bluuebunny/crossref_metadata_2025 huggingface dataset:
A Human Anti-CD1a Monoclonal Antibody for Targeted Immunotherapy in Langerhans Cell Histiocytosis and Hematologic Malignancies.
Authors: Bechan Gitanjali , Lee David W. , Egeler R. Maarten , Arceci Robert J.
Published: April 2020

Langerhans Cell Histiocytosis (LCH) is characterized by the clonal proliferative of immature Langerhans cells resulting in a disorder with a wide variety of clinical manifestations and outcomes. Current therapies are based on the use of chemotherapeutic and immunosuppressive agents, usually in combination. Treatments are relatively non-specific, associated with significant adverse side effects and are ineffective in a significant number of patients. More effective and less toxic treatments are therefore needed. The selective expression of the nonclassical MHC protein, CD1a, on the lesional Langerhans cells in LCH makes this antigen an excellent immunotherapeutic target. To this end, we have generated a completely human monoclonal antibody (Mab) that specifically binds to a native, external epitope of CD1a using a human spleen phage display library. Epitope binding regions were selected for recognition of external domains of native CD1a and sequences showing specific binding were cloned into a human IgG1 backbone. The anti-CD1a Mab, termed 2113, shows high affinity and specificity for CD1a and is internalized after binding to the surface of CD1a expressing cells. The 2113 Mab also demonstrates significant complement dependent lysis (CDC) and antibody dependent cell cytotoxicity (ADCC) against CD1a expressing cells. No direct antibody mediated apoptosis was observed for 2113 binding to CD1a. A preclinical murine CD1a positive tumor model is being used to test the 2113 Mab for selective localization and tumor cytotoxicity. This Mab should provide an excellent reagent for therapeutic targeting of LCH and CD1a positive hematologic malignancies as well as possibly having application in the treatment of autoimmune disorders, prevention of graft-versus-host disease and augmenting cancer vaccines through direct antigen delivery to Langerhans cells.
Etodolac Inhibits EBER Expression and Induces Bcl-2-Regulated Apoptosis in Burkitt’s Lymphoma Cells.
Authors: Kobayashi Miki , Nakamura Satoki , Shibata Kiyoshi , Sahara Naohi , Shigeno Kazuyuki , Shinjo Kaori , Naito Kensuke , Ohnishi Kazunori
Published: April 2020

Cyclooxygenase-2 (COX-2) is reported to be an important cellular target for therapy in malignancies. The growth inhibitory effects of COX-2 inhibitors on malignancies have been demonstrated to be through not only COX-2 dependent, but also independent mechanisms. In this study, we showed that etodolac, COX-2 inhibitor, induced apoptosis via COX-2 independent pathway, and investigated the molecular details of etodolac-induced apoptosis in Burkitt’s lymphoma cells. In Daudi and Raji Burkitt’s lymphoma cell lines, which expressed no COX-2 enzyme, etodolac more strongly induced apoptosis compared to meloxicam. Moreover, Etodolac did not induce apoptosis to normal B lymphocytes. For the pathway of etodolac-induced apoptosis, reduction of anti-apoptotic bcl-2 mRNA and Bcl-2 protein, activation of Caspase-9 and -3, down-regulation of caspase inhibitors, c-IAP-1 and Survivin were involved. Moreover, EBER-1 and -2 expression in EBV positive Daudi and Raji cells were reduced to result in down-regulation of Bcl-2 by treatment with etodolac. It has been reported that etodolac has stereoisomers, R- and S- etodolac. We found that racemate of etodolac more strongly induced apoptosis in Daudi and Raji cells compared to R- or S- etodolac. In conclusion, our findings indicated etodolac inhibited EBERs expression and induced apoptosis via a Bcl-2-regulated pathway. Moreover, racemate of etodolac more effectively induced apoptosis than R- and/or S- etodolac. Therefore, these activities of etodolac potentially extend to the treatment of patients with Burkitt’s lymphoma resistant to chemotherapy.
BCR/ABL Induces Expression of Histidine Decarboxylase and Synthesis of Histamine in CML Cells.
Authors: Aichberger Karl J. , Mayerhofer Matthias , Krauth Maria-Theresa , Vales Anna , Derdak Sophia , Pickl Winfried F. , Sillaber Christian , Valent Peter
Published: April 2020

Basophil numbers are typically elevated in patients with chronic myeloid leukemia (CML) and characteristically increase during disease progression. As a consequence, blood histamine levels are highly upregulated in CML. We examined the biochemical basis of production of histamine in CML cells and analyzed the effects of the CML-related oncoprotein BCR/ABL on the generation of this mediator. Expression of histamine and of histidine decarboxylase (HDC), the major enzyme involved in histamine synthesis, were examined in primary CML cells obtained from patients with chronic phase (CP) CML or accelerated phase (AP) CML with basophilia, in the CML-derived cell lines K562 and KU812, and in Ba/F3 cells inducibly expressing BCR/ABL on exposure to doxycycline (TonB.210-X cells). In all patients with AP-CML and basophilia, high levels of HDC mRNA and of histamine were detected, whereas in CP-CML cells only expressed low amounts of HDC and of histamine. HDC mRNA and histamine were detectable in the basophil-committed CML cell line KU812, but not in K562 cells. Exposure of primary CML cells or KU812 cells to the BCR/ABL tyrosine kinase inhibitors imatinib (1 μM; Novartis Pharma AG) or AMN107 (100 nM; Novartis Pharma AG) decreased the levels of histamine and expression of HDC in BCR/ABL-transformed cells. Moreover, BCR/ABL was found to promote the expression of HDC mRNA and to increase the levels of histamine in TonB.210-X cells. The BCR/ABL-induced synthesis of histamine in these cells was blocked by the PI3-kinase inhibitor LY294002, whereas neither the MEK inhibitor PD98059, nor the mTOR inhibitor rapamycin affected histamine levels. In conclusion, our data show that the CML-specific oncoprotein BCR/ABL induces expression of HDC and synthesis of histamine in leukemic cells through a pathway involving the PI3-kinase.
The Early Referral for Reduced-Intensity Stem Cell Transplantation in Patients with Ph1 (+) Chronic Myelogenous Leukemia in Chronic Phase in the Imatinib Era: Results of the Latin American Cooperative Oncohematology Group (LACOHG) Prospective, Multicenter Study.
Authors: Ruiz-Arguelles Guillermo J. , Gomez-Almaguer David , Morales-Toquero Amelia , Gutierrez-Aguirre Cesar H. , Vela-Ojeda Jorge , Garcia-Ruiz-Esparza Miriam A. , Manzano Carlos , Karduss Amado , Sumoza Abraham , de-Souza Carmino , Miranda Eliana , Giralt Sergio
Published: April 2020

Using a reduced intensity stem cell transplantation (RIST) schedule, 24 patients with Ph1 (+) chronic myelogenous leukemia (CML) in first chronic phase were prospectively allografted in four Latin American countries: Mexico, Brasil, Colombia and Venezuela, using HLA-identical siblings as donors. Median age of the patients was 41 years (range 10 to 71); there were 8 females. Patients received a median of 4.4 x 106/ Kg CD34 cells. Median time to achieve above 0.5 x 109/L granulocytes was 12 days, range 0–41, whereas median time to achieve above 20 x 109/L platelets was also 12 days, range 0–45. Twenty two patients are alive 81 to 830 (median 497) days after the RIST. The 830-day probability of survival is 92%, whereas median survival has not been reached, being above 830 days. Eleven patients (46%) developed acute graft versus-host disease (GVHD), whereas 7 of 23 (30%) developed chronic GVHD. Two patients died 43 and 210 days after the RIST, one as a result of sepsis and the other one of chronic GVHD. The 100-day mortality was 4.4 %, whereas the transplant-related mortality was 8%. RIST for patients with CML in chronic phase seems as an adequate therapeutic option.
Denaturing- HPLC and HR-1 Melter-Based Detection of ABL Mutations in Patients with Chronic Myelogenous Leukemia (CML).
Authors: Lee Il-Kwon , Yun Ju Hyun , Kim Nan Young , Choi Jeong-Hwa , Kim Yeo-Kyeoung , Kim Hee Nam , Park Kyeong-Soo , Lee Je-Jung , Shin Myung Geun , Choi Chan , Kook Hoon , Hwang Tai Ju , Park Moo Rim , Jo Deog Yeon , Won Jong Ho , Kim Hyeoung-Joon
Published: April 2020

Imatinib mesylate is the first line of drug for CML since its ability to induce durable remissions in most patients with Philadelphia-positive leukemias and cytogenetic responses in most patients with chronic-phase CML. Although Imatinib mesylate has revolutionized current chronic myeloid leukemia therapy the phenomenon of relapse because of the emergence of resistance were observed. There were well-known mutations in kinase domain that confer resistance despite continued Imatinib mesylate chemotherapy and number of resistance-conferring mutation is increasing. In this study we systematically scanned kinase domain-encoding exon 5, 7, 8, 9, 10 and 11 from genomic DNA of 79 patients with CML to identify novel mutation using denaturing high-performance liquid chromatography (dHPLC) and HR-1 melter (Idaho Technology). In total 15 mutations in the regions were identified and three mutations are found to be novel and two were located in the catalytic domain region (F359I and E374K). Since these two mutations were identified in patients with primary and secondary resistance respectively, it might be classified into resistance-conferring mutation conditional on further functional evaluation. Also we found P499L mutation in the C-terminus and interestingly we detected E478Q in six patients with CML. Analysis with dHPLC and HR-1 screening after PCR amplification proved to be accurate and effective methods to isolate mutation. In addition dHPLC and HR-1 more sensitive compared to PCR direct sequencing methods since two mutations which were not detected by direct sequencing were confirmed only after subcloning of PCR products and sequencing. Taken together, we designed six PCR amplicons encompassing the kinase domain of abl kinase and screened for novel mutations patients with CML. Out of fourteen mutations identified two novel mutations in the catalytic domain need further investigation to understand if they contribute to the acquirance of imatinib mesylate resistance
Elevated Expression of C/EBPα/ε and c-myb Is Required for Myeloid Differentiation in CML.
Authors: Kobayashi Shinichi , Kimura Fumihiko , Ikeda Takashi , Torikai Hiroki , Osawa-Nakata Yukiko , Sato Ken , Motoyoshi Kazuo
Published: April 2020

The BCR/ABL fusion protein transforms hematopoietic stem cells and causes chronic myeloid leukemia (CML). However, the mechanism leading to the expansion of mature myeloid cells in CML remains obscure. To investigate this mechanism, we compared the gene expression profile of CML neutrophils with that of normal neutrophils by microarray analysis. The genes upregulated in CML neutrophils include critical transcription factors that regulate granulocytic differentiation and proliferation, as well as genes encoding neutrophil granule proteins (Table 1). The protein levels of these upregulated genes were also elevated in the CML neutrophils. To investigate whether BCR/ABL regulates the expression of these upregulated genes, primary CML neutrophils were exposed to 1 μM imatinib (a BCR/ABL tyrosine kinase inhibitor) for 12–16 hours. C/EBPα protein expression was downregulated in primary CML neutrophils after treatment with imatinib, compared to control cultures. We further explored the function of these upregulated genes in KCL22 cells, which are derived from CML blastic crisis. To investigate the effect of C/EBPα or C/EBPε on myeloid differentiation, we established KCL22 cells in which C/EBPα or C/EBPε expression was inducible (KCL22/α or KCL22/ε, respectively). Overexpression of either C/EBP protein resulted in morphological changes, such as reduction of the nuclear to cytoplasmic ratio, more condensed nuclear chromatin, and segmented nuclei, as well as the expression of differentiation specific markers including the G-CSF receptor and macrophage adhesion molecule-1 (Mac-1). These data indicate that C/EBPα /ε expression is sufficient to induce myeloid differentiation in BCR/ABL positive CML cells. We examined whether BCR/ABL regulates the protein expression of c-Myb. The expression of c-Myb in KCL22 cells treated with 1 μM imatinib was downregulated and growth in these cells decreased in parallel. Knock-down of c-Myb expression with siRNA inhibited the growth of KCL22 cells and these data indicate that c-Myb expression is important for the growth of CML cells. To test the effect of c-Myb on differentiation of KCL22 cells, we knocked down c-Myb expression with siRNA in KCL22/α or KCL22/ε cells after induction of C/EBP protein. The knock-down of c-Myb in KCL22α /ε cells resulted in downregulation of differentiation specific genes, including G-CSF receptor and neutophil elastase. Morphologically, the fraction of KCL22α /ε cells with segmented nuclei decreased when c-Myb expression was knocked down. Our results suggest that elevated expression of C/EBPα /ε and c-Myb play a key role in proliferation and differentiation in CML cells.

Table 1. Genes Upregulated in CML Neutrophils. Description Gene transcription factor C/EBP α transcription factor C/EBP ε transcription factor c-Myb neutrophil granule protein lactoferrin neutrophil granule protein neutrophil elastase neutrophil granule protein lysozyme
Hematologic Remission of Chronic Myelogenous Leukemia after Treatment with Imatinib Includes Normalization of the Red Cell Distribution Width.
Authors: Bessman Joel David , Montoya Maria
Published: April 2020

After treatment with imatinib, the majority of patients with chronic myelogenous leukemia (CML) achieve hematologic remission. This includes normalization of blood counts (red cells, white cells, and platelets) normalization of the white cell differential, and disappearance of abnormal immature cells from the peripheral blood. However, abnormal erythropoiesis may be more subtle than abnormal red cell counts. We reviewed two other indices of erythropoiesis, the mean cell volume (MCV) and the red cell distribution width (RDW). We studied 15 patients with a diagnosis of CML confirmed by BCR-ABL, in whom the first-line therapy was imatinib. Treatment dosage varied from 400 – 800 mg/day. Pre-treatment, all patients had an elevated white cell count, 14 of 15 had a low hemoglobin, and 11 of 15 had an abnormal high platelet count. None had an abnormal MCV (mean, 91.3, range 82 – 97), and 13 of 15 had an abnormal RDW (mean 16.2, range 14.1 – 19.2). After treatment was started, all 15 achieved a normal white cell count, hemoglobin, and platelet count within 3 months. All continued to have a normal MCV, with no apparent change (mean 92.1, range 82 – 98). However, after treatment 14 of 15 developed a normal RDW (mean 13.7, range 12.0 – 15.2). The RDW progressively declined during the first 3 months after treatment and reached an asymptote at that time. We conclude that hematopoiesis normalizes qualitatively as well as quantitatively after successful imatinib therapy of CML. Future study will determine whether an isolated elevated RDW will predict relapse or briefer duration of remission.
Addition of GM-CSF to Imatinib Results in Major Cytogenetic Response in Patient with Chronic Myeloid Leukemia (CML).
Authors: Connor Rebecca , Hurd David , Molnar Istvan
Published: April 2020

Imatinib mesylate, an inhibitor of BCR/ABL tyrosine kinase, has remarkable activity in CML resulting in up to 87% major cytogenetic response, but further treatment is needed for the remaining 13% of patients and others who progress on therapy. Studies suggest that hematopoietic growth factors (HGF) induce the selective terminal differentiation of CML progenitors at concentrations that allow optimal growth of normal progenitors. Treatment with HGF may be able to stop the expansion of BCR/ABL positive cells while promoting the expansion of non-leukemic myeloid cells, thereby encouraging a cytogenetic response. Initially, these HGF were used to improve the safety of chemotherapies but had not been studied specifically to improve cytogenetic response. Our institution has an open phase II study of GM-CSF in patients with chronic phase CML who have not achieved a complete cytogenetic remission after at least 9 months of therapy. Our first patient on study was a woman originally diagnosed in 4/1996 with a variant Philadelphia chromosome [t(9;9;22)] positive CML. She was initially started on hydroxyurea and was then switched to interferon-α for approximately 5 ½ years. When imatinib became available her interferon was discontinued and she was treated with imatinib at 400 mg daily. Bone marrow biopsy 5/2003 was morphologically normal but on cytogenetic analysis all cells contained the same aberrant karyotype. Her imatinib therapy was complicated by pancytopenia requiring frequent transfusions. She had received approximately 22 units of packed red blood cells over 11 months. Her white count had been running in the 1,500 to 2,500/μL range and her platelet count had dropped to 64,000/μL. She presented to us 1/2004 at the age of 56 and was evaluated for a reduced-intensity allogeneic transplant but did not have any donors available in her family. She was not felt to be a candidate for an unrelated donor transplant because of co-morbid risks. Given her continued cytogenetic abnormalities and lack of transplant candidacy she was enrolled in our GM-CSF trial March 2004. At the time of entry onto study she had been on imatinib for 2 1/2 years with no cytogenetic improvement. She began treatment with daily GM-CSF 100 μg/m2 in addition to her imatinib. Cytogenetic studies revealed a gradual increase in percentage of normal cells from start, 4 months, 9 months, and 15 months at 0%, 10%, 55%, and 85% respectively. She also had reversal of her pancytopenia and has not required transfusion of blood or platelets since enrollment. She developed an eosinophilia which we attribute to GM-CSF. Clinical studies have examined the use of GM-CSF in addition to interferon alpha. However, with imatinib becoming the new standard of care it is more relevant to examine the combined effect of imatinib and GM-CSF. The use of GM-CSF with imatinib in CML still needs more evaluation, but it appears to be a promising approach to improve cytogenetic response.
Results of a Phase 2 Trial of HGS-ETR1 (Agonistic Human Monoclonal Antibody to TRAIL Receptor 1) in Subjects with Relapsed/Refractory Non-Hodgkin’s Lymphoma (NHL).
Authors: Younes Anas , Vose Julie M. , Zelenetz Andrew D. , Smith Mitchell R. , Burris Howard , Ansell Stephen , Klein Jerry , Kumm Elizabeth , Czuczman Myron
Published: April 2020

Two death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), expressed in a wide variety of cancer cell types including lymphoma, enable TRAIL (TNF related apoptosis inducing ligand), a member of the TNF ligand superfamily, to induce apoptosis. HGS-ETR1 (TRM-1, mapatumumab) is a fully human monoclonal antibody agonistic to TRAIL-R1 with preclinical evidence of anti-tumor activity in vitro and in vivo. We conducted a phase 2 multicenter study of HGS-ETR1 in 40 subjects with relapsed or refractory NHL. Patients were included on the study if they had relapsed/refractory NHL of any histologic subtype, irrespective of the number of previous treatment regimens. HGS-ETR1 was administered at 2 dose levels: 3 mg/kg (8 subjects) or 10 mg/kg (32 subjects) every 21 days in the absence of disease progression or prohibitive toxicity for up to 6 cycles. The primary endpoint was tumor response evaluated with the International Working Group Criteria after every third treatment. Responses were confirmed between 4 and 8 weeks after initial documentation of response. Secondary endpoints included safety and tolerability. The median age of subjects was 62 years (range 32–81) (24M:16F) with an ECOG performance status 0–2. Subjects had received up to 12 previous therapeutic regimens (69% received 3 or more prior regimens). Original histologic classification is presently available for 34 subjects (14 follicular, 7 diffuse large B-cell, 6 mantle cell, 1 small lymphocytic, 2 peripheral T-cell, 1 anaplastic large cell, 3 lymphomas not classified). Preliminary response data are available for all 40 subjects. 3 subjects (8%), all with follicular lymphoma, had clinical responses (1CR, 2PR). Additionally, 12/40 subjects (30%) had stable disease, and the remainder had progressive disease at the time of first evaluation. 8 subjects (7 follicular) remain on study without disease progression for >5 to >13 months. One subject with a PR (special permission) has continued to receive HGS-ETR1 (10 cycles to date). Subjects tolerated therapy well with no subjects discontinuing therapy due to toxicity related to drug. There have been 10 reported SAEs, 2 of which were considered possibly/probably related to HGS-ETR1 administration (shingles, fever). In conclusion, HGS-ETR1 can be safely administered to heavily treated NHL patients. Clinical responses were seen in 3/14 follicular lymphoma subjects to date. Final data for all subjects will be reported at the meeting. These data suggest that HGS-ETR1 has activity in heavily pretreated lymphoma patients and further studies in combination with agents active in lymphoma are justified.
Flow Cytometric Characteristics of CD34+ Cells in Refractory Anemia: Their Diagnostic Value.
Authors: Ogata Kiyoyuki , Kishikawa Yoshifumi , Shimazaki Tomoko , Satoh Chikako , Tamura Hideto , Otsuka Yoshihiko , Dan Kazuo , Hayashi Akio , Shimazu Mitsunobu
Published: April 2020

(INTRODUCTION) The diagnosis of refractory anemia (RA), a subtype of myelodysplastic syndromes (MDS), is often problematic because RA often does not show cytogenetic abnormalities and dysplastic hematopoietic cells are not specific to MDS. Recent studies have suggested that the abnormalities of hematopoietic cells detected by flow cytometry (FCM) might be useful for MDS diagnosis. However, this concept has not yet been widely accepted. We reported that MDS blasts often show aberrant antigen expression. In this study, we examined the diagnostic utility of the flow cytometric characteristics of CD34+ cells in RA.

(METHODS) Whole bone marrow cells obtained from RA patients and controls (e.g., cytopenic individuals other than RA and other malignancies) were analyzed using four-color FCM. The cells were stained with CD45 and CD34, for gating CD34+ B-cell precursors (hematogones) and CD34+ myeloblasts, in combination with various other antibodies. We determined percentages of hematogones and CD34+ myeloblasts and relative mean florescence intensity (RMFI: MFI of each antigen staining ÷ MFI of isotype-matched negative control staining) of various antigens on CD34+ myeloblasts.

(RESULTS) From the data on 50 control subjects in the first subject cohort, we determined the reference range (RR) for each flow cytometric parameter. We scored data on each subject by giving 1 point to each of 10 parameters if the data of such parameters were outside RRs. These 10 parameters, which were adopted after comparing data from control and RA subjects in the first cohort, were percentages of hematogones and CD34+ myeloblasts and RMFI of CD4, CD56, CD11b, CD13, CD15, CD33, CD117 and CD133. In the first cohort, scores of all control subjects were less than 3 (40 subjects had a score of 0) and scores of 7/12 RA patients were 3 or higher. Using the same parameters and same RRs, we then prospectively analyzed the second subject cohort. Again, this approach identified 8/13 RA patients (score 3 or higher) with 100% specificity. The degree of dysplasia and presence or absence of cytogenetic abberations were not related to RA identification.

(CONCLUSION) Our method is reproducible and helpful in RA diagnosis.
Aberrant Cell Cycle and Expression Profiles of Cell Cycle Regulatory Genes in Myelodysplastic Syndrome.
Authors: Shao Zonghong , Shi Jun , Liu Hong , Sun Juan , Jia Hairong , Bai Jie , He Guangsheng , Cao Yanran , Tu Meifeng , Wang Huaquan , Xing Liming
Published: April 2020

Deregulation of the cell cycle machinery, especially G1/S phase deregulation, is known to contribute to the uncontrolled cell proliferation and malignant transformation in haematological malignances. However, little is known about the aberrant bone marrow cell cycle and its molecular mechanisms in myelodysplastic syndromes (MDS). In this study, we conducted the cell cycle analysis of bone marrow mononuclear cells (BMMC) before and after exposure to haematopoietic growth factors (HGFs) cocktail, in combination with the analysis of the expression of Ki-67 antigen in BMMC and CD34+ cells in twenty-seven MDS patients. In addition, we examined the expression profiles of cell cycle regulatory genes. We found that MDS patients had higher percentage of BMMC in G0/G1 phase and lower ratio of cells in S+G2/M, and higher proportion of CD34+ Ki-67+ cells. After exposure to HGFs cocktail in vitro, high proportion of G0/G1 phase cells decreased significantly in MDS. All types of four cyclins (cyclin D2, D3, E, and A1) were at higher level had high amount of mRNA transcripts in MDS, together with higher activation of CDK2, CDK6, and RB genes. Furthermore, we also demonstrated higher activation of p21 and p27 genes. These results demonstrated that G1 arrest occurred in MDS Ki-67+ hematopoietic cells. High activation of cyclins, CDKs, and RB genes are the molecular bases of large proportion of Ki-67+ proliferate BMMC population. High activation of p21 and p27 genes in MDS blocked hematopoietic cells entry into the following cell cycle.
Bone Marrow Erythroid Maturation and Cytokine Production in Refractory Anemia Patients with Positive Mitogen-Stimulated-Direct Antiglobulin Test.
Authors: Zaninoni Anna , Imperiali Francesca G. , Iurlo Alessandra , Zanella Alberto , Barcellini Wilma
Published: April 2020

Autoimmune phenomena, particularly directed against RBC, are described in MDS. We already reported that the amount of anti-erythroblast antibodies was significantly greater in BM cultures of MDS patients versus controls, as detected by a new method named mitogen-stimulated-direct antiglobulin test (MS-DAT). We compared BM erythroid maturation and cytokine production in patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS), with or without MS-DAT positive BM cultures. MS-DAT was performed by stimulating marrow cultures with PMA and PHA and anti-erythroblast antibodies were detected by competitive solid phase ELISA. Cytokine production (IL-2, IL-4, IL-10, TNF-alpha, TGF-beta and IFN-gamma) was investigated in BM cultures by ELISA assay. Eleven out of 23 patients showed positive MS-DAT in BM (cut off value 200 ng/ml+3SD), whereas none of them was positive in PB. By comparing patients with MS-DAT positive in BM versus negative patients, we found increased erythroblast counts and peripheral signs of hemolysis (i.e higher reticulocytes, indirect bilirubin, and LDH, and lower haptoglobin) in the former, in spite of a negative MS-DAT in BP. In BM MS-DAT positive patients TNF-alpha, IL-4, and IL-10 production was lower compared with MS-DAT negative patients. BM MS-DAT positivity suggests the existence of an anti-erythroblast autoimmunity and could support steroid therapy in some early MDS patients.

Anti-erythroblast antibodies (ng/mL) Retics (%) LDH (U/L) Haptoglobin (mg/dL) Erythroblasts (%) NS PMA PHA *denotes p<0.05 MS-DAT positive vs. negative MS-DAT positive 565±130* 591±113* 696±159* 1.9±0.2* 404±22* 67.7±17 44.8±13.6* MS-DAT negative 114±18 114±17 104±17 1.2±0.1 332±18 130.4±7 26.6±3.5
Megakaryocytic Progenitors in Long Term Cultures from MDS Patients.
Authors: Majado Maria Juliana , Macizo María I. , González-García Consuelo , Salido Eduardo , Molina José A. , Monserrat Jorge , Morales Alfonso
Published: April 2020

Introduction: bone marrow long-term cultures (LTC) have been reported to produce very poor adherent stromal layer in MDS, but there are not many studies about the behaviour of the progenitor cells in supernatant culture medium. In this study we report the results of megakaryocytic progenitors (CFU-MK) in the supernatant of LTC of patients with refractory anemia (RA).

Material and method: LTCs were performed to 11 RA patients and to 13 normal multiorgan donors as control. Bone marrow red cells were sedimented with 1% methyl-cellulose and seeded for LTC in 10ml flasks (Nalge Nunc International), with Iscover medium supplemented with horse serum, foetal calf serum, hydrocortisone and carbonic air, at a final concentration of 1x106 per ml. Flasks were placed in an incubator for 8 weeks. Half culture medium volume was renovated weekly, cell counts and assays of CFU-MK (Megacult C, Stem Cell Technologies), were performed. CFU-MK colonies were separated in three groups, containing 3–20 cells, 21–50 cells and more than 50 cells. Student t-test was used for statistical comparisons.

Results: Are in the following table, expressed as mean + standard desviation. After the third week no colony growth was observed in normal such as in MDS. Growth of CFU-MK colonies containing more than 50 cells was higher in basal control bone marrow, no statistical differences were found in the rest of the cultures.

Results CFU-MK(3–30 Cells) CFU-MK(20–50 Cells) CFU-MK (>50 Cells) CFU-MK total w: week Control basal 15.75±15.79 3.33±5.42 4.88±7.44 22.42±26.43 RA basal 7.85±8.66 0.80±1.01 0.70±0.98 9.45±9.46 t-St ns ns 0.047 ns Control w 1 3.54±4.42 0.7±1.44 0.74±1.48 4.98±6.62 RA w 1 3.29±4.52 0.29±0.93 0.25±0.67 3.82±5.65 t-St ns ns ns ns Control w 2 0.55±1.14 0.07±0.24 0.05±0.25 0.67±1.43 RA w 2 0.18±0.32 0.00±0.00 0.04±0.13 0.21±0.38 t-St ns ns ns ns Control w 3 0.18±0.53 0.00±0.00 0.00±0.00 0.18±0.53 RA w 3 0.00±0.21 0.00±0.00 0.00±0.00 0.00±0.21 t-St ns ns

Discussion: No important difference was found in LTC supernatant CFU-MK in our RA patients, and this supports the idea that the stromal damage is more important, in the pathophysyology of MDS, than that of the stem cells.
Marked Elliptocytosis in Myelodysplastic Syndromes (MDS) Is Associated to Deletion of Chromosome 20q.
Authors: Baweja Mansoina , Moreno-Aspitia Alvaro , Menke David M. , Roy Vivek , Zubair Abba
Published: April 2020

Most common abnormalities in MDS involve the loss of chromosomal material resulting from the loss of an entire chromosome or a part of it, due to deletions or unbalanced translocations. Among partial chromosome losses, del (5q) was found to be the most common followed by del (20q). MDS can also be associated with multiple morphologic RBC abnormalities including macrocytes, ovalocytes, dacrocytes, fragmented cells and basophilic stippling. Limited cases of MDS with elliptocytosis have been reported in the literature. Elliptocytosis encompasses a heterogeneous group of erythrocyte disorders. Although acquired cases of elliptocytosis have been reported, these are most often hereditary disorders with a variable clinical presentation from asymptomatic carriers to patients with severe hemolytic anemia. This disorder is quite rare in the Unites States (US). Mutations in protein 4.1, alpha spectrin, beta spectrin, glycophorin C and band 3 have been reported in hereditary elliptocytosis. Only 7 well reported cases of marked elliptocytosis associated with MDS have been reported in the literature. Of those 7 cases, 4 of them were associated to del(20q). All of them were elderly males and all of them had ≤ 5% bone marrow blasts. Anemia is usually moderate (median 9.4 g/dL; range 8.1–12) and thrombocytopenia is mild to moderate (median platelet count 116 x 109/L; range 46–149 x 109/L). All cases were described either in Europe (n= 1; Rummens et al. Acta Haematologica1986; 75:174–177) or in Asia (n= 3; Ideguchi et al. Br J Haematol.1993, 75:387–92; Ishida et al. Cancer Genetics and Cytogenetics1997, 108:162–165; Hur et al. Clin Lab Haematology, 2004, 26: 69–72). Of these 7 cases, 3 of them were associated with protein 4.1 deficiency. To the best of our knowledge these are the first 2 cases of patients with marked elliptocytosis and MDS with del 20q abnormality reported in the US:

Patient 1 Patient 2 Age/sex 82/male 79/male FAB classification RA RA Year of diagnosis of MDS 1998 1999 WBC 3.0 x 109/L 11.5 x 109/L Platelets 82 x 109/L 79 x 109/L Hemoglobin 11.8 g/dL 10.7 g/dL Hematocrit 34.4% 31.9% Reticulocyte 4.3% NA LDH 350 u/L NA Direct Coombs Negative NA BM Cytogenetics 46 XY, del (20)(q11.2) 46 XY, del (20)(q11.2) Prior Therapy supportive care only Erythropoeitin

Evaluation for protein 4.1 deficiency in one of these 2 patients is currently pending.

The elliptocytosis noted in these MDS patients were most likely derived from acquired clones with abnormal development of the erythroid lineage and were associated with deletion of chromosome 20q. Patients with acquired elliptocytosis and associated MDS with del 20q chromosomal abnormality seem to have an indolent course with prolonged history of mild to moderate blood cytopenias and low risk to transformation to acute leukemia. Further research is needed to deduce the clinical significance of this subtype of MDS with this unusual RBC morphology
Pericyte Coverage of Abnormal Blood Vessels in Myelofibrosis Bone Marrows.
Authors: Zetterberg Eva , Vannucchi Alessandro M. , Migliaccio Anna Rita , Vainchenker William , Tulliez Micheline , Dickie Renee , Rogers Rick , Palmblad Jan
Published: April 2020

Neoangiogenesis, manifested as, for example high microvascular density (MVD), is an important feature of most solid tumors and also in the bone marrow of hematological malignancies. Very high MVD has been demonstrated in chronic myeloid leukemia and myelofibrosis (MF). In addition, MF marrows, as well as solid tumor vessels, display highly tortuous and branched vessels with caliber changes. However, the pathogenic mechanisms are poorly understood. Since pericyte (PC) deficiency has been described on similarly abnormal solid tumor vessels we studied vessel morphology and PC coverage in bone marrows from nine MF patients and nine controls as well as in three mouse models of MF. - In human MF marrows mean MVD was 6.2-fold higher compared to controls (P<0.001) and MF vessels displayed larger mean perimeters (89±61 μm vs. 19±9 μm; P<0.001). The cellular expression of VEGF-A was 6.6-fold higher in MF marrows than in controls (P<0.001). Unexpectedly, we found that 99±3 % of all MF vessels were PC-coated (i.e. postive for α-smooth muscle actin) compared with only 51±20 % in controls (P<0.001). To study whether these findings could be attributed to the abnormal megakaryocytopoiesis, typical for MF, we used mouse models of MF with genetic alterations affecting megakaryocyte differentiation:

low expression of the transcription factor GATA-1 (GATA-1low), over-expression of thrombopoietin, or TPO over-expressing mice lacking expression of TGF-β1 (however, fibrosis does not develop in model 3).

In all 3 models wide and morphologically aberrant vessels were detected and microvascular density was significantly higher in marrows as well as in spleens (P<0.001 compared with wild type animals). All models displayed PC-coated bone marrow vessels, similar to the findings in human MF. Thus, processes leading to fibrosis, including TGF-β1, are not sufficient to deregulate angiogenesis - We conclude that human and mice MF marrow vessels display similar pathologic blood vessel architecture, with intense PC coverage, and genetic modifications of two molecules, viz GATA-1 and TPO, important for the regulation of megakaryocytopoiesis, might be of significance for this process.
Essential Thrombocythemia: Analysis of Risk Factors for Thrombotic Events in a Series of 306 Patients.
Authors: Radaelli Franca , Bramanti Stefania , Colombi Mariangela , Iurlo Alessandra , Zanella Alberto
Published: April 2020

Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by peripheral thrombocytosis and abnormal proliferation of megakariocytes in the bone marrow. Even thought thrombosis is frequently associated to ET, the risk factors of this clinical complication are still controversial. The aim of this retrospective, single institution study was to investigate clinical and laboratory characteristics associated with the occurrence of thrombotic events, with the purpose of identifying subgroups of patients who could benefit from antiaggregant and/or cytostatic treatment. 306 consecutive ET patients (109 men and 197 females, median age 58 yr) diagnosed between January 1979 and December 2002 were included in the study. At the time of analysis, 196 patients were still alive with a median follow up of 96 months. The following variables were investigated for the association with thrombotic complications: age, platelet count, previous history of thrombotic events, time from diagnosis, treatment with antiaggregant/cytostatic drugs, and cardiovascular risk factors such as arterial hypertension, obesity, hypercolesterolemia, diabetes, cigarette smoking. At the time of last follow up, 46 patients (15%) experienced at least one thrombotic event. The occurrence of thrombotic events was observed in 26/64 (40.6%) patients with previous history of thrombosis and in 20/242 (8.3%) patients with no previous history of thrombosis (p<0.0001 Fisher’s exact test, odd ratio 7.6). A significant difference between the two groups of patients was also confirmed when Kaplan Meier estimates of thrombosis-free survival were compared by log-rank test (p<0.0001). By logistic regression, platelet number at diagnosis did not associate with occurrence of thrombosis in the whole patient population. When patients without previous history of thrombosis were stratified according to the number of cardiovascular risk factors (none vs one vs more than one), a significant correlation with occurrence of thrombotic events was observed (Mantel-Haenszel Chi-square 5.47, p<0.05). This study confirms that history of thrombosis is strongly related with risk of further thrombotic events in patients with ET, whereas platelet number at diagnosis does not seem to represent a prognostic factor. In patients with no previous history of thrombosis, the presence of other cardiovascular risk factors has to be taken into account when establishing the therapeutic approach.
Combined Muliparameter Approach to the Diagnosis of Polycythemia Vera and Essential Thrombocythemia.
Authors: Teofili Luciana , Martini Maurizio , Cenci Tonia , Larocca Luigi M. , Leone Giuseppe
Published: April 2020

The diagnosis of Polycythemia Vera (PV) and Essential Thrombocythemia (ET) is currently performed by excluding possible causes of secondary erythrocytosis and thrombocythosis. Recently, new typical findings of Ph-negative myeloprolipherative syndromes have been described, such as the PRV-1 RNA over-expression and the Val617Phe JAK-2 mutation. In this study we evaluated the diagnostic value of endogenous erythroid colony-assay (EECs), PRV-1 RNA over-expression and JAK-2 mutation, in the screening of patients with thrombocytosys and erythrocytosis which are candidate to a more extensive diagnostic approach including bone marrow biopsy and red cell mass evaluation. Peripheral blood samples were obtained from 56 patients referring to the hematological day hospital of our institution for erythrocythosis or thrombocythosis. EECs assay and PRV-1 expression were evaluated as prevoiusly described (Teofili L. et al, JCO 2002); Jak-2 mutation was investigated by the method described by Baxter et al. (Lancet 2005). The diagnosis of PV and ET was performed according to the PV and ET Study Group criteria. Twenty-five patients were affected by PV (19 males and 6 females, median age 68 years, range 34–80), 20 by ET, (5 males and 15 females, median age 64 years, range 31–85) 5 patients (3 males and 2 females, median age 56 years, range 35–69) and 6 patients (1 male and 5 females, median age 31 years, range 12–83) were diagnosed affected by secondary erythrocytosis and thrombocytosis, respectively. Results obtained are shown in Table 1.

In our series all PV cases were identified by means at least one positive test, whilst 20% of ET patients resulted negative to all three assays. Moreover, among PV patients, only 1 patient was positive at one single assay (JAK-2 mutation), and the vast majority of them resulted positive for two or three assays. On the contrary, among patients affected by ET, a high proportion of patients (35 %) showed only Jak-2 mutation (10%) or PRV-1 over-expression (25%).. Importantly, no false positive cases were detectable among patients with secondary erythrocytosis and thrombocythosis. In conclusion, these data confirm that PRV-1 over-expression is a feature typical not only of PV but also of ET. Nevertheless the combination of different used assays is more usefull in the differential diagnosis of erythrocythosis than of thrombocythosis.

TABLE 1 PV (%) ET (%) SE (%) ST (%) EECs pos. 18 (72) 8 (40) 0 0 PRV-1 pos. 24 (96) 14 (70) 0 0 Jak-2 mutated 17 (68) 7 (35) 0 0 Full positive 14 (56) 4 (20) 0 0 Full negative 0 4 (20) 5 (100) 6 (100)
Anagrelide Treatment and Cardiovascular Evaluation in 130 Patients with Essential Thrombocythemia (ET): Preliminary Report of the Registro Italiano Trombocitemia (RIT).
Authors: Gugliotta Luigi , Tieghi A. , Bulgarelli S. , Tortorella G. , Ciancia R. , Scalzulli P. R. , Cacciola E. , Cacciola R. , Rossi D. , Usala E. , Crugnola M. , Candoni A. , Giordano M. , Andriani A. , Santoro C. , Vimercati R. , Liberati M. , Sciorio A. , Specchia G. , Martinelli V. , Gaidano G. , Mazzucconi M. G.
Published: April 2020

Anagrelide treatment is considered potentially responsible of cardiovascular toxicity and side effects. For this reason, we have retrospectively evaluated 130 patients (pts) with Essential Thrombocythemia (ET) treated with Anagrelide in 10 Italian Hematological Centres belonging to the GIMEMA Group. The pts, 55 males and 75 females, had at diagnosis a mean age of 43 years (range 13–82) and a mean platelet (PLT) count of 1086x10(e)9/L. The Anagrelide treatment was started after a mean of 47 months from diagnosis(range 0–196). A previous treatment with cytoreductive drugs (mainly HU and IFN) and antiplatelet agents (mainly ASA), was performed in 65% and 98% of cases, respectively. The mean Anagrelide dose was 1 mg/day at start and 1.7 mg/day in the maintenance phase (mean PLT count below 500x10(e)9/L). Twenty-five patients (19%) discontinued the treatment after a mean of 27 months (range 1–85) for the following reasons: compliance loss (n8), no satisfactory response (n7), palpitations and tachycardia (n6), other side effects (n4), anemia (n1), renal toxicity (n1), thromboembolism (n1). During the follow-up (median 28 months) 4 thrombotic complications (1.15/100 pt-yrs) and 2 minor hemorrhages (0.57/100 pt-yrs) were registered. Moreover, 15 pts showed non lethal cardiovascular events: angina (n6), cardiomiopathy (n4), arrhythmia (n3), AMI (n2). An instrumental cardiovascular evaluation with Echocardiography (ECHO) and ECG was performed at Anagrelide start in 58 pts: the Ejection Fraction (EF) was abnormal in only one pt (EF 47%). The instrumental monitoring was done in 28 pts every 6–12 months. Four out of these 28 pts showed a decrease of the EF below the level of 50% (43%,45%,47%, and 49%), and only 1 pt discontinued Anagrelide for this reason (see table). The clinical and instrumental cardiovascular evaluation in ET patients receiving Anagrelide is becaming of routinary application and an observational prospective study of the Registro Italiano Trombocitemia (RIT) is now in progress.

ECHO EF<50% IN 4 OUT OF THE 28 ET PATIENTS MONITORED DURING ANAGRELIDE TREATMENT CASES AGE EF % BASELINE EF % 1ST YEAR EF% 2ND YEAR *Anagrelide discontinuation because tachycardia and EF<50% 1 44 60 45 61 2 50 50 43 50 3 51 51 49* / 4 47 47 47 /
Clinical and Molecular Findings in Childhood Polycythemia Vera.
Authors: Cario Holger , Pahl Heike L. , Milen Minkov , Harald Reinhard , Kohne Elisabeth , Debatin Klaus-Michael
Published: April 2020

Background: Polycythemia vera (PV) and congenital erythrocytoses constitute extremely rare diseases in pediatric and juvenile patients. Systematic data on clinical and laboratory evaluations as well as on treatment modalities are sparse.

Aims/Methods: We developed a protocol (PV-ERY-KA 03) for the systematic collection of clinical, hematological, biological as well as treatment data of children and adolescents with PV or congenital erythrocytoses. The collaborative trial was initiated in March 2004.

Results: To date, 5 (2 male / 3 female) pediatric patients with PV have been enrolled. The median age at onset was 14 years (7–18.5 years). 4/5 patients are of German, 1/5 of Turkish origin. Only 3 patients presented with PV-related symptoms (Budd-Chiari syndrome, isolated headache, or headache together with tinnitus, dizziness and weakness). At first presentation, the median hemoglobin content was 18 g/dl (15.5–21.1 g/dl), the median hematocrit 0.60 (0.59–0.73). Thrombocytosis was present in 4/5, leukocytosis in 2/5 patients. All patients presented with mild to marked splenomegaly. Granulocytic PRV-1-mRNA expression and JAK2V617F mutation were assessed in 4/5 patients. PRV-1-mRNA expression was significantly elevated in 3 of them and borderline the 4th patient. 3 patients including the patient with borderline PRV-1-mRNA expression presented with a heterozygous JAK2V617F mutation. Treatment strategies included phlebotomy (4/5), hydroxyurea (1/5), IFN-alpha (1/5), and aspirin (3/5). Stem cell transplantation from a matched unrelated donor was performed in one patient (now in complete remission). Because of progressive liver failure, the patient with Budd-Chiari syndrome underwent orthotopic liver transplantation.

Conclusions: The activating JAK2V617F mutation previously reported to be present in up to 90 percent of adult PV patients, is also involved in childhood PV. The age of onset and clinical presentations in the rare cases of childhood PV are varied. Different treatment strategies are applied. A collaborative assessment of children with PV may help to optimise treatment in these patients, but may in addition contribute to a better general understanding of the disease’s pathogenesis.
Bone Marrow Evaluation According to the PVSG and WHO Criteria in 90 Essential Thrombocythemia (ET) Patients Treated with PEG Interferon alpha-2b. Preliminary Results.
Authors: Gugliotta Luigi , Bulgarelli S. , Asioli S. , Tieghi A. , Gardini G. , Sabattini E. , Franco V. , Boveri E. , Kvasnicka H. M. , Vianelli N. , Candoni A. , Latagliata R. , Passamonti F. , Rupoli S. , Martinelli V. , Zaccaria A. , Lauria F. , De Biasi E. , Sacchi S. , Gobbi M. , Tabilio A. , Cacciola E. , Cacciola R. , Grossi A. , Mazza P. , Bonifazi F. , Favini P. , Mandelli F. , Baccarani M. , Thiele J.
Published: April 2020

Ninety ET patients diagnosed according to the PVSG criteria were enrolled in a phase II study (sponsored by the Schering-Plough Company) designed to evaluate the efficacy, safety and tolerability of a two years treatment with PEG Interferon alpha-2 b (PEG Intron). The patients, observed in 16 Italian Centres belonging to the GIMEMA Cooperative Group and judged at high risk, had been previously treated with cytoreductive (97%) and antiplatelet (91%) drugs. At the study start the patients, 60 F and 30 M, mean age 45 years, showed splenomegaly in 22% of cases. The Hematological Response (HR: PLT<500 x109/L) was observed in 64/81 (79%) and 48/55 (87%) of the patients on PEG Intron treatment at the end of the first and second year, respectively; the spleen enlargement disappeared in 75% of cases. The Bone Marrow data reported in the study CRFs document that the baseline increase of cellularity, of granulopoyesis, of megakaryocytes (MK) number, size and ploidy significantly decreased during PEG Intron treatment, while the MK displasia and fibrosis rate globally increased. In these patients a revision of the Bone Marrow biopsy slides was blindly performed by an Expert Pathologist Panel by applying the WHO criteria. The evaluation of two-thirds of cases showed the following distribution at the baseline: true ET (23%), IMF-0 (pre-MF 17%), IMF-1 (early MF 40%), IMF-2/3 (classical MF 3%), MPD-U (17%). Interestingly, the true ET patients never showed spleen enlargement that, otherwise, was present in IMF-0 (20%) and IMF-1/2/3 (42%) patients. The patients with true ET did not show any bone marrow evolution during the PEG Intron treatment, while the IMF-1/2/3 picture significantly increased. Moreover, the rate of the HR at the end of the first year was higher in true ET(93%) respect to the IMF-0 (60%) and IMF-1/2(71%) patients. These preliminary data show that the patients classified as true ET (WHO Criteria) are not splenomegalic, have no myelofibrotic evolution and reach a better response to the IFN treatment.