bluuebunny/crossref_metadata_2025 huggingface dataset:
TOP-M Chemotherapy (Thiotepa, Oncovin [Vincristine], Prednisone, Mitoxantrone) Plus Rituximab for Marginal Zone Lymphoma.
Authors: Coleman Morton , Furman Richard R. , Niesvizky Ruben , Ashe Michelle , Mathews Lena , Fiore Jennifer , Ketas Jamie , Leonard John P.
Published: April 2020

Although considered an indolent disease, marginal zone lymphoma (MZL) often requires treatment. No standard therapy has been clearly established. Mitoxantrone has been effective in combination for indolent lymphomas, and is associated with considerably less cardiotoxicity and alopecia than doxorubicin. Thiotepa likewise does not induce alopecia, and does not display the immunosuppressive properties of fludarabine, which is also frequently employed as therapy for indolent lymphoma. The combination of thiotepa, vincristine, prednisone and mitoxantrone chemotherapy, with a scope similar to CHOP, has been demonstrated to be useful in elderly patients with diffuse large cell lymphoma (Lichtman, et al, Am J Clin Oncol, 24(4):360–2, 2001). The TOP-M regimen consists of thiotepa 7.5 mg/m2, mitoxantrone 7.5 mg/m2, vincristine and prednisone as per the CHOP regimen, with rituximab 375 mg/m2 all given on day 1 with a 21–28 day cycle (in accordance with resolution of cytopenias). Thiotepa and mitoxantrone doses were escalated by a total of 2.5 mg/m2 to a maximum of 20 mg/m2, respectively, based on nadir blood counts. We have retrospectively reviewed data on 12 patients with disseminated MZL treated with TOP-M either alone (n=4) or with rituximab (n=8). All subjects had advanced stage disease (4 with stage III, 8 with stage IV), 4 had B symptoms, and all patients clinically required treatment. Median age was 69, and 4 (33%) had an elevated LDH. Myeloid growth factor support was routinely applied, and anti-emetics were generally unnecessary. Central venous access was not required since neither thiotepa nor mitoxantrone are associated with tissue injury in the event of extravasation. Treatment was well tolerated, with only two episodes of hospitalization for fever. There was no cardiac toxicity. Eleven of 12 patients (92%) demonstrated clinical responses, with 9 CR/CRu (75%) and 3 PR (17%). Median duration of response has not been reached at with a range of 11 to 40 months of followup. TOP-M with rituximab is an easily administered, well tolerated and active regimen for disseminated marginal zone lymphoma, and may have utility for other indolent lymphomas.
Quantitative Pharmacogenetics of Cyclophosphamide in Patients with Hematological Malignancies.
Authors: Hassan Moustapha , Xie Hanjing , Ståhle Lars , Hassan Zuzana , Kimby Eva
Published: April 2020

A high degree of interindividual variation in cyclophosphamide (CPA) pharmacokinetics was reported in certain cancer patient groups. To better understand the mechanisms underlying the variation in CPA metabolism, the pharmacokinetics of CPA and its active metabolite 4-OH-CPA were estimated in patients with hematological tumors and related to the genotype of CYP2B6, CYP2C9 and CYP2C19. The influence of liver function on CPA metabolism was also evaluated.

Twenty-nine patients with hematological malignancies (MM, ALL or NHL) treated with a conventional CPA dose (1 g/m²) were recruited to this study. Blood samples were collected before, during and after CPA treatment. HPLC was used to measure plasma concentrations of CPA and 4-OH-CPA. Patients were genotyped for the CYP2B6 G516T, CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3 alleles. Serum bilirubin levels were measured before the treatment. Data was analyzed individually and by population pharmacokinetic methods, using non-linear mixed effect modeling. The interindividual variability in exposure to CPA, 4-OHCPA and 4-OH-CPA/CPA was 5.8-, 3.3- and 10.3- fold, respectively. A positive correlation was found between half-lives of CPA and 4-OH-CPA (p<0.05) and there was a significant negative correlation between AUCs of CPA and 4-OH-CPA. In the population analysis the clearance contribution of the CYP2B6 G516T variant allele was about twice the wild type gene while the genotype of CYP2C9 did not influence clearance. A negative correlation was observed between bilirubin level and CPA bioactivation. There is a considerable variation in CPA formation of 4-OH-CPA among patients. The liver function influences CPA metabolism. Presence of the CYP2B6 G516T mutation increases the rate of 4-OH-CPA formation, a substantial variation among patients remain to be explained.
Prognostic Molecular Features in Diffuse Large B-Cell Lymphoma from Saudi Arabia.
Authors: Al-Kuraya Khawla S. , Siraj Abdul Khalid , Nayanappa Rajeswari , Al-Dayel Fouad , Ezzat Adnan , Al-Jommah Naif , Sauter Guido , Simon Ronald
Published: April 2020

Molecular features that can classify diffuse large B cell lymphoma (DLBCL) in clinically relevant subgroups have recently been demonstrated in studies investigating Western Patients. Different etiologic factors and potentially also varying genetic predispositions have been demonstrated for lymphoma patients from developing countries. To investigate whether proposed molecular classifiers are equally important for Middle East as for Western populations, we analyzed coexpression of CD10/bcl6 (by immunohistochemistry), IgH/Cyclin D1 translocations (by fluorescence in situ hybridization) and methylation of the gene encoding the DNA repair enzyme O6 methyguanine -DNA methytransferase gene (MGMT) in a series of 236 DLBCL patients from Saudi Arabia. Clinical follow up data were available from 190 patients. Coexpression of CD10/bcl6 was found in 21%, IgH/Cyclin D1 translocations in 18.3%, and MGMT methylation in 70% of cases. With the exception of MGMT methylation which was reported in only 20–35% of European patients, these numbers were in the range expected from the Western literature. Despite the markedly higher number of MGMT methylation in Saudi cases, its proposed positive effect on DLBCL prognosis could be confirmed (p<0.05). IgH/Cyclin D1 translocations were significantly more frequent in DLBCL with CD10/bcl6 coexpression (12 of 41) than in DLBCL without CD10/bcl6 coexpression (4 of 36; p<0.05). However, neither CD10/bcl6 coexpression nor IgH/Cyclin D1 translocation was related to clinical outcome in our study. In summary, our data do not suggest major molecular differences between Saudi and European DLBCL. Methylation of MGMT gene is a strong prognostic factor in DLBCL. IgH/Cyclin D1 translocations are predominantly found in DLBCL of germinal center (CD10/bcl6 positive).
Impaired Clonogenic Capacity Contributes to Bone Marrow Hypoplasia and Late Hematological Recovery after Zevalin in the Low-Grade NHL Patients.
Authors: Jurczak Wojciech , Szostek Marta , Rudzki Zbigniew , Krochmalczyk Dorota , Wegrzyn Joanna , Czyz Jaroslaw , Skotnicki Aleksander B.
Published: April 2020

Chemoimmunotherapy with Zevalin (anty CD20,90Y) is a new therapeutic approach in the resistant or relapsed low grade malignant lymphoma patients. The most important side effect observed in the up to date studies is related to transient peripheral blood cytopenias.

In an attempt to evaluate the involved pathogenetic mechanisms of the above findings, the blood and bone marrow samples from 8 low grade NHL patients were prospectively tested for the peripheral blood count, the clonogenic capacity and the histopathology abnormalities (trephine biopsies) before and every 14 days after Zevalin administration until recovery. In the majority evaluated patients the mild thrombocytopenia (30-55000/μl) was observed 4 weeks after Zevalin therapy. Bone marrow mononuclear cells were cultured in methylcellulose assay using combination of recombinant CSF (IL-3, IL-6, stem cell factor, erythropoietin; Methocult, StemCell, Canada), at a concentration of 1x10^5 cells, in 35 mm gridded Petri dish and incubated at 37°C, in a humidified atmosphere of 5% CO2. The hematopoietic colonies (CFU-GM, BFU-E and CFU-GEMM) were evaluated on 14th d. of the culture. The in vitro CFU-Meg formation in the serum-free collagen-based semisolid culture (Megacult, StemCell, Canada) were also studied, stained after 12 days with a monoclonal antibody against CD 41.

In the results of our studies the significant inhibition of colony formation was found already in 7th day for CFU-GM and BFU-E and starting from 14th day after Zevalin administration for CFU-Meg. In the subsequent cultures evaluated during 4–8 weeks after Zevalin therapy the three lineages hematopoietic clonogenic capacity returned to the pre-radiotherapy values. In the repeated trephine biopsis several morphological changes were observed: oedema of stromal cells, the relative increase of erythropoietic components with inverted proportion of granulo- to erythropoiesis.

In conclusion, chemoimmunotherapy with Zevalin resulted in the reversible peripheral cytopenia parallely with transient damage to bone marrow hematopoiesis and in vitro clonogenic capacity.
Rituximab Plus CHOP Followed by Maintenance Rituximab as Initial Therapy for Advanced Stage Indolent Non-Hodgkin’s Lymphoma (NHL); Initial Results of Induction Therapy in a Phase II Study.
Authors: Huang Jane E. , Hart Lowell , Polikoff Jonathon , Langmuir Virginia , Zhang Fan , Fehrenbacher Louis
Published: April 2020

Background: This study was designed to investigate the activity and safety of rituximab (Rituxan®, R) plus CHOP induction therapy followed by maintenance R in patients with previously untreated advanced stage indolent NHL. The initial results of R-CHOP induction treatment are reported here.

Methods: 102 patients with Ann Arbor Stage III or IV indolent NHL (follicle center/follicular grade I/II or nodal marginal zone B-cell by REAL; Type B or C by IWF) were enrolled into this open-label, multi-center, community-based, Phase II, single-arm trial. Patients received 6 cycles of standard R-CHOP induction therapy (cyclophosphamide 750 mg/m2, vincristine 1.4 mg/m2, doxorubicin 50 mg/m2 all IV on Day 1; prednisone 100 mg/day Days 1–5). R 375 mg/m2 was given on Day 1 of each cycle except for cycle 1, when it was administered 2–3 days prior to CHOP chemotherapy. Subjects with an ongoing response (CR/CRu or PR) after induction receive maintenance R (4 weekly infusions) within 28 days after the completion of induction and repeated every 6 months for 2 years for a total of 16 maintenance R doses. The primary endpoints were CR/CRu after 6 cycles of induction and the incidence of R infusion-related toxicity during induction and maintenance therapy. Secondary endpoints included overall response rates (ORR), infusion times, serious adverse events (SAE) in induction and maintenance, and rituximab pharmacokinetics in maintenance therapy.

Results: Baseline characteristics were: median age 57y (33.3% ≥60y); 40.2% female; ECOG performance status 0: 70.6%; Ann Arbor stage III: 28.4% and IV: 71.6%. Serum β2-microglobulin and LDH at baseline were above normal in 66.3% and 20.6%, respectively. CR/CRu at the end of induction in 102 patients was 51.0% (ORR 86.3%). 1% had progressive disease (PD) during induction. 2 subjects died during the induction period, both due to SAEs (probable pulmonary embolus, acute respiratory distress). SAEs occurred in 22.5% of subjects during induction with the most common event being febrile neutropenia (7.8%). Grade 3–4 R infusion-related toxicity occurred in 4.9% of patients in cycle 1; this decreased to 1% by cycle 2 of induction. Two subjects discontinued R for infusion-related toxicity. 44% of patients were able to receive their R dose within 3 hrs at cycle 2 and 69.2% of patients at cycle 6.

Conclusion: Patients with advanced stage indolent NHL treated in the community setting with R-CHOP induction tolerate therapy well and have an excellent response rate. The outcomes of maintenance therapy and rituximab pharmacokinetics in maintenance await further follow-up.
MTOR (Mammalian Target of Rapamycin) Inhibition with RAD001 Induces Cell Cycle Arrest in Diffuse Large B Cell Lymphoma (DLBCL) Cells and Sensitizes DLBCL Cells to Rituximab Therapy.
Authors: Wanner Kathrin , Hipp Susanne , Peschel Christian , Decker Thomas
Published: April 2020

Purpose: DLBCL is a common lymphoma entity. Although a significant amount of patients can be cured with modern chemotherapeutic regimen, a substantial proportion of patients die due to progressive disease. Therefore, new therapeutic strategies are clearly needed. Inhibitors of MTOR (mammalian target of rapamycin) represent a new class of antiproliferative drugs with applications as immunosuppressive and anticancer agents. Extensive safety data exist on RAD001 which is already approved as an immunosuppressant in organ transplant recipients.

Methods: We have analyzed the effects of MTOR inhibition in three DLBCL cell lines: SUDHL-4, DB, DOOH-2. Propidium Iodide staining, MTT- and Annexin-V assays were performed to test the effects on viability, cell cycle distribution and apoptosis. Expression of cell cycle regulatory and phosphorylation of p70 s6 kinase and 4-EBP-1 molecules was revealed in western blot experiments

Results: Rapamycin and RAD001 inhibited cell cycle progression in DLBCL cells by inducing a G1 arrest without inducing apoptosis. Phosphorylation of the main targets of MTOR, p70s6 kinase and 4-EBP-1 was reduced in cells cultured in the presence of RAD001.Cell cycle arrest was accompanied by reduced phosphorylation of the Retinoblastoma protein (RB) as well as reduced expression of cyclin D3, E and A in all cell lines. Although the effect of chemotherapeutic agents like Vincristin or Doxorubicin was not enhance by Rad001, Rituximab induced cytotoxicity was augemted in both Rituximab sensitive cell lines.

Conclusion: MTOR Inhibition is a promising therapeutic strategy in DLBCL of the germinal center like subtype by inducing a G1 arrest and augments Rituximab induced cytotoxicity. Therefore, combination of these drugs might be an interesting new therapeutic approach in DLBCL patients.
In Search for the Specificity of Clonal CTL in T-LGL Leukemia - Generation of Soluble LGL-Derived T Cell Receptor.
Authors: Wlodarski Marcin , Howe Evan , Maciejewski Jaroslaw P.
Published: April 2020

Large granular lymphocyte leukemia (LGL) is a clonal lymphoproliferation of CTL associated with immune cytopenias. Hematopoietic progenitor cells may be targets for the immune attack by clonal CTL. The rearranged unique portion of the T cell receptor (TCR), the complementarity determining region 3 (CDR3) of the variable beta chain can serve as molecular marker of the clonal process. Previously, we applied molecular analysis of TCR repertoire to the analysis of CDR3 regions of the variable beta-chain in LGL leukemia and obtained 70 individually specific CDR3 clonotypes. Moreover, using clonotype-specific PCR clonal size was determined. Shared or highly homologous clonotypic CDR3 sequences in some patients provide evidence for the non-random nature of the clonal transformation in LGL and the presence of a common antigenic target. Such an antigen may constitute a drive for the expansion of LGL and its distribution may explain lineage-specificity of the LGL CTL. Identification of the target antigen is essential for understanding of immune-mediated hematologic disorders, including autoimmune cytopenias such as those observed in LGL. T-LGL has inherent advantage of the availability of clonal CTL for experimentation. We hypothesized here that generation of a soluble TCR (sTCR) derived from T-LGL clone could facilitate identification of target cells and analysis of the cellular distribution of peptide presentation. For cloning of sTCR we selected 4 patients with extreme clonal CTL expansion and profound neutropenia. Flow cytometry with VB specific antibodies was applied to determine VB family utilization by the clonal TCR. B-chains were amplified with specific primers so that C region is truncated and the transmembrane and intracellular portions are deleted. Due to the lack of VA specific antibodies, a 5′RACE RT-PCR with a constant A primer was applied. Expanded LGL-specific A chain was determined and cloned in its truncated form as described for B-chain. For example, clonal TCR for patient #1 had a structure VB1/JB1.6/CB1 and VA9-2/JA41*01/CA. To construct a sTCR truncated VA and VB products were subcloned into a dual promoter baculovirus expression vector containing the k and H-chain cassettes (pAC-k-CH3 or alternatively pAC-Fc). This vector allows for expression of a bivalent TCR:Ig protein that can be produced in a baculovirus system. A flow cytometry on fluorochrome conjugated sTCR after co-culture with HLA matched normal bone marrow cells allows for the detection of cells expressing target peptides. Therapeutically, malignant T cell clone-specific sTCR may be used as a potential idiotypic vaccine for T cell lymphomas/leukemias expressing TCR.

Figure Figure
Percentage of CD34 + Cells in a Minimum 500-Cell Count in Bone Marrow Trephines of Patients with Chronic Myeloid Leukaemia Provides the Best Correlation with Aspirate Blast Count.
Authors: Elliot Victoria J. , Marin David , Horncastle Donna , Elderfield Kay , Apperly Jane F. , Lampert Irvin A. , Naresh Kikkeri N.
Published: April 2020

We studied the expression of CD34 in 169 bone marrow trephines of 53 patients with CML in chronic, accelerated and blastic phases. The bone marrow blast count ranged from 0–82% (median: 4.5%). All were receiving imatinib treatment. We aimed to establish which counting method in bone marrow trephines provided the best correlation with the blast count of the corresponding bone marrow aspirate in all phases of the disease. Paraffin embedded sections were stained with the monoclonal antibody QBEND10.

CD34 expression in each marrow trephine was evaluated by three counting methods. The first method, established in our department for several years, expressed the percentage of CD34 positive cells vs. all nucleated cells in a minimum 500-cell count (% CD34+/500 cells). The second recorded the total number of CD34 positive cells in ten high power fields (CD34+/10hpf) using x 40 magnification /numerical aperture 0.65. The third method evaluated the highest number of CD34 positive cells present in a single high power field (CD34+/hpf), this value being taken from the previous 10 hpf count. The two latter methods were employed because they may be more applicable for general use by practicing histopathologists. In addition, counting over 10 hpf provides a much larger sample size than a 500-cell count. CD34 expression in endothelial cells served as a positive internal control.

All three methods showed significant positive correlation with blast count (% CD34+/500 cells: Pearson’s Correlation - 0.723, sig - <0.001; CD34+/10hpf: Pearson’s Correlation - 0.434, sig - <0.001; CD34+/hpf: Pearson’s Correlation - 0.372, sig - <0.001). In conclusion, our previously established method of recording the percentage of CD 34 positive cells vs. all nucleated cells in a minimum 500-cell count provided the best correlation with the blast count.
Total Number of CD34 + Cells Per Ten High Power Fields in Bone Marrow Trephines of Patients with Chronic Myeloid Leukaemia Correlates with Probability of Complete Cytogenetic Response during the First Year of Treatment with Imatinib.
Authors: Elliot Victoria J. , Marin D. , Horncastle D. , Elderfield K. , Howard J. , Apperly J. F. , Lampert I. A. , Naresh K. N.
Published: April 2020

We studied expression of CD34 positive cells in the bone marrow trephines of two groups of patients with CML treated with imatinib. In the first group (n=17), patients went on to achieve sustained complete cytogenetic response during their disease course. In the second group (n =33) complete cytogenetic response was not achieved and patients later developed either accelerated or blastic phase disease. We studied paraffin embedded bone marrow trephines stained with the monoclonal antibody QBEND10 during an eighteen month period following initiation of imatinib therapy and recorded the highest CD34 positive cell counts and aspirate blast counts in three six-month intervals. CD34 positive cells were evaluated using three counting methods. The first method, established in our department for several years, expressed the percentage of CD34 positive cells vs. all nucleated cells in a minimum 500-cell count (% CD34+/500 cells). The second recorded the total number of CD34 positive cells in ten high power fields (CD34+/10hpf) using x 40 magnification /numerical aperture 0.65 and the third method utilised the highest number of CD34 positive cells expressed in a single high power field (CD34+/hpf), the value being taken from the previous CD34+/10 hpf count.

We hypothesised that the overall CD34 positive cell burden and absolute CD34 positive numbers would be more important in predicting the likelihood of complete cytogenetic response and this would be better assessed by volumetric evaluation than percentage calculation relative to other nucleated cells.

CD34 expression in endothelial cells was taken as a positive internal control. We aimed to establish which variable: % blast count, % CD34+/500 cells, CD34+/10hpf and CD34+/hpf provided better correlation with subsequent cytogenetic response to treatment. Values for each variable were dichotomised at the following levels; aspirate blast count <5 and > 5, % CD34+/500 cells < 2 and > 2%, CD34+/10hpf < 60 and > 60 cells and CD34+/hpf < 10 and > 10 cells. Univariate analysis was performed on samples within the first six months, seven to twelve months and thirteen to eighteen months. Only CD34+/10hpf showed significant discriminatory ability throughout the entire eighteen month period (Pearson’s Chi-square 2-sided sig: 0.031, 0.003 and 0.007 in the first six months, seven to twelve months and thirteen to eighteen months samples respectively). By logistic regression, we confirmed that CD34+/10hpf had the best correlation with cytogenetic response during the first year following initiation of imatinib therapy. Larger studies are required to confirm these initial findings and to establish volumetric assessment of CD34 positive cells as a predictor for cytogenetic response.
Response of Elevated Serum Soluble Interleukin-2 Receptor (sIL-2R) and Overexpression of Glucose-6-Phosphate Dehydrogenase (G6PD) in Patients with Chronic Myelogenous Leukemia Treated with Imatinib Mesylate.
Authors: McPherson Eugene , Shanmughan M. , Huang S.Y. , Kleinfeld J. , Hazel E. , Tassy P.
Published: April 2020

Regulation of intracellular redox potential is important for regulation of cell growth. G6PD functions to catalyze the first step in the pentose phosphate pathway physiologically providing the NADPH required for reductive biosynthesis and detoxification of free-radicals and peroxides in mature red blood cells. We present two cases of patients (pts) with Chronic Myelogenous Leukemia-chronic phase (CML-cp) with significant overexpression of sIL-2R and G6PD. Serum elevated levels of sIL-2R and G6PD correlated to CML stage and response to therapy with imatinib; one male pt and one female pt with white blood cell counts of 80 and 21,000 X 109 per liter and a mean sIL-2R and G6PD levels of 26.975 X 103 pg/ml and 14.10 units/gHb respectively. Both pts had BCR/abl-RT-PCR positive gene rearrangements with b2a2 in peripheral blood and bone marrow. Chromosomal abnormalities of female pt was consistent with CML-cp (t 9; 22) ( q34;q11.2) philadelphia chromosome. Treatment of both pts with imatinib 400 mg orally daily resulted in reduction toward normal levels of sIL-2R and G6PD. Our female pt had to have therapy interrupted due to severe gastrointestinal and myelosuppression with cardiac irritation, edema, headaches and arthralgia. She was changed to 300 mg of imatinib with a proton-pump inhibitor, growth factor G-CSF, and a cox-2 inhibitor with excellent tolerance and response to therapy ( see table I).

Response of sIL-2R and G6PD To Treatment With Imatinib Mesylate

MONTHS LAP-SCORE sIL-2R (pg/ml) CRP (mg/dL) G6PD (units/gHb) LDH (U/L) Lap Score(NL 40-130); sIL-2R (NL 1,770-9,753); G6PD (3.5–5.0) Zero 4 26,975 16.0 11.1 386 Three 12 5,629 2.75 4.1 241 Six 17 12,868 0.75 3.6 266 Twelve 143 6,111 3.75 3.9 203

CONCLUSIONS: Overexpression of sIL-2R and G6PD in CML-cp pts treated with tyrosine kinase inhibitor imatinib mesylate were significantly reduced to levels within normal limits after twelve months of therapy. Peripheral blood sera sIL-2R and erythrocyte G6PD levels in CML-cp pts may be useful clinical and prognostic indicators of leukemic cell burden.
Defective Production of Myeloid Dendritic Cells Results from Skewing of Chronic Myelogenous Leukemia Progenitors.
Authors: Guimond Martin , Boumedine Radia Sidi , Perreault Claude , Roy Denis-Claude
Published: April 2020

The robustness of the immunostimulatory response generated by dendritic cells (DCs) has positioned these cells as central and determining cellular mediators of T cell activation. Their production in sufficient number from hematopoietic precursors is crucial in order to drive immune responses. To determine the capacity of CML clonogenic cells to generate dendritic cells expressing endogenous CML-specific epitopes, we have evaluated the generation of DCs from CD34+ progenitor cells and CD14+ monocytes isolated from patients with CML (n=28). We found that the kinetics of production of DCs, in GM-CSF 800U/ml, TNF-a 50U/ml and IL-4 10U/ml, from CD34+ progenitors of CML patients were significantly delayed in comparison to healthy controls (p<0.05). Even after 20 day-cultures, DC numbers remained decreased (P<0.01). A proliferative defect of CML blasts cannot explain this finding since CD34+ cells divided normally according to total cell culture count and CFSE staining. Flow cytometry analysis of the few DCs produced from CML CD34+ cells showed the same phenotypic characteristics of expression of MHC (class I and II), costimulatory (CD80, CD86) and adhesion molecules (CD11a, CD11b and CD58), as DCs generated from control CD34+ cells, except for late downregulation of CD11a. Normal maturation of DCs from CML CD14+ cells and a sustained proportion of the bcr-abl+ DCs before and after DC expansion excluded a 9:22 translocation-induced differentiation defect. However, retarded down-modulation of the CD34 antigen at the surface of CML blasts, independently of IL-4 receptor synthesis, rather suggested decreased susceptibility of CML progenitors to IL-4 and GM-CSF-mediated DC growth conditions. Indeed, CML progenitors, plated in semi-solid media, showed an important skewing toward more mature progenitors (BFU-E and late CFU-GM) with a significant decrease in immature CFU-GM (> 500 cells) (CML = 29 vs normal = 95 colonies, P<0.001) and CFU-Mix (CML = 0.6 vs normal = 5.6 colonies, P=0.0006). Moreover, the number of DCs obtained after 10 and 15 day-cultures showed a strong correlation with the number of immature CFU-GM and CFU-Mix colonies (R=0.95). Thus, our study identifies a selective impairment in DC production from CD34+ progenitors of CML patients corresponding to a skewing of the progenitor cell compartment toward mature myeloid and/or erythroid progenitors. The lack of DCs derived from leukemia progenitors may explain the immunologic escape of CML clonogenic cells and provides clues to enhance their immune recognition.
Anti-Erythroblast Antibodies and Defective Apoptosis in Bone Marrow of Early Myelodysplastic Syndrome Patients.
Authors: Zaninoni Anna , Imperiali Francesca G. , Fermo Elisa , Zanella Alberto , Barcellini Wilma
Published: April 2020

Autoimmune phenomena, particularly directed against RBC, and alteration of apoptosis have been reported in MDS. We recently described a new method for the detection of anti-RBC antibodies in mitogen-stimulated whole blood cultures, named mitogen-stimulated-DAT (MS-DAT), which is able to disclose a latent anti-RBC autoimmunity in different diseases (autoimmune hemolytic anemia in clinical remission and in B-CLL). We investigated MS-DAT positivity and apoptosis in peripheral blood and bone marrow cultures from 14 patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS), compared with controls (healthy volunteers and miscellaneous haematological conditions). MS-DAT was performed by stimulating whole blood and marrow cultures with PMA, and anti-RBC or anti-erythroblast antibodies were detected by competitive solid phase ELISA. As apoptotic markers we evaluated NF-kB and Caspase-3 activity by ELISA and enzymatic assay, respectively. As shown in table, the amount of anti-erythroblast antibodies was significantly greater in BM cultures of MDS versus controls, and the test was clearly positive in 8/14 patients (not shown). The anti-apoptotic marker NF-kB was significantly increased in MDS versus controls, and consistently, caspase-3 activity decreased, although not significantly. On the contrary, PB of MDS displayed no MS-DAT positivity and no significant alterations of apoptotic and anti-apoptotic markers investigated (not shown). These findings suggest the existence of an anti-erythroblast autoimmunity in bone marrow of early MDS patients. This could be related to the observed defective apoptosis, which in turn determines survival of auto-reactive marrow effectors.

MDS Controls Medium PMA Medium PMA *denotes statistical significance versus controls. Values are mean±SE Anti-erythroblasts (pg/ml) 407±116* 487±146* 76±15 99±20 NF-kB (OD units) 294±50* 358±83 59±20 67±21 Caspase-3 (OD units) 132±24 145±26 209±53 186±46
Arsenic Trioxide (Trisenox®) with/without Thalidomide in Patients with Myelodysplastic Syndromes (MDS) Produces Hematologic Improvement (HI).
Authors: Raza Azra , Lisak Laurie , Billmeier Jennifer , Akbar Samreen , Washeed Khalid , Mumtaz Mohammad , Reddy Poluru , Galili Naomi
Published: April 2020

In a previous clinical trial, we have shown that 7/28 MDS patients, belonging to IPSS intermediate and high risk categories, responded to a combination of Trisenox® and thalidomide including a complete hematologic and cytogenetic remission. Two trilineage responses were seen in patients with inv(3) abnormality. It was not clear whether the responses were due to the combination or to either drug alone. In the present study, we treated 30 MDS patients with Trisenox® (5 days of loading dose at 0.25mg/kg IV over 2 hours followed by twice weekly dose) for 6 months followed by the addition of thalidomide (100mg po qday x 3 months). The Median age was 71 years (19 males, 11 females). FAB types: RA 3 patients, RARS 2, RAEB 14, RAEB-t 8, CMMoL 2, Unclassified 1. IPSS: Low 0 patients, Int-1 10, Int-2 7, High 13. Two, 1,1 and 4 patients had abnormalities affecting chromosomes 5, 7, 8, and 20, while 12 patients showed a complex karyotype. Therapy was fairly well tolerated and median number of cycles was 3. Five patients showed a variety of hematologic responses as judged by the IWG criteria (1 trilineage, 1 cytogenetic and change in FAB type due to decrease in blasts, 1 ANC and reduction in blasts, and 2 with 50% reduction in blasts). Among the responders, 1 had 47 XX, +8(16 metaphases), del(X)(q22q25),del(3)(q25q27)(4 metaphases), 3 had a normal karyotype, and 1 had del(20q). Three of the partial responders were started on thalidomide, but all stopped within 2 months. Following response, the first patient showed disappearance of the clone bearing the chromosome 3 abnormality, confirming the sensitivity of these cells to Trisenox® as reported in our previous trial. Interestingly, 3/5 responders showed an increase in WBC/ANC and 1 in platelets as well (12, 22 and 90,000/ul), without any evidence of a differentiation syndrome. One also had an increase in platelets from ~50,000/ul to 966,000/ul. Blasts were 11, 24 and 28% in these 3 patients prior to therapy and decreased to 4, 6 and 24% respectively after treatment. It was concluded that the twice-daily dose of arsenic trioxide was well tolerated, and some patients converted their disease to a more myeloproliferative type picture.
Myelodysplastic Syndrome Due to Somatic GATA1 Mutation.
Authors: Freson Kathleen , Vyas Paresh , Thys Chantal , Wittevrongel Christine , De Vos Rita , Van Geet Chris
Published: April 2020

Inappropriate hematopoietic differentiation resulting in trilineage dysplasia is a diagnostic hallmark of myelodysplastic syndrome (MDS). Understanding the molecular basis of MDS will not only shed insight into this common hematological disease, but also Acute Myeloid Leukemia (as MDS often progresses to AML) and normal hematopoiesis. The genetic basis of MDS is unclear and in particular the role of deregulated transcription factor activity, which is often seen in AML, has not been clearly defined. For the first time we describe a novel somatic GATA1 mutation in a 13-year-old boy with MDS who has 10-year history of anemia, neutropenia, moderate thrombocytopenia, bone pain and hepatomegaly. Bone marrow biopsy demonstrated trilineage dysplasia with a normal karyotype of bone marrow mononuclear cells. Sequence analysis showed an A to G mutation in the X-linked GATA1 gene at residue 598 in exon 4 in platelets and bone marrow mononuclear cell cDNA. The mutation was also detected in genomic DNA from purified myeloid progenitors but not peripheral blood leucocytes. This discrepancy may be due to selection of peripheral blood leucocytes that do not carry the GATA1 mutation. The mutation was also not seen in skin fibroblasts. The predicted amino acid substitution E200G is located in a highly conserved residue at the beginning of the N-terminal zinc finger (Nf). This did not perturb DNA binding to GATA sites by EMSA analysis nor reduce interaction with FOG1. Importantly, immunoblot analysis of the patient’s platelets showed strongly reduced protein levels of GATA1, normal FOG1 and increased RUNX1. The index case had an increased Ivy bleeding time (>15 min) and a significant prolongation of the PFA-100 occlusion time (collagen/ADP and collagen/epinephrine). Platelet ATP secretion and platelet aggregation (epinephrine, ADP and U46) were decreased compared to control platelets. In contrast to patients with an inherited GATA1 mutation, the platelet phenotype in the MDS patient is completely different. Platelets from the patient are smaller in size (MPV 8.6fL) and electron microscopy revealed a markedly reduced number of dense granules. His platelets have a normal GPIbα expression and normal ristocetin-induced platelet agglutination. The previously described patients with inherited Nf GATA1 mutations (D218G GATA1 Blood,2001,98,85) have giant platelets with characteristic membrane complexes and reduced GPIbα expression with abnormal ristocetin-induced platelet agglutination. Further study of the index patient will provide important detailed insight into GATA1 function and help unravel how perturbed activity of key myeloid transcription factors results in MDS.
Evidence for T-Cell Involvement in MDS: Similar Patterns of Hypermethylation of Key Genes in Purified CD34+ and CD3+ Cell Fractions in an RAEB Patient.
Authors: Aggerholm Anni , Rethmeier Anita , Guldberg Per , Carlsen Ingrid , Brændstrup Karin , Hokland Peter
Published: April 2020

Aims: Myelodysplastic syndromes (MDS) constitute a heterogeneous group of disorders with hematopoiesis in disarray and with preponderance to transformation to AML. In contrast to the situation in de novo AML patients, the hematopoiesis in MDS patients is often though to encompass most cell lineages with few non-malignant cells in the bone marrow due to displacement by the MDS clone. While the involvement of the myeloid sublineages has been relatively easy to verify in semi-solid CFU assays, that of the lymphoid lineages has required advanced flow sorting techniques in combination with FISH assays in the minority of MDS patients with a well-known genetic aberration such as 5q-, -7 or +8. Moreover, the latter studies have usually been restricted to the description of the involvement of the B-cell lineage. Transcriptional silencing of tumor suppressor genes by promoter hypermethylation is associated with hematological malignancy, including MDS. In a patient with a very immature MDS that was found to be hypermethylated in the p15, HIC, E-cadherin, and Estrogen receptor genes, we wanted to elucidate the methylation status in both myeloid and lymphoid cells.

Material and methods: Mononuclear cells from a 69-year-old female MDS patient with RAEB was analyzed. Using magnetic cell sorting, CD34 and CD3 positive cells were isolated from BM-MNC and PB-MNC, respectively. The purity of the isolated cell fractions was determined by flow cytometry (CD34+: 95% and CD3+: 84%). DNA isolated from the cells was treated with sodium bisulfite and analyzed for promoter hypermethylation in the p15, HIC, E-cadherin, and Estrogen receptor genes by means of Bisulfite-Denaturing Gradient Gel Electrophoresis (Bisulfite-DGGE).

Results: All cell fractions were found to be hypermethylated in the four genes analyzed. Since the Bisulfite-DGGE allows for the detection of heterogeneous methylation patterns, we were able to compare these patterns. Similar Bisulfite-DGGE band patterns were observed in total BM-MNC, total PB-MNC, CD34+ cells and CD3+ cells.

Conclusion: Because the methylation band patterns were the same in all cell fractions, it seems unlikely that methylation has occurred independently in progenitors committed for myeloid and lymphoid maturation, respectively. We therefore conclude that promoter hypermethylation in this patient has occurred in a stem cell with potential for both myeloid and lymphoid maturation. This methodology should provide an easy way to assess the extent of clonality of hematopoiesis in MDS patients.
Clinical Trial of Paricalcitol in Myelodysplastic Syndrome.
Authors: Koeffler H. Phillip , O’Kelly James , Aslanian Noune ,   The Southern California Leukemia Group
Published: April 2020

Myelodysplastic syndrome is often a pernicious disorder associated with pancytopenia in the elderly. Therapeutic approaches need to balance their toxicities versus the side-effects of the disease. 1,25(OH)2-vitamin-D3 inhibits proliferation and induces differentiation of leukemic cells in vitro. Small clinical trials have shown slight efficacy in MDS. Hypercalcemia prevents the administration of doses of this seco-steroid, which have been shown to be effective in vitro. This has provided a stimulus to identify vitamin D analogs that have anti-leukemic activity with minimal hypercalcemic effects. Paricalcitol (19-nor-1,25(OH)2D2, Zemplar) has been approved by the FDA for the treatment of secondary hyperparathyroidism; the drug is unique because it has little hypercalcemic potential; but in vitro, it has strong antileukemic effects. We conducted a clinical trial of oral paricalcitol to twelve MDS patients whose disease varied between an IPSS of low to high. Therapy began at 8 μg per day and increased at two week intervals until serum calcium was slightly above normal level; at which point, dose was decreased by 4-8 μg qd. The amount of paricalcitol taken varied between 8 μg qod to 54 μg qd (average 16 μg qd). We confirmed that the drug was having biologic activity in vivo by examining a target of the activated vitamin D3 receptor, 1,25-(OH)2-vitamin-D3-24(OH)ase mRNA. Each patient had prominent induction of this transcript in his or her white blood cells. Furthermore, in selected patients serum paricalcitol was measured and confirmed to be prominently present. The drug was well tolerated in all patients. Two of the 12 patients showed a clinical response. One patient’s platelet counts rose from 50,000 to 120,000/ul blood over 5 weeks; however, the patient succumbed to a fatal fungal infection. The second patient responded by a decrease in RBC transfusions associated with a rise in his hemoglobin, which lasted for about 5 months. Eventually, his hemoglobin began to fall, and erythropoietin therapy was substituted for paricalcitol. In summary, high doses of paricalcitol were well tolerated in all patients. Two patients had a partial clinical response. In general, paricalcitol given as a single agent to individuals with MDS is not therapeutically very efficacious; further trials should examine it in combination with other approaches.
Phase II Study of Gemtuzumab Ozogamycin (GO) and Cytarabine (ARA-C) in Patients with High Risk MDS and AML..
Authors: Kang Peter , Seiter Karen , Liu Delong , Arshad Muhammad , Qureshi Anila , Nelson John , Ahmed Tauseef
Published: April 2020

To evaluate the efficacy of GO + ara-c in high risk pts, we treated 22 pts with MDS (10) and AML (12) with cytarabine 100 mg/m2/d x 7 d and GO 9 mg/m2 x 1 on d 4. Pts with MDS were eligible if they had [1] RAEB-1 and either hgb < 8 gm/dl, platelet < 50,000/mm3, neutrophils < 1000/mm3, or cytogenetics other than 5q-, 20q-, -y, or normal, [2] RAEB-2, or [3] CMML. Pts with AML (newly diagnosed or relapsed) were eligible if they were ineligible for anthracycline-based therapy (poor performance status: 2 pts; low ejection fraction or high cumulative dose of anthracyclines: 6 pts, both reasons: 4 pts). The median age was 66, M:12, F:10. Diagnoses: RAEB-1: 4, RAEB-2: 5, CMML: 1, AML, newly dx’d: 7, AML relapsed: 5. Cytogenetics were high risk: 10, intermediate risk: 11 pts, low risk: 1 pt. Overall, 18% had a complete response (CR) after one cycle of therapy. Three pts (14%) had a partial response (PR), of which one had a CR after a second course of therapy. Of the pts with AML: CR: 2/12, PR: 2/12, Failure (F): 5/12, Toxic Death (TD): 3/12. For pts with MDS: CR: 2/10, PR: 1/10, F: 5/10, TD: 2/10. Toxicities included neutropenic fever/sepsis, mucositis, diarrhea, increased LFTs, hemorrhage. One pt with a history of ABVD and RT to chest for HD developed direct pulmonary toxicity due to chemotherapy (diffuse pulmonary infiltrates, biopsy proven toxic lung damage). Although overall response rate is modest, some pts had remarkable responses: One pt with RAEB-1, transfusion dependent and multiple high risk cytogenetics (−5, −7, multiple others) remains in CR (including cytogenetic CR) 7 months post treatment. A second pt with RAEB-1, transfusion dependent and multiple high risk cytogenetics (−5, −7, multiple others) had a PR after one course, but was unable to receive further chemotherapy due to toxicity. The latter pts suggest that this regimen should be studied further in pts with MDS, poor risk cytogenetics and low blast counts.
Acute Myeloid Leukemia (AML) - Type Chemotherapy for Newly Diagnosed Children with Myelodysplastic Syndrome (MDS): A Japanese Childhood MDS Study Group Trial MDS99.
Authors: Ohtsuka Yoshitoshi , Manabe Atsushi , Okamura Jun , Kojima Seiji , Ohara Akira , Tuchida Masahiro , Ikuta Koichiro , Hibi Shigeyoshi , Kawa Keisei , Yabe Miharu , Watanabe Arata , Ueda Kazuhiro , Nakazawa Shinpei , Miyazaki Sumio , Nakahata Tatsutoshi
Published: April 2020

Background The role of intensive chemotherapy in treatment of childhood MDS has not been clarified yet. We conducted a prospective trial which employed intensive AML-type chemotherapy for children with de novo MDS.

Methods Fifteen children aged 1 to 15 years who met diagnostic criteria of the FAB refractory anemia with excess of blasts (RAEB) with more than 10% of blasts in the bone marrow (BM) (RAEB-II) and RAEB in transformation (RAEBT) were prospectively enrolled in the study. Abnormal karyotype was documented in 11 patients including 3 with monosomy 7, 2 with complex karyotype, 1 with trisomy 8. Induction therapy consisted of etoposide (150mg/m2, on days 1–5), cytarabine (200mg/m2/12hr, on days 6–12), and mitoxantrone (5mg/m2, on days 6–10) in all but 1 patient, who received another regimen of AML induction therapy. The use of granulocyte-colony stimulating factor was allowed. Allogeneic stem cell transplantation (SCT) was scheduled after remission induction therapy. The median follow-up for 13 surviving patients was 685 days (58 to 1,476 days).

Findings Eleven patients (73%) including 3 with monosomy 7 achieved a complete remission after induction therapy at a median of 40 days (31 to 61 days). Remission was not obtained in the other 4 patients.

There was no significant difference in the frequency and severity of toxicity between in patients with RAEB-II/RAEBT and in those with de novo AML. Allogeneic SCT was performed in 13 patients including 9 in the remission at a median of 4 months (2 to 6 months) after the beginning of the therapy. None of nine patients who underwent SCT in the remission relapsed. Although two patients died of the complications of SCT. Overall survival for all the 15 patients at 2 years was 66.8 ± 14.1% (s.e.).

Interpretation AML-type induction therapy appears to be safe and effective for children with RAEB with more than 10% of blasts in the BM and RAEBT.

Figure Figure
Erythropoiesis Is Highly Stimulated in CD34+ Cells in Low-Risk Myelodysplastic Syndromes (MDS) with an Improper Mitochondrial Function.
Authors: Tehranchi Ramin , Invernizzi Rosangela , Zhivotovsky Boris , Fadeel Bengt , Forsblom Ann-Mari , Travaglino Erica , Samuelsson Jan , Hast Robert , Nilsson Lars , Wibom Rolf , Grandien Alf , Cazzola Mario , Hellstrom-Lindberg Eva
Published: April 2020

The apoptosis of early erythroblasts from patients with low-risk MDS, refractory anemia (RA) and RA with ringed sideroblasts (RARS), is mediated through a constitutive cytochrome c (cyt c) release from mitochondria (Tehranchi etal, 2003). Moreover, mature erythroblasts in RARS, but not in RA, show mitochondrial accumulation of aberrant ferritin (MtF) (Cazzola etal, 2003). This study aimed at further describing the pathophysiology of ineffective hematopoiesis in low-risk MDS, by studying cyt c release and MtF expression during erythroid differentiation and mitochondria ATP production in MDS bone marrow cells. We assessed freshly isolated CD34+ cells and day 4–14 erythroblasts from RARS, RA and normal bone marrow (NBM). CD34+ cells from all individuals were negative for MtF. NBM showed only few positive cells (0–4%, d4–14), and RA erythroblasts a median of 3% (0–8%) MtF+ cells. RARS erythroblasts, on the contrary, showed an early increase in MtF+ cells and a continuous increase during the culture period (d4=10%, d7=17%, d14=19%). There was a positive correlation between MtF expression and cyt c at day 14 ( r2=0.8). There was no significant difference in mitochondria ATP production between RARS, RA and NBM (all complexes or cyt -dependent complex IV). We found a significant over-expression (mRNA) of the pro-apoptotic genes for cyt c, Bid and Bax at day 0. Moreover, genes involved in erythroid differentiation were significantly up-regulated in MDS CD34+ cells: 6-fold for GATA-1 and 23-fold for β-globin; p<,0005 for both. GATA-1 and β-globin expression increased during normal erythroid maturation, but in MDS erythroblasts GATA-1 declined and β-globin showed only a weak increase. Comparing RARS with RA, the former showed both higher expression of the β-globin and GATA-1 genes, and a higher degree of cyt c release and MtF expression. This indicates that the cellular abnormalities leading to erythroid apoptosis as well as efforts to compensate for these defects are present at the stem cell level in RARS. G-CSF that reduces cyt c release in MDS erythroblasts (RARS>RA) showed no effect at all on ATP production or cyt c mRNA. Moreover, G-CSF tended to increase MtF expression in some RARS erythroblast cultures, indicating that it allows survival of pro-apoptotic MDS erythroblasts rather than addressing the cause of apoptosis. In conclusion, the aberrant MtF expression of RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in erythroid differentiation. Alternative mechanisms may be involved in RA pathogenesis, since these cells show cyt c release but only moderate MtF expression, and less gene up-regulation.