bluuebunny/crossref_metadata_2025 huggingface dataset:
The Use of 2nd generation Tyrosine Kinase Inhibitors (TKI) after Failure to 2 Prior TKI: Long-Term Follow-up.
Authors: Garg Ravi n Jain , Kantarjian Hagop M , O’Brien Susan , Quintas-Cardama Alfonso , Faderl Stefan , Estrov Zeev , Cortes Jorge
Published: April 2020

The 2nd generation tyrosine kinase inhibitors (TKI), nilotinib and dasatinib, are effective after imatinib failure. These agents are now also being used as 3rd line therapy after failure to 2 prior TKI. Preliminary data suggests responses can be achieved with this strategy, but the long-term benefit is unknown.

To investigate the long-term benefit of using a 2nd generation TKI after having failed imatinib and another TKI.

Patients with CML who were sequentially treated with 3 different TKI’s at M.D. Anderson Cancer Center were reviewed. Response to the 3rd TKI were scored according to standard definitions. Event-free survival (EFS) was considered from the time the 3rd TKI was started to loss of major hematologic response, loss of cytogenetic response, transformation to accelerated (AP) or blast (BP) phase, or death. Failure-free survival (FFS) was considered from start of 3rd TKI to event as defined for EFS, or loss of complete cytogenetic response (CCyR) or discontinuation because of toxicity.

37 patients were treated: 29 with dasatinib after imatinib and nilotinib failure, and 8 with nilotinib after imatinib and dasatinib failure. The median age was 61 years (yrs) (range, 19 to 70). The median time on imatinib was 33 months (mo) and 15 (41%) had transformation before starting 2nd TKI. Median time on 2nd TKI was 7.7 mo. At the start of 3rd TKI, 16 pts (43%) were in chronic phase (CP), 9 (24%) in AP, and 12 (32%) in BP. Best response to 3rd TKI in CP was 1 major molecular response (MMR), 2 CCyR, 1 partial cytogenetic response (PCyR), 5 minor cytogenetic response (mCyR), 2 complete hematologic response (CHR), and 5 no responses (NR); in AP, there was 1 CCyR, 2 PCyR, 1 mCyR, 4 CHR, and 1 NR; in BP, 2 CCyR, 1 PCyR, 1 mCyR, 2 return to CP (RCP), and 6 NR. Four patients discontinued treatment because of toxicity despite an acceptable response. The median CCyR duration was 28 mo; only 2 pts in CP who achieved CCyR (1 had MMR) had a sustained response lasting 20 and 24 mo, respectively. Both received dasatinib after imatinib and nilotinib failure, and had G250E and H396R at start of dasatinib, respectively. The table shows median (in months) OS, EFS and FFS by disease stage.

CP AP BP OVERALL No. 16 9 12 37 OS NR 19.6 4.8 18 EFS NR 4.7 4 8 FFS 20 4.7 2 4

7 pts continue on therapy (all CP): 2 sustained CCyR (1 MMR), 1 PCyR (lost CCyR), 2 mCyR, 2 no cytogenetic response (1 lost mCyR).

Use of 2nd generation TKI after failure to 2 TKI may induce responses in some pts but these are usually not durable except in some pts in CP. New treatment options are needed for these pts.
The Prevalence of 25-Hydroxyvitamin D Deficiency in Post-HSCT Pediatric Patients.
Authors: Duncan Christine , Vrooman Lynda , Bechard Lori , Barry Elly , Lehmann Leslie E.
Published: April 2020

Children undergoing HSCT are at risk for vitamin D deficiency due to lack of sun exposure, the recommended use of sunscreen, dietary insufficiency, and the effects of medications such as glucocorticoids and calcineurin inhibitors. We assessed the prevalence of 25-hydroxyvitamin D (25-OH vitamin D) deficiency in pediatric post-HSCT patients in an outpatient oncology clinic during 4 weeks in May 2008. Patients found to have low 25-OH vitamin D levels were referred for dietary counseling and given supplementation or repletion as needed. 25-OH vitamin D and parathyroid hormone (PTH) levels were measured in 62 (88.6%) of 70 eligible patients. 83.8% of patients had a 25-OH vitamin D level less than the institutional lower limit of normal, 30 ng/mL. 29% of patients were 25-OH vitamin D insufficient with levels 20–29 ng/mL (range of 20–29). 54.8% of patients were 25-OH vitamin D deficient with levels <20 ng/mL (range 5–19). The prevalence of insufficiency and deficiency was similar between male (87.8%; 57.6%) and female patients (57.6%; 55.2%).The mean duration of days following transplant was 532.6 days (median 251.5 days). The mean age at transplant was 3.7 years (median 3.5 years). 47% of patients were female. 75.8% were Caucasian. 90.3% received allogeneic transplants. The underlying diseases were as follows: ALL (27.4%), AML/MDS (24.2%), bone marrow failure (11.3%), nonmalignant hematologic diagnosis (8.1%), solid tumor (8.1%), immunodeficiency (6.5%), lymphoma (6.5%), and other diagnoses (8.1%). 8 patients regularly took either an over-the-counter multivitamin or vitamin D supplement and all 8 patients had 25-OH levels less than 30 ng/mL. There was a negative inverse correlation of (r= −0.3, p=0.029) between PTH and 25-OH vitamin D. There were no significant associations between 25-OH vitamin D level and any of the following: corticosteroid or calcineurin inhibitor use in the preceding year, time from transplant, age at transplant, current age, or graft-versus-host disease. 25-OH vitamin D insufficiency and deficiency are common following pediatric HSCT. We recommend vitamin D screening for all post-HSCT pediatric patients. Further investigation is needed to identify potential risk factors for vitamin D deficiency and the long-term effects of deficiency on bone health and development.
Clinical Significance of Autoantibody Expression in Allogeneic Stem Cell Recipients.
Authors: Moon Joon Ho , Lee Soo Jung , Kim Jong Gwang , Chae Yee Soo , Kim Shi Nae , Kim Yun Jeong , Rok Young , Suh Jang Soo , Lee Kun Soo , Sohn Sang Kyun
Published: April 2020

Some allogeneic stem cell recipients manifest autoimmune disease-like symptoms associated with the occurrence of various autoantibodies. We studied the association of autoantibodies with chronic graft-versus-host disease (cGVHD) and survival in allogeneic stem cell recipients. From Nov. 2001 to Mar. 2008, 121 patients with hematological diseases who had survived at least 3 months after allogeneic stem cell transplantation (SCT) were enrolled in the current study. Forty-seven patients (38.8%) expressed at least one of various autoantibodies after allogeneic SCT. ANA was expressed most commonly in recipients (n=22, 18.2%). Anti-dsDNA antibody was positive in 7 (5.8%), anti-Sm antibody in 6 (5%), rheumatoid factor (RF) in 17 (14.0%), and positive Coombs test in 12 (9.9%). The FANA pattern revealed 11 nucleolar, 6 speckled, and others (n=5). Positive rate of autoantibody was higher in patients with cGVHD (47.8 vs. 27.8%; p=0.025) and in patients with extensive cGVHD (62.5 vs. 27.2%; p<0.001). The expression of autoantibodies had a statistical correlation to specific organ involvement of cGVHD: ANA to sicca syndrome (p=0.010), anti-Sm antibody to hepatic GVHD (p=0.003), and RF to sicca syndrome (p=0.039) and musculoskeletal GVHD (p=0.026). The 5-year survival of autoantibody-positive patients was 63.8% and that of negative patients was 47.9% (p=0.003). The incidence of underlying disease relapse was lower in autoantibody-positive patients (20.2% vs. 39.3%; p=0.011). However, GVHD-specific survival was not related with autoantibody expression. Advanced age group (p=0.005, OR=0.246), unrelated donor (p=0.005, OR=0.218), and the presence of acute GVHD (p=0.029, OR=0.315) were significantly associated with a lower incidence of autoantibody expression. In conclusion, the expression of autoantibodies after allogeneic SCT was related with cGVHD. Patients expressing autoantibodies had a better survival and low incidence of underlying disease relapse.
The Influence of Cytochrome P450 3A Gene Polymorphisms on Tacrolimus Concentrations in Hematopoetic Stem Cell Transplantation Patients.
Authors: Ogasawara Masahiro , Kosugi Mizuha , Matsuoka Satomi , Asamuma Shinsuke , Noguchi Shinsuke , Fujii Sirou , Wada Akinori , Ota Shuichi , Nakata Masanobu , Imai Kiyotoshi , Kobayashi Naoki , Kiyama Yoshio , Kasai Masaharu
Published: April 2020

Objective: Tacrolimus is a commonly used immunosuppressive drug in hematopoetic stem cell transplantation (SCT). It is characterized by a relatively narrow therapeutic range and a large individual variation in concentrations. It is metabolized mainly by cytochrome P450 (CYP) 3A which has genetic polymorphisms influencing enzymatic activities; CYP3AP1*1 and *3 genotypes representexpressor and non-expressor, respectively. The objective of this study is to investigate whether the tacrolimusconcentration per dose and renal toxicity by tacrolimus is associated with CYP3AP1 gene polymorphisms.

Methods: 105 Japanese SCT patients (55 AML, 19 ALL, 11 CML, 6 MDS, 4 NHL, 1 HL, 2 ATL, 2 MM, 4 AA, 1 MF), receiving tacrolimus were studied. DNA was extracted from peripheral blood samples. CYP3AP1 genotype was analyzed by PCR-restriction fragment length polymorphism using specific primers and a restriction enzyme, AciI. Blood tacrolimus concentrations were measured twice a week by a commercially available EISA kit.

Results: The frequency of the CYP3AP1 *1/*1, *1/*3 and *3/*3 genotype was 8, 31, and 61% of 105 patients, respectively, and the genotype distribution was not different significantly from that of 121 Japanese healthy volunteers (3, 39, and 58%, respectively). The frequency of CYP3AP1 genotypes of white Americans is reported to be 2, 12 and 86%, respectively, indicating the ethnic difference of distribution. The dose-adjusted concentrations of tacrolimus 5 to 7 days following SCT were significantly higher in CYP3AP1 *3/*3 patients than in CYP3AP1 *1/*1 and *1/*3 patients (665.0±148.0 ng/ml per mg/kg vs. 409.0±116.2 ng/ml per mg/kg, P<0.001). The dose needed to achieve the target concentration (10 to 15 ng/ml) between 40 to 60 days following SCT, when the blood concentrations of tacrolimus tended to be stable in most patients, is significantly lower in CYP3AP1 *3/*3 patients than in CYP3AP1 *1/*1 and *1/*3 patients (0.008±0.005 mg/kg vs.0.018±0.005 mg/kg, P<0.001). Above grade two adverse events related to tacrolimus were observed only in CYP3AP1 *3/*3 patients (3 patients in grade 4 and 6 patient in grade2), suggesting the contribution of a higher concentration to renal toxicity by tacrolimus.

Conclusion: The tacrolimus concentration per dose in SCT patients is influenced by the CYP3AP1 gene polymorphism. This is the first report showing the relationship between CYP3AP1 gene polymorphism and the tacrolimus concentration in SCT patients to the best of our knowledge. Information of CYP3AP1 genotype may be helpful to determine the initial tacrolimus dose adequate for each patient.
The FcγRllla Polymorphism Correlates with Chronic Graft-Versus-Host Disease and Treatment Related Mortality.
Authors: Takami Akiyoshi , Espinoza J. Luis , Ishiyama Ken , Ohyachi Satomi , Kato Yuki , Kawamura Yoshimi , Kondo Yukio , Yasue Shizuka , Ohtake Shigeki , Nakao Shinji
Published: April 2020

The FcγRllla (FCGR3A) 158V/V allotype displays a higher antibody-dependent cellular cytotoxicity compared to the FCGR3A-158V/F or 158F/F allotype, leading to susceptibility to rheumatoid arthritis, better clinical response to rituximab in NHL, and lower risk of periodontitis. We tested the hypothesis that FCGR3A polymorphism influences the development of GVHD and GVL, and TRM. The FCGR3A-158V/F genotype was determined in 49 recipient and donor pairs who underwent HLA-matched SCT in our institute. Subjects with the recipient FCGR3A-158V/V allotype and the donor FCGRA-158V/V allotype were almost one-third and twice as likely to develop chronic GVHD respectively (Table 1). The recipients with the FCGR3A-158V/V allotype had a 60% increase in probability of 3-year TRM. Multivariate analysis showed that the recipient FCGR3A-158V/V allotype and the donor FCGRA-158V/V allotype were independent risk factors for chronic GVHD (HR = 0.8 and 1.2, respectively), as well as the unrelated donor BMT (HR = 1.6) and the related donor PBSCT (HR = 2.4). The recipient FCGR3A-158V/V was an independent predictor for 3-year TRM (HR = 4.0) with the high risk disease (HR = 3.0) and the cord blood transplantation (HR = 5.0). We conclude that the recipient and donor FCGR3A polymorphism is associated with the development of chronic GVHD and TRM after HLA-matched SCT. These findings could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic SCT. Besides, these raise the question of whether the genetic risk factor in a recipient might also be involved in the mechanisms of chronic GVHD.

Table 1

FCGR3A allotype 2–4 acute GVHD(HR) Chronic GVHD (HR) 3 YR TRM (HR) *P<0.05 Recipient FCGR3A-158V/V 46% (1.2) 17%* (0.8*) 72%* (4.0*) Recipient FCGR3A-158V/F or 158F/F 39% 57% 44% Donor FCGR3A-158V/V 46% (1.1) 67%* (1.2*) 43% (0.5) Donor FCGR3A-158V/F or 158F/F 38% 30% 50%
Anti Von Willebrand Factor Aptamer ARC 1779 for Refractory Thrombotic Thrombocytopenic Purpura.
Authors: Firbas Chri sta , Jilma Bernd , Wagner Patricia G , Hutabarat Renta , Schaub Robert G , Gilbert James C. , Knoebl Paul
Published: April 2020

Background: The investigational anti von Willebrand Factor (vWF) aptamer ARC1779 effectively inhibits vWF activity in blood samples of controls and of patients suffering from thrombotic thrombocytopenic purpura (TTP) (Jilma et al, Blood2007;110:279a, Gilbert et al. Circulation2007;116:2678–2686).

Methods: A 39 year old comatose male patient with acute (TTP) was treated with daily plasma exchange. Further, the patient received rituximab (375mg/m2 first treatment on day 8, and weekly thereafter for 8 weeks) and was splenectomized on day 18. Due to the refractory nature of his TTP, the patient received a concomitant intravenous infusion of ARC1779 at a rate of 2 μg/kg/min beginning on day 30.

Results: ARC1779 increased the platelet count slightly from 7 to a maximum of 30/nL; during this period septicaemia and DIC may have blunted the rise in platelet counts. However, platelet counts dropped to 5/nL by 16h after cessation of infusion (day 34). The infusion of ARC1779 was re-started on day 37, and platelet counts increased from 9 to 45/nL. (Figure) Due to a temporary lack of drug, the dose of ARC1779 was stopped at 78h, and platelet counts fell to 12/nL by 12 h after interruption of the infusion. (circle in the Figure) When ARC1779 was re-started, platelet counts increased to a maximum of 97/nL and the patient’s neurologic status improved to near normal under therapy with ARC1779 over the next week.

Conclusions: ARC1779 was well-tolerated, and caused a reproducible rise in platelet counts, which alleviated severe thrombocytopenia, in an otherwise refractory TTP case. This effect was reproducible under serial “re-challenge”. Together with the observed improvement in neurologic function, the data provide clinical proof-of-concept and suggest that ARC1779 treatment might improve the organ dysfunction which typically occurs in acute TTP. These data provide a rational basis for ongoing and planned phase II trials of ARC1779.

Figure Figure
Relative Impact of Psychosocial Factors, Comorbidities, and Disease Risk on the Utilization of Resources and Overall Outcome in Patients Undergoing Hematopoietic Stem Cell Transplantation
Authors: Speckhart Dawn S , Holland H. Kent , Solomon Scott R. , Morris Lawrence E. , Bashey Asad , Kerns Leslie , Berry Tina , Manion Karen
Published: April 2020

Several studies have begun to examine the relationship between psychosocial variables and outcome in hematopoietic stem cell transplantation (HSCT). Previous studies have also looked at the relationship between the Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) and overall survival in HSCT which demonstrated a negative relationship between HCT-CI and overall survival. In this study, data was collected on 128 patients undergoing HSCT. Based on the patient’s TERS score, each patient was stratified into one of two groups (low/moderate risk (n=96) vs. high risk (n=32)) based on their predicted psychosocial risk for problems during transplant. Patients were also stratified into two categories on the HCT-CI (low (n= 102) vs. high (n=26)) based on the number of comorbid features present pre-transplant. Patients were also grouped into disease risk categories as classified by the CIBMTR criteria. Only the TERS was found to be a significant predictor of utilization of resources (length of hospitalization) between low/moderate risk (26 days) versus high risk (40 days) patients (p<.02). The HCT-CI (p=.014) and CIBMTR disease risk status (p=.02) were found to be significantly correlated with overall survival. Alone, the TERS showed a similar trend but did not appear to have a significant impact on overall survival. Those patients who scored lowest on all three measures (TERS, HCT-CI and CIBMTR disease risk) had the best overall survival rate, nearing 75% survival at 2 years post HSCT. One would conclude that although psychosocial factors impact the utilization of resources, well equipped transplant centers can provide a relatively safe method for high risk psychosocial patients to undergo HSCT. These results reinforce the impact of the HCT-CI and disease risk on transplant outcome previously found at other large transplant centers.
Acute Lymphoblastic Leukemia Blast Cells Stimulate the Autocrine Production of CCL2 and IL-8 in Bone Marrow Stromal Cells
Authors: de Vasconcellos Jaira Ferreira , Zanchin Nilson Ivo Tonin , Cardoso Angelo A. , Brandalise Silvia Regina , Yunes José Andrés
Published: April 2020

The interactions of Acute Lymphoblastic Leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival and resistance to chemotherapy. ALL stimulates BM stromal cells, which reciprocally promotes leukemia cell survival. To identify molecules critically involved in leukemia–microenvironment crosstalk, we performed gene expression profiling analyses of primary BM endothelial cells (BMEC) and BM mesenchymal stem cells (BMMSC) following stimulation by primary ALL cells. Leukemia stimulation of BM stromal cells upregulates the expression of several inflammatory chemokines, including CCL2 and IL-8/CXCL8. Secretion of these molecules was confirmed by ELISA assays of in vitro co-culture experiments and in BM plasma samples from pediatric ALL patients. Most primary ALL samples were found to express mRNA for CCR2 and CXCR1/CXCR2, which are the cognate receptors for CCL2 and IL-8, respectively. Primary ALL cells expressing at least one myeloid marker (CD13, CD15 or CD33) exhibited increased mRNA expression of CCR2 (p = 0.02). Leukemia cells from most patients express CCL2 and IL-8 chemokines (ELISA test) but at lower levels than that of BMEC and BMMSC. In vitro functional studies revealed that the proliferation, survival and migration of primary ALL cells co-cultured with BM stromal cells were not affected by addition of CCL2, IL-8 or of neutralizing antibodies to these chemokines. On the other hand, both chemokines were found to enhance BMEC and BMMSC survival in serum-free medium and to increase their proliferation in serum-starved conditions. Interestingly, CCL2 and IL-8 affected endothelial morphogenesis as shown in Matrigel assays. Since CCL2 and IL-8 have suppressive effects in normal hematopoiesis but do not seem to affect primary ALL cells, it is possible that these chemokines may contribute to the establishment of survival/proliferative selective advantage for ALL cells in the leukemic BM microenvironment. In addition, CCL2 and IL-8 seems indirectly to contribute to ALL cell survival by stimulating the supporting BM stromal cells. Finally, preliminary results showed that standard risk pediatric ALL patients with BM plasma levels below 577pg/ml have better survival rates than those with higher CCL2 levels (p = 0.08). In conclusion, this work suggests a significant role for the chemokines CCL2 and IL-8 in the leukemia/microenvironment crosstalk in human ALL, and suggests that these molecules may represent valuable targets for therapeutic intervention in this cancer. Supported by: CNPq, FAPESP.
The Histone Acetyl-Transferase MOZ Cooperates with the Histone Methyl-Transferase MLL to Regulate HOX Gene Expression in Human Hematopoietic Stem Cells
Authors: Paggetti Jérôme , Aucagne Romain , Jacquel Arnaud , Largeot Anne , Lagrange Brice , Solary Eric , Bastie Jean-Noël , Delva Laurent
Published: April 2020

MOZ (MOnocytic leukaemia Zinc finger protein) (also called MYST3 or KAT6A) is a member of the MYST family of HATs which likely acetylate H4K16. The MLL (MixedLineageLeukemia) gene is a frequent target for recurrent chromosomal translocations found in AML and ALL. MLL (KMT2A) is a methyl-transferase targeting H3K4. It was shown that MOZ/CBP leukemia, as observed in MLL-rearranged leukemias, harbors abnormal levels of homeobox (HOX) genes expression. HOX transcription factors have a crucial function in hematopoiesis regulation. In addition, HOXA5, HOXA7, and HOXA9 are often considered to be pivotal HOX genes for MLL transformation, constituting downstream targets of MLL. In our study, we characterize an in vivo cooperation between MOZ and MLL. MOZ co-localizes and interacts with MLL in vivo. We also demonstrate that WDR5, an adaptor protein essential for trimethylation of H3K4, belonging to the MLL complex, co-localizes and interacts with MOZ. Moreover, by in vitro pull-down assays, we show that MOZ can interact with the lysine 4 of histone H3 methylated by MLL, as WDR5. Hence, MLL, WDR5 and MOZ cooperate together on the same targets corresponding to the trimethylated H3K4. MOZ may couple histone acetylation to histone methylation throughout its recruitment on lysine methylated by MLL. Furthermore, ChIP experiments in HSCs indicate that MLL and MOZ are both recruited to HOXA5, HOXA7, and HOXA9 genes promoters in correlation with the presence of H3K9K14ac, H4K16ac, H3K4me2 and H3K4me3, specific epigenetic marks of transcription activation. Remarkably, knockdown of MLL or MOZ inhibits MOZ or MLL recruitment and their specific epigenetic marks from HOX genes promoters, respectively. These alterations are associated with abnormalities in hematopoietic differentiation of HSCs. Our data suggest that the histone methyl-transferase MLL and the MYST family histone acetyl-transferase MOZ functionally cooperate to modulate HOX genes expression through specific epigenetic modifications in HSCs. In conclusion, we provide an example of a mechanism involving a direct cross-talk between two histone modifying enzymes, facilitating the rapid remodeling of chromatin in order to favor the commitment of human HSCs via HOX genes regulation.
Extensively Self-Renewing Erythroid Precursors Emerge from the Embryo but Not from the Adult
Authors: England Samantha , McGrath Kathleen E. , Palis James
Published: April 2020

The only cell in the hematopoietic hierarchy thought to be capable of long-term selfrenewal is the stem cell. An erythroid progenitor derived from mammalian hematopoietic tissue, fetal or adult, is capable of limited proliferation (103–106 fold expansion; Bauer, 1999; Panzenböck, 1998; von Lindern, 1999). Here we report that an erythroid precursor derived from the mouse embryo is capable not only of limited, but also of extensive proliferation (~1030 fold expansion). These cells resemble proerythroblasts and basophilic erythroblasts based on their morphology, their globin gene expression profile, and their immunophenotype. While aneuploidy is not necessary for extensive proliferation, it sporadically begins to accumulate after prolonged culture. These cells are capable of massive (>100 days) daily proliferation in vitro in the presence of Epo, SCF, IGF-1, and dexamethasone. Examination of cultures lacking each of these factors support that glucocorticoids play an important role in this expansion by uncoupling erythroid precursor proliferation from maturation. Despite prolonged in vitro culture, these cells preserve their potential to fully differentiate into enucleated red blood cells with the removal of dexamethasone. Differentiation occurs over 2–3 days and is characterized by the accumulation of adult (α, β1, and β2), but not embryonic (ζ, εy, and βH1), globins. The retention of full differentiation potential despite >1030 fold expansion indicates that this proliferation represents self-renewal. To determine the developmental origin of these extensively self-renewing erythroblasts (ESREs), we initiated in vitro cultures from staged mouse embryos as well as adult tissues. E7.5 embryos, that contain primitive but not definitive erythroid progenitors, failed to generate ESREs. In contrast, ESREs can be derived from E8.5–E10.5 yolk sac and E11.5–E14.5 fetal liver. These findings along with the globin expression pattern indicate that erythroblast self-renewal is associated with definitive, but not primitive, erythropoiesis. Surprisingly, marrow from adult steady-state hematopoiesis failed to yield ESREs. Furthermore, despite the characteristics shared by stress erythropoiesis (adult spleen) and fetal erythropoiesis (liver), stress erythropoiesis only yielded erythroblasts with limited, and not extensive, self-renewal capacity. This result suggests that extensive self-renewal potential is linked either to the transient yolk sac-derived definitive erythroid lineage or to the fetal hematopoietic microenvironment. We are currently investigating the mechanisms responsible for the extensive self-renewal capacity of such lineage-restricted and mature hematopoietic precursors. Our findings raise the possibility that the expansive cellular output of the erythron within the midgestation mammalian embryo may be regulated, in part, at the level of late stage erythroid precursors.
Notch1 Expression Is Regulated at Post-Transcriptional Level by 3′UTR Region in Hematopoietic Stem Cell Development
Authors: Mizuno Shin-ichi , Ozawa Hidetoshi , Iino Tadafumi , Arinobu Yojiro , Yong Chong , Shigematsu Hirokazu , Akashi Koichi
Published: April 2020

In hematopoietic stem cell development, the expression of critical genes is precisely regulated in a stage specific manner, which supports normal hematopoietic development through adequately regulating timing of cell division, self-renewal, and lineage commitment. Regulation of gene expression is known to take place at least at transcriptional level. In addition to the transcriptional regulation, there are growing evidences that post-transcriptional control of critical genes may play an important role, suggesting an interesting possibility that post-transcriptional control may also play a role in hematopoiesis. Here, we provide the evidence that the expression of Notch1, a key factor in lymphoid development, is controlled at post-transcriptional level in hematopoietic stem cell (HSC). By quantitative PCR, Notch1 mRNA is substantially expressed at HSCs as well as common lymphoid progenitors (CLPs) or double negative (DN) thymocytes. However, Notch1 protein is detected at very low level in HSCs compared to CLPs or DN thymocytes, suggesting that Notch1 expression is regulated at post-transcriptional level in HSC. To investigate the effect of 3′UTR (untranslated region) on post-transcriptional regulation, we prepared a retrovirus sensor vector, in which 3′UTR of target gene is placed between the GFP coding region and the retrovirus 3′LTR, and found that induction of the sensor vector with the 3′UTR sequence of Notch1 showed marked suppression of the GFP intensity at the HSC stage. This effect was not observed when we introduced the vector into DN thymocytes. Suppression of Notch1 by its 3′UTR was further confirmed by using a retrovirus vector which has two distinct markers of YFP and GFP-3′UTR fusion genes under bi-directional EF1 promoter. Deletion mutant analysis showed that the responsible region required for this post-transcriptional suppression is confined to 120-bp sequence within Notch1 3′UTR so far. These data suggest that the expression of Notch1 should be regulated at post-transcriptional level by its 3′UTR at the HSC stage and our data provide the first evidence that the stage-specific translational regulation can play an important role in organization of hematopoietic development.
Human Cytomegalovirus: Degranulation Is a Prominent Feature of Functionally Impaired pp65- and IE-1-Specific T-Cells in Patients after Allogeneic Stem Cell Transplantation
Authors: Fuhrmann Stephan , Ganepola Susanne , Uharek Lutz , Thiel Eckhard , Ludwig Wolf-Dieter , Volk Hans-Dieter , Kern Florian
Published: April 2020

Human cytomegalovirus (CMV) reactivation and disease is still a frequent complication after allogeneic stem cell transplantation (allo SCT). It is well accepted that T-cell immunity is mandatory to control CMV infection and disease and much effort has been put into the development of cell-based monitoring assays. Nevertheless, no reliable marker for protective immunity has been established to date. Most studies use one CMV model antigen (pp65) to compare the frequencies of cytokine producers (mainly IFNg) or multimer-specific T-cells. Methods: In total, we recruited 16 patients after allo SCT, (7 high risk, 9 standard risk pts.). We used 8-colour flow cytometry to detect degranulation (mobilized CD107a/b), intracellular IFNg, TNFa, IL-2 production and CD28-expression in peptide pool stimulated pp65 and IE-1 specific CD8 T-cells. Results were compared to 7 healthy CMV exposed donors. Results: Degranulation identifies the highest percentage of CMV-specific T-cells in allo-transplanted patients (pp65: 0,94% degranulation and 0,31% IFNg; IE-1: 1,44% degranulation and 0,87% IFNg, mean frequency). These T-cells are relatively cytokine deficient compared to those in healthy donors (cytokine-production/degranulation ratio: SCT=0,42, healthy=0,72 for pp65, p=0,048; SCT=0,61, healthy= 1,00 for IE-1, p=0,133, U-test). The cytokine expression pattern differs between antigens used for stimulation, for example more IL-2-producers could be detected in the pp65 specific compartment (12,5% for pp65 and 4,5% for IE-1 of all activated CD8 T-cells, p=0,015).

Conclusion: This study demonstrates that degranulation is the most prominent marker of CMV-specific T-cells (pp65 and IE-1) in allo SCT patients. Looking at IFN-g producers only may underestimate the frequencies of CMV specific T-cells in this setting. Furthermore, these subsets have a divergent functionality in transplant recipients compared to healthy individuals. Our data challenge the concept of enumerating CMV specific T-cells to estimate immunity. We rather propose measuring functional differences in the T-cell response may help to identify patients with a high risk of CMV reactivation. A careful dissection of these differences is a prerequisite for the development of monitoring tools and adoptive T-cell transfer.
FTY720 Abrogates the Therapeutic Potential of Adoptively Transferred Treg Via Inhibition of IL-2 Induced in Vivo Expansion
Authors: Wolf Anna Maria , Hochegger Kathrin , Zeiser Robert , Duerr Christoph , Sixt Michael , Rosenkranz Alexander , Gastl Gunther , Wolf Dominik
Published: April 2020

CD4+CD25+ T cells (Treg) entry into secondary lymphoid organs (SLO) and local expansion after activation is at least in part responsible for their immunosuppressive action. Thus we hypothesized that trapping of adoptively transferred Treg in SLO would be an effective means to tip the balance towards a more immunosuppressive milieu within the LN microenvironment. Systemic application of the sphingosine-phosphate receptor agonist FTY720 has been proven to trap harmful effector T cells in SLO, thereby inhibiting their migration and destruction of target tissue. Here we provide first evidence that selective entrapment of adoptively transferred Treg in inflammatory LN can be achieved by blockade of SP-receptors upon ex vivo exposure of Treg to FTY720 before adoptive transfer. FTY720 exposure did not interfere with proper Treg localization within the T-cell areas of SLO as determined by immunofluorescent microscopy after co-transfer of either FTY720- or solvent exposed and subsequently differentially labelled Treg. However, despite the fact that the in vitro phenotype (including expression of adhesion and chemokine receptors), function (including anergy and suppressive activity) and survival (determined by Annexin/PI staining) of Treg remained unaltered by FTY720, it abrogated their protective effect after adoptive transfer in a murine model of acute experimental glomerulonephritis (determined by quantification of proteinuria and histological analysis) as well as in an acute GvHD model (determined by survival analysis and quantification of the in vivo expansion of luciferase-transgenic effector T cells by bioluminiscence technology). Notably, adoptive transfer of CFSE-labelled Treg revealed a markedly impaired proliferation of Treg in inflammatory SLO when pre-exposed to FTY720 ex vivo. Accordingly, FTY720 blocked Treg-proliferation induced by TCR-stimulation in combination with IL-2 in vitro. In line with this observation, FTY720 completely abolishes IL-2 induced phosphorylation of STAT-5. Thus, SP-1P receptors induce Treg trapping in inflammatory SLO but abrogate their in vivo immunosuppressive potential by inhibition of local Treg expansion.
A Prospectively Randomized Placebo-Controlled Trial of Epoetin-α in Patients with Advanced-Stage Hodgkin Lymphoma: Final Analysis of the GHSG HD15-EPO Trial
Authors: Engert Andreas , Borchmann Peter , Fuchs Michael , Josting Andreas , Haverkamp Heinz , Diehl Volker
Published: April 2020

Anemia is frequent among cancer patients and impacts organ function and quality of life of those affected. Anemia has been shown to be a negative prognostic factor in several malignancies including Hodgkin Lymphoma (HL). In addition, the advent of more aggressive treatment regimen such as BEACOPP escalated has resulted in more HL patients needing red-blood-cell transfusion. Thus, apart from the chemotherapy-related question of the GHSG HD15 trial, ie 8 cycles of BEACOPP escalated or 6 cycles of BEACOPP escalated or 8 cycles of BEACOPP14, patients in HD15 were randomized between Epoetin-α and placebo. Study drug was given at 40.000 I.E. weekly with a hemoglobin target level of 12–13g/dl. Primary endpoint was fatigue after the end of chemotherapy (CT) and 6 months later measured with the EORTC QLQ-C30 questionnaire (0–100 scale). With respect to the more recent safety discussion related to erythropoetins (EPOs) in general, we also analyzed efficacy and safety endpoints although the trial was not sufficiently powered for time-to-event analyses. Between1/2003 and 12/2006 1.379 patients were randomized for the EPO question of whom 1.329 were eligible. Of these 1.286 (96.8%) are fully evaluable. Patient characteristics were balanced between the placebo and the EPO arm. In those 688 patients who were randomized before 7/2005 and eligible for analysis, the CR/CRu rate was 90, 6% with a median follow-up of 28 months, 58 progressions/relapses (8.4%) and 33 deaths (4.8%) without differences between study arms. There was also no difference in terms of freedom from treatment failure (hazard ratio (HR) 0.9, 95%-confidence interval (CI) 0.6–1.4) and overall survival (HR 1.1, 95% CI 0.6–2.3). 51 serious adverse (7.4%) and 35 thrombovascular events (5.1%) were observed with no significant difference between arms. The median number of red blood cell units given significantly favored patients receiving Epoetin-α (2 vs 4; test for trend p<0.001). The interim analysis for fatigue gave an average value of 61+/−25.8 (mean +/−SD) at the end of treatment and a reduced fatigue score of 33+/−25.3 at six months after CT.

Conclusion: This is the largest prospectively randomized, placebo-controlled trial with EPO in cancer patients. So far we found no difference in terms of fatigue, response, relapse, or side effects between those advanced-stage HL patients receiving Epoetin-α or placebo. The number of RBC units given was significantly reduced in the EPO group. However, all cases are needed for a final conclusion and will be presented.
PKC412, a Small Molecule Tyrosine Kinase Inhibitor, Regulates Survival and Proliferation Pathways in Waldenström Macroglobulinemia
Authors: Melhem Molly R. , Roccaro Aldo M. , Azab Abdelkareem , Ngo Hai T. , Sacco Antonio , Jia Xiaoying , Azab Feda , Runnels Judith , Burwick Nicolas , Quang Phong , Husu Emanuel N. , Leleu Xavier , Anderson Kenneth C. , Ghobrial Irene M.
Published: April 2020

Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase C (PKC) is a multigene family that that plays an important role in the regulation of cell growth and tumor progression. PKC412, a derivate of staurosporine, is a competitive inhibitor of ATP binding to PKC subtypes. This PKC inhibitor was used to examine its role in WM.

Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay. Cell signaling of HSP response, VEGF signaling and AKT pathways were determined by Western Blot analysis. Cell cycle and apoptosis analysis were obtained through flow cytometry.

Results: We showed that PKC412 induced cytotoxicity in WM cell lines tested (IC50 of 1uM) with no cytotoxic effect on healthy donor peripheral blood mononuclear cells at concentrations as high as 5uM. Cell proliferation and apoptosis analysis showed similar results, in a time independent manner. A G2/M arrest was observed upon cell cycle analysis, suggesting a possible mechanism for apoptosis. Similarly, the Akt pathway was investigated and was found to show a decrease in its activity in a dose dependent manner. Finally, PKC412 regulated VEGF levels in WM samples indicating an anti-angiogenic activity.

Conclusion: PKC412 has a significant effect on the survival and proliferation of WM cells, including regulation of angiogenesis. These studies provide the framework for clinical evaluation of this novel PKC inhibitor in Waldenström Macroglobulinemia.
Regulation of Histone Deacetylase in Waldenström Macroglobulinemia
Authors: Jia Xiaoying , Roccaro Aldo M. , Azab Abdel Kareem , Ngo Hai T. , Sacco Antonio , Melhem Molly R. , Azab Feda , Runnels Judith , Burwick Nicolas , Quang Phong , Husu Emanuel N. , Leleu Xavier , Anderson Kenneth C. , Bradner James , Ghobrial Irene M.
Published: April 2020

Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Histone deacetylase (HDAC) inhibitors represent promising new treatment strategy in B cell malignancies. We therefore investigated the in vitro effect of the novel hydroxamic acid derivative HDAC inhibitor LBH589 in WM.

Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTS assay and thymidine uptake assay. Cell signaling and apoptotic pathways were determined by Western Blot and immunofluorescence.

Results: LBH589 induced a significant decrease of proliferation and triggered cytotoxicity in all cell lines tested and primary CD19+ WM cells (IC50 of 20–40nM), even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, LBH589 did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells. LBH589 induced both intrinsic and extrinsic apoptotic pathways, with caspase-9, caspase-8, caspase-3, and PARP cleavage in a dose-dependent manner. We also demonstrated significant upregulation of the proapoptotic transcription factor p53 and down-regulation of the anti-apoptotic proteins BclxL, Mcl-1 and c-myc. We then demonstrated that LBH589 induced apoptosis in WM cells in a caspase-independent manner through induction of autophagy, as shown by upregulation of LC3B and Rab7 expression. We further determined the mechanism of action of LBH589 in WM, investigating the effect of LBH589 on histone acetylation and NF-kB pathways. We found that LBH589 induced a dose-dependent increase in histone H3-H4 acetylation; and inhibited both canonical and non-canonical pathways of NF-κB, as shown by western blot and immunofluorescence. In addition, LBH589 augmented rituximab, fludarabine, bortezomib, and perifosine-induced cyotoxicity in WM cells.

Conclusion: LBH589 has significant antitumor activity in WM in vitro, providing the framework for clinical trials evaluating LBH589 as a new therapeutic agent in patients with WM.
5-Azacytosine (Azacytidine) and 2′-Deoxy-5-Azacytidine (Decitabine) Mediate Opposite Effects on NK Cell Anti-Tumor Reactivity
Authors: Schmiedel Benjamin J , Arélin Viktor , Krusch Matthias , Kanz Lothar , Salih Helmut R
Published: April 2020

The cytosine analogues 5-azacytosine (azacytidine) and 2′-deoxy-5-azacytidine (decitabine) display substantial therapeutic potential in patients with AML and MDS. Besides causing DNA demethylation, azanucleosides also mediate cytotoxic effects, and it appears that clinical responses are influenced by both epigenetic alterations and by apoptosis induction. However, the molecular changes induced by these drugs are still poorly understood. NK cells play an important role in tumor-immunosurveillance by confining development and progression of hematopoietic malignancies and are also important after therapeutic intervention like e.g. haploidentical stem cell transplantation. Thus it is important to define how a given therapeutic agent influences NK cell reactivity. Here we studied the effect of pharmacological concentrations of azacytidine and decitabine on NK cell effector functions. After preincubation with the azanucleosides (12h or more), NK cell cytotoxicity was found to be significantly enhanced by decitabine while, in contrast, azacytidine nearly completely abolished NK cell lysis of K562 and Raji target cells (up to 50% increase and 88% reduction, respectively; E:T ratio 10:1). Of note, modulation of NK cell cytotoxicity was also observed when the compounds were, after NK cell pretreatment, absent during the cytotoxicity assays. In contrast, neither the presence of either agent in cytotoxicity assays without preincubation nor pretreatment of target cells with either agent for 4h (corresponding to the time of the cytotoxicity assay) altered NK cell reactivity. These results indicate that azanucleosides are capable to modulate NK cell responsiveness to activating stimuli. In line, after pretreatment with the compounds NK cell IFN-γ production upon stimulation with IL-2 and IL-15 or in cocultures with target cells was found to be enhanced by decitabine but inhibited by azacytidine (up to 46% increase and 85% reduction, respectively). NK cell effector functions were not affected by deoxycytidine and cytidine, the physiological counterparts of the azanucleosides. While azacytidine treatment substantially induced NK cell apoptosis (about 30% after 24h) which may explain its inhibitory effect, no induction of apoptosis by decitabine was observed. Our data demonstrate that azacytidine and decitabine differentially affect NK cell anti-tumor reactivity and suggest that, while azacytidine causes NK cell apoptosis, decitabine enhances NK cell responsiveness via a yet unknown mechanism which is presently under study.
WP-1130 Induces Apoptosis in Multiple Myeloma Cells by Inactivation of Jak2 and Syk Tyrosine Kinases Via a Novel Mechanism of Selective Protein Trafficking
Authors: Lebovic Daniel , Kapuria Vaibhav , Hari Malathi , Talpaz Moshe , Jakubowiak Andrzej , Donato Nicholas
Published: April 2020

Multiple myeloma is a plasma cell malignancy affecting more than 50,000 people in the United States. The disease remains incurable despite significant recent advances in treatment, including the immunomodulatory agents, thalidomide and lenalidomide, and the proteasome inhibitor, bortezomib. Thus the search for additional agents is warranted. We have previously shown that WP1130, a tyrphostin-like compound, has activity in a number of hematological malignancies including CML, mantle cell lymphoma and multiple myeloma. In this study, we investigated the activity and mechanism of action of WP1130 on multiple myeloma cells and compared these activities to bortezomib. Both WP1130 and bortezomib demonstrated significant cytotoxicity in MM1S cells with IC50s of 1 mM and 2 nM, respectively and both compounds induced similar levels of apoptosis (67% vs 73% cell death with WP1130 and bortezomib, respectively). Both WP1130 and bortezomib increased cellular ubiquitination levels in distinct cellular compartments but we were unable to detect 20S proteasome inhibition by WP1130. The effect of WP1130 on signaling modules in MM cells was also investigated. WP1130 altered the subcellular distribution of a subset of tyrosine kinases, including Jak2 and Syk which were irreversibly displaced from the cytoplasm to detergent insoluble complexes within the cell. Although Jak2 remained stable in this fraction, Syk was subject to proteolysis and destruction in this compartment. WP1130-induced translocation of these kinases through association with the cytoskeletal network and was associated with loss of cytokine and B-cell receptor signal transduction. WP1130 did not alter the cellular distribution of other tyrosine kinase or phosphoproteins including Lyn, STAT3, STAT5, PI3K, AKT, ERK, suggesting some selective protein translocation and signal modulation by WP1130. These results support a novel mechanism of protein trafficking, selective kinase destruction and apoptosis by WP1130 in MM cells.
Repetitive DNA Hypomethylation in Multiple Myeloma
Authors: Fabris Sonia , Bollati Valentina , Mosca Laura , Pegoraro Valeria , Ronchetti Domenica , Ciceri Gabriella , Baldini Luca , Morabito Fortunato , Deliliers Giorgio Lambertenghi , Bertazzi Pier Alberto , Baccarelli Andrea , Neri Antonino
Published: April 2020

Multiple myeloma (MM) is a malignant proliferation of bone marrow plasma cells characterized by a wide spectrum of genetic and epigenetic changes. Global hypomethylation of repetitive genomic sequences such as long interspersed nuclear elements-1 (LINE-1) and Alu repetitive elements (approximately 500.000 and 1.4 million in the human genome) has been associated with chromosomal instability. Additionally, satellite alfa DNA (SAT-alpha DNA) hypomethylation has been reported to be associated to karyotypic instability in human cancer, possibly playing a role in centromere function. So far, the LINE-1/Alu and centromeric SAT-alpha DNA methylation patterns have not been investigated in the context of the different clinical and molecular MM subtypes. Global DNA methylation changes were investigated in a panel of 53 newly diagnosed, untreated MMs, 7 plasma cell leukemias (PCL) and 11 healthy subjects as controls. DNA was extracted from purified plasma cells, treated with bisulfite and analyzed by bisulfite-PCR and Pyrosequencing. Methylation of LINE-1 and Alu elements was shown to correlate with total 5mC content and thus used to estimate global DNA methylation. MMs showed a decrease of Alu (21.1%) and LINE-1 (70.0%) methylation average levels compared with controls (25.2% and 79.5% respectively). Lower median methylation levels were also found in centromeric SAT-alpha DNA of MMs (77.95%) compared to controls (89.5%). The median methylation level of PCLs was lower than MMs (16.7% versus 21.1% for Alu; 45.5% versus 70.0% for LINE-1; and 33.3% versus 77.9% for SATalpha DNA). Notably, a statistically significant association between SAT-alpha DNA and LINE-1 methylation (Spearman’s rank correlation, ρ = 0.94; P < 0.001) was found in MM. The comparison between methylation pattern and different molecular MM subgroups by means of non parametric tests, revealed that LINE-1 and SAT-alpha DNA methylation was significantly lower in the nonhyperdiploid versus hyperdiploid (HD) tumors (P = 0.01 and 0.02 respectively). Alu and SAT-alpha were significantly lower in the MMs with t(4;14) (P = 0.02 and 0.004 respectively). Finally, in the context of translocation/cyclin D (TC) classification, a statistically significant differences inside the five different groups were found for SAT-alpha DNA methylation (P = 0.008, Kruskal-Wallis test). These findings may provide insights into the molecular mechanisms of MM pathogenesis and suggest that our approach may contribute toward a more exhaustive stratification of the disease.