bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Unique In Vitro and In Vivo Model System of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph-ALL) with Emphasis on Cell to Cell Interaction.
Authors: Tojo Arinobu , Izawa Kiyoko , Sekine Rieko , Nagamura-Inoue Tokiko , Kobayashi Seiichiro
Published: October 2019

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, readily acquires resistance to chemotherapeutic drugs including imatinib mesylate (IM), and shows a high relapse rate even after allogeneic stem cell transplantation. Nevertheless, primary blast cells are generally susceptible to apoptotic cell death in sort-term suspension culture after isolation from patients with Ph-ALL. We established two Ph-ALL cell lines and characterized their growth properties supported by adhesive interaction with a murine bone marrow stromal cell line, HESS-5. IMS-PhL1 (L1) cells mainly expressed p210-type BCR-ABL mRNA with wild type sequences in the ABL kinase domain and were weakly positive for p190-type mRNA. IMS-PhL2 (L2) cells exclusively expressed p190-type transcripts with Y253H mutation and showed much lower sensitivity to imatinib than L1 cells. The growth of L1 cells was slowly autonomous in suspension culture, but became more vigorous and their apoptosis was prevented by co-culture with HESS-5 cells. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5 cells and did not respond to soluble cytokines including SCF, IL3and IL7. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas. This migration was significantly inhibited by the pretreatment of those with a neutralizing antibody against α4-integrin. While non-adherent L1 cells were eradicated by 1 mM IM, a portion of adherent L1 cells could survive even at 10 mM IM. Similarly, adherent L2 cells considerably resisted prolonged exposure to 10 mM IM. Intravenous injection of both cell lines caused leukemia in NOD-SCID mice after distinct latent periods. Leukemia cells appeared in peripheral blood, bone marrow as well as spleen. Interestingly, expression of α5-integrin was significantly down-regulated in both leukemia cells collected from those tissues, but was restored after co-culture with HESS-5. The study of L1 and L2 cells in vitro and in vivo will not only contribute to further insights into microenvironmental regulation of clonal maintenance and progression of Ph-ALL but also provide a unique model for experimental therapeutics against Ph-ALL.

Figure Figure
The Wnt Pathway Modulates Expression of Growth and Survival Genes in Acute Lymphoblastic Leukemia Cells.
Authors: Khan Naveed I. , Bradstock Kenneth Francis , Bendall Linda J.
Published: October 2019

Wnt proteins are important bone marrow-derived growth factors known to support normal hematopoietic progenitor and stem cell development. Here we report that B cell progenitor acute lymphoblastic leukemia (pre-B ALL) cells express Wnt proteins, including Wnt-2b in 33%, Wnt-5a in 42%, Wnt-10b in 58% and Wnt-16b in 25% of cases. The Wnt receptors, Frizzled (Fz)-7 and -8 were also expressed in most cases while Fz-3, -4 and -9 were occasionally detected. Stimulation of pre-B ALL cells with Wnt-3a activated canonical Wnt signaling with increased expression and nuclear translocation of β-catenin. This resulted in a 1.7 to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. Wnt-3a also significantly increased the survival of pre-B ALL cells under conditions of serum deprivation. To determine the mechanisms involved we examined the effects of Wnt-3a on gene expression using the leukemic pre-B ALL cell line NALM6 and a cancer specific microarray (GEArray® OHS-802), which contains 440 known cancer genes. Expression of 83 genes (19%) could be detected on the array. Exposure to Wnt-3a for 24 hours resulted in increased (>1.5 fold) expression of 29 genes and reduced (<50% of control) expression of 3 genes. The most highly regulated genes in response to Wnt-3a were MYBL2, E2F1, CD10, VDAC1, CDC25B (upregulated) and TRAIL-R2 (downregulated). Using qRT-PCR, we confirmed regulation of these genes in NALM6 cells and/or in another leukemic cell line LK63. These genes play important roles in the control of cell cycle (MYBL2, E2F1 and CDC25B), apoptosis (VDAC1 and TRAIL-R2) and motility (CD10) in cancer cells. Our results suggest that Wnt signalling regulates cell growth and proliferation in leukemic cells by modulating the expression of a number of genes. To our knowledge this is the first study examining the gene expression profile following Wnt stimulation in leukemic cells and potentially identifies new therapeutic targets for treatment.
BIOV-111 a European Phase II Trial of Clofarabine (Evoltra®) in Refractory and Relapsed Childhood Acute Lymphoblastic Leukemia.
Authors: Kearns Pamela , Michel Gerard , Nelken Brigitte , Joel Simon , Al-Ghazaly Essam , Beishuizen Auke , Conter Valentino , Gadner Helmut , Henze Gunter , Smith Hazel , Baruchel Andre , Saha Vaskar
Published: October 2019

BIOV-111 is a European phase II non-randomised, open-label study of a next generation purine nucleoside analogue, clofarabine (Evoltra®), in pediatric patients with relapsed/refractory ALL. We report on the efficacy and safety data. Eligible patients (pts) had either primary refractory (2 pts) or relapsed/refractory (51 pts with ≥ 2 prior lines of treatment) ALL. Clofarabine 52mg/m2 daily × 5 days was given every 28–42 days (1 course). The primary endpoint is overall response rate (ORR) defined as either a complete response (CR) or CR without platelet recovery (CRp) after 2 courses. Adverse events (AEs) were graded according to NCI CTC (v3). Plasma, urine and intracellular clofarabine pharmacokinetics were also investigated. To date, 96 courses have been administered to 53 pts from 25 centres. The median number of prior treatments was 2 (range 1–5) and 20 pts (38%) had been previously transplanted. 8/29 pts receiving ≥2 courses responded (1CR, 7CRp) giving an ORR of 28%. Responses were observed in 14/53 (26%) pts receiving at least one course of clofarabine (6CR, 7CRp, 1 PR ). Eight (57%) responders had a prior transplant and 1 of these patients was transplanted post clofarabine. One pt with a prior transplant remains in remission at 20+ months. Four pts (1CR, 3CRp) have proceeded to transplantation. Serious adverse events (n=103) included febrile neutropenia (51/103), seizures (4/103), streptococcal septicaemia (3/103), palmo-plantar erythrodysaesthesia (2/103) and bone pain (2/103). Three hepatic events occurred (raised bilirubin, raised ALT/AST), 1 renal failure and 1 cardiac failure. AEs occurred in 4 pts. The renal and cardiac failure AE occurred in a pt with known anthracycline cardiac myopathy and renal impairment at study entry. Median end of infusion plasma clofarabine concentration (n=25) on days 1 and 2 were 1.5 (0.5–3.2) uM vs 2.0 (0.1–5.1) uM respectively (p=0.01) and were not different in pts achieving a response. Urinary clofarabine recovery on days 1, 5 and 6 (drug -free) was 48±18 %, 46±12 % and 5±3 %. Clofarabine TP analyses are ongoing. Response rates in this ongoing BIOV-111 study are consistent with the pivotal clofarabine study (CLO-212) in relapsed/refractory pediatric ALL. Notably, BIOV-111 has a lower incidence of AEs, including hepatic and renal AEs; (4% and 1% respectively vs 10% and 8% in CLO-212), possibly attributable to fewer prior treatments compared to CLO-212 (median 2 vs 3 respectively). Clofarabine achieves a significant response rate in this heavily pre-treated patient population and durable responses have been observed which may confer a survival advantage with longer follow-up.
Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.
Authors: Collins Natalie B. , Tomashevski Andrei , Kupfer Gary M.
Published: October 2019

Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.
Assessment of Anti-Asparaginase (ASNase) Antibodies (Ab) and ASNase Activity after Suspected Clinical Allergy.
Authors: Tiwari Prakash N. , Zielinski Monica , Quinn John J. , Siegel Stuart E. , Gaynon Paul S. , Wakamatsu Peter , Seibel Nita L. , Avramis Vassilios I.
Published: October 2019

Background: ASNase is a critical treatment component in childhood acute lymphoblastic leukemia (ALL). As ASNase is a bacterial protein, production of anti-ASNase Ab, often associated with clinical allergy, poses a frequent problem during ASNase therapy. Sixty percent of higher risk ALL patients, who received native ASNase in induction, were Ab(+) by 5 months into therapy (Panosyan et al., 2004). Moreover, 22%–29% of these patients had no clinical allergy (silent hypersensitivity) and were at increased risk for relapse.

Patients and Methods: Physicians from 13 institutions submitted samples for 48 children in first remission, aged 14 to 252 months, on various SR or HR ALL protocols with suspected ASNase allergy. The male/female ratio was 1.4. The serum samples were assessed for ASNase activity and anti-ASNase Ab as reported earlier (Avramis, et al., Blood 2002). Ab titer is expressed as the numeric ratio relative to an Ab negative control.

Results: Allergic symptoms ranged from localized skin rash and swelling to respiratory difficulties and/or anaphylaxis. Neutralizing anti-PEG-ASNase Ab’s were seen in 41 patients (85%). The results were examined with respect to age, gender, ASNase formulation received, time of post-ASNase administration, and the physician’s clinical observations. Higher Ab ratios > 1.1 correlated with low or no ASNase activity post-PEG-ASNase (46/48 patients) or post-Native ASNase dosing (2 patients). Fourteen patients had cutaneous manifestations (rash, hives, urticaria). All had neutralizing Ab and no enzymatic activity. Nine had higher Ab ratios and 5 had lower Ab ratios. Eight additional patients had localized or generalized swelling; all had higher Ab ratios. Similarly, over the entire CCG-1961 study, 526/1100 patients had a clinical reaction; 476/526 had higher Ab ratios (90%).

Conclusions: We found neutralizing Ab in 85% of patients with apparent clinical ASNase allergy. Neutralizing Ab was correlated with lower or absent ASNase activity. Lower Ab ratios may be associated with earlier time points in the anamnestic response. In the remaining 15% of patients with an apparent clinical reaction, we found no Ab and substantial ASNase activity. Monitoring ASNase activity and Ab may be useful for guiding ASNase therapy. Patients with no Ab and substantial activity, might be rechallenged with the same product despite an apparent allergic reaction.
Type II HMG-CoA Reductase Inhibitors (Statins) Provide Acute-Graft-Versus-Host Disease Protection by Th-2 Cytokine Induction While Sparing Graft-Versus-Leukemia Activity.
Authors: Zeiser Robert , Youssef Sawsan , Baker Jeanette , Steinman Lawrence , Negrin Robert
Published: October 2019

Recent studies have shown the beneficial impact of 3-hydroxy-3-methyl-CoA (HMG-CoA) reductase inhibitors (statins) on autoimmunity and allograft rejection. To investigate whether statins are capable of protecting from acute graft vs host disease (aGVHD) we utilized an established murine model (FVB/N->Balb/c) across major MHC barriers. Type II statins were potent inducers of a T-helper cell (Th)-2 cytokine profile in the adoptively transferred T cells upon exposure in vitro or in vivo. Expansion of alloreactive luciferase transgenic T cells as measured by total body light emission was significantly reduced by donor pre-treatment (10mg/kg bodyweight) or in vitro T cell treatment (10nM) with atorvastatin (AT vs PBS; p=0.0008) and fluvastatin (FLU vs PBS; p=0.0007). The beneficial effect of statin treatment translated into significantly reduced aGvHD lethality in statin as compared to PBS treated animals. Th-2 biased donor T cells could be tracked until day 15 after BMT by intracellular cytokine staining and FACS analysis. Host treatment prior to transplantation induced down-regulation of the costimulatory molecules CD40, CD80 and CD86 and MHC class II on recipient APCs and enhanced the protective statin effect when donor and recipient were treated (survival AT vs PBS, p=0.0012). Induction of the Th-2 cytokine profile was still permissive for in vivo graft vs leukemia effects by cytolytic effector function of CD8+ T cells against A20 B-cell lymphoma cells as assessed by bioluminescence imaging, FACS, histology and survival. In vitro CD8+ T cells derived from AT treated animals displayed equal cytolytic activity against Yac1 T-cell lymphoma cells as compared to T cells derived from PBS treated animals. Mechanistically we identified a role for the STAT-6 pathway in statin induced Th-2 induction as aGvHD protection was partially reversed with STAT-6−/− donors. The in vivo statin effect could be antagonized by L-mevalonate indicating the relevance of the mevalonate pathway for statin mediated aGvHD protection. Since L-mevalonate produced by the HMG-CoA reductase is the precursor of non steroidal isoprenoid compounds, such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, the statin impact on prenylation status of critical signaling proteins in alloreactive T cells was determined by Western blot. In vitro AT treatment reduced the levels of RAP-1, RhoB and Ras prenylation in allo-antigen exposed T cells and abrogated expression of T-bet which is critical for Th1 induction.

We conclude that type II statins have significant protective impact on aGvHD lethality by Th-2 cytokine induction and inhibition of an uncontrolled Th-1 response, which is of great clinical relevance given the widespread use and well defined toxicity profile of these agents.
Overexpression of CXCR4 in Acute Myeloid Leukemia Predicts Adverse Overall and Progression-Free Survival Independently of FLT3 Gene Mutation.
Authors: Konoplev Sergej , Rassidakis George Z. , Estey Elihu H. , Kantarjian Hagop M. , Huang Xuelin , Xiao Lianchun , Andreeff Michael , Konopleva Marina , Medeiros L. Jeffrey
Published: October 2019

CXC chemokine receptor 4 (CXCR4) expression in acute myeloid leukemia (AML) is reported to correlate with FLT3 gene mutation and poorer prognosis. We analyzed the prognostic significance of CXCR4 expression in 122 AML patients with diploid karyotype and no evidence of FLT3 gene mutation treated at our institution between 1997 and 2003. All patients received intensive chemotherapy according to institutional protocols; 84% received cytarabine-containing regimens. Bone marrow biopsy specimens obtained at diagnosis from untreated patients were immunostained with an antibody specific for CXCR4 (Abcam, clone 2074; Cambridge, MA). Statistical analysis included the following variables: CXCR4 expression, age, sex, race, incorporation of cytarabine in the therapeutic regimen, presence of multilineage dysplasia, history of antecedent hematologic disorder, history of prior chemotherapy (for either hematological or non-hematological disorder), performance status, leukocytosis, hemoglobin, platelet count, bilirubin, and creatinine. There were 70 men and 52 women with a median age of 62 years (range, 22–82 years). Median follow-up was 18 months (range, <1–97 months). Seventy-six patients achieved complete remission (CR); 39 relapsed. Sixty-six patients died, including 9 with no evidence of disease. CXCR4 was positive in 70 and negative in 52 patients, with CR rates of 58% and 71%, respectively (p=0.09). In the univariate Cox model, CXCR4 expression was associated with shorter overall survival (OS), p=0.008, and disease-free survival (DFS), p=0.012, figure1.

Figure Figure

Shorter OS was also associated with failure to achieve CR (p=<0.0001), age (p=0.01), presence of multilineage dysplasia (p=0.006), poorer performance status (p=0.049), higher creatinine level (p=0.00002), and lower hemoglobin level (p=0.042). Shorter DFS was associated with failure to achieve CR (p=0.0002), presence of multilineage dysplasia (p=0.02), history of prior chemotherapy (p=0.04), and higher creatinine level (p=0.0009).

In multivariate analysis, shorter OS was associated with CXCR4 expression (p=0.047), age (p=0.029), presence of multilineage dysplasia (p=0.042), and high creatinine level (p=0.016). Shorter DFS was associated with CXCR4 expression (p=0.036), history of prior chemotherapy (p=0.044), presence of multilineage dysplasia (p=0.007), and high creatinine level (p=0.034). These results suggest that CXCR4 expression is associated with poor prognosis in AML patients and is independent of FLT3 gene mutation.
Expression of Angiopoietins and Their Receptor Tie2 in the Bone Marrow of Patients with Acute Myeloid Leukemia.
Authors: Schliemann Christoph , Bieker Ralf , Padro Teresa , Kessler Torsten , Hintelmann Heike , Buchner Thomas , Berdel Wolfgang E. , Mesters Rolf M.
Published: October 2019

Angiopoietin-1 (Ang-1) and its natural antagonist Angiopoietin-2 (Ang-2), both ligands for the receptor tyrosine kinase Tie2, are known to play an essential role in normal and pathological angiogenesis. However, the importance of angiopoietin signaling in the pathophysiology of hematologic neoplasias such as acute myeloid leukemia (AML) remains to be elucidated. We investigated the expression of Ang-1, Ang-2 and Tie2 by immunohistochemical analyses in bone marrow biopsies of 64 adult patients with newly diagnosed AML and correlated angiogenic factor expression with clinicopathological variables and long-term survival. Expression of Ang-2 was significantly increased in the bone marrow of AML patients (median [interquartile ranges]: 4.7 [3.3 – 5.7] AU [arbitrary units]) as compared with 16 control patients (1.5 [1.5 – 1.8] AU; P < 0.0001). In contrast, Ang-1 expression levels in AML patients did not differ from those found in controls. Thus, we observed a reversal of the Ang-1 and Ang-2 expression balance in the neoplastic bone marrow (Ang-2:Ang-1 ratio: 1.73) as compared with normal bone marrow (0.51; P < 0.0001). Furthermore, the angiopoietin receptor Tie2 was significantly overexpressed in leukemic blasts (3.8 [2.8 – 4.9] AU vs. 1.8 [1.6 – 2.3] AU; P < 0.0001). Patients expressing high levels of Ang-2 showed significantly longer overall survival (OS) than those with low Ang-2 levels (52.7 vs. 14.7 months; P = 0.039). The impact of Ang-2 expression on OS was especially evident in AML patients simultaneously expressing low levels of Ang-1 (P = 0.0298). Multivariate Cox regression analysis revealed karyotype and Ang-2 expression as independent prognostic factors for OS (hazard ratio [CI]: 3.06 [1.39 – 6.70] and 0.31 [0.14 – 0.69], respectively; P < 0.01). In conclusion, these data provide evidence that the alteration of angiopoietin balance in favor of Ang-2 may play a critical role in the pathophysiology of AML. Furthermore, high pre-therapeutic bone marrow Ang-2 levels indicate a favorable prognosis in polychemotherapy treated AML by a yet unknown mechanism.
Chimeric Leukemic Protein PLZF/RARα Functions as an Adoptor for Cul3-Based E3 Ubiquitin Ligase To Regulate Proteosomal Degradation of RXRα.
Authors: Onozawa Masahiro , Kondo Takeshi , Kanamori Hiroe , Kahata Kaoru , Hashino Satoshi , Asaka Masahiro
Published: October 2019

Typical acute promyelocytic leukemia (APL) is associated with expression of the PML/RARα fusion protein resulting from chromosomal translocation t(15;17) and responsiveness to treatment with all-trans retinoic acid (ATRA). A few population of APL is associated with variant chromosomal translocations, t(11;17), t(5;17) and t(17;17). PLZF/RARα is the chimeric fusion protein resulting from the chromosomal translocation, t(11;17)(q23;q21). APL cells with PLZF/RARα have been reported to be unresponsive to ATRA-induced terminal differentiation clinically and experimentally. The molecular basis of unresponsiveness in PLZF/RARα-derived APL cells against ATRA explained by a rigid interaction between BTB/POZ domain of PLZF and a transcriptional co-repressor, N-CoR. PLZF/RARα contains BTB/POZ domain in its N-terminus. BTB/POZ domain is developmentally conserved among various species, and recently several BTB/POZ-containing molecules have been reported to function as substrate-specific adaptors for Cul3-based E3 ubiquitin ligase. Here we examined the possibility that PLZF/RARα functions as an E3 ligase.

We performed the series of transient transfection analysis. By immunoprecipitation assay, PLZF/RARα associated with Cul3, and PLZF/RARα also associated with RXRα. PLZF/RARα accelerated an ubiquitin-dependent degradation of RXRα, and resulted in the decreased expression of RXRα. This degradation of RXRα was dependent on the expression level of PLZF/RARα. On the contrary, co-expression of dominant negative form of Cul3 with PLZF/RARα resulted in the restored expression of RXRα. When we expressed PML/RARα, PLZF or PLZF/RARαΔBTB, the accelerated degradation of RXRα was not observed.

In RARα-responsive luciferase assay, PLZF/RARα repressed ATRA response. Consistent with the result that PLZF/RARαΔBTB did not down-regulate the expression of RXRα, PLZF/RARαΔBTB did not repress ATRA response. In addition, the transduction of recombinant RXRα molecule into PLZF/RARα expressing cells partially restored ATRA-responsiveness. Collectively, we suggest that ATRA resistance in PLZF/RARα-positive cells is explained by the novel function of PLZF/RARα molecule.
Physical and Functional Interactions of Acute Myeloid Leukemia-1 with Hypoxia Inducible Factor-1α Protein.
Authors: Yu Yun , Peng Zhen-Gang , Zhou Mei-Yi , Liao Shi-Hua , Huang Ying , Qiu Ji-Hui , Wang Li-Shun , Dong Shuo , Chen Guo-Qiang
Published: October 2019

Hypoxia inducible factor-1α (HIF-1α), a critical transcription factor for cellular responses to hypoxia, was shown to regulate the differentiation of some kinds of tissue cells including acute myeloid leukemic (AML) cells. Based on the important role of AML1 protein in hematopoietic development, this work tries to investigate the potential relationship of AML1 as well as its abnormal leukemogenic fusion protein AML1-ETO with HIF-1α protein. The results showed that HIF-1α protein physically and directly interacts with ectopically and constitutively expressed AML1 or AML1-ETO protein, as evidenced by integrated Lac operator array, co-immunoprecipitation and GST pull-down assay. It appears that AML1-ETO has better binding ability with HIF-1α protein. With this interaction, either AML1 or AML1-ETO attenuates the transcriptional activity of HIF-1α protein with the inhibition of expressions of vascular endothelial growth factor and glucose transporter-1, two well-known HIF-1α-targeted genes. On the contrary, hypoxia-stabilized HIF-1α protein potentiates transcriptional ability of AML1 and AML1-ETO, as assessed by AML1-binding sites-driven luciferase assay. According to these results, a speculative hypothesis is proposed for the dual roles of HIF-1α in leukemic cell differentiation and angiogenesis. The further investigations would open another avenue of attacking against cancer.
MEIS1 Is a Required Transcription Factor for AML Cell Proliferation.
Authors: Heinrichs Stefan , Brenner Chad J. , Kulkarni Rima V. , Thomas Look A.
Published: October 2019

MEIS proteins constitute an important subgroup of three amino acid loop extension (TALE) class transcription factors, which are characterized by an atypical homeodomain. MEIS1 has been shown to act as an important cofactor in HOXA9-mediated leukemogenesis in bone marrow transduction experiments. In human leukemia, MEIS1 was identified as a gene consistently upregulated in leukemias with MLL translocations. However, MEIS1 overexpression is also frequently found in AMLs with normal or complex karyotypes. To investigate the role of MEIS1 in AML pathogenesis, we measured the expression of MEIS1 and MEIS2 in 26 different established human AML cell lines, and documented high levels of expression of one or both genes in 20 of these lines. Despite the prevalence of MEIS1 expression in AML blasts, the downstream pathways and their contribution to the proliferation or self-renewal of the malignant clone are not well understood. To identify gene expression programs controlled by MEIS1, we targeted MEIS1 expression by RNAi in two AML cell lines that express HOXA9 and MEIS1, but only low levels of MEIS2. In comparison to a control shRNA, cells expressing two different MEIS1-specific shRNAs were markedly deficient in thymidine incorporation indicating very low levels of cellular proliferation. The RNAi-mediated knock-down of MEIS1 expression resulted in down-regulation of the tyrosine kinase receptor FLT3 by quantitative RT-PCR. Thus our data in human AML is consistent with results of Wang et al. (Blood, 106(1):254–64) implicating MEIS1 in the regulation of FLT3 tyrosine kinase expression, based on overexpression studies in mouse bone marrow progenitors. Our results in human AML suggest that MEIS1 is required for high levels of FLT3 expression, which is significant because FLT3 is mutated and activated in these two myeloid leukemia cell lines, suggesting the hypothesis that FLT3 is an important component of the pathway through which MEIS1 promotes proliferation. Programmed re-expression of activated FLT3 in AML lines with MEIS knock-down is underway to test whether FLT3 is the essential component acting downstream of MEIS1 expression in AML pathogenesis.
All-Trans Retinoic Acid and Gemtuzumab Ozogamicin as Adjunct To Salvage Therapy in Primary Refractory Acute Myeloid Leukemia: Results of Consecutive Phase II Studies of the AMLSG.
Authors: Schlenk Richard F. , Dohner Konstanze , Kerz Martina , del Valle Francisco , Heydrich Björn , Schmidt-Wolf Ingo , Pralle Hans , Götze Katharina , Koller Elisabeth , Nachbaur David , Mergenthaler Hans G. , Fiedler Walter , Haase Detlef , Kremers Stephan , Theobald Matthias , Matzdorff Axel , Ganser Arnold , Dohner Hartmut
Published: October 2019

Background: In primary refractory patients the achievement of a response to salvage therapy is of enormous prognostic impact.

Aims: To evaluate the impact of all-trans retinoic acid (ATRA) and gemtuzumab ozogamicin (GO) given as adjunct to high-dose cytarabine based intensive salvage therapy in primary refractory younger patients on clinical outcome.

Methods: Between 1993 and 2006 264 consecutive patients (median age: 48 yrs) were evaluated. All patients had primary refractory AML after one cycle of ICE (idarubicine, cytarabine, etoposide). The different salvage therapies were as follows: from 1993–1998: S-HAM for patients <55 years of age [cytarabine 3g/m2 bid. days 1,2,8,9, mitoxantrone 10mg/m2 days 3,4,10,11] and HAM for patients >=55 years of age [cytarabine 3g/m2 bid., days 1–3, mitoxantrone 12mg/m2 days 2,3]; from 1998 to 2004 A-HAM [HAM with ATRA 45mg/m2 days 3–5, 15mg/m2 days 6–28]; from 2004 up to date: GO-A-HAM [A-HAM with gemtuzumab ozogamicin 3g/m2 day 1].

Results: The distribution of the different salvage therapies was HAM n=21, S-HAM n=22, A-HAM n=118, GO-A-HAM n=62, other n=31, or no further therapy n=10. CR-rate according to salvage therapy was GO-A-HAM 49%, A-HAM 34% S-HAM 23%, HAM 14%. Treatment related mortality did not differ between regimens. No CTC-grade 3–5 liver toxicity was seen in patients receiving GO-A-HAM. Logistic regression on the achievement of CR revealed that regimens containing ATRA (odds ratio 2.0, p=0.05) and GO (odds ratio 1.9 p=0.05) were associated with response. 151 of 264 patients have received a stem cell transplantation as consolidation therapy (n=59 MRD, n=79 MUD, n=6 haplo-identical donor, n=7 autologous). The rates of severe veno occlusive disease after transplant was identical (5%) for patients receiving a salvage therapy including GO and those who did not. Survival analysis revealed a significant difference (p= 0.00178) with a median survival of 16.2 months for GO+ATRA, 12.5 months for ATRA versus 7.2 months for the others.

Conclusions: Although retrospective in nature our study suggests that ATRA and GO as adjunct to salvage therapy improves CR rates and survival in primary refractory AML patients.
A Phase 1 Dose-Escalation Study of the Novel Cell Cycle Active Agent SNS-595 in Advanced Leukemias.
Authors: Lancet Jeffrey E. , Karp Judith E. , Havrilla Nancy , Hoch Ute , Silverman Jeffrey , Adelman Daniel , Giles Francis J.
Published: October 2019

SNS-595 is a novel, cell cycle active cytotoxic naphthyridine analog that induces G2 arrest in a variety of preclinical tumor models. We initiated an escalating-dose phase 1 trial of SNS-595, administered as a weekly x 3 (arm A) or twice weekly x 2 bolus (arm B), in patients with advanced or refractory acute leukemias.

Objectives: The primary objectives were to:establish safety, tolerability, and MTD of SNS-595 given on each schedule,characterize pharmacokinetics (PK) of SNS-595 when given on these schedules.

Secondary objectives were:assessment of clinical activity,exploration of potential biomarkers.

Methods: SNS-595 was administered as a slow IV push on days 1, 8, 15 (arm A) or days 1, 4, 8, 11 (arm B). Minimum cycle length was 42 days (arm A) and 39 days (arm B). Additional cycles were permitted if patients achieved stable disease or better. The starting dose was 18 mg/m2/d on arm A, and 9 mg/m2/d on arm B and escalated by cohort using a modified Fibonacci schema. PK analyses for SNS-595 were performed on plasma samples collected during cycle 1. Pretreatment peripheral blood and bone marrow aspirate samples were collected for exploratory analyses of the level and functional activity of the DNA damage repair proteins DNA-PK and MSH2.

Results: To date, 21 patients have been enrolled and are evaluable in the live database, including 13 patients assigned to arm A and 8 assigned to arm B, 12 males and 9 females with a median age of 64 years. Diagnoses included AML (19 patients) and ALL (2 patients). All patients had disease refractory to or relapsed from prior therapy (median 3 prior regimens (range 1–6)). Dose escalation has proceeded to 50 mg/m2/d (arm A) and 19 mg/m2/d (arm B). No dose-limiting toxicities have been observed to date. Non-dose limiting toxicities included nausea/vomiting, diarrhea, and mucositis . Grade 4 neutropenic fever was observed in only one patient. Plasma exposures at the first two dose levels in each arm increased linearly, resulting in AUCs of 5.5 – 17.8 ughr/mL for 9–27 mg/m2 doses. CL, Vss, and terminal half-lives were similar to those reported previously in solid tumor patients, and averaged ~2 L/hr/m2, 58 L/m2, and 23 hr, respectively. No patients have achieved complete response to date, although 6 patients (distributed across all dosing groups) experienced >50% reductions in peripheral blasts following cycle 1.

Conclusion: SNS-595 appears to be well-tolerated in patients with advanced leukemias, with preliminary and promising signs of clinical activity as measured by decreases in leukemic blasts. Bone marrow ablation has not yet been achieved; patient accrual and dose-escalation are ongoing.
Phase-1 Study of ZIO-101: A New Organic Arsenic Active in Acute Myelogenous Leukemia (AML) and Multiple Myeloma (MM).
Authors: Berenson James R. , Boccia Ralph Vincent V. , Hussein Mohamad A. , Belch Andrew , Boise Lawrence , Schwartz Brian , Gale Robert Peter , Kornblau Steven M.
Published: October 2019

Background: Although arsenic trioxide (As2O3) is active in vitro against diverse hematological cancers, clinical data show activity only in acute progranulocytic leukemia (APL); there is little if any single-agent activity in multiple myeloma and other hematological cancers. This discordance may be because relatively little As2O3 can be given consequent to its substantial toxicity (especially QTc-prolongation) or because the mode of action (MOA) in APL (differentiation) is inoperative in other hematological cancers (or both). ZIO-101 (S-dimethylarsino-glutathione), a new organic arsenic, is active against diverse cancers in vitro and in animal models including AML and MM. Cell-killing by ZIO-101 is mediated by mitochondrial-disruption and apoptosis-induction rather than the differentiation MOA of As2O3. ZIO-101 can be given at doses ≥ 50-fold higher than As2O3 and achieves 5–10-fold higher intracellular concentrations at equimolar extracellular As concentrations. Gene-expression profiling data suggest different cellular responses to ZIO-101 and As2O3. These features make ZIO-101 attractive for evaluation in AML and MM.

Methods: 2 phase-1 studies evaluating safety, activity and pharmacokinetic (PK) profile of ZIO-101 in 21 subjects with advanced AML (N=8) or MM (N=13). Median age is 58 y (range, 41–85 y). Median N of prior therapies is 5 (range, 2–12) including 4 subjects failing prior As2O3 therapy. Starting dose was 78 mg/me2/d IV for 5 consecutive d every 4 w.

Results: Subjects received a median of 2 cycles (range, 1–6). Therapy was well-tolerated; adverse events ≥ grade-2 occurring in > 25% of subjects included neutropenia, hypokalemia and hyperglycemia. There was no clinically-important renal, liver or heart toxicity nor QTc-prolongation. Maximum tolerated dose (MTD) was 420 mg/me2/d. Pharmacokinetic (PK) studies at this dose showed a tmax=1 h (SD±0.9), Cmax=1.06 μg/mL (SD±0.07 μg/mL), t1/2=17.8 h (SD±1.4 h) and AUC0-∞=25.9 μg·h/mL (SD±0.8 μg·h/mL). 4 subjects with AML had stable disease (SD) after 1 cycle and received 2–4 more cycles before progressing. Blood myeloblasts decreased substantially in 1 subject and completely resolved in 2. Bone marrow myeloblasts decreased in 1. 1 subject with prior myelodysplastic syndrome (MDS) stopped requiring frequent RBC transfusions. 1 subject with rapidly-progressing As2O3- and bortezumib-resistant MM has stable disease (SD) >6 mo.

Conclusions: These early data suggest activity of ZIO-101 in advanced AML and MM. The MTD is 420 mg/me2/d, ≥ 50-fold higher than As2O3. Plasma levels exceed the IC50 for AML and MM cell in vitro and animal models. Clinically-important QTc-prolongation was not seen. Some subjects failing As2O3 responded to ZIO-101 indicating efficacy of a higher dose, different MOA or both. Because of these favorable results phase-2 studies in hematological cancers are in progress.
Intensification of Induction by High-Dose AraC and Outcome in Older Patients with De-Novo Acute Myeloid Leukemia (AML) and Subsets.
Authors: Buchner Thomas , Berdel Wolfgang E. , Schoch Claudia , Haferlach Torsten , Tchinda Joelle , Schnittger Susanne , Kern Wolfgang , Reichle Albrecht , Staib Peter , Serve Hubert L. , Ludwig Wolf-Dieter , Sauerland Maria Cristina , Heinecke Achim , Josef Wormann Bernhard , Hiddemann Wolfgang
Published: October 2019

As recently reported modifications, dose or duration of treatment had no impact on outcome of AML in older patients (Burnett et al. Blood 106: 162a, 2005). We therefore evaluated 764 patients 60 years of age or older and their various prognostic subgroups in the 1992 and 1999 mulitcenter randomized trials by the German AMLCG where patients received uniform postremission consolidation by one course of TAD (standard dose thioguanine, araC, daunorubicin) and maintenance by monthly 5 day courses of reduced TAD. For maximum homogeneity this analysis was restricted to de-novo AML and to patients with known karyotype. Before treatment started, patients were assigned to induction treatment by either TAD followed by HAM (HAM, high-dose araC 1g/m2x6 / mitoxantrone 10mg/m2x3), or to induction by two courses of HAM. The second induction course (HAM) was given to only patients with 5% or more residual bone marrow blasts. Therapy administered to the two randomized groups differed by a factor 2 in their araC dose. Despite this difference in treatment intensity patients in the two arms show similar response rate, overall survival (OS), ongoing CR, and relapse-free survival (see table). The same similarity in outcome as for de-novo AML overall is true for established prognostic subgroups as listed below.

Conclusion:

Intensification of induction therapy by high-dose araC has no effect on outcome in older age de-novo AML. Since potential influences other than the randomized treatment were minimized, present results do not support a risk adapted intensification strategy.

TAD-HAM HAM-HAM P TAD-HAM HAM-HAM P OS 4y % OS 4y % CR 4y % CR 4y % Total 18 13 .28 22 22 .53 Favorable Karyotype 17 16 .48 41 33 .82 Intermediate Karyotype 24 18 .31 24 26 .38 Unfavorable Karyotype 4 - .81 - - .37 LDH ≤ 700U/L 21 13 .86 23 24 .47 LDH > 700U/L 12 10 .07 - 16 .27 WBC ≤ 20x103/ccm 21 11 .91 22 21 .81 WBC > 20x103/ccm 15 16 .08 22 26 .19 Day 16 Blasts < 10% 26 16 .87 23 22 .81 Day 16 Blasts ≥ 10% 14 8 .28 18 19 .26
Prospective Multicenter Study for Patients with Therapy - Related Acute Myeloid Leukemia (AML) Using Intensive Induction and Postremission Therapy.
Authors: Wormann Bernhard Josef , Schoch Claudia , Reichle Albrecht , Staib Peter , Ludwig Wolf-Dieter , Sauerland Maria Cristina , Heinecke Achim , Berdel Wolfgang E. , Hiddemann Wolfgang , Buchner Thomas
Published: October 2019

Therapy - related acute myeloid leukemia (t-AML) is one of the most severe long - term complications of successful cancer treatment using chemo- and/or radiotherapy. Its frequency is increasing, also in patients with autoimmune disease after cytostatic therapy. Cytogenetic and molecular biological analysis have identified several subgroups, however large prospective trials on optimal treatment are lacking. In 1999 the German AMLCG started a prospective multicenter randomized trial including patients with t-AML. Patients received induction treatment, randomized to either TAD (standard dose thioguanine, araC, daunorubicin) followed by HAM (HAM, high-dose araC 1 or 3 g/m2x6/mitoxantrone 10mg/m2x3), or to induction by two courses of HAM. Above the age of 60 years, the second induction course was given only to patients with 5 % or more residual bone marrow blasts. Postremission therapy was randomized to either TAD followed by three year maintenance, or to autologous stem cell transplantation. Patients under the age of 60 years with a suitable donor received an allogeneic stem cell transplantation. 137 patients were included. The most frequent primary diagnoses were breast cancer (n = 43), Non Hodgkin’s lymphoma (n = 18), Hodgkin’s lymphoma (n = 9), autoimmune disease (n = 9), multiple myeloma (n = 5), germ cell tumor (n = 5) and ovarian cancer (n = 5). The median age was 57 years (23 – 77). 64 of 119 currently evaluable patients achieved CR (53,8 %), 34 (29 %) had persistent leukaemia, 20 (17,1 %) were classified as early death without evidence of disease. The CR rate was significantly lower than in 1532 patients with de novo AML (65,6 %), but higher than in patients with AML after MDS (46,8 %). Cytogenetic analysis was routinely performed in all patients with t-AML. 21 (17,7 %) had a favourable karyotype, 47 (39,5 %) an unfavourable karyotype, 51 (42,9 %) were classified as intermediate. Patients with favourable karyotype had a median survival of 25 months and an estimated survival rate at 5 yrs of 47,4 %. Median survival was 3 months for patients with unfavourable karyotype with an estimated survival rate of 12,5 %, while the intermediate group had a median survival of 19 months and an estimated survival rate of 24,2 %. This is one of the largest prospective studies on the therapy of patients with t-AML. The CR rate of all patients was inferior to patients with de novo AML. However, this difference was mainly due to the high number of patients with unfavourable karyotype. Within cytogenetically defined subgroups, the prognosis of t-AML patients does not differ significantly from patients with de novo AML.
All-Trans-Retinoic Acid (ATRA) Induces Autophagy in Acute Promyelocytic Leukemia Cells.
Authors: Ozpolat Bulent , Akar Ugur , Kondo Yasuko , Lopez-Berestein Gabriel
Published: October 2019

All-trans-retinoic acid (ATRA), a naturally occurring derivative of vitamin A (retinol), is a potent inducer of cellular differentiation, growth arrest, and apoptosis in various tumor cell lines. ATRA induces terminal differentiation of immature leukemic promyelocytes into normal mature granulocytes in vitro and in vivo, leading to a complete remission in majority of acute promyelocytic leukemia (APL) patients. Thus, this system provides an excellent in vitro model for studying the molecular events taking place during the terminal differentiation of myeloid cells. Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in such physiological processes as the development, homeostasis and elimination of unwanted cells. In contrast to apoptosis, autophagic cell death is caspase-independent and does not involve classic DNA laddering. Recent studies suggest that cancer cells in general tend to undergo less autophagy than their normal counterparts, supporting the contention that defective autophagic cell death plays a role in the process of carcinogenesis. The present study provides the first evidence that ATRA induces autophagy in acute promyelocytic leukemia cells. We demonstrated that treatment of APL (NB4) cells with ATRA resulted in a marked increase in formation of the acidic vesicular organelles demonstrated by acridine orange staining detected by flow cytometry. ATRA treatment caused about 12-fold increase in number of acridine orange positive cells compared to untreated group. Furthermore, ATRA induced marked upregulation of microtubule-associated protein light chain 3 (LC3-II), an important hallmark of autophagy, by western blot analysis. To determine the mechanim by which ATRA induces autophagy we examined expression of Beclin-1, an autophagy-inducing protein, and Bcl-2, an inhibitor of Beclin-1 as well as levels of mTOR, a suppressor of autophagy, upon ATRA treatment. ATRA inhibited Bcl-2, upregulated Beclin-1 expression and reduced induction of activation/phosphorylation of mTOR in NB4 cells. However, rapamycin, a specific inhibitor of mTOR failed to induce autophagy, suggesting that ATRA-mediated autophagy was independent of the mTOR pathway. We are currently testing whether inhibition of Beclin-1 can prevent ATRA-induced autophagy in APL cells. In conclusion, our results revealed that ATRA plays a role in regulation of autophagy by reducing Bcl-2 and inducing protein Beclin-1, which in turn induces autophagy, suggesting a novel action and mechanism of ATRA in regulation of autophagy in APL cells.
Isolated Brain Parenchyma Relapse of Non-Hodgkin’s Lymphoma (NHL): A Descriptive Analysis from the International Primary CNS Lymphoma Collaborative Group (IPCG).
Authors: Doolittle Nancy D. , Abrey Lauren , Shenkier Tamara , Siegal Tali , Bromberg Jacoline , Neuwelt Edward , Soussain Carole , Johnston Patrick , Illerhaus Gerald , Schiff David , Batchelor Tracy , Montoto Silvia , Zucca Emanuele , Kraemer Dale
Published: October 2019

Background: Isolated brain parenchyma relapse as initial site of relapse is a rare complication of NHL and carries a poor prognosis. Few large series focus on treatment characteristics and outcomes of isolated brain parenchyma relapse of NHL.

Methods: The IPCG conducted a retrospective review of patient and treatment characteristics and outcomes of this complication. Following initial diagnosis and treatment of NHL (1980–2004), cases with brain parenchyma relapse as initial relapse site, with no evidence of lymphoma elsewhere in the body at the time of brain relapse, were eligible. Cases with brain, spine or leptomeningeal involvement at NHL diagnosis were not eligible.

Results: 113 cases were assembled from 13 investigators in 8 countries. Preliminary data summaries are: 94 (83%) cases had diffuse large B-cell NHL, 5 (4%) follicular lymphoma, 3 (3%) Burkitt’s lymphoma, other NHL subtypes (11) . Median age at NHL diagnosis was 61yrs (16–85 yrs). 55% were male. Median ECOG at NHL diagnosis was 1. Median time from NHL diagnosis to isolated brain relapse was 1.8 yrs (3 months–15.9 yrs). 76 (67%) relapsed in brain less than 3 yrs after NHL diagnosis. Symptoms at brain relapse included mental status changes in 37%, gait/balance disturbance (27%), motor/sensory symptoms (23%). Median ECOG at brain relapse was 2. Parenchyma relapse was documented by brain imaging plus biopsy in 54 (48%), or imaging without biopsy in 58 (52%); not reported (1). 53 (48%) cases had one brain lesion; 56 (50%) had two or more lesions; not reported (4). Site of relapse was cerebral hemispheres in 53 (48%) cases, deep brain structures (brain stem/cerebellum) in 30 (27%), cerebral hemispheres and deep brain structures in 23 (21%); not reported (7). At brain relapse, CSF was positive in 11 (10%) cases, negative in 56 (50%); not reported (46[40%]). Treatment for brain relapse was chemotherapy alone in 52 (46%) cases, WBRT alone in 34 (30%), chemotherapy followed by RT in 26 (23%), and brain surgery alone (1). 78 (69%) cases are deceased. Median survival from brain parenchyma relapse to death was 1.6 yrs (95% CI: 11 months–2.6 yrs). Effect of treatment type at brain relapse on survival, will be reported. Brain lymphoma was the cause of death in 49 (63%) cases; CNS toxicity was the cause in 6 (8%) cases.

Conclusion: Though a rare relapse site, prospective studies are needed to improve understanding and outcomes of isolated brain parenchyma relapse of NHL.