bluuebunny/crossref_metadata_2025 huggingface dataset:
Approach to Establish Long Term Anti-Acute Myeloid Leukemia (AML) Immune Responses by Inducing Central Memory AML Specific CD4 T Cells.
Authors: Zhong Ruikun , Lane Thomas A. , Ball Edward D
Published: April 2020

The efficacy of adoptive cell therapy of cancer and leukemia is often limited by the failure of cultured T cells, particularly cloned CD8+ T cells, to persist in vivo, and insight into the basis for the poor survival of the transferred cells is lacking. We previously reported a novel culture method that induces AML dendritic cell differentiation and primes in situ AML-reactive T cells (AMLDC culture) (Zhong et al. Exp Hematol2008, 36:486). Highly reactive anti-AML T-cell lines were generated. These T-cell lines caused specific lysis of autologous AML cells, but not autologous LCL or allogeneic AML cells, and they depleted autologous AML colony-forming cells (CFC), but not normal CFC. The culture procedure has been further optimized in this study. We found that by the addition of the TLR-4 agonist LPS,(1–100 ng/ml) in the last 24 hours of AMLDC culture (day 6), CD80, CD86, CD53, CD83 or HLA-Dr expression of AML cells pre-induced by cytokine combination GM-CSF/IL-4 or GM-CSF/IL4/IL2/IL7/IL12 could be significantly enhanced (n=6, P<0.005–0.04; n=8, P<0.0001–0.005 respectively). Addition of LPS increased the IFN-gamma secretion by T cells generated from AMLDC culture in response to autologous AML cells 4–10 fold (3 of 3; P<0.001). Addition of TNF-alpha, (10–20 ng/ml) in the last 24 hours of culture could also significantly enhance the expression of above surface molecules. However, LPS induced significantly higher expression of CD80 and CD86 on AMLDC compared with TNF-alpha. T cells generated from AMLDC culture with TNF-alpha led to differential effects on autologous AML reactivity. IFN-gamma secretion was enhanced in 4 of 7 studies and suppressed in 3 of 7. The significantly enhanced AML cell reactivity of autologous T cells generated from AMLDC culture by adding LPS was also demonstrated in limiting dilution AMLDC culture in 96 well plates. The majority of T cell lines selected and expanded from AMLDC culture with LPS were CD3+CD4+ (12 of 18), and 18 T cell lines tested expressed moderate to high level of CD62L, implying the central memory phenotype.

Conclusions: Timely exposure of AMLDC culture to TLR-4 agonists, followed by T cell expansion, may promote the generation of AML-reactive T cells and differentiation toward the central memory phenotype. Theoretically, this should promote long-term maintenance and potential of regulation of both humoral and cellular immune responses against AML upon infusion of AML reactive autologous T cells derived from such cultures and may therefore enhance the therapeutic efficacy of these cells.
Donor Characteristics Affecting Graft Failure and Survival after Unrelated Donor Transplantation with Reduced Intensity Conditioning Regimens (RIC) for Hematologic Malignancies.
Authors: Passweg Jakob R. , Kan Fangyu , Zhang Mei-Jie , Rocha Vanderson , Isola Luis M. , Gee Adrian P. , Gibson John , Lazarus Hillard M. , Laughlin Mary J. , Loren Alison , Marks David , Gratwohl Alois , Eapen Mary
Published: April 2020

Impact of donor characteristics is well described for standard intensity unrelated donor and matched sibling donor transplants but may differ in recipients of unrelated donor RIC transplants. Less immunosuppressive regimens at transplantation may lead to higher graft failure rates. We examined risk factors affecting graft failure, acute and chronic graft-versus-host disease (GVHD) and survival after RIC unrelated donor transplants in 715 patients with acute (n=394) and chronic leukemia (n=74), myelodysplastic syndrome (n=70) and non-Hodgkin lymphoma (n=177). Graft failure was defined as <5% donor chimerism within 3 months after transplantation. 159 patients received bone marrow (BM) and 556 peripheral blood (PB) grafts. All transplantations occurred in 1999–2006 in the US. Median follow-up of surviving patients was 36 months (range 6–92). All donors and recipients were typed for HLA A, B, C and DRB1 using high resolution molecular methods. Mismatches at low resolution (antigen) and high resolution (allele) were considered together and are described collectively as mismatches. The day-28 incidence of neutrophil recovery (≥ 0.5 x 109/L) was 96%. After initial neutrophil recovery most patients (n=506) had >95% donor chimerism. 63 patients had <5% donor cells and the remaining 146 patients, 5–95% by 3-months post-transplant. In multivariate analysis, the only factor associated with graft failure was transplantation of BM vs. PB grafts (odds ratio 2.36, p=0.002). We specifically looked for an effect of donor-recipient sex match on graft failure and female donor parity on GvHD and found none. As expected risks of acute graft-versus-host disease (GVHD) were higher after mismatched transplants (p=0.015). No donor characteristic was associated with chronic GVHD. The only donor characteristic affecting overall survival was donor-recipient HLA disparity: 3-year overall survival rates were significantly lower at 23% after mismatched transplants compared to 42% after HLA-matched transplants (p=0.008). Additionally, mortality rates were significantly higher in patients older than 50 years (p=0.005), performance score <90 (p=0.002) and when transplantation occurred with active disease (p<0.001). As seen in recipients of myeloablative conditioning regimens, the only donor characteristic associated with survival is donor-recipient HLA disparity. Donor age, donor-recipient sex match, donor parity and donor cytomegalovirus serostatus were not associated graft failure or survival after unrelated donor RIC transplants.
Potential Use of miRNAs as Platelet Biomarkers of Storage.
Authors: Kannan Meganathan , Mohan Ketha V. K. , Kulkarni Sandhya , Atreya Chintamani D
Published: April 2020

MicroRNAs (miRNAs) are a class of small endogenous noncoding regulatory RNAs, which control cellular gene expression in eukaryotes at post-transcriptional level. MicroRNAs have been shown to play a role in cell differentiation and apoptosis by down regulating the translation of their target mRNAs. Differential expression profiles of miRNAs often help in identifying specific miRNAs as biomarkers of interest to a variety of cellular states, such as cancer. One of the key cellular components of blood that is of interest to transfusion medicine to control bleeding is ‘platelet concentrates (PCs)’. Platelets are stored at room temperature (22 0C) for up to 5 days for transfusion and during storage platelets undergo physiological changes often termed as storage lesions that adversely affect their survival in vivo following transfusion. Therefore identifying markers that truly reflect the cellular status at any given time during storage would help in developing efficacy biomarkers for stored platelets. Since mature platelets do not have a nucleus, but they do contain numerous mRNAs and undergo translational regulation, we hypothesized that miRNAs as translational regulators could play a crucial role in platelet biology and therefore may serve as biomarkers of platelets during storage. In this study, we explored differential miRNA profiling of 52-apoptosis-associated miRNAs of platelets using PCs obtained from the National Institutes of Health (NIH) blood bank and were stored at room temperature (22 0C) for up to 9 days. Samples were collected at day 0, day 2, and day 9. Out of 52 miRNAs analyzed by microarray, we observed that while two miRNAs levels had increased, another two miRNA levels decreased drastically from day 0 to day 9 (Table 1).

miRNA Day-0 Day-2 Day-9 Let-7b 1.12 0.015 15.1 miR-16 0.045 0.26 23.5 miR-145 35.09 10.18 5.415 miR-7 24.435 9.145 1.51

The differential microarray analysis presented here clearly suggests that miRNAs certainly have the potential to be useful as biomarkers of platelets during storage. Further analysis of the targets of these miRNAs in platelets will help understand the underlying mechanisms of platelet lesions.

The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.
Generation of Blood Cells from Human Ips Cells in Vitro through the Hematopoietic Progenitors Concentrated within the Unique Structures, Ips-Sac.
Authors: Takayama Naoya , Eto Koji , Nakauchi Hiromitsu , Yamanaka Shinya
Published: April 2020

Human embryonic stem cells (hESCs) are proposed as an alternative source for transfusion therapy or studies of hematopoiesis. We have recently established an in vitro culture system whereby hESCs can be differentiated into hematopoietic progenitors within the ‘unique sac-like structures’ (ES-sacs), that are able to produce megakaryocytes and platelets (Takayama et al., Blood, 111, 5298–306, 2008). However there is a little concern that repetitive transfusion with same human ESC-derived platelets may induce immunological rejection against transfused platelets expressing allogenic HLA. Meanwhile, induced pluripotent stem (iPS) cells established from donor with identical HLA are well known as a potential and given source on platelet transfusion devoid of rejection. To examine if human iPS cells could generate platelets as well as from hESCs, we utilized 3 different human iPS cell lines; two were induced by transduction of 4 genes (Oct3/4, Klf4, Sox2, and c-Myc) in adult dermal fibroblasts, and one was by 3 genes without c-Myc. Sac-like structures (iPS-sac), inducible from 3 iPS cell lines, concentrated hematopoietic progenitors that expressed early hemato-endothelial markers, such as CD34, CD31, CD41a (integrin αIIb) and CD45. These progenitors were able to form hematopoietic colonies in semi-solid culture and differentiate into several blood cells including leukocytes, erythrocytes or platelets. Of these, obtained platelets responded to agonist stimulation, in which the function was as much as human ESC-derived platelets, as evidenced by PAC-1 binding with activated αIIbβ3 integrin or full spreading onto fibrinogen. These results collectively indicated that human dermal fibroblasts could generate functional and mature hematopoietic cells through the reprogramming process and this method may be useful for basic studies of hematopoietic disorders and clinical therapy in the future.
Prognostic Value of Early 18fluorodeoxyglucose PET Scan in Patients with Diffuse Large B Cell Lymphoma Treated with Rituximab Plus CHOP or High-Dose CHOP.
Authors: Dupuis Jehan , Itti Emmanuel , Fruchart Christophe , Bardet Stéphane , Rahmouni Alain , Vilque Jean-Pierre , Lin Chieh , Belhadj Karim , Gnaoui Taoufik El , Kuhnowski Frédérique , Meignan Michel , Haioun Corinne
Published: April 2020

The results of 18Fluorodeoxyglucose – Positron Emission Tomography scan (PET) performed early during treatment (after 1 – 3 courses of chemotherapy) have been shown to predict outcome in aggressive lymphoma (Jerusalem, Haematologica 2000; Spaepen, Ann Oncol 2002; Haioun, Blood 2005). However, studies on early PET have been performed mostly in patients (pts.) treated without Rituximab. Our goal was to explore if the powerful predictive value of early PET was also observed in pts. treated with Rituximab-chemotherapy combinations. Seventy-three pts. with diffuse large B-cell lymphoma from 2 institutions were treated with R-CHOP (n=47) or R-high-dose CHOP (R-ACVBP, n=26) and were evaluated by PET after the two first cycles. PETs were interpreted on the basis of visual assessment. PET results were not taken into account to guide the treatment strategy after the 2 first cycles, which was solely based upon the results of conventional response assessment. Among responders, 16 young poor-risk pts. received planned consolidative high-dose therapy with autotransplant, while the others received immunochemotherapy based consolidation. Median age was 63 years (range 26–79) and 68% pts. were male. Baseline characteristics were as follows: Advanced Stage (Ann Arbor III–IV) 75%; Elevated LDH 62%; Poor performance status (ECOG 2–4) 32%. The International Prognostic Index was as follows: Low risk 27%, Low-intermediate 21%, Intermediate-high 21%, High risk 31%. PET was performed after a median of 15 days following the second cycle. Forty-nine pts. had a negative PET, while 24 were positive. With a median follow-up of 49 months, 11/49 (22%) PET negative pts. showed an event (progression or death) after a median delay of 13 months, while this was the case in 14/24 (58%) PET-positive patients after a median of 8 months. The estimated 5 year event free survival rates were 75% (95% CI: 63%–88%) for PET negative pts. and 41% (95% CI: 22%–61%) (log-rank test, p=0.001). Regarding overall survival, the estimated 5 year rates were 85% (95% CI: 78%–95%) and 53% (95% CI: 34%–74%) (log-rank test, p=0.005) in PET negative and PET positive pts., respectively. The same differences were found at 4 years when analysing separately pts. treated with R-CHOP and R-ACVBP (except for a non significant difference in OS in R-ACVBP pts.). In conclusion, we confirm the strong prognostic value of PET after 2 cycles in pts. treated with Rituximab combined to chemotherapy. Prospective studies (including a GELA study) are ongoing to evaluate the potential interest of tailoring treatment strategy to the results of early PET.
Mapping Interactions Between B2GPI and the Lipoprotein Receptors by Solution NMR Spectroscopy.
Authors: Beglova Natalia , Lee Chang-Jin
Published: April 2020

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by venous or arterial thrombosis and recurrent pregnancy loss. Beta2 glycoprotein I (B2GPI) is the major target for these antibodies. Current evidence suggests that B2GPI acquires pathological properties leading to APS after association with antibodies. It was demonstrated that APS related antibodies induce activation of endothelial cells, monocytes and platelets, although the identity of a cell-surface receptors that transmit the antibody-induced signaling inside cells remain unclear. Biochemical studies have shown that the receptors of the low-density lipoprotein receptor (LDLR) family are all capable to bind B2GPI/antibody complexes. These receptors are abundantly expressed by many cell types. The ligand-binding activity of the lipoprotein receptors is confined primarily to structurally homologous LA modules, which are arranged in sequence to form a ligand-binding domain. We investigated at the atomic level the features of the ligand-binding LA modules required for interaction with B2GPI. We sought to determine whether the same residues that form the ligand-binding pocket on the LA modules in the crystal structure of a RAP/LA3-4 complex also recognize B2GPI. We demonstrated that the same residues that form the ligand-binding pocket on LA4 in the crystal structure of the RAP/LA3-4 complex comprise the contact site for B2GPI. The domain 5 from B2GPI (B2GPI-D5) was expressed and labeled for NMR studies. The residues of B2GPI-D5 that are perturbed when B2GPI-D5 was titrated with LA4 are located at the C-terminus of domain 5. Using NMR relaxation measurements, we determined that B2GPI-D5 binds LA4 with 1:1 stoichiometry. The estimated Kd of the B2GPI-D5/LA4 complex is about 30 uM. NMR experimental data was used to build a docking model of the complex.
Protease Activated Receptor-4 (PAR4) Uses Anionic Residues to Interact with α-Thrombin in the Absence or Presence of PAR1.
Authors: Nieman Marvin T
Published: April 2020

Thrombin activates protease activated receptor 1 (PAR1) faster than protease activated receptor 4 (PAR4) due to a hirudin-like sequence in the exodomain of PAR1 that binds thrombin’s exosite I. However, recombinant exodomain studies indicate that PAR4 does have extended contacts with α-thrombin that influence PAR4’s kinetics of cleavage. In the current report, the role of an anionic cluster (Asp57, Asp59, Glu62, Asp65) in the exodomain of PAR4 is examined for its influence on cleavage and activation of PAR4 on cells in the absence or presence of PAR1. α-Thrombin induces wild type PAR4 (PAR4-wt) calcium flux with an EC50 of 110 nM whereas mutation of the four anionic residues (PAR4-AAAA) increases the EC50 to 641 nM. In contrast, PAR4-wt and PAR4-AAAA are activated by γ-thrombin with a similar EC50 (588 nM and 449 nM, respectively, p = 0.48), indicating a role for α-thrombin’s exosite I in PAR4 activation. Coexpression of PAR1, lowered the EC50 of cleavage 10 fold for both PAR4-wt from 321 to 26 nM and PAR4-AAAA from 2.2 μM to 360 nM, respectively. Individual point mutations at Asp57, Asp59, Glu62 or Asp65 show that PAR4-D57A is activated by α-thrombin with the same EC50 as PAR4-wt (140 nM) whereas PAR4-D59A is the same as PAR4-AAAA (699 nM). Glu62 and Asp65 contribute to α-thrombin recognition, but to a lesser extent. The current report shows that PAR4 uses its anionic cluster to interact with α-thrombin and that this interaction is important even in the presence of PAR1.
Comparative Effects of the Direct Factor Xa Inhibitor, Apixaban, and the Direct Inhibitors of Factor VIIa, XIa and Thrombin on Tissue Factor- Induced Human Platelet Aggregation.
Authors: Jiang Xiaosui , Wong Pancras C.
Published: April 2020

Background: Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late-stage clinical development for the prevention and treatment of venous thromboembolism, stroke prevention in patients with atrial fibrillation, and secondary prevention in patients with acute coronary syndrome. By inhibiting FXa, apixaban has the potential to reduce thrombin generation and indirectly decrease thrombin-mediated platelet aggregation. This study evaluated the in vitro effect of apixaban on human platelet aggregation induced by thrombin derived via the extrinsic pathway. Direct inhibitors of FXa (rivaroxaban), FVIIa (BMS-593214), thrombin (dabigatran, argatroban) and FXIa (BMS-262084) were included for comparison.

Methods: Citrated human blood, mixed with corn trypsin inhibitor (50 μg/ml) to block the contact factor pathway, was centrifuged to obtain platelets-rich plasma (PRP). The latter were incubated for 2 min at 37°C with 3 mM H-Gly-Pro-Arg-Pro-OH-AcOH (to prevent fibrin polymerization) and with vehicle or increasing concentrations of inhibitors. Human tissue factor (TF) (Innovin®; dilution 1:900 to 1:1000) plus 7.5 mM CaCl2 was added to PRP to trigger platelet aggregation. Platelet aggregation response, measured turbidometrically with a platelet aggregometer, was expressed as AUC occurring 5 min after TF stimulation.

Results: TF produced 85±3% aggregation of human platelets in the vehicle-treated group (n=10). Apixaban and other factor inhibitors, except the FXIa inhibitor, inhibited TF-induced platelet aggregation in a concentration-dependent manner with IC50 (nM) values as follows: 3.6±1.3 (apixaban), 7.5±1.5 (rivaroxaban), 13±1 (BMS-593214), 46±1 (dabigatran) and 79±1 (argatroban). BMS-262084 (IC50 = 2.8 nM vs. human FXIa) had no effect on TF-induced platelet aggregation at 10 μM. These inhibitors at 10 μM had no effect on platelet aggregation induced by ADP and collagen, as expected from their mechanism of action.

Conclusions: This study demonstrates that inhibition of thrombin generation by blocking upstream proteases (FVIIa and FXa) in the blood coagulation cascade is as effective as direct thrombin inhibition in preventing TF-induced platelet aggregation. Under these experimental conditions, FXIa did not play a role in platelet aggregation. Further, the potent indirect antiplatelet effect of FXa inhibitors together with their anticoagulant effects suggest that apixaban may have therapeutic potential in patients with arterial thrombosis. Accordingly, apixaban is in development for secondary prevention in patients with acute coronary syndrome.
Detection of Minimal Residual Disease in Chronic Lymphocytic Leukemia with Monoclonal Antibodies Specific for CD5, CD10, CD19, and ROR1.
Authors: Broome H. Elizabeth , Rassenti Laura , Bui Jack D. , Meyer Lilly , Kipps Thomas J.
Published: April 2020

ROR1 is an oncofetal protein expressed in chronic lymphocytic leukemia (CLL). We generated a monoclonal antibody (mAb) specific for ROR1 (4A5) and used this to stain blood or marrow cells from patients with CLL and other adults. We found that 4A5 reacted with the CLL cells of all patients examined and also with some cells that had an immunophenotype of B lymphocyte precursors (BLP) (CD10+, CD19+, CD20-variable, dim CD45+, surface immunoglobulin-negative). 4A5 showed no reactivity with other blood or marrow cells. We speculated that this mAb could be used to detect CLL cells in blood or marrow of patients after therapy, providing a means with which to assess minimal residual disease (MRD). For this purpose, we evaluated a four-color combination of mAbs using CD10-FITC, CD19-PE, CD5-PerCP-Cy5.5, and 4A5-Alexa-647. MRD detection limits were established through reconstitution studies in which we made serial dilutions of CLL cells into normal blood or marrow samples. MRD was measured by whole blood lysis and acquisition of 100,000 events using a FACSCalibur and a gating strategy designed to exclude CD5-negative, CD10-positive, and 4A5-negative B lymphocytes (CD19+). CLL cells stained with 4A5 had a mean fluorescence intensity (MFI) of 31 (N=100) and a MFI ratio (MFIR) relative to that of isotype control stained CLL cells of more than 10:1. On average, 94% of the CLL cells in each sample had higher fluorescence intensity when stained with 4A5-Alexa-647 than when stained with an isotype-control mAb (range 88–98%). Marrow samples (N=9) from adults with diagnoses other than CLL and that had 1–10% BLP had CD5− negative, CD10+, CD19+ cells that reacted with the 4A5-Alexa 647. These cells in such samples had a MFI of 19 and a MFIR relative to that of isotype-control-stained BLP of 6.3. On average, 48% of such cells in each sample had higher fluorescence intensity when stained with 4A5-Alexa-647 than when stained with an isotype-control mAb (range 18–79%). Four color flow cytometric analysis with CD10, CD19, CD5, and 4A5 detected CLL cells present at less than 0.1% in reconstituted blood or marrow samples, including marrow with 3–5% BLP. Background was between 0.01% and 0.1% with whole blood (marrow) lysis when we acquired a total of 100,000 flow-cytometric events. These data indicate that anti-ROR1 mAbs can be used with mAbs specific for CD10, CD19, and CD5 for sensitive detection of MRD in CLL.
Prognostic Stratification of Patients with Chronic Lymphocytic Leukemia Based on Quantitative Flow Cytometry Evaluation of ZAP-70 Versus Homology of IgVH Plot.
Authors: Klabusay Marti n , Kuhrova Viera , Hrabcakova Viera , Coupek Petr , Mayer Jiri
Published: April 2020

B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in the adult population. It has a heterogeneous behavior and variable prognosis. While some patients experience indolent disease requiring no therapy for many years, others demonstrate a more aggressive type unresponsive to therapy. An accurate prognostic stratification is essential for optimizing the therapeutic strategy. Many prognostic factors are presently known, but their relationships and significance are often not clearly understood. Several markers have been identified, including IgVH mutation status and ZAP-70. Mutation of IgVH (less than 98% homology with the germline sequence) is correlated with better prognosis. Expression of ZAP-70, which is a cytoplasmic ζ-associated tyrosine-kinase essential for T-cell receptor signal transduction, is associated with more rapid disease progression and shorter survival. However, its detection by flow cytometry has many technical difficulties, resulting in high interlaboratory variability. The expression of intracellular ZAP-70 in 217 patients with diagnosis of B-CLL was determined using a new approach: quantitative flow cytometry on CD5+19+ tumor cells. Other laboratory and clinical parameters were evaluated, including gender, age, type and number of therapies, CD38 expression, cytogenetics and mutation status of IgVH. The expressions of ZAP-70 and CD38 were measured in molecules of equivalent soluble fluorochrome (MESF units). The results were correlated with mutation status of IgVH. Patients were divided into two groups by cluster analysis in a plot of IgVH homology versus ZAP-70 MESF (left panel). Overall survival curves for these groups are shown (right panel). It could follow that patients above the diagonal (triangles, group B) in the IgHV versus MESF plot have significantly poorer prognoses (p=0.0001) than do patients below this diagonal (circles, group A). Expression of CD38 above 15000 MESF showed worse prognosis for patients in the group B (p=0.035). Only three of 61 patients with chromosomal aberrations associated with poor prognosis (del11q and del17p) were found in the group A, but all of them had del13q present, as well. The authors introduced a new approach for evaluating ZAP-70 expression in B-CLL cells using quantitative flow cytometry that is easily standardized and not burdened with technical problems. Plotting IgVH homology (%) versus ZAP-70 quantitative expression (MESF) could clearly divide patients according to their prognoses.

Figure Figure
Nucleophosmin (NPM) Has Increased Expression and Shuttling Activity in Chronic Lymphocytic Leukaemia without Somatic Hypermutation of the Immunoglubulin Gene (UM-CLL) and Is Linked to Ribosomal Proteins: A Possible Role for Increased Protein biosynthesis in the Adverse Outcome of UM-CLL.
Authors: Rees-Unwin Karen S , Faragher Robin , Hutchinson Claire V , Unwin Richard D , Adams Julie , Pettitt Andrew , Banham Alison H , Pulford Karen , Burthem John
Published: April 2020

Chronic lymphocytic leukaemia (CLL) is a neoplastic proliferation of mature B-lymphocytes that has a marked variability of clinical outcome. Two distinct groups of CLL may be distinguished by the presence (M-CLL) or absence (UM-CLL) of somatic hypermutation of the immunoglobulin heavy-chain gene. UM-CLL has an adverse outcome, but the precise biological differences that underlie this more aggressive clinical-course are unclear. Using a proteomic approach, we have found that M-CLL and UM-CLL are consistently distinguished according to protein expression pattern. Of those proteins, the most important predictor of CLL class was the protein nucleophosmin (NPM). A significant increase in expression of NPM was found in UM-CLL compared with M-CLL (3.2 fold p<0.05 Mann Whitney U test). This increased expression did not reflect differences in apoptosis or proliferation, or gene mutation. However, co-immunoprecipitation experiments identified the ribosomal proteins 60s (L19 and L24) and 40s (S9) to be associated with NPM in CLL. Ribosomal proteins are known to shuttle between the nucleoli, nucleus, and cytoplasm, of cells. Standard- and confocal-immunofluorescence microscopy identified NPM to be present, as expected, in the nucleoli of all CLL cells. Additionally however, NPM was consistently detected in the nucleoplasm of CLL, and in a proportion of cells from all CLL cases, NPM was also shown to be present in the cytoplasm (range =7–52% of cells expressed cytoplasmic NPM). Morphometric analysis suggested no net transfer from the nucleolus to the cytoplasm; but did suggest trafficking from the nucleoplasmic compartment to the cytoplasm consistent with shuttling between these two cellular regions. In UM-CLL the fluorescent intensity of the cytoplasmic NPM was found to be higher than that of the M-CLL (t-test p=0.009), and there was an approximate two fold increased proportion of cytoplasmic NPM-expression in the UM-CLL subgroup (p value< 0.001). We propose therefore that NPM, in association with ribosomal proteins, displays high nucleocytoplasmic shuttling in CLL, and may reflect increased protein biosynthesis. In particular, the higher levels of NPM expression and shuttling in UM-CLL may play a role in the adverse outcome of this form of the disorder.
Predicting Time to Initial Treatment in Early Stage CLL - Integration of Classical and Novel Prognostic Factors.
Authors: Keating Michael J. , Lerner Susan , Wang Xuemei , Rassenti Laura Z. , O’Brien Susan , Kipps Thomas J. , Wierda William G.
Published: April 2020

CLL patients (pts) who are not eligible for initiation of treatment by the NCI Working Group criteria (1996) are traditionally monitored on a “watch and wait” approach. There is increasing interest in initiating therapy earlier in pts who are considered high risk. The most important advice pts require in the “watch and wait” category is whether they will need treatment and when. There are a number of traditional prognostic factors that potentially identify those “watch and wait” (W&W) pts most likely to need treatment. These have recently been amplified by the discovery of new prognostic factors such as the immunoglobulin (IgVH) mutation status, FISH cytogenetics, ZAP-70, and CD38 expression on CLL cells. Most reports have emphasized the new prognostic factors without consideration of traditional characteristics such as tumor burden, stage, etc. Since January 2004 FISH, IgVH mutation status, ZAP-70, and CD38 were characterized for most pts presenting to M. D. Anderson Cancer Center. We identified an early stage low tumor burden group of 826 pts (W&W) defined with as having lymph nodes < 3 cm in the longest dimension, splenomegaly < 5 cm, and Rai stage 0 – II. Twenty-four percent of these pts have received treatment in the first two years of follow-up. From the traditional prognostic factors, number of lymph node sites (cervical, axillary, and inguinal), absolute lymphocyte count (ALC), serum beta-2-microglobulin (B2M) levels were highly correlated with time-to-initial treatment (P<.001). The predictive power of these characteristics was also confirmed in an earlier historic patient population. When these three characteristics are evaluated in multivariate analysis, IgVH mutation status, ZAP-70, and FISH cytogenetics all add independent prognostic power. When these three novel characteristics were evaluated in multivariate model with the traditional characteristics, the most important were the number of node sites, ALC, B2M, FISH, IgVH mutation status, and then ZAP-70.

Clinical only +Mutation Status +ZAP 70 +FISH + All 3 No. Lymph Node Sites 1 1 1 1 1 ALC 2 2 2 2 2 Beta-2-microglobulin 3 4 4 4 4 Mutation Status -- 3 -- -- 5 ZAP-70 -- -- 3 -- 6 FISH -- -- -- 3 3

Integration of novel prognostic factors into readily clinically available characteristics will enable us to develop a nomogram for predicting probability of each patient requiring therapy at one year, two years, and eventually five years. This nomogram will be presented when completed and then will be available for validation in independent patient populations studied by other groups.
CFAR, An Active Frontline Regimen for High-Risk Patients with CLL, Including Those with Del 17p.
Authors: Wierda William G , O’Brien Susan M , Faderl Stefan H , Ferrajoli Alessandra , Koller Charles , Estrov Zeev , Burger Jan A , Lerner Susan , Kantarjian Hagop M. , Keating Michael J
Published: April 2020

High-risk patients (pts) with CLL can be identified by prognostic factors. Traditional prognostic factors that identify high-risk pts include Rai stage, absolute lymphocyte count (ALC), rapid lymphocyte doubling time, and serum beta-2 microglobulin (β2M). Del 17p is associated with loss of the tumor suppressor gene TP53. Patients with del 17p also tend to have mutations in TP53, thereby providing a second mechanism for complete TP53 knockout. Pts with del 17p have poor response to purine analogue-based treatments. β2M greater than twice the upper limit of normal (2XULN) was an independent high-risk feature for pts treated with the frontline fludarabine (F), cyclophosphamide (C), rituximab (R) regimen (FCR). Pts younger than 70 years with β2M >2XULN treated with FCR had a complete remission (CR) rate of 60% and median time to progression (TTP) of 70 months, compared to 84% and 86 months for pts <70 yrs old with β2M <2XULN. We evaluated the CFAR regimen (F 20mg/m2 d3–5; C 200mg/m2 d3–5; R 375–500mg/ m2 d2; and Alemtuzumab 30mg d1,3,5 of each 4-wk course) as frontline treatment in a high-risk group of pts with CLL. The trial completed planned enrollment of 60 pts; 48 are currently evaluable for response and follow-up. In this high-risk group, 28% of the 48 evaluable high-risk pts had del 17p prior to treatment by FISH analysis. Overall, the CR rate for the 48 pts was 69%, 54% CR for the 13 pts with del 17p. The overall response rates were 94% and 77% for the 48 pts and 13 pts with del 17p, respectively. CFAR was associated with more myelosuppression and fewer pts could receive all 6 intended courses compared with the historic high-risk group treated with FCR. However, a higher proportion of CFAR pts had no evidence of residual disease in the bone marrow by 2-color flow cytometry evaluation at response assessment. There is currently no difference in TTP or OS, comparing CFAR and FCR in this retrospective historical analysis with a short median follow-up time of 16 months for the CFAR group. There was no significant difference in incidence of infection during treatment with CFAR compared to FCR, with the exception of CMV reactivation. The CFAR regimen is active and tolerated in the highrisk group of pts with CLL, including those with del 17p. Follow-up continues for the pts treated on this trial in order to evaluate responses in all 60 enrolled pts and to evaluate time-to-event endpoints and compare with the historic FCR experience.
Phase 1 Study of ABT-263, a Bcl-2 Family Inhibitor, in Relapsed or Refractory Lymphoid Malignancies.
Authors: Wilson Wyndham H , O’Connor O. , Czuczman Myron S. , LaCasce Ann , Gerecitano J. , Leonard John P. , Tulpule Anil , Xiong Hao , Chiu Yi-Lin , Busman Todd , Knight Raymond A. , Enschede Sari , Krivoshik Andrew , Humerickhouse Rod
Published: April 2020

ABT-263 is a novel, orally bioavailable BH3 mimetic that binds with high affinity (Ki ≤1nM) and inhibits multiple antiapoptotic Bcl-2 family proteins. ABT-263 displays potent mechanism-based activity (EC50 ≤ 1μM) against human lymphoid and small cell lung cancer cell-lines. Mechanistic toxicities observed preclinically include inhibition of spermatogenesis, reduction in circulating lymphocytes and decreased survival of circulating platelets, presumably mediated by inhibition of Bcl-w, Bcl-2 and Bcl-XL, respectively. Study M06-814, a phase 1 dose-escalation study is employing a modified Fibonaci 3 + 3 design to assess ABT-263 safety, pharmacokinetics and anti-tumor activity of two dosing schedules in patients with relapsed or refractory lymphoid malignancies. Enrollment continues with 42 patients (39 on a 14/21 day and 3 on a 21/21 day dosing schedule) enrolled. ABT-263 given on a 14/21 day dosing schedule began at 10 mg and continued escalation to 440 mg, with 315 mg defined as tolerable. The pharmacokinetic profile of ABT-263 on the 14/21 day schedule was linear from 10 – 440 mg with a t1/2 of 14 – 20 h. Consistent with preclinical models, a dose-dependent reduction in circulating platelets was observed. Typically, platelet nadirs were transient, occurring on days 3–5 with subsequent recovery during continued dosing, consistent with accelerated platelet apoptosis and compensatory increased bone marrow release and production. Thrombocytopenia was predictable and manageable. A one week lead-in dosing period at 150mg ABT-263 and a 21/21 day dosing schedule beginning at 200 mg, are being evaluated as measures to ameliorate the platelet nadir. Dose limiting toxicities on cycle 1 in the 14/21 day dosing schedule occurred at 160 mg (bronchitis), 315 mg (elevated ALT and gr 4 thrombocytopenia), and 440 mg (dyspnea). Responses were observed in 3 patients including a 75% and 99% reduction of bulky masses in 2 CLL/SLL and a 75% reduction in a NK/T cell lymphoma. Minor nodal responses occurred in 4 patients with follicular lymphoma and 3 patients with CLL/SLL. Additionally, 2 patients with CLL/SLL had ≥50% decreases in leukemic cells for at least 2 months. ABT-263 is a well tolerated oral small molecular inhibitor of Bcl-2 family proteins with a linear PK, toxicity profile indicative of on-target activity and anti-tumor activity in patients with relapsed or refractory lymphoid malignancies. Accrual continues.
Plasma Alemtuzumab Levels at End of Treatment Correlate with Response and Response Duration in Patients with CLL Receiving Treatment for Residual Disease.
Authors: Wierda William G , Kipps Thomas J , Keating Michael J , Brown Jennifer R , Gribben John G , Rassenti Laura Z , Greaves Andrew W , Neuberg Donna , O’Brien Susan M
Published: April 2020

We conducted a phase II trial with alemtuzumab self-administered, subcutaneously (SQ) to eliminate residual disease after chemotherapy or chemoimmunotherapy for patients (pts) who were in partial remission (PR) or pts in complete remission (CR) with 2-color flow cytometry evidence of disease. Pts received alemtuzumab SQ 3mg, 10mg, 30mg on days 1,2,3 then 30mg thrice weekly for a total of 12 doses, including rampup. After the 12th dose, pts with residual disease could receive a second course of 12 additional doses. Responding pts were pts in PR who converted to nodular PR (NPR) or CR by NCI-WG ‘96 criteria or pts in CR who remained in CR and had no evidence of disease by 2-color flow cytometry. There were 26 pts enrolled in PR, 5 pts in CR. 7 pts received a second course of alemtuzumab. End of treatment plasma alemtuzumab levels were measured by a flow cytometry-based assay (Rebello and Hale, J Immunol Methods, 2002). The response rate in the intent-to-treat population was 74%. We retrospectively evaluated patients’ bone marrow for minimal residual disease by 4-color flow cytometry. A disappointing 18% of pts evaluated was MRD-negative in bone marrow by this method. Responding pts had mean plasma alemtuzumab level of 8μg/ml verses 3μg/ml for non-responders (p=.003). The median plasma alemtuzumab level for responders was 6μg/ml. Responders with end of treatment plasma alemtuzumab level greater than 6μg/ml had a significantly longer median response duration of 21.2 months versus 8.9 months for pts with less than 6μg/ml (p=.048). End of treatment plasma alemtuzumab level correlated with response to treatment and response duration for patients treated with alemtuzumab SQ. Future studies to optimize dose and schedule for alemtuzumab SQ as treatment for CLL, including for residual disease, should include measurement of end of treatment plasma alemtuzumab levels and evaluation of bone marrow for minimal residual disease by 4-color flow cytometry. Furthermore, consideration of these results should be given to optimize the effective duration of treatment with alemtuzumab SQ.
Dasatinib Is Associated with Rapid and Durable Complete Hematologic Responses in Patients with Chronic-Phase Chronic Myeloid Leukemia (CPCML).
Authors: Liu Delong , Matloub Yousif , Mukhopadhyay Jaydip , Liu David , Goldberg Stuart L
Published: April 2020

Achieving a quick and durable complete hematologic response (CHR) is a near-term treatment goal in CP-CML. This is particularly relevant in patients with late CP-CML who are receiving second-line tyrosine kinase inhibitors (TKIs) after failing imatinib therapy. The rapidity of the CHR provides an early assessment of sensitivity or resistance to second-line TKIs and should be used to guide treatment decision in this patient population. In the CA180-013 trial (START-C), 387 subjects resistant to or intolerant of imatinib were treated with dasatinib. At 2 years of follow-up, CHR was achieved in 91% of all subjects (90% of 288 resistant subjects, and 94% of 99 intolerant subjects). Of the 233 patients with abnormal peripheral blood counts at baseline, 203 (87%) achieved CHR (86% imatinib-resistant and 92% imatinib-intolerant). The time to achieve CHR (defined as the duration between the first dose of dasatinib until the first day CHR criteria were met) was rapid, with a median time to CHR of 2.1 weeks (C.I: 2.1 to 2.4) across all cohorts. Furthermore, most subjects who achieved CHR did so within the first two months of treatment. In addition, CHRs achieved with dasatinib were durable; the 12- and 24-month CHR were 92% and 83%, respectively. Moreover, in an optimization trial (CA180-034) comparing efficacy between 100mg and 140mg total dasatinib administered in one or two doses, the rates of, time to, and duration of CHR were comparable to that observed in START-C (Table 1). No difference in time to or duration of CHR was observed between the two schedules. In summary, dasatinib is associated with a rapid and durable CHR in a majority of subjects with CP-CML resistant to or intolerant of imatinib therapy.

Table 1: Percentage of Dasatinib-Treated Patients with CHR who are Imatinib Resistant (CA180-034 trial)

Number (%) of Subjects by Treatment Group QD BID Best Hematologic Response 100 mg N = 124 140 mg N = 123 Total N = 247 50 mg N = 124 70 mg N = 126 Total N = 250 CHR 110 (89) 106 (86) 216 (87) 114 (92) 112 (89) 226 (90) no CHR 14 (11) 17 (14) 31 (13) 10 (8) 14 (11) 24 (10)
Long Term Follow-up and Matched Pair Analysis of Adjuvant Donor Lymphocyte Transfusions Following Allogeneic Stem Cell Transplantation after Reduced Intensity Conditioning for High-Risk AML.
Authors: Schleuning Michael , Schmid Christoph , Koenecke Christian , Hertenstein Bernd , Baurmann Herrad , Schwerdtfeger Rainer , Kolb Hans-Jochem
Published: April 2020

Adjuvant transfusion of donor lymphocytes (aDLT) may reduce the risk of relapse after allogeneic stem cell transplantation. We report our data on aDLT in high-risk AML. Cells were given within a prospective protocol that contained a sequence of chemotherapy, reduced intensity conditioning for allogeneic transplantation, and aDLT. For aDLT, patients had to be in CR at least 120 days from transplantation, off immunosuppression and free of GvHD. 30% of patients (n=46) alive at day +120 fulfilled the criteria for aDLT. They had been transplanted for refractory (n=11) or relapsed leukemia (n=24) or in CR1 because of unfavourable cytogenetics (n=7) or other unfavourable criteria (n=2) or in CR2 with unfavourable cytogenetics (n=2). 24 patients had an unfavourable karyotype, 10 with complex aberrations. Thirty-one patients with similar disease characteristics and fulfilling similar selection criteria (being alive in CR at d +120, no cGVHD and no history of aGVHD> II°) transplanted during the same time period served as control. Of these, 10 pts. were transplanted at a different center not using aDLT and 21 at the center in Wiesbaden during 2000 and 2002 prior to the introduction of the protocol at this center. The median time from transplant to first aDLT was 160 days (range 71–303). Median follow up of the surviving transfused patients is 3.6 years (range 1.8–7.8). Seven patients received 1, 15 patients received 2, and 24 patients received 3 transfusions in escalating doses, containing a median of 1x106, 5x106 and 1x107 CD3+ cells/kg at aDLT 1, 2 and 3, respectively. Reasons for giving less than 3 transfusions were GvHD, relapse or patient’s refusal. Induction of GvHD was the main complication; grade II/III acute GvHD developed in 4, and chronic GvHD in 8 patients. So far, 9 (19.6%) patients have relapsed despite aDLT, as compared to 52.9% in the control group (Fisher-exact: p=0.004). Of these, 8 pts. finally died despite adoptive immunotherapy and only one patient is now surviving more than 3 years after relapse was treated by a second transplantation. Non relapse mortality post DLT was low with patients dying from infection, severe cGvHD, and secondary solid tumour one each. At the time of analysis, 35/46 patients are alive and all are in CR at a median of 3.2 years post first DLT. The actuarial overall survival four years after transplant is 79% as compared to 34% in the control group and at six years 63% vs. 31%, respectively (p=0.000). In conclusion, aDLT is safe, when given in escalating doses and to a selected group of patients. Results are encouraging, and improved long term leukemia-free survival can be achieved.
No Impact of Postremission Consolidation Chemotherapy Before Allogeneic Transplant in Patients with First Remission Acute Myeloid Leukemia with Intermediate or High Risk Cytogenetics.
Authors: Sproat Lisa O. , Bolwell Brian , Rybicki Lisa , Kalaycio Matt , Dean Robert , Sobecks Ronald , Pohlman Brad , Andresen Steve , Sungren Shawnda , Chan Josephine , Sweetenham John , Copelan Ed
Published: April 2020

Despite a lack of evidence that postremission consolidation chemotherapy improves outcome of allogeneic transplantation in patients with acute myeloid leukemia (AML) in first remission, high dose cytarabine and other regimens are commonly administered to these patients. We studied a consecutive cohort of 73 adult patients with AML at high risk of relapse by published criteria who underwent allogeneic transplantation in first remision to determine whether specific patient and treatment characteristics identified subgroups of patients who might benefit from consolidation therapy prior to allogeneic transplantation. Pretransplant cytogenetics were available for 56 patients. Transplantation occurred between 1988 and 2008. The primary analysis grouped patients according to cytogenetic risk (poor versus intermediate) and consolidation chemotherapy (yes versus no).

Consolidation Chemotherapy No Consolidation Chemotherapy None of the measured outcomes (relapse mortality (RM), non-relapse mortality (NRM), overall survival (OS)) differed significantly between groups. Poor Risk Cytogenetics 9 13 Intermediate Risk 18 16 Cytogenetics P Values for Consolidation Versus No Consolidation by Cytogenetic Risk Poor Risk Cytogenetics Intermediate Risk Cytogenetics When cytogenetic risk was not stratified the results between consolidation and no consolidation were also not significant. RM 0.95 0.74 NRM 0.73 0.19 OS 0.77 0.21 P Values for Consolidation Versus No Consolidation RM 0.70 NRM 0.12 OS 0.14

Neither this study nor others provide evidence that consolidation chemotherapy is beneficial to all patients with AML in first remission who undergo allogeneic transplant or to cytogenetic subgroups. Delays in referral to a transplant center, in tissue typing or physician preference to administer consolidation chemotherapy are not justified and expose patients to the inconvenience, risk, and cost of unnecessary treatment. Clinicians should avoid consolidation chemotherapy and expedite allogeneic transplantion in patients with AML in first remission in whom transplantation is the preferred treatment.