bluuebunny/crossref_metadata_2025 huggingface dataset:
Allogeneic Blood Stem Cell Transplantation in Patients with Hematologic Diseases after Conditioning with Treosulfan and Fludarabine.
Authors: Casper Jochen , Wilhelm Stefan , Steiner Beate , Wolff Daniel , Grobe Norbert , Haehling Detlev , Hartung Gernot , Hilgendorf Inken , Lueck Andreas , Junghanss Christian , Kahl Christoph , Pichlmeier Uwe , Freund Mathias
Published: April 2020

Background and objective:

Allogeneic transplantation can not be offered to many patients due to potential side-effects of conventional conditioning. Dose-reduced conditioning approaches improve tolerability, however, treatment intensity and efficacy might be reduced as well. We have, therefore, developed a dose intense, but toxicity reduced conditioning regimen based on treosulfan and fludarabine and report first results.

Patients and methods:

65 patients with a median age of 50 years were transplanted from related (n=21) or unrelated donors (n=44) after conditioning with treosulfan (3x10, 3x12 or 3x14g/m2 i.v.) and fludarabine (5x30mg/m2 i.v.). 21 patients were in complete remission (CR) and 44 patients had not reached a CR at the time of transplantation. 59 of 65 patients were considered unfit for a conventional conditioning regimen.

Results:

After a median follow up of 3.2 years an actuarial overall survival (OS) of 59.2 % (at 3 years) and an event-free survival (EFS) of 40,1% has been reached. Patients with related donor or transplantation in CR had a better OS (85,4 resp. 74,2%) and EFS (52,2% resp. 61,9%). The cumulative incidence of relapse at 3 years was 26,2%. Transplantation in CR resulted in a lower relapse incidence than transplantation not in CR (17,9 vs. 30%). Treatment-related mortality (TRM) at day 100 is 17,4% (cumulative incidence). In stepwise Cox regression analyses for OS, EFS and TRM only the covariables transplantation in CR vs. not in CR and the donor status were shown to be influential. No effects were detected for age (under 50 years vs. 50 years and older), concomitant disease (present vs. not present) or intensity of pretreatment.

The cumulative incidence for acute graft-versus-host disease (GvHD) °I-IV at day 100 was 43.3%. The cumulative incidence for chronic GvHD at 3 years is 36.1%.

Conclusions:

These results with a conditioning therapy of treosulfan and fludarabine indicate that patients despite higher age, concomitant disease or after intensive pretreatment can be successfully transplanted without increased treatment-related mortality.
Identification of Novel MHC Class I and Class II Epitopes of WT1 Using a Peptide Library Screen.
Authors: Rezvani Katayoun , Mielke Stephan , Kilical Yasemin , Grube Matthias , Fujiwara Hiroshi , Sconocchia Giuseppe , Lee Jongming , El-Ouriaghi Frank , Hensel Nancy , Barrett A. John
Published: April 2020

Although several HLA-A*0201-restricted immunodominant peptides from the leukemia-restricted protein WT-1 are characterized, T cell responses to peptide sequences binding to other common class I and II epitopes of WT-1 remain almost completely unexplored. A more comprehensive definition of the WT-1 antigen would extend peptide antigen vaccines to individuals lacking HLA-A*0201 and *2402 and improve vaccine potency by recruiting both CD4+ and CD8+ T cell responses. Here we used a WT1 peptide library to identify WT-1 peptide sequences inducing CD4+ and CD8+ T cell responses in normal individuals and patients with AML and other myeloid leukemias. Six cases were studied. The library consisted of 110 15mer peptides overlapping by 11aa covering the entire WT-1 protein in 21 pools. Monocytes were isolated by plastic adherence and pulsed with peptide pools for 3 hours. Autologous CD8+ and CD4+ T cells were then added. Pools of peptides were prepared in such a way that each peptide was represented in two different peptide pools, allowing the identification of the respective peptide by responses in the two corresponding pools. Cells were harvested for RNA extraction and reverse transcription. Real time PCR (RQ-PCR) was used to identify peptide-specific induction of IFN-γ and IL-2 in CD8+ and CD4+ T cells. The SYFPEITHI binding motif software was then used to predict the probable HLA restriction for the candidate epitopes. To confirm candidate peptide immunogenecity and HLA restriction, selected peptides were synthesized and tested individually. In addition to the known HLA-A*0201 peptides WT37, WT126, WT187 and WT235 we identified 20 new MHC class I and II epitopes of WT1. Four were restricted by more than one HLA allele, demonstrating the promiscuity of epitope binding. One epitope (VPGVAPTLV) was restricted to HLA-A*0201 and HLA-B*5101. One epitope (SGQFTGTAGACRYGP) was restricted by a class I HLA allele, namely HLA-*6801 and a class II HLA allele, DR*1501. Two epitopes (YGPFGPPPPSQASGQ and QKKFARSDELVRHHN) were restricted by multiple MHC class II alleles. The proliferative response of CD4+ and CD8+ T cells to candidate peptides was confirmed using CFSE labeling. We now plan to characterize the antileukemic effects of CD4+ and CD8+ T cells induced by these peptides with a view to designing broad-spectrum vaccines inducing leukemia-reactive T cells across a wide range of HLA types.
Regulation of Signal Transducers and Activators of Transcription by Src Family Kinase in AML.
Authors: Ozawa Yukiyasu , Williams Ann H. , Estes Myka , Sokol Lubomir , Jove Richard , List Alan F.
Published: April 2020

Specific Signal Transducers and Activators of Transcription (STAT) family members are constitutively activated in leukemia where they contribute to leukemia self-renewal and apoptosis resistance. STAT5 activation in chronic myeloid leukemia (CML) is driven by the Bcr-Abl and Lyn kinases, whereas in acute myelogenous leukemia (AML), STAT5 and STAT3 activation is demonstrable under the control of as yet undefined upstream regulators. To investigate the regulation of STAT activation in AML, we evaluated the role of Src family kinases in AML cell lines and primary patient specimens. Inhibition of leukemia cell prolifaration by Src inhibitors was evaluated in 7 cell lines (U937, HEL, KG-1a, THP-1, OCI-AML3, HL-60 and NB-4). Treatment with either PD180970 or SKI-606 significantly suppressed proliferation and decreased viability in a concentration-dependent manner in two cell lines. U937 cells displayed sensitivity comparable to K562 CML cells, whereas KG-1a cells demonstrated moderate inhibition. Corresponding DNA-STAT5 binding activity assessed by electrophoretic mobility shift assay (EMSA) showed constitutively STAT5 activation in 3 cell lines (U937, HEL and KG-1a), with inhibition of STAT5 DNA binding By Src inhibitors in U937 and KG-1a cells, while STAT5 activity was not impaired in HEL cells. STAT5 protein phosphorylation assessed by immunoblot was suppressed by the Src inhibitors in the sensitive cell lines. Moreover, Src inhibition decreased Bcl-XL expression, a transcriptional target of STAT5, and induced apoptosis in U937 cells. Immunoblot assessment of phosphorylation of the activation loop tyrosine in the two major src kinases, c-Src and Lyn, confirmed inhibition of autophosphorylation in U937 cells and K562 cells by PD180970 and SKI-606, while phosphorylation was unchanged in HEL cells. Inhibition of src kinases was rapid (< 30 min), and preceeded inhibition of STAT5 activation. Analysis of primary AML patient cells revealed that 29 % (5 of 17) of patient cells have constitutively-activated STAT5, and 47 % (8 of 17) have constitutively-activated STAT3. More importantly, some primary AML patient cells were sensitive to Src inhibitor. These data suggest that Src activation of STAT5 has a pathogenetic role in AML cells. Investigation of Src inhibitors merits clinical investigation in patients with AML.
Thromboprophylaxis after Orthopaedic Surgery with an Oral, Direct Factor Xa Inhibitor: Pooled Results of Two Phase Iib Clinical Trials.
Authors: Turpie Alexander G.G. , Bauer Kenneth Alan , Borris Lars , Dahl Ola E. , Fisher William D. , Gent Michael , Haas Sylvia , Huisman Menno V. , Kakkar Ajay K. , Kälebo Peter , Kwong Louis M. , Homering Martin , Misselwitz Frank , Eriksson Bengt I.
Published: April 2020

Thromboembolic events, such as deep vein thrombosis (DVT) and pulmonary embolism (PE), are a serious risk after surgical procedures, such as major orthopaedic surgery. BAY 59-7939 is an oral, direct Factor Xa inhibitor in clinical development for the prevention and treatment of thromboembolic disorders. The efficacy and safety of BAY 59-7939 for thromboprophylaxis have been determined relative to enoxaparin in two clinical trials, one after elective total hip replacement surgery, and one after elective total knee replacement surgery. Both trials were multicenter, multinational, double-blind, dose-ranging studies; the hip surgery trial was performed in Europe, and the knee surgery trial in North America. This pre-specified analysis combines data from both trials. Patients (n=1343) were randomized to oral BAY 59-7939 at 2.5, 5, 10, 20, or 30 mg twice daily (bid), or subcutaneous enoxaparin (40 mg once daily starting 12 hours before hip surgery, or 30 mg bid starting 12 hours after knee surgery). Treatment continued until mandatory bilateral venography was performed 5–9 days after surgery. The primary efficacy endpoint was a composite of DVT, PE, and all-cause mortality, and was analyzed in 914 per-protocol patients. The secondary efficacy endpoint was major venous thromboembolism (VTE) - proximal DVT, PE, and VTE-related death. The primary safety endpoint was major, post-operative bleeding, and was analyzed in 1317 patients. Observed pooled event rates are shown in the table. No significant dose-response relationship for efficacy was observed with BAY 59-7939 (P=0.39 and P=0.46 for primary and secondary endpoints, respectively) in logistic regression models using total daily dose and adjusting for study, age and gender effects; this was potentially due to the efficacy achieved with the lower BAY 59-7939 doses. A significant dose-response relationship was observed for major, post-operative bleeding with BAY 59-7939 (P<0.001). In conclusion, this analysis showed that for the prevention of VTE following major orthopaedic surgery, BAY 59-7939 has a wide therapeutic window and, at doses of 2.5 to 10 mg bid, has similar efficacy and safety to the enoxaparin regimens.

BAY 59-7939 (mg bid) Endpoint 2.5 5 10 20 30 Enoxaparin DVT, PE, all-cause mortality 36/167 (21.6%) 38/166 (22.9%) 26/161 (16.1%) 38/156 (24.4%) 17/88 (19.3%) 49/176 (27.8%) Major VTE 5/167 (3.0%) 4/166 (2.4%) 5/161 (3.1%) 5/156 (3.2%) 1/88 (1.1%) 8/176 (4.5%) Major, post-operative bleeding 2/232 (0.9%) 3/238 (1.3%) 5/236 (2.1%) 9/232 (3.9%) 10/143 (7.0%) 4/236 (1.7%)
NF Kappa B as a Therapeutic Target in AML.
Authors: Jenkins Christopher , Pepper Christopher J. , Mills Ken I. , Burnett Alan K.
Published: April 2020

NF Kappa B is an inducible eukaryotic transcription factor, which is highly conserved throughout nature, and regulates a large number of genes which are essential for immune and inflammatory responses. Inappropriate expression has been implicated in a number of inflammatory and malignant conditions. We have investigated the expression of NF Kappa B in normal mobilised peripheral blood cells, AML cell lines (HL60, NB4, K562, U937) and 27 primary AML cells, with a view with it being a target for NF Kappa B inhibition. The cells were also treated with a range of doses of LC1 (Leuchemix Inc) which includes NF-KB inhibition in its activity profile. The expression of NF Kappa B was determined by Affymetrix Gene Chip Array (22,283 genes) and confirmed by RT-PCR. Overall the leukaemic cells had a greater than a two-fold expression compared with normal cells. Expression was significantly greater in the FAB M2 and M4 subgroups and tended to increase with the degree of morphological differentiation, and with poor risk cytogenetics. There was no relationship between expression age, gender or FLT3 mutation status. Since increased NF Kappa B expression confers resistance to apoptosis, the level of spontaneous apoptosis of cells after 24 hours in vitro was assessed by annexin V expression. Apoptosis was inversely correlated with expression.

LC1 (Leuchemix Inc) is a novel agent with NF Kappa B inhibition among its properties. Cells were treated with LC1 in a dose range of 0.002 to 20μmol, and the LC50 determined in an MTS assay. For the cell lines HL60, NB4 and U937 the average LC50 was 6.1μmol. K562 cells have a lower expression of NF Kappa B (40%) compared with the other cell lines and this was reflected in a lower in vitro sensitivity (LC50 > 100μmol). The LC50s for the 27 primary AML stem cell samples ranged from 0.61μmol to > 100μmol with a mode of 7.1μmol. The majority of the cases (24/27) were between 0.61 and 13.2μmol with a mean of 5.9μmol while the 3 outliers have an LC50 which was 4–10 fold higher. In order to assess whether LC1 enhance the cytotoxicity of Ara-C co-incubation experiments were undertaken using the MTS assay. Significant synergy was observed in 8 or 27 cases, and antagonism in 12 of 27 cases.

These data suggest that NF Kappa B inhibition with LC1 merits further evaluation as targeted treatment alone, or in combination, in AML.
Phase II Clinical Trial of Flavopiridol (FL) Followed by ara-c and Mitoxantrone (AM) for Adults with Relapsed and Refractory Acute Leukemias.
Authors: Karp Judith E. , Smith B. Douglas , Greer Jacqueline , Briel Janet , Levis Mark J. , McDevitt Michael A. , Gore Steven D. , Colevas A. Dimitrios
Published: April 2020

The cell cycle inhibitor FL targets cyclin-dependent kinases, induces checkpoint arrest and interrupts transcriptional elongation. We demonstrated in an in vitro model that F causes apoptosis in leukemic blasts and increases A cytotoxicity in remaining blasts (Clin Cancer Res9: 307–315, 2003). Based on these observations, we have conducted a Phase II trial of timed sequential therapy (TST) with FL 50 mg/m2 daily x 3 days followed by A 2 gm/m2/72 hr infusion beginning day 6 and M 40 mg/m2 day 9 (FLAM). A total of 42 adults (median age 50, range 23–75) received FLAM for acute myelogenous leukemia (AML, 37 pts) or acute lymphoblastic leukemia (ALL, 5 pts). Four pts had new AML (myelodysplasia- or treatment-related), 19 were in relapse after first complete remission (CR; median duration 8 mos, range 1.5 – 20), and 19 had refractory leukemia (9 primary). Four pts had prior allo- or auto-stem cell transplants, 13 (31%) had secondary AML and 25 (60%) had adverse cytogenetics, including 2 with Ph+ ALL. FLAM was well-tolerated with death occurring in only 3/42 (7%, fungemia, respiratory distress, multi-organ failure). Grade ≥ 3 non-hematologic toxicities consisted of oral mucositis (2/42, 5%), diarrhea (1/42, 2%), cardiac arrhythmia or decreased ejection fraction (3/42, 7%). F induced tumor lysis in 14 (33%) within 24 hrs of first F dose and a ≥ 50% fall in WBC after 2 F doses in 18 (43%). Median time to neutrophils ≥ 500/mm3 was day 30 day (range 24–46) and platelets ≥50,000/mm3 was day 35 (range 24 – 55). CR was achieved after 1 course of FLAM in 15/37 (41%) AML: 3/4 new patients (including complex cytogenetics), 11/19 relapse and 1/9 primary refractory. CR was achieved in 1/5 (20%) ALL in a patient with relapsed Ph+ ALL. Two of 4 with prior transplant achieved CR, while no patient who was refractory to multiple chemotherapies achieved CR. Of the 15 achieving CR, 13 received additional therapy. Eight pts (7 CR, 1 partial response; median age 45, range 26–62; 1 new diagnosis, 6 relapse, 1 primary refractory) went on to receive allo-transplant in second CR, with CR 2 durations 4.5–12+ mos. An additional 2 pts (ages 45, 46) who had relapsed after allo-transplant in CR1 were able to receive donor lymphocyte infusions (DLI) after FLAM and remain in CR2 of 8+ mos duration. Three pts over age 65 received a second cycle of FLAM in CR, with CR durations of 8.5+, 10, and 10.5 mos. Induction chemotherapy with FLAM yields a significant CR rate in pts with newly diagnosed poor-risk AML or in pts with short CR1, and allows eligible pts to proceed to allo-transplant in CR2.
Differentiation Therapy for Poor Risk Myeloid Malignancies: Results of a Dose Finding Study of Bryostatin-1 (BRYO) + GM-CSF.
Authors: Smith B. Douglas , Matsui William H. , Gladstone Douglas E. , Karp Judith E. , Gore Steven D. , Huff Carol Ann , Vala Milada , Baker Sharyn , Zhao Ming , Piantadosi Steven , Jones Richard J.
Published: April 2020

Clinically effective differentiating therapy has been limited to the use of all-trans-retinoic acid in patients (pts) with acute promyelocytic leukemia. We recently demonstrated that the in vitro activity of many pharmacologic differentiating agents, including the protein kinase C agonist bryo, was dependent on their ability to block cell cycle and maximized by the addition of growth factors. We studied the safety and efficacy of continuous infusion bryo in combination with GM-CSF (125 ug/m2/day SC) in patients (pts) with refractory and poor risk myeloid malignancies. Bryo was dose escalated using the Continual Reassessment Method from 6ug/m2/day to 20ug/m2/day for 14 days monthly and followed by 16ug/m2/day to 20ug/m2/day over 21 days each month. The treatment plan called for 2 cycles of combination therapy prior to response evaluation. A total of 32 patients with refractory or poor risk myeloid malignancies (9 relapsed or refractory AML, 1 therapy-related AML, 2 poor risk AML age >70, 19 poor risk MDS, 1 progressive PNH) were treated. The median age of subjects was 65 (range 23–75) yrs and a total of 73 (58 full + 15 partial) cycles were administered. The dose-limiting toxicity of the combination mirrored results from previous trials of bryo; grade 3–4 myalgias and arthralgias were observed in 12 of 14 pts at the highest dose levels (18 and 20ug/m2/day x 14 days, 16ug/m2/day x 21 days). Three pts discontinued therapy within a week of cycle 1 and were not considered evaluable for response. Eighteen (62%) pts responded (2 CRs and 16 PRs), 6 (21%) had stable disease, and 5 (17%) progressed. The PRs included 15 lineage responses and 1 bone marrow response (<5% blasts) with persistent cytopenias. The 2 CRs included a 59 yr old with poor risk MDS treated @ 20ug/m2/day x 14 days followed by a 2nd cycle @ 18ug/m2/day x 14 days (dose-reduced due to toxicity) who achieved a CR and continues to show improving cytogenetics at 11 mos and a 74 year old with newly diagnosed primitive leukemia (>50% marrow blasts) who received a single cycle @ 16ug/m2/day x 21 days and remains in CR at 5 mos. Both pts refused further courses due to myalgias and arthralgias, and interestingly, the CRs were not achieved until 4–6 weeks after completion of therapy. Our newly developed assay (sensitivity 50 pg/ml) detected measurable bryo only at the highest doses which supported the correlative in vitro assays that detected in vivo differentiation at the same doses. Bryo + GM-CSF has remarkable clinical activity in refractory myeloid malignancies, but is intolerable at biologically active doses. These results do suggest that strategies utilizing differentiating agents in conjunction with growth factors warrant further investigation.
Treatment of Patients with Refractory AML with SU5416 Downregulates VEGF-A, STAT5 and Akt Expression in Leukemic Blasts.
Authors: Loges Sonja , Tinnefeld Heike , Metzner Anja , Jucker Manfred , Butzal Martin , Bruweleit Melanie , Fischer Uta , Draab Elena , Schuch Gunter , O’Farrell Anne M. , Hossfeld Dieter K. , Bokemeyer Carsten , Fiedler Walter
Published: April 2020

Autocrine or paracrine activation of receptor tyrosine kinases are important pathogenetic factors in AML. Signal transduction via c-kit and FLT3 increase proliferation and apoptosis resistance of leukemic blasts. Additionally bone marrow angiogenesis plays a significant role in disease progression.

This provided the rationale for a multicenter clinical trial in patients with refractory AML with SU5416, a small molecule kinase inhibitor which blocks phosphorylation of c-kit, FLT3, VEGFR-1, VEGFR-2 (KDR) and VEGFR-3.

In the study presented here, we analysed expression levels of VEGF and the downstream signal transduction intermediates STAT5 and Akt in patients before and during treatment with SU5416.

The levels of VEGF mRNA expression were investigated in peripheral blood leukemic blasts by means of quantitative Real-Time PCR. 74% (14 of 19) of analyzed patients had rapid and persistent down regulation of VEGF during therapy. Patients initially expressing high VEGF-levels had a stronger down regulation and a higher clinical response rate (mean 865-fold, n=9) than patients initially expressing low VEGF-levels (mean 4-fold, n=8). These results suggest that downregulation of high pretherapeutic VEGF expression by SU5416 treatment may serve as an early surrogate marker of therapeutic response to anti-angiogenetic therapy.

Additionally, protein expression of STAT5 and Akt was assessed by Western blotting. Incubation of the leukemic cell line M-07e in-vitro with SU5416 using concentrations which are achievable in patients was used as a model system. In the AML patient samples parallel downregulation of both STAT5 and Akt was observed in several cases (STAT5 in 4 of 15, Akt in 3 of 6 examined patients). Decreased intracellular levels of STAT5 and Akt were also found in the line M-07e after incubation with SU5416 in vitro.

In summary, our data provide evidence for suppression of expression of VEGF and of the key signal transduction intermediates STAT5 and Akt in AML blasts in-vivo during treatment with SU5416.
Prevention of Venous Thromboembolism after Total Hip Replacement with Once-Daily BAY 59-7939 - An Oral, Direct Factor Xa Inhibitor.
Authors: Eriksson Bengt I. , Borris Lars , Dahl Ola E. , Haas Sylvia , Huisman Menno V. , Kakkar Ajay K. , Misselwitz Frank , Muehlhofer Eva , Kälebo Peter
Published: April 2020

Venous thromboembolism is a serious risk after major surgery, and significant research is underway to develop novel, convenient anticoagulants. Patients undergoing hip replacement surgery are a suitable population for the study of novel anticoagulants because of a high rate of silent deep vein thromboses (DVTs), and the ability to quantify bleeding in a controlled environment. BAY 59-7939 is an oral, direct Factor Xa inhibitor in clinical development for the prevention and treatment of thromboembolic disorders. Dose-finding studies in hip and knee replacement patients showed that twice-daily, oral BAY 59-7939 initiated post-operatively had a wide therapeutic window, with similar efficacy and safety to enoxaparin (30 mg twice-daily or 40 mg once-daily) for the prevention of venous thromboembolism (VTE), and was unlikely to require monitoring. This European, randomized, double-blind, double-dummy, dose-finding study was performed to compare the efficacy and safety of once-daily, oral BAY 59-7939 with subcutaneous enoxaparin for VTE prevention in patients undergoing total hip replacement. Males over the age of 18 and postmenopausal females undergoing elective total hip replacement were randomized to oral BAY 59-7939 5, 10, 20, 30, or 40 mg once daily, or subcutaneous enoxaparin 40 mg once daily. Patients received enoxaparin on the evening before surgery and at least 6–8 hours after surgery, or BAY 59-7939 6–8 hours after surgery. Study drugs were then given every 24±2 hours. Each treatment group was to include 135 patients: a total of 810 patients. Mandatory bilateral ascending venography was performed 6–10 days after surgery; the last dose of study drug was given on the day before venography. Patients were followed for 30–60 days after the last study treatment. The primary efficacy endpoint was a composite of any DVT, objectively confirmed non-fatal pulmonary embolism (PE), and all-cause mortality; the secondary efficacy endpoint was major VTE - proximal DVT, PE, or VTE-related death. The primary safety endpoint was the incidence of major, post-operative bleeding not later than 2 days after the last intake of study drug; secondary safety endpoints included clinically relevant, non-major bleeding, and minor bleeding. An in-depth analysis of the study results will be presented here. Based on the study findings, the potential clinical impact of oral BAY 59-7939 administered once-daily for thromboprophylaxis in orthopaedic surgery will be discussed.
Valproic Acid as Adjunct to Induction Therapy in Patients with Acute Myeloid Leukemia: First Results of Two Randomized Studies of the AMLSG.
Authors: Schlenk Richard F. , Bommer Martin , Schmid Mathias , Fröhling Stefan , Lehmann Katrin , Groner Silja , Hann Rainer , Krauter Jürgen , Döhner Konstanze , Ganser Arnold , Göttlicher Martin , Döhner Hartmut
Published: April 2020

Differentiation arrest in acute myeloid leukemia (AML) results in part from dysregulation of histone acetylation and deacetylation, leading to transcriptional repression of differentiation-inducing genes. Transcriptional repression can be returned by histon deacetylation inhibitors, such as valproic acid (VPA). In September 2004, we initiated two randomized trials [AMLSG 07-04 (age 18–60 yrs), AMLSG 06-04 (age >60yrs)] to evaluate the impact of VPA as adjunct to standard induction therapy (idarubicin and cytarabine; additional etoposide in pts. <60 yrs) in combination with all-trans retinoic acid (ATRA). This report focuses on serum-levels of VPA achieved by mid-fast saturation, correlation between total and free unbound serum levels of VPA, and influence on hematological reconstitution and morphological abnormalities at remission control induced by VPA. Dose schedule of mid-fast saturation is VPA i.v. 400mg bid. day 1+2, followed by serum level adapted p.o. treatment with retard formulation 4 times a day with a target serum level of 50 to 150mg/l. Data on serum levels in correlation to VPA-dose from 42 patients are available. Median serum level after 2 days of intravenous treatment was 34 mg/l (range 1 to 80mg/l). Median serum level at day 10 of induction therapy was 57mg/l (range 1 to 135mg/l) with 65% of patients having a minimum serum level above 50mg/l. Side effects induced by VPA were tremor, confusion and fatigue necessitating dose reduction of VPA in 5 (12%) cases. A linear regression model revealed high correlation between the free unbound fraction of VPA, total VPA-serum level (coef. 0.19 p<0.0001) and albumin serum level (coef. −0.30, p<0.0001). Other variables such as creatinine or total protein serum levels as well as age did not show significant influence on the free unbound fraction of VPA. High total VPA-serum levels during and after induction therapy were associated with morphological particularities at the time of morphological bone marrow assessment between day 21 to 28. In these cases there was morphological synchronization predominantly at the promyelocyte stage and a slightly but not significantly delayed reconstitution of peripheral blood values. In conclusion, VPA as adjunct to intensive induction therapy is feasible and has possible influence on the kinetics and morphology during hematological regeneration.
Inhibition of GSTpi Activity Increases the Sensitivity of MCL Cell Lines to Cisplatin, Cytarabin and Bortezomib, but Not Doxorubicin.
Authors: Paret Aurelie , Rolland Delphine , Ribrag Vincent , Coiffier Bertrand , Thieblemont Catherine
Published: April 2020

Preliminary data suggest drug resistance plays a major role in mantle cell lymphoma (MCL) and is linked to the aberrant expression of molecules such as Glutathion S-Transferase pi (GST pi) that catalyzes the nuclear conjugation of a broad variety of reactive electrophiles to the glutathion. We investigated in vitro the effect of the inhibition of GSTpi activity on the chemosensitivity of MCL cell lines by inhibiting the nuclear transport of GSTpi with Agaricus bisporus leptine (ABL).

Methods. Four MCL cell lines (Granta, NCEB, REC and UPN1) were analyzed for GSTP1 transcript expression by RT-PCR analysis and for GSTP1 genetic Ile105Val polymorphism. The effect of ABL on the 4 MCL cell lines viability was examined using cells pre-treated with ABL (40μg/ml) for 10 h followed by treatment with various concentrations of doxorubicin (DOX), cisplatin (CDDP), cytarabin and bortezomib for 48 h. The cell viability was estimated by MTT assay repeated three times.

Results. All the 4 cell lines expressed GSTPI transcript. Two of them (UPN1 and REC) were heterozygous for Ile105Val polymorphism, whereas NCEB and GRANTA were homozygous GSTP1-105Ile which has been correlated with an enzyme of high activity. DOX, CDDP, cytarabin and bortezomib have cytotoxic effects compared to non-treated cells, without any difference between the 4 MCL cell lines when considering their genetic polymorphism status. Co-administration of ABL caused a significant increasis of the cytotoxicity of CDDP and bortezomib in all cell lines (Student test: p between 0.0004 to 0.02 for CDDP; and p between 0.013 and 0.05 for velcade, depending on the cell lines). Co-administration of ABL caused an increasis of the cytotoxicity of cytarabin in 3 cell lines, the 4th being actually tested. No influence of ABL was detected on the cytotoxic effect of DOX. The analysis of the nuclear and the cytoplasmic localizations of GSTpi is currently realized by immunohistochemistry.

Conclusion. These results suggest that inhibition of the nuclear transfer of GSTpi increases in vitro the MCL sensitivity to CDDP, cytarabin and velcade but not DOX.
Heat Shock Protein 90 (HSP90) Inhibition by the Small Molecule 17-AAG Induces Cell Cycle Arrest and Apoptosis in Anaplastic Large Cell Lymphoma (ALCL) Irrespective of ALK Expression: Role of Akt, ERK and ALK Kinases in the Molecular Mechanisms of 17-AAG Anti-Tumor Activity.
Authors: Georgakis Georgios V. , Li Yang , Rassidakis George Z. , Samaniego Felipe , Medeiros L. Jeffrey , Younes Anas
Published: April 2020

Anaplastic Large Cell Lymphoma (ALCL) is a CD30 positive T-cell/null non-Hodgkin lymphoma which in many cases features a chromosomal translocation between tyrosine kinase nucleophosmin and anaplastic lymphoma kinase (NPM-ALK). Several groups demonstrated that patients with ALCL that do not express ALK have poor prognosis when treated with conventional chemotherapy, suggesting that these patients may require novel therapeutic approaches. Although the heat shock protein 90 (HSP90) has been shown to chaperone NPM-ALK, the effect of HSP90 inhibition on ALCL survival is not known. Furthermore, the effect of HSP90 inhibition on ALCL that do not express ALK is unknown. In the present study, the expression of HSP90 was evaluated in both ALK+ and ALK- primary and cultured ALCL cells. All of the ALK+ ALCL cases (21/21) and 58% (7/12) of the ALK- ALCL cases overexpressed HSP90, as determined by immunohistochemistry. In addition, 3 cell lines, two ALK+ (Karpas299 and SUDHL1) and one ALK- (Mac2A) were evaluated and were found to overexpress HSP90, as determined by western blot. The small molecule inhibitor 17-AAG induced antiproliferative effect in both ALK+ and ALK- ALCL derived cell lines. Inhibition of HSP90 in ALCL induces apoptosis, which was in part mediated by activation of the caspase pathway. Furthermore, 17-AAG depleted ALK kinase in ALK+ ALCL cells, in addition to depletion of the pro-survival Finally, AKT kinase. 17-AAG also decreased ERK phosphorylation, without altering total ERK levels in both ALK+ and ALK− ALCL cells. Moreover, 17-AAG reduced cyclin D1 and MDM2 levels which was associated with cell cycle arrest at G0/G1 phase. Finally, 17-AAG demonstared synergy with doxorubicin chemotherapy in the ALCL cell lines. Taken together, our data provide an excellent proof-of-principle for the potential therapeutic value of HSP90 inhibitors in ALCL, especially for ALK-negative subtype. Furthermore, our data suggest that 17-AAG may enahnce the effect of conventional chemotherapy in ALCL.
Evaluation of a New Prognostic Index for Peripheral T-Cell Lymphoma, Unspecified (PTCL-US).
Authors: McArthur Heather L. , Chhanabhai Mukesh , Randy Gascoyne D. , Joseph Connors M. , Kerry Savage J.
Published: April 2020

Background: Peripheral T-cell lymphoma, unspecified (PTCL-US) represents the largest subtype of PTCLs among Western populations. The prognosis of this heterogeneous group is poor with a 5-year overall survival (OS) of approximately 30%. The biological diversity of this subtype has prompted various attempts at refining clinical risk groups. For instance, the International Prognostic Index (IPI), first validated in DLBCL, has also been validated in PTCL-US in several studies. Recently, however, a new Prognostic Index for PTCL-US (PIT) has been proposed (Gallamini et al Blood103 (7): 2004). The purpose of this study was to apply the PIT to all patients with PTCL-US diagnosed and treated at the British Columbia Cancer Agency (BCCA) and to compare its prognostic utility with the IPI.

Methods: The BCCA Lymphoid Cancer Database was screened to identify all patients over 18 y diagnosed with PTCL-US by the World Health Organization classification system between January 1981 and June 2004. Patients were excluded if the diagnosis occurred outside of British Columbia or if the pertinent prognostic information was incomplete. Five year OS estimates were calculated for each variable in both the IPI (age >60 y, LDH> normal, PS ≥2, Stage III/IV and >1 extranodal sites) and PIT models (age, LDH> normal, PS ≥2 and bone marrow involvement). Five year OS estimates were then calculated for IPI groups 1, 2 and 3 (0/1, 2/3 and 4/5 factors, respectively) as well as PIT groups 1 through 4 (0, 1, 2 and 3/4 factors, respectively).

Results: Of the 134 patients identified, the median age was 61 y and the male to female ratio was 1.6. The predominant sites of extranodal involvement were bone marrow (10%), bone (6%), liver (4%), skin (4%), soft tissue (4%), lung (3%) and GI (3%). As demonstrated in figure 1, 5 year OS estimates were 73%, 24% and 22% for IPI groups 1, 2 and 3 (p=10−4), respectively and 76%, 35%, 25% and 19% for PIT groups 1 through 4, respectively (p=10−4 ). There was no difference between the prognostic models in the subset of 107 (80%) patients treated with CHOP-based chemotherapy.

Conclusions: Although the PIT was as effective as the IPI in defining clinical risk groups among PTCL-US patients, it did not provide any additional information in this study. Since the IPI is a well-established, familiar tool with similar prognostic capacity, it is reasonable to continue to apply this model in PTCL-US. With ongoing advances in gene expression profiling, it is likely that new biological models will emerge which may be used in combination with the IPI to better prognosticate this heterogeneous group.

Figure Figure
Proteomic Analysis of Shed Membrane Microparticles from Malignant Lymphoid Cells: A New Tool for Membrane Proteins Studies.
Authors: Mauvieux Laurent , Miguet Laurent , Felden Claire , Freyssinet Jean-Marie , Herbrecht Raoul , Potier Noelle , van Dorsselaer Alain
Published: April 2020

Plasma membrane antigens are critical for the classification and diagnosis of chronic mature B-cell lymphocytic malignancies, illustrated by the extensive use of the Matutes score for CLL diagnosis. Nevertheless, new surface markers that could be detected using flow cytometry are still needed for the diagnostic and classification of a number of chronic lymphoid malignancies. In this aim, proteomic tools could be of great interest in order to detect such antigens, but analysis of plasma membrane proteins is limited by the difficulty of their purification and separation of membrane proteins with usual 2-dimensional electrophoresis have often been poor. For all these reasons, we proposed to take advantage of the physiological production of plasma membrane microvesicles in order to study plasma membrane proteins of malignant lymphoid cells. Indeed, when eukaryotic cells are submitted to stress conditions (including mitogenic activation or apoptosis), the constitutive asymmetry between the inner and outer leaflet of the plasma membrane is disrupted and fragments are released from the plasma membrane, constituting the so-called microparticles (MPs). Although MPs reflect at least in vitro the cell state, little is known on their protein composition. We describe here the first set of experiments aiming to characterize the MPs proteome. First, two ways of triggering MPs formation from a T-lymphocytic cell line were analyzed using a 1D-gel approach coupled with Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). 390 proteins were identified in MPs among which 34% were localized to the plasma membrane. Replicate experiments from independent samples identified only very few nucleus or organelles proteins, but revealed a broad representation of plasma membrane proteins including seventeen hematopoietic clusters of differentiation and numerous signal transduction proteins. This approach was then successfully applied to circulating cells of a small-lymphocytic B-cell lymphoma. 414 proteins were identified in MPs, including 118 membrane proteins. In addition to various CD antigens, annexins, immunoglobulin chains and HLA antigens, proteins involved in the B-cell receptor signal transduction were recovered, including tyrosine kinases LYN, SYK, BTK, CSK, but also phospho-inositide 3-kinase (PI3K) phospholipase Cγ (PLCγ 2), hemopoietic cell kinase (HCK) and JAK1.

Proteomic analysis of MPs may represent a new tool to study plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination and potentially applicable to most cell types. Extensive proteomic analysis of MPs may thus be a way to investigate differential expression of plasma membrane components and to identify new pathology markers that could help for diagnosis and classification.
Minimal Residual Disease (MRD) in Adult T-Cell Leukemia/Lymphoma (ATLL) Evaluated by HTLV-1 Proviral Load and Soluble IL-2 Receptor Level in Peripheral Blood.
Authors: Tsukasaki Kunihiro , Takemoto Shigeki , Taguchi Hirokuni , Masuda Masato , Ikeda Syuichi , Yoshida Shinichiro , Suzushima Hitoshi , Uike Naokuni , Saburi Yoshio , Kawano Fumio , Yamamura Masaomi , Sueoka Eizaburo , Miyahara Masaharu , Tomonaga Masao
Published: April 2020

Background: Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type-1 (HTLV-1). Age, advanced PS, high LDH value, hypercalcemia and multiple visceral involvements were reported as prognostic factors for ATLL. Although initial therapy is often successful for aggressive ATLL with response rate of 50–80%, relapse (Rel) or progressive disease (PD) is frequent with 2 year-survival of 15–45% after chemotherapy or allogeneic hematopoietic stem cell transplantation (allo-HSCT). That means clinical evaluation of response is not sufficient to predict the prognosis of ATLL. HTLV-1 proviral load (PL) and soluble IL-2 receptor (sIL-2R) level have been reported to be associated with disease activity in ATLL.

Method: We prospectively analyzed MRD in aggressive ATLL, acute(A)-, lymphoma(L)- or unfavorable chronic(C)-type, by measuring PL and sIL-2R level in peripheral blood, using real time PCR for Tax region of the virus and conventional ELISA, respectively.

Results: PL and sIL2-R were measured just before start of therapy and every 2 months for 1 year unless Rel/PD happens in 78 pts. Median survival time was 7.3 months. Before chemotherapy (n=62), PL was high in order of C, A, and L, and sIL-2 level was of A, L and C, respectively. Neither PL nor sIL2-R level at first point was prognostic factor for pts having chemotherapy, while sIL2-R level, but not PL, was a prognostic factor in trend for pts having allo-HSCT (n=16). Median of the follow up for MRD was 4 months in the former and 6 months in the latter, respectively. In general, sequential data of both reflected disease activity in each case. PL and sIL-2R level were lower during complete response (CR) than partial response (PR), and elevated at Rel/PD. In cases with CR after chemotherapy, PL decreased gradually in 2 to 4 months and was still detected at levels of 1 to 10% during CR. In CR cases after HSCT, PL was often not detected (<0.2%) in 2 months, while sIL-2R level was still high probably reflecting GVHD activity. In CR cases with chemotherapy as compared to PR cases, increase in PL and/or sIL-2R level frequently preceded clinical relapse.

Conclusions: These results in aggressive ATLL suggest that; 1) sIL2-R level, but not PL, was a potential prognostic factor for pts having HSCT. 2) PL was better than sIL-2R in estimation of MRD after HSCT. 3) Exacerbation of PL and/or sIL2-R was more useful for prediction of relapse in CR cases as compared to PR cases after chemotherapy.
Relative Risk of Cardiovascular Disease after Treatment for Aggressive Non-Hodgkin’s Lymphoma.
Authors: Moser E. C. , Noordijk E. M. , van Leeuwen F. E. , le Cessie S. , Baars J. W. , Thomas J. , Carde P. , van Glabbeke M. , Meerwaldt J. H. , Kluin-Nelemans J. C.
Published: April 2020

Cardiovascular complications after therapy for Hodgkin lymphoma have been related to radiotherapy on the mediastinum, but have only incidentally been studied in NHL. As cardiovascular disease occurs commonly in the normal population, it is important to realize that risk factors as age, hypertension and life-style (diet and smoking) may be more outspoken in patients with NHL, who are generally older than patients with Hodgkin lymphoma. Moreover, although most patients with aggressive NHL will initially receive only chemotherapy, many will be treated with more than one therapy modality, incorporating stem cell transplantation or radiotherapy, because of early failure or relapses. Therefore, risk estimation of cardiovascular disease in NHL patients requires comparison to population-based rates. Here, we evaluated whether patients with aggressive NHL treated in 4 EORTC trials between 1980–1999 have an increased excess cardiovascular risk, compared to Dutch population rates. Relative risks (RR) and absolute excessive risks (AER per 1000 person-years) of cardiovascular disease were determined in 476 (Dutch and Belgian) patients and compared to incidence rates from the Continuous Morbidity Registry Nijmegen. Analyses were restricted to those patients treated with at least 6 cycles of doxorubicin-based chemotherapy and with a minimal follow-up time of 0.5 years. Only serious late events requiring daily medication and/or clinical interventions were recorded. Cumulative incidences of cardiovascular disease were estimated in the competing risk model by Gray with death by any cause as competing event. The overall cumulative incidence of cardiovascular disease was 12% at 5 and 22% at 10 years. At a median follow-up of 8.4 years, 66 cases of chronic heart failure (RR 5.4, 95% CI 4.1–6.9, AER 20.8), 17 myocardial infarctions (RR 1.2; 0.8–1.8, AER 0.8), 12 strokes (RR 1.8; 1.1–2.4, AER 1.5) and 9 other large vessel occlusions were registered. The large vascular events including strokes occurred in 16/21 patients after radiotherapy given in the same area. Pre-existent hypertension, NHL at young age (<55 years) and (any) salvage treatment increased risk of cardiovascular disease. Excess risk for myocardial infarction or stroke after radiotherapy on respectively the mediastinum or neck depended on cumulative radiation dose and was only seen after more than 40 Gy. Excess risk for chronic heart failure was registerd in both non-irradiated (RR 4.4) and irradiated patients, with an extremely high RR of 32 (13.7–57.0) if >40 Gy had been given. In conclusion, NHL patients treated with doxorubicin-based chemotherapy, especially those who are young, have hypertension, or received salvage treatment or radiotherapy above 40 Gy, are at high risk of cardiovascular disease and need lifelong monitoring in this regard.
High Frequency of AML1 Mutations in Patients with Chronic Myelomonocytic Leukemia.
Authors: Shih Lee-Yung , Huang Chein-Fuang , Liang Der-Cherng , Shih Yu-Shu , Wu Jin-Hou , Dunn Po , Wang Po-Nan , Kuo Ming-Chung
Published: April 2020

Transcription factor AML1 is essential for normal hematopoiesis. AML1 mutations have been described in myelodysplastic syndrome (MDS), especially in therapy-related MDS but were rarely examined in patients with chronic myelomonocytic leukemia (CMML). We sought to determine the frequency of AML1 mutations in CMML and to assess their role in the progression of CMML to AML. Mutation analysis of AML1 was performed on bone marrow samples from 63 patients with CMML by direct sequencing of all RT-PCR products amplified with 3 overlapping primer pairs which cover the coding sequences of AML1 gene from exon 3 through exon 8. AML1 mutations were detected in 24 of 63 patients (38%) at initial diagnosis, 16 patients had mutations located in Runt homology domain (RHD) (exons 3–5), and 8 had mutations located in the C-terminal region (exons 6–8). One patient had 2 missense mutations in RHD and another one had 2 frameshift mutations in RHD. Taken together, the patterns of 26 mutations consisted of 7 missense mutations, 5 nonsense mutations, 2 silent mutations and 12 frameshift mutations. Ten of 24 patients (41.7%) with mutations progressed to AML compared to 13 of 39 patients (33.3%) without mutations, no difference was observed in the risk of AML transformation between the two groups (P=0.593). Of the 23 patients who progressed to AML, 17 also had bone marrow samples available at AML transformation for mutation analysis. AML1 mutations were detected in 8 of 17 patients at both diagnosis of CMML and AML transformation, all exhibited identical patterns at both phases, with no loss or gain of mutations during AML progression. There were no differences in age, sex, blood counts, percentage of blasts in bone marrow or peripheral blood, subtype of CMML (1 or 2), and cytogenetic risk group between patients with AML1 mutations and those without mutations. Time to AML transformation and overall survival of patients with AML1 mutations did not differ from those without AML1 mutations. Our study showed that AML1 mutations were frequently detected in CMML, with 70% located in RHD. All mutant clones remained identical at initial diagnosis and AML transformation, suggesting that AML1 mutations play an important role in the leukemogenesis in this subset of patients with CMML.