Franck Viguié

Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy preliminary localized in the bone marrow and characterized by its capacity to disseminate. IL-6 and IGF-1 have been shown to mediate proliferative and anti-apoptotic signals in plasmocytes. However, in primary plasma-cell leukemia (PCL) and in end-stage aggressive extramedullar disease, the cytokine requirement for both effects may be not mandatory. This suggests that constitutive activation of signaling pathways occurs. One of the signaling pathways whose deregulation may play an oncogenic role in MM is the phosphatidylinositol 3-kinase (PI 3-K) pathway. In human growth factor-independent MM cell lines OPM2 and RPMI8226, we show that the PI 3-K inhibitors LY294002 and Wortmannin strongly inhibited cell proliferation, whereas inhibition of the mammalian Target Of Rapamycin (mTOR)/P70-S6-kinase (P70S6K) pathway with rapamycin or of the Mitogen-Activated Protein Kinase (MAPK) pathway with PD98059 had minimal effect on proliferation. In both cell lines, constitutive activation of the PI 3-K/Akt/FKHRL-1, mTOR/P70S6K and MAPK pathways was detected. LY294002 inhibited phosphorylation of Akt, FKHRL-1 and P70S6K but had no effect on ERK1/2 phosphorylation, indicating that the PI 3-K and MAPK pathways are independent. IGF-1 but not IL-6 increased phosphorylation of Akt, FKHRL-1 and P70S6K. Purified plasmocytes from four patients with MM and two patients with primary PCL were studied. In three of them including the two patients with PCL, constitutive phosphorylation of Akt, FKHRL-1 and P70S6K was present, inhibited by LY294002 and enhanced by IGF-1. In these patients with constitutive Akt activation, normal PTEN expression was detected. PI 3-K inhibition induced caspase-dependent apoptosis as confirmed by inhibition with the large spectrum caspase inhibitor Z-VAD-FMK and cleavage of pro-caspase-3. Both cell lines spontaneously expressed Skp2 and cyclin D1 proteins at high levels but no p27Kip1 protein. In the presence of LY294002, cell-cycle arrest in G0/G1 was observed, p27Kip1 protein expression was up-regulated whereas the expression of both Skp2 and cyclin D1 dramatically diminished. PI 3-K-dependent GSK-3α/β constitutive phosphorylation was also detected in OPM2 cells that may contribute to high cyclin D1 expression. Overall, our results suggest that PI 3-K has a major role in the control of proliferation and apoptosis of growth factor-independent MM cell lines. Most of the biological effects of PI 3-K activation in these cell lines may be mediated by the opposite modulation of p27Kip1 and Skp2 protein expression. Moreover, constitutive activation of this pathway is a frequent event in the biology of MM in vivo and may be more frequently observed in PCL.

Myeloid cell differentiation arrest by miR-125b-1 in myelodysplasic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34+ cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

Adult biphenotypic acute leukaemia: an entity with poor prognosis which is related to unfavourable cytogenetics and P-glycoprotein over-expression.

Biphenotypic acute leukaemia (BAL) patients represented 8% of the 287 de novo consecutive adult acute leukaemias (23 BAL, 230 acute myeloid leukaemia (AML) and 34 acute lymphoblastic leukaemia (ALL)) referred to our department during the last 4‐year period. Of these 23 BAL patients, 14 patients showed myeloid morphology and nine cases lymphoid morphology according to FAB criteria. There were no differences between lymphoid and myeloid BAL according to clinical and biological presentation and treatment outcome. We confirm the poor prognosis of BAL when compared to AML or ALL seen during the same period of time, in terms of complete remission (47%, 62% and 82% respectively, BAL v AML, NS and BAL v ALL, P = 0.006) and 4‐year overall survival (8.1%, 25.8% and 23.8% respectively, BAL v AML, P = 0.05 and BAL v ALL, P = 0.003). Comparing adult BAL patients with AML patients, we found an increase in poor prognostic factors: CD34+ phenotype (82% v 60% respectively, P = 0.03), unfavourable karyotype (60% v 20%, P < 0.0001) and Pgp over‐expression by RT‐PCR (0.705 v 0.107, P < 0.0001) and flow cytometry (0.824 v 0.391, P = 0.0001). MRP and LRP were not found to be poor prognostic factors. Comparing BAL patients with ALL patients, we found also an increase in poor prognostic factors: age (51 v 39, P = 0.003) and CD34+ phenotype (82% v 50%, P = 0.02). We conclude that BAL patients need a more aggressive treatment procedure, including high‐dose AraC or the use of Pgp modulators for first‐line therapy.

Report of 34 patients with clonal chromosomal abnormalities in Philadelphia-negative cells during imatinib treatment of Philadelphia-positive chronic myeloid leukemia.

Imatinib mesylate (Gleevec®), an inhibitor of the BCR-ABL tyrosine kinase, was introduced recently into the therapy of chronic myeloid leukemia (CML). Several cases of emergence of clonal chromosomal abnormalities after therapy with imatinib have been reported, but their incidence, etiology and prognosis remain to be clarified. We report here a large series of 34 CML patients treated with imatinib who developed Philadelphia (Ph)-negative clones. Among 1001 patients with Ph-positive CML treated with imatinib, 34 (3.4%) developed clonal chromosomal abnormalities in Ph-negative cells. Three patients were treated with imatinib up-front. The most common cytogenetic abnormalities were trisomy 8 and monosomy 7 in twelve and seven patients, respectively. In 15 patients, fluorescent in situ hybridization with specific probes was performed in materials archived before the initiation of imatinib. The Ph-negative clone was related to previous therapy in three patients, and represented a minor pre-existing clone that expanded after the eradication of Ph-positive cells with imatinib in two others. However, in 11 patients, the new clonal chromosomal abnormalities were not detected and imatinib may have had a direct effect. No myelodysplasia was found in our cohort. With a median follow-up of 24 months, one patient showed CML acceleration and two relapsed.

Acute myelogenous leukaemia in the elderly: retrospective study of 235 consecutive patients

A retrospective analysis was performed on 235 elderly acute myelogenous leukaemia (AML) patients aged 60 years or more, consecutively admitted to a single haematological department during a 10‐year period from 1980 to 1989. 46% of patients received only conventional induction chemotherapy. The rate of inclusion in EORTC cooperative clinical trials was significantly lower than for younger patients despite specific protocols proposed for the elderly since 1983, thus confirming the important selection bias of most published series on elderly AML patients. Compared with treatment results in patients <60 years. complete remission (CR) rate was lower (33·3%v 65·4%, P<0·0001), with a marked drop in patients older than 70, and induction death rate was higher (21·3%v 12·5%, P= 0·04). Intrinsic characteristics of leukaemic cells, especially expression of the MDR1 gene, in vitro growth of the leukaemic clonogenic cells and sensitivity to daunorubicin+cytosine arabinoside, did not differ according to age, except that there was a higher incidence of previous myelodysplastic syndromes and a lower incidence of good prognostic cytogenetics in the elderly patients. Thus, treatment failure in elderly AML patients appears to be mainly due to host‐related factors (especially performance status and age < or ±70 years), and to inadequate treatments. Some elderly patients may have been undertreated because of the planned anthracycline dose reduction, resulting in a higher rate of ‘resistant’AML, i.e. patients surviving the induction period without entering into CR, than in younger patients (45·4%v 22·1%, P<0·0001). 11 patients (4·7%) with untreated or ‘resistant’AML survived more than 1 year, while receiving only supportive care. These slowly progressive AML patients were characterized by a good performance status, and lower circulating blast cells and bone marrow blast counts.

Common 4q24 deletion in four cases of hematopoietic malignancy: early stem cell involvement?

We determined bone marrow karyotype at diagnosis in four female acute myeloid leukemia (AML) or myelodysplasia patients, aged between 52 and 56 years. In each case, we observed chromosome rearrangement involving the same 4q24 band. Three patients had a balanced reciprocal translocation as the sole abnormality – t(3;4)(q26;q24), t(4;5)(q24;p16) and t(4;7)(q24;q21) – and the fourth had del(4)(q23q24), +4. We used a set of 4q BAC probes for fluorescent in situ hybridization (FISH) in these four cases. We found a 4q24 submicroscopic deletion in all three translocations, with a common deletion of approximately 0.5 Mb. In three cases, we concluded that rearrangement occurred in an early hematopoietic stem cell, as it was detected, in mosaic with a normal karyotype, in a fraction of remission bone marrow cells, peripheral T and B lymphocytes, malignant lymph node T-lymphoma cells in one case and B-lymphoblastoid cell lines established in two cases. Moreover, one of 10 additional AML patients tested by FISH had a normal karyotype and deletion of one of the commonly deleted probe sequences. A tumor suppressor gene may therefore be involved, especially as two patients developed malignant lymphoma at the same time as myeloid proliferation.

Sequential emergence of MRp- and MDR1-gene over-expression as well as MDR1-gene translocation in homoharringtonine-selected K562 human leukemia cell lines

To investigate the mechanism of resistance to an anti‐neoplastic natural product homoharringtonine (HHT) in leukemic cells, we have established 5 sub‐lines of human myeloid leukemia K562 cells, designated as K‐H30, K‐H100, K‐H200, K‐H300 and K‐H400, which showed progressive resistance to different concentrations of HHT. These sub‐lines were cross‐resistant to daunorubicin, vincristine, etoposide and mitoxantrone, but not to melphalan. Immunofluorescence with monoclonal anti‐Pgp antibody MRK16 and Northern‐blot analysis demonstrated that resistance to HHT is related to the sequential emergence of MRP‐ and MDR1‐gene over‐expression. In the low‐level‐resistant K‐H30 sub‐line, the MDR1 gene was not over‐expressed, but the MRP gene was over‐expressed 2.1‐fold. In the intermediate‐level‐resistant K‐H100 and K‐H200 sublines, both the MRP and the MDR1 genes were over‐expressed. However, in the high‐level‐resistant K‐H300 and K‐H400 sublines, MDR1‐gene over‐expression predominated (20‐ and 21‐fold respectively). On the other hand, GSTπ‐gene expression was decreased in all 5 sub‐lines. Southern‐blot analysis revealed no MRP‐gene amplification in any of the 5 sub‐lines, whereas the MDR1 gene was amplified in the high‐level‐resistant K‐H300 and K‐H400 sub‐lines. The most interesting observation is a homogeneously staining region (HSR) found in chromosome 2 of the K‐H300 and K‐H400 sub‐lines. Chromosome painting and in situ hybridization demonstrated that this HSR was translocated from chromosome 7 and consisted of the amplified MDR1 gene, suggesting that there is a relationship between MDR1‐gene translocation and MDR1‐gene amplification.

In vitro culture of clonogenic leukaemic cells in acute myeloid leukaemia: growth pattern and drug sensitivity

Summary. Bone marrow from 43 of 45 AML patients grew leukaemic colonies in culture with a technique using methyl‐cellulose semi‐solid medium and stimulation with PHA‐leucocyte conditioned medium. Plating efficiency was significantly greater in M4 FAB subtypes than in M1 or M2. The presence of Auer rods in cultured cells and the existence of cytogenetic abnormalities in both fresh and cultured blast cells in one patient confirmed the leukaemic origin of these colonies. These clonogenic cells were closely related to the growth fraction, as demonstrated by a high suicide index and a linear correlation between percentage of bone marrow blasts in S phase and plating efficiency. In vitro CFU‐L sensitivity to cytosine‐arabino‐side (ARA‐C) and to adriamycin (ADR) was tested in 22 patients treated with these two drugs. In the group sensitive in vitro to ARA‐C (10 patients), 70% entered complete remission. In the resistant group (12 patients), only 25% had complete remission while 75% had resistant disease. Eight of 14 patients sensitive to ADR in vitro achieved complete remission, while five were resistant to chemotherapy. On the other hand, six of eight patients resistant in vitro were resistant in vivo. When drug sensitivities to ARA‐C and ADR were cumulated, an excellent in vitro to in vivo correlation was found when the patient was sensitive or resistant to both drugs in vitro.

Mechanisms of genesis of variant translocation in chronic myeloid leukemia are not correlated with ABL1 or BCR deletion status or response to imatinib therapy

Many published studies have indicated that various mechanisms could be involved in the genesis of variant chronic myelogeneous leukemia (CML) translocations. These are mainly one-step or two-step mechanisms, associated or not with deletions adjacent to the translocation junction on der(9) or der(22) chromosomes (or both). Based on the mechanism of genesis, it has been suggested that the complexity may affect the occurrence of ABL1 and BCR deletions (either or both), or may be associated with the CML disease course, and thus could determine the response to imatinib therapy. Through a retrospective molecular cytogenetic study of 41 CML patients with variant Philadelphia chromosome (Ph), we explored the genesis of these variant rearrangements and analyzed the correlation with deletion status and imatinib efficiency. Our results confirmed that the one-step mechanism is the most frequent, evidenced in 30 of 41 patients (73%); 3 patients demonstrated other more complex multistep events and 8 patients (19.5%) harbored ABL1 or BCR deletions that are not significantly associated with the complexity of translocation genesis. We also found no association between one-step, two-step, or multistep mechanisms and the response to imatinib therapy.

Prognostic factors in elderly acute lymphoblastic leukaemia

A retrospective study was performed on 46 unselected acute lymphoblastic leukaemia (ALL) elderly patients aged 60 years or more. Only 50% of these patients were included in the EORTC cooperative clinical trials, thus confirming the important selection bias in most of the published series on elderly ALL patients. 43% of the elderly patients achieved a complete remission (CR). The median survival was 10 months and the 5‐year overall survival was only 7.6±4%. In multivariate analysis, W.H.O. performance status and peripheral blast counts at day 7 were found to significantly influence achievement of CR and survival. In patients with W.H.O. performance status 2, 35% died during induction treatment versus 4% in patients with W.H.O. performance status <2. Patients >70 years old showed a marked drop of the CR rate (27%) compared to those aged 60–69 (67%), and a very high death rate during the induction period (38% versus 4%). This suggests that ALL protocol treatments should be proposed until 70 years in patients with good‐performance status, whereas less intensive treatment should be offered to elderly patients with performance status 2 and/or age 70. Peripheral blast counts at day 7 may help to adjust the treatment during induction phase.