bluuebunny/crossref_metadata_2025 huggingface dataset:
An In Vitro and In Vivo Evidence That Downregulation of Leukemia Inhibitory Factor (LIF) Receptor (LIF-R) Decreases the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.
Authors: Wysoczynski Marcin , Miekus Katarzyna , Marcinkowska Anna , Janowska-Wieczorek Anna , Ratajczak Mariusz Z.
Published: October 2019

Rhabdomyosarcoma (RMS) and skeletal muscle-derived tumors frequently infiltrate bone marrow (BM). We have demonstrated that the stromal-derived factor (SDF)-1-CXCR4 receptor (Blood2002;100:2597) and hepatocyte growth factor (HGF)-c-Met receptor (Cancer Res. 2003;63:7926) play an important role in RMS metastasis to BM. Leukemia inhibitory factor (LIF) is a well known factor that plays an important role in skeletal muscle development/regeneration and similarly as SDF-1 and HGF is secreted by BM stroma. This prompted us to examine whether the LIF-LIF receptor (LIF-R) axis affects the biology/metastasis of RMS cells. We employed in our studies, human established RMS cell lines, as well as RMS samples isolated from patients and noticed that LIF-R was expressed not only on established human RMS cell lines (7/7) but more importantly, it was also detectable in patient samples (23/23). We also found that in RMS cells LIF stimulatesphosphorylation of MAPKp42/44, AKT and STAT3,chemotaxis and adhesion andincreases resistance to cytostatics (e.g., etoposide).

These LIF-mediated effects were inhibited after downregulating the LIF-R by siRNA. To learn more on the biological significance of the LIF-LIF-R axis in vivo we employed two models. First, human RMS cells (RH-30) were exposed or not exposed to LIF-R siRNA and subsequently injected into SCID™-Beige immunodeficient mice. To estimate the number of RMS cells that seed to BM and liver in these animals, we isolated DNA and using real- time RT-PCR, amplified human a-satellite sequences and murine b-actin. The number of human cells present in murine organs was subsequently calculated from a standard curve derived from mixing varying numbers of human cells with a constant number of murine cells. We noticed that downregulation of LIF-R by siRNA significantly decreased the number of human RMS cells in murine BM and liver (x4 and x2 respectively). In a second model, the RH30 cell line was selected by repetitive chemotaxis for cells that are highly responsive to LIF (RH-30 L) and subsequently the cells from parental RH-30 cell line and RH-30 L cells were injected intramuscularly. Six weeks after tumour inoculation, we detected more metastasis in bone marrow and lungs in mice injected with RH-30L cells as compared to parental RH-30 clone (x6 and x3 respectively). In conclusion, we present evidence for the first time that the inhibition of LIF-LIF-R axis may decrease the invasive potential of human RMS both in vitro and in vivo. Hence, molecular targeting of LIF-LIF-R axis could possibly become a more effective new strategy to control the progression and metastasis of RMS.
Co-Transplantation of Autologous Umbilical Cord Matrix Mesenchymal Stem Cells Improves Engraftment of Umbilical Cord Blood in NOD/SCID Mice.
Authors: Friedman Robb , Betancur Monica , Tuncer Hande , Boissel Laurent , Cetrulo Curtis , Klingemann Hans-Georg
Published: October 2019

Umbilical cord blood (UCB) is a viable source of hematopoietic stem cells for transplantation of children and adults undergoing treatment for hematological malignancies. However only 4% of adults 70kg and over have a UCB unit available which contains the widely accepted minimum cell dose of 1.5x107 mononuclear cells per kilogram. Co-transplantation of hematopoietic stem cells with mesenchymal stem cells may enhance engraftment and therefore decrease transplant-related morbidity and mortality from delayed leukocyte recovery associated with a low pre-transplant cell dose. Umbilical cord matrix (UCM) cells, found in the Wharton’s Jelly, were easily and reliably extracted from minced pieces of cord by culture in RPMI + 20% fetal bovine serum at 37° and 5% humidified CO2. UCM expand best in 20% FBS but can also be expanded in human serum, autologous serum, and X-VIVO10. Small (1–3mm) minced pieces of umbilical cord can be cyropreserved at the time of delivery in 10% DMSO solution. UCM cells exhibit a fibroblast morphology and express markers common to mesenchymal stem cells: CD73 (SH3), CD105 (SH2), CD 29, CD44, CD49b, CD117, CD166, STRO-1 and HLA-DR. UCM are negative for CD14, CD 19, CD34, and CD45.

Morphology and cell surface marker expression is stable after greater than fifteen passages. UCM cells grown in culture were shown to produce more GM-CSF and G-CSF than similar numbers of adult bone marrow mesenchymal stem cells, GM-CSF 178 pg/mL versus 77 pg/mL and G-CSF 82.6 pg/mL versus 7.9 pg/mL. NOD/SCID mice treated with anti-NK 1.1 antibodies and irradiated with 350 cGy were injected with suboptimal (1x104) numbers of cord blood CD34+ cells with and without 1x106 autologous UCM cells, extracted from the same umbilical cord as the cord blood CD34+ cells. Bone marrow was harvested at six weeks post transplant from both femurs and tibias and peripheral blood obtained via cardiac puncture. The percentage of human CD45+ cells in the bone marrow and the peripheral blood was assessed by flow cytometry. NOD/SCID mice transplanted with 1x104 cord blood CD34+ cells alone had 3.0% human CD45+ cell engraftment in the bone marrow and 3.6% human CD45+ cells in the peripheral blood, while NOD/SCID mice transplanted with 1x104 CD34+ cells and 1x106 UCM cells had an average of 27.3% human CD45+ cell engraftment in the bone marrow and 3.9% human CD45+ cells in the peripheral blood. These results indicate a trend towards improved engraftment in vivo with co-transplantation of suboptimal numbers of umbilical cord blood CD34+ cells and autologous umbilical cord matrix cells versus transplantation of suboptimal numbers of umbilical cord CD34+ cells alone.
Secondary Bortezomib-Resistance Is Characterized by Upregulation of Proteasomal Subunits and Their Insufficient Inhibition by Bortezomib, Which Can Be Overcome by Alternative Proteasome Inhibitors Combined with Induction of the ER Stress Response.
Authors: Ruckrich Thomas , Gogel Jeannette , Kraus Marianne , Ovaa Huib , Driessen Christoph
Published: October 2019

Resistance towards proteasome inhibition by Bortezomib (Velcade®) represents a challenge for myeloma therapy. Its biology has not yet been characterized in detail. We have demonstrated that Bortezomib-sensitive malignant haematopoetic cells can acquire secondary resistance to Bortezomib in vitro. We here present the first analysis of proteasome biology and activity, alternative proteolytic pathways, ubiquitin-specific proteases (USP) and the ER stress response (unfolded protein response, UPR) upstream of the proteasome, as well as in vitro cytotoxicity of conventional cytotoxic drugs, alternative proteasome inhibitors and agents that target the UPR in Bortezomib-resistant (BR) cells, compared to wild type (WT) controls. BR cells had higher activities of all subunits of the constitutive and the immunoproteasome, as deferred from turnover of fluorogenic substrates as well as affinity-labelling of active proteasome subunits in intact cells. This was mirrored by increased levels of proteasomal β1 and β2, but especially β5 polypeptides, implicating a homeostatic system that senses and corrects low proteasome activity in cells chronically exposed to Bortezomib. While the vinylsulfone-type proteasome inhibitor NLVS abrogated detectable proteasome activity in both BR and WT cells, Bortezomib at therapeutic concentrations eliminated proteasomal β1 and β5-type activity only in WT cells, while BR cells retained residual activity. These changes in proteasome biology appear to be the molecular hallmark of required Bortezomib resistance, since no changes were observed between WT and BR cells in alternative cytosolic or lysosomal proteolytic pathways, UPR activity as well as the gross activity pattern of USP. As expected, this translated into sensitivity against cytotoxic drugs in vitro: BR cells were less sensitive towards alternative proteasome inhibitors. However, while the IC50 for pan-proteasome inhibitors was only roughly doubled in BR cells, it was nearly tenfold elevated for the β5-preferring vinylsulfone inhibitor NLVS. By contrast, sensitivity towards anthracyclines or cytotoxicity induced by ER stressors as well as the synergy between proteasome inhibitors and UPR-activators remained unaffected in BR cells. Based on our data, proteasome inhibitors with activity profiles different from that of Bortezomib, alone or in combination with induction of the UPR, may represent an appropriate concept to overcome secondary Bortezomib resistance.
Effective Targeting of Leukemic Cells in Children with B-Precursor Acute Lymphoblastic Leukemia Treated with Anti-CD22 (Epratuzumab). A Children’s Oncology Group (COG) Study.
Authors: Borowitz Michael J. , Carroll William L. , Adamson Peter C. , Cairo Mitchell S. , Goldenberg David M. , Wegener William A. , Raetz Elizabeth
Published: October 2019

Identifying new approaches to treating relapsed ALL is a top priority because these patients fare poorly with current retrieval strategies. COG protocol ADVL04P2 is a phase I/II feasibility pilot study for children with relapsed B-precursor ALL that uses epratuzumab, a humanized anti-CD22 monoclonal antibody, in combination with conventional re-induction chemotherapy. Children whose blasts are CD22 positive first receive 4 doses of 360 mg/m2 epratuzumab as a single agent on days minus(−)14, −10, −6 and −2. Subsequently, they receive epratuzumab weekly in combination with standard re-induction chemotherapy starting on day 0. We assessed the effectiveness of epratuzumab targeting by by assaying CD22 expression on residual leukemic blasts. Peripheral blood was obtained from 15 patients 24 hours after administration of the first dose (day −13), and again at day −6 and day 0 and stained with the combination of CD10/CD22/CD45/CD19 in four color flow cytometry to permit assessment of CD22 on analytically isolated leukemic blasts. We determined quantitative expression of CD22 with a calibration kit (Quantibrite, BDBiosciences, San Jose, CA) using two PE-conjugated anti CD22 antibodies directed against different epitopes of the CD22 molecule: clone RFB4 (Caltag, Burlingame CA), directed against the same epitope as epratuzumab, and clone SHCL-1 (BDBiosciences), directed against a non-cross-reacting epitope of CD22. RFB4 binding was decreased by more than 99% within 24 hours after administration of the first dose in all patients, indicating rapid targeting of epratuzumab to leukemic cells. In all but 4 patients, levels remained low at all subsequent time points; in 2 patients expression was restored to about 15% of baseline levels by day 0, and 2 additional patients had very small (1–2%) subsets of blasts with significant expression, one at day −6 and one at day 0. In contrast to RFB4, SHCL-1 identified residual CD22 antigen expression, but SHCL-1 binding was decreased by an average of 70–75% in 14 of 15 patients at all three post-epratuzumab timepoints. The one patient with no change in SHCL-1 binding was a patient with MLL-rearranged ALL with very low levels of CD22 expression at diagnosis, and poor response to therapy. Because binding of epratuzumab to CD22 has been shown to result in antigen internalization in vitro, we interpret our findings as demonstrating in vivo internalization of CD22 with epratuzumab binding, though we cannot exclude either shedding of antigen or conformational change to block SHCL-1 sites. We conclude that epratuzumab rapidly targets CD22 on ALL blasts in vivo, though some blasts in some patients may escape over time. Targeting is associated with a physical change in CD22 antigen expression in most patients, most likely through internalization.
AML Progenitor/Stem Cells Overexpress XIAP and Are Sensitive to the Small Molecular XIAP Inhibitor 1396-11.
Authors: Carter Bing Z. , Kim Tae Kon , Mak Duncan H. , Hu Wei , Pinilla Clemencia , Reed John C. , Andreeff Michael
Published: October 2019

Chemotherapy is the primary treatment modality for AML patients. Although most patients show initial responses to chemotherapeutic agents, the majority of patients relapse, suggesting that more primitive cells in AML are resistant to chemotherapy-induced apoptosis. XIAP, a potent cellular caspase inhibitor, is highly expressed in various tumor cells and leukemic cells and contributes to chemoresistance. However, the expression levels of XIAP in early progenitor/stem cells of AML patient are unknown. To address this question, we collected blasts from bone marrow or peripheral blood of AML patients (n=11) and separated them into CD34+/CD38+ and CD34+/CD38− populations by fluorescent-activated cell sorter after immunostaining. RNA was isolated and XIAP mRNA levels were determined by Taq-Man quantitative RT-PCR in these two cell populations and compared with levels from normal CD34+/CD38+ and CD34+/CD38− cells (n=13). Our results demonstrated that XIAP is highly expressed in both progenitor (CD34+/CD38+) and stem cell (CD34+/CD38−) compartments in primary AML blasts, with XIAP mRNA levels higher in these two cell populations in AML than in the corresponding normal bone marrow progenitor/stem cells (p=0.05 and 0.08 for CD34+/CD38+ and CD34+/CD38− compartments, respectively). Our previous studies showed that inhibition of XIAP expression by antisense oligonucleotide and its suppression by small molecule chemicals induce apoptosis of AML cells. 1396–11 and 1396–34, polyphenylurea-based small molecule XIAP inhibitors bind to and inhibit the BIR2 domain of XIAP. 1396–34 was previously tested in AML (Carter, et al. Blood105: 4043, 2005). Previous studies of 1396–11 demonstrated anti-tumor activity against human solid tumor xenografts in mice with no apparent hematopoietic or liver toxicities (Schimmer, et al. Cancer Cell5: 25, 2004; Berezovskaya, et al. Cancer Research65: 2378, 2005). We treated OCI-AML3, HL-60, Jurkat, and U937 leukemia cells with 1396–11 and found it to be more potent than 1396–34 in all leukemia cell lines tested. In addition, we treated AML blasts from 5 patients with 1396–11 and its inactive control 1396–28. Apoptosis was determined in total blasts and in both the CD34+/CD38+ and CD34+/CD38− compartments. Our results showed that 1396–11 induced cell death equally in bulk blasts, CD34+/CD38+ progenitor, and CD34+/CD38− stem cells. Four out of 5 AML samples showed significant apoptosis with ≤ 10 μM 1396–11. The control compound 1396–28 had minimal effect on cell survival in all 5 patient samples tested. Our studies illustrated that XIAP is overexpressed in AML progenitor/stem cells and that these cells are sensitive to a small molecule XIAP inhibitor, suggesting XIAP is a target for AML therapy, with the potential of eradicating primitive leukemic progenitor/stem cells.
Application of Multiparameter Flow Cytometry for the Identification of Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes.
Authors: Kern Wolfgang , Schoch Claudia , Schnittger Susanne , Haferlach Torsten
Published: October 2019

The diagnosis and classification of myelodysplastic syndromes (MDS) are based on cytomorphology (CM) and cytogenetics. A high degree of experience in CM is required to allow the accurate identification of dysmyelopoiesis and quantification of bone marrow blasts. The identification of dysplastic features in all lineages by multiparameter flow cytometry (MFC) has been shown feasible. To further analyze the potential role of MFC in the diagnostic work-up of MDS we analyzed 224 bone marrow samples from patients with suspected of proven MDS by MFC, CM, and cytogenetics in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 5%) and from 0% to 33% (median, 4%; correlation: r=0.192, p=0.018). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 7), 1 for monocytes (0 to 5), and 0 for erythrocytes (0 to 2). The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (20.5%), CD15 (14.3%) and CD64 (14.3%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (71.4%), aberrant coexpression of CD56 (29.0%), aberrant pattern of CD13/CD16 expression (26.3%), aberrant pattern of CD11b/CD16 expression (25.9%), reduced expression of CD64 (17.0%), and aberrant expression of HLA-DR (14.7%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (31.3%), aberrant coexpression of CD16 (26.3%), an aberrant pattern of CD11b/HLA-DR expression (6.7%), and aberrant coexpression of CD2 (5.8%). The most frequent dysplastic features observed in the erythroid cell populations included an aberrantly strong expression of CD71 and CD235a (23.7%), a lack of CD71 expression (10.7%), and an aberratly homogeneous expression of CD71 (7.1%). The presence of dysplastic features by CM as well as the presence of cytogenetic aberrations tended to be associated with a higher number of dysplastic features by MFC. These data suggest that the identification of dysplastic features by MFC is feasible although there is a large heterogeneity in aberrantly expressed antigens. Thus, a comprehensive panel of antibodies must be applied to allow the detection of dysplasia. Future studies will define the role of MFC in optimizing the diagnosis of MDS in cooperation with CM and cytogenetics.
A BMT Model Mice for Myelodysplastic Syndromes (MDS) and Transformation to AML.
Authors: Watanabe-Okochi Naoko , Kitaura Jiro , Harada Hironori , Ono Ryoichi , Nakajima Hideaki , Nosaka Tetsuya , Inaba Toshiya , Kitamura Toshio
Published: October 2019

To understand myelodysplastic syndromes (MDS) at the molecular level, we used a mouse bone marrow transplant (BMT) model. Several leukemogenic fusion proteins were demonstrated to induce leukemia in mouse BMT models. However, analysis of molecular basis of MDS has been hampered by the lack of mouse MDS models. Mutations of a transcription factor AML-1/Runx-1 were identified in 15–40% of MDS-RAEB and MDS/AML and 5–10% of de novo AML. To test transforming abilities of the mutant AML-1, we transduced AML-1 with different mutations (#5 and #27) into mouse bone marrow cells using our highly efficient retrovirus-mediated gene transfer, transplanted the transduced cells to irradiated mice, and found that most mice developed leukemia within 4–13 months after the transplant (table1 and figure1). The leukemic mice exhibited marked hepato-splenomegaly, and morphological abnormalities of myeloid and erythroid lineages were frequently observed. Some mice developed to leukemia after a long latency with MDS-like symptoms, either with pancytopenia or leukocytosis with anemia, thus mimicking the human disease. To clarify the differences between the leukemic mice with early onsets and those that developed leukemia after a long period of MDS-like symptoms in the mutant AML-1-induced mouse diseases, the integration sites of the transduced retroviruses were investigated using a polymerase chain reaction (PCR)-based technique bubble PCR. Our preliminary results suggested that the integration sites of the retroviruses harboring the mutant AML-1 contribute to the early onset of leukemia. In conclusion, we have developed a useful mouse in vivo model of MDS/AML that should help understand the molecular basis of MDS and progression of MDS to leukemia.

penetrance and latency mutation number position form penetrance latency #5 D171N point mutation in RHD 69.6 % 4–13 months #27 S291fsX300 truncation type 100 % 4–9 months Figure Figure
Evaluation of Cardial Iron Deposition by T2* MRI in Transfusion Dependent Patients with Myelodysplastic Syndromes.
Authors: Deplano Simona , Di Tucci Anna , Matta Gildo , Agus Annalisa , Gabbas Attilio , Murru Roberta , Angelucci Emanuele
Published: October 2019

Cardiac T2* Magnetic Resonance Imaging (MRI) has been recently used to evaluate myocardial iron deposition in patients with transfusion dependent beta-thalassemia major. No comparable studies have been published for patients with myelodysplastic syndromes receiving chronic red blood cell transfusion. Therefore we measured cardiac-MRI T2* in 16 patients (10 male, 6 female) with myelodysplastic syndromes (aged 54 – 82 years, median age 67). All of them were transfusion dependent having received a median number of 60 (range 16–225) packed red blood cell transfusion equivalent to 3.2 – 45 (median 12) grams of iron. Nine have been irregularly and sporadically chelated by deferoxamine, seven were unchelated. Serum ferritin levels ranged from 1163 to 6241 mg/dl (median value 2086). None of the patients presented signs or symptoms of cardiac dysfunction at the time of the study.

Cardiac-MRI T2*values obtained ranged from 5.6 to 80 (median value 46.5) milliseconds (ms). Correlation between serum ferritin and cardiac T2* value was weak ( r= 0.43, r2 =0.18). According to D. Pennel we considered as significant of myocardial iron deposition a relaxation time ≤ 20ms. Cardiac T2* was < 20ms in 3 patients who had never used iron chelators (5.6, 12.4 and 8.5 ms, respectively). They had received 39, 101 and 200 units of red blood cell transfusion, corresponding to 7.8, 20 and 40 grams of iron, respectively. Of relevance 2 of them died within few months after the end of the study and one showed early signs of left ventricular dysfunction. None of the patients with a cardiac T2* value >20 ms showed instrumental nor clinical signs of cardiac deterioration in six months follow up. No patient who had received less than 39 transfusions presented cardiac T2* value ≤20 ms. Evaluation of myocardial iron deposition by T2* cardiac MRI could be recommendable in myelodyplasia patients who had received more than 30 packed red blood cells transfusisions.
Efficacy and Safety of Denileukin Diftitox (Ontak®) in the Treatment of Cutaneous T-Cell Lymphoma (CTCL) According to CD25 Status.
Authors: Foss Francine , Duvic Madeleine , Geskin Larisa , Anderson Joseph , Porcu Pierluigi , Hohl Raymond J. , Cooper Maureen , Zain Jasmine M. , Zic John , Hibberd Patrica , Acosta Mark
Published: October 2019

Denileukin diftitox, an interleukin-2 (IL-2)-diphtheria toxin fusion protein, binds to and intoxicates cells expressing the medium (CD122, CD132) and high affinity (CD25, CD122, CD132) IL-2 receptor. Because its role in patients whose tumors tested negative for the CD25 component of the receptor had not been prospectively studied, we initiated a prospective, open label, multi-center study to evaluate the safety and efficacy of denileukin diftitox in CTCL patients according to CD25 status (CD25+ and CD25-). Patients with pathologically proven, persistent or recurrent CTCL Stages IB-IVA were treated with denileukin diftitox at a dose of 18 mcg/kg/day x 5 days every 21 days for up to 8 cycles. Expression of CD25 by tumor cells in skin biopsies was determined by IHC or flow cytometry at a central laboratory and investigators were blinded to CD25 status. Response was based on improvement in skin involvement by weighted skin assessment for lesion type (patch, plaque, tumor) and blood involvement based on quantitation of Sezary cells. Safety was evaluated based on reports of Grade 3 and 4 toxicities (NCI CTC version 2.0). Sixty-one pts were enrolled and treated with denileukin diftitox. Disease stages were: I-IIA (n=14), IIB (n=22), III (n=11) and IV (n=14). Eighteen (29.5%) had blood involvement. Fifty-seven pts completed at least one cycle of treatment and were evaluable for response. Four pts were excluded because they had Stage IVB (visceral) disease (n=2), did not have a post baseline skin assessment recorded (n=1), or did not complete the first cycle (n=1). The median number of cycles of denileukin diftitox was 4 (range 1–13). The overall response rate (CR+PR) was 53% (30/57, 95% CI 40–66%), with 2 CR. Of the 57 evaluable pts, tumor was CD25+ in 34 (70%), CD25- in 14 (25%) and unknown in 9. Response rate was similar in patients whose tumor tested CD25+ (56%, 95% CI: 39–73%), and CD25- (43%, 95% CI: 17–69%) (p=0.41). For the 61 patients the most common grade 3/4 toxicities included constitutional symptoms (11%) and metabolic/laboratory abnormalities (36%). One patient with significant cardiopulmonary disease died of myocardial infarction while on therapy. The frequency of grade 3/4 toxicities was similar in CD25+ 64% (23/36), vs CD25- 75% (12/16) (p=0.62). In conclusion, we found that 70% of the CTCL pts evaluated for this study tested CD25+ and that the response rates and safety profile of denileukin diftitox in CD25+ and CD25- patients were similar. These data support a role for denileukin diftitox in the treatment of CTCL regardless of CD25 status.
Zanolimumab, a Fully Human Monoclonal Antibody: Preliminary Results of an Ongoing Clinical Trial in CD4+ Peripheral T-Cell Lymphomas (PTCL).
Authors: d’Amore Francesco , Relander Thomas , Jerkeman Mats , Hagberg Hans , Osterborg Anders , Johnson Peter , Radford John A. , Hancock Barry W. , Gramatzki Martin , Schmitz Norbert , Tilly Herve , Morschhauser Franck , Lisby STeen , Baadsgaard Ole
Published: October 2019

Zanolimumab (previously referred to as HuMax-CD4) is a fully human monoclonal IgG1k antibody. It is specific for the CD4 antigen expressed on a subset of T cells and has been shown to possess cytotoxic and anti-proliferative effects. We report initial clinical efficacy and safety results following treatment with 980 mg of zanolimumab in 15 patients diagnosed with biopsy-proven, treatment-refractory or relapsed CD4+ systemic PTCL. The following PTCL subtypes were included : enteropathy-type T-cell lymphoma (n=1), anaplastic large T-cell lymphoma (ALCL, n=3, 2 alk-negative, 1 alk-status unknown), angioimmunoblastic T-cell lymphoma (AIL, n=8) and PTCL-unspecified (PTCLu, n=3). The primary endpoint of the study was objective tumor response. Responses were based on CT scan and clinical examination and classified according to the Cheson criteria. Out of 15 patients enrolled, 4 responses have been seen; 2 PR (one patient later achieved a CRu) and 2 CRu verified by CT scan. Further, clinical improvements (marked decrease in the size of peripheral lymph nodes) were recorded in 2 additional patients. So far, a total of 21 SAEs has been reported. Of these, 4 were considered related to the study treatment: febrile neutropenia (n=1), thrombocytopenia (n=1), infusion related reaction (n=1) and hyperthermia (38.8° C) with hypotension (n=1). Eight unrelated deaths have been reported so far, all secondary to disease progression. In conclusion, in this heavily pretreated patient population, zanolimumab demonstrated a good tolerability profile and the present results indicate efficacy in the treatment of CD4-positive PTCL.
European Collaborative Paediatric Database on Cerebral Venous Thrombosis: Risk Factors for Recurrent Venous Thromboembolism.
Authors: Kenet Gili , Kirkham Fenella , Niederstadt Thomas , Kurnik Karin , Schobess Rosemarie , Knofler Ralf , Zieger Barbara , Nowak-Gottl Ulrike
Published: October 2019

Background: Risk factors for cerebral venous thrombosis (CVT) in children include local and systemic underlying conditions, drug toxicity, and hereditary prothrombotic risk factors. Their relevance to the risk of a second venous thrombosis (VT), compared with other clinical, neuroimaging and treatment variables, is unknown.

Methods: 330 of 407 consecutively enrolled CVT-patients aged newborn to <18 years (median 5.3 years; male 55%) were prospectively followed for a median (range) of 33 (12–84) months. In accordance to international treatment guidelines, 259 children (78.5%) received acute antithrombotic therapy (AT) with UFH or LMWH, followed by long-term AT with LMWH or warfarin in 218 cases (66.0%).

Findings: Recurrent VT was diagnosed in 19 of 330 children (5.8%) at a median (range) age of 6 (0.5–84) months following CVT, with no difference observed between the study centres (p=0.84). Multivariate Cox regression analysis revealed that the risk of recurrence was significantly higher in those with older age at first CVT onset (p=0.02), those in whom AT was not administered prior to recurrence (p=0.0003), those who did not have complete patency rates on repeat venography at 3–6 months (p=0.01), and those subjects carrying the factor II G20210A mutation (p=0.03).

Interpretation: Age at CVT onset, administration of AT, poor patency rates, and the factor II G20210A variant were of relevance to recurrent VT in children with CVT. Until evidence-based data derived from randomized treatment trials are available the message of this follow-up study is to administer any AT prophylaxis on an individual patient basis in paediatric CVT cases in newly identified VT risk situations.
Oct-4 Is Crucial for Stress-Induced Murine Embryonic Stem Cell Survival/Anti-Apoptosis Mediated by STAT3/Survivin.
Authors: Guo Ying , Mantel Charlie , Hromas Robert A. , Broxmeyer Hal E.
Published: October 2019

Murine embryonic stem (ES) cells are pluripotent, and may be of potential use for cell replacement and gene therapy. By understanding the mechanism of survival and anti-apoptosis of ES cells, it may be possible to enhance use of ES cells for clinical usage. Our previous studies indicated that undifferentiated ES cells converted into polyploidy instead of apoptosis under the stress. However, differentiated ES cells went through apoptosis in the same manner as mature cells. Oct-4 is a marker for undifferentiated ES cells. We wondered if Oct-4 might be a key player for choosing between polyploidy and apoptosis of ES cells under the stress. To address this question, Oct-4 tetracycline conditional knockout cell line ZHBtc4, received from Dr Austin Smith, was used. To evaluate whether Oct-4 was essential for stress-induced apoptosis of murine ES cells, we used three methods to induce stress: etoposide treatment, heat shock and UV exposure. Four treatment groups were studied, A: Oct-4 knock down ZHBtc4; B: Control ZHBtc4; C: Tetracycline treated CGR8 cell line; D: Control CGR8. CGR8 is the wild type parental cell line for ZHBtc4. These latter two groups were added to eliminate potential effects of tetracycline. After knockdown of Oct-4 for 24 hours, we induced apoptosis by etoposide, heat shock or UV exposure. Apoptosis for Oct-4 knocked down ES cells was significantly increased in response to all stress situations. This suggests that Oct-4 is important for modulating stress-induced apoptosis of ES cells. In order to address potential mechanisms for these effects, we focused on one member of the IAP family—Survivin. Survivin is a well known anti-apoptosis protein. In addition, Oct-4 and survivin are both specifically expressed in embryonic tissue, germ line and tumor tissues (such as breast, pancreatic and colon cancer), but not in other tissues or at low levels in these cells. We hypothesized that the mechanism of Oct-4 related anti-apoptosis may be mediated through Survivin. Towards this possibility, we transfected the Survivin promoter into ES cells after knockdown of Oct-4. Promoter activity of the group with knocked down Oct-4 expression was dramatically decreased, indicating that Oct-4 mediated anti-apoptosis may act through Survivin. Western blots showed that Oct-4 knocked down ES cells had decreased Survivin protein expression. STAT3 is a well known transcription factor. Studies have shown that LIF-induced STAT3 is responsible for ES cell survival. Also, it was reported that STAT3 regulated survivin in breast cancer. We tested whether Oct-4 regulated Survivin through STAT3. Western Blot analysis showed that down regulated Oct-4 induced decreased phosphorylation of STAT3 and expression of total STAT3 protein. This suggests that Oct-4 is essential for anti-apoptosis of ES cells in response to stress, and this effect may be mediated through the STAT3/Survivin pathway.
Evaluation of Minimal Residual Disease in Blood and Bone Marrow for CLL as Assessed by 6 Color Flow Cytometry.
Authors: Maloum Karim , Sutton Laurent , Garff-Tavernier Magali Le , Fiocconi Caroline , Bonnemye Patrick , Brissard Martine , Merle-Beral Helene
Published: October 2019

MRD evaluation is becoming critical in CLL patients since the new therapeutic approaches lead to higher and higher biological response. Multicolor flow cytometry is demonstrated to be one of the best technique for assessing MRD. In a recent work, Moreno et al (Blood, 2006) suggested that peripheral blood could be sufficient for MRD evaluation. We here report our experience which compares peripheral blood and bone marrow aspirate for MRD assessment using a 6 color flow cytometry (FC) method. Two months after the end of the treatment, blood and bone marrow were harvested at the same time for 64 CLL patients in complete remission according to the NCI criteria. Patients were treated by chemotherapy followed or not by autologous stem cell transplantation (ASCT). None of them received immunotherapy. Until now 30 out of the 64 couples of blood and marrow samples have been studied. Immunophenotyping was performed using the FacsCanto Device (Becton Dickinson) and the following monoclonal antibody (moAb) combinaisons: k-FITC / l-PE / CD19-PerCP-Cy5.5 / CD5-PE-Cy7 / CD79b-APC / CD20-APC-Cy7; FMC7-FITC / CD38-PE / CD19PerCP-Cy5.5 / CD5-PE-Cy7 / CD20-APC-Cy7 for peripheral blood assessment and k-FITC / l-PE / CD45-PerCP-Cy5.5 / CD5-PE-Cy7 / CD19-APC / CD20-APC-Cy7; FMC7-FITC / CD19-PE / CD45PerCP-Cy5.5 / CD5-PE-Cy7 / CD79b-APC / CD20APC-Cy7 ; CD38-FITC / CD23-PE / CD45-PerCP-Cy5.5 / CD5-PE-Cy7 / CD19-APC. In the bone marrow, we have discriminated hematogons from CLL cells by using moAb combinations containing CD45 and CD38. Between 4500 and 5500 lymphocytes were analyzed in each test. By using a method of serial dilutions, we demonstrated a sensitivity of 10−4 for our 6-color FC technique. CLL cells were defined as CD19+CD5+CD79b low CD20low lymphocytes with low expression of monotypic Ig light chain. Results were expressed as a percentage of leukemic cells among total B lymphocytes. We showed a complete correlation between blood and bone marrow for MRD evaluation using this technique. A complete phenotypic remission defined by the absence of leukemic cells in the blood and in the bone marrow, was observed in 17 out of 30 cases. In the other 13 patients, persistent leukemic cells were detected in the marrow as well as in the peripheral blood with a median rate of 1.56 % [0.15 – 41] and 0.56% [range 0.06 – 36] respectively. Our results showed that, by using 6 color FC, blood assessment for detecting MRD in CLL after chemotherapy or ASCT could provide a simple surrogate to bone marrow assessment. Analysis of the remaining couples of samples for the last 34 patients is ongoing.
CD38 Expression in B-CLL Is Dynamic and Changes Due to Contact with Activating Stimuli Found within the Leukaemic Microenvironment.
Authors: Patten Piers E.M. , Buggins Andrea G.S. , Richards Julie , Wotherspoon Andrew , Hamblin Terry John , Mufti Ghulam , Devereux Stephen
Published: October 2019

High levels of CD38 expression in B-cell chronic lymphocytic leukaemia (B-CLL) confer a poor prognosis. Although its role in B-CLL is unknown, signalling through CD38 has been implicated in cell survival, trafficking and proliferation. Since proliferation in B-CLL is thought to take place within both bone marrow (BM) and secondary lymphoid tissue, we investigated whether CD38 expression might vary in response to stimuli that occur in these tissue compartments.

Firstly, we compared the percentage CD38 expression of CD5/19 cells on 35 paired PB and BM aspirate B-CLL samples. The mean CD38% was significantly higher in BM than PB in all samples (27% vs 19%, p=0.009) including samples with a PB CD38 of 7% or more (33% vs 42%, p=0.047), indicating that factors present in the BM up regulate CD38 expression. Next, CD38 expression and cell division of B-CLL peripheral blood mononuclear cells (PBMCs) were examined in an in vitro system aimed at mimicking the proliferation centre microenvironment where leukaemic cells are situated in close proximity to activated T lymphocytes. Positively selected T cells from 15 B-CLL patients were activated overnight with CD3/28 beads and subsequently cultured with autologous B-CLL PBMCs. Both the percentage of CD19+ CD38+ cells (29.9% vs 59.9%, p=0.003) and CD38 mean fluorescence intensity (75.1 vs 830.8, p=0.005) increased over the 6 day culture period. B-CLL cell division was assessed using the dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in the same co-culture system. This showed that co-culture with autologous activated T-cells can result in B-CLL cell division, and is preceded by CD38 up regulation. In addition, significantly more B-CLL cells underwent at least one division from patients with an initial CD38 level of 7% or more, as compared to under 7% (24.6% vs 10.9%, p=0.031). To further investigate the relationship between B-CLL cell proliferation, CD38 expression and the role of T-cells we examined tissue sections known to contain paraimmunoblasts and other proliferating B-CLL cells. Four colour confocal microscopy using CD3, Ki67, CD38 and CD23 to label frozen B-CLL lymph nodes was employed. Large Ki67+ CD23+ cells were present in close proximity to CD3+ T-cells and these large B-CLL cells had higher CD38 expression than the surrounding small B-CLL lymphocytes.

These results support the proposal that CD38 expression in B-CLL is dynamic and may reflect exposure to T-cell derived stimuli which contribute to proliferation in the BM or LN microenvironment. A possible explanation for the poorer prognosis of patients with higher CD38 expression may be that their disease has more proliferative potential.
Expression of ZAP-70 in Chronic Lymphocytic Leukemia Cells Enhances CD79b Phosphorylation Following Surface IgM Ligation.
Authors: Chen Liguang , Apgar John , Tang Li , Kipps Thomas J.
Published: October 2019

CD79b is B-cell surface molecule that non-covalently associates with CD79a and surface immunoglobulin (sIg), which together serve as the B-cell receptor complex (BCR). Both CD79a and CD79b have cytosolic immunoreceptor tyrosine-based activation motifs (ITAMs) that can become phosphorylated following sIg ligation, thereby allowing for recruitment to the BCR complex of cytosolic kinases, such as p72Syk , which then can initiate downstream intracellular signaling events. Compared to normal B cells, chronic lymphocytic leukemia (CLL) B cells typically expresses low levels of CD79b, which is speculated to contribute to the relatively poor capacity of CLL cells to initiate intracellular signaling following BCR ligation despite having apparently adequate levels of p72Syk. BCR signaling in CLL cells can be enhanced by expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that initially was identified in T cells, where it plays a critical role in the phosphorylation of ITAMs of the accessory molecules of the T-cell receptor (TCR) complex for antigen following TCR ligation. We investigated for phosphorylation of CD79b following BCR ligation with F(ab)2 anti- μ antibody in CLL cell samples that did or did not express ZAP-70. All CLL cell samples expressed similar amounts of surface IgM and p72Syk, as assessed via flow cytometry and immunoblot analysis. Within 10 minutes after treatment with anti-μ the CLL cell samples that expressed ZAP-70 (n = 28) experienced a mean increase in phosphorylation of CD79b of 21.5% (± 14.0% S.D.), which was significantly greater than the 7.5% increase (± 7.9% S.D.) experienced by similarly treated CLL cell samples that did not express ZAP-70 (n = 19) (P< 0.01). Immune precipitation studies demonstrated association of CD79b with p72Syk in CLL B cells. CLL cell samples (n = 5) lacking expression of ZAP-70 were transfected with a control vector or an expression vector encoding ZAP-70, allowing us to examine the effect that engineered-expression of ZAP-70 has on CD79 phosphorylation following treatment with anti-μ. Anti-μ treatment induced significantly higher mean levels of CD79b phosphorylation in CLL samples made to express ZAP-70 (33% ± 16%) than in control mock-transfected CLL cells (4% ± 2%). This also was associated with enhanced anti-μ induced phosphorylation of p72Syk. We conclude that expression of ZAP-70 in CLL B cells enhances phosphorylation of the accessory molecules in the BCR complex following sIg ligation, potentially allowing for improved recruitment of cytosolic kinases and adapter proteins to these accessory molecules for enhanced BCR signaling.
The Vascular Endothelial Growth Factor (VEGF) Autocrine Survival Signaling Pathway in Chronic Lymphocytic Leukemia (CLL) Is Regulated by Lysophosphatidic Acid (LPA).
Authors: Hu Xiaojie , Sun Jinmie , Johnston James B. , Gibson Spencer B.
Published: October 2019

Vascular endothelial growth factor (VEGF) is a survival and angiogenesis factor that has been the target of chemotherapy in a variety of cancers including chronic lymphocytic leukemia (CLL). The VEGF survival response is regulated by an autocrine loop where cells constitutively express VEGF and activate the VEGF receptors leading to up-regulation of anti-apoptotic Bcl-2 members and prevention of apoptosis. Herein, we have demonstrated that VEGF is elevated in the plasma of CLL patients compared to normal individuals. We have previously shown that the growth factor, lysophospatidic acid (LPA), is a survival factor in CLL and its receptor LPA1 is up-regulated in CLL cells (J Biol Chem, 280:9498, 2005). Now, we demonstrate that inhibiting VEGF receptor activation blocks the survival response seen with LPA in CLL. LPA treatment was found to induce VEGF mRNA and protein levels within 1 hour. LPA receptor inhibitor Ki16425 blocked the VEGF production and reversed the protective effect of LPA against apoptosis. Combining LPA and VEGF failed to increase survival responses in these cells. In addition, LPA protection against fludarabine and TRAIL induced apoptosis was eliminated using the VEGF receptor kinase inhibitor, epigallocatechin-3-gallate (EGCG), which is present in green tea, or VEGFR:Fc, that inhibits VEGF receptor activation. Furthermore, LPA induced AKT activation and up-regulation of the expression of the Bcl-2 family member, Mcl-1, were eliminated by the VEGF receptor inhibitor. Thus, the protective effect of LPA against apoptosis in CLL is related to increased production of VEGF. Targeting the LPA pathway similar to the VEGF pathway may thus be a novel approach for the treatment of CLL.
Long Term Outcome of Patients with Steroid Refractory Acute Intestinal Graft Versus Host Disease after Treatment with Pentostatin.
Authors: Klein Stefan A. , Mousset Sabine , Bug Gesine , Waßmann Barbara , Hoelzer Dieter , Martin Hans
Published: October 2019

Steroid refractory intestinal acute Graft versus Host Disease (aGvHD) is a mayor complication after allogeneic hematopoietic cell transplantation. Survival rate of severe intestinal aGvHD is low. No therapy has been proven to increase the survival rate in steroid refractory aGvHD so far. A promising strategy is the use of pentostatin, a purine nucleotide analogue and inhibitor of adenosine deaminase, which is known to decrease function and number of lymphocytes. Here we report the six year follow-up of a pilot study on salvage therapy of aGvHD by pentostatin. We treated 18 patients with steroid refractory intestinal aGvHD stage III or IV with Pentostatin (8 female and 10 male patients). The mean age was 45 (range: 25–61 years). The underlying diseases were ALL (4), AML (10), Multiple Myeloma (2), Hodgkin’s Disease (1) and CML (1). Eight patients were allografted with HLA-identical sibling donors, three with related donors with a single HLA class I mismatch, seven with matched unrelated donors. All patients were allografted with peripheral blood stem cells. As GvHD prophylaxis ciclosporine A in combination with mycophenolate mofetil or methotrexate were used. Eleven patients had aGvHD stage III, seven stage IV. All patients had a severe gastrointestinal-tract involvement (grade III or IV). After failure of steroid treatment (prednisolone >2 mg/kg for at least 3 days) pentostatin was applied as a salvage therapy (1 mg/m2 for three consecutive days). Eight patients received between one and three further courses of pentostatin every three to four weeks. Therapy was well tolerated. No severe neutropenia was observed. Except one case of hemolytic uremic syndrome (HUS) no impairment of renal function was observed. Eleven patients achieved a complete, five a partial remission of aGvHD. Time until a clinical improvement could be observed was 12–14 days. Two patients died before day 12 without improvement of GvHD symptoms. Seven patients (39%) are alive (510–2060 days post first cycle of pentostatin), two with extensive chronic GvHD. All one-year-survivors stayed alive. Eleven patients died (two due to relapse of leukemia, nine transplant related [1 x interstitial pneumonitis, 1 x pneumonia, 1 x chronic GvHD, 4 x aGvHD and multiple organ failure (MOF), 1 x parvo B19 infection and MOF, 1 x HUS and MOF]). In conclusion, in this pilot study pentostatin has been demonstrated to be a highly effective and well tolerated drug for salvage therapy of steroid refractory acute GvHD with intestinal involvement. Moreover, compared with earlier studies on steroid refractory aGvHD a high rate of long term survivors could be observed. By a multicenter prospective randomised study we are currently investigating whether pentostatin can improve the outcome in steroid refractory intestinal aGvHD.
Deleterious Roles of the KIR Ligand Mismatch on Acute Graft Versus Host Disease in Unmanipulated HLA-Mismatched/Haploidentical Blood and Marrow Transplantation.
Authors: Huang Xiao Jun , Zhao Xiang Yu , Liu Dai Hong , Liu Kai Yan , Xu Lan Ping , Chen Yu Hong , Han Wei
Published: October 2019

The beneficial effect of KIR ligand mismatch with a very low incidence of rejection, acute graft versus host disease (aGVHD), and leukemia relapse had been demonstrated by Perugia’s group in the Haploidentical mismatched Hematopoitic cell transplantation (HCT) with extensive T cells depletion in vitro. However, many analytical results of the haploidentical and unrelated mismatched transplantation appeared to be inconsistent with Perugia’s work. The disputes in the inconsistent roles of KIR ligand mismatch seem to be caused by the different transplant protocols with different extent of T cells depletion in vitro or in vivo. In recent years, we successfully established a novel protocol—conditioning including antithymocyte globulin followed by un-manipulated HLA-mismatched/haploidentical blood and marrow transplantation, which can achieve comparable outcomes to HLA-identical sibling transplantation. Following the contradictory results about the KIR ligand mismatch in the haploidentical related and mismatched unrelated HCT, we have evaluated the roles of the KIR ligand mismatch in 94 leukemia patients undergoing unmanipulated HLA-mismatched/haploidentical blood and marrow transplantation. Multivariate analysis showed that both KIR ligand mismatch (HR 2.833, CI, 1.286–6.241, p=0.01) and doses of T cells (HR 3.059, CI, 1.292–7.246, p=0.011) were independent risk factors causing the acute graft versus host disease (aGVHD). In addition, compared to patients without KIR ligand mismatch, KIR ligand mismatch worsened the adverse effect of ‘high’ dose T cells (>1.48×108/kg) on aGVHD (100% vs 63.3%, p=0.036), and increased the incidence of aGVHD in patients with HLA-C mismatch (80% vs 57.4, p=0.056). Since multivariate analysis demonstrated that high risk leukemia is the only predictor for TRM, relapse and OS, we further analyzed the effect of KIR ligand mismatch on prognosis in standard and high risk patients. The differences in TRM (50% vs 7.6%, p=0.005) and OS (50% vs 88.4%, p=0.014) between patients with and without KIR ligand mismatch were most striking for standard risk. Therefore we conclude that due to the presence of large dose T cells in the allograft, the alloreactivity of NK cells had been inhibited and KIR ligand mismatch directed alloreactive T cells played crucial roles in our model.