bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloid Nuclear Differentiation Antigen (MNDA) Protein Expression in Myelodysplastic Syndrome (MDS) Is Reduced in Maturing Myeloid Cells.
Authors: Shults Keith , Head David R. , Flye Leanne , Jagasia Madan H. , Goodman Stacey A. , McClintock-Treep Sara A. , Boulos Fouad I. , Jacobberger James W. , Stelzer Greg , Briggs Robert C.
Published: April 2020

Expression of the human MNDA protein is restricted to hematopoietic cells, and occurs at uniformly high level only in the normal granulocyte/monocyte lineage. Myeloblasts and all later stages of the lineage express MNDA, while stem cells do not (Hum Pathol1999;30:1040). Gene expression analyses of MDS cases have shown that MNDA mRNA is significantly down regulated in familial MDS and in high-risk sporadic MDS cases (Blood2000;100:3553 and Leukemia2004;18:449). We evaluated MNDA protein expression in patients with MDS.

Methods: We directly conjugated Alexa 488 (Molecular Probes) to a rat monoclonal antibody specific for MNDA for use in multiparameter cytometric analysis with CD45/CD34 and DRAQ5 (DNA content). Patients’ (n=17) clinical histories, laboratory data, cytogenetics, and morphology were reviewed for confirmation of diagnoses and blinded to study results. Patients lacking primary marrow disease (n=9) were used as controls. Analysis was performed using Winlist 5.0 software (Verity Software) with DDE links to ModFitLT 3.0 using modifications of published methods along with Esoterix Center for Innovation generated algorithms. Analyses were blinded to clinical results.

Results: The normal dataset revealed MNDA expression in expected cell populations, with only a small subset of CD34+ cells exhibiting MNDA (7.5% on average). Expression of MNDA protein in maturing myeloid forms in normals was consistently greater than in MDS samples (MFI=802 vs. 569 in MDS; MFI=mean fluorescence intensity defined by the linear mean of each population). Six of 17 MDS cases displayed greater than 10% maturing myeloid forms lacking MNDA protein expression (never observed in normals). The % MNDA+/CD34+ cells was identical in the two datasets (7.5% in normal vs. 7.6% in MDS).

Discussion: Our results suggest that MNDA protein expression in MDS patients follows a pattern similar to that in gene expression analyses. We additionally demonstrate that MNDA protein expression at the cellular level is variable in MDS, versus uniform expression in normals; parallel data for MNDA message expression at the cellular level is not available. The finding of MNDA-negative myeloid forms in MDS may have diagnostic utility, and may provide insight into both impaired myeloid differentiation in MDS and the biologic role of MNDA.
A Nation-Wide Survey of Primary Chronic Myelofibrosis in Japan.
Authors: Shimoda Kazuya , Okamura Takashi , Tanimoto Tetsuya , Mizoguchi Hideaki , Omine Mitsuhiro , Niho Yoshiyuki , Harada Mine ,   Reseach Committee for Idiopathic Hematopoietic Disorder
Published: April 2020

We retrospectively analyzed 336 patients with primary chronic myelofibrosis from 203 medical institutes in Japan. Average age at onset was 60.7 years. Men were affected 1.4 times more frequently than women. The median survival in 298 patients that could be evaluated was 10.0 years. The factors associated with shorter survival included anemia, leukocytosis/leukocytopenia, thrombocytopenia, and increased blasts in the peripheral blood, and sex (male), age (>60), and the presence of symptoms. Chromosomal abnormalities were found in 58 of 154 patients (38%), although these did not influence survival. In this retrospective study, the median survival in Japanese patients is much longer than that in French patients reported by Dupriez (Blood 1996) (120 mo in Japan versus 42 mo in France). Then we are now doing prospective analysis of newly diagnosed myelofibrosis patients from 1999. In these 5 years, 175 patients were diagnosed as myelofibrosis. Median age was 64 yr (range: 28–86), and men/women ratio is 1.66. Sixty-six patients (38 %) presented anemic symptoms, 19 patients (11 %) showed bleeding tendency. Chromosomal abnormalities were found in 67 of 115 patients (58%). In these five years, 11 of the patients have died. The most frequent causes of death were pneumonia and disseminated infections (7 patients). Three patients developed leukemia.
Investigation of Platelet Functions in Chronic Myeloproliferative Disorders by Optical and Luminesance Method.
Authors: Akay Meltem O. , Mutlu Fezan S. , Gulbas Zafer
Published: April 2020

Background: Bleeding and thrombosis are common causes of morbidity and mortality in patients with myeloproliferative disorders (MPD). Qualitative platelet abnormalities are frequently found in these patients and range from platelet hypofunction as demonstrated by defective invitro platelet aggregation, acquired storage pool disease and/or platelet membrane defects, in addition to enhanced platelet aggregation, increased plasma beta thromboglobulin levels or shortened platelet survival. In this study we aimed to perform platelet aggregation studies by optical method (on platelet rich plasma=PRP) and luminesance method (on whole blood) in chronic myeloproliferative disorders.

Methods:A total of twenty-five patients with MPD (17 chronic myeloid leukemia, 6 polycythemia vera, 2 essential thrombocytosis) were enrolled. Median age was 54,4 (29–76). Platelet aggregation was measured using the optic and luminesance method. The agonists used were adenosine diphosphate (ADP), arachidonic acid (AA) ristocetin and collagen. Platelets were considered to be hyperactive if at least one result (i.e. aggregation or ATP release with one agonist) was above the reference range, and hypoactive if at least one result (i.e. aggregation or ATP release) was below the reference range. Mixed hypo- and hyperactive platelets were considered present when at least one result (i.e. aggregation or ATP release) was below and above the reference range respectively.

Results:Platelet aggregation test results by two methods in myeloproliferative disorders were shown in Table 1. The percent for detection of platelet function abnormality by luminesance method was found to be higher than the optic method and a significant difference was shown between two methods (p<0,05).

Conclusion Our findings suggest that;1. Luminesance platelet aggregation study is more valuable than optic platelet aggregation study for invitro assessment of platelet function in patients with MPD. 2. The use of luminesance platelet aggregation study appears useful to select patients for antiplatelet therapy.

Platelet aggregation test results in myeloproliferative disorders (n=25)

Normal Hypofunction Hyperfunction Mix Total abnormality Luminesance method 1(%4) 4(%16) 8(%32) 11(%44) 24(%96) Optic method 9(%36) 11(%44) 2(%8) 3(%12) 16(%64)
Zap-70 on Bone Marrow Biopsy Is a Good Prognostic Indicator in B-CLL.
Authors: Deliliers Giorgio Lambertenghi , Vener Claudia , Gianelli Umberto , Bosari Silvano , Savi Federica , Cortelezzi Agostino , Somalvico Franco , Soligo Davide , Fracchiolla Nicola S.
Published: April 2020

Bone marrow biopsies (BMB) taken at the time of diagnosis of 108 patients with chronic lymphocytic leukemia (B-CLL) (median age: 62 years, range: 36–81; M/F ratio: 2/1) were collected between 1985 and 2004, and tested immunohistochemically for the expression of ZAP-70 (zeta-associated protein 70), “Anti-ZAP-70 (human), clone 2F3.2 (mouse monoclonal IgG2a)”. There were degrees of ZAP-70 expression: “negative (a)” (58 patients), “weak (b)” (20 patients) and “intense (c)” (30 patients)” in comparison with natural killer (NK) and T-cell ZAP-70 expression (BMB positive when 20% of B-CLL cells express ZAP-70). The cases were analysed separately by three different experts.

In terms of BMB morphology, proliferative centres were 36% in (a) 35% in (b) and 47% in (c); a diffuse pattern: 8.6% in (a), 10% in (b) and 13% in (c); a nodular pattern: 66% in (a), 55% in (b) and 37% in (c): (a) vs (b+c) (p=0.024), (a+b) vs (c) (p=0.014). The different degrees of ZAP-70 expression correlated with two groups of patients according to the Rai and Binet criteria: “0-I-II, A” and “II-III-IV, B, C”. Cases (a) correspond more frequently to the first, and (b+c) to the second (p=0.013); moreover, cases (a+b) correspond more frequently to the first group, and (c) to the second (p=0.0026).

A lymphocyte doubling time (LDT) of less than six months and less than one year was most frequent in (c) (p=0.00044) (p=0.0065). An LDT of less than one year was more frequent in (b+c) vs (a) (p=0,01). Patients receiving no therapy were more frequent in (a) or (a+b) (p=0.00011) (p=0.00045). Patients receiving no therapy or only one line of treatment were more frequent in (a) or (a+b) (p=3.2x10–5) (p=0.0073). Cytogenetic abnormalities were more frequent in (a) or (a+b) (p=0.017) (p=0.022), and complex caryotypes more frequent in (a+b), (p=0.016). The incidence of death was lower in (a+b) than (c) (8% vs 27%) (p=0.041).

Mann-Whitney analysis showed a statistical difference between (a+b) and (c) for the following variables at diagnosis: PLT (p=0.02); WBC (p=0.05); Ly (p=0.055); LDH (p=0.015); B2m (p=0.0005); splenomegaly (p=0.03); and a statistical difference between (a) vs (b+c) for the following variables at diagnosis: PLT (p=0.044); LDH (p=0.006); B2m (p=0.003); percentage of bone marrow infiltrate (p=0.055); CD 38 flow-cytometry (p=0.0009). Kruskall-Wallis analysis of (a) vs (b) vs (c) showed a statistical difference for the following variables at diagnosis: LDH (p=0.01); B2m (p=0.0017); CD 38 flow-cytometry (p=0.0051). These data confirm the prognostic significance of ZAP-70 expression in patients with B-CLL, and its good correlation with clinical and cytogenetic parameters and the Rai-Binet classification.
Normal and Chronic Lymphocytic Leukemia (CLL) Lymphocytes Can Be Distinguished in Flow-Cytometry-Based Automated Differential Cell Counts by Their Positional Parameters.
Authors: Millá Fuensanta , Barberá Angels , Oriol Albert , Navarro Tomas , Juncà Jordi , Rodriguez Lluis , Ribera Josep-Maria
Published: April 2020

Introduction and objectives. Flow-cytometry based automated blood cell counters measure the volume (V), conductivity (C) and light scatter (S) of cells and make available these values for each subpopulation of white blood cells. Although differences in V, C and S among several lymphoproliferative disorders, reactive lymphocytes and normal lymphocytes have been described, the ability to use this this information to detect lymphoproliferative disorders has not been defined.

Methods. We registered VCS data of all consecutive cases of lymphocytosis (lymphocytes > 3.5x10e9/l) detected in routine laboratory testing during 50 non-consecutive days. Lymphocytes were evaluated morphologically, immunophenotypically and by conventional cytogenetics if necessary and the patients were evaluated clinically to document diagnosis adequately. Discriminant analisys modelling was used to assess the ability to predict diagnosis of VCS data.

Results. 202 cases of lymphocytosis were detected in 120 males and 82 females, mean age 53 years (SD 16). Patients were considered to have normal lymphocytes (N=102, 50%), to present a reactive lymphocytosis (N=43, 21%, 20 of them HIV infected patients) or achieved a final diagnosis of lymphoproliferative disorder (N=57, 28%, 43 of them CLL).

The mean volume of CLL lymphocytes was significantly lower than that of normal and reactive lymphocytes (p=0.009) and so was light scatter (p=0.018). When mean VCS values and their standard deviations were included in a discriminant analysis model, a unique function adequately classified 166/189 cases (87%) as CLL versus reactive or normal. Sensitivity and specificity were 73% and 92% respectively and false positive and false negative cases were 26% and 8% respectively. Inclusion of a second discriminant function did not improve significantly the predictive power of the model.

Conclusion, VCS data from flow-cytometry based automated blood cell counters can be used to differentiate CLL from normal or reactive lymphocytes by simple discriminant analysis classification.
CD5 and CD23 Positive Mantle Cell Lymphoma Detected by Flow Cytometry and Confirmed by FISH Study t(11;14).
Authors: Zhang John , Chin David , Anthony Adam , Bolton Heather , Phillips Cheri , Hii Anselm , Chen Sing-Tsung
Published: April 2020

The differential diagnoses of CD5 positive B-cell lymphoproliferative disorders mainly include chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and mantle cell lymphoma. Occasionally large cell and marginal zone lymphomas may also be CD5 positive. An accurate diagnosis effects patient management.

The classical immunophenotype for chronic lymphocytic leukemia/small lymphocytic lymphoma is CD19/CD5/CD23 positive FMC-7 negative cells with dim CD20 and dim light chain expressions, while mantle cell lymphoma is CD19/CD5/FMC-7 positive with bright CD20 and bright light chain expressions. The diagnosis of mantle cell lymphoma is usually confirmed by either immunostain for cyclin D1 or FISH study for t(11;14). In reality, immunostaining for cyclin D1 can be difficult and may show variable results in different laboratories and FISH study may not be readily available. Generally, when it comes to the diagnosis of lymphoma, immunohistochemical positivity of both CD5 and CD23 is almost pathognomic for chronic lymphocytic leukemia/small lymphocytic lymphoma if no fresh tissue is saved for flow cytometry analysis.

Flow cytometry analysis of 44 FISH-confirmed mantle cell lymphomas was reviewed in our lab. Among these, 37 showed the classical immunophenotype of mantle cell lymphoma. However, 7 cases (16%) were positive for both CD5 and CD23. The expression of CD23 varied from dim to bright. When compared to typical CLL, they showed FMC-7 expression and brighter than dim light chain expression. In one case, the light chain expression was dim.

In conclusion, CD23 expression which was thought to be a specific marker for CLL/SLL may also be seen with mantle cell lymphoma. Although FMC-7 expression is seen in all CD23 positive mantle cell lymphomas, bright light chain expression is not universal. We recommend that FISH or immunohistochemical studies for cyclin D1 be performed on CD5/CD19 clonal B cell proliferations with CD23 expression if morphology or immunophenotype is atypical for CLL/SLL.
Establishment of an In Vitro Culture Method for Multiple Myeloma Cells.
Authors: Cohen Yossi , Hardan Yzhar , Nagler Arnon , Zipori Dov
Published: April 2020

In July 2004 we have established an in vitro method for culturing and expansion of human primary myeloma cells in a cytokine free system. This method which was designed to optimize the cell signaling between myeloma cells and their stromal support is easilly reproducible and so far has been applied successfully in 15 of 15 myeloma patients. This progress allows, for the first time, long term maintenance and expansion of the tumor cells with day by day observation of how the myeloma clone propagates, as assessed by morphology, FACS analysis and by the FISH-Bioview system (see figures 1,2 showing a group of myeloma cells after 28 days in culture along with their single FISH signal for known del13q).

The protocol is currently under intensive procedure of standardization for general use.

Applications of this culture system have already yielded several new findings. One, relating to the peculiar nuclear changes appearing in myeloma cells during culture is being presented in detail in another abstract. An additional issue is the ability to manipulate the culture to detect very small numbers of myeloma cells in the range of MRD. Yet another use is in vitro drug assay. Preliminary results have revealed no apparent influence of pamidronate or erythropoietin on myeloma cell expansion up to 28 days in co-culture (4 cases). In contrast, thalidomide and simvastatin had deleterious effect on the stromal support (3 cases) but the former did not eliminate the myeloma cells whereas no myeloma cells were seen with simvastatin after 3–7 days in culture.

Figure Figure Figure Figure
Vascular Endothelial Growth Factor (VEGF) Is a Growth and Survival Factor in Waldenstrom’s Macroglobulinemia.
Authors: Tournilhac Oliver , Le Gouill Steven , Santos Daniel Ditzel , Podar Klaus , Xu Lian , Hatjiharissi Evdoxia , Tai Yu-Tzu , Hunter Zachary , Branagan Andrew , Anderson Kenneth C. , Treon Steven P.
Published: April 2020

Waldenstrom’s macroglobulinemia (WM) is a B-cell disorder characterized by excess of bone marrow (BM) IgM secreting lymphoplasmacytic cells (LPC). In previous studies, we and others have demonstrated that VEGF within the BM micro-environment plays a role in the growth, survival and migration of myeloma plasma cells. Conversely, we demonstrated that BM mast cells (MC), which produce and secrete VEGF (Boesiger, J Exp Med, 1998) support growth and survival of WM LPC (Tournilhac et al, JCO2004; 22:517S). In the present studies we therefore explored the role of VEGF in WM pathogenesis. Using RT-PCR analysis, we detected VEGF receptors R1 (Flt-1) and R2 (flk1/KDR) transcripts in sorted LPC from 4/4 (100%) and 15/16 (94%) WM patients respectively. Moreover, we showed functionality for VEGF as a growth and survival factor in WM. Specifically, recombinant human VEGF (rh-VEGF) at 25 ng/ml induced dose dependent proliferation of sorted LPC from WM patients and prevented serum starvation- induced apoptosis. Inhibition of VEGF signaling pathways both induced apoptosis and blocked mast cell- induced proliferation of sorted WM LPCs in 4 of 6 and 4 of 4 patients, respectively. Besides the known MC-derived WM LPC growth and survival factors CD40 ligand (CD40L) (JCO2004; 22;517S) and B-lymphocyte stimulator protein (BLYS) (Ditzel Santos et al, ASH 2004 submitted), the present study demonstrates also a role of VEGF in WM pathogenesis. We therefore suggest VEGF as an additional therapeutic target in WM treatment regimens.
Bortezomib Targets Multiple Myeloma Endothelial Cells.
Authors: Roccaro Aldo M. , Hideshima Teru , Raje Noopur , Kumar Shaji , Ishitsuka Kenji , Yasui Hiroshi , Shiraishi Norihiko , Hamasaki Makoto , Richardson Paul G. , Anderson Kenneth C.
Published: April 2020

Introduction: Bone marrow (BM) angiogenesis is an important hallmark of multiple myeloma (MM) which correlates with progression. Although MM remains incurable despite conventional and high-dose chemotherapy, the proteasome inhibitor Bortezomib (Velcade, formerly PS-341), can overcome conventional drug resistance in vitro and in vivo and it has recently been FDA approved for treatment of relapsed and refractory multiple myeloma. Here we evaluated whether anti-angiogenesis may contribute to the anti-MM activity of PS-341. We examined the effect of PS-341 on the angiogenic phenotype of endothelial cells (ECs) isolated from BM of patients with MM.

Methods: MMECs were extracted from BM of patients with active MM using a lectin-based method. The MMEC population contained >95% factor VIII-related antigen (FVIII-RA)+ and CD31+ cells, as assessed by fluorescence activated cell sorting (FACS). Contamination by macrophages and plasma cells was <5%, evaluated by FACS for CD14 and CD38 positivity, respectively, as well as by RT-PCR and Western blot for CD38. Viability, assessed by trypan blue was >90%. MTT assay and [3H] thymidine uptake were used to evaluate the effects of PS-341 on survival and proliferation, respectively, of MMECs. Proliferation of MM.1S cells cocoltured with MMECs was measured by [3H] thymidine uptake. Cytokine (IL-6, VEGF) levels were quantitated by ELISA. Other in vitro angiogenesis functions examined included chemotaxis, spreading on fibronectin (FN), and morphogenesis on Matrigel. Ongoing work is looking at the effect of PS-341 on angiogenesis in vivo by using a chick embryo chorioallantoic membrane (CAM) model.

Results: PS-341, at concentrations achievable in the plasma of patients, inhibited in vitro MMEC and HUVEC functions related to angiogenesis, including proliferation, chemotaxis, spreading on FN, and capillary formation on Matrigel. All these functions were affected in a dose-dependent fashion. A significant concentration-dependent reduction of VEGF and IL-6 production was observed in the presence of PS-341, as demonstrated by ELISA. Importantly, binding of MM.1S cells to MMECs triggers tumor cell proliferation, and PS-341 inhibits proliferation of adherent MM.1S cells in a dose-dependent fashion. Similar data were demonstrated in HUVECs.

Conclusions: These data therefore demonstrate that PS-341 acts both directly and indirectly against MMECs, another mechanism which may contribute to the anti-MM activity of PS-341.
Aldehyde Dehydrogenase Activity - the Better Marker for the Quality of Hematopoietic Stem Cell Transplants?!.
Authors: Lioznov Michael V. , Freiberger Petra , Zander Axel R. , Fehse Boris
Published: April 2020

The use of functional rather than cell surface markers to identify and isolate stem cells has several advantages. Recently, aldehyde dehydrogenase (ALDH) enzyme activity has been shown to be a metabolic marker for primitive hematopoietic progenitor cells. We compared 7-day-expression kinetics of ALDH vs. the standard marker in human hematopoietic stem cell transplantation, CD34, and related these data to the colony-forming ability of bone marrow and leukapheresis samples. Cell probes directly taken from whole transplants were first analyzed on the day of harvest (day 0) using flow cytometry (CD34, ALDEFLUOR®) and methyl cellulose (MC) assays. To resemble usual conditions during transport of clinical transplants, cell samples were then stored without additional modification at either +4°C or room temperature (RT). Relative CD34+, ALDH+ and CFU numbers were determined at various time points (days 1, 2, 3 and 7). During this time course we found that progenitor (CFU-GM) cell activity correlated very well with ALDH enzyme function but not with CD34 expression, the commonly used parameter to evaluate the quality of hematopoietic stem cell transplants. This indicates that ALDH expression may be a more reliable marker for quality control of HSC transplants. Based on our assay systems we also could show that PBSC transplants lose most of their CFU-activity (>80%) within just 48 hours if stored at room temperature, but are stable at +4°C. Thus, transport of PBSC transplants at RT should be strongly avoided. In conclusion, our data may have important implications for both quality control and transport organization of HSC transplants, especially given the permanent increase in numbers of PBSCT from matched unrelated donors.
Infections after CD34-Selected or Unmanipulated Autologous Hematopoietic Stem Cell Transplantation.
Authors: Frère Pascale , Pereira Maguy , Fillet Georges , Beguin Yves
Published: April 2020

Relapse remains the major cause of failure after high-dose chemotherapy and autologous peripheral blood stem cell transplantation (PBSCT). As tumor contamination of the graft may contribute to this, CD34 selection has been used as an attempt to decrease the relapse rate. However, immune reconstitution may be delayed with CD34 selection due to the removal of T cells, natural killer (NK) cells and monocytes. The result could be an increased incidence of infectious complications. Some small series of patients have reported a higher incidence of infections, and in particular of viral infections, but others have not. Therefore, we compared the incidence of infection in 25 recipients of CD34-selected PBSC (CD34 group) and 75 case-matched recipients of unmanipulated PBSC (PBSC group) transplants. There were 52 males and 48 females with an average age of 52±11 yrs. They included 32 patients with non-Hodgkin’s lymphoma, 8 with Hodgkin’s disease, 40 with multiple myeloma and 20 with breast cancer, with 42 in first-line therapy and 58 in relapse, receiving their 1st (N=63) or 2nd (N=37) transplant. The number of CD34 cells infused as well as speed of engraftment were comparable in the two groups. There were no significant increase in the CD34 compared to the PBSC group for: actuarial incidence of infection (56 vs 49% at day 30, 70 vs 64% at 1 yr); proportion of patients with at least 1 (68 vs 68%), 2 (24 vs 17%) or 3 (0 vs 3%) infections; number of infectious events per patient before (1.32 vs 1.04) or after (0.59 vs 0.71) disease progression, before day 30 (0.84 vs 0.64), within days 31–100 (0.00 vs 0.09, p<0.01) or after day 100 (0.48 vs 0.31); risk of VZV (27 vs 30% at 5 yr) or CMV infection (8 vs 6%) or disease (0 vs 3%); use of antibiotics (8 vs 5 days) or antifungal therapy (0 vs 0%) or days with fever (1 vs 1 day) in the initial hospitalisation. However, bacteremias tended to be more frequent in the CD34 group (0.44 vs 0.20 episode/patient). Bacteria accounted for the majority of infections in both groups (64 vs 72%). Infection was the main or a contributing cause of death in 0% and 20% of patients in the CD34 group compared to 1% and 10% in the PBSC group (NS). In conclusion, our results indicate only a trend towards an increased risk of infection after CD34-selected autologous PBSCT. The role of extended infection prophylaxis should be evaluated.
Clostridium Difficile-Associated Disease in Allogeneic Transplant Patients.
Authors: Sadhu Justin S. , Dubberke Erik R. , Gatti Robert , Devine Steven M. , Fraser Victoria J.
Published: April 2020

Allogeneic transplant (allo) patients (pts) are at high risk for Clostridium difficile-associated disease (CDAD), yet there are few studies evaluating CDAD in allo pts. We carried out a retrospective chart review of all allo pts who had CDAD on a transplant (tx) unit between 8/01 and 7/03. 37 pts were identified who had CDAD during tx hospitalization (hosp) or after tx during the study period. 17 (46%) were admitted for initial tx, 13 (35%) for infection, and 7 (19%) for other reasons. 15 (40%) had acute leukemia, 6 (16%) lymphoma, 4 (11%) myelodisplastic syndrome, and 12 (32%) other underlying diseases. On admission, 17 (46%) pts were in complete remission, 4 (11%) in partial remission, 7 (19%) in relapse, and 9 (24%) presented with refractory disease. 5 pts (14%) had CDAD before day 0 (median d-2, range d-9 to d-1) and 32 pts (86%) had CDAD after tx (median d+49, d0 to d+3185). In the 60 days prior to CDAD, pts had median 1 (0–4) prior hosp and 15 days (1–50) hospitalized; 36 pts (97%) received antibiotics, 35 (95%) immunosuppressants, 33 (89%) antimotility/narcotic agents, 31 (84%) gastric acid suppression, 23 (62%) chemo, and 14 (38%) TBI. Within 48 hours of CDAD, 31 (86%) pts had diarrhea, 12 (33%) fever, 10 (28%) abdominal tenderness, 7 (19%) abdominal distention, and 7 (19%) hypothermia. 22 pts (59%) had either moderate or severe CDAD. 25 (68%) pts were treated for their primary episode of CDAD with metronizadole (met), 2 (5%) with vancomycin (vanc), 8 (22%) with met and vanc, and 2 (5%) were not treated for CDAD. 32 (86%) pts responded to therapy (median 2 days, range 0–25 days). Of the 32 pts who had CDAD post allo, 3 (9%) had gut graft-versus-host disease (GVHD) before CDAD and 8/29 (28%) developed gut GVHD after CDAD. Those 8 pts developed CDAD median d+114 (range +1 to +278) and gut GVHD d+131 (+1 to +454), with gut GVHD coming median 11 days (0 to 176) after CDAD. 7 pts (19%) died before discharge. The median survival time for all pts was 79 days after CDAD and 205 days after tx. Of the 30 (81%) pts discharged alive, 10 (33%) had recurrent CDAD episodes (median 1, range 1–3). When compared to mild CDAD, patients with severe CDAD had an increased risk of death at one year after CDAD (OR 3.3; CI 1.4–7.6). CDAD in allo pts may be associated with an increased risk of gut GVHD, death, and other adverse outcomes.
Extracorporeal Photopheresis (ECP) for Acute and Chronic GVHD, a Report of 56 Patients Treated in a Single Institution.
Authors: Dann Eldad J. , Badian Alla , Stravets Tselestina , Kertsman Elena , Avivi Irit , Elhasid Ronit , Haddad Nuhad , Fineman Riva , Zuckerman Tsila , Oliven Ana , Bonstein Lilah , Rowe Jacob M.
Published: April 2020

Acute and chronic GvHD remain the leading cause of morbidity and mortality of allogenic stem cells transplantation. Immunosuppressive therapy is the mainstay of prophylaxis and therapy for GvHD; however, it heavily impacts on post transplant morbidity and mortality and new modalities are continually needed. Reported here is the five-year experience in a single institution of ECP for acute and chronic GvHD patients. Mononuclear cells were collected using Cobe -Spectra version 7 manual operation for adults and version 6 for pediatric patients. 1.5–2.0 blood volumes were circulated through the cell separator with a product of approximately 5X109 mononuclear cells. When product hematocrit was less then 3%, volume was added to 300ml. With plasmacyte electrolyte solution and when the hematocrit was more then 3%, volume was added to 500 ml. 8 methoxy psoralen (8MOP) was added to a final concentration of 0.2ug/ml. Cells were then transferred to irradiation bag, exposed to 2 Joule /cm2 of UVA (365nM) (Biogenetic Vilber Lourmat) and re-transfused. The treatment protocol consisted of 2 consecutive ECP performed at bi-weekly intervals until clinical improvement. Fifty-six patients, 34 males and 22 females, age 1 to 64y (median 31 years), were treated. Patients underwent allogeneic bone marrow transplantation for the following indications. AML -21, ALL- 19, CML-8, Thalassemia 4, MDS-2, MM-1 NHL-1. Sixteen were treated for acute GvHD and 40 for chronic GvHD. Diffuse GvHD of skin was diagnosed in 43, gastro-intestinal tract 10; joint involvement 12, and liver involvement 35 patients. Patients had from 4 to 66 ECP (median 21). Results: 15 pts succumbed,10 with acute GvHD and 5 with chronic GvHD. Disease relapse was the cause of death in two patients with acute GvHD. Overall response rate was 46/56 (82%). Response rate for chronic GvHD was 37/40 (92%) and for acute GvHD was 9/16 (56%). Complete resolution of GvHD, partial response and no response were seen respectively in 33%, 60%, 7% for patients with chronic GvHD and 0%, 56% and 44% for patients with acute GvHD. Conclusions: ECP is a useful therapeutic modality for GvHD and is most effective in patients with chronic GvHD. It is most effective for both liver and skin involvement and less effective for patients with scleroderma. It is suggested that this modality should be used in early management of these patients concomitantly with immune suppressive therapy in the order to improve quality of life and reduce long-term side effects.

No. of Patients Median ECP/pt No. of CR No. of PR No response Acute GvHD 16 8 (4–41) 0 9 (56%) 5 (44%) Chronic GvHD 40 26 (4–66) 13 (33%) 24 (60%) 3 (7%)
Reconstitution of NK Cell Receptor Repertoire after Pediatric Stem Cell Transplantation.
Authors: Kobayashi Michihiro , Hiramatsu Hidefumi , Adachi So-ichi , Nakahata Tatsutoshi
Published: April 2020

Interaction between killer immunoglobulin-like receptors (KIRs) and HLA class I ligands influence development of natural killer cell repertoire and response to allogeneic tissue. Recently several reports suggests donor/recipient KIR-ligand imcompatibility have great influence for outcome of allogeneic Hematopoietic Stem Cell transplantation (SCT). We examined recovery of the NK cell receptor after pediatric SCT to elucidate emergence of NKR repertoire and frequency after SCT is depend on whether donor or recipient type and effect of HLA disparity. [Patients and method] Nine patients receiving allogeneic SCT were examined. Expressions and frequency of KIR on NK cells were determined by flowcytometry using anti CD158a/h, 158b/j, 158e, 158i, 159a, 94, 56,and 3 antibodies. Donor and recipient KIR repertoire frequency prior to SCT were also examined. In cord blood transplantation, donor KIR repertoire was checked using residual thawed and infused cord blood. [Result] Among 9 patients, PBSCT were performed in 3 patients, CBT in 3, unrelated BMT in 3. HLA matched SCT was all 3 cases of unrelated BMT. KIR expression frequency was various in each patient. But in early phase, relatively both high percentage and absolute number of each KIR positive repertoire were observed, and then gradually decreased toward to 1 year after SCT. NK cell receptor repertoire reconstitution was generally well and rapid except for one patient who was treated with steroid pulse therapy for severe GVHD. KIR repertoire expression pattern at 1 year after SCT was similar to donor type compare to that of pre-SCT recipient. Donor/recipient HLA mismatch did not have obvious effect for NKR repertoire reconstitution. [Conclusion] NKR repertoire reconstitution after pediatric SCT depends on donor cell type.
Harvest of Autologous Peripheral Blood Stem Cells Using Ifosfamide/Etoposide Regimen Combined with Granulocyte Colony-Stimulating Factor.
Authors: Noguchi Mitsuho , Tashiro Haruko , Goto Moritaka , Kawasugi Kazuo , Shirafuji Naoki
Published: April 2020

Aim: To make clear the feasibility of Ifosfamide/Etoposide (IE) regimen combined with granulocyte colony-stimulating factor (G-CSF) for the collection of autologous peripheral blood stem cells (APBSC), we evaluated the number of the collected CD34 positive cells, and the regimen-related toxicities.

Patients’ characteristics and Methods: 19 Patients (male 12, female 7) were received APBSC harvest in our hospitals during Aug. 2000 to Dec. 2003 with age of 54.3 years on average (30–66). Diagnoses were included HD (n=2), NHL (n=13), MM (n=3), and malignant synovioma (n=1). IE regimen was included with Ifomide 2000 mg/m2 (day 1), and Etoposide 200 mg/m2 (day 1–3) (4cases) or 500 mg/m2 (day 1,2) (15 cases). For the prevention of hemorrhagic cystitis patients were administered with Mesna, and NaHCO3 during and after 1 day of the administration of Ifosfamide. G-CSF was administered at the dose of 10 ug/kg (lenograstim) or 300 ug/m2 (filgrastim) when blood absolute neutrophil count came down to 1000/mm3. When the suppression of bone marrow was recovered and blood WBC count came up to 5000/m3, CD34-positive cells were counted in blood, and APBSC was harvested when blood CD34-positive cells were determined above 0.1 %. APBSC was harvested with CS-3000 plus (Fenwall).

Result: The median duration from the start of the administration of G-CSF to the finish of APBSC harvest was 6.5 days (4–11). The median number of CD34-positive cells of the harvested was 4.32 x 106/kg (1.10–13.50). All cases were harvested. The toxicities during from the conditioning to the harvest were included with grade 1 headache (2 cases), grade 2 nausea (6 cases), grade 1 bleeding (1 case), grade 1 constipation (1 case), grade 1 fever (1 case), and grade 1 thrombocytopenia (1 case).

Conclusion: IE regimen combined with G-CSF was feasible to harvest APBSC on the yield of the collection of the transplantable stem cells, and on the regimen-related toxicities.
Total Therapy 3 (TT 3) for Newly Diagnosed Myeloma, Incorporating Velcade into Remission Induction with DT PACE: Early Results Regarding Efficacy, PBSC Mobilization and Toxicities.
Authors: Barlogie Bart , Hollmig Klaus , Zangari Maurizio , Stover Julie , Jackson Lisa , Milner Teresa , Fassas Athanasios , Tricot Guido
Published: April 2020

TT 3 represents a further development of the Total Therapy concept after completion of accrual to TT 2 with 668 patients. Based on the high salvage potential of the Velcade + Thal + Dex (VTD) regimen for endstage MM, VTD had been added to PACE (4 day continuous infusions of cisplatin 10 mg/m2, adriamycin 10 mg/m2, cyclophosphamide 400 mg/m2 and etoposide 40 mg/m2) x 2 cycles for induction with PBSC collection with first or second cycle; followed by MEL 200 mg/m2-based autotransplant 2–3 months apart and subsequent consolidation therapy with further VTD PACE and VTD maintenance. As of July 1, 2004, allowing for a 1-month time to evaluate initial response and toxicity, 57 patients have been accrued to TT 3. M protein responses by level demonstrated ≥ near-CR in 16% prior to cycle 2; 26% after cycle 2 prior to first transplant and nearly 40% after first transplant (patients not yet evaluable for response for a particular step were considered non-responders; intent-to-treat analysis). The PBSC collection target of 20 million CD 34 cells/kg was accomplished within 2 days of collection in all 10 patients mobilized on the first cycle; among 29 patients collected on the second cycle, 17 collected at least 20 million CD 34 cells/kg during 2–3 days, especially when only 2 or 3 doses of Vel 1.0 mg/m2 were applied, whereas in case of 4 doses (day −2, +1, +5, +8) 5 of 23 patients failed to collect on this cycle and 13 reached ≥ 20 x 106/kg procurement goal. There was only 1 treatment-related death in a setting of renal failure at diagnosis. Other toxicities were mainly myelosuppression whereas non-hematologic toxicities appeared to be similar to those seen with DT PACE previously. Laboratory correlates such as gene expression profiling, after a test dose of Velcade and after VTD PACE along with early suppression of FDG uptake on PET will also be presented. These preliminary data indicate remarkable activity and safety of a regimen that incorporates all available drugs with single agent activity in a multi-combination strategy, aimed at maximizing and accelerating tumor mass reduction in an effort to overcome the negative consequences especially of metaphase cytogenetic abnormalities.
Junb Regulates Hematopoietic Stem Cell Numbers in Normal and Leukemic Mice.
Authors: Passegue Emmanuelle , Wagner Erwin F. , Weissman Irving L.
Published: April 2020

JunB is expressed in hematopoietic stem cells (HSC) as a partner for Fos in the composition of the AP-1 transcription factor. Previously, we have shown that junB inactivation in postnatal mice results in the development of a myeloproliferative disorder (MPD) resembling early human chronic myelogenous leukemia (CML) (Passegue et al., 2001, Cell, 104, 21-32). Here, we demonstrate that JunB is a critical transcriptional regulator of HSC numbers both in normal and leukemic mice. Overexpression of junB in long-term HSC (LT-HSC) dramatically decreases the frequency of LT-HSC, while inactivation of junB specifically expands the numbers of LT-HSC, and of granulocyte/macrophage progenitors (GMP), resulting in the development of a chronic MPD with many features of human CML, including progression to blast crisis, and death. JunB effects are mediated, at least in part, via the regulation of effectors genes such as the cell cycle inhibitor p16/INK4a, which is a direct junB-target gene and a key regulator of stem cell proliferation/senescence, as well as the anti-apoptotic proteins bcl2 and bcl-xl, two critical regulators of stem cell death. Using several models of conditional deletion of junB in hematopoietic cells, we demonstrate that junB inactivation must take place in LT-HSC, and not at later stages of myelopoiesis, to induce MPD. Most importantly, we show that only junB-deficient LT-HSC, and no other myeloid progenitor populations, are capable of transplanting the MPD to recipient mice. These results indicate a stem cell-specific role for JunB in normal and leukemic hematopoiesis, and provide an experimental demonstration that leukemia stem cells (LSC) can reside at the LT-HSC stage of development in a mouse model of chronic MPD.
Factor VIIIa Decay Due to A2 Subunit Dissociation Is Regulated by the A1 Domain.
Authors: Parker Ernest T. , Doering Christopher B. , Lollar Pete
Published: April 2020

FVIII contains three homologous A domains, two homologous C domains and a single B domain. The B domain and an activation peptide between the B and A3 domains are released during the activation of fVIII by thrombin. The resulting fVIIIa molecule has a heterotrimeric A1/A2/A3-C1–C2 subunit structure. FVIIIa is unstable at physiological concentrations and pH due to dissociation of the A2 subunit from the A1/A3-C1–C2 dimer, which is three-fold faster in human fVIIIa than porcine fVIIIa. Fine regulation of fVIII activity appears to be important because plasma fVIII levels are positively correlated with a risk of arterial and/or venous thrombosis. Additionally, mutations in the fVIII gene have been identified that result in fast A2 dissociation and a hemophilia phenotype. To characterize structural determinants involved in A2 subunit dissociation, we constructed and purified recombinant human, porcine and hybrid human/porcine fVIII molecules corresponding to all eight combinations of human and porcine fVIII A1–A2–A3 domains (HHH, HHP, HPH, HPP, PHH, PHP, PPH and PPP). All of the constructs contained the human C1 and C2 domains. The rate of A2 subunit dissociation was studied at pH 7.4 by measuring decay of fVIIIa activity in an assay system using purified intrinsic fXase components and displayed first-order kinetics in all cases. Constructs containing the porcine A1 domain behaved similarly to porcine fVIIIa (Table 1). In contrast, constructs containing the human A1 domain behaved similarly to human fVIII, except for the HPH construct, which displayed intermediate behavior.

Table 1 A domain hybrid fVIIIIa decay rates Construct k (min− 1)a a mean and estimated s.d. from nonlinear least-squares analysis HHP 0.317 ± 0.018 HHH (human) 0.275 ± 0.013 HPP 0.262 ± 0.011 HPH 0.193 ± 0.012 PPH 0.128 ± 0.005 PHP 0.118 ± 0.003 PPP (porcine) 0.109 ± 0.003 PHH 0.105 ± 0.002

The fVIII A domains contain two azurin-like subdomains. Additionally, the A1 domain contains a COOH-terminal 37 amino acid acidic region. To further characterize the involvement of the A1 domain in fVIIIa decay, we prepared constructs that contained the combinations hhp, phh, pph, hpp or hph of the three A1 subdomains in an otherwise human background. Comparison of their fVIIIa decay rates indicated that the acidic region is not involved in A2 dissociation (Table 2, cf. hhp versus pph). Substitution of either azurin-like porcine subdomain produced relatively stable fVIIIa molecules.

Table 1 A1 subdomain hybrid fVIIIIa decay rates Construct k (min− 1)a a mean and estimated s.d. from nonlinear least-squares analysis hhp 0.263 ± 0.012 phh 0.167 ± 0.005 pph 0.157 ± 0.008 hpp 0.133 ± 0.005 hph 0.134 ± 0.010

These results indicate that porcine-specific amino acid side chains in both azurin-like A1 subdomains are responsible for differential fVIIIa decay rates. A1 domain mutations may represent adaptations to differences in hemostatic requirements between species.
Specific Antibodies to Moesin, a Membrane-Cytoskeleton Linker Protein, Are Frequently Detected in Patients with Immune-Mediated Aplastic Anemia.
Authors: Takamatsu Hiroyuki , Feng Xingmin , Lu Xuzhang , Sugimori Chiharu , Chuhjo Tatsuya , Takami Akiyoshi , Okawa Katsuya , Nakao Shinji
Published: April 2020

Although there is a large body of evidence that immune mechanisms play an essential role in the development of acquired aplastic anemia (AA), knowledge of autoantigens eliciting the immune system attack is limited. In an immune-mediated subset of AA patients characterized by the presence of HLA-DRB1*1501 and an increase in the proportion of paroxysmal nocturnal hemoglobinuria (PNH)-type blood cells, not only T-cell response but also B-cell response to antigens derived from hematopoietic stem cells may take place. To test this hypothesis, we screened sera of such immune-mediated AA patients for the presence of IgG antibodies recognizing a megakaryoblastic leukemia cell line, UT-7, using immunofluorescence staining. Since the sera from several patients stained cytoplasm of UT-7 cells, lysates of the cell line were subjected to Western blotting using the same sera. A clear band of 80 kDa protein was revealed by the sera from several patients. Peptide mass finger printing of the protein extracted from the single band identified this protein as moesin. Moesin is a membrane-cytoskeleton linker protein in cytoplasm and involved in the formation of microvilli, cell adhesion sites, and ruffling membranes. When sera of a large number of AA patients were screened for the presence of antibodies specific to recombinant moesin protein with Western blotting and ELISA, significantly high titer of the antibodies was detected in 35 of 110 (32%) AA patients. The prevalence of anti-moesin antibodies in AA patients showing increased PNH-type cells (41%) was significantly higher than those (12%) without PNH-type cells ( p =0.002). Anti-moesin antibodies were detectable in 15 of 39 (38%) recently diagnosed AA patients before therapy and in 20 of 71 (28%) patients who had a long history of AA. Four of 6 newly diagnosed patients showing anti-moesin antibodies responded to ATG+cyclosporine therapy and restored good hematopoietic function. Since B lymphocytes reportedly secrete moesin as a form of exosome (J Biol Chem, 2003), we examined supernatants of various leukemia cell lines incubated for 1 hour for the presence of moesin. The membrane-linker protein was detectable in the culture supernatant of 4 (UT-7, K562, OUN-1 and NB4) of 10 myeloid leukemia cell lines. These findings indicate that B-cell response to moesin may represent immune pathophysiology of AA and that moesin secreted from hematopoietic progenitor cells as a form of exosome may be efficiently processed by antigen-presenting cells, leading to induction of antibodies and possibly T cells specific to moesin in immune-mediated AA.
Pharmacogenetic Analysis of Polymorphisms in Pharmacological Pathway of Vincristine, Doxorubicine and Dexamethasone (VAD Regimen) To Predict Response in Patients with Multiple Myeloma.
Authors: Jamroziak Krzysztof , Balcerczak Ewa , Panczyk Mariusz , Piaskowski Sylwester , Janus Agnieszka , Cebula Barbara , Mirowski Marek , Smolewski Piotr , Urbanska-Rys Halina , Cichocki Piotr , Robak Tadeusz
Published: April 2020

Combination of vincristine, doxorubicin and dexamethasone (VAD regimen) is a widely-used, effective and economic initial treatment of multiple myeloma (MM). However, between 30 and 50% of MM patients do not respond to VAD, and exposure to VAD may induce multi-drug resistance. Therefore, in the era of emerging novel therapies for MM, there is a need for pre-treatment identification of potential responders to VAD. Importantly, all three VAD components have common steps in their pathways of transport and metabolism, thus even modest inherited alteration caused by single nucleotide polymorphisms (SNP) may influence VAD efficacy an toxicity. The aim of this study was to investigate whether analysis of common functional SNP in drug transporter P-glycoprotein gene (MDR1 or ABCB1), phase I cytochrome P450A metabolizing enzymes genes (CYP3A4 and CYP3A5), phase II glutathione S-transferase P metabolizing enzyme gene (GSTP1) as well as in glucocorticoid receptor gene NR3C1 can be useful to predict response to VAD in MM. Fifty-two uniformly treated MM patients were investigated for 8 common SNPs including MDR1 exon 12 C1236T, MDR1 exon 21 G2677T/A, MDR1 exon 26 C3435T, CYP3A4*1B, CYP3A5*3, GSTP1 A313G as well as NR3C1 bcl I and NR3C1 N363S polymorphisms. Genotypes were identified using direct sequencing or RFLP methods. The response to VAD was estimated after administration of 3 courses of VAD, and patients who achieved complete or partial remission of MM were assigned as responders to VAD. In the cohort of included MM patients there were 39/52 (75%) responders to VAD. The analysis of the prognostic impact of the genotyped SNPs showed that carriers of P-glycoprotein gene MDR1 2677G allele had a significantly greater probability of achievement of the response to VAD as compared to the non-carriers, odds ratio(OR)=8.0, 95% confidence interval (95%CI)= 1.2–52.9, Fisher exact test p=0.031). Moreover, the predictive effect of MDR1 2677G allele was further increased by the presence of glucocorticoid receptor NR3C1 gene 363N allele. The carriers of both MDR1 2677G and NR3C1 363N alleles had 8.4-fold greater probability to achieve complete or partial remission with 3 courses of VAD (OR=8.40, 95% CI=1.8–40.0, p=0.008). Taken together, these data support the hypothesis that analysis of common functional polymorphisms in drug transporters, metabolizing enzymes and receptors may be useful for individualization of the initial therapy of MM. Particularly MDR1 G2677T/A and NR3C1 N363S SNPs can be considered as determinants of response to VAD therapy.