bluuebunny/crossref_metadata_2025 huggingface dataset:
A Cross-Sectional MRI T2* Study of Pancreatic and Pituitary Hemosiderosis in 180 Thalassemia Major Patients in Hong Kong.
Authors: Au Wing Y. , Lam Wynnie , Chu Winnie , Yuen Hui-leung , Ling S.C. , Li S.H. , Chan M.H. , Lee K.K. , Law M.F. , Liu Herman , Liang Raymond , Ha Shau-yin
Published: October 2019

Background: The use of magnetic resonance imaging (MRI) for organ specific iron assessment has allowed better tailoring of chelation therapy. Since endocrine failure is common in thalassemia major (TM) patients, we explored the utility of rapid T2* MRI assessment of hemosiderosis in the pancreas and pituitary. The results were correlated with standard T2* heart and liver MRI assessments and clinical data.

Material and methods: A total of 180 TM patients (M:F = 91:89, median age, range 12–48) were scanned on a 1.5 T scanner. (Sonata, Siemens Medical, Erlanger, Germany). T2* myocardium was assessed by a cardiac gated single breath hold 8-echo sequence (CMRtools; London, UK). The T2* liver, pancreas and pituitary were performed by a breath hold 20-echo sequence. Subcutaneous deferoxamine was used for chelation, except for addition of deferiprone in 24 cases for 1 year.

Results: There was a high incidence of hemosiderosis of heart (severe T2*<12ms: 34%, mild-moderate <20ms, 15%) and liver (severe T2*<1.4ms, 14% mild-moderate <6.3ms, 63%). Iron overload above normal control was commonly found in the pancreas (T2*<23ms, 84%) and pituitary (T2*<5.9ms, 24%). Pancreatic T2* correlated with pituitary T2* (p=0.007, r=0.2), cardiac T2*(p<0.001, r=0.33), liver T2* (p<0.001, r=0.35), ferritin (p=0.004, r=−0.19) and age (p=0.033, r=0.16). Similarly pituitary T2* related to cardiac T2* (p<0.001, r=0.36) and liver T2* (p=0.026, r=0.17). On multivariate analysis, however, pancreatic T2* related to both heart T2* (p<0.001) and liver T2* (p=0.001), while pituitary T2* only related to heart T2* (p<0.001). Documented complications amongst the cases included heart failure (ejection fraction EF<55%, n=28, 16%), hypogonadism (n=84,47%), diabetes mellitus (n=44, 25%), hypoparathyroidism (n=16, 9%) and hypothyroidism (n=36, 20%), with hepatitis B and C carrier state in 2% and 25% respectively. On univariate and multivariate analysis, all 4 endocrine failures correlated with only cardiac T2* results (all p<0.001) and age (all p<0.001), but not with pituitary or pancreatic T2* results. The EF correlated with T2* of pituitary, pancreas and heart, but only MRI heart T2* correlation remained significant on multivariate analysis.

Conclusions: MRI pituitary and pancreatic evaluation is viable in a cohort of poorly chelated Chinese thalassemia major patients on subcutaneous deferoxamine treatment. However, an abnormal cardiac T2* result is a good surrogate for endocrine iron overload and appeared more relevant in predicting endocrine complications.
Inhibition of Akt/Survivin Pathway Synergizes the Antileukemia Effect of Nutlin-3 in Acute Lymphoblastic Leukemia Cells.
Authors: Zhu Ningxi , Gu Lubing , Zhou Muxiang
Published: October 2019

PI3k/Akt and p53 pathways are known to play anti- and pro-apoptotic roles in cell death, respectively. High level of PI3k/Akt activation by loss of PTEN expression and inactivation of p53 by overexpression of MDM2 are associated with cancer cell growth and progression. Here, we report that inhibition of PI3k/Akt either by PI3k inhibitor Ly294002 or by expression of PTEN synergizes the MDM2 antagonist nutlin-3 in inducing apoptosis in acute lymphoblastic leukemia (ALL). First, we tested the effect of nutlin-3 on induction of p53 and apoptosis in a set of ALL cell lines with wild-type (wt) p53 and MDM2 overexpression. The p53 was induced by nutlin-3 in all cell lines tested but induction of apoptosis was different in cells with distinct PTEN status. Nutlin-3 induced potent apoptosis in cell lines with PTEN expression but not in cell lines without PTEN expression. Consistent with the apoptotic effects, nutlin-3 significantly downregulated expression of survivin in PTEN-positive cells but not in PTEN-negative cells. When these nutlin-3 resistant cells were simultaneously treated with the PI3K inhibitor Ly294002 or pre-transfected with PTEN gene, their sensitivity to nutlin-3 was increased with a concomitant downregulation of survivin. Furthermore, direct silencing of survivin by siRNA increased the apoptotic effect of nutlin-3 on PTEN-negative ALL cells. Taken together, our results suggest that Akt-mediated survivin upregulation in PTEN-negative ALL cells attenuate nutlin-3 induced apoptosis, and combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach in the treatment of refractory ALL.
Defining the Role of Monocytes in Heparin-Induced Thrombocytopenia (HIT): Insights from a Murine HIT Model.
Authors: Rauova Lubica , Cines Douglas B. , Poncz Mortimer
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is an iatrogenic disorder seen in 1–5% of patients exposed to unfractionated heparin. One unusual feature of HIT is that affected patients often have only moderate thrombocytopenia yet suffer severe, life-threatening thrombosis. We have previously defined the importance of PF4:glycosaminoglycan (GAG) antigenic complexes on the surface of circulating platelets in the development of thrombocytopenia using mice that express varying levels of human PF4 (hPF4+/+) as well as Fc γRIIA on the platelet surface after infusing the animals with a monoclonal antibody (KKO) that demonstrates HIT-like properties. Because HIT antibodies have been reported to activate monocytes to produce tissue factor in vitro, we further examined their role both in vitro and in vivo in the murine HIT model. Monocytes bind PF4 onto their surface forming antigenic PF4:GAG complexes recognized by KKO. Monocytes express antigenic complexes at low levels of PF4, a setting in which none can be demonstrated on platelets. Furthermore, monocytes express antigenic complexes at heparin concentrations that completely dissociate antigen from the surface of platelets. We then investigated the role of monocytes in a murine model of HIT. Monocytes were depleted by IV injection of 200 μL of clodronate-containing liposomes (Encapsula Nano Sciences) 12 hrs prior to IP injection of 200 μg of KKO. Over 92% of circulating monocytes were depleted for >24 hrs with a return to near baseline by 72 hrs. In co-transgenic hPF4+/+/FcγRIIA mice, clodronate did not cause a significant fall in platelet count at 4 hrs, but counts fell to 30 ± 14% of baseline by 24 hrs vs. 70 ± 23% of baseline after injection of control liposomes or no liposomes (p<0.001), thrombocytopenia persisted for 72 hrs. Nevertheless, monocyte depletion inhibited thrombosis in the HIT model. Rose Bengal (500 mg/kg) was injected and the right carotid artery injury was exposed to 3 mW green (540 nm) light for 5 min followed by an IV injection of KKO (500 μg/kg). 4 of 5 mice that did not receive liposomes and 4 of 5 mice that received control liposomes 16–24 hrs prior to KKO developed thrombosis, in contrast to 1 of 5 clodronate liposome-treated mice (p<0.01 vs. all controls) in spite of having platelet counts of 860,000 ± 185,000/μL. These studies show that antibody mediated activation of monocytes contributes to the initiation of HIT, while intravascular activation of platelets contributes to the development of thrombocytopenia. These studies also suggest that depletion of monocytes may be a novel target for therapeutic intervention in the early stages of the disease.
Toxicities of Intravenous (IV) Pegasparaginase (ONCASPAR®) in Adults with Acute Lymphoblastic Leukemia (ALL).
Authors: Advani Anjali , Earl Marc , Douer Dan , Rytting Michael , Bleyer Archie
Published: October 2019

Background: With the multiple reports of asparaginase-containing regimens used in pediatric ALL therapy achieving a greater survival rate that non-asparaginase treatment regimens used in adult patients, asparaginase therapy is now being increasingly applied in chemotherapy regimens for adults with ALL. One reason for this resurgence is the availability of a long-acting form of the enzyme, pegylated asparaginase, and more recently, flexibility in administration of pegasparaginase via either intramuscular or intravenous routes (Oncaspar®). Given an initial impression in the 1970s that adults were more vulnerable to the toxicities of asparaginase than were children, we assessed the initial experience of intravenous asparaginase in adults with ALL.

Methods: The intial experience with pegylated asparaginase at the University of Southern California (USC), Cleveland Clinic, and University of Texas M.D. Anderson Cancer Center were compiled and compared between institutions and with published results in pediatric patients.

Results (Table): In 76 adult patients administered 192 doses of pegasparaginase in combination with other chemotherapy agents for ALL, hepatotoxicity was most common, with grade 3–4 elevation of serum liver enzymes and grade 3–4 hyperbilirubinemia in 36% and 14% of the patients, respectively. Hyperglycemia and chemical pancreatitis were next most common, having occurred at grade 3–4 levels in 25% and 5% of patients, respectively. Grade 3–4 toxicities in the 5–10% range were thrombosis, hypofibrinogenemia, nausea/vomiting, and fatigue. Grade 3–4 allergy/hypersensitivity, neuropathy, and CNS ischemia were reported in 1–5% of patients.

Conclusions: Intravenous pegasparaginase is hepatotoxic in ∼1/3 of adult patients and has a variety of other, non-hepatic toxicities in <10% of patients, of which the most common are pancreatitis, thrombosis, nausea/vomiting and fatigue. Intravenous pegasparaginase has a toxicity profile, in combination with other chemotherapy agents used in ALL therapy, in adult patients that similar to that in pediatric patients, and warrants increased use in adult patients with ALL.

Grade 3–4 Toxicities of IV Pegasparaginase in Adults USC Cleveland Clinic MD Anderson Total Median Age (Years) 28 37 20 33 Age Range (Years) 17–57 20–68 14–28 17–68 No. Doses / Patients 81 / 45 41 / 18 70 / 13 192 / 76 % Patients with Grade 3–4 Toxicity Elevated liver enzymes 31% 28% 62% 36% Hyperbilirubinemia 13% 22% 15% 14% Hyperglycemia 27% 17% 31% 25% Elevated serum amylase 0% 0%R 0% 5% Fatigue 7% 0% 0% 7% Thrombosis 4% 6% 6% 0% Hypofibrinogenemia 0% 28% 28% 0% Elevated PT/INR 0% 0% 0% 7% Bleeding 0% 0% 0% 8% Nausea/vomiting 2% 17% 17% 1% Allergy/hypersensitivity 0% 0% 0% 1% Neuropathy 2% 0% 0% 4% CNS ischemia 0% 0% 15% 3%
Nilotinib in Patients (pts) with Relapsed/Refractory Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL) Who Are Resistant or Intolerant to Imatinib.
Authors: Ottmann Oliver G. , Larson Richard A. , Kantarjian Hagop M. , le Coutre Philipp , Baccarani Michele , Haque Ariful , Gallagher Neil , Giles Francis
Published: October 2019

Background: Ph+ ALL occurs in about 15% of adults with ALL and is characterized by a poor prognosis. Imatinib in combination with systemic chemotherapy has become the standard therapy for adults with Ph+ ALL. Nilotinib is a highly specific BCR-ABL inhibitor, which is 30-fold more potent than imatinib and active in vitro against 32 of a panel of 33 BCR-ABL mutations. Nilotinib was demonstrated in a phase I study to be active against a small subset of imatinib-resistant Ph+ ALL adult pts. Based on this preliminary data, a phase II, open-label study was designed to further evaluate the safety and efficacy of nilotinib in pts with relapsed/refractory Ph+ ALL.

Methods: The initial dose of nilotinib was 400 mg twice daily (BID) with escalation to 600 mg BID, when there was an inadequate response and no safety concerns.

Results: The safety and efficacy data were reported for 41 Ph+ ALL pts in which 38 had relapsed and 3 were refractory [(37 had active disease; 4 had minimal residual disease (MRD)]. The median age at study entry was 46 (18–75) years and 54% were men. At study entry 31% of pts had >35% Ph+ metaphases; chromosomal abnormalities other than Ph+ were noted in 32% (13/41) pts, and extramedullary involvement was present in 3 pts. The median time since initial diagnosis of Ph+ ALL was 15 months. Treatment with nilotinib is ongoing for 2 pts (5%). Most discontinuations were due to disease progression (68%). The median treatment duration was 53 (1–563) days and the median dose intensity was 800 mg/day. Thirty-one percent of pts had >35% of Ph+ metaphases, chromosomal abnormalities other than Ph+ were noted in 32% of the pts, and extramedullary involvement was present in 7% of pts. Complete response (CR) was reported in 10 (24%) pts including 1 patient with MRD. Hematological improvement was observed in 1 (3%) pt, stable disease was observed in 9 pts, 14 pts had progressive disease. The most common Grade 3/4 laboratory abnormalities were thrombocytopenia (60%), neutropenia (43%), anemia (21%), and hypophosphatemia (22%).

Conclusions: Nilotinib as a monotherapy appears to have promising activity and a favorable safety profile in relapsed/refractory Ph+ ALL pts. The use of nilotinib in combination with chemotherapy for Ph+ ALL merits further investigation.
PR1 Peptide Vaccine-Induced Immune Response Is Associated with Better Event-Free Survival in Patients with Myeloid Leukemia.
Authors: Qazilbash Muzaffar H. , Wieder Eric D. , Thall Peter F. , Wang Xuemei , Rios Rosa L. , Lu Sijie , Kant Shreya , Giralt Sergio , Estey Elihu H. , Cortes Jorge , Komanduri Krishna , Champlin Richard E. , Molldrem Jeffrey J.
Published: October 2019

Background: PR1 is a nonomeric HLA-A2-restricted peptide derived from the myeloid leukemia-associated antigens, proteinase 3 and neutrophil elastase. PR1-specific cytotoxic T lymphocytes (PR1-CTL) selectively kill MDS, AML and CML and contribute to complete cytogenetic remission. We conducted a phase I/randomized phase II trial to evaluate the safety and efficacy of a PR1 peptide vaccine to elicit PR1-CTL in leukemia patients.

Methods: Sixty-six HLA-A2+ patients with acute myeloid leukemia (42), chronic myeloid leukemia (13) or myelodysplastic syndrome (11) were treated with PR1 peptide vaccine. The first 54 patient received three vaccinations, and the last 12 patients received 6 vaccinations. PR1 was injected with incomplete Freud’s adjuvant (Montanide ISA) subcutaneously, at 3 week intervals at one of three dose levels: 0.25, 0.5 or 1.0 mg per vaccination. GM-CSF at a dose of 75 mg was injected subcutaneously into the same site. Immune response to the vaccine was defined as a ≥ 2-fold increase in peripheral blood PR1-CTL. Fifty-three patients had measurable disease and 13 were in complete remission at enrollment.

Results: The vaccine was well tolerated, with toxicity limited to grade I and II injection site reactions. PR1-specific immune response was observed in 25/53 (47%) patients with measurable disease. Clinical responses were observed in 9/25 (36%) immune responders versus 3/28 (10%) immune non-responders (p=0.03). The PR1 vaccine-induced immune response was associated with a longer event-free survival, 8.7 months vs. 2.4 months (p = 0.03), and a trend towards longer overall survival. Among the 13 patients treated in complete remission, 4 have remained in remission for a median of 30.5 months (range 11–59 months). This includes continued complete remissions in 3/10 who were IRV+, and 1/3 who were IRV-. PR1-specific immune response lasted for up to 4 years in some patients. In a multivariate model, low bone marrow blast count (<10%) emerged as a significant predictor of an immune response and a longer event-free survival (p=0.001).

Conclusion: PR1 peptide vaccine-induced immune response is associated with a clinical response and better event-free survival in patients with myeloid leukemia, who had measurable disease at vaccination. Patients with ≤10% bone marrow blasts are likely to obtain the maximum benefit.

Figure Figure
Whole Genome Association Study in Leukemia: Frequent Loss of Heterozygosity (LOH) and Single Nucleotide Polymorphism (SNP) in Acute Myeloid Leukemia with a Normal Karyotype.
Authors: Lee Young Y. , Park Joowon , Yoon Sung-Soo , Ahn Kwang-Sung , Choi Jung H. , Park Byoung B. , Kim Byoung K.
Published: October 2019

Acute myeloid leukemia (AML) is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis is generally recognized as the single most valuable prognostic factor. However, using conventional cytogenetic techniques, karyotype abnormalities are detected in only half of all AML cases and the other half are commonly described as normal-karyotype AML. Patients with normal-karyotype AML are classified as an intermediate risk group with a 5-year overall survival of between 35% and 45%, but clinical outcome may vary greatly. Also, classical cytogenetic methods are limited in resolution and dependent on highly skilled labor. Therefore, the appropriate choice of additional treatment in patients who attained first remission (chemotherapy versus autologous transplantation versus allogeneic transplantation) is unclear for these patients. Thus, additional markers with prognostic significance are needed to identify clinically relevant subgroups among AML patients with a normal karyotype. Recently, human genome-wide studies gain popularity to identify the genetic basis of complex disorders such as AML. Altered transcript levels in AML genomes are often related to copy number changes, and genome-wide detection of allelic imbalance in AML cells by polymorphic genetic markers has become an important technique to identify genetic events involved in the progression of AML. By using high density single-nucleotide polymorphism (SNP) microarrays designed to genotype more than 300K SNPs in the human genome DNA, the resolution of the whole genome scanning technique has increased considerably and allowed accurate and reproducible determination of copy number changes in whole genome of AML. It became possible to distinguish between LOH regions with underlying homozygous deletions and those with copy-neutral events. In the present study, we performed genetic changes in untreated AML with normal cytogenetics with infinium 300K SNP chip. SNP-based mapping array data and fluorescence in situ hybridization (FISH) copy number data correlated well. The most frequently identified alterations are located at 3p, 6q, 8p, 13q, 21q and 22p. LOH is found in these large regions and also in smaller regions throughout the genome with a median size of 1 Mb. Alterations was correlated with response to chemotherapy. Twenty six candidate genes showed significant evidence of linkage in the presence of disequilibrium, and ten of these were expressed in AML patients who failed to attain remission. Three other genes showing statistical evidence were not expressed. Many of the genes reported here have not been previously reported in relation to progression of AML. We show that this panel of markers adds important prognostic information for this largest subgroup in AML.
Favourable Treatment Outcome in Childhood AML Is Determined by Intact Apoptosis Signalling in Myeloid Leukaemia Cells.
Authors: Meyer Lüder H. , Dammé Manon , Eckhoff Sarah M. , Creutzig Ursula , Reinhardt Dirk , Karawajew Leonid , Ludwig Wolf D. , Stahnke Karsten , Debatin Klaus M.
Published: October 2019

Defects in cell death signalling might be responsible for treatment failure and relapse in acute leukaemia. In a study on paediatric acute lymphoblastic leukaemia (ALL) we recently reported the importance of intact apoptosis signalling for favourable outcome. Here we addressed this issue in 45 paediatric acute myeloid leukaemia (AML) patient samples. Two key apoptogenic events: cytochrome c release and caspase-3 activation were analysed by flowcytometry in the leukaemia cells after induction of apoptosis by factor deprivation. Release of cytochrome c and consecutive apoptosome formation leads to activation of downstream effector caspases such as caspase-3. Both events were closely correlated to each other in good responding patients (less than 5% blasts in bone marrow or minimal residual disease negativity) and in patients achieving remission. No correlation was found in patients with poor treatment response or patients not reaching remission. Caspases other than caspase-3 acting upstream of mitochondria (e.g. caspase-8) are also involved in initiating or amplifying this complex. By incubation in presence or absence of the pan-caspase inhibitor zVADfmk cytochrome c release was observed to be dependent or independent on upstream caspases. Of note, only cytochrome c released in dependence on amplifying caspases was observed to correlate with activated caspase-3 in patients with good response to treatment or patients without relapse. This indicates that an intact apoptosome function and active amplifying caspases are elementary for favourable outcome in childhood AML. The functional integrity of this important apoptogenic checkpoint is subsumed by the parameter caspase dependent cytochrome c-related activation of caspase-3 (CRACdep). Division of our cohort according to this new paramenter resulted in two groups of 38 CRACdep -positive and 7 -negative patients, the distribution of the values being unrelated to features such as FAB subtype or risk groups. 30 of 32 patients sustaining complete remission for more than one year were CRACdep -positive (Fisher exact test, P= .015). Analysis of survival (N=45, log rank P= .014) and remission duration (N=40, log rank P= .001) revealed a superior outcome for CRACdep -positive patients. This was also observed when considering the high risk patients only. Notably, standard risk stratified patients merely displayed CRACdep -positivity. Thus, the propensity to undergo apoptosis as reflected by intact signalling in leukaemia cells is an important feature for favourable treatment outcome and may serve as additional stratification tool for paediatric AML patients.
TGF-β Receptor Kinase Inhibitor LY2109761 Reverses the Anti-Apoptotic Effects of TGF-β1 in Leukemic Cells Co-Cultured with Stromal Cells.
Authors: Tabe Yoko , Xu Yuanyuan , McQueen Teresa , Andreeff Michael , Konopleva Marina
Published: October 2019

Transforming growth factor β1 (TGF-β1) is an essential regulator of cell proliferation, survival, and apoptosis, depending on the cellular context. TGF-β1 is also known to affect cell-to-cell interactions between tumor and stromal cells through production of the extracellular matrix and stimulation of integrin receptors. We investigated the role of TGF-β1 in the survival of human leukemic cells growing in the context of bone marrow (BM) microenvironment, and the anti-leukemia effects of the novel TGF-β receptor inhibitor LY2109761. BM-derived mesenchymal stem cells (MSC) produced TGF-β in an autocrine fashion. Treatment with rhTGF-β1 (2ng/mL) inhibited the spontaneous and Ara-C-induced apoptosis in U937 cells (% AnnexinV(+), control 34.5±8.4; TGF-β1 18.4±4.5; Ara-C 88.6±3.0; Ara-C/TGF-β1 60.4±8.0, p=0.04). These effects were more prominent in U937 cells co-cultured with MSC (% AnnexinV(+), control 19.4±2.8; TGF-β1 3.5±1.0; Ara-C 69.0±3.6; Ara-C+TGF-β1 24.9±3.3; p=0.01). In U937 cells co-cultured with MSC, rhTGF-β1 conferred higher cell protective effects on leukemia cells attached to MSC than on floating cells. Conversely, the pro-survival effects of TGF-β1 were inhibited by 5mM LY2109761 (%AnnexinV(+); MSC(−), control 31.8±2.3, TGF-β1 19.5±3.0, LY 28.4±4.4, TGF-β1+LY 37.7±2.0; MSC(+), control 22.1±1.7, TGF-β1 7.8±0.9, LY16.1±2.6, TGF-β1+ LY 18.0±1.1, p<0.01). Similar results were obtained using TGF-β1 neutralizing antibody. TGF-β1 induced pro-survival phosphorylation of Akt in U937 cells cultured alone or co-cultured with MSC, which was abrogated by LY2109761. Further, rhTGF-β1 induced a moderate increase in C/EBPβ gene and LAP isoform (cell cycle arrest inducing) of C/EBPβ protein in U937 cells cultured without MSCs, while markedly upregulating C/EBPβ gene and protein, both LIP (cell proliferation inducing) and LAP isoforms, under MSCs co-culture condition, suggesting the novel role of C/EBPβ in TGF-β1-mediated U937 cell survival. In summary, these results indicate that TGF-β1-secreting BM stromal cells promote the survival of U937 leukemia cells via direct cell-to-cell interaction and promote chemoresistance of leukemia cells through the activation of Akt signaling and upregulation of C/EBPβ. The blockade of TGF-β signaling by LY2109761, which effectively inhibited the pro-survival signaling, may enhance the efficacy of chemotherapy against leukemia cells in the BM microenvironment.
Membrane Type-Matrix Metalloproteinases (MT-MMPs) Are Expressed in Acute Myeloid Leukemia Cells and Are Upregulated by TNF-α.
Authors: Shirvaikar Neeta , Jalili Ali , Ilnitsky Sara , Larratt Loree , Mirza Imran , Janowska-Wieczorek Anna
Published: October 2019

Membrane type-matrix metalloproteinases (MT-MMPs) play a key role in tumor cell progression and metastasis but their role in hematological malignancy and leukemic cell dissemination is still unclear. To date, six MT-MMPs have been identified, of which four (MT1-, MT2-, MT3- and MT5-MMP) possess a trans-membrane domain that tethers the enzyme to the plasma membrane and two (MT4- and MT6-MMP) are anchored to the cell surface via a glycophosphatidyl inositol domain. MT-MMPs are known to activate pro-forms of MMPs. We examined the expression of all six MT-MMPs in 13 myeloid and lymphoid cell lines using RT-PCR, Western blotting and FACS. We found that

MT1-MMP was expressed on most of the cell lines tested, MT2-MMP was expressed in myeloid cell lines such as THP-1, HEL, K562 and U937, and in T cell lines such as Jurkat and CEM, but not in any of the tested B cell lines, MT3-MMP was not expressed in any of the cell lines except for THP-1, MT4-MMP was strongly expressed in all myeloid but not lymphoid cell lines and MT5-MMP and MT6-MMP were expressed in most of the myeloid cell lines.

Next, we examined the expression of all six MT-MMPs in primary AML samples and found that MT1-MMP is expressed in 20 out of 24 AML patient samples as detected by RT-PCR and Western blotting, MT2-MMP and MT4-MMP were expressed in 21 and 22 out of 24 samples, respectively, and MT6-MMP was expressed in 17 out of the 17 samples tested. In contrast, MT3- and MT5-MMPs were not found in the primary AML samples. Zymographic analysis showed that the pro form of MMP-2 was secreted in media conditioned by AML blasts and became activated only when these AML blasts were co-cultured with BM stromal cells. Since TNF-α is endogenously secreted by AML blasts we next stimulated these cells with human recombinant TNF-α and found strong upregulation of MT1-MMP and MT6-MMP. Moreover, zymography demonstrated that TNF-α-stimulated AML blasts are more potent in activation of pro-MMP-2 than unstimulated cells, indicating that activation may occur via upregulation of MT-MMPs. Furthermore, we found that TNF-α stimulation led to stronger upregulation/induction of MT1- and MT2-MMPs in CD34+ cells than in CD34- cells isolated from AML patients. In conclusion, we report here for the first time that MT1-, 2-, 4- and 6-MMPs are expressed in AML blasts and suggest that these MT-MMPs may contribute to the invasive phenotype of this malignancy.
Maintenance Chemotherapy (MC) after Autotransplantation in First-Remission Acute Lymphoblastic Leukemia (ALL): Single-Center Experience of 100 Patients.
Authors: Sirohi Bhawna , Powles Ray , Treleaven Jennifer , Kulkarni Samar , Saso Radovan , Potter Mike , Ethell Mark , Morgan Gareth , Singhal Seema , Mehta Jayesh
Published: October 2019

Results of autotransplantation in adult ALL are poor. MC, a component of standard ALL therapy, is usually not employed after autografting. 100 adults with ALL in CR1 received melphalan 110 mg/m2 with TBI followed by marrow (n=35) or melphalan 200 mg/m2 followed by blood stem cells (n=65) between 7/1984 and 2/2003. Daily 6-MP 25 mg was started when WBC and platelets reached 3 and 100 × 109/L respectively, and its dose increased gradually. Weekly methotrexate (MTX) 5 mg was added when the 6-MP dose reached 75 mg. Until 1995, monthly vincristine (1.4 mg/m2) and prednisolone (40 mg/m2 daily for a week) (VP) were given if 6-MP and MTX were not tolerated. After 1995, VP was given routinely. Starting MC was delayed only by inadequate counts, relapse, or medical problems that could get exacerbated. 6-MP and MTX doses were adjusted to maintain ANC >1 × 109/L. MC was stopped 2 y after starting 6-MP. 6 patients, all TBI and marrow recipients, died of toxicity (P=0.001). The 7-y cumulative incidence (CI) of relapse was 45%. The 7-y DFS and OS probabilities were 45% and 48%.

Age ≥30 y (49 patients), >4 wks to attain CR (20 patients), and karyotypes t(4;11) and t(9;22) (14 patients) were associated with higher relapse and lower DFS/OS. Patients with 0 (standard-risk; 42 patients), 1 (intermediate-risk; 36 patients), and 2–3 (high-risk; 22 patients) adverse features had 7-y CI of relapse of 19%, 53%, and 82% (P<0.0001), and 7-y DFS probabilities of 73%, 36%, and 7% (P<0.0001). 70 patients received 6-MP, 53 received MTX and 40 received VP. 22 patients received no MC at all, 14 received 1 agent, 43 received 2 agents, and 21 received 3 agents. MC did not result is any life-threatening toxicity. Amongst the 86 patients alive in CR at 4 months, the 7-year CI of relapse was 58%, 64%, 41%, and 23% for those receiving 0, 1, 2, and 3 MC agents (P=0.015). The corresponding 7-y DFS was 15%, 29%, 58%, and 61% (P=0.014). Patients getting MC had better outcome than those getting none. Patients getting 2/3 agents had better outcome than those getting 0/1 agents. MC intensity was an independent determinant of outcome in Cox analysis. The curves below show the effect of MC intensity.

When analyzed by disease risk, the benefit of MC was confined to patients with low- or intermediate-risk disease. While we cannot rule out the possibility that the ability to tolerate MC may have been a surrogate marker for a better-quality remission, the safety of post-transplant MC and its results are encouraging. We conclude that MC reduces relapse and improves DFS in non-high-risk ALL after autotransplantation, and this strategy should be incorporated as standard practice if an autograft is performed in ALL.

Figure Figure Figure Figure
Autologous Peripheral Blood Stem Cell Transplantation (PBSCT) Combined with Haploidentical Lymphocytes Infusion Increases the Long Term Survival of Adult Acute Myeloid Leukemia with Standard Risk.
Authors: Wu Bingyi , Kunyuan Guo , Hu Haiyan , Yang Yuliang , Deng Lan , Wu Jing
Published: October 2019

OBJECTIVES: The long term survival of adult patients with acute myeloid leukemia with standard risk factor which treated by autologous peripheral blood stem cell transplantation is about 30–50%. We designed a new therapy to improve the long term survival of adult patients with acute myeloid leukemia with standard risk factor. After completely remission, patients underwent 2–3 courses of consolidation chemotherapy. Autologous peripheral blood stem cell transplantation (PBSCT) was performed. After hematology recovery, haploidentical lymphocytes irradiated with 7.5 Gy were infused into patients. Here we evaluate the outcome of this approach in adult patients with acute myeloid leukemia with standard risk.

METHODS: From July 1998 to July 2007, twenty-four patients including 17 males and 7 females, median age 36 year old (range 14–58) with acute myeloid leukemia with morphology M2 subtype and normal karotype received this approach therapy. Autologous peripheral blood stem cells were collected by aphereses and froze in −198° and storied after 2–3 courses of median dose cytarabine consolidation chemotherapy The autologous stem cells were grafted to patients following the conditioning with the BuCy regimen (Bu16mg/kg and CTX 120mg/kg). Haploidentical lymphocytes obtained by leukapheresis from sibling donors or other relatives were irradiated with 7.5 Gy and were infused into patients after hematopoietic reconstitution.

RESULTS: A median dose of 2.5×108/kg, nucleated cells/kg (range, 2.97×107/kg-6.0×108/kg)containing 5 × 106 CD34+ cells/kg (range,1.5– 4.8) were autografted. All patients were got hematopoietic reconstitution. The median time for granulocyte recovery >0.5 × 109 /L was 11 days and for platelets >20 × 109/L was 14 days following transplantation. All patients were administrated a median dose of 2.0×108/kg haploidentical lymphocytes irradiated with 7.5 Gy. Severe acute GVHD occurred in two patients. One died with severe acute GVHD. Hematopoietic reconstitution delayed in four patients. No other side effects were observed. For the whole group of 24 patients, the median overall survival rate (OSR) was 83.7%. The median disease-free survival (DFS) time was 28.3 moths (range 2–120 m) and the 5-year DFS rate was 50%. The 2-year OS rate was 71%. After a median follow-up of 5.0 years from transplantation, the median DFS and OS were 28.3 years respectively, and the 3-year rates were 50%. Nineteen patients were still in continuous complete remission, 4 patients had relapsed and 3 had died.

CONCLUSIONS: Autologous peripheral blood stem cell transplantation (PBSCT) combined with haploidentical lymphocytes infusion may prolong the long term survival of adult acute myeloid leukemia with standard risk.
The Declining Risk of ABO Incompatibilities: Twelve Years of Hemovigilance in France.
Authors: Renaudier Philippe , Mai Mai Phuong Vo , Schlanger Sylvie , Ounnoughene Nadra , Breton Pascal , Cheze Stephane , Girard Albane , Hauser Lisette , Legras Jean Francois , Rebibo Danielle , Waller Chantal , Lassale Bernard , Caldani Cyril
Published: October 2019

Background: ABO incompatibilities (ABOi) usually result from the failure to comply with Standard Operating Procedures (SOP). Continuous training (CT) is the main way to ensure their proper use in ultra-high security systems like civil aviation but little is known for transfusion. In France, SOP include a Beth-Vincent test at the bedside before the transfusion of Packed Red Cells (PRC). At the hospitals, Hemovigilance Officers (HO) are in charge of transfusion safety along with the settlement of traceability and the notification of all Adverse Effects (AE). If SOP and CT are requested by Law since 1993 for blood banks, they are only advised for hospitals even though the bedside Beth-Vincent test requires a special training. However, HO have more and more transposed the model of blood banks in hospitals especially since 2000. Objective: to describe time-trends of ABOi observed in e-fit, the French Hemovigilance database. Population and methods: All AE with “acute ABOi” as the final diagnostic were considered. Time-trends were studied according to the Box and Jenkins model (ARMA).

Results: From 07/01/1994 to 12/31/2005, 79 106 AE were registered in e-fit, corresponding to about 30,000,000 Labile Blood Products (LBP) transfused. 304 (0.4%) were ABOi (1/107,850 LBP transfused). There were 183 men and 121 women with 81.3% of whom more than 40 years old. 205 patients (67.4%) received only PRC, 73 (24.0%) Platelet Concentrates (PC), 22 (7.2%) Fresh Frozen Plasma (FFP) and 4 (1.3%) more than a single LBP. 18 ABOi were fatal, possibly (4 cases) probably (4 cases) or definitely (9 cases) associated with a PRC, and 1 doubtfully with a FFP. Time-trends displayed 3 periods: (1) from 1994 to 1997 ABOi increased concurrently with the frequency of all AE notifications, (2) from 1997 to 2000 they reached a steady-state with 35 ABOi/year on average (1/70,000 LBP), (3) since 2001 they regularly decreased of 14%/year on average (13 ABOi for 2005 = 1/198,450 LBP). The localization of the failure to comply with SOP (i.e. hospital, blood bank or both) was studied for PRC. From 1997 to 2005, the liability of blood banks alone or along with hospitals remained stable (resp 0.42 and 2.58/year on average) whereas those of hospitals dramatically decreased (16.25/year from 1997 to 2000 and 8.6/year from 2001 to 2005).

Conclusion: (1) ABOi are now 3 times less frequent than in 2000 ; (2) a notification bias is unlikely because both hospitals and blood banks are aware of traceability data that include the ABO phenotype for both the recipient and the LBP issued ; (3) CT applied on a nationwide basis seems to be the main factor to reduce ABOi in hospitals ; (4) a residual risk related to human errors seems to exist ; (5) the way to overcome that risk remains a matter of debate.
Sistematic Analysis of ABO Gene in A3 and A3B Individuals Reveals New ABO Variants.
Authors: Domingues Alexandre E. , Novaretti Marcia C.Z. , Dorlhiac-Llacer Pedro E. , Chamone Dalton A.F.
Published: October 2019

BACKGROUND: In addition to the common ABO phenotypes, numerous phenotypes with a weak expression of the A or B antigens on red blood cells have been found. DNA genotyping on A3 e A3B subgroups has been performed in small number of samples, mainly in family studies. To date, seven ABO alleles have been associated to A3 and A3B subgroups (ABO*A301 to ABO*A307) revealing molecular heterogeneity. Our purpose was to study molecular events on exon 7 of ABO gene in 100 A3 and A3 B blood donors samples.

STUDY DESIGN AND METHODS: ABO serologic analyses of 12,283 blood donors of blood groups A and AB allowed the detection of 13 A3 and 87 A3B samples. Initially, DNA amplification was performed using sequence specific polymerase chain reaction (PCR-SSP) to identify 467C>T, 829G>A, 871G>A and 1060delC polimorphisms of exon 7 of ABO gene, followed by direct DNA sequencing.

RESULTS: New ABO variants were detected in 5/13 (38.5%) of A3 and in 38/87 (43.7%) of A3B samples. ABO*A302/ABO*A301 genotype was found in 30.7% A3 and, ABO*A302/ABO*B104 in 17.1% of A3B samples. The 871G>A mutation previously reported only in A3B individuals was found in 9 blood samples tested as A3 in our study. For the first time, mutations between 441–450 nucleotides of exon 7 of ABO gene was detected in 39 samples (single nucleotide polimorphisms and deletions) of A3 and A3B subgroups.

CONCLUSION: This is the first large molecular study of A3 and A3B samples. Sistematic analysis of A3 and A3B samples reveals new ABO variants caused by different molecular events. Mutations between 441 and 450 nt. of exon 7 are critical for transferase A expression in individuals typed as A3 and A3B.
Rapid Duffy Genotyping in Sickle Cell Disease Patients Using PCR-SSP Is a Valuable Tool for Blood Component Selection.
Authors: Novaretti Marcia C.Z. , Bonifacio Silvia L. , Medeiros Vitor R. , Gualandro Sandra F.M. , Dorlhiac-Llacer Pedro E. , Chamone Dalton A.F.
Published: October 2019

Background: Sickle cell disease (SCD) patients are frequently transfused and, finding adequate and safe blood component for them has a great impact in transfusion services and in patients clinical follow-up. DNA genotyping for RBC antigens is a tool for the management of these patients to overcome the limitations of hemagglutination assays. However, the polymerase chain reaction (PCR) by restriction fragment length polymorphism (RFLP) is time consuming. The aim of this study was to develop a rapid PCR specific sequence primer (SSP) approach for Duffy genotyping in SCD patients.

STUDY DESIGN AND METHODS: Blood samples from 91 SCD patients were studied by hemagglutination and by PCR-SSP for Duffy system. All samples typed as Fy(b-) were submitted to adsorption and elution techniques to detect the weak expression of Fyb antigen. DNA was extracted by a commercial DNA extraction kit (QIAamp DNA, QIAGEN, Germany). Detection of FY*A, FY*B, FY*B-33, FY*B-265T and FY*B-298A were performed using specific primers by PCR-SSP. DNA amplification was carried out in a final volume of 20μL with identical reaction mixture and cycling conditions as follows: 0.2μg of genomic DNA, 0.2mM of each dNTP, 5% of glycerol, 1.5 units of platinum Taq polymerase (Invitrogen, Brazil) in the buffer supplied, 5.0mM of MgCl2 and 10.0 pmoles of each primer. PCR fragments were separated for 90 minutes at 100V using 0.5μg.mL-1 ethidium bromide-stained 2.0% agarose gel and visualized in a UV light apparatus (Eagle Eye II, EUA).

Results: Table 1 shows the results of Duffy phenotyping, Duffy genotyping and gene frequencies in SCD patients The most common phenotype found in our study was Fy(a–b+), 35.2%. In three samples (3.3%) we detected weak Fyb antigen and identified FY*B-265T alelle. All samples phenotyped as Fy(a–b–) genotyped as (FYB-33/FYB-33).

CONCLUSION: PCR-SSP is a method that contributes for easily and accurate Duffy genotyping for SCD patients. In our study, all SCD patients with Fy(a–b–) can be safely transfused with Fy(b+) red blood cell components with no risk for developing anti-Fyb saving rare Fy(a–b–) units.

Table 1 Phenotype Genotype n Genotype Frequency Fy(a+b+) FYA/FYB 16 0.176 Fy(a–b+) FYB/FYB 23 0.253 FYB/FYB-265T 9 0.099 Fy(a+b–) FYA/FYA 18 0.198 FYA/FYB-33 4 0.044 Fy(a–b–) FYB-33/FYB-33 21 0.231 TOTAL 91 1.00
Risk Factors for Blood Product Usage Following Sibling Allogeneic Haemopoietic Stem Cell Transplantation (allo-HCT).
Authors: Hurst Nicola , Mullighan Charles G. , Heatley Sue L. , Robinson Kathryn , Danner Silke , Hahn Uwe , Szer Jeff , Shuttleworth Peter , Hoyt Rosie , Schwarer Anthony P. , Muirhead Jenny , Bradstock Kenneth , Miller Lisa , Bardy Peter G.
Published: October 2019

Red cell and platelet transfusion requirements have been reported to be lower following reduced intensity conditioning allogeneic hematopoietic stem cell transplantation (RIC allo-HCT) when compared to myeloablative (MA) allo-HCT. However, previous studies examined RIC regimens with lower haemopoietic toxicity than many of the regimens in use today. We investigated risk factors for red cell and platelet transfusion in patients enrolled prospectively in an Australian study investigating the non-HLA immunogenetics of sibling allo-HCT. The transfusion requirements in the first year post transplant were reviewed for 122 patients transplanted between 2002 and 2006 in three Australian transplant centres. Seventy-one patients received MA and 51 RIC regimens. Using regression analysis, the outcome variables of total red cell and platelet units transfused were analysed. The factors age, transplant centre, disease, transplant type (RIC v MA), days of neutropenia, death within 12 months, disease risk (high risk (HR) v standard risk (SR)) and ABO mismatch underwent univariate analysis. Associated variables with p<0.2 were included in a multivariable analysis. Duration of neutropenia, disease risk, death within 12 months and transplant centre were significantly associated with higher red cell and platelet usage (p<0.0001). Transplant type was not associated with transfusion requirement. Each additional day of neutropenia resulted in a 9% increase in number of red cell units transfused and 11% of platelet units. HR MA patients used an estimated 17 units of red cells compared to 12 units for SR. HR RIC patients used an estimated 26 units compared to 10 for SR RIC patients. HR patients used an estimated 16.9 platelet units compared to 8.5 for low risk. These data highlight the importance of disease risk and degree of myelosuppression as key risk factors for blood product usage following allo-HSCT.
Protein Tyrosine Phosphatase Receptor Type O (PTPRO) Exhibits Tumor Suppressor Properties in K562 Cells by Dephosphorylating Bcr-Abl.
Authors: Majumder Sarmila , Motiwala Tasneem , Ghoshal Kalpana , Kutay Huban , Datta Jharna , Lucas David , Byrd John C. , Jacob Samson T.
Published: October 2019

Regulation of protein phosphorylation by concerted action of protein kinases and phosphatases is important for normal physiological processes. Altered function or expression of one or more components of these regulatory molecules leads to many pathological conditions including cancer. We have previously shown that the truncated form of the receptor-type protein tyrosine phosphatase PTPROt predominantly expressed in haematopoietic cells is suppressed in Chronic Lymphocytic Leukemia (B-CLL). A direct correlation of CpG island methylation and reduced expression of the gene was observed in primary CLL samples and in several leukemia cell lines. To assess the functional significance of loss of PTPROt function in leukemia, we selected K562 cells as a model system, as PTPROt expression is silenced in these cells and is reactivated upon treatment with DNA hypomethylating agents. Ectopic expression of the catalytically active PTPROt inhibited growth of K562 cells and their clonogenic survival in soft agar (a characteristic of cancer cells). Further, cells expressing PTPROt exhibited delayed entry into S-phase from G0/G1 phase. Induction of apoptosis increased significantly in K562 cells expressing functional phosphatase upon serum withdrawal or exposure to the apoptogenic agent camptothecin. Tumorigenic potential of K562 cells in athymic nude mice was also significantly reduced upon ectopic expression of PTPROt. Finally, we demonstrate that the Bcr-Abl fusion protein, product of abnormal chromosomal translocation [t(9;22)] in chronic myelogenous leukemia, is a substrate of PTPROt. Tyrosine phosphorylation of this potent kinase was markedly reduced in K562 cells expressing the catalytically active PTPROt. Enhanced dephosphorylation of Bcr-Abl by PTPROt both in vivo and in vitro explains the observed phenotypes of the PTPROt expressing K562 cells. These data taken together delineate the molecular mechanism of tumor suppressor function of PTPROt in leukemic cells characterized by Philadelphia chromosome.

(This work was supported by a grant CA101956 from the National Institutes of Health).
Protein Disulfide Isomerase Is Required for Fibrin Generation and Platelet Thrombus Formation In Vivo.
Authors: Cho Jaehyung , Furie Barbara C. , Coughlin Shaun R. , Furie Bruce
Published: October 2019

Thiol isomerases catalyze disulfide oxidation, reduction and isomerization, playing an important role during protein synthesis. Recent studies suggest a role for protein disulfide isomerase (PDI), a prototype of the thiol isomerase family, in platelet function and regulation of tissue factor activity (Essex and Li. Curr Drug Targets. 2006; Chen and Hogg. J Thromb Haemost. 2006). To determine the role of intravascular PDI during thrombus formation, PDI expression, platelet accumulation, and fibrin generation were monitored following laser-induced arteriolar injury in the mouse cremaster muscle by intravital fluorescence microscopy. PDI antigen exhibited a time-dependent increase in the developing thrombus after vessel wall injury and remained associated with the thrombus. Infusion of bacitracin, a non-specific inhibitor of thiol isomerases, into the circulation inhibited platelet thrombus formation and fibrin generation in a dose-dependent manner. Infusion of a function-blocking monoclonal antibody to PDI (RL90) into the circulation of a wild type mouse also resulted in dose-dependent inhibition of platelet accumulation and fibrin generation. To determine whether PDI inhibits fibrin formation by blocking tissue factor activation, or by preventing platelet activation and the development of the membrane surface that is required for assembly of the tenase and the prothrombinase complex in vivo, we explored fibrin formation in mice lacking protease-activated receptor-4 (Par4). Although there is no stable accumulation of platelets and no platelet activation, fibrin formation is normal in the Par4 null mouse (Vandendries et al, Proc Natl Acad Sci USA. 2007), suggesting that fibrin generation in the laser-induced vessel injury model is independent of platelet activation. Infusion of the function-blocking anti-PDI antibody (RL90) into the circulation of a Par4 null mouse prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required to generate tissue factor in a form that leads to thrombin generation and fibrin formation during thrombus development and is required for thrombus formation.