bluuebunny/crossref_metadata_2025 huggingface dataset:
A Critical Role for the Retinoblastoma Tumor Suppressor Gene in Hematopoietic Stem Cells.
Authors: Geiger Hartmut , Filippi Marie-Dominique , Kalfa Theodosia A. , Daria Deidre
Published: October 2019

The retinoblastoma tumor suppressor protein (RB) plays important roles in the control of the cell cycle, DNA-damage checkpoint, differentiation and apoptosis. It is estimated that RB is dysfunctional/inactivated in up to 40% of human leukemias. Positive as well as inhibitory signals are integrated into the phosphorylation of the RB protein to regulate the G1 to S-phase progression of the cell cycle. Despite the importance of RB in leukemia, the consequences of loss of RB on hematopoietic stem and progenitor cell (HSPC) function in vivo are still not clear and have been controversially discussed. Using Cre-enzyme expression driven by the hematopoietic specific Vav1-promotor, we generated mice that are constitutively deficient in RB (hemRb−/− animals) in HSPCs. HemRb−/− mice showed anemia with an increased number of reticulocytes in PB, consistent with a published role of RB in erythroid differentiation. In addition, the frequency of Mac-1 positive cells in BM was increased to 67% compared to 47% in control animals, whereas the frequency of B220 positive B-lymphoid cells was almost 10-fold reduced, without affecting the T-lymphoid compartment. HemRb−/− mice possessed a 3-fold enlarged spleen with a 5-fold increased number of colony-forming cells (CFCs) and severe extramedullary hematopoiesis, a phenotype also reported for animals transplanted with Rb−/− fetal liver cells.

BM of hemRb−/− mice showed an almost 3-fold reduction of HSC frequency, measured by the cobblestone-area forming cell assay (CAFC) assay, but not a decrease in the number of HSCs determined by cell surface staining and flow cytometry. Upon transplantation into NOD/SCID animals or upon competitive transplantation into C57BL/6. CD45.1 animals, HSPCs from hemRb−/− mice contributed 4 to 6-fold less to hematopoiesis. HSPCs from hemRb−/− animals were neither impaired in their ability to home to the BM, nor did they show increased apoptosis. Finally, we detected a significant 4-fold decrease in stem cell function/numbers upon stress caused by 5-FU treatment in hemRB−/− mice compared to control animals. We conclude that upon transplantation/stress, HSPCs from hemRb−/− animals are impaired in their self-renewal function. HemRb−/− animals also showed a 2-fold increase in the frequency of CFCs in peripheral blood. As we detected no increased leukemia incidence in the hemRb−/− animals (now up to 1 year of age), loss of the tumor suppressor RB in hematopoietic cells might be regarded as necessary, but not sufficient for causing early onset leukemia. In summary, loss of RB results in context/localization dependent phenotypes in the hematopoietic hierarchy, influencing stem and progenitor cells in function, localization and differentiation ability.
Differentiation of Non-Adherent Hematopoietic Stem Cells from Umbilical Cord Blood Cells into Adherent Hepatocytic Lineage.
Authors: Peters Rowayda Elias , Mock Melissa , Meier Peter , Renner Christoph , Knuth Alexander , Stieger Bruno
Published: October 2019

The liver consists of a variety of different cell types, the vast majority of which are hepatocytes. It has been shown that adult, as well as umbilical cord blood (UCB) mesenchymal stem cells (MSC) are capable of differentiation into mesenchymal and non-mesenchymal tissues including hepatocytes. Only a limited number of UCB units (20%) such as frozen, unfractionated and concentrate leukocytes induced by hydroxyethyl starch volume reduced UCB units can generate MSC. In light of this, we have developed the technology to generate potent MSC from all frozen UCB samples tested (3/3). In the present study, the incubation of thawed out UCB mononuclear cells (MNC) and non-selected CD34+ cells in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB hematopoietic stem cells (non-adherent) (Peters et al., 2004), but also long-term expansion of MSC (adherent cells). Adherent cells were enriched by medium changes followed by trypsinization and subsequent culture and passing on fibronectin in the presence of 10% human serum and 30ng/ml bFGF. The stroma-free liquid cultures were tested for the production of hematopoietic stem cells (HSC), as well as MSC over a period of 15 weeks; MSC increased throughout expansion of HSC. We used the CFU-Fibroblast assay to demonstrate MSC activity in stromal cell formation in vitro. Expanded MSC between passages 3–5 were plated using MesenCult™ (Stem Cell Technologies). After 14 days incubation at 37oC, 5% CO2, very large and small CFU-fibroblast colonies generated from 105 MNC/ml (initiated on Day 0 in stroma-free liquid culture) were continuously detected throughout expansion. In order to test the capacity of MSC to differentiate into hepatocytes, cells were cultured in DMEM supplemented with dexamethasone and 25ng/ml HGF, the latter of which was replaced with 50ng/ml oncostatin M after incubation day 14. The cells were able to expand to cell lines and took on the common hexagonal shape characteristic of hepatocytes; this parameter was used as a primary screening criterion. In addition, the hepatocyte-like cells were further assessed by reverse transcription-polymerase chain reaction and found to express mRNA for albumin, as well as two hepatocyte-specific bile salt transport proteins, basolateral bile transport system NTCP and the canalicular bile salt export pump BSEP. These data demonstrate the presence of pluripotent stem cells in UCB with the capacity to differentiate into a hepatocytic lineage.The research in this area has great potential, both with respect to future application in patients with end-stage liver disease, as well as for learning more about the role of MSC in new clinical cellular therapies.
Derivation of SSEA4-/CD73+ Mesenchymal Stem Cells from Human Embryonic Stem Cells.
Authors: Trivedi Parul , Hematti Peiman
Published: October 2019

Human embryonic stem cells (hESCs) could potentially provide a renewable source of different types of cells for cell therapy applications. Recently, mesenchymal stem cells (MSCs) have been derived from hESCs either through co-culturing with murine OP9 bone marrow stromal cell line or directly from hESCs without co-culturing with OP9 cells. Although the latter methodology is clinically advantageous over co-culturing with an animal cell layer those mesenchymal cells were reported to be positive for SSEA4. SSEA4 is a marker of undifferentiated hESCs and thus the presence of this marker on hESC-derived cells could potentially be problematic for clinical applications. We have recently achieved a novel and reproducible methodology for deriving a pure population of SSEA4-/CD73+ MSCs from federally approved hESC lines H1 and H9. To initiate the differentiation of hESCs to MSCs, we cultured undifferentiated hESCs on matrigel plates in murine embryonic fibroblast conditioned media with media changes every 3 days. Under these culture conditions a portion of embryonic stem cells differentiated into fibroblast looking cells. Through a multi-step process which involved the use of a culture methodology similar to what is being used to culture bone marrow (BM)-derived MSCs, and passaging cultured cells at defined time points we were able to derive a pure population of cells that were uniformly positive for MSC marker CD73 in about a 4-weeks period. These cells had fibroblast/mesenchymal looking morphology, and expressed cell surface marker antigens similar to what has been reported for adult human BM-derived MSCs: they are positive for CD29, CD44, CD54, CD71, CD90, glycophorin A, CD105, and were negative for hematopoietic markers such as CD34 and CD45. Similar to adult BM-derived MSCs these cells express HLA class-I antigens but not class-II antigens. Using established differentiation protocols we could differentiate the hESC-derived CD73+ MSCs into adipocytes, osteocytes, and chondrocytes as verified by immunohistochemistry and RT-PCR assays. So far we have grown these CD73+ MSCs up to passages 15–18. These cells retained their differentiation potential, and were karotypically normal when tested at passage 12. Most importantly, we did not observe any MSCs that were double positive for CD73 and SSEA4 antigen at any time point during our experiments. MSCs from a variety of fetal and adult sources are in various stages of clinical trials with some encouraging preliminary results. Our hESC-derived MSCs that are very similar to adult BM-derived MSCs regarding their growth and morphologic properties, immunophenotypic characteristics, differentiation potential, and importantly are devoid of hESC marker SSEA4 could potentially provide a novel source of MSCs for clinical applications.
Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhhibitor GX15-070 as a Novel Therapeutic Strategy in AML.
Authors: Watt Julie C. , Konopleva Marina , Contractor Rooha , Samudio Ismael , Harris David , Estrov Zeev , Viallet Jean , Andreeff Michael
Published: October 2019

GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in leukemias. In this study, we investigated the activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 cell lines at IC50’s of 0.1, 0.5, 0.5 and 2.5μM, respectively, at 72 hours. Neither overexpression of Bcl-2 or Bcl-XL nor loss of expression of Bax conferred resistance to GX15-070. GX15-070 inhibited Bim/Bcl-2 heterodimerization and induced association of activated Bak with Bax in OCI-AML3 cells, as demonstrated by co-immunoprecipitation studies using CHAPS buffer. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity, caspase activation and a decrease in mitochondrial inner membrane potential. Notably, GX15-070 induced cytochrome c release from isolated mitochondria of leukemic cells. GX15-070 synergized with both AraC and the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells, a notoriously chemoresistant cell line (GX15-070 and ABT-737 average CI value 0.3; GX15-070 and AraC average CI value 0.36). In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. GX15-070 potently inhibited clonogenic ability of AML blasts at sub-micromolar doses (58.5±10.6% CFU-Blast at 0.1μM and 38.1±10.5% at 0.25μM, n=7). In summary, BH3 inhibitor GX15-070 induces apoptosis in AML cells via inhibition of association of pro-survival Bcl-2 family proteins and BH3-only proteins, followed by Bax/Bak activation and initiation of the intrinsic apoptotic pathway. Hence, GX15-070 alone or in combination with chemotherapeutic agents may have utility in AML therapy.
Inhibition of Cellular Aminopeptidases as Novel Therapy for AML.
Authors: Jenkins Christopher , Pepper Chris , Mills Ken , Burnett Alan
Published: October 2019

CHR 2797 is one of a new class of enzyme inhibitors with a pleiotropic effect against a number of human cancer cells. It is thought to inhibit the M1 family of metalloenzymes that include aminopeptidases, and is under investigation for the treatment of acute myeloid leukemia. Aminopeptidases catalyse the hydrolysis of the terminal amino acids from short chain polypeptides and they are involved in the continuous cycle of protein formation and degradation in cells. As malignant cells are thought to be more highly dependant on this protein cycling, interrupting this pathway is therefore a potential therapeutic target for novel agents. The effects of the aminopeptidase inhibitor CHR 2797 were investigated in AML cells in-vitro. Leukemic cells and cell lines were treated with CHR 2797 at a range of 0.0002 – 20μM and IC50 values were calculated from the WST-1 proliferation experiments. The AML cell lines HL60, KG1, K562 and U937 had an average IC50 of 1μM with a range between 0.01 and 10μM. Primary diagnostic AML samples (n=40) were analysed and an IC50 range of between 0.01 and >40μM were detected, with a median of 0.8μM. The effects of CHR 2797 were also analysed on normal bone marrow samples (n=10). The IC50 range was between 6.2 and >40μM with a median of 15μM, demonstrating a potential therapeutic window between the treatment of the leukemic cells and toxicity to the normal samples. The level of synergy or antagonism with conventional therapeutic agents was calculated using a combination index. Synergy was demonstrated in 70% of cell samples in combination with ARA-C, and 80% with Velcade. Synergy was also shown in 60% of cells samples with ATRA, even in non-promyelocytic leukemia types. Annexin V and cell cycle analysis confirmed apoptosis after treatment with CHR 2797 in many cases. A degree of differentiation of acute promyelocytic cells to mature myeloid cells was also stimulated with the treatment. The effects of CHR 2797 on cellular aminopeptidases were also measured. CD13 is a cell surface protein which is expressed selectively on myeloid cells and is also classified as an aminopeptidase N. Its activity can be measured by the conversion of the substrate ala-MCA to the protein MCA that can be detected on a fluorometric plate reader. CHR 2797 was demonstrated to reduce CD13 activity in a time and dose responsive manner. The reduction in activity was demonstrated immediately following addition of the drug, and a persistent effect was shown over four days of cell culture. A reduction in CD13 activity was also shown with a concentration of CHR 2797 of under 0.5μM; and with a 10μM dose the activity in many AML samples was reduced by >90%. New treatments are needed for acute myeloid leukemia to improve survival and reduce the toxicity of conventional therapy. This study demonstrates that CHR 2797 might be an effective molecular therapy for AML, either alone or in combination with other chemotherapeutic agents.
Canonical NF-kB Pathway Is Not Constitutively Activated and May Not Be the Major Bortezomib Target in Mantle Cell Lymphoma (MCL).
Authors: Camara-Clayette Valerie , Lepelletier Yves , Moura Ivan , Vainchenker William , Hermine Olivier , Ribrag Vincent
Published: October 2019

Bortezomib, a proteasome inhibitor, has promising activity in MCL. Bortezomib mechanism of action is complex since multiple proteins and cellular pathways are regulated by proteasome degradation. By blocking the degradation of IKBa, the canonical NF-kB pathway is one of the major Bortezomib target in lymphoid malignancies such as multiple myeloma. NF-kB activation has not been extensively assessed in other lymphoid malignancies such as MCL. NF-kB complexes (heterodimers P50/P65) were assessed by EMSA assays in REC GRANTA 519, NCEB, JEKO, JUN and UPN1 MCL cell-lines. EBV was present in 3/6 cell lines. P50/P65 complexes were detected in all EBV positive cell lines and only in 1/3 EBV negative cell-lines (JEKO). No nuclear P65 protein was detected by immunofluorescence or Western-blot analysis in the 2 other negative EBV cell lines (REC and UPN1). NF-kB transcriptional activity was measured by a luciferase-based reporter gene assay. Spontaneous NF-kB activity was low in UPN1, REC and JEKO compared to GRANTA 519 EBV positive cell-line (3 to 4 time lower) but can be strongly activated (up to 10 fold) using MEKK cotransfection assay. This suggest that NF-kB is functional, but not constitutively activated. Stable infections with Migr1-IRES-GFP IkBM (dominant negative IKB) or empty vector, were performed in GRANTA 519 EBV positive as well as in UPN1 cell line. No differences in proliferation or apoptosis were observed in UPN1 stably infected with IkBM or with the empty vector but GRANTA 519 EBV positive showed increased apoptosis and proliferation inhibition when infected with IkBM. Likewise, no P50/P65 heterodimers complexes were detected by EMSA in 4 patients with MCL. In vitro assays showed that UPN1 and JEKO cell lines had comparable sensitivities to Bortezomib than Multiple myeloma cell lines reported in the literature (IC 50: 6 nM and 12 nM respectively). This was also true for the 3 patients lymphoma cells assessed in vitro (12 nM). Taken together these results suggest that canonical NF-kB activation pathway is not constitutively active in EBV negative MCL cell lines and patients samples and could be associated with EBV infection in some MCL cell-lines. Therefore, this strongly suggests that Bortezomib target other molecules in MCL.
Multicolour Flow Cytometric Finetuning of the Classification of Myelodysplastic Syndromes.
Authors: van de Loosdrecht Arjan A. , Westers Theresia M. , Westra Guus , Draeger Angelica , Van der Velden Vincent H. , Ossenkoppele Gert J.
Published: October 2019

The WHO classification of myeloid disorders contribute to a more refined classification and prognostication of myelodysplastic syndromes (MDS). The considerable differences in clinical behaviour of pure refractory anemia (RA) versus refractory cytopenia with multilineage dysplasia (RCMD) with or without ringsideroblasts are of importance in the management of patients with MDS. Flow cytometry may add additional diagnostic criteria to adequately discriminate RA from RCMD (+/− ringsideroblasts; (RS)) and may contribute in identifying Idiopathic Cytopenia of Undetermined Significance. We developed a 4-colour flow-cytometric procedure that comprises all differentiation stages of granulocytic, monocytic and erythroid lineages, instrumental for the recognition of various subpopulations within all three lineages in normal bone marrow samples. In 43 evaluable patients with MDS (RA, RARS, RCMD, RCMD-RS, MDS-U, RAEB-1 and 2), aberrant expression of differentiation antigens were demonstrated in 1 or more lineages. Flow-cytometry identified aberrancies in granulopoiesis and monocytopoiesis in 93% and 74% of the cases, respectively. In the majority of cases abnormal relations between CD13, CD16, CD11b, CD15 and HLA-DR were prominent in the granulopoiesis. In 34% of the cases a striking monocytopenia was detected, whereas in 59% abnormal surface expression of CD14, CD36 and CD33 indicating aberrant differentiation of monocytes. We defined aberrant myeloid blasts by a leukaemia associated phenotype (LAP) according to the definitions used in acute myeloid leukaemia. In 47% of the patients a LAP was detectable by demonstrating co-expression of CD5, CD7, CD19 and CD56 on CD34+ myeloid blasts. In all patients diagnosed as RA/RARS and MDS-U (n=12) according to WHO criteria, additional flow aberrancies were identified including a leukaemia associated phenotype of myeloid blasts in 41% of the cases. Only in 3 out of 28 cases with RCMD/RCMD-RS no erythroid aberrancies were detectable by flow-cytometry. In 9 normal control BM samples, no flow-cytometric abnormalities were present. It is concluded that flow-cytometry in MDS identifies aberrancies in the granulocytic and monocytic lineages and may classify patients with multi-lineage aberrancies not otherwise determined by cytology (WHO). Flow-cytometry may discriminate pure RA or MDS-U from RCMD. Since new drugs are emerging in low-risk MDS, the value of flow-cytometry might be of importance to further refine the classification in MDS. The exact role of these aberrant differentiation patterns on IPSS, clinical behaviour, impact on treatment decisions and as tool in disease monitoring have to be determined in future prospective studies.
An Erythroid Differentiation Gene Expression Signature Predicts Response to Lenalidomide in Myelodysplasia.
Authors: Ebert Benjamin L. , Galili Naomi , Tamayo Pablo , Mak Raymond , Pretz Jennifer , Ladd-Acosta Christine , Stone Richard , Golub Todd R. , Raza Azra
Published: October 2019

Lenalidomide is an effective new agent for the treatment of Myelodysplastic Syndrome (MDS). In phase II clinical trials, patients treated with Lenalidomide achieved unprecedented rates of hematologic and cytogenetic responses, particularly in patients with interstitial deletions of chromosome 5q. More than 80% of patients with MDS do not have 5q deletions, though approximately 25% of these patients respond to Lenalidomide.3–5 Neutropenia and thrombocytopenia are the primary toxicities of Lenalidomide, and are severe in some cases. MDS is a highly heterogeneous disorder, and no diagnostic assays predict which patients without 5q deletions will respond to Lenalidomide. Using gene expression profiling, we identified a molecular signature that predicts Lenalidomide response. Biologic characterization of the predictive genes demonstrated that the response signature is comprised of a cohesive set of erythroid-specific genes: patients who respond have a lower expression of the erythroid signature in bone marrow mononuclear cells. To facilitate clinical implementation of a gene expression-based assay, we evaluated the signature using two methods, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and a bead-based detection technology. Using both methods, the erythroid gene expression signature was expressed at lower levels in patients who responded to Lenalidomide. The bead-based methodology employs ligation mediated amplification (LMA), a methodology that is capable of highly multiplexed reactions with preservation of the relative abundance of transcripts, with amplicon detection on Luminex microspheres, already in use for clinical diagnostic assays. This assay is capable of detecting a large gene expression signature (currently up to 100 genes) in a single, well-controlled assay. With further validation, LMA with fluorescent microsphere detection may become the superior technology for clinical diagnostic assays of multi-gene signatures. We tested the erythroid signature in an independent set of 12 samples using LMA with fluorescent microsphere detection. Again, all responders had a lower expression of the erythroid genes than responders. Validation in a larger cohort of patients is underway. These studies suggest that Lenalidomide-responsive patients without 5q deletions have a defect in erythroid differentiation analogous to the ineffective erythropoiesis in patients with 5q deletions, and that an erythroid gene expression signature predicts Lenalidomide activity in MDS.
Genetic Profiling of Myeloproliferative Disorders by Single Nucleotide Polymorphism Oligonucleotide Microarray.
Authors: Kawamata Norihiko , Ogawa Seishi , Levine Ross L. , Pikman Yana , Nanya Toshiki , Sanada Masashi , Miller Carl W. , Gilliland D. Gary , Koeffler H. Phillip
Published: October 2019

Myeloproliferative disorders (MPD) are clonal hematopoietic diseases including polycythemia vera (PV), essential thrombocytosis (ET) and myelofibrosis with myeloid metaplasia (MMM). Single-nucleotide polymorphism DNA microarray (SNP-Chip) was used to analyze 43 MPDs. All five cases of PV having homozygous JAK2V617F had uniparental disomy (UPD), thus duplicating the allele with the mutated JAK2 and eliminateing the normal allele. Concerning MMM, either the Rb1 (13q14) or NF1 (17q11) gene was deleted in five of the 9 cases that had no Jak2 mutations. Two of these cases had UPD at 1p. Sequencing of the target genes within this region of UPD revealed a point mutation of the MPL gene (S240F or W515L). Each of these cases also had a deletion of Rb suggesting that the mutated MPL may co-operate with an aberrant Rb. The 9 cases of MMM with JAK2 mutations had only infrequent (2/9 cases) additional genomic alterations. Interestingly, No genomic alterations were noted in any of the cases (10) of ET having a normal Jak2. Two of 7 ET cases with JAK2 mutations had deletion of either 5q or trisomy 9. In summary, UPD is the predominant mechanism by which MPD develops homozygous JAK2 mutations. 1p UPD is a signpost that MPL is mutated in cases of MMM having wild-type JAK2; and these cells often have loss of Rb. ET cases with no JAK2 mutations are surprisingly free of genomic alterations.
Inducible hkRASG12D Expression in Zebrafish Hematopoietic Cells Leads to Myeloproliferative Disease.
Authors: Le Xiuning , Langenau David M. , Keefe Matthew D. , Kutok Jeffery L. , Zon Leonard I.
Published: October 2019

Activating mutations in RAS family members are common in myeloproliferative disease (MPD) and acute myeloid leukemia (AML). Because the zebrafish has proven to be an excellent leukemia model and can be used in forward genetic and chemical screens to identify modulators of disease pathways, we developed a transgenic zebrafish model of RAS-induced myeloproliferative disease. Stable transgenic zebrafish lines were created in which the ubiquitous B-actin promoter drives expression of a loxed GFP transgene. Upon CRE-mediated recombination, the loxed GFP cassette is excised and the human kRASG12D transgene is expressed. When mated to hsp70-CRE transgenic zebrafish that express CRE recombinase when the animals are heated to 37C for 1 hour, a cohort of 300 double transgenic embryos was generated. In the heat-shocked group, the medial survival time was 25 days, suggesting that oncogenic RAS expression is lethal in developing zebrafish larvae. Of those animals that survived past this time window, most developed sarcomatous muscle tumors. In contrast, most of the double transgenic zebrafish that did not receive heatshock survived to adulthood. Because the hsp70 promoter is active in development and can be induced by stress, the hsp70-CRE transgenic zebrafish line exhibits low levels of CRE expression even in the absence of heatshock. In the non-heat shocked cohort, 10 of 120 double transgenic zebrafish developed MPD by 66 days of life. Histological examination and fluorescence cytometry analysis revealed an expansion of myeloid cell populations within the kidney, comprising granulocytic and monocytic cells in various stages of differentiation, mimicking myeloproliferative diseases seen in both human and mouse. To specifically induce kRASG12D expression in hematopoietic cells, kidneys were dissected from healthy double transgenic animals; heat shocked ex vivo, and then transplanted into sub-lethally irradiated recipient fish. Upon analysis of transplant animals at three months of age, flow cytometry confirmed that MPD had developed in the recipient fish, however other tumor subtypes were not observed. When kidney marrow from transplant animals having MPD were introduced into sub-lethally irradiated secondary recipients, transplant efficiency was greatly reduced. Taken together these results suggest that, as is seen in MPD in mammals, myeloid cells are not fully transformed in our model. In summary, we show that inducible hkRASG12D expression in zebrafish hematopoietic cells leads to myeloproliferative disease and suggest that this model will allow for the identification of novel pathways responsible for full transformation leading to AML.
Safety and Tolerability of Anti-IL-5 Monoclonal Antibody (Mepolizumab) Therapy in Patients with HES: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.
Authors: Klion Amy D. , Rothenberg Marc E. , Murray John J. , Singh Anish , Simon Hans-Uwe
Published: October 2019

Mepolizumab is a fully humanized monoclonal IgG antibody which binds to interleukin 5 (IL-5) with high affinity and specificity, preventing it from binding to IL-5 receptors on the eosinophil cell surface. Since IL-5 is central in eosinophil proliferation, differentiation, mobilization, activation, and survival, yet has limited effects on other cell types, IL-5 inhibition should inhibit the production and activity of eosinophils without affecting the function of other immune cells. Reducing the blood eosinophil count is a critical therapeutic objective in patients with eosinophil-driven diseases, such as hypereosinophilic syndrome (HES). The safety and tolerability of mepolizumab has been evaluated, as a secondary endpoint, in a multicenter, randomized, double-blind, placebo-controlled, parallel-group trial. The trial enrolled 85 patients with HES (blood eosinophil count >1500/μl for ≥6 months with documented eosinophilia-related organ involvement or dysfunction and no known cause of eosinophilia), who were negative for the FIP1L1-PDGFRα gene rearrangement. Blood eosinophil levels were stabilized at <1000 cells/μL on 20–60 mg/day prednisone monotherapy during a run-in period of up to 6 weeks, then patients were randomized to intravenous mepolizumab 750 mg (n=43) or saline (placebo; n=42) every 4 weeks for 36 weeks (final infusion at Week 32). Overall, 98% of placebo-treated and 93% of mepolizumab-treated patients reported an adverse event during treatment. The most common of these were fatigue (26% on placebo vs 30% on mepolizumab), pruritus (21% vs 28%), headache (21% vs 23%), and arthralgia (17% vs 21%). The only significant differences between the treatment groups were in pharyngolaryngeal pain (14% on placebo vs 2% on mepolizumab; P=0.027) and pain in extremity (12% on placebo vs 2% on mepolizumab; P=0.047); none of the adverse events was significantly more frequent with mepolizumab than with placebo. A greater than 8% difference in adverse event reporting for the mepolizumab vs placebo arms was noted for upper respiratory tract infections, myalgia, rhinitis, bronchitis, and urticaria. Serious adverse events were reported in 5 patients on placebo (7 events) and 7 patients on mepolizumab (14 events, including 1 death due to a cardiac arrest), but none was considered treatment related. Withdrawals due to adverse events (4 patients on placebo; 1 patient on mepolizumab) were also considered unrelated to treatment. Laboratory tests (hematology, clinical chemistry, and urinalysis), vital signs, and ECG findings did not raise any major safety concerns. In conclusion, this study, which is currently the largest placebo-controlled trial involving exclusively patients with HES, indicates that mepolizumab is safe and well tolerated in this patient population.
Characteristics and Outcomes of 38 Patients with Acute Myeloid Leukemia Evolving from Previous Well Characterized Myeloproliferative Disorder.
Authors: Tam Constantine S. , Giles Francis , Cortes Jorge , Garcia-Manero Guillermo , Estey Elihu , Kantarjian Hagop , Verstovsek Srdan
Published: October 2019

Antecedent hematological disorder (AHD) is a known adverse prognostic factor for patients with AML. AHD, however, encompasses a range of diverse hematological disorders. The characteristics and outcome of AML evolving from myeloproliferative disorders (MPD) without features of dysplasia remain poorly defined. Between 08/73 and 01/06, 76 patients were treated at the MD Anderson Cancer Center for AML evolving from a previous MPD. Of these, 38 were excluded from further analyses: inadequate pathological information for confident diagnosis of MPD (n=15), concomitant myelodysplastic features (n=11), chronic myelomonocytic leukemia (n=4), and less than twelve months between MPD diagnosis and AML occurrence (n=8). Baseline characteristics of remaining 38 patients with well-characterized MPD: median age 51 years (range 24–70); male 61%; cytogenetics - diploid 86%, del20q 14%; splenomegaly 27%. Diagnoses by WHO classification: polycythemia vera 47%; myelofibrosis 24%; essential thrombocythemia 18%; chronic MPD unclassifiable 11%. MPD therapy: hydroxyurea in 87% (median 3 years exposure), alkylating agent in 16%, and radioactive phosphorus in 8% of patients. AML occurred a median of 111 months (range 22 – 376) after MPD diagnosis. Median age at AML diagnosis was 65 years (range 40–81); cytogenetics were poor risk (−5, −7, 11q23, or ≥3 abnormalities) in 50% and intermediate risk in others (including diploid in 28%). Median survival was 23 weeks after AML diagnosis, and was not significantly different between patients who received AML therapy and those who received supportive therapy only (p=0.33) (figure 1), or between patients with intermediate and poor risk cytogenetics (p=0.12). Twenty-four patients received standard or high-dose Ara-C based induction chemotherapy: complete response (CR) rate was 38%, but responses were short-lived with all patients relapsing within 37 weeks in the absence of stem cell transplantation. Two patients underwent non-myeloablative matched sibling stem cell transplant in first CR: one remain leukemia free at 70 weeks, and the second relapsed 22 weeks post transplant but responded to re-induction chemotherapy, and remains alive at 38 weeks. AML evolving from a previous MPD carries an adverse prognosis, with very poor results from conventional induction chemotherapy. Exploration of novel agents in this patient population as a first line therapy is warranted.

Figure Figure
CNTO 328, an Anti-Interleukin (IL)-6 Monoclonal Antibody (mAb) - Preliminary Results of Subjects with Castleman’s Disease from a Phase 1 Study in Selected Hematological Malignancies.
Authors: VanRhee Frits , Fayad Luis , Borghaei Hossein , Voorhees Peter M. , Orlowski Robert Z. , Furman Richard R. , Zaki Mohamed , Prabhakar Uma , Jiao Trina , Lihou Christine , Garay Carlos A. , Kurzrock Razelle
Published: October 2019

Background: IL-6 is a pleiotropic cytokine, the dysregulation of which plays an etiological role in many diseases. Multicentric Castleman’s disease (CD) is a polyclonal lymphoproliferative disorder in which IL-6, overproduced by enlarged lymph nodes, is responsible for B-cell proliferation, auto-immune phenomena, systemic inflammatory manifestations (eg, fever, fatigue, and weight loss) and abnormal laboratory findings, including anemia, hyper-γ-globulinemia, hypoalbuminemia, thrombocytosis and increases in acute-phase proteins (eg, CRP, ESR and fibrinogen). The inflammatory syndrome can be severe and refractory to steroids, chemotherapy and even autologous stem cell transplantation. CNTO 328 is a chimeric mAb that specifically binds and neutralizes human IL-6 with high affinity. A phase I trial is being conducted to assess the safety and PK of CNTO 328 in subjects with CD and other hematological diseases.

Methods: This trial enrolls subjects with non Hodgkin’s B-cell lymphoma, multiple myeloma, or CD in cohorts exploring doses ranging from 3 to 12mg/kg given intravenously repeated either weekly, or every 2, or 3 weeks. Dose limiting toxicity (DLT) is defined as a ≥Grade 3 event (excluding hematologic toxicity), or ≥Grade 2 allergic reaction/hypersensitivity by NCI CTCAE attributed to CNTO 328 after the first administration. Clinical benefit assessment is based on improvements in Hb, fatigue, anorexia, fever/night sweats, weight, and size of target lymph node.

Results: Results from the first 7 CD subjects enrolled in Cohorts 1 (n=1, CNTO 328 3mg/kg q 2 weeks), 2 (n=2, CNTO 328 6mg/kg q 2 weeks) and 3 (n=4, CNTO 328 12mg/kg q 3 weeks) are currently available. At present time, subjects have received a median number of 4 (range 3 to 21) doses. CNTO 328 alone was well tolerated and no DLTs were observed. Non-related to CNTO 328 Grade 3 AEs such as anemia, thrombocytosis, fatigue and one possibly-related AE (ie, vomiting, grade 3) were observed. In addition, 3 subjects experienced Grade 3 SAEs that were deemed either unrelated or unlikely related to CNTO 328. Six evaluable subjects with CD showed a response by clinical benefit assessment including improvement in Hb and in severe thrombocytosis, and fatigue, reduction of fever, resolution of rash, improvement in skin tumors, or decrease in lymphadenopathy. PK and PD analysis from these subjects is currently ongoing and data from these analyses will be presented.

Conclusions: Treatment with CNTO 328 has been well tolerated in CD subjects at doses up to 12mg/kg q 3 weeks, and no allergic reactions have been observed after repeated dosing. Preliminary results suggest that CNTO 328 can abrogate the effects of IL-6 and induce both clinical and biochemical responses in CD.
Outcome in Patients with Diffuse Large B-Cell Lymphoma (DLBCL) Treated with CHOP-R Can Be Predicted by Stage and Serum Lactate Dehydrogenase (LDH) Level.
Authors: Sehn Laurie H. , Chhanabhai Mukesh , Gill Karamjit , Hoskins Paul , Klasa Richard , Savage Kerry J. , Shenkier Tamara , Sutherland Judy , Gascoyne Randy D. , Connors Joseph M.
Published: October 2019

Background: DLBCL is a heterogeneous entity with patients exhibiting a wide range of clinical outcomes. A reliable prediction tool is vital for optimizing patient care. We have previously demonstrated that a revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI following treatment with rituximab and CHOP (CHOP-R). (Sehn, ASH 2005). Further exploration of clinical prognostic factors in an expanded cohort of patients yields a simplified prediction model based on stage and LDH alone.

Methods: We performed a retrospective analysis to assess the predictive value of recognized clinical prognostic factors in an unselected population of patients with DLBCL treated with CHOP-R. Patients were identified using the Lymphoid Cancer Database of the British Columbia (BC) Cancer Agency and included all patients >16 y of age who were newly diagnosed with DLBCL prior to Jan 15, 2006, treated in BC with a CHOP-R protocol. Patients were excluded if they were HIV positive, had evidence of an active second malignancy or an underlying indolent NHL.

Results: 523 patients were identified with the following characteristics: median age 61 y (16–93); male, 60%; stage III/IV, 55%; elevated LDH, 53%; performance status (PS) ≥2, 41%; ≥2 extranodal sites, 29%. Patients with advanced stage lymphoma (stage III/IV, B symptoms or bulky disease) or patients who were not candidates for radiation therapy were treated with 6–8 cycles of CHOP-R (91%) while those with limited stage were treated with CHOP-Rx3 and involved field radiation therapy (9%). Median follow-up for living patients is 24 months. On univariate analysis, stage, PS and LDH remain predictive for both PFS and OS. Number of extranodal sites is a borderline predictor of PFS (p=0.05) and no longer predictive of OS (p=0.276). Although age no longer predicts for PFS (p=0.17), it remains predictive of OS (p=0.001) since elderly patients are less likely to be treated successfully with secondary therapy and more likely to die from unrelated causes. On multivariate analysis, only stage (p<0.001) and LDH (p<0.001) remain significant predictors of PFS with similar predictive capacity (relative risk 2.7 and 3.0 respectively). A prediction model based on these two factors alone can predict outcome (see Table) as effectively as the full 5-factor IPI.

Conclusions: A prediction model based on stage of disease and serum LDH level at presentation provides an accurate estimate of outcome following primary therapy with CHOP-R for patients with DLBCL.
Breakthrough Fungal Infections after Allogeneic Hematopoietic Stem Cell Transplantation in Patients on Prophylactic Voriconazole.
Authors: Trifilio Steve , Singhal Seema , Williams Stephanie , Winter Jane , Tallman Martin , Gordon Leo , Frankfurt Olga , Evens Andrew , Mehta Jayesh
Published: October 2019

Voriconazole is a triazole anti-fungal agent with excellent activity against Aspergillus spp. We use liquid itraconazole 200 mg PO twice daily from day 0 to a month beyond discontinuation of all immunosuppression as standard anti-fungal prophylaxis after allogeneic HSCT in patients with no prior history of aspergillosis. This is changed to PO voriconazole 200 mg twice daily if corticosteroid therapy is started for GVHD. Voriconazole is continued even after steroid therapy is discontinued. Patients with a past history of aspergillosis get voriconazole from day 0. 71 allograft recipients who received voriconazole, and in whom complete clinical, microbiologic, and pharmacokinetic data were available were studied to determine the efficacy of voriconazole in preventing invasive fungal infections (IFI). 17 patients had not received itraconazole previously. The remainder had received itraconazole for 1–161 days (median 14). The length of voriconazole therapy was 6–956 days (median 133). The total number of patient-days on voriconazole was 13805 (~38 years). A total of 10 IFIs were seen in patients on voriconazole: Candida glabrata (n=5), Candida krusei (n=1), Cunninghamella (n=1), Rhizopus (n=2), and Mucor (n=1). The figure below shows the actuarial probability of IFI − 18% at 1 year.

Figure 1 Figure 1.

It is noteworthy that while 4 cases of zygomycosis were seen, no case of Aspergillus infection was seen. The figure below shows the actuarial probability of zygomycosis − 7% at 1 year.

Zygomycetes are generally not susceptible to voriconazole, and thus breakthrough infections are not surprising. However, C. glabrata and C. krusei are often susceptible to voriconazole with MICs of <2 μ g/mL (Spellberg et al. Clin Infect Dis2006;42:244–251). In that context, it is interesting that plasma steady-state trough voriconazole levels around the time the infection occurred were <0.2, <0.2, 0.33, 0.55, 0.63, and 1.78 μ g/mL in the 6 candidiasis cases. Excluding the 4 zygomycosis cases, all 6 candidiasis cases were seen amongst the 43 patients with voriconazole levels of ≤ 2 μ g/mL and none amongst the 24 with levels of >2 μ g/mL (P=0.061; Fisher’s exact test). This observation is in keeping with a recent report that showed correlation between voriconazole levels and therapeutic success in aspergillosis (Smith et al. Antimicrob Agents Chemother2006;50:1570–1572). We conclude that (1) voriconazole is extremely effective at preventing aspergillus infections, (2) zygomycosis is a concern in voriconazole-treated patients although the incidence appears low, and (3) therapeutic drug monitoring with dose adjustment may be indicated in patients on voriconazole to avoid breakthrough infections with fungi that are otherwise susceptible to the drug.

Figure 2 Figure 2.
Autologous Serum Containing Platelet Derived Growth Factors (PDGFs) as Topical Treatment for “Sicca Syndrome” Associated with Chronic GvHD.
Authors: Claudia Del Fante , Cesare Perotti , Gianluca Viarengo , Andrea Marchesi , Cristina Parisi , Paola Bergamaschi , Laura Salvaneschi
Published: October 2019

Introduction: Severe “sicca syndrome” after haemopoietic stem cell transplantation impairs the quality of life of transplanted patients and no valid therapeutic options are available. Autologous serum enriched with PDGFs is a new therapeutic option because of its high content in different growth factors and fibronectin able to restore the epithelial integrity of cornea and conjunctive.

Herein we present our experience on 16 allotransplanted patients (pts) undergoing extracorporeal photochemotherapy treatment for drug resistant chronic GvHD associated with severe eye dryness. All pts were treated with autologous serum eye drops prepared in our laboratory under gmp conditions.

Material and Methods: A peripheral blood sample of 30 ml was obtained from each pt in sterile conditions and centrifuged at 1500 rpm per 15 min. Serum was diluted with sterile saline to obtain a final concentration of 30%. No antibiotic was added. The solution was aliquoted in 1 ml plastic tubes (with the purpose of single use) and freezed at −20°C. The maximum storage time allowed was 30 days. At time of preparation PDGFs (PDGF, TGF b1, VEGF, EGF, IGF) content was assessed by ELISA technique and bacterial and fungal contamination was tested.

16 pts (11 M, 5 F), median age 41 yrs (range: 14–61), median plt count 170x103/mL (range: 110–530), median Hct% 36 (32–46) were enrolled and instructed to instill two autologous serum eye drops per eye four times/day. A written informed consent was always obtained. Schirmer’s test was performed immediately before treatment and bi-monthly. The median follow up was 12,5 months (4–16).

Results: 14/16 pts experienced a significative dry eye improvement that was mantained during the course of treatment. The need of topical therapy diminished progressively till two drops per eye once a day. The mean Schirmer’s test value improved from 1.5 to 4.6 mm/5 mins during the follow up.

Neither microbial contamination on the samples tested nor oftalmic infectious episodes were documented.

Conclusions: Autologous serum eye drops demonstrated to be a valid, safe and low cost new therapeutic option for treatment of “sicca syndrome” associated with chronic GVHD. Preparation under gmp conditions, storage in single use vials and detailed instructions to the patient demonstrated to be an effective approach to avoid the addition of antibiotic and microbial contamination of the solution.
Predicting Steroid Refractroy Acute Gastrointestinal (GI) Acute Graft Versus Host Disease (AGVHD) by Assessment of Crypt Loss at Diagnosis.
Authors: Melson Joshua , Keshavarzian Ali , Jakate Shriram , Arai Sally , Gregory Stephanie A. , Fung Henry C.
Published: October 2019

Acute graft versus host disease accounts for most of the treatment failures after an allogeneic stem cell transplantation. The prognosis of patients with steroid refractory acute graft versus host disease is poor with conventional treatment. Early identification of patients with steroid refractory acute graft versus host disease may improve the transplant outcomes. Crypt loss has been associated with the severity of disease in patients with inflammatory bowel disease. It is also found in patients with acute gastrointestinal graft versus host disease with unknown clinical significance. In an attempt to evaluate the significance of crypt loss in patients with acute gastrointestinal graft versus host disease, we conducted a retrospective study on 22 consecutive post allogeneic stem cell transplant patients in our institution that underwent colonoscopy and colonic biopsy. In this study, crypt loss was graded and correlated with clinical parameters of disease severity including stool volume, endoscopic appearance, response to graft versus host disease treatment and one year mortality attributed to graft versus host disease. All the biopsies were reviewed by one of the co-authors (Shriram Jakate, M.D.). Twenty two consecutive patients who were treated for gastrointestinal graft versus host disease following stem cell transplantation were studied. Crypt loss was present in 16/22 cases and in 10/22 cases crypt loss was determined to be severe by the presence of contiguous areas of crypt loss. In those with severe crypt loss 9/10 patients had daily stool volumes in excess of 1000 ml/day while only 3/6 in those with minimal crypt loss had this level of severe diarrhea. Patients with severe crypt loss were more likely to have a pathologic appearance at endoscopy and to require second-line therapy (i.e. steroid refractory acute graft versus host disease). The diarrhea of those with severe crypt loss was less likely to resolve even though it was more aggressively treated. Of the 10 patients with severe crypt loss, 5 (50%) died of graft versus host disease related causes. Conversely, only one of 12 patients (8%) with mild or no crypt loss had a death attributable to acute graft versus host disease. Our study shows that severe crypt loss predicts for severity of acute gastrointestinal graft versus host disease, response to treatment and more importantly steroid refractoriness. In addition, crypt loss severity is associated with an increased risk of death related to acute graft versus host disease. We conclude that the assessment of crypt loss at diagnosis may serve as a tool to identify patients with steroid refractory acute gastrointestinal graft versus host disease and could be used to select patients for therapeutic trial in acute graft versus host disease.