bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP Binding Domain Mutants of BCR-ABL in Patients with Chronic Myeloid Leukemia Treated with Imatinib.
Authors: Liu Xiaoli , Zhang Song , Du Qingfeng , Fan Wei , Wang Yu , Yao Feng
Published: October 2019

Objective:Imatinib (also called STI571 or Gleevec), a competitive inhibitor at the ATP binding site of BCR-ABL, has been shown remarkable clinical activity in patients with chronic myeloid leukemia (CML). However, a significant proportion of Imatinib-treated CML patients with advanced stage disease develop resistance. In this study, we try to detect ATP binding domain mutants of BCR-ABL in Imatinib-treated CML patients and explore the possibility that ATP binding domain mutants might confer resistance to imatinib.

Patients and Methods:We analyzed bone marrow samples from 16 Imatinib-treated CML patients, including 6 chronic phase, 2 accelerated phase (AP) and 8 blast crisis (BC) patients. 8 patients were sensitive to imatinib and 8 patients resistant. A polymerase chain reaction strategy was used to amplify and sequence the ATP binding domain of BCR-ABL.

Results:Point mutations were found in the ATP binding domain of BCR-ABL in 3 of 16 patients. In 2 of 8 imatinib-resistance patients, a single nucleotide change was detected and resulted in a threonine to isoleucine substitution at position 315(Thr315Ile) of ABL kinase. This point mutation has proved to interfere with drug binding and cause resistance to imatinib. In addition, we found a mutation (Lys357Arg) remain sensitive to imatinib.

Discussion:Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by the malignant expansion of bone marrow stem cells. Its malignant clonal marker is Philadelphia chromosome (Ph) which harbors the BCR-ABL fusion gene. The latter encodes a chimeric BCR-ABL protein, identified as having a central role in the pathogenesis of CML. As a competitor for ATP binding of BCR-ABL, Imatinib selectively induces apoptosis and blocks proliferation in BCR-ABL positive cells and has been shown to have high activity in CML. Clinical studies demonstrate that Many Imatinib-treated CML patients with advanced stage disease respond initially but then relapse. Till now, the resistant mechanisms of imatinib have not all been known. In this study, we find that ATP binding domain mutants of BCR-ABL is one of the potential mechanisms of resistance to imatinib, but not all mutants associate with imatinib resistance and some mutants remain sensitive to imatinib.
Hematopoietic Precursor Cell Cultures (CFU-C) in Diagnosis and Prognosis of Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML).
Authors: Bernimoulin Michael Pierre , Jotterand Martine , Tichelli Andre , Meyer-Monard Sandrine , Heim Dominik , Gratwohl Alois , Nissen Catherine
Published: October 2019

Background

Diagnosis and prognosis of patients with established MDS/CMML is currently based on WHO criteria and the IPSS prognostic Index. High variation of survival in subgroups warrants the search for additional criteria. A comparison of CFU-C cultures with WHO criteria and IPSS score has not yet been done in a large patient group.

Patients and methods

We analyzed in a single center retrospective cohort study 93 untreated consecutive patients (55 male/ 38 female; median age: 66 years; range: 13 – 88 years) admitted between July 1992 and June 2002 and diagnosed as MDS/CMML (RA/RARS(4), MDS-U (2), RCMD (26), RAEB I/II (44) and CMML I/II (17). All patients had an unequivocal diagnosis of MDS or CMML according to WHO criteria. Simultaneous examinations of blood, bone marrow (cytology and biopsy), BM-cytogenetics and BM-and PB cultures for CFU-GM and BFU-E were done. Culture results were scored blindly and classified either as “Low risk CFU-C“ including normal growth (N=2), no colony growth (N=6) or reduced growth of normal colonies (N=19) or as „High risk CFU-C’s“ including excess normal growth in PB termed “MPS pattern” (N=5), discrete leukemic cluster growth (N=22), abundant leukemic cluster growth (N=14) or the „CMML pattern“ defined as giant“ pseudonormal“ CFU-GM and strongly reduced BFU-E (N=23). Culture score was compared with the WHO diagnosis and with the IPSS score in all 93 patients and survival was assesed in 82 patients treated with conventional therapy (12 allografted patients were excluded) after minimal observation time of 3 years in July 2005.

Results

Comparison of WHO diagnosis with culture score Low risk CFU-C score High risk CFU-C score RA/RARS/RCMD/MDS-U 15 16 RAEBI/II 12 30 CMML-I/II 0 17

The typical CMML pattern was observed in 13 of 17 patients with CMML (Specificity 94%).

Comparison of IPSS Score with culture score low risk CFU-C score low risk CFU-C score High risk CFU-C score High risk CFU-C score IPSS Score alive/dead mean survival(d) alive/dead mean survival(d) Low and Int-1 4/4 2022+/−543 8/39 965+/−143 Int-2 and High 1/1 1024+/−701 1/26 565+/−92

Mean survival time for patients with a low/intermediate-1 IPSS score was less than half if they had a high risk CFU-score (p<0.01, ANOVA t-test). For patients with an intermediate-2/high IPSS score the prognostic information gained with the cultures was similar, but low patient number did not permit statistical analysis.

Conclusions

Hematopoietic precursor cultures yield additional information in patients with MDS / CMML. For diagnosis, CMML has a typical growth pattern, that may help distinction of clonal from reactive monocytosis. For prognosis, the culture pattern adds information to the IPPS score, leukemic growth being strongly predictive of short survival.
Telomerase and Telomeres in Polycythemia Vera and Essential Thrombocythemia.
Authors: Gallamini Andrea , Ferraris Anna Maria , Mattei Daniele , Pujic Natalija , Mangerini Rosa , Rapezzi Davide , Tavera Sivia , Gaetani Gian Franco
Published: October 2019

Polycythemia Vera (PV) and Essential Thrombocythemia (ET) are chronic myeloproliferative disorders (CMPD) with a relatively favorable prognosis and a long natural history. They originate from a multipotent progenitor cell with dominance of the transformed clone over normal hematopoiesis. However, the heterogeneity of these diseases with respect to clonal development from a common progenitor has been established, and no specific molecular or cytogenetic markers are so far considered useful prognostic indicators. In the current study we correlate telomerase activity (TA) and telomeres [terminal restriction fragment (TRF)] lengths in granulocytes (PMN) and mononuclear cells (MNC), with the results of the clonality status, as determined by investigation of X chromosome inactivation patterns (XCIPs), from 22 informative female patients with ET and PV. All patients were in chronic phase without immature myeloid cells in the peripheral blood. After testing for heterozygosity at the HUMARA locus PMN expressed a monoclonal XCIP in 9 out of 13 subjects with ET (69%), and in 6 out of 9 with PV (67%). MNC were always polyclonal. The amount of TA for each reaction was expressed in Total Product Generated (TPG) units. ΔTRF length was calculated by subtracting the telomer length of PMNs from that of MNCs and expressed as kilobases. TA and ΔTRF results were blindly compared with those from XCIP analysis. Monoclonal PMN displayed a TA ranging between 47.6 and 212.5 TPG units (mean 118.2 ± 45.5 SD), whereas polyclonal PMN had a TA between 1.0 and 33.3 TPG units (mean 14.4 ± 11.9 SD) (p<0.001). MNC had a TA similar to that of polyclonal PMN. Mean ΔTRF of clonal PMNs was 4.6 kilobases, while that of polyclonal PMNs was 0.44 kilobases (p<0.001). PMNs from normal control subjects had a mean ΔTRF of 0.1 kilobases, similar to that of polyclonal PMNs (p= n.s.). The results of our study clearly show the existence of two distinct subgroups of CMPD, with polyclonal or monoclonal granulopoiesis, each of them correlating with a peculiar pattern of TA and telomere lengths. Although none of the patients analyzed showed any difference in clinical characteristics with respect to the parameters considered, we believe that TA and ΔTRF analysis and their relationship with clonality status may represent an useful tool for further investigation, to predict survival in larger cohorts of patients with PV and ET, over an extended period of follow up.
A Comparison of Zap-70 by Immunohistochemistry and Flow Cytometry Using Two Different Flow Analysis Methods.
Authors: Hertzberg Lawrence , Baggianni Karina , Tamayo Rosalba , Browne Patrick , Oldaker Teri
Published: October 2019

Zap-70 expression by flow cytometry has been shown to be a surrogate marker for un-mutated/germline IgH status in chronic lymphocytic leukemia (CLL).1 Reproducibility and confidence in the accuracy of results can make this assay problematic. We compared our current single-parameter/one-color, immunohistochemistry (IHC) Zap-70 assay with our flow cytometry Zap-70 assay. Twenty-one (21) patient samples submitted to our lab for Zap-70 testing were assayed by IHC and flow cytometry (FC). In addition, two FC analysis methods were used. One analysis method uses isotype controls to establish negative regions and a 20% cut-off for positivity1, with BD CellQuestTM software (CQ). The other analysis method uses internal negative and positive mean channel fluorescence (MCF) differences using BD Paint-A-GateTM software (PAG). The IHC assay resulted in nine positive, six negative and six equivocal results for Zap-70. The equivocal results were due to very low proportions of CLL cells (three cases) or other problems identified on review of stained slides (three cases). The FC assay resulted in six positive, thirteen negative and two equivocal results for both analysis methods. When comparing IHC Zap-70 and FC Zap-70, there was 29% (6/21) discordance using the CQ analysis and 24% discordance (5/21) using the PAG analysis (equivocal cases included in the denominator only). Comparison of the CQ and PAG analysis demonstrated 16% (3/19) discordance (equivocal cases excluded from the denominator). The FC Zap-70 PAG analysis method correlated somewhat better with the IHC Zap-70 results than did the FC Zap-70 CQ analysis method. This may be due to the variability introduced by user-defined/isotype cursor settings. We conclude that Zap-70 expression can be tested for using either IHC or FC, and that IHC testing may have higher sensitivity. Correlation between the results utilizing these different methods is not ideal, and improvement in both assays, likely including flow analysis methods, is needed. We plan additional, comparative evaluation of more cases.

CellQuest™ and Paint-A-Gate™ are trademarks of Becton, Dickinson and Company.
The Role of Proteasome Inhibitors and the Trail Apoptotic Pathway in the Treatment of Chronic Lymphocytic Leukemia.
Authors: McCrea Kristin E. , Kabore Albert , Johnston James B. , Gibson Spencer B.
Published: October 2019

TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) triggers the TRAIL apoptotic pathway selectively in tumour cells by binding to two death receptors, DR4 and DR5, that are present on the surface of many cancer cells. It is effective at inducing apoptosis in a variety of haematological malignancies, such as multiple myeloma. However, chronic lymphocytic leukemia (CLL) cells are relatively resistant to TRAIL-induced apoptosis. We have previously shown that the chemotherapeutic drugs, fludarabine and chlorambucil, increase the cell surface expressions of DR4 and DR5, and give synergistic apoptotic responses when combined with TRAIL. Proteasome inhibitors, that are used in the treatment of multiple myeloma, also upregulate TRAIL and its death receptors in CLL cells but not in normal B cells. Herein we have determined that proteasome inhibitors are effective at inducing apoptosis in CLL cells, and that the activation of the TRAIL apoptotic pathway contributes significantly to proteasome inhibitor cytotoxicity. Combining proteasome inhibitors with TRAIL enhanced apoptosis in CLL cells by approximately 15% over treatment with the proteasome inhibitor alone. Another novel approach to trigger the TRAIL apoptotic pathway is to use activating monoclonal antibodies directed against DR4 and DR5. Similar to TRAIL, we demonstrated that when used alone, the monoclonal antibodies were minimally cytotoxic against CLL cells. Proteasome inhibitors in combination with activating monoclonal antibodies against DR4 or DR5 increased the amount of apoptosis in CLL cells. Cell death was enhanced by 23% and 17% when proteasome inhibitors were combined with monoclonal antibodies against DR4 and DR5, respectively. While proteasome inhibitors may have a potential role in CLL treatment as single therapy, they are also cytotoxic to normal B cells. However, when activating monoclonal antibodies against TRAIL death receptors are given in combination with proteasome inhibitors, that upregulate DR4 and DR5 expression, the anti-tumor specificity and cytotoxicity of these agents may be increased in CLL.
Monoclonal Gammopathy of Undetermined Significance (MGUS): Predictors of Outcome and Recognition of an Evolving Type.
Authors: Rosiñol Laura , Bladé Joan , Cibeira Ma Teresa , Montoto Silvia , Esteve Jordi , Rozman Maria , Fililla Xabier , Montserrat Emili
Published: October 2019

MGUS occurs in up to 3% of persons older than 50 years and the probability of malignant evolution is 1% per year. The risk of progression from MGUS to a symptomatic monoclonal gammopathy has been investigated in a number of series. However, only the presenting features have been considered while the evolutive pattern during the first years of follow-up has not been taken into account. The aim of our study was to investigate the predictors of outcome considering both the initial features and the pattern of evolution of the M-protein size during the first three years in a large series of patients with MGUS with long follow-up. Three-hundred and fifty nine patients (160 M, 199 F; median age 66 yrs) diagnosed with MGUS at a single institution were included in the study. Patients who showed an inequivocal increase in their M-protein size at the serum electrophoresis during the first three years of follow-up were considered as evolving while the remainders were considered as non-evolving. In the overall series the median values for serum M-protein level and for the proportion of bone marrow plasma cells (BMPC) were 14.8 g/L and 4%, respectively. Three hundred an thirty patients had a non-evolving MGUS while 29 fulfilled the criteria for the evolving type. The M-protein size and the proportion of BMPC at diagnosis were similar in both types of MGUS. The IgG type was more frequent in the non-evolving (65% vs. 45%, p=0.005) whereas the IgA and IgM type were more frequent in the evolving type (55% vs. 32%, p=0.04). Overall, 32 patients developed malignant transformation after a median follow-up of 93 months. Fourteen of the 29 (48%) evolving patients developed symptomatic multiple myeloma while only 18 out of the 330 (5%) remaining patients with non-evolving MGUS progressed to a malignant condition. The progression rate at 10 and 20 years of follow-up for the evolving and the non-evolving types was 55% vs. 10% and 80% vs. 13%, respectively. No patient in the non-evolving type evolved to a malignant condition after the first 12 years of follow-up. The predictors for malignant transformation at the univariate analysis were: the type of MGUS (evolving vs. non-evolving, p<0.001), a proportion of BMPC higher than 5% (p=0.001), the immunoglobulin isotype (IgA vs. IgG, p=0.007) and the M-protein size greater than 15 g/L (p=0.05). At the multivariate analysis the features significantly associated to a higher risk of progression were: the evolving type (RR 12.1, p<0.001,), the IgA type (RR 2.9, p=0.006) and the M-protein concentration (RR 2.2, p=0.044).

Conclusions: The evolutive pattern of the serum M-protein during the first years of follow-up is the most important risk factor for progression in patients with MGUS.
Anti-Thymocyte Globulin (ATG) Induces Apoptosis in Myeloma Cells: A Basis for Myeloma Sero-Therapy?.
Authors: Ayuk Francis A. , Zander Axel R. , Kroeger Nicolaus
Published: October 2019

Allogeneic stem cell transplantation is a potentially curative therapy for patients with multiple myeloma. Polyclonal ATG is included in conditioning regimens to enhance engraftment and reduce the risk of severe graft-versus-host disease. Since ATG has been reported to induce depletion of T-, B- and dendritic cells, we sought to investigate its cytotoxic activity on myeloma cells. Complement-mediated and complement-independent activity of ATG-Fresenius was investigated on 4 myeloma cell lines (RPMI-8226, U266, KMS-12-BM and EJM) and bone marrow samples from 6 myeloma patients. Cytotoxicity was determined by staining with annexin V-FITC and 7AAD followed by flow cytometry. ATG at clinically relevant concentrations induced up to 100% and 85% complement-dependent killing of myeloma cell lines and primary myeloma samples respectively. In the absence of complement ATG still induced up to 50% and 80% apoptosis in myeloma cell lines and primary myeloma samples respectively. Preincubation of myeloma cells with a general caspase inhibitor (ZVAD-fmk) abrogated ATG-induced complement-independent cell death. Absorption assays indicate that ATG induced cytotoxicity is mediated by specific antibodies and antigens whose further elucidation may pave the way for antibody-based myeloma therapy.
BCR-ABL1 Disrupts SDF-1-Dependent Hematopoietic Cell Migration and Adhesion through the LFA-1 Integrin-Mediated Mechanism.
Authors: Chen Ying-Yu , Malik Mobeen , Tomkowicz Brian E. , Collman Ronald G. , Ptasznik Andrzej
Published: October 2019

Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, are essential for normal stem/progenitor cell movement, adherence, and retention within the bone marrow environment. Two mechanisms through which BCR-ABL1 are thought to disrupt CXCR4-mediated chemotactic responses have been described in leukemia:

the inhibition of CXCR4 expression (Geay et al. 2005, Cancer Res.) and intra-cellular signaling defects without modification of CXCR4 expression (Salgia et al. 1999, Blood; Ptasznik et al. 2002, J. Exp. Med.).

These opposing mechanisms suggest that the actual situation is more complex and that new signaling paradigms are needed. To address this, we studied the effects of BCR-ABL1 on SDF-1-dependent, integrin-mediated, migration and adhesion of hematopoietic precursors. Stimulation of BCR-ABL1(−) hematopoietic cells with SDF-1 showed reduced cell adherence to surfaces coated with ICAM-1 (a ligand for the LFA-1 integrin), which was associated with down-regulated expression of activation-dependent epitopes of the β2 integrin, LFA-1, on hematopoietic cells. Inhibition of Lyn expression with siRNA prevented the SDF-1-triggered down-regulation of LFA-1 and cell adherence, indicating that CXCR4 inhibited the function of LFA-1 through Lyn. Expression of BCR-ABL1 in these cells resulted in increased expression of activation-dependent epitopes of LFA-1 and prevented SDF-1-dependent regulatory effects on both LFA-1 affinity and ICAM-1 adherence. Also, expression of BCR-ABL1 prevented Lyn-mediated regulation of cell adhesion to ICAM-1 as well as Lyn-mediated regulation of LFA-1 affinity. These results indicate that BCR-ABL1 constitutively increases the affinity of the LFA-1 integrin to its ligand ICAM-1, locking the integrin into an “active” conformation. The net result is the loss of responsiveness of LFA-1 to SDF-1-induced ‘inside-out’ signaling involving CXCR4 and Lyn kinase. Because in our experiments BCR-ABL1 had no significant effect on the expression of CXCR4 in Mo7e cells, transfected with low and high amounts of p210-BCR-ABL, or in primary BCR-ABL(+) cells from CML blast crisis patients (n=3), we conclude that BCR-ABL1 inhibits CXCR4-triggered ‘inside-out’ integrin signaling rather than CXCR4 expression. Taken together, we propose that BCR-ABL1 disrupts the signaling link between the chemokine receptor, CXCR4 and the β2 integrin LFA-1 so as to inhibit normal SDF-1-mediated chemotaxis and adhesion in hematopoietic cells.
Gain-of-Function Mutations of GATA-2 in Acute Myeloid Transformation of Chronic Myeloid Leukemia.
Authors: Zhang Su-Jiang , Ma Li-Yuan , Huang Qiu-Hua , Li Guo , Gu Bai-Wei , Gao Xiao-Dong , Shi Jing-Yi , Wang Yue-Ying , Gao Li , Cai Xun , Ren Rui-Bao , Chen Zhu , Chen Sai-Juan
Published: October 2019

Acquisition of additional genetic and/or epigenetic abnormalities other than BCR/ABL fusion gene is believed to cause disease progression in chronic myeloid leukemia (CML) from chronic phase to blast phase. To gain insights into the underlying mechanisms, we screened DNA samples from CML patients during acute transformation for alterations in a number of transcription factor genes crucial to myeloid-lymphoid development. In 85 cases of CML blast transformation, we identified two new mutations in the coding region of GATA-2, a negative regulator of hematopoietic stem/progenitor cell differentiation. L359V within zinc finger domain (ZF) 2 of GATA-2 was found in 8 cases with myelo-monoblastic features, while an in-frame deletion of six amino acids (D341–346) across the border of ZF1 was detected in 1 patient at blast crisis with eosinophilia. Further studies showed that L359V not only increased transactivation activity, but also enhanced inhibitory effects on the major myelopoietic regulator PU.1. Consistent with the myelo-monoblastic features of CML patients with GATA-2 L359V mutant, transduction of GATA-2 L359V mutant into HL-60 cells or BCR/ABL-harboring mouse model disturbed myelo-monocytic differentiation/proliferation in vitro and in vivo, respectively. These data suggest that GATA-2 mutations may be involved in acute myeloid transformation in some CML patients.
The Plk-1 Inhibitor BI 2536 Counteracts Proliferation and Viability of CML Cells and Synergizes with Imatinib and Nilotinib (AMN107) in Producing Growth Inhibition.
Authors: Gleixner Karoline V. , Ferenc Veronika , Gruze Alexander , Kneidinger Michael , Baumgartner Christian , Mayerhofer Matthias , Pickl Winfried F. , Sillaber Christian , Valent Peter
Published: October 2019

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease in which BCR/ABL enhances growth and survival of leukemic cells. In most patients, the disease can be kept under control by the BCR/ABL tyrosine kinase inhibitor imatinib (STI571; Novartis Basel, Switzerland). However, resistance or intolerance against imatinib may occur during therapy. Therefore, current research is focusing on novel targets and targeted drugs in CML. Polo-like kinase 1 (Plk-1) is a serine/threonine kinase that plays an essential role in mitosis and is expressed in activated/phosphorylated form in various malignancies including acute myeloid leukemia (AML). BI 2536 (Boehringer Ingelheim GmbH, Germany) is a novel selective inhibitor of Plk-1, that is currently tested in AML-trials. In this study, we have evaluated expression and the potential role of Plk-1 as a novel target in CML cells. As assessed by PCR, Plk-1 mRNA was found to be expressed abundantly in primary CML cells and in the CML cell line K562, whereas normal peripheral blood cells did not express detectable levels of Plk-1 mRNA. The Plk-1 protein was detected in primary CML cells and K562 cells by immunocytochemistry. In consecutive experiments, we were able to show that CML cells display phosphorylated Plk-1. As assessed by 3H-thymidine-uptake experiments, BI 2536 was found to inhibit the proliferation of K562 cells in a dose-dependent manner (IC50 5–15 nM). Moreover, BI 2536 was found to inhibit the proliferation of both imatinib-naive (n=6) and imatinib-resistant (n=3) primary CML cells (IC50: 1–15 nM). The growth-inhibitory effect of BI 2536 on CML cells was found to be associated with mitotic arrest, a G2-M cell cycle arrest, and consecutive apoptosis. In normal bone marrow or peripheral blood mononuclear cells, neither mitotic cell arrest nor apoptosis were observed after exposure to BI 2536. In further experiments, primary CML cells were coincubated with BI 2536 plus imatinib or with BI 2536 plus nilotinib (AMN107; Novartis) at fixed ratio of drug concentrations. In these experiments, BI 2536 was found to synergize with both tyrosine kinase inhibitors in counteracting the proliferation of CML cells. In conclusion, our data show that Plk-1 is expressed in activated form in CML cells and plays a role in cell cycle progression and cell viability. Targeting Plk-1 with BI 2536 leads to mitotic arrest, growth inhibition, and apoptosis in imatinib-naive and imatinib-resistant leukemic cells. Moreover, BI 2536 synergizes with imatinib and nilotinib in counteracting the growth of neoplastic cells in CML. Targeting of Plk-1 may be a novel interesting pharmacologic approach to counteract growth of CML cells.
Safety and Efficacy Study of Subcutaneous Homoharringtonine (SC HHT) in Imatinib (IM)-Resistant Chronic Myeloid Leukemia (CML) with the T315I Mutation - Initial Report of a Phase II Trial.
Authors: Khoury H. Jean , Michallet Mauricette , Corm Selim , Roy Lydia , Jones Dan , Hochhaus Andreas , Kantarjian Hagop , Benichou Annie-Claude , Schwartz Richard , Cortes Jorge
Published: October 2019

HHT [USAN/INN designation - omacetaxine mepesuccinate] is an ester of cephalotaxine with activity in CML independent from tyrosine kinase (TK) inhibition. HHT is also active against Ba/F3 cell lines transfected with various BCR-ABL kinase domain (KD) mutants, including the T315I mutation. BCR-ABL positive cells with the T315I mutation are resistant, both in vitro and in vivo, to IM, as well as 2nd generation TK inhibitors (TKIs), such as dasatinib, nilotinib and bosutinib. Given the lack of active agents in this unique patient (Pt) population, we are conducting a multicenter open-label phase II trial evaluating safety and efficacy of SC HHT in Pts with IM-resistant T315I+ CML in all phases of the disease. The presence of T315I is confirmed at 2 reference labs. Peripheral blood BCR-ABL transcript levels are determined during HHT therapy by qRT-PCR and BCR-ABL KD mutation analyses. Induction consists of repeated cycles of HHT, 1.25 mg/m2 twice daily SC for 14 consecutive days every (q) 28 days until complete hematologic response (CHR) or hematologic improvement. Pts can receive maintenance HHT, 1.25 mg/m2 twice daily SC for 7 days q 28 days, until progression or for up to 24 mo. To date, 19 Pts with Ph+ CML with T315I have been enrolled, 11 in chronic (CP), 4 in accelerated (AP), 4 in blast (BP) phase. Median age is 56 yrs (23 to 79). Pts have failed a median 2 (1 to 8+) prior therapies, including IM (19), dasatinib (10), nilotinib (8), MK-0457 (3), ara-C (5), anthracyclines (3), interferon (2). Median T315I transcript level at baseline is 50% (20–100%). All Pts have completed at least one induction course of HHT, 10 Pts completed 2 courses, 2 Pts 3, and 1 Pt 4. Five Pts are on maintenance HHT (1–7+ mo). SC HHT is well tolerated; no Pt has discontinued treatment due to toxicity. Hyperglycemia, cardiac arrhythmia, and hypotension have not been reported, and local injection reactions are uncommon. One Pt experienced acute coronary syndrome due to HHT-induced anemia. Cycle 2 treatment delays due to neutropenia and/or thrombocytopenia have occurred in 7 of 14 Pts (1–41 days). 4 Pts have experienced progressive disease [1 in CP (splenomegaly, ↑BCR-ABL), 1 AP, 2 BP] after 1–2 induction cycles and were taken off study. T315I transcript levels are no longer detectable in 1 AP and 4 CP Pts, 2 of whom have achieved CHR (1 recently achieved, 1 maintained 7+ mo). In summary, current data from this phase II trial indicate that HHT has an acceptable safety profile and activity in Pts with IM-resistant CML. The SC route offers a convenient dosing format for self administration. Additional enrollment and longer follow-up will determine whether HHT provides a therapeutic option in highly-resistant CML Pts with T315I BCR-ABL KD mutation who have failed one or more TKIs.
Reduced Incidence of Severe Acute GVHD in Patients Transplanted from Donors with IL23R (Arg381Gln) Gene Variant.
Authors: Elmaagacli Ahmet H. , Koldehoff Michael , Steckel Nina K. , Beelen Dietrich
Published: October 2019

Background. Recently, a gene polymorphism of the IL23R gene (Arg381Gln), which is a subunit of the receptor of the pro-inflammatory cytokine interleukin-23, was found to be strongly associated with Crohn’s disease (Duerr et al.Science 2006). The exchange of arginine with glutamine at position 381 of IL23R causes a blockade of the IL-23 signaling pathway, which confers strong protection against Crohn’s disease. Because IL-23 activity is participated in autoimmune diseases, we hypothesized that it might also be important in the development of GVHD reaction post allogeneic transplantation.

Methods .Here we evaluated 401 patients with their donors for the occurrence of the IL23R gene variant (Arg381Gln, 1142 G>A) by real-time PCR who underwent allogeneic transplantation.

Results. We analyzed the IL23 gene variant 1142 G>A in a cohort of 221 transplant recipients and their HLA-identical sibling donors and in a second cohort of 186 transplant recipients and their HLA- identical unrelated donors. A heterozygous Arg381Gln gene variant was found in 13,1% of patients and in 11,3% of donors. The donor’s IL-23R genotype was significantly associated with a reduced risk of severe acute GVHD in both cohorts for patients after transplant. Analysis of all 407 transplant recipients showed that IL-23R (1142G>A, Arg381Gln) genotype of the donor were associated with a decreased risk of grade II-IV acute GVHD (31.6% compared to 51.0%, p=0.02) and severe acute GVHD III or IV (3.91% compared 23.4%, p=0.003). Death in remission was significantly lower in patients transplanted from donors with variante of IL23-R (11.7% versus 27.7%, p=0.028), whereas overall survival or relapse rates were not influenced significantly by the IL-23R genotype. The multivariate analysis including factors which influence the occurrence of severe acute GVHD as disease stage, age of patients (>or <40 years, gender constellation of recipient and donor, graft source, HLA-status (HLA-identical versus non-identical), donor constellation (sibling versus unrelated) confirmed that the gene variant of IL23R at the donor site confers protection against the occurrence of acute GVHD grade II-IV or severe acute GVHD grade III-IV. The association of IL23R mutation to GVHD was stronger than NOD2 mutations.

Conclusions. The results presented here suggest that IL-23 signaling pathway may play an important role in development of acute GVHD after transplant. Blockade of IL-23 signaling could be a rational therapeutic strategy for GVHD.
Impact of Conditioning on the Outcome of Allografting in Myelofibrosis with Myeloid Metaplasia: Better Survival with Reduced Intensity Approach in Patients ≥50 Years.
Authors: Gupta Vikas , Kroger Nicolaus , Aschan Johan , Leber Brian , Dalley Christopher , Sabloff Mitchell , Lipton Jeffrey H. , Messner Hans , Xu Wei , Brune Mats
Published: October 2019

We analyzed the impact of intensity of conditioning therapy on the outcomes of allogeneic blood or marrow transplantation (BMT) in patients (pts) with myelofibrosis with myeloid metaplasia (MMM). The study included 46 consecutive patients treated at 7 transplant centers between 1998 and 2005. The indications for BMT were intermediate/high risk disease (n=41) or low risk disease with constitutional symptoms (n=5). Donors were matched sibling/family donor (n=25); mismatched family donor, 2; and unrelated, 19. Twenty-three pts received conventional myeloablative (MY) conditioning: Cy/TBI (n=22); Bu/Cy (n=1), and 23 received non-myeloablative (NMA) conditioning [fludarabine (Flu)/PO or IV Bu (n=16); Flu/TBI 200cGy (n=6); others(n=1)]. Median age at BMT of the MY and NMA group was 47 (31–60) and 54 (38–74) years, respectively. NMA group had a higher proportion of pts ≥50 years (74% vs 30%, p=0.003), a longer interval from diagnosis to BMT (p=0.05) and more transplants using blood stem cells (p=0.0002). At a median follow-up of 31 (1–89) months, 8 and 16 patients were alive in MY and NMA groups, respectively. The transplant related outcomes of the two groups are summarized in the table. Serial pre and post BMT bone marrow biopsy results were available in 24 patients. Median days to resolution of fibrosis were 167 and 363 in the MY and NMA groups (p=0.16). In a multivariate analysis, after adjusting for age the survival was better for pts ≥50 years treated with NMA approach (hazard ratio 2.98, 95% CI 1.09–8.12, p=0.03). In conclusion, both MY and NMA approaches have curative potential for pts with MMM. NMA approach is particularly promising in pts ≥50 years.

Table: Comparison of BMT related outcomes for MY and NMA conditioning for MMM Outcomes MY Group (95% C.I.) NMA Group (95% C.I.) P value Neutrophil engraftment (days) 21 (13–46) 18 (1–30) 0.01 Cumulative incidence of graft failure (primary and secondary) at 2-years % 30 (16–58) 23 (10–50) 0.51 Median number of hospital days (Day 0 to +100) 41 (26–85) 27 (8–58) 0.0006 Cumulative incidence of acute GvHD grade II–IV (%) 78 (62–98) 18 (7–45) 0.0002 Cumulative incidence of chronic GVHD at 2-years % 94 (79–100) 45 (27–74) 0.0009 Cumulative incidence of TRM at 2-years % 43 (27–70) 27 (14–55) 0.08 Progression free survival at 2-years % 48 (27–66) 64 (40–80) 0.11 Overall survival at 2-years % 57 (34–74) 68 (45–83) 0.08
A European Phase II Study (BIOV-111) of Clofarabine (Evoltra®) in Refractory and Relapsed Childhood Acute Lymphoblastic Leukemia: Final Results.
Authors: Kearns Pamela , Michel Gerard , Nelken Brigitte , Joel Simon , Al-Ghazaly Essam , Beishuizen Auke , Conter Valentino , Henze Gunter , Baruchel Andre , Saha Vaskar
Published: October 2019

The BIOV-111 study was a European multicentre phase II non-randomised, open-label study of the novel purine nucleoside analogue, clofarabine (Evoltra ®). The study recruited patients ≤ 21 years old at initial diagnosis with primary refractory or relapsed/refractory acute lymphoblastic leukemia (ALL). The dose of clofarabine was 52mg/m2 daily x 5 days, every 28–42 days. Patients were evaluable after one complete course. The primary endpoint was overall response rate (OR) defined as either a complete response (CR) or CR without platelet recovery (CRp). Pharmacokinetic parameters and molecular responses were assessed in a sub-group of cases. The study enrolled a total of 74 patients. 65 were evaluable for response. We report the final efficacy and safety data on these evaluable patients. In total 120 courses of clofarabine were administered to 65 patients from 25 centres. The median age was 10 yrs (range 0.5–23 yrs). The median number of prior treatments was 2 (range 1–5) and 22 patients (34%) had been previously transplanted. The OR was 26% (6 CR, 11 CRp). In addition 1 PR was observed. 11/18 (61%) responders had a prior transplant and 3 of these patients received a further transplant post clofarabine. Of the 7 patients proceeding to transplant post-clofarabine; 3 patients had achieved a CR and 4 achieved a CRp. The updated median duration of response and survival will be presented. The pharamockinetic analyses showed plasma clofarabine concentration did not correlate with treatment outcome, however the ratio of day2/day 1 end of infusion intracellular clofarabine triphosphate (cloTP) levels were higher in responding pts. Serious adverse events included febrile neutropenia 50.8%, hepatic events (raised bilirubin, raised ALT/AST) 6.2%, renal failure 6.2%, palmar-plantar erythrodysaesthesia 4.6% and bone pain 4.6%, seizures 7.7% and cardiac failure 1%. In conclusion, this study demonstrated that clofarabine can achieve significant, durable response rates in heavily pre-treated paediatric patients with relapsed/refractory ALL and intracellular cloTP accumulation may be predictive of response. The response rates in this study are consistent with a previous clofarabine study (CLO-212) in relapsed/refractory pediatric ALL, however in BIOV-111 a lower incidence of adverse events was observed, including hepatic and renal adverse events, possibly attributable to fewer prior treatments compared to CLO-212 (median 2 vs 3 respectively). The reported episodes of seizure and cardiac failure were associated with pre-existing co-morbidities and/or sepsis in the majority of cases. The safety profile of clofarabine was manageable and acceptable and does not preclude subsequent HSCT.
Nasal Epithelial Cells of Donor Origin after Allogeneic HCT.
Authors: Khan Faisal , Lee Robyn , Storek Jan
Published: October 2019

Allogeneic HCT leads to hematolymphatic chimeras. Multiple studies in recent years have reported that stem cells can differentiate into muscle, skin, liver, lung, neurons and epithelium of GI tract after HCT. These studies were performed on recipients of sex-mismatched HCT using XY FISH and are often subject to criticism as XY discrimination may lead to overestimation or underestimation of donor cells due to reasons ranging from fetomaternal chimerism; male blood transfusions and loss of Y chromosome. Moreover, most recipients in these studies were ≤ 1 year post HCT. In the present investigation we determined whether donor epithelial nasal cells can be detected in patients ≥ 5 years post HCT. To avoid above limitations, we used the combination of immunocytochemistry, Laser capture microscopy (LCM) and STR based automated DNA fragment analysis for chimerism quantitation. We collected nasal scrapings and whole blood from 12 allo-HCT survivors (5–22 yrs after HCT; median = 10 yrs). Nasal cells were cytospinned; stained with cytokeratin (CK) and CD45 antibodies and true epithelial cells (CK+CD45−) were laser captured. DNA extracted from the captured cells and blood leukocytes was PCR amplified for a panel of 16 STR markers (ABI-Identifiler™). Genescan analysis of the amplicons was done to quantify chimerism. In all 12 recipients, epithelial cells of donor origin were identified accounting for 3.8% to 12.20% (mean = 7.25%) of the nasal epithelial cells. In conclusion, even with a method obviating the artifacts of XY based differentiation; donor origin epithelial cells were identified. We cannot conclude, whether these cells originate from

hematopoietic stem cells, or epithelial cell precursors transferred with the graft, or recipient epithelial cell fused with donor hematolymphatic cells.

However, as we found only up to 12.20% donor epithelial cells at 5–22 yrs post HCT, it is likely that none of the three mechanisms contribute to the generation of epithelial cells significantly.
Bleeding Events and Factor IX Levels in Female Carriers from a Large Kindred of Hemophilia B.
Authors: Salter John T. , Hardesty Brandon , Heiman Meadow , Sulkin Matthew , Shapiro Amy D. , Greist Anne
Published: October 2019

Introduction: Factor IX (FIX) deficiency (FIXdef) is an X-linked disorder where affected males manifest various hemorrhagic tendencies based on the severity of deficiency. Women who carry the causal mutation exhibit a range of FIX activity levels (FIXact) and may experience bleeding when the FIXact is below the minimum hemostatic level. There is a paucity of reported data regarding the range of FIXact and bleeding events in FIX carriers. We report the FIXact and associated bleeding symptoms in the female carriers of a large kindred of moderate FIXdef caused by a common point mutation (31008 C>T [thr296met]).

Methods: Since 2000, our facility has compiled a database containing information on over 1300 patients with bleeding disorders, including 402 males with FIXdef representing 98 families, 469 confirmed FIX carriers, and 406 at-risk females not yet tested for carrier status. Overall there are 121 FIX carriers with FIX act; this kindred represents 108 documented carriers of which 83 have had FIXact. In this kindred, data on FIXact and bleeding symptoms were collected on carriers. A bleeding symptom questionnaire was developed specific to this population and administered to the target population; at this time questionnaires on 42 of the known carriers is available. Responses on the questionnaire were compared to the FIXact to determine if a correlation existed between reported symptoms/events to FIXact.

Data: For 83 female carriers included in this kindred’s analysis, the mean FIXact was 52% (range 14–114%, median 46%). Of the 42 women completing the bleeding questionnaire, the mean FIXact was 50.2% (range 21–114% median 41%). Typical bleeding events reported included epistaxis, postpartum bleeding, menorrhagia, and easy bruising. By comparing reported bleeding events to FIXact we found that women with FIXact <40% reported increased rate of easy bruising/hematoma formation (9.8% vs. 2.4%) and postoperative bleeding (14.6% vs. 2.4%) compared to women with FIXact >40%. While other bleeding events demonstrated no direct correlation with FIXact, our limited sample available at this time likely impacted this association.

Conclusions: This effort represents the largest report of data from a single kindred of FIX deficiency to date. Female carriers in this kindred have an identical mutation causing moderate FIXdef, yet exhibit a wide range of FIXact; the etiology for this variability is not known and requires further investigation. Ongoing data collection in our FIX carrier population will provide additional information to determine an association between the range of FIXact and bleeding symptoms. FIX carriers require documentation of levels (regardless of the level of deficiency for which the specific mutation codes) and monitoring of symptoms, particularly related to childbirth or surgery, as treatment with exogenous FIX may be required for prevention of hemorrhage and optimal care.
Gene Dosage of the Cell Cycle Regulatory Phosphatases Cdc25C and PP2A Determines Sensitivity to Lenalidomide in del(5q) MDS.
Authors: Wei Sheng , Rocha Kathy , Williams Ann , Chen Xianhong , Burnette P.K. , Djeu Julie Y. , Liu Qing , Byrd John , Sokol Lubomir , Lawrence Nick , Pireddu Roberta , Dewald Gordon , Maciejewski Jaroslaw , List Alan
Published: October 2019

MDS with chromosome 5q deletion [del(5q)] is uniquely sensitive to clonal suppression by lenalidomide (LEN), yielding high rates of cytogenetic and erythroid response. Although haploinsufficiency for a candidate tumor suppressor gene within the common deleted segment (CDS) has been implicated in the pathogenesis of del(5q) MDS, no specific gene products account for the karyotype-dependent LEN sensitivity. Using primary AML cells, we showed that LEN is selectively cytotoxic to del(5q) cells, inducing cell cycle arrest at the G2 > G1 junctures and apoptosis in a concentration-dependent fashion. Now we show that the CDS-encoded Cdc25C and PP2A phosphatases (PPTs) serve as molecular targets for LEN. Cdc25C and PP2A are dual specificity PPTs that regulate cell cycle. The genes encoding the catalytic subunit alpha (PP2ACa), the dominant catalytic isoform, and the regulatory subunit B (beta isoform PR 52) of the PP2A holoenzyme reside within the del(5q) CDS. Real-time RT-PCR showed that Cdc25C and PP2ACa gene expression is significantly reduced in primary del(5q) AML and MDS BM-MNC compared to non-del5q and normal controls. A Cdc25C FISH probe confirmed presence of only a single allele in del(5q) specimens [n=5] vs. normal karyotype [n=5] with no overlap with the EGR1 locus. We found that LEN inhibited rHu-Cdc25C activity with IC90 inhibitory concentrations ≤10nM. Similarly, PP2A PPT activity was inhibited by LEN in enzyme immunoprecipitates from Ramos cells at sub-micromolar concentrations. In U937 cells, we confirmed intracellular Cdc25C and PP2A inhibition, showing concentration-dependent retention of the inhibitory p-Tyr15 -Cdc2 substrates for Cdc25C with corresponding phosphorylation of the PP2A substrate Ser216 Cdc25C, findings reproduced by treatment with the PP2A selective antagonist, fostreicin. Cdc25C immunoprecipitation showed that phosphorylation of Ser216 Cdc25C increased binding to the 14-3-3 protein with consequent cytoplasmic sequestration confirmed by fluorescence microscopy. Phosphorylation of histone H2A.X Ser139 was unchanged after LEN treatment, indicating that phosphorylation of the inhibitory Cdc25C serine does not arise from upstream DNA damage activated kinases. Importantly, siRNA suppression of Cdc25C gene expression in U937 cells promoted LEN-specific apoptosis in 45% of cells, which increased to 68% after fostreicin inhibition of PP2A. These data indicate that haplodeficiency for the Cdc25C and PP2ACa genes promotes selective sensitivity of del(5q) cells to LEN-induced apoptosis. Haplodeficiency of critical drug-able targets may represent a novel strategy for tumor selective therapy.
Erythropoietin (Epo) Secreting Mesenchymal Stromal Cells and Autocrine Signalling Via the Epo Receptor. A Novel, Cell-Based Paracrine Epo Delivery System for Cardiovascular Therapy.
Authors: Copland Ian B. , Jolicoeur Marc E. , Gillis Marc-Antoine , Cuerquis Jessica , Eliopoulos Nicoletta , Annabi Borhane , Calderone Angelo , Tanguay Jean-Francois , Ducharme Anique , Galipeau Jacques
Published: October 2019

Separately, the roles of MSCs and erythropoietin (Epo) in cardiovascular regenerative therapy have expanded rapidly. However, poor in vivo survival/engraftment of MSCs and Epo’s short half-life and bioavailability, limit their individual effectiveness. We propose that a combinatorial approach would be synergisitic. We therefore determined whether administration of Epo-secreting Mesenchymal stromal cells (MSCs) could represent a better therapeutic platform in cardiovascular medicine than MSCs in their primeval form. MSCs from C57Bl/6 mice were retrovirally transduced to express murine Epo (Epo+MSCs) and compared to wild type (WT) MSCs in relation to their abilities to promote positive heart tissue remodelling following myocardial infarction (MI). Relative to WT-MSCs, we found that via an Epo/EpoR autocrine loop, Epo+MSCs have an enhanced proliferative capacity and are more resilient to apoptotic stimuli. In vivo, Epo+MSCs also have enhanced survival and can initiate a more robust host-derived angiogenic response. Injection of WT-MSCs or Epo+MSCs into the borderzone of the MI following ligation of the left coronary artery demonstrated engraftment within the borderzone seven days post-implantation, while sequential (7 and 14 day post-MI) echocardiograms and invasive hemodynamic measurements (14 days post-MI) showed that the Epo+MSC group exhibited significantly improved LV systolic and diastolic function compared to WT-MSCs injected animals and PBS controls (Fractional shortening 29.7% ± 8.5 versus 19.9% ± 6.6 and 19.1% ± 7.4 respectively, p=0.02). Myocardial capillary density was significantly higher in the Epo+MSC group and was coupled to reduced neutrophilic infiltration by direct actions of Epo on endothelial and neutrophil chemotaxis. In conclusion, we show that Epo overexpression enhances the cellular regenerative properties of MSCs by both autocrine and paracrine pathways.