Decreased Expression of CD62L and CD54 Correlates with Poor Cytogenetic Risk Group In Myelodysplastic Syndromes

Abstract Abstract 2915 Interactions in the bone marrow (BM) between haematopoietic progenitor cells (HPC) and the BM micro environment are important for the regulation of cell adhesion, proliferation, differentiation and survival. Expression of both CD62L (L-selectin) and CD54 (ICAM-1) on HPC demonstrated to play a role in signal transduction routes for proliferation and growth regulation. Especially CD54 is involved in uncontrolled proliferation and block of apoptosis. Previously, it was described that decreased expression of CD62L in acute myeloid leukemia (AML) was associated with a poor cytogenetic risk profile and an adverse clinical outcome (Graf M et al, Eur J Haematol 2003) Myelodysplastic syndromes are a group of clonal HPC disorders characterized by ineffective hematopoiesis and a propensity to evolve into AML. The International Prognostic Scoring System (IPSS) provides information on both survival and risk of development of an AML. The purpose of our study was to evaluate CD62L and CD54 expression on CD34+ cells in MDS patients by flow cytometry and to assess the value of a CD62L/CD54 ratio for prognostication. Bone marrow samples of 30 newly diagnosed MDS patients (3 RA(RS)/18 RCMD(RS), the <5% blasts group; 5 RAEB-1, 4 RAEB-2, the >5% blasts group), 16 AML patients with prior MDS and 26 healthy volunteers were analyzed for CD62L and CD54 expression on CD34+ cells by using flow cytometry. An adhesion index was calculated as a ratio of the percentage and MFI of CD62L and CD54 positive cells (as was reported by Buccisano et al, Eur J Haematol 2007). The CD62L/CD54 ratio was significantly decreased in MDS with <5% blasts (median 79.09 p<0.0001) as compared to healthy volunteers (median 480.4) and even more decreased in high risk MDS (median 14.67 p<0.0001 and p=0.001 as compared to healthy volunteers and MDS with <5% blasts, respectively) and AML with prior MDS (median 12.54, p<0.0001 and p=0.009 as compared to healthy volunteers and MDS with <5% blasts, respectively). The MDS patients were assigned to the good, intermediate or poor IPSS cytogenetic risk category. Cytogenetics was available for 22 MDS patients. The CD62L/CD54 ratio was significantly lower in the cytogenetic poor risk category compared with the good risk category (median 5.4 and median 70.79 respectively, p=0.018). Moreover, a low CD62L/CD54 ratio correlated significantly with poor cytogenetics, p=0.006. In the group of MDS patients with <5% blasts, 4 developed a refractory anemia with excess of blasts or AML within a follow up period of 12 months. There was a trend for a lower CD62L/CD54 ratio for MDS patients who developed an AML compared with patients who did not. In conclusion, the CD62L/CD54 ratio is significantly decreased in MDS compared with healthy volunteers and even more decreased in AML with prior MDS. Both CD62L and CD54 are involved in regulation of proliferation and apoptosis of the HPC. A decreased adhesion ratio in low risk MDS patients might reflect HPC damage at an early stage of the disease with an increased proliferative capacity and a decreased apoptotic profile. Interestingly, a low CD62L/CD54 ratio showed a significant inverse correlation with the IPSS cytogenetic risk category. Due to an absence of metaphases in a proportion of MDS patients, cytogenetics is not always available. The CD62L/CD54 ratio might serve as a surrogate marker for poor prognosis cytogenetics in case no karyotype is available. Low risk MDS patients who developed an AML within 12 months tended to have a lower CD62L/CD54 ratio. Although these results are promising, sample size and follow up period needs to be extended. The CD62L/CD54 ratio might add to prognostication of MDS patients and might identify MDS patients with <5% blasts who are at risk for development of an AML. Disclosures: Ossenkoppele: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.2424.2424 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2424-2424

Author(s):

Yang Liu ◽

Yong Zhang ◽

Phong Quang ◽

Hai T Ngo ◽

Feda Azab ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Flow Cytometry ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Cpg Island ◽

Advisory Committees ◽

Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Observational Prospective Registry for the Assessment of the Clinical Impact of Starting Anti-Myeloma Treatment at Biological Relapse

Blood ◽

10.1182/blood.v124.21.4765.4765 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4765-4765

Author(s):

Adrian Alegre ◽

Merche Gironella ◽

Juan Miguel Bergua ◽

Esther Gonzalez ◽

Fernando Escalante ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Median Time ◽

Research Funding ◽

Marrow Transplant ◽

Clinical Relapse ◽

First Line ◽

Advisory Committees ◽

Free Survival

Abstract Introduction: Despite the great medical advances associated with the introduction of thalidomide, bortezomib (BORT), and lenalidomide (LEN) for the treatment of multiple myeloma (MM), it remains an incurable disease. Most patients (pts) show disease progression, consistent with the clinical evolution of MM, and only a low percentage achieve long-term responses and extended progression-free survival (PFS). The heterogeneous nature of MM in both the clinical and biological setting is reflected in the heterogeneity of MM relapses. The International Myeloma Workshop Consensus Panel (Rajkumar, Blood 2011) states that treatment (Tx) shall begin either at clinical relapse with symptoms (clinR), or in the event of asymptomatic relapse with significant paraprotein relapse, biological relapse (BR). The purpose of this Spanish registry is to describe MM relapse patterns comparing the impact of Tx decisions in pts who meet the criteria for biological relapse (BR) according to IMWG criteria with those in whom Tx was delayed until clinical relapse (clinR). Here, the preliminary results of this study are presented. Methods: MM pts in (or previous to) first or second BR who have achieved ≥ PR since their last Tx are eligible for inclusion in this observational prospective registry at the time BR is detected. Evaluations performed at least bi-monthly are mandatory. A total of 41 Spanish sites participated in the registry following approval from their independent ethics committees, with 410 pts expected to be included, without physician’s decision of prescribing Tx affecting the inclusion. The main objective of the registry is to assess the time to progression (TTP) from the start of anti-MM Tx at the onset of asymptomatic BR vs. the start of Tx at the time of clinR. Secondary objectives are to describe demographics of BR; to assess the median time elapsing from BR to clinR; to assess overall response rate (ORR), event-free survival (EFS), PFS, overall survival (OS) at BR and at clinR (if appropriate); to asses safety and quality of life (QoL) using 2 validated questionnaires (European Organisation for Research and Treatment of Cancer [EORTC] QLQ-C30 and QLQ-MY24); to document the tolerability profile of the Tx; and to describe the use of associated resources. Here, we summarize baseline characteristics and preliminary results from 83 pts (out of 126 registered pts) who had basal data in the registry at the time of this report. Results: Overall, 79% of pts presented with a BR and 21% were in a bi-monthly watchful waiting follow up. The mean age of pts was 67 years, 53% were female, 57% were in first relapse, 43% and 27% had an ECOG performance status (PS) of 0 and 1, respectively, while the ECOG PS was unknown in 30% of pts at the time of this report. In total, 30% of pts had ISS stage I, 26% had ISS stage II, and 22% had ISS stage III, while ISS stage data were not available or unknown for 12% and 10% of pts, respectively. MM types were IgG Κ (37% of pts), IgG λ (23%), IgA Κ (13%), IgA λ (9%), and type was unknown in 17% of pts. 28% of IgG/IgA MM types were Bence-Jones. Cytogenetic risk assessments were available in 66% of pts. Among those pts with a BR, 51% received active Tx without waiting for a ClinR. First-line Tx was BORT-based in 70% of pts. Overall, 55% of pts had undergone autologous stem cell transplantation, 15% had received consolidation Tx and 34% had received maintenance Tx. After first-line Tx, 17% of pts achieved a stringent complete response (sCR), 31% achieved a CR, 24% achieved a very good partial response (VGPR), and 10% achieved a PR. The median time to BR was 24.53 months. Most (63%) pts who registered after second relapse received LEN-based Tx. Conclusions: To our knowledge, this is the first prospective study in MM to evaluate BR as well as the effects of Tx based on the decision to start Tx at BR vs. clinR. In this preliminary cohort, the physicians’ decision to start active Tx at BR, before the onset of clinR in 50% of cases, was noteworthy. Further follow-up is needed to identify the differences between these two strategies. Updated clinical results will be presented at the meeting. MM-BR Study, Spanish Myeloma Group-GEM/PETHEMA Bibliography Alegre A, et al. Haematologica. 2002;87:609-14. Brioli A, et al. Blood. 2014;123:3414-9. Fernández de Larrea C, et al. Bone Marrow Transplant. 2014;49:223-7. Lenhoff S, et al. Haematologica. 2006;91:1228-33. Rajkumar SV, et al. Blood. 2011;117:4691-5. Zamarin D, et al. Bone Marrow Transplant. 2013;48:419-24. Disclosures Alegre: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lahuerta:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ruiz:Celgene: Celgene Stock options as part of the employee's compensation plan Other, Employment. Vilanova:Celgene: Contracted by Celgene Other.

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Factors Influencing Long-Term Hematopoietic Function Following Autologous Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.2186.2186 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2186-2186

Author(s):

Alissa Visram ◽

Natasha Kekre ◽

Christopher N. Bredeson ◽

Jason Tay ◽

Lothar B. Huebsch ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Infection Rates ◽

Advisory Committees ◽

Cd34 Cells ◽

Significant Difference

Abstract Background/Objective: Mobilized peripheral blood hematopoietic progenitor cells are the most common stem cell source for autologous hematopoietic stem cell transplantation (auto-HSCT). Successful short-term stem cell engraftment requires collection of at least 2x106 CD34+ cells/kg. The American Society of Bone Marrow Transplantation (ASBMT) recommends a stem cell infusion target of 3-5 x106 cells/kg (Giralt et al. 2014). However, the number of CD34+ cells to reinfuse to ensure long-term engraftment has not been established. Plerixafor, a reversible CXCR4 antagonist, increases CD34+ cell yield at collection even in patients who are predicted poor mobilizers (PPM). Although plerixafor could be used universally for all collections, this may not be the most cost-effective strategy (Veltri et al. 2012). This study sought to determine the minimum number of CD34+ cells/kg required for adequate long-term hematopoiesis, identify factors associated with poor long-term hematopoiesis, and determine if plerixafor mobilization improved long-term peripheral blood counts. Methods: A retrospective chart review was conducted on patients who underwent auto-HSCT between January 2004 and September 2013 at The Ottawa Hospital, for management of hematological malignancies. Peripheral blood cell counts were collected from 1 to 5 years after auto-HSCT, or until disease relapse. Poor long-term hematopoiesis was defined as an ANC <1 x109/L, hemoglobin <100 g/L, or platelets <100 x109/L. Patients were stratified into groups based on the infused CD34+ concentration (in cells/kg), and the proportion of patients with poor long-term hematopoiesis at 1, 2, 3, 4, and 5 years post auto-HSCT was compared with chi square tests. Long-term clinical outcomes (platelet and packed red blood cell transfusions, and post auto-HSCT infection rates) were compared between plerixafor-mobilized patients and PPM (defined as patients with pre-collection CD34+ <2 x 106 cells/kg) with standard mobilization regimens. Results: This study included 560 patients who underwent auto-HSCT, 210 with multiple myeloma and 350 with lymphoma. At 1 and 5 years post auto-HSCT 377 and 104 patients were included, respectively. A dose dependent improvement 1 year after auto-HSCT was seen in patients who received 0-2.99 x 106 CD34+ cells/kg (24.4%, n= 41) compared to patients who received 5-9.99 x 106 CD34+ cells/kg (11%, n=154, p=0.051) and ³10 x 106 CD34+ cells/kg (4.5%, n=66, p=0.006). Though there was a trend towards lower CD34+ infusions and poorer hematopoietic function (see table 1), there was no statistically significant difference in hematopoietic function based on CD34+ infusion concentrations after 1 year post auto-HSCT. 10 patients received <2 x106 CD34+ cells/kg, of whom the rate of inadequate hematopoiesis was 33% at 1 year (n=6) and 0% (n=1) at 5 years post auto-HSCT. Factors that increased the risk of poor hematopoiesis over the course of study follow up, based on a univariate analysis, included advanced age (OR 1.189, p=0.05), multiple prior collections (OR 2.978, p=0.035), and prior treatment with more than two chemotherapy lines (OR 2.571, p=0.02). Plerixafor-mobilized patients (n=25), compared to PPM (n=197), had a significantly higher median CD34+ cell collection (4.048 x109/L and 2.996 x109/L cells/kg, respectively, p=0.005). There was no significant difference in overall cytopenias, transfusion requirements, or infection rates between plerixafor-mobilized and PPM patients over the course of the study follow up. Conclusion: Low pre-collection CD34+ counts, advanced age, multiple prior collections, and more than two prior chemotherapy treatments adversely affected long-term hematopoiesis post auto-HSCT. We support the transfusion target of 3-5 x 106 cells/kg, as proposed by the ASBMT, given that at 5 years post auto-HSCT there was no statistical or clinically significant difference in hematopoietic function with higher CD34+ infusion targets. While mobilization with plerixafor significantly increased overall CD34+ cell collection when compared with PPM, long-term hematopoietic function and clinical outcomes were not different. This finding supports the practise of limiting plerixafor use only to patients who are PPM, thereby facilitating adequate stem cell collection and early engraftment, as opposed to universal plerixafor mobilization. Disclosures Sabloff: Lundbeck: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Canada: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Alexion: Honoraria.

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Interim Results from a Phase 1/2 Clinical Study of Lentiglobin Gene Therapy for Severe Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.1176.1176 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1176-1176 ◽

Cited By ~ 19

Author(s):

Julie Kanter ◽

Mark C. Walters ◽

Matthew M. Hsieh ◽

Lakshmanan Krishnamurti ◽

Janet Kwiatkowski ◽

...

Keyword(s):

Gene Therapy ◽

Board Of Directors ◽

Research Funding ◽

Phase 1 ◽

Globin Gene ◽

Advisory Committees ◽

Cell Dose ◽

Cd34 Cells ◽

Equity Ownership

Abstract β-globin gene transfer into hematopoietic stem cells (HSCs) has the potential to reduce or eliminate the symptoms and long-term complications of severe sickle cell disease (SCD). LentiGlobin Drug Product (DP) is a gene therapy product containing autologous CD34+ cells transduced with the BB305 lentiviral vector. BB305 encodes a human β-globin gene containing a single point mutation (AT87Q) designed to confer anti-sickling properties similar to those observed in fetal hemoglobin (γ-globin). In two ongoing studies, subjects with transfusion-dependent β-thalassemia (Studies HGB-204 and HGB-205) or SCD (Study HGB-205) receiving LentiGlobin DP have demonstrated sustained expression of 3-9 g/dL therapeutic hemoglobin (HbAT87Q) and have shown marked improvements in clinical symptoms 1 year post-treatment. Study HGB-206 is a multi-center, Phase 1/2 safety and efficacy study of LentiGlobin DP in adults with severe SCD. We previously (ASH 2015) presented results from 2 subjects, who had 3 and 6 months of follow-up after LentiGlobin treatment. We now present data from 7 treated subjects, 4 of whom have ≥6 months of follow-up data. Subjects (≥18 years of age) with severe SCD (history of recurrent vaso-occlusive crisis [VOC], acute chest syndrome, stroke, or tricuspid regurgitant jet velocity of >2.5 m/s) were screened for eligibility. Following bone marrow harvest (BMH), CD34+ cells were transduced with the BB305 vector. Subjects underwent myeloablative conditioning with busulfan prior to infusion of the transduced cells. Safety assessments include adverse events (AEs), integration site analysis (ISA) and surveillance for replication competent lentivirus (RCL). After infusion, subjects are monitored for hematologic engraftment, vector copy number (VCN), HbAT87Q expression, and other laboratory and clinical parameters. As of July 2016, 7 subjects with severe SCD (median age: 26 years, range 18-42 years) have received LentiGlobin DP in this study. All subjects successfully underwent BMH, with a median of 2 harvests required (range 1-4). Fifteen Grade 3 AEs in 5 subjects were attributed to BMH: pain (n=10), anemia (n=3) and VOC (n=2); all resolved with standard measures. Table 1 summarizes cell harvest, DP characteristics, and lab results. The median LentiGlobin DP cell dose was 2.1x10e6 CD34+ cells/kg (range 1.6-5.1) and DP VCN was 0.6 (0.3-1.3) copies/diploid genome. Median post-infusion follow-up as of July 2016 is 7.1 months (3.7-12.7 months). All subjects successfully engrafted after receiving LentiGlobin DP, with a median time to neutrophil engraftment of 22 days (17-29 days). The toxicity profile observed from start of conditioning to latest follow-up was consistent with myeloablative conditioning with single-agent busulfan. To date, there have been no DP-related ≥Grade 3 AEs or serious AEs, and no evidence of clonal dominance or RCL. The BB305 vector remains detectable at low levels in the peripheral blood of all subjects infused, with median VCN 0.08 (0.05-0.13, n=7) at last measurement. All subjects express HbAT87Q, with a median of 0.4g/dL (0.1-1.0 g/dL, n=7) at 3 months; most subjects demonstrated modest increases over time, and the 2 subjects with the longest follow-up expressed 0.31 and 1.2 g/dL HbAT87Q at 9 months. All 4 subjects with ≥6 months of follow-up experienced multiple VOCs in the 2 years prior to study entry (2-27.5 VOCs annually). Since LentiGlobin DP infusion, 3 of these 4 subjects have had fewer VOCs, although this trend may be confounded by the short follow-up, the effects of transplant conditioning, and/or post-transplant RBC transfusions. The decrease in VCN between DP and peripheral cells contrasts with previous reports of successful LentiGlobin gene therapy in ongoing studies HGB-204 and HGB-205. The relatively low in vivo VCN in this study appears to result in the lower HbAT87Q expression seen to date. We are exploring multiple hypotheses as to the etiology of the VCN drop between DP and peripheral blood, including the adverse impact of sickle marrow pathology on HSCs, the adequacy of myeloablation, and the magnitude of the transduced cell dose. We will provide an update on study data and ongoing efforts to increase in vivo VCN in patients with SCD, such as increasing the transduced cell dose through alternate HSC procurement methods or enhancing the DP VCN through manufacturing improvements. Disclosures Kanter: Novartis: Consultancy. Walters:Bayer HealthCare: Honoraria; AllCells, Inc./LeukoLab: Other: Medical Director ; ViaCord Processing Laboratory: Other: Medical Director ; Leerink Partners, LLC: Consultancy; Kiadis Pharma: Honoraria; bluebirdBio, Inc: Honoraria. Kwiatkowski:Ionis pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Consultancy; Sideris Pharmaceuticals: Consultancy; Apopharma: Research Funding; Luitpold Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. von Kalle:bluebird bio: Consultancy; GeneWerk: Equity Ownership. Kuypers:Children's Hospital Oakland Research Institute: Employment; bluebird bio: Consultancy. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Joseney-Antoine:bluebird bio: Employment, Equity Ownership. Asmal:bluebird bio: Employment, Equity Ownership. Thompson:bluebird bio: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Amgen: Research Funding; Baxalta (now part of Shire): Research Funding; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mast: Research Funding; Eli Lily: Research Funding.

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Safety and Efficacy of Romiplostim in over 200 Children with Immune Thrombocytopenia (ITP): Results of an Integrated Database of 5 Clinical Trials

Blood ◽

10.1182/blood-2018-99-109763 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2428-2428 ◽

Cited By ~ 1

Author(s):

Michael D. Tarantino ◽

Jenny M. Despotovic ◽

John Roy ◽

John Grainger ◽

Nichola Cooper ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Platelet Response ◽

Open Label ◽

Advisory Committees ◽

Baseline Platelet Count ◽

Platelet Counts

Abstract Background: Romiplostim is approved globally for use in adults with ITP and in the EU for children with ITP. More comprehensive data are needed on the use of romiplostim in children with ITP. Objective: To examine the safety and efficacy of romiplostim in trials in children with ITP. Methods: Data were combined from 5 romiplostim trials in children with ITP, both placebo-controlled (a phase 1/2 and a phase 3 trial) and open-label (a 3-year trial and 2 extension trials); trial data have been reported previously (Bussel Blood 2011, Bussel PBC 2014, Tarantino Lancet 2016, Tarantino ASH 2017, Grainger ASH 2017). Platelet counts in the 4 weeks after use of rescue medication were excluded from analyses. Descriptive statistics were used. Number (n), mean, standard deviation (SD), median, quartile range (Q1, Q3), minimum (min), and maximum (max) for continuous variables, and number and percentage for categorical variables were provided. Results: Patients (N=286, 24 initially placebo and 262 initially romiplostim) had median (Q1, Q3) age of 10 (6, 13) years, ITP duration of 1.9 (1, 4) years, and baseline platelet count of 14 (8, 23)×109/L. Previously, 88% had received corticosteroids, 87% IVIg, and 21% rituximab; 23% had received >3 prior treatments and 7% had prior splenectomy. Of the 282 patients exposed to romiplostim (20 initially received placebo), the median (min, max) duration of treatment was 65 (8, 471) weeks, with a median (min, max) average weekly dose of 6.6 (0.1, 9.7) μg/kg; total exposure was 468 patient-years. The most common reasons for discontinuing the parent study for romiplostim-treated patients were per protocol (19%; eg, sponsor decision, death, lost to follow-up), consent withdrawn (3%), noncompliance (1%), and administrative decision (1%). Of romiplostim-treated patients, 24% had serious adverse events (SAEs), most commonly epistaxis, low platelet counts, and headache (Table). There were 7 cases of postbaseline neutralizing antibody against romiplostim: 2 transient and 5 persistent. There were no neutralizing antibodies against endogenous TPO. For patients undergoing bone marrow biopsies in the 3-year open-label trial, there were no findings of collagen or bone marrow abnormalities (Year 1 n=27, Year 2 n=5, vs. baseline) (Grainger et al, ASH 2017). One patient had an increase in modified Bauermeister bone marrow grade from 0 to 2 (fine reticulin fiber network) with no associated AEs (the only AEs were a cold and injection site pain); per protocol, there was no follow-up biopsy. Once at a steady dose of 10 μg/kg, most (11/16) of this patient's platelet counts were ≥30×109/L. Investigators reported thrombocytosis AEs; 1 patient had a platelet count of 1462×109/L at Week 14 for 1 week and another had elevated platelet counts 10 times between Weeks 20-172 (max of 872×109/L); there were no associated thrombotic events. Median platelet counts rose quickly and were over 50×109/L from Week 12 on (Figure). Platelet response rates also rose quickly. Overall, 89% of romiplostim-treated patients (vs 8% of placebo) had a platelet response (platelet counts ≥50×109/L; Figure). For romiplostim-treated patients, the first platelet responses occurred after a median of 6 weeks. The median % (Q1, Q3) of months responding was 76% (25%, 93%) and # of months responding was 11 (3, 20); from time of first monthly response, the median (Q1, Q3) % of months responding was 92% (75%, 100%) and # of months responding was 14 (7, 23). Nineteen romiplostim-treated patients discontinued all ITP therapies including romiplostim for ≥6 months while maintaining platelet counts ≥50×109/L (here defined as remission). These treatment-free periods lasted a median (Q1, Q3) of 12 (8, 14) months; no placebo patients remained free of treatment. There were no clear differences between those who did and did not enter remission (ie, age, sex, race, past treatment, ITP duration, baseline platelet count). Bleeding was reported for most (68%) patients: mostly grade 1/2, with 10% having grade 3 bleeding (most commonly epistaxis in 13 patients) and 2 patients having grade 4 bleeding (both reported as "ITP"). Conclusions: In this comprehensive database of romiplostim ITP trials in 286 children with 468 patient-years of romiplostim exposure, romiplostim was well tolerated. With romiplostim, the vast majority (89%) of patients had a platelet response, with some children able to discontinue all ITP treatments for ≥6 months. Disclosures Tarantino: Health Resources and Services Administration: Research Funding; Centers for Disease Control and Prevention: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Other: Reviews grants; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Grifols: Research Funding, Speakers Bureau. Despotovic:AmGen: Research Funding; Sanofi: Consultancy; Novartis: Research Funding. Grainger:Biotest: Consultancy; Ono Pharmaceuticals: Consultancy; Amgen: Consultancy, Honoraria, Other: Educational grant; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cooper:Amgen, Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kim:Amgen Inc.: Employment, Equity Ownership. Eisen:Amgen Inc.: Employment, Equity Ownership.

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Dissecting Signaling Network Responses to PAK4 Allosteric Modulators (PAMs) in Cell Subsets within Primary Human Acute Myeloid Leukemia Samples

Blood ◽

10.1182/blood.v126.23.3686.3686 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3686-3686

Author(s):

Paul Brent Ferrell ◽

William Senapedis ◽

Alexander Cook ◽

Erkan Baloglu ◽

Yosef Landesman ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemia Stem Cells ◽

Allosteric Modulators ◽

Advisory Committees ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28). Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21). Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK. Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. Disclosures Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Blood ◽

10.1182/blood-2021-154332 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3597-3597

Author(s):

Denis Tvorogov ◽

Chloe AL Thompson-Peach ◽

Johannes Foßelteder ◽

Mara Dottore ◽

Frank Stomski ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Liquid Culture ◽

Jak Inhibitor ◽

Advisory Committees ◽

Nsg Mice ◽

Cd34 Cells ◽

Calr Mutations ◽

Bone Marrow Engraftment

Abstract Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells. Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation. Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG control Conclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. Disclosures Ross: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

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IgM-Gammopathies Transformed into Aggressive Lymphoma: Incidence, Basal Clinical Features and Outcome of a Registry Based, Spanish Retrospective Series

Blood ◽

10.1182/blood-2019-123353 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4011-4011

Author(s):

Irene Dogliotti ◽

Cristina Jiménez ◽

Federica Cavallo ◽

Noemi Puig ◽

Gian Maria Zaccaria ◽

...

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

B Lymphocytes ◽

Clinical Features ◽

Research Funding ◽

Initial Diagnosis ◽

Aggressive Lymphoma ◽

Advisory Committees ◽

Histological Transformation

Background Transformation into aggressive lymphoma (AL) is a rare complication of indolent lymphoproliferative disorders (LPDs) and is characterized by poor outcome. Immunoglobulin M (IgM) gammopathies are a spectrum of conditions, from monoclonal gammopathy of undetermined significance (MGUS) to Asymptomatic Waldenstroem Macroglobulinemia (AWM) and Symptomatic WM (SWM) that can eventually evolve to transformed WM (tWM). Actually, tWM represents a clinical challenge, mainly because of its poor characterization. Aims This registry study aims to better characterize tWM, focusing on prognostic factors heralding transformation to AL. Methods Two registries of IgM-MGUS, AWM and SWM [Owen, Semin Oncol 2003] based in Salamanca and in the region of Castilla and Leon (Spain) were investigated to identify cases with histological transformation. IgM-secreting patients with other LPDs (e.g. chronic lymphocytic leukemia, marginal zone lymphoma, IgM-multiple myeloma) were excluded from the analysis. All patients provided written informed consent in accordance to Helsinki's declaration. Statistical analysis was carried out using R v 3.3.3. tool; survival analyses were performed with Log-Rank method, while group comparison was performed with t-student for continuous variables and Chi-square tests for categorical variables. Results Data from 903 patients with IgM-secreting disorders diagnosed between 1976 and 2019 were analyzed; 587 cases with confirmed diagnosis of IgM-MGUS, AWM or SWM were selected. Out of 587 IgM-gammopathies, 22 cases with histological transformation to AL were identified. Cumulative incidence of tWM was: 1.4% at 5, 3.4% at 10 and 5.3% at 12 years, respectively (figure 1). Clinical features at first diagnosis of patients subsequently developing tWM where then analyzed: 3/22 tWM evolved from previous IgM-MGUS, while the remaining patients originally presented with AWM (6/22) or SWM (13/22). IPSS-WM prognostic score was LR for 5, IR for 12 and HR for 3/20 patients, respectively [Morel, Blood 2009]. Glancing on distributions between groups according to the outcome, tWM differed from not transformed (NT) cases for: lower median age at diagnosis (66 vs 72 years, p=0.018), lower platelets levels (median 188 vs 235 x 10^9/mmc, p=0.017), higher LDH ratio (0.8 vs 0.67, p=0.015), higher incidence of chromosome 6q deletion by FISH (40 vs 14%, p=0.021) and higher clonal B lymphocytes infiltration on marrow aspirate by flow cytometry (15 vs 4.5%, p= 0.022). Moreover, 13/22 patients received anti-WM treatment within 3 months from initial diagnosis, mainly chlorambucil-based; 5/22 patients received rituximab in first line and 13 in second line. From the whole series, after a median follow-up of 80 months, median transformation-free survival was 61 months from initial diagnosis (range: 0-228). Among these, Only 1/22 of tWM patient is still alive; 19/21 deaths were thus related to AL/WM, with a median survival after transformation of 12 months (0-53). In the whole series (n=587), median OS from initial diagnosis of IgM gammopathy was 76 months for the tWM group (6-225), that is shorter than the NT group (128 months, p=0.012, figure 2). Focusing only on patients treated at initial diagnosis, median survival after first treatment (SAFTI) was 62 vs 90 months for tWM vs NT (p=0.011, figure 3), and median time to next treatment was 28 vs 46 months, respectively (p=0.13). Overall, 10/22 tWM patients received ≥3 treatment lines, and median number of lines prior to transformation was 2 (0-3). Finally, in the whole series IPSS-WM score at diagnosis confirmed to impact on survival (median OS=151, 119 and 56 months for LR, IR and HR groups, respectively, p <0.001). However, this was not the case for tWM cases only, where OS was no longer different between groups. Conclusions In this retrospective study, we confirmed dismal outcome for tWM patients; incidence of transformation was comparable to expectations at 5 years, but higher at subsequent follow-up. At initial diagnosis of IgM gammopathy, younger age, low platelets level, high LDH ratio, high B lymphocytes infiltration by flow cytometry and presence of 6q deletion were significantly enriched among patients subsequently developing tWM. IPSS-WM score looked less predictive among tWM patients probably given to the limited numbers of tWM series. Novel prognostic tools are eagerly awaited for tWM patients. Figure Disclosures Cavallo: Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Puig:The Binding Site: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria. Ferrero:Gilead: Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Speakers Bureau; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees. Boccadoro:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Sanofi: Honoraria, Research Funding.

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Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma

Blood ◽

10.1182/blood-2019-123838 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3060-3060

Author(s):

Marion Eveillard ◽

Even H Rustad ◽

Mikhail Roshal ◽

Yanming Zhang ◽

Amanda Kathryn Ciardiello ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

M Protein ◽

Advisory Committees ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Active Disease ◽

Tof Ms

Introduction In multiple myeloma (MM), the absence of measurable residual disease (MRD) after completed therapy is associated with longer progression free survival. Different techniques are available to detect low levels of plasma cells in bone marrow (BM) either by flow cytometry or by next-generation sequencing as a gold standard of molecular methods. But these techniques are limited because they require a representative bone marrow sample obtained by an invasive procedure. Therefore, detecting low levels of disease in blood would be ideal, because serial sampling is much easier and fully representative, and it would allow for the detection of extramedullary disease. Mass spectrometry-based methods have been shown to be more sensitive for detecting monoclonal proteins (M-protein) in serum. In this study, we were motivated to evaluate MALDI-TOF mass spectrometry (MALDI-TOF MS) head-to-head with an established BM-based MRD assays. Patients and Methods This cohort included 71 patients treated at Memorial Sloan Kettering Cancer Center (MSKCC) who had serum samples available at 2 timepoints including during active disease and within 60 days of MRD results as determined by flow cytometry of bone marrow aspirates (Flow-BM-MRD). The cohort enrolled 26 females and 45 males with a median age of 61 years (range 37-78 years). Twenty-seven patients had high-risk cytogenetics at baseline. The median time between diagnosis and the MRD timepoint was 13.4 months (3.4-91 months). MALDI-TOF MS analysis was performed according to the method published by Mills et al. Immunoglobulins were purified from serum samples using CaptureSelect beads specific of each isotype and were then eluted from the beads. Light chains and heavy chains were separated by the addition of a reducing agent. Purified samples were mixed in matrix and spotted onto a stainless steel MALDI plate and were analyzed using a Microflex LT MALDI-TOF mass spectrometer (Bruker). Samples taken during active disease were used to identify the mass to charge ratio (m/z) of the M-protein and served as a surrogate marker in the analysis of subsequent samples. MALDI-TOF MS results were compared to the Flow-BM-MRD assay, performed using the MSKCC's ten-color, single-tube method. Results MALDI-TOF MS detected an M-protein in all 71 active disease samples and in 25 MRD samples. MALDI-TOF-MS results at the MRD timepoint were concordant with Flow-BM-MRD for 44/71 patients (p=0.342, chi-square test). Eight patients were positive and 36 negative by both techniques. Twenty-seven patients were discordant, including 10 patients detectable only by Flow-BM-MRD and 17 detectable only by MALDI-TOF MS. Among the 10 patients detectable by flow cytometry but not by MALDI, the median MRD level was 0.00092% (+<0.0001% - 0.011%). The M-protein could have been present but below the polyclonal background. Regarding the 17 patients positive only by MALDI-TOF-MS, the BM sample for flow analysis was not suitable for 3 patients due to hemodilution. The others 14 samples reached the target of sensitivity with a limit of detection of 0.0001%. Alternatively, the MALDI-TOF result could be a false positive in terms of disease detection. MS is likely not falsely detecting M-proteins and indeed, immunofixation was also positive in 11/17 of these samples. However, low levels of M-protein may not indicate the presence of active disease. Indeed, a confounding factor is that immunoglobulins have a long half-life in serum. To determine the clinical utility of more sensitive M-protein detection, we evaluated the clinical outcome for the 48 newly diagnosed MM patients in CR at the MRD timepoint with a median follow-up of 11 months. Of these 48 patients, 2 of the 3 that were positive by both techniques relapsed during follow-up. One out of 27 patients that were negative by both techniques relapsed. None of the 10 patients who were positive only by MALDI-TOF relapsed and 1 of the 8 patients who were positive only by Flow-BM-MRD relapsed. Conclusions This study is important because it is a first step in understanding how to use a more sensitive blood test for the follow-up of MM patients. MALDI-TOF MS analysis may provide complementary results to Flow-BM-MRD especially for the follow-up of patients in CR and during maintenance therapy to detect poor responders that would be positive by both techniques. In summary, our results suggest that MALDI-TOF may be quite useful for early detection of relapse. Disclosures Roshal: Physicians' Education Resource: Other: Provision of services; Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services. Hassoun:Celgene: Research Funding; Janssen: Research Funding; Novartis: Consultancy. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy. Lesokhin:Takeda: Consultancy, Honoraria; Serametrix Inc.: Patents & Royalties; Genentech: Research Funding; GenMab: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Juno: Consultancy, Honoraria. Mailankody:Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Takeda Oncology: Research Funding; CME activity by Physician Education Resource: Honoraria. Landgren:Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Other: IDMC; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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A Retrospective Multicenter Case Study Evaluating the Characteristics, Management and Outcome of Acquired Amegakaryocytic Thrombocytopenia

Blood ◽

10.1182/blood-2021-153388 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3167-3167

Author(s):

Anais Roeser ◽

Guillaume Moulis ◽

Mikael Ebbo ◽

Louis Terriou ◽

Elsa Poullot ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Aplastic Anemia ◽

Board Of Directors ◽

Bone Marrow Biopsy ◽

Research Funding ◽

Advisory Committees ◽

Overall Response ◽

Acquired Amegakaryocytic Thrombocytopenia

Abstract Introduction Acquired amegakaryocytic thrombocytopenia (AAT) is an extremely rare disease characterized by acquired megakaryocytic aplasia or hypoplasia with no other lineage abnormalities. Given limited evidence, the first aim of this study was to describe the characteristics, management and outcome of patients with AAT, the second aim was to examine the therapeutic response through a systematic review of published case reports. Patients and Methods We carried out a retrospective multicenter study through the French Reference Network for Adult Autoimmune Cytopenias, including patients aged > 18 years with acquired thrombocytopenia with a platelet count < 50 x 10 9/L, associated with a megakaryocytes / granulocytes ratio < 50 % on bone marrow, diagnosed from July 2007 to February 2020. Exclusion criteria were: abnormal granular lineage, evidence of dysplasia, bone marrow infiltration by tumor cells or hematologic malignancy, significant karyotype abnormality, and significant paroxysmal nocturnal hemoglobinuria clone. Bone marrow biopsy were centrally reviewed. Patients' medical charts were collected using the standardized form of the referral center for adult immune thrombocytopenia (ITP). Response to treatment was defined according to standardized international criteria for ITP: response (R) and complete response (CR) were respectively defined as platelet count of > 30 × 10 9/L with at least a doubling of the baseline value, and platelet count of > 100 × 10 9/L ; overall response as either R or CR. We performed a systematic review conducted through Medline and Scopus databases from 1970 to April 2021. Cases were included in the analysis if initial platelet count was < 50 x 10 9/L and bone marrow examination was available, demonstrating a megakaryocyte hypoplasia or aplasia with no alternate diagnosis. Results We screened 23 patients reported as thrombocytopenia with absence or decreased megakaryocytes. Eleven patients were excluded because of: presence of megakaryocytes on bone marrow biopsy despite megakaryocytic aplasia on bone marrow aspirate (n=2), absence of bone marrow biopsy (n=4), aplastic or hypoplastic bone marrow (n=3), moderate thrombocytopenia > 50 x 10 9/L (n=1), lack of data (n=1). Twelve patients were included in the analysis. AAT patients had a median age of 52.5 years, 5/12 (41.7%) were female, 6/12 (50%) had a preexisting autoimmune disease (Table 1). All bone marrow biopsies reviewed to date contained CD8+ T-cell infiltrates. Eight patients received a first line treatment with corticosteroids and/or intravenous immunoglobulins (IVIg), a single response was observed. Ten patients received cyclosporine in monotherapy resulting in 4CR, and 1R or in combination with diverse agents with heterogenous responses. Six had received a single therapy with thrombopoietin receptor agonists (TPO-RAs) inducing 4 CR. Eventually, 9 patients (75%) achieved a CR under therapy, obtained with ciclosporin alone in 3 cases, ciclosporin in association with TPO-RA or ATG in 2 cases, cyclophosphamide followed-up by mycophenolate mofetil in 1 case, and TPO-RAs alone in 4 patients (of whom 3 had previously received at least on immunosuppressive therapy). After a median follow up time of 4.0 years (range 1.2 - 11.9), 2 (16%) patients eventually developed an aplastic anemia, 7 and 41.5 months respectively after initial AAT diagnosis. The literature search yielded 108 articles, of which 75 articles reporting 85 cases were included in the final analysis. The pooled analysis of newly reported and historic cases included 97 cases. Overall response rates to corticosteroids and IVIg were respectively 22.4 % and 5.3 % (Table 2). Ciclosporin was used as single agent in 37.1 % of patients, with an overall response rate of 66.7 %. TPO-RAs were used in 9 cases, with a CR in 7 patients (77.8%). Overall, 9/97 patients (9.3 %) experienced an aplastic anemia during the follow-up. The presence of a thymoma was associated with a higher risk of aplastic anemia (OR 6.83 (95%CI 1.22-34.00, p=0.020)). Conclusion Distinguishing AAT from ITP is of significance as the outcome and response to therapy strongly differ. Aplastic anemia may occur in the follow-up but remain rare. Corticosteroids and IVIg are inefficient in most cases, ciclosporin appear to be very effective, TPO-RA could also be an option, as single therapy or in associations. Further data will be needed to define the respective place of these treatments. Figure 1 Figure 1. Disclosures Moulis: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenix: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees. Ebbo: Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Other: Attendance Grant; Amgen: Honoraria; Sobi: Other: Attendance Grant; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Terriou: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Haioun: Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding; Servier: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Miltenyi: Honoraria, Research Funding. Michel: Amgen,Novartis,UCB,Argenx,Rigel: Honoraria. Godeau: Amgen: Consultancy; Novartis: Consultancy; Grifols: Consultancy; Sobi: Consultancy. Mahevas: GSK: Research Funding; Amgen: Honoraria.

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