bluuebunny/crossref_metadata_2025 huggingface dataset:
The Addition of Plerixafor Allows Adequate PBSC Collection in Multiple Myeloma and Lymphoma Patients Poor –mobilizers After Chemotherapy and G-CSF.
Authors: D'Addio Alessandra , Curti Antonio , Giudice Valeria , Motta Maria Rosa , Rizzi Simona , Dan Elisa , Taioli Simona , Baccarani Michele , Lemoli Roberto M
Published: October 2019

Abstract 3233

Poster Board III-170

Peripheral blood stem cell (PBSC) mobilization and subsequent autologous stem cell transplantation (ASCT) is the most effective strategy in many patients with Multiple Myeloma (MM), Hodgkin's Disease (HD) or non-Hodgkin's Lymphoma (NHL). However, a significant proportion of patients are not able to mobilize an adequate number of cells after conventional stem cell mobilization strategies [i.e. chemotherapy and granulocyte colony-stimulating factor (G-CSF) or G-CSF alone], and can not proceed to ASCT.

In the present work, we report, for the first time, of five heavily pre-treated or treatment refractory MM or lymphoma patients (3 males and 2 females, median age 51 years) classified as “poor-mobilizers”, or “no mobilizers” at all, after chemotherapy (DHAP, =2; Cyclophosphamide, =3) and G-CSF mobilization strategy, who received Plerixafor to successfully collect PBSCs. Poor mobilization was defined when the concentration of PB CD34+ cells was consistently lower than 10 cells/μL during the recovery phase after chemotherapy and/or the collection of PBSCs was predicted to be inadequate to perform single or double ASCT. The timing for Plerixafor administration was not planned in advance. The rapid biological activity of Plerixafor (i.e. peak time of CD34+ cell mobilization occurring 10-12 hours after injection) allowed its administration “on demand”, that is when PBSC mobilization was felt to be inadequate, considering WBCs and CD34+ cell kinetics. Plerixafor, at 0.24 mg/Kg, was administered subcutaneously for a maximum of 3 consecutive days, while continuing G-CSF, the evening before the planned leukapheresis.

Overall, the drug proven to be safe and no adverse events were recorded. In all patients we observed a significant fold-increase (mean value= 7.96) in the number of circulating CD34+ cells following Plerixafor administration as compared to CD34+ cell concentration evaluated daily after chemotherapy and G-CSF. All patients were able to collect the minimum required dose for ASCT (2×106 CD34+ cells/Kg) and 4/5 patients collected the target cell dose (4×106 CD34+ cells/Kg for single ASCT in NHL and 6×106 CD34+ cells/Kg for tandem ASCT in MM). Three of 5 patients have already undergone ASCT with Plerixafor-mobilized PBSCs showing a rapid and durable hematological recovery.

Our results demonstrate that the addition of Plerixafor to G-CSF after chemotherapy is safe and allows the rescue of heavily pre-treated or poor-prognosis NHL and MM patients, who need to perform ASCT as consolidation therapy, but are failing PBSC mobilization.

Disclosures

No relevant conflicts of interest to declare.
Deletions of the Normal ABL1 at the Non-Translocated Chromosome 9 in CML Associated with TKI Resistance.
Authors: Virgili Anna , Brazma Diana , Grace Colin , Nacheva Elisabeth P
Published: October 2019

Abstract 3254

Poster Board III-1

Chronic myeloid leukaemia (CML) is characterized by the expression of the BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase that commonly results from the formation of the Philadelphia chromosome (Ph) after a t(9;22)(q34;q11) or variant rearrangement. Some 10% to 15% of CML patients present with cryptic deletions adjacent to the translocation breakpoint junction on the derivative chromosome 9. These deletions are believed to occur during the formation of the Ph translocation and are associated with a worse prognosis for CML patients treated with hydroxyurea or interferon. However there is still conflicting data regarding the significance of these deletions in patients receiving imatinib therapy, which appears to overcome the poor prognosis associated with the genome loss. Here we present data of three BCR/ABL1 positive CML patients on tyrosine kinase inhibitors (TKI) who developed therapy resistance. In all patients loss of the ABL1 sequences was identified. Surprisingly, these deletions affected not the ABL1 sequences of the ABL1/BCR fusion on the der(9) chromosome but the normal ABL1 allele on the non- translocated homologue of chromosome 9. This loss was not present at the time of diagnosis but was a new event developed during disease progression and found along with other additional chromosome rearrangements in the BCR/ABL1 positive cells. Bone marrow chromosome suspensions were studied by array comparative genomic hybridization (aCGH) analysis and/or fluorescent in situ hybridization (FISH) using and a range of commercial and bacterial artificial chromosomes (BAC) probes. While one patient carried a large deletion del(9)(q31q34) that spanned several megabases and was detectable by G-banding analysis, the other two patients had small cryptic losses (from 1.8 Mbp to 2.6 Mbp). The loss involved 3' ABL1 sequences in one patient whereas it affected the whole gene in the other two cases. Since the negative control exercised by the normal ABL1 allele over the BCR/ABL1 expression has been documented, the sensitivity to the TKI might be affected in the BCR/ABL1 positive cell clone bearing the genome loss. In summary, we report a new type of ABL1 loss in Ph positive cells - deletions at the 9q34 associated with TKI resistance in CML patients.

Disclosures:

No relevant conflicts of interest to declare.
Treatment with mTOR Inhibitor, Everolimus (RAD001) Overcomes Resistance to Imatinib in Ph-Leukemia Quiescent or T315I-Mutated Cells.
Authors: Minami Yosuke , Minami Miho , Kuwatsuka Yachiyo , Tanizaki Ryohei , Nomura Yuka , Abe Akihiro , Kiyoi Hitoshi , Naoe Tomoki
Published: October 2019

Abstract 3277

Poster Board III-1

Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. Aberrant activation of mTOR signaling has also been reported to be involved in LSCs. In order to examine mechanisms of drug resistance in Ph-positive (Ph+) LSCs and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., Proc Natl Acad Sci USA, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells derived from leukemic NOG mice were co-cultured with S17 stromal cells and treated with imatinib and the mTOR inhibitor, everolimus (RAD001, Novartis Pharmaceuticals). While quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib in spite of BCR-ABL- and CrkL-dephosphorylation, substantial cell death including CD34+ population was induced with nM level of everolimus. In imatinib-resistant Ph+ leukemia cell lines harboring T315I-mutation (Baf3p210/T315I and TCC-Y/T315I), everolimus induced cell death with low IC50 values in PI-exclusion assays. We are also investigating detailed biomarkers in the cell death (such as phosphorylation of 4E-BP1 or p70 S6K) and effects of theses drugs in the leukemic NOG mice systems. These results imply that treatment with everolimus can overcome the resistance to imatinib in Ph+ LSCs or T315I-mutated cells.

Disclosures:

Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe:Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.
Ubiquitin-Mediated Binding and Stabilization of β-Catenin by p210 BCR/ABL.
Authors: Chen Ru , Mahon Gwen , Whitehead Ian P
Published: October 2019

Abstract 3278

Poster Board III-1

Chronic myelogenous leukemia (CML) is a malignant hematopoietic stem cell disorder that is invariably associated with a balanced reciprocal translocation between chromosomes 22 and 9. The major product of the rearrangement is a 210 kD in-frame fusion protein (p210 BCR/ABL) that contains amino-terminal sequences from BCR, and carboxy-terminal sequences from the non-receptor tyrosine kinase ABL. This fusion event leads to the constitutive activation of the ABL-encoded kinase activity, which is the principal driving force behind CML. Thus, inhibitors that target the kinase activity produce durable clinical remissions. Although the ABL-encoded kinase activity is essential for disease progression, several studies have shown that BCR encoded sequences are also necessary for BCR/ABL-mediated leukemogenesis. In this current study we have identified ubiquitin as a binding partner for BCR, and demonstrated that binding is retained in p210 BCR/ABL. We have localized the docking site for ubiquitin to residues 180-191 which is present in all fusion variants of BCR/ABL (p190, p210 and p230). This putative ubiquitin binding domain (UBD) does not conform to any known consensus sequence, and is unique to BCR. Deletion of the UBD does not impair the auto-or trans-kinase activity of p210 BCR/ABL, nor does it impair the interaction between p210 BCR/ABL and GRB2, or the ability of p210 BCR/ABL to activate ERK1/2. A mutation at residue tyr-177 of p210 BCR/ABL also does not impair the interaction with ubiquitin suggesting that the GRB2 and ubiquitin binding sites are adjacent, but separable. β-catenin has been identified as a binding partner for BCR and BCR/ABL, and the docking site has been mapped to a region of BCR/ABL that contains the UBD. Over-expression of p210 BCR/ABL, but not the ubiquitin binding mutant, leads to an accumulation of β-catenin that is phosphorylated on the serine residues that normally trigger ubiquitin-mediated turnover. This difference cannot be attributed to a difference in the activation status of GSK-3β or Pin-1. These results suggest that either β-catenin and ubiquitin share a docking site on p210 BCR/ABL, or that the interaction between β-catenin and p210 BCR/ABL is ubiquitination dependent. The latter possibility is consistent with a previous study which showed that glycine residue in β-catenin is required for Bcr interaction. We propose a model in which p210 BCR/ABL may influence the Wnt signaling pathway by binding ubiquitinated β-catenin, and stabilizing it against degradation.

Disclosures:

No relevant conflicts of interest to declare.
WP1130 Inhibits Signaling through BCR-ABL Ubiquitination and Cytoplasmic to Aggresome Trafficking to Induce Apoptosis of CML Cells.
Authors: Donato Nicholas J , Sun Hanshi , Kapuria Vaibhav , Peterson Luke F , Bartholomeusz Geoffrey , Bornmann William G. , Talpaz Moshe
Published: October 2019

Abstract 3303

Poster Board III-191

BCR-ABL remains the primary target for chronic myelogenous leukemia (CML) therapy but longstanding control and complete suppression of the disease may require additional strategies beyond kinase inhibition. We previously described a small molecule, WP1130, that mediated down-regulation of BCR-ABL, induced apoptosis of CML cells with limited toxicity against normal myeloid progenitors and decreased CML tumor growth in mice (Blood. 2007;109(8):3470-8). In this report we further investigated the BCR-ABL targeting activity and mechanism of action of WP1130 in CML cells. WP1130 rapidly (within 30 to 60 min) reduced cytoplasmic levels of BCR-ABL protein and suppressed Stat5 tyrosine phosphorylation without affecting Jak2, c-Abl, BCR, Erk, Akt, Lyn, Hck or gp130. Down-regulation of BCR-ABL by WP1130 was not affected by point mutations that alter imatinib or dasatinib binding or BCR-ABL kinase inhibitory activity. Examination of the cellular distribution of BCR-ABL in treated cells revealed that BCR-ABL was sequestered into compact, cytoskeletal-enriched aggresomal structures that were recovered in the detergent-insoluble fraction of the cell. WP1130-mediated translocation of BCR-ABL was associated with increased cellular poly-ubiquitination without 20S proteosome inhibition, disruption of Hsp90/BCR-ABL complexes or increased reactive oxygen species. WP1130 induced BCR-ABL trafficking into aggresomes correlated with its poly-ubiquitination (primarily K63) and lysates from WP1130 treated cells demonstrated reduced capacity to hydrolyze ubiquitinated substrate. Together, these results suggest that WP1130 induces poly-ubiquitination and trafficking of BCR-ABL into signaling incompetent aggresomes in CML cells through a unique mechanism that may involve inhibition of deubiquitinase activity and define a unique pathway to suppress BCR-ABL signaling and induce CML cell destruction.

Disclosures

No relevant conflicts of interest to declare.
Allogeneic Stem Cell Transplantation (allo-SCT) for Secondary Acute Myeloid Leukemia (AML) Following Breast Carcinoma.
Authors: Ayari Sameh , Mohty Mohamad , Bilger Karin , Guillerm Gaelle , Guyotat Denis , Michallet Mauricette , Ifrah Norbert , Chevallier Patrice
Published: October 2019

Abstract 3305

Poster Board III-193

Patients with breast carcinoma who received Radio and/or chemotherapy, have an increased risk for developing therapy-related myelodysplastic syndromes/acute myeloid leukemia (1-5%). Such secondary AMLs have often poor prognosis when treated with conventional chemotherapy.

This retrospective series assessed the outcome of 29 female patients who were reported to the Societe Francaise de Greffe de Moelle et de Therapie Cellulaire Registry, and who were treated with allo-SCT for secondary MDS/AML developing after initial therapy for breast cancer. The median age of patients at time of breast cancer treatment was 51 (range, 31-62) years. The median age at time of allo-SCT was 53 (range 31-63) years. Diagnosis included 21 AML and 8 MDS. Cytogenetics: four patients had CBF abnormalities, 10 had 11q23 abnormalities, 5 had an intermediate caryotype and 3 had unfavourable one. At time of allo-SCT, 21 patients were in complete remission (CR), while 8 had a refractory/relapsed disease. PBSCs were used as stem cell source in 18 patients, while 9 patients received classical bone marrow and one patient received an unrelated umbilical cord blood. Patients received a median of 5.2 ×10e6/Kg CD34+ cells. A matched related donor was used in 23 cases (82%) and an unrelated donor in 5 cases (18%). Conditioning regimen was myeloablative (Cy-TBI or Bu-Cy) in 7 cases (24%) and reduced-intensity in 22 cases (76%). Twenty four patients engrafted with a median time of 18 (range, 9-32) days for ANC>500/μL. Seven (24%) patients experienced grade 2-4 acute GVHD. Also, 7 patients (24%) experienced chronic GVHD (5 extensive and 2 limited).

With a median follow-up of 24 (range, 3-129) months, 16 patients (55%) were still alive. Disease progression accounted for 6 deaths while transplant related causes (infection n=4, GVHD n=1, MOF n=1, cardiac failure n=1) occurred in 7 cases. The Kaplan-Meier estimates of overall and disease-free survival at 2 years were at 54 and 38% respectively.

These results highlight the poor outcome of secondary leukemia occurring after therapy for breast cancer, even with the use of allo-SCT. Innovative maintenance approaches such as early use of hypomethylating or immunomodulatory agents after allo-SCT aiming to decrease the relapse are warranted.

Disclosures

No relevant conflicts of interest to declare.
Factors Predicting the Outcome of the Blood and Marrow Transplant Patients Admitted to Intensive Care Unit.
Authors: Pati Nalini K , George Biju , Kerridge Ian , Gilroy Nicole , Nayyar Vineet , Stachowski Eddie , McGurgan Mary , Huang Gillian , Bradstock Kenneth F , Gottlieb David J , Hertzberg Mark
Published: October 2019

Abstract 3340

Poster Board III-228

Aim:

To identify factors predicting outcome of patients admitted to intensive care (ICU) following allogeneic haematopoietic stem cell transplantation (allo-HSCT).

Methods:

Retrospective audit of all allo-HSCT patients requiring ICU admission.

Results:

Between 2000 and 2009, 392 patients underwent allo-HSCT. Of these, 106 (27%) had 129 ICU admissions. The median age was 47 (range 16-65) with myeloablative transplant in 89 and reduced intensity in 40 patients. Respiratory failure was the main reason for admission (54.6%) followed by sepsis (41.5%).

During the period of ICU admission, 29.2% demonstrated improvement in organ failures, 39.2% remained stable and 28.4% deteriorated. Sixty-seven patients (51.9%) were discharged from ICU but only 48 (37%) were subsequently discharged from the hospital (ICU). Univariate analysis identified ICU admission within 30 days post HSCT, number of organ failures at admission, progression of organ failure during ICU admission, APACHE II score at admission, steroid refractory GVHD, and requirement for inotropic support or dialysis as significant predictors for survival in ICU. Patients requiring intubation and mechanical ventilation had a poorer outcome than the group who did not (84.4% Vs 20.0%, p=0.001). Those who required only non-invasive ventilation generally had a good outcome with 84.4% surviving til ICU discharge. While bacterial infection prior to ICU admission did not alter the outcome (p=0.221), the onset of a new infection in ICU was associated with a poor outcome (p=0.0001). Logistic regression analysis identified steroid refractory GvHD (P=0.027; 95% CI of 1.17-14.8), APACHE II score > 30 (p=0.003; 95% CI 1.5-10.5), admission <30 days post HSCT (p = 0.015; 95% CI 0.12-0.8), requirement of invasive ventilatory support (p = 0.005, 95% CI 2.58 – 223.83) and dialysis (p = 0.011; 95%CI 1.401 – 13.20) as significant factors for a poor outcome.

Conclusion:

More than 50% of patients admitted to ICU following allogeneic HSCT survive. A high APACHE II score, steroid refractory GVHD, admission into ICU within 30 days of HSCT, multiorgan failure, progression of organ failure during ICU stay, and the need for ventilation or dialysis, carries a dismal prognosis. Identification of risk factors associated with a poor outcome will assist in clinical management and may ultimately improve the outcome of patients requiring ICU admission following allogeneic HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Pretransplant Factors That Influence Intravenous Busulfan Kinetics in Adult Patients.
Authors: McBride Ali , Fancher Karen , Kim Jongphil , Davis Donn , Conwell Michelle , Field Teresa , Anasetti Claudio , Perkins Janelle
Published: October 2019

Abstract 3350

Poster Board III-238

We performed a retrospective review of intravenous busulfan (Bu) pharmacokinetic (PK) parameters and pretransplant characteristics on patient receiving Bu and fludarabine (40mg/m2/day x 4) (BuFlu) prior to allogeneic hematopoietic cell transplant (HCT) in order to determine the predictive value of pretransplant characteristics on Bu area under the concentration time curve (AUC). Included in this analysis are 265 pts transplanted between 7/04 and 12/08 who received 130 or 145mg/m2/day for 2 doses then had doses 3 and 4 adjusted based on first dose PK parameters (from 5 plasma concentrations) to achieve a target AUC of 5300 micromole*min/L. Pretransplant variables collected included: gender, age, ethnicity, height, actual body weight (ABW), body surface area (BSA), body mass index (BMI), underlying diseases states (hypertension, diabetes mellitus, coronary artery disease and liver disease), transplant diagnoses, prior chemotherapy, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB), serum creatinine (SCr), measured creatinine clearance (CrCl) and concomitant medications administered up to 24 hours prior to first Bu dose. Median first dose AUC was 5252 micromole*min/L (2633-21400 micromole*min/L). Univariable and multivariable analyses were utilized to identify prognostic factors that may affect AUC levels based on first dose kinetics. In univariable analyses, BMI and CrCl were negatively correlated with Bu AUC while AST and SCr were positively correlated. In addition, the use of azole antifungals, metronidazole proton pump inhibitors, doxycycline, and choice of antiseizure prophylactic medication also had a significant effect on Bu AUC. Multivariable analysis demonstrated BMI and CrCl were negatively associated with AUC (p=0.008 and 0.005, respectively) while AST was positively associated (p=0.035). The use of fluconazole, metronidazole and doxycyline was associated with higher AUCs (p=0.011, 0.021, and 0.004, respectively) while concurrent use of voriconazole was associated with lower AUC (p=0.011). Based on these data, pts with large BMI, increased CrCl, or on concomitant voriconazole may require larger doses of busulfan to achieve a target AUC whereas pts with an elevated AST or taking fluconazole, metronidazole and doxycyline may require less. In order to validate these findings, we are developing a population based dosing model that will require testing in a prospective pt sample.

Disclosures:

Field: PDL BioPharma: Research Funding. Perkins:PDL BioPharma: Research Funding. Off Label Use: IV busulfan and fludarabine for pretransplant conditioning.
Inhibition of p38 MAPK Promotes Hematopoietic Stem Cells Self-Renewal and Ex Vivo Expansion.
Authors: Wang Yong , Kellner Joshua , Zhou Daohong
Published: October 2019

Abstract 34

Activation of the p38 mitogen-activated protein kinase (p38 MAPK) is implicated in the inhibitory effects of TNF-α, TGF-β, interferons and reactive oxygen species (ROS) on hematopoiesis and self-renewal of hematopoietic stem cells (HSCs). Clinically, overactivated p38 MAPK contributes to the pathogenesis of myelodysplastic syndromes (MDS) and Fanconi anemia. Inhibition of p38 MAPK with pharmacological agents improves hematopoietic progenitors' function in MDS. However, it has yet to be determined if p38 MAPK plays a role in regulation of normal HSC self-renewal and whether inhibition of p38 MAPK can improve HSC ex vivo expansion. In the present study, we found that sorted mouse bone marrow HSCs (Lin− Sca1+ c-kit+ cells or LSK+ cells) exhibited specific activation of p38 MAPK after seven days culture in serum-free medium supplemented with stem cell growth factors (SCF, Tpo and Flt3 ligand). The activation of p38 MAPK was associated with rapid differentiation of HSCs and induction of cellular senescence. Addition of SB203580 (SB, a specific p38 MAPK inhibitor) to the culture abrogated the activation of p38 MAPK, inhibited the induction of cellular senescence and increased the expression of several HSC self-renewing genes (such as CXCR4, HoxB4 and Gfi1). Moreover, HSCs cultured with SB resulted in a significantly greater HSC expansion than HSCs cultured without SB as assessed by flow cytometry and cobblestone area-forming cell (CAFC) assay. Finally, competitive repopulation assays revealed that HSCs expanded with SB produced a dramatic increase in donor-derived engraftments after transplantation to irradiated recipients. These findings suggest that p38 MAPK plays an important role in the regulation of HSC self-renewal and its inhibitors (e.g. SB203580) may be clinically useful in the ex vivo expansion of HSCs.

Disclosures:

No relevant conflicts of interest to declare.
Y90 Plus High Dose BEAM with Autologous Stem Cell Transplantation for Chemorefractory Non Hodgkin Lymphoma.
Authors: Krishnan Amrita , Raubitschek Andrew , Palmer Joycelynne , Nademanee Auayporn P. , Kogut N. , Yamauchi Dave , Popplewell Leslie , Federico Laura , Simpson Jennifer , Tsai Ni-Chun , Forman Stephen J
Published: October 2019

Abstract 3423

Poster Board III-311

Long term survival for patients with chemo-refractory non-Hodgkin lymphoma (NHL) is poor even with high dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT). Strategies such as novel transplant conditioning regimens and post transplant maintenance therapy are being evaluated to improve outcomes. Herein we report on the results of a phase II study designed to assess the impact of a novel radioimmunotherapy (RIT) based conditioning regimen of Y90 ibritumomab tiuxetan in conjunction with high dose BEAM (BCNU, Etoposide, Ara–C, Melphalan) in patients with poor risk NHL. Between May 2002 and Jan 2009, 31 patients who were either primary induction failure (n=18) or in partial remission (n=13) underwent HDC with Y90 plus BEAM. Patients received an imaging dose of Indium 111 on day -21 followed by a treatment dose of Y90 (0.4mCi/kg) on day-14. HDC with BEAM was started on day -7. The median age at ASCT was 57 (range: 19–70). The majority of patients were male (n=23, 74%) and had advanced stage disease (III or IV) at the time of diagnosis, (n=23, 74%). The histologies included Follicular n=6, Diffuse Large B-cell n=13, Mantle cell n=8, and Transformed n=4. The median number of prior regimens was 2 (range: 1-5). All patients engrafted at a median of 11 days. At the time of analysis, 23 (74%) patients were alive, and eight had expired (seven due to relapse/progressive disease, one due to COPD). The non-relapse mortality was 3.2%. The median length of follow-up for surviving patients is 46.9 months (range: 3.3-75.0). The two-year overall survival and disease-free survival was 77.9% (95%CI: 62.7-87.6%) and 50.9% (95%CI: 40.5-60.3%) respectively for the entire cohort. However, the relapse rate in the subset of MCL pts was strikingly high and approached 80% at 4 yrs. The other subgroups had a stable DFS (figure 1). In conclusion in this poor risk group of patients Y90 plus high dose BEAM can induce responses and long term remissions, however, for patients with chemorefractory MCL other treatment approaches may be warranted.

Disclosures

Off Label Use: yttrium 90 in combination with high dose BEAM chemotherapy.
A Phase 1, Open-Label, Multi-Center, Multiple-Dose, Dose-Escalation Study of MDX-1342 in Patients with CD19-Positive Refractory/Relapsed Chronic Lymphocytic Leukemia.
Authors: Camacho Luis H , Joyce Robin , Brown Jennifer R , Chanan-Khan Asher , Amrein Philip C. , Assad Albert , Carrigan Marion
Published: October 2019

Abstract 3425

Poster Board III-313

MDX-1342 is a fully human monoclonal antibody (HuMAb) with enhanced antibody-dependent cellular cytotoxicity (ADCC) effector function, targeting CD19-membrane receptor which is highly expressed on malignant chronic lymphocytic leukemia (CLL) cells. An open-label, multi-center, multiple-dose, dose-escalating Phase 1 study is being conducted to determine the safety and tolerability profile of MDX-1342 in subjects with CD19-positive relapsed or refractory CLL. To date, MDX-1342 has been administered intravenously (iv) weekly for 4 weeks to 12 subjects (8 Male, 4 Female) - in cohorts of 3 subjects each - at doses of 0.7, 7, 40, and 200 mg/dose. Overall, MDX-1342 has been well tolerated. No drug-related serious adverse events have been reported among the 12 subjects treated. Grade 1 and 2 infusion reactions (including rigors, chills, and wheezing) have been observed in 9 subjects. This has been adequately managed with the use of antihistamines and corticosteroids. Dose escalation continues and a maximum tolerated dose has not yet been identified. Of the 9 currently evaluable subjects, 1 has experienced a partial response (40 mg/dose cohort), 6 have stable disease and 2 have discontinued due to progressive disease. Preliminary results indicate antileukemic signals with dose-correlative reductions in both white blood counts (WBC) and circulating CD20+ cells after one 4-week cycle of weekly i.v. infusions of MDX-1342. In the 40 mg/dose cohort, the median reduction in WBC was 62% and the median reduction in circulating CD20+ cells was 74%. Additional subjects are being accrued to higher dose cohorts to more accurately characterize the safety and clinical activity of MDX-1342 and to determine if there is a consistent cumulative drop in WBC and circulating CD20+ cells as the dose increases.

Disclosures

Assad: Medarex, Inc.: Employment. Carrigan:Medarex, Inc: Employment.
Cell of Origin Influences Leukemia Stem Cell Phenotype.
Authors: Krivtsov Andrei V. , Sinha Amit U. , Stubbs Matthew C. , Kung Andrew , Armstrong Scott
Published: October 2019

Abstract 3459

Poster Board III-347

MLL-fusion proteins can transform either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP) into leukemia stem cells (LSC). However, the leukemogenic process in HSC may differ from that in GMP. We transduced HSC and GMP with MLL-AF9 or control retroviruses. Single-cell sorted MLL-AF9 expressing HSC or GMP could be serially replated for over 9 passages. Upon transplantation into syngeneic mice, 86.3% (n=22) of HSC:MLL-AF9 single cell derived clones (SCC) induced AML with a median latency of 61 days, while 33.3% of GMP:MLL-AF9 SCC induced AMLs with median latency of 100 days. Immunophenotype analysis of the resultant leukemias demonstrated that long-term repopulating HSC (LT-HSC) and GMP-derived leukemias were quite similar, with a GMP-like (LGMP) population enriched in LSC in both cases. Gene expression analysis demonstrated that globally the LGMP isolated from HSC derived AMLs (AML:HSC) and GMP derived AMLs (AML:GMP) were similar to each other but possessed specific genetic programs reminiscent of the cell of origin (HSC or GMP). For example Evi1, Jun, and Fos oncogenes were highly expressed in HSC and AML:HSC, but expressed at low level in GMP or AML:GMP. The genetic program that distinguished LGMP:HSC from LGMP:GMP was found to be enriched in hematopoietic stem cells compared to more differentiated myeloid progenitors and correlate with genetic programs in and human MLL-rearranged AML associated with a poor clinical outcome in two independent MLL-rearranged AML data sets. In order to directly assess differences in treatment response for leukemias derived from different cells of origin, we treated leukemic mice with a chemotherapeutic agent often used to treatment human AMLs. Treatment of leukemic mice with Etoposide reduced the spleen weights in mice transplanted with AML:HSC to a lesser extent (28%) than in mice transplanted with AML:GMP (88%). Altogether, these data indicate that cell of origin of AML can influence the genetic program of fully developed leukemia, and thus could account for some of the heterogeneity in human leukemias and perhaps outcome.

Disclosures

No relevant conflicts of interest to declare.
Hypermethylation of CD44 Is Characteristic of Specific Lymphoma Subtypes.
Authors: Röhrs Sonja , Romani Julia , Zaborski Margarete , Rosenwald Andreas , Drexler Hans G. , Quentmeier Hilmar
Published: October 2019

Abstract 3469

Poster Board III-357

Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a common hallmark of cancer. It is generally agreed, that CpG island hypermethylation profiles are specific for different tumor types [Costello et al., 2000, Nat Genet 25:132-138]. Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification), methylation of 24 different TSG was analyzed in 40 lymphoma cell lines representing Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). On average 8 ± 2.8 TSG out of the 24 analyzed were methylated per lymphoma cell line, whereas 0/24 TSG were methylated in healthy donor tonsils. Methylation frequencies decreased from HL, ALCL, BL, FL, DLBCL to MCL cell lines. The TSG methylation status of the most relevant genes was verified by methylation-specific PCR. Moreover, TSG hypermethylation correlated with transcriptional silencing as assessed by quantitative real time PCR. While our studies on the methylation status of TSG in lymphoma cell lines support previous methylation analyses performed on primary lymphoma patient material for many of the genes analyzed here, MS-MLPA screening also revealed a new interesting candidate: CD44. Hypermethylation of CD44 was characteristic of ALCL, BL, FL and DLBCL cell lines and allowed their discrimination from MCL and HL cell lines. In CD44 hypermethylated cell lines expression of CD44 was re-inducible at mRNA and protein levels by treatment with the demethylating agent 5-Aza-2'-deoxycytidine, confirming its epigenetic regulation. Furthermore, CD44 ligation with an anti-CD44 antibody induced apoptosis in CD44+ (CD44 unmethylated) lymphoma cell lines whereas CD44 hypermethylated cell lines showed no response. Thus, CD44 might be an interesting new epigenetic marker and a potential molecular target for the diagnosis and treatment of specific lymphoma subtypes.

Disclosures

No relevant conflicts of interest to declare.
Class I HLA-Specific Antibodies Can Cause “false-positive” Test Results in the Serotonin Release Assay for Heparin-Induced Thrombocytopenia (HIT).
Authors: Rocky Shiu-Ki Hui , Ellis Thomas M , Aster Richard H. , Curtis Brian R.
Published: October 2019

Abstract 3506

Poster Board III-443

A diagnosis of heparin-induced thrombocytopenia (HIT) is confirmed with support from the laboratory. Antibodies associated with HIT are specific for complexes made up of heparin and platelet factor 4 (PF4) and can be detected in a solid phase ELISA (PF4 ELISA). However, a functional test, the serotonin release assay (SRA) is regarded by many to be the “gold standard” for laboratory investigation of this disorder. In our facility, the SRA is performed by incubating serotonin-labeled platelets (pooled from three different group O donors) with test serum and low dose (0.1 units/ml) or high dose (100 units/ml) heparin and determining the percentage of total serotonin released. Release of serotonin (20-100%) with low dose heparin and inhibition of this release with high dose heparin is considered to be “positive” for platelet-activating HIT antibodies. Sera from some patients cause serotonin release with low dose heparin that is not inhibited with high dose heparin. The significance for these “indeterminate” reactions is unclear, but they are considered not to reflect the presence of “true” HIT antibodies.

We studied selected serum samples from 238 patients referred for HIT testing. Of these, 119 tested “true” positive and 117 produced “indeterminate” reactions in the SRA. The same samples were tested for the presence of antibodies reactive with beads coated with various Class I HLA antigens using a flow cytometric bead assay (Flow PRA, One Lambda). Sera producing at least 30% release in SRA and reactive with at least 20% of the bead panel were selected for analysis. As shown in Figure 1, there was a high correlation between the likelihood of an “indeterminate” SRA test result and the presence of Class I HLA antibodies (p << 0.0001).

Figure 1 SRA Category No. with PRA >/=20% No. with PRA <20% Total Sample SRA “Indeterminate” (>/=30% Release and Uninhibited by High Dose Heparin) 91 19 110 SRA “True Positive” (>/=30% Release and Inhibited by High Dose Heparin) 36 72 108

As expected, there was a significant correlation between the strength of reactions produced by individual “true positive” sera in the SRA (% release) and in the PF4/heparin ELISA (O.D. value). However, in analyzing 38 sera with “true positive” test results in the SRA, we identified two that were negative in the PF4 ELISA and contained broad Class I HLA reactivity (reactive with 100% and 41% of the panel, respectively).

We conclude 1) Class I HLA antibodies are the major cause of “indeterminate” reactions in the serotonin release assay and 2) A subset of these antibodies can be inhibited by high dose heparin and therefore mimics the behavior of “true” HIT antibodies in the SRA. Unless the PF4 ELISA test is used together with the SRA, this type of reaction could lead to an erroneous diagnosis of HIT.

Disclosures:

No relevant conflicts of interest to declare.
Poster Board III-457 Surface Plasmon Resonance (SPR) Analysis Detects Clinically Significant Low Avidity HPA-1a Antibodies Not Identified by Conventional Serology.
Authors: Peterson Julie A. , Kanack Adam , Gitter Maria L. , Rathore Vipul , McFarland Janice , Curtis Brian R , Aster Richard H.
Published: October 2019

Abstract 3520

Poster Board III-457

Neonatal alloimmune thrombocytopenia (NATP) is caused by passively transmitted maternal antibodies specific for fetal platelet antigens inherited from the father. The diagnosis is made by demonstrating a maternal antibody specific for a paternal platelet alloantigen inherited by the affected infant. It is not unusual for mothers of suspected NATP cases to be antibody-negative using standard serologic methods. Assays routinely used to detect alloantibodies (flow cytometry, glycoprotein-specific solid phase assays) require repeated washing of the target cell or antigen to remove unbound antibody, followed by detection of bound antibody with a labeled secondary probe. Low avidity antibodies could dissociate from their targets during these washing steps and might, therefore, not be detected; however, the extent to which such antibodies cause human disease is controversial. Surface plasmon resonance (SPR) directly measures the accretion of ligand on an immobilized receptor in real time and could therefore lend itself to detection of low avidity, clinically significant human antibodies. In fact, Socher et al recently described use of SPR to detect maternal antibodies specific for the HPA-1a (PlA1) platelet antigen in two NATP cases not diagnosed by standard serology (Transf 49:943, 2009).

We have also studied the utility of SPR for detection of clinically important human antibodies. HPA-1a/a-positive and -negative GPIIb/IIIa was isolated from platelets and was immobilized in the lanes of a CM5 Biacore chip. IgG used for testing was doubly purified from serum to remove vitronectin and other proteins that might react with the target integrin through RGD binding sites, thereby masking antibody-specific signals. The range for binding of IgG from normal subjects to these targets was defined with IgG from 30 healthy donors.

In preliminary studies, we found that SPR analysis is about 5 times as sensitive as standard serology for detection of known HPA-1a-specific antibodies. We then studied maternal sera from 45 cases of suspected NATP in which the mother was HPA-1a-negative but no maternal antibody was detected. Seven of these sera were found to contain IgG that recognizes HPA-1a-positive, but not HPA-1a-negative GPIIb/IIIa. A comparison of the reaction kinetics of these antibodies with those of “conventional” HPA-1a-specific antibodies showed that the former have a significantly higher off rate, as would be expected of low avidity antibodies. We conclude that low avidity antibodies specific for HPA-1a are capable of causing NATP and may not be rare and that SPR may provide a unique tool for their detection. The extent to which SPR can be useful in studying other antibody-mediated human disorders deserves exploration.

Disclosures:

No relevant conflicts of interest to declare.
Increased Copy Number of Interleukin-6 Receptor Gene Is Associated with Adverse Survival In Multiple Myeloma Patients Treated with Autologous Stem Cell Transplantation
Authors: Kim Seon Young , Min Hyun Jung , Park Hyun Kyung , Oh Bora , Kim Tae Young , Kim Hyun Kyung , Kim Inho , Yoon Sung-Soo , Park Seonyang , Kim Byoung Kook , Lee Dong Soon
Published: October 2019

Abstract 1895

Background:

Interleukin-6 (IL-6) is a potent pleiotropic cytokine that regulates plasma cell (PC) growth via IL-6 receptor (IL-6R). We hypothesized that upregulation of IL-6R in myeloma cells might confer the growth privilege to myeloma cells over bone marrow (BM) hematopoietic cells. We investigated the frequency and prognostic implication of increased copy number of IL-6R gene by fluorescence in situ hybridization (FISH) in patients with newly diagnosed multiple myeloma (MM). To confirm the role of IL-6R, the change of proliferative potentials after blocking of IL-6R by monoclonal antibody was observed in a myeloma cell line.

Methods:

102 patients with newly diagnosed MM were enrolled. FISH study for IL-6R was performed using home-made probe for the locus. The BAC (bacterial artificial chromosome) clone RP11 350G8 (BACPAC Resources, Oakland, CA) was used as the IL-6R gene-positive clone and the FISH probe was labeled by a nick translation reaction. FISH signals were counted among BM plasma cells sorted by immunoglobulin light chain staining (cIg FISH). U266 myeloma cell line was treated with IL-6R monoclonal antibody (MRA, Chugai, Japan) at concentration of 0.06 to 1.25 μM and then cell viability test were performed.

Results:

The amplification of IL-6R was detected in 53/102 patients (52.0%). The 5-year overall survival (OS) rate of patients with IL-6R gene amplification was 41.3% versus 44.8% for those with a normal IL-6R (P = 0.425). In 44 patients treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT), patients with 3.1 or more mean IL-6R gene copy number per PC showed worse 5-year OS compared to those who had less than 2.1 mean copies of IL-6R gene (44.4% versus 78.0%, P = 0.024). In multivariate analysis, the increase of IL-6R copy numbers (mean copy/PC ≥ 3.1) could be considered as an independent prognostic factor for MM patients underwent ASCT (hazard ratio, 30.9; 95% CI, 1.74–548.6; P = 0.020). Treatment of IL-6R monoclonal antibody on the U266 cell line induced marked reduction of cell viability compared with control, ranged from 7.1% to 98.7% according to the increase in treated antibody level.

Conclusions:

The gain of IL-6R gene is frequent in myeloma, showing an association with adverse prognosis in MM patients treated with ASCT. In vitro suppression of cell proliferation by IL-6R blocker suggests the potential role of IL-6R in myeloma cell growth and therapeutic implication of IL-6R blocker in the future.

Disclosures:

No relevant conflicts of interest to declare.
Valuation of MCP-1 and VEGF Chemokines In Serum of Patients with AL Amyloidosis
Authors: Moscetti Alessandro , Saltarelli Francesca , Bianchi Maria Paola , Bruno Guglielmo , Salerno Gerardo , Corsetti Maria Teresa , Cardelli Patrizia , Ferranti Giovanna , La Verde Giacinto
Published: October 2019

Abstract 1922

AL amyloidosis is a pathology characterised by the deposition of fibrillary aggregates of immunoglobuline light chains with β-sheet conformation. The light chains are synthetized by neoplastic plasma cell and fibrils deposition can infiltrate tissues leading to multi systemic organ damage. To evaluate if vascular modifications are involved in AL amyloidosis, inflammatory activity of cytokines as MCP-1 and VEGF was investigated. MCP-1 is a chemokine that activates mononuclear phagocytes by promoting leukocyte-endothelium binding and migration to sites of inflammation, while VEGF is an endothelial cell mitogen and permeability factor that is potently angiogenic in bone marrow of AL amyloidosis patients. Aim of this study is to evaluate serum cytokines MCP-1 and VEGF levels in patients with systemic or localized AL amyloidosis at presentation to find out potential differences useful to define a characteristic inflammatory pattern. Blood samples were collected from 8 patients with systemic amyloidosis and from 4 patients with localized amyloidosis and analyzed for serum MCP-1 and VEGF levels. Mann-Whitney test and Spearman correlation were used to compare results. MCP-1 level was significantly higher in the serum of patients with systemic disease (350.52±58.70 pg/ml) compared to the group of patients with localized amyloidosis (147.82±26.03; p=0.004); VEGF was also significantly increased in systemic disease group (p= 0.028). In addition, a positive correlation between MCP-1 and VEGF (r2= 0.755; p=0.031) has been found in the group of patients with systemic amyloidosis. Results seems to suggest a difference in serum cytokine MCP-1 and VEGF levels between AL systemic and localized amyloidosis. In systemic amyloidosis the neoplastic plasma cells interact with bone marrow microenvironment resulting in VEGF release leading to a new angiogenesis also supported by an inflammatory cells increase. The MCP-1 activates and promotes leukocyte-endothelium binding increasing the inflammatory process. The high correlation between MCP-1 and VEGF suggests a positive relationship between a new angiogenesis and a migration of inflammatory cells in the bone marrow stroma. On the basis of our results, MCP-1 and VEGF chemokines can be used to evaluate the inflammatory process in patient with systemic or localized AL amyloidosis.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Maintenance with Lenalidomide Improves Progression Free Survival In Myeloma Patients with High-Risk Cytogenetics: An IFM Study
Authors: Avet-Loiseau Hervé , Caillot Denis , Marit Gerald , Lauwers-Cances Valerie , Roussel Murielle , Facon Thierry , Hulin Cyrille , Lamy Thierry , Pegourie Brigitte , Maisonneuve Herve , Garderet Laurent , Moreau Philippe , Stoppa Anne-Marie , Dib Mamoun , Jaubert Jerome , Casassus Philippe , Mathiot Claire , Harousseau Jean-Luc , Moreau Philippe , Attal Michel
Published: October 2019

Abstract 1944

Cytogenetic abnormalities have been associated with specific outcome in myeloma, as in many other hematologic malignancies. Among the large spectrum of chromosomal changes observed in myeloma, at least two of them are clearly associated with a specific poor outcome, both in terms of PFS and OS, i.e., t(4;14) and del(17p). Recent data suggested that bortezomib can at least partially overcome the prognostic value of t(4;14). However, t(4;14) remains a prognostic factor in the IFM series. Deletion 17p is rare (< 10%), but is associated with a very poor outcome, whatever the treatment proposed to these patients. In 2005, the IFM designed a clinical trial aiming to test the role of lenalidomide maintenance until relapse in patients under 65 years of age treated with a VAD or bortezomib/dexamethasone induction, followed by high-dose melphalan. Six hundred and fourteen patients have been enrolled, and the clinical results of this trial are presented in another abstract. Chromosomal data focused on t(4;14) and del(17p) were available for 488 of those patients, as analyzed at diagnosis by FISH on sorted plasma cells. The t(4;14) was observed in 13.3% of the patients and del(17p) (defined by presence in at least 60% of the plasma cells) was present in 6.6% of them, incidences that are in agreement with previous reports. The median PFS (calculated from the date of randomization) for patients with t(4;14) were 15 months and 27 months for patients in the placebo and lenalidomide arms, respectively. The median for patients with del(17p) were 14 months and 29 months for patients in the placebo and lenalidomide arms, respectively. The comparison of PFS in the lenalidomide arm for patients with t(4;14) or del(17p), as compared with those lacking the chromosomal abnormalities shows a significant difference, in favor of the “no abnormality” group. In conclusion, lenalidomide maintenance very significantly improves the PFS of patients with high-risk cytogenetics, albeit these chromosomal changes retain their prognostic value.

Disclosures:

Facon: celgene: Consultancy, Research Funding; johnson and johnson: Consultancy. Attal:celgene: Consultancy, Research Funding; johnson and johnson: Consultancy, Research Funding.