bluuebunny/crossref_metadata_2025 huggingface dataset:
MRD-Detection by Multiparametric Flow Cytometry in Childhood Precursor B-Cell All:Predictive Impact of Early Blast Reduction Kinetics in Peripheral Blood (PB) on MRD-Level in Bone Marrow (BM) at Day 33 of the All-BFM2000 Potocol.
Authors: Ratei Richard , Martin Mathilde , Basso Guiseppe , Gaipa Guiseppe , Schrappe Martin , Dworzak5 Michael , Ludwig Wolf-Dieter , Karawajew Leonid
Published: October 2019

Detection of minimal residual disease in childhood acute lymphoblastic leukemia (ALL) has been proven to be of crucial importance for relapse prediction. However, MRD assessment, usually performed at postinduction time points, does not allow early stratification of induction therapy. Here, we address the predictive impact of early blast reduction in pB on BM MRD-levels at the end of induction therapy phase I (d33), quantified by multiparametric flow cytometry (MFC-MRD). Absolute blast cell counts (BCC, blasts/μl) in pB at diagnosis and day 8 as well as MRD-levels in BM and pB at day 15 and 33 were determined in a series of 77 consecutive patients (pts.) enrolled in the AIEOP-BFM-FCM-MRD pilot study (BM d33:MRD+ n=29, MRD- n=48). Blast cells were detected using a two platform method and a 4-color antibody panel with three follow-up tubes:1) CD20-FITC/CD10-PE/CD34-PC5/CD19-PC7, 2) CD58-FITC/CD10-PE/ CD34-PC5/CD19-PC7, 3) CD10-FITC/CD11a-PE/CD45-PC5/CD19-PC7. Additionally, staining with the cell permeant live-cell nucleic acid fluorochrome SYTO 16 combined with CD19 or CD3 and CD45 were used to exclude residual non-nucleated erythroid cells, platelets or debris from absolute blast calculations. We found that BCC in BM and pB at d15 but not at diagnosis and after prednisone prephase at day 8 correlated with MRD-levels in BM at d33 (Spearman correlation coefficient rs=0.41, p<0.001 and rs=0,30, p<0,01). In order to further investigate the impact of early blast reduction between day 0 and day 8 we calculated the relative blast reduction rate on day 8 (BRRd8 = 1-BCCd8/BCCd0). In the group of MRD-negative patients the mean values of BRRd8 were found to be significantly higher than in the group of MRD-positive pts. (98,9% vs. 88,0%, p< 0,05). Moreover, the BRRd8 correlated with MRD-levels in BM at day 33 (Spearman correlation coefficient rs=−0.25, p<0.05). In the current series of pts. maximally selected log-rank statistics were performed for the BRRd8 and the MRD-level in BM at d33 and provided a cut-off value of 97% separating MRD-positive and MRD-negative pts. In conclusion, the assessment of the dynamics of early blast reduction with the parameter of BRRd8 is highly predictive for the outcome of induction phase I of the ALL-BFM2000 protocol.
CD28 Fails to Costimulate Tumor-Specific Activation of Epstein Barr-Virus-Specific Memory Effector T Cells.
Authors: Altvater Bianca , Pscherer Sibylle , Juergens Heribert , Rossig Claudia
Published: October 2019

Genetic modification of cytotoxic T cells with tumor antigen-specific chimeric receptors (chRec) specifically redirects their effector functions towards tumor cells. Integration of the signaling domains of the costimulatory molecule CD28 into chRec enhances antigen-specific proliferation of peripheral blood-derived polyclonal human T cell populations. While CD28 plays an essential role in the priming of naïve CD4+ T cells, its contribution to effector memory cytotoxic T cell (CTL) responses is controversial. We investigated the function of chRec containing the signaling domains of CD28 in vitro expanded T cells with specificity for Epstein-Barr-virus (EBV). Chimeric T cell receptors containing an extracellular single-chain antibody domain directed against the tumor ganglioside antigen GD2 fused to the intracellular signaling domains of both the CD28 and the T cell receptor (TCR) ζ chain (14.G2a-CD28ζ), or TCRζ alone (14.G2a-ζ) were expressed in EBV-specific cytotoxic T cell (CTL) lines from three seropositive donors and in peripheral blood T cells preactivated by CD3-/CD28-specific antibody crosslinking by retroviral gene transfer. Following transduction with the chRec genes, 14.G2a-ζ and 14.G2a-CD28ζ EBV-CTL had comparable levels of chRec surface expression (21–28% versus 26–40%). EBV-CTL had an immunophenotype characteristic of memory effector T cells, coexpressing CD3 and CD45RO in the absence of CD45RA and CD27 surface expression. The transduced CTL maintained their capacity to specifically lyse autologous EBV targets in 4 hr 51Cr release assays and to proliferate after stimulation with autologous EBV targets. Intracellular cytokine staining demonstrated efficient and comparable antigen-specific IFN-γ secretion by both 14.G2a-ζ and 14.G2a-CD28ζ-transduced CTL in response to EBV and tumor targets. Furthermore, GD2+ neuroblastoma targets were lysed in a comparable and antigen-specific manner. However, while antigen engagement by 14.G2a-CD28ζ efficiently induced expansion of non-specifically activated polyclonal peripheral blood lymphocytes in an antigen-dependent manner, CD28 signaling failed to overcome the failure of transduced EBV-CTL to specifically proliferate in response to GD2+ tumor cells. Thus, the costimulatory requirement for the highly efficient proliferative activation response of EBV-specific CTL to viral antigen can not be mimicked by combined CD28 and ζ signaling. Exploring the mechanisms and costimulatory requirements of effector memory T cell reactivation may help to exploit the full potential of this highly cytolytic T cell population for adoptive immunotherapy of cancer.
Expression and Activation of the Farnsesoid X Receptor in Human Dendritic Cells.
Authors: Ziros Panos , Micheva Ilina , Habeos Ioannis , Papavasiliou Athanasios , Zoumbos Nicholas
Published: October 2019

Baground: The farnesoid X receptor/bile acid receptor (FXR, NR1H4) is a member of the nuclear hormone superfamily that regulates bile acid as well as lipoprotein and glucose metabolism after heterodimerization with retinoid X receptor. Dendritic cells (DCs) are potent antigen presenting cells capable of regulating immune responses. It has previously been reported that spleen, a tissue rich in DCs, expresses FXR. Taking this into account, we wanted to investigate the possible involvement of FXR in DCs biology.

Material and Methods: Immature DCs were generated from peripheral human blood monocytes (CD14+ cells) by culturing them with GM-CSF and IL4 for 7 days. For maturation, immature DCs were cultured with the addition of TNFα, and LPS for another 24 hours. The expression and activity of FXR in DCs was assessed with RT-PCR, western immunoblotting, immunofluoresence and EMSA analysis.

Results and Discusion:We have shown with RT-PCR that FXR is marginally expressed in human monocytes (CD14+ cells) and its expression is elevated during the differentiation process of these to dendritic cells. Moreover, using specific primers we have found that only FXR -alpha and not FXR-beta is expressed in DCs. Western blot analysis with a specific anti-FXR antibody confirmed the FXR expression in DCs. Immunofluoresence microscopy with a specific anti-FXR antibody showed that FXR, in contrast with the classical FXR-expressing tissues (liver, kidney), has strong cytoplasmic and perinuclear localization and weak nuclear localization in DCs. The nuclear localization is potentiated upon treatment of the immature dendritic cells with LPS or TNFα. Finally, using a DNA probe that contains consensus FXR binding sites, we have shown with EMSA analysis that FXR is transcriptional active in immature DCs but not in CD14 monocytes, and is strongly activated upon LPS or TNFα treatment for 48 h of immature DCs. Overall our study reveals, for the first time, that FXR, a nuclear hormone receptor with a limited tissue expression, might have a new role in DCs biology, since it is not only expressed in human dendritic cells but moreover its transcriptional activity is affected by signals (TNFα, LPS) that influence the function of DCs.
Rituximab Maintenance Therapy in CD20+ B-Cell Non-Hodgkin’s Lymphoma.
Authors: Trajkova Sanja A. , Cevreska Lidija A. , Panovska-Stavridis Irina Z. , Pivkova Aleksandra , Georgievski Borce , Karanfilski Oliver B. , Stojanovic Aleksandar T.
Published: October 2019

Purpose: Clinical and pharmacokinetic data suggest that high serum concentrations of rituximab and prolonged exposure to rituximab are associated with higher response rates and improved quality of response. We used rituximab-maintenance therapy to target minimal residual disease, with the aim of increasing the duration of remissions achieved following initial treatment.

Methods: During 2001–2005, 14 patients with CD20+ B-cell non-Hodgkin’s lymphoma (seven with diffuse large B cell lymphoma [DLBCL] and seven with follicular lymphoma [FL]) were enrolled in the study. Patients with DLBCL and FL were required to have a CR or PR, respectively, following initial treatment. The most common initial treatments were R-CHOP, R-FC or CHOP. Rituximab-maintenance therapy was administered as 375mg/m2 doses every 3 months for 2 years. Three patients with aggressive DLBCL were treated with high-dose chemotherapy/autologous stem-cell transplantation (HDT/ASCT) and received rituximab-maintenance therapy every 2 months for four doses in total.

Results: Most patients had disease of Ann Arbor stage ≥ 3 (two FL patients stage I). In patients with DLBCL, median event-free and overall survivals were 22.1 months and 22.3 months, respectively, with the corresponding values being 20.6 months and 26.1 months in patients with FL. To date, 13 patients are in continuous clinical remission and only one patient has relapsed.

Conclusions: Rituximab-maintenance therapy is effective and well tolerated in this setting. Recruitment for this study is ongoing and evaluation of a larger patient population, together with a longer follow-up, will determine whether this treatment approach has curative potential.
Concomitant Administration of Recombinant Human Erythropoietin (rHuEPO) and Oral Iron Chelator Deferiprone May Stabilize Iron Balance in Transfusion Dependent Patients with Early Myelodysplastic Syndrome (MDS).
Authors: Cermak Jaroslav
Published: October 2019

Eight transfusion dependent patients with refractory anemia (RA) or refractory anemia with ringed sideroblasts (RARS) subtypes of MDS (according to the FAB classification) with serum ferritin ranging from 1630 to 4227 μg/l were treated with oral iron chelator deferiprone (Ferriprox, Apotex Ltd., Canada) in a daily dose of 75–80 mg/kg for at least 12 months. Beginning the second month of therapy, rHuEPO (Eprex, Cilag AG, Switzerland or NeoRecormon, Roche Diagnostic, Germany) was administered concomitantly with deferiprone in a dose of 10 000 IU 3 times per week. A significant increase in urinary iron excrection after addition of rHuEPO compared to treatment with deferiprone as a single agent was observed in all patients and the amount of excreted iron ranged from 7.5mg to almost 20mg per day. In one patient with complete response to rHuEPO (Hb > 115 g/l + transfusion independence) and in another patient with partial response resulting in transfusion independence, serum ferritin level decreased from 3034 to 1872 μg/l and from 2086 to 879 μg/l, respectively. The results corresponded with a moderate decrease in liver iron documented by NMR imaging. In 6 patients who did not respond to rHuEPO and remained transfusion dependent a simultaneous administration of rHuEPO and deferiprone enabled to stablize serum ferritin level despite continuing iron load from transfusions. The treatment was well tolerated except one patient, in whom adverse GIT symptoms led to cessation of deferiprone administration after 4 months of treatment. The ability of deferiprone to bind intracellular iron that may be either excreted from the body or donated to partially saturated transferrin for utilization in bone marrow for rHuEPO stimulated erythropoiesis on one hand and an increased excretion of deferiprone chelated non transferrin bound iron originating from an increased amount of destroyed red blood cells on the other hand may be a possible explanation of observed phenomenon. A concomitant administration of rHuEPO and deferiprone might be an effective tool for maintaining iron balance in transfusion dependent iron overloaded patients with early MDS who are not indicated for intensive treatment.
Adoptive Immunotherapy with Donor Lymphocyte Infusion after Allogeneic Nonmyeloablative Stem Cell Transplantation.
Authors: Qiao Zhenhua , Zheng Zhuanzhen , Ye Fang , Ma Liangming , Gong Rong
Published: October 2019

Purpose: To explore the efficacy and toxicity of adoptive immunotherapy with donor lymphocyte infusion(DLI) after allogeneic nonmyeloablative stem cell transplantation (allo-NST).

Methods: We retrospectively sum up the clinical data of 28 patients who had been performed with allo-NST among 41 patients and had been treated with DLI for 72 times between October 1999 and October 2004. Among these patients, there are 20 chronic myeloid leukemia (CML), 16 acute leukemia (AL), 2 multiple myeloma (MM) and 3 solid tumor. We used two different preparative regimens with or without Fludarabin. Cyclosporin A and MMF were used in the prevention of graft versus host disease (GVHD). The median dose of DLI was positive CD3 cells 2.7×107/kg (1.0×107/kg–2.0×108/kg). According to the patients’ situation and chimerism, we divided these patients into two groups, one group included 15 patients whose chimerism decreased for the reason of disease relapsed or advanced, the other included 13 patients whose chimerism decreased because of disease remission or stable. We analysis the change of chimerism and patients’ situation after DLI and the side effects of DLI.

Results: Among 28 patients, there are 16 CML, 6 acute myeloid leukemia (AML), 3 acute lymphocytic leukemia (ALL), 1 MM and 2 solid tumor. 13 of them were with increased donor chimerism and 11 of them were with full chimerism. In 11 of the 16 CML patients, whose chimerism decreased because of relapsed or advanced, only one patient who achieved partial remission (PR) and other 10 died for the sake of no effect after DLI. With DLI, 5 patients whose chimerism decreased during the course of disease remission got increasing donor chimerism, 3 got complete remission (CR), 2 got PR, 4 survived and 1 PR patient died of GVHD. Among 6 AML patients with DLI, 1 died due to disease relapsed or advanced and decreased chimerism, other 5 who were with disease remission or stable situation and decreased chimerism obtained CR and survival according to the increased donor chimerism after DLI. Among 3 B-ALL patients post DLI, one alive and the other two died. One MM patient died of no effect. Two late phrase lung cancer patients got PR. One has survived for 14 months and another died of lung virus infection. Their rates of donor chimerism were higher than 80 percent and 96 percent. Among all these 28 patients, their average following up time was 30 months (10–66 months). 12 of them survived (42.6 percent) and total efficient rate was 57.14 percent. 3 developed grade II aGVHD that was DLI-associated. 1 died and 5 was complicated with grade II to IV cGVHD (3 extensive and 2 local). Given active treatment, they were alive. 2 patients had ever been with transient complete blood cell decreasing and had recovered after active surppoting treatment.

Conclusion: DLI is a potential therapy that can make chimerism increase and prevent disease relapse post allo-NST. Its toxicity and side effects can be accepted.
Bruton’s Tyrosine Kinase (BTK) Is Not Required for BCR-ABL-Mediated Transformation of Hematopoietic Cells.
Authors: MacPartlin Mary , Honigberg Lee A. , Druker Brian J. , Deininger Michael W.
Published: October 2019

Background: Bruton’s tyrosine kinase (BTK) is a member of the Tec family of protein tyrosine kinases. Mutations of BTK have been associated with a block in B cell development, and are causal to X-linked agammaglobulinemia (XLA) in humans. Bcr-Abl is a constitutively active tyrosine kinase that is essential for the transforming capacity of Bcr-Abl. Using phosphoproteomics we have shown that BTK is consistently tyrosine phosphorylated in Bcr-Abl expressing Ba/F3 cells [Griswold et al. Mol Cell Biol.2006;26(16):6082–93.]. Since BTK has also been implicated in resistance to imatinib [Hofmann et al. Lancet2002;359(9305):481–6] and one study [Feldhahn et al. J Exp Med.2005; 201(11):1837–52] had suggested a role of BTK in Bcr-Abl-induced transformation we decided to test whether BTK may be a critical node in Bcr-Abl transformation and potential drug target in imatinib-resistant Bcr-Abl-positive cells.

Results: We depleted BTK in Ba/F3 and 32D cells expressing native and kinase domain (KD) mutant (E255K and T315I) Bcr-Abl, using shRNA. BTK levels were reduced to <10% of controls, but no differences in viability and cell proliferation were observed. Additionally, responses to imatinib were not affected by BTK depletion. We further tested the effects of reversible (PCI-33918) and irreversible (PCI-31523) small molecule inhibitors of BTK on the above cell lines as well as human Ph+ B-lymphoblastic lines. Selective BTK inhibition did not impact cell proliferation. Lastly, BTK inhibition had no effect on Bcr-Abl-mediated transformation of primary murine hematopoietic cells in colony forming assays.

Conclusion: Despite the fact the BTK is consistently tyrosine phosophorylated in Bcr-Abl expressing cells, our data suggests it is not essential for Bcr-Abl-mediated transformation of lymphoid or myeloid cells.
Multicenter, Randomized, Phase III Study Comparing Imatinib (Glivec) Standard Dose (400 mg/d) with Imatinib High Dose Induction (800 mg/d) Followed by Imatinib Maintenance (400 mg/d) in Patients with Pretreated Ph+/BCR-ABL+ CML in Chronic Phase - Results from the First Planned Interim Analysis (CELSG-CML 11 ISTAHIT Study).
Authors: Petzer Andreas L. , Wolf Dominik , Fong Dominik , Lion Thomas , Dyagil Irina , Masliak Zvenyslava , Boskovic Darinka , Griskevicius Laimonas , Lejniece Sandra , Spasov Emil , Gercheva Liana , Stojanovic Aleksandar , Peytchev Dontcho , Tzvetkov Nikolay , Griniute Rasa , Stanchev Atanas , Kwakkelstein Martin , Ulmer Hanno , Gastl Guenther A.
Published: October 2019

227 patients with pretreated Ph+/BCR-ABL+ CML were randomized into this 2-arm phase III trial comparing standard dose Imatinib (400 mg/d; arm A) with high dose (HD) Imatinib (800 mg/d for 6 months) for induction followed by Imatinib 400 mg/d for maintenance (400 mg/d; arm B). The interim analysis presented here was performed on all patients after 50% of the patients had been treated for 12 months since randomisation. There were no significant differences between treatment arms regarding sex, age and different pretreatments. Rates of complete hematological responses did not differ significantly between both groups at 3, 6 and 12 months (82% arm A, 90% arm B). However, significantly* more patients achieved a major (MCR) and a complete cytogenetic response (CCR) at 3 months (MCR: 21% arm A, 37% arm B; CCR: 6% arm A, 25% arm B) and 6 months (MCR: 34% arm A, 54% arm B; CCR: 20% arm A, 44% arm B). Moreover, significantly* more patients achieved a major molecular response (MMR) at 6 months in the Imatinib HD arm B compared to arm A (20% vs 7%). At 12 months, following dose reduction of Imatinib to 400 mg/d for maintenance at month 6 in the HD arm B, the rates of MCR (the primary endpoint of the study) were comparable (57% arm A, 59% arm B). Nevertheless, there was still a clear trend to higher rates of CCR (37% arm A, 48% arm B) and MMR (16% arm A, 21% arm B) in the HD arm, but at the time of the interim analysis these values did not reach statistical significance. In contrast to non-hematological toxicities, grade 3/4 hematological toxicities were significantly* more common in the HD arm B [anemia: 2% (A), 12% (B); leukopenia: 23% (A), 41% (B), thrombopenia: 14% (A), 34% (B)]. In conclusion, this is the first randomized phase III trial demonstrating significantly* higher rates of MCR, CCR and MMR in chronic phase CML during therapy with HD Imatinib. *p<0.05
Genetic Polymorphisms in the Inflammasomes Are Associated with Relapse and Survival in HLA-Identical Sibling Donor Allogeneic Stem Cell Transplantation.
Authors: Granell Miquel , Urbano-Ispizua Alvaro , Pons Aina , Arostegui Juan Ignacio , Navarro Alfons , Jansa Sonia , Artells Rosa , Gaya Anna , Fernandez-Aviles Francesc , Martinez Carmen , Rovira Montserrat , Carreras Enric , Yague Jordi , Montserrat Emili , Monzo Mariano
Published: October 2019

The inflammasomes are intracellular multiprotein complexes responsible of the secretion of IL-1β in response to the recognition of microbial patterns. Polymorphisms of genes codifying for inflammasomes proteins might influence the clinical outcome of patients receiving allogeneic stem cell transplantation (allo-SCT). Fourteen single nucleotide polymorphisms (SNPs) in NALP1, NALP2, NALP3, CARDINAL and CASPASE5 were genotyped by allelic discrimination in 133 adult donor-patient pairs undergoing sibling donor allo-SCT in a single institution. Patient median age was 41 years (range 17–64) and 61% were males. All patients were diagnosed with hematological malignancies; 71% of them were in advanced phase at transplantation. Median follow-up of the surviving patients was 39 months (range 1–118). Actuarial probabilities of relapse, non-relapse mortality (NRM) and overall survival (OS) were 40% (95% CI 39–41), 35% (95% CI 34–36) and 48% (95% CI 47–49), respectively. Genotype frequencies were similar to those published at NCBI database. NALP2 rs1043684 and rs1043673 (p<0.0001) as well as NALP3 rs10925027 and rs10754558 (p<0.0001) were inherited in linkage disequilibrium. NALP2 rs1043684 (GG 25% vs. GA/AA 12%, p=0.09) was the only factor weakly associated with severe acute GVHD. At multivariate analysis, donor NALP3 rs10925027 TT genotype was associated with relapse (RR 4.22, p=0.000001). Patient NALP1 rs5862 AA genotype and donor NALP2 rs1043684 GG were associated with NRM (RR 5.11, p=0.0002 and RR 2.97, p=0.005, respectively), and OS (RR 2.15, p=0.011 and RR 2.81, p=0.003, respectively). Inflammasome genetic SNPs of both donor and patient influence the outcome of allo-SCT. If these findings are validated in other series, genotype of NALP genes would become an important factor in donor selection.
Alemtuzumab Reduces Chronic Graft Versus Host Disease (cGVHD) and Treatment Related Mortality (TRM) after Reduced Intensity Conditioning for AML and MDS.
Authors: van Besien Koen , de Lima Marcos , Artz Andrew , Oran Betul , Stock Wendy , Giralt Sergio
Published: October 2019

In vivo T-cell depletion with alemtuzumab has been used to reduce acute and chronic GVHD. In order to evaluate its overall effect on transplant outcomes in AML and MDS we compared 90 pts who received fludarabine/melphalan/alemtuzumab (FMA) conditioning and post-transplant tacrolimus at the University of Chicago, with 112 who received fludarabine/melphalan (FM) and post-transplant tacrolimus/methotrexate at MD Anderson Cancer Center. Pt and transplant characteristics were well balanced except for a higher proportion of MDS in the FM group. Median age, proportion unrelated donor tx and proportion high/intermediate and low risk by ASBMT criteria were balanced between the groups. With median follow up of 28 months in both groups, one year progression free survival and overall survival are identical. TRM is significantly higher after FM, but relapse is higher in FMA. 19/103 d 28 survivors after FM vs 7/84 after FMA developed gr III–IV acute GVHD (p=0.04). 46/77 d100 survivors after FM developed ext cGVHD vs 7/63 after FMA (p=0.0000). 43 patients remain alive after FM and 27 have ext cGVHD. 41 remain alive after FMA and 1 has ext cGVHD. Alemtuzumab results in a considerable reduction in acute and particularly chronic GVHD. TRM is reduced compared with standard GVHD prophylaxis. Low incidence of chronic GVHD and reduced TRM may be the major benefit of this strategy. Relapse rates are increased, because of reduced GVL effects or because of improved early survival of high risk patients. Other approaches are necessary for improving long term outcomes.

Patient Characteristics and Outcome FMA FM P-value N 90 102 Age (range) 54 (22–74) 51 (17–77) 0.6 AML/MDS 13/77 29/83 0.04 MUD/related 42/48 59/63 0.5 High./Intermediate/LowRisk 48/13/28 76/10/26 0.12 Median Follow up mths (range) 28 (3–89) 28 (1–66) 0.07 TRM@ 100 days 13% + 7% 24% + 8% 0.04 TRM@ 1 year 30% + 12% 42% + 10% 0.04 Relapse @ 1 year 40% + 12% 26% + 10% 0.01 PFS @ 2 years 33% + 11% 37% + 9% 0.9 OS @ 2 year 42% + 11% 44% + 9% 0.5 AGVD gr III–IV 7/84 19/103 0.04 Ext cGVHD 7/63 46/77 0.0000 Ext cGVHD in survivors 1/41 27/43 0.0000
Kinetics of Circulating Endothelial Progenitors in Patients with Sclerodermatous Chronic Graft-Versus-Host Disease.
Authors: Ashihara Eishi , Shimura Kazuho , Shimazaki Chihiro , Matsunaga Shinsaku , Tanaka Ruriko , Yokota Asumi , Murotani Yoshihide , Takeuchi Miki , Kawata Eri , Kamitsuji Yuri , Kuroda Junya , Taniwaki Masafumi , Kimura Shinya , Maekawa Taira
Published: October 2019

Chronic graft-versus-host disease (cGVHD) is a common late complications of allogeneic stem cell transplantation (allo-SCT). Some cGVHD patients develop skin lesions. This may be partly because cytotoxic T cells injure skin endothelial cells in GVHD patients. Since the skin lesions in sclerodermatous cGVHD (s-cGVHD) patients resemble those in progressive systemic sclerosis, which is characterized by impaired production of circulating endothelial progenitor cells (EPCs). We investigated whether low EPC production may help to promote the development of skin lesions in s-cGVHD patients. Peripheral blood (PB) was obtained from 14 healthy volunteers and 27 allo-SCT patients. Five patients developed s-cGVHD. In accordance with the Declaration of Helsinki recommendations, all procedures were approved by the institutional review board, and written informed consent was obtained from every participant. First, we assesed the absolute number of CD34+ cells by using a flow cytometric gating strategy on the basis of the International Society of Hematotherapy and Graft Engineering guidelines. The median CD34+ cell frequencies of the healthy volunteers, the 14 non-s-cGVHD, and the s-cGVHD patients were 1.74 /μL PB [IQR 1.34–2.01], 0.85 [0.73–1.62], and 0.41 [0.17–0.42], respectively. The non-s-cGVHD patients had significantly lower frequencies than control (p=0.018) while the s-cGVHD patients had significantly lower frequencies when compared to either the healthy volunteers (p=0.0012) or non-s-cGVHD (p=0.014) groups. Next, the CD34+/CD133+/vascular endothelial growth factor (VEGF) receptor-2+ EPCs were quantified after CD34+ cells were purified by using the magnetic cell sorting separation system. The s-cGVHD patients had significantly lower median circulating EPCs frequencies than non-s-cGVHD patients or control (median 145/20 mL [107–193] vs 1083.5 [669.3–2151]; p=0.0023, and vs 1530.5 [IQR 961.3–2158]; p=0.0012, respectively, Figure 1). We next assessed the ability of endothelial colony in PBMNCs of each group by using the standard CFU-EC assay. The s-cGVHD patients also had impaired median endothelial-forming ability compared to non-s-cGVHD patients or controls (3.8 [IQR 1.0–4.3] vs 12.8 [IQR 8.8–28.8], and vs 26.4 [IQR 23.6–30.6] respectively; p=0.0012, Figure 2). Lastly, the endothelial cell colony-formation potential was evaluated. Serum VEGF and basic fibroblastic factor (b-FGF) concentrations were measured by ELISA. Their VEGF and b-FGF serum levels were also higher than in controls. In conclusion, s-cGVHD patients show impaired vasculogenesis that may limit blood perfusion and could be responsible for the development of sclerodermatous lesions. Administration of EPCs to the skin lesions of s-cGVHD patients may be a novel therapy.
Updated Analysis of Allogeneic HSCT after RIC for Hematological Malignancies from the Société Française de Greffe de Moelle Osseuse et de Thérapie Cellulaire (SFGM-TC) Registry.
Authors: Michallet Mauricette , Le Quoc-Hung , Mohty Mohamad , Nicolini Franck E. , Boiron Jean-Michel , Espérou Hélène , Attal Michel , Milpied Noel , Lioure Bernard , Bordigoni Pierre , Yackoub-Agha Ibrahim , Bourhis Jean-Henri , Rio Bernard , Deconninck Eric , Renaud Marc , Sobh Mohamad , Raus Nicole , Blaise Didier
Published: October 2019

This report updates a retrospective study from SFGM-TC registry concerning 1108 patients who underwent allogeneic hematopoeitic stem cell transplantation (HSCT) after reduced intensity conditioning (RIC) from HLA identical siblings (84%) and unrelated donors (16%) for hematological malignancies. At time of conditioning, 442 patients were in CR, 337 in PR, 107 in stable disease (SD) and 222 in progressive disease (PD). As conditioning, 255 patients received fludarabine and TBI (2 grays), 465 patients fludarabine, busulfan and ATG and 388 patients an other regimen. After transplant, 336 patients (30%) developed an acute GVHD ≥ grade II (grade II: 178, III: 80 and IV: 78). A chronic GVHD was present in 388 patients (35%) (185 limited and 203 extensive). With a median follow-up of 30 months, the 3 and 5-year probability of overall survival (OS) were 43.5% (40–47) and 32%(29–35) respectively and the 3 and 5-year probability of event-free survival (EFS) were 35%(31–39) and 28% (24.5–31) respectively. The TRM at 1 year, 2 years and 3 years was 15% (13–17), 18% (15.5–21) and 20% (17–23). A mixture model, gfcure with Splus statistical package determined the percentages of long-term survivors and its adequacy was verified graphically. The probability to be a long-survivor was 24% (17.5–32.5) (Fig.1) and to be a long event-free survivor was 23% (19–28) (Fig. 2). The multivariate analysis has tested recipient and donor age, disease status pre-transplant, number of transplants before RICT, HSC source, sex matching, HLA matching, CMV status and ABO compatibility. The only factor which had a significant impact on long-term survival after RICT was the disease status just prior conditioning: PR versus CR: HR: 3.63 [1.14–9.18] p<0.001 and PD versus CR: HR: 4.35 [2.22–8.51] p<0.0001. In conclusion, these updated data demonstrate that allogeneic HSCT after RIC was able to possibly cure 23% of patients with haematological malignancies and the most important factor to take into account remains to be in CR pre-transplant.

Figure 1 Figure 1. Figure 2 Figure 2.
Allogeneic HSCT for Hematologic Malignancies in 145 Patients - Identical Biologic Prognostic Factors Affect Outcome Despite Different Conditioning Regimen Intensities.
Authors: Rondelli Damiano , Chunduri Sandeep , Dobogai Lisa , Mehta Jayesh
Published: October 2019

Analysis of pre-transplant variables affecting the outcome of allogeneic HSCT after 100 mg/m2 melphalan (± 50 mg/kg cyclophosphamide depending upon prior autograft) in 91 adult patients with hematologic malignancies identified refractory disease, poor performance status (ECOG 2/3), elevated LDH, and donor age >45 years as significant adverse prognostic factors affecting overall survival (OS) (Mehta et al. ASH 2006, BMT 2006). GVHD prophylaxis comprised cyclosporine-mycophenolate (HLA-matched siblings) or tacrolimus-mycophenolate (other donors). The patients were treated at NU. The outcome of 54 patients with hematologic malignancies treated at UIC with more intensive conditioning regimens was studied to see if the prognostic factors identified at NU were applicable to them. The conditioning regimens at UIC comprised 12.8 mg/kg IV busulfan + 120–160 mg/m2 fludarabine (n=36) or 140 mg/m2 melphalan + 150 mg/m2 fludarabine (n=18). Tacrolimus and methotrexate were used for GVHD prophylaxis. While the characteristics of the two patient populations were significantly different (especially diagnosis), transplant-related mortality and relapse rates were not significantly different for the two centers. As the table below shows, chemorefractory disease, poor performance status, and elevated LDH were adverse risk factors for OS in both groups of patients.

Variable Mel100 (NU) BuFlu/Mel140 (UIC) (OS) RR (95% CI) P RR (95% CI) P *Note the disparity - unfavorable in the NU group and favorable in the UIC group Refractory disease 3.0 (1.7–5.0) 0.0001 4.5 (1.8–11.4) 0.002 Performance status 2/3 3.9 (2.2–6.8) <0.0001 6.0 (2.0–17.8) 0.001 Elevated LDH 2.1 (1.3–3.5) 0.004 4.2 (1.7–10.8) 0.003 Donor age >45 1.9 (1.2–3.2) * 0.009 0.24 (0.06–1.03) * 0.06

However, donor age >45, an adverse risk factor in the NU population, appeared to be a borderline favorable factor in the UIC population. HLA mismatch was found to be an additional significant adverse risk factor in the combined population when it did not reach statistical significance in either group individually. The figures below shows the effect of the number of adverse risk factors (excluding donor age) on DFS and OS in the entire group of 145 patients (P<0.0001 for each). We conclude that biologic disease-related risk factors such as chemorefractoriness and elevated LDH, and patient-related risk factors such as poor performance status appear to be applicable to allograft recipients irrespective of the intensity of the underlying transplant procedure. The reason for the disparate effect of donor age in the two groups of patients is unclear, and needs to be studied further.

Figure Figure Figure Figure
Reduced Intensity (RI) Allogeneic Hematopoietic Cell Transplantation (HCT) Improves Outcomes for Older (≥ 60 Yrs) Patients (Pts) with Acute Myeloid Leukemia (AML).
Authors: O’Donnell Margaret R. , Stein Anthony S. , Nakamura Ryotaro , Slovak Marilyn L. , Tsai Nicole , Palmer Joycelynne , Rodriguez Roberto , Spielberger Ricardo , Smith Eileen P. , Snyder David S. , Parker Pablo M. , Popplewell Leslie , Pullarkat Vinod , Karanes Chatchada , Kogut Neil , Somlo George , Forman Stephen J.
Published: October 2019

The current paradigm of cytarabine-based induction chemotherapy followed by repetitive cycles of similar consolidation has yielded 2-year disease-free survival (DFS) of no more than 20% in older pts (≥60 yrs) who achieve remission. Induction failure (IF) is also high in this population (20–35%), depending on cytogenetic risk. Regimen-related toxicities have been a barrier to allogeneic HCT in older pts. RI conditioning provides a means to harness graft vs. leukemia in this group. Between 2/2000 and 3/1/2007, 46 pts ≥ 60 yrs (median 63 yr, range 60–71) received RI HCT from either related (26) or unrelated (20) HLA matched donors. In 23 pts, RI HCT was used as consolidation of remission (CR1 [19 pts] + CR2 [4 pts]) while 23 had active disease, including 10 IF. Karyotype was favorable in 9%, intermediate in 48% and poor in 41%. The median % marrow blasts for non-remission pts was 31% (6–80%) and peripheral blood blast was 5% (0–78%). The majority (41 pts) received fludarabine (FLU)/melphalan (MEL) for conditioning. Four pts received FLU with either 200G total body irradiation (TBI) (3 pts) or busulfan (1 pt) and one received TBI alone. Graft vs. host disease (GVHD) prophylaxis was based on either cyclosporine/mycophenolate mofetil alone or with methotrexate (MTX) (20/10) or tacrolimus/sirolimus +/− MTX (2/14 pts). The graft source was peripheral blood in 43 pts and marrow in 3 pts. Engraftment occurred at a median of 15 days (0–27 days) in 98% of recipients; graft failure occurred in one sibling HCT. Donor engraftment based on DNA analysis ranged from 30–100% at 4–6 wks post-HCT (median 100%) in the 33 analyzed pts. Mortality at day 100 was 10.8%. Acute GVHD (Grade 2–4) occurred in 61% of recipients and chronic GVHD has occurred in 23/33 evaluable pts. With a median follow-up of 24.5 months (m) for surviving pts (4–83 m), 25/46 (64.5%) pts are alive. For remission pts, the 2 yr DFS is 65% (CI 51–76%). For pts who received RI HCT as “salvage”, 34% are DF at 2 yrs. (CI 27–41%). Relapse rates were 11% for remission pts and 60% for non-remission pts. Deaths post HCT were attributed to relapse in 12 pts and treatment related mortality due to GVHD (6 pts) or infection (3 pts).

Conclusion: DFS can be improved for older patients with the use of RI HCT as remission consolidation. RIC also provides a meaningful salvage option for pts with IF or early relapse. Evaluation of donor options including both siblings and unrelated donors should be considered during induction for patients with good performance status.
Expression of CD38 and High Levels of CD5 Identifies Members of CLL Clones That Have Progressed beyond G1 and Proliferated.
Authors: Calissano Carlo , Damle Rajendra N. , Hellerstein Marc , Murphy Elisabeth J. , Hayes Gregory M. , Matthew Kaufman , Allen Steven , Rai Kanti , Chiorazzi Nicholas
Published: October 2019

Chronic lymphocytic leukemia (CLL) is characterized by the monoclonal expansion of CD5+ B cells with the morphology of small mature lymphocytes. Although the great majority of circulating CLL B cells appears as quiescent/non-proliferating lymphocytes, a small percentage of cells in peripheral blood appear to be cycling. Furthermore, within these clones, the CD38+ subset is enriched in those cells that entered G1, as evidenced by a larger number of Ki67-expressing cells. In an attempt to further identify submembers of CLL clones with different proliferative capacities, we measured, by flow cytometry, expression of Ki67 along with mcm6 and Cyclin D1, expressed from G1 to M, and Cyclin B1, expressed in G2-M. Since the expression of CD5 is characteristic of CLL cells and increases in vitro upon cellular activation, the intensity of CD5 expression was used to further divide CD38+ and CD38− cells into CD5bright and CD5dim fractions. The leukemic clones of eighteen IgVH unmutated CLL (U-CLL) and 16 IgVH mutated CLL (M-CLL) patients were analyzed. A similar pattern of expression for each cell cycle related marker studied was found. The presence of CD38 and high expression of CD5 was correlated with increased percent of cells expressing the various cell cycle markers (CD38−CD5dim<CD38−CD5bright; CD38+CD5dim<CD38+CD5bright). Strikingly, significant differences characterized the intraclonal comparisons between the 4 subpopulations (Table). To confirm these findings, the cells of 4 CLL patients involved in a deuterated “heavy” water protocol were sorted using CD38 and CD5 expression. In two of these cases, because of limiting cell numbers, CD5bright and CD5dim cells could be obtained only among CD38− cells. CD38−CD5bright cells contained more deuterium in their DNA than CD38−CD5dim cells indicating that more CD38−CD5bright cells had divided than CD38−CD5dim cells. A similar pattern was found in two patients for whom all four subfractions could be sorted. Thus, lowest deuterium enrichment in DNA was found in CD38−CD5dim cells and highest in CD38+CD5bright cells. The combined use of surface and intracellular markers, together with the 2H labeling, effectively further subdivides the proliferative leukemic compartment in CLL.

Expression of Cell Cycle related markers in CLL B cells defined by surface expression of CD38 and CD5 N=34 mcm6 % CyclinD1 % Ki67 % CyclinB1 % For every marker studied: all intraclonal comparisons (1 vs. 2, 1 vs. 3 etc.) are statistically significant (P<0.05) with the exception of 2 vs. 3 and, for cyclinB1, 3 vs. 4 CD38−CD5dim (1) 4.0±0.8 9.9±2.6 3.8±0.8 0.8±0.2 CD38−CD5bright (2) 9.8±1.9 17±4.2 10.2±1.9 2.1±0.5 CD38+CD5dim (3) 8.5±1.4 13.7±2.8 7.8±1.1 2.3±0.6 CD38+CD5bright (4) 19.5±2.3 24.3±4.0 15.2±1.8 2.8±0.6
Constitutively Serine Phosphorylated STAT3 Provides Chronic Lymphocytic Leukemia (CLL) Cells with Survival Advantage and Promotes Their Proliferation.
Authors: Hazan-Halevy Inbal , Ferrajoli Alessandra , Harris David , Mandal Pijus K. , McMurray John S. , Keating Michael , Estrov Zeev
Published: October 2019

Because signal transducer and activator of transcription (STAT) 3 is constitutively phosphorylated in several malignant disorders and plays a role in the pathophysiology of lymphoid malignancies we sought to investigate the activity of STAT3 in CLL. Using Western immunoblotting we studied peripheral blood (PB) cells from 40 patients with CLL and found that in all patients, regardless of disease stage or PB cell count, STAT3 is constitutively phosphorylated (p) on serine 727, but not on tyrosine, residues. Because it is thought that phosphorylation of tyrosine residues is requires for activation of STAT3 we asked whether the serine phosphoryalted form of STAT3 is biologically active in CLL. First we explored whether serine pSTAT3 translocates to the nucleus. Using confocal microscopy and Western blot analysis of cytoplasmic and nuclear cellular fractions we demonstrated that serine pSTAT3 is present both in the cytoplasm and nucleus of CLL cells. Then we asked whether serine pSTAT3 binds to DNA. Using the electrophoretic mobility shift assay (EMSA) we identified both a ‘shift’ and a ‘super-shift’ with anti-STAT3 and anti-serine 727 pSTAT3 antibodies, indicating that serine pSTAT3 binds to DNA. To further elucidate the role of serine pSTAT3 in CLL we inhibited its activity. We used PM, an analog of the STAT3 inhibitor Ac-Tyr(PO3H2)-Leu-Pro-Gly-Thr-Val-NH2 (Coleman DR et al., J Med Chem48:6661, 2005). Previous studies demonstrated that PM binds to the SH2 domain of STAT3 (but not to that of STAT5) and specifically inhibits STAT3 activity. Therefore, we incubated CLL cells with increasing concentrations of PM and found that PM inhibited the phosphorylation of STAT3 on serine 727 residues and downregulated Bcl-2 protein levels in a dose-dependent fashion. Because the Bcl-2 gene is activated by STAT3 it is conceivable that serine pSTAT3 activates its transcription in CLL cells. Furthermore, PM suppressed CLL colony-forming cell proliferation and induced apoptosis of CLL cells as determined by Annexin V, suggesting that constitutive serine phosphorylation of STAT3 induces both proliferative and antiapoptotic effects in CLL cells. Taken together, our data suggest that STAT3 is constitutively phosphorylated on serine 727 residues and is biologically active in CLL. Therefore, the activity of STAT3 inhibitors should be investigated in this disease.
Regulation and Function of NOTCH2 in B-CLL Cells.
Authors: Hubmann Rainer , Hilgarth Martin , Schnabl Susanne , Demirtas Dita , Schwarzmeier Josef D. , Jaeger Ulrich , Shehata Medhat
Published: October 2019

B-cell chronic lymphocytic leukemia (B-CLL) represents a clonal expansion of self-reactive CD5+ B-lymphocytes which seems to be resistant to apoptosis in vivo. One of the characteristics of B-CLL lymphocytes is the high expression of the B-cell differentiation/activation marker CD23 which we recently identified as a target gene for NOTCH2 signaling. NOTCH2 is implicated in the development/homeostasis of murine CD5+ (B-1a) B-cells, suggesting a function for human NOTCH2 in B-CLL leukemogenesis. Here we show that peripheral B-CLL lymphocytes overexpress a transcriptionally active form of NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 expression, and cytogenetics). Although the majority of unstimulated B-CLL samples downregulate their N2IC activity in vitro, DNA-bound N2IC complexes could be maintained by the protein kinase C (PKC) activator TPA (12-O-tetradecanoylphorbol 13-acetate) accompanied by an upregulation of the NOTCH2 target gene CD23 and increased B-CLL cell viability. These effects are sensitive to the PKC-δ selective inhibitor Rottlerin. In 80% of B-CLL cases, NOTCH2 signaling was found to be resistant to the γ-secretase inhibitors (GSI’s) Dapt and compound E, indicative for the expression of truncated forms of NOTCH2 which do not require γ-secretase for processing and function. Inhibition of NOTCH2 either by Dapt in GSI sensitive B-CLL cases or, more specifically, by RNA interference downregulates CD23 expression on the mRNA and protein level and sensitizes B-CLL cells for apoptosis. Since self-reactive B-cells are normally eliminated either by chronic (anergy) or apoptotic (negative selection) B-cell receptor (BCR) signaling, we asked whether NOTCH2 modulates B-cell fate decisions triggered by the BCR. For this reason, we stably transduced the human B-cell line BL41 with constitutive active forms of NOTCH2 and found that NOTCH2 inhibits BCR mediated apoptosis induced by surface-IgM cross-linking. In summary, the data demonstrate that NOTCH2 signaling is deregulated in B-CLL cells and might be critically involved in the PKC-dependent maintenance of their malignant phenotype.
Histone H1.2 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).
Authors: Gine Eva , Crespo Marta , Muntañola Ana , Calpe Eva , Gomes Maria Joao , Villamor Neus , Montserrat Emili , Bosch Francesc
Published: October 2019

Cytosolic release of histone H1.2 has been described as a new apoptogenic mechanism induced by DNA damage that results in cytochrome C release and activation of the apoptotic mitochondrial pathway. Primary tumoral CLL cells from 25 patients were investigated for histone H1.2 cytosolic release after treatment with genotoxic (fludarabine, mitoxantrone, etoposide, or X-ray radiation) and non-genotoxic (dexamethasone) agents. Cases were analyzed for the presence of poor-risk genetic alterations, particularly deletions at 17p13 and 11q22. Histone H1.2 release was correlated with the presence of genetic abnormalities and with the best clinical response obtained with standard treatments. FISH analysis, cell viability measured by annexin V binding, Western Blot studies and inmunofluorescence techniques with confocal spectral microscopy were also employed. DNA-damaging agents induced H1.2 release in a p53-dependent manner, which was confirmed by the lack of H1.2 release in p53-deleted cases. Non DNA-damaging agents induced release of H1.2 in both p53-deleted and non-deleted CLL cases. Moreover, nuclear H1.2 release was observed after genotoxic and non-genotoxic treatment independently of ATM function. From the clinical standpoint, the lack of histone H1.2 release correlated with resistance to genotoxic treatment. In CLL cells, histone H1.2 traffic was dependent on the p53-status after genotoxic treatment, but was also inducible after treatments acting independently of p53. In contrast, histone H1.2 release seemed not to be dependent on ATM function. Nuclear histone H1.2 release appears to be an important element in apoptosis induction in CLL, particularly in cases with abnormal p53 function resistant to conventional treatment.