bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Antigen Mismatched Related Donor Allogeneic Stem Transplants Have Similar Outcomes as Matched Unrelated Donor Allogeneic Stem Cell Transplants: A Single Center’s Experience.
Authors: Newton Benjamin , Sprague Kellie A. , Klein Andreas K. , Klingemann Hans G. , Chan Geoffrey W.
Published: October 2019

We retrospectively reviewed transplant outcomes of 32 patients who received an allogeneic stem cell transplant from single human leukocyte antigen (HLA) mismatched related family donors (MMRD), and compared them to 45 case-matched controls who received transplants from matched unrelated donors (MUD). Controls were matched for age, sex, disease type, disease status, CMV seropositivity, and type of conditioning regimen (full versus reduced intensity). Full donor neutrophil engraftment for MMRD and MUD transplants were achieved in 81% and 93% of patients respectively (p=0.1). Grade 2–4 acute graft-versus-host disease (GVHD) developed in 32% of MMRD transplants versus 42% for MUD transplants (p=0.43). Limited and extensive chronic GVHD developed in 22% and 19% of MMRD transplants versus 14% and 30% of MUD transplants (p=0.61). Day 100 transplant related mortality (TRM) for MMRD and MUD transplants were 31% and 33% respectively (p=0.69). Disease relapse occurred in 16% of MMRD transplants versus 13% in MUD transplants (p=0.87). Overall survival at 1 and 3 years for MMRD transplants were 53% and 40% versus 49% and 32% for MUD transplants (p=0.61). There were no statistically significant differences among HLA-A, HLA-B, and HLA-DR MMRD transplants in terms of neutrophil engraftment and grade 2–4 acute GVHD, but day 100 TRM among HLA-DR MMRD transplants were higher than among HLA-A and HLA-B MMRD transplants (50% versus 0% and 17% respectively, p=0.01). Overall survival at 3 years was 53% for HLA-A and HLA-B MMRD transplants, 30% for HLA-DR MMRD transplants, and 32% for MUD transplants (p=0.42). In conclusion, single antigen mismatched related donor allogeneic stem cell transplants have similar transplant outcomes as matched unrelated donor allogeneic stem cell transplants and usually takes a shorter amount of time to stem cell donation. HLA-DR mismatch transplants have higher day 100 TRM than Class I mismatch transplants. Single antigen mismatched related donors should be considered as donors for allogeneic stem cell transplantation.

MMRD vs MUD Transplants MMRD MUD P-value MMRD=mismatch related donor; MUD=matched unrelated donor N n=32 n=45 Median age 44 years 41 years P=0.14 High-Risk Disease 63% 71% P=0.51 CMV Seropositivity 45% 42% P=0.93 Full Conditioning 72% 76% P=0.72 Neutrophil Engraftment 81% 93% P=0.1 Grade 2–4 aGVHD 32% 42% P=0.43 Day 100 TRM 31% 33% P=0.69 Disease Relapse 16% 13% P=0.87 3-year OS 40% 32% P=0.6 Figure Figure
Autologous Stem Cell Transplantation as First-Line Therapy in Peripheral T-Cell Lymphomas. A Prospective Multicenter Study.
Authors: Reimer Peter , Ruediger Thomas , Schertlin Tobias , Geissinger Eva , Weissinger Florian , Einsele Hermann , Mueller-Hermelink Hans Konrad , Wilhelm Martin
Published: October 2019

Peripheral T-cell lymphomas (PTCL) are rare diseases representing only 10–15% of all non Hodgkin’s lymphomas and show a poor outcome following conventional chemotherapy. Long-term remissions are achieved in only 15 to 35% of patients following conventional chemotherapy. However, the impact of more aggressive therapeutic approaches such as high-dose therapy with autologous stem cell transplantation (ASCT) as first-line therapy is poorly defined mainly due to the lack of prospective PTCL-restricted studies. Therefore, in 2000 we initiated the first prospective PTCL-restricted multicenter study in PTCL. The results of the first 30 patients (pts) have recently been published. Here we update our data on all pts. entering the study.

Study design: Pts. < 65 years with PTCL of all subtypes without primary cutaneous lymphoma and ALK1+ anaplastic large cell lymphoma were included. Treatment consisted of 4–6 courses of CHOP followed by DexaBEAM or ESHAP regimens before collection of stem cells. Subsequently, pts. underwent total body irradiation (TBI) and high dose cyclophosphamide (60 mg/kg body weight) chemotherapy with ASCT.

Patient characteristics: From 6/00 to 7/05 75 pts. (65% male) with a median age of 50 years were enrolled. Main subtypes were Peripheral T-cell lymphoma not otherwise specified (NOS, 41%) and Angioimmunoblastic T-cell lymphoma (AIL, n= 31%). According to the Ann Arbor classification, 75% of the pts had stage III/IV disease. The International Prognostic Index (IPI) was low/low intermediate in 51% and high intermediate/high in 49% of pts, respectively.

Results: So far 65 pts are eligible for evaluation. Forty pts could be transplanted (62%). After a median follow-up of 10 months post-transplant 22 pts (42%) are in sustained remission and 8 pts (15%) have relapsed. Treatment-related mortality was 2/65 (3%, one secondary AML, one multiorgan failure). Twenty-five pts (38%) did not proceed to ASCT mainly due to progressive disease (n= 18). Treatment-related toxicity was comparable to other high-dose studies in B-cell lymphomas. The IPI does not seem to have a significant impact on response to therapy, progression during CHOP therapy, or relapse rate following ASCT.

Conclusion: Our data confirm the feasibility and efficacy of first line ASCT following myeloablative radiochemotherapy in PTCL. A sustained remission seems achievable for a significant proportion of pts. However, additional treatment strategies are required to prevent early progression before myeloablative therapy. Longer follow up is necessary to confirm long term remission rates.
Efficacy of High-Dose Therapy and Autologous Stem Cell Transplantation for Chemoresistant Hodgkin’s Lymphoma.
Authors: Metcalfe Tracee L. , Gooley Ted A. , Press Oliver W. , Pagel John M. , Petersdorf Stephen H. , Bensinger William , Holmberg Leona , Maloney David G. , Gopal Ajay K.
Published: October 2019

High-dose therapy (HDT) with autologous stem cell transplantation (ASCT) is the standard treatment for patients (pts) with chemosensitive relapsed or refractory Hodgkins lymphoma (HL). Unfortunately, there are few established options for HL pts with chemoresistant disease and data regarding the efficacy of HDT and ASCT in this setting are limited. One hundred and seventy-two patients underwent HDT followed by ASCT for HL at our centers between 1971 and 2005. Of these, 66 pts (38.6%) had chemoresistant disease defined as < 50% reduction in the size of the tumor with the chemotherapy regimen immediately preceding HDT. The baseline characteristics of these pts included: median age = 34 years (yrs) (range, 14–59 yrs), male = 39 (59%), stage I/II= 15 (23%), stage III = 17 (26%), Stage IV = 34 (51.5%), ≥ 5cm tumor bulk = 23 (35%), > 1 extranodal site of disease (ENS) = 13 (20%), median # of prior regimens = 2 (range 1–5), and prior radiation (RT) = 34 (52%). Histologies included nodular sclerosis (NS) in 53 (80%), mixed-cellularity in 11 (17%), and lymphocyte predominant in 2 (2%). Twenty-seven pts (41%) received TBI-based preparative regimens and 39 (59%) underwent non-TBI conditioning with the most common chemo-only regimen of bulsulfan, melphalan, thiotepa used in 19 pts.The estimated 5-year overall (OS) and progression-free survival (PFS) of this cohort was 31% and 18%, respectively with a median follow-up of 2.6 yrs among surviving pts (Figure). Twenty-two (56%) of the 39 deaths were due to relapse and 17 (44%) resulted from non-relapse causes including: 7 infections, 5 pulmonary, 1 cardiac, 1 hepatic, 1 renal, and 2 (5%) unknown causes. Multivariable modeling utilizing the above baseline criteria identified factors that were statistically significantly associated with inferior OS and PFS. These included: NS histology (OS: HR=3.8, p=0.03; PFS: HR=2.5, p=.03), non-TBI conditioning (OS: HR=2.6, p=0.009; PFS: HR=2.1, p=.02), 2 or more ENS (OS: HR=4.1, p=.0005; PFS: HR=3.7, p=0.0008); and male gender (OS: HR=2.0, p=.05; PFS: HR=2.0, p=.03) These data suggest that HDT and ASCT may result in prolonged remissions and survival for a significant minority of chemoresistant HD pts, with improved outcomes observed in those treated with TBI-based regimens, non-NS histology, female gender, and fewer ENS, though novel improved therapies for these patients are still required.

Figure Figure
Differential Expression of Hem1 and CTLA-4 in B-CLL Patients with High and Low CD38 Expression.
Authors: Joshi Avadhut D. , Dickinson John D. , Lynch James C. , Bierman Philip J. , Bociek Gregory R. , Devetten Marcel P. , Armitage James O. , Joshi Shantaram S.
Published: October 2019

B chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the elderly with heterogeneous clinical outcome. Rai stage, immunoglobulin heavy chain mutation status, Zap-70 expression, CD38 expression and chromosome abnormalities are used as prognostic markers. Among these, CD38, a differentiation marker of T lymphocytes, has been reported to have a role in B-CLL pathogenesis. High CD38 expression is associated with an unfavorable clinical course with advanced disease stage, poor responsiveness to therapy, short time to first treatment, and shorter survival, whereas B-CLL patients with low CD38 expression required minimal or no treatment, remained treatment free for longer time and had better survival. However, the molecular basis for the association of CD38 expression and clinical course of B-CLL patients is not known. Therefore, in this study we classified 35 B-CLL patients into CD38 high (>30%) and CD38 low (<30%) expressions, and the clinical course was correlated with gene expression profile using oligonucleotide DNA microarrays consisting 10,000 genes. There was a significant difference in CD38 expression levels in patients with good clinical outcome (mean CD38 ~ 20%) compared to poor clinical outcome (mean CD38 ~ 46%). Median time to treatment with high CD38 expression was 30 months compared to 69 months for low CD38 expressing patients. Significance analysis of microarray (SAM) identified 52 differentially expressed genes. From these genes, Hem1 and CTLA-4 were further studied because the Hem1 expression is limited to hematopoietic cells and CTLA-4 is involved in modulation of immune response. Also, CTLA-4 is a member of the immunoglobulin superfamily involved in the cell cycle regulation and prolongs the progression through the G1 phase of the cell cycle. The differential expression of Hem1 and CTLA-4 was also confirmed by semi-quantitative RT-PCR. Hem1 was found to be over expressed in B-CLL patients with higher CD38 expression compared to lower CD38 expression, whereas CTLA-4, was over expressed in B-CLL patients with lower CD38 expression compared to patients with high CD38 expression thus confirming the microarray results. Furthermore, to determine whether the expression levels of these two genes are associated with disease progression in B-CLL, log rank test was applied across the expression of Hem1 and CTLA-4 and disease progression. The higher expression of Hem1 was associated with shorter time to treatment (Figure 1), whereas lower expression of CTLA-4 was associated with shorter time to treatment (Figure 2). Thus, these results while confirming the prognostic value of CD38 expression, demonstrate the possible molecular basis of CD38 mediated clinical course in B-CLL patients involving key genes such as Hem1 and CTLA-4.

Figure 1 Figure 1. Figure 2 Figure 2.
Congenital Dysfibrinogenemia - Clinical Manifestations in Relation to the Fibrinogen Gene Mutation.
Authors: Miesbach Wolfgang A. , Galanakis D. K. , Scharrer Inge
Published: October 2019

The congenital dysfibrinogenemia is a rare clotting disorder which is based on a structural defect in the fibrinogen molecule. Approximately 350 cases of unrelated families with dysfibrinogenemia have been reported. Of these, the structural defects have been determined in nearly 170 families.

Patients and methods:

We describe the clinical manifestations in 50 patients from 19 families from Frankfurt. Up to now the underlying structural defect could be found in 43/50 (86 %) of the patients and 14/19 (74 %) of the families (Galanakis, New York, von Depka, Hannover).

Results:

In 19 families, 50 patients were found with dysfibrinogenemia (52% female, age median: 52 years-old, 9 - 89 years old). The fibrinogen level, according to Clauss decreased in median with 51 mg% (15 – 86 mg/dl, norm: 150 – 450 mg/dl). The determination of the other fibrinogen levels resulted in the normal range. Fibrinogen according to Schulz: 240 mg/dl, immunological fibrinogen: 244 mg/dl. The Reptilase time was prolonged to 55 sec (median, norm: − 20 sec.). 50% of the patients stated a distinct bleeding tendency and mostly a tendency for haematoma (59%) and secondary haemorrhaging (45%). 13 patients had a thrombotic disease, of which 80% were arterially caused. 12 % of the patients stated a tendency for bleeding as well as for thrombosis. 19% of the patients had miscarriages, partly recurrent and 18/50 (36 %) of the patients were asymptomatic. Furthermore, it was observed that the clinical symptoms are often similar in the affected families. By administration of fibrinogen concentrates, the pregnancy in 4 patients who suffered from abortions in the first weeks of pregnancy resulted in healthy children. Fibrinogen was administered from the beginning of the pregnancy till the delivery (aiming fibrinogen levels of Clauss up to 100 mg%). In 11 of the families, an Aalpha 16 Arg-Cys mutation was found. In the other three families there were found gamma mutations, e.g., gamma 356 Ala-Thr, gamma 318–319 del and gamma 318 Ap-Gly. The clinical manifestations in the families with Aalpha 16 Arg-Cys mutation were bleeding (65 %), thrombosis (22 %) and abortions (16 %). 25 % were asymptomatic and 12 % of the patients experienced both, bleeding and thrombosis.

Conclusion:

The investigations of the Frankfurt patient group with dysfibrinogenemia showed a high rate of clinical manifestations, mostly the tendency for haematoma and secondary haemorrhaging. The tendency for thrombosis is concerned rather with the arterial vessels. The high tendency for miscarriage is striking, which makes the administration of fibrinogen concentrate during pregnancy necessary. The most common found fibrinogen mutation was at the Aaplha chain in position 16. In patients with this mutation mostly both, bleeding and thrombosis occurred. Thus, management of patients with this mutation may be complicated by the variablility of clinical manifestations of congenital dysfibrinogenemia.
The Prothrombin C>T Mutation at Position 20209 (F2 20209*T) Promotes 3′end mRNA Processing and Thus Contributes to Thrombophilia through Gain-of-Function.
Authors: Danckwardt Sven , Hartmann Kathrin , Ben-Tal Ofira , Hentze Matthias W. , Kulozik Andreas E.
Published: October 2019

The functional analysis of the common prothrombin (F2) 20210*A allele has recently revealed gain-of-function of 3′end processing as a novel genetic mechanism predisposing to human disease. Based on these observations we sought for the functional impact of a novel C>T mutation at position 20209 (F2 20209*T). This mutation affects the penultimate nucleotide of the 3′ untranslated region (UTR) of the F2 mRNA and has been reported to be associated with fetal loss, intrauterine growth retardation and a history of venous thrombosis. We have therefore systematically analyzed the functional effect of this polymorphism and show that the F2 20209*T mutation up-modulates the F2 mRNA expression and protein expression by ~1.4 fold which corresponds to the observation that the reported patients with the F2 20209*T allele tend to have moderate F2 level elevations, slightly above - but also within - the upper normal-range.

Furthermore, because of the critical position of the F2 20209*T allele we have analyzed the putative impact of this mutation on 3′end mRNA formation and show that this mutation represents a gain-of-function mutation, causing increased cleavage site recognition, increased 3′ end processing and increased mRNA accumulation neither affecting the poly(A) tail length nor the site of cleavage. Thus, this novel mutation falls into a general category of mutations in the F2 gene which contribute to thrombophilia by interfering with a tightly balanced architecture of non-canonical 3′end formation signals. This, together with previous results, demonstrates that the F2 cleavage site and adjacent parts of the 3′ flanking sequence represent an error-prone “vulnerable region” in which N>T mutations may give rise to clinically relevant gain-of-function phenotypes.
Proteomic Analysis of Gray Platelet Syndrome by iTRAQ Labelling and Mass Spectroscopy: A Potential New Diagnostic Strategy for Platelet Disorders.
Authors: Perez-Pujol Silvia , Anderson Lorraine B. , Martinez Michael B. , Higgins LeeAnn , White James G. , Nelsestuen Gary L. , Key Nigel S.
Published: October 2019

The recent introduction of novel proteomic techniques to evaluate normal human platelet proteins and their modifications following activation may also help to elucidate the causes and improve diagnoses of platelet-related bleeding disorders. Previous studies from this and other laboratories have demonstrated the sensitivity and reliability of this new technique to detect minor changes (+/−20%) in the protein content of platelets. More than 600 proteins shared by normal platelets were identified, and >200 in platelet-derived microparticles (MPs) generated by ionophore or Thrombin Receptor Activating Peptide (TRAP) activation. As a prelude to our ultimate goal of establishing this new technology, we applied the iTRAQ method (multiplexed relative protein quantitation by mass spectrometry) in the study of two unrelated patients with the Gray Platelet Syndrome (GPS).

Megakaryocytes of patients with GPS can synthesize alpha-granule proteins and enclose them within membranes. However, the membranes lack structure linked latency, and enclosed proteins leak out of the organelles before platelets are delivered to circulating blood. Thus, GPS platelets contain empty alpha-granule vacuoles instead of alpha-granules. Due to large changes in platelet properties, it was surprising to find that cytoskeletal proteins of GPS were present in similar abundance to normal platelets (i.e. in the range of 0.80–1.20). Receptor proteins showed a similar distribution. As expected, the content of most alpha-granule proteins in the platelets from the 2 GPS patients was markedly reduced, with individual protein abundance ratios of 0.10 to 0.52 compared to normal platelets. However, certain trans-membrane proteins of alpha-granules (such as vesicle-associated proteins and P-selectin) were better preserved in GPS platelets, with ratios in the range of 0.73 to 1.16 compared to normal platelets. These results showed that many structural components (such as cytoskeleton and receptor proteins) are unchanged in GPS platelets. The results support our previous data indicating that detection of a change of as little as 20% may indicate a pathological change in the platelet proteome that may impact normal function. Our results therefore demonstrate that highly sensitive methods, capable of detecting small protein changes, may be needed to fully appreciate pathological disorders. The iTRAQ technique is able to detect low level changes and may be a leading tool that can generate new insights into the molecular pathophysiology and genetic variations of platelet disorders.
Clinical Characteristics of Autosomal Dominat Giant Platelet Syndromes (GPS) and Mutation Analysis of MYH9 Encoding Non-Muscle Myosin Heavy Chain from 6 Unrelated Korean Patients.
Authors: Kook Hoon , Yang Jung Mi , Kee Hae Jin , Eom Gwang Hyeon , Baek Hee Jo , Hwang Tai Ju , Cho Duck , Shin Myung-Geun , Lee Je Jung , Kim Hyeoung Joon , Lee Jun-Hyuk , Eom Soo Hyun , Kook Hyun
Published: October 2019

Autosomal dominant GPS is characterized by thrombocytopenia, giant platelets and Doehle-like inclusion bodies. Studies have indicated that platelet structure and function are normal, suggesting a defect in megakaryocyte fragmentation. This study was aimed to identify Korean patients with GPS and define clinical findings and molecular characteristics on them. Twenty-two patients from 6 unrelated families were identified. Giant platelets, measuring greater than red cells on blood smear were found to be 3.1% (1–11%). The median platelet count was 61,000/μL. Doehle-like inclusion bodies were found in 4 families, while renal impairment in 2. MYH9 in each family was analyzed by direct sequencing of exons or cDNAs. Among 6 families, 5 families were found to have the mutations: Arg1933Ter, Trp33Cys, Lys373Asn, Ile1816Val, Arg1165Cys located in 41st, 2nd, 11th, 38th, 27th exons, respectively. All of the mutations are found only in the affected patients, but not in the normal siblings. Arg1933Ter and Lys373Asn were reportedly associated with GPS and Arg1933Ter is known to make the protein unstable. The novel mutations, Trp33Cys and Ile1816Val, were placed in highly conserved amino acids in mammals and Drosophila, and even in other types of myosin heavy chain. Computer-aided modeling analysis shows that Trp33Cys is located in the proximal portion of the myosin head, serving as a dock for actin binding, while Ile1816Val is in the distal portion of myosin tail, which may act to form polymer with other myosin heavy chains. Isolated mononuclear cells or the stable cell lines originated from Trp33Cys, Lys373Asn and Arg1165Cys patients were subjected to immunoblot. Compared to their normal siblings, the 220 kDa bands in the soluble fraction were decreased in those patients, while the bands were increased in the insoluble pellet. However, Northern blot analysis revealed that transcript levels were not altered in the samples from patients. Trp33Cys, Lys373Asn, and Arg1165Cys also produced aberrant 140 kDa bands in addition to normal 220 kDa MYH9 band in the insoluble fractions. After immunoprecipitation with MYH9 antibody, the 140 kDa bands were analyzed and turned to be actinin. These results suggest that novel mutations found in GPS are directly associated with the disease and that the phenotype might be caused by the solubility or instability of the proteins.
A New Gene Which Regulates Platelet Dense Granules.
Authors: Gautam Rashi , Rusiniak Michael E. , Chintala Sreenivasulu , Tan Jian , Li Wei , Novak Edward K. , Reddington Madonna , Swank Richard T. , McGarry Michael P.
Published: October 2019

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disease characterized by defects in trafficking of intracellular vesicles to lysosome-related organelles such as melanosomes, lung lamellar bodies and platelet dense granules. The ashen (rab27a) mouse coat color mutant maintained at Roswell Park Cancer Institute (ash-Roswell) has been proposed as an animal model of HPS based upon prolonged bleeding times, decreased platelet aggregation and greatly decreased platelet dense granule components, including serotonin and ADP. However, human patients with Rab27a deficiency (Griscelli Syndrome) and other isolates of mouse ashen mutants do not exhibit prolonged bleeding suggesting an additional mutation in a gene affecting platelets but not melanocytes in the ash-Roswell mouse. In order to identify this mutation, a backcross between ash-Roswell and the PWK control strain was analyzed. In 390 backcross progeny, deficient platelet serotonin and diluted coat color (Rab27a) phenotypes segregated independently and in the proportions expected for single gene traits. The gene for serotonin deficiency was narrowed to a 2.1Mb region of mouse chromosome 10 containing 15 genes. None of these genes have been implicated previously in platelet storage pool deficiency. These studies indicate this genomic region contains a gene which uniquely controls platelet dense granules and is a candidate gene for a new form of human HPS.
S1P1 Overexpression Inhibits SDF1/CXCR4 Dependent Migration and In Vivo Homing of Hematopoietic Stem Cells.
Authors: Ryser Martin F. , Ugarte Fernando A. , Lehmann Romy , Malech Harry L. , Roesler Joachim , Roesen-Wolff Angela , Brenner Sebastian
Published: October 2019

Hematopoietic stem cell (HSC) migration to stromal cell-derived factor (SDF) -1 correlates with CXC chemokine receptor 4 (CXCR4) expression and bone marrow homing. Activation of Sphingosine-1-Phophate (S1P) receptors (S1P1–5) modulate chemokine induced migration of lymphocytes and HSC. S1P1 partial activation and internalisation after binding of FTY720, a S1P analogue, leads to trapping of lymphocytes in lymphoid tissue. To analyze the influence of S1P1 on SDF-1 dependent migration in HSC we cloned S1P1 cDNA into an oncoretroviral vector containing an EMCV-IRES GFP reporter gene (MFGS-S1P1-IRES-EGFP). Transduction of mobilized peripheral blood CD34+ hematopoietic stem cells (PBSC) and Jurkat cells with MFGS-S1P1-IRES-EGFP derived virus particles resulted in transgene overexpression in more than 95% of target cells. A significantly higher amount of S1P1 transgene positive PBSCs acquired an adherent phenotype compared to GFP control-transduced cells. Flow cytometric analyses revealed a 3 fold downregulation of CXCR4 expression on S1P1 overexpressing PBSCs and Jurkat cells. SDF-1 dependent migration of S1P1 overexpressing PBSCs or Jurkat-cells in transwell assays was inhibited by more than 90% compared to control transduced cells. In contrast retrovirus mediated overexpression of S1P2 or S1P3 in Jurkat cells did not show a significant influence on SDF-1/CXCR4 dependent migration. SDF-1 dependent ERK1/2 phosphorylation was abolished in S1P1 overexpressing PBSCs, suggesting that Gi-protein signaling pathways are involved in the inhibition of migration. Sublethally irradiated NOD/SCID mice were transplanted with ex vivo cultured 6 day old PBSC which were either transduced with MFGS-S1P1-IRES-EGFP to 95% or with MFGS-EGFP to 85%. Screening for GFP positive human cells in the mouse bone marrow 18 hours after transplantation revealed a 5 to 10 fold reduction (p<0,01) in homing of MFGS-S1P1-IRES-EGFP transduced cells compared to MFGS-EGFP control transduced cells. Our data suggest that high levels of S1P1 act as a downregulator of CXCR4 expression and as a strong inhibitor of CXCR4 dependent migration in HSCs.
Vaccination with MAGE-3 Protein Can Induce a Potent Immune Response in a Healthy Donor Which Can Be Adoptively Transferred Via Stem Cell Transplant (Tx) to a Multiple Myeloma (MM) Patient.
Authors: Gnjatic Sacha , Szmania Susann , Moreno Amberly , Cottler-Fox M. , Shaughnessy John , Barlogie Bart , Old Lloyd , Tricot Guido , van Rhee Frits
Published: October 2019

A MAGE-3 positive, stage IIIA MM patient with a twin donor provided a unique opportunity to study if vaccination with MAGE-3 protein induces immune responses in the healthy twin, and if these responses can be adoptively transferred to the patient and expanded by booster vaccines. The patient was treated with MEL 200 tandem Tx (Tx1: auto, Tx2: syngeneic) and MAGE-3 recombinant protein in ASO2B adjuvant. The MAGE-3 negative, identical twin donor received 3 vaccines and was leukapheresed (LP1) one week after the 3rd vaccine to store vaccine-induced immune cells. Donor G-CSF mobilized stem cells (Tx2) were transfused fresh to the patient after MEL conditioning. Donor LP1 was transfused to the patient 21 days post Tx2 and followed by 8 booster vaccines. The donor continued vaccinations and LP2 was collected one week after the 8th vaccine and transfused to the patient who had received 4 of 8 planned booster vaccines. Ab responses to MAGE-3 and controls NY-ESO-1, LAGE-1, MAGE-1, MAGE-4, and p-53 were assessed by ELISA. MAGE-3 specific CD8+ T-cell responses to autologous EBV transformed cells pulsed with pooled 20-mer overlapping peptides from MAGE-3 were assessed by ELISPOT. Neither twin had pre-existing anti-MAGE-3 humoral immunity. The donor twin developed MAGE-3 ab up to 1/6400. The patient had a detectable anti-MAGE-3 titer post donor Tx2 (1/400) which was boosted by subsequent vaccinations (1/1600). After the transfer of LP2 and vaccines 5–8, the patient had MAGE-3 ab titers similar to those in the donor. There was low but significant cross-reactivity only to MAGE-4. A vaccine-induced CD8+ cellular response to a previously undescribed MAGE-3115–123, HLA-A68 restricted epitope was observed in the donor and patient. MAGE-3115–123 is in the same region as (but is distinct from) two other known HLA-A2 and -B40 epitopes. Further studies with recombinant MAGE-3 protein and tetramers are in progress to study if this epitope is indeed naturally processed and presented. The patient is in CR nearly 1 yr post Tx2. We show for the first time that vaccinating a healthy donor with a defined cancer-testis antigen can induce both cellular and humoral responses and that such responses can be transferred and expanded post Tx in the recipient. Close inspection of the ab curves shows that humoral immunity was transferred with the donor stem cells (Tx2) and expanded in the patient by further booster vaccines. We could not establish with certainty that LP1 and LP2 contributed further to transferring humoral or cellular immunity. It is hoped that this approach may be used to induce a specific anti-MM immune response after autologous or allogeneic Tx and reduce MM relapse.

Figure Figure
The Role of Integrin αDβ2 in Macrophage Migration.
Authors: Yakubenko Valentin P. , Ugarova Tatiana P.
Published: October 2019

Integrin αDβ2 (CD11d/CD18), the most recently discovered member of the β2 sub-family of adhesion receptors, is strongly upregulated on macrophage foam cells which underscores its potential role in atherosclerosis. However, the contribution of αDβ2 to monocyte/macrophage adhesive reactions and the significance of its overexpression on these cells remain unknown. Recently we characterized αDβ2 as a multiligand receptor capable of binding many extracellular matrix proteins with the recognition specificity overlapping that of the major myeloid-specific integrin αMβ2 (Mac-1). We hypothesized that the αDβ2 ability to bind numerous ligands in the extracellular matrix and its capacity to be upregulated to high density on the surface of macrophages may modulate cell adhesiveness and, thus, affect migration. To evaluate the role of αDβ2 in migration, we generated model and natural cells expressing different densities of αDβ2 and tested their migration to different extracellular matrix proteins. In vitro studies demonstrated that αDβ2 expressed at low densities, either on the surface of HEK293 cells or the mouse macrophage cell line IC-21, supported migration which was partially inhibited by anti-αD function-blocking antibodies. Furthermore, β1 and β3 integrins expressed on HEK293 cells and IC-21 macrophages, respectively, contributed to migration because anti-β1 and anti-β3 antibodies inhibited migration. Increased expression of αDβ2 on the surface of HEK293 cells and its upregulation by PMA on IC-21 macrophages resulted in the inhibition of cell migration. Ligation of αDβ2 with anti-αD antibodies restored β1- and β3-driven cell migration by means of removing restraints imposed by the excess of the αDβ2-ligand adhesive bonds. To test the possibility that progressive upregulation of αDβ2 can block macrophage migration in vivo, we assessed the effect of anti-αD function blocking antibodies using the thioglycollate-induced peritonitis model. More than 4-fold upregulation of αDβ2 was detected on macrophages in 72 h after thioglycollate stimulation and, similar to in vitro studies, the numbers of migration macrophages increased in the presence of anti-αD antibodies. These results demonstrate that the density of αDβ2 can modulate cell migration and suggest that low levels of αDβ2 can contribute to monocyte migration while αDβ2 upregulation on differentiated macrophages might facilitate their retention at the site of inflammation.
mafB Deficient Mouse Results in Impaired Macrophage Differentiation.
Authors: Hamada Michito , Moriguchi Takashi , Morito Naoki , Zhang Chuan , Hasegawa Kazuteru , Yoh Keigyou , Kuroda Etsushi , Yokomizo Tomomasa , Engel James D. , Yamamoto Masayuki , Takahashi Satoru
Published: October 2019

MafB is one of the large Maf (musculoaponeurotic fibrosarcoma) proteins sharing a structurally similar basic region/leucine zipper DNA binding motif and a putative activation domain at the N-terminus. MafB is known to be expressed in the myelomonocytic lineages of hematopoietic cells. The recent in vitro experiments showed that the forced MafB expression induced monocytic differentiation of myeloblast progenitors, suggesting MafB could be the critical determinant in monocyte/macrophage differentiation. Nevertheless, the precise expression pattern and the physiological function of MafB in hematopoietic cells have never been analyzed in vivo setting. To address these issues, we have generated mafB locus GFP knock-in mouse. In E14.5 mafB GFP knock-in heterozygous embryos, GFP expression was specifically observed in F4/80 (differentiated macrophage marker) positive fetal liver cells. In homozygous mutant, expression of F4/80+ was significantly decreased in the neonatal liver, spleen and peripheral blood. Interestingly, the colony assay using E14.5 fetal liver cells revealed that the number of macrophage colony was significantly increased in homozygous mutant under the M-CSF supplemented condition, whereas the colony size was dramatically reduced, suggesting MafB deficiency prevent the terminal macrophage differentiation of fetal liver cells, then evoked the abnormal accumulation of premature progenitors. In the macrophages of homozygous mutant obtained by in vitro fetal liver differentiation, F4/80 expression and phagocytotic activity was dramatically suppressed compared to the wild type control. These observations strongly suggest that MafB is indispensable for the differentiation and the functional maturation of macrophages.
Outcome of a Risk-Adapted Treatment for Childhood Acute Lymphoblastic Leukemia in Thailand.
Authors: Nuchprayoon Issarang , Seksarn Panya , Vanichsetakul Preeda
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most curable cancer in children. In developing country with limited resource and manpower like Thailand, where 3 pediatris hematologists take care of 100 new cancer children per year, it is a challenge to achieve satisfactory outcome. We treated ALL according to a risk-adapted modified CCG-105 protocols (standard or intensive induction of remission, consolidation, 1800 cGy CNS radiation, and maintenance therapy with or without delayed intensification, DI) since 1988. We reported here the outcome of childhood ALL treated with this protocol between 1997–2004. 181 children with newly-diagnosed ALL in this period were classified into B-precursor, or T-cell ALL by immunophenotyping with flow cytometry. B-precursor ALL were classified as low risk group (age 1–10 yr and initial white cell count (WBC) <20,000/μl), standard-risk (age 1–10 yr and WBC between 20,000–50,000/μl) and high-risk (age >10 yr or WBC >50,000/μl). The low-risk group were assigned to protocol B (CCG-105 standard induction without DI, n=69). The standard- and high-risk group were assigned to protocol C (CCG-105 intensive induction with DI, n=71). All T-cell ALL (n=21) were assigned to a T-cell protocol (DFCI 85-01 high-risk group) which includes high-dose methotrexate and asparaginase. Infantile ALL (n=9), mature B-cell ALL (n=3), a child with t(1;19), and 5 children who refused treatment or received other protocols of treatment were excluded from analysis. The remission rates were 98.5% for protocol B, 92.4% for protocol C, and 83.3% for T-cell disease. All remission failure were attributed to early deaths. The 5-yr event-free survival (EFS ±95% confidence interval) was 83.1 ± .7% for low-risk group treated with protocol B, 75.9 ± 6.0% for protocol C, and 68.6 ± 12.1% for T-cell ALL. These outcomes are significantly higher than our previous report. (Nuchprayoon I, Songnui T, Vanichsetakul P, Seksarn P. Treatment of childhood acute lymphoblastic leukemia in Thailand- outcome and cost analysis. Blood 96 (11 suppl 1): 435a) and can be attributed to a better risk classification, more intensified treatment for standard-risk group and T-cell ALL, and better supportive care. Satisfactory outcome of childhood ALL can be achieved in developing country with risk-adapted treatment strategy.

Figure Figure
Outcome of Patients with Secondary Leukemias Managed with Different Treatment Strategies at a Single Center.
Authors: Abdelhameed Arwa A.N. , Brandwein Joseph M. , Chun Kathy , Gupta Vikas , Kamel-Reid Suzanne , Lipton Jeffrey H. , Minden Mark D. , Schimmer Aaron D. , Schuh Andre C. , Messner Hans A.
Published: October 2019

134 patients with a prior diagnosis of a malignant solid tumor presented between 1 Jan 95 and 31 Dec 04 to the leukemia service at PMH for management of a secondary hemopoietic malignancy or aplasia (AML n=99, MDS n=22, ALL n=11, AA n=2). The cohort included 65 males (median age 70 years, ranging from 18–94 years) and 69 females (median age 60 years, ranging from 25–87 years). The tumor types showed the expected gender based variation. Based on age, performance status and patient preference 81 individuals received remission induction therapy, while 53 patients were managed with supportive care. 49 of the patients undergoing induction therapy met the age criteria (70 years) for a blood and marrow transplant (BMT) and survived at least 70 days post initiation of induction therapy. 25 of the 49 patients underwent allogeneic BMT from related (n=20) or unrelated donors (n=5). The median survival of all 134 patients amounted to 314 days with an overall survival (OS) at 2 and 3 years of 25% and 20%. The respective values for patients undergoing induction therapy and supportive care were 413 days,36%,28% and 192 days, 9%,7% (p= 0.0002). Survival was strongly influenced by age (p= 0.0017), while gender (p=0.1436) and FAB subtype (p=0.219) did not contribute significantly to outcome. Subset analysis of the 25 BMT recipients showed a median survival of 922 days, with a 51% and 39.5 % OS at 2 and 3 years. The respective data for the remaining 24 not transplanted patients were not significantly different (p= 0.7508) with 522 days, 42% and 35%. Both subgroups however differed significantly in their causes of death favoring a lower relapse rate for transplant recipients and lower treatment related mortality (TRM) for non-transplanted patients (p=0.0012). In conclusion, this single center study confirms the relatively poor outcome for patients with secondary leukemias but is consistent with the view that patients able to undergo intensive therapy including transplantation may derive a significant survival benefit. The low relapse rate observed for BMT patients compared to patients not transplanted may translate into a survival benefit provided TRM can be reduced
Identification of Target Gene at 2p15 Amplification in DLBCL Using Contig Array CGH.
Authors: Fukuhara Noriko , Tagawa Hiroyuki , Kameoka Yasuhiro , Kasugai Yumiko , Karnan Sivasundaram , Morishima Yasuo , Nakamura Shigeo , Seto Masao
Published: October 2019

Genomic amplification of the 2p arm has been identified as a recurrent alteration in classical Hodgkin lymphoma, follicular lymphoma, primary mediastinal large B cell lymphoma and diffuse large B-cell lymphoma (DLBCL). We previously reported that 2p15 was gained in 25 out of 100 DLBCL patients by use of a genome-wide array-comparative genomic hybridization (array-CGH). In DLBCL with 2p amplification, genomic co-amplification of REL and BCL11A has been observed. Recent studies suggest that REL amplification is infrequently associated with nuclear REL expression and NFkB activation. In an attempt to identify the target gene at 2p15 amplification, we made BAC contig array CGH glasses for 2p15 region with 33 BAC clones covering 4.5Mb, and found that seven samples of the DLBCL with 2p amplification displayed alterations. REL and BCL11A were located within majority of the gained regions. The minimal common region of amplification was mapped to 0.5 Mb and we found that this region did not include BCL11A. To investigate the relationship between genomic gains and gene expression, we performed real-time quantitative polymerase chain reaction (RQ-PCR) analysis. The results indicated that REL, rather than BCL11A, is the target of 2p15 alterations in DLBCL.
Expression of the Wilms’ Tumor 1 (WT1) Gene Renders Acute Promyelocytic Leukemia Cells Resistant to All-Trans-Retinoic-Acid-Induced Differentiation.
Authors: Menke Aschwin L. , Knops Ruth H.J.N. , Marteijn Jurgen A.F. , Wissink Willemijn , Smeets Josie , van Piekeren Arnold , Vierwinden Gerty , de Witte Theo , van der Reijden Bert A. , Jansen Joop H.
Published: October 2019

Acute myeloid leukemia is characterized by the uncontrolled proliferation of immature cells that have lost their ability to differentiate. In the case of acute promyelocytic leukemia (AML-M3), the cells can be forced to differentiate by pharmacological dosages of all-trans retinoic acid (ATRA), a phenomenon that is successfully used in the treatment of APL patients. About 70% of the patients, suffering from PML-RARa -positive acute promyelocytic leukemia, can be cured with a combination of ATRA and anthracycline - based chemotherapy. However, relapse remains a major problem.

The molecular mechanisms by which the retinoic acid receptors mediate their biological functions have been studied extensively and although various retinoic acid-responsive genes have been identified, the target genes that are crucially involved in leukemogenesis are unknown. The Wilms’ Tumor 1 gene, has been implicated in the development of leukemia. WT1 overexpression can be detected in most acute leukemias and is particularly highly expressed in APL cells. Several groups have found an inverse correlation between the expression levels of WT1 and the overall survival of leukemia patients. The underlying mechanism, however, remains to be elucidated.

We have shown that the Wilms’ Tumor 1 (WT1) is strongly downregulated in APL cells, during ATRA-induced differentiation. Using a newly developed realtime RT-PCR method we have found that the expression levels of all four major WT1 isoforms are downregulated. To study the biological activity of each WT1-isoform, we have retrovirally transduced the APL cell line NB4, with the 4 major WT1 isoforms and analyzed the effect on ATRA-induced differentiation. Using flowcytometry and NBT staining, we show that ectopic expression of the different WT1-isoform inhibited ATRA-induced differentiation and subsequently, the apoptosis of APL cells, albeit with different potential. WT1-transduced cells survived pharmacological dosages of ATRA for more than 14 days and in some cases even continued to grow. These data indicate that downregulation of WT1 is essential for ATRA-induced differentiation of APL cells and provide an explanation why AML patients with high WT1 expression levels have worse overall survival in comparison to patients with low WT1 expression levels.
Study on DNA Methylation Status of WT1 Gene in Its Promoter Region in Hematologic Neoplasm Cell Lines.
Authors: Zhao Ye , Chen Zi-xing , Hu Shao-yan , Cen Jian-nong
Published: October 2019

The methylation at CpG island in the promoter region of a gene is one of the important epigenetic mechanism which regulates the gene activity. To study the DNA methylation pattern of WT1 gene promoter region within hematologic neoplastic cell lines and its correlation with WT1 gene expression by using the PCR-based methods. RT-PCR and Methylation-specific PCR were performed to study the WT1 gene expression in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines and the DNA methylation status in promoter region of WT1 gene. After treatment of U937 cell line by 5-aza-CdR, a demethylation inducing agent, the changes of WT1 gene expression level and the methylation status in its promter region in U937 cells was determined. Our Results showed that HL-60, K562, KG-1, NB4, SHI-1 cell lines demonstrated higher level of WT1 expression, while extremely low level was found in 8226, Jurkat, Raji, U266 and U937. The DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cell lines. The WT1 gene expression in U937 was markedly enhanced after treatment with 5-aza-CdR in company with the decrease of methylated level and the increase of unmethylated level in its promoter region. These results indicate that modulation of the DNA methylation in WT1 promoter region is one of the epigenetic mechanisms to regulate its expression.
In-Depth Mutation Scanning of Signaling Pathway Genes Involved in the Development and Targeted Treatment of Hematopoietic Malignancies.
Authors: Lilleberg Stan L. , Durocher Jeffrey , Hempel Jill , Wasserburger Carrie , Koul Manika , Troy John , Nickerson Mike L.
Published: October 2019

The Raf/MEK/ERK, Wnt/beta-catenin, JAK/STAT and PI3K/Akt signal transduction pathways have key roles in the regulation of cell cycle progression and apoptosis, and are current focal points of therapeutic development and intervention strategies for hematopoietic neoplasias. These pathways have several regulatory components that keep proliferative and anti-apoptotic mechanisms in check, but which can also drive neoplastic processes when their functions are altered by genetic and epigenetic events. Links between critical pathways are also being established and, although only partially understood, may provide important clues for the development of more efficacious therapies that target multiple pathways. Although mutations in several pathway-associated genes known to contribute to the malignant phenotype have been discovered, the mutation status of the pathway as a whole in a particular blood cancer type remains to be determined. In this study, we applied novel mutation detection technologies (DHPLC, Surveyor Nuclease, and fluorescent DNA sequencing) in a combinatorial fashion to achieve high throughput, accuracy, and sensitivity for a comprehensive screening of signal transduction pathway genes. Key targets for the analysis included genes encoding receptor tyrosine kinases, cytoplasmic kinases, GTPases, transcription factors and tumor suppressors, with emphasis on Raf/MEK/ERK and PI3K/AKT pathways. We present both somatic alterations and inherited polymorphisms (SNPs) in these genes from the analyses of AML and CML sample sets. Functional analyses will confirm the causative nature of these genetic variants and their contributions to tumorigenesis. Other groups have focused on gene families for analysis of somatic mutations in tumors, such as kinases and phosphatases. This study takes a signaling pathway approach to group gene targets for analysis. We believe that pathway-based mutation analysis strategies offer significant potential to enhance understanding of cancer initiation, disease progression, response to therapy, and mechanisms of drug resistance.