bluuebunny/crossref_metadata_2025 huggingface dataset:
Benefit of Targeted Intensification for NCI High Risk Childhood Lymphoblastic Leukaemia: Results of the United Kingdom Medical Research Council Trial ALL97 and ALL97/99.
Authors: Vora Ajay , Ward Rachel , Payne Jeanette , Mitchell Chris , Eden Tim , Kinsey Sally , Lilleyman John , Richards Sue
Published: October 2019

In November1999, the UK childhood ALL trial, ALL 97, adopted CCG risk stratification and treatment regimens in favour of the UKALL approach due to concerns over lower event-free survival (EFS) in the UK compared to the US. A key new feature in the amended trial, ALL97/99, was NCI risk group and early marrow response targeted intensification. Of 1935 patients registered in the trial between January 1997 and June 2002, 997 were treated on ALL97 and 938 on ALL97/99. EFS was better in ALL97/99 compared with ALL 97 (5 year EFS: ALL 97 = 74.1%, 95% CI 71.4%–76.8% vs ALL 97/99 = 78.9%, 76.0%–81.8%, p=0.002). To investigate whether particular sub-groups benefited more or less with the CCG approach, we compared outcomes for different risk groups within the two parts of the trial in regard to EFS, overall survival (OS) and CNS relapse risk. All p values quoted are two-sided. EFS and OS were significantly better in ALL97/99 compared with ALL97 for NCI high risk (HR) patients (5 year EFS: ALL 97 = 61.8%, 95% CI: 56.9–66.7%, ALL 97/99 = 71.8% 95% CI: 66.7–76.9%, p = 0.0006. 5 year OS: ALL97 = 71.3%, 95% CI: 66.8–75.8%, ALL97/99 = 80.3% 95% CI: 76.0–84.6%, p = 0.005), but did not differ significantly for NCI standard risk (SR) patients (5 year EFS: ALL 97 = 81.7% 95% CI: 78.6–84.8%, ALL 97/99 = 83.3% 95% CI: 79.8–86.8%, p = 0.3. 5 year OS: ALL97 = 91.2% 95% CI: 89.0–93.4%, ALL97/99 = 92.6% 95% CI: 90.4–94.8%, p = 0.5). The incidence of isolated CNS relapse was also significantly lower in ALL97/99 for NCI HR (ALL97 = 8% vs ALL97/99 = 3.5%, p = 0.01) but not NCI SR patients (ALL97 = 3.5% vs ALL97/99 = 2.7%, p = 0.6). Despite restricting the use of cranial radiotherapy to patients with overt CNS disease (CNS 3, <5% of all patients), the incidence of isolated CNS relapse in ALL97/99 was reassuringly low, even for sub-groups at high risk of CNS relapse such as those with WCC > 100 × 10 9/l (4.8%) or T cell phenotype (3.8%). Isolated CNS relapse risk in ALL97/99 patients randomised to dexamethasone (which was compared with prednisolone in the trial) was 1.8%, similar to that reported with use of cranial radiotherapy for a higher proportion of patients. A targeted intensification approach improves EFS for NCI HR patients and, especially when combined with systemic dexamethasone, results in a low incidence of isolated CNS relapse for high risk sub-groups without use of cranial radiotherapy.
Outcomes in Children and Adolescents with a Markedly Elevated White Blood Cell Count (>200,000) at Diagnosis of High Risk Acute Lymphoblastic Leukemia (ALL): A Report from the Children’s Oncology Group.
Authors: Hastings Caroline , Sather Harland N. , Seibel Nita L. , Nachman James , Steinherz Peter G. , Bertolone Kathy , DeLaat Cynthia , Ettinger Lawrence J. , Franklin Janet , Robert Freyer David , Mattano Leonard A. , Mitchell Torrey L. , Rubin Charles M. , Pyesmany Allan F. , Heerema Nyla A. , Edens Cheryl , Gaynon Paul S.
Published: October 2019

In prior studies, children and adolescents with markedly elevated (ME) WBC≥200,000 had a poor outcome. We examined the outcome of these patients enrolled on CCG l961 (n=250) and compared them to other patients with WBC<200,000 (n=1800). Criteria for CCG-1961 enrollment included age 1–9 years and WBC ≥ 50,000; or age ≥ 10 years with any WBC. Two-thirds of ME WBC patients were male; one third were ≥ 10 years of age, and 7% had CNS disease at diagnosis. Among patients with evaluable immunophenotypes (n=223), 46% had B precursor and 55% had T cell ALL. Event free survival (EFS) at 5 years for ME vs lower WBC patients was 60% and 73%, respectively (p<.0001). Survival (S) at 5 years was 73% vs 82% for ME and lower WBC patients, respectively (p=.0015). EFS for ME B precursor patients was 45% compared to 72% for lower WBC B precursor patients (p<.0001). EFS for ME and lower WBC T cell patients was 71% and 74%. On CCG 1961, rapid early response (RER) patients were randomized to receive stronger or standard intensity therapy and standard or longer intensification. Slow early response (SER) patients received increased intensity therapy and were randomized to receive sequential idarubicin/cyclophosphamide or weekly doxorubicin in intensification. All SER patients received 2 delayed intensifications. EFS for RER ME WBC patients (n=143) was 73% for stronger intensification and 60% for standard intensification. EFS for RER ME WBC was 72% for standard duration and 59% for longer duration intensification. EFS was 61% for SER patients (n=83) with no difference among regimens. Eleven of 103 B lineage ME WBC patients had Ph+ ALL of those there were 8 subsequent events. Patients with CNS 2 had significantly worse outcome (4 year EFS 52%) compared to patients with CNS 3 (4 year EFS 59%) or CNS 1(4 year EFS 68%) status at diagnosis, (p=.04). In conclusion, a ME WBC appears to have no prognostic significance for T cell patients while a ME WBC is highly prognostic for B precursor patients. Stronger intensification improves EFS for RER ME WBC patients as compared to standard intensification. Although the numbers are small, the magnitude of this difference is similar to the outcome of the entire study.
Post-Remission Therapy with Imatinib and HAM Improve MRD before Tansplant for Patients with Philadelphia-Positive Acute Lymphoblastic Leukemia (Ph+ALL): Results of the GRAALL AFR03 Study.
Authors: Leguay Thibaut , Witz Francis , De Botton Stéphane , Gabert Jean , Cayuela Jean-Michel , Macintyre Elizabeth , Preudhomme Claude , Fegueux Nathalie , Thomas Xavier , Cahn Jean-Yves , Escoffre Martine , Delannoy Andre , Ifrah Norbert , Dombret Herve , Pigneux Arnaud
Published: October 2019

Combination of Imatinib with chemotherapy has been reported as very promising for patients with Ph+ ALL. In this study, we attempted to minimize the MRD (Minimal Residual Disease) level before transplant for Ph+ ALL by combining Imatinib with HAM (HAMI). This regimen consisted to Cytarabine 2g/m2/12 hours between day1 and day4, Mitoxantrone 10mg/m2/day between day1 and day3 combinated with Imatinib (3 different doses) between day1 and day45. MRD was studied by RT-PCR at day1 (before treatment) and day45 (after treatment) and was exprimed by BCR-ABL/ABL levels. Between April 2002 and September 2005, 36 patients in complete remission (CR) after induction chemotherapy have been analysed. This group consisted of 13 females and 23 males with a median age of 42 years (19–60). Three doses of Imatinib (400 mg, 600 mg and 800 mg/day) have been tested for six patients in each cohort (the first eighteen patients). After an intermediate analysis, the dose of 800 mg per day has been shown to be toxic (one grade IV hypokaliemia), and a last cohort of 18 patients were treated with 600 mg/day. No toxic-death related to HAMI regimen was reported. Thirthy-one patients could be studied for MRD before and after HAMI consolidation. Four patients were in molecular remission (level of BCR-ABL/ABL<10−5) before treatment and 12 after. No difference between 3 cohorts of dose was observed. The median level of MRD was 10−3 (0–0.7) and 3 × 10−5 (0–0.004) before and after treatment respectively (p=0.0015). After a median follow-up of two years and for all patients, the estimated overall survival (OS) and disease free survival were respectively of 71% (51%–84%) and 52% (32%–69%). The cumulative incidence of relapse and the cumulative incidence of death in CR were respectively of 15% (6%–32%) and 31% (17%–52%). Twenty patients had allogenic matched transplantation (thirteen related, seven unrelated) and composed the group with donor. In this first group, seven patients obtained MRD negativity after HAMI treatment. The others sixteen patients composed the second group of patients without donor or older than 55 years. In this second group, eleven patients received autologous stem-cell transplant followed by imatinib for nine of them, and five received imatinib in maintenance with or without chemotherapy. In this group, six patients had obtained MRD negativity after HAMI. The two-years OS was identical for patients with or without donor (71%). In conclusion, imatinib combined with high dose chemotherapy improved molecular remission before transplantation and lead to an improve outcome.
Prognostic Factors for Relapsed Childhood Acute Lymphoblastic Leukemia (ALL): A Report from Tokyo Children’s Cancer Study Group (TCCSG) Study L95-14 and L-99-15.
Authors: Ogawa Chitose , Ohara Akira , Manabe Atsushi , Koh Katsuyoshi , Kaneko Takashi , Goto Hiroaki , Ota Setsuo , Hanada Ryoji , Tsuchida Masahiro
Published: October 2019

More than 70% of children with ALL enjoy a long-term event-free survival (EFS), however, the rest of them still suffer from a relapse and the treatment for relapsed ALL is not always successful. The treatment results of childhood ALL with a first relapse were retrospectively analyzed to determine prognostic factors.

Of the 1336 children enrolled on the TCCSG L95-14 and L99-15 studies, diagnosed between 1995 and 2003, 224 suffered from a relapse before March 2005. The relapsed patients were treated differently according to the protocol of each institution. The potential prognostic factors examined were : the age (low; 1–9 years, high; over 10 years), white blood cell count (low; <50,000/μl, high; others) and immunophenotype of leukemic blasts at initial diagnosis (B-lineage or T), gender, initial risk groups, the site of relapse, the timing of relapse (very early: within 18 months after diagnosis, early: other than either very early or late relapse, late: later than 6 months after the discontinuation of the front-line chemotherapy), and the treatment after relapse (chemotherapy alone or chemotherapy plus SCT).

The sites of relapse were: isolated bone marrow (BM, n=165), BM with other sites (n=21) and extramedullary only (n=38). Ninety-nine relapses occurred in very early stage, 43 in early stage and 82 in late stage. Twenty-three patients received SCT before relapse according to the protocol.

A second complete remission (2CR) was achieved in 175/220 patients (79%) and 74 of them are still in 2CR. Five-year EFS for all the relapsers was of 29±3% (mean±s.e.) and 5-year overall survival rate was 38±4%. In a subset of patients (n=126) with B-lineage phenotypes and with isolated BM relapse, who were treated with chemotherapy alone before relapse, we found 2 significant prognostic factors identified by a multivariate analysis : the age at initial diagnosis (EFS: age low; 35±5%, high; 8±8%, p=0.0016) and the timing of relapse (EFS: late; 39±8%, early; 24±9%, very early; 16±5%, p=0.0051). Gender, initial leukocyte count, or the use of SCT after obtaining 2CR was not related with patient’s outcome in a multivariate analysis.

The importance of the timing of relapse was overwhelmingly shown, however, the significance of the age at the diagnosis was not demonstrated before. The results will be incorporated into our next protocols to relapsed patients with ALL.
Targeting the mTOR Pathway Suppresses the Growth of Acute Myeloid Leukemia (AML) Progenitors and Enhances Arsenic Trioxide Induced Antileukemic Responses.
Authors: Altman Jessica K. , Sassano Antonella , Yoon Patrick J. , Redig Amanda J. , Katsoulidis Efstratios , Tallman Martin S. , Platanias Leonidas C.
Published: October 2019

-Background: A limiting factor for the use of arsenic trioxide (As2O3) in the treatment of non-APL subtypes of AML is the requirement of very high concentrations for the induction of leukemic cell death. The mammalian target of rapamycin (mTOR) is a key cellular regulator of multiple signaling events that control initiation of mRNA translation in leukemia cells. We examined the effects of As2O3 on mTOR-dependent cellular pathways in AML cells and determined whether modulation of the mTOR pathway sensitizes them to the antileukemic properties of As2O3.

Methods: The human AML cell lines KG1, U937, or MM6 were treated with increasing concentrations of As2O3. Activation of p70 S6 kinase (p70S6K) was evaluated by immune-complex kinase assays in anti-p70S6K immunoprecipitates, while phosphorylation of various downstream mTOR effectors was assessed by immunoblotting with specific antibodies against phosphorylated S6 ribosomal protein (rpS6) and the translational repressor 4E-BP1. MTT assays were used to assess proliferation of the various cell lines, while apoptosis was determined by annexin V-propidium iodide staining. Finally, bone marrow mononuclear cells from patients with AML were used in clonogenic assays in methylcellulose to assess the effects of As2O3 and/or rapamycin on leukemic progenitor cell growth.

Results: Treatment of AML cells with As2O3 resulted in activation of the p70 S6K and phosphorylation of its downstream effector, rpS6, on Ser240/244, and such events were mTOR-dependent. In addition, the translational repressor 4E-BP1 was phosphorylated on Thr37/46 and Thr70, sites whose phosphorylation is required for 4E-BP1 de-activation and dissociation from eIF4E. Pharmacological inhibition of mTOR activity blocked the growth of AML cell lines as determined by MTT assays, while the addition of As2O3 to the cultures further enhanced the inhibitory effects of rapamycin. On the other hand, AS2O3-dependent induction of apoptosis was not enhanced by rapamycin in AML cells, suggesting selective effects on cell cycle and growth inhibition. Rapamycin also inhibited leukemic CFU-GM progenitor colony formation from 6 AML patients and enhanced the suppressive effects of AS2O3 on leukemic progenitor cell growth.

Conclusions: Paradoxically, As2O3 activates the pro-growth and pro-survival mTOR pathway in AML cells. Such activation appears to occur in a negative feedback regulatory manner to compensate for AS2O3 responses. Thus, combinations of AS2O3 with mTOR inhibitors may be an effective approach to overcome AS2O3-resistance in AML cells.
Acute Myeloid Leukemia-Derived Dendritic Cells Express the Immunoregulatory Enzyme Indoleamine 2,3-dioxygenase.
Authors: Curti Antonio , Pandolfi Simona , Aluigi Michela , Ferri Elisa , Ottaviani Emanuela , Durelli Ilaria , Horenstein Alberto L. , Fiore Francesca , Massaia Massimo , Piccioli Milena , Pileri Stefano A. , Martinelli Giovanni , Baccarani Michele , Lemoli Roberto M.
Published: October 2019

Acute myeloid leukemia (AML) cells may be differentiated into dendritic cells (DC) which have increased immunogenicity, but retain some immunosuppressive features of leukemic cells. Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed AML patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. We, then, in vitro generated DCs from 7 AML samples (AML-DCs) in the presence of GM-CSF, IL-4 and TNF-α. The cells we obtained were morphologically and phenotypically semi-mature DCs expressing CD40, CD80, CD86, HLA-DR and CD1a molecules and they were more efficient to induce T-cell proliferation and type 1 cytokine production than primary AML blasts. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. These data identify IDO-mediated catabolism as a tolerogenic mechanism in AML-DCs and have clinical implications for the use of AML-DCs as cellular vaccine against leukemia.
Targeting Mutant Nucleophosmin (NPM)1: A Promising Approach To Induce Growth Inhibition and Differentiation in Human AML.
Authors: Herger Bryan , Fiskus Warren , Rao Rekha , Bhalla Kapil
Published: October 2019

NPM1 is a nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids and is capable of histone and nucleosome assembly. NPM1 shuttles between nucleus and cytoplasm, but is mutated and aberrantly localized to the cytoplasm in 35% of patients with AML. NPM1 is involved in regulating the activity of p53 and p14ARF, such that cells lacking NPM1 show decreased p14ARF. Mutant (m) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal (NES). This causes mNPM1 to remain in the cytoplasm. In AML, mNPM1 has been correlated with the presence of FLT3-ITD and normal karyotype. NPM1 maps to 5q35 locus, which is often deleted in myelodysplastic syndrome. The role of the cytosolic mNPM1 in the pathogenesis of AML is not clear. In the present studies we determined the expression and biologic effects of mNPM1 in cultured and primary AML cells. Mutant nucleophosmin can be readily identified by reverse transcription followed by amplification with PCR primers specific for the mutant sequence. Sub-cellular fractionation and immunoblot analysis and/or immunoflourescent microscopy, as well as RT-PCR to detect the mNPM1 transcripts, confirmed that mNPM1 is localized in the cytosol in the cultured AML OCI-AML3 but is unmutated and nuclear in HL-60 cells. As compared to control siRNA, treatment with siRNA to NPM1 for 72 hours significantly induced p21 expression, inhibited cell proliferation and induced morphologic differentiation of OCI-AML3 cells. NPM1 siRNA did not induce significant apoptosis or loss of cell viability as determined by trypan blue uptake assay. OCI-AML3 cells co-express HoxA9 and Meis1 proteins, which are known to be leukemogenic in mouse myeloid progenitor cells. Notably, treatment with NPM1 siRNA depleted Meis1 and HoxA9 expressions in OCI-AML3 cells. Treatment with leptomycin B (5 nM for 48 hours), an inhibitor of the NES receptor CRM1/exportin-1, shifted the localization of mNPM1 from the cytosol to nucleus and also depleted the levels of Meis1 and HoxA9 in OCI-AML3 cells. Meis1 is known to transactivate FLT3 tyrosine kinase in AML progenitor cells, and a significant association of FLT-3-ITD with the presence mNPM1 has been documented. In the present studies, of the 16 primary AML samples, four that expressed FLT3-ITD also demonstrated expression of mNPM1. Taken together, these findings suggest that abrogation of the levels or reversing the aberrant cytosolic localization of mNPM1 may be a promising anti-leukemia treatment strategy, especially for targeting AML that co-express FLT3-ITD and mNPM1.
Replicative Senescence in Good-Risk Acute Myeloid Leukemia.
Authors: Zijlmans Mark , Swiggers Susan , Rife Soler Maria , Beverloo Berna
Published: October 2019

Immortal cell growth is considered the hallmark of tumor cells. In contrast, normal cells have a limited proliferative capacity of 40–60 cell divisions, also known as the Hayflick limit. The limited proliferative capacity of normal cells relates to gradual telomere shortening as a consequence of the end-replication problem. Upon critical telomere shortening, cells enter a non-replicative but viable state referred to as replicative senescence. These replicative senescent cells stain blue in a beta-Galactosidase assay and activate DNA double-strand break repair pathways at telomeres (e.g. gamma-H2AX foci). In human fibroblast models, escape from senescence results from loss of p53 and Rb function. Escape is associated with reactivation of telomerase. High levels of telomerase, as observed in germ cells and most tumor cells, allow for immortal cell growth. Recently, we demonstrated low levels of telomerase in AML patients with t(8;21) or inv(16) (Swiggers et al, G.C.C. 2006). Interestingly, levels of telomerase in these AML samples were similar to levels of telomerase in normal bone marrow progenitor cells. We hypothesized that AML without re-activated telomerase may still have intact senescence pathways that limit the proliferative capacity of normal cells. This hypothesis was addressed by studying AML patient samples without telomerase re-activation, i.e., t(8;21), t(15;17) or inv(16) (n=10), and a control group of AML with telomerase re-activation (multiple gains/losses of genetic material, n=8). AML samples werelong-time cultured in vitro in the presence of hematopoietic growth factors (range 3–6 weeks),analyzed in vivo following transplantation in NOD-SCID mice andin patients at time of relapse.

Cells with all characteristics of replicative senescence, i.e. enlarged, viable, non-proliferating, blue-coloring in beta-Galactosidase assay, critical short telomeres and gamma-H2AX foci at telomeres, were clearly observed in all AML samples with t(8;21), t(15;17) or inv(16). Gradual telomere shortening was observed in these AML cells in vitro upon long-term culture, in vivo after transplantation in NOD-SCID mice and in vivo in patients at relapse compared to time of diagnosis, indicating that these AML cells do not have an adequate telomere maintenance mechanism. We conclude that AML cells with t(8;21), t(15;17) or inv(16) are characterized by intact pathways that induce replicative senescence. Intact pathways that limit proliferative lifespan may be critical to the high cure rates following chemotherapy treatment of patients with good-risk AML.
The JAK2-V617F Mutant Found in Myeloproliferative Disorders and Acute Myeloid Leukemia Cooperates with the EpoR, but Not with FLT3 To Promote Proliferation of IL-3 Dependent Cells.
Authors: Kaza Seshu K. , Vempati Sridhar , Carola Reindl , Hiddemann Wolfgang , Spiekermann Karsten
Published: October 2019

The Janus Kinase (JAK) 2 mutation V617F can be detected in patients with polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) as well as at low percentages in other myeloproliferative disorders (MPDs) and myelodysplastic syndromes (MDS) and in 5–6% of de novo AML. Moreover, a recent study showed that JAK2 complemented by an additional signal of KIT or FLT3 confers self renewal capacity to hematopoietic cells. Since the JAK2V617F mutation is found in AML, we asked whether JAK2 might cooperate with FLT3, a receptor tyrosine kinase that is overexpressed in most patients with AML to induce proliferation.

JAK2-WT and JAK2V617F did not confer factor independent growth when expressed in IL-3 dependent Ba/F3 cells. Further, exploring the coordination of JAK2 and its mutant with the type I cytokine receptor EpoR, Ba/F3 cells co-expressing both JAK2 and EpoR showed proliferation in the presence of Epo but not in the absence of Epo. In contrast Ba/F3 cells co-expressing JAK2V617F and EpoR have a profound proliferation advantage and are able to grow in the absence of Epo. These data provide strong evidence that JAK2V617F has transforming potential in the presence of a type I cytokine receptor. The coexpression of the JAK2V617F mutant with a class III cytokine receptor like FLT3, which is involved in the pathogenesis of acute myeloid leukemia (AML), does not result in an increased proliferative response. Both, JAK2-WT and JAK2V617F expressing Ba/F3 cells were able to survive in the presence of the FLT3 receptor, but did not show autonomous growth.

Analysis of signaling pathways showed that the JAK2-V617F mutant, but not JAK2-WT is able to activate STAT5 in cooperation with the EpoR and FLT3.

Our data show that the JAK2-V617F mutant which is found in 5–6% of AML patients cooperates with the EpoR, but not with FLT3 to promote proliferation in IL-3 dependent cells.
Destabilization of the Factor V B-Domain Results in Procofactor Activation.
Authors: Camire Rodney M. , Zhu Hua , Bos Mettine H.A. , Toso Raffaella
Published: October 2019

A hallmark of hemostasis is that proteins involved in the formation of a blood clot remain in a quiescent state and are only activated following an appropriate stimulus. Blood coagulation factor V (FV), which is structurally homologous to FVIII, cannot function in the prothrombinase complex and is thus considered a procofactor. Thrombin catalyzes the conversion of FV to FVa following three cleavages (Arg709, Arg1018, and Arg1545) releasing a large heavily glycosylated central B-domain (836 amino acids). Explanations as to how bond cleavage or B-domain release facilitates the transition to the active species are incomplete. Recent studies using a partial B-domainless form of FV (FV-810 des811–1491) support a model in which removal of B-domain sequences from FV rather than specific proteolysis underlies the mechanism by which cofactor function is realized. This single-chain derivative is functionally equivalent to FVa suggesting that the deleted B-domain sequences somehow suppress cofactor activity. To investigate this further, we have expressed and purified several single-chain derivatives of FV that vary in B-domain length from 155 to 497 residues. Functional activity assays as well as direct binding fluorescent measurements revealed that elimination of most of the C-terminal half of the B-domain (residues 1034–1491; 458 out of 836 a.a. deleted) had no influence on maintaining the procofactor state. However, deletion of sequences from the N-terminal half of the B-domain resulted in derivatives with cofactor-like properties. Using progressively finer deletion variants we were able to demonstrate that either a B-domain length of at least 378 amino acids or specific sequences contained within residues 902–1033 is sufficient to suppress cofactor activity. To examine these possibilities, we constructed additional FV variants in which a B-domain length of 378 amino acids was maintained, but specific portions of 902–1033 were exchanged with FVIII B-domain. Using the FV-1033 derivative (residues 1034–1491 of B-domain deleted) as a scaffold, three constructs were prepared, s-131, s-104, and s-46, representing 131, 104, and 46 amino acids from the FV B-domain exchanged with FVIII B-domain. In activity assays and direct binding measurements, each of these variants had properties consistent with the cofactor-like form indicating that a length of ~375 residues is not sufficient to maintain the procofactor state. These findings demonstrate for the first time that there are indeed specific FV-B domain sequences between 902–1033 that directly or indirectly stabilize the procofactor state. Remarkably, simply replacing these sequences in FV-1033 resulted in activation of the proteins in the absence of proteolysis. These observations change existing ideas about FV activation and provide insight into specific regions of the B-domain that assist in preserving the procofactor state.
Phase II Study of Low-Dose Decitabine for the Front-Line Treatment of Older Patients with Acute Myeloid Leukemia (AML).
Authors: Cashen Amanda , Schiller Gary J. , Larsen Julie S. , Cullen Michael T. , DiPersio John F.
Published: October 2019

Decitabine (Dacogen™) was recently approved for the treatment of patients with myelodysplastic syndromes (MDS). Response in MDS is associated with reversal of the hypermethylated phenotype of silenced genes. The prevalence of hypermethylated genes contributing to AML progression, coupled with the challenges involved in the treatment of AML in the elderly, is a basis for exploring the activity of decitabine in this patient population. The objective of this phase II trial was to estimate the rate of morphologic complete response (CR) in patients with AML. To be eligible for the study, patients had to be at least 60 years old with newly diagnosed, untreated AML with intermediate- or poor-risk cytogenetics, and they could not be candidates for standard induction chemotherapy. Decitabine was given at a dose of 20 mg/m2 over 1 hour for 5 consecutive days every 4 weeks. At the time of the analysis, 27 patients were enrolled in the trial (14 men, 13 women; median age = 69 years, range = 61–87) and received at least 1 cycle of treatment (median = 4, range = 1–10). Seventeen patients (63%) received 3 or more cycles of treatment. Seven of 27 patients (26%) responded, 2 patients (7%) with a morphologic CR, 1 patient (4%) with a complete cytogenetic response (CRc), and 4 patients (15%) with a morphologic CR with incomplete blood count recovery (CRi) according to AML response criteria (Cheson, JCO 2003). In addition to these defined responses, 11 other patients demonstrated clinical benefit by achieving stable disease, with 4 of these patients having a >25% reduction in bone marrow blasts. Median time to response was 3 treatment cycles. Of the 13 patients with a prior history of MDS, 6 patients had a response (CR, CRc, or CRi, 46%). Two of 2 patients with leukemia cutis exhibited complete resolution of skin lesions and responded (CR or CRi). Eight patients discontinued treatment prior to receiving 3 cycles because of death, progressive disease, or patient decision. All patients were evaluated for safety. Besides myelosuppression, the most commonly reported adverse events considered related to decitabine treatment were fatigue, nausea, febrile neutropenia, vomiting, and asthenia. Decitabine given daily for 5 days showed activity in patients with newly diagnosed AML, with a toxicity profile that was manageable in this older patient population. The results to date are comparable to those from previous studies in AML patients using a variety of decitabine schedules, which have reported CR rates from 14% to 33% (Blood2004;103:1635, Blood2005;106:527a, Cancer2006;106:1794). Updated response and adverse event data will be available at the time of presentation.
Homoharringtonine Based Triple-Drug Regimen as Induction Chemotherapy for De Novo Acute Myelogenous Leukemia.
Authors: Jin Jie , Qian Wenbin , Liu Hui , Jian Daozi , Mai Wenyuan , Tong Hongyan , Men Haitao
Published: October 2019

HAA regimen, consisting of homoharringtonine (HHT), aclarubicin and cytarabine (Ara-C), is an efficacious chemotherapy regimen for induction treatment of acute myelogenous leukemia (AML). HHT, a plant alkaloid that is derived from a Chinese evergreen tree, has been shown to inhibit protein, DNA, and RNA synthesis by inhibition of chain initiation. HAA regimen consists of HHT administered at a dose of 4 mg/m2/day by continuous infusion over 4 hours or 2 mg/m2 intramuscular injection twice daily on days 1–3, aclarubicin administered at a dose of 12 mg/m2/day by continuous infusion over 2 hours on days 1–7, and cytarabine (Ara-C) given at a dose of 75 mg/m2 twice daily on days 1–7. Granulocyte colony-stimulating factor (Lenograstim) 5 μg/kg/day subcutaneously was started from the day neutrophil count <0.5×109/l, and continued until the day neutrophil count >1.0×109/l on 3 successive days. For patients with partial remission (PR) after the evaluation of the first course of the therapy, another same induction HAA regimen was administered. Between May 1999 and June 2006, 80 patients consisted of 31 male and 49 female patients were enrolled. All patients had newly diagnosed with de novo AML and had not received any induction treatments before. Out of them, 3 were M1, 44 M2, 2 M4, and 31 M5 according to FAB classification criteria. The median age was 36 (14–59) years. Of all the 76 patients with cytogenetic analysis available, 13 had favorable karyotype, 58 intermediate karyotype, and 5 unfavorable karyotype. In all, 68 (85%) patients achieved complete remission (CR), and the first single course of this induction regimen resulted in a CR rate of 75%. The CR rate of 100%, 88% and 20% were achieved in patients with favorable, intermediate and unfavorable cytogenetics, respectively. We also found that patients with M5 achieved a CR rate of 74% (23/31), while patients with M1 or M2 94% (44/47). The toxicities associated with this regimen were no more than those expected with standard chemotherapy, and the most common non-hematological toxicity was infection. This study suggested that HAA regimen is a safe regimen and it is efficacious, well-tolerable induction therapy for newly diagnosed de novo AML, the use of G-CSF (Lenograstim) appears to be safe, with little risk of accelerating leukemic relapse. A high CR rate can be achieved with only one or two courses of this regimen. Besides, cytogenetics is an important prognostic factor.
Impact of Pegfilgrastim on Hematological Reconstitution and Incidence of Neutropenic Fever after Consolidation Therapy with High-Dose Cytarabine in Acute Myeloid Leukemia: Comparative Analysis between AMLSG 07-04 and the German AML Intergroup Trial.
Authors: Schlenk Richard F. , Dohner Konstanze , Groner Silia , Hartmann Peer C. , Krauter Jürgen , Ganser Arnold , Dohner Hartmut
Published: October 2019

Therapy using high-dose cytarabine (HiDAC) according to the CALGB scheme (3g/m2 bid. days 1,3,5) is recognized as a standard consolidation treatment for younger adult patients with acute myeloid leukemia (AML). Pegfilgrastim has been shown to be effective in reducing the duration of neutropenia in the treatment of solid tumors and it seems to be even more effective than Filgrastim in reducing the incidence of neutropenic fever. The objective of the current study is to investigate the effect of pegfilgrastim given after HiDAC consolidation within our AMLSG 07-04 clinical trial. The AMLSG 07-04 trial was initiated in September 2004 (age 18–60 yrs). Consolidation therapy consists of 3 cycles of HiDAC (3g/m2 bid. days 1,3,5) with pegfilgrastim 6mg given at day 10 after start of chemotherapy. As a control group, patients randomized from AMLSG into the German AML Intergroup protocol using the same scheme for consolidation therapy with permitted interventional application of G-CSF were used. Data from 127 patients and a total of 285 cycles are available, including 104 patients of the AMLSG 07-04 trial and 23 patients of the German AML Intergroup trial. Data from all three cycles were pooled for the comparison between AMLSG 07-04 (prophylactic pegfilgrastim) and German AML Intergroup (interventional G-CSF) trials. The duration of leukopenia and neutropenia was significantly shorter in patients receiving prophylactic pegfilgrastim (p=0.01 and p=0.008, respectively) compared to patients with interventional G-CSF, which was given in 16% of the pts. This was paralleled by a lower incidence of neutropenic fever with 53% in patients receiving prophylactic pegfilgrastim compared to 77% in patients with interventional G-CSF (p=0.0004). Data from 12 patients and 23 cycles were available for pharmocokinetics of pegfilgrastim. The median peak G-CSF serum level was 221 ng/ml (range 57–553 ng/ml) measured between 24 and 48 hours after the administration of pegfilgrastim and median terminal half-life was 5 days (range 3–7 days). G-CSF serum levels returned to normal in all patients after neutrophil reconstitution. Multivariable regression analysis for longitudinal data on G-CSF serum levels revealed total leukocyte count (p=0.02) and presence of neutropenic fever (p=0.03) as statistically significant variables. In conclusion, the administration of pegfilgrastim shortened the duration of leuko- as well as neutropenia and reduced the rate of neutropenic fever in pts. with AML receiving consolidation therapy with high-dose cytarabine.
Chromosomal Aberration Patterns of Aggressive B-Cell Lymphomas Closely Correlate with Molecularly Defined Disease Subtypes and Regional mRNA Expression. An Analysis of 213 Tumor Samples within the German MMML Network.
Authors: Wessendorf Swen
Published: October 2019

Recently, it has been demonstrated that aggressive B-cell non-Hodgkin lymphomas (B-NHL) split up into distinct subgroups such as molecular Burkitt (mBL) and molecular non-Burkitt lymphomas (non-mBL). The non-mBL group can further be devided in activated B-cell (ABC) and germinal center B-cell (GCB) like lymphomas. In the present investigation we aimed at unravelling correlations of single or multiple chromosomal imbalances, their respective regional mRNA gene expression levels and the above mentioned molecular subgroups. Aggressive B-NHL samples (n=255) were investigated within the German network project Molecular Mechanisms in Malignant Lymphomas (MMML). Data from genomic arrayCGH analysis were evaluable for 213 lymphoma cases. Consensus review diagnoses according to WHO criteria were: DLBCL (n=154), atypical BL (n=20), typical BL (n=15) and other aggressive B-NHL (n=24). For all cases gene expression analysis was done using the Affymetrix U133A GeneChip. FISH data were available for translocations involving the MYC locus and for t(14;18). Using arrayCGH, chromosomal gains in more than 10% of all cases have been identified on 18q(24.4%), 3q(19.8%), 1q(19.1%), 12q(16.7%) 2p(16.3 %), 7p and 7q(16.3%), 6p(13.2%) and 11q(12.5%). Deletions occurred most frequently on chromosome arm 6q(20.3%), 17p(15.4%), 9p(14.8%) and 8p(10%). Molecular BLs had significantly less chromosomal aberrations than non-mBLs but frequently show gains of chromosome arm 1q. Non-mBLs show higher genomic complexity and strikingly differing aberration patterns for GCB (more than 20% gains on 1q, 2p, 7q, 11q, 12q) and ABC (more than 20% gains on 3q, 9p, 18q, loss on 6q) type lymphomas. These findings were also supported by results of genomic clustering algorithms such as non negative matrix factorization. For a large number of recurrent chromosomal aberrations we were able to delineate minimal aberrant chromosomal regions and to correlate respective regional mRNA gene expression levels. Strong proportional correlations were found for gains of 18q21.1-q21.33 and losses of 6q21-q24.3. In conclusion, genomic aberration patterns closely correlate with distinct molecular subtypes of aggressive B-NHL. These aberration patterns might serve as surrogate markers providing diagnostic and prognostic information as well as a basis for targeted gene investigations and risk adapted therapy studies.
Gene Expression Profiling of Diffuse Large B-Cell Lymphomas Supervised by CD21 Expression.
Authors: Miyazaki Kana , Yamaguchi Motoko , Kimura Miho , Ogawa Shoko , Ueno Satoshi , Katayama Naoyuki , Shiku Hiroshi , Kobayashi Tohru
Published: October 2019

We have reported that CD21 expression in tumor cells is a favorable prognostic factor in diffuse large B-cell lymphomas (DLBCLs)(Ogawa S, et al. Br J Haematol, 2004; 125: 180–86). In the present study, we investigated the relationship between CD21 expression and the groups of germinal center B-cell-like (GCB) DLBCLs and activated B-cell-like (ABC) DLBCLs. The diagnosis of DLBCL was made according to the WHO Classification. CD21 in tumor cells was examined by means of immunohistochemistry using frozen sections. Forty cases of DLBCLs were investigated using Agilent 44K human oligo-microarrays (Agilent Technologies, Palo Alto, CA). GCB and ABC DLBCLs were identified based on Rosenwald’s gene set (Rosenwald A, et al. N Engl J Med, 2002; 346: 1937–47). Four cases (10 %) of 40 DLBCLs were found to be GCB DLBCLs, and 36 cases ABC DLBCLs. Incidence of CD21-positive cases was 50% (2/4) in GCB DLBCLs and 39% (14/36) in ABC DLBCLs. Overall survival of 36 patient with ABC DLBCLs were shown in Fig.1.

Fig. 1 Fig. 1.

Signature genes to distinguish between CD21-positive and CD21-negative DLBCLs were as follows; IgM (P=0.00017), CCR6 (P=0.0011), IL7 (P=0.0017), IgK (P=0.0037), EBI2 (P=0.0066), CD24 (P=0.0099) etc. were overexpressed in CD21-negative DLBCLs, and CR2 (CD21) (P=0.0010), MKNK2 (P=0.0028), LMO2 (P=0.00478), CDKN2A (P=0.0059), PDE8B (P=0.0066) etc. were overexpressed in CD21-positive DLBCLs. In 40 cases of DLBCLs, overall survival of patients with sIgM-positive DLBCL was significantly worse than that of patients with sIgM-negative DLBCL (P=0.023). Furthermore, in 216 consecutive cases of DLBCLs analyzed by immunohistochemistry using frozen sections from 1987 to 2004, overall survival according to sIgM expression was significantly different (P=0.013).

In conclusion,CD21 expression was a favorable prognostic factor in ABC DLBCLs.A top feature gene to distinguish between CD21-positive and CD21-negative DLBCLs was IgM. CD21 expression seemed to be related to the differentiation level of lymphoma cells.sIgM expression itself was a poor prognostic factor in ABC DLBCLs and in whole DLBCLs.
Spindle Checkpoint Insufficiency and Unscheduled APC-Dependent Proteolysis in Acute Myeloid Leukemia.
Authors: Schnerch Dominik , Felthaus Julia , Engelhardt Monika , Waesch Ralph M.
Published: October 2019

Genetic instability including aneuploidy is frequent in most cancers. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism responsible for accurate chromosome segregation. Unattached chromosomes or lack of spindle tension are sensed by the SAC. The activated SAC inhibits the ubiquitin-ligase anaphase-promoting complex (APC), which prevents the proteolysis of cell cycle regulators in order to delay progression through mitosis and allow cells to recover from defective mitotic spindle attachment. Spindle checkpoint malfunction proved to favor the generation of aneuploidy. In our recent work we investigated the roles of essential SAC proteins in acute myeloid leukemia (AML). We found the SAC-protein Bub1 to be posttranscriptionally downregulated in all investigated AML cell lines. As a consequence, after exposure to the microtubule disrupting agent nocodazole we observed a defective mitotic delay mechanism in comparison to SAC-competent cell lines and increased apoptosis consistent with the effects of Bub1 downregulation by RNA interference. At the molecular level we found a dramatic decline in mitotic regulator levels such as cyclin B1 and securin despite lasting spindle disruption. Additional data showed that the levels of these regulator proteins can be efficiently restored by exposure to the proteasome inhibitor MG-132 indicating that APC-dependent proteolysis is directly involved in SAC insufficiency. Thus, continuous activation of the APC triggers degradation of essential regulator proteins even in leukemic cells faced to mitotic stress such as complete spindle disruption. Such defects can lead to establishment of aneuploidy in vivo. Our findings emphasize a role of SAC insufficiency and unscheduled proteolysis in rise and progression of AML with complex karyotype.
Array-CGH Based Genomic Profiling in B-Cell Chronic Lymphocytic Leukemia Reveals Specific Correlations of Genomic Imbalances and Prognostic Subgroups and Underlines the Consistency of Chromosomal Aberration Patterns within This Disease.
Authors: Schwaenen Carsten , Kienle Dirk , Krober Alexander , Ruf Sandra , Winkler Dirk , Kohlhammer Holger , Kestler Hans A. , Lichter Peter , Wessendorf Swen , Dohner Hartmut , Stilgenbauer Stephan
Published: October 2019

Chronic lymphocytic leukemia of B-cell type (B-CLL) is characterized by a number of typical genomic aberrations. In comparison to patients with normal karyotypes or 13q deletions patients with high risk imbalances such as deletions of 11q, 17p or unmutated IgVH status have a higher risk for advanced disease and a significantly shorter survival.

For a precise mapping of chromosomal imbalances as well as to verify the number of aberrations per case in different genetic subgroups of B-CLL (e.g. del11q, del13q, del17p, +12 or unmutated IgVH status) we performed high resolution genomic profiling using a genomic DNA-chip containing 2.800 probes. Target clones compriseda large genome-wide cluster of clones covering the genome at a distance of approx. 1.5Mb andclones mapping to genomic regions or genes of possible pathogenetic relevance in lymphoma.

This chip covers approximately 10% of the human genome.

In 93 (70%) of 133 analyzed B-CLL cases 171 genomic imbalances were identified (between 1–7 aberrations/case). Besides the confirmation of known recurrent chromosomal aberrations, previously unknown recurrent imbalances were detectable on 2p (8%), 4p (4%), 7p-q (5%), 10q (5%) and 20p (3%). Most of these imbalances were of larger extension (> 10 Mb) and therefore impeded a further delineation of minimal aberrant regions and the identification of possible candidate genes. The mean number of chromosomal aberrations per case (= genomic complexity) in IgVH unmutated CLLs was approx. 2 times higher than in mutated cases (0,77 vs. 1,58 per case). 84% of samples with > 2 aberrations showed an unmutated IgVH status. Moreover, most of the previously unknown imbalances were identified within this group. A higher genomic complexity was also shown for samples with gain on 2p vs. balanced 2p status (2.2 times higher; 2.5 vs. 1.2) and in samples with del17p vs. balanced 17p status (3.7 times higher; 3.52 vs. 0.95). 11q aberrations had no impact on the number of genomic aberrations per case (1.6 vs. 1.2). Moreover, we found a strong association of 2p gains and an unmutated IgVH status (100%).

Array based genomic profiling confirmes the chromosomal aberration structure and underlines the consistency of chromosomal aberration patterns of B-CLL. The biological and prognostic relevance of 2p gains and unmutated IgVH mutational status have to be further investigated.
Occurrence of Chromosomal Translocations as Independent Prognostic Factor in Chronic Lymphocytic Leukemia.
Authors: Mayr Christine , Schulz Cathrine , Stilgenbauer Stephan , Kröber Alexander , Döhner Hartmut , Jäger Ulrich , Kainz Birgit , Hopfinger Georg , Hoffmann Rüdiger , Wendtner Clemens , Hallek Michael , Speicher Michael R.
Published: October 2019

Background: The course of chronic lymphocytic leukemia (CLL) is highly variable. Therefore, there is a need for prognostic factors that are readily performed and have a high predictive power.

Methods: The occurrence of translocations, a recently identified prognostic factor in CLL (Blood2006;107:742–751), was studied in 148 previously untreated, mostly early-stage patients and compared with respect to treatment-free survival (TFS) to several prognostic factors (Binet stage, mutational status of immunoglobulin genes, CD38, thymidine kinase serum concentration and cytogenetic aberrations detected by interphase FISH). To investigate chromosomal translocations, we applied a new method, CpG oligodeoxynucleotide stimulation that allows efficient preparation of metaphase spreads from CLL cells.

Results: The occurrence of translocations classified the majority of patients with poor prognosis. If translocations were investigated in addition to the currently used prognostic factors they identified those patients who were classified to be in a low-risk group based on traditionally used criteria, who had in fact a high risk for progression. Vice versa, patients in the high-risk groups for progression who did not have translocations had a long TFS. There was a substantial overlap of patients who had translocations and additional risk factors. But when we omitted patients who had translocations in addition to a given risk factor, we found that the respective risk factor lost its prognostic significance for the remaining patients. The two factors that retained their prognostic power in these patients were translocations and the Binet stage. This could suggest that the prognostic significance of the currently used factors derives from their frequent co-occurrence with translocations. Finally, multivariate analyses demonstrated that Binet stage (p=0.02) and translocations (p=0.0005) are the factors with the highest impact on TFS in our study cohort.

Conclusion: We present a method for efficient preparation of metaphase spreads in CLL cells in order to investigate chromosomal translocations. The occurrence of translocations is an independent prognostic marker in CLL. Finally, translocations occur not as a late event in the course of the disease and may define a new biological subgroup in this disease entity.