Dominique Penther

CD38 expression and secondary 17p deletion are important prognostic factors in chronic lymphocytic leukaemia.

Summary. CD38 expression and chromosomal abnormalities are novel prognostic factors in chronic lymphocytic leukaemia (CLL). However, their value remains undetermined. CD38 was evaluated in 123 patients and chromosomal aberrations in 111 cases with fluorescence in situ hybridization (FISH). CD38 expression was found in 27% ofthe cases. In addition, seven out of 32 CD38– patients became CD38+ during evolution of the disease. Chromosomal abnormalities included isolated 13q deletion (40%), 12q trisomy (14%), 11q deletion (without 17p deletion) (14%) and 17p deletion (7%). CD38 expression was significantly associated with Binet stages B and C, atypical morphology and 11q deletion. On univariate analysis of survival estimates, advanced Binet stages, CD38+ phenotype, atypical morphology and 11q or 17p deletions were associated with shorter event‐free survival (EFS), treatment‐free interval (TFI) and overall survival (OS). Multivariate analysis identified both Binet stages and CD38 as independent prognostic factors with regard to EFS and TFI. However, CD38 appeared as an independent factor for OS when restricted to Binet stage A. Chromosomal aberrations were re‐evaluated during evolution in 31 cases. The 17p deletion was the most frequent new chromosomal abnormality (35%) and significantly associated with death (64%). In conclusion, CD38 expression and secondary 17p deletion are important poor prognostic indicators, especially in Binet stage A CLL.

Wide diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogénétique Hématologique study

PAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

Chromosomal aberrations and their prognostic value in a series of 174 untreated patients with Waldenström's macroglobulinemia

Waldenströms macroglobulinemia is a disease of mature B cells, the genetic basis of which is poorly understood. Few recurrent chromosomal abnormalities have been reported, and their prognostic value is not known. We conducted a prospective cytogenetic study of Waldenströms macroglobulinemia and examined the prognostic value of chromosomal aberrations in an international randomized trial. The main aberrations were 6q deletions (30%), trisomy 18 (15%), 13q deletions (13%), 17p (TP53) deletions (8%), trisomy 4 (8%), and 11q (ATM) deletions (7%). There was a significant association between trisomy of chromosome 4 and trisomy of chromosome 18. Translocations involving the IGH genes were rare (<5%). Deletion of 6q and 11q, and trisomy 4, were significantly associated with adverse clinical and biological parameters. Patients with TP53 deletion had short progression-free survival and short disease-free survival. Although rare (<5%), trisomy 12 was associated with short progression-free survival. In conclusion, the cytogenetic profile of Waldenströms macroglobulinemia appears to differ from that of other B-cell lymphomas. Chromosomal abnormalities may help with diagnosis and prognostication, in conjunction with other clinical and biological characteristics. This trial is registered with Clinicaltrials.gov, numbers NCT00566332 and NCT00608374.

Amplification of AML1 gene is present in childhood acute lymphoblastic leukemia but not in adult, and is not associated with AML1 gene mutation.

The AML1/CBFA2/RUNX1 gene is the target of many recurrent translocations seen in different leukemia subtypes. The t(12;21)(p13;q22) is the most frequent translocation observed in childhood B acute lymphoblastic leukemia (ALL), occurring in 20% to 25% of cases. In adult ALL this rearrangement is scarce. Another route of AML1deregulation could be point mutations in the runt domain. We now report on AML1amplification in two cases of childhood ALL, found in a series of 107 consecutive children with B-lineage ALL analyzed by fluorescence in situ hybridization (FISH). A parallel analysis of 42 adult B-ALL failed to detect any AML1 rearrangement by FISH. The two patients with AML1 amplification were further analyzed using molecular techniques. SSCP analysis did not detect any mutation. Furthermore, direct sequencing of the cDNA did not reveal any mutation. In conclusion, AML1amplification seems to be observed only in childhood ALL and is not associated with AML1 gene mutation. Other mechanisms, such as gene dosage effects could be hypothesized.

Analyses of TET2 mutations in post-myeloproliferative neoplasm acute myeloid leukemias.

Classic myeloproliferative neoplasms (MPN) include essential thrombocythemia, polycythemia vera and primitive myelofibrosis. These diseases are characterized by an over production of myeloid cells and may transform into acute myeloblastic leukemia (AML). An acquired point mutation that leads to the expression of a constitutively active form of the Janus kinase 2 (JAK2) tyrosine kinase, JAK2V617F, is observed in almost all polycythemia vera cases, and in 50–60% of essential thrombocythemia and primitive myelofibrosis. In experimental models, JAK2V617F recapitulates most of the features of MPN, establishing its essential role in the clinical phenotype of the diseases. It is, however, unclear how a single mutation gives rise to three related but distinct disorders. Moreover, when AML develops in a patient with a JAK2V617F-positive myeloproliferative neoplasm, the fully transformed clone is often JAK2V617F negative. These and other observations suggested that, in some patients, another unknown mutation has preceded the JAK2V617F mutation in the development of MPN. Recent publications have reported acquired mutations of the TET2 gene in various hematopoietic malignancies. The most frequent type of

Genome profiling of acute myelomonocytic leukemia: alteration of the MYB locus in MYST3-linked cases

The t(8;16)(p11;p13) is a rare translocation involved in de novo and therapy-related myelomonocytic and monocytic acute leukemia. It fuses two genes encoding histone acetyltransferases (HATs), MYST3 located at 8p11 to CREBBP located at 16p13. Variant translocations involve other HAT-encoding genes such as EP300, MYST4, NCOA2 or NCOA3. MYST3-linked acute myeloid leukemias (AMLs) share specific clinical and biological features and a poor prognosis. Because of its rarity, the molecular biology of MYST3-linked AMLs remains poorly understood. We have established the genome and gene expression profiles of a multicentric series of 61 M4/M5 AMLs including 18 MYST3-linked AMLs by using array comparative genome hybridization (aCGH) (n=52) and DNA microarrays (n=44), respectively. We show that M4/5 AMLs have a variety of rare genomic alterations. One alteration, a gain of the MYB locus, was found recurrently and only in the MYST3-linked AMLs (7/18 vs 0/34). MYST3-AMLs have also a specific a gene expression profile, which includes overexpression of MYB, CD4 and HOXA genes. These features, reminiscent of T-cell acute lymphoid leukemia (ALL), suggest the targeting of a common T-myeloid progenitor.

Incidence of 17p deletions and TP53 mutation in myelodysplastic syndrome and acute myeloid leukemia with 5q deletion.

TP53 mutations are frequent in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) with complex karyotype that include del(5q) and are often associated with deletion of 17p. They have also recently been observed in MDS with isolated del(5q). We assessed the incidence of 17p deletion detected by fluorescence in situ hybridization (FISH) and of TP53 mutations detected by direct sequencing and their correlation and prognostic value in 26 MDS and 17 AML with del(5q). In the 20 cases with isolated del(5q) or one additional abnormality, no 17p deletion was found and 3 of the 18 cases analyzed (17%) had TP53 mutation. In the 23 patients with complex karyotype, 17p deletion was suspected by conventional cytogenetics in 15 cases and confirmed by FISH in 10 of them, while TP53 mutation was found in 8 of the 15 patients tested (53%), only five of whom had 17p deletion. In the whole patient series, TP53 mutations were associated with shorter survival (P = 0.07). We confirm the existence of TP53 mutations in 17% of MDS with isolated del(5q). In patients with del(5q) and complex karyotype, FISH and direct sequencing are complementary techniques to analyze TP53 abnormalities. Our findings also suggest that sequencing of the TP53 gene should be included in the study of patients with del(5q) as a single abnormality or in complex karyotype before lenalidomide treatment.

TET2 and TP53 mutations are frequently observed in blastic plasmacytoid dendritic cell neoplasm

918. Dunleavy, K., Hakim, F., Kim, H.K., Janik, J.E., Grant, N., Nakayama, T., White, T., Wright, G., Kwak, L., Gress, R., Tosato, G. & Wilson, W.H. (2005) B-cell recovery following rituximab-based therapy is associated with perturbations in stromal derived factor-1 and granulocyte homeostasis. Blood, 106, 795–802. Hirayama, Y., Kohda, K., Konuma, Y., Hirata, Y., Kuroda, H., Fujimi, Y., Shirao, S., Kobune, M., Takimoto, R., Matsunaga, T. & Kato, J. (2009) Late onset neutropenia and immunoglobulin suppression of the patients with malignant lymphoma following autologous stem cell transplantation with rituximab. Internal Medicine, 48, 57– 60. Lai, G.G., Lim, S.T., Tao, M., Chan, A., Li, H. & Quek, R. (2009) Late-onset neutropenia following RCHOP chemotherapy in diffuse large B-cell lymphoma. American Journal of Hematology, 84, 414–417. Lemieux, B., Tartas, S., Traulle, C., Espinouse, D., Thieblemont, C., Bouafia, F., Alhusein, Q., Antal, D., Salles, G. & Coiffier, B. (2004) Rituximab-related late-onset neutropenia after autologous stem cell transplantation for aggressive non-Hodgkin’s lymphoma. Bone Marrow Transplantation, 33, 921–923. Nitta, E., Izutsu, K., Sato, T., Ota, Y., Takeuchi, K., Kamijo, A., Takahashi, K., Oshima, K., Kanda, Y., Chiba, S., Motokura, T. & Kurokawa, M. (2007) A high incidence of late-onset neutropenia following rituximab-containing chemotherapy as a primary treatment of CD20-positive B-cell lymphoma: a single-institution study. Annals of Oncology, 18, 364–369. Voog, E., Morschhauser, F. & Solal-Céligny, P. (2003) Neutropenia in patients treated with rituximab. New England Journal of Medicine, 348, 2691–2694. Wolach, O., Bairey, O. & Lahav, M. (2010) Late-onset neutropenia after rituximab treatment; case series and comprehensive review of the literature. Medicine (Baltimore), 89, 308–318.

Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method

ZAP-70 expression appears to be a promising prognostic factor in B-cell chronic lymphocytic leukemia. This article describes an optimized flow cytometry method for direct detection of ZAP-70. Background Flow cytometry allows specific assessment of the expression of ZAP-70, a promising new prognostic factor in B-cell chronic lymphocytic leukemia (B-CLL), but suffers from a lack of multicenter standardization. Design and Methods An optimized method for direct detection of ZAP-70 in flow cytometry was tested in a multicenter fashion. Adapted for frozen cells, this method includes a normalization step by addition of B cells from a pool of peripheral blood mononuclear cells collected from normal donors. ZAP-70 expression levels were assessed for 153 patients with typical B-cell chronic lymphocytic leukemia chronic lymphocytic leukemia. Results were expressed as the ratio of ZAP-70 mean fluorescence intensity between B-CLL cells and normal B cells. Results The statistically optimized cut-off of ZAP-70 positivity was a ratio of 1.4. Concordance between ZAP-70 and CD38 expression was 67%. Concordance between the mutational status of IgVH genes and ZAP-70 or CD38 expression was 87% and 65%, respectively. ZAP-70 was significantly expressed in 28%, 54% and 61% of patients with Binet stages A, B and C B-cell chronic lymphocytic leukemia, respectively (p=0.008). The absence of ZAP-70 expression was associated with isolated del(13q14), a cytogenetic abnormality with a good prognosis, while most patients with the del(17p13) poor prognosis cytogenetic marker expressed ZAP-70 (p<10−5). ZAP-70 expression was not related to the other poor prognosis cytogenetic abnormality del(11q22.3) nor to trisomy 12. Conclusions This new technique provides highly reliable results well correlated with the mutational status of IgVH genes, CD38 expression, Binet stage and cytogenetic abnormalities. This robust discriminative technique appears of particular interest for routine diagnosis and assessment of ZAP-70 expression in large, prospective, multicenter therapeutic trials.

Complex karyotype in mantle cell lymphoma is a strong prognostic factor for the time to treatment and overall survival, independent of the MCL international prognostic index

Mantle cell lymphoma (MCL) is usually an aggressive disease. However, a few patients do have an “indolent” evolution (iMCL) defined by a long survival time without intensive therapy. Many studies highlight the prognostic role of additional genetic abnormalities, but these abnormalities are not routinely tested for and do not yet influence the treatment decision. We aimed to evaluate the prognostic impact of these additional abnormalities detected by conventional cytogenetic testing, as well as their relationships with the clinical characteristics and their value in identifying iMCL. All consecutive MCL cases diagnosed between 1995 and 2011 at four institutions were retrospectively selected on the basis of an informative karyotype with a t(11;14) translocation at the time of diagnosis. A total of 125 patients were included and followed for an actual median time of 35 months. The median overall survival (OS) and survival without treatment (TFS) were 73.7 and 1.3 months, respectively. In multivariable Cox models, a high mantle cell lymphoma international prognostic index score, a complex karyotype, and blastoid morphology were independently associated with a shortened OS. Spleen enlargement, nodal presentation, extra‐hematological involvement, and complex karyotypes were associated with shorter TFS. A score based on these factors allowed for the identification of “indolent” patients (median TFS 107 months) from other patients (median TFS: 1 month). In conclusion, in this multicentric cohort of MCL patients, a complex karyotype was associated with a shorter survival time and allowed for the identification of iMCL at the time of diagnosis.