bluuebunny/crossref_metadata_2025 huggingface dataset:
A Phase I Study of the Farnesyltransferase Inhibitor Tipifarnib in a Week-On Week-Off Dose Schedule in Acute Myelogenous Leukemia.
Authors: Kirschbaum Mark H. , Stein Anthony Selwyn , Tuscano Joseph , Zain Jasmine M. , Popplewell Leslie , O’Donnell Margaret R. , Karanes Chatchada , Wright John , Pulone Bernadette , Rincon Amalia , Frankel Paul , Forman Stephen J. , Newman Edward
Published: October 2019

Tipifarnib (R115777, Zarnestra) is a methylquinolone analogue that is a potent inhibitor of farnesylation. The initial phase I study in leukemia by Karp and Lancet (Blood, 2001) showed clinical responses in 10/34 evaluable patients with AML on a 21 day schedule, with 600 mg bid identified as the MTD. Preclinical work shows that farnesyltransferase remains inhibited for seven days after tipifarnib, suggesting an alternate week dosing schedule. We report the results of a tipifarnib phase I dose escalation trial in AML on a week on week off schedule. Dose levels were 400, 600, 800, 1200, 1400, and 1600 mg BID on a standard 3+3 design. Eligibility was defined as relapsed or refractory AML after one to three prior induction regimens. A total of 37 patients were accrued, with 32 evaluable for toxicity. Median age at treatment is 62 (range 33–77), with 15 F, 22 M.

RESULTS: The MTD on this schedule was 1200 mg B.I.D., with the major dose limiting toxicity determined by course one being creatinine elevation. At the 6 pts treated at the MTD there was one DLT (grade 3 creatinine), one grade 3 nausea (not a DLT), and grade 2 toxicities including liver enzyme abnormalities, nausea/vomiting, and rash. At dose level 1, metabolic acidosis and hepatic failure in a patient with progressive leukemia who had previous hepatic toxicity during chemotherapy was observed. Two patients treated at the MTD had a complete remission: a 69-yr old male who relapsed after 231 days, and a 66-yr old male who relapsed after 258 days, but who responded to re-treatment with tipifarnib and is disease-free 548 days (18 months) since enrolling in the study. At 1000mg BID dose, a 69-yr old woman with relapsed AML after autologous transplant had a CR after 2 cycles and went on receive a successful allogeneic transplant and is in remission 2yrs from her first cycle of tipifarnib. One patient with AML evolving out of CMMoL had disappearance of the AML blasts but persistence of the CMMoL after two cycles at the 800 mg bid level, and one patient at the 1400 dose had a platelet rise to 140 from a starting platelet count below 40 despite continued presence of blasts. Higher doses resulted in a higher incidence of dose limiting renal toxicity, fewer courses, and no complete remissions. Additional patients are accruing at the MTD to further understand the toxicities on this schedule. In AML patients, a two-fold increase in tipifarnib dosing can be tolerated on this dosing schedule with responses seen at the higher doses. A monotherapy phase 2 or tipifarnib combination study in AML using this schedule appears warranted.
Phase I Study Demonstrates Activity and Tolerability of Diphtheria Toxin Interleukin-3 Fusion Protein in Patients with AML and MDS.
Authors: Frankel Arthur E. , Smith E.R. , George T.A. , Liu J.S. , Lee J. , Park S.K. , Rizzieri David A. , Hogge Donna E.
Published: October 2019

Interleukin-3 receptor (IL-3R) proteins are over-expressed on blasts and cancer stem cells of multiple hematologic malignancies. The recombinant diphtheria toxin fusion protein, DT388IL3, specifically targets IL-3R and is composed of the catalytic and translocation domains of diphtheria toxin (DT388) fused to human interleukin-3 (IL3). DT388IL3 demonstrated selective toxicity to acute myeloid leukemia (AML) stem cells both in vitro and in vivo, and was prepared for a Phase I clinical study in AML and myelodysplasia (MDS) patients. FDA approval (BB IND#11314) and IRB approvals were obtained. 80 AML and MDS patients were screened and 38 AML and 3 MDS patients were treated. The median age of treated patients was 62 years (range, 25–81 years). There were 22 males and 19 females. AML disease was de novo in 5, 1st relapse in 12, 2nd relapse in 8, and refractory in 12. MDS disease was high-grade in one and intermediate-grade in 2. Five AML patients had a history of MDS, and one had a history of secondary AML. One patient each had previously received an autologous or allogeneic stem cell transplant. AML cytogenetics was unfavorable in 15, intermediate in 21, and not done in 2. Seven patients were treated with 4 ug/kg, eight patients with 5.3 ug/kg, thirteen patients with 7.1 ug/kg, seven with 9.4 ug/kg, and six with 12.5 ug/kg DT388IL3 for up to six treatments (one cycle). Of note, patients have not yet received a second cycle. Drug-related toxicities were mild to moderate and transient including fever, chills, hypotension, transaminasemia, hypoxemia, and hypoalbuminemia. Pretreatment anti-DT antibody levels ranged from 0 – 9.6ug/mL (mean = 2.3ug/mL); day 15 post-treatment antibody levels were 0 – 600ug/mL (mean = 92ug/mL); day 30 antibody levels were 1.1 – 306ug/mL (mean = 82ug/mL). Day 1 Cmax ranged from 0 – 350 ng/mL and correlated with dose; Day 12 Cmax ranged from 0 – 179 ng/mL and correlated with Day 12 anti-DT antibody levels. Development of anti-DT antibody levels at Day 15 and 30 did not interfere with response. Consistent with an absence of toxicity to normal hematopoietic progenitors, responses occurred in the absence of prolonged myelosuppression. Among 38 evaluable AML patients, we have observed one CR of 8 months duration, 2 partial remissions (PRs) lasting one and three months, and 3 minimal responses with clearance of peripheral blasts and marrow blast cytoreductions of 89%, 90%, and 90% lasting one to two months. In one evaluable MDS patient, we achieved a PR lasting four months with improvement in hematologic parameters and conversion to transfusion independence. Patient accrual is proceeding with correlative IL3R content of blasts and cancer stem cells. These results of activity and tolerability are encouraging and suggest that DT388IL3 may achieve a role in the management of patients with myeloid malignancies.
Phase I Study of Suberoylanilide Hydroxamic Acid (SAHA) and Decitabine in Patients with Relapsed, Refractory or Poor Prognosis Leukemia.
Authors: Ravandi Farhad , Faderl Stefan , Thomas Deborah , Burger Jan , Koller Charles , Garcia-Manero Guillermo , Morris Gail , Torma Ritva , Kantarjian Hagop , Issa Jean-Pierre
Published: October 2019

Few effective treatment strategies are available for patients with relapsed and refractory leukemias and their prognosis remains poor. Epigenetic therapy by targeting epigenetic pathways is currently under investigation as a potential strategy to reverse transcriptional silencing of tumor suppressor genes leading to the reversal of the neoplastic phenotype. 5-Aza-2′-deoxycytidine (DAC, Decitabine) and suberoylanilide hydroxamic acid (SAHA, Vorinostat) can affect epigenetic change in leukemic cells through promoter CpG island hypomethylation and inhibition of histone deacetylation, respectively. We have conducted a phase I study of combination of DAC plus SAHA (using a sequential dosing schedule) in patients with relapsed, refractory, or high risk leukemias. Five cohorts (Dose levels 0 – 4) of 6 patients each received escalating doses of decitabine (10, 10, 15, 20 and 25 mg/m2 IV daily x 5) followed by SAHA (100 mg PO tid x 14 in the first cohort and 200 mg PO tid x 14 in all subsequent cohorts). From 7/06 to 8/07, 31 patients (21 M, 10 F) have been accrued with the last cohort receiving DAC 25 mg/m2 daily x 5 followed by SAHA 200 mg tid x 14. Median age was 62 years (range, 22 – 82). Twenty one patients (68%) had refractory AML (5 with prior MDS), 1 (3%) refractory acute biphenotypic leukemia, 3 (10%) relapsed MDS, 3 (10%) refractory ALL, 1 (3%) untreated high risk MDS, and 2 (6%) Philadelphia chromosome-negative myeloproliferative disease. Median number of prior regimens was 2 (range, 0 – 8). One patient did not receive therapy due to rapid disease progression and 30 were evaluable for assessment of toxicity. Median number of cycles of DAC + SAHA administered was 2 (range, 0 – 6). One patient had pulmonary embolism and one grade 3 diarrhea, both considered to be dose limiting. Other adverse events included syncope, neutropenic fever, diarrhea, fatigue, renal failure, rash, nausea, thrombosis, and angioedema. Of the 30 evaluable pts, 1 patient achieved complete remission lasting 5.5 weeks, 4 had significant reductions in the bone marrow blasts, 4 had stable disease, 7 are too early for response evaluation, and 14 had no response/disease progression. We conclude that the sequential combination of DAC and SAHA is safe and has activity in advanced leukemia. Correlative studies evaluating DNA promoter methylation and histone acetylation will be presented.
A Phase I Study with CP-4055 In Patients with Hematologic Malignancies.
Authors: Giles Francis , Kantarjian Hagop M. , Vey Norbert , Rizzieri David , Ravandi Farhad , Prebet Thomas , Jacobsen Tove Flem , Nilsson Bo I. , Staudacher Karin , O’Brien Susan
Published: October 2019

Background: CP-4055 (ELACYT™, cytarabine 5′-elaidic acid ester) is a novel cytotoxic nucleoside analogue. While CP-4055 has similar mechanisms of action to cytarabine, it is, unlike cytarabine independent of nucleoside transporters for cellular uptake. This is the first study in patients with haematologic malignancies. CP-4055 is currently in Phase II studies in patients with solid tumors. Aims: Determination of the maximum tolerated dose and the preferred infusion time in patients with haematologic malignancies.

Methods: The study was initiated in May 2006. Patients (pts) received IV CP-4055 over 2 hours (2 hr IV, Arm A) or 24 hours (CIV, Arm B) in a day 1–5 q3w schedule. The starting dose in Arm A was 300 mg/m²/day and in Arm B 200 mg/m²/day, with standard definitions of DLT for haematologic malignancies. Dosing increments were by 50% until non-haematologic toxicities, and thereafter increments were by 30%.

Results: In the ongoing study, 40 pts [25 male, median age 60.5 yrs (range 25–77); ECOG PS 0–1: 32 pts, ECOG PS 2: 8 pts; AML 35 pts, BAL 1 pt, MDS 1 pt, ALL 2 pts, CML-BP 1pt, average 2–3 lines prior chemotherapy (range 0–7)] have been treated at 2 US and 1 European centres. Five patients are ongoing. Two treatment-related serious adverse events (SAE) have been observed. One patient experienced fever and one patient had transaminase increase. Both patients recovered from their SAE while still on the study. No DLT has been observed and dose escalation is ongoing at 1950 mg/m²/day in Arm A and at 1500 mg/m²/day in Arm B. Nausea, anemia, diarrhea and thrombocytopenia were the most common possibly related AEs. One patient (875 mg/m²/day Arm B) with CRp received one course of consolidation therapy. Five patients with stable disease/mild myelosuppression received more than 2 cycles of CP-4055: one pt (AML) received 4 cycles and four patients (3 AML, 1 CML-BP) received three cycles. One of these patients was referred to stem cell transplantation. Fifteen patients received 2 cycles of therapy.

Summary/Conclusion: Patients with haematologic malignancies tolerated CP-4055 well up to a dose of 1500 mg/m²/day (2 hr IV) and 1150 mg/m2/day (CIV) in a d1–5 q3w schedule. Myelosuppression has been observed with minimal toxicity. Accrual is ongoing.
In Vivo Correlates of Response for Phase I Trial of Oral Tipifarnib (T) Combined with Oral Etoposide (E) for Adults Age ≥ 70 with Newly Diagnosed Acute Myelogenous Leukemia (AML) Who Are Not Candidates for Traditional Cytotoxic Chemotherapy (TCC).
Authors: Ricklis Rebecca M. , Karp Judith E. , Feldman Eric J. , Galkin Suzanne , Greer Jacqueline M. , Ironside Valerie , Mirjafary Kim , Talbot Timothy D. , Morris Lawrence E. , Wright John J. , Kaufmann Scott H.
Published: October 2019

T is a farnesyltransferase inhibitor with clinical activity in elderly adults with newly diagnosed AML who are not candidates for TCC. For adults age ≥ 65 with poor-risk AML, T 600 mg BID for 21 out of 28–63 days induced complete remissions (CR) in 14%, median duration 7.3 mos, median survival 18.3 mos (Lancet JE, et al, Blood109:1387–1394,2007). In vitro, T+E exhibits synergistic anti-AML activity. In an attempt to increase CR rates in elderly AML pts, we conducted a Phase I trial of oral T+E, with escalation of both drugs and 14 vs. 21 days of T every 28–63 days (median 31 days, range 29–50). T+E resulted in a 25% CR rate (21/84 pts), duration 2 – 26+ mos, with best rates achieved with T dose ≥ 400 mg BID for 14 days (Karp JE, et al, Blood108:130a,2006). In the context of this trial, we have conducted longitudinal laboratory studies of changes in DNA damage and apoptosis in AML marrow blasts obtained from 84 patients prior to T+E (Day 0) and on Day 8 of T+E. Blasts were enriched by Ficoll density gradient separation and fixed in 70% ethanol. Persistent, unrepaired DNA damage was quantified by flow cytometry of cells stained with antibody to phosphorylated histone H2AX (γH2AX). Apoptosis was measured by flow cytometry of propidium iodide-stained cells with sub2n DNA content as observed in cell cycle distribution. To date, we have analyzed changes in in vivo γH2AX from 10 pts (4 CR, 6 stable disease or no response, SD/NR). CR pts had a 2-fold increase (range 1.91–2.22) in DNA damage induced by Day 8 of T+E relative to Day 0 values, while patients with SD/NR had no such increase (0–1.06) in DNA damage. Similarly, the apoptotic fraction of the cell cycle distributions of CR patients increase by 2-fold (range 1.2–5.3) for Day 8 marrow blasts. In contrast, changes in apoptosis were widely divergent for SD/NR patients (range 0.2–7.0). Taken together, these preliminary studies on AML marrow blasts obtained prior to and during T+E therapy suggest a possible linkage among induction of DNA damage, apoptotic response, and clinical outcome. Full analysis of all acquired specimens is ongoing.
Arsenic Trioxide with Ascorbic Acid and High-Dose Melphalan Is Safe and Effective for Autotransplantation for Multiple Myeloma.
Authors: Qazilbash Muzaffar H. , Saliba Rima M. , Parikh Gaurav , Hosing Chitra , Mendoza Floralyn , Qureshi Suhail P. , Weber Donna M. , Wang Michael , Flosser Thuy , Couriel Daniel R. , Kebriaei Partow , Popat Uday , Alousi Amin , de Lima Marcos , Champlin Richard E. , Giralt Sergio
Published: October 2019

Background: Arsenic trioxide (ATO) has been shown to be synergistic with melphalan both in vitro and in vivo. We conducted a randomized phase II trial to determine the safety and efficacy of a combination of ATO, melphalan, and ascorbic acid (AA) as a preparative regimen in patients undergoing autologous hematopoietic progenitor cell transplantation for multiple myeloma (MM). We also assessed the impact of ATO levels on melphalan pharmacokinetics, engraftment, and toxicity.

Methods: Forty-eight patients with secretory myeloma (23 females, 25 males; median age, 54; range 35–70) were treated between 4/04 and 8/05. All patients received melphalan 100 mg/m2 IV on days -4 and -3 and AA 1000 mg/day IV on days -9 to -3. Patients were randomized to 3 arms: no ATO (arm 1), ATO 0.15 mg/kg IV on days -9 to -3 (arm 2), and ATO 0.25 mg/kg IV on days -9 to -3 (arm 3). Twelve patients had received a prior autograft, with a median 4.5 x 106/kg (range 2.3–10.9) CD34+ cells infused.

Results: Patients in all 3 arms were evenly matched. Twenty-nine patients (60%) were transplanted for consolidation of first remission and 19 patients (40%) for relapsed disease. With a median follow-up of 26 months (range 10–37) post-autograft, no dose-limiting toxicity, or nonrelapse mortality was reported. Toxicity was limited to grade 1 or 2 nausea, vomiting, and diarrhea and was comparable in all 3 arms. Melphalan pharmacokinetics was not altered by ATO pretreatment. Median time to neutrophil engraftment (absolute neutrophil count >500/dL) was 9 days, with no engraftment failures or delays in either the control or ATO arms. The complete response (CR) rate for all patients was 25% (12/48), and the partial response rate was 60% (29/48) for an overall response rate (ORR = CR + PR) of 85%. Progression-free survival (PFS) and overall survival (OS) after 24 months of follow-up were 59% and 91%, respectively. Median PFS was 29 months; median OS has not been reached. There was no significant difference in CR, ORR, PFS, or OS among the 3 arms (P =.9, .9, .5, and .6, respectively). PFS and OS were comparable among patients with chromosomal abnormalities or relapsed disease at transplant and patients undergoing a second autotransplant for salvage.

Conclusions: Addition of ATO + AA to high-dose melphalan is safe and well tolerated as a preparative regimen for autotransplants in patients with MM, including high-risk patients. There was no adverse impact of ATO on engraftment. Longer follow-up is necessary to assess the efficacy of this combination on PFS and OS.
Multiple Myeloma with IGH-Involving del(14q): Report of 34 Cases.
Authors: Wlodarska Iwona , Ross Fiona M. , Russell Lisa J. , Pospisilova Helena , Chiecchio Laura , Van Caillie Marie-Astrid , Dagrada GianPaolo , De Wolf-Peeters Christiane , Harrison Christine J. , De Paepe Pascale , Vandenberghe Peter , Michaux Lucienne
Published: October 2019

Chromosomal translocations involving the IGH locus on 14q32 are a hallmark of B-cell malignancies. These translocations are particularly frequent in non-hyperdiploid (NHD) multiple myeloma (MM), representing approximately 50% of myeloma cases. MM-associated primary t(14q32) target at least 7 partner genes including cyclins D (CCND1, −D3), MAF transcription factors (CMAF, MAF B and A) and MMSET/FGFR3. Some of the translocations are predictive of clinical outcome. Recently, we identified a novel interstitial del(14q) involving IGH and recurrent in chronic lymphocytic leukaemia and MM (Pospisilova et al, Leukemia, 2007). In spite of extensive studies, the mechanism(s) and molecular consequences of del(14q) remain unknown. We report here 34 cases of plasma cell (PC) malignancies with del(14q) involving IGH, as proven by FISH. Cases were collected in UK and Belgium. The estimated incidence of these aberrations in PC malignancies was 1.4%. There were 13 female and 21 male patients ranging in age from 49 to 86 years (average 68). Twenty seven patients had MM, one had SMM and 6 had MGUS. In almost all cases, the del(14q) was detected at diagnosis. Clinical data of the reported cases have been collected. The del(14q) were roughly mapped by FISH and grouped into 3 categories according to the proximal breakpoint:

del(14)(q24.1q32.33) involving the ZFP36L1 region (11 cases); deletions proximal to ZFP36L1 (14 cases) and deletions distal to ZFP36L1 (7 cases).

The size of del(14q) was not determined in 2 cases. Biallelic deletion of TRAF3/14q32.33 recurrently occurring in MM, was detected by FISH in 1 out of 9 analyzed cases. Additional FISH analysis showed that the del(14q) was predominantly associated with NHD tumors (62% vs 38% with hyperdiploid karyotypes) and frequently (60%) accompanied by del(13q), regarded as a poor prognostic factor. All reported cases were negative for t(4;14) and t(11;14); they also showed a normal status of CCND3, CMAF, MAFB and CMYC, when examined. A gain of 1q/CKS1B was found in 57% (8/14) of analyzed cases. The expression pattern of cyclin D1−D3 has been examined.
Oxidative-Stress Specific Interaction Between FANCD2 and FOXO3a.
Authors: Li Jie , Maynard Suzette , Andreassen Paul R. , Pang Qishen
Published: October 2019

Abstract 1089

Poster Board I-111

Fanconi anemia (FA) is a human genomic instability syndrome that is uniquely sensitive to oxidative stress. Members of the FA protein family are involved in repair of genetic damage caused by DNA cross-linkers. The molecular pathway in which the FA proteins function in oxidative stress response has not been defined. Here we report functional interaction between the FA protein FANCD2 and the forkhead transcription factor FOXO3a in response to oxidative stress. FOXO3a was colocalized to FANCD2 foci in cells subjected to oxidative stress. The FANCD2-FOXO3a complex was not detected in cells deficient for the FA core complex component FANCA, but could be restored after complementation with a functional FANCA. Consistent with this, a non-monoubiquitinated FANCD2 mutant failed to bind FOXO3a. While both DNA cross-linker mitomycin C and ionizing radiation induced monoubiquitination of FANCD2, neither was able to induce the association of FANCD2 and FOXO3a. This indicates that the FOXO3a-FANCD2 interaction is oxidative stress specific. Overexpression of FOXO3a reduced abnormal accumulation of reactive oxygen species, enhanced cellular resistance to oxidative stress, and increased antioxidant gene expression in corrected but not mutant FA-D2 cells. The novel oxidative stress response pathway converging FANCD2 and FOXO3a identified in this study is likely to contribute to cellular anti-oxidant defense.

Disclosures

No relevant conflicts of interest to declare.
Comprehensive Protein Expression Analysis of Chronic Lymphocytic Leukemia Uncovers a Correlation of CDK4, P27 and P53 with Hierarchical Risk and Suggests a Dichotomy of Apoptosis and Proliferation.
Authors: Winkler Dirk , Schneider Christof , Nitsch Daniela , Schulze Kerstin , Zenz Thorsten , Mohr Julia , Buehler Andreas , Habermann Annett , Zucknick Manuela , Benner Axel , Döhner Hartmut , Stilgenbauer Stephan , Mertens Daniel
Published: October 2019

Abstract 1255

Poster Board I-277

Chronic lymphocytic leukemia (CLL) has a variable clinical course. Genomic aberrations identify prognostic subgroups, pointing towards distinct underlying biological mechanisms. However, for a subset of aberrations, the affected cellular pathways are unknown. Even though the importance of posttranscriptional regulation, e.g. by micro RNAs, has become evident, the proteome of CLL remains little characterized. Therefore, the expression of 23 proteins involved in cell cycle, DNA damage, apoptosis and signaling was investigated in a total of 206 cytogenetically well characterized CLL cases by Western blotting. CLL cases were categorized according to the hierarchical model by Döhner et al. (NEJM 2000) into del(17p) (N=41), del(11q) (N=39), del(13q) (N=39) as sole abnormality, trisomy 12 (N=58) or normal karyotype (N=28). VH mutation status was available in 198 of 206 cases. For statistical analyses, protein expression was quantified by densitometry in 185 patients. Interestingly, in CLL subgroups with a good prognosis, differentially expressed proteins were rather involved in apoptosis. In contrast, in cytogenetic subgroups with a worse prognosis, deregulated proteins were associated with DNA damage and cell cycle control (see figure). In addition, higher levels of CDK4, P27 and P53 significantly correlated with increasing hierarchical risk as defined by genetic subgroups. Inter-protein correlations pointed to either STAT6-signaling or AKT1-signaling to be affected in CLL, but the low number of significant partial correlations between proteins underlines the major influence of genetic aberrations on the cellular CLL phenotype. In summary, differences in protein levels define distinct molecular phenotypes with a dichotomy of apoptosis and proliferation in CLL associated with prognostic subgroups. These findings open new therapeutic options for patient-tailored treatment of CLL.

Figure:

Overview of protein regulations in hierarchical subgroups of CLL suggesting a dichotomy of apoptosis and proliferation in CLL associated with benign and aggressive subgroups, respectively. The figure illustrates an increasing number of deregulated proteins associated with proliferation/DNA damage and a decreasing number of deregulated proteins that control apoptosis with higher genetic risk category (arrows indicate upregulation or downregulation when compared to other risk categories; two arrows: proteins with significant differential expression; single arrows: significant before adjustment for multiple testing (trend)).

Disclosures

No relevant conflicts of interest to declare.
Evaluation of Potential role for Valproic Acid in IL-3-Mediated Megakaryopoiesis and Erythropoiesis in Serum-Free and Serum-Containing Cultures.
Authors: Ohishi Kohshi , Liu Bing , Masuya Masahiro , Heike Yuji , Takaue Yoichi , Katayama Naoyuki
Published: October 2019

Abstract 2746

Poster Board II-722

Although attention has been paid to the antitumor activity of histone deacetylase (HDAC) inhibitors in hematologic disease, a role for HDAC in normal hematopoiesis has not been clearly defined. Previous study have shown that the potent HDAC inhibitor FK228 (romidepsin, depsipeptide) stimulates IL-3-mediated erythropoiesis in serum-free cultures. Here, we precisely examined the effects of well-tolerated antiepileptic drug valproic acid (VPA) on IL-3-mediated megakaryopoiesis and erythropoiesis in serum-free and serum-containing cultures. After incubation of CD34+ cells in serum-free medium containing IL-3+SCF, CD61+GPA− megakaryocytic, a CD61+GPA+ mixture of megakaryocytic and erythroid, and CD61−GPA+ erythroid precursors were generated. Pharmacological concentration of VPA (100 μg/ml) and FK228 (0.45 ng/ml) remarkably increased the number of CD61+GPA+ cells by 7-fold and 9-fold, respectively. Other populations were also increased by FK228 and VPA. VPA and FK228 enhanced the generation of megakaryocytic and erythroid precursors from more mature CD34+ cell-derived CD36+ erythroid and megakaryocytic precursors. Thus, VPA was found to be as potent as FK228 in stimulating the IL-3-mediated megakaryopoiesis and erythropoiesis. In the presence of GM-CSF+SCF, CD61−GPA+ erythroid precursors were mostly developed. Addition of VPA to the cultures mainly promoted the generation of CD61−GPA+ erythroid precursors, while the generation of megakaryocytic precursors was also moderately enhanced. In serum-containing cultures, there were only low numbers of CD61+ or GPA+ cells detected even in the presence of IL-3+SCF. Nevertheless, addition of VPA to the cultures containing IL-3+SCF increased the numbers of CD61+GPA−, CD61+GPA+, and CD61−GPA+ cells by 3-fold, 18-fold, and 4-fold, respectively from three independent experiments. In megakaryopoiesis and erythropoiesis, GATA-1 and GATA-2 are shown to play important roles in the proliferation and differentiation process, respectively. By quantitative RT-PCR analysis, after incubation of CD34+ cells with IL-3+SCF, GATA-2 m-RNA expression was increased at day1 but returned to initial levels at day3. VPA increased the expression level of GATA-2 gene by 1.6-fold and 1.7-fold at day1 and day3, respectively. While GATA-1 gene expression was remarkably elevated at day1 and day3, its expression was not affected by VPA. Our data reveal a potential for VPA to promote the ability of IL-3 to stimulate megakaryopoiesis and erythropoiesis in serum-free and serum-containing cultures and thus implicate a novel therapeutic approach of epigenetic therapy for treatment of hematologic disease such as myelodysplastic syndrome.

Disclosures:

No relevant conflicts of interest to declare.
Prediction of Response to Treatment with Lenalidomide in Patients with Non-Del 5q Myelodysplastic Syndrome by Gene Expression Profiling Remains Difficult.
Authors: Hofmann Wolf-Karsten , Nolte Florian , Benlasfer Ouidad , Thiel Eckhard , Ehninger Gerhard , Platzbecker Uwe
Published: October 2019

Abstract 2781

Poster Board II-757

Lenalidomide belongs to a proprietary class of immunmodulatory drugs showing therapeutic activity in patients with myelodysplastic syndrome (MDS), in particular in those having the 5q-abnormality, but also in patients not showing this cytogenetical aberration. In 2008, Ebert et al. (PLos Med. 2, e35) could demonstrate that there is a specific gene expression profile in bone marrow cells collected from MDS-patients either with 5q- syndrome as well as MDS-patients having no 5q-abnormality which is strongly correlated with the clinical response to treatment with lenalidomide. Whereas this finding is not of clinical importance in patients with MDS del 5q (overall response 75 %) it may play a pivotal role for prediction of clinical response to lenalidomide in non-del 5q MDS-patients. Therefore, we have studied gene expression profile (HG-U133plus2.0, Affymetrix, Santa Clara, CA) of routinely isolated low-density mononuclear bone marrow cells from 8 patients with IPSS low/int-1 risk MDS having no deletion on chromosome 5 but were subsequently treated with lenalidomide 5 mg/day. All of the patients were transfusion dependent for red blood cells. The median duration of treatment with lenalidomide was 22 weeks. RNA was extracted by Trizol and quality controlled by using a Bioanalyzer 2100 system (Agilent, Waldborn, Germany) to exclude RNA degradation. Microarray hybridization was performed according to the standard Affymetrix protocol. Data were analyzed by Microarray Analysis Suites 5.0 (MAS 5.0; Affymetrix) and GeneSpring (Agilent Technologies, Santa Clara, CA). For clustering analysis we utilized the gene list of 68 discriminating genes as published by Ebert et al. the molecular analysis did clearly separate two groups of patients having specific gene expression profiles according to the responder/non-responder group as published previously. Furthermore, single sample prediction could discriminate three out of 8 patients to be possible responders to lenalidomide but this was not correlated to the clinical course of those patients while on treatment with lenalidomide. However, none of the MDS-patients receiving lenalidomide did show significant clinical response as defined by reduction of transfusion requirement by 50 % or transfusion independence. In conclusion, prediction of response to lenalidomide in non-del 5q patients by gene expression profiling so far remains critical. Prospective analysis of molecular changes including DNA analysis in larger clinical trials using lenalidomide in non-del 5q MDS-patients are required to establish reliable predictive markers in MDS.

Disclosures:

Hofmann: Celgene: Research Funding. Platzbecker:Celgene: Research Funding.
Incidence of Centrosome Amplification in Multiple Myeloma.
Authors: Smejkalova Jana , Dementyeva Elena , Nμmec Pavel , Fedor Kryukov , Raja Karthick Raja Muthu , Grešlíková Henrieta , Smetana Jan , Zaoralova Romana , Kupska Renata , Kuglik Petr , Hajek Roman
Published: October 2019

Abstract 2827

Poster Board II-803

Centrosome amplification (CA) has been previously detected in hematological malignancies including multiple myeloma (MM) and is usually associated with disease progression. CA leads to the formation of multipolar mitotic spindles that may lead to chromosome segregation errors and genomic instability. In this pilot study, we have evaluated the occurrence of CA in two populations of B-lineage cells including B-lymphocytes and plasma cells (PCs) of MM patients. We have analyzed possible associations of CA with established prognostic factors including the most common chromosomal abnormalities in malignant PCs.

Immunofluorescence labeling was used for the evaluation of centrosome amplification (CA) in B-cells (CD19+) and PCs (CD138+) of MM patients. The centrin (centrosome protein) copy numbers were used to define three cellular subpopulations: (1) no centrin signal (Non-CS), (2) 1-4 centrin signals (1-4CS) or (3) more than 4 signals of centrin (CA). Samples with ≥11% of B-cells or ≥10% of PCs with >4 fluorescence signals of centrin were considered CA positive. A total of 70 patients were evaluated for CA in PCs and/or B-cells, including 18 patients who had analysis of both cell types. The patient population characteristics were as follows: males/females 34/36, median age of 65 years (range, 40-84 years). Most patients had advanced stage of MM (DS II/III n = 48; ISS II/III; n = 21). Peripheral blood samples from 20 healthy donors were used as controls and for the estimation of CA positivity threshold for B-cells (Mean + 3SD).

There was a statistically significant difference between the percentage of B-cells subpopulations with centrosome amplification in MM patients and that in healthy donors ([Mean ± SD] 9.9 ± 7.9% versus 3.2 ± 2.5%; P<0.0001). The frequency of MM cases positive for CA was 34% (17/50) and 37% (14/38) based the analysis of PC samples and B-cell samples, respectively. Overall, 22% (4/18) MM patients were double-positive. No significant correlation was detected between B-cells and PCs (r=0.387; P=0.113) obtained from patients with both available samples. No significant associations were identified between CA status and the following common cytogenetic abnormalities in PCs: del(13)(q14) (p= 1.000); del(17)(p13) (p=0.132); gain(1)(q21) (p= 1.000), hyperdiploidy (p= 1.000).

In summary, we have confirmed the presence of centrosome amplification in B-cells of MM patients. Immunofluorescence staining is a sensitive method for the detection of abnormal subpopulations of B-cells that probably represent a reservoir of clonogenic cells in MM.

This study was supported by grants NR 8945-4/2006, MSM 0021622434, MZ LC 06027 and IGA NR 9317 from the Departments of Education and Health of the Czech Republic.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy of the Combination of Bortezomib and Dexamethasone in Systemic AL Amyloidosis.
Authors: Lamm Wolfgang , Willenbacher Wolfgang , Zojer Niklas , Müldür Ercan , Ludwig Heinz , Lang Alois , Zielinski Christoph C , Drach Johannes
Published: October 2019

Abstract 2871

Poster Board II-847

Introduction:

AL amyloidosis is characterized by misfolding of structurally unstable light chains that form deposits in various organs thereby causing impaired organ function. Treatment of AL amyloidosis remains challenging and is primarily directed towards the underlying abnormal plasma cell clone. Novel agents with proven efficacy in multiple myeloma like the proteasome inhibitor bortezomib have also shown initial promising results in patients with AL amyloidosis, but its value needs to be further established. In this retrospective analysis, we have collected data regarding efficacy and tolerability of treatment with bortezomib plus dexamethasone in patients with AL amyloidosis.

Patients and Methods:

25 patients with histologically proven systemic AL amyloidosis were included in this analysis. All patients received bortezomib at a standard dose of 1.3mg/m2 (days 1, 4, 8, 11 of a 3-week cycle) in combination with dexamethasone (8mg to 20mg administered on the day of bortezomib and the day after). Routine clinical and laboratory parameters including evaluation were obtained on a monthly basis.

Results:

Patients (male n=13, female n=12) were at a median age of 57 years (range 42-83), and 17 patients (68%) received bortezomib/dexamethasone as their first line treatment. The majority of patients had an ECOG performance status of < 2. Twelve patients (48%) had only one organ involved, whereas > 2 organs were involved in 6 patients (24%). Organs most frequently involved were kidneys (100%), heart (28%), liver (12%) and the gastrointestinal tract (12%). At the time of analysis, a median of 3 cycles of bortezomib/dexamethasone (range, 1 to 8) have been administered. A hematologic response was observed in 14 patients (56%) including 5 patients (20%) qualifying for a complete response (CR). All CR patients received bortezomib/dexamethasone as their first line treatment, and parameters of organ function also improved in these patients. Median overall survival has not yet been reached after a median follow-up of 12 months. Grade 3 and 4 toxicities were rare and consisted predominantly of transient thrombocytopenia. Grade 2 neurotoxicity was observed in 24% of patients. Other grade 1/2 toxicities observed at higher frequencies included hypotension (16%), edema (16%), and fatigue (12%).

Conclusion:

Our results confirm the activity of bortezomib/dexamethasone in patients with AL amyloidosis (hematologic response rate 56% including a 20% CR rate).

Disclosures:

Ludwig: Celgene: Honoraria; Mundipharma: Honoraria; AMGEN: Honoraria; Ortho-Biotech : Honoraria; Janssen-Cilag: Research Funding; Roche: Honoraria.
Integrative Analysis of MicroRNA and Gene Expression Profiling Contributes to Understand Mantle Cell Lymphoma Pathogenesis.
Authors: Di Lisio Lorena , Gómez-López Gonzalo , Sáanchez-Beato Margarita , Rodríguez Maria Elena , Abad Cristina Gómez , Villuendas Raquel , Mollejo Manuela , Martínez Miguel A. , Menarguez Javier , Diaz-Alderete Andrea , Gil Juana , Cigudosa Juan , Pisano David G. , Piris Miguel A. , Martínez Nerea
Published: October 2019

Abstract 2936

Poster Board II-912

Mantle cell lymphoma (MCL) pathogenesis is still partially unexplained. Although the overexpression of CyclinD1 dependent of t(11;14) is a distinctive molecular hallmark of this neoplasm, this event alone cannot account for the increased survival signaling that characterizes this lymphoma type. Here we investigate whether microRNA (miRNA) expression profile may help to explain the changes in the expression of gene pathways that are characteristic of MCL.

Twenty-three frozen MCL samples, 11 frozen control tissues (7 lymph nodes and 4 tonsils), 8 MCL cell lines and 3 samples of CD19+IgD+CD27- cells obtained from tonsils, were studied for miRNAs and gene expression. MiRNA one color microarray data for 470 human miRNA were analyzed using SAM (Significance Analysis of Microarrays) algorithm. MiRNA targets were predicted by miRanda and TargetScan methods. Pathways identification and analysis was carried out by GSEA (Gene Set Enrichment Analysis) online resource.

The analysis of 23 MCL cases compared to 11 control tissues showed a miRNA signature that included 117 miRNAs with FDR<0.05, 85 of which downregulated and 32 upregulated. Combined analysis of these miRNAs and changes in the gene expression profile, paired with bioinformatic target prediction, revealed a group of genes and pathways potentially targeted by the miRNAs, including essential pathways for lymphoma survival. An interesting correspondence was found between the simultaneous increase in CD40, MAPK, NFKB and others pathway signaling with the downregulation of 15 miRNA predicted to target genes belonging to these pathways. Functional validation in MCL cell lines demonstrated NF-kB nuclear translocation to be regulated by the expression of one of these miRNAs. Most of the MCL cell lines exhibit a strong expression of the mir-17-92 polycistron (Oncomir-1).

MiRNAs were used also for the identification of survival prognostic markers; using different analysis (22 frozen specimens) and validation (54 paraffin embedded cases) series. A single miRNA distinguished a group of MCL cases with a 72.2% survival at 60 m.

This study identifies a set of miRNAS involved in MCL pathogenesis, which could be used in MCL recognition and clinical prognostication.

Disclosures:

No relevant conflicts of interest to declare.
Incidence and Clinical Outcomes of Occult Malignancies Detected by Computed Tomographic Pulmonary Angiography in Patients with Acute Pulmonary Embolism.
Authors: Shen Yi-Hao , Wells Phil , Dennie Carole , Carrier Marc
Published: October 2019

Abstract 2983

Poster Board II-959

Introduction:

Venous thromboembolism (VTE) can be the earliest sign of malignancy. Approximately 10% of patients with unprovoked VTE will be diagnosed with cancer within the next 12 months. Diagnosis of pulmonary embolism (PE) using computed tomographic pulmonary angiograhy (CTPA) allows the visualization of anatomy in addition to thoracic vasculature. Hence, CTPA might be useful for detecting occult cancers in patients with PE.

Objective:

To evaluate the incidence and clinical outcomes of occult cancers detected by CTPA in patients with acute PE.

Methods:

This is a retrospective cohort study of consecutive patients with suspected PE undergoing CTPA at the Ottawa Hospital from Jan 1, 2007 to Dec 31, 2008. PE was defined as a subsegmental or larger pulmonary artery filling defect on CTPA. Occult malignancy was defined as any new cancer first detected by CTPA with index PE diagnosis in patients with no known history of malignancy. All patients were followed for a minimum of 6 months after the index PE.

Results:

A total of 4410 CTPA were reviewed and 748 (17%) were positive for acute PE. Of these, 57 (7.6%; 95% CI: 5.7 to 9.5) revealed abnormalities suggestive of possible occult cancers. Twenty-two (2.9%; 95% CI: 1.7 to 4.2) patients were diagnosed with occult cancers. Among these 22 patients with occult cancers, 20 (91%) had unprovoked PE. Thirteen (59%) patients had occult lung cancer and 16 (73%) had advanced stage (stage 3 or 4) cancers. Sixteen (73%) patients have died following the diagnosis of occult cancer, and their median survival following the diagnosis of PE was 51 days.

Conclusion:

CTPA detected occult cancers in approximately 3% of patients with acute PE. Most of these cancers were detected at an advanced stage and were associated with high mortality and short survival.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of An Automatic Washing Method for DMSO Cryopreserved Peripheral Blood Hematopoietic Progenitors Using the Sepax S-100.
Authors: Blanquer Miguel , Cabañas-Perianes Valentín , Majado Maria Juliana , Ibáñez Victoria Sánchez , Sánchez Pilar Menchón , Monserrat Jorge , Lázaro Alfonso Morales , Moraleda Jose Maria
Published: October 2019

Abstract 3219

Poster Board III-156

Introduction

Dimethylsulfoxide (DMSO) containing cryoprotective solutions are routinely used for the storage of hematopoietic progenitors (HP). At room temperature, DMSO is toxic for the cells and may produce severe adverse reactions during their infusion, especially in the pediatric patients. These problems can be avoided by washing the cells prior to the infusion. Our objective was to test if an automatic washing method (Sepax S-100, Biosafe) allowed us to preserve the CD34+ cell numbers with an adequate viability and engraftment potential.

Material and Methods

Forty five peripheral blood HP apheresis that have been cryopreserved using autologous plasma plus 9% DMSO were studied. After rapid thawing in a water bath at 37° C, an automatic wash with the Sepax S-100 (2 washes cycle) was performed. Nucleated cell levels determined by an hematology analyzer, flow cytometry CD34+ cell counts and Trypan Blue cell viability test were performed on aliquots collected prior to and after the washing technique. The paired Student's t-test and the Pearson's correlation coefficient were used for the statistical analysis.

Results

The mean total nucleated cell (TNC) and CD34+ cell recovery was 75,47% ± 3, and 94,66% ± 4,62 respectively. In spite of the TNC significant loss (p<0,001), there were no significant differences between the pre and post-washing CD34+ cell numbers (p=0,08) or viability (p=0,19). The TNC loss had no correlation with the pre or post-washing number of platelets (p=0,18 and p=0,16 respectively), neither with the loss of platelets during the procedure (p=0,31). In contrast with the 40% of untoward reactions recorded in our historical data of 226 DMSO containing cell infusions, we observed just one adverse effect (2,22%) with the washed cells. One patient reported a transient mild abdominal pain with nausea and one vomit. Median time to neutrophil engraftment (>500 cells /mL) and platelet engraftment (>50.000 cells/mL) were 11 ± 0,2 and 20 ± 1,4 days respectively.

Conclusions

The Sepax S-100 automatic wash protocol of DMSO containing peripheral blood progenitor cells determines a good CD34+ cell recovery and preserves their viability and engraftment potential. This method avoids the DMSO infusion related adverse events and it constitutes a closed and easy to do procedure.

Disclosures

No relevant conflicts of interest to declare.
Diagnosis of Invasive Fungal Infections by a Real-Time Panfungal PCR Assay in Pediatric Patients Undergoing Intensive Chemotherapy or Allogeneic Stem Cell Transplantion.
Authors: Landlinger Christine , Baskova Lenka , Preuner Sandra , Grotel Martine Van , Hartwig Nico G. , Heuvel Marry M. van den , Lion Thomas
Published: October 2019

Abstract 343

Invasive fungal infections are life threatening events in severely immunocompromised patients, and there is urgent need for reliable screening methods facilitating rapid and broad detection of pathogenic fungi. We have established a two-reaction real-time PCR assay permitting highly sensitive detection and quantitative monitoring of more than 80 fungal pathogens, covering a large spectrum of moulds, yeasts and Zygomycetes (European patent No. 06817468.9). To assess the clinical potential of the panfungal real-time PCR assay, more than 600 consecutive specimens from 126 pediatric patients carrying a high risk of invasive fungal infections were analyzed. The results revealed an excellent correlation between PCR positivity and the presence of proven, probable or possible fungal infection according to the criteria of the European Organization for Research and Treatment of Cancer (EORTC), indicating a sensitivity of the assay of 96% (95%CI: 81-99.3%). Hence, the negative predictive value of the panfungal PCR assay presented is very high, and our current data indicate that molecular screening of patients during febrile neutropenic episodes by the assay can help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. The specificity of the assay in the test cohort of patients was in the range of 76% (95%CI:62-87%), and the observations indicate that rapid species identification may be required to assess the positive predictive value for impending fungus-related disease. The availabel data provide a basis for appropriately designed clinical studies addressing the full diagnostic potential of fungal screening by highly sensitive, broad- spectrum molecular assays in severely immunocompromised patients.

Disclosures:

No relevant conflicts of interest to declare.
Long-Acting Interleukin-11 Shows Improved Activity to Increase Platelet Numbers.
Authors: Jung Yuni , Ahn Haesook , Kim Dong-Sik , Hwang Yu Ri , Ho Seong-Hyun , Kim Sujeong , Kim Jong-Mook
Published: October 2019

Abstract 3512

Poster Board III-449

Often the clinical efficacy of a therapeutic protein relies on the structural stability, bioavailability, and clearance rate of the protein. The current work presents greatly improved efficacy of human interleukin-11 mutein conjugated with single polyethylene glycol polymer (PEG-mIL-11). The interleukin-11 mutein (mIL-11) is newly redesigned human interleukin-11 to endure chemical and proteolytic stresses while retaining all the biological activities, which reduces chance of causing side effects in clinics. The interleukin-11 mutein is currently under phase II clinical trial in China to evaluate ability to increase platelet counts after chemotherapy. Further, single methoxy-polyethylene glycol succinimidyl carbonate was attached onto mIL-11 via surface exposed amines to prolong the circulation time in vivo. Unlike often observed cases with other PEGylated proteins, the attached bulky PEG provided no hindrance to the receptor binding of IL-11, when in vitro cell proliferation activity was monitored on Ba/F3 cells expressing IL-11 receptors. Interestingly, single subcutaneous administration of PEG-mIL-11 showed higher efficacy to increase platelet counts than daily subcutaneous administration of unmodified mIL-11 of same dosage for 7 days in rats. The pharmacokinetic profile of PEG-mIL-11 was monitored after single subcutaneous injection in rats. The half-time (t1/2), time to reach the maximum level (Tmax), and bioavailability (area-under-curve) of PEG-mIL-11 increased 5-, 6- and 5-fold as compared to the values of unmodified mIL-11 of same dosage, respectively. Mono-PEGylated IL-11 mutein demonstrated not only the prolonged circulation time but also the retained biological activity, thereby the possibility for clinical applications such as the treatment or prevention of the chemotherapy-induced thrombocytopenia.

Disclosures:

No relevant conflicts of interest to declare.