bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Mechanisms Involved in Homing and Migration of B-Chronic Lymphocytic Leukemia (CLL) in Response to CXCR4 Stimulation and Downstream Activation of the PI3K Pathway.
Authors: Ghobrial Irene M. , Ghobrial Joanna M. , Bramati Patricia , Timm Michael M. , Kumar Ashock , Witzig Thomas E. , Kay Neil , Hedin Karen E.
Published: October 2019

Malignant B-cells characteristically home to the bone marrow and lymph nodes. However, the mechanisms by which cells are recruited into and mobilized from the bone marrow/lymph nodes into the peripheral blood are not well understood. Chemokines such as SDF-1 and it receptor CXCR4 play a central role for lymphocyte trafficking and homing. Downstream activation of the PI3K pathway by chemokines has been implicated in the migration of many cell types. We recently demonstrated that CXCR4 expression correlates significantly with disease progression in CLL. Patients with Rai stage 4 had a higher expression of CXCR4 when compared to patients with Rai stage 0. The expression of CXCR4 was not found on CLL B cells resident within the lymph nodes implying that there may have been downregulation of CXCR4. In this study, we explore the molecular mechanisms involved in homing and migration of B-CLL cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of CLL cells in response to SDF-1. Boyden chamber in vitro migration assays were used with primary CLL peripheral blood cells and the MEK1 CLL cell line. In addition, we expressed fluorescent YFP-CXCR4 in the cells and used confocal microscopy to visualize changes in the subcellular location of the fluorescent CXCR4 before and after SDF-1 stimulation. Inhibitors of the PI3K pathway such as the PI3K inhibitor LY294002 (10uM, one hour pretreatment) and the mTOR inhibitor, rapamycin (200nM overnight) were used. SDF-1 induced a dose dependent migration of primary CLL cells and MEK1 cells, indicating functional CXCR4 receptors. MEK 1 cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping and internalization subcellularly. Pretreatment of the cells with PI3K pathway inhibitors led to inhibition of migration with LY294002 but not with rapamycin. Confocal microscopy demonstrated abrogation of subcellular localization in the presence of LY294002 but not with rapamycin with the addition of SDF-1. These results demonstrate that CLL cells express functional CXCR4 receptora and that the PI3K pathway is essential for SDF-1 dependent CLL migration. In addition, we demonstrate that the CXCR4 receptor is internalized in response to SDF-1 stimulation indicating that CLL cells downregulate the CXCR4 receptor once they home to the bone marrow or lymph nodes where there is abundant SDF-1 in the microenvironment. This leads to the confinement of the cells in the microenvironment preventing further migration of the cells outside of the bone marrow/lymph nodes. Supported in part by CA97274
Inhibition of Tumor Growth by RGD Peptide Directed Delivery of Truncated Tissue Factor to the Tumor Vasculature.
Authors: Kessler Torsten , Bieker Ralf , Padro Teresa , Herrera Federico , Ruiz Sandra , Serve Hubert , Kreuter Micheal , Berdel Wolfgang E. , Mesters Rolf M.
Published: October 2019

Selective activation of blood coagulation in tumor vessels with subsequent tumor infarction is a promising anticancer strategy. To this end, a fusion protein consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) was fused to the peptide GRGDSP selectively targeting avb3 and avb5 integrins on tumor endothelial cells. The fusion protein tTF-RGD retained its thrombogenic and integrin binding activity as demonstrated by coagulation assays and binding assays with purified avb3 and endothelial cells. In vivo studies in mice bearing established human adenocarcinomas (CCL185), human melanoma (M21) and human fibrosarcoma (HT1080) revealed that i.v. administration of tTF-RGD induced partial or complete thrombotic occlusion of tumor vessels as indicated by histological analysis. Furthermore, treatment studies showed that tTF-RGD but not untargeted tTF induced significant tumor growth retardation or regression in all three types of solid tumors in mice without apparent side effects such as thrombosis in liver, kidney, heart or lung.

Thus, selective thrombosis in the tumor vasculature induced by tTF-RGD may be a promising strategy for the treatment of cancer.
Functional Pan-Universality of the Age-Related Regulatory Mechanisms of Gene Expression.
Authors: Kurachi Kotoku , Fontaine Jean-Marc , Abe Takahiro , Kurachi Sumiko
Published: October 2019

Through analyses of human factor IX (hFIX) and protein C (hPC) gene expressions in transgenic mice, we recently discovered the first molecular mechanism of age-dimension gene regulation, involving two critical genetic elements ASE and AIE required for age-related stable and increase patterns of gene expression, respectively. The next most critical issue to be tested was whether or not these elements, particularly ASE due to its possible utility, similarly functions with grossly different genes. We constructed hFIX expression vectors carrying a CMV promoter with or without ASE, and tested them in transgenic mice. Vectors with no ASE showed an age-dependent gradual decrease in hFIX expression, reaching the background levels in 3–9 months. Vectors with ASE, however, showed age-stable hFIX expression over the same time period. These findings supported the pan-universality of ASE function. As we previously reported, ASE regulates gene expression not only temporally but also spacially. With ASE, CMV-driven expression showed the highest in the heart muscle, whereas without ASE, the highest in the skeletal muscle. These results led to development of the Age Dimension Technology, a new field for exploring the unique applications of the age-related knowledge.
Intermediate Dose Methotrexate in Low-Risk Acute Lymphoblastic Leukemia. Results of the FRALLE 93A Protocol.
Authors: Perel Yves , Chambost Hervé , Gandemer Virginie , Leblanc Thierry , Pascale Schneider , Claudine Schmitt , Debré Mariane , Vermylen Christiane , Couillault Gérard , Lamagnere Jean-Paul , Auclerd Marie-Françoise , Leverger Guy , Baruchel André ,   the FRALLE group
Published: October 2019

The FRALLE 93A protocol aimed to identify a very low-risk ALL subgroup, to limit treatment morbidity and to investigate the value of intermediate dose methotrexate. Inclusion criteria were : B-cell precursor ALL, age 2-7 years, leukocyte count <10G/l ; patients with CNS or testicular leukemia, bulky disease, t(9;22), t(4;11), t(1;19) or hypodiploïdy (<44 chromosomes) were not included.

181 children were included (13.5% of all the ALL included in the FRALLE 93 protocol) between June 1993 and Dec 1999. After a predisone prophase and induction phase (4-week daily prednisone, 4 weekly vincristine, thrice weekly 10,000 units of E coli asparaginase for 6 doses), during the 12-week antimetabolite consolidation therapy, patients were randomized to receive either low-dose methotrexate (25 mg/m2 p.o.) (lmtx) or 6 doses of intermediate dose methotrexate (IMTX) at 1500 mg/m2 by 24 hours infusion. Further therapy included a CCG type delayed intensification (dexamethasone, weekly vindesine, 3 doses of weekly doxorubicin and asparaginase, cytarabine) but without cyclophosphamide and a two years maintenance treatment. All pts received 16 triple intrathecal injections. The complete remission rate was 99.5%. The overall survival and event-free survival at 5 years were 93 % ± 4 % and 82 % ± 6 % (point date : 01.01.04). Only 1pt was considered prednisone-resistant (0.5%) D21 M1 marrow response is associated to a better 5 y EFS (86 + 5% vs 44 + 21% for M2/M3 pts (18 pts); p=0.001). Among 158 randomized patients, 9 testicular/meningeal relapses (6 isolated, 3 combined) occurred in the lmtx arm versus 1 in the IMTX arm (p=0.03) ; six BM relapses occurred in the lmtx group vs 9 in the IMTX group (p= NS). The 5-year DFS is not different 83% ± 8% and 85% ± 8% (p=0.47).

Conclusion: a high cure rate could be achieved in low-risk ALL without cranial radiation, alkylating agents or epipodophyllotoxin and with a low cumulative dose of anthracyclins (75 mg/m2). Prednisone response is of no help in this subgroup of BCP-ALL. D21 marrow response allows to identify a very low-risk subgroup in addition to initial criteria. IMTX proved superior to lmtx in preventing testicular or meningeal relapse in low-risk ALL.
Somatic Mutations and Polymorphisms of the Glucocorticoid Receptor Gene Are Not Related to Glucocorticoid Resistance in Childhood ALL.
Authors: Tissing Wim , Meijerink Jules P.P. , Den Boer Monique L. , Brinkhof Bas , van Rossum Liesbeth , van Wering Elisabeth R. , Koper Jan Willem , Sonneveld Pieter , Pieters Rob
Published: October 2019

In vivo and in vitro response to glucocorticoids (GC) are important prognostic factors in childhood ALL. To induce apoptosis, GC bind to the intracellular GC receptor (GR), which subsequently triggers transactivation and repression of downstream genes. As somatic mutations and polymorphisms have been linked to both increased and decreased GC sensitivity in healthy individuals, we studied the relationship between these gene alterations and GC sensitivity in childhood ALL.

Methods: We analysed leukemic cells taken at diagnosis and normal lymphocytes at time of complete remission (CR) from 57 children. DNA mutations were screened for using a PCR - SSCP technique. PCR products with abnormal SSCP patterns were sequenced. In vivo response to prednisone was determined according to BFM criteria as prednisone good response (PGR) or prednisone poor response (PPR). The in vitro prednisolone sensitivity was determined by MTT assay.

Results: The polymorphism ER22/23EK was found in 4% of cases, N363S in 12%, Bcl1 in 55%, intron mutation 16 basepairs upstream of exon 5 in 40%, H588H in 2% and N766N in 21%. They were proven to be polymorphisms as they were found in normal lymphocytes of the same patients at CR as well. The incidence of the polymorphisms was comparable to reports in the literature in the healthy population, except for N363S, which was found at a 2–4 fold higher incidence in ALL patients. Using the Chi-square test and the Mann-Whitney U test, no relation could be found between the occurrence of polymorphisms and in vivo prednisone response or in vitro sensitivity to prednisolone.

Conclusion: GC resistance in childhood ALL can not be attributed to polymorphisms of the hGR. The association between a higher incidence of the N363S polymorphism in childhood ALL as compared to the normal population needs further investigation.
Chromosomal Rearrangements Induced by DNA Double-Strand Breaks.
Authors: Varga Tamas , Aplan Peter D.
Published: October 2019

It has been suggested that introduction of double-strand DNA breaks into human chromosomes can lead to gross chromosomal rearrangements (GCRs) through improper repair mechanisms, and that these GCRs may be leukemogenic through the loss of tumor suppressor genes or the creation of fusion oncogenes. Using the human histiocytic lymphoma cell line U937, we developed a model system in which we were able to introduce a double-strand DNA break (DSB) in vivo at a predetermined location, and subsequently examine the ensuing chromosomal changes flanking the breakage site. In this model, U937 cells are stably transfected with a construct that contains the 18 bp recognition site for a rare cutting endonuclease, I-SceI, whose recognition sequence is not normally present in the human genome. The I-SceI site is placed between a strong constitutive promoter (EF1alpha) and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker by conferring ganciclovir (GCV) sensitivity to the cells. Following transfection of an I-SceI expression vector, double-strand DNA breakage occurs and abrogatesHSV-tk expression, leading to a dramatic increase in GCV resistant cells. We showed that, even in the abscence of GCV selection, the IsceI enzyme was able to cleave its recognition sequence in at least half of the transfected cells. The predominant type of rearrangement detected following the appearance of a double-strand break was an interstitial deletion. These deletions typically displayed uneven recession of free DNA ends flanking the cleavage site, generally resulting in either very short (<10 bp) or very long (>1000) bp deletions extending into the EF1alpha promoter and homogeneous deletions of 100–1700 bp into the HSV-tk gene. Moreover, in many clones, nucleotide microhomologies of 1–6 basepairs, palindromes, or both were detected at the breakpoint junctions, suggesting repair via a non-homologous end joining (NHEJ) mechanism.. We also showed that in 25 % of the recovered clones the DSB was repaired by formation of direct or inverted repeats derived from the breakpoint junction. Several clones showed similarly complex rearrangements, including short nucleotide insertions, inversions and duplications. Finally, approximately 4.5% (7/150) of the clones demonstrated insertion events that involved integration of nucleotide sequences derived from distant genomic regions, including LINE elements, coding regions of endogenous genes and sequences exhibiting known genomic instability. These insertions may have been derived via reverse transcription of mRNA sequences; alternatively the distant genomic regions may have served as templates for the inserted sequences, or genomic DNA fragments may have been directly incorporated into the I-SceI-induced break. A step-by-step modification of this loss of function selection scheme will provide a platform for studying DNA rearrangements induced by faulty dsDNA repair.
Synergistic Cytotoxic Effects of a Combination of a Novel Tyrosine Kinase Inhibitor AMN107 and Histone Deacetylase Inhibitor LBH589 Against Bcr-Abl Expressing Human Leukemia Cells.
Authors: Scuto Anna , Annavarapu Srinivas , Bali Purva , McCullers Molly , Fiskus Warren , Mouttaki Abdelmoughite , Guo Fei , Sigua Celia , Sondarva Gautam , Atadja Peter , Manley Paul , Bhalla Kapil N.
Published: October 2019

AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) is a potent aminopyrimidine tyrosine kinase (TK) inhibitor, which is active at nanomolar concentrations against c-Kit, PDGFR as well as the wild type Bcr-Abl and common variety of mutant Bcr-Abl (e.g., M351T, F317L and E255V) TK. In a previous report, we demonstrated that treatment with the hydroxamic acid analogue histone deacetylase inhibitor LBH589 (Novartis) (20 to 100 nM) induces hsp90 acetylation and promotes polyubiquitylation and proteasomal degradation of Bcr-Abl, which is associated with apoptosis of the cultured Bcr-Abl expressing human chronic myeloid leukemia-blast crisis (CML-BC) K562 and LAMA-84 cells, as well as of primary CML-BC cells. In the present studies, we determined the cell cycle and apoptotic effects of AMN107 and/or LBH589 in K562, LAMA-84 and primary CML-BC cells. Treatment with AMN107 (20 to 100 nM) induced cell cycle G1 phase accumulation and apoptosis, exerting 10 to 20 fold more potent effect than imatinib mesylate (IM) in K562 and LAMA-84 cells. This was associated with marked induction of p27, inhibition of Bcr-Abl TK activity, as well as attenuation of the levels of p-STAT5, p-AKT, Bcl-xL, and c-Myc levels in a dose dependent manner. Co-treatment with LBH589 (50 nM) and AMN107 (100 nM) induced more G1 phase accumulation than either agent alone. The combination of AMN107 and LBH589 also exerted synergistic apoptotic effects in K562 cells, as determined by the median effect isobologram analysis. This was associated with more attenuation of the levels of pro-growth and pro-survival proteins, e.g., p-STAT5, p-ERK1/2, c-Myc and Bcl-xL, as well as greater induction of the levels of pro-death p27 and Bim proteins. Treatment of human AML HL-60 cells containing ectopic expression of the IM-refractory, mutant Bcr-AblT315I with LBH589 (50 nM for 24 hours) attenuated the mutant Bcr-Abl levels and induced apoptosis of HL-60/Bcr-AblT315I cells. Treatment with AMN107 (up to 2.0 μM) alone was ineffective in inducing loss of viability of HL-60/Bcr-AblT315I cells, and co-treatment with LBH589 (50 nM) and AMN107 did not induce more loss of cell viability of HL-60/Bcr-AblT315I cells than treatment with LBH589 alone. AMN107 levels ≥ 200 nM significantly inhibited survival of HL-60/Bcr-AblE255K and HL-60/Bcr-AblM351T cells. Consistent with this, the combined treatment with LBH589 and AMN107 also induced more loss of viability than treatment with either agent alone in 4 samples of IM-refractory primary CML-BC cells. These studies demonstrate that both AMN107 and LBH589 are active against IM-resistant CML-BC and the combination of the two exerts superior activity against IM-sensitive and IM-resistant, wild type and mutant Bcr-Abl expressing cultured and primary CML cells.
STI571 Suppresses Proliferation by Restoring Nuclear Cyclin Dependent Kinase Inhibitors (CDKIs) while STI571+TRAIL Promotes Cell Death by Decreasing Cytoplasmic CDKIs.
Authors: Dao Mo A. , Verfaillie Catherine M.
Published: October 2019

Cyclin dependent kinase inhibitors (CDKIs), p27Kip1 and p21Cip1, function as cell cycle inhibitors when located in the nucleus. When localized to the cytoplasm, these CDKIs can function as anti-apoptotic molecules by sequestering/preventing the activation of pro-apoptotic proteins such as ASK1 and procaspase-3. Our lab has reported elevated cytoplasmic CDKIs and decreased nuclear CDKIs in hematopoietic cells expressing BCR/ABL, the oncogene found in more than 90% of cases of chronic myeloid leukemia. Within the past decade, STI571 has been shown highly promising for CML treatment. However, there is increasing evidence suggesting that the drug might function more as a suppressor of proliferation and less as a promoter of cell death. In the current studies, we differentiate the effect of STI571 on proliferation vs. survival by tracking the subcellular increase/decrease in CDKIs using MO7e cells engineered to express BCR/ABL. To determine if a correlation exists between STI571 resistance and levels of cytoplasmic anti-apoptotic CDKIs, we also investigated changes in levels of nuclear vs. cytoplasmic CDKIs in LAMA84 -S (sensitive to STI571) vs. LAMA84-R (resistant to STI571). And lastly, we tested whether activation of TRAIL would enhance cell death in STI571-resistant cells. STI571 treatment increases nuclear CDKIs, correlating directly with a decrease in proliferation of MO7e/p210 cells. However, the high levels of cytoplasmic CDKIs in MO7e/p210bcr/abl was not modulated following STI571 treatment and cell death was not prominent, unless growth factors were removed. Moreover, cytoplasmic p21Cip co-immunoprecipitated with ASK1 and procaspase 3. When compared with LAMA-S cells, LAMA-R cells expressed even higher levels of cytoplasmic CDKIs. Treatment with STI571 decreased cytoplasmic CDKIs in LAMA84-S cells and resulted in cell death. As hypothesized, LAMA84-R cells did not show reduction in cytoplasmic CDKIs and did not enter apoptosis. However, when treated with STI571 and TRAIL, LAMA84-R cells showed a decrease in cytoplasmic CDKIs, and increase in apoptosis. Based on these observations, we conclude that: 1. BCR/ABL expression reduces nuclear CDKIs but increases cytoplasmic CDKIs. 2. STI571 treatment restores nuclear CDKIs and reduces cell proliferation of BCR/ABL expressing cells under physiological conditions. 3. Treatment of STI571+TRAIL reduces cytoplasmic CDKIs and increases cell death of BCR/ABL expressing cells, most notably, the STI571-resistant cells. In conclusion, we show that the imbalance between nuclear (cell cycle inhibitor) and cytoplasmic (cell survival enhancer) CDKIs exist in BCR/ABL-hematopoietic cells.
Enhanced Killing of Chronic Myelogenous Leukemia Cells by Rapamycin and Imatinib Is Associated with Differential Inhibition of 4E-BP1 and eIF4E Phosphorylation and Decreased Protein Expression by Non-Overlapping Mechanisms.
Authors: Moore James C. , Ly Chi , Luke Halbur , Ong S. Tiong
Published: October 2019

The Bcr-abl tyrosine kinase is known to promote transformation by dysregulating gene transcription, but its role in dysregulating translation is less well documented. Our recent work has implicated the mammalian target of rapamycin (mTOR) signaling as a downstream target of Bcr-Abl, since we find that the mTOR effectors, 4E-BP1 and S6, are phosphorylated in a Bcr-Abl kinase-dependent manner (Ly et al., Cancer Research, 2003). Because mTOR is a central regulator of eukaryotic translation, and inhibitors of mTOR act synergistically with imatinib mesylate (imatinib) to kill CML cells, these results suggest that, like transcription, translation may be a general cellular process dysregulated by Bcr-Abl activity. If this were so, then components of the cellular apparatus co-opted by Bcr-Abl to increase translation would constitute rational therapeutic targets. These would include signaling pathways mediating increased translation, components of the cap-binding complex (eIF4E, eIF4GI, and eIF4A) that regulate cap-dependent mRNA translation, as well as proteins whose translation is increased by Bcr-Abl kinase activity. Here we identify eIF4E as well as cyclin D3 as potential therapeutic targets in CML. Since eIF4E is essential for cap-dependent translation, and increased translation parallels eIF4E phosphorylation at Ser209, we determined the status of eIF4E phosphorylation in murine hematopoietic Ba/F3 cells expressing Bcr-Abl (Ba/F3-Bcr-Abl), and its dependence on Bcr-Abl kinase activity. Using phosphospecific antibodies to eIF4E, we found that Bcr-Abl kinase activity was essential for phosphorylation of eIF4E at Ser209, but had no effects on total levels of the protein. In contrast, rapamycin had no effect on the degree of eIF4E phosphorylation, although it was able to inhibit phosphorylation of 4E-BP1 completely (unlike imatinib). By examining total mRNA and protein levels of known targets of Bcr-Abl, we determined that cyclin D3, but not cyclin D2, was post-transcriptionally regulated by Bcr-Abl. Metabolic labeling studies were also conducted in Ba/F3-Bcr-Abl cells treated with media alone, imatinib, rapamycin, or both. Our results demonstrated that translation of cyclin D3 protein is regulated by the mTOR kinase in Bcr-Abl-expressing cells, and that combined inhibition of mTOR and Bcr-Abl resulted in an additional decrease in protein levels. Together, these results demonstrate that Bcr-Abl promotes protein translation of specific genes via mTOR, and that the activity of both Bcr-Abl and mTOR kinases contribute to dysregulated protein expression via non-overlapping mechanisms in CML cells. Ongoing studies are being conducted to determine the role of both eIF4E and cyclin D3 in the pathogenesis of CML.
Is Family History of Breast Cancer Associated with Therapy-Related Myelodysplastic Syndrome and Therapy-Related Leukemia? .
Authors: Li Loretta S. , Huo Dezheng , Karrison Theodore , Larson Richard A. , Olopade Olufunmilayo
Published: October 2019

Therapy-related myelodysplastic syndrome (t-MDS) and therapy-related acute myeloid leukemia (t-AML) occur in a subset of patients treated with chemotherapy and/or radiation for various primary malignancies. Whether or not a patient develops t-MDS/t-AML can be influenced by a combination of genetic and environmental factors. We conducted a retrospective analysis of t-MDS/t-AML patients who had cytogenetic analyses performed at the University of Chicago Hospitals between 1972 and 2001 to determine if a family history of cancer could be associated with t-MDS/t-AML development. These included 141 males and 165 females whose median age was 51 years (range was from 3–83 years) at the time of primary diagnosis and 58 years (range was from 6–86 years) at the time of t-MDS/t-AML presentation. Out of 306 patients, data on first-degree family history of cancer was documented in 160 patients, but there was no statistically significant difference in the characteristics examined below between patients with self-reported family history of cancer and patients with undocumented family histories. A first-degree family history of any cancer was reported in 89 patients (55.6%). The prevalence of family history of specific sites were: 25 (15.6%) of breast cancer, 23 (14.4%) of lung cancer, 13 (8.1%) of colorectal cancer, 11 (6.9%) of hematologic malignancy, 10 (2.3%) of prostate cancer, 6 (3.8%) of stomach cancer, 5 (3.1%) of uterine cancer, 5 (3.1%) of CNS cancer, 5 (3.1%) of pancreas cancer, 4 (2.5%) of melanoma, 3 (1.9%) of oral-pharyngeal cancer, 3 (1.9%) of ovarian cancer, 3 (1.9%) of cervical cancer, 2 (1.3%) of vulvar cancer, 1 (0.6%) each of bladder cancer, sarcoma, renal cancer, or esophageal cancer. Of note, only a family history of breast cancer was associated with clinical presentation as show in Table 1 below. Patients with a family history of breast cancer were also more likely to have a chromosome 5 and/or 7 abnormality (84%) than patients without (62%, p=0.04). There was no significant association between family history of cancer at other sites and clonal cytogenetic abnormalities. A subgroup analysis was conducted in patients with a primary diagnosis of breast or ovarian cancer. Of the 24 patients who had data on family history of cancer, 16.7% reported having a first degree relative with breast cancer. These findings suggest a possible genetic component linking family history of breast cancer and development of t-MDS/t-AML, as was previously reported by Rauscher et al. (2002) for de novo adult leukemia incidence. Further studies of family histories of cancer, in particular breast cancer among patients receiving chemotherapy and/or radiation, could potentially elucidate the genetic basis of leukemia and ultimately lead to new tools for risk assessment, early detection or prevention of secondary leukemia in cancer survivors.
The Role of DNA Methylation of HoxA5, a Regulator of Haematopoiesis, in Progression to Myeloid Blast Crisis in CML.
Authors: Strathdee Gordon R. , Holyoake Tessa L. , Sim Alyson , Brown Robert
Published: October 2019

Hox (homeobox) genes are known to be key regulators of development and haematopoiesis and several have also been implicated in leukaemogenesis. Overexpression of HoxA5 in human haematopoietic progenitors leads to an increase in myelopoiesis, suggesting a role for this hox gene during induction of myeloid differentiation. Inactivation of genes by CpG island DNA methylation is known to be important in the development and progression of leukaemia, and inhibitors of DNA methylation are currently of great interest as novel therapeutics for a number of haematopoietic malignancies. Here we show that in peripheral blood from healthy volunteers HoxA5 exhibits methylation of 50% of alleles across an extensive CpG island covering the promoter region/1st exon of HoxA5. In patients with chronic myeloid leukaemia, 33% (15/45 patients) exhibited increased methylation of HoxA5 (80–100% of alleles methylated) in the chronic phase of the disease. However, such hypermethylation of HoxA5 was invariably present in samples from patients in myeloid blast crisis (15/15 patients). In contrast, patients in lymphoid blast crisis did not exhibit increased levels of hypermethylation. Analysis of patients in chronic phase demonstrated a statistically significant correlation (p < 0.006) between hypermethylation of the HoxA5 gene and other prognostic factors associated with high risk of progression to blast crisis (high Hasford/Sokal score, incomplete response to Imatinib, known early progression, del 9), suggesting that methylation of HoxA5 may be a clinically useful prognostic indicator. The results are also compatible with a direct role for hypermethylation of HoxA5, and consequent loss of HoxA5 expression, in inhibition of myeloid differentiation during progression to blast crisis and implicate HoxA5 as a therapeutic target for inhibitors of DNA methylation in the treatment of leukaemia.
Modelling Molecular Consequences of Leukemia Initiation by TEL-AML1 Fusion.
Authors: Ford Anthony M. , Cardus Penny , Greaves Mel F.
Published: October 2019

We have previously shown that TEL-AML1 (ETV6-RUNX1) fusions in acute lymphoblastic leukemia commonly arise pre-natally. The fusion protein initiates, presumably via transcriptional deregulation, a pre-leukemia condition that infrequently (~1%) converts over a 10 year period to overt leukaemia in the presence of post-natal, secondary genetic changes including TEL deletion.

One challenge is to identify the molecular mechanisms by which TEL-AML1 elicits and sustains stable pre-leukemic clones which are then vulnerable to additional genetic alterations in the context of particular etiological exposures (e.g. infection). We have established cell lines of murine lymphoid progenitor cells that stably express a regulatory, truncated human progesterone receptor ligand-binding domain (pSwitch, Invitrogen) that undergoes a conformational change in the presence of the steroid agonist, mifepristone and conversion to an active form. The active binding domain is then able to bind to and activate transcription from an otherwise silent TEL-AML1 gene that we have also stably introduced into these cells. In the presence of sub-physiological levels of mifepristone, TEL-AML1 becomes abundantly expressed in the nucleus within a few hours. Microarray analysis comparing uninduced (time 0) cDNA with cDNA from both control and TEL-AML1 induced cells grown for 4 days in the presence of mifepristone showed changes in the expression pattern for over 250 genes. The majority of these genes were down regulated in the presence of TEL-AML1, in keeping with a repressive activity for the fusion gene. A number of apoptotic and cell cycle genes were down regulated, while anti-apoptotic genes were up regulated in the presence of the fusion protein. These genes are currently being examined by both in-silico and laboratory-based validation techniques and may provide key insights into the pathways that are deregulated by TEL-AML1 in ALL.
Lack of Value of Clinical Predictors of Heparin Induced Thrombocytopenia (HIT) in Complex Patients.
Authors: Baker Jessica , DeLoughery Thomas G. , Boshkov Lynn
Published: October 2019

HIT is a devastating complication of heparin use that can result in thrombosis and death. However, it can be difficult to diagnose. Although the clinical hallmark of HIT is a drop in platelet counts in patients receiving heparin, in complex patients there may be many confounding reasons for thrombocytopenia and thrombosis. One proposed diagnostic tool for HIT is to quantitate the percent drop in platelet count from the highest level. Also Warkentin has proposed the “4T’s” scoring system where 4 clinical indicators (% drop in platelet count, timing of heparin dosing, presence of thrombosis, and lack of other causes of thrombocytopenia) are used to derive a score to indicate High, Intermediate, or Low probability of HIT.(Curr Hem Rep, 2, 148–157) We sought to verify the predictive value of these clinical systems. Over a 30 month period, we reviewed the charts of 544 patients who were suspected of having HIT. 61% of these patients were in intensive care units, 47% were peri-operative, and 22% were cardiac patients. HIT was diagnosed by a combination of review of the clinical course plus results of platelet activation assay, and with at least 2 faculty hematologist agreeing on the diagnosis. Data was abstracted concerning platelet count, timing and use of heparin, presence of thrombosis, and other causes of thrombocytopenia including infection and major surgery. 18 out of 544 patients (3.3%) had heparin induced thrombocytopenia. 16 of 18 patients received therapeutic standard heparin and 2 patients received only prophylactic doses of LMWH. There was no difference in nadir platelet count nor percent drop in HIT (75±79,000/Ul, 65±26%) vs those without HIT (90±71,000/uL, 53±24%). Two major causes of thrombocytopenia in this population were infections and surgery. 175 patients (32%) had positive blood cultures with a mean platelet drop of 55±23% preceding the positive cultures. 47% had major surgery with a mean platelet drop afterwards of 60±22%. A drop in platelets of over 50% had a 82% sensitivity for HIT but only a 43% specificity. Using different cut-offs did not reveal a value with better predictability. Interestingly, only 2 (11%) of the HIT patients scored on the 4T’s as High probability with 39% being Intermediate and 50% as Low. This was due to presence of other causes of thrombocytopenia in patients and the fact that 30% of the HIT patients were on heparin longer that 10 days. 4% of the non-HIT group scored as High and 28% as Intermediate but close review of their subsequent clinic course did not reveal evidence for HIT. Simple clinical tools for diagnosing HIT are of limited utility in ill and complex patients due to other competing causes of thrombocytopenia and the potential for long duration of heparin exposure.
Clinical Features and Effects of Eradication on Helicobacter Pylori Positive 207 ITP Cases in Japan.
Authors: Fujimura Kingo , Kuwana Masataka , Kurata Yoshiyuki , Imamura Masahiro , Harada Hiroshi , Sakamaki Hisashi , Teramura Masanao , Koda Kyuhei , Nomura Shosaku , Sugihara Sayaka , Shimomura Takeshi , Fujimoto Tetsuro Takahiro , Oyashiki Kazuma , Ikeda Yasuo
Published: October 2019

In 1998, Gasbarrini et al reported that in ITP cases with Helicobacter pylori (H.pylori) infection, elevation of platelet counts was observed by eradication of this bacterium. Since then, several reports from Italy and Japan confirmed the elevation of platelet counts after eradication. However, the characteristic background in the H.pylori positive ITP and eradication effects on platelet counts is unclear. On the other hand, reports from Spain, North Europe and USA could not show the evidence that eradication is effective on elevating platelet counts in H.pylori positive ITP.

Therefore, we designed a nationwide retrospective study in Japan to evaluate the incidence of H.pylori positive ITP cases and the effects of eradication on platelet counts and to clear above problems.

Four hundred and thirty-five ITP cases were enrolled over a period of one and half years (2002. 7~2003.12) from 12 hospitals. H. pylori infection was found in 300 cases(65%), who were significantly (P<0.005) older and showed hyperplastic megakaryocyte in bone marrow (P=0.011) comparison with negative cases. Eradication to H. pylori was performed in 207 H. pylori positive ITP cases and as a whole, the platelet count response was observed in 63% of eradication succeeded group. In the successful group, CR and PR rate were 23% and 42% respectively at 12 months after eradication. The platelet count response was significant in the successfully eradicated group (P<0.005) and the increased platelet count was maintained without ITP treatment for over 12 months. In conclusion, H. pylori infection was involved in most ITP patients over 40 years old in Japan and eradication therapy proved effective for increasing platelet counts even in splenectomy non-responsive cases and the platelet count response appeared one month after eradication. This evidence suggests that eradication therapy is the first line of treatment in H. pylori positive ITP patients.
Mabthera (Rituximab) in the Treatment of 92 Patients with Refractory Immune Thrombocytopenic Purpura.
Authors: Cabrera JRafael , Penalver F. Javier , Millan Isabel , Jimenez-Yuste Victor , Almagro Manuel , Alvarez-Larran Alberto , Rodriguez Lluis ,   other 36 participants in the multicentric retrospective Spanish Study on use of Rituximab in Refractory ITP
Published: October 2019

Objectives: Rituximab (RB) is an anti-CD20 murine/human chimeric antibody that has been used in the treatment of auto-immune diseases, including immune thrombocytopenic purpura (ITP). The effectiveness of RB in the treatment of patients with ITP has been evaluated.

Patients and methods: A multicentric retrospective study of the patients with ITP treated with RB has been carried out. A questionnaire about clinical characteristics and response to RB was sent to the participating centers. These questionnaires were filled out by the physicians in charge of each patient. We received questionnaires from 43 centers The response criteria were those published by Stasi R et al (Blood2001; 98:952): Complete Response (CR): platelets >100 x 109/l; Partial Response (PR): 50–100 x 109/l; Minimum Response (MR): 30–50 x 109/l. Data from 99 patients were analyzed. Ninety two out of 99 patients were evaluated. The median age at diagnosis was 54 years (4–98), 56% were women. 55.4% of the cases were idiopathic ITP, 14.1% were associated with B-CLL, 8.7% with auto-immune diseases and 3.3% with lymphoma. Before RB they have received several treatment schemes including steroids (98%), IVIG (88%) and cyclophosphamide (26%). 52.2% were splenectomized. Median of platelets before treatment with RB was 8 x 109/l (1–30), 66.3% had less than 10 x 109/l. 87% of the patients were treated with 4 doses of RB (1–6), while 34 received it in association with other treatments, mainly steroids.

Results: 42 patients obtained CR (46.2%) and 8 patients obtained PR (8.8%), a total of 50 global responses (55%). The majority of the responses took place in the first week (26% out of the total, 40% of the responders). The maximum response was also soon achieved, median of 5 weeks, with platelets of 141 x 109/l (10–651). There were no differences in the response between splenectomized or not splenectomized patients. 29 patients maintained the CR (69% of responders), with a monitoring median of 9 months (2–42). The toxicity of the treatment was minimum: 2 fever episodes with the infusion and 2 skin eruptions.

Conclusions: This is the widest series of patients with ITP treated with RB. These results suggest that RB can be considered as a rescue treatment for patients with refractory ITP to steroids and/or splenectomy, with an excellent tolerance. Further monitoring is needed in order to establish the real role of RB as a rescue treatment for refractory ITP.
Direct Intramarrow Injection of CD34+ Cells May Improve Long-Term Engraftment in Nonhuman Primates.
Authors: Jung Chul W. , Morris Julia C. , Beard Brian , Neff Tobias , Beebe Kate , Kiem Hans-Peter
Published: October 2019

Recent studies in the NOD/SCID model have shown improved engraftment of SCID-repopulating cells and higher levels of engraftment in the secondary transplantation when cells were administered by intramarrow (IM) versus intravenous (IV) injection suggesting that direct injection into the marrow cavity may be beneficial for stem cell engraftment in a clinical setting. To study whether IM injection was feasible and would result in improved engraftment in a clinically relevant large animal model, we compared IM vs IV injection in our competitive repopulation assay in baboons. Enriched CD34+ cells were split into 2 equal fractions and transduced with either a GFP- or YFP-expressing vector. Pretransplant transduction efficiencies and expansion of CD 34+ cells were similar in both fractions. One fraction was then infused into the marrow cavity of the right femur and the other fraction was given intravenously. Three baboons received gene-modified CD34+ enriched autologous bone marrow cells after myeloablative radiation. Peripheral blood granulocyte marking levels showed peaks at 2–3 weeks after transplantation and decreased thereafter. In all three monkeys, marking levels of IM injected cells (GFP) were lower than marking levels of IV injected cells (YFP) early after transplantation up to 7 weeks. However, in two of the three monkeys, GFP marking increased steadily after 2 months resulting in higher marking levels from IM injected cells. The trend sustained up to the last follow-up of nine months after transplantation, marking levels being 25.5% and 7.4% from IM and IV injected cells, respectively, in M00228. This pattern was recapitulated in the marking of bone marrow cells of the two animals. GFP (IM) and YFP (IV) marking levels of bone marrow cells from non-injected bone were 24.2% and 33.9%, respectively, at 1 month, 7.9% and 4.6% at 3 months, 19.1% and 12.6% at 6 months after transplantation in M00228. In addition, the GFP marking of the bone marrow cells from the injected bone was higher than that of the BM cells from non-injected bone while YFP marking level was similar. In conclusion, our data suggest that direct intramarrow injection of CD34+ cells may lead to improved engraftment of long-term repopulating cells. Clonal analysis is currently under way to determine the clonal pattern of the differentially marked repopulating cells.
New Aspects on the Safety of Multidrug-Resistance 1 Gene Transfers: No Indication for Clonal Dominance after Long-Term Follow-Up in a Primate Transplantation Model.
Authors: Bozorgmehr Farastuk , Buss Eike C. , Laufs Stephanie , Nagy K. Zsuzsanna , Sellers Stephanie , Zeller W. Jens , Dunbar Cynthia E. , Fruehauf Stefan
Published: October 2019

It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking.

One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites.

In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive.

By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector.

We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.
PDC-E2, a Common Auto Antigen in Primary Biliary Cirrhosis (PBC) Is Also a Target of an Antibody Response in Patients Who Achieve Complete Remission after Donor Lymphocyte Infusion.
Authors: Bellucci Roberto , Gallagher Meagan , Nguyen Hong-Nam , Alyea Edwin P. , Zorn Emmanuel , Anderson Kenneth C. , Munshi Nikhil C. , Ritz Jerome
Published: October 2019

The ability of donor lymphocyte infusions (DLI) to induce complete responses (CR) in patients with relapsed multiple myeloma (MM) following allogeneic bone marrow transplantation provides clear evidence of graft-versus-myeloma (GVM) immunity. To identify GVM associated target antigens, we previously screened an MM cDNA expression library with post DLI serum from 5 MM patients who achieved CR after DLI. One of the antigens identified in the screening was dihydrolipoamide acetyltranferase the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Using a phage plate assay, antibody reactivity against PDC-E2 was found in 2 of 9 MM and 1 of 5 chronic myeloid leukemia (CML) patients who achieved a CR after DLI. No antibodies were found before DLI, in 20 normal donors, 10 patients who underwent T-cell depleted allogeneic BMT, 6 MM DLI non-responders and 20 patients with chronic GVHD. Primary biliary cirrhosis (PBC) is an autoimmune liver disease in which more than 80% of patients have autoantibodies against PDC-E2. In patients with PBC the major immunogenic epitope of PDC-E2 has been mapped to the region associated with the inner lipoyl domain. To better characterize the antibody response in MM patients who had a CR after DLI a GST-PDC-E2 fusion protein was purified and used to quantify the antibody response by ELISA. Using this sensitive assay we found 1 additional MM DLI responder who had antibody reactivity against PDC-E2. Analysis performed at serial time points after DLI showed that reactivity persisted for 1.5 and 3 years after DLI in two MM patients and 3 years after DLI in the CML DLI responder. To map the antibody response after DLI and compare this to antibody reactivity in patients with PBC, we synthesized a series of 85 overlapping peptides covering the entire length of PDC-E2 and analyzed the specificity of the antibody response by ELISA. Using this assay, post-DLI serum was found to be reactive against 4 peptides located in the E2 catalytic domain of PDC-E2 but no reactivity was detected against peptides located in the inner lipoyl domain of the protein, the region commonly recognized by auto-antibodies in PBC. Analysis of serial samples showed that the antibody response persisted against these peptides up to 2–3 years after DLI but no reactivity was found pre-DLI and in normal serum. In conclusion we show that PDC-E2, a common auto-antigen in the autoimmune disease PBC, is also the target of an antibody response in patients with MM and CML who achieve a CR after DLI. The antibody response found after DLI is directed against a different region of the protein. Further studies characterizing T cell epitopes in these patients are underway and they will help to better characterize the immune response against PDC-E2 in patients after DLI.
A Comparison of Acute GVHD after HLA-Haploidentical and HLA-Identical Stem Cell Transplantation.
Authors: Hill Wolfgang , Tischer Johanna , Bigalke Iris , Rieger Christina , Ledderose Georg , Kolb Hans-Jochem
Published: October 2019

Acute graft-versus-host disease (aGVHD) is a major obstacle of allogeneic stem cell transplantation (SCT). We compared a cohort of 58 patients with HLA-haploidentical transplants (haplo) and a contemporary group of 229 patients with HLA-identical transplants (id) for the manifestation of aGVHD. Haplo-patients were given unmodified marrow (bm) and CD6- depleted mobilized blood cells (mbc) 6 days after marrow transplantation. The combination of cyclosporin A (CSA) and a short course of methotrexate (sMTX) was given post-grafting. Standard intensity conditioning was given to 34 and reduced intensity conditioning to 24 patients. Id-patients were given bm in 140, mbc in 79 and the combination of bm and mbc in 6 cases. Post grafting immunosuppression consisted of CSA either alone (N=10) or in combination with sMTX (N=158), and/or mycophenolate mofetil (MMF) (N=57). Conditioning was of reduced intensity in 50 patients and standard in 175 patients. Haplo-patients were younger in age (34 vs. 44 yrs.), more frequently male and in a more advanced stage of their disease. Manifestations of aGVHD, microangiopathy characterized by schistocytes and elevated LDH and virus infections were evaluated. Haplo-patients had more severe aGVHD of the skin than id-patients (IBMTR index B – D: 53% vs. 37%; p<0.02). Response to the treatment with corticosteroids was better in haplo-patients than in id-patients (74% vs. 52%; p =0.048). Extensive chronic GVHD was less frequent in haplo-patients than id-patients (14% vs. 39%; p=0.005). However virus infections requiring virostatic therapy were more frequent in haplo-patients (60% vs. 40%; p=0.03). Modification of aGVHD has been achieved by transfusion of CD6-depleted mbc 6 days after transplantation of unmodified bm. CD6-depleted mbc contain non-specific suppressor cells. Improved depletion has eliminated severe aGVHD (IBMTR C & D) completely. However immune deficiency and recurrent viral infections remain a problem.