bluuebunny/crossref_metadata_2025 huggingface dataset:
Clofarabine Combinations in Acute Myeloid Leukemia (AML) Salvage: A Dose-Finding Phase I Study of Clofarabine Plus Idarubicin and Clofarabine/Idarubicin Plus Cytarabine (ara-C).
Authors: Faderl Stefan , Ferrajoli Alessandra , Wierda William , Ravandi Farhad , Estrov Zeev , Verstovsek Srdan , Thomas Deborah A. , Garcia-Manero Guillermo , Kwari Monica , Kantarjian Hagop M.
Published: April 2020

Clofarabine is a second-generation nucleoside analog with single agent activity in acute leukemias. To try and improve efficacy, various combination trials are being conducted. In studies of clofarabine plus ara-C we reported overall response rates of 41% (CR 24%) in AML salvage and 60% (CR 52%) in untreated elderly AML with acceptable toxicity profile. To explore additional clofarabine combinations in AML we conducted a phase I study of clofarabine (C) with idarubicin (I) [CI] alone and with ara-C (A) [CIA] in pts with relapsed AML and high-grade MDS. Dose-limiting toxicities (DLT) were defined as ≥ grade 3 drug-related toxicities. Maximum tolerated dose (MTD) was determined by “3+3” method. Thirty-three patients (18 on CI and 15 on CIA) have been treated and are evaluable. Of 18 pts on CI, 6 were primary refractory and 12 in first relapse (median first remission duration [CRD1] 2 mos. [range 0–9]. Eleven pts had abnormal cytogenetics. Fourteen pts received prior ara-C-based regimens, 2 relapsed from allogeneic transplant (SCT). Median age: 57 yrs (range 24–71). Four dose levels have been explored. When C was given at 22.5mg/m2 i.v. daily x 5d and I at 12mg/m2 i.v. daily x 3d, 2 ≥ gr. 3 toxicities (diarrhea, rash, ↑ bili) occurred necessitating dose de-escalation. Subsequent levels included C at 15mg/m2 x 5d/I at 8mg/m2 x 3d (6 pts, 1 ≥ gr.3 toxicity [↑ bili]), C at 18 mg/m2 x 5d, I at 10mg/m2 x 3 d (3 pts, no DLT), and C at 22.5mg/m2 x 5d, I at 10mg/m2 x 3d (3 pts, no DLT). Three (17%) responses (2 CRp, 1 CR) occurred. Of 15 pts on CIA, 4 were primary refractory and 11 in first relapse. Median CRD1 was 9 mos (0–24). Eight pts had an abnormal karyotype. Seven pts received prior ara-C-based regimens and 2 failed unrelated donor SCT. Median age: 58 yrs (23–78). Three dose levels were evaluated. At C 22.5mg/m2 i.v. daily x 5d, I 8mg/m2 i.v. daily x 3d, A 1g/m2 i.v. daily x 5d, 2 of 3 pts developed ≥ gr.3 toxicities (↑ bili, diarrhea) necessitating dose de-escalation. Subsequent levels included C at 15mg/m2 x 5d, I at 6mg/m2 x 3d, A at 0.75g/m2 x 5d (6 pts, 1 with ≥ gr. 3 rash, ↑ bili), and C at 22.5mg/m2 x 5d, I at 6mg/m2 x 3d, and A at 0.75g/m2 x 5d (6 pts, 1 with ≥ gr. 3 ↑ bili). Nine (60%) responses (8 CR, 1 CRp) occurred. Further dose escalation of clofarabine is planned in both trials. The preliminary results indicate feasibility of the combinations. The higher response rates with CIA need to be evaluated in view of different pt. characteristics between the two trials.
Valproic Acid Induces Cell Cycle Arrest and Differentiation of t(15;17) Positive Leukemic Cells Independent from ATRA Sensitivity and Has No Effect on CD34pos Cells.
Authors: Drescher Bettina , Goerlich Kerstin , Reile Dagmar , Doehring Axel , Ganser Arnold , Heil Gerhard , Wagner Katharina , Krauter Juergen
Published: April 2020

The translocation t(15;17) in acute promyelocytic leukemia results in the PML/RARα fusion protein. PML/RARα recruits histone deacetylases (HDAC) and represses target genes of wild-type RARα. Recently, the anticonvulsant drug valproic acid (VPA) has been described as an HDAC inhibitor. In order to evaluate the role of VPA in the treatment of acute promyelocytic leukemia, the effect of VPA on the t(15;17) positive cell line NB4 was examined. To exclude toxicity on non malignant stem cells, CD34pos cells were analyzed. In NB4 cells, VPA led to an increase of acetylated histone H4. Histone acetylation was associated with a dose dependent inhibition of proliferation. Furthermore, inhibition of NB4 clonogenic growth was observed. In contrast, clonogenic growth of CD34pos cells was not affected by the presence of VPA. The VPA induced inhibition of cell growth in malignant cells was not caused by altered apoptosis. Rather an arrest at the G1/G0 phase was observed in the presence of VPA. In agreement with p21 expression of leukemic cell lines not harboring the t(15;17) translocation, VPA induced p21 mRNA expression also in t(15;17) positive cells. However, no induction of p21 was observed in normal CD34pos cells. VPA triggered the differentiation of NB4 cells measured by NBT test and surface expression of CD11b and CD11c. Flow cytometry of colonies from CD34pos cells showed an increased fraction of CD34pos cells in the presence of VPA. Thus, VPA does not induce differentiation in non malignant hematopoiesis. To study whether VPA and ATRA induced differentiation are mediated via identical pathways, the process of differentiation was studied in NB4 cells and ATRA resistant NB4-R2 cells. While differentiation in the presence of ATRA led to induction of C/EBPβ and C/EBPε, no change in the expression of these transcription factors was observed after VPA treatment. Downregulation of c-myc was detected upon ATRA as well as VPA treatment, an additive effect was seen after the combination. Resistance to ATRA did not interfere with effects of VPA on cell differentiation. However, upon ATRA treatment no c-myc downregulation was observed in the NB4-R2 cell line. Taken together, VPA acts as an HDAC inhibitor in t(15;17) positive cells and induces myeloid differentiation by mechanisms distinct from ATRA and independent of ATRA-resistance. Moreover, VPA does not interfere with normal hematopoiesis. Therefore, this substance might be helpful in patients with APL - especially in ATRA-resistant disease.
Downregulation of Bub1 and a Defective Spindle Checkpoint in Acute Myeloid Leukemia.
Authors: Waesch Ralph , Schnerch Dominik
Published: April 2020

Chromosomal aberrations are common in leukemia. The spindle assembly checkpoint (SAC) is a surveillance mechanism responsible to ensure proper chromosome segregation. Unattached chromosomes to the mitotic spindle or lack of spindle tension are sensed by the SAC leading to metaphase arrest. The activated SAC inhibits the ordered proteolysis of regulatory proteins and thereby prevents mitotic progression providing time for the repair of spindle defects. Ubiquitin-dependent proteolysis of cell cycle regulators is a central mechanism of cell cycle control. The anaphase-promoting complex (APC) is an essential ubiquitin-ligase responsible for mitotic progression. The activated SAC inhibits the APC preventing degradation of cyclin B1 and the anaphase inhibitor securin and thereby delaying chromosome separation. We found this mitotic delay mechanism to be defective in acute myeloid leukemia (AML). Leukemia cells that were exposed to the microtubule disrupting agent nocodazole were not capable of preventing anaphase onset. This is indicated by a low mitotic index and degradation of the APC targets securin and cyclin B1 in nocodazole treated Kasumi-1 cells compared to the SAC competent burkitt lymphoma cell line DG-75. Thus, unattached chromosomes were unable to induce prolonged metaphase arrest in these cells. They undergo apoptosis instead due to the complete absence of the mitotic spindle. Similar results with defective mitotic arrest and enhanced apoptosis were obtained for several other leukemia cell lines. In vivo SAC malfunction in AML can favor the generation of aneuploidy, consistent with the numerical aberrations observed in these cells. In order to identify the underlying defect we screened for mutations or altered expression of checkpoint proteins and identified the SAC component Bub1 to be posttranscriptionally downregulated in all tested AML cell lines. Our studies suggest that impaired SAC function is an important mechanism to create genetic instability and thus may contribute to rise and progression of AML.
High Resolution Genome-Wide Array-Based Comparative Genome Hybridization Reveals Cryptic Chromosome Changes in AML and MDS Cases with Trisomy 8 as the Sole Cytogenetic Aberration.
Authors: Paulsson Kajsa , Heidenblad Markus , Strombeck Bodil , Borg Ake , Fioretos Thoas , Johansson Bertil
Published: April 2020

Although trisomy 8 as the sole chromosome aberration is the most common numerical abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), next to nothing is known about its pathogenetic effects. Considering that +8 is a frequent secondary change in AML and MDS, cryptic - and possibly primary - genetic aberrations may occur in cases with trisomy 8 as the apparently single anomaly. To date, however, no such hidden anomalies have been reported. We performed a high resolution genome-wide array-based comparative genome hybridization (array CGH) analysis of ten AML/MDS cases with isolated +8, using an array set containing >30,000 BAC and PAC clones. Array CGH revealed intra-chromosomal imbalances, not corresponding to known genomic polymorphisms, in 5/10 cases, comprising ten duplications and hemizygous deletions ranging in size from 0.5 to 2.2 Mb. Most notably, a 1.8 Mb hemizygous deletion at 7p14.1, which had occurred prior to the +8, was identified in one MDS transforming to AML. Furthermore, a hemizygous deletion at 12p13.2, including ETV6, was present in one case. The remaining eight imbalances involved more than 40 genes. The present results show that cryptic genetic abnormalities are frequent in trisomy 8-positive AML and MDS cases and that +8 as the sole cytogenetic aberration is not always the primary genetic event.
A LDI-PCR Based Method Allows the Identification of Any MLL Rearrangement.
Authors: Meyer Claus , Schneider Bjoern , Dingermann Theo , Klingebiel Thomas , Marschalek Rolf
Published: April 2020

Chromosomal rearrangements of the MLL gene are responsible for 5–10% of all acute leukemias, biphenotypic leukemias and myelodysplastic syndromes. 5–10% of these MLL aberrations are therapy-related leukemias with the frequent fusion partners ELL, MLLT3, MLLT10 and MLLT2. The large number of known and unknown MLL fusions (>80) renders a precise diagnosis a demanding task. Even though all MLL rearrangements are associated with high risk acute leukemias, the outcome (favorable or poor) depends on the partner gene. Thus, the identification of MLL gene fusions is necessary for rapid clinical de-ci-sions resulting in specific therapy regimens. After their cytogenetic identification, only the most common MLL fusions MLLT2, MLLT3, MLLT1, MLLT4, ELL and MLLT10 (80%) are investigated by RT-PCR analysis, whereas infrequent or unknown MLL rearrangements (20%) are more or less excluded from further analysis. Therefore we established a LDI-PCR (long-distance inverse PCR) based method that uses small amounts of genomic DNA to determine any type of MLL associated chromosomal abnormality. With this diagnostic tool more than 400 prescreened und unscreened adult and pediatric samples (AML/ALL) from different European diagnostic centers have been analyzed. During this study more than 200 MLL rearrangements were characterized for their precise localization of genomic break-points. The identified MLL rearrangements consist of 25 different fusion genes including the following eight novel MLL partner genes: ACACA, ARGHEF17, BCL9L, MAML2, SELB, SMAP1, and TIRAP. The identified MLL aberrations are basically reciprocal translocations, but also deletions, inversions, insertions and interstitial duplications have been determined. Combining the data of our study and data retrieved from the literature, a total of 88 partner genes can now be annotated. 52 fusion partners (60%) have been characterized on the molecular level whereas at least 36 (40%) remain to be identified. These results demonstrate that this new diagnostic tool in combination with split-signal FISH is qualified for the rapid analysis of known as well as unknown MLL partner genes. Furthermore, the determined patient specific fusion sequences are useful for minimal residual disease (MRD) monitoring of MLL associated acute leukemias. The use of these MRD markers will contribute to improve the treatment and outcome of acute leukemia patients. A first prospective study was already initiated by the German ALL study group and verified the reliability of these genomic markers for MRD monitoring.
Activation of a Novel Proteasomal Independent Bcr/Abl Degradation Pathway by WP1130 Induces Apoptosis in CML Cells.
Authors: Bartholomeusz Geoffrey A. , Donato Nichalos , Estrov Zeev , Priebe Waldemar , Talpaz Moshe
Published: April 2020

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells caused by 9:22 reciprocal chromosomal translocation resulting in expression of a highly stable, constitutively active tyrosine kinase, Bcr/Abl. Inhibition of Bcr/Abl with imatinib mesylate, a potent Abl-specific tyrosine kinase inhibitor, is a highly effective therapy for this disease. However, clinical resistance occurs particularly in the later stages of the disease due mainly to the occurrence of point mutations in the Abl kinase domain and continual Bcr/Abl signaling. Alternative and supplemental therapies are still needed and of great clinical interest. Screening a library of small molecular weight compounds for their ability to induce the degradation of critical transcription factors and tyrosine kinases led to the design and synthesis of WP1130, a compound that reduces Jak2 and Bcr/Abl protein stability. Treatment with WP1130 caused the rapid destruction of Bcr/Abl in human CML cell lines (K562, BV173) as well as in clinical CML specimens obtained from patients. This treatment also resulted in dephosphorylation of pSTAT5, pHck and pCrkL, three known targets of Bcr/Abl. Loss of Bcr/Abl protein following WP1130 treatment was similar to that caused by Bcr/Abl silencing (siRNA), and both treatments lead to the induction of apoptosis. Treatment with WP1130 caused rapid degradation of both wild-type and mutant (T315I) Bcr/Abl protein in BaF3 cell expressing these proteins and in a CML cell line expressing wildtype (BV173) and T315I mutant BCR-ABL (BV-173R). Treatment with WP1130 also strongly inhibited colony formation in soft agar of human CML cells from imatinib resistant patients expressing the T315I mutation. The degradation of Bcr/Abl in cells treated with WP1130 was rapid, (1h) and independent of the proteasomal system and HSP90, a chaperone associated with Bcr/Abl stability. Interestingly, WP1130-induced Bcr/Abl degradation was blocked in the presence of vanadate, a tyrosine phosphatase inhibitor. Together, these observations suggest that WP1130-induced Bcr/Abl degradation is mediated by a unique, proteasomal independent pathway in CML cells. Further development of WP1130 is underway with the goal of developing an effective therapy for treating CML by targeting Bcr/Abl protein expressed in early CML progenitors or stem cell populations.
Interferon Regulatory Factor 4 Is Not Required for Induction of Chronic Myeloid Leukaemia-Like Myeloproliferative Disease by Bcr/Abl in Mice.
Authors: Illert Anna Lena , Miething Cornelius , Grundler Rebekka , Schmidt Manuel , Burchert Andreas , Neubauer Andreas , Mugler Claudia , Peschel Christian , Duyster Justus
Published: April 2020

Interferon regulatory factors (IRF) are activating and/or repressing transcription factors induced by treatment with type I and II Interferon (IFN), other cytokines, receptor cross-linking and viral infection. In contrast to IRF-1 and IRF-2, which are widely expressed, IRF-4 and IRF-8 are tissue-restricted factors. IRF-8 is expressed mainly in cells of haematopoietic origin and has recently been shown to inhibit mitogenic activity of p210 Bcr/Abl-transformed myeloid progenitor cells by activating several genes that interfere with the c-Myc pathway. IRF-4 is most homologous with IRF-8 (approximately 70% overall homology) and its expression is highly restricted to lymphocytes of the B-cell type (pre-B, B, and plasma cells), mature T-cells and macrophages. Furthermore IRF-4 expression is significantly impaired in CML and AML patient samples predominately in T-cells. To examine a potential role of IRF-4 in Bcr/Abl mediated transformation we used a bone marrow transplant model (BMT). We transduced IRF-4 knockout (KO) bone marrow with retrovirus expressing p210 Bcr/Abl and transplanted it into lethally irradiated recipient C57/bl6 mice. For proper control we transplanted also wildtype (WT) bone marrow transduced with Bcr/Abl and mock transfected IRF-4 KO bone marrow (BM). All recipients transplanted with Bcr/Abl transduced BM (regardless of which IRF-4 KO or WT) developed rapidly a myeloproliferative disorder characterized by leukocytosis and expression of the myeloid lineage markers CD11b and Gr1. Surprisingly, IRF-4 KO Bcr/Abl infected BM recipient mice survived slightly longer than the control group transplanted with WT p210 BM (12 vs. 19 days). Histopathologic studies of the affected organs (spleen/lung) revealed extramedullary haematopoiesis in the spleens of both groups and a distinct infiltration of the tumor cells in the lung of WT Bcr/Abl transduced BM recipient mice, resulting in massive punctuated bleedings. Interestingly, preliminary analysis suggest a significantly reduced lung infiltration with almost no pulmonary bleedings in IRF-4 KO Bcr/Abl infected BM recipient mice, which we assume to be the reason for the differences in the overall survival. Taken together our data demonstrate that IRF-4 is not required for the induction of a myeloproliferative disorder by Bcr/Abl in vivo and for its ability to transform BM cells in vitro, but IRF-4 deficiency seems to have an impact on the fulminant pulmonary haemorrhage occurring in the murine CML-like disease.
Enhanced Phosphorylation of Nbs1, a Member of the DNA Repair/Checkpoint Activation Complex Rad50/Mre11/Nbs1, Prolongs Cell Cycle S Phase and Contributes to Drug Resistance in BCR/ABL-Positive Leukemias.
Authors: Rink Lori , Stoklosa Tomasz , Skorski Tomasz
Published: April 2020

Nbs1, a member of the DNA repair/checkpoint activation complex Mre11/Rad50/Nbs1, is phosphorylated by ATM in response to the presence of DNA double strand breaks (DSBs) resulting in the activation of the S phase checkpoint. Here we show that the BCR/ABL tyrosine kinase, as well as growth factors (IL-3, GM-CSF), stimulate the expression of Mre11 and Nbs1, but not Rad50. This effect is dependent on the kinase activity of BCR/ABL protecting Mre11 and Nbs1 from caspase-dependent, but not proteasome-dependent degradation. Cells expressing the BCR/ABL kinase are resistant to chemotherapeutic agents, including mitomycin C (MMC). Western analysis showed enhanced phosphorylation of Nbs1 on Serine 343 in MMC-treated BCR/ABL leukemia cells (CML patient cells and leukemic cell lines) in comparison to normal cells in the presence of growth factors. Immunofluoresence studies demonstrated an increase of γ-H2AX nuclear foci (DSBs indicator) and pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. In addition, BCR/ABL-positive cells displayed a higher percentage of colocalization of γ-H2AX and p-Nbs1 implicating the presence of p-Nbs1 at the DSBs. Clonogenic assays performed after down regulation of Nbs1 in BCR/ABL positive cells using siRNA showed increased sensitivity to MMC. Specifically, the expression of the Nbs1-S343A phosphorylation-less mutant also decreased resistance in BCR/ABL cells to MMC. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells, BCR/ABL-transformed cell lines and normal counterparts displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase. Expression of Nbs1-S343A mutant caused a significant decrease in the accumulation of BCR/ABL leukemia cells in S phase after MMC treatment, whereas cells transfected with both the empty construct and wild-type Nbs1 displayed S phase accumulation. Surprisingly, Nbs1-S343A mutant did not affect the ability of normal cells to accumulate in S phase in response to MMC. Altogether, we hypothesize that enhanced phosphorylation of Nbs1 on S343 leads to increased resistance to genotoxic agents in BCR/ABL leukemia cells by prolonging the S phase checkpoint and allowing longer time for the repair of excessive DNA damage.
BCR/ABL Regulates the Expression and Interacts with Werner Syndrome Helicase/Exonuclease To Modulate Its Biochemical Properties.
Authors: Slupianek Artur , Jozwiakowski Stanislaw , Gurdek Ewa , Nowicki Michal O. , Skorski Tomasz
Published: April 2020

A genome-wide screen suggested that BCR/ABL kinase might stimulate WRN, a member of the RecQ-like DNA helicases family. The Werner syndrome protein (WRN) exerts DNA helicase and 3′-5′ exonuclease activities. Inactivating mutations in the WRN gene causes Werner syndrome, characterized by premature aging, genomic instability and cancer predisposition. The WRN helicase unwinds unusual DNA structures, which can occur physiologically, or can be accidentally generated during DNA repair (double-stranded DNA with mismatched tails, bimolecular G4 quartets and Holliday junctions). In addition, WRN physically interacts with components of two major systems for DNA double-strand breaks (DSBs) repair: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here we demonstrated that BCR/ABL regulates the expression of WRN mRNA and protein in CML primary cells and BCR/ABL-transformed cell lines. BCR/ABL kinase-induced WRN expression is mediated by c-MYC, but not STAT5 - dependent transcription as well as by inhibition of caspases-dependent cleavage. In addition, immunoprecipitation and pull-down studies indicated that BCR/ABL interacts directly with WRN resulting in its tyrosine phosphorylation. Mutation analysis revealed that multiple domains/amino acid residues of BCR/ABL and WRN are involved in the interaction. BCR/ABL-positive leukemia cells exerted an enhanced WRN-dependent helicase activity. In addition, immunoprecipitation and double-immunofluorescence co-localization studies demonstrated an elevated interaction between WRN and RAD51 in BCR/ABL cells undergoing genotoxic stress in comparison to parental counterparts. Altogether, it is likely that WRN is involved in DSBs repair by HR in leukemia cells. More detailed studies are underway to pinpoint the role of WRN in DNA damage response in BCR/ABL-transformed cells.
Peptides from NM23B - A Transcription Factor with NDP Kinase Activity - Are Expressed on the Surface of Leukemic Cells and Are Recognized by T-Lymphocytes.
Authors: Tschiedel Sabine , Reinhardt Annette , Ruschpler Peter , Woelfel T. , Woelfel C. , Dietger Niederwieser , Chiara Gentilini
Published: April 2020

Objective: During the last years a growing number of MHC-restricted antigens were recognized using autologous or HLA-matched cytotoxic T-cell lines (TCL). Molecules were isolated by HPLC or identified using cDNA expression cloning from normal or malignant target cells and found to derive from normal proteins or from mutated tumor-specific proteins. The majority of the tumor-specific peptides were derived from melanoma cells. The aim of this project was to search for immunogenic peptides on leukemia cells with the help of TCLs obtained from a stem cell donor against chronic myelogenous leukaemia (CML) recipient cells and to identify the immunogenic peptides by cDNA expression cloning.

Methods: Irradiated leukemic cells of a patient with Philadelphia-chromosome positive CML were used to stimulate PBMC of his HLA-identical related donor. TCLs were expanded to >200x106 cells with antigen specific stimulation and their specificity tested in in vitro assays. Restriction of the TCLs was tested in an Elispot assay using a panel of normal and malignant autologous and allogeneic cells and mAb blocking experiments (mAb W6.32, B1.23.2, SRF8-B6, GAP-A3). Subsequently cDNA libraries from leukemic cells were established in the eukaryontic expression vector pcDNA3.1/V5HISTOPO and cDNA of 100 bacterial colonies were pooled. 293T cells were then co-transfected with the cloned HLA-cDNA of the restricting HLA and with pools of cDNA (pools of 100) obtained from the leukemic cells. Transfected cells were tested with the TCL in the Elispot assay. Positive cDNA pools were further diluted in pools of 10 colonies and tested again. Positive pools were finally diluted in single clones and screened again. cDNA from the positive single clones was sequenced with different primers.

Results: The TCL PaHe recognized bcr/abl positive PBMC, CD14+ and CD34+ subpopulations and EBV-LCL but not autologous CD4+ and CD8+ subpopulations or bcr/abl negative PHA blasts. Blocking experiments showed that the recognized peptides were restricted by HLA-A32, HLA-B44 and HLA-C03. Therefore 293T cells were transfected with HLA-A32 and with the cDNA pools of the tumor cells. After testing one thousand 100-cDNA pools and four hundred 10 cDNA pools a total of three single cDNA clones were identified, which after transfection were recognized by the TCL. All three positive cDNAs clones were sequenced and found to be identical with the sequence of NM23B. No mutations of the molecule were detected even if different primers were used.

Conclusions: Peptides from NM23B, a transcription factor for c-myc with nucleosid diphosphate kinase (NDP) activity, are recognized by TCLs in an HLA-A32 restricted way. This is the first report of NM23B as an immunogenic molecule, which might play an important role in graft-versus-tumor reactions but also in the regulation of hematopoiesis.
Reduced Intensity Cord Blood Transplantation Using Mycophenolate Mofetil and Cyclosporine for Adult Patients with Advanced Hematologic Malignancies.
Authors: Hamaki Tamae , Kajiwara Koichi , Hoshi Keiko , Kogure Katsuhiro , Harima Akane , Yamada Kazunari , Kodo Hideki , Kozai Yasuji
Published: April 2020

Objectives: To evaluate the feasibility of reduced-intensity unrelated cord-blood transplantation (RI-UCBT) using cyclosporine and mycofenolate mofetil as graft-versus-host disease (GVHD) prophylaxis for adult patients with advanced hematological diseases

Methods: Twenty-one patients (median age, 54; range, 30–76) with advanced hematological diseases underwent RI-UCBT at Metropolitan Fuchu Hospital between July 2002 and September 2004. Underlying disease included AML (n=7), non-Hodgkin lymphoma (n=10), Hodgkin lymphoma (n=2), and aplastic anemia (n=1). Preparative regimen comprised fludarabine (Flu) and total body irradiation (TBI), combined with busulfan (BU) or cyclophosphamide (CY). GVHD prophylaxis was cyclosporine and mycophenolate mofetil. Primary endpoint of this study was engraftment. Secondary endpoints were to describe clinical features of immune reactions following RI-CBT. Immune reactions included high-grade fever (> 38 degree centigrade), elevation of serum C-reactive protein (CRP) levels, skin rash or erythema, diarrhea, hyperbilirubinemia, symptoms of central nerve systems, and body weight gains. They were classified according to their onset after transplant: pre-engraftment immune reactions, engraftment syndrome, and GvHD.

Results: Ten patients achieved primary neutrophil engraftment at a median of day 22 (range, 11–33). Five patients died of disease progression before engraftment. Primary graft failure was diagnosed in the remaining 6 patients. Among them, 3 patients received second transplant, and the other 3 patients developed autologous hematopoiesis recovery. Seven of 10 patients who achieved engraftment developed pre-engraftment immune reactions including fever, an increase in CRP, skin rash, diarrhea, and body weight gain. Similar manifestations were observed in six patients who had not achieved primary engraftment. Six patients developed engraftment syndrome. Nine patients developed Grade II to IV GVHD: skin rash (n=9), diarrhea (n=7), and hyperbilirubinemia (n=1). Seven of 10 patients who achieved engraftment survived without disease progression at a median follow-up of 17.5 months (range, 8.4–29.0).

Conclusions: This study demonstrated the feasibility of RI-UCBT in adults, indicating that acute GVHD prophylaxis with cyclosporine and mycophenolate mofetil is worth considering for further intense evaluation.
Nonmyeloablative Unrelated Donor (URD) Hematopoietic Cell Transplantation (HCT) for the Treatment of Patients (pts) with Poor-Risk, Relapsed or Refractory Multiple Myeloma.
Authors: Georges George E. , Maris Michael B. , Maloney David G. , Sandmaier Brenda M. , Sorror Mohamed L. , Shizuru Judith A. , Niederwieser Dietger W. , Agura Edward D. , Bruno Benedetto , McSweeney Peter A. , Pulsipher Michael A. , Chauncey Thomas R. , Storer Barry E. , Storb Rainer F.
Published: April 2020

We carried out HLA-matched URD peripheral blood stem cell (PBSC) transplant in 24 pts with poor-risk multiple myeloma after nonmyeloablative conditioning with fludarabine (90 mg/m2) and 2 Gy total body irradiation. Postgrafting immunosuppression consisted of cyclosporine and mycophenolate mofetil. The median age of the 19 men and 5 women was 53 (23 – 66) years. Conventional metaphase cytogenetics obtained in 14 pts showed Δ13 in 6 pts and complex abnormalities in 9 pts. Stage III disease was present in 83% and 17% had stage II disease. The median time from diagnosis to URD HCT was 25 (range, 8–130) months, and 96% were beyond 1st complete remission (CR) or had never achieved CR1, despite multiple lines of chemotherapy (median 4.5, range 2–10). At study entry, 17 pts (71%) had chemotherapy-refractory disease and 14 pts (58%) had failed autologous HCT. Thirteen pts had planned autologous-URD tandem HCT, while 11 proceeded directly to URD HCT. The median follow-up was 2.5 years after allografting. One pt experienced non-fatal graft rejection. The incidences of acute grades II, III and chronic graft-versus-host disease were 54%, 13% and 75%, respectively. Non-relapse mortality was 22% at 2.5 years. CRs were observed in 11 pts (46%) and partial remissions (PR) in 3 (13%). Best disease responses were seen in pts given tandem autologous-URD HCT with CR in 8 pts and PR in 2 pts (77% CR+PR rate). The estimated overall survival (OS) at 2.5 years for all 24 pts was 65% and progression-free survival (PFS) 41%. Pts receiving tandem autologous-URD HCT had superior OS and PFS, 76% and 63% (Fig. A), compared to pts proceeding directly to URD HCT, 52% and 14% (Fig. B), respectively (PFS p-value=.03). Risk factors for worse OS included pts with significant medical comorbidities (p=.03), chemotherapy-refractory disease prior to HCT (p=.03), and pts who had failed autologous HCT (p=.07). Pts who failed autologous HCT had 48% OS and 30% PFS at 2.5 years. For pts with poor-risk, relapsed or refractory multiple myeloma, cytoreductive autologous HCT followed with nonmyeloablative conditioning and URD HCT is very promising treatment with low non-relapse mortality, high complete remission rates and prolonged PFS. The results also suggest that URD HCT may provide improved graft-versus-myeloma effect compared with HLA-matched sibling HCT without an increase in the risk of non-relapse mortality.

Figure Figure
Partially HLA Matched, Non-Myeloablative Allogeneic Transplantation.
Authors: Rizzieri David A. , Koh Liang Piu , Long Gwynn D. , Gasparetto Cristina , Sullivan Keith M. , Horwitz Mitchell , Chute John , Gong Jerald Z. , Lagoo Anand S. , Niedzwiecki Donna , Lu Cong Xiao , Marshall Dawn , Dowell Jeanette M. , Chao Nelson J.
Published: April 2020

We have investigated the use of partially matched family member donors in combination with alemtuzumab and a non-myeloablatvie preparative regimen in an effort to expand the population of patients who may benefit from allogeneic immunotherapy.

Methods: Sixty three patients received fludarabine 30mg/sq m, cyclophosphamide 500mg/sq m IV qd x 4 days and alemtuzumab 20mg IV qd x 5 days followed by infusion of partially matched family member donor stem cells. Mycophenolate alone or in combination with cyclosporine was given for 6–8 weeks following transplantation.

Results: Patient diagnoses included lymphoma/myeloma n= 16, leukemias/MDS n=33, myelofibrosis/aplasia n=3 and metastatic solid tumours n=11. The median age was 48 (range 17–66) with a median follow up of 28 months. The donor matching was 5/6 in 8, 4/6 in 22, and 3/6 in 33 cases. The median CD34+ cell dose collected was 13.47 x 106/kg (SD= 5.10) with nearly 2 logs of T cells depletion. Engraftment occurred in 92% of patients with a median of 87% donor cells responsible for patient hematopoiesis by 6 weeks following therapy. Twenty patients also received a DLI (range 105–107 CD3+ cells/kg). Grade III–IV acute GVHD occurred in only 8/63 (13%) with 14 (22%) experiencing grade II–IV. Nine patients developed chronic GVHD or failure to thrive (15%). The transplant regimen was well tolerated with 10% TRM. In those with hematologic malignancies, only 6 (12%) started in remission, though 38 (73%) attained a CR. The most common cause of death in this group was progressive disease (33%), followed by infections (19%), and GVHD (12%). Overall 1 year survival for this high risk group of 63 patients was 26%. A subgroup had aplastic anemia/myelofibrosis or leukemia/lymphoma in second or greater CR, PR and had a median disease free and overall survival of 3 years.

Figure Figure

Phenotypic analysis of lymphocyte subsets (measured by flow), and T cell VBeta family recovery (spectratype analysis using PCR) revealed robust recovery by 6 months in those without GVHD (dark bars) compared to those with GVHD (gray bars), despite T depletion.

Figure Figure

Further, the measured T cell recovery is from peripheral expansion of residual transplanted T cells as newly educated T cells are not measurable (using TRECs analysis) for at least 1 year, if at all.

Conclusions: The results demonstrate reasonable tolerance and reliable engraftment using T cell depleted, partially matched family member donors in a non-myeloablative setting with low treatment related mortality and severe GVHD. The future challenge will to be to develop strategies to improve immune recovery to enhance immune-mediated graft-versus-tumour effect and to minimize the risk of infections.
Treatment of Myeloid and Lymphoid Malignancies with Highly Purified Alloreactive CD56+CD3− NK-Cells and Haploidentical Stem Cell Transplantation: Promising Results of a Phase I Study.
Authors: Gentilini Chiara , Hilbers Urte , Hartung Goetz , Lange Thoralf , Kliem Constanze , Hegenbart Ute , Zeis Mathias , Huppert Volker , Glass Bertram , Uhrberg Marcus , Schmitz Norbert , Thiel Eckhard , Niederwieser Dietger W. , Uharek Lutz
Published: April 2020

NK cell alloreactivity can mediate strong graft versus leukemia (GvL) effects following haploidentical hematopoietic stem cell transplantation (HSCT). In an attempt to further improve the antileukemic effectiveness of this approach, we have adoptively transferred high numbers of alloreactive donor NK cells during the early phase after transplantation.

Method: In a phase-II study, 10 patients (6 AML, 1 MDS, 1 HD, 1 CML, 1 ALL, median age 38 yrs, range 17–48 yrs) were transplanted in late phases of their disease (5 pts. as 2nd transplantation) and received purified NK cells from their haploidentical donors at day +2 after HSCT. Conditioning consisted of 12 Gy fTBI, Thiotepa (10mg/kg), Fludarabine (5 x 30 mg/qm) and OKT3 (day −4 to +2). Two patients received a reduced conditioning with Fludarabine and OKT3 alone. NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection.

Results: No severe technical problems occurred and a mean of 12,1 x 10E8 (16,74 x10E6/kg, range 6,12 to 27,2 x10E6/kg) CD34+ cells was selected in high purity (95,9 %) with a very low T-cell content (mean 1,83 x10E4/kg CD3+ cells, 4,5 Log depletion). A mean of 5,7 x10E8 (6,7 x 10E6/kg) CD56+CD3− NK cells was transferred (yield 70,36%, purity 70%). The mean number of contaminating CD3+ cells was 3,2 x10E4/kg (3,6 Log depletion).No severe acute toxicity attributable to NK cell infusion was observed. Hematopoietic recovery was fast with leukocytes > 1/nl between day 5 and 11. Seven patients developed early grade II GvHD of the skin which promptly resolved after CSA and steroids. One patient developed late graft rejection five months after reduced conditioning, received a 2nd graft from a different donor, engrafted but unfortunately died due to pneumonia one month after the second transplantation. One pt with CML died due to adenovirus infection at day 140. One pt. with HD in 5th CR died due to pneumonia at day 85. One patient showed a relapse of the AML three months after transplant. She received a DLI with NK cells form the donor but died due to disease progression one month after. Interestingly, leukemic cells from this patient proved to be resistant to donor NK cell mediated lysis. Five of 9 patients are alive and in CCR with a median follow up of 192 days, the two patients with the longest follow up are in very good condition and free of GvHD at day +1271 and +1019. Our data show for the first time that the early adoptive transfer of high numbers of HLA-mismatched NK cells is safe and feasible.
Characterization of CD6-Negative Suppressor Cells Facilitating HLA-Haploidentical Stem Cell Transplantation.
Authors: Bigalke Iris , Falk Christine , ter Meer Dominik , Leeping Marina , Prevalsek Dusan , Tischer Johanna , Kolb Hans-Jochem
Published: April 2020

HLA- haploidentical stem cell transplantation (SCT) has been shown to be a suitable alternative for treatment of hematopoietic malignancies if no matched donor is available. Haploidentical family members sharing one HLA-haplotype may differ in one or more HLA-antigens of the second haplotype. The risks are rejection of the graft and severe graft-versus-host disease (GVHD). In particular unmodified grafts may cause severe GVHD, and T-cell depleted grafts may be rejected. We have designed a protocol of unmodified marrow transplantation supplemented by G-CSF mobilized blood cells (MBC) 6 days after marrow transplantation from which CD6-positive cells are depleted. These cells facilitate engraftment and modify GVHD. Patients were conditioned with 12 Gy total body irradiation (TBI), donor leukocytes, antithymocyte globulin and cyclophosphamide, unmodified marrow was given on day 0 and CD6-depleted MBC on day 6. In a first series of 53 patients CD6 cells were depleted by indirect loading and depletion with goat-anti-mouse antibody coupled to Dynabeads. The second series comprised 22 patients whose MBC were depleted using the CliniMacs system with CD6-antibody directly bound to immunomagnetic beads.

Table I shows the median and the range () of the cell numbers transplanted after CD6 depletion comparing the two methods.

Table II shows the number of patients with acute GVHD in relation to the median of all given transplants.

Rejection of the transplant was not observed in 71 evaluable patients. GVHD >= 2 occurred in 47 %, GVHD >= 3 in 21% of 70 evaluable patients. The incidence of acute GVHD was lower in patients given less CD4 cells (p=0.06), it was significantly lower in patients given MBC separated by immunomagnetic beads charged with the specific antibody (p < 0.02). The content of CD8-positive, NK-, B- and CD34-positive cells had no effect on the incidence of GVHD.

We conclude from these results that transfusion of CD6-depleted MBC on day 6 facilitates engraftment of marrow from HLA-haploidentical donors without T cell depletion without jeopardizing engraftment and without severe GVHD, if CD4 cells are depleted. A particular advantage is the content of NK cells and CD8 T cells maintaining a potential GVL-effect.

Tab.I CD34 x 10e6/kg CD56 x 10e6/kg CD19 x 10e6/kg CD8 x 10e6/kg CD4 x 10e6/kg Indirect labelling (N=53) 7.3 (0.4–35.8) 19.8 (4.8–65.4) 36.1(5.6–117.3) 5.8 (0.75–55) 3.9 (0.12–65.4) Direct labelling (N=22) 13.4(2.5–29.1) 17.1 (1.7–57.3) 48.7 (5.1–175) 2.0 (0.12–13.2) 0.47 (0.05–2.5) Table II aGVHD 0 aGVHD I–II aGVHD III–IV total CD4 <2.29 x 10e6/kg 16 10 4 30 CD4 >2.28 x 10e6/kg 11 16 11 38 CD8 <4.62 x 10e6/kg 11 13 7 31 CD8 >4.61 x 10e6/kg 16 13 8 37
Harvesting for “A Rainy Day”: A Retrospective Analysis of Its Cost-Effectivenes and Ethical Implications.
Authors: Ortin Miguel , Bricknell Sue , Kimber Jane , Stirrup Lynn , Hofer Dietmar , Dyer Martin J. , Hunter Ann
Published: April 2020

Background: Peripheral blood stem cell (PBSC) harvesting from patients with haematological malignancies in complete remission is a frequently undertaken cautionary measure. Its aim is to provide a back-up of stem cells should the patient require high dose therapy at a later date, when harvesting may prove to be more difficult. This approach has economic and ethical implications if such harvests are not regularly used. We present a retrospective study on the use of these collections in patients with haematological malignancies.

Method: We retrospectively analysed our Stem Cell Laboratory database. All collections performed between January 1980 and December 2003 were reviewed. Endpoints included number of collections per condition group and number of collections used in each group.

Results: Over 13 years there were 1665 autologous collections. 396 (23.8 %) were collections from 158 patients who did not have need of high dose therapy at the time but in whom the potential need in the future existed. The majority of collections were from patients with Non Hodgkin Lymphoma (NHL) (34%) and Multiple Myeloma (MM) (27%). Only 69 (21%) of collections have been used, the rest remain stored. Collections from MM patients were the most frequently used (34.3%), followed by Hodgkin’s Disease (HD) and acute myeloid and chronic myeloid leukaemia (AML, CML), Only 8% of the collections from NHL patients have been used, the last infusion dating from 1995.

Conclusion: The regular, systematic collection of back-up harvests for all patients in remission is common practice. For patients with MM there is good evidence that this provides increased treatment options and is of value. The low percentage of collections subsequently used in NHL does not justify the cost or resources required and this practice should be reviewed.

Total Stored Used % Used NHL 139 128 11 8 HD 19 14 5 26 MM 105 69 36 34.3 CLL 18 18 0 0 AML 41 31 10 24.4 CML 28 22 6 21.4 ALL 16 15 1 6 APML 4 4 0 0 MDS 2 2 0 0 Miscellaneous 24 24 0 0
Plasma Is a Reliable Source of mRNA for Testing IgVH Mutation Status in Patients with Chronic Lymphocytic Leukemia.
Authors: Ma W. , Jilani I. , Gorre M. , Keating M. , Chan H. , Tseng R. , Kantarjian H. , O’Brien S. , Giles F. J. , Albitar M.
Published: April 2020

The mutational status of the immunoglobulin heavy-chain variable-region (IgVH) gene in the leukemic cells of patients with chronic lymphocytic leukemia (CLL) is an important prognostic marker. Lack of IgVH mutation is associated with rapid disease progression and shorter survival. The assay used to determine IgVH mutation status requires specific amplification of the mRNA of the expressed clonal IgVH gene in CLL. However, in some patients with CLL or lymphocytic lymphoma, the bulk of the disease resides in the lymph nodes or bone marrow, with few circulating leukemic cells. In addition, contamination of leukemic clonal cells by reactive polyclonal plasma cells can cause difficulty in obtaining homogenous IgVH mRNA for sequencing, and may therefore lead to failed sequencing or sequencing of the wrong IgVH mRNA. We have reported that plasma is enriched with leukemia-specific DNA, RNA, and protein because of the high turnover of leukemic cells relative to non-neoplastic cells. We thus reasoned that plasma might be a more reliable source of IgVH mRNA than cells from peripheral blood or bone marrow, where mixed populations of leukemic cells and non-neoplastic lymphocytes or plasma cells could hinder sequencing. We tested the plasma from 8 patients in whom routine (cell-based) testing for IgVH mutation status failed to yield results due to a paucity of leukemic cells (<10% of total cells). The plasma of all the 8 samples showed unique, easily defined amplification products of the IgVH mRNA. Sequencing of these products showed the presence of mutated IgVH in 5 patients and unmutated IgVH in 3. Testing paired plasma and cell samples from 19 patients with CLL showed identical mutation rates and family types of the expressed IgVH gene. In contrast, plasma from 20 normal individuals showed no IgVH amplification products that could be sequenced. This data suggest that plasma can be used as an alternative to cells for testing IgVH mutation status. More importantly, plasma is more reliable when few leukemic cells are in circulation and the bulk of the tumor is in the bone marrow or lymph nodes.
Expression of Lymphocyte Activation Gene 3 (LAG-3/CD223) by Chronic Lymphocytic Leukemia B Cells.
Authors: Chuang Han-Yu , Liu Yu-Tsueng , Rassenti Laura Z. , Huynh Lang , Carson Dennis , Kipps Thomas J.
Published: April 2020

The clinical course of chronic lymphocytic leukemia (CLL) is variable. DNA-microarray studies have shown that the gene-expression patterns of CLL cells with unmutated IgVH genes are similar to those of cells with mutated IgVH genes, but that the patterns of both are distinct from those of other leukemias and lymphomas. Nevertheless, the two subtypes of CLL can be distinguished by the differential expression of a small number of genes, one of which encodes ZAP-70, an intracellular tyrosine kinase with a critical role in T-cell receptor signaling. Further analysis of the mutation status of IgVH genes and ZAP-70 expression revealed that CLL patients with B cells expressing ZAP-70 and unmutated IgVH genes had a more aggressive disease. LAG-3 (CD223) is thought to play a role in immune responses mediated by T and NK cells. LAG-3, a CD4 homolog, is a ligand for MHC class II antigens. Similar to ZAP-70, LAG-3 is selectively expressed on activated T and NK cells and has recently been shown to be expressed on T-cell activated B cells. We compared the gene expression profiles of the B cells purified from 15 CLL patients using the Affymetrix HG-U133 plus 2.0. This analysis revealed LAG-3 and parathymosin differentially expressed at higher levels by the CLL cells expressing ZAP-70 and unmutated IgVH genes. LAG-3 and parathymosin are located on chromosome 12p13 with head-to-tail orientation. To examine for surface expression of LAG-3, we performed flow cytometry on these same 15 CLL samples using an anti LAG-3 mAb (CD223). We found LAG-3 expressed at high levels by the CD5/CD19 B cells of 4/8 (50%) cases that expressed ZAP-70 and unmutated IgVH genes. Conversely, the samples with B cells lacking ZAP-70 and with mutated IgVH genes did not express LAG-3 (0/7). All samples (15/15) expressed high level of MHC class II antigens, as assessed by flow cytometry. LAG-3 may interact with MHC class II molecules expressed by CLL cells to form an autocrine loop that may further enhance the activation of ZAP-70-expressing CLL B cells. Further analysis is needed to delineate the role of LAG-3 in the pathogenesis and/or progression of this disease.