bluuebunny/crossref_metadata_2025 huggingface dataset:
The IRON2 Study. A Retrospective Observational Study to Describe the Evolution of Iron Overload in Patients with Low-Risk Myelodysplastic Syndrome
Authors: Remacha Angel , Arrizabalaga Beatriz , Villegas Ana , Duran Maria Soledad , Hermosin Lourdes , de Paz Raquel , García Marta , García Regina , del Cañizo Consuelo , Sanz Silvia , Sanz Guillermo
Published: October 2019

Abstract 1723

Introduction and Objective:

A large number of patients with low-risk myelodysplastic syndrome (MDS) need blood transfusions, being iron overload an inevitable consequence that may cause hepatic fibrosis and cirrhosis, diabetes or myocardiopathy unless it is not treated. The main objective of this study is to describe the evolution of iron overload in transfusion-dependent patients with low-risk MDS. Secondary objectives include the evaluation of the impact of iron chelation therapy on Event Free Survival (EFS) (including infections, arthropathy, diabetes mellitus, hepatic and cardiac complications), Overall Survival (OS) and Leukemia Free Survival (LFS).

Methods and Patients:

Retrospective observational study carried out in Haematology Departments of 47 Spanish hospitals between March 2010 and March 2011. Transfusion-dependent patients with low/intermediate-1 IPSS risk who had received ≥10 red blood cell (RBC) transfusion during at least 12 months previous to study entry were eligible.

Results:

A total of 263 patients were evaluated [age at diagnosis, 74 ± 10.5 years]. IPSS classification was available in 82.9% included patients (lack of cytogenetics in 37 patients), classified as low risk (86%) and int-1 risk (14%). At diagnosis, 25.5% of patients had serum ferritin (SF) levels >500ng/ml, 8.7% SF>1,000ng/ml, and 22% TSI>50%. The median number of RBC transfusions per month was 2.35 RBC/month; during the course of the disease, 82.4% of patients reached SF levels >1,000ng/ml. Cardiac complications worsened/appeared in 24.4% of patients, having received a median RBC transfusions of 22 (7–92), and showing a median SF levels of 1,365 (735–3,025) ng/ml. One-hundred forty-seven (55.9%) patients started iron chelation therapy (85.5% with deferasirox) with a median number of RBC transfused of 23 (14–38) and a median SF levels of 1,570 (1,231.5–2,195) ng/ml. 71% of patients with SF>1,000ng/ml were on iron chelation therapy. Table 1 shows the results of univariate analysis of EFS, OS and LFS. As part of this study, we are currently performing a multivariate analysis whose results will be presented in forthcoming congress.

Conclusions:

The results of this study show that a high percentage of transfusion-dependent patients with low-risk MDS (82.4%) reach SF levels >1,000ng/ml during the course of their disease. Patients on iron chelation therapy show a higher cardiac EFS, OS and LFS compared with those not treated, being deferasirox the most frequently used chelating agent in clinical practice.

Disclosures:

Sanz: Novartis Farmaceutica S.A.: Employment.
Outcomes and Treatments of Relapsed AL Amyloidosis Following Stem Cell Transplant
Authors: Warsame Rahma , Bang Soo-Mee , Kumar Shaji K. , Lacy Martha Q , Buadi Francis K , Dingli David , Hayman Suzanne R , Lust John A , Russell Stephen , Witzig Thomas E. , Kyle Robert A. , Leung Nelson , Zeldenrust Steven R , Rajkumar S. Vincent , Gertz Morie A , Dispenzieri Angela
Published: October 2019

Abstract 1858

Systemic light chain amyloidosis (AL amyloidosis) is a condition where clonal plasma cells produce misfolded insoluble immunoglobulin light chains that deposit in various organs causing progressive organ dysfunction. Chemotherapy and autologous stem cell transplant (ASCT) when eligible is the standard treatment options for patients with AL amyloidosis. There are several studies who report long term outcomes of patient post ASCT. However, there is a paucity of literature describing the outcomes of patients who have received ASCT but have relapsed. We performed a retrospective study to assess the outcomes and treatment regimens employed following relapse after ASCT. Between 1996 and 2009, 410 patients received ASCT at the Mayo Clinic as first line therapy. Of those 410 patients 42 patients died within 3 months of transplant, 64 patients died without documented relapse, 158 patients were alive without documented progression, and 146 patients had documented progression. Those 146 patients are the subject of our study. The median time to hematologic relapse was 2 years (range: 0.2–15.5 years). At relapse, 59 patients were treated with IMiD based therapy, 36 with alkylator based therapy, 24 with bortezomib, 15 with steroids, and 5 with second ASCT. The respective hematologic response rates were 58%, 33%, 50%, 53%, and 60%. The remaining six patients were not evaluable for response for one other following reasons: organ transplants; no further therapy; inevaluable disease. With a median post relapse follow up of 3.6 years, the median overall survival (OS) from the first post ASCT relapse was 4.6 years. The median post transplant follow up was 6.1 years, the median OS for these patients was 7.3 years from the time of transplant. These data provide novel information about outcomes after SCT relapse, which should be useful not only for patients and doctors but also for investigators designing studies for salvage therapies post-transplant.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy of 5% Albumin and Fresh Frozen Plasma (AFFP) Versus Fresh Frozen Plasma (FFP) only as Replacement fluid in Plasma Exchange for Thrombotic Thrombocytopenic Purpura (TTP)
Authors: Louie James , Boctor Fouad , Chiang Jason , Anwar Zeeshan , Wittenberg Amanda , Gogas Elena
Published: October 2019

Abstract 272

The treatment of Thrombotic Thrombocytopenic Purpura (TTP) with therapeutic plasma exchange (TPE) with Fresh Frozen Plasma (FFP) and/or Cryopoor Plasma (CPP) replacement is well established. This process adds plasma ADAMTS-13 activity and removes plasma ADAMTS-13 inhibitor. Limited data has indicated combination of 5% albumin in the first half of the procedure followed by FFP in the second half of the procedure is effective in treating TTP. Theoretical calculation of addition of exogenous ADAMTS-13 is still substantial because ADAMTS-13 infused initially is removed as the plasma exchange procedure progresses. A 50% reduction of FFP would decrease the number of donor exposures and transfusion reactions associated with human plasma. Further, plasma of limited supply, such as AB plasma, can be more effectively used.

We report a retrospective comparison of TPE for TTP using all FFP versus 5% Albumin/FFP (AFFP) replacement fluid. One health system with two closely connected tertiary campuses with common Hematology/Oncology services and Transfusion Medicine services treated 17 idiopathic TTP patients (March/2009 – June/2012) with documented very low ADAMTS-13 levels (<10) and high inhibitor levels (≥0.4). All other thrombotic microangiopathies were excluded from this comparison. One campus treated 10 patients with FFP and the other campus treated 7 patients with AFFP. Daily one plasma volume TPE was performed until target platelet count was achieved and the patient was then tapered off TPE. For AFFP replacement, 50% of the replacement fluid was 5% albumin given in the first half of the procedure followed by FFP for the second half. Additional medical therapy was managed by the same Hematology/Oncology service.

Table 1 shows that the FFP versus the AFFP groups at presentations are comparable. Table 2 indicated FFP versus AFFP treatment is comparable. For the FFP and AFFP groups respectively, mean number of total procedures: 16 vs. 14, length of time from first TPE to last TPE 19 vs. 22 days, Relapses 2 vs. 2, and deaths 1 vs. 0.

Conclusion:

The use of AFFP as replacement fluid for idiopathic TTP does not appear to be inferior to 100% FFP replacement. Length of treatment, rate of recovery, and relapse rate are comparable. There were more deaths in the FFP cohort but the low number of observations limits the significance. AFFP fluid replacement should be considered for TPE in idiopathic TTP.

Disclosures:

No relevant conflicts of interest to declare.
Subanalysis of the StiL NHL 1–2003 Study: Achievement of Complete Response with Bendamustine-Rituximab (B-R) and CHOP-R in the First-Line Treatment of Indolent and Mantle Cell Lymphomas Results in Superior Survival Compared to Partial Response.
Authors: Rummel Mathias J. , Niederle Norbert , Maschmeyer Georg , Banat G.-Andre , von Grünhagen Ulrich , Losem Chrisoph , Kofahl-Krause Dorothea , Heil Gerhard , Welslau Manfred , Balser Christina , Kaiser Ulrich , Weidmann Eckhart , Duerk Heinz A. , Ballo Harald , Stauch Martina Beate , Roller Fritz , Barth Juergen , Hinke Axel , Burchardt Alexander C. , Brugger Wolfram
Published: October 2019

Abstract 2724

Background:

The NHL 1 study, a prospective, multicenter, randomized, phase 3 study which compared B-R and CHOP-R as first-line treatment in indolent lymphomas and mantle cell lymphoma (MCL), demonstrated a significant benefit in progression-free survival (PFS) as well as improved tolerability for B-R compared with CHOP-R. Here we present an analysis of the impact of response quality on outcome.

Methods:

514 patients (pts) with indolent or MCL were randomized to receive B-R or CHOP-R for a maximum of 6 cycles.

Results:

The overall response rate in the 514 pts (261 B-R; 253 CHOP-R) was 92.7% and 91.3% in the B-R and CHOP-R arms, respectively (as presented at the last ASCO meeting, J Clin Oncol 30, 2012 (suppl; abstr 3). A complete response (CR) was observed in 39.8% in the B-R arm and in 30% in the CHOP-R arm (p=0.021). The achievement of CR was associated with a significantly prolonged PFS and overall survival (OS) (Table 1). Analysis by treatment arm revealed a trend for superior PFS and a significantly improved OS for patients achieving CR following treatment with B-R. In the CHOP-R arm, patients in CR had a significantly superior PFS compared to those in PR with a trend to superior OS.

Regardless of the quality of response, PFS was superior with B-R versus CHOP-R: For patients in CR, the median PFS was not reached with B-R, whereas for CHOP-R it was 53.7 months (p=0.0204). In patients achieving PR, treatment with B-R resulted in a median PFS of 57.2 months, and this was 30.9 months with CHOP-R (p=0.0002).

We noted a statistically significant difference in CR rates between male (n=272, median age 63 years) and female (n=242, median age 64 years) patients. The CR rate was 28.6% in male patients and 42.1% in female patients (p=0.0016). Female patients had a longer median PFS (51.4 months) compared to male patients (38.6 months), however, this difference was not statistically significant (p=0.0866).

Conclusions:

Patients in CR following first-line treatment in our study had a significantly longer PFS and OS compared to those achieving a PR. Therefore, our results strongly suggest an association between quality of response and outcome.

Disclosures:

No relevant conflicts of interest to declare.
L-Asparaginase Monotherapy in Patients with Relapsed or Refractory Extranodal NK/T-Cell Lymphoma, Nasal Type: Results of A Phase II Study.
Authors: Guo Ye , Ma Xuejun , Xia Zuguang , Xue Kai , Zhang Qunling , Hong Xiaonan
Published: October 2019

Abstract 2730

Introduction:

Recently, L-asparaginase-based combination chemotherapy was found to be effective in salvage treatment in patients with relapsed or refractory extranodal NK/T-cell lymphoma, nasal type. To explore the single-agent activity of L-asparaginase, we conducted a single-institute, prospective phase II study.

Methods:

Patients with relapsed or refractory extranodal NK/T-cell lymphoma, nasal type were eligible for enrollment regardless of prior treatment. L-asparaginase monotherapy (6000 U/m2 on days 1 to 7) was administered as the protocol treatment and repeated every 3 weeks for at most 8 cycles. For responding patients, the decision to proceed with hematopoietic stem-cell transplantation was made at the discretion of treating physicians. The primary endpoint was the best objective response after L-asparaginase.

Results:

A total of 40 patients were enrolled and treated with L-asparaginase for a median of 5 cycles (range, 1 – 8). The patient characteristics were shown in Table 1. Half of the patients had stage IV disease at enrollment and the vast majority (18 patients) presented with disseminated cutaneous and soft-tissue involvement. Thirty-seven patients (92.5%) had prior exposure to systemic chemotherapy and 14 of them (37.8%) received more than 1 line. The overall response rate was 82.5%. The complete response (CR) and partial response (PR) rates were 40% and 42.5%, respectively. The incidence of adverse events was shown in Table 2. In short, anemia, neutropenia, hypoalbuminemia, nausea and liver-related disorders were common toxicities, which were usually mild and manageable. No grade 4 adverse events and treatment-related mortality were observed. Five patients (12.5%) developed allergic reaction to L-asparaginase and 3 of them had to withdraw from the study since L-asparaginase re-challenge with prophylactic antiallergic agents was unsuccessful. After a median follow-up time of 31.6 months (range, 21.9 – 41.3), the median progression-free survival (PFS) was 12.8 months and median overall survival (OS) was not reached. Response status (CR, PR or no response) after L-asparaginase had a significant impact on either PFS (Figure 1) or OS (Figure 2). Moreover, its prognostic value was confirmed in the multivariate analysis.

Conclusions:

L-asparaginase demonstrated a high single-agent activity in salvage setting for patients with extranodal NK/T-cell lymphoma, nasal type. The first-line L-asparaginase-containing chemotherapy regimen warrants urgent investigation.

Disclosures:

Off Label Use: L-asparaginase, which was used in our study for NK/T-cell lymphoma, is approved to treat acute lymphocytic leukemia by US and Chinese FDA.
Impact of Rituximab On the Treatment of Waldenström's Macroglobulinemia (WM).
Authors: Thomas Sheeba K. , Delasalle Kay B , Shah Jatin J. , Wang Luhua , Orlowski Robert Z. , Weber Donna M.
Published: October 2019

Abstract 2734

Background:

Rituximab was first approved by the FDA for use in relapsed and/or refractory low grade or follicular B cell non-Hodgkin's lymphoma in 11/1997. Since that time it has been widely used in WM, and is the backbone of drug combinations developed for treatment of this disease.

Methods:

To evaluate the impact of rituximab on overall survival (OS), we retrospectively evaluated patients (pts) treated at our center who received primary therapy for symptomatic WM with combinations of either alkylating agents (AA), nucleoside analogs (NA) +/− AA, or bortezomib (B), each with or without rituximab (R). The effect of primary therapy on OS was compared between patients receiving primary therapy before and after rituximab use for WM began at our center (11/11/1998). OS was calculated from the date of initial treatment until date of death or last follow-up. Due to small patient numbers, those who received vincristine-doxorubicin-dexamethasone (2 pts), single agent rituximab (3 pts), or rituximab-alkylating agent combinations (11 pts) as primary therapy, were excluded from the analysis.

Results:

Among 315 patients with previously untreated symptomatic WM treated since 3/1966, 118 received AA alone; 137 received NA +/− AA alone (85 pts) or with R (52 pts), and 44 received B + R. Median age, hemoglobin, platelet count and β2 microglobulin were comparable between all treatment groups (AA alone, NA +/− AA alone, NA +/− AA + R, B + R). Only median albumin differed significantly, with levels of 3.7 g/dL (AA alone), 4.1 g/dL (NA+/− AA alone), 4.3 g/dL (NA+/− AA + R) and 4.4 g/dL (B + R), respectively (p < 0.01). The median OS of pts treated with AA was 4.5 years, and 6.5 years with NA +/− AA, while the median OS of pts treated with NA+/− AA + R (median follow-up: 8 years) and B + R (median follow-up: 2.6 years) has not been reached. Comparing all patients treated with vs. without R, median OS is >13 years vs. 5.6 years (p<0.01).

Conclusions:

While pts with WM have most certainly benefitted from improvements in supportive care and the greater availability of salvage therapy options in the modern era, the addition of rituximab to available therapies appears to have significantly improved the OS of patients with WM compared with AA or NA +/− AA alone. Our data supports the role of rituximab as an indispensable component of modern therapy for this disease.

Disclosures:

No relevant conflicts of interest to declare.
Overexpression of Eukaryotic Initiation Factor 4E Induces Critical Transcription Factors Such As c-Myc in Multiple Myeloma Resulting in Enhanced Clonogenic Tumor Growth in Vitro and in Vivo.
Authors: Li Shirong , Fu Jing , Schecter Jordan M. , Lu Caisheng , Mapara Markus , Lentzsch Suzanne
Published: October 2019

Abstract 2908

Introduction:

The eukaryotic initiation factor 4E (eIF4E) is a critical regulator in protein synthesis. It has been shown that overexpression and/or activation of eIF4E is critical for oncogenic protein synthesis. The precise role of protein translation in multiple myeloma (MM) is less clear. Recently it has been shown that eIF4E protein levels are higher in primary CD138+ MM cells than in normal plasma cells (Li, Jin et al. 2011) and that mutations in genes related to mRNA translation are involved in the pathogenesis of multiple (Chapman, Lawrence et al. 2011). Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. In this study, we analyzed the effects of introduction of ectopic eIF4E in MM cell lines compared with their parent cells in vitro and in vivo.

Results:

To examine the effect of overexpressed eIF4E in MM, we transduced MM cell lines with lentiviral particles encoding human eIF4E with GFP as selection marker. Introduction of ectopic eIF4E significantly increased critical factors for myeloma cell growth such as myc, cyclin D1, C/EBP beta and IRF4 as detected by western blotting. Overexpression of eIF4E resulted in a significant (p<0.001) increase of DNA synthesis compared to empty vector control (EV) cells. Cell cycle analysis revealed a decreasing number of cells in G0/G1 phase (62% vs 49%) and cells arresting in the G2/M phase (14% vs 23%), not affecting cell apoptosis. Overexpression of eIF4E further led to the significant increase (p=0.004) of clonogenic tumor growth with expansion of clonogenic colony numbers (22.3 ± 2.5 vs 40.3 ± 2.1) and size. To determine whether overexpressed eIF4E also affects MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the EV and eIF4E-OE-U266 cells. In contrast to EV-U266 tumors, animals bearing eIF4E-OE-U266 xenografts showed a significant increase (p<0.001) of tumor growth by 180% after 13 days.

Conclusion:

Here we show that eIF4E, a key player in translational machinery, promotes multiple myeloma cell growth both in vitro and in vivo. When eIF4E is overexpressed, it enhances protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation.

Disclosures:

Lentzsch: Celgene: Consultancy, Research Funding.
Organotypic Epigenetic Signature Predicts Bone and Marrow Niche Forming Capacity of Stromal Progenitors in a Novel Mouse Model in Vivo.
Authors: Reinisch Andreas , Etchart Nathalie , Hofmann Nicole A , Frühwirth Margareta , Ortner Anna , Thaler Daniela , Feilhauer Birgit , Hartwig Udo F , Beham-Schmid Christine , Trajanosky Slave , Wagner Wolfgang , Alves Frauke , Schallmoser Katharina , Andreeff Michael , Strunk Dirk
Published: October 2019

Abstract 2987

Mesenchymal stem/progenitor cells (MSPCs) from numerous tissues are currently tested in clinical trials despite a limited understanding of their in vivo behavior. In this study we used MSPCs from adult and fetal tissues to select the appropriate source for clinical application. We asked whether MSPCs derived from human bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC), compared to skin fibroblasts, bear an equivalent bone and marrow niche formation potential with of in vivo. Furthermore we evaluated attraction and engraftment of murine as well as human hematopoietic stem/progenitor cells (HSPCs) into newly formed MSPC-derived niches. To elucidate potential mechanisms responsible for a tissue-specific MSPC potential after transplantation gene expression profiling and DNA methylation analysis on a novel high resolution 450K-CpG methylation array were employed.

MSPCs were transplanted subcutaneously to test for their spontaneous bone and marrow niche formation potential in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). BM-derived MSPC transplantation reproducibly led to the development of mature bone (17/17 donors) through an endochondral ossification process leading to subsequent marrow niche formation. Additionally, these newly formed hematopoietic microenvironments attracted complete mouse hematopoiesis including immature lineage negative, Sca-1 positive, c-kit positive (LSK) HSPCs. Non-BM derived MSPCs completely lacked bone and marrow niche-forming potential and did not attract hematopoietic cells (0/9 donors). Induction of human hematopoietic chimerism through transplantation of umbilical cord blood (UCB)-derived human CD34+ HSPCs in advance of subcutaneous ectopic bone and marrow development resulted in immigration of re-transplantable human hematopoiesis into extra-medullary ossicles. Comparative 450K-CpG methylation array profiling of MSPCs revealed a tissue-specific epigenetic signature virtually corresponding to the in vivo differentiation. MSPCs from BM but neither fibroblasts nor MSPCs from WAT or UC showed epigenetically imprinted human bone and marrow niche (HuNiche) formation capacity favoring BM-MSPCs for skeletal regeneration. This novel HuNiche model should be ideally suited for studying normal and malignant hematopoiesis regulation in an ectopic human marrow with subsequent human hematopoietic engraftment that mimics clinical BM transplantation reality.

Disclosures:

No relevant conflicts of interest to declare.
Identification of a Human Endogenous Retrovirus Type-E (HERV-E) Envelope with Selective Expression in Clear Cell Kidney Cancer That Is Immunogenic in Vitro.
Authors: Cherkasova Elena , Weisman Quinn , Kajigaya Sachiko , Takahashi Yoshiyuki , Reger Robert N , Harashima Nanae
Published: October 2019

Abstract 3015

Recently, we isolated and expanded a CD8+ T-cell clone from the blood of a patient with regressing renal cell carcinoma following an allogeneic hematopoietic stem cell transplant (HSCT) that killed patient tumor cells in vitro. This CTL clone was found to have tumor specific cytotoxicity, recognizing an HLA-A11-restricted 10-mer peptide named CT-RCC-1. The transcripts encoding this antigen, CT-RCC-8 and CT-RCC-9 were found to be derived from a novel human endogenous retrovirus type E (named CT-RCC HERV-E) located on chromosome 6q. Both transcripts present splice variants that share a common sequence region encoded in the retroviral 5'LTR that is spliced to non-shared regions derived from the protease and polymerase genes, respectively. Remarkably, these transcripts were found to be selectively expressed in the clear cell variant of renal cell carcinoma (ccRCC) with no expression observed in normal tissues or any other type of tumor cell. We have now discovered transcripts encoding the entire envelope gene (env) of the CT-RCC HERV-E provirus are also expressed in ccRCC. Expression of these unique HERV-E env transcripts was detected in kidney cancer cells by RT-PCR, Northern blot, and sequence analysis. The proviral env was found to be expressed concurrent with the previously identified CT-RCC-8 and -9 transcripts, and was only observed in ccRCC cells with no expression observed in any other tumors or normal tissues. We generated 2 peptides derived from the env surface region and 2 peptides derived from the env transmembrane region that were predicted to have a high binding affinity for HLA-A2. Dendritic cells generated from the PBMC of healthy HLA A2+ donors were pulsed with one of these 4 env peptides then were used to stimulate autologous T-cells in vitro. After three stimulations, 3 of these peptides were found to expand CD8+ T-cells that secreted IFN-γ when co-cultured with a HERV-E Env expressing ccRCC tumor cell line transfected to express HLA A2+ but did not secrete IFN-γ against the wild type tumor that was HLA A2 negative.

Conclusion: The envelope of the CT-RCC HERV-E provirus is expressed selectively in ccRCC and encodes highly immunogenic antigens that can be targeted by cytotoxic T-cells. Our data suggests antigens derived from this newly discovered HERV-E envelope could represent excellent targets for T-cell based immunotherapy for kidney cancer.

Disclosures:

No relevant conflicts of interest to declare.
Detection of Th1 Driven T-Cell Responses in Peripheral Could Guide Individualized Immunosuppression and Risk of Viral Reactivation Under GvHD Prophylaxis Following Allogeneic Stem Cell Transplantation.
Authors: Feucht Judith , Opherk Kathrin , Neinhaus Cornelia , Kayser Simone , Bethge Wolfgang A. , Handgretinger Rupert , Lang Peter , Feuchtinger Tobias F.
Published: October 2019

Abstract 3047

Allogeneic stem cell transplantation (SCT) can expose patients to a transient but marked immunosuppression, during which viral infections are an important cause of morbidity and mortality. The control of these infections will ultimately depend on the restoration of adequate T-cell immunity. Most viral infections after SCT are caused by endogenous reactivation of persistent pathogens such as cytomegalovirus (CMV), adenovirus (ADV) and Epstein-Barr-virus (EBV). Risk of viral complications is even higher under GvHD treatment or prophylaxis like calcineurin inhibitors and steroids. Post transplant often the immunosuppression needs to be reduced to improve viral complications with the risk of GvHD. The virus-specific T-cell responses in peripheral blood have been shown to be a good marker of immunological protection, but has not been used for clinical decision making and the guidance of drug plasma levels. Therefore, we performed a prospective clinical trial in 33 adult and pediatric patients after allogeneic stem cell transplantation receiving pharmacologic immunosuppression with steroids, Cyclosporin A, Tacrolimus, Everolimus or Mycophenolate. Median Age was 16 years. T-cell responses were analyzed ex vivo against Cytomegalovirus (pp65), Adenovirus (hexon antigen) and Epstein-Barr Virus (EBNA, LMP) using intracellular cytokine staining. In addition in vitro analysis of the proliferation responses using CFSE were performed. Responses were compared to healthy donors. The T-cell responses in vitro under low, high and supraphysiologic plasma concentrations of the respective drugs were investigated. Under the direct influence of steroids, activated, virus-specific T-cells underwent apoptosis. Among the Calcineurin inhibitors, Tacrolimus had the strongest inhibition on virus-specific T-cell immunity, followed by Cyclosporin A. But, under low therapeutic levels, Virus speciffic T-cell responses have been able to develop in PBMCs. Mycophenolate had only in high concentrations a strong effect on the T-cell response against viral pathogens. Relevant differences in the frequency of virus-specific T-cells secreting IFN-g could be detected within the CD4 compartment in correlation to the level of immunosuppression. In conclusion we could show that detection of virus-specific T-cells could be used to guide the level of immunosuppression in case of viral complications after allogeneic stem cell transplantation, since emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load.

Disclosures:

No relevant conflicts of interest to declare.
Epidermal Growth Factor-like Domain 7 Promotes Hematopoietic Stem Cell Expansion and Increases Myeloid-Megakaryocytic Lineage Priming through beta3 Integrin
Authors: Nishida Chiemi , Salama Yousef , Gritli Ismael , Umemoto Terumasa , Nakauchi Hiromitsu , Hattori Koichi , Beate Heissig
Published: October 2019

Interactions between stem cells and their surrounding microenvironment, or niche, are critical for the establishment and maintenance of stem-cell properties. Niche cells within the bone marrow (BM) are contacted by the hematopoietic stem cells (HSCs), which retain their stem-cell character through the direct association with these niche cells. Within the BM microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that the extracellular matrix protein epidermal growth factor-like domain protein 7 (Egfl7) induced HSCs to enter the cell cycle and to undergo myelo-megakaryocytic differentiation both under steady state conditions and after myelosuppression in vivo. Mechanistically, we show that Egfl7 binds to the beta3 integrin expressed on HSCs, thereby activating the Akt pathway in HSCs. In beta3-/- mice, Egfl7-mediated HSC expansion, cell cycle progression and myelo-megakaryocytic differentiation did not occur. We propose that the ECM protein Egfl7 recruits dormant HSCs into active cell cycle, and can govern stress-induced hematopoiesis by beta3 integrin-dependent anchoring to the stem cell niche.

Disclosures

No relevant conflicts of interest to declare.
Comparative Analysis of in Vitro Chemosensitivity to Four Proteasome Inhibitors in Human Myeloma Cell Lines
Authors: Mitra Amit Kumar , Harding Taylor S , Van Ness Brian
Published: October 2019

Proteasome inhibitors (PI) are effective chemotherapeutic agents in the treatment of multiple myeloma (MM), used alone or in combination with other anti-cancer agents, such as alkylating agents, topoisomerase inhibitors, corticosteroids, histone deacetylase inhibitors (HDACis) and immunomodulatory drugs (IMiDs). Bortezomib (Velcade/Bz) was the first PI to be approved by US-FDA for the treatment of relapsed and refractory MM. Other second generation PIs include carfilzomib (Kyprolis/Cz), ixazomib/Iz and oprozomib (Opz). Wide inter-individual variation in response to treatment with PIs is a major limitation in achieving consistent therapeutic effect in MM. Yet few studies have compared the efficacy of all four PIs in a range of myeloma subtypes.

In our current study, we performed comprehensive in vitro chemosensitivity profiling of response to four (4) PIs (Bz, Cz, Ix and Opz) in a panel of forty-five (45) human myeloma cells lines (HMCLs) generated through the immortalization of primary multiple myeloma cells (MMCs) and representing the biological and genetic heterogeneity of MM with regards to chromosomal abnormalities, oncogene mutations (e.g. Ras), tumor suppressor variations (e.g. p53), cell surface phenotypes, or growth factor response. Cells were treated with increasing concentrations of Bz, Cz, Ix and Opz as single agents and cell viability assays were performed using CellTiter-Glo luminescent cell viability assay to generate survival curves and determine the half maximal inhibitory concentration (IC50) values by calculating the nonlinear regression using sigmoidal dose-response equation (variable slope). Our results in comparing the cellular responses to PI treatment among HMCLs showed wide range of variability in IC50 values identifying some lines which were highly sensitive and some lines relatively refractory to PI treatment. Pearson product-moment correlation (PPMC) test demonstrated statistically significant (adjusted p values < 0.001) positive correlation between IC50 values of the following drug pairs: Bz vs Opz (r = 0.82); and Ix vs Opz (r = 0.88); Bz vs Ix (r = 0.65); Cz vs Opz (r = 0.69) and Cz vs Ix (r = 0.63). Subgroup analysis revealed significant correlation between carfizomib IC50 and chromosome number (p < 0.05). Furthermore, it was interesting to note that although all 4 drugs belong to the same drug class (PI), not all cell lines responded the same across all PI treatments. This demonstrates tumor heterogeneity even in response to inhibitors of the same class, and further demonstrates tumors refractory to one PI may still respond to another. We are currently examining genetic characteristics that are associated with response among the four PIs, and analysis of these characteristics will be presented.

Disclosures

No relevant conflicts of interest to declare.
Genomic and Expression Profiling of Hairy Cell Leukemia Revealed Multiple Mechanisms of NFkB Activation
Authors: Nagel Stefan , Ehrentraut Stefan , Meyer Corinna , Kaufmann Maren , Drexler Hans G , MacLeod Roderick AF
Published: October 2019

Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder where the malignant B-cells manifest a “hairy” appearance under the microscope. Recent studies have identified BRAF V600E mutations in most HCL patients, highlighting this abnormality as a molecular hallmark for this disease. However, mutated BRAF occurs widely – already described in several solid tumors, including melanoma, thyroid and colorectal carcinomas, indicating that BRAF V600E is not pathognomonic in HCL. Cell lines originating from HCL patients lack BRAF mutations but retain the typical piliferous morphology and the appropriate HCL immunophenotype (CD19, CD11c, CD103, CD25, CD123), thus constituting tools for identifying alternative mechanisms of leukemogenesis in this disease entity. Genomic aberrations in hematopoietic tumors recurrently target loci bearing genes involved in malignant transformation. These genes may include both candidate biomarkers and potential therapeutic targets. To identify such genes in HCL we here combined genomic profiling and gene expression quantification of a well characterized HCL cell line containing several chromosomal aberrations. The expression levels of genomically targeted genes were compared to HCL control cell lines, identifying 91 deregulated genes. Gene set enrichment analysis of which indicated apoptosis, cell cycle regulation and DNA damage response (DDR) as altered processes in HCL. Accordingly, REL (NFkB and apoptosis), CDK6 and BRAF (cell cycle), ATM and CUTL1 (DDR) comprised prominent target genes overexpressed in this cell line. The same genes were found to be conspicuously expressed in HCL patient samples in silico (Fernandez et al., 2010; Gene Expression Omnibus GSE16455), supporting their clinical significance. Treatments of HCL cell lines for particular siRNA-mediated gene knockdowns and with selective pharmacological inhibitors helped to reveal a regulatory network highlighting NFkB at a central position. Consistently, focused analysis of expression profiling data of several cell lines supported elevated NFkB-pathway activity in HCL and ABC-DLBCL when compared to GC-DLBCL. In conclusion, we identified deregulated genes and multiple mechanisms which contribute to aberrantly activated NFkB-pathway in HCL. Therefore, NFkB may represent a B-cell specific hallmark of HCL and a promising novel therapeutic target most notably in patients lacking BRAF mutations in this entity.

Disclosures

No relevant conflicts of interest to declare.
OncoHist®, an rh Histone 1.3, Is Cytotoxic to Acute Myeloid Leukemia Cells and Results in Altered Downstream Signaling
Authors: Liu Suiyang , Kharbanda Surender , Stone Richard M.
Published: October 2019

Acute myeloid leukemia (AML) is a heterogeneous myeloid stem cell disorder; subtypes, which can be defined histologically, cytogenetically, and genetically, have varying prognoses and clinical characteristics. However, all but those younger patients with favorable biological features are inherently resistant to available cytotoxic chemotherapeutic agents; novel and less toxic therapies are required. Studies have shown that exposure of cancer cells, including leukemic blasts, to histone H1 produce cytotoxicity. The mechanism of cytotoxicity is believed to be an increase in focal membrane permeability induced by histone H1 binding to phosphotidyl serine moieties. However, the precise mechanism and functional effects of histone H1 on AML cells is currently unclear. The present study was aimed at investigating the effect of recombinant human Histone 1.3, OncoHist® (Xenetic Biosciences), on the proliferation of AML cell lines and on primary AML cells. We demonstrate that purified OncoHist® exerts growth inhibition and induces necrosis by flow cytometry assessment of staining for propidium iodide (PI) of AML cell lines (MOLM14, MV4-11, U937, HL60) as well as primary AML (n=3, including one with a p53 mutation) cells independent of lineage, stage and maturation with an IC50 of 2-5 mM. An AML cell line (MOLM13) with a FLT3 activating mutation (length or internal tandem duplication) which was developed to express resistance to the FLT3 inhibitor midostaurin/PKC412 was sensitive to OncoHist®-induced growth arrest measured by trypan blue staining and death (flow for PI) with about 70-80% necrosis at 5mM. Furthermore, 2uM OncoHist® treatment of MV4-11 and MOLM-14 cells for 15 minutes was associated with inhibition of the FLT3 downstream effectors phospho-AKT and phospho-extra cellular regulated kinase (phospho-ERK) as assessed by immunoblotting. Moreover, treatment of MOLM14 cells with OncoHist® in combination with cytarabine was associated with a significant synergistic inhibition of growth as measured by Alamar blue staining with a CI index of 0.35. Our findings support the development of OncoHist® alone and in combination with chemotherapy for the treatment of AML. A Phase I trial of OncoHist® for the treatment of refractory/relapsed AML is planned.

Disclosures

Kharbanda: Xenetic: Equity Ownership. Stone:Xenetic: Consultancy.
Early T Acute Lymphoblastic Leukemia in Childhood: Prevalence and Clinical Characteristics
Authors: Rossi Jorge Gabriel , Carrara Carolina , Rubio Patricia L. , Alonso Cristina N. , Bernasconi Andrea , Sajaroff Elisa , Cocce Mariela , Gallego Marta , Eandi Silvia , Ibarrola Paz , Luna Patricia , Vides Marisol , Pedrini Cesy Cristina , Felice Maria Sara
Published: October 2019

Background: Early T precursor ALL has been defined as a poor prognosis subtype, characterized phenotypically by absence of CD1a and CD8, CD5 weak expression and presence of at least one myeloid and progenitor cell marker.

Objectives: Our aims were to identify Early T-ALL among our T ALL cases, based on the characteristic phenotype and thus evaluate the prevalence, biological characteristics and outcome of this particular subset of T-ALL

Methods: From April ’94 to December ’13, 197 T-ALL cases were diagnosed and 20 of them showed the typical Early T marker profile by flow cytometry (FC). Conventional cytogenetics, FISH and RT-PCR studies were performed according to standard techniques. Clonality assessment for TRG, TRB, TRD, IGH and IGK was performed by PCR (Biomed-2), heteroduplex and sequencing.

Results: Sex distribution was: 16 males and 4 females; median of age: 7.9 (range: 0.3-16.6) years; median WBC: 14.9 (range: 0.6-254.0) x109/L. All but one case expressed CD34, 70% HLA-DR, 61% CD117 and 50% TdT. The most frequently expressed myeloid markers were: CD33 (85%) and CD13 (55%). Three cases expressed g/d TCR. Chromosomal abnormalities were detected in 14 of 16 evaluated cases, in 5 of them compromising 12p13 region. TCR/IgH rearrangements were detected in 63% (10/16). FLT3-ITD mutations were found in 14 % (2/14).

Patients were treated with BFM-based ALL schedules and were stratified as Intermediate Risk ALL (n: 7) and High Risk ALL (n: 13) according to protocol criteria. Early response to treatment was poor in 16 cases: 6 presented poor response to prednisone (day 8), 3 MRD >10%, 5 presented bone marrow M2-M3 and 2 non response. Seventeen cases (85%) achieved CR on day 33 and 2 achieved CR later. Five patients underwent HSCT in first CR. Three patients relapsed at 5, 6 and 65 months from diagnosis and one showed lineage switch to M5-AML at 12 months from CR. Three pts died in CR (2 after HSCT and 1 patient with primary immunodeficiency due to pneumonia) and one is in palliative care. Twelve patients remain in CR with a median follow-up of 54 (r: 8-144) months.

Conclusions: The prevalence of Early T precursor phenotype within T ALL was 10.5% in our setting. The most frequent progenitor marker was CD34 and CD33 among myeloid markers. Of note, translocations involving 12p13 region were found in 5 patients. Sixty percent of patients remain disease free, although longer follow-up is needed in order to define prognosis of this group in our cohort of patients. The lineage switch case supports the notion of the myeloid differentiation potential of these blasts.

Disclosures

No relevant conflicts of interest to declare.
Age and Population-Based Early Mortality Rate (EMR) in Acute Myeloid Leukemia (AML) Versus the EMR Reported By SWOG
Authors: Yaghmour George , Hahn Andrew , Martin Michael G
Published: October 2019

Background: In their seminal paper SWOG described the expected EMR for patients by age enrolled on clinical trials in AML (Appelbaum et al Blood 2006; 107: 3481-5). We sought to contrast these to population based outcomes.

Methods: Using case listing session of SEER 18 (1973-2010) we identified patients (pt) 18 years of age and older diagnosed (dx) from 1990-2005 with AML (Site recode ICD-O-3/WHO 2008 Acute Myeloid Leukemia which includes ICD-0-3 9861/3, 9873/3, 9920/3, 9910/3, 9840/3, 9891/3, 9867/3, and 9895/3 and includes all FAB and WHO subtypes except M3 ). This was done in order to match the accrual periods of the SWOG studies. We then examined the EMR of pt with non-M3 AML and compared them to those reported by SWOG. EMR in SEER was defined as death within 0 to 1 month from dx; EMR in the SWOG studies was defined as death within 30 days of initiation of induction chemotherapy. Analyses were conducted with SEER*Stat 8.1.2, Microsoft Excel 2007 and GraphPad Prism 6. All p-values were 2-sided.

Results: 26,272 pt were identified within SEER and 955 pt were in the SWOG EMR cohort. 54% of patients were male in SEER and 55% were male in SWOG; 85% of patients were white in SEER and 89% were white in SWOG. The EMR was 38.6% (10,130) in the SEER cohort; EMR was 12.2% (116) in the SWOG cohort (chi-squared with Yates correction p<0.001). Age distribution and mortality by age are presented in the table below. In general the SEER patients were older and experienced a two-fold greater EMR than SWOG patients in each age subgroup.

Conclusion: EMR in pt with non-M3 AML is much higher across all studied age groups in population based data than reported in SWOG studies. TableSEERSWOGEMR Fisher’s exact p-value% of ptEMR% of ptEMR<562616383P<0.00156-6516282511P<0.00166-7526412820P<0.001>753260832P<0.001

Disclosures

No relevant conflicts of interest to declare.
Myelodysplastic Syndromes: A Multidisciplinary Integrated Diagnostic Work-up for Patients' Risk Stratification
Authors: Ciabatti Elena , Ferreri Maria Immacolata , Valetto Angelo , Guazzelli Alice , Bertini Veronica , Grassi Susanna , Guerrini Francesca , Azzarà Antonio , Metelli Maria Rita , Petrini Iacopo , Simi Paolo , Petrini Mario , Galimberti Sara
Published: October 2019

Conventional cytogenetics continues to have a fundamental role in the classification and risk scoring of myelodysplastic syndromes (MDS). Nevertheless, non-informative karyotypes represent up to 20% of cases. Some different molecular methods, not included in the routinary diagnostic workup, such as aCGH or mutational analysis, could be able to detect new abnormalities and improve the subtyping of MDS.

The aim of this study was to propose a new diagnostic workup to determine the eventual adjunctive value offered by FISH, aCGH, and somatic mutation assays in respect of the the conventional cytogenetics only.

In this study, we analyzed 50 patients: 29% female and 71% male, median age 71 (range 30-88 years), 66% at low/int1 IPSS risk, 54% at very-low/low R-IPSS risk, 33% with RCMD, 15% with RA, 14% with RARS, 14% with RAEB, 8% 5q-, and 16% with MMCL.

We assessed these new MDS cases by different techniques: i) conventional cytogenetics; ii) FISH for chromosome 5, 7, PDGFRa, and PDGFRb; iii) aCGH, and iiii) specific RT-PCR for ASXL1, EZH2, TP53, and TET2 mutations.

Conventional cytogenetics showed 42% of patients with at least one chromosomal aberration, including +8, del(11), del(7), del(5), -Y, +6, del(13), +14, del(20), and complex karyotypes (6% of cases). After FISH analysis, we were able to correctly classify as affected by the 5q- syndrome 2 cases who then received lenalidomide.

The aGCH allowed to detect quantitative chromosomal aberrations in 44% of cases (del(13), -7, del(12), del(16), del(17), del(11), del(8), dupl(14), 5q-), including 10 cases (20%) showing a normal karyotype. After the RT-PCR, 32% of patients resulted mutated, with highest frequency for TP53 (22%). Four of these TP53-mutated patients showed normal karyotype, and resulted unmutated also by FISH and aCGH; in a case TP53 mutated we added treatment with steroid. Other 3 patients TP53-mutated did not respond to azacitidine Four low-risk patients (8%) showed ASXL1 gene mutation, three of them not earlier detected by cytogenetics or aCGH. One of these patients died after progression into acute leukemia. The identification of TP53 or ASXL1 mutations after RT-PCR and of dupl(14) by the aCGH prompted us to strictly follow patients at high risk of transformation. Only one case showed TET2 mutation; although TET2 mutations have been related to a better survival in patients receiving 5-azacitidine, this patient resulted not-responsive after 9 cycles.

In conclusion, these results sustain the necessity of an integrated work-up for the diagnosis and the correct risk scoring of MDS patients.

Disclosures

No relevant conflicts of interest to declare.
Contribution of IPSS-R Cytogenetics to Predict Outcome after Allogeneic Stem Cell Transplantation for Myelodysplastic Syndromes: A Study from the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC)
Authors: Gauthier Jordan , Gandhi Damaj , Langlois Carole , Robin Marie , Chevallier Patrice , Beguin Yves , Nguyen Stephanie , Bories Pierre , Blaise Didier , Cornillon Jerome , Clavert Aline , Mohty Mohamad , Huynh Anne , Thiebaut Anne , Vigouroux Stephane , Duhamel Alain , Yakoub-Agha Ibrahim
Published: October 2019

The prognosis of myelodysplastic syndromes (MDS) after allogeneic stem cell transplantation (allo-SCT) is critically determined by cytogenetic abnormalities. In this study, we assessed the impact of the IPSS-R cytogenetic score (C-IPSS-R) on the outcome of 367 patients with MDS transplanted from HLA-identical siblings or HLA allele-matched unrelated donors. According to the C-IPSS-R 178 patients (48%) fell in the good risk, 102 (28%) in the intermediate risk, 77 (21%) in the poor risk and 10 (3%) in the very poor risk group. In multivariate analysis, the poor and very poor-risk categories correlated with shorter overall survival (HR = 1.59, P = 0.009 and HR = 3.18, P = 0.002, respectively) and higher relapse rates (HR = 1.82, P = 0.004 and HR = 2.44, P = 0.060, respectively), after a median follow-up of 4 years. Overall, the C-IPSS-R changed the IPSS cytogenetic risk only in 8% of cases but identified a new risk group with dismal prognosis. These findings urge the investigation of new post-transplant therapeutic approaches for patients with very poor C-IPSS-R karyotypes, including maintenance therapy.

Disclosures

No relevant conflicts of interest to declare.