bluuebunny/crossref_metadata_2025 huggingface dataset:
Disclosure of Copy Number Alterations in AML with Normal Karyotype Using Matrix-Based Comparative Genomic Hybridization.
Authors: Rucker Frank G. , Bullinger Lars , Kestler Hans A. , Lichter Peter , Dohner Konstanze , Dohner Hartmut
Published: April 2020

Clonal chromosome abnormalities represent one of the most important prognostic factors in adult acute myeloid leukemia (AML), and cytogenetic data are used for risk-adapted treatment strategies. By conventional cytogenetic analysis, approximately 50% of patients lack clonal chromosome aberrations, and normal cytogenetics are associated with an intermediate clinical outcome. This clinically heterogeneous group seems to be in part characterized by molecular markers, such as MLL, FLT3, CEBPA, and NPM1 mutations.

In order to identify novel candidate regions of genomic imbalances, we applied comparative genomic hybridization to microarrays (matrix-CGH). Using this high-resolution genome-wide screening approach we analyzed 49 normal karyotype AML cases characterized for the most common clinically relevant molecular markers (MLL-PTD n=13, FLT3-ITD n=7, FLT3-ITD/NPM1+ n=4, MLL-PTD/FLT3-ITD n=3, CEBPA+ n=12, CEBPA+/FLT3-ITD n=1; CEBPA+/NPM1+ n=1; no molecular markers n=8) with a microarray platform consisting of 2799 different BAC or PAC clones. A set of 1500 of these clones covers the whole human genome with a physical distance of approximately 2 Mb. The remaining 1299 clones either contiguously span genomic regions known to be frequently involved in hematologic malignancies (e.g., 1p, 2p, 3q, 7q, 9p, 11q, 12q, 13q, 17p, 18q) (n=600) or contain oncogenes or tumor suppressor genes (n=699).

In addition to known copy number polymorphisms in 5q11, 7q22, 7q35, 14q32, and 15q11, the CLuster Along Chromosomes method (CLAC; http://www-stat.stanford.edu/~wp57/CGH-Miner) disclosed copy number alterations (CNAs) in terms of gains in 1p, 11q, 12q, and 17p. CNAs in terms of losses were identified in 9p, 11q, 12p, 12q, and 13q. Two-class supervised analyses using the significance analysis of microarrays (SAM) method identified for the MLL-PTD cases a gain of a single clone harboring the MLL gene.

While the significance of these findings, which are currently validated using fluorescence in-situ hybridization (FISH), still remains to be determined, our preliminary results already demonstrate the power and reliablity of this microarray-based technique allowing genome-wide screens of genomic imbalances as the MLL aberration was detected in all cases known to have a MLL-PTD. Furthermore, ongoing correlation of high-resolution genomic profiling with global gene expression studies will help to disclose pathways underlying normal karyotype AML, thereby leading to new insights of leukemogenesis.
Extended Follow-Up and Analysis with Central Radiological Review of Patients Receiving FavId® (Id/KLH) Vaccine Following Rituximab.
Authors: Koc Omer , Redfern Charles , Wiernik Peter , Rosenfelt Fred , Winter Jane , Guthrie Troy , Kaplan Lawrence , Holman Peter , Densmore John , Hainsworth John , Lin Thomas , Castillo Rene , Janakiraman Nalini , Gold Dan , Gutheil John , Bender John
Published: April 2020

Background: Idiotype vaccination (Id/KLH) is a promising approach to the treatment of patients (pts) with B-cell malignancies. Our experience suggests that Id/KLH may function best in pts with lower tumor burden who are less heavily pretreated. We have conducted a phase II trial of Id/KLH following cytoreduction with rituximab in patients with treatment naive (TN) or relapsed/refractory (R/R) follicular NHL (FL).

Treatment: Pts received rituximab, 375mg/m2, IV, weekly x 4, wk 1–4. Pts with without progression received Id/KLH, 1 mg, SQ, monthly x 6, starting on wk 12, with GM-CSF, 250 mcg, SQ, on days 1–4. Pts could receive booster Id/KLH on a reduced schedule until disease progression.

Results: One hundred three pts were enrolled and received rituximab; 89 pts received Id/KLH. The last patient was registered in Dec 2003. Among these 89 evaluable pts, 55 were R/R to prior therapy, and 34 were TN. The overall response rate (ORR) following rituximab (assessed at month 3 based on a centralized read of CT scans) was 49% (44/89). Following Id/KLH, the ORR improved to 64% (57/89). The median time for improved response following initiation of Id/KLH treatment was 6.6 months (range 5.8 – 23.4 months) which was consistent with the time required to demonstrate an immune response in those pts tested. Prognostic factors for time to tumor progression (TTP) were evaluated and demonstrated a significant negative relationship for liver involvement (hazard ratio; HR = 9.3, p=0.0017) and 3 or more prior regimens (HR=4.7, p=0.0035). Median TTP has not been reached in the TN group (N=34) and in pts who relapsed after prior chemotherapy (N=26), with an actuarial 18 month progression free survival rate of 69% and 70%, respectively. The group of pts who would be eligible for the ongoing phase 3 randomized registration study (N=71) of rituximab followed by Id/KLH have also not reached median TTP. T-cell responses to both Id and KLH were observed in 80% and 86% of pts respectively (N=15) and were seen after a median of 2 doses of Id/KLH. Likewise, antibody responses to Id and KLH were seen in 13% and 69% respectively (N=23) and developed after a median of 5 to 6 doses of Id/KLH.

Conclusion: Id/KLH, administered following rituximab to pts with FL, appears to result in an increase in the response rate and TTP compared to that reported for rituximab alone. We also observed a high rate of cellular immunity against idiotype following Id/KLH. These data support the design of an ongoing PIII clinical study of Id/KLH following rituximab which excluded pts with poor prognostic indicators.
Expression of Tropomodulin1 (Tmod1) in the Heart Rescues Embryonic Lethality of Tmod1 Null Mice and Results in a Mild Hemolytic Anemia Due to Absence of Tmod1 in Red Blood Cells.
Authors: Fowler Velia M. , Moyer Jeannette , Nowak Roberta , Fritz-Six Kimberly , Ferreira-Mota Carmela , Sussman Mark A.
Published: April 2020

Tropomodulin1 (Tmod1) caps the pointed ends of actin filaments in the red blood cell (RBC) membrane skeleton and is proposed to regulate actin filament lengths and organization in the membrane skeleton. Tmod1 is first expressed at E7.5 of mouse embryogenesis in blood islands of the yolk sac and in the cardiac myocytes of the developing heart tube. Targeted deletion of the Tmod1 gene in mice leads to abnormal cardiac development and defective vasculogenesis and hematopoesis in the yolk sac, followed by embryonic lethality between E9.5–10. To investigate the function of Tmod1 in RBCs we rescued the embryonic lethality of the Tmod1 null mouse by crossing with a TOT mouse that expresses a Tmod1 transgene in the heart under the control of the α-myosin heavy chain promoter. Genotyping of litters from crosses of Tmod1-/+;TOT+ mice with Tmod1+/− mice demonstrates that embryos with no endogenous Tmod1 but expressing the Tmod1 transgene (Tmod1−/−;TOT+) develop to term with no apparent defects in vasculogenesis or hematopoeisis. Western blotting and immunofluorescence staining demonstrates that Tmod1 protein is present in the heart but absent from RBCs in Tmod1−/−;TOT+ rescued embryos or adult mice. Instead, the Tmod3 isoform that is normally present in embryonic RBCs, but not detected in adult RBCs of wild-type mice, is increased on the membranes of Tmod1-deficient adult RBCs. Hematological analyses reveal that these Tmod1-deficient mice exhibit a compensated mild hemolytic anemia, with decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), increased red cell distribution width (RDW), and reticulocytosis. Quantitative western blotting demonstrates that major RBC membrane proteins are not altered in the absence of Tmod1, but that there is about a two-fold increase in levels of actin and tropomyosin on the membrane, suggesting that actin filaments may be longer than in normal RBCs. We conclude that 1) Tmod1 in RBCs is not required for embryonic development or viability, 2) yolk sac and hematopoetic defects in Tmod1 null mice are secondary to cardiac defects, 3) Tmod’s function in embryonic RBCs may be performed by the Tmod3 isoform that is normally expressed in these RBCs, and 4) the absence of Tmod1 in adult RBCs is incompletely compensated by up-regulation of Tmod3, leading to altered actin filaments, decreased membrane stability and a mild hemolytic anemia.
Four ABVD and Involved-Field Radiotherapy in Unfavorable Supradiaphragmatic Clinical Stages (CS) I-II Hodgkin’s Lymphoma (HL): Preliminary Results of the EORTC-GELA H9-U Trial.
Authors: Ferme Christophe , Diviné Marine , Vranovsky Andrej , Morschhauser Frank , Bouabdallah Réda , Gabarre Jean , Bastard-Stamatoullas Aspasia , Delarue Richard , Zagonel Vittorina , Jaubert Jérôme , Hagenbeek Anton , Kramer Mark Henri H. , Rieux Chantal , Thomas José , Henry-Amar Michel
Published: April 2020

The aim of the trial was to compare three modalities of chemotherapy and involved-field radiotherapy (IF-RT) in adult patients with supradiaphragmatic CS I-II HL with risk factors. Patients were randomized if they presented with age ≥ 50, or CS II4–5, or A + ESR ≥ 50, or B + ESR ≥ 30, or MT ratio ≥ 0.35. The trial compared ABVD x 6 cycles and IF-RT (36–40 Gy) vs ABVD x 4 cycles and IF-RT vs BEACOPP baseline x 4 cycles and IF-RT. From October 1998 to September 2002, 808 patients were enrolled in 111 institutions from 10 European countries. The proportions of grade 3–4 chemotherapy-related hematological toxicity (mainly WBC) were 74%, 70% and 63%, respectively; That of grade 1–3 RT-related hematological toxicity were 10%, 12% and 17%, respectively. Ten (2+3+5, 1%) patients stopped chemotherapy because of toxicity and 8 (2+2+4, 1%) refused the treatment; Six (2+2+2, 1%) patients stopped radiotherapy because of toxicity and 9 (3+1+5, 1%) refused the treatment. The proportions of patients in CR/CRu were 74%, 71% and 59% after 6, 4 ABVD and 4 BEACOPP, respectively. After a median follow-up of 57 months (range 33–81), 78 events (26 progressions, 37 relapses, 15 deaths) were observed. At July 2005, the 4-year event-free survival (EFS) and overall survival (OS) rates are as follows:

Treatment No. Pts CR-CRu /PR /NC-PD 4-yr EFS 4-yr OS 6 ABVD + IF-RT 276 87% /8% /5% 91% 95% 4 ABVD + IF-RT 277 86% /11% /3% 87% 94% 4 BEACOPP + IF-RT 255 84% /12% /4% 90% 93% P value 0.607 0.380 0.978

Overall, 42 patients have died of progressive disease (5, 7 and 7 patients), treatment-related complication (7, 5 and 2), intercurrent disease (1, 0 and 2), second malignancy (1 NHL, 0 and 1 AML) or cause unspecified (0, 3 and 1). These preliminary results indicate that a combination of 4 cycles of ABVD and IF-RT is sufficient to cure a large majority of HL patients with unfavorable early stage disease and that BEACOPP baseline has no advantage over ABVD in these patients.
Silencing Reversed: Coordinated Expression of Embryonic and Fetal Globin Genes in Adult Human Erythroblasts.
Authors: Bhanu Natarajan V. , Lee Y. Terry , Oneal Patricia A. , Rognerud Cheryl L. , Ou Ching-Nan , Miller Jeffery L.
Published: April 2020

It was recently demonstrated that erythropoietin combined with stem cell factor and transforming growth factor beta (EST; EPO+SCF+TGF-B) signals a pancellular reversal of gamma-globin gene silencing among ex vivo cultures of CD34+ cells when compared to cultures supplemented with erythropoietin (EPO) alone (Bhanu et al., Blood.2005; 105(1):387–93). This primary human erythroblast culture model is now being utilized to study the molecular basis of globin gene regulation. In this study, we sought to determine whether EST-signaled effects are restricted to the gamma-globin gene(s). For this purpose, quantitative PCR (QPCR) assays were developed for the alpha-globin (zeta, mu, alpha, and theta) and beta-globin (epsilon, gamma, delta, and beta) gene clusters. EPO versus EST matched cultures from a total of 15 donors (3 single donors and two pools, 6 donors each) were investigated using culture day 7 proerythroblasts and day 14 hemoglobinized precursors. The QPCR products were quantified by comparison with plasmid-generated standard curves (20,000,000 to 200 copies). The patterns of mu-, alpha- and theta-globin gene expression were unchanged under all culture conditions. The delta- and beta-globin transcripts showed a significant reduction in levels in response to EST. In contrast, zeta-, epsilon- and gamma-globins all demonstrated significant increases in response to EST. The increases were consistently detected in the individual and pooled samples. The most dramatic increase in the zeta-, epsilon- and gamma-globins occurred at the proerythroblast stage of differentiation. On day 7, zeta-globin levels showed 190-fold increase (EPO: 6.9E+01±5.5E+01;EST: 1.3E+04±7.7E+03; p=0.01), epsilon-globin levels, a 27-fold increase (EPO: 1.0E+03±5.8E+02; EST: 2.7E+04±1.1E+04; p=0.001), and gamma-globin levels, a 13-fold increase (EPO: 9.1E+05±4.1E+05; EST: 1.2E+07±1.8E+06; p=0.001). Consistent with the 3–4 log difference between the levels of zeta- and epsilon-, versus gamma-globin copy numbers and the low sensitivity of HPLC compared with QPCR, no embryonic proteins were detected by reverse-phase HPLC (day 14 samples studied; 9 donors). However, G-gamma and A-gamma globin peaks confirmed EST-signaled activation of both gamma-globins genes (G-gamma/A-gamma= 1.0 in EPO versus 1.9 in EST; (total gamma)/(total gamma + beta-globin) = 3.7±2.4% in EPO versus 42.4 ±5.8% in EST; p=1.6E-08). These novel data suggest that cytokines can reverse the coordinated developmental silencing of human embryonic and fetal genes in both globin loci.
Identification of zfp148 (ZBP-89) as a Novel GATA-1 Associated Transcription Factor Involved in Megakaryopoiesis and Definitive Erythropoiesis.
Authors: Woo Andrew J. , Moran Tyler B. , Choe Seong-Kyu , Schindler Yocheved L. , Sullivan Matthew R. , Fujiwara Yuko , Paw Barry H. , Cantor Alan B.
Published: April 2020

The zinc finger transcription factor GATA-1 plays an essential role in megakaryocytic and erythroid terminal maturation. Yet the molecular mechanisms that control GATA-1 function are incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. Here, we show that GATA-1 participates in stable multiprotein complexes ranging from ~100 kDa to greater than 600 kDa in murine L8057 megakaryoblastic cells. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and performed tandem anti-FLAG immunoaffinity and streptavidin affinity purification of GATA-1 associated complexes. Mass spectrometry (LC/MS/MS) of co-purified proteins identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β, SP1 and all components of the NuRD complex. In addition, we reproducibly co-purified zfp148 (also called ZBP-89/BERF-1/BFCOL-1, mtβ), a ubiquitously expressed Kruppel-type zinc finger transcription factor that binds GC-rich DNA elements. Zfp148 also co-purified with GATA-1 in erythroid (MEL) cells. Physical interaction between GATA-1 and zfp148 was confirmed by co-immunoprecipitation experiments using an anti-zfp148 antibody, and their interaction domains mapped to a C-terminal region of zfp148 and the zinc finger domain of GATA-1. Morpholino-mediated knock-down of zfp148 expression in zebrafish embryos ablates thrombocyte (equivalent to mammalian megakaryocyte/platelet) development, but does not significantly affect erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development). In vitro differentiation of murine ES cells containing homozygous disruption zfp148 by genetrap insertion (zfp148gt/gt) shows essentially normal primitive erythropoiesis, but markedly impaired megakaryocytic and definitive erythropoiesis. Preliminary chimeric studies in adult mice demonstrate a failure of zfp148gt/gt ES cells to contribute to mature erythrocytes. These findings suggest that zfp148 cooperates with GATA-1 to control differentiation of erythroid and megakaryocytic lineages. The differential requirement of zfp148 for definitive, but not primitive, erythropoiesis raises the possibility of a role in developmental hemoglobin switching.
T-bet Over-Expression in T Cells from Patients with Aplastic Anemia.
Authors: Solomou Elena E. , Keyvanfar Keyvan , Sloand Elaine M. , Weinstein Barbara , Nunez Olga , Young Neal S.
Published: April 2020

Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by immune-mediated destruction of hematopoietic stem cells. T cells from patients with AA overproduce IFN-γ, a cytokine that inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. In T cells, regulation of IFN-γ production occurs primarily at the level of transcription. A proximal site of the IFN-γ gene (−75 to −45bp of the IFN-γ promoter) is a binding site for different transcription factors including NFAT, AP-1, ATF, CREB, and T-bet. T-bet is a member of the T-box family of transcription factors, this family contains a highly conserved DNA binding domain, the T-box, that binds to a specific sequence in the promoter of different genes, including the IFN-γ promoter. T-bet is found in Th1 but not in Th2 cells and is the key regulator of Th1 development and function (Rengarajan et al., Immunol Today2000; 21: 479). The inducible expression of T-bet is mediated in part by Itk kinase (Sjabo et al., Science2005; 307: 430). In the present study, we examined T-bet protein levels in T cells from patients with AA. Samples from 17 of 20 patients examined (85%) by immunoblot showed increased T-bet protein levels in unstimulated T cells compared to normal controls (p = 0.0001). Normal controls showed undetectable T-bet protein levels in unstimulated T cells but T-bet expression was induced after 24 hrs of stimulation with PMA and ionomycin. In electrophoretical mobility shift assays, we observed increased T-bet binding to the proximal site of the IFN-γ promoter in T cells from patients with AA; no binding was detected in unstimulated T cells from healthy controls, but binding was present after stimulation for at least 24 hrs. T-bet protein levels correlated with disease activity. Patients with increased T-bet protein levels showed increased intracellular IFN-g levels compared to controls, as detected by flow cytometry (p<0.05). Patients that expressed increased T-bet protein levels also showed increased levels of the Itk kinase (p=0.02). We examined if other kinases that lie downstream of Itk in the signal transduction activation cascade in T cells affected the inducible expression of T-bet. In normal T cells, rottlerin, a PKC-theta (PKC-𝛉) inhibitor, decreased T-bet protein levels by 50%; in AA T cells, rottlerin also decreased T-bet protein levels and IFN-γ intracellular levels by 50%. Our results suggest that the increased IFN-γ levels observed in AA are the result of activation of transcription of the IFN-γ gene by the regulator T-bet. Blocking of transcription of the IFN-γ gene by kinase inhibitors might represent a therapuetic strategy for AA and other human T cell-mediated autoimmune diseases.
Otx1 Enhances Erythroid Differenciation through Activation of the SCL Gene.
Authors: Sincennes Marie-Claude , Sanguin-Gendreau Virginie , Martin Richard , Grondin Benoit , Tremblay Mathieu , Cerisoli Francesco , Magli Christina , Hoang Trang
Published: April 2020

Members of the paired class of homeobox proteins are critical determinants of left-right asymmetry and establish the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that mice lacking Otx1, a bicoid homeodomain-containing gene, exhibit an impairment of the erythroid compartment, associated with decreased SCL levels. In the present study, we show that Otx1 is coexpressed with SCL in yolk sac during embryonic development; in differentiating embryonic stem cells, Otx1 is upregulated with SCL in both primitive and definitive erythroid colonies, while Otx expression is absent in cardiomyocytes and skeletomyocytes. To address the role of Otx1 in hematopoiesis, we overexpressed Otx1 in primary hematopoietic cells using the MSCV retrovirus. The gain of Otx1 function gives rise to a 6-fold increase in endogenous SCL levels together with an increase in TER119-positive erythroid cells. Strikingly, the generation of CD11b-positive myeloid cells was almost abrogated by ectopic Otx1 expression, suggesting that Otx1 favours the erythroid lineage at the expense of the myeloid lineage. Furthermore, we took several approaches to provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. Indeed, Otx1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity is dependent on the proximal GATA site of the SCL promoter. Next, we show by chromatin immunoprecipitation that Otx1 and GATA-1 occupy the SCL proximal promoter in vivo in erythroid cells. At the molecular level, we show that Otx1 physically interacts with GATA-1 in erythroid cells, and the homeodomain of Otx1 is sufficient for this interaction. Finally, a gain of function of SCL rescues the erythroid deficiency of Otx1−/− mice, consistent with the model in which SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL are involved in the same genetic pathway to specify the erythroid fate during hematopoiesis.
Characterization of a Megakaryocyte-Specific Enhancer of the Key Hemopoietic Transcription Factor GATA1.
Authors: Guyot Boris , Murai Kasumi , Fujiwara Yuko , Valverde-Garduno Veronica , Hammett Michele , Wells Sara , Dear Neil , Orkin Stuart H. , Porcher Catherine , Vyas Paresh
Published: April 2020

Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well-studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the transcription factor GATA1, that is required for normal maturation of these two lineages. Megakaryocyte- and erythroid-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, we previously showed that the 3′ 179 base pairs (bp) of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show that mHS-3.5 binds key hemopoietic transcription factors in vivo (GATA1, SCL/TAL-1) and is required to maintain histone acetylation in the mGata1 locus in primary megakaryocytes. When deletional constructs containing mHS-3.5 were used to direct GATA1-LacZ reporter gene expression in transgenic mice, a 25 bp element within the 3′ 179bp in mHS-3.5, was critical for megakaryocyte expression. In vitro three uncharacterized DNA-binding activities A, B and C bind to the core of the 25 bp element, and these binding sites are conserved through evolution. Of these, only activity B is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals that activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows that sequence corresponding to the binding site for activity B is a previously unrecognised motif present in the cis-elements of other megakaryocyte-specific genes. In summary, we have identified a motif and a DNA-binding activity that are likely to be important in directing a megakaryocyte gene expression program distinct from that in red cells.
Severe Mucositis Adversely Affects Survival after Autologous Stem Cell Transplantation (ABMT) for Lymphoid Malignancies.
Authors: Bolwell Brian , Kalaycio Matt , Sobecks Ronald , Andresen Steven , Rybicki Lisa , Kuczkowski Elizabeth , Mendiola Sherry , Kohuth Joe , Bell Kimberly , Curtis Julie , Brown Stacey , Pohlman Brad
Published: April 2020

Mucositis is a well known short term complication of ABMT. Many studies of mucositis in ABMT, however, are flawed due to heterogeneity of both mobilizing regimens and high-dose preparative regimens. There is also sparse data on the long term consequences of mucositis after ABMT. This study retrospectively reviews all patients with lymphoid malignancies undergoing ABMT from 5/2000 to 2/2005 at our institution that received a uniform mobilizing regimen of VP-16 plus G-CSF and a uniform high dose preparative regimen of busulfan, cyclophosphamide and VP-16. 191 patients fit the entry criteria into this study. Mucositis was graded using the OMAS mucositis grading scale, which has a scoring range of 0–2.0. Severe mucositis for the purposes of this study was designated as having a maximal OMAS scale of ≥ 1.0. 87 patients experienced severe mucositis and 104 did not. Median age for patients was 52 years; underlying diagnoses were non-Hodgkin’s Lymphoma (n=155) and Hodgkin’s Disease (n=36). The two groups (developing severe mucositis vs not) were balanced with respect to disease status at transplant, number of courses of prior chemotherapy, prior exposure to radiation therapy of any kind, and elevated LDH at the time of transplant. Median CD34+ cell dose collected for the entire group was 8.5 x 106 /kg; there was no difference between the 2 groups. Severe mucositis correlated with mortality within 100 days of transplant. Patients with a maximal OMAS scale of < 1.0 had 99% 100-day survival (103/104) as compared to 92% (80/87) for those with an OMAS scale ≥ 1.0 (p=0.015). To date, 30% of patients have relapsed with no difference in the two groups. However, Kaplan-Meier estimates of survival reveal that severe mucositis has a profound negative impact on survival (p=0.002) as shown graphically below:

Figure Figure

One of the key drivers of this difference in survival was the fact the patients with severe mucositis were more likely to experience pulmonary or multi-organ failure as a cause of death: 8 patients with severe mucositis died of these toxicities, as compared to zero in the less severe mucositis group. Multivariable Cox analysis was performed to analyze risk factors for mortality. Three variables were found to be significant: elevated LDH at transplant (p=0.03), number of prior chemotherapy regimens (for one regimen increase, p=0.024) and severe mucositis (p=0.003). In conclusion, patients that develop severe mucositis have a higher risk of mortality after ABMT. The etiology is likely multi-factorial; mucositis may directly contribute to mortality risk, and may also be a surrogate marker of other organ toxicities.
Melphalan 200mg/m2 (MEL 200) and MEL 140/DT-PACE Are Equally Effective in Multiple Myeloma (MM), While the Latter Is Less Toxic.
Authors: Tricot Guido , Reiner Maureen , Zangari Maurizio , van Rhee Frits , Barlogie Bart
Published: April 2020

MEL 200 is now generally accepted to represent the best preparative regimen for autotransplantation in MM patients. We have previously reported that DT-PACE is very effective in inducing rapid responses even in high risk myeloma (JCO 2003, 31: 73–80). The present study aimed at comparing outcome and toxicity of previously treated (≥ 2 cycles of prior therapy) MM patients, who were randomized to tandem transplants with either MEL 200 or MEL 140 plus full dose DT-PACE administered over two days (MEL/DT-PACE). The treatment in both arms consisted of one induction cycle with DT-PACE, followed by tandem transplants (randomized), one consolidation cycle with DT-PACE and 2 years of maintenance therapy with dexamethasone (20mg/d, days 1–4, every 3 weeks) plus thalidomide 100mg/day. 97 patients were enrolled; 93 collected sufficient stem cells for transplantation and were randomized to MEL 200 (N=48) or MEL140/DT-PACE (N=45). Event-free (EFS) and overall survival (OS) were analyzed using Kaplan-Meier plots. Graphs were compared with logrank statistics. Median age was 57 years. Prior to study enrollment β2M was ≥ 4mg/L in 28%; CRP ≥ 4 mg/L in 59%; LDH ≥ 250 U/L in 16%. 16% had > 12 months preceding therapy and 27% showed abnormal metaphase cytogenetics. Baseline characteristics were similar in both groups, except for a trend of more patients with β2M ≥ 4mg/: in the MEL 200 arm (p=0.09). 100% of randomized patients proceeded to the first transplant and 82% to a second; 87% in the MEL/DT-PACE and 77% in the MEL 200 arm (p=0.23). The CR rates were identical (44% and 42%, respectively). EF/OS at 3 years were 51%/71% in the MEL/DT-PACE arm and 55%/79% in the MEL 200 arm (p=.89/.73) (Figure 1). Recovery of ANC > 500μl plus platelets > 20,000/μl untransfused, was comparable after the first (p=0.21) and second transplant (p=0.17). Grade 3–5 toxicity was lower in the MEL/DT-PACE arm in terms of mucositis (p=.006) and febrile neutropenia (p=.001). We conclude that both preparative regimens, MEL 200 and 140 with the addition of DT-PACE, appear equally effective in previously treated MM patients. However, the Mel 140 resulted in a significantly lower incidence of mucositis and febrile neutropenia and may thus be preferred in patients not in excellent clinical condition prio to transplantation.

Figure Figure
A Critical Role of Blockade of Cell Differentiation in BCR-ABL-Induced B-Cell Acute Lymphoblastic Leukemia (B-ALL): Basis for Superb Therapeutic Effect on B-ALL in Mice by Promotion of Pro-B Leukemic Cell Differentiation and Treatment with Imatinib.
Authors: Hu Yiguo , Kong Linghong , Staples Kevin , Mills Kevin , Monroe John G. , Li Shaoguang
Published: April 2020

The BCR-ABL oncogene induces human Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) that advances to acute phase of CML called blast crisis. In this acute phase, CML patients can develop either B-ALL or acute myeloid leukemia. In B-ALL, differentiation of leukemic cells are blocked at pro-/pre-B stage, and the underlying mechanism is unknown. We hypothesize that this blockade of B-cell differentiation may be important for the development of B-ALL induced by BCR-ABL, and if so, promotion of B-leukemic cell differentiation would create a novel therapeutic strategy for B-ALL. To test this hypothesis, we first compared the percentages of IgM+ B-leukemic cells in BALB/c and C57BL/6 (B6) mice with BCR-ABL-induced B-ALL, because we have previously found that B-ALL develops more quickly in BALB/c mice than in B6 mice (Li et al, J. Exp. Med.189:1399–1412, 1999). We expressed BCR-ABL in bone marrow (BM) using retroviral transduction and transplantation in these two different strains of inbred mice to induce B-ALL. There were significantly more peripheral blood B220+ B cells in BALB/c B-ALL mice than those in B6 mice, correlating to faster B-ALL in BALB/c mice than in B6 mice. Among these B220+ cells, IgM+ cells were much less in BALB/c mice than in B6 mice. We also compared rearrangement of the B cell antigen receptor (BCR) heavy chains (m chains) between BALB/c and B6 backgrounds using BCR-ABL-expressing pro-B cell lines isolated from the B-ALL mice. Normal m chains rearrangement was found in B6 leukemic cells, but not in BALB/c leukemic cells. These results indicate that more differentiated B-leukemic cells are associated with less aggressive disease. To further demonstrate the role of blockade of B-cell differentiation in B-ALL development, we induced B-leukemic cell differentiation by co-expression of BCR-ABL and intact immunoregulatory tyrosine activation motifs (ITAM) contained in immunoglobulin (Ig)_/Igß complexes in BM cells of B-ALL mice, comparing to expression of BCR-ABL alone. We treated these mice with imatinib (orally, 100 mg/kg, twice a day). The treated mice with B-ALL induced by co-expression of BCR-ABL and ITAM lived three-week longer than those with B-ALL induced by BCR-ABL only, with some mice in long-term remission. Prolonged survival was associated with 50% increased B220+/IgM+ B-leukemic cells in peripheral blood of the mice. Taken together, our results demonstrate that blockade of B-cell differentiation is critical for the development of B-ALL induced by BCR-ABL, and provide a rationale for combination therapy of B-ALL with imatinib and induction of leukemic cell differentiation.
WP1066: A Novel PI-3K Inhibitor with Antileukemic Activity in Philadelphia Chromosome-Positive ALL.
Authors: Ferrajoli Alessandra , Faderl Stefan , Wang Tony , Priebe Waldemar , Kantarjian Hagop , Talpaz Moshe , Estrov Zeev
Published: April 2020

Prognosis for patients with Philadelphia chromosome (Ph) positive ALL remains dismal. Ph+ ALL is characterized by the activation of several tyrosine kinases that provide the neoplastic clone with its proliferative capacity and survival advantage. We found that WP1066, a novel derivative of the tyrphostin AG490, inhibits the JAK-STAT pathway and cytokine-dependent and independent signaling pathways. Therefore, we sought to investigate the activity of WP1066 in Ph+ ALL. To do this, we first studied the effect of WP1066 on the Ph+ ALL cell lines Z-119 and Z-181 (Estrov Z et al. J. Cell Physiol.166(3):18, 1996). Using the MTT assay we found that WP1066 inhibited the growth of both Z-119 and Z-181 cells in a concentration-dependent manner with only 8% and 4% of the cells surviving at a concentration of 4 mM, respectively. Similarly, the clonogenic growth of both Z-119 and Z-181 cells was effectively inhibited by WP1066 with more than 90% reduction in colony numbers at concentration of 4 mM. Using Western Immunoblott analysis of cell lysates, we found that 4 mM of WP1066 induced caspase-3 cleavage in a time- and dose-dependent manner in both Z-119 and Z-181 cells. In addition, WP-1066 downregulated uncleaved poly (adenosine diphosphate-ribose) polymerase (PARP) and upregulated cleaved PARP protein levels in a time-dependent manner after 2 hours of exposure to 4 mM. We further evaluated induction of apoptosis using the annexin V-FITC assay and showed a dose dependent increase of the fraction of apoptotic cells in both Z-119 and Z-181 cells. After 24 hour of exposure to 4 mM of WP1066 the fraction of apoptotic cells increased by 23% and 43%, respectively. To elucidate the mechanisms by which WP1066 induces growth inhibition and apoptosis in Ph+ ALL cells, we investigated the effect of this agent on the phosphatidylinositol 3-kinase (PI-3K) pathway because the PI-3K pathway is constitutively activated in Ph+ leukemias. We found that WP1066 inhibited the phosphorylation of AKT in a time-dependent fashion in both cell lines and that this inhibitory effect lasted for 24 hours.

In conclusion, our data suggest that exposure to WP1066 induces caspase-dependent apoptosis, is associated with PI3-K inhibition and reduces the growth of the Ph+ cell lines Z-119 and Z-181. The activity of WP1066 in Ph+ ALL should be further studied.
Coculture of Pediatric Acute Lymphoblastic Leukemia (ALL) Blasts with CD40 Ligand Transfected Cells Leads to Changes of Surface Antigens and RNA Expression.
Authors: Stachel Daniel K. , Eickelmann Uta , Meilbeck Rita , Albert Michael H. , Buhmann Raymund , Hallek Michael , Schmid Irene
Published: April 2020

Although multi-agent chemotherapy is remarkably successful in the treatment of pediatric acute lymphoblastic leukemia (ALL), about 25% of patients experience relapse. A possible way to increase the cure rate for leukemia could be to modulate the immune response against leukemic cells.

It is known that ALL blasts are poor stimulators of cellular immune responses. This is believed to be mainly the result of a reduced presentation of costimulatory molecules on the surface of the ALL blasts.

For B cells, CD40 is an essential molecule which can be targeted therapeutically through its ligand, CD40 ligand or CD152. Normal B cells can induce cytotoxic T cell responses following stimulation with a soluble trimeric CD40 ligand.

We therefore hypothesized that by coculture with CD40 ligand transfected HeLa cells for four days, pediatric ALL blasts could be induced to upregulate costimulatory molecules, apoptosis inducing ligands and cytokines. The expression of the surface antigens CD40, CD40 ligand, CD80, CD86, CD95/fas, HLA-ABC and HLA-DR was evaluated by flow cytometry and the RNA expression of CD40, CD80, CD86, Fas ligand (FasL), TRAIL, IL-4, IL-6, IL-10, TGF-β was assessed by semiquantitative RT-PCR.

We found that coculture with CD40 ligand transfected HeLa cells significantly increased the expression of CD40 ligand, CD80, CD86 and CD95/fas while the expression of HLA-ABC, HLA-DR and CD40 remained unchanged. We found a significantly increased RNA expression of IL-6, TGF-β and CD80, a small but not significant increase of CD86, but no change in expression for IL-4, IL-10, FasL, TRAIL and CD40 after coculture. Addition of IL-4 to the culture did not influence the results.

In summary CD40 ligand mediated stimulation lead to increased expression of costimulatory molecules CD80 and CD86 and the pro-apoptotic CD95/fas, while the anti-apoptotic cytokines IL-6 and TGF-β were also upregulated.

Our results show that stimulation through CD40 ligation upregulates costimulatory molecules on pediatric ALL blasts, while it has differential effects on apoptosis inducers. This might have functional consequences which could be therapeutically exploited to modulate the immune system in an attempt to increase cure rates in pediatric ALL.
Vitamin K3 Selectively Induces Apoptosis in Acute Lymphoblastic Leukemia Cells with Constitutive Activation of IKKα/NF-kB Activation.
Authors: Zhu Ningxi , Gu Lubing , Findley Harry W. , Chiang Kuang-Yueh , Zhou Muxiang
Published: April 2020

Although the cytotoxic effect of vitamin K3 (VK3) on human cancer cells has been repeatedly reported, no clear conclusions from either in vitro or in vivo tests have so far been made for VK3 as an anticancer agent due to marked inter-tumor variability of efficacy in response to VK3 treatment. Here, we report that sensitivity of neoplastic cells to VK3-induced killing depends on IKKα expression/NF-kB activation in the cells. We tested the sensitivity to VK3 of 14 leukemic cell lines established from children with acute lymphoblastic leukemia (ALL). The 14 lines were classified into three groups: IKKα +/NF-kB+, IKKα +/NF-kB−, IKKα−/NF-kB−. IKKα +/NFkB+ cell lines that are generally resistant to doxorubicin are more sensitive to VK3 induced cell death than are the IKKα +/NFkB− lines that are usually sensitive to doxorubicin. The median of IC 50 values of VK3 and doxorubicin as tested by WST analysis for IKKα +/NFkB+ cells were 3.92 mM and 1.58 mM, respectively, compared to IKKα +/NFkB− cells (7.3 mM of VK3 and 0.71 mM of doxorubicin, p<0.01, t-test). Assays by testing activation of caspase and cleavage of death substrate PARP as well as flow cytometry showed that apoptosis was induced in a line with high levels of IKKα/NF-kB activation at 2 h after VK3 treatment. In contrast, apoptosis was not induced by VK3 even at 48 h post-treatment in two lines that lack IKKa expression and NF-kB activation. To test if IKKα/NF-kB is a molecular target of VK3 inducing apoptosis in ALL, we examined the expression and activation of IKKα/NF-kB in VK3-treated cells. VK3 specifically reduced IKKα expression and inhibited NF-kB activation, resulting in downregulation of NF-kB-mediated gene expression and apoptosis. These results suggest that inhibition of IKKα/NF-kB signaling pathway is essential for VK3 to induce cell death, and that VK3, a dietary factor with no cytotoxic effect on normal cells, would be a useful adjuvant in the treatment of ALL and other cancer patients whose neoplastic cells express constitutive NF-kB and are resistant to chemotherapy.
ERBB Expression in Philadelphia Chromosome-Positive ALL as a Potential Therapeutic Target.
Authors: Hughes Dennis P.M. , Tracy Jeannette L. , Lee Kenneth E. , Zhang Pingyu , Estrov Zeev , Chandra Joya
Published: April 2020

Philadelphia chromosome-positive ALL (Ph+ALL) strikes older children and adults, is difficult to treat and often has poor outcomes with conventional therapies. Tremendous advances have been made in other cancers through identification of essential signaling molecules that can be inhibited by small molecule medicines. ERBB proteins are such targets for several adult carcinomas, including breast and lung cancer. Expression of one member of the ERBB family, Her-2, is associated with chemotherapy resistance in one report of adult B-ALL. We examined the expression, activation status and contribution to growth and survival of ERBB receptors in Ph+ALL cell lines Z33, Z119 and Z181. Phosphorylated Her-2 was detected by western blot in all 3 lines, as was trace amounts of Her-3. Western-blot identified expression of the p80 form of Her-4 in Z119, which correlated with flow cytometry assessment of Her-4. Expression in Z33 of EGFR, detected by western blot, correlated with down-regulation of both EGFR and Her-2 from the cell surface, as assessed by flow cytometry. Internalization of EGFR homo- and hetero-dimers is associated with receptor phosphorylation and active signaling. To assess the physiologic importance of ERBB signaling in Ph+ALL, cells were cultured in the pan-ERBB inhibitor CI-1033 (Pfizer Global Research and Development), which is specific for ERBB family receptors in tissue culture up to 20 μM concentrations. All lines demonstrated reduced cell yield after CI-1033 exposure, with average IC50 of 0.783 for Z119, 1.178 for Z181 and 1.737 for Z33. The increased sensitivity of Z119 to CI-1033 correlated with an increased rate of apoptosis: 3 μM CI-1033 caused a 26% increase in the fraction of sub-diploid Z119 cells at 24 hours, compared to 13% for Z181 and 16% for Z33. Higher sensitivity to ERBB inhibition also correlated with growth factor resistance, since Z33 and Z181 are dependent upon autocrine GM-CSF for growth while Z119 grows independent of known autocrine secretion of hematopoietic growth factors. These data suggest that Ph+ALL cells may acquire GM-CSF independence by relying more on ERBB-derived signals, since these receptors are capable of activating similar second messenger pathways. Our data suggest that, surprisingly, the ERBB family of receptor tyrosine kinases are intact and functioning in Ph+ALL, and inhibition of these signals may provide benefit to Ph+ALL patients, either in combination with imatinib or in patients who develop imatinib resistance.
Anti-Leukemic Effect of Maintenance Chemotherapy Is Mediated by Induction of Differentiation.
Authors: Lin Tara L. , Matsui William , Vala Milada S. , Brennan Sarah , Smith B. Douglas , Jones Richard J.
Published: April 2020

The clinical benefit of maintenance therapy (prolonged post-remission low-dose therapy) has been limited to two diseases, acute lymphocytic leukemia (ALL) and acute promyelocytic leukemia (APL), despite extensive study in many malignancies. Moreover, the mechanisms responsible for the anti-leukemic effects of maintenance therapy in both ALL and APL are unclear. Maintenance therapy in ALL consists of the low-dose cytotoxic agents methotrexate (MTX) and 6-mercaptopurine (6MP), while maintenance therapy in APL has primarily been all-trans-retinoic acid (ATRA). Interestingly, the addition of low-dose MTX and 6MP to maintenance ATRA has recently been shown to improve disease-free survival in APL. These clinical results, combined with our recent data and that from others showing that low-dose cytotoxic agents are potent differentiating agents, suggest that these agents may induce the differentiation of leukemic progenitors responsible for disease relapse. Therefore, we examined the differentiating effects of ATRA and low-dose MTX and 6MP on human APL and ALL cells. The APL cell line NB4 was treated with ATRA (1 μM), MTX (0.01 μM) and 6MP (1 μM) for 48 hours and then analyzed for apoptosis, clonogenic growth and surface expression of the myeloid differentiation antigens CD11b and CD15 by flow cytometry. As expected, ATRA did not induce immediate apoptosis, but inhibited clonogenic growth associated with markedly increased expression of both myeloid antigens, consistent with induction of terminal differentiation. Both MTX and 6MP had similar effects on the NB4 cells, and, thus, similarly appeared to induce terminal differentiation rather than cytoxicity. We then examined the effects of these agents on two ALL cell lines: REH, which is derived from pediatric ALL and contains the t(12;21) chromosomal translocation producing the TEL-AML1 fusion protein, and RS4;11, which is derived from adult ALL and contains the t(4;11) chromosomal translocation producing the MLL-AF4 fusion gene. ATRA, MTX and 6MP induced upregulation of CD19 and CD38 surface antigen expression. Similar to their effects on the NB4 APL line, the agents also inhibited clonogenic growth of the ALL cells without induction of immediate apoptosis. ATRA, MTX, and 6MP produce similar effects on APL and ALL cells in vitro. These results suggest that low-dose MTX and 6MP used as maintenance therapy improve clinical outcomes in ALL and APL by inducing the terminal differentiation of leukemic progenitors responsible for disease relapse. As the addition of low-dose MTX and 6MP has improved the results of maintenance ATRA in APL, the addition of pharmacologic differentiating agents such as ATRA to MTX and 6MP may improve ALL therapy, particularly for adults.
The Effect of Early High Dose Ara-C in Children with High-Risk Acute Lymphoblastic Leukemia (ALL): Tokyo Children’s Cancer Study Group (TCCSG) Study L99-15.
Authors: Manabe Atsushi , Ohara Akira , Ikuta Kouichiro , Hanada Ryouji , Yabe Hiromasa , Kinoshita Akitoshi , Koike Kenichi , Okimoto Yuri , Noguchi Yasushi , Kajiwara Michiko , Takahashi Hiroyuki , Sugita Kanji , Fukushima Takashi , Isoyama Keiichi , Saito Masahiro , Kikuchi Akira , Ota Setsuo , Kumagai Masaaki , Hayashi Yasuhide , Saito Tomohiro , Tsuchida Masahiro
Published: April 2020

High dose Ara-C is employed in the treatment of acute myeloid leukemia; however, its effect on ALL has not been evaluated systematically. We tested high dose Ara-C incorporated in an early phase with a randomized fashion in children with high-risk ALL. Patients diagnosed as ALL between 1999 and 2003 were consecutively enrolled on TCCSG L99-15 study. Risk stratification was based on the age, initial white blood cell count, immunophenotype, cytogenetics, and the response to prednisolone monotherapy. Totally, 317 out of 770 children (41%) were categorized in a high-risk group. A standard-risk group constituted 35% and a very high-risk group constituted 24% of all the patients. Induction treatment consisted of a standard 4-drug regimen plus cyclophosphamide. Remission was obtained in 310 patients (98%). After remission was obtained, patients were randomized to receive either early intensification regimen A or B: regimen A consisted of high-dose cytarabine (2g/m2 x 8 times with L-asparaginase); regimen B consisted of a combination of cyclophosphamide, cytarabine (75mg/m2 x 15 times), and 6-mercaptopurine. Then all the patients were given an identical treatment including high-dose methotrexate (3g/m2 x 3 times), reinduction therapy, late intensifications, and maintenance therapy. The treatment was completed 24 months after diagnosis. The number of patients randomized in each arm was 155 cases for each arm. Clinical features including immunophenotype, specific karyotype, and the response to prednisolone monotherapy were not different in patients allocated in each arm. The median follow-up for patients who survived at the time of the analysis was 3.2 years. Event-free survival (EFS) (SE) and overall survival (OS) (SE) at 4 years for the 310 patients were 78.4% (2.9%) and 88.9% (2.3%), respectively. EFS (SE) at 4 years for patients in regimen A and B were 77.0% (4.1%) and 79.7% (4.0%) while OS (SE) at 4 years in regimen A and B were 88.3% (3.2%) and 89.7% (3.2%). The site of relapse was not different in two arms. In 23 cases with T-cell phenotype, patients treated with regimen A did not have better EFS than the others. Similarly, in 25 cases with E2A-PBX1 rearrangement, patients treated with regimen A did not have better EFS than the others. Although no patients in either regimen died of infections, more patients in regimen A experienced bacterial sepsis: 13 cases in regimen A vs. 8 cases in regimen B. In conclusion, we did not find any benefit of employing early high-dose Ara-C in children with high-risk ALL.