Efficacy and safety of autologous hematopoietic cell transplantation in elderly patients with multiple myeloma: a retrospective national multi-site cohort study.

We aimed to test the efficacy and toxicity of autologous hematopoietic cell transplant (HCT) in Multiple Myeloma (MM) patients aged ≥65 years compared to patients aged 60-64. Two hundred twenty consecutive patients (age ≥65, n = 87) with MM aged 60 and above, who underwent HCT as part of an upfront MM treatment, at four Israeli centers between 2000 and 2014 were included. A melphalan dose of 200 mg/m2 was more frequent in the 60-64 age group vs. the ≥65 age group (77 vs. 57%, p = 0.002). There were no differences between groups in median day of neutrophil engraftment, incidence of infections, grades 3-4 mucositis, cardiovascular events, or non-relapse mortality at 100 days post HCT (4.7, vs. 5%, p = 0.9). A similar rate of improvement in response level was observed (36, vs. 35%, p = 0.87). At 3 years post HCT progression-free survival (PFS) was higher in the 60-64 age group (42 vs. 29%, p = 0.04); however, it was no longer so after adjustment for disease status prior to HCT (p = 0.49). In a Multivariate analysis, melphalan doses and age did not predict PFS. There was no difference in overall survival (OS) between age groups (p = 0.2). We conclude that toxicity profile, response, PFS, and OS of HCT in aged ≥65 patients with myeloma is similar to patients aged 60-64.

Divergence in CD19-mediated signaling unfolds intraclonal diversity in chronic lymphocytic leukemia, which correlates with disease progression.

Emerging data on intraclonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. In this study, we report intraclonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into CD19-responsive (CD19-R) and -nonresponive subpopulations. Engagement of CD19 by anti-CD19 Ab rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA, exhibited distinct morphological features, and were preferentially abolished in rituximab-treated patients. Both subpopulations reacted to sIgM stimulation in a similar manner and exhibited similar levels of Akt and Erk phosphorylation, pointing to functional signaling divergence within the BCR. CD19 unresponsiveness was partially reversible, where nonresponding CD19 cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19-signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow-derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with >15.25% of the CD19-R cell fraction had a shorter median time to treatment compared with patients with <15.25% of CD19-R cell fraction. In conclusion, divergence in CD19-mediated signaling unfolds both interpatient and intraclonal diversity in CLL. This signaling diversity is associated with physiological implications, including the location of the cells, their responses to anti-CLL therapeutics, and disease progression.

Imatinib plasma trough levels in chronic myeloid leukaemia: results of a multicentre study CSTI571AIL11TGLIVEC.

Imatinib has been accepted as frontline treatment for patients with chronic myeloid leukaemia (CML), and patients generally receive doses ranging from 400 to 800 mg/day. Previous studies have demonstrated that maintaining imatinib plasma levels (IMPLs) >1000 ng/mL leads to improved responses and long-term outcomes. However, IMPLs vary among patients because of factors such as drug-drug interactions, adherence, toxicity and differential levels of expression of cellular efflux/influx proteins. In this study, IMPLs were analysed in 191 patients with CML and were compared with achievement of molecular and cytogenetic responses (CyR). IMPLs were also correlated with renal and hepatic dysfunction. Additionally, self-reported adherence was monitored. The median and mean IMPLs were 994 ng/mL and 1070 ± 686 ng/mL, respectively, with 96 patients (50%) achieving plasma levels >1000 ng/mL. Self-reported patient compliance was 98%. Patients who achieved a complete CyR (CCyR) had significantly higher IMPLs (1078 ± 545 ng/mL) than those without CyR (827 ± 323 ng/mL, p = 0.045). When grouped together, patients who achieved a CCyR or partial CyR had significantly higher IMPLs than patients who achieved a minimal CyR or did not achieve a CyR (1066 ng/mL vs 814 ng/mL, p = 0.002). There was no significant difference observed in the IMPLs between patients who achieved molecular responses (n = 177) on treatment (major molecular response, 976 ± 385 ng/mL versus complete molecular response, 1138 ± 809 ng/mL, p = 0.387). Mean IMPLs were similar in patients with or without renal or hepatic impairment. Overall, this study confirmed previous reports that higher IMPLs correlate with clinical responses and demonstrated that imatinib exposure did not differ in patients with or without liver and/or renal dysfunction. The use of IMPL testing and patient diaries may be practical tools for the management of imatinib therapy in patients with CML.

Increased CD39 expression on CD4(+) T lymphocytes has clinical and prognostic significance in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes proinflammatory extracellular ATP, generates anti-inflammatory adenosine, and also protects regulatory T cells from ATP-induced cell death. In this study, we investigated the clinical significance of CD39 expression on CD4(+) T cells in 62 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4(+)CD39(+) lymphocytes were increased in the peripheral blood of patients with CLL and correlated with the advanced stage of disease. CD4(+)CD39(+) cells were also higher in patients with CLL, who needed therapeutic intervention, and in those who had unmutated immunoglobulin heavy chain variable region gene, were ZAP70(+) or had ß2-microglobulin levels of >3 g/L. There were more CD4(+)CD39(+) lymphocytes in the bone marrow compartment than in the peripheral blood, and in vitro studies showed that CD39 can be induced on CD4(+) cells by exposure to ATP or indirectly, following B cell receptor engagement. This may support the notion that the leukemic cells contribute to create an immune-subversive environment, and perhaps to a poorer prognosis. CD39(+) may also serve as a future target for the development of novel therapies with immune-modulating antitumor agents in CLL.

Mimicking the haematopoietic niche microenvironment provides a novel strategy for expansion of haematopoietic and megakaryocyte-progenitor cells from cord blood.

Severe neutropenia and protracted thrombocytopenia remain serious clinical problems following cord blood transplantation (CBT) due to the paucity of stem and progenitor cells in the grafts. Administration of ex-vivo expanded megakaryocyte progenitor cells may facilitate platelet production. We propose a novel strategy to expand these rare cells ex-vivo, from a small portion of the cord blood (CB) unit, using fibronectin (FN), a major component of hematopoietic niches, combined with cytokines, including thrombopoietin and the hematopoietic stress-associated acetylcholinesterase readthrough peptide (ARP). Application of multiple gates and high definition flow cytometry enabled clear resolution of expanded hematopoietic stem/precursor cells (HSPC) and megakaryocyte progenitors (Mk-p) and their early subsets while eliminating positively stained non-relevant cells. FN increased viability, expansion of all CD34(+) HSPC populations and Mk-p. The combination of FN + thrombopoietin + ARP maintained and expanded very early myeloid and thrombopoietic precursors, increased the proliferation of megakaryocyte, granulocyte-macrophage and multilineage colony-forming progenitors and supported Mk maturation as measured by ploidy and glycoprotein IIb/IIIa expression by quantiative reverse transcription polymerase chain reaction. This approach, which involves expanding HSPC and Mk precursors from a small portion of the CB unit, without sacrificing the coveted stem cells, may lead to improved cell therapy modalities to facilitate earlier myelopoiesis and platelet production post-CBT.

Mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.

Multiple myeloma is characterized by the malignant growth of immunoglobulin producing plasma cells, predominantly in the bone marrow. The effects of primary human mesenchymal stromal cells on the differentiation phenotype of multiple myeloma cells were studied by co-culture experiments. The incubation of multiple myeloma cells with bone marrow-derived mesenchymal stromal cells resulted in significant reduction of the expression of the predominant plasma cell differentiation markers CD38 and CD138, and cell surface immunoglobulin light chain. While the down-regulation of CD138 by stromal cells was completely dependent on their adhesive interactions with the multiple myeloma cells, interleukin-6 induced specific down-regulation of CD38. Mesenchymal stromal cells or their conditioned media inhibited the growth of multiple myeloma cell line, thereby reducing the overall amounts of secreted light chains. Analysis of primary multiple myeloma bone marrow samples reveled that the expression of CD38 on multiple myeloma cells was not affected by adhesive interactions. The ex vivo propagation of primary multiple myeloma cells resulted in significant increase in their differentiation markers. Overall, the data indicate that the bone marrow-derived mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.

Tumorigenic potential and disease manifestations of malignant B-cell variants differing in their fibronectin adhesiveness.

OBJECTIVE: Microenvironmental interactions of malignant B cells can modulate various in vitro physiological responses, including proliferation, migration, apoptosis, and drug resistance. Disease manifestations of human malignant B-cell variants, isolated based on their differential interactions with fibronectin, were examined in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. MATERIALS AND METHODS: Disease manifestations were assessed by pathological examinations and skeletal imaging of NOD/SCID mice injected with malignant B-cell variants. Dissemination patterns were analyzed by whole-body real-time imaging of mice injected with fluorescence-labeled malignant cells. RESULTS: Initial dissemination patterns and dynamics of both high (type A) and low (type F)-adherent variants, following intravenous inoculation, were similar. Both cell types reached the spleen and liver within 30 minutes after injection, then increasingly accumulated within the bone marrow. Mice injected with type-A cells developed multiple myeloma-like disease within the bone marrow, with multiple lytic bone lesions. In contrast, type-F cells displayed low tumorigenic capacity in spite of their efficient homing to the bone marrow niche. In addition, type-A cells grew as extramedullary tumors in some of the intravenous-inoculated mice, and formed solid tumors following subcutaneous injection. Both cell variants retained their characteristics surface markers following in vivo outgrowth as tumors, indicating that at least some of their properties are relatively stable. CONCLUSION: Data suggest that the differential tumorigenicity of B-cell adhesive variants is attributable to the capacity of type-A cells to survive and proliferate within the bone marrow, rather than to different initial dissemination of the two cell populations.

Functional CXCR4-expressing microparticles and SDF-1 correlate with circulating acute myelogenous leukemia cells.

Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of acute myelogenous leukemia (AML). Cellular microparticles, submicron vesicles shed from the plasma membrane of various cells, are also associated with human pathology. In the present study, we investigated the putative relationships between the SDF-1/CXCR4 axis and microparticles in AML. We detected CXCR4-expressing microparticles (CXCR4(+) microparticles) in the peripheral blood and bone marrow plasma samples of normal donors and newly diagnosed adult AML patients. In samples from AML patients, levels of CXCR4(+) microparticles and total SDF-1 were elevated compared with normal individuals. The majority of CXCR4(+) microparticles in AML patients were CD45(+), whereas in normal individuals, they were mostly CD41(+). Importantly, we found a strong correlation between the levels of CXCR4(+) microparticle and WBC count in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, levels of functional, noncleaved SDF-1 were reduced in these patients compared with normal individuals and also strongly correlated with the WBC count. Furthermore, our data indicate NH(2)-terminal truncation of the CXCR4 molecule in the microparticles of AML patients. However, such microparticles were capable of transferring the CXCR4 molecule to AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID/beta2m(null) mice. The CXCR4 antagonist AMD3100 reduced these effects. Our findings suggest that functional CXCR4(+) microparticles and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML variable.

Heparanase modulates heparinoids anticoagulant activities via non-enzymatic mechanisms.

A key element for the physiological restriction of blood coagulation at the endothelial cell surface is its non-thrombogenic property, mainly attributed to cell surface heparan sulfate proteoglycans. Heparanase is an endo-beta-D-glucuronidase with specific heparan sulfate degrading activity, which is produced and stored in platelets, and is released upon their activation. We examined the effects of heparanase pro-enzyme on coagulation functions, predominantly under physiological conditions. While heparanase pro-enzyme does not directly affect coagulation protein activities, it has profound effects on heparinoid-mediated regulation of coagulation responses, apparently via mechanisms that do not involve its enzymatic activity. Heparanase pro-enzyme reverses the anti-coagulant activity of unfractionated heparin on the coagulation pathway as well as on thrombin activity. In addition, heparanase pro-enzyme abrogated the factor X inhibitory activity of low-molecular-weight heparin (LMWH). The pro-coagulant effects of the non-active heparanase were also exerted by its major functional heparin-binding peptide. Finally, the effects of heparanase on the activity of factor VII activating protease that is auto-activated by heparinoids indicated a complete antagonistic action of heparanase in this system. Altogether, heparanase pro-coagulant activities that were also demonstrated in plasma samples from patients under LMWH treatment, point to a possible use of this molecule as antagonist for heparinoid treatment.

Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome.

BACKGROUND: ZAP-70 has emerged as a potential pivotal prognostic marker for patients with chronic lymphocytic leukemia (CLL), which could replace immunoglobulin heavy chain mutation status. Although several flow cytometry assays have been described for assessing ZAP-70 in CLL, certain technical and scientific issues remain unsolved, which have prevented results of this crucial test from being reported, even in the best routine flow cytometry laboratories. In this report, we aimed to solve some of these issues by providing a computerized quantitative flow cytometric assay for ZAP-70 within the entire CLL population, which would be easy to perform and enable standardization between laboratories. METHODS: Intracellular ZAP-70 levels in CLL and normal B cells were assessed by molecules of equivalent soluble fluorochrome (MESF), employing Quantum FITC MESF calibration beads to establish a standard curve relating channel value to fluorescence intensity in MESF units and the QuickCal v. 2.2 program (www.bangslabs.com) and clinical relevance of the data was determined. RESULTS: The average ZAP-70 expression value in the CD19(+)/CD5(+) cells from 35 CLL patients was 103,701 MESF when compared with 12,621 MESF in B cells from 20 normal blood samples. "Low" and "high" ZAP-70 CLL subgroups were defined. Patients with "high ZAP-70 MESF" CLL had a shorter time to disease progression (P = 0.0005) and a more advanced clinical stage (P = 0.0018) when compared with patients in the "low ZAP-70 MESF" CLL subgroup. CONCLUSIONS: This quantitative analysis method can be employed to obtain a more specific and highly accurate assessment of ZAP-70 levels in CLL cells. The method can easily be standardized, in any routine flow laboratory, thereby improving reproducibility and reliability of ZAP-70 analysis.

Fatal interstitial pneumonitis related to rituximab-containing regimen.

Rituximab, a chimeric anti-CD20 monoclonal antibody, is commonly being used to treat indolent and aggressive B-cell non-Hodgkin's lymphoma. Rituximab is considered a relatively safe drug, but recently, severe and fatal adverse effects related to this drug have been reported. In this regard, we report an 80-year-old patient with follicular grade 3 non-Hodgkin's lymphoma who developed a fatal interstitial pneumonitis related to treatment with a rituximab/CHOP (cyclophosphamide/doxorubicin/vincristine/prednisone) regimen. The pneumonitis was diagnosed on a routine midtreatment positron emission tomography/computed tomography scan when the patient was almost asymptomatic. Pulmonary deterioration occurred as the treatment with rituximab/CHOP was continued. In this article, we also review the literature on rituximab-associated pneumonitis, and we discuss the differential diagnosis with cyclophosphamide-induced lung injury.

The role of the anti-CD20 antibody rituximab in hematopoietic stem cell transplantation for non-Hodgkin's lymphoma.

Autologous hematopoietic stem cell transplantation is widely accepted as effective therapy for patients with relapsed aggressive B-cell non-Hodgkin's lymphoma, and to a lesser extent, for indolent and mantle cell lymphoma, resulting in prolonged disease-free survival. Despite these advances, disease recurrence remains a problem and a major clinical challenge. Allogeneic transplantation has also been increasingly utilized in patients with relapsed aggressive and indolent lymphoma but is associated with high toxicity and graft-versus-host disease. Recently, nonmyeloablative preparatory regimens have shown encouraging results, attributed to graft-versus-lymphoma effects. Rituximab, a monoclonal antibody targeted against the CD20 antigen, is a potent therapeutic tool with documented efficacy in B-cell lymphomas. It is effective when used alone or in combination with chemotherapy, resulting in a significantly improved response rate compared with chemotherapy alone, in both aggressive and indolent lymphomas. Increasing evidence suggests that rituximab is also effective at in vivo purging prior to transplantation and may prevent relapse by eradication of residual disease when administered after transplantation. This review summarizes the available data on the use of rituximab and discusses the current evidence for its role in conjunction with auto- and allotransplantation.

Integration of automated morphological features resolves a distinct group of atypical chronic lymphocytic leukemias with chromosomal aberrations.

Automated morphological assessment of peripheral blood slides has become an important modality facilitating characterization and quantification of cells in a uniform, fast and robust manner. In this study, we evaluated the morphological diversity in peripheral blood films of 94 chronic lymphocytic leukemia (CLL) patients using the DM1200 CellaVision automated microscopy system. Aberrant lymphocytes and smudge cells were enumerated and correlated with CLL immunophenotype, chromosomal aberrations and prognostic parameters. Herein, we show that the percentages of aberrant and smudge cells was highly variable between patients and did not correlate with each other. Increased aberrant lymphocytes and fewer smudge cells were associated with an atypical immunophenotype including low expression of CD23, higher levels of FMC7 and bright surface levels of CD20. High fraction of aberrant lymphocytes also was associated with trisomy 12. These cells were predominantly of small/medium size, sometimes with cleft nuclei. No correlation was noted between aberrant or smudge cells and clinical stage, CD38, ZA70 or time to first treatment. Taken together, automated morphological analysis of peripheral blood leukocytes emerged as a powerful and robust tool for the quantitative morphological stratification of CLL. Integration of the automated morphological features discriminates between different CLL phenotypes and distinct chromosomal aberrations.

Absolute monocyte count trichotomizes chronic lymphocytic leukemia into high risk patients with immune dysregulation, disease progression and poor survival.

Peripheral absolute monocyte count (AMC) has been reported to correlate with clinical outcome in different types of cancers. This association may relate to alteration in circulating monocytic subpopulations and tumor infiltrating macrophages. In this study we evaluated the clinical significance of peripheral AMC in 80 treatment naive patients with CLL. Measurement of AMC was based on direct morphological enumeration, due to our findings that complete blood count data may yield incorrect monocytes enumeration values in CLL. The median AMC in patients with CLL was within normal limits, however the AMC range exceeded the values of healthy individuals. The AMC trichotomized patients into 3 distinct sub-groups with different characteristics and outcomes. High AMC patients were younger and had higher absolute lymphocytes count, while patients with low AMC had prominent immune dysregulation (lower serum IgA levels, susceptibility to infections and a tendency for positive direct anti-globulin test). The low and high AMC patients had a shorter time to treatment compared to the intermediates AMC subgroups, whereas low AMC was associated with increased mortality caused by infectious complications. In conclusion, AMC quantification during the disease course classifies CLL patients into subgroups with unique clinical features and outcomes.

High response rate for treatment with gemtuzumab ozogamicin and cytarabine in elderly patients with acute myeloid leukemia and favorable and intermediate-I cytogenetic risk.

UNLABELLED: Recent studies have reevaluated whether gemtuzumab ozogamicin (GO) improves the outcome of acute myeloid leukemia (AML) in elderly patients. Over 5 years, we treated 16 elderly patients with AML with GO and cytarabine. A high response rate, prolonged survival, and low toxicity were observed in the favorable and intermediate-I genetic groups of AML. Our study raises the issue about the optimal protocol for these patients. BACKGROUND: The benefit of gemtuzumab ozogamicin (GO) in combination with chemotherapy as frontline therapy in patients with acute myeloid leukemia (AML) is still debated. PATIENTS AND METHODS: We evaluated the safety and efficacy of low-dose GO with cytarabine in elderly patients with newly diagnosed AML. Over the past 5 years, we have treated 16 elderly patients with AML (64-82 years) with GO (3 mg/m(2)) followed by continuous infusion of cytarabine (100 mg/m(2)) for 7 days. RESULTS: Complete remission (CR) was achieved in 68.8% of patients; however, this was true only in patients in the favorable or intermediate-I cytogenetic risk groups. Of the 12 patients with AML in the favorable and intermediate-I genetic groups, 11 (91.7%) achieved CR. By comparison, of all 4 patients in the intermediate-II or adverse genetic groups, none of the patients achieved CR (P = .003). The median disease-free survival and overall survival (OS) was 10.9 and 18.8 months, respectively, for patients who achieved CR. The estimated median survival was 15 months in the favorable and intermediate-I cytogenetic groups and only 4.4 months in the intermediate-II and unfavorable risk groups (P = .008). The toxicity profile was also manageable in patients with AML who were mainly older than 70 years with good performance status (PS). The 8-week mortality rate was 6.25%, which is relatively low in this high-risk group of patients. These data are in line with results from 2 randomized trials suggesting that the addition of low-dose GO should be further investigated to reevaluate its role in selected elderly patients with AML and raises the issue of the optimal protocol.

Predictive parameters for a diagnostic bone marrow biopsy specimen in the work-up of fever of unknown origin.

OBJECTIVE: To determine the role of bone marrow biopsy (BMBX), performed in association with comprehensive blood and imaging tests, in the evaluation of patients with fever of unknown origin (FUO). PATIENTS AND METHODS: We reviewed the medical records of 475 hospitalized patients who underwent BMBX in our medical center from January 1, 2005, to April 30, 2010. We identified 75 patients who fulfilled the accepted classic Petersdorf criteria for FUO. All patients underwent in-hospital investigation for fever, including chest and abdominal computed tomography. RESULTS: In 20 patients (26.7%), BMBX established the final diagnosis. Sixteen patients had hematologic disorders, including 8 patients with non-Hodgkin lymphoma, 2 with acute leukemia, 1 with multiple myeloma, 1 with myelodysplastic syndrome, and 4 with myeloproliferative disorders. The remaining patients with diagnostic BMBX specimens had solid tumors (2 patients), granulomatous disease (1 patient), and hemophagocytic syndrome (1 patient). Multivariate analysis revealed the following as the significant positive predictive parameters for a diagnostic BMBX specimen: male sex (odds ratio [OR], 7.35; 95% confidence interval [CI], 1.19-45.45), clinical lymphadenopathy (OR, 21.98; 95% CI, 1.97-245.66), anemia (OR, 2.21; 95% CI, 1.28-3.80), and increased lactate dehydrogenase levels (OR, 1.003; 95% CI, 1.001-1.006). CONCLUSION: Bone marrow biopsy is still a useful ancillary procedure for establishing the diagnosis of FUO, particularly if used in the appropriate clinical setting. Clinical and laboratory parameters associated with hematologic disease are predictive of a diagnostic BMBX specimen in patients with FUO.

Tyrosine kinase inhibitors induced immune thrombocytopenia in chronic myeloid leukemia?

The outcome and quality of life of chronic myeloid leukemia (CML) patients has remarkably changed with the treatment of tyrosine kinase inhibitors (TKIs). Currently, hematopoietic stem cell transplantation (HSCT) is considered mainly as a third line salvage therapy in cases of TKIs resistance or intolerance. Here we describe a patient with chronic phase CML who developed both resistance and late occurrence of s severe thrombo-cytopenia on first and second generation TKIs and eventually underwent HSCT. Although the mechanism of the myelosuppression is not fully understood, we showed for the first time the development of dose dependent platelet antibodies in the presence of TKIs, suggesting the possibility of TKIs induced thrombocytopenia. Our case emphasizes that late development of severe myelosuppression during imatinib treatment is probably an important indication for consideration of early HSCT.