Bone Marrow Lymphoid Infiltrates and Aplasia after CD19-Directed CAR T Cell Therapy in Pediatric B-Lymphoblastic Leukemia

Introduction Chimeric Antigen Receptor (CAR) T cell therapy is used to treated relapsed/refractory B-Acute Lymphoblastic Leukemia (B-ALL) patients. Long term side effects include B cell aplasia and cytopenia that are treated with supportive therapy. Prolonged cytopenia is seen in 16% of CAR T-cell treated B-ALL patients. The etiology of cytopenia is not clear, and could be attributed to the impact of chemotherapy, CAR T cells and disease or patient-specific factors. The impact of CAR T cell infusion on bone marrow cellularity, lymphocyte compartment and its correlation with cytopenia has not been investigated. Methods We analyzed pre- and post-CAR bone marrow biopsies in 178 B-ALL patients who received a CD19-directed CAR T-cell product between 2012-2017 and followed for at least 12 months. Responses were categorized into sustained responders, non-responders, CD19-positive relapses and CD19-negative relapses as previously described. Bone Marrow biopsy (BMB) overall cellularity, complete blood counts (CBC) in a 12-month post infusion period were analyzed. BMB were considered mildly, moderately and severely hypocellular based on cellularity of 50, 25 and 5% respectively. Hypocellularity was further stratified by CBC per aplastic anemia (AA) classification guidelines: Non-Severe Aplastic Anemia (NSAA) and Very Severe Aplastic Anemia (VSAA). Immunohistochemistry (IHC) for CD3, CD4, CD8, CD163, granzyme and perforin were performed on pre- and post-CAR BMB. CD3 IHC was digitally scanned and analyzed quantitatively (Leica Aperio ImageScope) and qualitatively. Targeted RNA sequencing-based gene expression profiling (EdgeSeq Immuno-Oncology Panel, HTG Diagnostics) was performed on pre-and post-treatment biopsies, with differential expression assessed by DESeq2 within HTG Reveal software. RNAseq gene expression was deconvoluted to impute relative expression of immune cell subsets. Results 31% of patients were mildly hypocellular but none were severely hypocellular at baseline pre-CAR timepoints. The highest proportion of hypocellularity was at the 1-month time point. 81% of patients were mildly hypocellular (≤50%), 42% were markedly hypocellular (≤25%), and 13% were severely hypocellular (≤5%) at the 1-month time point. By month 12, the proportion of mildly hypocellular patients was 74%, markedly hypocellular patients was 26%, and severely hypocellular patients was 7% (Figure 1). The proportion of NSAA and VSAA was 57% and 10% in moderately hypocellular BMB. The proportion of NSAA and VSAA was 64% and 18% in severely hypocellular BMB. Severely hypocellular patients had a higher average day minus 1 disease burden (33% involvement) compared to their moderate (24%) and non-hypocellular patients (16%). VSAA patients had a lower baseline BMB cellularity (47%) compared to NSAA patients (71%) and those without AA (60%) Increased CD3+ T cells were noted in the post-CAR BMB compared to the pre-CAR BMB (Figure 1B). Lymphocyte were singly scattered or formed loose aggregates and tight lymphohistiocytic clusters. IHC and RNAseq analysis showed increased CD8+ granzyme+ cells in the post-CAR BMB compared to pre-CAR BMB. Sustained responders showed higher T cell infiltration compared to other categories of patients (Figure 1C and D). Lymphoid infiltrates did not correlate with hypocellularity or cytopenia. Conclusion We describe for the first time the changes in bone marrow cellularity and lymphocyte compartment after CD19-directed CAR T-cell infusion in B-ALL. A subset of patients were moderately or severely hypocellular and met criteria for aplastic anemia. However, most of them recovered bone marrow cellularity and CBC. Hypocellularity and AA were correlated with pre-CAR disease burden and baseline cellularity rather than post-CAR lymphocyte infiltration. We also show for the first time that sustained responders to CD19-CAR showed increased CD3+ CD8+ T cell infiltrates compared to CD19-positive relapses and non-responders. Figure 1. A. Bone marrow hypocellularity post-CAR T-cell infusion. Proportion of patients who were mildly hypocellular, markedly hypocellular, and severely hypocellular in the 12month follow up period. B. Increased CD3+ T cells and aggregates after CAR T cell infusion. C and D. Sustained responders show significantly greater CD3+ T cells after CAR T cell infusion compared to other categories. *P<0.05. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Immunomodulatory Activity of Punarnavine Alkaloid from Boerhaavia diffusa

Background: Immunomodulators could alter the activity of the immune system. Most of the synthetic chemotherapeutic agents are immune suppressants and produce side effects. Immunomodulators derived from plants are used as general adaptogens and immune strengtheners without undesired effects. Methods: Present study established the mode of immunostimulatory action of punarnavine alkaloid (PA) isolated from Boerhaavia diffusa Linn. root by examining various parameters. PA was separated from B. diffusa and its acute toxicity was studied by up-down regulation method. Immunomodulatory activity of PA was determined in Albino mice by observing its effect on organ weight (liver, spleen, thymus and kidney), expression of cytokines, bone marrow cellularity and alpha-esterase positive cells. Plaque Forming Assay (PFA), Delayed Type Hypersensitivity (DTH), and phagocytosis activity were also carried out to support the effect of PA. PA did not exhibit any toxic effect in mice. Results: In DTH study, the foot pad thickness due to influx of mononuclear cells at the site of inoculation was distinctly increased in PA treated mice. PA enhanced the phagocytic activity of the polymorphonuclear cells by increasing the engulfment of the Candida cells thereby stimulating a non-specific immune response. PFA confirmed that PA treatment could elevate the humoral immune response due to the synthesis of antibody which in turn is responsible for the enhancement of macrophages and Blymphocyte subsets. The significant increase in the number of α-esterase positive cells and bone marrow cellularity due to immunomodulatory effect indicated the proliferation of stem cells. Different organ weight was also markedly improved by PA treatment compared to SRBC sensitized group. Further, in real time PCR studies treatment of PA significantly increased IL- 7, IL- 10, IL-12a and IL-12b mRNA gene expression. Conclusion: From the above findings it can be concluded that PA could be developed as a potent immunomodulatory agent.

Differences of Bone Marrow Features and BCR-ABL Variants in Chronic Granulocytic Leukemia Post Tyrosine Kinase Inhibitor Therapy

Chronic Granulocytic Leukemia (CGL) occurs due to chromosomal translocation (9;22) known as Philadelphia chromosome. p210 BCR-ABL1 oncogenes are classified into b2a2 and b3a2 transcripts which possibly lead to different clinical manifestations and response to therapy. This study was aimed to prove that there is a difference of bone marrow features and BCR-ABL between remissive and resistant CGL after Tyrosine Kinase Inhibitor (TKI) therapy. This research was an observational study with a cross-sectional design carried out at Ulin Hospital Banjarmasin on 32 subjects. BCR ABL was detected by using PCR and bone marrow features were assessed by using bone marrow aspiration technique. The difference of bone marrow features and BCR-ABL variants was analyzed by using T-test (p < 0.005) and Chi-Square (p < 0.005), respectively. There was a difference of BCR-ABL variants with p=0.091 and characterized by M:E ratio (p=0.124), myeloblast count (p=0.063), and eosinophil count (p=0.055). In addition, there was a difference of bone marrow cellularity (p=0.000) and basophil count (p=0.016) between remissive CGL and resistant CGL patients. There was no difference of BCR ABL variants, myeloblast count and eosinophil count between remissive CGL and resistant CGL patients. However, there was different of bone marrow cellularity and basophil count between remissive CGL and resistant CGL patients.

Prognostic Significance of Bone Marrow Cellularity in the Outcome of Patients with Myelodysplastic Syndromes Treated with Azacyitidine: A Retrospective Analysis from the Hellenic MDS Study Group

Introduction: Clinical trials in patients with high risk myelodysplastic syndromes (MDS) have shown that these patients benefit from the available hypomethylating agents 5-azacytidine and decitabine. The majority of these patients display hypercellular bone marrow, but a small proportion despite the excess of blasts, exhibit marrow hypocellularity (<30% cellularity). Data are limited for the efficacy and safety of treatment with hypomethylating agents in this patient subgroup. In the present study we examined the effect of bone marrow cellularity in the overall survival in patients with MDS treated with azacitidine. Patients & Methods: This is a retrospective multicenter study from the Hellenic National MDS Registry (EAKMYS) on behalf of the Hellenic MDS Study Group. Between 1.1.2009 and 31.12.2018 a total of 1161 MDS patients who have received treatment with azacytidine have been registered. Complete patient information and follow-up were available for 989 patients, and all these have been included in the final analysis. Statistical analysis was performed and overall survival (OS) was evaluated, using Kaplan-Meier estimates (GraphPad Prism software, CA). A p value less than 0.05 was considered statistically significant. Results: Forty nine patients had a hypocellular bone marrow (hMDS), representing the 4.95% of the whole patient population. Of these patients 39 were men (5.3% of all men included in the study) and 10 were women representing the 2.98% of all women enrolled (male to female ratio 3.9). In the non-hypoplastic group, 750 were men and 358 were women (male to female ratio 2.09). The median age at diagnosis for the hMDS group was 70.8 years, compared to 72.8 years in the non-hypoplastic group. The IPSS-R prognostic risk categorization included 15 hMDS patients in the low group, 9 in the intermediate, 14 in the high and 11 in the very high risk group. Twenty-six patients (53%) of the hMDS group had bone marrow blasts between 10 and 20%, and the remaining 23 (47%) had 5-10% blasts. The patients with hMDS received an average of 10 cycles of azacytidine treatment during the follow-up period (range 2-29 cycles). The outcomes tested were overall survival and progression to AML. The median overall survival of patients with hMDS, following azacytidine treatment start, was not significantly different from the median survival of patients with non-hypoplastic MDS [20 months versus 16 months in the non-hypoplastic group (95% CI of ratio: 0.839 to 1.863). The survival curves were not significantly different between the hMDS and non-hypoplastic MDS group (p=0.32, Figure 1). Progression to AML was also evaluated. Eleven (22.4 %) hMDS patients showed disease progression to AML. Patients with hMDS had significantly prolonged estimated median time to AML transformation, compared to the non-hypoplastic MDS population (31.7 versus 22 months respectively, p<0.001). There were not any major safety issues among patients with hMDS, despite the increased RBC and Platelet transfusion needs. The infectious episodes and the hospitalization courses did not differ significantly between the hMDS and the non-hypoplastic group. Discussion and Conclusive remarks: In this retrospective study, in which a large number of MDS patients was analyzed, we showed that bone marrow cellularity does not affect the outcome in patients treated with azacyitidine. Patients with hMDS show statistically significant slower AML progression compared to non-hypoplastic MDS. Bone marrow cellularity should not be a contraindication for using hypomethylating agents as a therapeutic option, and this type of treatment can be used safely, when indicated, also in patients with hMDS. Disclosures Pappa: Amgen: Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Vassilakopoulos:Merck: Honoraria; Takeda Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Winmedica: Honoraria; Servier: Membership on an entity's Board of Directors or advisory committees. Symeonidis:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.

MPL-Mutated Essential Thrombocythemia: A Morphologic Reappraisal

Background : Myeloproliferative leukemia virus oncogene (MPL)-mutated essential thrombocythemia (ET) is uncommon, with a cited incidence of less than 5%, while that of MPL-mutated primary myelofibrosis (PMF) is at least twice as frequent (Blood. 2006;108:3472;Blood.2008;112:141). MPL-mutated ET cohorts also have higher reported rates of fibrotic progression than their MPL wild type counterparts (Blood. 2014;124:2507). These observations suggest the possibility that some instances of MPL-mutated ET might actually represent prefibrotic PMF. Methods : Patients were recruited from institutional databases of the Mayo Clinic, Rochester, MN, USA. Diagnoses were according to the 2016 World Health Organization (WHO) criteria (Blood. 2016;127:2391). Study inclusion criteria required the availability of bone marrow biopsy slides for central review by one of the authors (C.A.H.). Central pathology review included assessment of bone marrow cellularity and extent of tri-lineage proliferation, megakaryocyte morphology and grading of reticulin fibrosis. Data was additionally collected from MPL-mutated patients with PMF, for comparison. Standard statistical methods were used for statistical analysis, including calculation of survival data (JMP® Pro 13.0.0, SAS Institute, Cary, NC, USA). Results: A total of 665 patients with ET were annotated for their driver mutational status; 18 (2.7%) were reported out as being MPL-mutated; by comparison, among 867 patients with PMF, 47 (5.4%) were signed out as MPL-mutated. Among the 18 cases with MPL-mutated ET, bone marrow slides were available for central pathology review in 14 patients (Table 1). The latter were subsequently reassigned the diagnosis of either prefibrotic PMF (n=8; 57%) or were felt to be morphologically consistent with true WHO-defined ET (n=6; 43%). Comparison of these two distinct histopathological patterns, i.e. true ET vs reassigned prefibrotic PMF, was respectively characterized by lower (median 35%, range 30-50) vs higher (median 65%, range 40-80) bone marrow cellularity (P<0.001), ET (n=6) vs PMF (n=8) consistent megakaryocyte morphology (P<0.001) and presence of tri-lineage proliferation (0% vs 100%; P<0.001); in contrast the degree of reticulin fibrosis was similar between the two (P=0.1) (Table 2). The reassigned prefibrotic PMF (n=8), vs confirmed ET (n=6), cases also displayed a higher frequency of increased serum levels of lactate dehydrogenase (60% vs 0%; P=0.02), higher likelihood of displaying hemoglobin levels below the sex-adjusted reference range values (29% vs 0%; P=0.1), leukoerythroblastosis (14% vs 0%; P=0.2), constitutional symptoms (13% vs 0%; P=0.2) and a higher incidence of thrombosis history at presentation (38% vs 0%; P=0.04) (Table 2). Interestingly, reassigned prefibrotic PMF also displayed a narrower MPL mutational spectrum compared to those confirmed as ET (MPLW515L/K incidence 100% vs 60%; P=0.04). The incidences of abnormal karyotype and high risk molecular mutations (ASXL1, SRSF2 and U2AF1) were similar between the two. We documented a higher incidence of post-diagnosis thrombosis in prefibrotic PMF (25% vs 0%; P=0.1) but similar rates of leukemic transformation (0% for both) and fibrotic progression (38% vs 33%); when all 665 ET patients were assessed for myelofibrosis-free survival, MPL-mutated cases (prior to central review) displayed significantly worse outcome, compared to patients with other driver mutations (Figure 1a). Median overall survival in confirmed MPL-mutated ET, ET re-classified as PMF, and MPL-mutated PMF was not reached, 11.6 and 5.3 years, respectively (confirmed ET vs PMF, P=0.01; ET re-classified vs PMF, P=0.04; confirmed ET vs ET re-classified as PMF, P=0.54) (Figure 1b). Conclusions: The current study suggests that the majority of routinely-assigned MPL-mutated ET probably represents prefibrotic PMF, when morphologically scrutinized. Prior to central pathology review, we show a higher rate of fibrotic progression in MPL-mutated ET, compared to patients with other driver mutations; accordingly, after central pathology review, the similar rate of fibrotic progression between morphologically confirmed MPL-mutated ET and those reassigned as prefibrotic PMF further suggests the latter to be biologically more akin to PMF, despite its ET-consistent morphologic features. Disclosures No relevant conflicts of interest to declare.

Effects of a Novel Orally Bioavailable Small Molecule (Imidazolyl Ethanamide Pentandioic Acid) on Acute Radiation Syndrome

Introduction Acute radiation syndrome (ARS) develops within 24 hours of exposure to ionizing radiation. Leukocyte growth factors have been used to reduce mortality and mitigate the hematopoietic symptoms of ARS. Three subcutaneously applied radiomitigators G-CSF, Peg-G-CSF and GM-CSF have been approved by the FDA as medical countermeasures but few others are under development. Imidazolyl ethanamide pentandioic acid (IEPA, Myelo001) is a novel small molecule for the treatment of ARS. Preclinical and clinical studies have shown that IEPA applied orally or intraperitoneally was effective in reducing hematopoietic symptoms caused by radiation and chemotherapy. Objective To investigate the effects of IEPA as a radioprotector (prophylactic) and radiomitigator (therapeutic) for ARS and hematopoietic syndrome of ARS (H-ARS). Methods Multiple oral or intraperitoneal administrations of IEPA (25 or 50 mg/kg doses) and radiation levels of 5.8 Gy (estimated LD25/30) and 6.0 Gy (estimated LD50/30) on mortality, body weight and bone marrow cellularity were assessed in a mouse model. 205 C57BL/6 mice were subdivided into 1 unirradiated group, 6 groups exposed to 5.8 Gy, and 2 groups exposed to 6.0 Gy. Prophylactic treatment (25 or 50 mg/kg) was started 3 days before total-body irradiation, while therapeutic treatment (50 mg/kg) was begun 24 h post exposure. The 6 LD25/30 groups consisted of a vehicle control group (VL; 2), twice daily intraperitoneally administered IEPA (ML; 3), orally twice a day (ML; 4) or once a day (ML; 5), G-CSF positive control subcutaneously administered once a day (GL; 6) or in combination with IEPA (M/GL; 7). The 2 LD50/30 groups consisted of a vehicle control group (VH; 8) and a group administered IEPA orally once a day (MH; 9). The experiments assessed mortality using Kaplan-Meier estimator, body weight and bone marrow cellularity over the course of 30 days with prescheduled sacrifices of subgroups on days 7, 14 and 30. Results No significant benefit of prophylactic and therapeutic treatment on survival in the lower (5.8 Gy) irradiation group was detected. Groups ML; 3 and ML; 4 had a dose reduction factor (DFR) < 1 vs VL; 2 whereas ML; 5, GL; 6 and M/GL; 7 had a DRFs > 1. In the high radiation group (6.0 Gy), the Kaplan-Meier estimator revealed an increase in survival (85 %) after therapy compared to controls (56 %). The dose reduction factor in group MH; 9 compared to the controls (VH; 8) was 1.5. The highest protective effect on body weight was observed in the therapeutic regimen (MH; 9) used for 6.0 Gy exposure, which showed a positive effect on days 15, 21 and 30. Therapeutic IEPA treatment mitigated the impact of radiation on bone marrow cellularity. A pronounced effect on peripheral hematology was neither observed in the prophylactic, therapeutic IEPA nor the positive control G-CSF treated groups. Conclusions Different routes of administration and doses of IEPA in the prophylactic groups did not alter ARS symptoms. However, therapeutic treatment in the LD25/30 setting with IEPA and G-CSF, and the LD50/30 setting with IEPA at a dose of 50 mg/kg showed a reduction in mortality and weight loss compared to the controls. Additionally, IEPA treatment mitigated the impact of radiation on bone marrow cellularity. Analysis of peripheral blood did not reveal significant differences across the treatment groups probably due to no optimal time point analysis. Limitations included the small size of the prophylactically treated groups exposed to a low radiation dose. Disclosures Pleimes: Bayer: Consultancy, Equity Ownership; Myelo Therapeutics: Employment, Equity Ownership. Bunker:Myelo Therapeutics: Other: Contract Research via SNBL USA on behalf and in account of Myelo Therapeutics GmbH; SNBL USA: Employment. Meyer:Myelo Therapeutics GmbH: Consultancy. Czajkowski:Myelo Therapeutics GmbH: Employment.