bluuebunny/crossref_metadata_2025 huggingface dataset:
The Natural History of Mutated, Stage AO Chronic Lymphocytic Leukemia.
Authors: Oscier David Graham , Gardiner Anne C. , Mould Sarah J. , Glide Sharron , Davis Zadie A. , Corcoran Martin , Wiestner Adrian , Staudt Louis M. , Thomas Peter , Orchard Jenny A.
Published: October 2019

Patients who present with stage AO CLL and have mutated IgVH genes are generally considered to represent a benign sub-set of CLL with prolonged survival .However, within this subgroup there is considerable heterogeneity with regard to disease progression and the need for treatment. From a cohort of 260 patients studied at a single centre, we identified 70 who presented with stage AO disease as an incidental finding on a routine blood count and who had mutated IgVH genes. 47 were followed up for over 10 years without receiving treatment. 31/47 remained stage AO with a lymphocyte count of <20 x 109/l and were considered to have stable disease (SD). 16/41 remained Stage A with a gradually rising lymphocyte count and were designated as having slowly progressive disease (SPD). 23/70 had progressive disease (PD), defined by one or more of the following parameters: an increase in stage, the appearance of lymphadenopathy > 1cm or splenomegaly, a persistent fall in Hb or platelet count, LDT of <1 year or the need for treatment. There was no difference in mean ZAP 70 expression, CD38 expression using 7% and 30% cut-off’s, % of IgVH gene mutations, IgVH gene usage, p2 X7 polymorphisms or presenting serum immunoglobulin levels among the three subgroups. Gene expression profiling using the lymphochip microarray was performed on 14 cases with PD, 8 with SPD and 12 with SD. No significant differences in gene expression were found. Karyotypic abnormalities at presentation were found in 68% of patients with PD, 31% with SPD and 27% SD (p= 0.007). The most frequent abnormality was (t)/del 13q14, found in 43%,0% and 16% of patients with PD, SPD and SD respectively. When interphase FISH and Southern analysis were also used to detect del13q14, the incidence of loss in the 3 groups rose to 91%,68% and 70% respectively, indicating both the high frequency of 13q loss and the greater responsiveness of leukemic cells to mitogens in patients destined to have PD. Loss of the ATM or p53 genes were found at presentation or on sequential testing in 4 patients with PD but not in SPD or SD. Patients with SD and SPD remained asymptomatic on prolonged follow-up and were not prone to recurrent infections. In contrast, 12 patients with PD had recurrent infections and 8 received regular immunoglobulin infusions. There was no significant difference between serum IgG, IgA and IgM levels between patients with PD and those with SPD or SD at presentation. However serum IgG but not IgA or IgM levels were significantly lower on follow-up in patients with PD compared to SPD and SD (p <0.0001). A subset of patients with good prognosis CLL develop progressive disease which is associated with significant morbidity due to immuno-deficiency. Apart from differences in the frequency of cytogenetic abnormalities, these patients are not readily identified at presentation using currently available prognostic markers.
B Cell Receptor (BCR) Ligation Enhances Cell Survival in Progressive Chronic Lymphocytic Leukemia (CLL).
Authors: Deglesne Pierre-Antoine , Chevallier Natalie , Letestu Remi , Salanoubat Celia , Sanhes Laurence , Nataf Joelle , Varin-Blank Nadine , Ajchenbaum-Cymbalista Florence
Published: October 2019

Recent gene profiling studies demonstrated that signalisation through the BCR plays a central role during CLL disease progression. However, incidence of BCR engagement on cell survival remains unclear as both increased and decreased apoptosis have been reported after BCR ligation by anti-mu antibodies. We investigated the effect of anti-mu exposure on fresh cells from 32 untreated CLL cases. Cell viability was assessed both by MTT assay and Annexin V/ IP labelling. Culture conditions were set after time and dose experiments. A 25% increase of viability, obtained by MTT assay after 72h of culture, was considered as significant. When a coated rabbit anti-human IgH mu specific antibody was used, it significantly prevented apoptosis in 20/32 cases (named responders) and never increased it. On the same CLL cases, soluble goat F(ab’)2 anti-human IgH mu specific antibody increased apoptosis in all but one case. However, coating of this latter antibody to the culture plate cancelled the pro-apoptotic effect and restored cell survival promoting effect in 13/14 cases tested. Unspecific effects were ruled out using control antibodies. Cytometry experiments and confocal microscopy demonstrated that uncoated antibody was rapidly internalized and degraded. In responders cases, enhanced survival was supported by increased expression of early cell cycle protein cyclin D2 and cdk4. There was no subsequent cell cycle progression as shown by IP staining and absence of pRB phosphorylation most likely related to the lack of p27 down regulation. There was a strong correlation between the extent of apoptosis prevention after anti-mu exposure and disease progression. Indeed, the 12 over 32 cases who were found to have no IgVH somatic hypermutations, happened to be all responders. Conversely among the 20 mutated cases, 12/20 were non responders and had a clinically stable disease. As for the 8 responders with somatic mutations, four had detectable levels of ZAP 70 protein and 2 others had a clinically progressive disease. In conclusion, coated anti-mu antibody is likely to mimic in vivo conditions of stimulation, suggesting that BCR ligation could play a major role in the pathophysiology of the progressive disease, giving a survival advantage to these CLL cells
Individualized Once-Daily Dosing of Intravenous Busulfan: A Pharmacokinetic Study in Patients Undergoing Conditioning for Allogeneic Stem Cell Transplantation.
Authors: Kangarloo Shabal B. , Naveed Farrukh , Andersson Borje S. , Quinlan Diana , Russell James A.
Published: October 2019

Background: Busulfan (Bu) given in myeloablative doses is a frequent component of preparative regimens for hematopoietic stem cell transplantation. Variations in the area under the concentration/time curve (AUC) for oral Bu may result in over/under dosing, which may increase the risk of toxicity or reduce efficacy. The availability of an IV formulation of busulfan reduces this by eliminating variability in absorption but inter-patient differences in metabolism remain. A pharmacokinetically guided test dose strategy before the high dose Bu may be used to achieve a specific target AUC.

Purpose: To establish a reliable model to predict a therapeutic dose of IV Bu from a small test dose in order to improve inter-patient variability in AUC.

Methods: Pharmacokinetic (PK) analysis was performed on 35 paired patient samples, comparing a limited sampling test dose to the therapeutic high dose. For the test dose, an AimPlus infusion pump was used to administer 12 mg of IV Bu over a 20-minute infusion on day −6. High dose Bu was given at a dose of 3.2mg/kg daily over 4 hours on days −5 to −2. The test dose PK parameters were compared to the high dose IV Bu PK parameters on day −3. Busulfan concentrations in plasma were determined by UV-HPLC. All concentration-time plasma Bu data were analyzed by non-compartmental analysis using WinNonlin Version 4.1 software (Pharsight Corporation, Mountain View, CA, USA).

Results: The mean (CV%) PK parameters for the 12 mg test dose patients were as follows: Cmax, 0.36 ug/mL (30.4%); clearance (CL), 16.3 L/hr (21.4%); volume of distribution Vd, 57.4 L (26.1%); elimination half-life, 2.4 hr (16.1%); and AUC, 199 μM.min (23.6%). The mean (CV%) Cmax, CL, Vd, half-life, and AUC for the once daily high dose were 3.95 ug/mL (22%), 11.7 L/hr (25.4%), 46.3 L (25.3%), 2.8 hr (15.6%), and 5190 μM.min (22.7%) respectively. The range of AUC for the high dose was 2832 to 7354 μM.min. The ratio of the high dose over the test dose required a correction factor “k” of 1.45 to be equivalent to the ratio of the AUC high dose over the AUC test dose.

Conclusion: Dosing based on patient weight results in 2–3 fold variability of AUC. The fact that a correction factor based on the test dose is needed is most likely due to the statistically significantly higher CL of busulfan during the test dose as compared to the high dose CL. This raises questions concerning the proposed linear kinetics of IV busulfan. However using the above correction factor and given a target therapeutic AUC, it is possible to individualize the therapeutic dose for IV busulfan based on this test dose strategy.
Expression of P73 Splice Variants Correlates to Resistance to DNA Damaging Drugs in Childhood T-Lineage Acute Lymphoblastic Leukemia.
Authors: Meier Marrit , Den Boer Monique L. , Meijerink Jules P.P. , Passier Monique , Van Wering Elisabeth R. , Janka-Schaub Gritta E. , Pieters Rob
Published: October 2019

Children with T-lineage Acute Lymphoblastic Leukemia (T-ALL) have a higher relapse-risk and are in-vitro more resistant to therapeutic drugs compared to ALL patients with a precursor-B phenotype. Cellular resistance to anti-cancer agents has previously shown to be associated with failure of P53 family member signaling by abrogation of P53 function due to loss-of-function mutations or dominant-negative inhibition by isoforms of P73 lacking (part of) the N-terminal transactivation domain (P73ΔEX2, P73ΔEX2/3, ΔN-P73 and ΔN’-P73). Since p53 mutations are not commonly found in T-ALL, we investigated the expression levels of p73 splice variants in relation to drug resistance in children with T-ALL. Splice variants were quantitatively measured at the mRNA level in leukemic cells of 55 T-ALL patients and mononuclear cells of 12 non-leukemic controls. TA-p73 (transactivation competent), p73Δex2, p73Δex2/3, ΔN-p73 and ΔN’-p73 were all found to be present at a relatively higher mRNA level in T-ALL patients than controls (P < 0.05 for all), suggesting that expression of the TP73 gene is deregulated in T-ALL. Resistance of T-ALL cells to the DNA damaging drug daunorubicin correlated with mRNA levels of the dominant-negative variants of p73, i.e. ΔN-p73 and ΔN’-p73 (Rs = 0.38, P = 0.03). In contrast, expression of none of the variants, including ΔN-p73 and ΔN’-p73, was related to resistance of T-ALL cells to non-DNA damaging drugs (prednisolone, vincristine and L-asparaginase). In conclusion, high expression of ΔN-p73 and ΔN’-p73 variants possibly contributes to resistance to DNA damaging drugs in childhood T-ALL.
Oncogenic TLS-ERG Fusion Protein Promotes Differentiation of Myeloid Progenitors into Myeloblasts but Blocks Their Terminal Differentiation.
Authors: Zou Junhui , Zielinska-Kwiatkowska Anna , Blackburn Michael L. , Ichikawa Hitoshi , Yang Liu
Published: October 2019

In a subset of acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) in blast crisis, the TLS (translocation liposarcoma) gene is fused to the ERG (ets-related gene) gene through a recurrent t(16;21) translocation. This chromosomal translocation results in the generation of the TLS-ERG fusion protein. More recently, the same TLS-ERG fusion protein has also been identified in Ewing’s sarcoma in children. To investigate the oncogenic mechanism of TLS-ERG, we stably expressed TLS-ERG or its mutant TLS-ERGΔets in mouse L-G myeloid progenitor cells through retroviral transduction. L-G cells normally proliferate in the presence of IL-3 and undergo terminal differentiation into hypersegmented neutrophils when treated with G-CSF. In contrast, expression of TLS-ERG in L-G cells led to spontaneous differentiation into immature myeloblasts but G-CSF treatment of these cells failed to induce terminal differentiation. L-G myeloblasts harboring TLS-ERG were characterized by high levels of myeloperoxidase (MPO), a phenomenon also observed in immature myeloblasts isolated from human leukemia patients with the t(16;21) translocation. As deletion of the ets DNA-binding domain from TLS-ERG abolished its ability to promote spontaneous myeloblast differentiation or the ability to up-regulate the MPO gene, we further examined how TLS-ERG fusion protein might affect the functions of other regulators such as PU.1, an ets transcription factor known to play a critical role in the differentiation of myeloid progenitors. Though both proteins have similar DNA-binding property, TLS-ERG differs from PU.1 through binding directly to RNA polymerase II as well as through escaping repression by histone modification enzymes. Together, these findings suggest that the TLS-ERG fusion protein can cause blockage of myeloid differentiation by interfering with the gene expression program normally controlled by key regulators.
Single or Tandem Autologous Stem-Cell Transplantation Given According to Prognostic Factors at Relapse for Hodgkin Lymphoma.
Authors: Brice Pauline , Morschhauser Franck , Divine Marine , Ferme Christophe , Salles Gilles
Published: October 2019

Patients with relapsed hodgkin lymphoma (HL)have a different prognostic after high-dose therapy (HDT)according to time to relapse and extend of relapse. From 1995 to 1997, 48 patients with early relapse or refractory HL were included in a pilot study of tandem transplantation to evaluate the feasibility before the protocol. From 1998 to 2002, 200 patients with refractory disease or first relapse of HL were prospectively treated with induction chemotherapy followed by HDT and autologous stem-cell transplantation (ASCT). Patients were stratified in 2 groups according to prognostic factors at relapse: groupe 1 (unfavorable relapse: primary refractory disease or early/disseminated relapse) and group 2 (favorable relapse: patients with either early or disseminated relapse). Induction chemotherapy consisted of ifosfamide/etoposide with doxorubicin (IVA) in 70% of patients or with vinorelbine and mitoguazone (MINE)for the remainings. Group 1, patients received 2 cycles of chemotherapy, PBSC collection and tandem ASCT, with a CBV mitoxantrone (30 mg/m2) and 2 months later cytarabine (6g/m2), melphalan (140mg/m2)with total body irradiation (40%) or busulfan (12 mg/kg) followed by the second ASCT.

Group 2, patients received 3 cycles of chemotherapy, PBSC collection and a BEAM regimen followed by ASCT. Final results, updated, January 2005 are presented with 245 evaluable patients.

Results: after induction chemotherapy overall response rate was at 61% in group 1 and 96% in group 2. In group 1, 70% received the two ASCT, the major reason not to receive the procedure was disease progression after induction chemotherapy (10%) or after the first ASCT (15%), than low stem-cell collection (4%) or toxicity (2%). In group 2, 97% of patients received ASCT and 1 patient received a tandem ASCT for refractory relapse. 5 patients died from toxicity in group1 and none in group2, but 2 secondary leukemia were observed in this group. In intent to treat analysis, at a 3 years median follow-up from the relapse, the EFS was at 45% in group 1 versus 75% in group 2 and the survival at 55% in group 1 versus 80% in group 2. Despite a different consolidation between group 1 and 2, results remained better in group 2. In conclusion, these results confirmed the importance of prognostic factors at relapse of HL.No differences were found in the unfavorable group 1 between refractory patients and early/disseminated relapse. HL patients with adverse prognostic factors (group 1) but responding to second line chemotherapy and eligible for tandem ASCT may benefit from this procedure with an EFS at 70%. The major cause of failure was chemoresistance either at induction or after high-dose therapy.
Killer Cell Immunoglobulin-Like Receptor Genotype and HTLV-1 Associated Disease’s Susceptibilities.
Authors: Obama Kosuke , Furukawa Yoshitaka , Tara Mitsutoshi , Marshall Sara , Levoguer Alison , Saito Mineki , Bangham Charles , Osame Mitsuhiro
Published: October 2019

[Introduction] Natural killer (NK) cells show cytotoxicity against virus-infected cells and some tumor cells. The highly polymorphic killer cell immunoglobulin-like receptors (KIRs) which recognize HLA class 1 molecules play a major role in these cytotoxic activities, and also express on some T-cells. Human T-lymphotrophic virus-1 (HTLV-1) is a caucassive agent for leukemia and some autoimmune diseases. We investigated the correlations between the KIR genotype and susceptibilities to HTLV-1 associated diseases i.e. adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy (HAM).

[Materials and Methods] Fifty six HTLV-1 carriers, fifty one ATL patients and fifty seven HAM patients were recruited in this study. Genotyping of six KIR genes (3DL1, 2DL5, 2DS1, 2DS2, 2DS3 and 3DS1) was performed using PCR amplification with sequence specific primers (Martin et al. J Immunol 2002).

[Results and Conclusion] Frequencies of each KIR and genotype were analysed as described in Table. Genotype means the pattern of positive KIR combinations. Significant differences between carrier vs. HAM in genotype 2 (p=0.01), and ATL vs. HAM in genotype 4 (p=0.01) were observed. Increased frequencies of genotype 3 and 2DS3 allele in HAM compared to carrier were also observed although these tendencies were not statistically significant. These preliminary results suggest that KIR gene polymorphism may play a role in determining susceptibility to HTLV-1 associated diseases.

KIR genetype and HTLV-1 associated diseases Genotype 3DL1 / 2DL5 2DS1 / 2DS2 2DS3 / 3DS1 Carrier (%) * ATL (%) * HAM (%) * * this measns the genotype frequency; Y, positive; N, negative 1 Y / N N / N N / N 51.8 47.1 63.2 2 Y / Y Y / N N / Y 26.8 21.6 8.8 3 Y / Y Y / N Y / Y 1.8 7.8 10.5 4 Y / N N / Y N / N 5.4 11.8 0 5 Y / Y N / Y Y / N 1.8 2.0 7.0 6 N / Y Y / N N / Y 1.8 2.0 3.5 7 Y / Y Y / Y N / Y 1.8 3.9 1.8 Others 8.9 3.9 3.5 Carrier (%) 96.5 / 42.9 39.3 / 16.1 10.7 / 35.7 ATL (%) 96.1 / 41.2 39.2 / 21.6 9.8 / 39.2 HAM (%) 94.7 / 36.8 29.8 / 14.0 22.8 / 28.1
Plasma Brain Natriuretic Peptide, Serum Troponin T and Echocardiographic Evaluation in Children Treated with Doxorubicin*.
Authors: Jackowska Teresa , Wasilewski Robert , Wasik Maria , Golabek Malgorzata , Matysiak Michal
Published: October 2019

Background: Doxorubicin (DOX) is one of the most effective agents in treatment of acute lymphoblastic leukaemia (ALL). However, a potential heart damage caused by DOX in patients who have been cured of ALL may lessen the success of their cure. Plasma brain natriuretic peptide levels and cardiac Troponin T are highly sensitive biochemical markers for myocardial damage.

Objectives of the study: We aimed to measure N-terminal proBNP (8–29) (Nt-proBNP) and serum Troponin T(cTnT) levels in 125 children (98 with ALL after completion of treatment with DOX and 27 healthy children). Median age at diagnosis was 5.5 years.

Methods used: Concentration of plasma N-terminal proBNP was measured in children by enzyme immunoassay (Biomedica). cTnT levels were measured by an electrochemiluminescence immunoassay process (Third generation) on the Elecsys 1010 System (Roche Diagnostic). Echocardiograms were performed by paediatric cardiologists. Using two-dimensional M-mode echocardiography shortening fraction (%SF) and ejection fraction (%EF) were determined as systolic function.

Results: The cumulative DOX doses were 240mg/m2. None of the patients had clinical signs or symptoms of cardiotoxicity. All of the patients had normal systolic function parameters. Mean Nt-proBNP plasma levels were normal − 64 fmol/ml (10.3–597.3 fmol/ml) in all examined children. Mean Nt-proBNP both in ALL patients (63.8 fmol/ml) and in healthy (58.4 fmol/ml) were within the normal range. Out of the 98 patients 45 (46%) had slightly elevated Nt-proBNP levels in comparison with healthy subjects. Nt-proBNP plasma levels were not significantly different in boys (55.8 fmol/mL) in comparison with girls (56.3 fmol/mL. In all patients the serum levels of cTnT were below the detection limit (<0.010 ng/ml).

Conclusion: Our preliminary results suggest that measurement of Nt-proBNP plasma levels is useful in the detection of subclinical left ventricular dysfunction in children with ALL receiving DOX therapy.
Hematological Responses to Megesterol Acetate in Patients with Myelodysplastic Syndrome Involving an Abnormal Chromosome 6.
Authors: Bourg Wilson C. , Launder Theresa M. , Mughal Tariq I.
Published: October 2019

The presence of an abnormal chromosome (chr) 6 often confers a poor prognosis on patients with myelodysplastic syndrome (MDS), particularly therapy related-MDS (t-MDS). We report two patients with MDS and an abnormal chr 6 who achieved significant improvements in their hematological parameters after being subjected to megesterol actetate at 80mg/d; one patient achieved a complete cytogenetic remission, albeit based on a limited study. The first patient, a 73 y female presented with a monocytopenia (thrombocytopenia; 43K/dl), a bone marrow consistent with MDS, blasts 6% and an abnormal chr 6 (sole cytogenetic abnormality) in February 2003. In July 2005 she was asymptomatic with a platelet count of 353K/dl and is due to have cytogenetics shortly. The second patient, a 72 y female presented in December 2004 with trilineage cytopenias, a bone marrow consistent with MDS and a complex karyotype, including an abnormal chr 2, 5, 6, 7 & 16. She had had a history of primary (essential) thrombocythemia of 25 y duration and had received P 32 and intermittent courses of hydroxyurea and was on hydroxyurea at the penultimate presentation and probably had t-MDS. This therapy was promptly discontinued and megesteral acetate commenced at 80mg/d. In July 2005 her hematological parameters had improved significantly (W 3.3K/dl; Hb 11.3g/dl;Platelets 135K/dl); her bone marrow remained hypercellular with reticulin fibrosis and cytogenetic studies revealed a 46XX kayotype in a limited sample. At the time of this report, both patients remain well on megesterol acetate with normal or near normal hematological parameters. MDS is a heterogeneous stem cell disorder and hematological and possible cytogenetic responses are noteworthy. The patient with the sole abnormal chr 6 has remained in hematological remission for over 18 months; the second patient with the complex karyotype is also, remarkably, maintaining near normal hematological parameters. We speculate that megesterol acetate, a relatively inexpensive progestational agent with a defined palce in the management of malignancy-related anorexia may have a role in selected patients with MDS. Updated observations and suggested hypotheses will be presented.
Velcade an Active Agent for Multiple Myeloma Patients. Experience of a Single Center.
Authors: Petrucci Maria Teresa , Martini Vincenza , Gallucci Cristiano , Finolezzi Erica , Levi Anna , Del Bianco Patrizia , Mandelli Franco , Foà Robert
Published: October 2019

Velcade (PS-341, Bortezemib), a proteasome inhibitor, has recently been approved for the treatment of relapsed and refractory multiple myeloma (MM). We hereby report the results obtained using this new drug at our center. Between April 2003 and December 2004 we treated 23 patients with MM. The patient population consists of 13 males and 10 females, with a median age of 53 years (range 32–72); 16 were IgG MM, 6 IgA, 1 light chain. All patients were in stage III of their disease, with a median time of observation (from diagnosis to Velcade therapy) of 59 months (range 11–120), were pretreated with at least two lines of therapies and were refractory or in relapse after the last treatment. Velcade 1.3 mg/m2 was scheduled on days 1, 4, 8 and 11 of a 21-day treatment cycle for 8 cycles according to the tolerability and response. All cycles were delivered in day hospital. A median of 6 cycles (range 3–8) were administered to the overall population. Eleven patients concluded their program, while 12 discontinued the treatment: 1 because she received an allogeneic stem cell transplantation, 7 for adverse events and 4 due to disease progression. In this heavily pretreated population, our primary end point was to obtain a decline in the monoclonal component (MC) of at least 25%. All patients were considered evaluable for response because treated with at least 3 cycles of therapy. Thirteen patients responded to treatment: 1 (4%) obtained a disappearance of the MC, 4 (17%) achieved a reduction of the MC level >75% and 8 (34%) >50%. Six further patients (26%) obtained a decline in the MC <25%. The median number of cycles required to achieve a response was 3 (range 1–8) and the median duration of response was 6 months, with 8 patients presently still responsive. Among the responding patients, 5 relapsed and 4 of them have died due to disease progression. Among the 4 patients (17%) who did not respond to Velcade, 1 died. The majority of adverse events, which resolved with the discontinuation of treatment, were nausea, vomiting, diarrhea, fatigue, thrombocytopenia and peripheral neuropathy. Taking into consideration the very poor prognostic likelihood of our patients, this study adds further evidence con the efficacy of this new drug in MM. Velcade can be considered an effective anti-myeloma drug even though its toxicity must be taken into account in designing new clinical trials.
Identification of RPS14 as the 5q-Syndrome Gene by RNA Interference Screen.
Authors: Ebert Benjamin L. , Pretz Jennifer , Bosco Jocelyn , Chang Cindy Y. , Tamayo Pablo , Galili Naomi , Raza Azra , Root David , Attar Eyal , Ellis Steven R. , Golub Todd R.
Published: October 2019

We report here that haploinsufficiency of the ribosomal protein encoding RPS14 gene causes the characteristic hematologic phenotype that defines the 5q- syndrome. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), was first described in 1974 as a distinct clinical entity associated with deletions of the long arm of chromosome 5. Since that time, the common deleted region has been narrowed in search of the causal gene within the region. However, no biallelic inactivation events have been identified for any of the 41 genes within the critical region, suggesting that a haploinsufficiency mechanism may explain the disease phenotype. We addressed this question through a functional genomic approach, utilizing systematic RNA interference (RNAi) to interrogate the function of each gene in the common deleted region. We introduced 3–5 unique, lentivirally expressed short hairpin RNAs (shRNAs) targeting each of the 41 genes in the region into normal CD34+ human bone marrow hematopoietic progenitor cells and assayed the effects of each shRNA on hematopoietic differentiation. We found that knock-down of a single gene, RPS14, recapitulated the phenotype of the 5q- syndrome: namely, a block in erythroid differentiation (leading to erythroid cell apoptosis) with relative preservation of megakaryocyte differentiation as measured by FACS analysis. Importantly, forced expression of an RPS14 cDNA in primary bone marrow cells from patients with the 5q- syndrome rescued the phenotype, yet had no effect on cells from MDS patients without 5q deletions. In addition, we found that RPS14 haploinsufficiency caused a block in the processing of pre-ribosomal RNA and in the formation of the 40S ribosomal subunit. This ribosomal processing defect is highly analogous to the functional defect seen in Diamond Blackfan Anemia (also characterized by an erythroid differentiation defect and predisposition to AML), thereby establishing an unexpected link between the molecular pathophysiology of acquired 5q- syndrome and congenital bone marrow failure syndromes. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function, highlighting the importance of translational control in hematologic malignancy. The results further suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.
PHA-739358, an Aurora Kinase Inhibitor, Induces Clinical Responses in Chronic Myeloid Leukemia Harboring T315I Mutations of BCR-ABL.
Authors: Paquette Ronald L. , Shah Neil P. , Sawyers Charles L. , Martinelli Giovanni , John Nicoll , Chalukya Meenal , Rocchetti Maurizio , Fiocchi Carina , Comis Sylvia , Capolongo Laura , Laffranchi Bernard
Published: October 2019

Background. The T315I BCR-ABL mutation confers high level resistance to clinically available ABL kinase inhibitors (i.e., imatinib, nilotinib, dasatinib, bosutinib, INNO-406). PHA-739358 is an aurora kinase inhibitor that selectively inhibits the ATP site of Aurora-A, Aurora-B and Aurora-C kinases, and binds with high affinity to both wild type Abl and Abl/T315I in vitro. A multicenter phase II study of PHA-739358 is being conducted in patients with chronic myeloid leukemia (CML) relapsing on imatinib or other c-ABL therapy.

Methods. Seven consenting CML patients (1 in chronic phase, 1 in accelerated phase, 5 in blast phase) were initially entered in this trial and received PHA-739358 dosed at 250 or 330 mg/m2/day administered by a once-weekly 6-hour infusion for 3 consecutive weeks, every 4 weeks. Six out of seven patients had the BCR-ABL T315I mutation. Pharmacokinetic and pharmacodynamic (PD) samples were collected and analyzed during cycle 1. The PD analyses evaluated BCR-ABL inhibition (phospho-CRKL) and Aurora kinase inhibition (phospho-histone H3).

Results. Two patients with T315I mutated BCR-ABL achieved a complete hematologic response (CHR) to PHA-739358. One of these patients treated in accelerated phase achieved a complete cytogenetic response (CCyR) and a complete molecular response after 3 months on the 330 mg/m2 dose level. The CCyR is ongoing after 6 months of treatment. The second patient who achieved a CHR initiated therapy in chronic phase. A minor cytogenetic response was achieved after 3 cycles of therapy at the 330 mg/m2, and a minimal cytogenetic response persists after nine months of treatment. Uncomplicated grade 4 neutropenia and an infusion-related reaction requiring acetaminophen, benadryl and hydrocortisone premedication was observed in one patient. No other grade 3/4 non-hematologic toxicity was observed. Patients with blast crisis did not have CHRs at the dose and schedule employed, although suppression of peripheral blood blasts was evident. PHA-739358 pharmacokinetics were in good agreement with those observed in other phase I - II studies. Average exposure, AUC0-t(168h), observed at 330 mg/m2/day was about 45 uM/h with Cmax values around 4 – 6 uM/h. Pharmacodynamic analyses demonstrated treatment-associated decreases of CRKL phosphorylation in 6 out of 7 patients, including both responders. Modulation of histone H3 phosphorylation was observed in 3 of 5 evaluable patients.

Conclusions. Objective clinical responses to PHA-739358 have been observed in two CML patients with T315I mutations of BCR-ABL, with an acceptable tolerability and safety profile. Additional doses and schedules of PHA-739358 are being investigated in patients with advanced phase CML.
The Predictive Value of Gene Expression Profiles for Acute Graft-Versus-Host Disease after Hematopoietic Cell Transplantation with Nonmyeloablative Conditioning for Hematological Malignancy.
Authors: Masmas Tania N. , Friis-Hansen Lennart , Johansen Jens Vilstrup , Petersen Soren Lykke , Kornblit Brian T. , Winther Ole , Nielsen Finn Cilius , Vindelov Lars
Published: October 2019

Purpose: To test the hypothesis that global gene expression profiles of peripheral blood mononuclear cells (PBMNC) day +14 after hematopoietic cell transplantation with nonmyeloablative conditioning could predict the later occurrence of acute graft-versus-host disease (GVHD) grade II–IV.

Material: Between March 2000 and Marts 2006, 100 patients with hematological malignancies received peripheral blood stem cells from an human leukocyte antigen identical sibling/mother donor or from a matched unrelated donor following nonmyeloablative conditioning with low dose fludarabine and 2 Gy of total body irradiation. Post-transplant immunosuppression consisted of cyclosporine and mycophenolate mofetil. Only patients with sustained engraftment, who did not experience late-onset acute GVHD after day +100 were included; eight patients were excluded due to graft rejection, three patients due to suboptimal RNA or lacking PBMNC samples, and further 15 patients due to late-onset acute GVHD. Seventy-four patients were then eligible for microarray analysis.

Methods: RNA was precipitated from frozen PBMNC from day +14 post-transplant and gene profiling analyses were performed using Human Genome U133 Plus 2.0 GeneChip Array. The array data were normalized, RMA modelled and asinh transformed in R. The differentially regulated gene expression between the group of patients developing acute GVHD before day +40, +56 and +84 post-transplant compared to the patients never experiencing acute GVHD was identified and formed the basis for the subsequent principal component analysis (PCA) and classifying models. No patients experienced acute GVHD between day +85 and +100 post-transplant.

Results: The patients experiencing acute GVHD by different time points were separated from the patients never experiencing acute GVHD by the PCA plot. Furthermore the classifying models could separate the groups correctly in up to 93% of cases in the best of the classifying model. In addition, differentially regulated genes between the two groups were identified.

Conclusion: These data suggest that the pattern of gene expression profiles early post-transplant is able to predict patients with a high risk of later occurrence of acute GVHD from those never experiencing acute GVHD. This knowledge could be exploited to increase the immunosupression and thus prevent acute GVHD in patients at risk. Furthermore, candidate genes of interest for the pathogenesis of acute GVHD have been identified.
Impact of FLT3 Internal Tandem Duplication (FLT3/ITD) on the Outcome of HLA-Identical Sibling Hematopoietic Transplantation for Adult Acute Myeloid Leukemia (AML) in First Remission: Substantial Probability of Leukemia-Free Survival Despite Increased Relapse Risk. A Retrospective Analysis of the EBMT-ALWP.
Authors: Brunet Salut , Labopin Myriam , Cornelissen Jan J. , Socié Gérard , Iori Ana Paola , Verdonck Leo F. , Ruutu Tapani , Gratwohl Alois , Harousseau Jean-Luc , Sierra Jorge , Rocha Vanderson
Published: October 2019

The prognosis of patients with AML and FLT3 internal tandem duplication (FLT3/ITD) is poor. It is unclear whether this mutation has impact on the outcome of allogeneic stem cell transplantation (Allo-SCT) for AML. Different cooperative groups have obtained controversial results. For this reason, registry data including large numbers of patients are of interest. We analysed the predictive value of FLT3/ITD on relapse incidence (RI), non-relapse mortality (NRM) and leukemia-free survival (LFS) in patients with AML in 1st CR who underwent a myeloablative allo-SCT from HLA-identical siblings and were reported to the EBMT. The series included 682 patients, 610 negative ITD/FLT3 and 72 FLT3/ITD positive reported between 1998 and 2007 with a median follow up time of 24 and 11 months, respectively. No significant differences were observed between the two groups regarding gender, median age (38 vs 40 yrs), FAB classification, cytogenetic distribution, number of induction courses to achieve CR, interval diagnosis to transplant (134 vs 133 days), source of stem cells (PB or BM) and CMV serostatus. In contrast, patients with FLT3/ITD had higher leukocyte counts at diagnosis (14 vs 65 × 109/L, p<0.0001), were transplanted with T-cell replete progenitors (78% vs 97%, p<0.0001) and more frequently a female donor to male recipient (yes: 31% vs 20%, p=0.04). Univariate (Table1) and multivariate analyses demonstrated the adverse impact of FLT3/ITD for LFS (HR 0.56, p=0.009) and relapse incidence (HR=2.9, p<0.001) but not for NRM (p=0.83). It is remarkable, however, 53% of FLT3/ITD positive patients transplanted for AML in first CR remain alive and disease-free at 2 years. Other independent prognostic factors for LFS and relapse were: T-cell depleted graft, poor cytogenetics and more than one induction course to achieve first CR. For NRM the only prognostic factor was age ≥ 45 years (HR=1.9, p=0.004). In summary, this analysis shows an adverse impact of FLT3/ITD on transplantation outcome. Despite this finding, half of FLT3/ITD positive AML patients in first CR remain disease-free at 2 years, a proportion that seems higher than with other treatment options.

Table 1. Two years outcomes of HSCT in adults patients with AML in CR1 according to the presence of FLT3/ITD. LFS Relapse NRM FLT3/ITD+ (n=70) 53±7% 36±7% 17±7% FLT3/ITD-(n=610) 64±2% 20±2% 19±2% P (log rank test) P=0.06 P<0.0001 P=0.49
Allogeneic Stem Cell Transplantation Can Be an Effective Post-Remission Strategy for Acute Myeloid Leukemia Patients Aged Less Than 60 Years with Core Binding Factor: A Nation-Wide Retrospective Study in Korea.
Authors: Shin Ho-Jin , Chung Joo Seop , Kim Hyioung-Joon , Sohn Sang Kyun , Min Yoo Hong , Won Jong-Ho , Kim Inho , Lee Je-Hwan , Lee Jae Hoon , Jung Chul Won , Ahn Jin Seok , Lee Kyoo-Hyung , Joo Young Don
Published: October 2019

High-dose cytarabine (HDAC)-based postremission therapy has been generally considered to be more beneficial than allogeneic stem cell transplantation (alloSCT) for AML patients with core binding factor (CBF). However, advancements in methods and supportive care in SCT over decades have led to gradual improvement in the treatment-related mortality rate after alloSCT. Therefore, it could be considered that alloSCT after the first CR in AML with CBF patients may show better results than HDAC chemotherapy alone compared with past. We conducted a retrospective study in 169 patients with CBF AML aged less than 60 years collected in 18 institutions in Korea between 1994 and 2006, with comparing alloSCT (n= 60), autoSCT (n= 31) and HDAC chemotherapy (n= 78) as postremission therapy during first remission. With a median follow-up of 46 months, the probability of disease-free survival (DFS) and overall survival (OS) rates at 5 years were 67.8% and 66.6%, retrospectively. AlloSCT (83.1% ± 5.2%) shows a significantly improved 5-year probability of DFS over autoSCT (59.9% ± 9.6%) or HDAC (60.8% ± 6.2%; p=0.048). There was no differences in 5-year probability of OS from complete remission (CR) between 3 treatment arms. In comparison of DFS and OS according to period of enrollment of patients, 4-year probability of DFS or OS of the three groups prior to 2003 was not different, nonetheless, after 2003, 4-year probability of DFS was better in the group underwent alloSCT in comparison with the group without performing it (86.8% vs 70.0%; p=0.035), and in the group underwent SCT (alloSCT plus autoSCT) in comparison with the group without performing it (89.0% vs 67.3%; p=0.047). 4-year probability of OS has a tendency to be improved slightly in the group underwent SCT than the group without performing it (89.0% vs 67.3%; p=0.08). In comparison with prior to 2003, cumulative treatment-related mortality and relapse risk of alloSCT after 2003 was decreased to 13.5% vs 6.7% and 26.2% vs 6.9%, respectively, and depending on the result, 4-year probability of DFS and OS of the alloSCT showed the improvement from 73.9% and 63.3% prior to 2003 to 93.1% and 90.0% after 2003, respectively. This study has the limitation of retrospective study, nonetheless, it could suggest the possibility that alloSCT as a postremission therapy in AML with CBF patients after the first CR may be appropriate if a HLA matched sibling donor were available.
Description of a Large Duplication in F8 Gene Responsible for Severe Hemophilia A.
Authors: Vinciguerra Christine , Goudemand Jenny , Zawadzki Christophe , Pellecchia Dorothee , Negrier Claude
Published: October 2019

Hemophilia A is a common inherited bleeding disorder, caused by factor VIII (FVIII) deficiency as a result of mutations in the factor VIII gene (F8 gene). Intron 1 and 22 inversions, small insertions/deletions and point mutations are the most common genetic defects responsible for severe hemophilia A. However, a F8 gene mutation is not found in 2–5% of patients with severe hemophilia A (FVIII:C<1IU/dL). Large rearrangements are frequent in other genetic disorders and only one case of exon 13 duplication was described in a patient with mild hemophilia (Casula et al, Blood 1990). We described here a large duplication in F8 gene in a CRM− patient with severe hemophilia A: (FVIII:C < 1 UI/dL, FVIII:Ag < 1%) who developed a high-titer inhibitor (peak 10 BU). A previous investigation did not find intron 1 and 22 inversion or exons /splice sites sequencing abnormalities. The promoter region was also sequenced, but no genetic mutation was then characterized. The duplication was detected by MP/LC (Multiplex PCR/Liquid chromatography) which is a quantitative method able to detect large rearrangements. Initially described by Dehainault et al (Nucl Acids Res 2004) in retinoblastoma patients, this method showed that a duplication affected a large part of F8 gene, e.g. the 3′ part of intron 10, exons 11 to 14 and a part of intron 14. To our knowledge, this is the first duplication responsible for severe hemophila A described so far. This finding suggests that the detection of large rearrangements with quantitative method as MP/LC, QMPSF or MLPA would be useful in hemophilic patients where the screening for inversions or genetic events in the coding regions are unsuccessful. This kind of large gene modifications may indeed account for at least some of the 2–5% cases where the coding sequence does not appeared to be altered. In this case, the truncation of the primary polypeptide sequence did not result in any secretion of the FVIII protein, that may have increased the risk of inhibitor development.
Decreased Bone Mineral Density among Patients with Hemophilia.
Authors: Gerstner Gregory , Damiano Mary Lou , Tom Angela , Worman Christina , Schultz Wendy , Recht Michael , Stopeck Alison T.
Published: October 2019

Background: Osteoporosis among adult males is a major and under-recognized problem in the United States. Patients with hemophilia have several predisposing risks for developing decreased bone mineral density (BMD) and osteoporosis, and may represent an important group to target for screening and treatment for fracture prevention.

Patients and Methods: Patients over the age of 18 with moderate or severe hemophilia A or B (as defined by factor activity < 5%) and no history of prophylactic factor use prior to age 10 were eligible. Bone mineral densities were obtained using DEXA scans (DXA) along with measurements of joint mobility and physical activity and laboratory parameters.

Results: Twenty-eight patients have been consented with accrual ongoing. 21 have undergone DXA scans. Median age of 39 (range 18–61), 86% HCV positive, 26% HIV positive. Median T-score for all sites (lumbar, femoral neck, hip, and other) was −1.7 (−5.8–0.6), with the most effected area being the femoral neck, T-score −1.7 (−5.8–0.8). Based on WHO criteria, 76% of patients had decreased BMD, 33% (n=7) with osteoporosis, and 43% (n=9) with osteopenia. Trends associated with decreased BMD included decreased serum 25-hydroxy-vitamin D levels, increased alkaline phosphatase, and decreased weight. All patients with osteoporosis were HCV positive, and all HIV positive patients had decreased BMD. Median activity scores were lower among osteoporotic patients vs normal BMD. Joint range-of-motion in the lower extremities was limited to 59.5% of predicted values in patients with osteoporosis, 84% in osteopenia, and 93% in patients with normal BMD.

Summary: Patients with hemophilia are at markedly increased risk for developing osteoporosis and osteopenia. Potential predictors of risk for decreased BMD are concurrent HCV and HIV infection, low vitamin D levels, elevated alkaline phosphatase, lower weight, decreased range of motion and lower activity scores. More aggressive screening for decreased BMD among moderate and severe hemophilia patients with initiation of therapy is appropriate.

Median Values Worst T-Score Activity Score (1–5) Joint ROM (% Pred) Weight (kg) 25-Hydroxy-D (ng/mL) Alk Phos (IU/L) Normal BMD 0.1 5 94.0 91.6 28.0 59 Osteopenia −1.6 4 85.0 80.7 23.0 86 Osteoporosis −3.0 3 68.5 73.0 21.8 99
Phase I Study of AVE1642 Anti IGF-1R Monoclonal Antibody in Patients with Advanced Multiple Myeloma.
Authors: Moreau Philippe , Hulin Cyrille , Facon Thierry , Boccadoro Mario , Mery-Mignard Dominique , Deslandes Antoine , Harousseau Jean-Luc
Published: October 2019

Rationale: CD221 (IGF-1R) is aberrantly expressed in multiple myeloma (MM) and is associated with disease severity (Bataille et al, Haematologica2005;90:706). IGF-1R is thus an attractive therapeutic target in patients with advanced disease.

Patients and methods: We have conducted an open-label dose escalation phase I study of AVE1642, anti IGF-1R monoclonal antibody, in patients with advanced MM. The primary objective was to determine the selected dose of AVE1642 administered every 3 weeks (q3w) based on pharmacokinetic (PK), pharmacodynamic (PD) parameters and dose limiting toxicities. The secondary objectives were to assess the safety profile, the biological activity (saturation of receptors) on peripheral granulocytes, the potential immunogenicity and preliminary clinical activity of AVE1642.

Results: 14 patients have been treated with AVE1642 as IV infusion administered q3w (day 1 = day 22) at 3 different dose levels: 3 (n = 4), 6 (n = 6) and 12 mg/kg (n = 4). A median number of 2 infusions (1–8) were administered. AVE1642 was well tolerated, except reversible grade 3 hyperglycemia observed in 2 diabetic patients. No hypersensitivity during infusion was reported. No human antibody anti AVE1642 was detected. One patient with Bence-Jones MM experienced a decrease in proteinuria and relief of bone pain. Based on PK/PD results, the dose of 12 mg/kg of AVE1642 has been selected for further clinical evaluation in MM patients. Based on the in vitro synergistic activity of AVE1642 + bortezomib (Descamps et al, ASH2006, 845a), we have started a combination trial of AVE1642, 12 mg/kg q3w + bortezomib (1.3 mg/m2 at d1, d4, d8 and d11 q3w) in patients with advanced MM.