Chronic Lymphocitic Leukemia Research Articles

Role of ZNF224 in cell growth and chemoresistance of chronic lymphocitic leukemia.

Chronic lymphocytic leukaemia (CLL) is associated with apoptosis resistance and defective control of cell growth. Our study describes for the first time a critical role in CLL for the KRAB-zinc finger protein ZNF224. High ZNF224 transcript levels were detected in CLL patients with respect to control cells. Moreover, ZNF224 expression was significantly lowered after conventional chemotherapy treatment in a subset of CLL patients. By in vitro experiments we confirmed that ZNF224 expression is suppressed by fludarabine and demonstrated that ZNF224 is involved in apoptosis resistance in CLL cells. Moreover, we showed that ZNF224 positively modulates cyclin D3 gene expression. Consistently, we observed that alteration of ZNF224 expression leads to defects in cell cycle control. All together, our results strongly suggest that in CLL cells high expression level of ZNF224 can lead to inappropriate cell growth and apoptosis resistance, thus contributing to CLL progression. Targeting ZNF224 could thus improve CLL response to therapy.

Combined Inhibition of mTOR and DNA-PK Blocks Survival, Adhesion, Proliferation and Chemoresistance in Primary Chronic Lymphocytic Leukemia (CLL) Cells

CLL progression and chemoresistance can result from signals from the lymph node (LN) microenvironment and from acquired aberrations in the DNA damage repair (DDR) pathway. Clinical targeting of kinases upstream in the B cell receptor (BCR) activation pathway, such as Btk or PI3Kδ results in egress of cells from the LN microenvironment [1,2]. Such prolonged lymphocytosis during kinase-inhibitor treatment appears to pose no clinical disadvantage [3]. However, it enhances the chance of accumulating resistance-inducing mutations, and therefore drugs that combine LN egress with direct cytoxicity could provide an improved therapeutic strategy for CLL.The mTOR complex, consisting of mTOR1 and 2, is the main downstream kinase of the PI3K/Akt pathway and contributes to proliferation and survival. DNA-PK is a kinase required for non-homologous end joining (NHEJ) of the DNA repair pathway. Inhibitors of crucial components of the DDR pathway might be active in CLL, especially in patients harboring mutations in DNA repair molecules such as Ataxia telangiectasia mutated (ATM). In this study the potency of a novel dual mTOR1,2 and DNA-PK inhibitor (CC-115) was studied in primary CLL samples of different prognostic subgroups with respect to induction of cytotoxicity, and inhibition of adhesion, CD40-mediated chemoresistance and proliferation.In vitro, CC-115 inhibited mTOR1 and 2 and also affected the DDR reflected by inhibition of irradiation-induced γH2AX, not only in ATM-mutated but also in ATM-wild type CLL cells. CC-115 showed induction of caspase-dependent cell killing (IC50=0.625µM) which was more robust than selective kinase inhibitors (table 1), irrespective of p53 or ATM status. This cytotoxic effect was not observed in the T cells from CLL patients. BCR-mediated adhesion to fibronectin [4] was inhibited by CC-115 to a similar extent as PI3Kδ inhibitor (idelalisib) (table 1). CD40-mediated chemoresistance [5] could be reverted completely by CC-115 while more specific inhibitors had only modest effects. CLL proliferation induced by CD40L+IL-21 treatment [6] was completely blocked by both CC-115 and a dual mTOR1,2 inhibitor but not by inhibitor of the more upstream kinase PI3Kδ (table 1).In conclusion, these data show that CC-115 induces direct cytotoxicity and inhibits several clinically relevant biological features of CLL, and provide a rationale for clinical trials with CC-115 in CLL patients.[1] Hoellenriegel J, Meadows SA, Sivina M et al. The phospoinositide 3’-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011;(118):3603-3612[2] Burger JA. Bruton’s Tyrosine Kinase (BTK) Inhibitors in Clinical Trials. Curr Hematol Malig Rep (2014) 9:44–49[3] Herman SE, et al. Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study. Leukemia. 2014 Apr 4. doi: 10.1038/leu.2014.122. [Epub ahead of print][4] de Rooij MF, Kuil A, Geest CR et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;(119):2590-2594.[5] Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;(109):1660-1668.[6] Pascutti MF, Jak M, Tromp JM et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood 2013;(122):3010-3019.Smit LA, Hallaert DY, Spijker R et al.Table 1The effect of the mTOR1,2 + DNA-PK inhibitor (CC-115), mTOR1,2 inhibitor (CC-214), DNA-PK inhibitor (NU7441) and PI3Kδ inhibitor (idelalisib) on CLL cells in functional assays.CC-115CC-214NU7441Idelalisib (CAL-101)TargetmTOR1,2DNA-PKmTOR1,2DNA-PKPI3KδCytotoxicity (IC50)0.625 µ M>10µM>10µM>10µMInhibition of adhesion40%**0%22%48%**ActivationInhibition of CD40-mediated resistance to fludarabine (6.25 µ M)92%**42%*n.d.21%Inhibition of CD40L+IL21 induced proliferation98%**98%*28%51%**n.d. = not done The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p <0,05;** p<0,01 DisclosuresKersten:Celgene: Research Funding. Kater:Celgene: Research Funding.

Combined Inhibition of Phosphatidylinositol 3-Kinase (PI3K) Isoform α and δ By the Pan-Class I PI3K Inhibitor SAR245409 (XL765) in Primary Chronic Lymphocytic Leukemia Cells Blocks Survival, Adhesion and Proliferation

CLL cells are highly dependent on B- cell receptor (BCR) signaling and on stimuli from the microenvironment for survival and proliferation. New drugs targeting PI3K downstream of BCR signaling have emerged as promising treatment options for patients with CLL. Among four PI3K catalytic subunits, the PI3Kd isoform is crucial for downstream BCR signaling, but the relative importance of the PI3Kα isoform in CLL is less clear. Impressive clinical activity of idelalisib in CLL and indolent NHL patients was recently reported. Idelalisib, a PI3Kd specific inhibitor, inhibits chemotaxis and adhesion of leukemia cells, resulting in rapid lymphocytosis followed by a decrease in lymphadenopathy. However, idelalisib has no direct impact on leukemic cell survival [1], raising the potential risk of residual clones responsible for the development of resistance. In this study, we evaluated the impact of a pan-class I PI3K inhibitor (SAR245409/XL765), a PI3Kα-specific inhibitor (BYL719) and a PI3Kd specific inhibitor (idelalisib) on PI3K/mTOR signaling, apoptosis, cell adhesion, CD40-induced survival and proliferation in primary patient derived leukemic cells.Phosphorylation of the downstream effector of mTOR, S6RP, was completely blocked by SAR245409 but not by BYL719 or idelalisib. SAR245409 induced apoptosis in unstimulated CLL cells (IC50= 0.86µM) in contrast to BYL719 or idelalisib (IC50 >10µM), demonstrating that targeting multiple PI3K isoforms is required to completely block the PI3K/Akt/mTOR pathway (table 1). Importantly, SAR245409 also induced apoptosis in p53 or ATM dysfunctional CLL samples. SAR245409, as well as idelalisib, and in contrast to BYL719 completely inhibited BCR-mediated adhesion to fibronectin [2]. Similarly, SAR245409 inhibited CD40L-mediated survival [3], and induced upregulation of the pro-apoptotic protein BIM. All 3 PI3K inhibitors inhibited CD40 ligation + IL-21-mediated CLL proliferation [4].This study revealed that the pan-class I PI3K inhibitor SAR245409 is more cytotoxic to primary CLL cells than PI3Kα or PI3Kd specific inhibitors. Furthermore, combined inhibition of PI3Kα and d can block signaling pathways that are critical for CLL survival, adhesion and proliferation in the LN microenvironment (see table 1). This work provides a rationale for the evaluation of SAR245409 in CLL patients either as monotherapy or in combination therapies.[1] Hoellenriegel et al. The phospoinositide 3′-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011;(118):3603-3612[2] de Rooij et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;(119):2590-2594.[3] Smit et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;(109):1660-1668.[4] Pascutti et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood 2013;(122):3010-3019.Table 1The effect of the PI3Kd inhibitor idelalisib, PI3Kα inhibitor BYL719 or pan PI3K inhibitor SAR245409 on CLL cells in functional assaysPI3Kd inhibitorPI3Kα inhibitorpan PI3K inhibitorCytotoxicity (IC50)1>10µM>10µM0.86µMInhibition of adhesion248%**21%43%**ActivationInhibition of CD40L-induced survival314%0%54%*Inhibition of CD40L+IL21 induced proliferation447%*35%*51%*1 CLL cells were incubated with 0.001-10 μM idelalisib (n=18), BYL719 (n=6) or SAR245409 (n=28) for 48 hours. Viability was assessed by DiOC6/PI staining.2 CLLcells pretreated with 1 µM idelalisib, BYL719, or SAR245409 were stimulated with α-IgM and allowed to adhere to fibronectin-coated surfaces (n=5). 3 CLL cells were cultured on fibroblast expressing CD40L in the absence or presence of 1 µM of idelalisib, BYL719, or SAR245409 for 3 days. Apoptosis was assessed by DiOC6/PI staining (n=8).4 CFSE labelledCLL cells were cultured on fibroblast expressing CD40L with IL-21 and co-treated with 1 µM idelalisib, BYL719, or SAR245409. After 4 days, CFSE was measured by FACS (n=11)2-4 The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p <0,05;** p<0,01 DisclosuresEgile:Sanofi: Employment. Kersten:Sanofi: Research Funding. Kater:Sanofi: Research Funding.

Possible Mechanisms Of Resistance To The Novel BH3-Mimetic ABT-199 In In Vitro Lymph Node Models Of CLL – The Role Of Abl and Btk

The new BH3-mimetic ABT-199 antagonizes Bcl-2 and avoids the thrombocytopenia associated with clinical application of its predecessor ABT-263 (navitoclax). Chronic lymphocytic leukemia (CLL) cells are highly sensitive to ABT-199 and the first clinical results show clear reductions in peripheral and bone marrow CLL cells and in lymph node size. In the lymph node, CLL cells receive pro-survival signals that upregulate Bcl-XL, Mcl-1 and Bfl-11. These Bcl-2 family members are not targeted by ABT-199, which poses the potential risk of remaining clones with residual viability. Here, we aimed to define the signals that determine sensitivity for ABT-199 and ABT-737 in an in vitro lymph node model of CLL.We applied CD40 and cytokine stimulation in combination with kinase inhibitors that are known to change microenviroenmental signals and increase drug resistance in CLL. Stimulation via CD40 plus IL-4 or IL-21 differentially affected the expression of Mcl-1, Bcl-XL, Bfl-1 and Noxa and this correlated with strong alterations in sensitivity to ABT-737 and ABT-199 (see table 1 for LC 50 values). As reported before2, in vitro CD40 stimulation reduced sensitivity to ABT-737 by 100-fold, and this was further decreased by IL-4. Strikingly, CD40+IL-4 stimulation in primary CLL cells resulted in full resistance to 10 μM ABT-199, probably due to very high levels of Bcl-XL.Table 1The LC50 of ABT-737 or ABT-199 for CLL cells stimulated with CD40L and IL-21 or IL-4 (averaged values n=8)StimulationLC50 (μM)ABT-737ABT-1993T3 (control)0.0050.0013T40L0.781> 103T40L + IL-210.1950.2103T40L + IL-46.772> 103T40L + IL-21 + IL-40.4269.121We next sought ways to circumvent resistance against ABT-199 induced in our in vitro model. We showed previously that the broad spectrum kinase inhibitor dasatinib prevented CD40-mediated resistance to various drugs, including ABT-7373. We therefore first characterized the targets of dasatinib in primary CLL by solid-phase pull-down, mass-spectrometry and competition binding. Abl and Btk were identified as dominant and specific interactors of dasatinib. Importantly, resistance for BH3-mimetics could be overcome by dasatinib (see table 2) and the Abl inhibitor imatinib, but not by the more selective Btk inhibitor ibrutinib. Conversely, BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, Abl and Btk function in two key pro-survival arms; chemoresistance and localization in the protective environment.Table 2The LC50 of ABT-737 or ABT-199 for CLL cells stimulated with CD40L in combination with Dasatinib (averaged values n=4)StimulationLC50 (μM)ABT-737ABT-1993T30.0050.0013T40L0.781> 103T40L + 100 nM Dasatinib0.0810.0663T40L + 1000 nM Dasatinib0.0370.020The observed resistance to ABT-199 induced in our in vitro a co-culture system designed to simulate the CLL microenvironment does not reflect the observations from clinical trials in patients. Nevertheless, long-term clinical application of ABT-199 in CLL might select for resistant clones at protective niches. Our data suggest that this may be overcome by combination treatment with kinase inhibitors that either directly abrogate anti-apoptotic signals or cause egress from lymph node sites and prevent the resistance mechanism from coming into play.1. Smit LA, Hallaert DY, Spijker R et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;109:1660-1668.2. Vogler M, Butterworth M, Majid A et al. Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia. Blood 2009;113:4403-4413.3. Hallaert DY, Jaspers A, van Noesel CJ et al. c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL; Implications for therapeutic targeting of chemoresistant niches. Blood 2008;112:5141-5149. Disclosures:No relevant conflicts of interest to declare.

New Potential Therapeutic Approach for the Treatment of B-Cell Malignancies Using Chlorambucil/Hydroxychloroquine-Loaded Anti-CD20 Nanoparticles

Current B-cell disorder treatments take advantage of dose-intensive chemotherapy regimens and immunotherapy via use of monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents, and often cause adverse effects on patients. In this contribution, we propose a novel therapeutic approach in which relatively high doses of Hydroxychloroquine and Chlorambucil were loaded into biodegradable nanoparticles coated with an anti-CD20 antibody. We demonstrate their ability to effectively target and internalize in tumor B-cells. Moreover, these nanoparticles were able to kill not only p53 mutated/deleted lymphoma cell lines expressing a low amount of CD20, but also circulating primary cells purified from chronic lymphocitic leukemia patients. Their safety was demonstrated in healthy mice, and their therapeutic effects in a new model of Burkitt's lymphoma. The latter serves as a prototype of an aggressive lympho-proliferative disease. In vitro and in vivo data showed the ability of anti-CD20 nanoparticles loaded with Hydroxychloroquine and Chlorambucil to increase tumor cell killing in comparison to free cytotoxic agents or Rituximab. These results shed light on the potential of anti-CD20 nanoparticles carrying Hydroxychloroquine and Chlorambucil for controlling a disseminated model of aggressive lymphoma, and lend credence to the idea of adopting this therapeutic approach for the treatment of B-cell disorders.

Mature Circulating Endothelial Cells and Progenitors in Patients with Chronic Gvhd

Abstract 4700Acute and chronic graft-versus-host disease are a common complication of allogeneic stem cell transplantation. In animal models acute GVHD (aGVHD) is associated with increased neovascularization and number of circulating endothelial cells (CECs), while patients with sclerodermatous chronic GVHD (cGVHD) show a significant decrease in the number of circulating endothelial progenitor cells (EPCs) in peripheral blood as compared to patients with non sclerodermatous cGVHD or controls.In an attempt to evaluate the role of CECs and EPCs in patients with cGVHD, we analysed a total of 15 patients affected by hematological malignancies (3 Non-Hodgkin Lymphoma, 4 Hodgkin Disease, 4 Multiple Myeloma, 2 Myelodisplastic Syndrome, 2 Chronic Lymphocitic Leukemia) having undergone allogeneic stem cell transplantation following reduced intensity conditioning. Donors were HLA identical in 14 patients (12 sibling and 2 matched unrelated donors) and HLA aploidentical in 1.Acute GVHD and cGVHD were defined on the basis of time of manifestation, ≤100 days for aGVHD and >100 days for cGVHD. At the time of the blood sample collection, 8 patients, median age 42 years (28-51), with a median time after transplant of 177 days (21-1373), had no evidence of GVHD; of those 5/8 were evaluable for aGVHD and cGVHD, 2 only for aGVHD; 4/8 were on post-transplant calcineurin inhibitors immunosuppressive therapy; 7 other patients, median age 51 years (38-64), with a median time after transplant of 844 days (314-1779), were all evaluable for acute and cGVHD and had evidence of cGVHD as follows: sclerodermatous in 3 patients requiring systemic immunosuppressive therapy, oral mucosa lichen in 1 patient taking oral corticosteroid and cutaneous erythematous and dischromic cGVHD in the other 3 patients, with only 1 patient on systemic immunosuppressive therapy. Viable and apoptotic CECs and EPCs were evaluated by six color flow cytometry (Mancuso et al, Clin Cancer Res, 2009). Briefly, CECs were defined as DNA+CD45-CD31+ CD146+, EPCs as CD45- CD34+. The combination of Syto16 and 7-AAD was used to discriminate between viable (syto16bright/7-AAD-) and apoptotic (syto16weakly pos/7-AAD+) endothelial cells, and to exclude from analysis, platelets and endothelial macroparticles.The results, expressed as median of cells/mL, are summarized in the following table:Total CECsViable CECsApoptotic CECsEPCsHealthy Subject103 (33–322)21 (3–67)77* (28–303)31 (0–56)Patients with cGVHD46 (29–94)41 (25–68)5* (3–26)30 (0–213)Patients without GVHD138 (30–179)39 (10–153)68* (5–136)49 (0–355)*p<0.017These preliminary data indicate a significant reduction in apoptotic circulating mature endothelial cells, likely reflecting a poor vascularization of multiple organs and tissues targeted by cGVHD and a trend towards a decreased number of EPCs in patients with cGVHD. A multicentric study is now planned to confirm these hypotheses and investigate a possible predictive/prognostic role of CEC and EPC counts in cGVHD. Disclosures:No relevant conflicts of interest to declare.

IGH Repertoire Analysis In Multiple Myeloma (MM): Lack of Intra-Disease Homology and Occasional Clustering with Sequences of Other B-Cell Neoplasms Sharing Identical Geographical Origin

AbstractAbstract 2951 Background:The identification of stereotyped immunoglobulin (IG) receptors has improved our knowledge on the pathogenesis of several B-cell malignancies, suggesting the role of antigen-driven stimulation in chronic lymphocitic leukemia (CLL), marginal-zone lymphoma (MZL) and mantle-cell lymphoma (MCL). Multiple myeloma (MM) is a terminally-differentiated neoplasm no longer expressing surface IG; however some reports suggest the existence of early B-lymphocyte precursors which could be susceptible to antigen-driven stimulation. IG heavy chain (IGH) repertoire has not been extensively investigated in MM, with the largest available reports containing less than 80 complete sequences. Aims:To address this issue we created a database of MM IGH sequences including our institutional records (mostly derived from minimal residual disease studies) and sequences available from the literature. We planned a two-step analysis: a) first we characterized the MM repertoire and performed intra-MM clustering analysis; b) then we compared our MM series to a large public database of IGH sequences from neoplastic and non-neoplastic B-cells in search of similarities between MM sequences and other normal or neoplastic IGH repertoires. Patients and methods:131 MM IGH genes were amplified and sequenced at our Institutions and belonged to Italian patients, while 214 MM IGH sequences from non-Italian patients were derived from published databases (NCBI-EMBL-IMGT/LIGM-DB) for a total of 345 fully interpretable MM sequences (out of 396). 28590 IGH sequences from other malignant and non-malignant B-cells were retrieved from the same public databases, including approximately 4500 CLL/Non-Hodgkin lymphoma (NHL) sequences and comprising 500 sequences from Italian patients. All sequences were analyzed using the IMGT database and tools (Lefranc et al., Nucleic Acid Res. 2005; http://imgt.cines.fr/) to identify IGHV-D-J gene usage, to assess the somatic hypermutation (SHM) rate and to identify HCDR3. HCDR3 aminoacidic sequences were aligned together using the ClustalX 2.0 software (Larkin et al., Bioinformatics, 2007; http://www.clustal.org/). Subsets of stereotyped IGH receptors were defined according to Stamatopoulos et al. (Blood, 2007). Result:IGHV analysis in MM was almost in keeping with the normal B-cell repertoire, showing a less remarkably biased IGH usage compared to CLL, MCL and MZL (with seven genes accounting for 40% of cases, compared to respectively five, three and two genes). However, a modest but significant over-representation of IGHV1-69, 2–5, 2–70, 3–21, 3–30-3, 3–43, 5–51 and 6-1 genes and under-representation of the IGHV1-18, 1–8, 3–30, 3–53 and 4–34 was noticed. The rate of somatic hypermutation in MM followed a Gaussian distribution with a median value of 7.8%. Intra-MM search for HCDR3 similarities never met minimal requirements for stereotyped receptors. When MM sequences were compared to non-MM database, only a minority of MM sequences (2.6%, n=9) clustered with sequences from lymphoid tumors and normal B-cells (figure 1A). In particular two non-Italian MM sequences clustered with previously characterized, uncommon CLL subsets (n.37 and n.71 according to Murray et al., Blood 2008). Moreover, novel provisional clusters were observed including three MM-CLL subsets, one MM-NHL subset, and three MM-normal B-cell subsets. While the MM-normal B-cell clusters involved non-Italian patients, we unexpectedly noticed that the four MM-CLL/MM-NHL clusters were composed exclusively of Italian patients, as shown in figure 1B, although Italian subjects represented less than 12% of the entire CLL-NHL database. Conclusion:The analysis of the largest currently available database of MM IGH sequences indicates the following: 1) MM IGH repertoire is closer to physiological distribution than that of CLL, MCL and MZL; 2) MM specific clusters do not occur to a frequency detectable with currently available databases; 3) 98% of MM sequences are not related to other “highly-clustered” lymphoproliferative disorders; 4) Uncommon clustering phenomena may follow a geographical rather than a disease-related pattern. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Regulatory T Cell Content in the Bone Marrow Graft Does Not Predict the Occurrence of Acute GVHD

The subpopulation of regulatory T cells (Treg) was shown to play a key role in alloreactive responses. In allogeneic hematopoietic stem cell transplantation, several groups tested whether Treg content in transplants correlates with graft-versus-host disease (GVHD) with controversial results. In a retrospective study of 49 consecutive HLA-matched sibling transplantations, we studied the relationship between Treg content in bone marrow transplants and acute GVHD (aGVHD) occurrence. We observed a large variability in Treg in bone marrow grafts. However, contrary to previous observations in peripheral blood stem cells transplantation, we report that the Treg content of allogeneic bone marrow transplantation did not predict the occurrence of aGVHD.

90 y-Ibritumomab Tiuxetan or Purine Analogues Severely Affect Peripheral Blood Stem Cell Mobilization: An Analysis On 248 Patients.

Abstract 3210Poster Board III-147 BACKGROUNDHigh-dose chemotherapy followed by autologous Peripheral Blood Stem Cell (PBSC) transplantation represents an effective option in relapsing/refractory malignant lymphoma as well as in selected solid tumors. Collection of a sufficient amount of stem cells (CD34+ ≥ 2.0×106/kg) by apheresis is mandatory for the procedure. However many factors could influence the mobilization of PBSC and about 30% of patients eligible for this therapeutic option fail stem cell mobilization. Peripheral CD34+ counts of 20/μL is conventionally considered the cut-off for a successful collection. METHODSWith the aim to identify those factors affecting PBSC mobilization, we retrospectively reviewed data about our experience at European Institute of Oncology from 01/2005 to 05/2009. By evaluating the number of CD34+cells after mobilization, patients were considered as good mobilizers (peripheral CD34+ ≥ 20/μL, group A), relative poor mobilizers (peripheral CD34+ counts <20 and ≥8/ μL, group B) and absolute poor mobilizers (peripheral CD34+ counts <8/μL, group C). A total of 248 patients were enrolled into a mobilizing PBSC program; apheresis was performed when the CD34+ cell count was >5/μL. Patients characteristics were: 140 male, 108 female; median age was 51 yrs; diagnosis included: 124 Non Hodgkin Lymphoma (NHL), 50 Hodgkin Lymphoma (HL), 35 Multiple Myeloma (MM), 5 Acute Leukemia (AL) and 33 solid tumors, 1 Chronic Lymphocitic Leukemia (CLL); mean number of previous chemotherapy lines was 1 (0-9). The main mobilization regimens in hematological patients were cyclophosphamide 4g/mq (n=118) and ESHAP either followed by G-CSF (n=27) or pegylated G-CSF (n=48); the majority of patients affected by solid tumors received ICE regimen plus G-CSF (n=26). RESULTSBy evaluating the number of CD34+cells after mobilization, 163 (65.7%) patients resulted good mobilizers, 43 (17.3%) patients relative poor mobilizers and 42 (17%) patients absolute poor mobilizers. All patients in group A, 31 patients in group B (72%) and 8 patients in group C (19%) collected >2.0×106 CD34+cells/kg. According the Two-sided Fisher's exact test, more than 3 previous chemotherapy lines before mobilization (p<0.001), pretreatment with purine analogues (p=0.007) or 90Y-Ibritumomab Tiuxetan (p=0.002) were found to be independent factors able to affect PBSC mobilization. In a multivariate analysis, these factors were confirmed as detrimental (purine analogs p=0.019, 90Y-Ibritumomab Tiuxetan p=0.003). CONCLUSIONSThese results could help to identify those factors affecting PBSC mobilization. To reduce poor mobilizers we suggest to anticipate mobilization program and to evaluate the opportunity to reserve purine analogs or radioimmunotherapy after collection. About 20% of patients defined as absolute poor mobilizers might successfully mobilize PBSC by lowering the CD34+ cut-off before apheresis. DisclosuresNo relevant conflicts of interest to declare.

Disease and Comorbidity Status Predict Outcome after Nonmyeloablative Allografting for Advanced Haematological Malignancies.

Nonmyeloablative (NM) regimens extended the application of allogeneic transplantation (AT) to older and/or medically unfit patients ineligible for high-dose conditionings. Moreover, several patients are offered AT after failure of prior lines of therapy. We retrospectively evaluated 108 patients, median age 51 (16–67), with advanced or heavily pre-treated hematological cancers [19 acute myeloblastic (AML) and 1 lymphoblastic leukemia (ALL); 8 myelodysplastic syndrome (MDS); 9 chronic myeloid (CML) and 9 chronic lymphocitic leukemia; 3 myelofibrosis (MF); 48 myeloma beyond first line treatment; 8 Hodgkin's and 8 non-Hodgkin's lymphoma) who underwent NM-AT at 12 Italian transplant Centres. Median time between diagnosis and transplant was 20 months. Sixty-one% of patients had failed at least one prior line of therapy, including 36 refractory or relapsed patients after 1 or more autologous transplants. At allografting, 15 patients had disease at lower risk of relapse (AML and ALL in first complete remission (CR), MDS without blast excess, CML in first chronic phase, untreated MF). Forty-two patients had active disease. The comorbidity score was evaluated with an AT-specific comorbidity index (CI) as described by Sorror et al: 51 patients had CI=0, 42 CI=1–2 and 15 CI≥3. Patients were conditioned with fludarabine 90 mg/m2 and 2 Gy total body irradiation. GVHD prophylaxis consisted of cyclosporine and mycophenolate mophetile. Graft failure was observed in 1 patient and rejection after disease recurrence in 4. After a median follow-up of 28 months post-allografting, 58 (54%) had refractory/progressive disease and 36 (33%) were in CR, including 23 who were not in CR at transplant (p<0,001). Cumulative incidences of grade II–IV acute GVHD and extensive chronic GVHD were 35% and 60% respectively. Fourteen patients (13%) died of transplant-related causes (TRM) and 36 (33%) of disease progression (DRM). Two-year TRM was 12% and was higher in patients with CI≥3 (HR 2.84, p=0.08) whereas DRM was 31% and was significantly influenced by disease status at transplant (HR 0.37 for patients in partial (PR) or CR, p=0.003). Median event-free (EFS) and overall survivals (OS) were 11 (95% Cl: 8–15) and 35 months (95% Cl: 21–42) respectively. OS was significantly better in patients in remission (HR 0.44, p=0.004) and a trend for better OS was also observed in patients with CI<3 (HR 0.73, p=n.s.). By multivariate analysis, disease remission at transplant was associated with better OS and EFS (HR 0.39, p=0.001 and HR 0.41, p<0.001 respectively) and there was a trend for worse EFS in patients with CI≥3 (HR 1.88, p=0.08). Age, number of prior therapies and time between diagnosis and AT did not affect OS and EFS. In summary, though retrospective, this report confirms that disease-status and comorbidity scores predict transplant outcome. The use of AT specific comorbidity scores in prospective studies is imperative to select patients who most benefit from this procedure.

Rapid Infusion of Rituximab (RIR) Is Well Tolerated and Can Safely Be Delivered in Patients with Lymphoproliferative Diseases.

It has been proved that Rituximab is a useful drug for the treatment of patients with lymphoma. Being a humanized antibody, it presents high specificity. Rituximab is a well tolerated medicine, although a slow infusion rate given in many hours is recommended to avoid reactions derived from the liberation of cytokines. As a consequence, patients need admissions for a treatment that can take hours. The recent demostration of its usefulness in the maintenance of the response of patients with follicular lymphomas has concluded in the revision of hospitalization time, proposing a faster infusion. We are presenting our experience with RIR, on a group of patients with lymphoproliferative diseases. We used RIR with the aim to observe the toxicity and to reduce the time of hospitalization in patients with lymphoma. We evaluated the adverse events and tested the feasibility of a 90 minutes infusion schedule for rituximab (20% of the dose administered in the first 30 minutes, remaining administered over 60 minutes). Premedication included oral paracetamol and intravenous hidrocortisone and diphenhydramine. Sixty seven RIR were used to treated 31 patients (27 non-Hodgkin Lymphoma and 4 Chronic Lymphocitic Leukemia). All of them had previously received one or more rituximab conventional infusion without grade 3 or 4 toxicity. Twenty nine patients received also CHOP or CHOP-like chemotherapy and the other two Rituximab alone as maintenance therapy. Overall RIR was well tolerated. There were 5 adverse events. Four patients developed grade one adverse events: one patient had an hypotension episode, one presented chest pain and the other two had abdominal pain. Only one patient developed a grade 3 adverse event (abdominal pain) and withdrew the RIR schedule. We conclude that rapid infusion rituximab is safe and well tolerated. The adoption of this schedule in clinical practice reduces in many hours the time of hospitalization for patients with lymphoma and may be a cost-effective strategy.

Early and Late Complications in 247 Adult Autologous Stem Cell Transplant Recipients.

High-dose therapy supported by autologous stem cell transplantation (ASCT) has became a widely applied therapy in many haematological malignancies during two decades.ASCT may be curative and even more commonly leads to prolonged survival.Along with the use of mobilised blood progenitor cells and improved supportive care after ASCT, the early treatment-related mortality (<100 d) has declined and is now in the order of 2–3%. Although relapse or progression of underlying malignancy is the most common cause of death in ASCT recipient, also late (>100 d) non-relapse mortality (NRM) occurs.We have analysed a cohort of 191 patients (108 male and 83 female) who received ASCT (247 transplant) in 1994–2005 in Bone Marrow Transplant Unit of San Gerardo Hospital.The median age was 49 years (16–70).The most common diagnoses included lymphoma (88), multiple myeloma (67), acute leukemia (32), chronic lymphocitic leukemia (2) and myelodysplastic syndrome (2).At the time of transplant (247 transplant) 93 patients was in complete remission, 67 patients in partial remission and 86 patients in persistent/progression malignancies.The most common conditioning treatment ASCT included: Melphalan (200 mg/m2 or 140 mg/m2) in 55% of case, BEAM in 30%, Busulfan-Cyclophosphamide in 4%, others in 11% (Thiotepa- Cyclophosphamide, Busulfan-melpalan, Mitoxantrone-melphalan, TBI- Cyclophosphamide).The median dose of CD34+ reinfused was 5.21 × 10^6/kg (1.2–15 × 10^6/kg).The median time to achieve an absolute neutrophil count >0.5 × 10^9/l was 12 days (range 0–49) and to achieve a platelet count > 20 × 10^9/l was 19 (range 0–36).The median time of hospitalisation was 22 days (range 10–93).During hospitalisation 158 patients (63%) developed oral mucositis: 100 (53%) of them developed mucosites of grade 1–2, 58 (37%) of grade 3–4. High dose chemotherapy with BEAM was more toxic on the oral mucose than high dose of Melphalan alone. In 41% of patients total parenteral nutrition support was necessary.During hospitalisation 180 patients (73%) developed sepsis: possible infection 61%, Gram + 20%, Gram − 12%, miscellaneous 3%, fungi 2%, viral 1%, Pneumocistis Carinii 1%.Regimens including cyclophosphamide was responsible for most sepsis.44 patients presented a positive antigenemia of Cytomegalovirus; all of them were seropositive for CMV before ASCT; only one case of primary infection was seen and this patient died after 55 days after ASCT.At 100 days from ASCT 173 patients were in complete remission, 53 of them had a relapse of hematologic malignancies in a median time of 1 year (range 51 days-6 year), 32 patients in partial remission and 35 patients in persistent/progression malignancies.The late complications were: cardiac (30%), hepatic (16%), renal (3%), gastrointestinal (12%), CNS (8%), other (MGUS, cataract, aseptic necrosis of bone, diabete)(31%).The most important cardiac complications were: atrial fibrillation, sinusal tachycardia, arrhythmia, pericarditis. 61 patients (32%) died after ASCT.Death was due to complications related to disease progression in 62% of patients. 25% of patients died from infections.Secondary malignancies (acute myeloid leukemia) were the cause of late non relapse mortality in 2 patients.Fatal cardiovascular complications were observed in 4 patients (acute myocardial infarction or cardiomyopatia most probably due to previous anthracycline therapy).

An unusual pathological finding of chronic lymphocitic leukemia and adenocarcinoma of the prostate after transurethral resection for complete urinary retention: case report

BackgroundWe describe a patient who underwent transurethral resection of the prostate for urinary obstructive symptoms and had histological findings of adenocarcinoma of the prostate with prostatic localization of chronic lymphocitic leukemia (CLL).The contemporary presence of CLL, adenocarcinoma of the prostate and residual prostatic gland after transurethral resection has never been reported before and the authors illustrate how they managed this unusual patient.Case presentationA 79-years-old white man, presented with acute urinary retention, had a peripheral blood count with an elevated lymphocytosis (21.250/mL) with a differential of 65.3% lymphocytes and the prostate-specific antigen (PSA) value was 3.38 ng/mL with a percent free PSA of 8.28%. The transrectal ultrasound (TRUS) indicated an isoechonic and homogenic enlarged prostate of 42 cm3 and the abdomen ultrasound found a modest splenomegaly and no peripheral lymphadenophaty. The patient underwent transurethral resection of the prostate and had a pathological finding of adenocarcinoma in the prostate with a Gleason Score 4 (2+2) of less than 5% of the material (clinical stage T1a), associated with a diffused infiltration of chronic lymphocitic leukemia elements.ConclusionsThe incidental finding of a prostatic localization of a low-grade non-Hodgkin's lymphoma does not modify eventually further treatments for neither prostate cancer nor lymphoma. The presence of a low-grade and low-stage lymphoma, confirmed by a hematological evaluation, and the simultaneous evidence of an adenocarcinoma after transurethral resection of the prostate for acute urinary retention do not require any immediate treatment due to its long-term survival rate and the follow-up remains based on periodical PSA evaluation and complete blood count.

Moléstia de chagas crônica associada a leucemia linfática: ocorrência de encefalite aguda como alteração do estado imunitário

É relatado um caso de paciente apresentando a forma crônica da doença de Chagas, no qual foi observada a presença de encefalite chagásica aguda, com demonstração de leishmanias no sistema nervoso central. O caso é único na literatura, sendo interpretado como devido a agravamento da forma crônica da doença de Chagas pela presença de leucemia linfática crônica associada. A existência de leucemia linfática pode levar à extinção de clones linfocitários responsáveis por fenômenos imunitários, criando, assim, condições para o agravamento e agudização da forma crônica da doença de Chagas.