 cases of acute myeloblastic leukemia with maturation and t(8;21) translocation is reported. Despite a certain polymorphic appearance, the characteristic cytologic picture, consisting essentially of large myeloblasts with an abundant cytoplasma containing a large Auer rod, allowed the presence of the chromosome anomaly to be predicted. t(8;21) translocation was attended by the loss of a sex chromosome in 7 of 10 cases. The comparative study of mitoses using cytologic and cytogenetic techniques showed that cells exhibiting normal karyotypes were essentially erythroblasts. This finding suggests that the chromosome anomaly does not affect all the bone marrow cell lines. After short-term culture, the percentage of normal karyotype mitoses diminished, as did the number of mitoses in erythroblasts.

A diagnosis of hairy cell leukemia was made by optic microscopy, phase- contrast microscopy, electron microscopy, scanning microscopy, and histochemistry of the abnormal blood cells. In vivo these cells were found to have a half-time in the blood of approximately 150 hr. In vitro they had the capacity to adhere firmly to plastic, making it possible to obtain a pure population of hairy cells. Neither T-rosette formation nor phytohemagglutinin (PHA) transformation could be demonstrated in these cells. On the other hand, the presence of immunoglobulins on the surface of the hairy cells (HC) by immunofluorescence, and the synthesis and secretion by these cells of IgM type lambda-chains shown by radioimmunodiffusion, were in favor of their B-type lymphocyte origin. Similarities to chronic lymphocytic leukemia were apparent.