Alicia Domingo

Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)]

Most patients with acute myeloid leukemia (AML) and t(8;21) or inv(16) have a good prognosis with current anthracycline- and cytarabine-based protocols. Tandem analysis with flow cytometry (FC) and real-time RT-PCR (RQ-PCR) was applied to 55 patients, 28 harboring a t(8;21) and 27 an inv(16), including one case with a novel CBFbeta/MYH11 transcript. A total of 31% (n=17) of CR patients relapsed: seven with t(8;21) and 10 with inv(16). The mean amount of minimal residual disease (MRD) detected by FC in relapsed and nonrelapsed patients was markedly different: 0.3 vs 0.08% (P=0.002) at the end of treatment. The mean number of fusion transcript copies/ABLx104 also differed between relapsed and non-relapsed patients: 2385 vs 122 (P=0.001) after induction, 56 vs 7.6 after intensification (P=0.0001) and 75 vs 3.3 (P=0.0001) at the end of chemotherapy. Relapses were more common in patients with FC MRD level >0.1% at the end of treatment than in patients with ⩽0.1%: cumulative incidence of relapse (CIR) was 67 and 21% (P=0.03), respectively. Likewise, using RQ-PCR, a cutoff level of >10 copies at the end of treatment correlated with a high risk of relapse: CIR was 75% for patients with RQ-PCR >10 compared to 21% for patients with RQ-PCR levels ⩽10 (P=0.04). Combined use of FC and RQ-PCR may improve MRD detection, and provide useful clinical information on relapse kinetics in AML patients.

Type I MOZ/CBP (MYST3/CREBBP) is the most common chimeric transcript in acute myeloid leukemia with t(8;16)(p11;p13) translocation

The t(8;16)(p11;p13) fuses the MOZ (MYST3) gene at 8p11 with CBP (CREBBP) at 16p13 and is associated with an infrequent but well‐defined type of acute myeloid leukemia (AML) that has unique morphocytochemical findings (monocytoid blast morphology with erythrophagocytosis and simultaneously positive for myeloperoxidase and nonspecific esterases). RT‐PCR amplification of MOZ/CBP (MYST3/CREBBP) chimera has proved difficult, with four different transcripts found in four reported cases. We studied 7 AML‐t(8;16) patients, 5 with cytogenetically demonstrated t(8;16) and 2 with similar morphocytochemical and immunophenotypical characteristics. Clinically, 3 cases presented as therapy‐related leukemia. Extramedullar involvement was observed at presentation in 2 patients and coagulopathy in 4. The clinicobiological findings confirmed the distinctiveness of this entity. Of note is the erythrophagocytosis in 5 of 7 cases and the immunological negativity for CD34 and CD117 and positivity for CD56. Using a new RT‐PCR strategy, we were able to amplify a specific band of 212 bp in six cases in which sequence analysis confirmed the presence of the previously described MOZ/CBP fusion transcript type I. This is the largest molecularly studied AML‐t(8;16) series, which demonstrates that MOZ/CBP breakpoints are usually clustered in intron 16 of MOZ and intron 2 of CBP. The newly designed single‐round PCR provides a simple tool for the molecular confirmation of MOZ/CBP rearrangement.

Incidence and Prognostic Impact of Secondary Cytogenetic Aberrations in a Series of 145 Patients with Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a mature B‐cell neoplasm with an aggressive behavior, characterized by the t(11;14)(q13;q32). Several secondary genetic abnormalities with a potential role in the oncogenic process have been described. Studies of large MCL series using conventional cytogenetics, and correlating with proliferation and survival, are scarce. We selected 145 MCL cases at diagnosis, displaying an aberrant karyotype, from centers belonging to the Spanish Cooperative Group for Hematological Cytogenetics. Histological subtype, proliferative index and survival data were ascertained. Combined cytogenetic and molecular analyses detected CCND1 translocations in all cases, mostly t(11;14)(q13;q32). Secondary aberrations were present in 58% of patients, the most frequent being deletions of 1p, 13q and 17p, 10p alterations and 3q gains. The most recurrent breakpoints were identified at 1p31‐32, 1p21‐22, 17p13, and 1p36. Aggressive blastoid/pleomorphic variants displayed a higher karyotypic complexity, a higher frequency of 1p and 17p deletions and 10p alterations, a higher proliferation index and poor survival. Gains of 3q and 13q and 17p13 losses were associated with reduced survival times. Interestingly, gains of 3q and 17p losses added prognostic significance to the morphology in a multivariate analysis. Our findings confirm previous observations indicating that proliferation index, morphology and several secondary genetic alterations (3q gains and 13q and 17p losses) have prognostic value in patients with MCL. Additionally, we observed that 3q gains and 17p losses detected by conventional cytogenetics are proliferation‐independent prognostic markers indicating poor outcome.

Regulation of the proapoptotic BH3-only protein BIM by glucocorticoids, survival signals and proteasome in chronic lymphocytic leukemia cells.

Glucocorticoids induce apoptosis in chronic lymphocytic leukemia (CLL) cells through a caspase-dependent mechanism. However, their mechanism of action remains unknown. We have studied the regulation of the proapoptotic BH3-only Bcl-2 interacting mediator of cell death (BIM) in CLL cells. We demonstrate that glucocorticoids upregulate BIM at protein and mRNA levels. We have investigated the ability of different survival signals, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), stromal cell-derived factor-1α (SDF-1α), interleukin 4 (IL-4) and B-cell receptor (BCR) activation, to influence the levels of BIM and its induction by glucocorticoids. TPA downregulates BIMEL by extracellular signal-regulated kinase (ERK)-mediated BIM phosphorylation and further proteasome-mediated degradation. However, SDF-1α and BCR activation induce transient BIM phosphorylation, without protein degradation. Proteasome inhibitors do not modify the levels of BIM with respect to untreated cells. However, they induce apoptosis and inhibit TPA-induced BIMEL degradation, leading to its accumulation. In conclusion, the results implicate BIM in glucocorticoid-induced apoptosis in CLL cells. BIMEL phosphorylation through the ERK pathway targets the protein for proteasomal degradation.

Regulation of Akt/PKB by phosphatidylinositol 3-kinase-dependent and -independent pathways in B-cell chronic lymphocytic leukemia cells: role of protein kinase Cβ

Apoptosis of B cell chronic lymphocytic leukemia (B‐CLL) cells is regulated by the PI‐3K‐Akt pathway. In the present work, we have analyzed the mechanisms of Akt phosphorylation in B‐CLL cells. Freshly isolated cells present basal Akt phosphorylation, which is PI‐3K‐dependent, as incubation with the PI‐3K inhibitor LY294002 decreased Ser‐473 and Thr‐308 phosphorylation in most samples analyzed (seven out of 10). In three out of 10 cases, inhibition of protein kinase C (PKC) inhibited basal Akt phosphorylation. Stromal cell‐derived factor‐1α, IL‐4, and B cell receptor activation induced PI‐3K‐dependent Akt phosphorylation. PMA induced the phosphorylation of Akt at Ser‐473 and Thr‐308 and the phosphorylation of Akt substrates, independently of PI‐3K in B‐CLL cells. In contrast, PKC‐mediated phosphorylation of Akt was PI‐3K‐dependent in normal B cells. Finally, a specific inhibitor of PKCβ blocked the phosphorylation and activation of Akt by PMA in B‐CLL cells. Taken together, these results suggest a model in which Akt could be activated by two different pathways (PI‐3K and PKCβ) in B‐CLL cells.

Multiplex ligation‐dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is the commonest form of leukaemia in adults in Western countries. We performed multiplex ligation‐dependent probe amplification (MLPA) analysis in 50 CLL patients to identify multiple genomic CLL‐specific targets, including genes located at 13q14, 17p13 (TP53), 11q23 (ATM) and chromosome 12, and compared the results with those obtained with fluorescence in situ hybridization (FISH). There was a good correlation between MLPA and FISH results, as most alterations (89%) were detected by both techniques. Only three cases with a low percentage (<25%) of cells carrying the alterations were not detected by MLPA. On the other hand, as MLPA uses multiple probes it identified intragenic or small alterations undetected by FISH in three cases. MLPA also detected alterations in 8q24 (MYC) and 6q25–26. In summary, unlike interphase FISH, MLPA enabled the simultaneous analysis of many samples with automated data processing at a low cost. Therefore, the combination of robust multiplexing and high throughput makes MLPA a useful technique for the analysis of genomic alterations in CLL.

Expression of adhesion molecules in 113 patients with B-cell chronic lymphocytic leukemia: Relationship with clinico-prognostic features

The B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease, its clinical and biological behavior possibly being influenced by surface molecules expressed in B-lymphocytes. These molecules mediate cell adhesion, mobility and homing. Expression of surface adhesion molecules of the integrin family (CD11a/CD18 or LFA-1, CD11c/CD18), of the immunoglobulin-related family (CD54), of the selectin family (CD62L or LAM-1) and the lymphocyte homing receptor (CD44) were analyzed in peripheral cells from 113 B-CLL patients. The association with three prognosis-related parameters (Rai stage, bone marrow pattern and doubling time) was determined. The study included only patients with B-CLL lymphocytes of typical morphology, which always expressed CD5 and CD23. Low expression of integrins, particularly CD18, was associated with advanced disease (Rai stages III-IV) and diffuse bone marrow pattern, even after adjusting for other prognosis-related variables. Expression of CD54 was associated independently with rapid doubling time (less than 12 months). The association persisted after adjusting for stage and bone marrow pattern; CD44 was expressed in all patients. No correlations were found between expression of CD62L and the prognostic variables analyzed. In conclusion, CD54 expression and low CD18 expression are both significantly associated with poor prognostic features.

Incidence and characteristics of lymphoid malignancies in untreated myelodysplastic syndromes.

We have analyzed 1,198 patients with untreated myelodysplastic syndromes (MDS) with two main objectives: (1) to determine the prevalence of lymphoid malignancies (LM) in MDS patients; and (2) to ascertain whether there is some relationship between the MDS subtype and the LM type. In fourteen of 1,198 primary MDS patients (1%) (4 with refractory anemia, 3 with refractory anemia with ring sideroblasts, 2 with refractory anemia with excess of blasts and 5 with chronic myelomonocytic leukemia) a LM was detected. In all cases, the LM was of the B-cell type: 6 cases of chronic lymphocytic leukemia, 5 cases of lymphoplasmacytoid lymphoma, and 3 cases of multiple myeloma. B-cell malignancy did not prevail in any MDS subtype and no correlation was observed between the different varieties of both diseases. In conclusion, in this large series, 1% of the untreated patients with MDS had B-cell malignancy, an association that in most cases is likely to be merely coincidental.

Simultaneous occurrence of cutaneous T cell lymphoma and low-grade B cell lymphoproliferative diseases: A report of two cases

Two previously unreported patients with cutaneous T cell lymphoma associated with systemic low-grade B cell proliferations are presented. The first patient had Waldenströms macroglobulinemia and the second had simultaneous chronic lymphocytic leukemia. The use of immunosuppressive drugs or a genetic predisposition may have contributed to the second malignancy. However, the possibility that malignant helper/inducer T lymphocytes were a factor in the promotion of the proliferation of B cells cannot be excluded.

The potential anticancer agent PK11195 induces apoptosis irrespective of p53 and ATM status in chronic lymphocytic leukemia cells

Background and Objectives The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein (18KDa) (TSPO) ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents. Primary chronic lymphocytic leukemia (CLL) cells overexpress TSPO. The aim of this study was to examine the effects of PK11195 on CLL cells. Design and Methods Using cytometric analysis, we studied the cytotoxic effects of PK11195 on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Western blot and cytometric analyses were used to study the mitochondrial effects of PK11195 on CLL cells. Moreover, we analyzed the cytotoxic effect of PK11195 in patients’ cells with mutated p53 or ATM. Results PK11195 induces apoptosis and had additive effects with chemotherapeutic drugs in primary CLL cells. Other TSPO ligands such as RO 5-4864 and FGIN-1-27 also induce apoptosis in CLL cells. PK11195 induces mitochondrial depolarization and cytochrome c release upstream of caspase activation, and dithiocyana-tostilbene-2,2- disulfonic acid (DIDS), a voltage-dependent anion channel (VDAC) inhibitor, inhibits PK11195-induced apoptosis, demonstrating a direct involvement of mitochondria. CLL cells and normal B cells are more sensitive than T cells to PK11195-induced apoptosis. Interestingly, PK11195 induced apoptosis in CLL cells irrespective of their p53 or ATM status. Interpretation and Conclusions These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL.