bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Splicing Factor RBM25 in Erythroid Differentiation.
Authors: Zhou Anyu , Cho Aeri , Huang Shu-Ching , Benz Edward J.
Published: April 2020

Many protein factors that guide pre-mRNA modification pathways are composed of RNA recognition motif (RRM) domains. In an effort to identify splicing regulators in erythroid cells, we cloned a RRM-containing protein RBM25 and characterized its role in alternative splicing. RBM25 consists of a proline-rich region and a RRM domain at the amino-terminal end, an ER rich domain at the central region, and a PWI domain at the carboxyl terminal end. RBM25 partially co-localized with splicing factor SC35 in nuclear speckles. While both the RRM and PWI domains are diffusely distributed in the nucleoplasm, the ER domain is highly concentrated in nuclear speckles. We examined cellular mRNA targets of RBM25 in HeLa cells and demonstrated that it binds Bcl-x mRNA and affects its alternative splicing. Depletion of RBM25 by RNA interference caused accumulation of anti-apoptotic Bcl-x(L), whereas its up-regulation increased the levels of pro-apoptotic Bcl-x(s). The expression level of RBM25 also correlated with the degree of cell death, further suggesting that it functions in regulating apoptotic factor(s) expression. Apoptosis plays an important role in red cell development; earlier erythroid progenitors are more sensitive to apoptosis while mature erythroblasts are resistant to apoptosis. A significant decrease in RBM25 expression occurs during erythroid differentiation and correlates with resistance to apoptosis. Our results suggest that erythroblasts may acquire resistance to apoptosis during maturation through differential expression of crucial splicing regulators of the apoptotic machinery.
Localization of Factor VIII Interactive Site within Plasmin/Plasminogen Which Is Responsible for Plasmin-Catalyzed Activation/Inactivation of Factor VIII.
Authors: Nishiya Katsumi , Nogami Keiji , Okada Kiyotaka , Matsuo Osamu , Takeyama Masahiro , Ogiwara Kenichi , Tanaka Ichiro , Yoshioka Akira , Shima Midori
Published: April 2020

Plasmin (Plm), an active form of plasminogen (Plg), not only functions as a key enzyme in the fibrinolytic system, but also directly inactivates several coagulation factors. Especially, factor VIII is inactivated by Plm immediately after the activation by limited proteolytic cleavage at Lys36, Arg336, Arg372, and Arg740 in the heavy chain, and at Arg1689 and Arg1721 in the light chain (Nogami et al. J. Biol. Chem. 2007, 282, 5287). We recently have identified the plasmin-interactive sites on the A2 domain responsible for cleavages at Arg336 and Arg372, and on the light chain responsible for cleavage at Lys36 (Abst #1991/1709, BLOOD 102/108, 2005/2006). In the present study, we attempted to localize a factor VIII-interactive site on Plm (and Plg). Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of lysine-binding site (LBS) of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the light chain binding to active-site modified Plm (anhydro-Plm), whilst inhibited weakly the A2 binding (by ∼30%). These results suggested that the light chain interaction with Plm was mainly dependent upon LBS, but the A2 interaction was independent. The addition of monoclonal antibody (mAb) against Plg kringle 5-catalytic domain (K5-CD) significantly inhibited Plm-catalyzed activation/inactivation of factor VIII or VIIIa with an ∼4-fold lower rate constant. On the other hand, anti-K1-3 and anti-K4 mAbs any little affected. SDS-PAGE analysis revealed that only anti-K5-CD mAb blocked Plm-catalyzed cleavages at Arg336 and Arg372 by ∼90% in dose-dependent manners (IC50: ∼20 nM). Surface plasmon resonance-based assays showed that the isolated K5-CD bound to factor VIII with an ∼50-fold higher affinity (Kd: 3 nM) compared to the K1-3 and K4, similar to the affinity obtained with anhydro-Plm (Kd: 4 nM). In particular, the K5-CD bound to the A2 domain with an ∼5-fold higher affinity (Kd: 42 nM) than those obtained with the K1-3 and K4. In contrast, both the K1-3 and K4 bound to the light chain predominantly (Kd: 43 and 87 nM, respectively), whilst the K5-CD failed to bind. Furthermore, the addition of a goat antibody against the CD (C-14; Santa Cruz Biotechnology) completely blocked the A2 and K5-CD interaction (by ∼95%). These findings suggest that the CD of Plm (and Plg) interacts with the factor VIII A2 domain through the LBS-independent mechanisms, whilst the K1-3 (and/or K4) interacts with the light chain through the LBS-dependent mechanisms. Furthermore, the CD and A2 interaction would regulate the activation/inactivation of factor VIII by proteolytic cleavages of Arg336 and Arg372.
Autologous Stem Cell Transplantation (ASCT) for Elderly Patients (≥ 60 Years) with Non-Hodgkin’s Lymphoma (NHL): An Analysis Based on EBMT Registry.
Authors: Jantunen Esa , Canals Carmen , Blaise Didier , Rambaldi Alessandro , Tilly Herve , Conde Eulogio , Gisselbrecht Christian , Dreger Peter , Ossenkoppele G.J. , Allione Bernardino , Pfreudschuh M. , Craddock Charles , Sureda Anna
Published: April 2020

Limited data is available on feasibility and efficacy of ASCT in elderly patients with NHL.

Patients: In 2000–2005 15869 NHL patients with ASCT were reported to EBMT database, 3133 (20%) were ≥ 60 years. Only patients with MED-B dataset and those with either diffuse large B-cell lymphoma (DLBCL), mantle cell (MCL) or follicular lymphoma (FL) were subjected to more detailed analysis. This group included 906 elderly NHL patients (median age 63 years, range 60–75) (DLBCL, n = 463; MCL, n = 208; FL, n = 235) who were compared with 3661 patients < 60 years (DLBCL, n = 2149; MCL, n = 435; FL, n = 1077) regarding outcome. Bulky disease was more common in younger patients (26% vs. 15%, p < 0.001) as well as B-symptoms at diagnosis (42% vs. 36%, p = 0.02). Elderly patients had received more often at least two treatment lines before ASCT (70% vs. 59%, p<0.001). The median follow-up for the surviving patients was 14 months.

Results: Non-relapse mortality (NRM) was higher in patients ≥ 60 years of age: 3.8% vs.2.3% at 100 days, 6.9% vs. 3.9% at 1 year and 9.4% vs. 5.8% at 3 years (p<0.001). No differences in NRM were observed between patients aged 60–64 years (n = 633) and those aged 65–69 (n = 240). A higher NRM was observed in DLBCL and MCL patients compared to FL patients (p=0.001and p=0.002, respectively). Other variables associated with a higher NRM were an elevated LDH at diagnosis (p=0.04), ≥ 2 treatment lines before ASCT (p<0.001); a poor performance status at ASCT (p<0.001); not being in CR1 at ASCT (chemosensitive disease vs. CR1, p=0.02; chemorefractory disease vs. CR1, p<0.001) and BM as stem cell source (p=0.02). In multivariate analysis, elderly patients showed a higher NRM [RR = 1.6 (CI 1.2–2.1), p=0.001]. In patients with DLBCL, age ≥ 60 years at ASCT was associated with a trend to a higher risk of relapse or progression (p =0.07) and a worse PFS (p=0.008). PFS at 2 years was 69% vs. 79% for patients in CR1 and 52% vs. 60% for patients with sensitive disease at ASCT, respectively. In MCL, elderly patients had worse PFS (p=0.008). PFS at 2 years was 78 vs. 81% for MCL patients in CR1 and 52% vs. 67%, respectively for those patients autografted with sensitive disease. Older age was not a significant prognostic factor either for relapse rate or for PFS in patients with FL. PFS at 2 years was 69% and 81% for FL patients in CR1, and 69% and 69% for FL patients with sensitive disease, respectively.

Conclusions: ASCT is feasible in selected NHL patients aged 60–69 years. The outcome is promising taking into account the generally poorer prognosis of lymphomas in elderly population.
Germinal Center B Cell and Non-Germinal Center B Cell-Like DLBCL Have Similar Clinical Features at the Time of Progression and Comparable Outcome Following Autologous HSC Transplantation.
Authors: Costa Luciano J. , Feldman Andrew L. , Micallef Ivana N. , Inward David J. , Johnston Patrick B. , Porrata Luis F. , Litzow Mark R. , Ansell Stephen M.
Published: April 2020

Background: Germinal center B cell-like (GCB) DLBCL, as determined by gene expression profiling or immunohistochemistry, is more likely to be cured by initial conventional chemoimmunotherapy than non-germinal center B cell-like (non-GCB) DLBCL. For patients with relapsed or refractory chemosensitive DLBCL, high-dose chemotherapy and autologous hematopoietic stem cell (HSC) transplant is considered standard-of-care treatment, but it is unknown whether the outcome of these patients is similarly influenced by the subtype of DLBCL. We therefore explored the differences between patients with GCB and non-GCB DLBCL as regards their clinical features at relapse after first line therapy and their outcome following salvage autologous HSC transplant.

Methods: Patients undergoing BEAM conditioning and autologous HSC transplantation for relapsed or refractory chemosensitive DLBCL at Mayo Clinic, Rochester, MN between 2001 and 2006 were included. Immunohistochemical analysis was performed for CD10, BCL-6 and MUM1 allowing classification in GCB and non-GCB-like DLBCL, as well as for BCL-2. GCB and non-GCB groups were compared in terms of known prognostic factors at time of progression and outcome after HSC transplant

Results: Fifty-nine patients were included and had retrievable tumor samples to allow immunohistochemical analysis. Median follow-up of survivors was 25 months; median age at the time of transplant was 60 years (range 17–77); All patients had failed at least one previous anthracycline-based regimen (15 had refractory disease). Overall, 25/59 cases (42%) were positive for CD10, 32/58 (55%) for BCL-6, and 19/58 (32%) for MUM1. Thirty-two patients (54%) were classified as having GCB and 27 (46%) non-GCB-like DLBCL. Patients in the GCB and non-GCB group had similar time to progression (TTP) (median 12.5 months vs. 11 months, Wilcoxon P=0.81) after first line therapy and similar IPI-R scores (Chi-square, P=0.38). In univariate analysis, GCB and non-GCB did not differ in time to relapse after HSC transplant (log rank test P=0.77) or survival (log rank test P=0.48; figure). The lack of demonstrable difference in survival persisted even after correction for IPI-R and TTP, factors know to affect transplant outcome (Cox regression, RR=0.80 for GCB; P=0.28). BCL-2 was highly expressed in both GCB (81%) and non-GCB (96%) and did not correlate with outcome in the entire population nor in any of the two groups.

Conclusion: Patients with chemosensitive relapsed or refractory GCB and non-GCB-like DLBCL derive similar benefit from autologous HSC transplant.

Figure Figure
No Significance of Derivative Chromosome 9 Deletion on the Clearance Kinetics of BCR/ABL Fusion Transcripts, Cytogenetic or Molecular Response, Loss of Response or Treatment Failure to Imatinib Mesylate Therapy for Chronic Myeloid Leukemia.
Authors: Kim Dong Hwan (Dennis) , Sriharsha Lakshmi , Kamel-Reid Suzanne , Chang Hong , Messner Hans , Lipton Jeffrey
Published: April 2020

Background: Deletion of the derivative chromosome 9 (der 9; del-der9) is a poor prognostic factor in chronic myeloid leukemia (CML) patients treated with hydroxyurea or interferon. However, its prognostic implication in imatinib mesylate (IM) treated patients is unclear. One study reported that IM improved but did not fully reverse the poor prognosis of CML patients with del-der 9, while another suggested that IM might overcome the poor prognostic significance of del-der 9. The purpose of current study is to evaluate the prognostic implication of del-der 9 in CML patients in terms of hematologic, cytogenetic and molecular responses, loss of response to IM and IM treatment failure rate in addition to BCR/ABL mRNA quantitative PCR monitoring.

Methods: The current study included 163 CML patients with available FISH results using the LSI-BCR/ABL-(ES) probe (Vysis, IL, USA) treated at the Princess Margaret Hospital (Toronto, Canada). End points of the study included hematologic response (HR), major or complete cytogenetic response (CyR; MCyR/CCyR), major or complete molecular response (MoR; MMoR/CMoR), loss of response (LOR), treatment failure (including primary hematologic or cytogenetic resistance and LOR), progression to accelerated phase or blast crisis (AP/BC), or time to IM dose escalation to achieve further response or to overcome LOR.

Results: Of 163 patients, 22 (13.5%) had del-der 9 prior to initiation of IM. No differences were noted between those with and without del-der 9 in terms of disease phase or additional cytogenetic abnormalities. No differences were noted for the time to HR (p=0.598), MCyR (p=0.281), CCyR (p=0.883), MMoR (p=0.125), or CMoR (p=0.834). Times to LOR (p=0.974), treatment failure (p=0.455), progression to AP/BC (p=0.276), or dose escalation of IM (p=0.816) were not significantly different between those with and without del-der 9. The serial monitoring of BCR/ABL mRNA quantitative PCR showed similar patterns of BCR/ABL mRNA reduction between the 2 groups.

Conclusion: The presence of del-der 9 in CML patients prior to IM therapy does not influence:

the response to IM therapy in terms of HR, CyR and MoR; LOR, treatment failure or progression to AP/BC; and time to dose escalation of IM.

Therefore, from the clinical point of view, the presence of del-der 9 does not impact on the management of CML patients treated with IM.

Figure 1. No differences of complete cytogenetic response rate (A) or major molecular response rate between patients with versus without deletion of derivative chromosome 9 after imatinib mesylate therapy Figure 1. No differences of complete cytogenetic response rate (A) or major molecular response rate between patients with versus without deletion of derivative chromosome 9 after imatinib mesylate therapy
Prognostic Impact of Deletions of Derivative Chromosome 9 on Patients (pts) with Chronic Myelogenous Leukemia (CML) in Chronic Phase Treated with Nilotinib or Dasatinib.
Authors: Quintás-Cardama Alfonso , Kantarjian Hagop , Thomas Deborah , Koller Charles , Shan Jenny , Cortes Jorge
Published: April 2020

Background: Submicroscopic deletions of the derivative chromosome 9 [del der(9)] mapping to regions adjacent to the translocation breakpoints occur in 9% to 15% of patients with CML. Deletion of der(9) is powerful prognostic indicator associated with an unfavorable prognosis in patients treated with interferon-alpha (IFN-α)-based therapies. Imatinib is currently the standard treatment for patients with CML and it appears to overcome the adverse prognostic impact imparted by deletions of der(9).

Methods: We tested the prognostic impact of the deletions of der(9) in 323 patients with CML treated at our institution with the 2nd generation tyrosine kinase inhibitors (TKIs) nilotinib (n=144) or dasatinib (n=179). The presence of deletions of der(9) was investigated by FISH analysis using the LSI-BCR/ABL-(ES) probe (Vysis, Downers Grove, IL) in 244 patients. The median age was 55 years (range, 15–83) and the median follow-up was 18 months (range, 1–44). The primary endpoints evaluated were survival, complete hematologic response (CHR) and cytogenetic responses.

Results: Twenty-eight (12%) patients were found to have del der(9) and 216 an intact der(9). Among patients with deletions, 16 were in chronic phase (CP), 8 in accelerated phase (AP), and 4 in blast phase (BP) at the start of nilotinib or dasatinib therapy. In the group of patients without deletions, 112 were in CP, 54 in AP and 50 in blastic phase (BP). Overall, 222 (91%) of 244 patients were assessable for response. There was no significant difference between patients with or without deletions regarding the rate of CHR, major (MCyR) or complete (CCyR) cytogenetic responses, and event-free (EFS), and overall (OS) survival at 12 months for the total population.

CML Phase deletion der(9) [No. Patients] CCyR (%) p EFS (12 months) p OS (12 months) p No (112) 72 1 84 0.2 97 0.01 Chronic Yes (16) 73 78 92 Not Done (30) No (54) 31 0.7 54 0.22 69 0.62 Accelerated Yes (8) 38 75 88 Not Done (27) No (50) 24 1 9 0.56 30 0.56 Blastic Yes (4) 25 25 25 Not Done (22)

Among pts in CP there was no difference in response rate or EFS, but pts without del der(9) had an improved OS at 12 months compared with those carrying der(9) (97% vs 92%; p=0.01). There appears to be no significant difference between pts treated with nilotinib and those treated with dasatinib.

Conclusion: Our results suggest that although the outcome of patients with CML treated in AP or BP with either nilotinib or dasatinib does not differ regardless of their deletion status, those in CP carrying del der(9) deletions may have a worse overall survival than their counterparts without deletions. This is in contrast to what has been reported with imatinib therapy. The reason for this difference is unclear.
Long-Term Ultra-Low-Dose Acyclovir Against Varicella-Zoster Virus Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Asano-Mori Yuki , Kanda Yoshinobu , Oshima Kumi , Kako Shinichi , Shinohara Akihito , Nakasone Hideki , Sato Hiroyuki , Watanabe Takuro , Hosoya Noriko , Izutsu Koji , Asai Takashi , Hangaishi Akira , Motokura Toru , Chiba Shigeru , Kurokawa Mineo
Published: April 2020

Varicella-zoster virus (VZV) infection remains a common complication after hematopoietic stem cell transplantation (HSCT). The introduction of long-term prophylaxis with low-dose acyclovir against VZV reactivation has been investigated, because VZV-related complications including post-herpetic neuralgia and secondary infection significantly affect the patient’s quality of life. We started long-term oral acyclovir at 200 mg/day in July 2001. Acyclovir was continued until the end of immunosuppressive therapy and at least one year after transplantation. To evaluate the efficacy of this long-term prophylaxis with ultra low-dose acyclovir against VZV reactivation, we analyzed the records of 242 Japanese adult patients who underwent allogeneic HSCT for the first time from June, 1995 to November, 2006 at University of Tokyo Hospital. Sixty-six patients developed VZV reactivation at a median of 248 days after HSCT, with a cumulative incidence of 34.7%. There was no VZV-related death. Only one breakthrough reactivation occurred during long-term acyclovir, responding well to the therapeutic dose of valacyclovir. The use of long-term acyclovir was the only independent determinant that significantly decreased the overall incidence of VZV reactivation (20.4% vs 50.5%, P<0.0001). As for VZV-related complications, only post-herpetic neuralgia was seen in three patients with long-term prophylaxis, whereas serious complications including CNS involvement, motor neuropathy and ophthalmic complications were involved in 14 without long-term prophylaxis. Eleven of 16 patients with long-term prophylaxis were treated as outpatients, whereas 39 of 50 patients without long-term prophylaxis required hospitalization (hospitalization 31% vs 78%, P=0.0015). Fifteen of the 57 patients who discontinued long-term acyclovir developed VZV reactivation at a median of 147 days after the discontinuation of acyclovir, with a cumulative incidence of 32.1%. VZV reactivation following discontinuation predominantly occurred in patients who were receiving immunosuppressive therapy at the cessation of acyclovir (44% vs 20%, P=0.12). These findings suggest that long-term prophylaxis of ultra-low-dose acyclovir resulted in a successful prevention of severe VZV-related symptoms and death, with a significantly decreased overall incidence of VZV reactivation. With this prophylaxis, visceral dissemination and serious complications other than post-herpetic neuralgia were completely eliminated, and thereby need for hospitalization was significantly reduced. To continue acyclovir for patients with profound immunosuppression might be important to further decrease the overall incidence of VZV reactivation.

Figure Figure
Prospective Measurement of BK Virus Blood and Urine Levels, and Associations with Morbidity, in Recipients of Allogeneic Hematopoetic Stem Cell Transplants.
Authors: O’Donnell Peter H. , Swanson Kate , Josephson Michelle A. , Artz Andrew S. , Rich Elizabeth , Stock Wendy , van Besien Koen
Published: April 2020

BK virus (BKV) is an important pathogen and cause of nephropathy in recipients of renal transplants, but its clinical significance in patients following hematopoetic stem cell transplantation (HSCT) is less well described. Over a 10 month period we prospectively measured BKV (by quantitative PCR) in the blood and urine of 103 patients who had undergone previous allogeneic HSCT [HLA-identical donor (n = 58), alternative donor (HLA-mismatched-related, matched-unrelated, or cord) (n = 45)] and who were receiving inpatient or outpatient post-transplant care. The median time from transplantation to the last follow-up virus sampling was 422 days (range = 12 to 3456 days), and the cohort included patients undergoing transplants as early as 1998 through 2007. BK viruria was manifest at any time during the period of measurement in 62% of the patients. Only 16% (16/103) developed BK viremia. All patients with BK viremia also had detectable BK viruria. In 38 patients who underwent BKV monitoring beginning immediately after transplant, development of BK viruria was an early phenomenon (median 26 days post-transplant, range = 4 to 274 days), while BK viremia occurred later (median 157 days post-transplant, range = 62 to 323 days). More recipients of alternative donor transplants compared to HLA-identical transplants developed BK viruria (75% vs 53%, P = 0.03) and BK viremia (24% vs 9%, P = 0.02). Given the tropism of BKV for the genitourinary tract and its association with hemorrhagic cystitis, urinalyses from all patients were analyzed for the presence and number of red blood cells (RBCs). Those patients with microscopic hematuria (81/103) had significantly higher levels of urinary BKV than patients without hematuria (P < 0.001), and there was a suggestion that the degree of hematuria was directly related to the amount of urinary virus, since a subset of patients with more pronounced hematuria (> 5 RBCs per high-powered microscopic field) had higher levels of urinary BKV than those with only small numbers of urinary RBCs (P = 0.06). Blood BKV levels were not associated with hematuria. The presence of BK viremia and/or viruria were not significantly associated with renal function, as measured by the mean serum creatinine, although 2/16 patients with BK viremia developed biopsy proven BKV interstitial nephritis, with one requiring hemodialysis. In summary, BK viruria is a common early finding in patients undergoing allogeneic HSCT, especially in those who do not have HLA-identical donors. Our data support the claim that BKV urinary infection is associated with hematuria post-HSCT. BK viremia occurs later and less commonly. Investigation of a larger number of patients is warranted to establish the incidence of and risk factors for BKV nephropathy in the allogeneic HSCT setting.
Once Daily Busulfan Cyclophosphamide Is Well Tolerated and Effective Prior to Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: LeMaistre Julia A. , Bachier Carlos , Smith Brad , LeMaistre C.F. , Shaughnessy Paul J.
Published: April 2020

The development of intravenous busulfan (Bu) allowed for the delivery of a standard daily dose of Bu to be given in one dose instead of dividing 4 times per day. Pharmacokinetic (PK) studies of Bu support the feasibility of once daily dosing and prior clinical studies found no unexpected toxicity of once daily Bu when combined with fludarabine. Based on this data we began an institutional protocol of delivering Bu once daily follwed by standard cyclophosphamide (Cy) and allogeneic hematopoietic stem cell transplantation (HCT). Only limited data in the literature describes toxicity and clinical outcomes of once daily BuCy. We report a retrospective review of our institutional data using once daily BuCy vs 4x daily BuCy and total body irradiation (TBI)/Cy in pts who received allogeneic HCT from January 2000 to December 2006. Bu 3.2mg/kg daily × 4 days followed by Cy 60mg/kg daily × 2 days was given to 42 patients (pts). Bu 0.8mg/kg/dose given 4x daily for 4 days followed by Cy 60mg/kg was given to 15 pts. Cy 60mg/kg daily for 2 days and fractionated TBI 1200 cGy delivered over 3 days was given to 60 pts. All donors were HLA matched at A, B, C, DR, and DQ and were related/unrelated in 23/19, 11/4, and 21/39 in the once daily BuCy, 4x daily BuCy, and TBI/Cy, respectively. Significantly more pts with myeloid leukemias received a BuCy regimen and significantly more pts with lymphoid malignancies received TBI/Cy. Median follow up for all pts was 370 days. VOD developed in 2 pts in the once daily BuCy group, 1 pt in the 4x daily BuCy group, and in no pts in the TBI/Cy group. Acute GVHD grade II-IV occured in 33% of the once daily BuCy pts, 53% of the 4x daily BuCy pts, and 32% of the TBI/Cy pts. Estimated actuarial transplant related mortality (TRM) and survival are described in the table below. There was no statistical difference in TRM or survival between the once daily and 4x daily BuCy groups or the total BuCy group and TBI/Cy group. The once daily BuCy group had significantly less TRM than the TBI/Cy group at 100 days (p=0.04) and at 1 year (p=0.01). The once daily BuCy group also had significantly better survival at one year compared to the TBI/Cy group (p=0.01), but this became non-significant at 3 years. The significant differences in the pt populations treated in the BuCy and TBI/Cy groups, as well as the retrospective nature of this study, limit the ability to draw conclusions comparing the groups. However, this review does demonstrate once daily BuCy and allogeneic HCT is well tolerated with no unexpected TRM, and provides good long term survival in pts with myeloid malignancies.

Survival and TRM 1xBuCy (95%CI) 4xBuCy (95%CI) Cy/TBI (95%CI) 100 day TRM 7% (1–16%) 21% (7–51) 22% (15–35) 1 year TRM 21% (11–38) 37% (17–69) 45% (33–59) 1 year Survival 70% (53–82) 47% (21–69) 49% (35–61) 3 year Survival 43% (24–59) 27% (8–50) 37% (24–49)
Comparison of Outcomes of Bone Marrow and Umbilical Cord Blood Transplants from Unrelated Donors in Adult Patients with Acute Myeloid Leukemia/Myelodysplastic Syndrome.
Authors: Masuoka Kazuhiro , Miyakoshi Shigesaburo , Ishiwata Kazuya , Tsuji Masanori , Takagi Shinsuke , Yamamoto Hisashi , Kato Daisuke , Matsuhashi Keiko , Seo Sachiko , Matsuno Naofumi , Uchida Naoyuki , Wake Atsushi , Yoneyama Akiko , Taniguchi Shuichi
Published: April 2020

 Promising results of cord blood transplants from unrelated donors have been reported in adults. To compare of outcomes of bone marrow transplants (BMT, n = 51), and umbilical cord blood transplants (UCBT, n = 110) from unrelated donors in adult patients with acute myeloid leukemia (AML) / myelodysplastic syndrome (MDS), we analyzed retrospectively the results of 161 adult patients with AML and MDS in our hospital.

 We reviewed medical records of 161 patients with AML/MDS who had received a hematopoietic stem cell transplant from an unrelated donor between August 2000 and April 2007 at Toranomon Hospital, Tokyo, Japan.

 Patient’s median age was 55 years (17–71). Diagnoses include de novo AML (n =85), MDS overt AML (n=48), refractory anemia (RA) (n=13), and refractory anemia with excess of blasts (RAEB) (n=15). Disease status consisted of standard (CR1 of AML and RA, n=30) and advanced (other status, n=131). Recipients of UCBT had more advanced disease than recipients of BMT at the time of transplantation (89 percent vs. 65 percent, P<0.001). The median number of nucleated cells that were infused was 0.26×108 per kilogram of the recipient’s body weight for cord blood and 2.5×108 per kilogram for bone marrow (P<0.001). The major difference were higher number in the UCBT group of HLA mismatches (defined by serology for class 1 and molecular typing for DRB1).The donor was HLA mismatched in 96% of UCBT recipients, and in 41% of BMT recipients (P<0.001). Other significant differences were observed in preparative regimens, and graft-versus-host disease (GVHD) prophylaxis. Nonadjusted estimates of 2-year OS and DFS rates were 53% and 48% in the BMT group, and 33% and 25% in the UCBT group (P<0.001). However, 2-year OS and DFS rates in the standard group were not significantly different in the two groups (63% and 63% in the BMT group, and 75% and 58% in the UCBT group; p=0.98 and 0.32). Compared with BMT recipients, UCBT recipients had delayed hematopoietic recovery (Hazard ratio [HR]= 0.52; 95% confidence interval [95CI]: 0.36–0.75; p<0.001), increased 100 day TRM (HR=3.07; 95CI 1.45–6.51; p<0.01) and decreased grade II–IV acute graft-versus-host disease (aGVHD) (HR=0.58; 95CI 0.35–0.96; p=0.03). Two-year relapse rate was not significantly different in the two groups.

 We conclude that UCBT from an unrelated donor is a therapeutic option for adult AML/MDS patients who lack an HLA-identical donors. Higher mortality, especially from non-relapse causes, is the biggest problem to be solved to increase the feasibility of this approach.
A Worldwide Meta-Analysis on the Use of Zidovudine and Interferon-alpha for the Treatment of Adult T–Cell Leukemia/Lymphoma.
Authors: Bazarbachi Ali , Panelatti Gerard , Ramos Juan Carlos , Tortevoye Patricia , Otrock Zaher , Taylor Graham , Gessain Antoine , Harrington William , Plumelle Yves , Hermine Olivier
Published: April 2020

HTLV-I associated adult T cell leukemia/lymphoma (ATL) is an aggressive T cells malignancy, with poor prognosis due to chemotherapy resistance. Multiple small phase II studies using zidovudine (ZDV) and interferon-alpha (IFN) showed response in ATL patients. However, the impact of this therapy on ATL prognosis remains to be determined. Here, we report a worldwide meta-analysis on the use of ZDV/IFN treatment for ATL in 209 patients treated from 1994 to 2006. Patients were recruited in the USA (27 patients), the UK (13 patients), Martinique (102 patients) and France metropolitaine (67 patients). Collected data included geographic origin, age, sex, type of the disease, clinical presentation, LDH levels, calcemia, and lymphocytes number. Median age was 49 years (range 16 to 95). According to Shimoyama classification, there were 98 acute ATL, 20 chronic ATL, 9 smouldering ATL, 75 ATL lymphoma, and 7 patients with an unknown subtype. Hypercalcemia was present in 60% of patients. The data concerning the course of the disease were also collected, particularly the response status, the length of the response, the duration of ZDV+IFN therapy, as well as previous and post chemotherapy treatments. One hundred patients received first line ZDV+IFN therapy. In these patients, response rate was 66%, including 43% of patients achieving complete remission (CR). Median overall survival and 5 year overall survival rate were 24 months and 50% for patients who received first line ZDV+IFN therapy, versus 7 months and 20% for patients who received first line chemotherapy. When analysis was performed by ATL subtype, patients with acute, chronic, and smouldering ATL significantly benefited from first line ZDV+IFN therapy, whereas no additional benefit was achieved in patients with ATL lymphoma. Achievement of CR with first line ZDV+IFN therapy resulted in prolonged survival of more than 10 years in 70% of the study population, and 75% of the acute ATL subgroup. Finally, first line ZDV+IFN therapy in chronic and smouldering ATL resulted in 100% overall survival at 10 years. In conclusion, these results confirm that treatment of ATL using ZDV and IFN results in a high response and CR rates particularly in acute, chronic and smouldering ATL, resulting in impressive prolonged survival and hence should be considered as gold standard first line therapy.
Generation of alpha-Type-1 Polarized Dendritic Cells as a Potent Immunogen in Patients with Chronic Lymphocytic Leukemia.
Authors: Lee Je-Jung , Mailliard Robbie , Muthuswamy Ravikumar , Foon Kenneth A. , Kalinski Pawel
Published: April 2020

We tested the feasibility of generating alpha-type-1 polarized DC (αDC1) from the blood of chronic lymphocytic leukemia (CLL) patients and the in vitro effectiveness of autologous tumor-loaded αDC1s in inducing CTLs against CLL. CD14+ cells were isolated from the peripheral blood of CLL patients and were cultured in rhu GM-CSF and IL-4. On day 6, DC maturation was induced using either a standard cytokine cocktail (in IL-1β, TNFα, IL-6, and PGE2) or αDC1-polarizing cocktail (in IL-1β, TNFα, IFNα, IFNγ, and poly-I:C). In either case, the maturing DCs were loaded with gamma-irradiated autologous CLL cells. αDC1s from CLL patients expressed substantially higher levels of several costimulatory molecules (CD83, CD86, CD80, CD11c and CD40) than standard DCs (sDCs) while CCR7 showed lower expression. Gamma-irradiated CLL cells were more efficienly taken up by DCs compared to UVC-irradiated CLL cells, but the ability of tumor antigen uptake was similar between αDC1 and sDCs. The capacity of IL-12p70 secretion in DCs (baseline and after stimulation with CD40L-transfected J558 cells) was significantly elevated in αDC1s (10–60 times) compared to sDCs. αDC1s also induced significantly higher numbers of functional CD8+ T cells against CLL cells, as determined by IFN-γ ELISPOT, using anti-MHC class I blocking mAb (W6/32) to verify the specificity of CTL responses. Our data demonstrate that αDC1s are a potent alternative to standard DCs as inducers of T cells for adoptive immunotherapy in patients with CLL and a clinical trial will be initiated.
Gene Conversion Is a Late Event in Evolution of High Risk AML.
Authors: Meshinchi Soheil , Pollard Jessica A. , Tsuchiya Karen D. , Stirewalt Derek A. , Bernstein Irwin D. , Radich Jerald
Published: April 2020

Evolution and progression of AML is considered a result of cooperation of multiple genetic and epigenetic alterations. FLT3 internal tandem duplication (ITD) is a common mutation in AML and is considered a secondary, proliferative event in the pathogenesis of AML. We used SNP/CGH microarrays to evaluate the mechanism of disease evolution in FLT3/ITD-positive AML. Diagnostic DNA from patients with FLT3/ITD (N=25), FLT3 activation loop mutation (FLT3/ALM, N=15) or with no FLT3 mutations (FLT3/WT, N=55) was evaluated for copy number and LOH alterations. 12/25 (48%) FLT3/ITD-positive patients had loss of heterozygosity (LOH) of a large segment of 13q arm downstream of the FLT3 gene without an associated chromosome loss, demonstrating a copy neutral LOH (CN-LOH) state. In contrast, CN-LOH was not detected in those with FLT3/ALM or FLT3/WT suggesting that FLT3/ITD is causally associated with evolution of CN-LOH. Single cell analysis of the FACS purified leukemic blasts from FLT3/ITD-positive samples that were heterozygous for 13q (no CN-LOH) by SNP/CGH arrays demonstrated the presence of 3 distinct molecular subtypes that included a subpopulation with CN-LOH, as well as heterozygous ITD and FLT3/WT, demonstrating oligoclonal nature of the leukemic blast population which prevented identification of CN-LOH. In patient samples with CN-LOH, CFU-GM and BFU-E colonies derived from CD34+/CD33- progenitors demonstrated heterozygous ITD, with no LOH or copy number alterations. In contrast, more differentiated, CD33+ leukemic blasts as well as CFU-GM colonies derived from CD34+/CD33+ progenitors had homozygous ITD and CN-LOH. This cumulative data demonstrates that CN-LOH of the 13q, as a result of gene conversion-type event mediates the pathogenesis of FLT3/ITD and is a secondary event in the evolution of high risk FLT3/ITD. Gene conversion, leading to haplo-insufficiency may be an important event in the evolution of AML.
Integrated Genomic Profiling of Chronic Lymphocytic Leukemia Identifies Subtypes of Deletion 13q14 with Differential Expression of LATS2.
Authors: Malek Sami N. , Erba Harry , Mark Kaminski S. , Kerby Shedden , Peter Ouillette
Published: April 2020

Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous illness with a variable clinical course. Loss of chromosomal material on chromosome 13 at cytoband 13q14 is the most frequent genetic abnormality in CLL but the molecular aberrations underlying del13q14 in CLL remain incompletely characterized. We analyzed 171 CLL cases for LOH and sub-chromosomal copy loss on chromosome 13 in DNA from FACS-sorted CD19+ cells and paired buccal cells using the Affymetrix XbaI 50K SNP array platform. Comparative analysis of SNP-array-derived detection of del13q14 using copy number analysis compared with clinical FISH results for del13q14 demonstrated strong agreement between both methods: FISH detected 91/171=53% overall del13q14 incidence and 77/171=45% del13q14 cases with greater than 25% of cells involved. SNP-arrays detected 82/171=48% del13q14 incidence. SNP-arrays detected 74/77=96% of all CLL cases that were greater than 25% FISH positive and 82/91=90% of all CLL cases that were FISH positive (% positive range for FISH was 2–97%). Finally, SNP-arrays detected 2 CLL cases not previously known to be del13q14 by FISH. The resulting high-resolution genomic maps, together with array-based measurements of expression levels of RNA in CLL cases with and without del13q14 and Q-PCR-based expression analysis of selected genes, support the following conclusions:

del13q14 is heterogeneous and composed of multiple subtypes (types Ia, Ib and II) with deletion of Rb or the miR15a/16 loci serving as anatomic landmarks, respectively; del13q14 type Ia deletions are relatively uniform in length and extend from breakpoints close to the miR15a/16 cluster to a newly identified telomeric breakpoint cluster at ∼50.2–50.5 Mb physical position; LATS2 RNA levels are ∼2.6–2.8 fold lower in cases with del13q14 type I that do not delete Rb, as opposed to del13q14 type II lesions or all other CLL cases; ∼15% of CLL cases display marked reductions in miR15a/16 expression that are often but not invariably associated with bi-allelic miR15a/16 loss; and Bcl-2 levels and miR16/15a locus loss or miR16/15a expression are not correlated.

This data should aid future investigations into biological differences imparted on CLL by different del13q14 subtypes, including investigations into LATS2 as one of the genes found deregulated as part of del13q14 type I and investigations into additional miR16/15a targets.
Eph Receptor Tyrosine Kinases and Ephrin Ligands Are Epigenetically Inactivated in Acute Lymphoblastic Leukemia and Are Potential New Tumor Suppressor Genes in Human Leukemia.
Authors: Kuang Shao-qing , Fang Zhi-Hong , Lopez Gonzalo , Tong Weigang , Yang Hui , Garcia-Manero Guillermo
Published: April 2020

The Eph (erythroprotein-producing hepatoma amplified sequence) family receptor tyrosine kinases and their ephrin ligands (ephrins) are involved in a variety of functions in normal cell development and cancer. We have identified several members of this family as potential targets of aberrant DNA methylation using Methylated CpG Island Amplification (MCA) / DNA promoter microarray technology. This is of importance as there are no prior reports of potential Eph receptor or Ephrin epigenetic inactivation in human leukemia. To further investigate the role of Eph receptor and ephrin family genes in leukemia, we have analyzed their DNA methylation status in a panel of 23 leukemia cell lines and 65 primary ALL patient samples. Aberrant DNA methylation of 9 of these genes (EPHA4, EPHA5, EPHA6, EPHB2, EPHB3, EPHB4, EphrinA5, Ephrin B2, and EphrinB3) was detected in multiple leukemia cell lines but not in normal samples by bisulfite pyrosequencing. In ALL patient samples, the frequencies of DNA methylation detected in the promoter regions of these genes ranged from 23% to 87% for EPHA4, EPHA5, EPHA6, EPHB2, EPHB3, EPHB4, EphrinA5, Ephrin B2, and EphrinB3. Expression analysis of 3 of these genes (EPHA5, EPHB4 and Ephrin B2) in leukemia cell lines by real-time PCR further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with DNA methyltransferase inhibitor 5′-aza-2′-deoxycytidine resulted in gene re-expression. Forced overexpression of EPHB4 using a lentivirus transduction system in Raji cell lines resulted in decreased cell proliferation and adhesion-independent cell growth, as well as in an increase in staurosporine induction of apoptosis. In addition, EPHB4 overexpression resulted in a significant downregulation of phosphorylated Akt pathway but had no effect on mitogen-activated protein kinase pathway. In summary, we describe for the first time the epigenetic suppression of Ephrin receptors and their ligands in human leukemia, indicating that these genes may be potential tumor suppressors in leukemia. Targeting of these pathways may result in the development of new potential therapies and biomarkers for patients with ALL.
Hazard of Subsequent Hematological Cancer in Patients with Incident Venous Thromboembolism (VTE): A Population-Based Cohort Study.
Authors: Gangat Naseema , Ashrani Aneel A. , Petterson Tanya M. , Schmoll Jeffery , Heit John A.
Published: April 2020

Background: While currently-diagnosable hematological cancer is a potent VTE risk factor (RR range =12–32), whether undiagnosable hematological cancer is a risk factor is uncertain.

Objective: To estimate the hazard of subsequent hematological cancer among incident VTE patients with no apparent active hematological cancer within ± 3 months of the VTE event.

Methods: In a population-based cohort study, we followed Olmsted County, MN residents with incident VTE over the 27-year period, 1973–1999, and previously matched Olmsted County residents without VTE, who survived with no apparent active hematological cancer within ± 3 months of the VTE event or a routine medical visit date, respectively, forward in time through their complete medical records in the community for hematological cancer. Data were abstracted on baseline demographic and clinical characteristics, type and stage of hematological cancer, and any prior solid tumor treatments. For cases and controls, hematological cancer-free survival from the event date was estimated by the Kaplan Meier method, and the homogeneity of survival curves was examined by the log-rank test. Cox proportional hazards regression was used to adjust for covariates.

Results: 1860 VTE cases (56% females) and 1890 controls (53% females) were followed for 16,497 and 15,320 person-years, respectively, conditional on surviving 3-months free of hematological cancer. 43 VTE cases and 24 controls developed hematological cancer at a median of 63.5 months (range 0.7 – 334 months). The median age for cases was 63.2 years compared to 67.8 years for the controls (p<0.001). Hematological cancer types included non-Hodgkin’s lymphoma (n=19), chronic lymphocytic leukemia (n=11), acute myeloid leukemia (n=1), multiple myeloma (n=9), myeloproliferative disorders (n=10), and myelodysplastic syndromes (n=14). Case status was not univariately associated with subsequent hematologic cancer (HR=1.42; 95% CI: 0.85, 2.39; p=0.18). After adjusting for age, also associated with cancer, case status was significantly associated with hematological cancer (HR=1.90; 95% CI: 1.13, 3.20; p=0.015).

Conclusions: Incident VTE patients have nearly a two-fold increased risk of subsequent hematological cancer. We hypothesize that VTE can be the initial manifestation of an occult hematological cancer clone.
Phosphorylation of AML1/Runx1 Is Essential for T Cell Differentiation and Early Hematopoietic Development.
Authors: Yoshimi Mayumi , Goyama Susumu , Nakagawa Masahiro , Asai Takashi , Ichikawa Motoshi , Kurokawa Mineo
Published: April 2020

AML1/Runx1 is one of the most frequent targets of gene aberrations associated with human acute myelogenous leukemia and myelodysplastic syndrome. Furthermore, gene-targeting studies in mice demonstrated that AML1 is a critical regulator for T-cell differentiation and early development of definitive hematopoiesis. It has been shown that phosphorylation of AML1 at specific serine/threonine residues (276, 293, 300, 303) controls both transcriptional activity and rate of degradation. However, the biological consequences of phosphorylation with respect to AML1 function are unclear. In this study, we evaluated hematopoietic activities of AML1 mutants which harbor serine/threonine-to-alanine (A) or serine/threonine-to-asparatic acid (D) mutations at these phosphorylation sites using primary culture systems. We first evaluated the biologic effects of AML1 phosphorylation on T-cell differentiation using the fetal liver (FL)/OP9-DL1 coculture system. In this system, AML1-excised FL cells failed to undergo normal T-cell differentiation, which is successfully restored by the reintroduction of wild-type AML1. AML1-4A, which cannot be phosphorylated at any of the four serine/threonine residues, showed a severely impaired capacity to rescue the defective T-cell differentiation of AML1-deficient cells. We also demonstrate that blocking the ERK-mediated phosphorylation of AML1 by the MEK inhibitor significantly suppressed the AML1-induced T-cell differentiation. Next, we assessed the effect of AML1 phosphorylation on early hematopoietic development using para-aortic splanchnopleural (P-Sp) region/OP9 coculture system. In this system, AML1-deficient P-Sp cells failed to produce any hematopoietic cells and this hematopoietic defect can be rescued by retrovirally transferred AML1. AML1–4A could rescue the hematopoietic defect of AML1-deficient P-Sp cells, but the emergence of hematopoietic cells in cultures infected with AML1–4A was reproducibly delayed two or more days compared with those infected with wild-type AML1. Because it was shown that serine 462 is phosphorylated upon phorbol ester treatment together with the four phosphorylation sites, we then generated AML1 mutants in which serine 462 is substituted with alanine (A) or aspartic acid (D) in AML1–4A or AML1–4D respectively (AML1–5A or AML1-5D). Interestingly, introduction of AML1–5A into AML1-deficient P-Sp cells did not generate any hematopoietic cells. Moreover, AML1–5A completely lost its T-cell differentiation activity. In contrast, the phospho-mimic protein, AML1–5D rescued the hematopoietic defects of AML1-deficient cells as efficiently as wild-type AML1. Taken together, these results suggest that phosphorylation of AML1 at the five serine/threonine residues (276, 293, 300, 303 and 462) is essential for T cell differentiation and early hematopoietic development.
Indian Hedgehog Is Essential for Definitive Haematopoiesis in the Fetal Liver.
Authors: Perkins Andrew C. , Cridland Simon
Published: April 2020

Indian hedgehog (Ihh) is a member of the hedgehog family of secreted proteins which play diverse roles in development. Members of this family have established roles in craniofacial development, long bone formation and spermatogenesis. Mammals have three hedgehog proteins which all act through the Patched receptor to activate smoothened and downstream zinc finger transcription factors of the Gli family. Ihh can induce differentiation of primitive red blood cells during primitive streak formation, and definitive red blood cells from CD34+ cord blood stem cells. Both Ihh and the Patched receptor are expressed in the fetal liver. In this report we show Ihh knockout mice display a partially penetrant defect in fetal liver haematopoiesis characterised by severe anaemia and apoptosis of the fetal liver erythroid compartment. Primitive haematopoiesis is normal, so mutant embryos survive until ∼E14. Components of the hedgehog signalling pathway are expressed in stromal, stem cell and progenitor cell components of the haematopoietic system. Lastly, fetal liver progenitor cells (CFU-e, BFU-e, and myeloid CFUs) are normal in Ihh null mice, suggesting a critical requirement for Ihh in the fetal liver stem cell niche.