bluuebunny/crossref_metadata_2025 huggingface dataset:
Pre-Clinical Trial on the Co-Transplantation of MSCs and Umbilical Cord Blood Stem Cells.
Authors: Shao-liang Huang , Ke Huang , Dun-hua Zhou , Jian-pei Fang , Yang Li , Chun Chen , Xiang Peng
Published: October 2019

Objective Cotransplantion of Mesenchymal stem cells(MSCs)with UCB-HSC promote the engraftment, accelerate the recovery of hematopoiesis and alleviate the severity of graft versus host disease(GVHD). Cases and methods 18 cases were treated with UD-UCBT (15 cases) and RD-UCBT (3 cases) .of them, acute lymphoblastic leukemia (high risk) 2 cases, acute myeloblastic leukemia 4 cases, chronic myeloblastic leukemia 3 cases, β-thalassemia major 8 cases and, mucopolysaccharidosis 1 case. HLA matched (6/6) in 3 cases, one locus (5/6) were dismatched in 12 cases and two HLA loci (4/6) in 3 cases. UCB was transfused into recipients according to the regular method. The MSCs were from auto-BM in 3 cases, from allogenic bone marrow of parents 4 cases, from allogenic related bone marrow 10 cases, and cord blood MSCs 1 case. The MSCs were transfused by IV (14 cases) or IBM (4 cases) injection. The conditioning regimens modified on the basis of Bu/CY + Fluda + ATG. GVHD was prevented according to the regular method in our department. Infused NCs dose of UCB was 6.77×107/kg, CD34+ÆÞsG5.58×105/kg; allo-MSCs 4.03×106/kg, auto-MSCs 8.83×106/kg. MSCs origin were detected.

Results

Ten cases were full donor engrafted, the time to get ANC ≥0.5×109/L and PLT ≥2.0×1010/L were on the 13.9th day and the 30.8th day. CFU-MIX, MSCs of recipients in Co-transplantation groups recovered gradually from 14th day to 28th day. MSCs origin were detected in 4 cases, all were recipients.

Conclusion

MSCs co-transplantation accelerated HSCs engraftment and hematopoiesis recovery, the time to get ANC ≥0.5×109/L and PLT ≥2.0×1010/L were faster than those of single CBT and UD-BMT. MSCs co-transplantation cannot promote engraftment in CBT of β-thalassemia major. Co-transplantation of MSCs accelerated reconstitution of hematopoietic cells and MSCs.
Vaccination of Leukemia Patients with Autologous In Vitro Cultured Leukemia Dendritic Cells.
Authors: Yue Lu , Geng Qi-Rong , Xia Jian-Chuan , Fan Hui-jie , Xu Hai- , Chen PengXiao-qing
Published: October 2019

OBJECTIVE:To investigate the safety and immunomodulation effect of dendritic cells(DCs)vaccine to leukemia patients cultured in vitro from autologous leukemia cells.

METHOD:Leukemic-dendritic cells were cultured from autologous bone marrow leukemia cells in vitro using combined cytokines. Patients received four administrations of autologous in vitro cultured leukemia-DCs by intravenous injection during chemotherapy intervals when obtain complete remission (CR), once a week for four times. Peripheral blood were collected to analyze the serum Th1/Th2 cytokines as IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α by cytokine bead array(CBA) analysis and T cell subpopulations by flow cytometric method (FCM).

RESULTS:A total of 31 patients who are hematologically confirmed as leukemia received the DC vaccination therapy from December 2002 to October 2005. 20 patients can be evaluated. Firstly, autologous leukemic-dendritic cells were cultured in vitro successfully. Secondly vaccinations were well tolerated. No major toxicity occurred in any of the patients. Thirdly, serum cytokines were all elevated 24hs after the first administration of DC vaccination compared to those before that. And IL-2, IFN-γ, IL-4 and IL-6 have statistically significant increases with Z values −2.576, −2.033, −2.777, −2.053 and p values 0.010,0.042,0.005,0.040, respectively. Relative increase of the cytokine levels induced by DC vaccination show a trend of TH1 cytokines advantage, suggesting an enhancing of cellular immune response. T cell subsets significantly increase in CD4/CD8 ratio and CD56 proportion with Z values −2.040, −1.988 and P values 0.041, 0.043, respectively.

CONCLUSIONS:Autologous leukemic-DCs could be generated in vitro from leukemia cells. And the intravenous injection of autologous L-DC vaccine is safe and feasible, and it could enhance the immune response in vivo to a certain degree.
Construction of Wif1-IgG1/Fc Recombinant Vector and Characterization of Its Expression in CHO Cells.
Authors: Cai Zhen , Hu Jie
Published: October 2019

Wnt signalling dysregulation has been implicated in cancers, including lymphoma. The Initiation of Wnt signalling modulated by soluble Wnt ant agonists, including soluble frizzled related proteins, dickkopf proteins, and Wnt inhibitory factor-1 (Wif1). These natural inhibitors can be used directly as theraputic reagents for cancers. But the recombinant antagonist only give very low blood levels, are cleared rapidly from the body (only several hours) and must be administered daily via subcutaneous injection. Therefore, development of new forms of Wnts inhibitors with prolonged circulating half-life is of significant interest. Human IgG immunoglobulins have longer circulating half-life (21 days in humans). When fused with a Wnt inhibitor, its dimeric structure further increases the effective size and circulating half-life. Thus administration of vector plasmid may be every 2~3 week. Moreover, IgG Fc can interact with FcR of CTL, NK, MΦ, and complements and the target cells can be killed through ADCC and CDCC. We constructed a Wif1-IgG1/Fc expressing vector and expressed it in Chinese hamster ovum cells in order to seek effective methods to inhibit lymphoma growth. Wif1 and IgG/Fc cDNAs were amplified by RT-PCR from human lymphocyte and cloned into the eukaryotic expression vector pcDNA3.0 by direct cloning and the resultant recombinant plasmid pcDNA Wif1-IgG1/Fc was transfected into 293 cells with lipofectmin. The constitutively expressing cells were obtained by G418 screening. Expression of the fusion protein was confirmed by Western blot, the fusion protein was purified with affinity chromatography, and its’ biological activity was examined by flow cytometry, MTT colorimetry and ELISA. Our results show that the fusion protein significantly upregulated Wif1 density and promoted molt3 cell apoptosis, which was time dependent. The molt3 cell apoptosis induced by the fusion protein increased in the presence of NK cells or complements. Thus, we have successfully constructed and functionally expressed Wif1-IgG1/Fc fusion protein and shown that the purified fusion protein could kill molt3 cells through apoptosis and by ADCC and CDCC.
Stem Cell Transplantation in Children with Infantile Osteopetrosis Is Associated with a High Incidence of VOD, Which Could Be Prevented with Defibrotide.
Authors: Corbacioglu Selim , Manfred Hoenig , Lahr Georgia , Stoehr Susanne , Berry Gaurav , Friedrich Wilhelm , Schulz Ansgar S.
Published: October 2019

Background: Malignant infantile osteopetrosis (MIOP) is a rare hereditary disorder of osteoclast function, which can be reversed by hematopoietic stem cell transplantation (SCT). In recent studies defibrotide (DF) demonstrated superior efficacy in the treatment of hepatic veno-occlusive disease (VOD).

Methods: Twenty children with MIOP were consecutively transplanted in our centre between 1996 and 2005. Eleven of these patients were transplanted between 1996 and 2001 without any VOD prophylaxis. Thereafter nine patients were transplanted between 2001 and 2005 with prophylactic DF from the first day of conditioning until 30 days after SCT because we observed a high incidence of VOD in transplanted patients and explored the prevention of this complication by using DF as a prophylaxis.

Results: Patients without DF experienced an overall incidence of VOD of 63.6% (7/11). VOD was severe in three patients and one patient succumbed to VOD related multiorgan failure (MOF). In nine patients consecutively transplanted between 2001 and 2005 only one patient (11.1%) was diagnosed with moderate VOD.

Conclusion: SCT in patients with MIOP is associated with a very high risk to develop VOD. Prophylactic DF was implemented in our current transplant protocol and reduced the VOD rate dramatically in this high risk population.
Alemtuzumab in the Treatment of Steroid Refractory Acute Graft-Versus-Host Disease.
Authors: Gomez-Almaguer David , Ruiz-Arguelles Guillermo , Gonzalez-Llano Oscar , Cantu Olga G. , Gutierrez Cesar H. , Jaime-Perez Jose Carlos , Tarin-Arzaga Luz , Giralt Sergio
Published: October 2019

Corticosteroids therapy is the mainstay of treatment for GVHD, however, it heavily impacts on post transplant morbidity and new modalities are continually needed. Alemtuzumab a humanized monoclonal antibody to CD52 has been used mainly as GVHD prophylaxis. Only a few patients have been treated with this antibody. From December 2004 to May 2006, we recruited 13 steroid refractory acute GvHD patients in a prospective trial evaluating the efficacy of alemtuzumab (Campath 1H) after exclusion of other severe HST-related complications. Primary endpoints were response to treatment after 14 and 28 days. Secondary endpoints were side effects and incidence of infectious complications. Treatment consisted of Campath 1H 10mg given s.c. on days 1–5. Median age was 33 years old (range:1–59) years, a fludarabine-based reduce intensive conditioning (RIC) regimen was used and the hematopoietic cells were obtained from HLA-identical siblings in 12 cases and one patient received stem cell from umbilical cord blood. All but one received CSA and MTX for GvHD prophylaxis. GvHD affected gut in 6 cases, skin in 3 liver in 4, and combination of gut and skin in 6 patients. In 6 of the 13 patients the clinical manifestations of GVHD were noticed after the first 100 days of HSCT. Complete resolution of GvHD, partial response and no response were seen respectively in 23%, 62% and 15%. Six over the 13 patients were able to decrease steroid use. Five patients developed CMV (pp65) reactivation and 3 of them were successfully treated with valganciclovir. All patients maintained complete chimerism during and after alemtuzumab therapy, and after a median follow-up of 4 months (range, 1– 17months), 8 remain alive, 3 without evidence of GVHD. Five patients died, 3 due to GvHD and the others due to infectious complications. This preliminary study suggests that alemtuzumab is a well-tolerated agent and has a beneficial effect in the treatment of refractory GvHD. It is only a pilot study and more studies are needed, but we suggest that this modality could be used early in the management of these patients in order to improve quality of life and reduce the long-term side effects of corticosteroids.
Hepatic Injury Following Reduced-Intensity Unrelated Cord Blood Transplantation for Adult Patients with Hematological Diseases.
Authors: Kusumi Eiji , Kami Masahiro , Kanda Yoshinobu , Seki Kunihiko , Fujiwara Masayo , Koyama Rikako , Komatsu Tsunehiko , Kodama Yuko , Matsumura Tomoko , Yuji Koichiro , Miyakoshi Shigesaburo , Taniguchi Shuichi
Published: October 2019

Liver injury is a common complication in allogeneic hematopoietic stem cell transplantation. Its major causes comprise graft-versus-host disease (GVHD), infection, and toxicities of preparative regimens and immunosuppressants; however, we have little information on liver injuries following reduced-intensity cord blood transplantation (RICBT). We reviewed medical records of 104 recipients who underwent RICBT between March 2002 and May 2004 at Toranomon Hospital. Preparative regimen and GVHD prophylaxis comprised fludarabine/melphalan/total body irradiation, and cyclosporine or tacrolimus. We assessed the etiology of liver injuries based on the clinical presentation, laboratory results, comorbid events, imaging studies in 85 patients who achieved primary engraftment. The severity of liver dysfunction was assessed according to the National Cancer Institute Common Toxicity Criteria version 2.0. Hyperbilirubinemia was graded according to the report by Hogan et al. (Blood.2004;103:78–84). Moderate to very severe liver injuries were observed in 36 patients. Their causes included cholestatic liver disease related to GVHD or sepsis (CLD) (n=15), GVHD (n=7), cholangitis lenta (n=5) and others (n=9). Median onsets of CLD, GVHD and cholangitis lenta were day 37, 40 and 22, respectively. Frequencies of grade 3–4 ALT elevation were comparable among the three types of hepatic injuries. Serum GTP was not elevated in any patients with cholangitis lenta, while 27% and 40% of patients with CLD and GVHD, respectively, developed grade 3–4 GTP elevation. Multivariate analysis identified two risk factors for hyperbilirubinemia; grade II–IV acute GVHD (relative risk 2.23, 95% confidential interval (95% CI) 1.11–4.47, p=0.024) and blood stream infection (relative risk 3.77, 95% CI 1.91–7.44, p=0.00013). In conclusion, the present study has demonstrated that the hepatic injuries are significant problems following RICBT, and that GVHD and blood stream infection contribute to their pathogenesis.
The Relationship between Anemia and Chronic Kidney Disease (CKD) in Older Nursing Facility (NF) Residents.
Authors: Artz Andrew , Robinson Bruce , Culleton Bruce , Critchlow Cathy , Sciarra Angela , Audhya Paul
Published: October 2019

Anemia is a common condition among older NF residents that often has unidentified causes. CKD is common in older adults and frequently contributes to anemia. However, the association between declining kidney function and the prevalence and severity of anemia in NF residents has not been well characterized. We retrospectively analyzed the independent association of CKD and anemia among older residents in 372 NFs across the US. Any resident who was admitted to a NF between 01 Jan 2002 and 31 Dec 2003, was still active as of 31 Jan 2004, was ≥ 65 years of age, and had more than one serum creatinine (SCr) measurement and more than one hemoglobin (Hb) documented during the admittance period, was considered. Residents who had end-stage disease, were comatose, or were receiving dialysis were excluded. CKD was conservatively defined as an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2 (MDRD equation). Anemia was defined using the WHO criteria for anemia (< 13 g/dL for men and < 12 g/dL for women). Regression analysis was used to determine the association between kidney function and Hb levels after adjusting for age, race, and gender. Of the 10315 residents identified, 6200 were eligible for this study: 85% (5291/6200) were white, 70% (4354/6200) were female, and the mean ± SD age was 83.1 ± 7.9 years. Anemia was found in 59.6% (3697/6200) residents as defined by the WHO criteria and CKD was present in 43.1% (2671/6200) residents. The odds of having anemia was 1.47 times greater (95% CI: 1.33, 1.63) for residents with CKD than those without CKD. The severity of anemia also increased with the presence and severity of CKD. The odds ratio increased to 1.65 (95% CI: 1.49, 1.84) after adjusting for age, race, and gender. Of those with CKD, 36.4% (972/2671) had a Hb < 11 g/dL, however, only 5.1% (50/972) and 25.4% (247/972) received erythropoiesis-stimulating protein (ESP) and iron therapies, respectively. Our data suggest CKD contributes to the high prevalence of anemia in older NF residents. These results reinforce the importance of evaluating kidney function and Hb level in older NF residents. Future studies evaluating the impact of anemia and benefits of anemia therapy are warranted in this setting.
Timing of Second Autologous Transplantations in Multiple Myeloma: Results of a Multicenter Sequential Randomized Clinical Trial.
Authors: Abdelkefi Abderrahman , Ladeb Saloua , Ben Othman Tarek , Torjman Lamia , Lakhal Amel , Ben Romdhane Neila , El Omri Halima , Elloumi Moez , Bellaj Hatem , Jeddi Ramzi , Aissaoui Lamia , Msadek Fahmi , Saad Ali , Hsairi Mohamed , Boukef Kamel , Amouri Ahlem , Louzir Hechmi , Dellagi Koussay , Ben Abdeladhim Abdeladhim
Published: October 2019

Background: Autologous stem cell transplantation (ASCT) is now considered standard therapy in young patients (<65 years) with multiple myeloma (MM). The Intergroupe Francophone du Myelome conducted a randomized trial of the treatment of MM with high-dose chemotherapy followed by either one or two successive ASCTs. The probabilities of event-free-survival and overall survival were doubled with a double transplant. However, no randomized trial has compared tandem transplant up-front with a strategy including planned second ASCT at relapse or progression. Therefore, we performed a multicenter, sequential, randomized trial designed to assess the optimal timing of a second ASCT.

Methods: From May 2003 to April 2006, 140 patients with symptomatic MM (de novo) and less than 60 years of age, were randomly assigned to receive either tandem transplantation up-front (within 6 months of the first transplantation) [Arm A, n=69] or one ASCT followed by a consolidation therapy with thalidomide (day +90, 100 mg/per day during 5 months) [Arm B, n=71]. Patients included in the arm B received a second transplant in case of disease progression on consolidation therapy, or in case of relapse in responders. Clinical characteristics of each group were similar. In both arms of the study, ASCT was preceded by first-line therapy with thalidomide-dexamethasone and subsequent collection of peripheral blood stem cells with high-dose cyclophosphamide (4 g/m2) and G-CSF. Data were analyzed on an intent-to-treat basis.

Results: With a median follow-up of 23 months (range: 6–34), the 2-year overall survival was 55% in the arm A and 75% in the arm B. Survival curves were not different (P=0.28, log-rank test). The 2-year event-free survival was 41% in the arm A and 60% in the arm B (P=0.4, log-rank-test). In the arm B, relapse-free survival of ≥ 16 months following the first transplantation was an important predictor of overall survival (p< 0.001).

Conclusion: Data from the present study suggest that up-front single ASCT followed by a consolidation therapy with thalidomide and a second ASCT after relapse or progression is a safe and effective global strategy to treat MM patients. Longer follow-up is needed before definite conclusions can be given concerning the optimal timing of second autologous transplantations in patients with MM.
A Humanized Anti-IGF-1R Monoclonal Antibody (AVE1642) Synergizes with Bortezomib To Induce Apoptosis in CD45neg Myeloma Cells.
Authors: Descamps Geraldine , Wuilleme-Toumi Soraya , Gomez-Bougie Patricia , Venot Corinne , Bladt Friedhelm Bladt , Bataille Regis , Amiot Martine
Published: October 2019

IGF-1 is a well-known growth factor for human myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for the treatment of multiple myeloma. In this study, we investigated the effect of a humanized antagonistic monoclonal antibody to the IGF-1R (AVE1642). We showed that AVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines (HMCL). Since we had previously shown the functional relevance of CD45 expression in the growth of myeloma cells, and the association of CD45 negative (CD45neg) status with a less favorable clinical outcome, both CD45 positive (CD45pos) and CD45neg HMCL were studied. We found that AVE1642 strongly inhibited the growth of CD45neg HMCL, leading to a G1 growth arrest, whereas it had almost no effect on the growth of CD45pos HMCL. Indeed, by cell counting over four days in standard culture conditions, AVE1642 induced a significant growth inhibition of the CD45neg LP-1 HMCL of 90% whereas it had no effect on the growth of the CD45pos XG-1 HMCL. Furthermore, AVE1642 binding induced a significant reduction of IGF-1R expression. In western blot, a decrease of 60% the IGF-1R expression of the LP-1 CD45neg HMCL was observed. This decrease was also confirmed by flow cytometry analysis. Finally, a stable CD45 silencing XG-1 cell line was generated and became sensitive to AVE1642. By cell counting over four days in standard culture conditions, AVE1642 inhibited the growth of the shCD45neg XG-1 HMCL of 56% whereas it had almost no effect on the growth of the shLuci-XG-1 HMCL. Thus, for the first time, we provided direct evidence that the expression of CD45 renders myeloma cells resistant to IGF1R inhibition. Furthermore, AVE1642 in combination with Bortezomib strongly increased the apoptosis induced by Bortezomib alone on CD45negHMCL. The CD45neg LP-1 HMCL was considered as a modestly sensitive cell line since 10nM of Bortezomib, which is in the range of therapeutic concentrations, induced 40±16% of apoptosis. Bortezomib in combination with AVE1642 was found to induce 80±1% of apoptosis in the CD45neg LP-1 HMCL. Apoptosis induction by AVE1642/Bortezomib combination was associated with an important induction of Noxa, a BH3-only pro-apoptotic protein that was increased by the addition of AVE1642. We also found a strong increase of caspase-3, -8 activation under this combination compared to Bortezomib alone. Taken together, these results support that therapy directed against IGF-1R associated with Bortezomib, could be beneficial in treating CD45neg patients, a population known for poor clinical outcome.
Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.
Authors: Neumann Frank , Bruennert Daniela C. , Koch Anne-Marie , Bruns Ingmar , Gattermann Norbert , Kronenwett Ralf , Haas Rainer
Published: October 2019

Introduction: Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Particular emphasis was put on 1539 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in first chronic phase in comparison to CD34+ cells from normal bone marrow (Diaz-Blanco et al., Leukemia 2006).

Methods: Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5x107 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration, IC 50). FISH analysis confirmed the K562 cell line to be BCR-ABL positive. Gene expression data of the treated cells were compared with the data of untreated cells. In addition, proliferation (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. A colony assay was performed to see differences in cell growth.

Results: Looking at those 1539 differentially expressed genes in K562 cells which distinguish patients with CML from healthy donors, we found that Imatinib led to a significant downregulation of 187 and upregulation of 45 genes. In general, Nilotinib had a more pronounced effect than Imatinib regarding the number of genes affected and the degree of suppression. It caused downregulation of 418 and upregulation of 41 genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib such as those related to bcr-abl signalling (Lyn, BCL2, Myc, PIK3CB, G3BP2). Downregulation of genes involved in cell cycle (CDK2, ORC5L, MCM3, POLE2, CCNG1) was only observed following Nilotinib exposure. The stronger effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5 fold greater following treatment with Nilotinib in comparison to Imatinib after 24 hours. Treatment with either Imatinib or Nilotinib produced a similar apoptotic rate and similar decrease in cell numbers after 96 hours. In the colony forming assay, the controls (K562 cells incubated with DMSO only) displayed strong leukemic growth which was inhibited by both Nilotinib and Imatinib, allowing only small clusters to appear.

Conclusion: Nilotinib is apparently more potent than Imatinib with regard to the number of genes downregulated and the degree of their suppression. Many of the suppressed genes are associated with bcr-abl signalling and cell cycle.
High-Resolution Assessment of Gains and Losses of Chromosomes in Patients with Multiple Myeloma Treated with Bortezomib.
Authors: Danaee Hadi , Bryant Barb , Roels Steve , Lichter Dave , Koening Eric , Trepicchio William L. , Boral Anthony , Richardson Paul , Meng Yan , Bergsagel Peter Leif , Sonneveld Pieter , Broyl Annemiek , Mitsiades Constantine , Chng Wee-Joo , Anderson Kenneth , Mulligan George
Published: October 2019

Multiple myeloma is a disease of the antibody producing plasma cells in the bone marrow. It has been characterized by frequent genomic alterations including gains and losses of chromosomes. Some of these alterations result in poor clinical prognosis. Bortezomib, a first in class proteasome inhibitor, is approved for the treatment of relapsed multiple myeloma. To better understand how the genome wide changes observed in myeloma relate to prognosis and treatment response, we have used single nucleotide polymorphism (SNP) array technology to assess genotypes and DNA copy number at up to 100,000 genomic loci. DNA from 147 bone marrow tumor biopsies collected in multi-center phase II and III clinical trials of relapsed multiple myeloma patients prior to treatment with Bortezomib were hybridized on SNP arrays to assess genomic aberrations. Commonly seen genomic alterations in myeloma were observed, including deletions of chromosome 13 (37%), 1p (25%), 6q (23%), and amplifications on 1q (35%). We found that 35% of this relapsed myeloma population exhibited a hyperdiploid genome characterized by gains of chromosomes 3, 5, 7, 9, 11, 15, and 19. Other notable genomic deletions included 17p (22%), 8p (27%) and 10p (16%). A correlation of response to bortezomib therapy in patients with differential gains and losses at the chromosomal level is ongoing. Consistent with previous reports, response to bortezomib was observed in patients with poor prognostic chr 13 deletions. A more in-depth analysis of specific loci associated with clinical outcome will be discussed. In summary, this data will help further define genomic aberrations in multiple myeloma and their association with treatment outcome.
The Obligate Role of TAK1 in the Survival of Hematopoietic Stem Cells and Progenitors in Mice.
Authors: Tang Minghui , Xia Zhenbiao , Zhang Shubin , Zhang Shanshan , Wei Xudong , Breslin Peter , Wei Wei , Diaz Manuel , Akira Shizuo , Zhang Jiwang
Published: October 2019

TGFβ1-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of stress and proinflammatory signals. TAK1 can be activated by inflammation-mediating cytokines, including tumor necrosis factor-α (TNF-α and interleukin-1b (IL-1β), as well as by T- and B- cell receptors (TCR/BCR), and Toll-like receptors (TLRs) signals. Activated TAK1 induces the nuclear localization of NF-kB and the activation of JNK/AP1 by stimulating IKKβ and MKK3/MKK6 phosphorylation respectively. TAK1 has been found to play an important role in inflammation, immunity, T- and B-cell activation, and epithelial cell survival. The TAK1−/ − phenotype is lethal in mice at the early embryonic stage. We found higher levels of TAK1 expression and activity in hematopoietic stem cells and progenitors (HSC/Ps), and reduced expression and activity in differentiated mature hematopoietic cells. To study the role of TAK1 in bone marrow hematopoiesis, we generated inducible-TAK1 knockout mice by crossing TAK1loxp mice with Mx1Cre mice, the latter being an interferon-inducible Cre mouse line. After injection of polyI:C to induce the knockout, we found that all the TAK1 knockout mice died within 8 to 10 days after the first polyI:C injection, showing severe hematopoietic and other defects; heterozygotes were phenotypically comparable to wild-type control animals. The TAK1 deletion in these mice resulted in ablation of bone marrow hematopoiesis due to the loss of C-Kit+ HSC/Ps. Annexin-V staining showed a 3-fold increase in apoptosis in the C-Kit+ HSC/Ps from TAK1 mutant mice compared to those from littermate control mice. Almost all of the mutant animals showed intestinal bleeding as well as other hemorrhaging due to the significant reductions in platelet counts. In reciprocal bone marrow transplantation experiments, we found that the TAK1-mutant bone marrow microenvironment was able to support the growth and function of wild-type HSC/Ps, while HSC/Ps from TAK1−/ − mice failed to grow within the wild-type bone marrow microenvironment. These observations suggest that the bone marrow ablation phenotype which develops in TAK1-mutant mice is the result of intrinsic defects in HSC/P’s. We propose that TAK1-mutant HSC/Ps might mediate a survival signal for HSC/Ps stimulated by hematopoietic growth factors and cytokines, such as stem cell factor (SCF). The details of possible mechanisms by which this phenomenon might occur is currently under active investigation by our group.
Targeting Cdk4/6 in Combination Therapy Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma through Synergistic Mitochondria Depolarization.
Authors: Huang Xiangao , Louie Tracey , Di Liberto Maurizio , Rice Jun , Jayabalan David , Zhang Kang , Palladino Michael , Chen Isan , Toogood Peter , Niesvizky Ruben , Moore Malcolm A.S. , Chen-Kiang Selina
Published: October 2019

Deregulation of Cdk4 or Cdk6 is central to the loss of cell cycle control in cancer. In myeloma, over-expression of Cdk4-cyclin D1 or Cdk6-cyclin D2 predisposes primary bone marrow (BM) myeloma cells to proliferation in vivo1. Conversely, silencing Cdk4/6 by its physiologic inhibitor, p18INK4c, is required for G1 cell cycle arrest during the generation of normal plasma cells2. Targeting Cdk4/6, therefore, is critical for the control of disease progression and drug resistance in myeloma, but no Cdk4/6-specific inhibitor was available. We have now targeted Cdk4/6 in combination therapy in myeloma using PD 0332991 (Pfizer), a novel orally bioactive, small molecule Cdk4/6-specific inhibitor. We demonstrate that as a single agent, PD 0332991 rapidly inhibits Cdk4/6 (IC50 ∼ 60 nM) and induces exclusive G1 cell cycle arrest in primary human myeloma cells in the presence of BM stromal cells. Non-invasive whole body imaging reveals that PD 0332991 nearly completely prevents tumor growth in a rapidly disseminated xenograft human myeloma model without overt toxicity3. Because PD 0332991 is reversible and does not induce apoptosis when used as a specific Cdk4/6 inhibitor, we have further developed two strategies to target Cdk4/6 in combination therapy. In the first, the release of PD 0332991-induced G1 arrest leads to synchronous S phase reentry and preferential killing of cycling myeloma cells by cytotoxic agents. In the second, prolonging PD 0332991-induced G1 arrest markedly sensitizes primary myeloma cells to killing by cytotoxic agents including the proteasome inhibitors bortezomib and NPI-0052, as well as the steroid dexamethasone. The Cdk4/6-based combination therapy rapidly reduces tumor load in animal models. More importantly, it overcomes drug resistance in primary myeloma cells isolated from refractory relapse patients despite the protection from BM stromal cells. Synergistic killing of myeloma cells lies in the synergistic induction of mitochondria depolarization. Based on this study and the favorable outcome of a PD 0332991 Phase I clinical trial, a Phase I/II PD 0332991-bortezomib clinical trial for myeloma is underway. Targeting Cdk4/6 by PD 0332991 in combination with a selective cytotoxic agent, therefore, represents the first promising cell cycle-based therapy in myeloma, and possibly other cancers.
Relocation of Cell Iron. A Mode of Chelation with Application to Diseases of Regional Iron Accumulation.
Authors: Cabantchik Zvi I. , Sohn Yan S. , Breuer William , Munnich Arnold
Published: October 2019

Iron raises to toxic levels in mitochondria of excitable cells in some forms of neuro-degeneration with brain accumulation (NBIA), in sideroblastic anemia and in Friedreich’s ataxia (FA), often leaving the cytosol iron-depleted. In anemia of chronic disease (ACD) iron is withheld by macrophages, while iron levels in extracellular fluids (e.g. plasma) are drastically reduced. Although excessive iron deposition occurring in organs of iron overloaded (IO) patients can be reduced with iron chelators, it is uncertain whether this is applicable to conditions where iron accumulates within selected tissues/cells in the absence of systemic IO.

Objective. We assessed whether deferiprone (DFP), a membrane-permeant bidentate chelator in clinical use for treating systemic IO, might serve as an iron relocating agent in settings of regional iron accumulation by a. capturing labile iron accumulated in cell compartments and b. conveying the chelated iron either to other cell locations for metabolic integration or to transferrin for systemic reutilization.

Methods. DFP capacity to shuttle iron intracellularly and transcellularly was assessed in macrophages (J774) and heart (H9c2) cell models using organelle-targeted fluorescent iron-sensors in conjunction with fluorescence microscopy imaging. We employed pairs of sensors targeted to different cell compartments and iron-evoked quenching and chelator-evoked dequenching as means to trace DFP mediated iron transfer. Mitochondrial iron accumulation was generated with succinylacetone or by silencing genes affecting mitochondrial iron metabolism.

Results. DFP facilitated iron transfer: a. from iron-laden cell organelles to other cell compartments or to medium (and vice versa) and b. from iron loaded macrophages to pre-erythroid MEL cells (for chemically induced hemoglobin synthesis) either directly or via transfer to extracellular transferrin.

Discussion The results of this study indicate that relocation of cell accumulated iron can be used as a modality of chelation for treating conditions of regional iron accumulation by applying chelators able to permeate into cell compartments but also to transfer the chelated iron to cell acceptors or to the extracellular iron carrier transferrin.
Enhanced Anticoagulant Activity of Factor VIII Inhibitors Due to Positive Cooperativity between Two Classes of Anti-Factor VIII C2 Antibodies.
Authors: Meeks Shannon L. , Healey John F. , Barrow Rachel T. , Parker Ernest T. , Lollar Pete
Published: October 2019

We recently identified 5 distinct structural classes of anti-human factor VIII (fVIII) C2 domain monoclonal antibodies (MAbs) in a murine model of hemophilia A. Three classes, Groups A, B, and C, consist of MAbs with non-overlapping epitopes. Group AB MAbs overlap epitopes of Group A and B MAbs, and Group BC MAbs overlap epitopes of Group B and C MAbs. Concentration dependent binding of biotinylated Group A MAbs to fVIII bound to a microtiter plate was studied in the presence and absence of Group BC MAbs by ELISA. Surprisingly the binding of all 3 Group A antibodies tested, ESH4, 3E6, and I54, was increased in the presence of Group BC antibodies. Group A antibodies block the binding of fVIII to von Willebrand factor (VWF) and phospholipid. Group BC antibodies inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to VWF and phospholipid. To establish functional relevance, the effect of Group BC MAbs on the ability of Group A MAbs to block fVIII binding to VWF was determined by ELISA. Group A MAbs were serially diluted into fixed concentrations of Group BC MAbs and then incubated with fVIII in solution for 2 hours. The fVIII - antibody mixtures were added to microtiter wells coated with VWF, and fVIII binding to VWF was measured. Group BC MAbs increased the ability of ESH4 and 3E6 to inhibit the binding of fVIII to VWF, consistent with the observation that the binding of Group A and Group BC antibodies is positively cooperative. We previously have shown that antibody-mediated inhibition of fVIII binding to VWF is correlated with inhibition of fVIII binding to phospholipid and with fVIII inhibitor titers. Although most Group BC MAbs have high inhibitory titers against fVIII, Group BC MAb D105 is only minimally inhibitory at 0.8 Bethesda units/mg IgG. Because D105 increased the binding of ESH4 to fVIII like other Group BC MAbs, the effect of D105 on the inhibitor titer of ESH4 was investigated using the Bethesda assay. The inhibition of fVIII was significantly increased by the presence of D105 (Fig. 1). This study identifies increased binding of Group A MAbs to fVIII in the presence of Group BC MAbs using 3 different assays. Our results suggest that the cooperative interaction of fVIII antibody epitopes affects the overall inhibitor patient phenotype and that inhibitors with very low inhibitor titers can significantly increase the inhibitory effects of other anti-fVIII antibodies.

Figure Figure
Burdern of Long-Term Morbidity after Hematopoietic Cell Transplantation: A Report from the Bone Marrow Transplant Survivor Study (BMTSS).
Authors: Sun Can-Lan , Francisco Liton , Kawashima Toana , Robison Leslie L. , Baker K.S. , Weisdorf Daniel , Forman Stephen J. , Bhatia Smita
Published: October 2019

Long-term survival is an expected outcome after HCT. However, no study has assessed the burden of long-term morbidity in these survivors, or attempted to identify subpopulations at highest risk for severe, debilitating conditions. In this study, we determined the prevalence and severity of chronic health conditions in a large population of long-term HCT survivors and compared these outcomes with their siblings. BMTSS, a collaborative effort between City of Hope National Medical Center and University of Minnesota, examined self-reported chronic health conditions in individuals who underwent HCT between 1976 and 1998, and survived two or more years. A severity score (grade 1 through 4, ranging from mild to life-threatening or disabling) was assigned to each health condition according to the Common Terminology Criteria for Adverse Events (version 3). A partial list of conditions graded as severe or life-threatening (grade 3 or 4) included congestive heart failure, second malignant neoplasms, coronary artery disease, cerebrovascular accident, renal failure/dialysis/renal transplant, and active chronic graft vs. host disease. Adverse psychosocial outcomes were not included. Cox proportional-hazard models were used to estimate hazard ratios and their 95% confidence intervals. We compared the prevalence and severity of chronic conditions in 1013 HCT survivors (455 autologous, 460 related donor, and 98 unrelated donor HCT survivors) with 309 siblings. The median age at study participation was 44 (range 18–73) and 45 years (range 17–79) for survivors and siblings, and the median follow-up for the survivors was 7.3 years (range 2–28) from HCT. Among the 1013 survivors, 69% had at least one chronic condition, and 29% had a severe or life-threatening condition (grade 3 or 4). The comparable figures in siblings were 39% and 7%, respectively (p<0.001 compared to survivors). After adjustment for age at HCT, sex and race/ethnicity, survivors were 2.4 times as likely as their siblings to develop any chronic health conditions (95%CI, 2.0–2.9), and 4.5 times more likely to develop severe/life threatening conditions (95%CI, 3.0–6.7). Groups at highest risk for a severe or life-threatening condition are summarized in the Table. Among survivors, the cumulative incidence of a chronic health condition reached 84% at 20 years post HCT, with a cumulative incidence of 55% for severe/life threatening conditions at 15 years after HCT. The chronic health burden of this population is significant, and life-long follow-up of patients who receive transplantation is recommended.

Table: Groups at highest risk for severe or life-threatening condition Risk Factors Relative Risk 95% CI Siblings 1.0 __ CML 5.8 3.8–8.9 AML 4.9 3.2–7.5 ALL 5.2 3.3–8.3 Allogeneic sibling donor 5.9 3.9–8.7 Unrelated donor 7.4 4.9–11.1 TBI 5.0 3.4–7.5
High-Throughput Sequence Analysis of the Tyrosine Kinome in Acute Myeloid Leukemia.
Authors: Loriaux Marc , Levine Ross , Tyner Jeffrey , Frohling Stephan , Scholl Claudia , Stoffregen Eric , Wernig Gerlinde , Erickson Heidi , Eide Chris , Berger Roland , Bernard Olivier , Griffin James , Stone Richard , Meyerson Matthew , Heinrich Michael , Deininger Michael , Gilliland Gary , Druker Brian
Published: October 2019

Mutations that result in constitutive tyrosine kinase activation occur in a wide spectrum of human malignancies, including acute myeloid leukemia (AML). Although activating mutations in the tyrosine kinases FLT3 and c-KIT occur in a significant proportion of patients with AML, the genomic events responsible for oncogenic signaling in most patients with AML have not been identified. To determine whether other aberrantly activated tyrosine kinases contribute to the pathogenesis of AML, we employed high throughput (HT) DNA sequence analysis to screen the exons encoding domains implicated in kinase activation (activation loop), and autoinhibition (juxtamembrane domains) in 85 tyrosine kinase genes in 192 AML patients without FLT3 or c-KIT mutations. The screen identified 34 non-synonymous sequence variations in 25 different kinases that had not been reported in single-nucleotide polymorphism (SNP) databases. These included a novel activating allele in FLT3, and a previously described activating mutation in MET (METT1010I). The majority of the novel sequence variants were cloned into their respective tyrosine kinase cDNA, and stably expressed in the factor-dependent Ba/F3 hematopoietic cell line. Apart from one FLT3 allele, none of the other novel variants showed evidence of constitutive phosphorylation by immunoblot analysis, and none transformed Ba/F3 cell lines to factor independent growth. These findings indicate that the majority of these alleles are not potent activators of tyrosine kinase activity in this cellular context, and that a significant proportion of non-synonymous sequence variants identified in HT DNA sequencing screens may not have functional significance. Although several explanations are possible for this observation, these data are consistent with recent reports that a significant fraction of such sequence variants are “passenger” rather than “driver” alleles, in cancer, and underscore the importance of functional assessment of candidate disease alleles.
TG101348: A Dual-Acting JAK2/FLT3 Small Molecule Kinase Inhibitor for the Treatment of AML.
Authors: Tam Betty , Mak Chi Ching , Stoughton Silva , Virata Cyrus , Lohse Dan , Hanna Ehab , Alomia Marcela , Jamieson Catriona , Doukas John , Noronha Glenn , Martin Michael , Soll Richard , Hood John
Published: October 2019

A number of genetic mutations have been identified in the development of acute myeloid leukemia (AML), including mutations in the proto-oncogene N-RAS, p53, c-Kit and the FMS-like receptor tyrosine kinase 3 (FLT3). FLT3 plays an important role in the formation of early stem/progenitor cells during hematopoietic development. FLT3 is mutated in ∼30% of AMLs, with internal tandem duplications (ITD) in the juxtamembrane region being the most common (FLT3-ITD). This results in constitutive activation of FLT3 kinase activity and patients harboring such mutations do not respond well to conventional treatments and demonstrate poor clinical prognosis. There is growing evidence that the JAK/STAT pathway may also contribute to the etiology of AML. For instance, the JAK2 V617F point mutation driving various chronic myeloproliferative disorders (MPDs) has recently been detected in de novo AMLs and STAT3/5 phosphorylation is commonly elevated in AML patients. We have developed TG101348, a selective JAK2/FLT3 small molecule inhibitor which has the potential to treat both MPDs and AML. In in vitro based assays, TG101348 was capable of inhibiting the proliferation of human AML cell lines representing a variety of mutations. In AML cell lines carrying mutations in N-RAS such as KG-1, HL-60, and THP-1, EC50s ranged from ∼300 nM-1000 nM. In TF-1a AML cells, which harbor a mutation in p53, TG101348 demonstrated an EC50 of 493 nM. Most interestingly, for these cell lines, FLT3/VEGFR2 specific small molecule inhibitors such SU5416 were significantly less effective, demonstrating EC50s that were generally >10-fold higher. TG101348 was most potent against the FLT3-ITD positive cells lines, MV411 (EC50=57 nM) and MOLM13 (EC50=69 nM). In MV411 cells, TG101348 functionally inhibited FLT3 autophosphorylation, and phosphorylation of downstream intermediates including STAT5, ERK, and AKT. In a human MV411 xenograft models, oral administration (120mg/kg, BID) of TG101348 resulted in complete regression of the subcutaneously implanted MV411 tumors. Taken together, these data suggest that the dual-acting JAK2/FLT3 kinase inhibitor TG101348 may have a therapeutic advantage in that it has the capacity to block multiple pathways critical for dysregulated cell proliferation and survival in AML.
A Phase I Trial of Two Sequence-Specific Schedules of Decitabine and Vorinostat in Patients with Acute Myeloid Leukemia (AML).
Authors: Yee Karen W.L. , Minden Mark D. , Brandwein Joseph , Schimmer Aaron , Schuh Andre , Gupta Vikas , Messner Hans A. , Foley Ronan , Wasi Parveen , Zwiebel James A. , Leber Brian
Published: October 2019

Background: Epigenetic silencing of genes has been documented in AML. This phase I trial evaluates the safety, tolerability, and maximum tolerated dose (MTD) of two schedules of administration of the hypomethylating agent decitabine in combination with the pan-selective histone deacetylase inhibitor vorinostat.

Methods: Patients receive escalating doses of oral vorinostat administered either sequentially [100 mg bid (n=4), 200 mg bid (n=4), or 200 mg tid (n=8) Days 6–21] or concurrently [100 mg (n=3) or 200 mg (n=6) bid Days 1–21 or 200 mg tid (n=2) Days 1–14] with decitabine (20 mg/m2/d IV Days 1–5) every 28 days.

Results: Twenty-seven patients with AML have been treated. Median age was 67 years (range, 32–82 years). Median ECOG status 1 (range, 0 to 2). Eighteen patients (67%) had received prior therapy (median, 1 regimen; range, 0 to 4 regimens); 3 had received a prior allogeneic stem cell transplant. A total of 85 cycles have been administered, with a median of 2 cycles (range, 1 to 13 cycles); 10 patients (37%) have received 3 or more cycles of therapy. One of 7 patients treated at dose level 3 of the sequential schedule developed dose-limiting toxicities (DLT), consisting of grade 3 fatigue, weakness, and mucositis. Therefore, the MTD was not reached in the 3 planned dose levels of the sequential schedule. One DLT (grade 3 fatigue) occurred in 6 patients treated at dose level 2 of the concurrent schedule. Most common drug-related non-hematological toxicities of any grade (all CTCAE grades 1 or 2) were nausea (71%), fatigue (54%), diarrhea (54%), vomiting (42%), anorexia (25%), constipation (13%), abdominal pain (13%), dehydration (13%), and headache (13%). No other non-hematological grade 3 or 4 toxicities were observed. Of the 25 evaluable patients, one patient achieved an incomplete CR (without neutrophil recovery), one a morphologic leukemia-free state (without blood count recovery), and three partial remissions (1 achieved red cell transfusion independency and a second normalization of platelet counts). Seven of these patients remain on study for 2.7 to 13.5+ months. Correlative studies examining histone acetylation and gene promoter methylation in leukemic cells at baseline and after treatment, as well as plasma pharmacokinetic levels for both decitabine and vorinostat are being evaluated.

Conclusions: The combination of decitabine and vorinostat is safe, well-tolerated, and has clinical activity in patients with AML. Enrollment is ongoing.