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.

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.

New chromosomal alterations in a series of 23 splenic marginal zone lymphoma patients revealed by Spectral Karyotyping (SKY).

Splenic marginal zone lymphoma (SMZL) is a B-cell lymphoproliferative disorder with characteristic clinical, immunophenotypic, cytological and histological features. Some karyotypic abnormalities have been related to this disorder and most of them are usually complex and difficult to define. The aim of present study was to characterize new chromosomal aberrations involved in this disease. We performed conventional banding cytogenetics and Spectral Karyotyping (SKY) technique in 23 patients diagnosed with SMZL having a complex karyotype among a series of 160 SMZL cases. Del(7)(q22-q32) and trisomy 3/3q were the most common chromosomal aberrations. In addition, new translocations involving chromosomes 3, 6, 8, 9, 12 and 14q32 region were detected.

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. Table 1. Characteristics of the patients with chronic lymphocytic leukemia. 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.

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

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-1alpha, 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 PKCbeta 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 PKCbeta) in B-CLL cells.

Bcl-2 inhibitors induce apoptosis in chronic lymphocytic leukemia cells.

OBJECTIVE: Antiapoptotic Bcl-2 is overexpressed in most cases of chronic lymphocytic leukemia (CLL). The inhibition of the antiapoptotic Bcl-2 proteins is an attractive strategy for either restoring normal apoptotic process in cancer cells or making these cells more susceptible to conventional chemotherapy. We studied the effect of Bcl-2 inhibitors on the viability of cells from CLL and other mature B-cell neoplasms. MATERIALS AND METHODS: We studied the cytotoxic effects of four nonpeptidic cell-permeable Bcl-2 inhibitors (HA14-1, antimycin A, GX15-003, and GX15-070) on B cells from patients with CLL, mantle cell lymphoma (MCL), and splenic marginal zone lymphoma (SMZL). Moreover, we analyzed the effect of these inhibitors in combination with fludarabine or chlorambucil. RESULTS: HA14-1 induced apoptosis with an EC50 lower than 50 microM in 26 of the 36 CLL samples analyzed. The mean EC50 for these sensitive patients was 23 +/- 2 microM. Antimycin A induced apoptosis in 13 of the 18 CLL samples analyzed. Both HA14-1 and antimycin A induced cytochrome c release from mitochondria and caspase-3 activation. Moreover, HA14-1 induced apoptosis in peripheral cells from MCL and SMZL. HA14-1 also induced apoptosis in CLL samples with alterations in p53 or ATM. Finally, GX compounds induced apoptosis in B cells from 9 of the 11 CLL samples tested. The combination of either HA14-1, antimycin A, or GX compounds with fludarabine or chlorambucil had additive cytotoxic effects on CLL cells. CONCLUSION: Bcl-2 inhibitors induce apoptosis in CLL cells ex vivo and could be used in CLL as monotherapy or given in combination with current chemotherapy.

Translocation t(9;14)(p13;q32) in cases of splenic marginal zone lymphoma.

Translocation t(9;14)(p13;q32) involving PAX5 and IGH genes was first described in lymphoplasmacytic lymphoma. New data suggest that this translocation is not restricted to a specific morphologic subtype but occurs in other B-cell lymphomas. We present three cases with a diagnosis of splenic marginal zone lymphoma and t(9;14) confirmed by fluorescent in situ hybridization.

Clonal cytogenetic abnormalities in patients with chronic myeloid leukemia in complete cytogenetic response to imatinib mesylate.

The emergence of clonal chromosomal abnormalities in Philadelphia-negative cells during treatment with imatinib in patients with Philadelphia-positive chronic myeloid leukemia has been reported. We add information to this issue presenting a series of 29 patients in complete cytogenetic response after imatinib treatment, three of whom developed clonal aberrations.

Meningeal lymphomatosis as the first manifestation of splenic marginal zone lymphoma.

Meningeal lymphomatosis (ML) as the first manifestation of a splenic marginal zone lymphoma (SMZL) is rare. The descriptions of only 2 cases with this complication, one of which had ML as the first manifestation, have been published to date. We describe a 53-year-old man, an ex-smoker, who presented with transitory episodes of bilateral loss of visual acuity. On examination, only papilledema and splenomegalia were observed. The hemogram showed a predominance of lymphocytes with a villous morphology. Cytochemical staining and an immunophenotypic analysis revealed a positive reaction to tartrate-sensitive acid phosphatase and B-lineage markers (CD19+, CD20+, CD79b+, surface immunoglobulin 3 expression, immunoglobulin D+, CD5-, CD23-, CD10-, CD25-, CD103-, and CD11c-). Magnetic resonance imaging of the brain showed tumoral infiltration in both optic nerves and in the cervicodorsal meninges. The cerebrospinal fluid examination revealed significant pleocytosis, and all lymphocytes had a phenotype identical to that of the peripheral blood, confirming the presence of ML. The bone marrow section also showed lymphocytes with an immunophenotype identical to that of the peripheral blood.A splenectomy confirmed the SMZL diagnosis. Treatment with corticosteroids and intrathecal chemotherapy was administrated; however, the response was not good, and the patient died. In this report, we discuss the other 2 cases and ML in B-cell chronic lymphoproliferative disorders.

Changes in biochemical, hematological and immunological profiles after low-dose intravenous immunoglobulin administration in patients with hypogammaglobulinemia.

We assessed the immediate effect of intravenous immunoglobulins (IVIG) on the biochemical, immunological and hematological profiles in patients with hypogammaglobulinemia. Over a period of three months, patients with antibody deficiencies, who had been established on stable IVIG treatment as replacement therapy in our hospital, were enrolled in the study. Participants underwent pre-therapy determinations of their biochemical, immunological and hematological profiles. Laboratory determinations were repeated after completion of IVIG infusions. Over the study period, fourteen patients were enrolled and a total of 34 pre- and post-IVIG infusion determinations were performed and results compared. We found that low-dose IVIG treatment in patients with hypogammaglobulinemia results in post-infusion biochemical and hematological changes, as follows: an increase in total protein concentration and a reduction in albumin, total cholesterol, sodium and alkaline phosphatase concentrations as well as lymphocyte and platelet counts. All these biochemical and cellular changes seems to be transient, since they were not observed in the subsequent pre-infusion determination. However, in other patient populations, some of these changes might differ, depending on the dose of IVIG administered and the baseline condition and immunological status of the patient.

Acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): Clinical and biological features and comparison with other acute myeloid leukemias with cytogenetic aberrations involving long arm of chromosome 3.

OBJECTIVES: To compare, from a biological and clinical perspective, a significant group of patients with AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) with another group of AML carrying different abnormalities of 3q at q21 or q26, the latter named as the AML abn(3q) group. METHODS: We developed a national survey with the participation of 13 Spanish hospitals, and retrospectively reviewed (from 1990 to 2010) these subtypes of AML. Fifty-five patients were collected: 35 with AML inv(3)/t(3;3) and 20 with AML abn(3q). A data collecting page that included main features at diagnosis, therapeutic approach and response, and survival variables, was distributed and completed. RESULTS: We did not find significant differences in sex, age, history of myelodysplastic syndrome or chemo-/radiotherapy, clinical presentation, WBC and platelet counts, hemoglobin level, blasts immunophenotype, serum lactatedehydrogenase, peripheral blood and bone marrow cellular dysplasia, and bone marrow biopsy findings. Although the association with monosomy 7 was significantly more frequent in AML inv(3)/t(3;3), this did not seem to influence outcome. The lack of response to the different modalities of treatment and the aggressive course of the disease were the standard in both cohorts of patients. DISCUSSION: Although not yet recognized by the World Health Organization classification, our results are in agreement with the findings of other authors, who include both subsets of AML together in the same group of adverse prognosis. CONCLUSION: In an attempt to simplify and bound entities with similar genetic background and clinical behavior, it would be desirable to bring together both subgroups of AML in a single section.

Immunophenotypic findings in acute myeloid leukemia with FLT3 internal tandem duplication.

BACKGROUND AND OBJECTIVES: The biological characteristics and the prognostic significance of the internal tandem duplication of the FLT3 (FLT3/ITD) were investigated in a series of de novo acute myeloid leukemia (AML) patients. One hundred and fifty-six adult patients with AML were included in the study. FLT3/ITD was detected in 41 (26%) patients (FLT3/ITD(+)). DESIGN AND METHODS: The main differences observed between the groups with and without FLT3/ITD: a higher leukocyte count, a raised percentage of a normal karyotype and a more frequent M5 FAB diagnosis in the FLT3/ITD(+) patients. As regards the immunophenotype characteristics the FLT3/ITD(+) group very often expressed monocytic markers (CD36 and CD11b) and less commonly immature markers (CD34 and CD117). A promyelocytic-like immunophenotype pattern was also detected in a minority of these patients(4/36). RESULTS: The FLT3/ITD(+) patients had a shorter overall survival, a shorter event-free survival and a higher probability of relapse. Minimal residual disease (MRD) was investigated in the FLT3/ITD(+) patients using flow cytometry. This technique had a sensitivity of 62% and a specificity of 83% in relapse prediction. Minimal residual disease analysis was hampered by the low number of patients with detectable aberrant immunophenotype. INTERPRETATION AND CONCLUSIONS: A high frequency of changes in the phenotype and/or genotype pattern between diagnosis and relapse was detected (5/6). FLT3/ITD is a frequent molecular lesion in de novo adult AML and seems to be associated with a monocytic differentiation, a high leukocyte count and a poor prognosis. Immunophenotype and genotype patterns observed at relapse suggest that the FLT3/ITD(+) blasts may be genetically unstable and prone to clonal evolution. FLT3/ITD may not be a suitable target for minimal residual disease studies.

Genetic characterization of Sézary's syndrome by conventional cytogenetics and cross-species color banding fluorescent in situhybridization.

BACKGROUND AND OBJECTIVES: Sezary's syndrome is a peripheral T-cell neoplasm characterized by a pruritic exfoliative or infiltrated erythroderma, lymphadenopathies, and atypical T lymphocytes in the peripheral blood. Cytogenetic studies are scarce. This study was designed to increase cytogenetic information on this disorder. DESIGN AND METHODS: Peripheral blood samples were collected from 21 patients with Sezary's syndrome (10 men, 11 women, mean age 64 years) and analyzed by conventional cytogenetics (72-hr cultures with phytohemagglutinin). For a better characterization of multiple chromosomal rearrangements, cross-species color banding (RxFISH) was used in four cases. RESULTS: Fifteen (71.4%) of the 21 cases showed cytogenetic aberrations, with the karyotype being complex in 14. Among the 15 patients with an abnormal karyotype, 8 presented a diploid/near-diploid karyotype and 7 a near-tetraploid karyotype. The chromosomes most frequently involved were 1, 6, 8, 9, 10, 11, and 17. The most common structural rearrangements affected 1q, 2q, 6q23-27, and 8q22. Monosomies of chromosomes 9 and 10 and trisomies of chromosome 18 were recurrently observed. A statistical trend between abnormal and complex karyotypes, the presence of monosomy 10, the number of Sezary cells, and a decreased overall survival was observed. RxFISH technology allowed the description of 27 previously undetected chromosomal abnormalities. INTERPRETATION AND CONCLUSIONS: Abnormal karyotypes, particularly complex karyotypes, were frequently detected in patients with Sezary's syndrome. Monosomy 10 was the most frequent recurrent cytogenetic marker (73% in abnormal cases). There was a high diversity of chromosomal breakpoints. RxFISH is a useful novel technology for redefining complex karyotypes.

Prospective study of clinical and biological prognostic factors at diagnosis in patients with early stage B-cell chronic lymphocytic leukemia.

Retrospective series have reported many clinical and biological significant prognostic factors in chronic lymphocytic leukemia (CLL). We describe a prospective cohort of 135 patients with CLL homogeneously studied at diagnosis for prognostic factors. Biological variables analyzed were CD38 and ZAP-70 expression, fluorescence in situ hybridization (FISH) for 13q-, +12, 11q-, and 17p-, and conventional cytogenetics. Univariate and multivariate analysis for progression-free survival (PFS) were performed in patients with early stage (Rai 0-1) CLL. CD38 was positive in 42 (31.6%) patients and ZAP-70 in 47 (35.9%). The most frequent FISH finding was isolated 13q- in 50 (38.5%) patients, and 17p- -was found in 11 (8.4%). Among 135 patients, 114 (84.4%) were Rai 0-1 at diagnosis and 39 (28.9%) presented adenopathies. With a median follow-up of 39 months, the presence of lymphadenopathy in patients with Rai 0-1 stage CLL was the only significant variable for predicting PFS in multivariate analysis (odds ratio [OR] 7, 95% confidence interval [CI] 2.2-22, p = 0.001). When only biological factors were analyzed, CD38 expression (OR 3.2, 95% CI 1.1-9.3, p = 0.03) and 17p- (OR 3.5, 95% CI 0.95-13.1, p = 0.05) correlated with worse PFS. A longer follow-up is necessary to analyze the prognostic value of these variables regarding overall survival.

[Persistent polyclonal B-cell lymphocytosis: study of 35 cases]. / Linfocitosis B policlonal persistente: estudio de 35 casos.

BACKGROUND AND OBJECTIVES: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare entity, presenting especially in adult smoker women. It is characterized by an increase of serum IgM, DR7-HLA haplotype, cytogenetic abnormalities and multiple IgH/BCL-2 rearrangements. To date, it has not been elucidated whether this is a benign or premalignant disorder. We analyzed the PPBL characteristics with especial attention to its evolution. PATIENTS AND METHODS: Thirty-five PPBL patients from 5 hospitals in Catalonia were retrospectively analyzed. A simultaneous morphologic review of the blood smears was performed by members of the GCCH in a 16 multiple-observer optic microscope. Clinical and biological data were also analyzed. RESULTS: PPBL presents in the majority of cases with persistent polyclonal B-cell lymphocytosis and affects primarily smoker women. The morphologic hallmark, in absence of viral infections, is the presence of activated lymphocytes with bilobulated and/or cleaved nuclei, and nuclear pockets in the ultrastructural study. Increased serum IgM, HLA-DR7 haplotype, chromosomal abnormalities such as i(3)(q10) and multiple IgH/BCL-2 rearrangements were detected. Thirty-four out of 35 patients are alive after a median follow up of 70.7 months. One patient died because of lung adenocarcinoma and another developed a follicular lymphoma without relation to PPBL. CONCLUSIONS: PPBL has an asymptomatic and stable evolution, although it frequently presents genetic abnormalities. It remains unknown whether it is a premalignant entity, similar to monoclonal gammopathies of unknown significance. Hence, accurate cytologic diagnosis and follow-up are essential.

MDM2 antagonists activate p53 and synergize with genotoxic drugs in B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. Several drugs currently used in the therapy of B-CLL act, at least partially, through activation of the p53 pathway. Recently, nongenotoxic small-molecule activators of p53, the nutlins, have been developed that inhibit p53-MDM2 binding. We have investigated the antitumor potential of nutlin-3 in B-CLL and find that it can activate the p53 pathway and effectively induce apoptosis in cells with wild-type p53, including cells with dysfunctional ataxia telangiectasia mutated, but not mutant p53. Nutlin-3 stabilized p53 and induced p53 target genes, including MDM2, p21(CIP1), PUMA, BAX, PIG3, and WIG1. Nutlin-3 synergized with the genotoxic drugs doxorubicin, chlorambucil, and fludarabine, but not with acadesine, which induces p53-independent apoptosis. Normal human T cells showed lower sensitivity to nutlin-3 than B-CLL cells and no synergism with the genotoxic drugs. These results suggest that MDM2 antagonists alone or in combination with chemotherapeutic drugs may offer a new treatment option for B-CLL.

Use of reagent strips for the rapid diagnosis of spontaneous bacterial empyema.

GOALS: To assess the utility of reagent strips for rapid diagnosis of spontaneous bacterial empyema in cirrhotic patients with hepatic hydrothorax. BACKGROUND: Analysis of ascitic fluid using reagent strips is a useful diagnostic test for spontaneous bacterial peritonitis. METHODS: A reagent strip for leukocyte esterase designed for the testing of urine was used to evaluate pleural fluid analysis in 47 nonselected thoracenteses in 28 cirrhotic patients with hepatic hydrothorax. RESULTS: Twelve spontaneous bacterial empyemas were diagnosed. Simultaneous spontaneous bacterial peritonitis was present in 7 of 10 cases in which ascites fluid was analyzed. When a test result of 3 or 4 was considered positive, sensitivity was 83% (10 of 12), specificity was 100% (35 of 35), and positive predictive value was 100%. When result of 2 or more was considered positive, sensitivity was 92% (11 of 12), specificity was 80% (28 of 35), and negative predictive value was 97%. CONCLUSION: Analysis of pleural fluid with reagent strips is a rapid, easy to use, and inexpensive tool for the diagnosis of spontaneous bacterial empyema in cirrhotic patients. A positive result should be considered an indication for antibiotic therapy.

A new translocation t(11;13)(q13;q14) in a mature B-cell neoplasm.

We present the case of a man affected by an unclassified mature B-cell neoplasm with a bone marrow culture stimulated with TPA showing a 46,XY, t(11;13)(q13;q14)[14]/46,XY [6] karyotype. Fluorescent in situ hybridization demonstrated that the BCL1 oncogene is translocated (not rearranged) to chromosome band 13q14 and that a copy of D13S319 locus is deleted. To our knowledge, this is the first reported case with this novel cytogenetic aberration.

Rapid diagnosis of spontaneous bacterial peritonitis by use of reagent strips.

We studied the use of reagent strips for diagnosis of spontaneous bacterial peritonitis (SBP) in cirrhotic patients with ascites. A reagent strip for leukocyte esterase designed for the testing of urine with a colorimetric 5-grade scale (0 to 4) was used to evaluate ascitic fluid in 228 nonselected paracentesis performed in 128 cirrhotic patients. We diagnosed 52 SBP and 5 secondary bacterial peritonitis by means of polymorphonuclear cell count and classical criteria. When we considered positive a reagent strip result of 3 or 4, sensitivity was 89% (51 of 57), specificity was 99% (170 of 171), and positive predictive value was 98%. When we considered positive a reagent strip result of 2 or more, sensitivity was 96% (55 of 57), specificity was 89% (152 of 171), and negative predictive value was 99%. In conclusion, the use of reagent strips is a rapid, easy to use, and inexpensive tool for diagnosis of ascitic fluid infection. A positive result should be an indication for empirical antibiotic therapy, and a negative result may be useful as a screening test to exclude SBP.