bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cytofluorimetric/FISH Diagnostic Approach Define a B-Cell Variant-CLL with Peculiar Clinico-Biological Features.
Authors: Cro Lilla , Baldini Luca , Nobili Lucia , Neri Antonino , Zucal Nadia , Ferrario Andrea , Lionetti Marta , Bertoni Francesco , Morabito Fortunato , Guffanti Andrea , Goldaniga Maria , Cutrona Giovanna , Cortelezzi Agostino , Luminari8 Stefano , Ferrarini Manlio , Deliliers Giorgio Lambertenghi
Published: October 2019

In the present study, we describe the clinico-biological features of 63 cases of variant B-CLL (v-B-CLL) defined according to a previously described diagnostic algorithm for CD5+ mature B-cell leukemias, based on the combined use of Cytofluorimetric (CFM) analysis and t(11;14)(q13;q32) detection by means of fluorescence in situ hybridisation (FISH). These leukemias were characterized by SIgbright/CD23+ or CD23+/− , CD79b/CD20 bright, and negativity for t(11;14)(q13;q32). A historical series of 112 classical B-CLL was used as comparison. The mean age was 61 years (33–85) and M/F ratio 1.9. The v-B-CLL cases were significantly different from the B-CLL cases in terms of the following clinico-hematological variables: age <70 yrs, lymphocytosis <20 ×109/L (P <.0001), lymphocyte doubling time ≤ 12 months (P=.04), high serum ß2-microglobulin levels (P=.003) and presence of splenomegaly (P=.007). No differences were found in terms of sex, Hb levels, platelets count, presence of serum monoclonal component, serum LDH levels and HCV-Ab positivity. In v-B-CLL and B-CLL pts the median follow-up was respectively 56 and 84 months. The median overall survival was 80 months in v-B-CLL pts while it was not reached in B-CLL pts; 37 pts (58.7%) in the first group started chemotherapy vs 84 pts (56.4%) in the latter. The analysis of leukemic cell biological characteristics related to the prognosis was also made in part of the cases. A classic or mixed-CLL cytomorphology was seen only in 22/61 pts (36.1%). The percentage of CD38 positive cases was higher in v-B-CLL (60.3) than in B-CLL (44.6; P=.028); the IgVH mutation status, evaluated in 30/63 cases, was more frequently hypermutated in v-B-CLL group (23/30) than B-CLL (P=.03); the percentage of CFM Zap-70 positivity, evaluated in 42/63 cases, was similar in two groups (47.6 vs 57.4%, respectively). Interestingly, significant differences were found about the frequence of the recurrent chromosome alterations, evaluated in 48/63 cases, by means of FISH analysis: trisomy 12 was more frequent in v-B-CLL (37.5 vs 18.4%, p=0.02), while del13q14 was more frequent in B-CLL (14.5 vs 37.8%, P=0.007). In both groups, del 11q22.3 and del 17p13.1 were relatively rare (4 vs 8%, and 2 vs 7.7%, respectively). In conclusion, our study identifies, on the basis of a defined CFM-FISH diagnostic approach, a variant form of B-CLL that shows significant differences in terms of genetic and clinical features.
Epigenetic Profiling Identifies EGLN3 as a Frequent Target for Transcriptional Silencing in Plasma Cell Neoplasias.
Authors: Shah Reshma , Hatzimichael Eleftheria , Syed Nelofer , Bourantas Konstantinos L. , Crook Tim
Published: October 2019

Background: Multiple myeloma (MM) results from an excess of monoclonal plasma cells in the bone marrow. MM cells are interleukin (IL)-6 and stromal cell dependent. Although the molecular signature of MM cells has been identified, the signature of the stromal component of the BM microenviroment is not yet fully determined and holds important clues to the understanding of disease pathobiology and progression. It has been suggested that bone marrow hypoxia is lessened during myeloma progression and that myeloma-associated angiogenesis is functional. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor and a master regulator of hypoxia responsive genes in cancer, which promote angiogenesis, vasodilation and glycolysis. As part of a programme to seek novel genes subject to epigenetic regulation in B cell neoplasia, we have identified the egl9 homologue 3 (EGLN3) as a frequent target for methylation-dependent transcriptional silencing in multiple myeloma (MM) and Waldenström’s macroglobulinemia (WM). EGLN3 is a prolyl hydroxylase (POH) that functions as a cellular oxygen sensor that targets HIF-1 alpha for proteasomal degradation via the von Hippel-Lindau (VHL) complex.

Patients and Methods: Thirty six MM patients (18 male, 18 female, age range 50–87 years), 4 patients with WM (2 male and 2 female, age range 47–75 years) and two patients with monoclonal gammopathy of undetermined significance (MGUS; 2 male age 56 and 67 years old) were included in the study. Twenty out of 36 MM patients had advanced stage disease (Durie Salmon stage≥II, IPI≥II). All samples were taken at diagnosis except for 5 that were taken when progression occurred from plateau phase. The Durie-Salmon staging system and the International Prognostic Index (IPI) were used for clinical and prognostic discrimination of patients. Bone marrow aspiration was performed with aseptic technique. Genomic DNA was isolated with a commercially available kit (QIAmp DNA mini kit, Qiagen).

Results: We identified a CpG island in the 5′ sequences of the EGLN3 gene. Using bisulphite sequencing and methylation specific PCR, we observed aberrant CpG methylation in 17/36 cases of MM and 1/4 cases of WM. Expression of EGLN3, assessed by quantitative PCR (qPCR) correlated inversely with methylation. In contrast, we detected no methylation in the related EGLN1 or EGLN2 in either MM or WM. Cases with methylated EGLN3 were more likely to have bone lytic lesions (r=0.39, p≤0.05), but there was no correlation with extramedullary disease, hemoglobin<10 mg/dl or advanced disease stage.

Conlusions: EGLN3 is frequently methylated in patients with multiple myeloma. We suggest that methylation of EGLN3 leading to decreased HIF-1a degradation could be a possible mechanism of increased angiogenesis and altered bone marrow microenviroment that is more supportive for survival and growth of MM cells. The role of POH in MM merits additional study.
GA101, a Novel Humanized Type II CD20 Antibody with Glycoengineered Fc and Enhanced Cell Death Induction, Mediates Superior Efficacy in a Variety of NHL Xenograft Models in Comparison to Rituximab.
Authors: Friess Thomas , Gerdes Christian , Nopora Adam , Patre Monika , Preiss Susanne , van Puijenbroek Erwin , Schuell Christine , Bauer Sabine , Umana Pablo , Klein Christian
Published: October 2019

GA101 is a novel humanized and glycoengineered CD20 antibody that was derived by humanization of the parental B-Ly1 mouse antibody and subsequent glycoengineering leading to the following characteristics: high affinity binding to the CD20 type II epitope; low complement dependent cytotoxicity (CDC) activity; high direct cell death induction; and high antibody-dependent cellular cytotoxicity (ADCC) potency. In vivo studies examined the consequences of GA101 on the growth of aggressive NHL xenograft models in SCID beige mice and the subsequent effects on survival. These studies demonstrated an outstanding dose-dependent anti-tumor activity of GA101. In particular, GA101 mediated superior anti-tumor efficacy in staged s.c. Z138 mantle cell lymphoma (MCL) and SU-DHL4 diffuse large B cell lymphoma (DLBCL) xenograft models in direct comparison to rituximab. Weekly dosing of GA101 at 10 mg/kg (Z138 model) or 30 mg/kg (SU-DHL4) was able to induce complete tumor remission and long-term survival (cures) in all animals. Consistent anti-tumor efficacy against established tumors was also observed in a number of additional NHL models including Raji Burkitt’s lymphoma or OCI-LY18 DLBCL models. In addition GA101 was evaluated in an orthotopic disseminated Z138 model. After i.v. injection of Z138 cells SCID beige mice developed large intraperitoneal lymphoid tumors in the ovary. Several experiments showed that GA101 mediated increased overall and median survival in this model in direct comparison to rituximab. All xenograft studies were performed in SCID beige mice that are incompetent for NK-mediated ADCC. Indeed, control experiments with non-glycoengineered GA101 in s.c. xenograft models suggest that the superiority of GA101 does not depend on enhanced interactions of the engineered Fc region with murine effector cells such as macrophages/monocytes or granulocytes. Thus, the anti-tumor effects of GA101 in s.c. xenograft models can be primarily attributed to non-effector cell mediated activities. Currently, experiments are ongoing to evaluate the efficacy of GA101 in combination with classical chemotherapy regimens and novel targeted agents. First results from these studies will be presented during the 2007 ASH Annual Meeting. In summary it can be assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.
Structural Determinants for Heparin Binding in Human Coagulation Factor XI.
Authors: Shikov Sergei , Wu Wenman , Walsh Peter N.
Published: October 2019

Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD ∼110 nM) affinity via residues (K252, K253 and K255) located in the Apple 3 (A3) domain. In contrast, the enzyme, FXIa, was shown to bind to heparin (Biochemistry40: 7569–7580, 2001) with significantly higher affinity (KD ∼9 nM by SPR and ∼1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). This interaction potentiates by ∼10-fold the inhibition of FXIa by protease nexin-2. Also, polyanions heparin and dextran sulfate inhibit the catalytic activity of the enzyme factor XIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI to heparin binding. Mutational analysis of full-length FXI expressed in HEK293 cells was based on the following criteria:

Conservation of the positively charged residues in FXI among various species; Surface exposure of the residues based on the X-ray crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J,Nat. Struct. & Mol. Biol. 2006; 13:557–558); and comparison with human plasma prekallikrein (PK), which does not bind heparin.

Two positively charged residues Arg507 (147, chymotrypsin numbering) and Arg532 (173) are conserved in FXI genes of all species for which sequences are available. In human PK, Arg507 is replaced by lysine, while Arg532 is replaced by a neutral glutamine. We have expressed and purified wtFXI, R507A, R532A as well as monomeric C321S/K331A and C321A/I290A. While wtFXI, R507A and R532A demonstrated normal activity in APTT assays; monomeric FXI mutants retained 60-70% activity. The R532A and R507A mutants demonstrated ∼75% decrease in total number of heparin binding sites based on the solid phase ELISA assay using 5F7 monoclonal antibody. Also, the apparent dissociation constants for R507 (11 nM) and R532A (22 nM) were 7 and 11-fold increased respectively compared with 1.6 nM for the wtFXI. We also characterized monomeric FXI C321S/K331A and C321A/I290A proteins for their ability to bind to heparin compared with wtFXI using surface plasmon resonance (SPR). Surprisingly, the monomeric FXI mutants, C321S/K331A and C321A/I290A, which had no mutations in any heparin-binding regions, displayed major defects in binding to heparin by SPR. Although kinetic analysis is challenging due to complex binding kinetics, while Rmax is about 10-fold lower, the off-rate for the binding of the monomeric FXI mutants is drastically increased when compared to that of wtFXI. These results suggest the possibility that the unique dimeric structure of FXI is required for cooperative binding to heparin. Thus, the dimeric structure of FXI and basic residues R507 and R532 in the catalytic domain of factor XI are both necessary for high-affinity heparin binding.
ThaDD-V Treatment for Patients with Relapsed/Refractory Multiple Myeloma: A Feasibility/Activity Study.
Authors: Offidani Massimo , Polloni Claudia , Corvatta Laura , Piersantelli Maria-Novella , Mele Anna , Catarini Massimo , Brunori Marino , Ferranti Mario , Amoroso Barbara , Leoni Pietro
Published: October 2019

ThaDD regimen has provided significant results in recurrent/relapsed multiple myeloma (MM) patients (Offidani et al, 2006). In order to further improve those results without significantly increasing toxicity, we decided to include Velcade, synergic as per activity but not toxicity with the other drugs of ThaDD regimen. ThaDD-V was scheduled as follows: Thalidomide 100 mg/day, pegylated liposomal Doxorubicin 30 mg/sm iv days 4; Dexamethasone 20 mg days 1–2, 4–5, 8–9, 11–12 and Velcade 1.3 mg/sm iv days 1, 4, 8, 11 every 28 days (induction therapy). Patients received bortezomib for alternate cycles as following: 1 mg/sm day 1, 8, 15 and dexamethasone 20 mg days 1–2, 8–9, 15–16 and thalidomide 100 mg/day and dexamethasone 40 mg/day for 4 days monthly for a total of six cycles as consolidation therapy. Then patients received thalidomide 100 mg/day until relapse (maintenance therapy). Actually 20 patients (7 M, 13 F; median age 62.5 yrs, range 31–75) are assessable for response and toxicity. Five pts (25%) showed WHO performance status (PS) > 1, 9 pts (45%) presented refractory disease, 8 pts (40%) were priorily administered ≥ 2 lines of a treatment and 14 patients (70%) were submitted to one previous autologous stem cell procedure. Seven patients (35%) had extramedullary disease and 7 had unfavourable cytogenetics. Twelve patients scored an ISS ≥ 2 and 11 (55%) were in first remission for ≤ 12 months median duration. No patients were previously treated with Velcade, whereas six patients had received short-term Thalidomide treatment. Response was assessed according to IMVG uniform response criteria. Seventeen of 20 patients responded after at least one chemotherapy cycle reporting 5 (25%) sCR, 3 CR (15%), 8 VGPR (40%) and 1 stable disease. Three patients (15%) had extramedullary progressive disease. In a median follow-up of 12 months, 2 (10%) patients progressed and 1 (5%) died from cardiac infarction. Time to progression and overall survival were 73% and 95% at 12 months. We observed 4 grade 3 thrombocytopenia, 2 grade 3 DVT, 1 grade 3 diarrhoea, 1 grade 3 asthenia, 1 grade 4 infection and 1 grade 3 dermatological toxicity. Six patients developed grade 2 peripheral neuropathy and other three grade 3. In conclusion, ThaDD-V is extremely active in advanced MM patients as demonstrated by the elevated precentage of high quality remission. Nevertheless, patients are at substantial risk of developing neurotoxicity so the protocol was amended as per Velcade dose intensity and Thalidomide dose.
Early Normalization of Serum Free Light Chain Is Associated with Prolonged Time to Progression Following Bortezomib ± Pegylated Liposomal Doxorubicin Treatment of Relapsed/Refractory Multiple Myeloma.
Authors: Orlowski Robert , Sutherland Heather , Bladé Joan , Miguel Jesús San , Hájek Roman , Nagler Arnon , Sonneveld Pieter , Spencer Andrew , Robak Tadeusz , Mundle Suneel , Luo Donghan , Zhuang Sen Hong , Harousseau Jean-Luc
Published: October 2019

Multiple myeloma (MM), like other monoclonal gammopathies, frequently causes impairment of the κ/λ serum free light chain (sFLC) ratio. Based on a short serum half-life of FLC as compared to the complete immunoglobulin (hours versus days), early normalization of κ/λ could predict for response to treatment. As part of a randomized, controlled study, we examined the association between normalization of the κ/λ ratio after one or two 21-day cycles of treatment with bortezomib (B) ± pegylated liposomal doxorubicin (PLD) among patients with relapsed/refractory multiple myeloma. Patients with ≥1 prior therapy were randomized to receive PLD 30 mg/m2 on day 4 and B 1.3 mg/m2 on days 1, 4, 8, and 11, or B alone for up to eight 21-day cycles, or at least 2 cycles beyond complete response (CR) or optimal response, unless disease progression or unacceptable toxicities occurred (Orlowski, JCO 2007). κ/λ measurements were carried out prior to the start of therapy and at the end of each cycle through the entire study period using an immunoassay (Freelite, The Binding Sites, Birmingham, UK). Serial sFLC κ/λ measurements were available on sera from 487 patients with baseline values out of a total of 646 study patients. At baseline, 458/487 patients (94%) had an abnormal κ/λ ratio (<0.26 or >1.65). The percentage of patients with normal κ/λ ratio increased from 6% at baseline to 12% after cycle 1, 17% after cycle 2 and 23% by the end of the study. Among patients with a normal κ/λ ratio after cycle 1 (n=54), the median time to progression (TTP) was 345 days compared to 225 days in patients with abnormal ratios (n=395, p=0.0005, HR=0.47 favoring normalization). Following cycle 2, TTP was 325 days vs. 224 days (p≤0.001) in patients with normal (n=72) vs. abnormal (n=348) ratios, respectively. Additionally, patients with normalized sFLC ratios showed significantly higher overall response rates as compared to those with persistently abnormal ratios (≥ partial response [PR] 73% vs. 47%, p=0.001 following cycle 1, and 77% vs. 48%, p<0.0001 following cycle 2, respectively). Similarly, CR + very good PR rates were significantly higher among patients with normalized sFLC ratios after cycles 1 and 2 (p<0.0001 and p<0.001 respectively). As reported previously, both treatments, B + PLD and B alone, were well tolerated (Orlowski, JCO 2007). Normalization of the sFLC ratio as early as after cycle 1 of B + PLD, or B alone, was associated with a prolonged TTP and higher response rates. Additional analyses are undertaken to evaluate whether Freelite data:

predict treatment outcome earlier than electrophoresis, and correlate with 24-hour urine M-protein.
USF1 Recruits hSET1 Complex and Is Important for Maintaining Active Chromatin Domains in the β-Globin Locus.
Authors: Li Xingguo , Hu Xin , Felsenfeld Gary , Huang Suming
Published: October 2019

The chicken β-globin insulator element acts as a barrier to the encroachment of chromosomal silencing. This barrier insulator prevents the spread of a 16 Kb domain of condensed chromatin which lies immediately upstream of the β-globin genes in the chicken genome. We have previously shown that the transcription factors USF1/2 mediate the barrier activity by binding to the 5′HS4 insulator element of the chicken β-globin locus and maintaining a local active chromatin structure. In the mouse β-globin locus, USFs also play a critical role in regulating the developmental stage-specific transcription of the β-major globin gene. To further understand the diverse roles of USF proteins, we undertook the biochemical isolation of USF1-containing multiprotein complexes. We found that USF1 associates with the hSET1 complex, which is an H3K4-specific methyltransferase. This complex exhibits histone H3 methyltransferase enzymatic activity. Consistent with the role of USF1 in recruiting histone modifying enzyme hSET1 to the 5′HS4 insulator, knockdown of USF1 expression in erythroid leukemia cell line 6C2 leads to the loss of histone H3K4 dimethylation at 5′HS4 of the chicken β-globin locus. In addition, USF1, hSET1, as well as H3K4 methylation are colocalized on HS2 of the locus control region (LCR) and the promoter of the β-major globin gene. Furthermore, the double ChIP analysis demonstrates that the histone methyltransferase hSET1 is recruited by transcription factor USF1 onto the mouse β-globin locus. The results suggest that hSET1 may play an important role in establishing H3K4 methylations on the active β-globin locus and reveal the mechanisms of USF and its associated proteins in maintaining the open chromatin domain and in regulation of globin gene transcription.
Preferentially Expressed Antigen of Melanoma (PRAME)-Specific Cytotoxic T-Lymphocytes (CTLs) and Transgenic T Cells To Target Chronic Myelogenous Leukemia (CML).
Authors: Quintarelli C. , Dotti G. , De Angelis B. , Hoyos V. , Mims M. , Pane F. , Heslop Helen E. , Rooney C.M. , Brenner M.K. , Savoldo B.
Published: October 2019

Following allogeneic hematopoietic stem cell (HSC) transplantation, a graft versus leukemia (GVL) effect mediated by donor T cells contributes to the maintenance of long-term remission. GVL is likely mediated not only by alloreactive T cells, but also by donor lymphocytes recognizing tumor associated antigens overexpressed by leukemic cells. Selective expansion of such tumor-specific CTLs could augment GVL without increasing the risk of graft versus host disease (GVHD). PRAME is a cancer testis antigen (CTA) that is over-expressed by many hematological malignancies, including CML. We therefore generated PRAME-specific CTLs and evaluated their activity against primary CML tumor cells. CD8+ cells from 9 HLA-A*02 healthy donors and 5 CML patients were primed with autologous CD40L-activated B blasts loaded with HLA-A*02 restricted PRAME-peptides in the presence of low concentrations of IL-12, IL-7 and IL-15. Cells were then re-stimulated once a week using an artificial antigen presenting cell line (aAPC), consisting of K562 cell line that was genetically modified to stably express HLA-A*02, CD80 and CD40L and loaded with the same PRAME-peptides. In 8 out of 9 HLA A*02 healthy donors, we expanded (22±7 fold) T cells specific for the ALY-PRAME peptide. Similarly, PRAME-specific CTLs could be expanded from 3 out of 5 CML pts. When assessed by Elispot assay, these CTLs released IFNγ in response to ALY-peptide (511±260 SFC/105 cells) compared to 30±10 IFNγ SFC/105 against an irrelevant peptide. They also lysed autologous-PHA blasts loaded with ALY (84%±12% specific 51Cr release at 20:1 E:T ratio), but not with an irrelevant peptide (<10%). Both IFNγ release and cytotoxic activity were blocked by MHC class I antibodies, indicating that these effects were MHC-restricted. PRAME-specific CTLs also recognized primary CML cells since they produced IFNγ (320±31 SFC/105) in response to CD33+ CML blasts overexpressing PRAME (as assessed by Real Time PCR) isolated from HLA-A*02+ CML patients. Since routine expansion of PRAME-specific CTLs for clinical trials of adoptive T-cell transfer may remain problematic, we next cloned the αβT cell receptor (TCR) from a CTL clone with high affinity for the ALY-peptide (621 IFNγ SCF/105 cells at 0.2 nM peptide). The α- and β-chains were cloned in the SFG retroviral vector using an IRES sequence, and the transgenic αβTCR was expressed in primary T lymphocytes. T cells expressing the transgenic PRAME-specific αβTCR produced IFNγ on exposure to PRAME+ CML blasts (448± 69 IFNγ SFC/105 cells) and killed PHA blasts loaded with the ALY-peptide (42%±7% at 20:1 E:T ratio). In conclusion, our data show that PRAME is a promising target antigen on CML blasts and that adoptive transfer of CTLs or of T cells expressing a transgenic αβTCR targeting this antigen may be of value for patients with the disease.
Pharmacokinetic and Toxicological Results of a 28-Day Multiple Dose Study of Starch-Conjugated Deferoxamine (S-DFO, 40SD02) in Iron-Loaded Dogs.
Authors: Grady Robert W. , Bakke James , Steinmetz Karen , Bishop Terry R. , Hedlund Bo E.
Published: October 2019

Starch-conjugated deferoxamine (S-DFO) is a long-acting, polymeric iron chelator designed to achieve iron balance in patients requiring frequent transfusions. Results from an ascending single dose study in 16 patients with beta-thalassemia showed that S-DFO is capable of achieving up to 7 days of iron balance (Br. J. Haematol. 138, 374–381, 2007). The multiple dose toxicology study described here was conducted to support extended clinical studies in patients with thalassemia and other iron overload disorders. The study was designed to evaluate physiological effects of S-DFO in iron-loaded animals;to seek evidence of cardiovascular side-effects;to find evidence of drug accumulation in plasma and other tissues; andto determine the No Observable Adverse Effect Level (NOAEL) following biweekly intravenous infusion for 4 weeks.

Iron loading was achieved by multiple biweekly infusions of iron-dextran so as to give a cumulative dose of 500 mg of iron/kg body weight. Equilibration of iron was then allowed to proceed for 10 weeks before initiation of treatment. The study included four groups of 10 iron-loaded animals (5M/5F):

saline controls; 210 and 420 mg/kg of deferoxamine (DFO) equivalents as S-DFO; 105 mg/kg of non-conjugated DFO;

and two groups of 10 normoferremic animals (5M/5F):

saline controls; and 210 mg/kg of DFO equivalents as S-DFO.

All animals received 8 doses of either S-DFO, DFO or saline over 28 days. Six animals in each group (3M/3F) were euthanized on Day 29 for histological examination, the remaining 4 animals in each group on Day 43. Safety parameters for cardiovascular pharmacology, hematology and clinical chemistry showed no changes attributable to S-DFO treatment. The polymeric chelator is cleaved/metabolized by serum amylase(s). While trough concentrations of S-DFO fragments prior to the final drug infusion on Day 25 showed some accumulation, there was no evidence of resulting pathological changes. Thus, the NOAEL is at least 420 mg/kg of DFO equivalents. The hepatic iron content of the iron-loaded animals was approximately 20 times greater than that of the normoferremic controls. Treatment with 420 mg/kg of DFO equivalents led to a 13% reduction in the liver iron content. Neither iron loading nor treatment with S-DFO had a significant effect upon the cardiac iron status. The lack of toxicity in normoferremic animals suggests that S-DFO may have applications in clinical situations where increased iron stores are not characteristic of the disease. Furthermore, the ability to safely infuse up to 420 mg/kg of DFO equivalents without concern for hypotension indicates that S-DFO may also be well suited for treatment of acute iron poisoning. Finally, these results support initiation of multidose studies of S-DFO in patients with thalassemia and other iron overload disorders.

Supported by NIDDK Contract Number N01-DK-32624.
The Early Treatment Response of the Clinically Challenging Group of Childhood T-ALL without NOTCH1 Mutations Is Signified by a Specific mRNA Gene Profile.
Authors: Kulozik Andreas , Breit Stephen , Pfister Stefan , Liu Shangyou , Stanulla Martin , Schrappe Martin , Ludwig Wolf-Dieter , Köhl Ulrike , Borkhardt Arndt , Kirschner-Schwabe Renate , Muckenthaler Martina U.
Published: October 2019

Activating mutations within the NOTCH1 gene occur in more than 50% of childhood precursor T-cell lymphoblastic leukemia (T-ALL, Weng et al. Science 2004). Our previous work has shown that in the context of the ALL-BFM 2000 treatment strategy, activating NOTCH1 mutations significantly correlate with a good early treatment response and specified a subgroup of patients with an exceptionally favorable long term outcome. By contrast, most of the relapses occurred in those patients without NOTCH1 mutations, which thus represents a clinically important and challenging subgroup (Breit et al., Blood 2006). We now aimed to further differentiate this subgroup without NOTCH1 mutations by analyzing the mRNA expression profile of primary pediatric T-ALL bone marrow samples. We first validated the biological significance of these analyses by comparing samples with and without activating NOTCH1 mutations and confirmed that the presence of activating NOTCH1 mutations correlates with differential expression of multiple downstream signaling pathways that are known to be activated by NOTCH1. Importantly, the comparison of patients without NOTCH1 mutations and good or poor prednisolone response revealed a specific gene signature that differentiates wild-type NOTCH1 T-ALL with a favourable from those with an unfavourable early treatment response. These discriminatory signatures significantly enrich for specific gene ontology categories (e.g. cell death, cellular growth and proliferation, and molecular transport), suggesting functional relevance of these differentially expressed genes. The specific comparison of different T-ALL subgroups thus reveals prognostic gene expression signatures particularly in the clinically difficult patients without NOTCH1 mutations.
PAX5 Mutations Occur Frequently in Adult B-Cell Acute Lymphoblastic Leukemia (B-ALL) and Is Significantly Associated with BCR-ABL1 Fusion Gene.
Authors: Familiades Julien , Bousquet Marina , Dastugue Nicole , Lafage-Pochitaloff Marina , Marie-Christine Béné , Beldjord Kheira , Cayuela Jean-Michel , Grardel Nathalie , Preudhomme Claude , Cavé Hélène , Lhéritier Véronique , Delannoy André , Chalandon Yves , Ifrah Norbert , Pigneux Arnaud , Dombret Hervé , Macintyre Elizabeth A. , Huguet Françoise , Brousset Pierre , Broccardo Cyril , Delabesse Eric
Published: October 2019

PAX5 is a transcription factor central to B-cell differentiation, frequently mutated in pediatric B-ALL (38.9% in 192 B-ALL, Nature,2007;446:758). PAX5 mutations consist in whole or partial gene deletions or amplifications, fusion gene or point mutations. Adult B-ALL differs in many regards from pediatric B-ALL as demonstrated by differences in prognosis and genetic abnormalities (ETV6-RUNX1 almost absent from adult; BCR-ABL1 rare in children). We screened the occurrence of PAX5 mutations in a series of 119 adult patients with B-lineage ALL prospectively treated in the GRAALL-2003 (pediatric-like protocol for BCR-ABL1 negative patients) or GRAAPH-2003 (imatinib-containing protocol for BCR-ABL1 positive patients), with a median follow-up of 667 days. PAX5 copy number was evaluated using quantitative PCR of each exon performed in hexaplicate (10 exons). PAX5 point mutations of the paired and transactivation domains (exons 2, 3, 7, 8 and 9) were evaluated by sequence analysis. PAX5 rearrangement was evaluated by caryotype and FISH analysis. Globally, PAX5 mutations were identified in 35 cases (30%). Isolated complete deletion of 1 allele was found in 13 cases (11%). Hypomorphic PAX5 alleles were detected in 22 cases (19%) consisting either of partial deletion (13 cases), partial amplification (1), point mutations (7 cases, frequently associated with complete deletion of the remaining allele in 5 cases) or fusion gene (1 PAX5-ELN). In addition, 2 cases of whole PAX5 amplification were found (2%). The remaining 82 cases had two normal PAX5 alleles (68%). BCR-ABL1 fusion gene was significantly associated with PAX5 mutations (Pearson Chi2, p=0.045) occurring in 40% of patients with PAX5 mutations and 18.3% without. PAX5 mutations were rare in the early stages of B-ALL as estimated by the immunological EGIL classification (only 2/35 PAX5 mutant cases [5.7%] vs. 16/82 [19.5%] in normal PAX5 B-ALL cases blocked at the B-I stage). Frequencies of CD10+ and CD20+ blasts were significantly higher in PAX5 mutant cases (Mann-Whitney, p=0.022 and p=0.038, respectively). As PAX5 is essential to the immunoglobulin heavy chain DHJH to VHDHJH transition, we analyzed IGH rearrangement. No difference was detected regarding the frequency of IGH rearrangement (78% in normal vs. 74% in mutants) or the rearranged VH. Survival was not significantly different between the two groups of patients (log rank, p=0.45). In conclusion, PAX5 mutations were a frequent event in adult B-ALL, associated with BCR-ABL1 fusion gene but were not associated with a significant clinical evolution.
A Tamoxifen-Dependent Conditional Model of MYC-Induced T Cell Acute Lymphoblastic Leukemia in the Zebrafish.
Authors: Gutierrez Alejandro , Feng Hui , Edward Prochownik , Kanki John , Look A. Thomas
Published: October 2019

The MYC oncogene plays a central role in the pathogenesis of human T cell acute lymphoblastic leukemia (T-ALL), and our laboratory has previously developed a zebrafish model of Myc-induced T-ALL. The primary strength of the zebrafish as a model system for human disease lies in is its suitability for unbiased forward genetic and small molecule screens. Our central hypothesis is that forward screens performed using our zebrafish model of MYC-induced T-ALL will lead to the identification of entirely novel genes and pathways that play critical roles in MYC-induced leukemogenesis. However, zebrafish from our original line develop rapidly progressive T-ALL prior to achieving reproductive maturity, making this line poorly suited for the performance of large-scale screens. Therefore, a conditional model was required. We have now generated a transgenic zebrafish line that expresses a human MYC-estrogen receptor fusion construct under the control of the zebrafish recombination activating gene 2 (Rag2) promoter, which is lymphocyte-specific. When mated against fish transgenic for a Rag2-GFP transgene, the development and progression of T-ALL can be readily tracked in live fish by fluorescent microscopy. Upon treatment with 4-hydroxytamoxifen (4HT), zebrafish from this line develop fully penetrant T-ALL, with a mean time to tumor onset of 8 weeks. Additionally, removal from 4HT invariably led to complete morphologic remission in leukemic zebrafish from this line, and all of these fish remained alive and were able to mate successfully for greater than 6 months after removal from 4HT. This conditional zebrafish model of MYC-induced T-ALL will now allow the successful performance of forward genetic and small molecule screens to identify known and novel genes and pathways that play critical roles in T-ALL leukemogenesis and MYC-induced transformation.

Figure Figure
Efficacy of Dasatinib in Patients with Philadelphia-Chromosome-Positive Acute Lymphoblastic Leukemia Who Are Resistant or Intolerant to Imatinib: 2-Year Follow-Up Data from START-L (CA180-015).
Authors: Porkka Kimmo , Simonsson Bengt , Dombret Herve , Martinelli Giovanni , Ottmann Oliver G. , Zhu Chao , Van Tornout Jan M.A. , Garzon Felix T. , Larson Richard A.
Published: October 2019

Patients with Philadelphia-chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) generally have a rapid disease course and a poor overall prognosis that is compounded by resistance to imatinib. Dasatinib is a novel multi-targeted kinase inhibitor of BCR-ABL and SRC family kinases, with in vitro potency some 325-fold greater than imatinib against BCR-ABL and proven efficacy in this patient population. START-L is an open-label, international, multicenter study in which patients with imatinib-resistant or -intolerant Ph+ ALL enrolled from January through July 2005 were treated with dasatinib 70 mg BID. Dose escalation to 100 mg BID and reduction to 50 or 40 mg BID were allowed for inadequate response and toxicity, respectively. At the time of this update, all 46 treated patients (median age 48 years; 59% male) had a minimum follow-up of 12 mo. 96% of patients were imatinib-resistant; 46% had received doses of imatinib >600 mg/d, and 52% had received imatinib therapy for >12 mo. Median time from the initial Ph+ ALL diagnosis was 18 mo (range 3–163) and 37% had undergone prior stem-cell transplantation (SCT). BCR-ABL mutations were present at baseline in 78% of patients. The overall major hematologic response rate (MaHR) was 41% with a complete HR rate of 33%. Major cytogenetic response (MCyR) was achieved in 57% of patients with a complete CyR attained in all but one of these patients (54%). Response rates were consistent irrespective of pre-existing BCR-ABL mutations. The median duration of MCyR was 6.9 mo. Median overall survival (OS) was 8.0 mo and 22% of patients remained alive and progression-free after 1 year of treatment. Outcome was favorable for patients with prior SCT: median OS of 9.0 mo (5.8 mo for patients without prior SCT). As expected in these pre-treated patients, grade 3–4 cytopenias were significant with 78% of patients experiencing severe thrombocytopenia and neutropenia. Non-hematologic side-effects included diarrhea in 33% of patients (grade 3–4 in 9%), pleural effusion in 24% (grade 3–4 in 7%), nausea in 22% (no grade 3-4), and pyrexia in 22% (grade 3–4 in 2%). The dose of dasatinib was reduced in 30% of patients and interrupted in 43%; this was primarily attributable to non-hematologic toxicities. This resulted in a median of the average daily dose of 143 mg. Dasatinib continues to show impressive efficacy in this poor-prognosis patient population with Ph+ ALL after the failure of imatinib. Updated analyses corresponding to a minimum follow-up of 2 years will be presented.
CpG Oligonucleotides Induce Anti-Leukemia Activity in a Syngeneic Murine Model of Acute Lymphoblastic Leukemia.
Authors: Seif Alix E. , Bruno Marlo D. , Hall Junior , Brown Valerie I. , Grupp Stephan A. , Reid Gregor S.D.
Published: October 2019

Acute lymphoblastic leukemia (ALL) accounts for 80% of all pediatric leukemias and is the most common form of childhood cancer. While most children with ALL are cured by current therapies, refractory and relapsed ALL comprise a significant proportion of all pediatric cancers. Furthermore, nearly half of all ALL diagnoses occur in adults, who carry a much poorer prognosis, with the majority dying of relapsed disease. Relapsed ALL generally requires intensive therapy with significant associated morbidity and mortality. Development of novel therapies is essential to improving outcomes. DNA oligodeoxynucleotides containing CpG motifs (CpG ODN) stimulate anti-tumor immune activity via Toll-like receptor 9 (TLR9) activation and are currently in clinical trials for a variety of solid tumors. We have previously reported that CpG ODN stimulation alters antigen presentation by human ALL cells, enhancing allogeneic Th1 responses. In addition, we have shown that CpG ODN administration in vivo reduces the leukemic burden of primary human ALL xenografts in Nod-SCID mice, and that this activity is mediated in part by NK cells. To further the development of CpG ODN as a novel therapeutic agent for ALL, we have investigated the induction of anti-ALL activity by CpG ODN in a syngeneic ALL setting. CpG ODN did not exhibit direct toxicity against cell lines derived from leukemic Eμ-ret transgenic mice in vitro, nor did it alter CD40 or CD86 expression or cytokine production. However, using a flow cytometry-based in vitro killing assay we observed CpG ODN-induced elimination of leukemia cells when cultured with splenocytes or bone marrow cells from Eμ-ret transgene-negative mice (P=0.0388). The difference between CpG ODN-treated and untreated controls became more pronounced with increasing effector:target ratios (P<0.0001). Preliminary data show that depletion of NK cells markedly decreases the magnitude of the observed effect, supporting the hypothesis that this cell type is involved in targeted control of ALL in this model. The ability of CpG ODN to exert anti-leukemia activity in a syngeneic setting suggests that it may have utility as an adjuvant therapy. To test this hypothesis we administered CpG ODN (or PBS) to syngeneic leukemia-bearing mice 2 days after completion of a chemotherapy regimen used to reduce leukemia burden. When mice were sacrificed 3 weeks after treatment, we found significantly reduced leukemia burden in bone marrow (P=0.0019), spleen (P<0.00001) and blood (P=0.00028) of CpG ODN-treated mice. Cell-depletion and cytokine-neutralization assays are currently ongoing to define the mechanism of action of CpG ODN in these settings. To our knowledge, this is the first demonstration of CpG ODN-induced anti-ALL activity in a post-chemotherapy syngeneic model, suggesting that this agent has the potential to treat minimal residual disease and to reduce the incidence of relapse.
CLL-1 Is a Potential Target for the Treatment of Acute Myeloid Leukemia.
Authors: Korver Wouter , Zhao Xiaoxian , Singh Shweta , Hsi Eric D. , Abo Arie
Published: October 2019

CLL-1 (C-type Lectin-Like Molecule-1) is an inhibitory receptor expressed on myeloid cells. We have generated a series of monoclonal antibodies (mAbs) against CLL-1 and used these mAbs to assess expression of the receptor on normal and AML cells and their therapeutic potential in in vitro and ex vivo cytotoxicity assays. By Immunohistochemistry, normal tissues lacked expression of CLL-1, with the exception of spleen. Using flow cytometry, expression was demonstrated on monocytes, granulocytes and dendritic cells, but not on lymphocytes and platelets. Tissue micro-arrays revealed CLL-1 expression in 97.3% (37/38) of AML cases. Flow cytometry of AML blasts demonstrated expression of CLL-1 on 81% (17/21) of patient samples. No detectable expression was detected in ALL blasts (n=5). Selected anti-CLL-1 mAbs mediated dose-dependent Complement Dependent Cytotoxicity (CDC) in various AML-derived cell lines, while no cytotoxicity was observed in CLL-1 negative K562 (CML) or CA46 (B lymphoma) cells. Human embryonic kidney 293 cells only became susceptible to anti-CLL-1 mAb mediated killing after transfection with CLL-1, demonstrating specificity. Furthermore, anti-CLL-1 mAbs showed CDC activity against all AML blasts tested in ex vivo assays (n=10), while no activity was observed against ALL blasts. Our results demonstrate restricted expression of CLL-1 on cells from myeloid origin and AML blasts and specific cytotoxic activity in in vitro and ex vivo assays. We are currently undertaking xenograft models to evaluate the therapeutic potential of these mAbs in vivo.
Interactions of Midostaurin (PKC412), Rapamycin and Ara-C in AML Cell Lines: Role of FLT3 Signalling.
Authors: Seedhouse Claire H. , Shang Shili , Russell Nigel , Das-Gupta Emma
Published: October 2019

Treatment options for patients with AML who are unfit for intensive chemotherapy are currently very limited. Commonly used treatment regimens such as low dose Ara-C result in complete remission rates of 17% and these remissions are not sustained (Burnett et al 2007). There is a rationale to examine the effect of combining small molecule inhibitors of signalling pathways with low dose Ara-C with the aim to improve its antileukaemic activity. To this end we have studied the cytotoxic interactions of the FLT3 inhibitor PKC412 and the mTOR inhibitor rapamycin with Ara-C in isobologram analysis (CalcuSyn). 4 cell lines were studied: 2 with FLT3 ITDs (MV4-11 and MOLM13) and 2 wild type for FLT3 (HL-60 and BV173). As expected synergy was seen between Ara-C and PKC412 in the FLT3 ITD positive cell lines (CI50: 0.47 MOLM13 and 0.68 MV4-11), but not in the wild type cell lines where antagonism was noted (CI50: 1.21 BV173 and 1.7 HL-60). In contrast, the Ara-C and rapamycin combination was synergistic in all cell lines, irrespective of FLT3 status. When the combination of PKC412 and rapamycin was studied, high levels of synergy were observed in the FLT3 ITD cell lines (CI50: 0.16 MOLM13 and 0.26 MV4-11) and in the BV173 cell line (CI50: 0.37) which has been shown to have autocrine activation of wild type FLT3 (Zheng et al, 2004) but not in HL-60 cells, where the combination was antagonistic (CI50: 3.4). FLT3 signalling pathways converge with the mTOR pathway via AKT and therefore our results suggest that a double hit on FLT3 signalling is likely to be beneficial in the treatment of FLT3 positive leukemias. In contrast rapamycin augments the cytotoxicity of AraC independently of Flt3 status. The combination of all 3 drugs produced no further benefit compared to the use of Ara-C and rapamycin alone. These promising drug combinations warrant further investigation in clinical studies.
A First-In-Human Clinical Trial of Adoptive Therapy Using CD19-Specific Chimeric Antigen Receptor Re-Directed T-Cells for Recurrent/Refractory Follicular Lymphoma.
Authors: Jensen Michael C. , Popplewell Leslie , DiGiusto David , Kalos Michael , Raubitschek Andrew , Forman Stephen J.
Published: October 2019

The immunotherapeutic ablation of lymphoma is a conceptually attractive treatment modality that is the subject of intense translational research activities. One approach being piloted through the City of Hope Lymphoma SPORE is the application of cellular immunotherapy employing cytolytic T-lymphocyte (CTL) grafts genetically engineered for CD19-specific effector functioning. This has been achieved by the construction of a chimeric T-cell antigen receptor (CAR) composed of a CD19-specific single chain immunoglobulin variable fragment (scFv) extracellular targeting domain molecularly coupled to the CD3 complex zeta chain. Cytolytic T-cells that are genetically modified to express this CAR display HLA-independent CD19-specific recognition and killing of lymphoma target cells. Using clinically applicable plasmid gene transfer/selection systems our group has demonstrated that anti-CD19 CAR+/HyTK+ CD8+ effectors can be generated from CTL precursors present in the peripheral blood and expanded to clinically relevant cell doses. We have designed an FDA-authorized (BB-IND#11411) clinical trial in which patients with recurrent/refractory follicular lymphoma undergo leukapheresis and a polyclonal CTL product derived that a.) expresses the anti-CD19 CAR, b.) kills CD19+ lymphoma targets, c) remains dependent on exogenous IL-2 for survival and proliferation, and d.) is sensitive to ganciclovir ablation by virtue of co-expression of the selection-suicide transgene HyTK. The trial is designed to assess the safety of intra-patient dose escalation beginning at a cell dose of 100x106 cells/m2 escalating to a dose of 2,000x106 cells/m2. In addition, this trial employs the use of rituximab to clear circulating B-cells prior to the first T-cell dose and fludarabine following the first T-cell infusion to both induce temporary lymphopenia and limit anti-transgene rejection responses. The protocol employs low-dose subcutaneous rhuIL-2 to support the numerical and temporal persistence of transferred CTLs. Treated patients are monitored with immunologic correlative studies that include Q-PCR for tracking persistence of transferred CTLs, RT-QPCR for following the levels of CD19+ B-cells in the peripheral blood, bone marrow, and lymph nodes, as well as, CT and PET imaging studies to follow disease responses. Here, we report on our initial clinical experience treating patients on this trial.