bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical and Immunological Activity of WT1 Peptide Vaccination in Patients with Acute Myeloid Leukemia and Myelodysplasia: Results of a Phase II Trial.
Authors: Keilholz Ulrich , Letsch Anne , Busse Antonia , Asemissen Anne M. , Schmittel Alexander , Hofmann Wolf K. , Uharek Lutz , Blau Igor W. , Thiel Eckhard , Scheibenbogen Carmen
Published: October 2019

The transcription factor Wilms tumor protein (WT) 1 belongs to a new generation of tumor antigens, which are essential for tumor cell proliferation. WT1 is highly expressed in AML and in MDS upon appearance of blasts. A phase II trial of vaccination with the HLA-A2-restricted WT1.126–134 peptide was performed in patients with AML and MDS and overexpression of WT1 to determine immunogenicity and clinical activity. Patients received vaccinations with 0.2 mg WT1.126–134 peptide (day 3), 62.5 mcg dendritic cell-stimulating adjuvant GM-CSF (days 1–4) and 1 mg T helper protein keyhole limpet hemocyanin (day 3). The initial 13 patients were to receive 4 biweekly and subsequent 4-weekly vaccinations, the subsequent 13 patients were continuously vaccinated biweekly. Vaccination was continued in absence of overt disease progression. WT1 levels were assessed by quantitative RT-PCR and WT1-specific T cell responses by tetramer analyses and cytokine flow cytometry. Response assessment following IWG-MDS criteria was used, capturing stable disease and hematologic improvement. A duration of 8 weeks was required for stable disease. Enrolment was completed in June 2006 with 24 patients with AML and 2 with MDS (RAEB). Of the 24 AML patients, 16 had > 5% marrow blasts at study onset (8 without prior chemotherapy, 4 with disease persistence following chemotherapy, 4 with PR), and 8 were in CR at high risk for relapse. A median of 10 (range 4 – 23) vaccinations was administered with 8 patients currently still under treatment. No significant toxicity occurred. To date, 22 patients are evaluable for clinical response. Overall, 8/16 patients with > 5% marrow blasts at study onset displayed clinical efficacy of vaccine treatment (SD or better). One AML patient achieved CR for 12 months after brief initial progression, and 7 patients had disease stabilization (2, 2+, 3, 3, 6, 10+, 14 months). One of these patients with RAEBII had a major response of neutrophils and platelets, and one AML patient had initial progression and subsequent transient complete clearance of peripheral blasts. WT1 transcripts as molecular disease marker decreased at least 3-fold (range 3-fold - >50-fold, median >10-fold) in 12 of 20 currently evaluated patients, including all 8 patients with evidence of clinical efficacy and 4 of 5 AML patients vaccinated in CR. The generation of a WT1-specific T cell response in peripheral blood and bone marrow was detected in 12 of 16 evaluated patients including all 6 of these 16 patients with evidence for clinical efficacy. This study shows that WT1 vaccination has promising antileukemia activity. A multicenter comparative WT1 vaccination study in CR patients at high risk of relapse is currently initiated.
A Phase II Randomized, Double-Blind, Eight-Arm, Parallel-Group, Dose-Response Study of Apixaban, a New Oral Factor Xa Inhibitor for the Prevention of Deep Vein Thrombosis in Knee Replacement Surgery - On Behalf of the Apixaban Investigators.
Authors: Lassen Michael R. , Davidson Bruce L. , Gallus Alexander , Pineo Graham , Ansell Jack , Deitchman David
Published: October 2019

Background: Apixaban is an orally active, highly selective, reversible inhibitor of Factor Xa. It binds directly to the active site of Factor Xa without requiring anti-thrombin. Antithrombotic activity was evaluated in a dose-ranging study in patients undergoing total knee replacement.

Methods: Patients were allocated randomly to 1 of 6 double-blind doses of apixaban (5, 10, or 20 mg) given as a single or a twice-daily divided dose, or enoxaparin 30 mg bid, or open-label warfarin titrated to an INR of 1.8–3.0. Apixaban and enoxaparin were started 12 to 24 hours after completing surgery; warfarin the evening of the day of surgery. Treatment continued for 10–14 days. Mandatory bilateral venography was performed at the end of treatment. Patients could then be treated with non-study drug prophylaxis according to the attending physician’s discretion. All patients were seen at follow-up on day 42. The primary efficacy outcome was a composite of all venous thromboembolic events (VTE) comprising asymptomatic and symptomatic deep vein thrombosis (DVT), pulmonary embolism (PE) and all-cause death during the treatment period. A central independent committee whose members were unaware of treatment assignments adjudicated all efficacy outcomes and bleeding events.

Results: Of 1217 subjects treated and included in the safety analysis, 856 were eligible for the efficacy analysis. The composite VTE plus all cause death rate in the apixaban groups combined was significantly lower than the rate in the enoxaparin (p<.02) and warfarin (<.001) groups (8.6%; 15.6%; 26.6%, respectively). A trend for a dose-related decrease (p=.09) in the composite VTE plus all cause death rates of 10.6%, 8.6%, and 6.8% was apparent when QD and BID apixaban doses were combined as total daily doses of 5, 10, and 20 mg, respectively. When QD and BID apixaban doses were assessed separately, dose-dependent decreases in the composite VTE plus all cause death rates were observed for each but did not reach statistical significance. The composite VTE plus all cause death rates for apixaban 2.5 mg BID and 5 mg QD were 9.9% (95% CI:5.1–17.0) and 11.3% (95% CI:5.8–19.4) respectively, compared with a rate of 15.6% (95% CI: 9.4–23.8) in the enoxaparin group. For the composite outcome of proximal DVT+PE+all-cause death, each apixaban group had a lower event rate (0–2.7%) than the enoxaparin rate (4.6%). The incidence of major bleeding for apixaban ranged from 0.0% (2.5 mg BID) to 3.3% (20 mg QD); no major bleeding was observed in either the enoxaparin or warfarin groups

Conclusions: Apixaban, an oral Factor Xa inhibitor, demonstrates a favorable efficacy and an acceptable bleeding safety profile at doses of 5 to 20 mg per day in the prevention of VTE after knee replacement surgery.
Prestorage Leukoreduction of Transfused Red Cells Is Associated with Significant Ongoing 2–12 Month Survival Benefit in Cardiac Surgery Patients.
Authors: Boshkov Lynn , Chien Grace , VanWinkle Donna , Furnary Antony P. , Wu Yingxing , Grunkemeier Gary , Morris Cynthia D.
Published: October 2019

Introduction: Red cell (RBC) transfusion (vs no transfusion) in cardiac surgery is associated with higer operative mortality and shortened long-term survival. Studies have suggested use of leukoreduced RBCs vs standard RBCs in transfused cardiac surgery patients is associated with improved short term (60 day) survival. It is unclear if this benefit persists longer term. Study Aim: This prospective, randomized, double-blind, 3-center trial compared short term and intermediate-term mortality (2–12 mos) in adult cardiac surgery patients undergoing cardiopulmonary bypass (CPB) randomized to receive either prestorage leukoreduced RBCs (LR-RBCs) vs standard RBCs (S-RBCs)

Methods: 1226 cardiac surgery patients (undergoing coronary artery bypass grafting, cardiac valve replacement, or a combination of the two) were pre-operatively randomized to receive either LR- or S-RBCs. Patients were operated on by the same group of surgeons. Patients and clinicians were blinded as to product type. All deaths were verified by National Death Index records.

Results: 562 patients (45.8%) were transfused: 304 received LR-RBCs and 258 S-RBCs. Groups were statistically equivalent demographically and by all Society of Thoracic Surgery risk criteria. Operative mortality was not affected. However a survival benefit for the LR-RBC group was evident at 60 days which persisted for 12 months (see Table 1). Mortality for non-transfused patients was significantly lower than for patients receiving either LR- or S-RBCs at all time points (p<0.0001).

Conclusion: In adult cardiac surgery patients undergoing CPB who require transfusion, use of LR-RBCs vs S-RBCs is associated with significant improvement in survival at 2 months, and this survival advantage persists for up to 1 year.

Table 1: Percent (%) Mortality--Operative (Op) and Cumulative by Month (mo) Post-operative Op 2 mo 4 mo 6 mo* 9 mo* 12 mo* *Kaplan-Meier (KM) analysis LD-RBCs 4.9 4.9 5.3 5.9 5.9 7.0 S-RBCs 7.0 9.7 9.7 10.1 11.3 11.7 p-value 0.305 0.029 0.044 0.070 0.025 0.053
Modification of Anemia Parameters after Blood Transfusion.
Authors: Berz David , Singh Himani , McCormack Elise , Mazur Eric
Published: October 2019

Background: Optimally the necessary laboratory evaluation for the diagnosis of the underlying causes for anemia is done before blood transfusion. However, clinical and logistical demands often mandate the transfusion of packed red blood cells before all the necessary parameters are obtained. It is poorly defined which impact transfusion has on the parameters necessary for the diagnosis of the underlying anemia cause. We conducted a prospective study to investigate the effect of the transfusion of two units packed red blood cells on lactate dehydrogenase(LDH), ferritin, transferrin, haptoglobin, total iron binding capacity(TIBC), folate, cyanocobalamine(B12), hemoglobin, MCV and hematocrit by measuring those tests before and at 6, 12, 24 and 48 hrs after transfusion. Patients and

Methods: We included nineteen normovolemic, not actively bleeding consecutive patients; six of whom were diagnosed with anemia of chronic disease, one MDS and twelve iron deficiency anemias. Acutely bleeding patients, patients not able to give informed consent, prisoners under hospitalization and patients younger than 18 years old were excluded. All patients received transfusion of two units of packed red blood cells. The statistical analysis was performed using a repeated measurement ANOVA algorithm.

Results: The transfusion of two units of packed red blood cells did not result in a significant change of ferritin, transferrin, haptoglobin, cyanocobalamin and lactate dehydrogenase throughout the observation period. Hemoglobin, MCV and hematocrit demonstrated an early change and equilibration without significant change after the first six hours post transfusion. Serum iron levels were transiently elevated in the severely iron deficient population, but returned back to baseline after 24hrs. Folate experienced a statistically significant, persistent change in the posttransfusion period. For the first 48 hrs post transfusion the following conclusions were made: First, the diagnosis of iron deficiency or chronic disease anemia is not confounded by blood transfusion, particularly when using ferritin as the predominant diagnostic parameter. Second, the diagnosis of hemolytic anemias, using haptoglobin and LDH is not interfered by transfusion of two units of packed red blood cells. Third, vitamin B12 deficiency can be diagnosed after transfusion, whilst folate deficiency on the base of red blood cell folate cannot.
Classification and Survival of 179 Patients with T- and NK-Cell Lymphomas at the Federal Research Hematology Center of Russian Academy of Medical Sciences: A Retrospective Study.
Authors: Vinogradova Yulia E. , Lutsenko I.N. , Samoilova R.S. , Kaplanskaya I.B. , Vorobiev Ivan A. , Zingerman Boris V. , Rijicova N.B. , Shklovskiy-Kordi Nikita E. , Giliazitdinova E.A. , Margolin Oleg V. , Maryin D.C. , Krementskaya Alexandra , Kravchenko S.K. , Varticovski Lyuba , Vorobiev Andrei I.
Published: October 2019

Introduction: According to the new WHO classification for Non-Hodgkin’s lymphomas developed in 2001, the diagnosis of many lymphoma patients has to be reevaluated. The existing information on therapeutic options for patients with these rare malignancies prior to development of WHO classification has been unreliable. The T-cell malignancies are associated with poor outcome, and it is important to identify patients that could benefit from novel intensive therapies.

Materials and Methods: We conducted retrospective analysis of patients with T- and NK-cell malignancies treated from 2000–2006 at the Federal Research Hematology Center of Russian Academy of Medical Sciences. The purpose of this study was to determine the frequency, clincopathological features, and outcomes of patients in predominantly Slavic population (European part of Russia).

Results: Because these malignancies present in a variety of nosological forms and their response to standard therapies is poor, multiple therapeutic regimens were employed as expected. Information on 151 patients with complete clinical and pathologic information that allowed appropriate diagnosis according to the WHO criteria was available for the analysis. Analysis of 188 patients required changes from the initial diagnosis after additional laboratory investigation: 25 cases (13.3%) previously considered to be T-cell lymphomas were excluded from the analysis, mostly because of an inadequate histopathological material. Similar to previously reported data, a significant number of T- and NK- cell lymphomas (12.4%) were excluded as compared to B-cell (3.1%) lymphomas, which emphasizes the difficulties in diagnosis of mature T- and NK-cell lymphomas. Twelve out of 163 patients with verified T-cell lymphomas after correction of diagnosis were treated and observed in other countries or regions, and were also excluded from the final analysis. The only difference in 151 remaining patients was an increased incidence of T-cell malignancies with mediastinal involvement, with overall survival similar to those reported elsewhere. Final analysis of 28 additional cases in 2006 for a total of 179 patients will be presented.

Conclusions: Our survey of patients with T-cell malignancies represents a systematic analysis of these rare malignancies and establishes the communality of clinical features and response to therapies.

Kaplan-Mayer Survival of all TCL Kaplan-Mayer Survival of all TCL
Leukemia-Derived Fibroblast Cells Produce Vascular Endothelial Growth Factor Significantly in Chronic Myelogenous Leukemia Cases.
Authors: Shirasaki Ryosuke , Tashiro Haruko , Noguchi Mitsuho , Kawasugi Kazuo , Shirafuji Naoki
Published: October 2019

Objective: We reported recently that myofibroblast cells in bone marrow cells of chronic myelogenous leukemia (CML)-patients were partly derived from CML clone, which was identified after long-term bone marrow cell-cultures. The generated BCR-ABL-positive fibroblast cells produced various kinds of cytokines more than that observed in the normal clone-derived fibroblast cells. Among these cytokines, we focus on vascular endothelial growth factor (VEGF)-system in this report.

Materials and Methods: Bone marrow cells were collected from chronic myelogenous leukemia cases (20 of chronic phase (CP), 3 of accelerated phase (AP), 8 of blast crisis phase (BC)), which were separated with gravity sedimentation. Obtained non-adherent mononuclear cells were cultured in DMEM with 10% FCS in the humidified 5% CO2 incubator. When cells showed morphological changes into fibroblastoid cells, cells were treated with trypsin and further cultured. The obtained stromal fibroblast cells were sub-cloned into culturing in 96 well plates, and from which RNAs were extracted. cDNAs were synthesized, and RT-PCR was performed to identify BCR-ABL fusion product. The selected clones were further analyzed on DNA levels with FISH to identify BCR-ABL. The concentration of VEGF in the patients’ sera and in the conditioned media of non-adherent mononuclear cells, of fibroblast cells derived from normal clone and of fibroblast cells with BCR-ABL was measured with ELISA kit (R&D Systems). The expression of VEGF, VEGF receptor type-1 and VEGF receptor type-2 was also compared at RNA levels. The effect of anti-VEGF antibody to the culturing CML cells was determined using 3H-incorporation assay system.

Results and Discussion: The levels of VEGF in sera from CML patients were elevated significantly. However, in patients with CP the expression of VEGF was not observed in non-adherent mononuclear cells. In contrast, VEGF production was observed in non-adherent mononuclear cells from the patients with AP and BC. Stromal fibroblasts produced VEGF in CP, AP and BC, in which BCR-ABL-positive fibroblast cells produced at the significant levels. The expression of VEGFR-1 and -2 was detected in non-adherent mononuclear cells from CP, AP and BC. When non-adherent mononuclear cells from CML patients were cultured onto the stromal fibroblast cells, significant additive effects were observed with 3H-thymidine incorporation assays. And when anti VEGF antibody was added to the cultures, the proliferation activity was significantly decreased. These observations indicate that in CML cases, especially in CP, VEGF plays an important role for the cellular proliferation with paracrine system.
Development of Renal Calculi and Imatinib Therapy in CML: Is There an Association?.
Authors: Saikia Tapan , Hazarika Nandini , Dhabhar Boman
Published: October 2019

Reports are accumulating regarding the altered bone and mineral metabolism caused by imatinib (IM) therapy (Berman E, et al 2006 NEJM). This probably occurs through in vivo inhibition of PDGF receptors. It has been proposed that prolonged use of IM may cause some potentially serious metabolic pathological disorders. Compensatory rise in parathyroid hormone level may cause renal pathologies in these patients. In this observational study we came across a few patients on imatinib therapy for CML who experienced prolonged dyspepsia, abdominal and bone pain (suggestive of groans, moans, bones and stones features) and abdominal ultrasonography revealed renal calculi. This has led to collection of clinical data. So far, no systematic evaluation with radiological or biochemical have been undertaken in our cohort of 177 of CML on imatinib therapy. At the time of this report we have noted development of renal calculi (all symptomatic) in 7 patients of CML on imatinib therapy. All these patients have been on imatinib therapy 400 mg daily for more than 1 month before symptoms developed. In all but one, no surgical intervention has been necessary so far. Biochemical evaluation for altered bone metabolism revealed hyperphosphatemia in one and normal in another. Analysis of renal stone in one showed a composition of calcium phosphate and oxalate. Imatinib was not discontinued but reduced to 300 mg in 3 patients. It is possible that prolonged use of IM may alter calcium and phosphate metabolism leading to renal calculi formation. The proposed mechanisms could be,

interference with bone and mineral metabolism coupled with parathyroid dysfunction which in turn leads to renal tubular abnormalities or availability of excess oxalate for intestinal absorption brought about by low calcium ingestion.

Well designed cohort studies may throw more light in this area.
The Effect of CAHB on Myelodysplastic Syndrome In Vitro and Its Application in a Prospective Clinical Trial.
Authors: Cai Zhen , He Jingsong , Shi Jiming , Li Li , Han Xiaoyan , Guo Qunyi , Zhou Hui , Zhang Buwen , Huang He , Lin Maofang
Published: October 2019

Introduction: Despite recent advances, myelodysplastic syndrome (MDS) is still a therapeutic challenge. CAHB is a novel differentiation inducer which showed early evidence of efficacy in a phase I clinical trial. We used this agent to treat cultured MDS cells in vitro in our research laboratory and then, in a prospective clinical trial in our clinic. Preclinical data: We first investigated the effect of CAHB in vitro in human MDS-RAEB cell line MUTZ-1 cells. Treatment with CAHB remarkably inhibited the growth of MUTZ-1 cells in a dose-dependent and time-dependent manner with IC50 at 5.6 mmol/L at 48 hours. CAHB treatment decreased S phase cells, increased G2/M phase cells, but did not change G0/G1 phase cells, which indicated arrest in G2 phase. When treated with different concentrations of CAHB (0, 2.5, 5, 10 mmol/L) for 24 hours, the expressions of survivin mRNA decreased in a dose-dependent fashion.

Patients and method: Eligible patients with MDS had organ functions and were without significant cormobidities. Data from 3 pancytopenic patients on this trial were analyzed. Their whole blood counts were 1.1, 2.3, and 2.5 k/uL, respectively. Their hemoglobins were 7, 9, and 7 g/dL, respectively. Their platelets counts were 10, 31, and 20 k/uL, respectively. The blasts form all 3 patients were of myeloid immunophenotype and diploid chromosomal karyotype. All 3 patients received 0.45g/m2 CAHB for eight hours daily for 10 days every 5 weeks as 1 cycle. Clinical

Results: Upon the completion of the two courses, all had fatigue. Their bone marrow blasts decreased from 13.5% to7% (48% decrease), 26.5% to12% (55% decrease), and 11% to 2% (82% decrease), respectively. No grade 3–4 toxicities were observed after 2 months. Grade 1–2 toxicities included nausea and vomiting. Two patients had EKG changes with ST-segment depressions, u wave and bundle branch block which disappeared after the drug was stopped. There was no significant change in the peripheral blood cell counts in all 3 patients.

Conclusion: CAHB inhibited cell growth and induced apoptosis of MUTZ-1 cells in vitro. CAHB reduced blast cells in 3 MDS patients. Two patients had transient EKG changes. Our preliminary clincial data showed early evidence of efficacy of CHAB in treating MDS with a tolerable toxicity profile. Its mechnism of action is under further investigation.
Investigations on the Importance and Prevalence of the Acquired von Willebrand Syndrome in Patients with Myeloproliferative Disease.
Authors: Scharrer Inge , Flick Caroline
Published: October 2019

The acquired von Willebrand syndrome (AVWS) is a bleeding disorder, which is amongst others associated with myeloproliferative diseases. Its prevalence varies from 0,04–0,13% in literature. Typical laboratory findings such as a low VWF:RCo/VWF:Ag- Ratio, a prolonged aPTT, PFA and bleeding time, a decrease of VWF:RCo, FactorVIII:, VWF:Ag, typical VWF multimeric structure and the absence of a family history of bleeding are the basis for the diagnosis of AVWS. Bleeding episodes are typical for AVWS and are mostly of the mucocutaneous type (epistaxis, heavy menstrual bleeding, gingival and postoperative bleedings). Myeloproliferative diseases, particularily essential thrombocythemia, are with 15–18% the third most frequent observed comorbidity of patients with AVWS, but the prevalence of AVWS in patients with MPS is -at our knowledge- still unknown. We investigated 54 patients with the diagnosis of myeloproliferative disease (44 ET, 9 PV, 1 IMF), which was established according to the WHO-criteria over a period of 6 years (2000–2006). By analysing the typical laboratory parameters for AVWS, the VWF multimeric structure and the own and family history of bleeding we detected that 67% (36/45) of these patients had an AVWS: 27/44 ET (61%), 8/9 PV (89%), 1/1 IMF. The two most sensitive parameters in this context were VWF:RC/vWF:Ag- Ratio and VWF:RCo. It is well known that AVWS can disappear by treating the MPD. We could also show that treating the MPD with Hydroxycarbamid platelets, VWF multimeric structure, VWF:RCo/VWF:Ag-Ratio and VWF:RCo and the bleeding tendency were normalized. The JAK2-V617F mutation is frequently found in myeloproliferative disorders (MPD): Up to 97% patients with polycythemia vera (PV) and about 50% of patients with essential thrombocythemia (ET) carry this mutation. A further goal in this investigation was to find out a correlation between JAK2-V617F mutation, MPD and AVWS. JAK2-V617F diagnosis was performed in 27 of the 54 patients with MPD. 10 of these 27 patients carried a mutated JAK2-allele. However we observed no significant difference between mutated and unmutated patients in correlation to AVWS. In our investigation the high prevalence of 67% AVWS in patients with MPD confirm the hypothesis that until now AVWS is an underdiagnosed disease. Because of this high prevalence, the possibility of regression by treating MPD and the importance of early diagnosis for prevention of unexpected, sometimes letal bleeding complications, it would be benefical to introduce the AVWS- diagnosis-procedure as a routine step for patients with MPD.
Risk-Adapted Treatment of Acute Promyelocytic Leukemia: Updated Results of the Spanish PETHEMA LPA99 Trial Using ATRA and Anthracycline Monochemotherapy.
Authors: Sanz Miguel A. , Montesinos Pau , Vellenga Edo , Rayon Chelo , Parody Ricardo , de la Serna Javier , Bergua Juan , Leon Angel , Negri Silvia , Rivas Concha , Brunet Salut , Gonzalez Marcos , Esteve Jordi , Lowenberg Bob
Published: October 2019

Background: A first report of the PETHEMA LPA99 trial in acute promyelocytic leukemia (APL) showed that a risk-adapted treatment strategy combining ATRA and anthracycline alone for induction and consolidation results in high antileukemic efficacy and low toxicity. We report here an updated analysis of this trial including a significantly higher number of patients and longer follow-up.

Methods: From November 1999 to July 2005, 564 patients (median age 40 years, range 2–83) with APL received induction with ATRA (45 mg/m2/d) until CR and idarubicin (12 mg/m2/d) on days 2, 4, 6 and 8. Patients in CR received 3 monthly courses of risk-adapted consolidation therapy as follows:

“low-risk” patients (WBC <10×109/l and platelets >40×109/l), idarubicin 5 mg/m2/d × 4 (course #1), mitoxantrone 10 mg/m2/d × 5 (course #2), and idarubicin 12 mg/m2/d × 1 (course #3); “intermediate-risk “ (WBC <10×109/l and platelet <40×109/l) and “high-risk” (WBC >10×109/l) patients received ATRA (45 mg/m2/d × 15) in combination with reinforced chemotherapy (Idarubicin 7 mg/m2/d in the course #1 and two days instead of one in the course #3).

Maintenance therapy consisted of 50 mg/m2/d mercaptopurine orally, 15 mg/m2/week methotrexate intramuscularly, and 45 mg/m2/d ATRA for 15 days every 3 months.

Results: CR was achieved in 511 patients (91%). Except for three cases labelled as resistant, of the remaining 50 patients 56%, 24%, 16% and 4% died due to hemorrhage, infection, retinoic acid syndrome, and acute myocardial infarction, respectively. Multivariate analysis showed that WBC >10×109/l, age >60 years, male gender, and serum creatinine >1.4 mg/dl at presentation had independent predictive value of death during induction. The median follow-up of the cohort was 57 months (range 20–94 months). Thirteen patients (median age 72 years, range 4–81) died in remission and 99% of patients completed the entire assigned therapy. Thirty-six patients presented haematological relapse, 16 molecular relapse, and 8 secondary myelodysplastic syndrome or acute myeloid leukemia. Overall, the 5-year cumulative incidence of relapse (CIR), disease-free survival, and overall survival were 11%, 85%, and 84%, respectively. The 5-year CIR for low-, intermediate- and high-risk patients were 4%, 7% and 28%, respectively.

Conclusions: A risk-adapted strategy combining ATRA and anthracycline monochemotherapy for induction and consolidation therapy results in high antileukemic efficacy, low toxicity and a high degree of compliance in newly diagnosed APL.
An 86-Probe Gene Expression Signature Can Predict Survival in AML with Normal Karyotype Independently of FLT3 ITD and NPM1 Mutation Status - A Collaborative Study from the AMLCG and CALGB Study Groups.
Authors: Metzeler Klaus H. , Hummel Manuela , Bloomfield Clara D. , Spiekermann Karsten , Sauerland Maria C. , Heinecke Achim , Radmacher Michael , Marcucci Guido , Whitman Susan P. , Maharry Kati , Paschka Peter , Larson Richard A. , Berdel Wolfgang E. , Büchner Thomas , Wörmann Bernhard , Mansmann Ulrich , Hiddemann Wolfgang , Bohlander Stefan K. , Buske Christian
Published: October 2019

Patients with acute myeloid leukemia and a normal karyotype (NK AML) comprise 50% of all AML cases and show heterogeneous treatment outcomes and survival. We used gene expression profiling to develop a prognostic gene signature that predicts survival in this clinically relevant AML subgroup. Our analysis was based on data from 163 patients with newly diagnosed NK AML treated in the German multicenter AMLCG 2000 trial, for whom pretreatment gene expression profiles were obtained using Affymetrix HG-U133 microarrays. We used supervised principal component analysis to identify 86 oligonucleotide probesets (corresponding to 66 different genes and ESTs) that were correlated with overall survival (OS), and to define a prognostic score based on these probesets. When applied to an independent test cohort of 79 NK AML cases from the same AMLCG trial, the continuous prognostic score was predictive of OS (P=0.002, hazard ratio [HR] for a change in prognostic score equal to the difference between the 75th and 25th percentiles of the score = 1.94) and event-free survival (EFS) (P = 0.001, HR=1.70). The score based on our gene signature showed a strong correlation with the presence of the FLT3 internal tandem duplication (ITD), but retained its prognostic value for OS in the test cohort even after adjustment for FLT3 ITD, NPM1 status and age (P=0.037, HR=1.65). When we defined a cut-off value in the training population and used it to dichotomize the gene expression score values in the test cohort, the resulting two subgroups had significantly different OS (median, 259 days vs. not reached, P=0.002) and event-free survival (EFS) (median, 72 vs. 300 days, P = 0.015). We subsequently confirmed our findings in a group of 64 NK AML patients (Blood2006;108:1677–83) treated on CALGB 9621. In this validation cohort, our continuous gene expression score was predictive of OS (P < 0.001, HR=4.11) and EFS (P < 0.001, HR=2.90). In multivariate analyses that adjusted for age, NPM1 and FLT3 ITD status, the gene expression score remained significant for OS (P = 0.007, HR=3.40). When we used the prognostic score to split the CALGB validation cohort into two groups, based on the same cut-off value as in the AMLCG test population, the two resulting subgroups differed in their OS (median, 375 days vs. not reached, P < 0.001) and EFS (median, 258 vs. 728 days, P = 0.027). In summary, we present a novel and robust gene expression signature that offers independent prognostic information for patients with normal karyotype AML.
SFRP1 Is Estrogen Inducible in Bone Marrow Stromal Cells and Suppresses the Earliest Events in Lymphopoiesis.
Authors: Yokota Takafumi , Oritani Kenji , Garrett Karla P. , Kouro Taku , Nishida Makoto , Takahashi Isao , Ichii Michiko , Matsumura Itaru , Kincade Paul W. , Kanakura Yuzuru
Published: October 2019

It has long been known that lymphopoiesis is transiently suppressed during pregnancy, and that this can be experimentally simulated by treatment with estrogen. Indeed, sensitivity to estrogen made it possible to identify very primitive lymphoid cells in the Lin- c-kitHi Sca-1+ fraction that is enriched with hematopoietic stem cells (HSC) and multipotent progenitors of mouse bone marrow. However, analyses showing how the earliest events in lymphopoiesis is suppressed in those circumstances have not been performed. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, very little has been known regarding mechanisms. In the present study, we performed high resolution analysis using RAG-1/GFP reporter mice and confirmed that early lymphoid progenitors (ELP) in the Lin-c-kitHi Sca-1+ fraction are particularly depressed in pregnancy or after estrogen injection. The Lin- c-kitLo fraction containing common lymphoid progenitors (CLP)/pro-lymphocytes was very sensitive to estrogen in stroma-free serum-free culture, but earlier stages of lymphopoiesis seemed to be blocked in vivo. Thus, we focused on identifying mechanisms involving bone marrow environment. Two independent strategies with macroarrays or differential display-PCR were used to isolate stromal cell genes that were both estrogen regulated and associated with inability to support B lymphopoiesis. We have identified secreted frizzled related protein 1 (sFRP1) as an estrogen inducible gene, and its expression corresponded to inability to support lymphopoiesis. This member of the complex Wnt family has been previously described as both an agonist and an antagonist of canonical, β-catenin dependent signaling. We found that sFRP1, like recombinant Wnt3a, stimulated the canonical pathway in the Lin- c-kitHi Sca-1+ fraction, and blocked progression in the B lymphocyte lineage even when exogenous Wnt ligands were not provided. The suppressive effects of sFRP1 and Wnt3a on B lymphopoiesis were canceled out when both were present simultaneously. To better understand the vulnerability of early progenitors to the Wnt signaling, we examined the expression pattern of Frizzled receptors. Interestingly, the HSC-enriched fraction (Lin- c-kitHi Sca-1+ IL-7Rα-RAG-1/GFP-) highly expressed 7 out of 9 Frizzled receptors, which markedly declined as they differentiated to ELP (Lin- c-kitHi Sca-1+ IL-7Rα- RAG-1/GFP+) and CLP (Lin- c-kitLo Sca-1Lo/- IL-7Rα+). Myelo-erythroid progenitors were less affected by sFRP1 in culture, suggesting that it is similar to estrogen with respect to lineage specificity. Bone-lining stromal cells express sFRP1 and the transcripts were elevated in bone marrow by pregnancy or estrogen injection. In summary, these observations implicate sFRP1 as a possible mediator in hormone regulation of the earliest events in lymphopoiesis.
Constitutive Activation of STAT5a in Myeloid Progenitors of Severe Congenital Neutropenia Patients Leads to Downregulation of LEF-1.
Authors: Skokowa Julia , Gupta Kshama , Brechlin Annette Mueller , Meyer Johann , Moore Malcolm A.S. , Welte Karl
Published: October 2019

Severe congenital neutropenia (CN) is a hematological disorder characterized by an early stage maturation arrest of granulopoiesis in bone marrow at promyelocytic stage. Previously we found that LEF-1 and its target gene C/EBPα are severely abrogated in promyelocytes of CN patients, which plays a decisive role in the pathomechanism of defective granulopoiesis (Skokowa J., et al., Nat. Med.2006;12:1191). Recently it has been shown that constitutively activated STAT5a inhibits myeloid differentiation of human CD34+ cells by downregulation of C/EBPα (Moore MA, et al., Exp. Hematol.2007;35:105). Therefore, we investigated whether the presence of constitutive activated STAT5a in CD34+ cells of healthy individuals leads to downregulation of LEF-1 and whether STAT5a is activated in myeloid progenitors of CN patients. A screen of the 10kb upstream region of LEF-1 gene revealed two putative STAT5a binding sites (−3913 bp to −3894 bp and −3728 bp to −3709 bp) and specificity of the STAT5a binding to the LEF-1 promoter in nuclear extracts of CD34+ cells was confirmed in a chromatin immunoprecipitation (ChIP) assay. Additionally, our data demonstrated that introduction of retroviral constructs expressing IRES-GFP and cDNA of constitutively activated STAT5a (mutant of STAT5a, (STAT5A[1*6]) into CD34+ cells of healthy individuals resulted in 20-fold downregulation of LEF-1 mRNA expression. To compare G-CSF- dependent phosphorylation of STAT5 in CD33+ bone marrow myeloid progenitors of CN patients and healthy individuals, we treated these cells with G-CSF in vitro and measured phosphorylation of STAT5 by intracellular staining of cells with phosphoSTAT5 (Y694) antibody. We found that G-CSF stimulation resulted in a significantly higher phosphorylation of STAT5 in myeloid progenitors of four CN patients, as compared to four healthy volunteers (mean fluorescence intensity (MFI) 436 ± 57 in CN patients vs 212 ± 30 in healthy controls). Interestingly, augmented phosphorylation of STAT5 has been observed in both groups of CN patients carrying either ELA2 or HAX1 mutations. These data confirm the hypothesis that downregulation of LEF-1 and LEF-1 target gene C/EBPα is at least in part a consequence of increased activation of STAT5a in both CD34+ cells of healthy individuals and in patients with CN.
Increased Risks of Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Myelofibrosis (MF) among 24577 First-Degree Relatives of 11039 Patients with Chronic Myeloproliferative Disorders (MPD) in Sweden.
Authors: Landgren Ola , Goldin Lynn R. , Kristinsson Sigurdur Y. , Samuelsson Jan , Bjorkholm Magnus
Published: October 2019

Background. Familial clustering of PV, ET, MF, and chronic myeloid leukemia (CML) has been reported through case reports and smaller case series. Recently, several studies reported that in families with multiple MPD patients, the JAK2 mutation is not an early germ line predisposing factor for MPD but rather a facilitator of proliferative advantages. However, the degree of familial clustering in the population has not been defined. We have conducted the first large population-based study to quantify risks of developing MPD and related malignancies in first-degree relatives of MPD patients.

Methods. Using high-quality central population-based registries, we identified 6217 PV, 2838 ET, and 1984 MF patients diagnosed in Swedish hospitals 1958–2005 (Cancer and local hospital-based registries), with linkable relatives; 43550 frequency-matched controls (Population registry); and first-degree relatives of cases (n=24577) and controls (n=99542) (Multigenerational registry). Relatives of MPD patients and controls were linked with the Cancer and local hospital-based registries to define occurrence of MPD and other related neoplasms. We used a marginal survival model to calculate relative risks (RR) and 95% confidence intervals (CI) as measures of familial aggregation.

Results. Compared with controls, relatives of MPD patients had significantly increased risks of PV (RR=5.70; 95% CI 3.55–9.14), ET (RR=7.37; 95% CI 3.67–14.81), and MF (RR=3.53; 95% CI 1.59–7.85). Risks were similar when we restricted the analyses to relatives of patients with the same MPD (PV-PV, ET-ET, and MF-MF) Also, risk-estimates were virtually the same for various types of first-degree relatives (parents, siblings, offspring); the same was true when we calculated risks by age at MPD of cases (above vs. below 65 yrs), and sex of relatives. Age at diagnosis of MPD was not different for case and control relatives. Furthermore, relatives of MPD patients (vs. controls) had a borderline increased risk of CML (RR=1.86; 95% CI 0.90–3.74; p=0.07). As expected, we found excess of subsequent acute myeloid leukemia (AML) (n=271; 2.5%) and myelodysplastic syndrome (MDS) (n=27; 0.2%) among MPD patients; however, there were no increased risks of AML or MDS among relatives of MPD patients.

Conclusions. In this first large population-based study including more than 11000 MPD patients and their almost 25000 linkable first-degree relatives, we found 3- to 7-fold elevated risks of developing MPDs among first-degree relatives of MPD patients. Our results support the hypothesis that there are common, strong, shared susceptibility genes that predispose to PV, ET, MF, and possibly CML.
NF1 Microdeletions in Pediatric MLL-Rearranged AML and T-ALL: A Novel Mechanism for RAS Activation.
Authors: Balgobind Brian V. , Van Vlierberghe Pieter , Hollink Iris H. , van den Ouweland Ans M.W. , Beverloo H. Berna , Terlouw-Kromosoeto Joan N.R. , van Wering Elisabeth R. , Kaspers Gertjan J.L. , Reinhardt Dirk , Horstmann Martin , Hahlen Karel , Pieters Rob , Zwaan Christian M. , Meijerink Jules P.P. , van den Heuvel-Eibrink Marry M.
Published: October 2019

Oligo array-comparative genomic hybridization (CGH) is a technique to detect DNA copy number changes (amplifications and/or deletions), and can be used to identify novel genetic abnormalities in cancer. Searching for such abnormalities in specific selected subgroups of pediatric leukemia, we analyzed array-CGH data obtained on 212 samples (71 with MLL-rearranged acute myeloid leukemia (AML), 38 with normal karyotype (NK) AML, and 103 with T-cell- acute lymphoblastic leukemia (T-ALL). Cryptic deletions involving the neurofibromatosis type 1 (NF1) locus, del(17)(q11.2), were identified in 2/71 MLL-rearranged AML and 3/103 T-cell ALL samples, all cases without clinical evidence of NF1, but were not observed in the NK AML samples. The single copy loss of the NF1 locus was confirmed by NF1 specific MLPA assay. Subsequent mutation analysis revealed mutations in the remaining NF1 allele in 3 patients, confirming its role as a tumor-suppressor gene in cancer, since both were restricted to the leukemic cells. Furthermore, NF1 expression in the del(17)(q11.2)-positive leukemias was significantly lower in the patients tested compared to the other patient samples without this deletion. NF1 is an autosomal dominant genetic disorder caused by mutations in the NF1 gene. A small proportion of NF1 patients show germline NF1 gene deletions, in contrast to the confirmed somatic deletions described here in our leukemia patients. Other mutations in NF1, such as inactivation of both alleles by point mutations, or loss of one wild-type allele as an effect of uniparental disomy for the affected chromosome 17 has been observed in NF1 patients with JMML, may have been missed in our cohort. NF1 is a negative regulator of the RAS pathway (RAS-RAF-MEK-ERK pathway). Other mutations in this pathway have previously been associated with myeloid malignancies, and result in RAS activation, apoptotic resistance and proliferation of leukemic cells. We identified 22% N- and K-RAS and 1.4% PTPN11 mutations in our MLL-rearranged AML subgroup. RAS activation may therefore play an important biological role in at least 25% of MLL-rearranged AML samples. In conclusion, our data show that NF1 microdeletions are involved in leukemogenesis in pediatric leukemias, and that RAS-activation may be especially involved in the underlying biology of MLL-rearranged AML.
Limited-Stage Diffuse Large B-Cell Lymphoma (DLBCL) Patients with a Negative Pet Scan Following Three Cycles of R-CHOP Can Be Effectively Treated with Abbreviated Chemoimmunotherapy Alone.
Authors: Sehn Laurie H. , Savage Kerry J. , Hoskins Paul , Klasa Richard , Shenkier Tamara , Voss Nicholas , Wilson Don , Connors Joseph M.
Published: October 2019

Four cycles of CHOP chemotherapy has been shown to be sufficient treatment for low-risk elderly patients with limited-stage DLBCL, eliminating the need for radiation therapy (Bonnet et al, JCO 2007). FDG-PET scanning is an effective prognostic tool that may identify chemo-sensitive patients (regardless of age or clinical risk factors) who can appropriately be treated with abbreviated chemoimmunotherapy alone. Patients: Beginning in 2005, we have recommended that all prospective patients with limited-stage DLBCL (stage I/II, no B-symptoms, mass < 10cm) treated in British Columbia (BC) undergo a PET scan following 3 cycles of R-CHOP; PET-negative patients should then receive one additional cycle of R-CHOP (total 4 cycles R-CHOP), while PET-positive patients receive involved-field radiation therapy (IFRT). Clinical characteristics of the first 50 patients are as follows: median age 67 y (range 31–88); 56% male; 62% stage I, 38% stage II; 4% PS>1; 6% elevated LDH; 58% at least 1 extranodal site, 10% >1 extranodal site. Stage-modified IPI risk factors: 22% 0; 70% 1–2; 8% 3–4. Median follow-up is 17 mos (range 4–26).

Results: 37 patients (74%) were PET-negative and 13 patients (26%) were PET-positive after 3 cycles of R-CHOP. No clinical factors were found to be predictive of PET status. Of the 37 PET-negative patients, 35 completed treatment with one additional cycle of chemoimmunotherapy, 1 received IFRT due to poor chemotherapy tolerance, and 1 died of toxicity before receiving any more treatment. Only 1/37 PET-negative patients has relapsed (alive with lymphoma after salvage therapy). All 13 PET-positive patients received IFRT, with 3 relapses and 2 deaths from lymphoma to date. Although longer follow-up is necessary, the 2-year estimated Kaplan-Meier progression-free survival is 91% overall (97% and 75% for PET-negative and PET-positive patients, respectively, p=0.09). (see figure) The 2-year estimated Kaplan-Meier overall survival is 97% for PET-negative and 69% for PET-positive patients, p=0.1.

Conclusion: Patients with limited-stage DLBCL who are PET-negative after 3 cycles of R-CHOP can be effectively treated with abbreviated chemoimmunotherapy alone (4 cycles R-CHOP), avoiding the long-term toxicity of radiation while preserving excellent lymphoma control.

Figure Figure
Novel Spliced MLL Fusions Have Been Identified Involving the MLL Partner Genes ELL, EPS15, MLLT3, and SEPT5.
Authors: Meyer Claus , Kowarz Eric , Brahim Wajih , Grümayer Renate , Ben-Abdelali Raouf , De Braekeleer Etienne , De Braekeleer Marc , Macintyre Elizabeth A. , Delabesse Eric , Dingermann Theo , Klingebiel Thomas , Marschalek Rolf
Published: October 2019

Chromosomal rearrangements of the human MLL gene are a genetic hallmark for aggressive acute leukemias. More than 60 partner genes have been characterized at the molecular level until now. The observed recombination mechanisms between MLL and its many recombination partner genes include reciprocal chromosomal translocations, insertions, and intrachromosomal 11q deletions and inversion. “Spliced MLL fusions”, have recently been described as a novel mechanism to generate functional chimeric fusion transcripts for 50% of t(11;19)(q23;p13.3) leukemia patients where the MLL gene is fused to the MLLT1 (ENL) gene and for two single leukemia patients. One patient had a t(11;15) involving the ZFYVE19 gene and the other patient had a 11q interstitial deletion involving the DCPS gene. Within these cases the disrupted MLL gene is fused only on RNA level to a functional transcription unit located in the vicinity of the breakpoint. Here we describe eight novel “spliced MLL fusions” involving the MLL partner genes ELL (1x), EPS15 (3x), MLLT3 (1x), and SEPT5 (1x). For ELL, MLLT3 and SEPT5 the identified spliced fusion seems to be a single or rare event. However 1/3 of the t(1;11)(p32;q23) leukemia cases exhibit “spliced MLL fusions”. I.e. approximately 1/3 of all breakpoints are located upstream of the EPS15 gene. Further studies will help to investigate the remaining question whether these two groups of t(1;11)(p32;q23) leukemia patients have any significant difference in outcome. Interestingly, for the t(11;22)(q23;q11.2) leukemia the major identified fusion transcript has an additional 57 bp exon derived from the 2 kb area up-stream of the SEPT5 gene. Furthermore this patient has a T-ALL and thus differs from all other cases where the patients show myeloid markers when the MLL gene is fused to one of the five members of the septine family. This is leading to the question whether or not this 57 bp big exon can be made responsible for the lineage shift. With these results the number of genes involved in „spliced MLL fusions” has increased from three to seven. Supported in part by grants Ma 1876/7-1, /8-1 and /9-1 from the DFG, grant N1KR-S12T13 from the BMBF and grant 102362 from the Deutsche Krebshilfe.