bluuebunny/crossref_metadata_2025 huggingface dataset:
Disturbed NK Cell Compartment in CML Patients with Anti-Leukemia Immune Responses Under Treatment with Imatinib Mesylate
Authors: Chen Christiane I , Maecker Holden T , Falkow Rhoda , Lee Peter P.
Published: April 2020

Imatinib mesylate has revolutionized the treatment of CML and, as an oral agent with few side effects, became the first-line therapy for patients with CML. However, patients who discontinue the drug invariably relapse. Currently, the only curative treatment for CML is allogeneic stem cell transplantation (ABMT). A major mechanism of ABMT is immunological as evidenced by the poor clinical outcome with T cell-depleted ABMT. To determine anti-leukemia immune responses in patients in remission on imatinib, blood and bone marrow samples before (12 samples) and serially after treatment (59 samples, range 10 months – 35 months) were collected and analyzed. The NK cell (CD56+CD3−) compartment in the 12 CML patients were significantly decreased before treatment (median 3,4 % vs 10 %, p<0.01) and remained low in patients in remission on imatinib (5 % vs 10 %, p< 0.01) compared to 32 age matched healthy donors. Within the CD8+ T cell compartment, naïve T cells (CD45+CD27+) were significantly higher before treatment (55.5 % vs 37.7 %, p=0.04) and decreased slowly to normal levels after successful treatment (41.5 % vs 37.7 %, p=0.28), whereas memory (CD45−CD27+) T cells presented the opposite way. They were significantly lower before treatment (21.7 % vs 31.1 %, p=0.03) and increased slowly to normal levels after successful treatment (27.5 % vs 31.1 %, p=0.39). However, the percentage of T cells (CD4+, CD8+, CD4+CD25+ regulatory T cells) and B cells were not significantly different. These results correlated (p<0.01) with T cell responses to autologous leukemia cells at the same time points, analyzed by IFN-g ELISPOT assay (median 36 SFCs vs 5 SFCs, p<0.01), and were confirmed by cytokine flow cytometry (IFN-g 3.6 % vs 0.29 % and TNF-a 6.6 vs 0.1 %). These results suggest that a significant portion of CML patients in remission with imatinib develop an anti-leukemia T cell response, whereas the NK cell compartment is disturbed. Mechanisms by which imatinib treatment leads to anti-leukemia immune responses, and the molecular targets to which these cells are directed, will be further investigated. This knowledge will be useful in the development of immunotherapy strategies against CML and may, in combination with imatinib, lead to eradication of residual leukemia cells for a durable cure.
Degrasyn (a novel tyrophostin) Impacts BCR-ABL Protein Level and Is Cytotoxic to Chronic Myeloid Leukemia Early Progenitors
Authors: Peterson Luke F , Sun Hanshi , Bornmann William , Peng Zhenghong , Maxwell David , Donato Nicholas , Talpaz Moshe
Published: April 2020

Despite the potent anti-proliferative and apoptotic effects of tyrosine kinase inhibitors (imatinib, dasatinib) on mature CML cells these compounds have limited activity on Chronic Myeloid Leukemia (CML) early progenitors (Lin-/CD38−/CD34+). Mechanisms of resistance to these agents in the early progenitor population are poorly understood but are distinct from BCR-ABL mutation induced resistance. Degrasyn (WP1130) was previously shown by our laboratory to inhibit CML cell growth and colony formation. Further structure-activity relationship studies led to the development of a more potent compound, CP2005 (CP). CP displays enhanced activity in suppressing growth and activating apoptosis in various CML and ALL cell lines (IC50 ~1 microM). Here we show that total CD34+ from CML patients are more effectively growth inhibited by CP2005 compared to WP1130. An anti-leukemic effect was also noted in primitive progenitors and was associated with the induction of apoptosis. BaF3 cells expressing an EGFP-tagged BCR-ABL protein treated with CP or WP1130 show that both facilitate translocation of BCR-ABL from the cytoplasm to a detergent insoluble compartment which subsequently leads to degradation of BCR-ABL. We observed that the Akt/mTOR pathway is activated by CP which could be partially suppressed by mTOR or PI3K/Akt inhibition (rapamycin and wortmannin, respectively). CP-induced BCR-ABL translocation was associated with a reduction in the level of tyrosine-phosphorylated Stat3 and Stat5. However, although there is activation of survival signals associated with Akt-signaling 2 microM CP is able to promote significant apoptosis (67%) as a single agent in CML early progenitors. Although imatinib was minimally effective in reducing the early progenitor cell survival, co-incubation of imatinib with CP2005 resulted in near complete induction of apoptosis in early progenitor cells (~ 93%), suggesting that the combined effects of both CP and imatinib on BCR-ABL protein degradation and kinase inhibition markedly impair the survival of CML early progenitors. The combination of BCR-ABL degradation and kinase inhibition may be effective in engaging pro-apoptotic signaling in CML early progenitor cells.
Study of Expression of Ku70 and NHEJ in CML Cells
Authors: Fu Chengcheng , Chen Peishuai , Depei Wu , Chen Zixing
Published: April 2020

Objective FThe DNA double strand breaks (DSB) in mammalian cells are predominantly repaired by a process called non-homologous DNA end joining (NHEJ). Ku70 played a pivotal role in NHEJ pathway. As a common hematological malignant disease with unique chromosomal translocation t (9;22), chronic myeloid leukemia (CML) can be considered as a paradigm for neoplasias that evolve through a multi-step process. As we reported before, protein Ku70 expressed significantly higher in CML than in normal BM cells. Meanwhile, its expression level in blast phase was markedly higher than that in chronic phase. The present study furthermore aims to investigate the expression of the gene Ku70 in CML cells at different clinical stage and reveal the correlation among the expression of the gene Ku70, the protein Ku70 and BCR-ABL in cells of CML. The NHEJ efficiency to repair DSB in CML was also investigated.

Methods: Bone marrow cells were collected from 24 cases of normal adults and 27 cases of de novo diagnosed CML patients. 15 CML patients were in chronic phase and 12 in blast phase. The expression of gene Ku70 was detected by RT-PCR and RQ-RT-PCR. The fusion gene BCR-ABL was detected by TaqMan probe Real Time PCR, using ABL as the internal control. Nucleic extracted proteins were used to determine NHEJ efficiency by an in vitro end-ligation system.

Results: The mean level of NHEJ activity of normal BM cells and CML cells were 18.6±13.1% vs 24.8±14.9%, .024. The gene Ku 70 expression in normal BM cells and CML cells were 31.08±8.41 vs 544.63±1185.71 copies/10,000 β-Actin copies, P=0.039. gene Ku 70 expressed significantly higher in blast phase (1103.31±1645.62 copies/10,000 β-Actin copies, P<0.01). There were positive correlations of BCR-ABL when compared with the expression of the gene Ku70 (r=0.573 P=0.002) and with the protein Ku70 (r=0.705 P<0.001) in CML cells. There was also significantly correlations between the expression of the gene Ku70 and the protein Ku70 (r=0. 808, P<0.001).

Conclusions: The gene and protein of Ku70 were expressed significantly higher in CML than in normal BM cells with NHEJ activity was also enhanced in CML cells. Meanwhile, Ku70 expression level in acute phase was markedly higher than that in chronic phase. There were significantly correlations among the expression of fusion gene BCR-ABL, the gene Ku70 and the protein Ku70 in cells of CML This Study illustrates DNA instability in CML cells which was mainly damaged by DSB, and this kind of DNA damage was repaired by NHEJ pathway predominantly. NHEJ pathway plays an important role in disease progression of CML.
Interim Positron Emission Tomography (PET) in Diffuse Large B-Cell Lymphoma: Independent Expert Nuclear Medicine Evaluation of ECOG 3404
Authors: Horning Sandra J. , Juweid Malik E. , Schoder Heiko , Wiseman Gregory , McMillan Alex , Swinnen Lode J. , Advani Ranjana , Gascoyne Randy D. , Quon Andrew
Published: April 2020

Background: Positive interim PET scans have been associated with inferior outcomes in DLBCL treated with chemotherapy, alone or with rituximab. In the ECOG 3404 study for bulky and advanced DLBCL, PET scans at baseline and after 3 R-CHOP are centrally reviewed by a single reader; those with positive scans cross-over to R-ICE after 4 R-CHOP, whereas those with negative scans continue on R-CHOP. The primary endpoint of E3404 is progression-free survival. To determine the reproducibility of interim PET scan interpretation, we convened an expert panel.

Methods: Three external nuclear medicine physicians visually scored baseline and interim PET scans independently and blinded to other clinical information or outcome. ECOG study criteria were binary (0,1) based on residual disease in initially involved sites with uptake greater than the liver. London criteria were on a scale of 0–5, where 4–5 was positive, based on increased uptake relative to the liver. Overall scores and agreement among experts were evaluated for both criteria, with application of the kappa statistic to correct for chance.

Results: Using the ECOG criteria, external reviewers were in complete agreement in 68% of 38 interim scans and completely agreed with the central review in the same 68% cases. Agreement among the experts was 71% employing the London criteria in these cases. The range of PET+ interim scans by reviewer was 16.8% to 34.2% (p=NS) by both ECOG and London criteria. The kappa statistic for overall pairwise correlation between readers was 0.445 (0.396–0.533) using ECOG and 0.502 (0.396–0.630) using London criteria – indicating moderate consistency. Areas of disagreement often, but not exclusively, related to bone disease, the shape and focality of residual uptake, splenic disease, and rare scans without CT-fusion.

Conclusions: These data show that visual criteria, either ECOG or London in this series, for interim PET results are moderately reproducible among individual nuclear medicine experts. Our finding of variability among experts indicates the need for caution in interpreting interim PET results in studies and in practice. Review of all E3404 cases is planned after accrual is completed (projected 12/08). Other ongoing studies evaluating interim PET after 1–3 cycles of therapy, the application of quantitative criteria, and consensus panels may provide further valuable information.
DNA-Dependent Protein Kinase Is a Therapeutic Target in Poor Prognosis B-Cell Chronic Lymphocytic Leukemia
Authors: Willmore Elaine , Elliott Sarah L. , Crawford Clark J. , Summerfield Geoffrey , Mainou-Fowler Tryfonia , Jackson Graham , Evans Paul , Newton Paula , Gowans Duncan , Durkacz Barbara W.
Published: April 2020

Poor prognosis B-cell chronic lymphocytic leukemia (CLL) is characterised by del(17p), del(11q) and unmutated IgVH genes. Mutational inactivation of p53 and ataxia telangiectasia-mutated kinase (ATM) are more frequent in these patients and confer drug-resistance. Over-expression of DNA-dependent protein kinase (DNA-PK), the enzyme that mediates DNA double strand break (DSB) repair via non homologous end joining (NHEJ), also correlates with chemo-resistance. Thus, alterations in DNA damage signalling pathways are associated with poor risk CLL. We have shown that DNA-PK is a new therapeutic target in CLL1, and are evaluating the efficacy of novel small molecule inhibitors of DNA-PK in ex vivo studies using leukemic lymphocytes from a well-characterised cohort of CLL patients (n=85). We hypothesised that targeting DNA-PK would inhibit NHEJ and thus sensitise CLL cells to drug-induced DNA damage.

NU7441 and KU-0060648 are potent small molecule inhibitors of DNA-PK, developed in collaboration with KuDOS Pharmaceuticals (Cambridge, UK). Lymphocytes were treated with fludarabine, chlorambucil, and Topoisomerase II poisons (mitoxantrone, etoposide, doxorubicin) in the presence or absence of NU7441 (1 μM) or KU-0060648 (0.2 mM). There was a concentration-dependent decrease in viability in response to single agent treatment (XTT/apoptosis assays) that was potentiated in the presence of a DNA-PK inhibitor. For example, 14/18 cases tested with mitoxantrone (currently in clinical trials) were sensitised by NU7441. Measurement of γH2AX foci formation (a surrogate marker for DSB) after Mitoxantrone treatment showed foci formation within 3 hr (n=4), which was maximally potentiated at 24hr following co-incubation with NU7441, implicating DNA-PK as a mediator of DSB repair following drug treatment. Stratification by karyotypic status demonstrated striking results. Although del(17p) cases were more resistant to mitoxantrone (mean LC50 1.2 mM ± 0.2) compared to del(13q) cases (mean LC50 0.4 mM ± 0.03), they had the greatest sensitization (7–13 fold) to Mitoxantrone by NU7441 (p=0.0006), indicating the particular effectiveness of this combination in del(17p) cases. Consistent with this observation, DNA-PK expression (Western blot and activity assays) was highest in del(17p) cases, confirming the utility of this novel drug combination. Whereas Topoisomerase IIα expression was negligible (Western blotting), Topoisomerase IIβ expression varied 3-fold. RT PCR analyses are underway to further study expression of DNA-PK and Topoisomerase II in this cohort. Taken together, these data show that use of a DNA-PK inhibitor increases the therapeutic index of drugs currently used to treat CLL and identify a targeted and novel approach for poor prognosis disease.
The Role of Heme Oxygenase 1 in Erythroid Differentiation.
Authors: Santos Daniel Garcia dos , Schranzhofer Matthias , Chies Jose Artur Bogo , Ponka Prem
Published: April 2020

Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75–80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is performed by heme oxygenases (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO-1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of overexpressed HO1 on heme and iron metabolism in stably transfected MEL cells (MEL-HO1) and their non-transfected counterparts. Compared to wild type cells, DMSO-treated MEL-HO1 cells displayed a reduction in heme stability (measured by the incorporation of 59Fe into heme) in addition to impairment of erythroid differentiation. Moreover, although wild type and transfected cells expressed similar levels of transferrin receptors in the uninduced state, MEL-HO1 cells, as compared to wild type MEL cells, showed only a small increase in transferrin receptors upon treatment with DMSO. Finally, we measured apoptosis using annexin-V and observed an increase in the number of apoptotic cells in HO1 transfectants, but not in wild type MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during physiological erythroid differentiation. In addition, our results showing that high levels of HO1 in erythroid cells cause heme catabolism and a defect in erythroid differentiation raise the possibility that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.
Use of Multiphoton Intravital Imaging and a Multiscale Computational Model of Thrombus Development to Study the Role of FVII in Thrombogenesis
Authors: Kamocka Malgorzata , Xu Zhiliang , Chen Nan , Alber Mark , Rosen Elliot D.
Published: April 2020

Using 2-photon intravital microscopy we have generated high resolution, near real-time 3-dimensional images of a developing thrombus. Following Titanium - Sapphire laser-induced injuries in mouse mesenteric vessels, the developing thrombus was monitored by collecting stacks of confocal images through the developing thrombus. Data were collected in 3 channels for fluorescently labeled platelets, fibrinogen and 70,000 MW dextran. By including fluorescently labeled dextran in the blood we were able to monitor flow (plasma) and not labeled cells (leukocytes and erythrocytes) forming black silhouettes in the plasma. Since each 3-D reconstruction involves a series of scans, we were able to generate approximately 2–3 reconstructions per minute. Thus, the system sacrifices temporal resolution for high resolution structural information revealing the changing, heterogeneous sub-domain structure of the developing thrombus. The imaging system has been used to study the consequences of FVII-deficiency. Following injury, of the luminal surface of the vessel, a thin layer of platelets and fibrin accumulated at the injury site. Unlike injuries in wild-type mice where the thrombus continues to grow, the injuries in FVII deficient mice failed to grow as a result of frequent embolization from the developing structure. Interestingly, in a model of ferric chloride induced injury of the carotid artery, thrombi in FVII deficient mice form large structures capable of reducing flow although, unlike wild type mice, the FVII deficient animals fail to form stable occlusive clots. In parallel with the modified experimental vascular injury model, we have begun development of a computational model of thrombus development. The modeling framework consists of a stochastic and discrete Cellular Potts Model (CPM) to describe platelet and cellular interactions and continuous submodels to describe hydrodynamic and biochemical reactions. Our multiscale model includes the vessel wall, platelets (in resting and activated states), blood cells, coagulation reactions, fibrin formation, and hydrodynamic parameters as components. By comparing the in vivo experimental results with those of simulations varying the concentration of FVII we are able to refine and validate the computational model of thrombogenesis.
Young Adults with Acute Lymphoblastic Leukemia (ALL) Treated with Adapted Augmented Berlin-Frankfurt-Munster (ABFM) Therapy
Authors: Rytting Michael , Thomas Deborah , Franklin Anna , Wierda William , Faderl Stefan , Ravandi Farhad , Muriera Brenda , Kantarjian Hagop M. , O’Brien Susan
Published: April 2020

The survival of teen-aged patients (pts) with acute lymphoblastic leukemia (ALL) has been analyzed by various groups, with most groups showing improved survival for these pts using pediatric ALL regimens compared to adult treatment regimens. ABFM therapy is an effective therapy for ALL pts up to age 21 and the therapy has acceptable toxicities. We designed a trial of modified ABFM therapy for adolescent/young adults (AYA) up to age 40 with acute lymphoblastic leukemia. Enrollment on the trial began in October 2006. Pts receive four drug induction with prednisone 60 mg/m2 daily for 28 days, daunorubicin 25 mg/m2 weekly for 4 doses, vincristine 2 mg weekly for 4 doses and a single dose of intravenous PEG-asparaginase in week one as well as intrathecal (IT) cytarabine on day one and IT methotrexate on day 8 and 29 depending on the presence of spinal fluid blasts. Induction is extended by two weeks for pts who do not achieve morphologic remission by day 29. Pts who have a morphologic remission by day 15 are classified as rapid early responders, and they receive one phase of delayed intensification. Patients who do not achieve morphologic remission by day 15 but who attain morphologic remission by day 29 or day 42 are slow early responders. Compared to preceding high risk ALL therapy in children, the first year of ABFM treatment uses more vincristine, asparaginase and intravenous methotrexate. As per prior pediatric protocols, central nervous system (CNS) radiation is recommended for pts with CNS involvement at diagnosis and for pts with T-cell ALL and a high presenting white blood cell count. 34 pts have been enrolled. 29 are evaluable (2 with prior relapse, 2 with lymphoblastic lymphoma, and 1 withdrew on day 1 of therapy). The median age is 20 years (range 14–34). 27 (90%) pts are in remission at day 28. Two pts are in remission with extended induction. One pt is refractory to induction. 26 (87%) have rapid early responses by marrow morphology. Minimal residual disease (MRD) testing shows 15/29 positive at day 15, 10/29 positive at day 29, and 3/24 positive at day 84. The most frequent grade 3–4 toxicities are infectious in 19 (65%), elevated transaminases in 17 (58%) patients, hyperglycemia in 13 (44%), and hyperbilirubinemia in 9 (31%). 7 (25%) have Grade 3–4 thrombosis or fatigue. Less frequent but significant toxicities include leukoencephalopathy, osteonecrosis and severe allergic reactions each seen in 3 (10%) pts. One pt had a prolonged delay in chemotherapy due to liver toxicity and relapsed. One pt has CNS relapse, and two have died of leukemia. ABFM therapy in young adults is an effective induction regimen but entails significant hepatic and infectious toxicities. The majority of these toxicities have resolved. Long-term follow-up is needed to determine if this treatment regimen offers an advantage to AYA patients with ALL.
Synergistic Anti-Leukemic Effect of Combination Therapy with Anti-FLT3 Antibody IMC-EB10 and Methotrexate
Authors: Surguladze David , Corcoran Erik , Tonra James , Li Yiwen
Published: April 2020

FLT3 is a class III receptor tyrosine kinase overexpressed by blast cells in approximately 90% of acute myeloid leukemia and acute lymphoid leukemia (ALL) patients. Recent findings testing a small molecule FLT3 inhibitor (PKC412) in combination with eight conventional antileukemic agents, reported that methotrexate in particular did not combine well with PKC412 against leukemia cells (Furukawa, Y. et al., Leukemia 2007, 21: 1005–1014). IMC-EB10 is a fully human neutralizing antibody that binds FLT3 with high affinity (Kd 158 pM) and blocks FLT3 ligand binding to FLT3 (IC50 ~10 nM), leading to inhibition of MAPK, STAT5, and PI3K/Akt signaling in leukemia cells. Here we test for benefits of adding methotrexate to IMC-EB10. In an SEMK2 model of ALL we evaluated survival effects of treatments administered intraperitoneally, beginning 1 day after after intravenous injection of 5 × 10e5 cells/mouse. In this model the maximum tolerated dose (MTD) of methotrexate administered once per week for 3 weeks, was determined to be 100 mg/kg. At this MTD, Methotrexate increased median survival to 44 days, compared to 30 days in the control group (p<0.0001 versus control by Log Rank Test). IMC-EB10 at its maximally efficacious dose (10 mg/kg, ip, 2×/week) increased survival in the SEMK2 model to 60 days (p<0.0001 versus control). When these two regimens were combined, survival significantly increased to 83 days (p<0.0001 versus all other treatment groups). Furthermore, 4 of 12 combination treated mice survived to 168 days after SEMK2 cell injection, but none of the mice in the other groups survived to this point. In this regard these agents were able to achieve an effect in combination that they could not achieve as monotherapies. These data support the significant therapeutic potential of this combination strategy in the treatment of leukemia.
Identification of Idiotype TCR Vα6, Vα10 and Vβ23 Relate to APL-Associated Antigen
Authors: Chen Shao hua , Li Yangqiu , Yang Li jian , Zhou Yu bing , Li Bo
Published: April 2020

Differentiation therapy with all-trans retinoic acid in combination with chemotherapy has significantly improved survival in patient with acute promyelocytic leukaemia (APL). However, relapse remains a major problem. In recent years, specific adoptive immunotherapy with tumor-specific T-cells was considered as a new approach to malignancy therapy. Recently, researches are focus on searching the generation of effective tumor antigen specific T-cells. The tumor antigen specific T cells could be determined by analysis of the T-cell receptor (TCR) Vα or Vβ repertoire, which display monoclonal or oligoclonal pattern. In order to develop the specific cellular immunotherapy in APL, in this present study, we analysis the clonality and the pattern of CDR3 sequence of TCR Vα and Vβ repertoire in peripheral blood T cells by RT-PCR and genescan technique, which was expected to define the APL associated TCR Vα or Vβ subfamily T cells. Oligoclonal T cells expressed TCR Vα6, Vα10 or Vβ23 genes were identified from one APL patient. The molecular characteristics of the CDR3 of Vα and Vβ genes rearrangement were Vα6-N-Jα17, Vα10-N-Jα35 and Vβ23-N-Dβ1-NJβ2.7. And the amino acid sequence of CDR3 region in TCR Vα6, Vα10 and Vβ23 subfamilies were CAMRENSAGNK, YLCAGDELLWECA or CASSSKWAGGTYEQY, respectively. These sequences were accepted by GenBank (Accession Number EU544946, EU544947 and EU647219). In conclusions, three idiotype Vα and Vβ sequences were identified in a case with APL, which was thought that may mediate the host specific immunity response for leukemia cells in APL. Farther investigation will be performed on construction the vector containing the antigen specific TCRs for gene transfer to establish TCR modified-T cells for specific immunotherapy of APL.
Feasibility of Inpatient Geriatric Assessment for Older Adults with Newly Diagnosed Acute Myelogenous Leukemia
Authors: Klepin Heidi D , Ellis Leslie Renee , Levitan Denise , Powell Bayard
Published: April 2020

Background: Acute myelogenous leukemia (AML) is a disease which largely affects older adults, for whom optimal therapy is unclear. Evidence based strategies are lacking to identify those older adults who may tolerate and benefit from standard therapies.

Objective: Test the feasibility of pre-treatment, inpatient geriatric assessment (GA) in older adults hospitalized with newly diagnosed AML.

Methods: Prospective evaluation of consecutive patients ≥60 years of age with newly diagnosed AML and planned induction chemotherapy admitted to a single institution from 6/2007–6/2008. Bedside GA was performed within 72 hours of diagnosis. In addition to demographics and routine labs, the GA measures obtained by a trained nurse included Mini-Mental Status Exam (MMSE), Center for Epidemiologic Studies Depression Scale (CES-D), Charlson Comorbidity Index (CCI), Vulnerable Elders Survey-13 (VES-13), Short Physical Performance Battery (SPPB, includes timed 4 meter walk, chair stands, standing balance), and grip strength.

Measures to assess feasibility included:

recruitment; time to complete the assessment, and proportion completing entire GA battery.

Results: Among 22 eligible inpatients, 11 enrolled (50%). The median age was 71 (range 63–78) and 72.7% were female. Poor risk cytogenetics were present in 27.3%. Laboratory measures included white blood cell count (mean=24.6×103/mm3, SD 26.2×103), hemoglobin (mean=8.5 g/dl, SD 1.6), platelet count (mean=60.3×103/mm3, SD=38.4×103), lactate dehydrogenase (LDH) (mean=340.0 U/L, SD=235.1), and albumin (mean=3.2 g/dl, SD=0.36). 66.6% of participants completed the entire GA battery; the remainder completed only self-report measures. Mean time for completion of the GA was 36.8 minutes (SD 9.8).

Mean scores for survey measures included: MMSE=26.1 (range 21–30, SD 3.2), CCI=1.6 (range 0–4, SD 1.1), CES-D=22.9 (range 8–37, SD 11.0), VES-13 survey=5.0 (range 1–8, SD 2.5). A wide range of objective physical performance was demonstrated including mean SPPB total score=7.4 (range 5–10; SD 2.1), mean timed 4 meter walk=12.9 seconds (range 6.6–22.1; SD 5.1), and mean grip strength=29.3 (range 12–62; SD 16.8).

Conclusions: Inpatient GA including physical performance assessment is feasible in older adults hospitalized for AML and our preliminary findings demonstrate significant variability in cognitive, emotional and physical status. These measures may represent valuable candidate predictors of outcomes, and ongoing studies will identify which measures are most predictive of treatment morbidity and response.
CLL-1, a Receptor Expressed on Myeloid Leukemic Stem Cells, Is a Potential Target for Immunotherapy of AML
Authors: Korver Wouter , Zhao Xiaoxian , Singh Shweta , Pardoux Cecile , Zhao Jingsong , Sen Siddhartha , Guzman Monica L. , Jordan Craig T. , Hsi Eric D , Abo Arie
Published: April 2020

CLL-1 (C-type Lectin-Like Molecule-1) is an inhibitory receptor expressed on myeloid cells which was previously shown to be expressed on AML cancer stem cells. To further validate the potential therapeutic against CLL-1, we generated a series of monoclonal antibodies (mAbs) against CLL-1 and assessed their cytotoxic and anti-cancer activities. While expression of CLL-1 was restricted to myeloid cells, it was expressed in 80% (37/46) of AML blasts but not in ALL blasts (n=5). Expression on AML CD34+/CD38- stem cells was detected in 71% (12/17) of cases. Selected anti-CLL-1 mAbs mediated dose-dependent. Complement Dependent Cytotoxicity (CDC) against various AML-derived cell lines with no detectable cytotoxic activity against lymphoid derived cell lines. Moreover, human embryonic kidney 293 cells became susceptible to anti-CLL-1 mAb mediated killing only after transfection with CLL-1, demonstrating that cytotoxic activity is mediated through a specific interaction with CLL-1. Furthermore, anti-CLL-1 mAbs showed CDC activity against all AML blasts tested in ex vivo assays (n=13), while no activity was observed against ALL blasts. CLL-1 efficiently internalizes upon antibody binding in both 293 transfected cells as well as AML cell lines, demonstrating the potential therapeutic opportunity for toxin-conjugation of anti-CLL-1 mAbs. In vivo anti-cancer activity of the lead chimeric mAb against CLL-1 was tested in an HL-60 xenograft model. In this model, growth of established tumors was reduced by 38% at 5 mg/kg twice weekly dosing. Our results demonstrate CLL-1 as an attractive target for AML with restricted expression on cells from myeloid origin, AML blasts and leukemic stem cells, as well as specific cytotoxic activity in in vitro, ex vivo and in vivo assays. We are currently undertaking additional xenograft models to evaluate the therapeutic potential of these mAbs against primary AML engrafted cells and in combination with chemotherapy in vivo.
Improvement of Remission Duration with Low-Dose Post-Remissional Gemtuzumab Ozogamicin (GO) in Elderly Patients with Acute Myeloid Leukemia
Authors: Marmont Filippo , Audisio Ernesta , D’Ardia Stefano , Salvi Flavia , Tonso Anna , Ferrero Dario , Levis Alessandro , Vitolo Umberto
Published: April 2020

The prognosis for elderly patients with acute myeloid leukaemia (AML) is dismal. Intensive chemotherapy is offered only to fit patients. The complete remission is obtained usually in 50% of the patients. The remission duration is short and the majority of patients will relapse within one year, with a poor quality of life. In this study we have evaluated the outcome of sixty-eight elderly patients (mean age 69.2 years) with AML or high-risk myelodysplasia (HR-MDS) treated with an induction program with fludarabine (30 mg/sqm/day for 4 days), cytarabine (1 gr/sqm/day for 4 days) and interspaced continuous infusion of idarubicin (12 mg/sqm/day on days 1,3,5), followed by lenograstim (FLAIG). Thirty-six patients (53%) were in CR after the first course. Thirty-four received a second reduced outpatient course. To account for the “guarantee time” required to survive the induction and retain the CR until the beginning of the post-remissional treatment, we used a landmark analysis at three months from CR to evaluate the disease-free survival (DFS), therefore excluding from the analysis early relapses and deaths in CR. In the first cohort, 13 patients received a post-remissional treatment with thioguanine and weekly cytarabine until relapse. In the second cohort, 14 patients received gemtuzumab ozogamicin (GO) at 3 mg/sqm every 3 months for 1 year or until relapse. The two cohorts were comparable for age, WHO classification, karyotype and CD33 expression. No difference in the median overall survival (OS) was observed between the two cohorts (7.4 vs. 12.3 months; p=0.1).. The landmark analysis showed an improvement of the DFS in patients receiving a post-remissional treatment with GO in confront to those treated with maintenance chemotherapy. (12.5 vs. 6.73 months; p=0.04). Although not influencing survival, the prolongation of DFS without hospitalisation or complications may improve the quality of life of elderly AML patients. The post-remissional therapy with low-dose GO deserves further evaluation in prospective randomized studies.
The Influence of Lepirudin, Bivalirudin and Dabigatran on the Calibrated Automated Thrombogram (CAT)
Authors: Woodhams Barry J , Carlo Audrey , Le Flem Lena , Guinet Celine , Depasse Francois , Samama Michel Meyer
Published: April 2020

In a two centre study (laboratories in Diagnostica Stago and Biomnis) we compared the in vitro effect on thrombin generation (TG) of Dabigatran and Bivalirudin (reversible direct anti-IIa inhibitors) with that of Lepirudin (an irreversible direct anti-IIa inhibitor) spiked into normal pool plasma. The effect of Lepirudin, Bivalirudin and Dabigatran were evaluated in both centres using the CAT (Diagnostica Stago, France) TG method in a concentration ranges up to 5, 20 and 1 μg/mL respectively. Testing was done in triplicate and repeated over 2 days. To reduce assay variability both centres used the same reagents lots and the same normal pool plasma (George King, USA). The range of each drug tested extended well above the therapeutic range concentrations normally found in patient plasma (0.5 to 1.0 μg/mL, 5 to 10 μg/mL and 0.1 to 0.3 μg/mL respectively for Lepirudin, Bivalirudin and Dabigatran). To see the effect of increasing activation forces, TG was performed at 3 different final concentrations of Tissue Factor (TF) - 1, 5 and 20 pM. All reagents were used as recommended by the manufacturer (Thrombinoscope, The Netherlands). A prolongation in the lag time (LT) is observed with all 3 drugs with all 3 concentrations of TF, but this is more marked for Lepirudin and Bivalirudin than it is for Dabigatran. In the therapeutic range Dabigatran (at 5pM TF) shows both an increase in LT and a decrease in peak thrombin and the ETP. At low concentration of Bivalirudin or Lepirudin, there is a paradoxical increase in peak height, which is even more pronounced at low TF concentration. At 1pM TF, this paradoxical peak increase is also observed with Dabigatran.

Results obtained in both laboratories are similar and complement our previous results and those reported elsewhere (1–4). The effect of Lepirudin and Bivalirudin on TG is different from that of Dabigatran. We also note that at lower TF concentration the anticoagulant effect on TG initiation is more intense but the test becomes less reproducible.
ANTI-hrs associated with the RHD-RHCE HAPLOTYPE DAR-ceAR in a patient with Sickle Cell Trait
Authors: Costa Sidneia Sanches , Chiba A. K , Martin F. O , Langhi Dante M , Chiattone C. S , Bordin Jose Orlando
Published: April 2020

Introduction: The RH blood group system is the most polymorphic and immunogenic among blood groups and some rare Rh phenotypes are found exclusively in the black population. The RH 18 phenotype (Hr-negative) that is one of these phenotypes is characterized by a high incidence antigen. The production of anti-RH 18 imposes to use equivalent rare antigen-negative red blood cells (RBCs), or deleted-Rh RBCs (Rhnull or D- -) for transfusion.

Case Report: A 24-year-old female patient with two previous abortions and one ectopic pregnancy and miscarriage had to be transfused with a RBC unit. She was typed as blood group A, Dccee phenotype with weak D, positive RBC antibody screening, auto-control negative and TAD negative. The antibody was anti-e (title 1/64) determined by testing serum against a commercial RBC panel (Fresenius-Kabi) by IgG indirect antiglobulin test tube (PEG-IAT), and nontreated (LissCoombs) and papain-treated RBCs on a gel matrix (DiaMed, Latino America). After adsorption of serum on Rhe-negative RBC (DccEE), the retrieving antibody was an anti-e-like antibody reacting with all normal Rhe-positive RBCs. Molecular and serologic Rh typing was also performed on blood samples obtained from five relatives of the patient and her husband.

Results: Patient and relatives cDNA sequence of gene RHD and RHCE transcripts were analyzed for RHCE exons 4 to 5 and exon 6; for RHD exons 4 to 5, 6 and 7. Sequencing of cDNA from the patient, her father and her sister showed in RHD the presence of the DAR allele point mutation 602 C>G in exon 4; 667 T>G and 744 C>T in exon 5; 957 G>A and 1025 T>C in exon 7; while on gene RHCE we found the ceAR allele carrying 712 A>G, 733 C>G e 787 A>G in exon 5. Hemoglobin tests revealed that the patient, her father and sister had a sickle cell trait (patient HbA1=55%; HbA2=2,8%; HbS=41%; HbF=1,2%; father HbA1=58,5%; HbA2=1,4%; HbS=37,7%; HbF=2,4%; sister HbA1=57,4%; HbA2=2,5%; HbS=38,6%; HbF=1,5%).

Discussion: The family study showed that the haplotype DAR-ceAR was inherited from her father. Individuals carrying the ceAR allele may produce anti-hrs antibody that is defined as a Rhe-like which does not react with rare Rhe positive variant RBCs. The antibody may be clinically significant and represents a great risk for those patients who are not transfused with a similar rare phenotype variant. Serologically, DAR has shown weaker reactions with anti-D, it is characterized by complete loss of at least 9 of 37 RhD epitopes, and may produce anti-D. Both mutated alleles show a replacement of RHD exons by RHCE counterparts.
An Investigation Report of Frozen Plasma Evidence-Based Transfusion in a Tertiary Care Teaching Hospital
Authors: Chen Qin-fen , Li Pei , Xie Yi , Chen Bao-An
Published: April 2020

Objective: To estimate the appropriateness of frozen plasma (FP) transfusions in a tertiary-care teaching hospital.

Methods: The medical records of all hospitalized patients transfused with FP between 1 April 2007 and 31 April 2008 were reviewed, analyzed retrospectively according to the Clinical Blood Transfusion Technical Specification from Ministry of Healthy.

Results: In 5084 times 13627 units FP transfusions, departments of surgery used 77.7%, and departments of internal medicine used 22.3%. The medical records showed that the reasons of FP transfusion included supply coagulation factors, supply intraoperative blood loss, postoperative supporting therapy for enhancement of wound healing, volume expansion, plasma exchange, nutritional support and massive transfusion. Overall, the 68.5% of FP transfusions were assessed as inappropriate. The inappropriateness rate in departments of surgery was 71.7%, and that in departments of internal medicine was 22.6%.

Conclusion: There is considerable inappropriate transfusion of FP in our hospital, for there has some important objective reasons. It is stated that the Technical Specification is not suitable to the condition of blood production supply and clinical practice. Clinicians should follow evidence-based transfusion guidelines for appropriate use of FP. So we suggest the Technical Specification should be renew periodly.
Assessment of SBDS Expression Patterns in Human Marrow Hematopoietic and Stromal Cells by Immunohistochemistry and Its Use as a Diagnostic Screen for Schwachman-Diamond Syndrome
Authors: Wong Trisha , Calicchio Monica , Fleming Mark D. , Shimamura Akiko , Harris Marian Hester
Published: April 2020

Objective. Shwachman Diamond Syndrome (SDS) is an autosomal recessive syndrome characterized clinically by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal abnormalities and a predisposition to leukemia. Molecularly, it is characterized by mutations in the Shwachman-Bodian-Diamond-Syndrome (SBDS) gene encoded on chromosome 7. The majority of mutant alleles (74%) associated with SDS are the result of gene conversion events with an adjacent SBDS pseudogene, leading to truncation, splice site, and frameshift mutations and a marked decrease in SBDS expression. The remaining pathogenic alleles include frameshift and, rarely, missense changes1. In this study, we examined the SBDS expression pattern in bone marrows from patients with SDS compared to normal controls or patients with other bone marrow failure syndromes using a new immunohistochemical (IHC) assay.

Methods. Archived bone marrow biopsy tissue sections from patients with SDS (21 specimens from 9 patients), idiopathic aplastic anemia (AA, 12 patients), Fanconi Anemia (FA, 4 patients), Diamond-Blackfan Anemia (DBA, 9 patients), Severe Congenital Neutropenia (SCN, 3 patients) and 13 normal controls were stained with an antibody directed against the C-terminus of SBDS. Four cell lineages (myeloid precursors, megakaryocytes, plasma cells and osteoblasts) were scored in a blinded fashion by two pathologists on a scale from 0 (negative) to 3 (strong). The scores from each cell lineage within each diagnostic group were averaged to yield an overall IHC score.

Results. We first characterized the SDS expression pattern in normal controls. Promyelocytes, early myelocytes, megakaryocytes, plasma cells, and osteoblasts all showed high levels of SBDS protein. In the myeloid lineage, SBDS expression decreased to undetectable levels in mature granulocytes, consistent with prior reports that SBDS is not necessary for terminal neutrophil differentiation. SBDS staining was not detectable in erythroid precursors or lymphocytes. We next compared SBDS IHC staining in these normal controls to that seen in SDS patients and patients with other bone marrow failure syndromes. The mean overall IHC score for SDS patients was 0.6 whereas it was 2.5 in normal controls, 2.23 in AA, 2.4 in DBA, 2.67 in FA and 2.38 in SCN. Four samples from two SDS patients with rare missense mutations in SBDS had moderate staining, consistent with previous studies that showed intermediate levels of SBDS expression in patients with missense mutations. When the analysis was restricted to patients harboring biallelic gene conversion SBDS mutations, the mean SBDS IHC score dropped to 0.14.

Conclusion. SBDS protein is highly expressed in early myeloid progenitors, megakaryocytes, and osteoblasts. This novel SBDS IHC assay may provide a rapid screen for the most common SBDS mutations.
Transfusion Independence Achieved in a Case of Diamond Blackfan Anaemia by Essential Amino Acid Supplementation
Authors: Wright Diana , Ball Sarah , de la Fuente Josu
Published: April 2020

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome usually presenting as pure red cell aplasia in the first few months of life, which can be associated with systemic features like short stature, skeletal, cardiac and renal abnormalities, and has an increased risk of aplasia and malignancy. The genetic defect is now known in 50% of the cases affecting ribosomal proteins and although the pathophysiological mechanism has not been elucidated, it is known that they have inefficient translation, which a recent report suggests that in some cases can be overcome by leucine supplementation both in vitro and in vivo.

We report an 8 years old boy diagnosed at 2 years of age with an Hb 3.4 g/dL requiring monthly blood transfusions. During the first year following the diagnosis he received trials of both steroids and ciclosporin, which he failed to respond to and remained transfusion dependent at the same frequency. At 3 years and 10 months of age the parents sought the advice of a nutritionist because his height was below the 2nd centile (92 cm) and weight in the 9th centile (13.9 kg). High performance liquid chromatography measurement of amino acids in blood revealed deficiency of isoleucin 40 μM (NR >44) and threonine 34 μM (NR >31). He received an essential aminoacid supplementation formula containing the following daily amounts: L-5-Hydroxytryptophan 0.01669 g/day, L-Arginine 0.3373 g/day, L-Histidine 0.3625 g/day, L-Isoleucine 0.6043 g/day, L-Leucine 0.6964 g/day, L-Lysine 0.3539 g/day, L-Methionine 0.5265 g/day, L-Phenylalanine 0.4628 g/day, L-Taurine 0.1669 g/day, L-Threonine 0.4908 g/day, L-Valine 0.6652 g/day, Pyridoxal-5-phosphate 0.0106 g/day, Alpha-ketoglutaric acid 0.3049 g/day. Six months after the initiation of the essential amino acid supplementation the interval between transfusions started to become longer and he became transfusion independent at 12 months. The Hb has increased steadily from 10 g/dL to 13.6 g/dL. His height has steadily improved to the 9th centile and the weight to the 25th–50th centile.

Whilst we cannot exclude that this child may have entered into spontaneous remission as it is known to happen in 5–10% of the patients with DBA, this case supports the recent report that essential amino acid supplementation may overcome the terminal differentiation defects found in the erythropoiesis of patients with DBA. A controlled trial of essential amino acid supplementation is warranted in patients with DBA.