bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Oral Administration of KRN383, a Novel Flt3 Inhibitor, Induces Eradication of the Xenograft Tumor Harboring Flt3 Mutation in Mice.
Authors: Nishiyama Uichi , Ozai Masako , Yoshioka Rieko , Yoshino Tetsuya , Ogasawara Yuko , Kitahori Miyuki , Yoshioka Eiji , Kubo Kazuo , Komeno Yukiko , Kurokawa Mineo , Ogawa Seishi , Chiba Shigeru , Osawa Tatsushi , Kuwaki Tomoaki , Hirai Hisamaru , Miwa Atsushi
Published: April 2020

Constitutively activating mutations of the Flt3 receptor tyrosine kinase including internal tandem duplication (ITD) mutations are the most common genetic abnormality and are associated with a poor prognosis in acute myeloid leukemia (AML) patients. Therefore, Flt3 is a potential therapeutic target in AML. We show here the results of pre-clinical studies on KRN383, a novel orally active quinoline-urea derivative.

KRN383 inhibited autophosphorylation of ITD mutant (IC50=1.3nM) and wild type (IC50=0.4nM) Flt3 in the leukemia cell line MV4-11 and THP-1, respectively. KRN383 induced cell cycle arrest, apoptosis and suppression of proliferation (IC50=0.8nM) of MV4-11 in vitro. Single (80mg/kg) or consecutive (20mg/kg/d X 28d) oral administration of KRN383 induced eradication (longer than 6mo) of tumor xenograft (MV4-11) subcutaneously implanted in all nude mice. All these effects were superior to those of SU11248, a precedent Flt3 inhibitor. In addition, single (80mg/kg) administration of KRN383 prolonged the survival of SCID mice to which MOLM-13 (ITD mutant positive leukemia cell line) was intravenously transplanted. The advantage of KRN383 over SU11248 was more obvious in in vitro studies in which the inhibition of Flt3 autophosphorylation and cellular proliferation induced by transient exposure with those drugs was determined. These results indicate that KRN383 has therapeutic potential in ITD mutant positive AML. Furthermore, eradication induced by single administration of KRN383 suggests that KRN383 provides feasibility to set wide variety of clinical regimens including multi-cycle and combination therapies. In vitro activity of Ki23819, hydrochloride salt of KRN383, is also to be reported in this ASH meeting (Komeno et al.).
PTPN11 Mutations E76K, D61V, and D61Y Induce Hypersensitivity of Hematopoietic Progenitors to GM-CSF and IL-3.
Authors: Chan Rebecca J. , Li Yanjun , Leedy Melissa B.
Published: April 2020

The childhood leukemia, juvenile myelomonocytic leukemia (JMML), is a lethal disease of young children characterized by spontaneous growth of peripheral blood hematopoietic progenitors and hypersensitivity of hematopoietic progenitors to the cytokine GM-CSF. Notably, hematopoietic progenitors from JMML patients do not typically demonstrate hypersensitivity to the cytokine IL-3. Mutations in the RAS and NF1 genes have long been recognized as pathogenic in this disease, and more recently, mutations in the PTPN11 gene, which encodes Shp-2, a non-receptor protein tyrosine phosphatase, have also been found commonly in leukemic cells from children with JMML. We hypothesized that the mutant Shp-2 molecules observed in JMML patients would induce hypersensitivity of hematopoietic progenitors to GM-CSF. To examine this hypothesis, we subcloned the WT Shp-2 cDNA and three mutant Shp-2 cDNAs found in leukemic cells from children with JMML (E76K, D61V, and D61Y), into the murine stem cell retroviral plasmid pMIEG3 in tandem with EGFP. Each vector was sequenced to verify the desired point mutation and to rule-out unwanted mutations. Murine bone marrow low density mononuclear cells were subjected to fibronectin-assisted retroviral transduction and sorted for EGFP positive cells using fluorescence activated cell sorting. EGFP positive cells were plated into progenitor assays with increasing concentrations of GM-CSF (0, 0.01 0.1, 1, and 10 ng/mL). As predicted, transduction with each of the Shp-2 mutations induced hypersensitivity to GM-CSF compared to vector alone or to WT Shp-2, as evidenced by significantly higher % maximal colony formation at each GM-CSF dose tested (Figure 1).

We next conducted progenitor assays using EGFP positive cells with increasing concentrations of IL-3 (0. 0.01, 0.1, 1, and 10 U/mL). Although hematopoietic progenitors from JMML patients have not been described to be hypersensitive to IL-3, surprisingly, we have observed in preliminary studies that introduction of each of the Shp-2 mutations induced hypersensitivity to IL-3 in a similar fashion to that observed with GM-CSF (Figure 2).

These data demonstrate that somatic PTPN11 mutations observed in children with JMML induce hematopoietic cell growth aberrancies in a manner overlapping with the classical description of JMML, yet also induce unique hematopoietic cell growth characteristics based on the observed hypersensitivity to IL-3. These findings suggest that signaling pathways in addition to the Ras-MAPK cascade may be dysregulated in PTPN11 mutation-bearing hematopoietic cells and provide a novel model for the investigation of new therapeutics in JMML. Current studies are ongoing to examine the effect of these mutations on in vivo hematopoiesis and leukemogenesis.
Oligomerization of Evi-1 Contributes to Recruitment of Transcriptional Corepressor CtBP and Repression of TGF-β Signaling.
Authors: Nitta Eriko , Izutsu Koji , Yamaguchi Yuko , Imai Yoichi , Ogawa Seishi , Chiba Shigeru , Kurokawa Mineo , Hirai Hisamaru
Published: April 2020

Aberrant oligomerization of transcription factors has recently emerged as a prevalent mechanism for activating their oncogenic potential in hematopoietic malignancies. Furthermore, for some chimeric transcription factors generated in leukemia-associated chromosomal translocations including PML-RARα and AML1-ETO, it has also been proposed that aberrant recruitment of corepressor complexes caused by oligomerization is critical for the development of leukemia. Among leukemia-associated transcription factors is Evi-1, which is frequently activated in cases with myeloid malignancies. Evi-1a and Evi-1c, with the latter also called MDS1-Evi-1, are two alternative forms derived from the Evi-1 gene. Evi-1a is a Zn finger transcription factor which recruits CtBP as a transcriptional corepressor and possesses the ability to repress TGF-β signaling. Amino-terminal to the Evi-1a, Evi-1c harbors an additional sequence similar to the PR domain, which was originally identified in PRDI-BF1 and RIZ1. In contrast to frequent upregulation of Evi-1a in myeloid malignancies, expression of Evi-1c widely varies depending on cases. Thus, although several lines of evidence suggest that Evi-1a is involved in leukemic transformation of hematopoietic cells, a role for Evi-1c in leukemogenesis has remained elusive. To elucidate the mechanism that underlies leukemogenesis by Evi-1, we investigated the ability of Evi-1 proteins to form oligomeric complexes. We found that Evi-1a forms a homo-oligomer in mammalian cells, whereas Evi-1c exclusively exists as a monomer. Remarkably, Evi-1c has lost the ability to interact with CtBP and failed to efficiently repress TGF-β signaling. We previously reported that AML1-Evi-1, a chimeric transcription factor generated in t(3;21) leukemia, interacts with CtBP as Evi-1a does. In the current study, we also found that AML1-Evi-1 forms a homo-oligomer, suggesting a close relationship between oligomer formation and CtBP binding. Taken together, these data indicate that homo-oligomerization may contribute to the oncogenic potential of the Evi-1 proteins by regulating the interaction with CtBP. These results also identify a novel function of PR domain to dictate oligomerization of transcription factors.
Effect of G-CSF and Intramyocardial Injection of Bone Marrow Cells on Cardiac Function in Mouse Model of Myocardial Infarction: A Word for Neo-Vasculogenesis.
Authors: Verda Larissa , Luo Kehuan , Han Xiaoqiang , Wasserstrom Andrew , Lomasney Jon , Kowalska Barbara , Burt Richard K.
Published: April 2020

Recent studies suggest that primitive stem cells derived from bone marrow (BM) possess greater functional plasticity that was expected previously. It has been shown that bone marrow stem cells (BMSC) promote repairing mechanisms within myocardium following ischemia/reperfusion models of myocardial infarction (MI). Although it remains unclear whether BMSC transdifferentiate into or just fuse with cardiomyocytes, hemodynamic improvement after intramyocardial BMSC injection as well as after G-CSF injection has been demonstrated. Here, we investigated the contribution of BMSC versus G-CSF administration in myocardial repair following MI. Ten weeks old C57BL/6J mice were irradiated and transplanted with green fluorescent protein (GFP) positive bone marrow cells. Three months later, these mice underwent ligation of left anterior descending branch (LAD) of coronary artery and subsequently divided into three groups. One group (n=7) received G-CSF administration at 200ug/kg for 10 consecutive days. Another group (n=9) was injected with GFP+ marrow cells directly into ischemic heart. The third group was held as control (n=7). One month after coronary ligation we found significant improvement in cardiac function determined as a cardiac output, maximum power and dP/dt, in the G-CSF group compared to control. We evaluated the phenotype of GFP+ cells within myocardium in each treatment group by 488 nm laser-scanning confocal miscroscopy (of whole heart and slides) 35 days after LAD ligation. We found no evidence of myocardial transdifferentiation or cardiomyocyte cell fusion. Instead GFP+ capillaries were present and exclusively located in infarct border zones in both the G-CSF and bone marrow implantation groups, confirmed by anti-factor VIII staining. G-CSF administration and to a lesser extent marrow injection resulted in improved post infarct cardiac function indices. This beneficial effect is not due to transdifferentiation but could be explained by marrow injected or G-CSF mobilized endothelial progenitor cells (EPC) and/or cytokine mediated neo-vasculogenesis.
Evidence That CXCR4+ Neural Tissue-Committed Stem Cells (TCSC) Reside/Hide out in the Bone Marrow and Are Mobilized into the Peripheral Blood during Stroke.
Authors: Kucia Magda , Ping Zhang Y. , Ratajczak Janina , Ildstad Suzanne T. , Shields Chris , Ratajczak Mariusz Z.
Published: April 2020

Several recent studies in animals as well as humans support the notion that bone marrow (BM)-derived cells participate in brain regeneration. However, the identity of the specific cell type responsible for regeneration remains unknown. Recent work from our laboratory revealed that BM contains a highly mobile population of CXCR4+ cells that express mRNA for various markers of early tissue-committed stem cells (TCSC) and which are distinct from hematopoietic stem cells (HSC) (Leukemia2004:18;29–40). In this study we investigated whether BM also contains a mobile pool of TCSC destined to differentiate into neural cells. The TCSC were isolated from bone marrow by employing chemotactic gradient to SDF-1 or by FACS sorting and subsequently evaluated for a presence of early neural markers by i) real time RT-PCR analysis (nestin, GFAP), ii) immunohistochemical staining (nestin, beta-III tubulin), and iii) by employing functional in vitro assays to study ability of these purified cells to form neurospheres. Our data demonstrate that TCSC for neural cells (i) are present in significant amounts in BM harvested from young (1–2 month-old) while being barely detectable in older (1-year-old) mice; ii) reside in populations of murine BM-derived non-adherent non-hematopoietic Sca-1+ CD45− cells and in population of human CXCR4+ CD34+ AC133+ CD45− BMMNC, iii) are mobilized from BM into peripheral blood (PB) during pharmacological mobilization or 24 hours after Bengal-rose induced stroke in mice; iv) SDF-1 is highly upregulated in damaged brain tissue, and v) TCSC are chemoattracted for potential brain regeneration in SDF-1-CXCR4, dependent manner. Thus, we conclude that bone marrow is a potential source of TCSC for brain repair and since purified CD45+ HSC neither express neuronal markers nor differentiate in vitro into neurospheres, we provide for a first time evidence that neural TCSC residing in bone marrow but not “plastic” HSC account for neural differentiation of BM-derived cells. Furthermore, our observation that the number of marrow derived mobile/circulating neural TCSC is the highest in BM of young animals and decreases with age provides a novel insight into aging and may explain why the brain regeneration process becomes less effective in older individuals. Finally, these observations provide rationale for further studies aimed at optimizing therapeutic brain regeneration by BM-derived neural TCSC.
Rapid Risk-Profiling Including Fast Donor Search within a Multi-Center Trial Allows for Early Allogeneic Stem Cell Transplantation during Aplasia in Patients with High-Risk Acute Myeloid Leukemia.
Authors: Schaich Markus Andreas , Fuessel Monika , Bornhaeuser Martin , Thiede Christian , Mohr Brigitte , Illmer Thomas , Schaekel Ulrike , Prange-Krex Gabriele , Oelschlaegel Uta , Ehninger Gerhard ,   on behalf of the DSIL AML2003 study group
Published: April 2020

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment option for most high-risk patients with acute myeloid leukemia (AML). However, many high-risk patients regenerate with blasts or relapse early after induction therapy. Thus, consolidation with allogeneic HSCT in first CR is often not possible. Performing allogeneic HSCT within induction therapy can circumvent these problems and may furthermore reduce cumulative toxicity in high risk patients.

Therefore, the prospective randomized treatment trial AML2003 for patients <= 60 years was set up, to investigate the feasibility and value of an intensified treatment strategy, i.e. early allogeneic stem cell transplantation in aplasia after induction therapy, for high risk AML patients in a multi-center setting. To achieve this goal, rapid analysis of cytogenetics, FLT3 status and HLA-types of the patient and possible family donors is of utmost importance. This fast search diagnostics together with routine analyses of morphology and immunophenotyping is accomplished centrally in all enclosed patients. Furthermore, in all patients the likelihood to find an unrelated donor is checked by an internet search in the BMDW database.

Within the first 8 months 107 AML patients with a median age of 48 (17–60) years were included in the study. Fast search diagnostics was complete within a median of 15 (range 5–31) days after arrival of the bone marrow samples for all patients. 57/107 patients were randomized into the intensified treatment arms. Out of these 25 (44%) patients with high risk characteristics have been identified. A suitable related or unrelated donor was found for 22 (88%) of those high-risk patients. Nine of those high risk patients with a donor (41%) received early allogeneic stem cell transplantation in aplasia after the first (n=4) or the second (n=5) induction therapy course on an intend to treat basis within the protocol. Three were transplanted with stem cells of related and six of unrelated donors. The preparative regimen consisted of melphalan 150mg/m2 and fludarabine 150mg/m2. So far no treatment associated death had to be recognized.

These encouraging preliminary results show that fast risk-profiling and early donor-search is feasible in a large multi-center study. This leads to a significant proportion of early allogeneic stem cell transplants in aplasia after induction therapy within the group of high risk AML patients, which may improve the disastrous prognosis of this group of patients in the future.
Identification of the Genomic Breakpoints at 1p36.3 and 3q21.3 in Five MDS/AML(M4) Patients with t(1;3)(p36;q21) Translocation. GATA2 Mutation May Be Involved in Leukemogenesis.
Authors: Xinh Phan Thi , Vu Hoang Anh , Taketazu Fumitoshi , Fujisawa Shin , Tanaka Hideo , Yagasaki Fumiharu , Toyoda Atsushi , Sakaki Yoshiyuki , Rowley Janet D. , Tokunaga Katsushi , Sato Yuko
Published: April 2020

Reciprocal translocation t(1;3)(p36;q21) is found in a subset of MDS/AML characterized by trilineage dysplasia, especially dysmegakaryopoiesis and a poor prognosis. In this leukemia, ectopic expression of MEL1 at 1p36.3 driven by the promoter of RPN1 at 3q21 is thought to be associated with the pathogenesis. Recently, we reported the identification of 2 kinds of the 1p36 breakpoint (BP) region in 3 patients with t(1;3), 1) a 70.7-kb region within the first intron of MEL1 in one patient, and 2) a 29.2-kb region in the 5′ region of MEL1 in the remaining two. However, the precise BP at 3q21 has not yet been determined. Here, we report that we have defined the 3q21 BP in 4 patients, and the 1p36.3 BP in 2 additional patients with FISH using bone marrow cells obtained from 4 patients (4 with MDS-related AML-M4 and one with CMML). To identify the BPs and deletions at 1p36.3 or at 3q21.3, we used 18 non-contig BAC/PAC probes and 7 contig BAC/PAC probes, respectively. FISH study revealed that the 3q21 BPs were clustered within RP11-475N22 probe in the 4 patients, in the 5′ region of GATA2. The 1p36.3 BP was found within the first intron of MEL1 in one patient, but in a 68.5-kb region which included a part of the first intron and a region of 5′ site of MEL1 in the other. Therefore, 3 BP regions at 1p36 were found among the 5 patients. No deletions were detected in the vicinity of the 1p36 BP or 3q21 BP in any of our 5 patients, although Lahortiga et al., detected a large deletion at the both BPs in their patients with MDS (RAEB) and t(1;3) (Oncogene 23:311–316, 2004). In Patient 1 (the 1p36 BP was in the first intron of MEL1) and Patient 3 (the 1p36 BP was in 5′ site of MEL1), the expression patterns of MEL1, ARPM2, and GATA2 which are located near the BPs were analyzed with RT-PCR. ARPM2 was not overexpressed in the two patients, while MEL1 was overexpressed in Patient 1, but not in Patient 3 when the expression level was compared with that of normal bone marrow cells. It is reported that GATA2, one of the zinc finger transcription factors, serves as a regulator of genes controlling proliferative capacity of early hematopoietic cells, and that two zinc finger domains of GATA2 are closely involved in inducing megakaryocytic differentiation. Interestingly, a deleted GATA2 transcript was detected in Patient 3 together with a wild type GATA2 transcript. The short GATA2 transcript contained 1009 nucleotides deletion from exon 3 to exon 6 with a frameshift, resulting in lacking the two zinc finger domains. Thus, there is the possibility that dysmegakaryopoiesis in this patient is associated with the expression of GATA2 without the zinc finger domains.
Improved Heart Function after the Combined Treatment of Patients Undergoing Coronary Artery Bypass Grafting through Stem Cell Therapy and Transmyocardial Laser Revascularisation.
Authors: Stockschlaeder Marcus , Ghodsizad Ali , Gams Emmeran , Poll L. , Stoldt Volker , Piechaczek E. R. , Feifel N. , Klein Michael , Scharf Ruediger E.
Published: April 2020

To improve tissue regeneration of ischemic myocardium, autologous bone marrow-derived stem cells have been injected intramyocardially in five patients undergoing coronary artery bypass grafting and transmyocardial laser revascularization. We have established an innovative method for the intraoperative isolation of CD133+-stem cells in less than 3 hours. After induction of general anesthesia, approx. 60 – 240 ml of bone marrow were harvested from the posterior iliac crest and processed in the operating room under GMP conditions using the automated cell selection device CliniMACS. Following standard coronary artery bypass grafting, laser channels were shot in predefined areas within the hibernating myocardium. Subsequently, autologous CD133+-stem cells (1.9–9.7 x 106 cells; purity up to 97%) were injected in a predefined pattern around the laser channels (0.5–1.0x106 cells/channel). Three months postoperatively, significant improvement of cardiac function as assessed by transthoracal echocardiography (ejection fraction before treatment: 25–30% - after treatment: 38–46%; left ventricular enddiastolic volume before treatment: 58 – 64 mm - after treatment: 40–44 mm; endsystolic wall thickness before treatment: 5–7 mm - after treatment 9–13 mm) and improvement of cardiac wall motion (electrocardiographically-triggered magnetic resonance imaging) were documented. This significant gain in heart function might be partly explained through the synergistic angiogenic effect of transmyocardial laser revascularization leading to a localized inflammatory response and the cellular effects of intramyocardially injected pluripotent bone marrow-derived stem cells promoting tissue repair. Through the intraoperative isolation of CD133+-cells, this effective treatment of ischemic myocardium can be applied to patients scheduled both for elective and for emergency revascularisation procedures.
Validation Study on the Prediction of Response to Imatinib Mesylate in Chronic Myeloid Leukemia (CML) Patients by Genome-Wide cDNA Microarray Analysis.
Authors: Kagami Yoshitoyo , Katagiri Toyomasa , Kaneta Yasuyuki , Tsuruo Takasi , Nakamura Yusuke , Ohno Ryuzou
Published: April 2020

Imatinib mesylate (IM) is highly effective in the first line therapy of CML. However, it remains unknown whether IM cures CML or not, and other therapeutic modalities like stem cell transplantation sometimes become necessary. Predicting the therapeutic effect of IM before the start of therapy would be useful, because other kinds of therapy could be planed during IM treatment. Previously, in order to predict the effect of IM therapy in CML, we established a prediction system by cDNA microarray containing 23,040 genes. According to the profiling data of RNA expression of peripheral blood mononuclear cells in 22 CML patients, who had previously failed in interferon-α therapy, 79 genes were identified that were expressed differentially between responders and non-responders to IM. By a prediction scoring system, 15 out of the 79 genes were selected which discriminated the two populations most clearly.(Kaneta Y et al. Jpn J Cancer Res 93, 849–856,2002) This time we applied this scoring system to another cohort of chronic or accerelated phase of CML patients who would receive IM as monotherapy. So far, 42 patients were analyzed. Of 40 patients who attained major cytogenetic response (MCR) by 6 months from the start of therapy, 38 (95%) had been predicted as the responder by the prediction system. On the other hand, two patients whose effect was less than MCR had been predicted as the non-responder. These data indicate that our prediction system for IM using cDNA microarray could effectively discriminate responders from non-responders before the start of therapy.
The Antileukemic Effect of NK and NK-T Cells Against Chronic Myelogenous Leukemia Is Dominated by Interactions between Activating Molecules and Their Ligands Which Overcome Inhibitory KIR-MHC Class I Interaction.
Authors: Sconocchia Giuseppe , Lau Michelle , Provenzano Maurizio , Rezvani Katayoun , Fujiwara Hiroshi , Ferrone Soldano , Barrett John
Published: April 2020

We compared the interactions of autologous, HLA-identical and mismatched CD56+ cells with chronic myelogenous leukemia (CML) CD34+ cells. In 14 donor-recipient pairs, HLA-matched donor CD56+ cells inhibited CML CFU-GM at an E/T ratio of 10:1 (mean inhibition 42±9%). This was comparable to the 39.5±7% inhibition observed with 14 HLA-mismatched unrelated CD56+ cell donors, indicating that KIR incompatibility was not required for the antileukemic effect. Both CD56+CD3- (NK) and CD56+CD3+(NK-T) subsets inhibited colony formation of CD34+ CML cells but not normal CD34+ cells from the stem cell donor, suggesting that CD56+ cells recognized surface molecules specific to CML cells. Using indirect immunofluorescence to detect NKG2D ligands, 11/12 CML CD34+ samples were found to be MICA/B+ positive, while normal CD34 cells were uniformly negative. Five of six MICA/B+ CML samples bound a NKG2D/FC+ chimeric molecule indicating the presence of functional ligands. CML CFU-GM inhibition was partially blocked by anti-NKG2D suggesting that NKG2D-MICA/B interactions contributed to the antileukemic activity of NK and NK-T cells. Colony formation of MICA/B+ CML cells was also inhibited by caspase-blocked HLA matched donor CD56+ NK cells indicating that the effect was not FAS-dependent. However, soluble MICA/B was detected in patient serum by immunoprecipitation and CML patient serum blocked anti-MICA/B binding to CML cells, suggesting that in vivo anti-leukemic activity of CD56+ cells is diminished. Furthermore NKG2D expression on CD3+ cells was found to be low in 3 of 4 CML patients, which was accompanied by a reduced IL-10 production upon NKG2D ligation by an anti-NKG2D moAb. These studies confirm early observations of KIR-independent antileukemic cytotoxicity by autologous and HLA matched NK cells, implicating pro-cytotoxic signaling via NKG2D and its ligand MICA/B. They also reveal mechanisms whereby CML cells may escape from NK regulation in vivo.
hTERT, the Catalytic Component of Telomerase, Is Down-Regulated in the Hematopoietic Stem Cells of Patients with Chronic Myeloid Leukemia.
Authors: Campbell Lisa J. , Eagleton Helen , Littlewood Tim J. , Peniket Andy , Kusec Rajko , Gal Shira , Fidler Carrie , Wainscoat James S. , Boutlwood Jacqueline
Published: April 2020

Telomere shortening and increased telomerase activity is associated with disease progression in chronic myeloid leukemia (CML). In order to gain insight into the biology of telomerase and its regulation in CML we have evaluated the expression levels of the components of telomerase, its regulators, and several telomeric associated proteins in the hematopoietic stem cells (HSC)/leukemic blast cells of 21 patients with this disorder, as compared to the HSC population of a group of healthy individuals, using quantitative real time PCR. In this study we have made the surprising observation that hTERT, the catalytic component of telomerase, is down-regulated in the CD34+/blast cells of the majority of CML patients in chronic phase (CP) (7 out of 9, p=0.0339) relative to CD34+ cells from normal controls. Furthermore hTERT was found to be significantly down-regulated in 2 out of 3 patients in accelerated phase (AP) and in 9 out of 9 patients in blast crisis (BC) relative to normal controls (p=0.0003). The expression levels of hTR, the telomerase RNA template, and the telomeric- associated proteins TEP1, TRF1, TRF2 and tankyrase were all up-regulated in the CD34+/blast cells of the majority of the CML patients in CP and AP, relative to normal control CD34+ cells. With the exception of TEP1, the expression levels of all these factors were highest in CP CML and, in the majority of patients, decreased during disease progression. We speculate that the up-regulation of tankyrase (positive regulator of telomere length) and TRF2 (protects critically shortened telomeres) may contribute to telomere maintenance in CML. The expression levels of hTERT and c-myc, a positive regulator of hTERT expression, correlate in all stages of CML (R2=0.623). The expression levels of c-myc decreased with disease progression, falling below the levels observed in normal CD34+ cells in the majority of patients in BC. The expression levels of hTERT were increased in the CD34+ cells of three additional CP CML patients in remission following successful treatment with imatinib, relative to both untreated CP CML patients (p=0.0126) and to normal individuals, reflecting a restoration of haematopoiesis towards normal. In CML the turnover of all types of Ph+ stem and progenitor cells is known to be increased. We suggest that the reduced (and presumably inadequate) expression levels of hTERT in CML CD34+/blast cells, and the consequent telomere shortening with each division, is the direct cause of the shortened telomeres frequently observed in CML patients. These data challenge the widely accepted view that hTERT expression is always increased in tumorigenesis. It is possible that the Bcr-Abl fusion product may play a role in the down-regulation of hTERT observed in the HSC of CML patients in this study.
The DCC Heterozygote Reduces the Latency Period and Changes the Disease Phenotype of Myeloproliferative Disease in p230BCR/ABL-Expressing Mice.
Authors: Inokuchi Koiti , Inami Mituharu , Wang Yazhu , Watanabe Ayako , Yamaguchi Hiroki , Tanosaki Sakae , Shimada Takashi , Dan Kazuo
Published: April 2020

Chronic neutrophilic leukemia (CNL) is a distinct disease form with less aggressive phenotype mimicking chronic myelogenous leukemia (CML). CNL sometimes has a unique p230BCR/ABL gene abnormality, as does Philadelphia-positive myeloproliferative disease (MPD) mimicking essential thrombocythemia (Ph+ET). The reason of the phenotypic difference between CNL and Ph+ET remains unclear. Recently, we have generated the p230BCR/ABL-transgenic mice, whose disease phenotype is MPD mimicking ET, to study the disease phenotype. In studies of human leukemia cells, loss of DCC (deleted colorectal carcinoma) gene expression is reported in about 20% of CML cases.

To investigate whether loss of DCC expression plays a role in the disease phenotype of p230BCR/ABL-expressing MPD, we crossmated p230BCR/ABL-transgenic (BCR/ABLTg/−) mice with DCC-heterogenous (DCC+/−) mice, thereby generating p230BCR/ABL-transgenic, DCC-heterogenous (BCR/ABLTg/− DCC+/−) mice.

BCR/ABLTg/− DCC+/− mice have a short latency (9 to 12 months) compared with BCR/ABLTg/− DCC+/+ mice (17 to 20 months) for developing overt MPD. Less thrombocytosis (91–140x104/mm3) was found in the BCR/ABLTg/− DCC+/− transgenic littermates compared with the BCR/ABLTg/− DCC+/+ transgenic littermates (115–170 x 104/mm3). Leukocytosis and the percentage of granulocytes in BCR/ABLTg/− DCC+/− mice were greatly increased compared with BCR/ABLTg/− DCC+/+ transgenic littermates (6.1–13.5 x 103/mm3 vs 5.0–11.2 x 103/mm3; 42–68 % vs 23–49 %). However, blasts, which were detected in the blastic crisis, were not found in the peripheral blood of either mouse line.

Thus, the DCC-heterozygote, in cooperation with p230BCR/ABL cDNA, shortened the latency period of overt MPD, and changed the disease phenotype from MPD mimicking ET to MPD mimicking CNL. However, pathological analysis of the bone marrow, spleen, liver, etc. did not reveal any difference between the BCR/ABLTg/− DCC+/− transgenic littermates and the BCR/ABLTg/− DCC+/+ transgenic littermates.
Promoter Methylation of RASSFIA, SHP1 and SOCS1 Genes in Acute Myeloid Leukaemia (AML) and Myelodysplastic Syndromes (MDS).
Authors: Johan M. Farid , Goodeve Anne C. , Bowen David T. , Reilly John T.
Published: April 2020

Mutations in the receptor tyrosine kinase (RTK)/RAS signalling pathway frequently provide a proliferative signal in AML, especially those with inv(16) and t(15;17). We have reported, for example, that approximately 70% of AML and inv(16) have mutations in FLT3, c-KIT and RAS. However, the role of RASSFIA, SHP1 and SOCS1, negative regulators of RTK/RAS signalling, has not been extensively investigated in AML. RASSFIA is a tumour suppressor gene that serves as a RAS effector that inhibits cell cycle progression and mediates apoptotic cell death, while SHP1 and SOCS1 are negative regulators of the Jak/STAT pathway. In many cancers, the CpG islands of RASSFIA, SHP1 and SOCS1 are aberrantly hypermethylated, resulting in gene inactivation due to repression of transcription. In this study, methylation-specific polymerase chain reaction (MS-PCR) was employed to determine if aberrant promoter methylation of these negative regulators are involved in the pathogenesis of AML and MDS. Patients with MDS (n=107), AML (n=154), JMML (n=5) were studied, together with 15 normal controls. Primers were located in the promoter region of each gene as well as within exon 2 of SOCS1 as previously reported. Methylation of RASSFIA was found in five of 55 (11%) MDS patients, but in none of 57 AML cases. RASSFIA methylation was present in one case (20%) of JMML, which lacked a RAS, NF1 or PTPN11 mutation, a finding that strengthens the concept of mutual exclusivity of genetic and epigenetic changes in this disorder. SHP1 methylation was identified in 13 of 121 (11%) AML cases, including 3 AML with inv(16) and c-KIT 419 mutations. In contrast, none of the 65 MDS or 5 JMML cases showed SHP1 methylation. MS-PCR for SOCS1 promoter and exon 2 gave discrepant results. Promoter analysis was negative in the 15 controls whereas 2 controls showed exon 2 positivity. SOCS1 promoter methylation was seen in eight of 74 (11%) MDS patients, but not in the five JMML cases. Methylation of RASSFIA and SOCS1 promoters in MDS were mutually exclusive. No case of AML showed SOCS1 promoter methylation whereas 19 of 47 (40%) cases exhibited exon 2 methylation. In conclusion, promoter methylation may lead to epigenetic silencing of RASSFIA and SOCS1 in MDS but not in AML, whereas SHP1 gene silencing may be important in the pathogenesis of AML. SHP1 methylation studies suggest that some patients with inv(16) AML and RTK mutations possess additional epigenetic events. Finally, SOCS1 exon 2 methylation does not appear to be pathogenetically important, since methylation may occur in normal samples and results do not correlate with promoter methylation.
Prevention of Murine Acute Graft-versus-Host Disease by Recipient-Derived TGFβ1-Treated Dendritic Cells.
Authors: Mou Haibo , Lin Maofang , Cen Hong , Huang He , Cai Zhen
Published: April 2020

Acute graft-versus-host disease (GVHD) remains the major barrier of allogeneic bone marrow transplantation (allo-BMT). In the current view, GVHD fundamentally depends on donor T cells interaction with host derived dendritic cells (DC) leading to their activation, proliferation, and differentiation. We and others have demonstrated that transforming growth factor β1 (TGF-β1) treated DC (TGFβ-DC) have regulatory characteristic and could induce allogeneic specific immune tolerance in vitro. In the current study, we focused on the effects of recipient-derived TGFβ-DC in a murine GVHD model. After total body irradiation, 5.0×106 recipient-derived TGFβ-DC were injected into C57BL/6 (H-2b) with bone marrow-splenocyte grafts from major histocompatibility complex (MHC) disparate BALB/c mice (H-2d). Survival analysis showed TGFβ-DC co-transplantation resulted in significant prolongation of allograft survival and mean survival time (MST) was 44.3±4.5 days (p<0.01), whereas MST of untreated BMT recipient was 9.5±0.6 days (p<0.01). However, mature DC aggravated the GVHD and the MST was 6.6±0.6 days (p<0.01). In addition, donor-derived or the third party-C3H mice derived TGFβ-DC could not enhance the survival rate indicating that the protection effect of TGFβ-DC was recipient-specific. Consistent with clinical outcomes, the histopathologic studies showed no or mild GVHD evidences were found in recipient-derived TGFB-DC co-transplantation mice, while specific severe GVHD alternations occurred in the liver, skin and intestine in other groups. Flow cytometry analysis of TGFβ-DC labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed approximately 5% of TGFβ-DC in spleen mononuclear cells the next day after transplantation and half-life of the injected TGFβ-DC was around 20 days after transplantation. Furthermore, serum Th1 type cytokines of IFN-γ, IL-12 and IL-18 levels in TGFβ-DC co-transplantation mouse reduced compared with untreated BMT mouse, while serum IL-10 level was not changed. Taken together, these preliminary results indicate that TGFβ-DC co-transplantation may induce MHC-specific tolerance and attenuate the severity of GVHD by suppressing Th1 responses. Further research should be carried out to explore the optimal transplantation regimen and elucidate the mechanism.
Cologne High-Dose Sequential Chemotherapy in Relapsed and Refractory Hodgkin Lymphoma - Results of a Large Multicenter Study of the German Hodgkin Lymphoma Study Group (GHSG).
Authors: Josting Andreas , Rudolph Christian , Mapara Markus , Glossmann Jan-Peter , Sieber Markus , Kirchner Hartmut H. , Dörken Bernd , Hossfeld Dieter K. , Kisro Jens , Metzner Bernd , Berdel Wolfgang E. , Diehl Volker , Engert Andreas
Published: April 2020

Purpose: Combination chemotherapy can cure patients (pts) with Hodgkin lymphoma (HD), but those with treatment failure or relapse still have a poor prognosis. We thus, designed a dose- and time-intensified high-dose sequential chemotherapy regimen with a final myeloablative course.

Patients and Methods: Eligibility criteria included age 18–65 years, histologically proven primary progressive (PD) or relapsed HD. Treatment consists of two cycles DHAP (dexamethasone 40mg d1-4, high-dose cytarabin 2g/m2 12q d2, cisplatinum 100mg/m2 d1); pts with partial (PR) or complete remission (CR) received cyclophosphamide 4g/m2, followed by peripheral blood stem cell (PBSC) harvest; methotrexate 8g/m2 plus vincristine 1,4mg/m2; and etoposide 2g/m2. The final myeloblative course was BEAM followed by PBSCT.

Results: 102 pts (median age 34 years, range 18–64) were enrolled. The response rate (RR) at the final evaluation (100 days posttransplantation) was 80% (72% CR, 8% PR). PBSC harvest was succesful in 96% of pts. Toxicity was tolerable. With a median follow-up of 30 months (range 3–61 months) freedom from second failure (FF2F) and overall survival (OS) were 59% and 78% for all patients, respectively. FF2F and OS for patients with early relapse were 62% and 81%, for late relapse 65% and 81%; for PD: 41% and 48% and for multiple relapse 39% and 48%, respectively. In multivariate analysis response after 2 cycles of DHAP (p < 0.0001) and duration of first remission (PD and multiple relapse vs. early and late relapse; p = 0.0127) were prognostic factors for FF2F. Response after DHAP (p < 0.0081), duration of first remission (p = 0.0017) and anemia (p = 0.019) were identified as prognostic factors for OS.

Conclusion: We conclude that this regimen is feasible, tolerable and highly effective in poor risk patients with relapsed and refractory HD. Based on these results a prospective randomized european intergoup study was started comparing this intensified regimen with two courses of DHAP followed by BEAM (HD-R2 protocol). First results of the second interim analysis of this study will be presented.
MAL Is Expressed in a Subset of Hodgkin Lymphomas and Identifies a Population of Patients with Poor Prognosis.
Authors: Hsi Eric D. , Sup Stephen J. , Alemany Carlos , Tso Elisa , Skacel Marek , Elson Paul , Alonso Miguel A. , Pohlman Brad
Published: April 2020

Recent evidence suggests that classical Hodgkin lymphoma (cHL) and primary mediastinal diffuse large B-cell lymphoma (PMLBL) have similarities that span clinical, histopathologic, and molecular genetic features. MAL is a gene that encodes a protein associated with lipid rafts in T-cells and epithelial cells. In T-cells, it is involved in the reorganization of lipid rafts during T-cell receptor activation and signaling. MAL is overexpressed in PMLBL and in a minority of cHL cases; however, data on its expression in a large series of cHL with clinical follow-up is lacking. In order to determine whether there might be some clinical significance associated with MAL expression, we evaluated MAL expression in a series of cHL by immunohistochemistry. Clinical information including age, sex, stage, bulky tumors (≥ 1/3 of the thoracic diameter or mass ≥ 10cm), hemoglobin, WBC, lymphocyte count, albumin, and outcome were collected for the cHL patients. Tissue microarrays containing diffuse large B-cell lymphoma (n=33), PMLBL (n=41), and cHL (n=87) were stained for MAL. 54% of PBLBL, 17% of cHL, and 3% of non-mediastinal diffuse large B-cell lymphomas were found to express MAL. Focussing on cHL, MAL expression correlated with the nodular sclerosis histologic subtype (P=0.03, Fisher exact). Overall, 22 HL patients have failed (relapse or death) and 15 patients have died. Median follow-up of patients alive and free of disease is 6.7 years (range 1.0–16.9 years). The 5-year failure-free and overall survival are 75 +/− 5% and 85 +/−4%, respectively. Using P<0.1 as significant, univariable analysis (Wilcoxon rank sum) showed that the following factors were associated with shorter failure free survival and overall survival: age ≥ 45 (P=0.01 and P<0.001, respectively), stage III/IV (P=0.09 and P=0.03, respectively), and MAL expression (P=0.05 and P=0.01, respectively). Cox proportional hazards modeling showed that the following were independent predictors of both time to treatment failure and overall survival: age (P=0.03 and 0.01, respectively), stage (P=0.06 and 0.04, respectively), and MAL expression (P=0.06 and 0.02, respectively). These data suggest that MAL expression in cHL identifies a biologically different subset of cHL compared to those cases that lack MAL and that MAL expression is a predictor of adverse outcome.
Epstein-Barr Virus Infection Is Not the Sole Cause of High Prevalence for Hodgkin’s Lymphoma in Saudi Arabia.
Authors: Al-Kuraya Khawla S. , Narayanappa Rajeswari , Al-Dayel Fouad , El-Solh Hassan , Ezzat Adnan , Ismail Hoda , Belgaumi Asim , Atizado Valerie , Bhatia Kishor , Sauter Guido , Simon Ronald
Published: April 2020

Age adjusted incidence of Hodgkin’s Lymphoma (HL) is markedly higher in Saudi Arabia than in the United States. For example, HL accounts for 10.5% of all neoplasia in children 15 years and younger in Saudi Arabia. EBV virus infection, which can induce HL transformation has been suspected to cause high HL incidence in developing countries. To investigate the role of EBV for the high frequency of HL in Saudi Arabia, we analyzed 265 HL from Saudi Arabia and 58 HL from Europe for EBV infection by in situ hybridization with fluoresce in-conjugated Epstein-Barr virus (EBER) on tissue microarray (TMA) sections. All Saudi and European HL were analyzed in one staining run under identical conditions. Unexpectedly, our data show only minor, statistically insignificant differences in EBV infection rates between Saudi (64 out of 150 informative cases; 42.6%) and European HL (11 out of 30 informative cases; 33%; p=0.5). Within the Arabian population, EBV positivity was more frequent in 79 children (53%) than among 133 adults (36%; p=0.015). EBV positivity was also linked to high stage with EBV positivity in 36% of 69 stage I/II and 64% of 73 stage III/IV tumors (p=0.009). EBV infection was most frequently seen in mixed cellularity HL (63% of 27 cases). This was significantly more frequent than in nodular sclerosing HL (39% of 136; p=0.02). Interestingly, EBV positivity was associated with good prognosis in Saudi childhood HL (p=0.016) but with poor prognosis in Saudi adulthood HL (p=0.0048). In conclusion, our data strongly suggest that EBV is not the main cause for the high prevalence of HL in Middle East countries. Among others, this would be consistent with a major role of genetic susceptibility genes for HL in these populations. Saudi Arabia with high consanguinity and large families would be ideal to search for HL susceptibility genes.
Immunohistochemical Markers Can Be of Predictive Value in Hodgkin’s Lymphoma Response to Treatment. A Preliminary Study of 23 Cases.
Authors: Canioni Danielle , Deau Benedicte , Taupin Pierre , Delarue Richard , Vasiliu Viorel , Brousse Nicole , Hermine Olivier
Published: April 2020

Hodgkin’s lymphoma (HL) belongs to the most curable tumor diseases in adults and about 80% of patients can be cured with modern treatment strategies. However, some HL are primary refractory to usual treatments including a first line of anthracyclins based regimen and have more than 50% of residual disease after treatment. Currently, clinical parameters are considered as main relevant factors for prognosis. Herein, in order to predict value of some other factors on treatment response, we evaluate immunohistochemical markers of apoptosis, proliferation and of environmental non neoplastic cells in HL. A retrospective study was performed on pre-treatment biopsy specimen of 23 patients presenting with HL treated with the same anthracyclin based regimen, 8 primary refractory HL (non responders or relapse occurring within one year) and 15 good responders (free of disease for 2 or 3 years) to chemotherapy. These patients ranged in age from 23 to 52 years old (median= 36.5 years) for refractory HL and from 21 to 75 years old (median= 37.9 years) for good responders (n.s). All refractory HL, except one for whom histological type could not be defined because of a too small biopsy, had a nodular sclerosis (NS) histological type. The histological type was NS in 13 responding patients and mixed cellularity for the 2 others (n.s). The semi-quantitative immunohistochemical study used bcl2, p53, Ki67, TiA1 and c-kit antibodies. The results were statistically evaluated using a Wilcoxon sum rank test. Ki67 was strongly expressed on Hodgkin (Hg) and Reed-Sternberg (RS) cells whatever patients were refractory or responders to chemotherapy. By contrast, p53 and bcl2 had a significantly higher expression on Hg or RS cells in refractory patients (median= 90%&60%) compared to responding HL (median= 42.5 & 8%)( p=0.0001 & p=0.026 respectively). The cytotoxic marker TiA1 stained a significantly higher number of small lymphocytes in refractory HL (median= 53 per high power field (hpf)) compared to responding patients (median= 24/hpf)(p=0.0003). C-kit antibody was negative on Hg or RS cells but stained significantly more mastocytes in refractory HL (median= 13/hpf) comparing to responders (median= 3.8/hpf) (p=0.001). These results indicate that some immunohistochemical markers may be useful for predicting the response to therapy of HL. A high expression of bcl2 and p53 in refractory HL supports the notion that an intact apoptosis cascade is essential for killing effect of chemotherapy. The increase of TiA1 and c-kit positive cells emphasizes the importance of the environmental non neoplastic cells in HL. A larger study is mandatory to confirm these preliminary results.
Regulation of NKT Cell Development by Sap, the Adaptor Mutated in X-Linked Lymphoproliferative Disease.
Authors: Nichols Kim E. , Hom Jamie , Ganguly Arupa , Ma Cindie S. , Tangye Stuart G. , Cannons Jennifer L. , Schwartzberg Pamela L. , Koretzky Gary A. , Stein Paul L.
Published: April 2020

The adaptor molecule SAP is expressed in T and natural killer (NK) cells, where it regulates cytokine production and cytotoxicity. Here we demonstrate that SAP also plays a critical role during NKT cell development. Sap−/− mice lack thymic and peripheral NKT cells and fail to produce cytokines in response to the NKT cell stimulus alpha-galactosyl ceramide. Infection of Sap−/− bone marrow cells with retroviruses expressing wild-type SAP permits NKT cell development after transfer into irradiated recipients. X-linked lymphoproliferative disease (XLP) patients, who harbor germline mutations in the gene encoding SAP, also lack NKT cells. Furthermore, a female XLP carrier demonstrates completely skewed X chromosome inactivation within peripheral blood NKT cells, but not T or B cells. Thus, SAP is a critical regulator of NKT cell ontogeny in humans and in mice. The absence of this cell lineage may contribute to the phenotypes of XLP, including abnormal anti-viral and anti-tumor immunity and hypogammaglobulinemia.