bluuebunny/crossref_metadata_2025 huggingface dataset:
Unbalanced Chromosome Rearrangements Involving the 20q11.2 Region in Myeloid Malignancy.
Authors: Howard-Reeves Julie , Valgañón Mikel , Mazzullo Helen , Grace Colin , Campbell Lynda , Nacheva Elisabeth
Published: April 2020

Deletions of the long arm of chromosome 20 (del 20q) are a recurring abnormality associated with haematological malignancy (Mitelman 1995, Sandberg 1991, Heim and Mitelman,1995, Asimakopoulos & Green, 1996). The 20q deletions are seen in approximately 10% of the cases with PV and IMF, 4% of cases with myelodyspalstic syndrome (MDS) (Fenaux et al., 1996), 1–2% of patients with acute myeloid leukaemia (AML) as well as other myeloproliferative disease (MPD) (Bench et al. 2000). Deletions of chromosome 20q are well recognised in myeloid disorders and have been seen alone or as part of a complex karyotype (Johansson et al, 1993, Schoch et al 2002). A common deleted region (CDR) of chromosome 20q was observed by fluorescence in situ hybridization (FISH) mapping within 20q11.2/q12 region (Roulston et al., 1993, Bench et al, 2000). The breakpoints within this CDR have been shown to be heterogeneous (Asimakopoulos et al., 1994) and more recently a considerable overlap of this region has been observed in myeloid malignancy (Douet-Guilbert et al, 2008). Previous studies have been performed on cases with simple deletions of chromosome 20q as assessed by G-banding and confirmed by FISH, to be interstitial and no other rearrangements involving 20q have been reported so far. Here we present 43 patients with non-random translocations of chromosome 20q that appear to have a common breakpoint within the 20q11.2 region. Karyotypically some of the cases presented with the classical del(20q) morphology while in other cases translocations were identified, such as t(1;20)(q21;q11.2). Of these cases 44% have been shown to carry an unbalanced chromosome rearrangement with loss of material at the region of the breakpoint at 20q11.2 both with and without loss material on the partner chromosome. An example of the latter is the rearrangement t(17;20)(p13;q11.2) where 20q11.2 loss was accompanied by a p53 deletion (17p13). FISH mapping located the 20q11.2 breakpoint to be within the BAC clone RP1-1J6, which covers only the sequences of one gene, Topoisomerase 1 (TOP1). This gene is reported to form a fusion with NUP98 in therapy related MDS. Indeed we confirmed by FISH the presence of this fusion in 6 of our cases, all of which carried the t(11;20)(p15;q11.2). Unexpectedly, the rest of the cases also showed breaks within the same BAC clone, where the partner chromosomes were Yp11, 1q21, 17p11 and 21q11. These findings highlight the consistent involvement of the TOP1 gene at 20q11.2 in rearrangements occurring in MDS and MPD.
Protein Kinase C-Epsilon Is An Important Component of the Lyn Kinase Signaling Pathway in the Apoptotic Resistance of B- Chronic Lymphocytic Leukemia Cells.
Authors: Velcheti Vamsidhar , Tamma Srikanth , Larned Zoe L , Fuloria Jyotsna , Li Li , Rodwig Francis R , Cole John T. , Samaniego Felipe , Prakash Om
Published: April 2020

Although several mechanisms of apoptotic defects have been implicated in B- chronic lymphocytic leukemia (CLL) cells, the exact signaling pathways underlying these defects remain unresolved. Lyn kinase and protein kinase C (PKC)-epsilon play a critical role in B-cell antigen receptor signaling. They are also known to be negative regulators of apoptosis, and are linked to chemo-resistance. Because activation of PKCs results in their translocation from the cytosol to distinct subcellular locations in which they perform their specific functions, we examined the role of Lyn kinase in the cellular localization of PKC-epsilon in B-CLL cells and its link to the apoptotic defects. Untouched peripheral blood B-CLL cells or nonmalignant B cells were isolated from PBMCs of B-CLL patients or normal blood donors. Cytoplasmic and nuclear fractions of B-CLL cells and nonmalignant cells were prepared and examined for PKC-epsilon localization by Western blot analysis. Unlike the cytoplasmic localization in nonmalignant cells, a large fraction of PKC-epsilon in B-CLL cells was located in the nuclear fraction. Nuclear localization of PKC-epsilon in B-CLL cells was linked to the transcriptional activation of the antiapoptotic genes such as Mcl-1, XIAP, and Bcl-2, as well as NF-kappaB activation and VEGF production., both strongly implicated in the apoptotic resistance of B-CLL cells. Treatment of B-CLL cells for 16-20 hours with 10 micromolar Lyn-specific inhibitor peptide targeting a unique interaction site within Lyn (Cancer Res.2004;64:1058) resulted in redistribution of PKC-epsilon from the nuclear to the cytoplasmic fraction, decreased expression of the antiapoptotic genes as much as 60%, and decreased the viability of the leukemic cells. In addition, the Lyn-specific inhibitor peptide induced more than 50% inhibition of NF-kappaB p65 nuclear translocation and VEGF production in B-CLL cells but not in nonmalignant B cells. Similar results were obtained when a selective inhibitor peptide of PKC-epsilon translocation but not the negative control peptide was used. Our results suggest that PKC-epsilon is a downstream target of Lyn kinase, and that inhibition of PKC-epsilon nuclear translocation may have therapeutic potential by targeting Lyn kinase signaling pathway for the treatment of B-CLL.
Rituximab, Fludarabine, Cyclophosphamide, and Mitoxantrone (R-FCM) Is a Highly Active Chemoimmunotherapy Regimen for Chronic Lymphocytic Leukemia.
Authors: Bosch Francesc , Abrisqueta Pau , Villamor Neus , Terol Maria Jose , Ribera Josep-Maria , Gonzalez-Barca Eva , Gonzalez Marcos , Abella Eugenia , Delgado Julio , Gonzalez Yolanda , Ferrer Secundino , Carbonell Felix , Escoda Lourdes , Jarque Isidro , Monzo Encarnacion , Muntañola Ana , Salamero Olga , Montserrat Emili
Published: April 2020

The addition of monoclonal antibodies to chemotherapy has significantly improved treatment of patients with CLL. Based upon the excellent results obtained with our chemotherapy-only regimen fludarabine, cyclophosphamide and mitoxantrone (FCM) (Bosch et al. Clin Cancer Res, 2008) we have build up a new chemoimunotherapy combination, R-FCM (rituximab plus FCM). In November 2005 we initiated a multicentric phase II clinical trial that includes R-FCM as initial treatment followed by a maintenance therapy phase consisting of rituximab (375 mg/m2 every there months for 2 years). We report here the final results of the initial phase of this study, namely R-FCM treatment. From November 2005 to November 2007, 72 patients under the age of 70 with active CLL according the NCI and IWCLL criteria (Cheson et al. Blood, 1996; Hallek et al. Blood, 2008) were treated. Patients received rituximab 500 mg/m2 on day 1 (375 mg/ m2 in the first cycle), fludarabine 25 mg/ m2 i.v. on days 1 to 3, cyclophosphamide 200 mg/ m2 on days 1 to 3, and mitoxantrone 6 mg/ m2 i.v. on day 1, given at 4-week intervals up to six cycles. Treatment outcome was correlated with clinical and biological parameters. Minimal residual disease (MRD) was evaluated by four-color flow cytometry (Rawstron et al. Leukemia, 2007). Median cycles of R-FCM administered were 5 (range, 3–6), with 91% of patients completing all planed treatment. Response was evaluated three months after finishing therapy. Altogether, the overall response, MRD-negative CR, MRD-positive CR, and PR rates were 93%, 46%, 36%, and 10%, respectively. Variables correlated with a lower CR rate were del(17p) (25% CR) and increased β2-M serum levels (72% CR). No differences in response were observed according to the age of the patients. Severe neutropenia developed in 13% of patients. Major and minor infections were reported in 8% and 5% of cycles, respectively. Treatment-related mortality was 1%. With a median follow up of 24 months no cases of secondary MDS/AML have been observed.. Although based in two different phase II studies that preclude a completely valid statistical comparison, the CR rate obtained with R-FCM (82%, of which 46% MRD-negative CRs) favorably compares with that achieved with FCM (CR 64%, MRD-negative CRs 38%). In summary the 82% CR rate obtained with R-FCM is among the highest ever reported for any form of therapy for CLL. Both high β2-M serum levels and del(17p) predicted lower CR rate. Treatment toxicity was acceptable and manageable. Based on these results, R-FCM warrants further investigation, particularly in randomized clinical trials.
Randomized Comparison of Cladribine Plus Cyclophosphamide with Fludarabine Plus Cyclophosphamde in Untreated Patients with Chronic Lymphocytic Leukemia: Report of the Polish Adult Leukemia Group (PALG-CLL3).
Authors: Robak Tadeusz , Blonski Jerzy Z , Jamroziak Krzysztof , Gora-Tybor Joanna , Stella-Holowiecka Beata , Konopka Lech , Ceglarek Bernadeta , Warzocha Krzysztof , Seferynska Ilona , Kloczko Janusz , Piszcz Jaroslaw , Calbecka Malgorzata , Kostyra Aleksandra , Dwilewicz-Trojaczek Jadwiga , Wiater Elzbieta , Dmoszynska Anna , Kowal Malgorzata , Zawilska Krystyna , Grzywacz Alina , Hellmann Andrzej , Mital Andrzej , Zdunczyk Andrzej , Dybowicz Jacek , Kuliczkowski Kazimierz , Potoczek Stanislaw , Skotnicki Aleksander , Nowakowska-Domagala Maria , Lewandowski Krzysztof , Sulek Kazimierz
Published: April 2020

Purine nucleoside analogues, cladribine(2-CdA) and fludarabine (FA), especially combined with cyclophosphamide (CY) are potent cytotoxic drugs for the treatment of chronic lymphocytic leukemia (CLL). In this randomized study we aimed to establish whether combination of 2-CdA plus CY (CC) with FA plus (FC) provide similar benefit to previously untreated patients with CLL. The trial was started in January 2004 and the recruitment was ended in May 2007. The study primary endpoints were overall response (OR) and complete response (CR). The secondary endpoints included progression free survival (PFS), overall survival (OS), minimal residual disease negativity (MRD/-/) and treatment related toxicity. Eligible patients were randomly assigned to receive 6 courses of either 2-CdA 0.12 mg/kg/d i.v. + CY 250 mg/m2/d i.v. or FA 25 mg/m2/d i.v. + CY 250 mg/m2/d, both combinations for 3 consecutive days. The treatment response and toxicity were evaluated according to NCI-WG guidelines. MRD was evaluated in patients with CR using four-color flow cytometry assay.

There were no significant difference in the rates of OR, CR, MRD negativity, grade 3/4 neutropenia, thrombocytopenia and infections. PFS and OS were also similar in both groups. In conclusion, CC and FC regimens are similarly active and toxic in previously untreated CLL, however trend of longer OS in CC group is observed.

Characteristic CC arm FC arm P value Pts enrolled 212 211 - Pts evaluated 184 187 - No of courses (median, range) 6 (2–6) 5 (2–6) 0.56 OR (%) 163 (88.6) 159 (85.0) 0.31 CR (%) 86 (46.7) 91 (48.7) 0.43 MRD/–/ (%) 33 (68.8) 44 (72.1) 0.70 PFS (median, years) 2.195 2.361 0.86 Thrombocytopania gr 3/4 (%) 23 (12.6) 22 (11.6) 0.77 Neutropenia gr 3/4 (%) 39 (21.4) 43 (22.8) 0.76 Infection gr 3/4 (%) 53 (29.1) 54 (28.6) 0.91 OS (median, years) 4.066 2.531 0.10 Death (%) 37 (20.2) 53 (27.9) -
Retention but Significant Reduction of BCR-ABL Transcript in Hematopoietic Stem Cells in Chronic Myelogenous Leukemia after Imatinib Therapy.
Authors: Abe Akihiro , Minami Yosuke , Kitamura Kunio , Hayakawa Fumihiko , Nomura Yuka , Murata Makoto , Katsumi Akira , Kiyoi Hitoshi , Jamieson Catriona H.M. , Wang Jean Y.J. , Naoe Tomoki
Published: April 2020

Chronic myeloid leukemia (CML) is effectively treated with imatinib mesylate (IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors in CML patients. While IM induces disease remission, it does not appear to eradicate BCR-ABL-positive stem cells. We analyzed the HSC/Progenitors profiles using fluorescence-activated cell sorting (FACS) and investigated the minimal residual disease (MRD) in HSC and myeloid progenitors from patients with CML chronic phase (CP) after IM-therapy. HSC identified as CD34+CD38–Lin–, were separated to Thy-1+ (HSC/Thy-1+) and Thy-1– (HSC/Thy-1–) cells. HSC/Thy-1+ (CD34+CD38–Thy-1+Lin–), HSC/Thy-1– (CD34+CD38–Thy-1–Lin–), common myeloid progenitors (CMP: CD34+CD38+IL-3Rα+CD45RA–Lin–), granulocyte–macrophage progenitors (GMP: CD34+CD38+IL-3Rα+CD45RA+Lin–), and megakaryocyte–erythroid progenitors (MEP: CD34+CD38+IL-3Rα–CD45RA– Lin–) were isolated by cell sorting and MRD was quantified with real-time polymerase chain reaction detecting BCR-ABL transcripts. FACS analysis revealed higher levels of the HSC/Thy-1– cells and progenitors (CD34+CD38+Lin– cells) in bone marrow from patients with CML CP than in normal bone marrow although the level of long-term HSC in CML CP was similar with normal bone marrow. After the IM-therapy the proportion of progenitor pools (CD34+Lin– cells) within Lin– were remarkably reduced, especially that of HSC/Thy-1– cells and progenitor cells. The proportion of MEP was increased and that of GMP was decreased in bone marrow from patients with CML CP as compared with their normal counterparts. BCR-ABL positive progenitors in bone marrow were eradicated within 12 month in 5 out of 9 patients. BCR-ABL positive cells, however, remained in the stem cell population. They were positive even after achieving undetectable levels of BCR-ABL transcript in total RNA isolated from the bone marrow. The ratio of BCR-ABL to BCR was significantly decreased with the continuation of imatinib, however the retention of BCR-ABL-positive cells was observed in HSC/Thy-1– or HSC/Thy-1+ populations except one out of 9 cases. These results indicated retention but significant reduction of BCR-ABL positive stem cells in CML during IM-therapy. They also implicate that the sorted and purified stem cells are useful for more sensitive quantification of BCR-ABLpositive MRD.
Pulmonary Function in Pediatric survivors of non-Malignant Disorders after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Motosue Megan S , Inaba Hiroto , Pan Jianmin , Stokes Dennis C. , Srivastava Deo Kumar , Kasow Kimberly A. , Hale Gregory A. , Leung Wing
Published: April 2020

Allogeneic hematopoietic stem cell transplantation (HSCT) is used to treat children with non-malignant disorders. As the number of survivors of non-malignant disorders after allogeneic HCST rises, monitoring late sequelae becomes increasingly important. However, no studies have solely targeted this population especially on pulmonary function. We therefore performed a long-term prospective study of pulmonary function in 38 pediatric survivors of non-malignant disorders after allogeneic HCST, including aplastic anemia (n = 15), sickle cell anemia (n = 13), immunodeficiency syndromes (n = 8), Hurler syndrome (n = 1), and osteopetrosis (n = 1). A total of 260 pulmonary function tests were conducted on the patients. Older age of recipients was associated with poorer pulmonary function; lower ratio of forced expiratory volume in 1 second and forced vital capacity (FEV1/FVC; p < 0.0001); lower percent predictives in FEV1 (p = 0.0178), forced expiratory flow (FEF25–75%; p < 0.0001), FVC (p = 0.0299), and diffusion capacity corrected for hemoglobin (DLCOcorr; p = 0.0079); higher ratio of residual volume and total lung capacity (RV/TLC; p = 0.0193); and higher percent predictives in RV (p = 0.0308) and functional residual capacity (FRC; p = 0.0489). The following percent predictive values declined over time (median follow-up: 8 years): FEF25–75% (p = 0.0064), RV (p = 0.0134), FRC (p = 0.003), FVC (p = 0.0004), TLC (p = 0.0248), and DLCOcorr (p = 0.0077). Also, busulfan use was associated with lower FEV1 (p = 0.026), FVC (p = 0.0194), and DLCOcorr (p = 0.0105). This study underscores the need to monitor risk-adapted long-term lung function and provide early interventions in this vulnerable population, especially for those who received HSCT at an older age or had been administered busulfan.
Long Term Molecular Remissions after Autologous Haematopoietic Stem Cell Transplant in Follicular Lymphoma.
Authors: Lissandre Séverine , Vourch Patrick , Desbois Isabelle , Benboubker Lotfi , Dartigeas Caroline , Delain Martine , Senecal Delphine , Colombat Philippe , Gyan Emmanuel
Published: April 2020

Background and aim of the study: Autologous stem cell transplant (ASCT) has been shown to be an effective treatment for first-line and relapsed follicular lymphomas (FL). But no long term molecular remission was described. The aim of this retrospective study was to determine the clinical and molecular outcome of patients with FL who received ASCT during a 12-year period.

Method: All patients who underwent ASCT for first-line or relapsed FL between January 1992 and December 2004 were included. Overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan-Meier method and cumulative incidence of nonrelapse mortality (NRM), with relapse as a competing event, by Fine and Gray method.

Patient characteristics: Seventy-one patients with a median age of 45 years and a median follow-up of 108 months were analysed. The majority were of the subtype grade 1 (57 %), had a high tumour burden (50 %) and were treated in firstline (52 %). After an anthracyclin-based induction regimen, 12 patients were in first complete remission (CR), 25 in first very good partial response (VGPR), 8 in second CR and 26 in second VGPR. They received BCNU, Etoposide, Aracytine, Melphalan (BEAM in 58 %) or Cyclophosphamide, total body irradiation (42 %) as conditioning for the ASCT. The majority of them received an unpurged graft (58%).

Results : Thirty-eight patients were alive, 24 without progression between 4 and 12 years; 31 patients had died, 7 without progression. A total of 38 patients (55 %) developed recurrent lymphoma. Median OS was estimated at 8 years and 4 months. The ten-year PFS and the ten-year OS were 33 % and 47 %, respectively and the tenyear molecular PFS was 37 %. There was an apparent plateau on the remission duration curve at 32 % at 72 months and on the molecular remission duration curve at 37 % at 80 months. A plateau on the OS curve seemed to emerge at 41 % from the tenth year. Patients who received a purged graft had better OS and better PFS (median OS not reached versus 50 months, p = 0.08; median PFS not reached versus 22 months, p = 0.035) Three patients developed a secondary neoplasm and two a secondary myelodysplastic syndrome. The 10-year non-relapse mortality (NRM) was 20 %.

Conclusion : This long follow-up study showed a plateau on the PFS and on the molecular PFS curves, suggesting that a selected group of patients might be cured by ASCT.
Increase in Proportion of Th-17 Lymphocytes in Blood Heralds Overt Acute Graft Versus Host Disease in HSCT Patients.
Authors: Lange Andrzej , Dlubek Dorota , Jaskula Emilia , Drabczak-Skrzypek Daria , Sedzimirska Mariola , Lange Janusz
Published: April 2020

IL-17 is involved in chronic inflammation and autoimmunity, this cytokine producing cells are well characterized in the mouse model. In man, situation is more complex due to the genetic variability, co-morbidities and environmental factors influencing the immune response. In patients post HSCT alloreactivity with the presence of aGvHD is a major factor affecting the outcome of transplantation. Understanding of the role of cells involved in regulation of the immune system is crucial for the treatment tailoring to favour tolerance but not making the recipient more prone to opportunistic infections and leukaemia relapse.

In this study 27 patients post HSCT were investigated for the presence of regulatory cells and those producing IFNgamma and IL-17 in blood. Blood was collected at the time of haematological recovery or when the first clinical symptoms of aGvHD became apparent and then in one week intervals until +60 day post transplantation. PBMC were stimulated for 4 hrs with BD Leukocyte Activation Cocktail in the presence of Golgi Stop (BD, Erembodegen, Belgium) and then stained extracellularly with anti-CD4, permeabilized with Fixation/Permeabilization Concentrate and Diluent (eBioscience, San Diego, CA) and finally stained with anti-IFNgamma (BD), anti-IL-17 (eBioscience) anti FoxP3 (eBioscience). CD4+ cells subpopulations were analysed according to the expression of the stained features.

It was found:

FoxP3+CD4+ cells were in higher proportions in pts with aGvHD (results from all time-points taken together) (9.93%±0.61 vs 8.2%±0.50, n=98, p=0.040, U Mann-Whitney test)

IFNgamma producing CD4+ lymphocytes were in higher proportions (0,34 vs 0.14, ns) in blood samples taken from patients lacking as compared to those having aGvHD.

CD4+IL-17+ lymphocytes proportions increased from 1.39%±0.42 to 5.33%±2.45 (p=0.04, Wilcoxon Test for pairs) one week before aGvHD.

Notably, at the time of full blown aGvHD the proportions of CD4+IL-17+ cells were lower as compared to the results of previous measurements (0.74%±0.29, p=0.008, Wilcoxon Test for pairs). Taking all results together the proportion of CD4+IL17+ lymphocytes were lower in patients having as compared to those lacking aGVHD (0.93%±0.27 vs 1,53%±0,41, p=0.05, U Mann-Whitney test).

It appears that:

FoxP3 positive cells expand during aGvHD likely as a response to alloreactive stimulation.

INFgamma+ CD4+ cells benefit the course post HSCT making the patients less susceptible to aGvHD.

CD4+IL17+ cells are likely involved at the early stage of aGvHD patho-mechanism heralding the clinical manifestation of this complication, but then they disappear from blood, possibly being marginalized in the inflamed tissues.
Apoptosis-Independent Activation of Caspase-3 in CD34–Positive (CD34+) Mobilized Cells.
Authors: Augeul-Meunier Karine , Crampé Carine , Farce Philippe , Mounier Christiane , Guyotat Denis , Campos Lydia
Published: April 2020

G-CSF mobilized peripheral blood CD34+ cells are now the preferred and major source of hematopoietic stem and progenitor cells harvested for both autologous and allogeneic transplantation. Several mechanisms, like SDF-1/CXCR4 interactions or degradation of adhesion molecules by proteolytic environnement, are involved in the mobilization process. However this phenomenon is still partially understood. Gene expression analysis has identified an overexpression of the caspase-3 gene in CD34+ mobilized cells, compared to CD34+ from normal bone marrow. Caspase-3 is the main effector of the terminal phase of apoptosis. However recent studies have provided evidence of its implication in non apoptotic cellular processes, such as differentiation, migration and cytoskeleton modelling. We evaluated by multicolour flow cytometry the expression of activated caspase-3 in G-CSF mobilized CD34+/CD45+ cells from blood (n=16), and from apheresis products (n=10). CD34+/CD45+ cells from normal bone marrow (n=4) served as control. Caspase-3 activity on fluorescent substrate (PhiPhiLux method) and apoptosis (Annexin V assay) were also evaluated. Finally we analysed the expression of anti apoptotic proteins Bcl-2, Bcl-Xl, and of Heat Shock Proteins HSP27, HSP70 and HSP90 in the same cell population. There was no significant difference for apoptosis between mobilized and bone marrow CD34+ cells (26% versus 33% apoptotic cells). Activated caspase-3 levels were significantly higher in mobilized CD34+ cells (mean fluorescence intensity 3.64 fold higher). This was consistent with cleavage of caspase-3 substrate observed in mobilized cells, but not in bone marrow CD34+ cells. An increased expression of HSP90 (of which caspase-3 is a client protein) was observed in peripheral CD34+ cells, but there was no variation of BCl-2 and Bcl-Xl expression. Our results show an activation of caspase-3 in the mobilized peripheral blood CD34+ cells, which appears to be independent of apoptosis induction. The role of this activation and possible control by HSPs warrants further analysis to establish its relationship with mobilization mechanisms.
The Use of Clinical Grade Magnetic Beads to Deplete Immune Suppressive Elements from Autologous Peripheral Blood Hematopoietic Stem Cell Grafts Enhances T Cell Immune Responses.
Authors: Al-Kadhimi Zaid S , Mahdi Haider , Abdalamir Moshrik , Paul Elyse , Ratanatharathorn Voravit , Uberti Joseph , Lum Lawrence G.
Published: April 2020

Background: Despite the demonstrated efficacy of high dose chemotherapy and autologous hematopoietic stem cell transplantation in Myeloma and relapsed Lymphoma, relapse continues to be a problem. Improving post transplant immune reconstitution in general and tumor specific immune response can reduce relapse rates. Pre-clinical and clinical data suggest that the abundance of CD4+/CD25+ regulatory T-cells (T-regs) and CD14+ monocytes in the autologous graft can contribute to the well described immune suppression state in the early months post transplant. We hypothesized that it is feasible to deplete both of these cellular inhibitory elements from the autologous grafts using clinical grade CD25 and CD14 magnetic beads from Miltenyi. We also hypothesized that the depletion of these populations would improve the proliferative capacity of T-cells in these grafts.

Methods: Samples of peripheral blood hematopoietic stem cell grafts from 9 patients (4 lymphomas and 5 myelomas) undergoing autologous stem cell transplantation, with G-CSF mobilized grafts. We depleted T regs and monocytes, using CD25 and CD14 beads from these graft samples. Then we compared T cell reactivity between the manipulated graft versus the unmanipulated graft samples, in a mixed lymphocyte reaction.

Results: We show that it is feasible to deplete both populations based on phenotype analysis that confirmed selective depletion of CD4+/CD25+/CD127-T reg population (70–80%) and CD14 population (74%.). The positively selected population was enriched for T regs CD4+CD25+CD127-(50–85%) and monocytes CD14+ (75%+). Furthermore in 9 out of 9 patients T cell proliferation measured by 3H thymidine uptake was close to two fold or higher in the double depleted grafts compared to the unmanipulated grafts (P=0.004 Wilcoxon Signed Rank Test).

Conclusion: Double depletion of T regs and monocytes from autologous grafts using clinical grade magnetic beads, leads to enhanced T cell proliferation. This work will lay the ground for a subsequent clinical trial of regulatory T-cell/Monocyte depleted autologous transplantation using the same strategy, to enhance immune recovery post transplant and achieve better anti-lymphoma, or anti-myeloma immune response.

Figure Figure
Ethnic Variations in Diagnosis, Treatment, Socioeconomic Status and Survival in a Large Population-Based Cohort of Elderly Patients with Non-Hodgkin’s Lymphoma
Authors: Song Shen , Burau Keith D , Fang Shenying , Wang Haijun , Du Xianglin L , Wang Luhua
Published: April 2020

Background: Little is known about ethnic disparities in survival among patients with Non-Hodgkin’s Lymphoma (NHL), although these disparities have been documented for patients with many solid tumors.

Methods: We studied a retrospective cohort of 13,321 patients diagnosed with incident NHL at age >=65 in 1992–1999, identified from the SEER (Surveillance, Epidemiology and End Results)-Medicare linked database with 11 years of follow-up. Of these patients, 11,868 were Caucasians, 533 were African-Americans, and 920 were other ethnicities. A time-to-event Cox regression model was used to examine the relative risk of all-cause and disease-specific mortality.

Results: A larger proportion (72.2%) of African-Americans were in the poorest quartile of socioeconomic status as measured by the poverty level compared to 21.8% of Caucasians, and 43.2% of African-Americans received chemotherapy compared to 52.4% of Caucasians (p<0.01).

Hazard ratio of all-cause and NHL-specific mortality increased significantly with age, advanced tumor stage, higher comorbidity scores, and poorer socioeconomic status. Patients receiving either chemotherapy or radiotherapy or both were significantly less likely to die. Compared to Caucasians, African-Americans were not statistically significant at risk of all-cause (Hazard ratio = 0.97, 95% confidence interval: 0.88–1.08) and disease-specific mortality (1.07, 0.92–1.25), after adjusting for treatment, comorbidity, and socioeconomic status.

Conclusions: The risk of mortality in patients with NH-lymphoma was associated with socioeconomic status and was reduced in patients receiving chemotherapy. No significant differences in the risk of mortality were observed between African-American and Caucasian patients after controlling for factors such as treatment and socioeconomic status.
Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist CTCE-9908 Enhances Chemotherapy-Induced Apoptosis in AML
Authors: Lu Hongbo , Zeng Zhihong , Shi Yuexi , Konoplev Sergej , Wong Donald , Konopleva Marina , Andreeff Michael
Published: April 2020

The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells towards the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva, Leukemia 2002). Using a peptide analog of SDF-1α designated CTCE-9908, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that CTCE-9908 significantly inhibits SDF-1α-induced migration of U937 (43% inhibition) and OCI-AML3 cells (40% inhibition) in a dose-dependent manner. In three of the four primary AML samples which expressed CXCR4 on cell surface and migrated in response to SDF-1α, 50 μg/ml CTCE-9908 reduced SDF-1α-induced migration of leukemic blasts (60%, 19% and 50% inhibition respectively). In in vitro co-culture systems, stromal cells significantly protected OCI-AML3 cells from chemotherapy induced apoptosis [no MS-5, 75.2±5.2% annexinV(+); with MS-5, 59±1.1% annexinV(+)]. Western blot analysis revealed that CTCE-9908 inhibits Akt and Erk phosphorylation in a dose-dependent manner in the OCI-AML3 cell line stimulated by SDF-1α. Blockade of CXCR4 expression with CTCE-9908 markedly abrogated the protective effects of stromal cells on OCI-AML3 [Ara-C, 59±1.1% annexinV(+); Ara-C + CTCE-9908, 76.9±1.35 annexinV(+)]. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in four out of five primary AML samples with surface expression of functional CXCR4 (mean increase, 25.1±9.3% compared to chemotherapy alone). In vivo, subcutaneous administration of 1.25mg CTCE-9908 induced mobilization of leukemic cells from primary AML patient transplanted into NOD/Scid-IL2Rγ-KO mice (from 15% to 27% circulating leukemic cells 1 hour post CTCE-9908 injection). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis via retention of leukemic cells in the bone marrow microenvironment niches. Disruption of these interactions by the potent CXCR4 inhibitor CTCE-9908 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interaction. Based on these observations, in vivo experiments are ongoing to characterize the efficacy of chemotherapy combined with CTCE-9908.
Disruption of the Osteoblast Niche by G-CSF Is Associated with Hematopoietic Stem Cell Quiescence and Loss of Long-Term Repopulating Activity: Role of Cdkn1a
Authors: Gopalan Priya K. , Christopher Matthew J. , Greenbaum Adam M. , Link Daniel C.
Published: April 2020

The bone marrow microenvironment plays a key role in regulating hematopoietic stem cell (HSC) function. In particular, bone marrow stromal signals contribute to the maintenance of HSC quiescence, a property that is thought to be associated with long-term repopulating activity. We previously reported that G-CSF treatment disrupts the osteoblast niche by inducing osteoblast apoptosis and inhibiting osteoblast differentiation. In this altered bone marrow microenvironment, we also showed that the number of HSCs in the bone marrow after G-CSF treatment (as defined by CD34− Kit+ Sca+ lineage-cells or CD150+ CD48− CD41− lineage-[SLAM] cells) was unchanged and that the HSCs were more quiescent than HSCs from untreated mice. However, despite the quiescent phenotype, there was a marked loss of HSC long-term repopulating activity. To define mechanisms for this phenotype, we first asked whether G-CSF acts directly on HSCs to inhibit their long-term repopulating activity. Bone marrow chimeras containing wild type and G-CSFR−/− cells were established and treated with G-CSF. The contribution of G-CSFR−/− cells to hematopoiesis remained stable for at least 3 months after G-CSF treatment, demonstrating that the effects of G-CSF on HSC function are not direct. We next performed RNA expression profiling on sorted SLAM cells, a cell population highly enriched for HSCs. These data showed that expression of Cdkn1a (p21cip1/waf1) was increased in HSCs harvested from G-CSF treated mice. To define the contribution of Cdkn1a to HSC quiescence and loss of repopulating activity following treatment with G-CSF, Cdkn1a−/− mice (inbred on a C57BL/6 background) were studied. Wild-type or Cdkn1a−/− mice were treated with G-CSF for 7 days and pulse labeled with bromo-deoxyuridine (BrdU), and the percentage of SLAM cells that labeled with BrdU was determined. Consistent with our previous observations, treatment of wild-type mice with G-CSF resulted in a significant decrease in the percentage of BrdU+ SLAM cells in the bone marrow. In contrast, in Cdkn1a−/− mice, no change in the percentage of BrdU+ SLAM cells after G-CSF treatment was observed [10.08 ± 2.26% (untreated); 10.96 ± 2.80% (G-CSF treated); p = NS]. To assess HSC function, competitive repopulation assays were performed using untreated or G-CSF treated bone marrow from wild type or Cdkn1a−/− mice. Surprisingly, G-CSF had a similar deleterious effect on HSC repopulating activity in both wild type and Cdkn1a−/− mice. Collectively, these data show G-CSF treatment, possibly through disruption of the osteoblast niche, induces HSC quiescence and loss of long-term repopulating activity. HSC quiescence, but not loss of repopulating activity, is dependent upon Cdkn1a−/−. The mechanisms by which G-CSF treatment results in a loss of HSC function are under investigation.
Red Blood Cell Scramblase Activation Is Induced by Phorbol Ester and Prevented by Inhibition of Protein Kinase C
Authors: Barber Latorya Arnold , Palascak Mary B , Joiner Clinton H. , Franco Robert S.
Published: April 2020

Phosphatidylserine (PS) is normally confined to the inner leaflet of the red blood cell (RBC) membrane, but is externalized in many sickle RBC. This external PS may contribute to decreased sickle RBC survival, increased thrombogenesis, and increased sickle RBC-endothelial interaction. Aminophospholipid Translocase (APLT) and Phospholipid Scramblase (PLSCR) are thought to regulate the distribution of PS between the inner and outer membrane leaflets. APLT maintains phospholipid (PL) asymmetry by returning externalized PS to the inner membrane leaflet. PLSCR disrupts PL asymmetry by causing nonspecific PL equilibration across the membrane. The mechanism(s) of PLSCR activation have not been defined, but previous studies have demonstrated PS externalization as a result of treatment with the potent PKC activator, PMA (phorbol-12-myristate-13-myristate). We examined the effect of PMA on PLSCR activation (measured by NBD-PC internalization) in normal RBC in the presence or absence of the PKC inhibitor, chelerythrine chloride. In response to PMA, PLSCR is significantly and rapidly activated as measured by the fraction of NBD-PC internalized after six minutes compared to a DMSO control (0.267±0.046 vs. 0.056±0.011, n=5, p<0.001). There was also a significant increase in PS externalization (Annexin V binding) in response to PMA stimulation after thirty minutes compared to a DMSO control (14.76±4.80% vs. 1.50±1.55%, n=5, p=0.021). PMA-dependent NBD-PC internalization and PS externalization were significantly inhibited in the presence of chelerythrine chloride (0.180±0.031 vs. 0.267±0.046, n=5, p=0.016 for NBD-PC internalization at six minutes and 3.51±0.92% vs. 14.76±4.80%, n=5, p=0.011 for PS externalization at thirty minutes). These data provide evidence that PKC is involved in the regulation of scramblase activity and thus the distribution of PL across the RBC membrane. Future treatment strategies for sickle cell disease could include PKC inhibitors that would prevent PS externalization and potentially result in decreased sickle RBC-endothelial interactions, decreased thrombogenesis, and prolonged sickle RBC survival.
Deficient Apoptosis Signaling in Primary Pediatric BCP-ALL Xenograft Cells Determines the Kinetic of Engraftment in Vivo in a NOD/SCID Model and Patient Outcome
Authors: Queudeville Manon , Eckhoff Sarah M. , Stahnke Karsten , Debatin Klaus-Michael , Meyer Lueder H.
Published: April 2020

We previously identified the importance of intact apoptosis signaling for treatment response in primary pediatric ALL and AML by analysing two key apoptogenic events, caspase-3 activation and cytochrome c release. A correlation of both events was exclusively found in the groups of patients with good response to treatment, showing that intact apoptosis signaling is a critical feature for favorable outcome. Using a NOD/SCID mouse model for pediatric B cell precursor ALL we found that rapid in vivo leukemia growth (Time to leukemia (TTL) short) in transplanted recipients determines poor patient outcome. In this study we now investigated the importance of deficient apoptosis signaling for leukemia engraftment in this model. We analyzed a total of 17 BCP-ALL xenografts, 5 of which had a short time (TTLshort) to clinical manifestation of the disease (< 10 weeks) and 12 with a significantly longer time (TTLlong) to leukemia (> 10 weeks). The apoptosis signaling parameters cytochrome c release and caspase-3 activation were evaluated flowcytometrically in directly isolated cells from the leukemia bearing mice before and after induction of apoptosis by factor deprivation in culture and related to TTL. Comparing TTLshort and TTLlong a significant correlation of both apoptosis parameters was found in the TTLlong group only (P= .017). Importantly, patients whose blasts engrafted in NOD/SCID-mice with TTLlong showed a superior outcome compared to the TTLshort group. Additionally, proficient apoptosis signaling as indicated by the correlation of cytochrome c release and caspase-3 activation, was only found in blast cells from recipients where the corresponding patient had favorable prognostic factors such as TEL/AML1-positivity (P= .023), absence of hyperleucocytosis at diagnosis (P= .012), absence of relapse (P= .038) and no early relapse (P= .017). In contrast, the correlation of cytochrome c release and caspase-3 activation was absent in the groups with poor prognosis. We also found a direct positive correlation between TTL and cytochrome c (P= .022) and TTLlong and caspase-3 activation (P= .048), indicating that a longer engraftment time may be due to proficient apoptosis signaling in the leukemic blasts. Our finding in the NOD/SCID human-ALL model matches our retrospective results in pediatric ALL and AML to conclude that the functional integrity of a downstream apoptotic checkpoint (cytochrome-c related caspase activation) is an important feature regulating leukemia biology. Thus, deficient apoptosis signaling appears to determine rapid engraftment of leukemia cells in the NOD/SCID model in vivo and consequently poor patient outcome.
The Role of Activation-Induced C-Type Lectin (AICL) in NK Cell Reactivity against Acute Leukemia
Authors: Baessler Tina , Kuttfruff Sabrina , Krusch Matthias , Kanz Lothar , Steinle Alexander , Salih Helmut R
Published: April 2020

Studies of haploidentical stem cell transplantation (SCT) demonstrate that NK cells play an important role in immunosurveillance of leukemia and indicate that acute lymphoblastic leukemias (ALL) are less susceptible to NK cell reactivity than acute myeloid leukemia (AML). The molecular mechanisms mediating NK cell reactivity against leukemia cells, especially those responsible for the differential susceptibility of AML and ALL to NK cell immunosurveillance, are yet largely unclear. Recently we have identified AICL as a myeloid-specific ligand for the activating NK cell receptor NKp80 (Welte et al., Nat. Immunol.2006, 7:1334). Here we studied the role of AICL and NKp80 in NK cell reactivity against acute leukemia. Substantial levels of AICL were expressed on blasts in 23 cases (40%) of 57 AML patients studied, while no AICL expression was detected on leukemic cells of 21 investigated ALL patients. Healthy CD34+ cells did also not express AICL, which suggests that AICL may be a marker for a malignant phenotype of myeloid hematopoietic precursor cells. Expression in AML was not restricted to a specific French-American-British (FAB) subtype, but was significantly associated with myelomonocytic differentiation (FAB M4 and M5). No correlation with a particular cytogenetic abnormality or expression levels of HLA class I was observed. Inhibition of AICL-NKp80 interaction by blocking NKp80 (Fab’)2 fragments significantly diminished both cellular cytotoxicity (>60% reduction, E:T ratio 40:1) and IFN-γ production (>60% reduction) of NK cells in cocultures with AICL-positive AML cells demonstrating that AICL expression renders AML blasts susceptible to NK cell reactivity. On NK cells of AML patients, expression of NKp80 was found to be significantly reduced compared to healthy donors. Cocultures of AICL- and control-transfectants with NK cells revealed that NKp80 expression is downregulated after interacting with its ligand, which provides a functional explanation how leukemia cells may evade NKp80-mediated NK cell immunosurveillance. Taken together, our data indicate that AICL-NKp80 interaction substantially contributes to NK cell reactivity against AML but not against ALL, which may explain the differential susceptibility of AML and ALL to NK cell reactivity. Our data further support the notion that NK cells play an important role in the pathophysiology of acute leukemia and provide useful information for therapeutic approaches in AML which, like e.g. haploidentical stem cell transplantation, rely on a sufficient NK cell response.
Rituximab in Combination with CHOP in Patients with Follicular Lymphoma: Analysis of Treatment Outcome of 552 Patients Treated in a Randomized Trial of the German Low Grade Lymphoma Study Group (GLSG) after a Follow up of 58 Months
Authors: Buske Christian , Hoster Eva , Dreyling Martin , Forstpointner Roswitha , Kneba Michael , Schmitz Norbert , Schmits Rudolf , Metzner Bernd , Reiser Marcel , Lengfelder Eva , Woermann Bernhard J , Harder Harriet , Hegewisch-Becker Susanna , Berdel Wolfgang E. , Hess Georg , Eimermacher Hartmut , Aldaoud Ali , Planker Manfred , Unterhalt Michael , Hiddemann Wolfgang
Published: April 2020

We have previously reported that compared to CHOP alone the addition of Rituximab (R) to CHOP significantly increases the response rate (RR), the time to treatment failure (TTF) and also the overall survival (OS) in patients with newly diagnosed advanced follicular lymphoma. However, in the previous report, the median observation time was short with 18 months and no data were reported on the outcome of different risk groups according to the FLIPI (Hiddemann et al., Blood 2005). We now report on the treatment outcome of 552 patients with advanced stage follicular lymphoma randomized between R-CHOP versus CHOP alone after a median follow up of 58 months. Responding patients < 60 yrs. of age underwent a second randomization in first remission for Interferon alpha maintenance (IFN) versus myeloablative radio-chemotherapy with subsequent stem cell transplantation (ASCT), whereas all older pts received IFN maintenance.

R-CHOP induced a significantly higher RR (97% vs. 91%, p=0.005). Furthermore immunochemotherapy resulted in significantly longer TTF (median not reached vs. 35 months, 5-years TTF 65% vs. 32%, p<0.0001), response duration (RD, 5-years RD 66% vs. 35%, p<0.0001), and OS (5-years OS R-CHOP 90% vs. CHOP 84%, p = 0.0493). To analyse the impact of R-chemotherapy in different risk groups we performed subgroup analyses for patients with low (LR), intermediate (IR) or high risk (HR) according to the FLIPI: R-CHOP prolonged significantly TTF in all risk groups compared to CHOP alone (5-years TTF: 84% vs. 46% for LR (p=0.0021); 73% vs. 37% for IR (p<0.0001); 49% vs. 23% for HR (p<0.0001). The superiority of R-CHOP vs. CHOP could be documented in the patient group not treated with ASCT: the RD was significantly prolonged by the immunochemotherapy (5-years probability 62% vs. 26%, respectively; p<0.0001). This could not be seen in the group of younger patients consolidated with ASCT (5-years probability 78% vs. 66%, p=0.43). However, the number of events was too low to draw any final conclusion.

These data with a mature follow-up demonstrate that first–line R-CHOP significantly improves response rate and TTF in patients with FL compared to the respective chemotherapy alone. However, they also demonstrate that R-chemotherapy might be not sufficient for a long-lasting improvement. This indicates the necessity to perform clinical trials that test whether adding consolidation and/or maintenance after initial cytoreduction further improves treatment outcome.
Lenalidomide Enhances Anti-Tumor Effect of γδ T Cells against Mantle Cell Lymphoma
Authors: Gaidarova Svetlana , Corral Laura G , Gleizer Emilia , Young David , Brady Helen , Bennett Brydon , Lopez-Girona Antonia
Published: April 2020

Introduction: Mantle cell lymphoma (MCL) has had one of the poorest prognoses of all NHL subtypes, and is characterized by a relatively short duration of response, progression-free survival, and time-to-progression, with most patients relapsing. The immunomodulatory drug lenalidomide has shown promising activity in MCL and currently is being investigated further in the clinic for treatment of this tumor. In previous experiments, lenalidomide stimulated proliferation as well as activation of γδ T cells in vitro. Potential mechanisms of lenalidomide’s anti-tumor activity in MCL were investigated here.

Methods: As γδ T lymphocytes mediate anti-tumor effects through antigen recognition and intracellular signaling, we assessed the effect of lenalidomide on tumor cell expression of co-stimulatory and antigen-presenting molecules, as well as other immune response molecules, using microarray analysis. We then assessed the impact of these tumor cell changes on tumor-cell recognition by γδ T lymphocytes. We treated peripheral blood mononuclear cells (PBMCs) from healthy donors with vehicle or lenalidomide. We next purified γδ T cells from control or lenalidomide treated PBMCs and co-cultured them with MCL cells.

Results: Lenalidomide upregulated the expression of several genes involved in the immune response, cell adhesion, cytokine-cytokine receptor interaction, cell cycle and apoptosis by at least 2-fold. In the immune response category, co-stimulatory and antigen-presenting molecules were strongly up regulated including CD86, CD40, CD58, and CD1c. The lenalidomide-induced up-regulation of CD1c was confirmed in MCL lines by quantitative RT-PCR as well as flow cytometry and was also observed in primary B-CLL cells and normal B cells treated with lenalidomide ex-vivo. CD1c, a member of the CD1 family of MHC-like molecules, is expressed mainly in professional antigen-presenting cells, such as B cells, and mediates the presentation of lipid antigens to γδ T cells. Lenalidomide strongly promoted expansion of the Vδ1 subpopulation of γδ T cells and enhanced production of interferon-g. Treatment of MCL cells and γδ T cells with lenalidomide induced changes in the organization of the actin cytoskeleton, re-localization of surface markers and enhanced the number of γδ T-MCL synapses. Furthermore, gd T cells, expanded in the presence of lenalidomide, had enhanced killing activity against MCL cells.

Conclusion: Our studies show that lenalidomide might provide dual activity against MCL cells by inducing expression of CD1c in the tumor cells and by enhancing γδ T cell-mediated cytotoxicity. The therapeutic implications, as well as the prospects for novel biomarker development will be discussed further.