bluuebunny/crossref_metadata_2025 huggingface dataset:
CLL Transcript and Serum Profiling Identifies Distinct Oncogenic Pathways in Rai Stage Progression.
Authors: Mahadevan Daruka , Klinkhammer Thomas , Cooke Laurence , Riley Christopher , Choi James , Sinari Shripad , Miller Thomas P. , Spier Catherine
Published: October 2019

Background: Chronic lymphocytic leukemia (CLL) is a common leukemia that is incurable. Survival curves for Rai stage 0/I and III/IV are distinct and a specific gene expression signature may be associated with stage progression (SP). However, distinct oncogenic pathways that differentiate stage progression are not well defined.

Methods: Mononuclear cells from Rai stage 0/I (N=8) and III/IV (N=8) CLL patients were obtained through an IRB approved protocol and analyzed utilizing the HG-U133A 2.0 Affymetrix array after isolating and purifying total RNA (Qiagen, RNAeasy). Control RNA samples are from 4 normal volunteers’ peripheral blood (PB) B-cells (AllCell, CA) and a normal reactive lymph node. Statistical significance of individual genes and pathways was analyzed using ‘BioConductor’. Differential gene expression was analyzed using ‘Limma’. Pathways and cytogenetics regions were analyzed for enrichment using ‘Category’. Real time RT-PCR on specific genes was performed to validate the GEP based on stage. A serum cytokine profile (120 cytokines, RayBiotech) on 8 CLL patients (N=4 low risk; N=4 high risk) and 4 normal volunteers was evaluated to identify a signature for diagnosis and prognosis.

Results: Data are presented as “robust” increases or decreases of relative gene expression common to 8 patients in the low or high risk stages. The Wnt/Ror-1 non-canonical, and TGFb pathways are over-expressed in Rai stage 0/I, implicating a program of cell survival and dysregulated migration. The Hedgehog, Proteasome, TNF/FAS/NF-kB, and MAPK pathways are over-expressed in Rai stage III/IV, suggestive of a tumor microenvironment driven program in SP. Genes located on chromosome 3q27.1 are also dysregulated in Rai stage III/IV. A distinct serum cytokine profile also differentiates low from high stage CLL.

Conclusions: GEP of CLL with RT-PCR validation has identified distinct oncogenic pathways that distinguish Rai stage 0/I from III/IV. A serum cytokine profile also differentiates between low and high risk CLL. These data together provide a ‘molecular signature’ for SP in CLL.
Phenotypic Analysis of Plasma Cells in Monoclonal Gammopathy and Multiple Myeloma Subjects.
Authors: Kovarova Lucie , Buresova Ivana , Pour Ludek , Suska Renata , Adam Zdenek , Krejci Marta , Vorlicek Jiri , Penka Miroslav , Hajek Roman
Published: October 2019

Introduction: Multiple myeloma (MM) is a B-cell neoplasia caused by the proliferation of clonal plasma cells (PCs). MM and benign monoclonal gammopathy of unknown significance (MGUS) are routinely distinguished on the basis of paraprotein concentration, level of PCs infiltration and the presence or absence of other clinical features. Flowcytometric detection of PCs according to the expression of CD38 and CD138 has limitation in discrimination between normal and abnormal PCs, but this is possible in multicolor phenotypic analysis. The aim of this study is to compare the numbers of normal and abnormal PCs in MGUS and MM subjects and to find some parameter useful for evaluation PCs distribution.

Materials and methods: 51 newly diagnosed untreated MM patients (63,9±10,0 years old) and 31 non-treated MGUS subjects (64,2±13,8 years old) were analysed. Lysed whole bone marrows were analysed by flowcytometric immunophenotyping and PCs were indentified by expression of CD38, CD138, CD45 and also CD56 and CD19.

Results: Discrimination between normal CD19+ PCs and abnormal CD56+ PCs was done on CD38+CD138+ population. Ratio of normal PCs count and abnormal PCs counts (normal/abnormal=N/A) was used to describe a distribution of PCs. Subjects with MGUS had 0,97±1,65% (average±SD) of CD38+CD138+ cells (median 0,45; range 0,03–7,47%) with average N/A ratio 2,91±3,94 (median 1,36; range 0,01–18,44). Newly diagnosed MM patients had 4,96±9,94% of CD38+CD138+ cells (median 0,85; range 0,02–41,80%) with average N/A ratio 1,70±3,03 (median 0,13; range 0–11,5). In 9,7% MGUS subjects evaluation of distibution of PCs showed transformation of MGUS into MM. In some newly diagnosed MM patients (31,4%) CD38+CD138+ plasma cells were positive for CD19 although they were aberrant and these PCs were mostly CD45+. In some patients (9,7% of MGUS; 17,6% of MM) PC did express neither CD19 nor CD56 and this fact may complicate further evaluation of PCs.

Conclusions: Results confirmed that in MGUS subjects we can find lower numbers of PCs which are mostly CD45+CD19+. A majority of plasma cells in newly diagnosed MM patients are abnormal CD56+ PCs and these plasma cells are usually CD45−. Further follow-up of patients can confirm the N/A ratio as a predictive factor for transformation of MGUS into MM and its value for evidence of relapse or progression to active myeloma.
Activity of ARRY-614, an Inhibitor of p38 Map Kinase and Angiogenic Targets, in Hematologic Malignancies.
Authors: Winski Shannon L. , Gross Stefan D. , Brown Suzy A. , Anderson Deborah , Garrison Augusta , Rodriguez Mareli , Lee Patrice , Munson Mark , Winkler James
Published: October 2019

Multiple Myeloma (MM) is a malignancy characterized by the clonal expansion of plasma cells within the bone marrow microenvironment. Within this compartment, the proliferative capacity of MM cells is enhanced by pro-inflammatory cytokines, and there is growing recognition that bone marrow stromal cells play an important role in the support of MM growth and chemo-resistance. We have identified a series of compounds, of which ARRY-614 is representative, that are potent inhibitors (EC50<10 nM) of cytokine synthesis through the inhibition of p38 MAPK. This compound is also a potent inhibitor of Abl tyrosine kinases and Tie2/Tek receptor tyrosine kinase. ARRY-614 is active against all of these targets on both the isolated enzymes and in cells. To confirm these activities, ARRY-614 was evaluated in relevant in vitro and in vivo models. ARRY-614 inhibited p38α in ex vivo stimulated human whole blood (EC50=2 nM) and the release of IL-6 and TNFα from SEA- or LPS-challenged mice (ED50<10 mg/kg). Further, ARRY-614 administered as a single agent was efficacious at inhibiting bFGF-driven angiogenesis in an in vivo matrigel invasion assay as well as inhibiting tumor growth in subcutaneous K562 (BCR-Abl dependent) and RPMI 8226 (multiple myeloma) tumor xenografts in mice, at doses ranging from 30 to 100 mg/kg qd, PO. In regulated safety studies, this compound was well-tolerated at doses up to 100 mg/kg qd and 30 mg/kg qd in rats and cynomolgus, respectively. Together, these data support the advancement of this agent into clinical trials for hematologic malignancies.
Prospective Evaluation of MRD-Kinetics in 274 Children with High-Risk ALL Treated in Trial ALL-BFM 2000: Insights into Development of Resistance and Impact on Further Refinement of Treatment Stratification Strategies.
Authors: Schrauder André , Stanulla Martin , Flohr Thomas , Cario Gunnar , Köhler Rolf , Panzer-Grümayer Renate , Schäfer Beat , Zimmermann Martin , Peters Christina , Bartram Claus R. , Schrappe Martin
Published: October 2019

In trial ALL-BFM 2000, high-risk (HR) acute lymphoblastic leukemia (ALL) is defined by inadequate initial response to induction treatment [poor prednisone response on treatment day eight, non remission on treatment day 33, and/or a high load of minimal residual disease (MRD, ≥10E-3) after 12 weeks of treatment (TP2)] and/or by cytogenetics [t(4;11 or t(9;22)]. Between August 1999 and November 2006, 494 (15%) out of 3255 study patients were stratified into the HR branch of trial ALL-BFM 2000. 431 (87%) of these HR patients underwent successful MRD monitoring at TP2 with 274 (56%) patients having received additional extensive prospective MRD monitoring subsequent to TP2. Patients with an indication for stem cell transplantation (SCT) and a suitable donor were scheduled for SCT within six weeks after the third HR block of the intensive consolidation phase. The estimated 4-years event-free-survival (4y-pEFS) for the entire HR group was 68%+/−3%, estimated survival was 74%+/−3%. Patients with MRD load of ≤10E-4 at TP2 (n=231) had a 4y-pEFS of 82%+/−3%, patients with MRD levels of 10E-3 at TP2 (n=84) had a 4y-pEFS of 74%+/−6%, and patients with MRD of >10E-3 at TP2 (n=116) had a 4y-pEFS of 35%+/−5%. MRD-kinetics subsequent to TP2 revealed, that 85% of all patients with an MRD level of 10E-3 at TP2 continued to decrease their load below 10E-3 during the pulsatile intensive consolidation phase, whereas this was observed in only 35% of patients with an MRD level of >10E-3 at TP2. The 4y-pEFS of patients with an MRD load persisting at 10E-2 after application of three intensive HR blocks after TP2 was 0% if no SCT was performed, and 33%+/−11% after SCT in CR1. Our data reflect that extensive MRD measurements in HR-ALL patients allow a dynamic insight into the development of resistance, and serve as valuable tool for further clinical treatment adjustment. “MRD non-response” after three BFM HR blocks identifies a patient group in urgent need of alternative treatment elements, closely monitored by MRD, before going into SCT in CR1.
Loss of FOXP3 Expression and Emergence of Cytokine-Producing Cells after In Vitro Expansion of Human CD4+CD25+CD127 - Regulatory T Cells.
Authors: Hoffmann Petra , Eder Ruediger , Boeld Tina J. , Huehn Jochen , Floess Stefan , Wieczorek Georg , Baron Udo , Olek Sven , Andreesen Reinhard , Edinger Matthias
Published: October 2019

We and others previously showed that the adoptive transfer of donor-type CD4+CD25+ regulatory T (Treg) cells protects from graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT) in animal models. Exploring this strategy in human SCT, we currently perform a first Phase I clinical trial using freshly isolated Treg cells. For future trials requiring large Treg cell numbers for repetitive treatments, we described in vitro culture conditions that permit a more than 3-log expansion of polyclonal Treg cells (Blood 104:895; 2004). A highly enriched starting population proved to be crucial for the generation of pure Treg cell products and we could show that the naive, CD45RA+ subpopulation of CD4+CD25high T cells fulfills this criterion (Blood108:4260; 2006). A recently proposed alternative approach for the isolation of pure Treg cells relies on the exclusion of CD127+ cells, as activated (i.e. CD25+) conventional T cells express high levels of CD127 while CD4+CD25+ Treg cells show no or only weak expression levels (Seddiki et al. and Liu et al., JEM203:1693; 2006). To directly compare these two approaches, we isolated CD4+CD25+CD127low/neg T cells (CD127-Treg) and CD45RA+CD4+CD25high T cells (RA+ Treg) from the same leukapheresis products and analyzed the cells after 2 and 3 weeks of expansion. Whereas both populations were > 94% FOXP3+ upon isolation, only RA+ Treg maintained this high level of FOXP3+ cells throughout the expansion period (93% (range: 78 to 97%; n=11) FOXP3+ after 2 and 87% (range: 71 to 97%; n=9) FOXP3+ after 3 weeks). In contrast, the proportion of FOXP3+ cells in CD127-Treg cultures was already reduced after 2 weeks (82% (range: 56 to 96%; n=11)) and highly variable and significantly lower than that of RA+ Treg cultures after 3 weeks (57% (range: 18 to 93%; n=9; p=0.006)). When further subdivided into CD45RA+ and CD45RA- subpopulations before expansion, cultures of CD45RA-CD127-Treg cells lost FOXP3 expression and comprised substantial numbers of FOXP3- cytokine producing cells (on average 54% and 38% IL-2 and IFN-γ producers, respectively) after 3 weeks, whereas CD45RA+ CD127- Treg behaved similar to RA+ Treg cells, as they maintained FOXP3 expression over time and contained only low numbers of cytokine producers. Furthermore, when we analyzed the DNA methylation status of Treg cells, we found the Treg-specific CpG demethylation pattern within the FOXP3 gene (EJI 37, 2007) in RA+ Treg cell lines, while CD127- Treg cell cultures showed increased methylation over time and even more so RA- Treg cell cultures. Based on these findings, we suggest that isolation and expansion of CD45RA+CD4+CD25high T cells at present represents the best strategy for adoptive cell therapies requiring in vitro expanded Treg cells.
Non-Classical Anti-Factor VIII C2 Domain Inhibitors Are Present in the Plasmas of Most Factor VIII Inhibitor Patients.
Authors: Meeks Shannon L. , Healey John F. , Barrow Rachel T. , Parker Ernest T. , Lollar Pete
Published: October 2019

Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. These inhibitors primarily are directed against the A2 or C2 domains of fVIII. The human response to the C2 domain of fVIII classically has been thought to inhibit fVIII activity by blocking its binding to phospholipid. We recently characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of antibodies. Groups A, AB, and B are classical anti-C2 antibodies. Groups BC and C consist of non-classical anti-C2 antibodies that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Most non-classical antibodies have inhibitor titers greater than 10,000 Bethesda units/mg IgG. To determine if non-classical antibodies are present in fVIII inhibitor patients, patient plasmas were tested in an ELISA for their ability to block the binding of representative antibodies from the different anti-human fVIII C2 antibody groups. Classical and non-classical monoclonal antibodies (MAbs) were biotinylated and serially diluted into either fVIII deficient plasma or patient inhibitor plasma and then added to microtiter wells coated with fVIII. The ability of patient plasma to block the binding of the murine MAbs to fVIII was determined. A total of 16 patient plasmas were assessed: 4 from patients with a C2 predominant response, 2 with a non-C2 predominant response, and 10 with unknown specificities. Three of the 4 patients with C2 predominant responses had non-classical anti-C2 antibodies, while the 2 with non-C2 predominant responses did not. In the unknown plasmas, 6 of 10 had evidence of non-classical antibodies. Figure 1 shows representative results of the effect of 3 patient plasmas on the binding of a biotinylated non-classical MAb to fVIII. Patient plasmas 1 and 2 blocked MAb binding while patient plasma 3 did not. This study indicates that the majority of patients with fVIII inhibitors have non-classical anti-C2 antibodies in their response to fVIII.

Figure Figure
RhoH Functions as an Adaptor Molecule for ZAP-70 in TCR Signaling Pathway.
Authors: Chae Hee-Don , Siefring Jamie E. , Hildeman David A. , Gu Yi , Williams David A.
Published: October 2019

RhoH is a hematopoietic-specific and GTPase-deficient Rho GTPase of the RhoE family. In a previous report, we showed that RhoH plays a critical role in T cell receptor (TCR) signaling via recruitment of ZAP-70 in T cell development (Gu et al., Nat. Immunol., 2006). RhoH-deficient T cells show impaired CD3ζ phosphorylation which is critical for the recruitment of ZAP-70 to the TCR complex. In this study, we investigated whether RhoH acts as an adaptor molecule for ZAP-70 membrane targeting or a regulator of the Lck activity. Although Lck activity was not significantly decreased in RhoH-deficient T cells as determined by the level of Lck phosphorylation and in vitro kinase assay, Lck protein did not translocate to the immunological synapse in these cells and the association between Lck and CD3ζ was impaired. ZAP-70 has been reported to mediate the recruitment of Lck into TCR complex with subsequent CD3ζ phosphorylation. In RhoH-deficient T cells, ZAP-70 did not translocate to TCR complex in the plasma membrane, especially the detergent-insoluble (cytoskeletal) fraction. To further determine if RhoH mediated membrane localization of ZAP-70 and if this localization was responsible for normal T cell differentiation, we generated a myristoylation sequence-fused ZAP-70 (Myr-ZAP-70). Myr-ZAP-70 localized to the membrane fraction and resulted in the partial rescue of RhoH−/ − T cell phenotype as measured by T cell reconstitution in Rag2−/ − recipient mice (Table 1). These results suggest that RhoH plays a key role in TCR signaling as an adaptor for ZAP-70 plasma membrane localization.

Table 1: Engraftment and T cell reconstitution of the transduced RhoH−/ − BM cells in Rag2−/ − recipient mice Donor cells (retroviral vector transduction) CD4+ CD8− cells (x 10e5) CD4− CD8+ cells (x 10e5) Spleen PB (ml) Spleen PB (ml) RhoH−/− (empty vector) 3.33 0.9 1.2 0.36 RhoH−/− (ZAP-70) 16.7 1.2 3.1 0.6 RhoH−/−(Myr-ZAP-70) 35.2 3.3 9.1 2.2 RhoH−/− (HA-RhoH) 65.0 3.9 13.6 1.3
Role of CD37SMIP a Novel Engineered Small Modular Immunopharmaceutical in the Treatment of CLL.
Authors: Lapalombella Rosa , Zhao Xiaobin B. , Baum Peter R. , Ledbetter Jeffrey A. , Muthusamy Natarajan , Byrd John C.
Published: October 2019

CD37 is a lineage-specific B-cell antigen highly expressed on both normal and transformed B-cells. The significant B-cell selective CD37 expression makes it a valuable target for therapy against B cell malignancies including chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), and non-Hodgkins lymphoma (B-NHL). A novel CD37-specific small modular immunopharmaceutical (CD37SMIP) was engineered to contain a single chain variable region (scFv) linked to a human IgG1 hinge, CH2 and CH3 domains. CD37SMIP induces potent apoptosis in the presence of a crosslinker and antibody dependent cellular cytotoxicity (ADCC) against primary CLL cells. Significant in vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model that is dependent upon NK cell function. The induced cytotoxicity in primary CLL cells was independent of activation of caspase cascade, and consistent with this, the pan-caspase inhibitor z-VAD-fmk failed to rescue CD37 SMIP drug induced cytotoxicity. In an attempt to define the CD37 mediated early signaling events and their role in cytotoxicity, we investigated protein tyrosine phosphorylation as a potential early activation event responsible for CD37 SMIP drug mediated cytotoxicity. Primary B CLL cells were stimulated with increasing concentration of CD37 SMIP with or without crosslinker. Western blot analysis of cellular lysates with anti-phosphotyrosine antibody revealed several tyrosine phosphorylated proteins including a predominant ∼65KDa protein in response to the cross-linking of CD37 SMIP within 10 minutes. Further, pre-treatment with the tyrosine kinase inhibitor herbimycin prevented tyrosine phosphorylation of the predominant 65KDa protein and inhibited CD37 SMIP induced apoptosis in a dose dependent manner. Attempts to identify the tyrosine phosphorylated proteins, using MALDI-TOF mass spectrometry, and to define the potential role of these target proteins in the apoptotic pathway are ongoing. To further examine the molecular mechanism whereby CD37 SMIP triggers cell death, we investigated the involvement of mitochondrial pathways including changes in mitochondrial potential (Δψm) and generation of reactive oxygen species. Treatment with CD37 SMIP induces a time dependent mitochondrial membrane depolarization and increased production of ROS. Further, primary B CLL cells cultured with CD37SMIP induced cleavage and mitochondrial translocation of Bax and upregulation of Bim in a time dependent manner indicating a potential role for Bim and Bax pro apoptotic proteins in CD37 induced apoptosis. These findings provide strong justification for CD37 as a therapeutic target aimed at modulation of pro apoptotic proteins and introduce small modular immunopharmaceuticals as a novel class of targeted therapies for B-cell malignancies. (SMIP trademark is owned by Trubion Pharmaceuticals).
PKC zeta-mTOR Pathway: A New Target for Rituximab Therapy in Follicular Lymphoma.
Authors: Laurent Guy , Leseux Ludivine , Laurent Camille , Bezombes Christine
Published: October 2019

Previous studies have documented that, in malignant B-cells, Rituximab elicits a complex and not yet totally understood signalling network contributing to its anti-tumour effect. In this context, we investigated the role of protein kinase C zeta, an atypical PKC isoform, in the cellular response to Rituximab. We found that follicular lymphoma cells displayed an increase in PKC zeta expression and activity levels, compared to non malignant B cells, and that this enzyme was a critical regulator of the classical MAPK module by stimulating Raf-1 kinase activity. PKC zeta appeared to be a significant contributor of abnormal mTOR regulation in follicular lymphoma cells through a MAPK-dependent mechanism. Rituximab was found to inhibit the PKC zeta/MAPK/mTOR module in these cells but not in other B-cell lymphomas. Importantly, the expression of a constitutively active form of PKC zeta resulted in an efficient protection of these cells towards Rituximab. Altogether, our study describes a new regulatory component of mTOR pathway in follicular cell lymphoma, and demonstrates that PKC zeta is a target for Rituximab. Therefore, PKC zeta could represent an important parameter for Rituximab efficacy and a promising target for future targeted therapy in follicular lymphoma.
Suppression of Cyclin D1 by on 01910.Na Is Associated with Decreased Survival of Trisomy 8 Myelodyplastic Bone Marrow Progenitors: A Potential Targetted Therapy.
Authors: Sloand Elaine M. , Pfannes Loretta , Reddy Ramana , Reddy Premkumar , Groopman Jerome S. , Young Neal S.
Published: October 2019

Trisomy 8 CD34 cells persist and even expand in patients with bone marrow failure despite a potent specific immune response against them(Sloand EM et al; Blood2005; 106(3):841). We previously demonstrated dramatic increases in c-myc, survivin, and CD1 in trisomy 8 CD34 cells by microarray analysis, realtime PCR, and immunoblot (Sloand et al; Blood2007; 109:2399) and postulated that upregulation of c-myc located on chromosome 8 was responsible for increases in CD1 and survivin, an anti-apoptotic protein. Knock-down of either survivin or c-myc resulted in selective trisomy 8 apoptosis and death. The styryl sulfones (Reddy, M et al Acta Chim Hungarica1984;115:269), are novel small molecule anticancer agents that inhibit cell cycle progression in cancer cells, and phase I studies for solid tumors have demonstrated little toxicity in humans (Donehower, R et al, J. Clin. Oncol. 24. 2006 abstract #13026). Initial studies in our laboratory demonstrated significant decreases in CD1 measured after trisomy 8 cells were co-cultured with increasing concentrations (10–30 nM). In this study, we examined the effect 01910.Na, a styryl sulfone, on trisomy 8 cell growth and survival. Short-term culture of mononuclear cells from four normal bone marrows with increasing concentrations of the drug (10–200nM) showed no adverse effect on hematopoietic colony formation (N=4). MDS bone marrow cells from ten trisomy 8 patients; 4 with RAEB and 6 with RA and two monosomy 7 patients with RAEB were studied. All patients with cytogenetics demonstrating trisomy 8 either had it as the sole karyotypic abnormality or with monosomy 7 in the same clone. Bone marrow was cultured with drug for 2 weeks at four different drug concentrations and fluorescent in situ hybridization performed by three blinded investigators counting 300 cells each. There was a substantial decline in the number and percent of aneuploid cells containing trisomy 8 (p=0.03; N=6) Fig 1. Other cells, including those with monosomy 7 as the sole abnormality and diploid cells were relatively unaffected. Continued treatment of the cells with a 100–250nM concentration for a 14 day period resulted in optimal killing of aneuploid cells when compared to one treatment at the beginning of the two week period. The number of blasts also decreased over the two week period when slides were prepared, stained with H&E, and read blindly by a pathologist; this effect was dose-dependent (N=3; p=0.02; Fig 2). Flow cytometric examination of a fourth patient confirmed these findings, demonstrating increased proportions of mature CD15 positive myeloid cells and decreased number of immature CD33 cells or blasts staining with CD34 concomitant with decreased numbers of trisomy 8 cells (Fig3). ON 01910.Na may prove a targeted therapy for patients with MDS and a trisomy 8 clone. A phase I/II trial is set to commence shortly.

Figure Figure
Targeting CD22 in Childhood B-Precursor Acute Lymphoblastic Leukemia (Pre-B ALL): Pre-Clinical Studies and Phase I Trial of the Anti-CD22 Immunotoxin CAT-3888 (BL22).
Authors: Wayne Alan S. , Findley Harry W. , Lew Glen , Ahuja Yachna , Gu Lubing , Stetler-Stevenson Maryalice , Kreitman Robert J. , Pastan Ira
Published: October 2019

Although most children with pre-B ALL are cured, treatment is associated with multiple toxicities and outcome after relapse is poor. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. We evaluated the B-lineage differentiation antigen CD22 and the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) in childhood ALL.

Methods: Blasts were obtained from children with pre-B ALL. Flow cytometry was used to determine CD22 expression and anti-CD22 antibody binding using the BD Quantibrite System. 72 hour in vitro protein synthesis cytotoxicity assays were conducted. Murine xenografts (human ALL cell line EU-1 into CB17 SCID mice) were treated with varying doses of CAT-3888. A pediatric Phase I trial of CAT-3888 was initiated.

Results: All cases (n=69) were CD22 positive. Median CD22 site density (studied in 28 cases) was 4,099 sites/cell (range, 451 – 9,380). In vitro cytotoxicity to CAT-3888 was assessed in 43 cases and the median IC50 was 10 ng/ml. Murine xenografts treated with CAT-3888 had significant prolongation of leukemia free survival in a dose responsive fashion (p<0.05). 21 subjects (18 ALL, 3 lymphoma; 3–22 years of age) were treated on a Phase I trial of CAT-3888 at doses of 10–40 mcg/kg QOD for 3–6 doses with cycles repeated every 21–28 days. Treatment was well tolerated and adverse events attributed to CAT-3888 were reversible. Pharmacokinetic parameters appeared to be influenced by disease burden consistent with rapid drug binding by CD22 positive blasts. All subjects were heavily pre-treated and had progressive high burden disease at the time of treatment. Transient clinical activity was observed in 17 of 21 subjects as evidenced by decreased peripheral blood blast count (8), decreased marrow infiltration (6), decreased extramedullary infiltration (3), increased platelet count (2), increased neutrophil count (1), increased reticulocyte count (1), improved PET scan (1), decreased tumor-associated pain (2), and/or peripheral blast count stabilization (5). A dose response was apparent with 6 of 12 (50%) achieving disease stabilization at or above 30 mcg/kg (vs. 0/9 at lower doses). Three subjects developed high-titer neutralizing antibodies, 1 of which had pre-existing low titer antibodies from prior pseudomonas infection.

Conclusions: CD22 represents a relevant target for childhood pre-B ALL. All cases tested were CD22+ and most blasts were highly sensitive to CAT-3888 in vitro at concentrations below clinically achievable levels. CAT-3888 showed a dose response effect and prolonged leukemia free survival in murine ALL xenografts. CAT-3888 is well tolerated in children and activity has been seen in most subjects treated on the Phase I trial. A new higher affinity anti-CD22 immunotoxin (CAT-8015 or HA22) is in development.
Prospective Study of Oral Valganciclovir for Pre-Emptive Therapy of Cytomegalovirus After Allogeneic Stem Cell Transplantation.
Authors: De La Camara Rafael , Vazquez Lourdes , Lopez Javier , Solano Carlos , Serrano David , Gracia-Noblejas Ana , Ribera Josep-Maria , Ferra Christelle
Published: October 2019

Abstract 1168

Poster Board I-190

Intravenous ganciclovir (iv-Gcv) is the standard drug for pre-emptive therapy of citomegalovirus (CMV) infection in allogeneic stem cell (allo-SCT) patients. Valganciclovir (Vgcv), an oral prodrug of ganciclovir, is used very often in practice although there is no approved indication for this specific use.

Methods:

We performed a prospective phase II multicentric study with Vgcv in adult patients to evaluate its efficacy and safety in the pre-emptive treatment of CMV-Ag or DNAemia within the first 180 days after SCT (ClinicalTrials.gov Identifier: NCT00386412). The results were compared with those obtained with iv-GCV in a retrospective control group. The primary study endpoint (success) was the achievement of negative CMV-AG/PCR on day 14 of treatment without changing the protocol antiviral (Vgcv or iv-gcv). Secondary end-points were negative CMV-AG/PCR, rate of neutropenia (<500 neutrophils/mm3) and nephrotoxicity, until 35 days after the beginning of treatment. A minimum patient weight of 50 kg was required. Vgcv was given at 900 mg/12h for 14 days (induction therapy) followed by 900 mg/day for another 14 days (maintenance treatment), in both cases adjusted according to renal function. If CMV was positive at day +21, the treatment was considered a failure and Vgcv was stopped.

Patients:

Here we presented the final results on 90 patients (60 prospective treated with Vgcv and 30 treated with iv-Gcv). There were no differences between Vgcv and iv-Gcv groups in median age (47 vs 49 years), median weight (72,5 vs 62kg), type of donor (55% vs 37% HLA identical siblings, 43% vs 56% unrelated and 2% vs 7% related mismatch), and conditioning regimen (mieloablative 42% vs 57%; reduced intensity in 58% vs 43%). More iv-Gcv patients had acute GVHD II-IV at CMV viremia presentation (12% vs 42%). The majority of the patients where receiving immunosuppression at CMV viremia presentation (97% vs 97%). Median time for CMV-Ag/PCR positivity was 46 days after SCT for both groups.

Results:

Two patients developed CMV disease during antiviral therapy: one in the Vgcv group (a proved gastric on day +9 of therapy) and one in the iv-gcv group. At 2 months of follow-up no more cases of CMV disease were diagnosed.

Nuetropenia (<500/mm3): 8 cases in Vgcv group (13.3%) vs 1 case in iv-gcv group (3.3%) (P ns). Only one patient in the iv-gcv group developed nephrotoxicity.

Comments:

Oral valganciclovir therapy given at a fixed dose in patients over 50 kg for the pre-emptive therapy of CMV in allogeneic patients was effective and with similar toxicity compared with iv-gcv.

Disclosures:

No relevant conflicts of interest to declare.
Defective Inhibitory Signaling in CLL B Cells and Increased Recruitment of PI3K by c-Cbl in Zap-70+ CLL.
Authors: Kai Xin , Chellappa Vasant , Moya Jesse , Taylor Kendra N. , Hochberg Ephraim P. , Amrein Philip C. , Pillai Shiv
Published: October 2019

Abstract 1258

Poster Board I-280

Background

An inhibitory signaling pathway involving sialic acid 9-O-acetyl esterase (SIAE), sialic acid binding lectins (Siglecs) particularly Siglec-2/CD22, the Lyn tyrosine kinase, and the SH2 domain containing tyrosine phosphatase, SHP-1, attenuates B cell receptor signaling and sets a threshold for B cell activation. A key step in the process is the requirement that SIAE access N-glycans on Siglec ligands and remove 9-O-acetyl groups from terminal αa2,6 linked sialic acid moieties. Siglec-ligand interaction is followed by phosphorylation of ITIM tyrosines on CD22 by Lyn, and the recruitment of SHP-1 by CD22 resulting in signal attenuation (Cariappa et al., J.Exp.Med.2009, 206, 125). While Lyn has both positive and negative signaling functions, knockout mice studies suggest that inhibitory functions are dominant. Previous studies have shown that CLL cells overexpress active Lyn at the protein level and that Lyn is localized to sites beyond the plasma membrane. Although cell surface expression of CD22 is reduced in CLL, it is not known if CD22 can be accessed in CLL cells by promiscuously active Lyn. We sought to ask if cancer progression in CLL involves the evolution of mechanisms to evade inhibitory signaling, thus tipping the balance towards positive, pro-proliferative signaling by Lyn.

Methods

CLL B cells from patient and control subjects were isolated. Immunoprecipitation and Western blot approaches were used to quantitate the total cellular levels of Lyn and CD22αa and β proteins at the protein level, the ratio of CD22 phosphorylated on an inhibitory tyrosine to total CD22, recruitment of SHP-1 by CD22, the activation of Syk, the expression of c-Cbl and the recruitment of PI3K by c-Cbl in CLL and control B cells.

Results

A modest decrease in total CD22αa and β proteins was observed in CLL but a dramatic reduction in the proportion of ITIM-phosphorylated CD22, and a reduction in the recruitment of SHP-1 by CD22 in CLL B cells. Decreased inhibitory signaling in CLL correlates with an increase in active Syk. An increase in c-Cbl protein levels was observed and an increased recruitment of p85PI3K was observed specifically in Zap-70 positive CLL.

Conclusions

Defective inhibitory signaling may contribute to disease progression in CLL. This defect probably results from the inability of CD22 to access 9-O-deacetylated ligands even in the presence of active Lyn. Enhanced constitutive BCR signaling prevails in all CLL patients but in Zap70+ CLL patients p85PI3K is more readily recruited by c-Cbl.

Disclosures

Hochberg: Biogen-Idec: Speakers Bureau; Genentech: Speakers Bureau; Amgen: Speakers Bureau; Enzon: Speakers Bureau.
Copy Number Alterations at Polymorphic Loci May Be Acquired Somatically in Patients with Myelodysplastic Syndromes.
Authors: Starczynowski Daniel T. , Vercauteren Suzanne , Sung Sandy , Brooks-Wilson Angela , Lam Wan L , Karsan Aly
Published: October 2019

Abstract 2782

Poster Board II-758

Myelodysplastic (MDS) clones are thought to arise from a primitive CD34+ hematopoietic cell as a result of disrupted genomic integrity. Although approximately 50% of low-risk MDS patients do not exhibit karyotypic alterations, recent studies have identified submegabase copy number alterations in a proportion (18–68%) of cytogenetically normal cases using genomic array platforms. Recently, we observed disease-associated copy number alterations in low-risk MDS patients that overlapped common loci known to be sites of copy number variants (CNV). These CNV or copy number polymorphisms are a source of structural variation in the genome of a normal population, and widely assumed to be constitutional and not relevant findings in malignant tissue. Here we show by whole genome array comparative genomic hybridization that copy number changes in the CD34+ MDS clone that occur at polymorphic loci are frequently somatic alterations rather than constitutional variants. Further, the extent of copy number variation is increased in CD34+ cells of MDS patients compared to CD34+ cells of age-matched controls. This increase in disease-associated copy number alterations at polymorphic loci shows a trend toward association with poorer overall survival. Our findings demonstrate that copy number alterations at known polymorphic loci in CD34+ cells from lower-risk MDS patients are frequently due to somatic changes rather than constitutional polymorphisms. Further, alterations in copy number at these sites are likely not benign, regardless of whether they represent constitutional or somatic alterations, and may contribute to disease progression.

Disclosures:

No relevant conflicts of interest to declare.
Compromised Proteolytic Cleavage of Von Willebrand Factor Type 2N Variants by ADAMTS13 in the Presence of Factor VIII (and Platelets) Under Fluid Shear Stress.
Authors: Skipwith Christopher G. , Haberichter Sandra L. , Cao Wenjing , Gehrand Ashley L. , Zheng X. Long
Published: October 2019

Abstract 28

von Willebrand Factor (VWF) is a large, multimeric adhesive glycoprotein that is involved in the formation of a platelet plug after vascular injury. In addition, VWF functions as a carrier protein for clotting factor VIII (FVIII) which prevents rapid clearance of plasma FVIII. Decreased levels of VWF or defects in VWF function are found in patients with von Willebrand disease (VWD). Quantitative defects of VWF protein in type 1 and 3 VWD affect plasma levels of both VWF and FVIII, whereas qualitative defects in type 2 VWD result in abnormal binding of VWF to platelets such as in type 2A, 2B, and 2M, or to FVIII such as in type 2N. Previous studies have demonstrated that FVIII binds VWF, which dramatically accelerates the proteolytic cleavage of multimeric VWF by ADAMTS13 under mechanically-induced shear stresses. This rate-enhancing effect of FVIII on VWF proteolysis appears to depend on the ability of FVIII to bind VWF, as a FVIII variant lacking the A3 acidic region fails to exhibit the cofactor activity that accelerates VWF proteolysis. To determine whether reduced FVIII binding in VWF type 2N variants affects VWF proteolysis, we examined the proteolytic cleavage of recombinant VWF type 2N variants in the presence of FVIII (and lyophilized platelets) for variants with a moderate VWD phenotype (Arg854Gln and His817Gln) or severe VWD phenotype (Arg763Gly, Arg782Trp, Thr791Met, and Arg782Trp + His817Gln). Recombinant VWF type 2N variants (37.5 μg/ml or 150 nM) were incubated with ADAMTS13 (25 nM) in the absence and the presence of various concentrations of FVIII (0-40 nM) with or without lyophilized platelets (0-600×103/μl) under fluid shear stress. The proteolytic cleavage products (350 kDa) were determined by 5% SDS-PAGE and Western blot under denaturing but non-reducing conditions. We show that the proteolytic cleavage of VWF type 2N variants by ADAMTS13 under these conditions was variably reduced as compared that of wild type VWF. The reduction in the cleavage rate was proportional to the degree of reduction in VWF FVIII binding activity, which was assessed with a microtiter assay, with the least cleavage by ADAMTS13 of the variants with the lowest FVIII binding activity. This reduced cleavage of the type 2N variants was not correlated with the binding affinity between the type 2N variants and ADAMTS13 protease. These results provide further evidence that binding of FVIII to VWF, which may alter VWF conformation, is necessary to accelerate VWF proteolysis by ADAMTS13 under fluid shear stress. This variability in ADAMTS13 cleavage may contribute to the heterogeneity of bleeding phenotype of type 2N VWD variants. The bleeding phenotype may be modulated not only by plasma FVIII levels, but also the extent of VWF proteolysis by ADAMTS13 under physiological fluid shear stress.

Disclosures:

No relevant conflicts of interest to declare.
Combination of ATRA with a COX2 Inhibitor Induces Synergistic Effects On RARalpha2-Expressing Multiple Myeloma (MM) Cells.
Authors: Wang Siqing , Shi Lei , Xu Hongwei , Xu Chunjiao , Zangari Maurizio , Tricot Guido J. , Zhan Fenghuang
Published: October 2019

Abstract 2809

Poster Board II-785

We recently observed that ATRA treatment selectively kills RARalpha2-expressing, while sparing RARalpha2-deficient MM cells. Previous investigations in the colon cancer cells have shown that ATRA inhibits WNT signaling through down-regulation of COX-2. Therefore, we wanted to evaluate the role of WNT signaling in ATRA-induced cell death and growth inhibition of RARalpha2-expressing myeloma cells. To our surprise, we found that ATRA treatment activated but not inhibited WNT signaling in RARalpha2-expressing myeloma cells, based on increased β-catenin levels in ATRA-treated cells. ATRA exerted minimal effects on activation of WNT signaling pathway in RARalpha2-deficient MM cells, and forced expression of RARalpha2 in RARalpha2-deficient cells restored the stimulatory activities of ATRA on the WNT signaling pathway, demonstrating that RARalpha2 expression is required for the ATRA-induced stimulation of WNT signaling in MM cells. Lithium chloride (LiCl) treatment, which activates WNT signaling, partially abrogated ATRA-induced cell death and growth inhibition in RARalpha2-expressing cells, indicating that ATRA-induced activation of WNT signaling resulted in ATRA-resistance and decreased killing of MM cells, suggesting that a combination of targeting WNT signaling pathway and ATRA treatment is necessary for ATRA-based therapy of RARalpha2-expressing myeloma. COX-2 inhibition blocks WNT signaling in colon cancer. Similarly, we found that a COX-2 inhibitor CAY10404 also blocked WNT signaling in RARalpha2-expressing cells as well as in ATRA-treated cells. Interestingly, CAY10404 activated MEK/ERK signaling pathway, while ATRA abrogated CAY10404-induced activation of MEK/ERK signaling pathway. These results demonstrate that the combination of ATRA and COX-2 inhibitor exerts synergistic inhibitory effects on both WNT and MEK/ERK signaling pathways. A combination of ATRA and the COX-2 inhibitor resulted in synergistic cytotoxicity of RARalpha2-expressing MM cells in-vitro. More importantly, the combination of ATRA and CAY10404 also resulted in a synergistic growth inhibition of established MM tumors in SCID mice. Our study demonstrates the importance of targeting WNT signaling in ATRA-based therapy in RARalpha2-expressing myeloma and provides a rationale for the combinational use of ATRA and COX-2 inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Factor XII Stimulates ERK1/2 and Akt through uPAR, Integrins, and the EGFR to Initiate Angiogenesis.
Authors: Larusch Gretchen , Mahdi Fakhri , Shariat-Madar Zia , Adams Gregory N , Sitrin Robert , Zhang Wanming , McCrae Keith R. , Schmaier Alvin H.
Published: October 2019

Abstract 3040

Poster Board II-1016

The physiologic function of factor XII (XII) is not known. New interest in XII has occurred because XII KO mice are protected from thrombosis. Exposed arterial collagen, aggregated protein, RNA, and platelet polysomes are recently recognized entities in developing thrombus that promote XII autoactivation to XIIa increasing thrombus formation, independent of a role in hemostasis. However, these observations do not indicate a constitutive, physiologic function for XII. We sought a physiologic function for zymogen XII. XII has been recognized to stimulate MAP kinase. Cleaved high molecular weight kininogen (HKa) is known to be antiangiogenic. Further, XII and HK mutually block each others binding to endothelial cells. Both XII and HKa bind urokinase plasminogen activator receptor (uPAR) at an overlapping site. We investigated if XII stimulates cells by interacting with uPAR and if this activity influences angiogenesis. XII (3-200 nM) with 0.05 mM Zn ion induces ERK1/2 (MAPK44 and 42) and Akt (Ser473) phosphorylation in endothelial cells. XII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. The uPAR signaling region for XII is on domain 2 adjacent to uPAR's integrin binding site. HKa or peptides from HKa's domain 5 inhibit XII-induced ERK1/2 and Akt phosphorylation. A beta-1-integrin peptide that binds uPAR, antibody 6S6 to beta-1-integrin, or the EGFR inhibitor AG1478 blocks XII-induced phosphorylation of ERK1/2 and Akt. XII induces endothelial cell proliferation and 5-bromo-2'deoxy-uridine incorporation. XII stimulates aortic sprouting in normal but not uPAR deficient mouse aorta and this mechanism is blocked by PD98059, LY294002, AG1478, or HKa. XII also induces angiogenesis in matrigel plugs. Finally, XII knockout mice have reduced constitutive and wound-induced blood vessel number on initial biopsy and 9 days after wound healing, respectively. In sum, XII initiates outside-in signaling mediated by uPAR, beta-1-integrin, and the EGFR leading to HUVEC proliferation, growth, and angiogenesis. XII is a constitutive proangiogenic protein.

Disclosures

No relevant conflicts of interest to declare.
Interferon Alpha Alone Is Able to Cure Chronic Myeloid Leukemia In a Small Subset of Patients Despite the Persistence of Leukemic Cells: Experience of Long Follow up After Treatment Discontinuation
Authors: Mahon Francois-Xavier , Fort Marie-Pierre , Etienne Gabriel , Marit Gerald , Milpied Noel , Pigneux Arnaud , Cony-Makhoul Pascale , Reiffers Josy
Published: October 2019

Abstract 2299

Before the imatinib era and besides allogeneic hematopoietic stem cell transplantation (HSCT), interferon alpha (IFN) was an alternative therapeutic option to treat chronic myeloid leukemia (CML). In very rare situations, some patients achieved a complete molecular remission (CMR) defined as the absence of detectable BCR-ABL transcript using qualitative PCR available at that time.We previously reported the outcome of patients with CML in complete cytogenetic remission after cessation of IFN (J Clin Oncol., 2002, 20:214-220.). Here, we update this experience with a long outcome after discontinuation of IFN. The main criteria for IFN discontinuation was the achievement of a sustained CMR lasting for at least two years. Twenty-one patients (9 males, 12 females; median age 39 years) were eligible for this study. One, 2 and 18 pts belonged to respectively high, intermediate and low risk group according to the Sokal score.All patients were treated with IFN (median dose 56ui/week) during a median time of 7.5 years (2.4-14) and 4 of them received an autologous HSCT. The median time to achieve CMR was 54 month (5-140). The median follow up after discontinuation of IFN treatment was 8 years (mean 9, range 5–18). Quantitative PCR was used to quantify BCR-ABL and evaluate residual disease. We distinguished two groups of patient according to the molecular pattern: group 1 (n=12) regards patients with sustained CMR confirmed by quantitative PCR. One patient belonging to this group relapsed and progressed suddenly after 5 years of IFN discontinuation and was treated with allogeneic HSCT. Group 2 (n=9) regards patients who are not in CMR with leukemic cells detectable by RQ-PCR. Six of them exhibit a fluctuation of BCR-ABL detection assessed by RQ-PCR. For the 3 other patients with a median follow up of 6 years after discontinuation, RQ-PCR became clearly positive after a median time of 2 years with a level of residual disease that corresponds to the definition of major molecular response (3 log reduction of BCR-ABL). No progression was observed in this group.

So we conclude that the persistence of leukemic cells at low level after discontinuation of IFN treatment does not automatically lead to CML relapse. This long term follow up after IFN discontinuation raises the question of the need of a complete eradication of residual leukemic cells to cure the disease

Disclosures:

No relevant conflicts of interest to declare.