bluuebunny/crossref_metadata_2025 huggingface dataset:
Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells
Authors: Hooper Kathryn , Havens Kyle , Krostag Anne-Rachel , Magee Michael S , Martin Unja , Gupta Ankit , Smurnyy Yegor , Pechilis Lisa , Rode Amanda , Chavkin Andrew , Grande Shannon , Morgan Richard A. , Jarjour Jordan , Astrakhan Alexander
Published: October 2019

Chimeric antigen receptor (CAR) T cell therapies continue to show excellent outcomes in hematological cancers. Achieving success in additional tumor indications, however, will likely require modulating inhibitory pathways that limit CAR T cell potency. We developed a megaTAL nuclease targeting the gene encoding Casitas B-lineage lymphoma proto-oncogene-b (CBLB), a ubiquitin ligase that serves as an intracellular checkpoint that negatively regulates T cell activation. The megaTAL nuclease platform has been previously shown to drive highly efficient genome editing in primary T cells. Electroporation of primary T cells with mRNA encoding the CBLB megaTAL resulted in >90% indels at the target locus and a concomitant reduction of Cbl-b protein levels. Specificity characterization studies revealed three detectable non-exonic off-target sites with near negligible indel frequencies. We next assessed the functional impact of CBLB disruption in CAR T cells engineered to target the epidermal growth factor receptor (EGFR). When co-cultured with EGFR+ target cells, CAR T cells with megaTAL-mediated CBLB gene knockout had a 2-fold increase in pro-inflammatory cytokine production compared with mock-treated CAR T cells. We developed an A549 tumor xenograft model to test the activity of CBLB megaTAL-treated CAR T cells in vivo. While mock-treated CAR T cells had a transient impact on tumor growth, we observed complete and durable tumor elimination in mice infused with the CBLB megaTAL-treated CAR T cells. Improved responses in the megaTAL treated animals were particularly pronounced at lower CAR T cell doses, suggesting that CBLB knockout enhances the potency of CAR T cells. In summary, the CBLB megaTAL is a highly efficient and specific gene editing nuclease that enhances CAR T cell anti-tumor responses in vitro and in vivo, and thus could potentially improve the efficacy of CAR T therapy.

Disclosures

Hooper: bluebird bio: Employment, Equity Ownership. Havens:bluebird bio: Employment, Equity Ownership. Krostag:bluebird bio: Employment, Equity Ownership. Magee:bluebird bio: Employment, Equity Ownership. Martin:bluebird bio: Employment, Equity Ownership. Gupta:bluebird bio: Employment, Equity Ownership. Smurnyy:bluebird bio: Employment, Equity Ownership. Pechilis:bluebird bio: Employment, Equity Ownership. Rode:bluebird bio: Employment, Equity Ownership. Chavkin:bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership. Jarjour:bluebird bio: Employment, Equity Ownership. Astrakhan:bluebird bio: Employment, Equity Ownership.
FIP1L1 Regulates Alternative Polyadenylation of Leukemia-Associated Genes in Acute Myeloid Leukemia
Authors: Davis Amanda G , Shima Takahiro , Wang Ruijia , Zheng Dinghai , Tian Bin , Zhang Dong-Er
Published: October 2019

Alternative polyadenylation (APA) is a prevalent mechanism of post-transcriptional gene regulation. APA can impact the protein coding portion of a gene or the length of the 3' untranslated region (UTR), a crucial mRNA region mediating stability, localization, and translation efficiency. Global shifts in APA usage have been reported during development and in cancer transformation; however, the impact of APA in acute myeloid leukemia (AML) is currently unexplored. Therefore, we sought to address whether global shifts in APA patterns can be seen in leukemic blasts and if APA changes contribute to leukemogenesis. Here we report that leukemic blasts exhibit global transcript shortening that contributes to the upregulation of leukemia-associated genes: NRAS, BAALC, and MAPKAPK3. We also implicate FIP1L1 as an important mediator of APA alterations in AML.

To identify whether global trends of APA are seen in leukemia patients, we performed a 3'RNA sequencing experiment comparing healthy CD34+ hematopoietic stem and progenitor cells (HSPCs) with CD34+ enriched leukemic blasts from primary patient samples. We observed a trend in whole transcript shortening, with an emphasis on 3'UTR shortening of affected genes. Importantly, genes with altered APA were among pathways associated with oncogenic transformation and specifically AML. We selected three genes for further functional validation (NRAS, BAALC, and MAPKAPK3) that exhibited a correlation of shortened 3'UTRs with higher mRNA expression. Using mRNA stability assays and luciferase assays, we confirmed that 3'UTR shortening contributes to the upregulation of these leukemia-associated genes in blood cells.

To probe upstream regulators of APA alterations in leukemia, we performed overexpression and knockdown studies of APA machinery members that have altered expression in leukemia patients. We identified FIP1L1 as a novel regulator of APA in AML. Overexpression of FIP1L1 resulted in 3'UTR lengthening and downregulation of our three selected genes. Conversely, knockdown led to 3' UTR shortening and upregulation. Interestingly, altering FIP1L1 expression (both up and downregulation) is detrimental to leukemia cell lines, suggesting that modest changes in gene expression can have a dramatic impact on hematopoietic cell fitness. We conclude that APA changes represent another dysregulated layer of gene expression that can contribute to AML development. Current studies aimed at identifying the role of FIP1L1 in healthy HSPCs will elucidate the feasibility of FIP1L1 as a novel therapeutic target in AML.

Disclosures

No relevant conflicts of interest to declare.
Flow Cytometry Assay to Monitor Effectiveness of Gene Therapy Correction in Peripheral Blood Mononuclear Cells of Gaucher Disease Type I Patients
Authors: Barese Cecilia , Waring Michael , Pfeifer Richard , Mason Chris
Published: October 2019

The most common lysosomal storage disorder is Gaucher disease type I (GD-I) which is caused by a genetic defect in the gene encoding the enzyme beta-glucocerebrosidase (GCase). In GD-I, deficient GCase activity results in accumulation of glucocerebrosidases within macrophages of the reticuloendothelial system.

We hypothesize that putative precursors for macrophages, monocytes, in peripheral blood of GD-I patients, represent an excellent target population to monitor the effectiveness of novel curative treatments such as lentiviral vector driven gene correction targeting hematopoietic stem and progenitor cells in clinical setting.

Here, we present an improved flow cytometry assay that takes advantage of selective uptake of pentafluorobenzoylamino fluorescein di-β-D-glucopyranoside by the CD14+ monocytes resulting in fluorescence when it is hydrolyzed by active GCase, in the presence of formaldehyde (FA), commonly used for intracellular staining and to inactivate pathogens.

Using this substrate uptake/FA fixation method our results show a mean of 33.7% ± 3.6% (X±SD) GCase+ cells in GD-1 patients (n=2) compared to 83.7% ± 9.6% (X±SD) in normal controls (n=5). The mean fluorescence intensity (MFI) of the enzymatic activity showed no overlap between untreated GD-1 patients (3480 ± 49) and controls (7187 ± 595.7). Method specificity was demonstrated by complete inhibition of enzyme activity and fluorescence using 400 mM conduritol-B-epoxide, a GCase specific inhibitor. Next, mixtures of normal donor peripheral blood mononuclear cells (PBMC) and GD-1 PBMC were created in vitro to assess the lower limit of detection. As low as 1% normal donor cells could be reliably distinguished within these mixtures. GCase+ monocytes in the mixtures could be identified and quantified based on a higher MFI compared to GD-1 patient cells.

Compared to the conventional 4-MU biochemical method, this approach requires only a small blood sample and minimal processing, is rapid and straightforward, and more importantly allows quantification of GCase enzyme activity at individual cells instead of in a bulk cell lysate. These findings suggest this novel flow cytometry method is sensitive and specific to quantify small increases in GCase activity at single cell level, and thus has potential to monitor in vivo the effectiveness of gene therapy for Gaucher disease Type 1 in patients in real time. Additional sample analysis from GD-1 patients and normal donors are in progress to confirm consistency of the method.

Disclosures

No relevant conflicts of interest to declare.
Localized- and advanced-stage follicular lymphomas differ in their gene expression profiles
Authors: Staiger Annette M. , Hoster Eva , Jurinovic Vindi , Winter Stefan , Leich Ellen , Kalla Claudia , Möller Peter , Bernd Heinz-Wolfram , Feller Alfred C. , Koch Karoline , Klapper Wolfram , Stein Harald , Hansmann Martin-Leo , Hartmann Sylvia , Dreyling Martin , Weigert Oliver , Hiddemann Wolfgang , Herfarth Klaus , Rosenwald Andreas , Engelhard Marianne , Ott German , Horn Heike
Published: October 2019

The genetic background of follicular lymphomas (FLs) diagnosed in advanced clinical stages III/IV, and which are frequently characterized by t(14;18), has been substantially unraveled. Molecular features, as exemplified in the clinicogenetic risk model m7FLIPI, are important tools in risk stratification. In contrast, little information is available concerning localized-stage FL (clinical stages I/II), which accounts for ∼20% of newly diagnosed FL in which the detection rate of t(14;18) is only ∼50%. To investigate the genetic background of localized-stage FL, patient cohorts with advanced-stage FL or localized-stage FL, uniformly treated within phase 3 trials of the German Low-Grade Lymphoma Study Group, were comparatively analyzed. Targeted gene expression (GE) profiling of 184 genes using nCounter technology was performed in 110 localized-stage and 556 advanced-stage FL patients. By penalized Cox regression, a prognostic GE signature could not be identified in patients with advanced-stage FL, consistent with results from global tests and univariate regression. In contrast, it was possible to define robust GE signatures discriminating localized-stage and advanced-stage FL (area under the curve, 0.98) by penalized logistic regression. Of note, 3% of samples harboring an “advanced-stage signature” in the localized-stage cohort exhibited inferior failure-free survival (hazard ratio [HR], 7.1; P = .0003). Likewise, in the advanced-stage cohort, 7% of samples with a “localized-stage signature” had prolonged failure-free survival (HR, 2.3; P = .017) and overall survival (HR, 3.4; P = .072). These data support the concept of a biological difference between localized-stage and advanced-stage FL that might contribute to the superior outcome of localized FL.
Platelet lamellipodium formation is not required for thrombus formation and stability
Authors: Schurr Yvonne , Sperr Andreas , Volz Julia , Beck Sarah , Reil Lucy , Kusch Charly , Eiring Patrick , Bryson Sheila , Sauer Markus , Nieswandt Bernhard , Machesky Laura , Bender Markus
Published: October 2019

Key Points

Cyfip1 plays a crucial role for branching of actin filaments and for lamellipodium formation. Lamellipodium formation is not required for the formation of a hemostatic plug or thrombus.
Bioengineered iPSC-derived megakaryocytes for the detection of platelet-specific patient alloantibodies
Authors: Zhang Nanyan , Santoso Sentot , Aster Richard H. , Curtis Brian R. , Newman Peter J.
Published: October 2019

Key Points

HPA allele–specific HLA class I–negative MKs can be differentiated from CRISPR-edited human iPSCs. Such cells can be stored frozen and thawed to use in whole-cell flow cytometric assays to detect anti-HPA-3a, -3b, and -9b alloantibodies.
Splenectomy prolongs in vivo survival of erythrocytes differently in spectrin/ankyrin- and band 3–deficient hereditary spherocytosis
Authors: Reliene Ramune , Mariani Mariagabriella , Zanella Alberto , Reinhart Walter H. , Ribeiro M. Leticia , del Giudice Emanuele Miraglia , Perrotta Silverio , Iolascon Achille , Eber Stefan , Lutz Hans U.
Published: October 2019

Red cell (RBC) deformability and membrane-bound immunoglobulin G (IgG) were studied to better understand premature clearance of erythrocytes in hereditary spherocytosis. Averaged deformability profiles from cells having comparable cell age revealed that splenectomy was more beneficial for spectrin/ankyrin-deficient than for band 3–deficient RBCs. Splenectomy prevented an early loss of young cells in both types of deficiencies. It had an additional beneficial effect on spectrin/ankyrin-deficient but not band 3–deficient RBCs. It prolonged the survival of mature spectrin/ankyrin-deficient RBCs such that they lost their deformability more slowly than RBCs from patients who had not undergone splenectomy. Band 3–deficient RBCs lost their deformability at the same rate before and after splenectomy. In HS patients with band 3 deficiency who underwent splenectomy, RBC deformability inversely correlated with the number of RBC-bound IgG (up to 140 molecules per cell). In spectrin/ankyrin deficiency, RBC-bound IgG remained at control levels (60 IgG or less per cell). It appears that spectrin/ankyrin-deficient RBCs escaped opsonization by releasing band 3–containing vesicles because their band 3 content and deformability dropped in parallel with increasing cell age. Band 3–deficient RBCs did not lose band 3 with increasing cell age. Hence, it is possible that band 3 clusters required for bivalent binding of low-affinity–IgG, naturally occurring antibodies were retained in band 3–deficient RBCs with a relative excess of skeletal proteins but were released from spectrin/ankyrin-deficient RBCs, in which vesicle budding was facilitated by an impaired skeleton.
Direct evidence for new T-cell generation by patients after either T-cell–depleted or unmodified allogeneic hematopoietic stem cell transplantations
Authors: Lewin Sharon R. , Heller Glenn , Zhang Linqi , Rodrigues Elaine , Skulsky Eva , van den Brink Marcel R. M. , Small Trudy N. , Kernan Nancy A. , O'Reilly Richard J. , Ho David D. , Young James W.
Published: October 2019

Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution of normal T-cell immunity. Recipient thymic activity, biologic features of the allograft, and preparative regimens all contribute to immune reconstitution. We evaluated circulating T-cell phenotypes and T-cell receptor rearrangement excision circles (TRECs) in 331 blood samples from 158 patients who had undergone allogeneic HSCTs. All patients had received myeloablative conditioning regimens and were full donor chimeras in remission. Younger patients exhibited more rapid recovery and higher TRECs (P = .02). Recipients of T-cell–depleted allografts initially had lower TRECs than unmodified allograft recipients (P < .01), but the difference abated beyond 9 months. TREC level disparities did not achieve significance among adults with respect to type of allograft. Measurable, albeit low, TREC values correlated strongly with severe opportunistic infections (P < .01). This finding was most notable during the first 6 months after transplantation, when patients are at greatest risk but before cytofluorography can detect circulating CD45RA+ T cells. Low TRECs also correlated strongly with extensive chronic graft-versus-host disease (P < .01). Recipients of all ages of either unmodified or T-cell–depleted allografts therefore actively generate new T cells. This generation is most notable among adult recipients of T-cell–depleted allografts, most of whom had also received antithymocyte globulin for rejection prophylaxis. Low TREC values are significantly associated with morbidity and mortality after transplantation. T-cell neogenesis, appropriate to age but delayed in adult recipients of T-cell– depleted allografts, justifies interventions to hasten this process and to stimulate desirable cellular immune responses.
Killer Immunoglobulin-Like Receptors (KIRs) Genotypes in a Belgian Population.
Authors: Hidajat Melanny , Selleslag Dominik , Van Hoof Achiel , Van Droogenbroeck Jan , Billiet Johan , Criel Arnold
Published: October 2019

KIRs (Killer cell Immunoglobulin-like Receptors) are expressed on NK (Natural Killer) cells and a subpopulation of T lymphocytes namely memory CD8+ T cells. The distribution of KIR genes varies among individuals and populations. These genes are encoded on chromosome 19 (19q13.4). Till now 17 KIR genes and pseudogenes have been identified. KIRs recognise groups of HLA class I alleles. NK activity is partially controlled through the interaction between KIRs and their HLA ligands. Several studies report that KIRs may affect the outcome of Hematopoietic Stem-Cell Transplantations.

We performed KIR typing of 17 genes and pseudogenes in 100 healthy Belgian unrelated individuals in “West-Vlaanderen” from Caucasoid origin using a PCR-SSP method. Three genes (KIR3DL3, 2DL4 and 3DL2), named frame-work genes, and the pseudogene 3DP1 were found in all individuals. KIR2DL1 and 2DP1 genes were present in a frequency of 99%. In addition, KIR3DL1 and 2DS4 genes represented a frequency of 97. The KIR2DL3 was found in 90% and the frequencies of other genes varied between 56% and 24%. The individual KIR gene content ranged from 8 to 17 genes. A total of 19 KIR locus profiles was observed. The most common KIR locus profile (32%) consisted of a combination of genes characterising A haplotypes (KIR2DL1 and 2DL3) without the presence of genes characteristic of B haplotypes (KIR2DL2 and 2DS2). The second most common KIR locus profile, accounting for 20% contained a combination of genes characteristics for both A and B haplotypes. The allele KIR2DS4*003 was found in 89% and KIR2DS4*00101/00102/002 only in 41%. KIR3DP1*00301/00302 was present in all individuals and KIR3DP1*001/002 only in 35%.

Our results show that frequencies of most KIR loci in our Belgian population were comparable to literature data of other Caucasian populations. Only KIR2DS1 was lower (27% vs 47.7% with a p-value of 0.0002).

In the future, KIR typing of donor and patient before a Hematopoietic Stem-cell Transplantation may be necessary, due to the diversity in KIR genotypes.
XmAb™2513, an Fc Engineered Humanized Anti-CD30 Monoclonal Antibody, Has Potent In Vitro and In Vivo Activities, and Has the Potential for Treating Hematologic Malignancies.
Authors: Lawrence Chris E. , Hammond Phil W. , Zalevsky Jonathan , Horton Holly , Chu Seung , Karki Sher , Desjarlais John R. , Carmichael David F.
Published: October 2019

XmAb2513 is a novel humanized monoclonal antibody (mAb) that binds to the human cell surface antigen CD30 and demonstrates anti-proliferative activity against CD30-positive (CD30+) cell lines. XmAb2513 also has an engineered Fc region to enhance cell killing activity via recruitment of effector cells through increased binding affinity to Fcγ receptors (FcγRs). Consequently, XmAb2513 exhibits superior antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP), when compared to a native IgG1 (unengineered) version of the antibody. To evaluate the potential clinical activity of XmAb2513 in CD30+ diseases such as Hodgkin Lymphoma (HL) and Anaplastic Large Cell Lymphoma (ALCL), XmAb2513 was tested in murine subcutaneous xenograft models of HL using the CD30+ L540 cell line. In the ICR-SCID mouse strain, intraperitoneal (ip) administration of XmAb2513 at 3 mg/kg every 4 days for 10 doses (q4d ×10), gave a statistically significant reduction in tumor growth and enhanced survival relative to the control. At doses of 10 and 30 mg/kg XmAb2513 (ip, q4d ×10) tumor growth was not only slowed, but elimination of established tumors was observed in 3/9 and 5/9 animals respectively. The treatment was well-tolerated. Preclinical studies were conducted to evaluate the safety and pharmacokinetics of XmAb2513 in large animals. In vitro studies demonstrated that the cynomolgus monkey was the appropriate species for study. Binding affinities of XmAb2513 to both human and cynomolgus monkey CD30 and FcγRs were evaluated by Biacore methods and were found to be similar. Additionally, fluorescein-XmAb2513 gave similar staining patterns in immunohistochemistry cross-reactivity studies with normal human and cynomolgus monkey tissue panels. As evidenced by in-life observations single (0, 1 and 100 mg/kg XmAb2513, intravenous [iv] infusion) and repeat dose (0, 10, 30 and 100 mg/kg XmAb2513, q5d ×6, iv infusion) treatment with XmAb2513 was well-tolerated. Serum cytokines showed no trend that was indicative of an XmAb2513-related effect following single dose administration. The pharmacokinetics of XmAb2513 was also determined after either single or repeat dose administration. In the repeat dose study (0, 3, 10 and 30 mg/kg XmAb2513, q5d ×6, iv infusion) exposure increased in a dose proportional manner, and terminal half-life (t1/2) ranged from 12–17 days. In the single dose study (0, 1 and 100 mg/kg XmAb2513, iv infusion) exposure was also proportional to dose. The exposure and t1/2 data support an every other week dosing interval in the clinic. These preclinical data provide a rationale for the clinical testing of XmAb2513 in patients with hematologic malignancies that express CD30, specifically HL and ALCL, and support the safety of repeat administration of XmAb2513 in humans.
Sequential Treatment with Rituximab and CHOP Chemotherapy in B-Cell PTLD - A New Standard in Therapy?.
Authors: Trappe Ralf , Choquet Sylvain , Oertel Stephan , LeBlond Veronique , Ekman Tor , Sender Monica , Molle Peter , Reinke Petra , Lehmkuhl Hans , Hummel Manfred , Jonas Sven , Neuhaus Ruth , Salles Gilles , Morschhauser Franck , Jaccard Arnaud , Dierickx Daan , Anagnostopoulos Ionannis , Raphael Martine , Riess Hanno
Published: October 2019

Purpose: This trial aimed to investigate the efficacy and safety of sequential treatment with rituximab and CHOP-21 in patients with PTLD unresponsive to reduction of immunosuppression.

Methods: An ongoing prospective multicenter phase II trial was initiated in January 2003. Patients were treated sequentially with rituximab at days 1, 8, 15 and 22 followed by four cycles of CHOP-21 combined with G-CSF support starting 4 weeks after the last dose of rituximab.

Results: In this 3rd interim analysis after enrolment of 75 patients 64 patients have finished the protocol. The median follow up is 19.6 months. 58 patients were diagnosed with monomorphic PTLD, 6 with polymorphic PTLD. 23 patients were kidney, 15 liver, 12 heart, 3 lung, 2 heart+lung, 3 kidney+pancreas, 1 bone marrow transplant recipients (5 others). Median age was 53 years (range 16 to 74). 59% had stage III or IV disease. 48% of tumors were EBV positive. 79% of patients had late PTLD (i.e. later than 1 year after transplantation). LDH was elevated in 67% of patients. The overall response rate of sequential therapy was 90% (CR 65%, PR 25%). Progression free survival (PFS) and disease free survival (DFS) were 71.4% and 81.2% at two years, respectively. Treatment response to rituximab (CR/PR versus SD/PD) was a significant factor predicting overall survival (OS) with OS rates of 91.3% and 56.5% at 1 year, respectively (p=0.0107). Following chemotherapy, WHO °3/4 leukopenia was observed in 38% of cycles and 16% of patients suffered from WHO °3/4 infections. There were four early therapy-associated deaths due to infections (7%). Fatal bleeding complications occurred in 3% and 5% of patients died from primary refractory disease.

Conclusions: This is one of the largest prospective studies in PTLD. Sequential treatment with rituximab and CHOP-21 + G-CSF is well tolerated and highly effective. Treatment response to rituximab is predictive for overall survival. As compared to rituximab monotherapy more patients achieve a CR with sequential therapy and PFS is very much prolonged.

Figure Figure
Disruption of Bcl-2 Heterodimerization by the BH3 Mimetic ABT-737 Restores Spontaneous Apoptosis and Induces Differentiation in High Risk MDS and AML.
Authors: Ades L. , Boehrer S. , Braun T. , Grosjean J. , Fabre C. , Fenaux Pierre , Kroemer Guido
Published: October 2019

Background : Increased spontaneous apoptosis is a hallmark of low risk MDS and its suppression by secondary events favors progression to AML. The ratio of Bcl-2 family members is related to progression in MDS, and high expression of proapoptotic Bax is associated with favorable outcome while high expression of antiapoptotic Bcl-2 is linked to poor prognosis.

Methods : we investigated the potential of ABT-737 - known to disrupt the Bcl-2/Bax heterodimer - on cell death, cell cycle progression and differentiation of AML and MDS cells, including AML and MDS cell lines (HL 60, U 937, P39) and cells from MDS and AML patients.

Results : In the cell lines studied

ABT-737 induced cell death in a time- (from 24 to 72 hours) and dose-dependent (from 100 nM to 1 mM) manner as evidenced by the loss of the mitochondrial transmembrane potential and expression of phosphatidylserine residues on the cell surface. In addition, incubation of HL60 and P39 cell lines with ABT-737 increased apoptosis (up to 80% at 48h with 500 nM of ABT 737) while U937 cells were resistant ; ABT-737 promoted concomittant release of mitochondrial protein cytochrome c and subsequent activation of caspase-3 ABT-737 caused caspase-dependent cell death, which was prevented by the pan-caspase inhibitor zVAD-fmk.

Evaluating the underlying molecular mechanisms by small interfering RNA, we found that knockdown of Bcl-2 recapitulated the apoptosis-inducing effect of ABT-737, whereas abrogation of its proapoptotic counterpart Bax remained without effect. Moreover, down-regulation of Mcl-1 rendered otherwise ABT-737-resistant blasts (U937 cells) sensitive to the drug. Noteworthy, although ABT-737 had no impact on the cell cycle distribution of blasts, it nevertheless enabled them to overcome the differentiation block. Indeed, incubation of HL60, P39 cells with ABT-737 increased cell surface expression of the differentiation marker CD11b, an event that could also be prevented by caspase inhibition. Finally, CD34-positive cells from 12 MDS and AML patients were treated ex vivo with ABT-737. ABT-737 induced apoptosis in the 8 samples of high risk MDS and AML (20–60% increase in cell death with 200 nM of ABT737, as compared to controls), whereas the 4 samples of low risk MDS proved to be resistant.

Conclusion, we provide novel evidence that ABT-737 is able to restore apoptosis and to overcome the differentiation block that characterizes malignant cells of high risk MDS and AML, suggesting potential therapeutic activity in those disorders.
Comparison of Cytochemistry, Flow Cytometry and Immunohistochemistry for the Detection of Myeloperoxidase in Acute Myeloid Leukemia.
Authors: Saravanan Linda , Juneja Surender
Published: October 2019

Myeloperoxidase (MPO) is an unequivocal marker of myeloid differentiation and is used routinely in the diagnosis of acute myeloid leukaemia (AML). Cytochemistry (CC), flow cytometry (FCM) and immunohistochemistry (IH) are three routine techniques used to demonstrate MPO positivity. As FCM techniques have improved, IH and CC are being performed less frequently. However, very few reports comparing combinations of these modalities have been published, and to our knowledge none comparing all three. We have reviewed 158 bone marrow biopsies of patients diagnosed with AML between 2000 and 2007 at the Royal Melbourne Hospital, Victoria, Australia. A comparison of MPO positivity using CC, FCM and IH was performed. In 101/158 biopsies all three methods were performed, while the remaining 59 cases had only 2 of the 3 performed. 76 (75%) of these 101 cases showed MPO positivity by all three modalities, defined as at least 3% MPO positive cells on cytochemistry or IH, and at least 10% MPO positive cells by FCM. A further 2 biopsies were negative for MPO by all three techniques. In 23/101 (23%) biopsies in which all three methods were performed, discrepancies occurred between the different modalities (Table 1). The largest group (11/23, 48%) was positive by FCM and IH but negative by CC. A further 7/23 (30%) cases were positive by IH only. Four (17%) cases were positive by CC and IH in the presence of negative FCM, and there was a single case in which FCM was positive when CC and IH were negative. In three cases of AML minimally differentiated (FAB-M0), IH was the only modality able to identify MPO positivity. CC was the least sensitive with no cases of detection of MPO positivity in the presence of negative results by FCM and IH. However there were still 4 cases where FCM was negative in the presence of a positive CC result. These results confirm the importance of employing more than one method of detecting MPO, particularly in cases where FCM and CC are negative or discrepant. In our study, IH was the most sensitive marker for demonstration of MPO. Despite the widespread use of FCM for typing AML, CC and IH still have an important role to play in the demonstration of MPO positivity in these cases.

Table 1. Detection of MPO by cytochemistry, flow cytometry and immunohistochemistry in discrepant cases. Cytochemistry Flow Cytometry Immunohistochemistry Number of cases (n=23) + MPO positive, − MPO negative − + + 11 − − + 7 + − + 4 − + − 1
Wilms Tumor 1 Gene (WT1) Isoforms in Leukemia and Lymphoma.
Authors: Li Aihong , Westman Ulla-Britt , Golovleva Irina , Kutok Jeffery L. , Silverman Lewis B. , Sallan Stephen E. , Roos Goran
Published: October 2019

The Wilms tumor 1 gene (WT1) acts as a transcriptional activator or repressor and is important for normal development. Evidence has accumulated to show that WT1 is oncogenic in acute leukemia and in some solid tumors. WT1 has 4 major isoforms and each encoded polypeptide is thought to make a contribution to normal gene function, but no data are available on their expression in hematologic malignancies. We have analyzed the expression of WT1 and its four isoforms (A, B, C, and D) in 187 diagnostic samples from patients with leukemia or lymphoma. WT1 RNA expression was detected in 9/10 (90%) chronic myeloid leukemias (CML), in 41/47 (87%) of acute myeloid leukemias (AML), in 38/56 (68%) acute lymphoblastic leukemias (ALL) and in 22/74 (30%) of malignant lymphomas. The highest expression levels were found in pre-B ALL and in AML samples. Aggressive lymphomas were more often WT1 positive and with higher levels compared to indolent cases (p < 0.01). Only 9% of AML and ALL bone marrows in complete remission (n=22) showed detectable WT1 expression and all benign lymph nodes studied (n=11) were WT1 negative. The different WT1 isoforms demonstrated highly variable expression levels with isoform A as the most common form in all tumor types. There was no difference in isoform expression between T-ALL and B-lineage ALL. However, AML-M3 demonstrated a significantly higher expression of isoform A compared to other AML subtypes (p<0.01). T-cell lymphoma had a higher level of all isoforms than other lymphoma types (p<0.01). We observed a high concordance regarding WT1 expression levels between matched samples from peripheral blood and bone marrow in T-ALL (R=0.982, P<0.01). A significant reduction in WT1 levels was demonstrated during chemotherapy in two pediatric AML patients. In conclusion, WT1 expression was coupled to malignancy in both bone marrow and lymph node samples and thus could be considered as a potential molecular marker for minimal residual disease detection in acute leukemia. All four WT1 isoforms could be detected in most WT1 positive samples, but their functional significance remains to be clarified.
Treatment of T Cell Large Granular Lymphocyte Leukemia with Reduced-Intensity Allogeneic Stem Cell Transplantation.
Authors: Donato Michele L. , Goldberg Stuart L. , Pecora Andrew L. , Rowley Scott D.
Published: October 2019

T cell large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder of clonal CD3-positive cytotoxic T cells. Clinical manifestations include neutropenia, anemia, thrombocytopenia, recurrent infections and splenomegaly. Two patients with refractory LGL leukemia were treated with an allogeneic stem cell transplantation following a reduced-intensity conditioning regimen. The first patient was a 54 yo female with transfusion dependent anemia and splenomegaly. Prior therapy included low dose methotrexate, cyclosporin A, equine antithymocyte globulin and alemtuzumab. The second patient was a 34 yo female with lymphocytosis and recurrent infections. Prior therapy included cyclosporin A and splenectomy. The hematopoietic cell source was related donor peripheral blood stem cells (5.79 x 106 CD34+ cells/kg) for the first patient, and unrelated donor peripheral blood stem cells (8.04 x 106 CD34+ cells/kg) for the second patient. The preparative regimen was fludarabine 30mg/m2 daily on days −5, −4, −3; melphalan 140 mg/m2 on day −2; the unrelated donor recipient also received rabbit antithymocyte globulin 6mg/kg in 3 divided doses on days −3, −2, −1. GVHD prophylaxis was provided with tacrolimus and methotrexate 5mg/m2 on days +1, 3, 6, 11. Granulocyte engraftment >0.5/μL occurred on day +20 and +11; platelet engraftment >100/μL occurred on day +26 and +116 for the first and second patient respectively. The patient receiving the unrelated donor developed stage 1 skin GVHD and did not require systemic treatment. Patients achieved 100% donor chimerism by day 100. There was no chronic GVHD in either patient. Both patients achieved a complete remission and remain in CR, off immunosuppressive therapy, at 13 and 14 months post transplantation. Allogeneic transplantation using a reduced-intensity preparative regimen is an effective therapy for T cell large granular cell leukemia in patients who have failed prior therapy.
Sodium Gluconate Containing Parenteral Nutrition Solution as a Cause of False Positive Aspergillus Galactomannan Assay in Paediatric Patients with Hematological Malignancies.
Authors: Ribaud Patricia , Sulahian Annie , Touratier Sophie , Brethon Benoit , de Latour Regis Peffault , Socie Gerard , Menotti Jean , Derouin Francis
Published: October 2019

Background: The detection of Aspergillus galactomannan antigenemia (GM-Ag) is a contributive method in the diagnosis of invasive aspergillosis (IA). Herein, we report 3 cases of false positive GM-Ag in paediatric patients (pts) receiving NP2 Enfants AP-HP® parenteral nutrition (Fresenius, France), and none of them had or developed IA.

Methods: In December 2006, the sera of these 3 pts (1 allogeneic transplant recipient and 2 neutropenic acute leukaemia pts) taken before, during, and after cessation of NP2 solution were tested for the presence of GM-Ag using the Platelia® Aspergillus kit. This solution, its 30 components and 8 other commercially available solutions for parenteral nutrition were tested for the presence of GM.

Results: GM became detectable within 1 day after IV administration of NP2 with indices up to 9.25, remained positive throughout treatment, and became negative (≤0.5) within 48 hours in 2/3 pts. The NP2 solution and a single one of its components, sodium gluconate (SG), tested positive with GM estimated at 55.4ng/ml. No Aspergillus DNA was detected by real-time PCR. Three NP2 and 6 SG batches from different origins yielded similar results. On the other hand, 6 different batches of calcium gluconate and 8 other commercially available SG-free parenteral nutrition solutions tested negative.

Conclusions: These results suggest a strong cross reactivity of Platelia® kit with SG-containing NP2, as recently described with Plasma-Lyte® (Baxter Distribution Centre Europe). We estimate that clinicians and biologists should become aware of this problem which might lead to inappropriate invasive investigations and/or treatments.
Effects of Anemia on the Rehabilitation Outcomes in Elderly Patients in the Post-Acute Care Setting.
Authors: Witkos Maciej , Uttaburanont Michelle , Vicenio Myra , Lang Christopher , Haddad Rami Y.
Published: October 2019

Background - More than 3 million people in the United States over the age of 65 are anemic. The evidence indicates that older people with anemia suffer hospitalizations, physical decline and disability more than people without anemia. Additionally there are reports that anemia is an independent risk factor for this decline in physical performance in those over the age of 65.

Objective - The goal of this study was to detail if anemia significantly affects patient’s rehabilitation outcomes by focusing on the ability to perform activities of daily living (ADL) as assessed by the physical therapist, the disposition upon discharge (meeting the therapy goals) and an analysis of length of stay (LOS) in relation to anemia.

Methods - We conducted a chart review of 260 medical records of patients who underwent rehabilitation therapy at the North Chicago Veterans Affairs Medical Center, and detailed the progress of 69 of these patients who received ADL therapy. The study is ongoing with a projected chart review of 437 medical records, and with an estimated final sample size of 150. Univariate and multivariate analysis was conducted in relation to our outcome measures.

Results - The anemic group had 43 patients and the non-anemic 26. Average values for anemic patients were: Hgb 11 Hct 32, MCV 88, MCH 29.2, RDW 15.6, Platelets 259, the non-anemic patients values were: Hgb 13.4, Hct 39, MCV 89, MCH 33.3, RDW 14.3, platelets 229. The patients with anemia had a median change in ADL scores of 9 versus non-anemic patients’ median change in scores of 12 (p=0.154). Length of stay was 51 days (median) for anemic patients and 36 days (median) for non-anemic patients (p=0.108) Of the non-anemic patients, 19 out of 26 (73%) met the goals of therapy and 21 out of 43 (49%) of the anemic patients met the goals of therapy (p=0.055). Surprisingly, only 3 out of the 43 (7%) anemic patients were treated for their anemia or had work up done to identify the cause of the anemia. The multivariate model for length of stay indicated that anemia was not significant (p=0.842), the morbidity of the patient was marginally not significant (p=0.089), however, the patients’ age was statistically significant at p=0.05. For the multivariate model of ADL improvement the results were: anemia p=0.186, sex p=0.298, Charlson index p=0.50.

Conclusion - There is a definite lack in the identification and treatment of anemia in patients undergoing rehabilitation. Anemia exerts an influence on the course of patient therapy/recovery and should be addressed as an individual problem. Anemic patients undergoing rehabilitation are scoring lower, and are admitted longer to the hospital.
Outcomes in Subcutaneous Panniculitis-Like T-Cell Lymphoma (STCL)
Authors: Alpdogan Onder , Ornstein Deborah , Subtil Tony , Seropian Stuart , Cooper Dennis L. , Foss Francine
Published: October 2019

Subcutanous panniculitis–like T cell lymphoma (STCL) is a rare clinical entity which simulates panniculitis and can present with an aggressive clinical course. STCL is divided two major subgroups (αβ-STCL and γδ-STCL), according the T cell receptor expression on the malignant T cells. γδ-STCL is often associated with a more aggressive course and poor prognosis with an 11% 5-yr survival in a retrospective study of 20 cases, only 6 of which were confirmed by TCRδ -1 staining.1 We report our results from 10 patients with STCL, 2 BetaF1+ (αβ-STCL), 8 γδ-STCL. The median age at presentation was 43 (25–63); 7 of 10 were female. Immunohistochemical studies and TCR gene rearrangements (TCRR) were performed on all patients. Cytotoxic T-cell markers were expressed in all pts (TIA-1 in 8 of 10 and Granzyme B in 5). Six were CD8+ and 3 were CD3+CD4−CD8−. CD56 expression was detected in 3. All demonstrated clonal TCRR. All pts presented with skin nodules or ulcerations, and 3 had visceral involvement (blood/bone marrow in 2, liver in 1). Three patients (2αβ-STCL and γδ-STCL) were treated initially with denileukin diftitox; one each with αβ- and γδ- disease had PR on therapy and have been maintained without PD. Seven patients were treated with cytotoxic chemotherapy regimens. Four of 7 achieved a remission after EPOCH (2), denileukin diftitox-CHOP (1) or pentostatin/cyclophosphamide followed by alemtuzumab (1). Four pts (1 with refractory ab-STCL, 2 with refractory gd-STCL, and 1 with γδ-STCL in first CR after denileukin diftitox-CHOP) underwent allogeneic hematopoietic stem cell (HSCT) from matched-related donors. Two patients are alive 6 and 13 months after HSCT with no evidence of disease; 1 patient died in CR from infectious complications of HSCT, and 1 relapsed 6 mo after HSCT and died from PD. At a median follow up of 3 yrs from diagnosis, 8 pts (80%) are alive, including the 2 pts with αβ-STCL and 6 of 8 pts with γδ-STCL. In summary, while γδ-STCL is reported in retrospective studies to have a poor prognosis, we demonstrate that aggressive therapies along with the incorporation of novel T-cell directed agents such as denileukin diftitox and alemtuzumab into treatment regimens and the use of allogeneic HSCT as a potentially curative therapy are promising approaches for these patients.
Is Diabetes Mellitus An Independent Risk Factor for Incident Venous Thromboembolism (VTE)? a Population-Based Case-Control Study
Authors: Heit John A. , Leibson Cynthia L. , Ashrani Aneel A. , Petterson Tanya M. , Bailey Kent R. , Melton L. Joseph
Published: October 2019

Introduction: Diabetes mellitus is often cited as a VTE risk factor. However, persons with diabetes are frequently hospitalized for medical illness or undergo surgery, both major VTE risk factors. Thus, the association of VTE with diabetes independent of surgery or hospitalization is uncertain.

Methods: Using longitudinal, population-based Rochester Epidemiology Project resources, we identified all Olmsted County, MN residents who met objective criteria for incident VTE over the 25-year period, 1976–2000 (n=1922), and one to two controls per case, matched on age, gender, Olmsted County residency, and length of medical history (n=2115). For cases and controls, we reviewed their complete medical history in the community for baseline characteristics previously identified as independent VTE risk factors, including clinically-diagnosed diabetes mellitus. We tested diabetes as a potential VTE risk factor both alone and after adjusting for other baseline characteristics, and in the subset of cases with idiopathic VTE, using conditional logistic regression.

Results: Among all cases and controls, 231 (12%) and 199 (9.4%) had diabetes, respectively. Univariately, diabetes was associated with overall VTE (odds ratio [OR]=1.32; 95% CI: 1.07, 1.62; p=0.009). However, after controlling for body mass index (OR=1.04, p<0.001), hospitalization with major surgery (OR=25.2, p<0.001) or hospitalization for medical illness (OR=7.3, p<0.001), active cancer (OR=8.3, p<0.001) and varicose veins (OR=1.3, p=0.003), diabetes was no longer associated with VTE (OR=1.09; 95% CI: 0.85, 1.41; p=0.49). Among 458 idiopathic VTE cases and 518 matched controls, 43 (9.4%) and 46 (8.9%) had diabetes, respectively. Diabetes was not associated with idiopathic VTE (OR=1.07, 95% CI: 0.69, 1.67; p=0.76).

Conclusions: Diabetes mellitus is not an independent risk factor for overall or idiopathic incident VTE.
Hemoglobin Blocks Von Willebrand Factor Proteolysis by ADAMTS-13: A Mechanism Associated with Acquired ADAMTS-13 Deficiency in Patients with Sickle Cell Disease
Authors: Zhou Zhou , Hyojeong Han , Cruz Miguel A. , Lopez Jose A. , Dong Jing-fei , Guchhait Prasenjit
Published: October 2019

One of the hallmark events of sickle cell disease (SCD) is vasoocclusion and episodic pain crisis. Although the mechanism of vascular occlusion is very complicated, processes like thrombosis and thromboembolism have been recognized to play an important role in the development of such clinical manifestation in SCD. Studies have shown that the von Willebrand factor (VWF), especially the ultra-large (UL) multimers play a major role in vasoocclusion, which clearly indicates a possible impairment of the VWF-cleaving metalloproteae ADAMTS-13 in these patients with SCD. In a recent work, indeed we have mentioned that the plasma ADAMTS-13 in patients with SCD having normal antigen level showed 35% less protease activity than the normal. There may be several plasma factors responsible for the acquired deficiency of ADAMTS-13 in SCD. Since, the increasing evidences suggest that the elevated level of extracellular hemoglobin (Hb) in plasma parallely associated with the pathogenesis of SCD, we investigated the effects of extracellular Hb on VWF proteolysis by ADAMTS-13. We observed that purified Hb dose-dependently inhibited the ADAMTS-13 cleavage of recombinant(r) VWF and endothelial ULVWF multimers under static and flow conditions. Hb bound to VWF multimers in a saturation-dependent manner and more potently to the rVWFA2 domain (affinity Kd~24nM), which contains the cleavage site for ADAMTS-13. Hb bound also to the ADAMTS-13 (Kd~65nM), with 2.7 times less affinity than to VWFA2. The bindings were neither calcium-dependent nor affected by haptoglobin. However, it is the Hb-binding to VWF that prevented the substrate from being cleaved by ADAMTS-13. These in vitro findings are consistent with the in vivo observations in patients with SCD. An elevated level of extracellular Hb in plasma was inversely correlated (linear regression, r2 =0.6354) with the low activity of ADAMTS-13 in a cohort of ten adult patients with SCD (mean±SE, Hb 346±138 mg/l; activity 33.3±30%) compared to age and gender-matched normal individuals (n=10; Hb 24±8 mg/l; activity 76.2±16%). The data together suggest that patients with SCD suffer from acquired ADAMTS-13 deficiency, primarily because Hb competitively binds and inhibits the proteolysis of VWF multimers, leading to ULVWF accumulation on vascular endothelium and in circulation. The Hb-VWF interaction may therefore be considered as a therapeutic target for reducing thrombotic and vasoocclusive complications in patients with severe hemolysis such as those with SCD.