Diagnostic Bone Marrow Aspirates Research Articles

Blinded Ex-Vivo testing in AML Reveals Differences between Hypomethylating Agents When Combined with Venetoclax

Introduction The therapeutic strategy of combining the hypomethylating agents (HMAs) Azacitidine (Aza) and Decitabine (Deci) with the BCL-2 inhibitor Venetoclax (Ven) has significantly improved the clinical outcomes of patients with Acute Myeloid Leukemia (AML), reaching overall response rates of up to 70% in clinical trials (Wei et al, JCO 2021). Still, on an individual basis, clinicians struggle to predict which patients will respond to Ven/HMA treatment. Furthermore, there is little data to guide selection of which HMA to use and the decision is generally based on physician preference and logistics. Functional ex-vivo testing using patient-derived primary cells has emerged as a promising approach to guide clinical decision (Letai et al, Cancer Cell 2022). This is particularly relevant for heme malignancies, given the ease of access to patient-derived cells from peripheral blood and/or bone marrow. We conducted a blinded study using OncoPrecision's proprietary Patient Micro-Avatars (PMAs) to predict clinical responses to Ven/HMA treatment. Methods De-identified diagnostic bone marrow aspirate samples from the leukemia program biobank at Weill Cornell Medicine that received Ven/HMA were provided to OncoPrecision for ex-vivo testing without information about the specific HMA used for treatment nor clinical response. PMAs are based on a triple co-culture platform that, by promoting the long term ex-vivo survival of Patient-Derived Cells (PDCs), allows testing the performance of different treatments. PDCs are co-cultured with two types of heterologous engineered cells: i) bone marrow stroma cells that act as a feed layer and as a control of non-specific toxicity (Tox-Control) and ii) a known outcome AML cell line that serves as an internal control of the system robustness and reproducibility in the response to drugs (System-Control). PMAs also leverage a multi-tagging approach coupled to high-throughput flow cytometry to track the differential response to treatments in subpopulations of pathological and healthy cells from each patient. This technology previously showed robust predictive power in observational studies in AML (García et al, Blood - 2022 ; Andino et al, Blood - 2023). Results PMAs were used to screen 39 AML patients who were treated with either Aza+Ven or Deci+Ven and from whom bone marrow or peripheral blood samples were stored prior to the start of treatment. OncoPrecision blindly tested these samples ex-vivo against a full dose-response curve of Aza+Ven and used their machine learning algorithm to predict clinical outcomes. When the predictions were matched against the clinical outcomes of the 27 patients of the cohort that received Aza+Ven at the clinic, the predictive accuracy (85%) was similar to the one previously reported in OncoPrecision's observational study (80%). Remarkably, however, when Aza+Ven predictions were matched to the clinical outcomes of the 12 patients in the cohort who received Deci+Ven, the predictive accuracy dropped to 50%. Interestingly, such a dramatic drop in the predictive power cannot be explained by a decrease in the negative predictive value (which remained >80%), but rather to a subpar positive predictive value (<30%), thus indicating that Aza may clinically outperform Deci in several of these patients. It is clear that ex-vivo testing using PMAs may have the ability to discern treatment sensitivity between Aza vs. Deci, and that there is potential to utilize this technology to tailor Ven/HMA therapy for individual patients. This is valuable not only to maximize potential disease response in a patient, but to also better prioritize patient quality of life during treatment, considering that Aza more commonly causes adverse effects like nausea and constipation than Deci. Conclusions Ex-vivo testing using PMAs has the potential to identify patients likely to respond to Ven/HMA and to discern treatment sensitivity between Aza vs. Deci. Our findings suggest that Aza and Deci may not always have equivalent efficacy for patients. This variation between Aza and Deci merits further exploration, especially since the therapeutic landscape for AML is likely to further expand with additional HMAs.

T-cell and Soluble Co-inhibitory Receptor Expression in Patients With Visceral Leishmaniasis Are Markers of Treatment Response and Clinical Outcome.

Co-inhibitory receptors (immune checkpoints) regulate activated immune cells. Their expression on T cells can limit host defense. We hypothesized that chronic Leishmania donovani infection in patients with visceral leishmaniasis (VL) leads to expression of co-inhibitory receptors that could be markers of treatment response and clinical outcome. A prospective cohort of 21 subjects with VL (7 with HIV coinfection) and 10 controls was established to measure T-cell expression of co-inhibitory receptors (PD-1, Tim-3, LAG-3, CTLA-4, and TIGIT) by flow cytometry in discarded remnants of diagnostic splenic or bone marrow aspirates and peripheral blood collected before and after treatment. Plasma levels of soluble co-inhibitory proteins (sPD-1, sTim-3, sLAG-3, and sCTLA-4) and selected cytokines were determined by immunoassay. Expression of co-inhibitory receptors in peripheral blood T cells generally reflected findings in spleen and bone marrow aspirates. PD-1 and Tim-3 were upregulated in CD4+ T cells in HIV-negative and HIV-positive subjects with VL compared to controls. CD8+ T cells from HIV-negative subjects with VL displayed a similar pattern. Plasma levels of sPD-1 and sTim-3 were also greater in VL patients than controls. CD8+ and CD4+ T cells coexpressing PD-1 and Tim-3 showed considerable decline with treatment. Mortality in HIV-negative VL patients was associated with increased CD8+ T cells coexpressing Tim-3 and PD-1, triple-positive CD4+ and CD8+ T cells (PD-1+Tim-3+LAG-3+), and elevated sLAG3. Tim-3 and PD-1 expression on CD4+ and CD8+ T cells, and increased plasma sLAG-3, were markers of treatment response and clinical outcome in patients with VL.

Clearing soluble MIC reverses the impaired function of natural killer cells from patients with multiple myeloma

BackgroundMajor histocompatibility complex (MHC) class I chain-related protein (MIC) is a stress-induced ligand released from multiple myeloma (MM) cells during progression, and soluble MIC impairs natural killer group 2D (NKG2D)...

Ex Vivo Drug Sensitivity Correlates with Clinical Response and Supports Personalized Therapy in Pediatric AML.

Acute myeloid leukemia (AML) is a heterogeneous disease that accounts for ~20% of all childhood leukemias, and more than 40% of children with AML relapse within three years of diagnosis. Although recent efforts have focused on developing a precise medicine-based approach towards treating AML in adults, there remains a critical gap in therapies designed specifically for children. Here, we present ex vivo drug sensitivity profiles for children with de novo AML using an automated flow cytometry platform. Fresh diagnostic blood or bone marrow aspirate samples were screened for sensitivity in response to 78 dose conditions by measuring the reduction in leukemic blasts relative to the control. In pediatric patients treated with conventional chemotherapy, comprising cytarabine, daunorubicin and etoposide (ADE), ex vivo drug sensitivity results correlated with minimal residual disease (r = 0.63) and one year relapse-free survival (r = 0.70; AUROC = 0.94). In the de novo ADE analysis cohort of 13 patients, AML cells showed greater sensitivity to bortezomib/panobinostat compared with ADE, and comparable sensitivity between venetoclax/azacitidine and ADE ex vivo. Two patients showed a differential response between ADE and bortezomib/panobinostat, thus supporting the incorporation of ex vivo drug sensitivity testing in clinical trials to further evaluate the predictive utility of this platform in children with AML.

Targeting IDH1-Mutated Pre-Leukemic Hematopoietic Stem Cells in Myeloid Disease, Including CCUS and AML

Introduction: Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been reported in clonal hematopoiesis and are recurrent, early events in the development of acute myeloid leukemia (AML). A subsequent stepwise acquisition of additional genetic aberrations leads to the development of frank leukemia. At the time of AML diagnosis, rare pre-leukemic hematopoietic stem cells (pHSC) can be detected containing some, but not all, of the mutations in the corresponding leukemia, although these pHSCs are phenotypically indistinguishable from residual wildtype hematopoietic stem cells (HSC). The frequency of pHSCs at the time of diagnosis is known to correlate to overall survival in AML patients (Corces et al, Nat Genet 2016), indicating the potential relevance of therapeutically targeting these cells to prevent relapse. Methods: To model these rare pre-leukemic cells, we introduced IDH1 R132H and IDH2 R140Q mutants or their wildtype counterparts into human CD34+ hematopoietic stem and progenitor cells (HSPC) using CRISPR/Cas9 and AAV6-mediated homology directed repair (HDR). Successfully edited cells were then sorted by FACS based on GFP expression for subsequent analyses including 2HG quantification, colony formation, differentiation capacity, RNA sequencing, and metabolic assays. Additionally, primary pHSCs from diagnostic bone marrow aspirates from AML patients were also isolated by FACS and subjected to treatment, colony formation assays, and genotyping by ddPCR. Results: Human CD34+ HSPCs engineered to express either IDH1 R132H or IDH2 R140Q produced 2HG and showed a striking reduction in colony formation and differentiation capacity, recapitulating the cytopenic phenotype observed in IDH-mutated clonal cytopenia of undetermined significance (CCUS). RNA-seq of these cells revealed large transcriptomic changes shared between IDH1 R132H and IDH2 R140Q mutations, but gene set enrichment analyses revealed the specific and significant downregulation of oxidative phosphorylation-related genes in R132H-mutated cells. These R132H cells were further confirmed to have lower basal metabolic activity coupled to a higher mitochondrial dependency as determined by SCENITH (Argüello et al, Cell Metab 2021). These metabolic alterations suggested that it might be possible to selectively therapeutically target R132H mutated cells. To evaluate this, we treated HSPCs engineered to express GFP, IDH1 wildtype, or IDH1 R132H, with the known mutant-specific inhibitor ivosidenib, the OXPHOS-inhibitor IACS-010759, or DMSO as a control. Strikingly, ivosidenib restored the defect in colony formation conferred by R132H and increased the total number of colonies whereas IACS-010759 effectively inhibited R132H colony formation (Figure 1A). To further investigate this vulnerability, we sorted pHSCs from diagnostic bone marrow samples from primary IDH1 and IDH2-mutated AML samples according to the phenotype CD45dimCD3-CD19-CD20-CD34+CD38-CD99-TIM3-. These cells were subjected to treatment with DMSO, ivosidenib, enasidenib, or IACS-010759 and subsequently plated for colony formation. Because the sorted cell population contained a mixture of mutant pHSCs and residual wildtype HSCs, all colonies were picked and genotyped for IDH mutations using ddPCR, revealing that only IDH1 wildtype colonies grew out after IACS-010759 treatment whereas all other conditions resulted in a mixture of wildtype and mutant colonies (Figure 1B). Conclusions: The OXPHOS-inhibitor IACS-010759 can eradicate IDH1-mutated pHSCs whereas ivosidenib cannot. This effect is specific for IDH1 mutations and the same effect is not seen in IDH2-mutated pHSCs. These results have potential clinical implications when considering therapeutic options in AML as well as IDH1-mutated pre-leukemic conditions such as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognosis of NF1 Mutations in Acute Myeloid Leukemia

Background: As molecular testing becomes more widely available, the prognostic and therapeutic implications of specific genetic mutations in acute myeloid leukemia (AML) continue to evolve. Mutations in the Neurofibromin 1 (NF1) gene are not well studied in AML. A limited number of studies have evaluated the presence and potential role of NF1 in AML (Eisfeld et al., 2018; Telfah et al., 2019). NF1 mutations were found in about 5% of AML patients and may carry a worse prognosis. In this IRB-approved retrospective case-control study, we analyzed specific clinical outcomes for AML patients with any NF1 mutation compared to patients with a wild-type NF1 gene, including complete remission (CR) rate and overall survival (OS). Methods: We reviewed electronic medical charts of newly diagnosed AML patients who had next-generation-sequencing (NGS) from bone marrow aspirates at diagnosis at our institution between 2017-2021. We identified patients with any pathogenic or likely pathogenic NF1 mutation. Control patients with a wild-type NF1 gene were matched to the NF1 positive group based on age, sex, race, and AML risk per 2017 ELN criteria. The primary outcome was overall survival; secondary outcomes included the rate of achieving a complete remission (CR), complete remission with incomplete hematologic recovery (CRi) at bone marrow evaluation after chemotherapy induction, allogeneic hematopoietic stem cell transplant rate for those who achieved CR, and finally overall survival (OS). CR, CRi, and transplant rates were compared using a Fisher's exact test and by calculating odds ratios (OR). OS was estimated by the Kaplan-Meier method and compared by using a log-rank test. Significance was defined by a P value <0.05. Results: We identified 34 AML patients with an NF1 mutation on NGS from initial diagnostic bone marrow aspiration between 2017-2021. We compared the outcomes with a matched control group (n=130) as described in the methods. The median age was 63 (27-84) and 64.5 (23-86) in the NF1 mutated and wild-type groups, respectively. There were no differences in sex or race between the two groups (Table 1). There were 20 patients (63%) with high-risk disease in the NF1 mutated group, similar to 74 patients (57%) in the control group. The co-occurring mutations varied between the two groups; RUNX1 mutations were more common in the NF1 mutated group, while DNMT3A and FLT3 were less common (Table 1). NF1 mutated patients were less likely to achieve CR/CRi at 44% compared to 65% of NF1 wild-type patients (OR 0.40; P 0.03; figure 1A). Rates of subsequent allogeneic transplant for patients who achieved CR/CRi were similar in NF1 mutated and wild-type patients (66% and 60%, respectively). The median OS for NF1 mutated patients was 474 days compared to 688 days for wild-type NF1 patients (hazard ratio (HR) for death, 1.45; 95% CI, 0.88 to 2.49; P 0.15; figure 1B). There was no significant difference in median OS between patients who received allogeneic transplants, but NF1 mutated patients who did not receive a transplant had a significantly worse median OS compared to the NF1 wild type patients (HR 1.63, P 0.03; figure 1C). Discussion: Somatic NF1 mutations are not well studied in AML. Prior limited studies are suggestive of worse outcomes, and we have observed similar results in this study. Patients with NF1 mutations were significantly less likely to achieve CR/CRi compared to patients with a wild-type NF1 gene, either with intensive or non-intensive therapies. OS was worse for NF1 mutated patients but did not reach statistical significance except for patients that did not receive an allogeneic transplant, who had significantly worse median OS. While the patients' characteristics, including the risk of AML, were similar between the NF1 mutated and wild-type groups, we observed differences in co-occurring mutations. The common DNMT3a and FLT3 mutations were significantly less common in NF1 mutated patients, while there were more RUNX1 mutations in this group. This distinctive co-occurring mutation pattern may suggest a role for NF-1 in leukemogenesis with a specific leukemia development pathway and provides an opportunity for personalized targeting. This retrospective study has a relatively small sample size. Nonetheless, it provides further evidence for the potentially adverse prognosis of NF1 mutations in AML, which can guide conversations between treating physicians and patients about individualized prognostic risks. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of Nurses Knowledge about Diagnostic Bone Marrow Aspiration and Biopsy at Blood Diseases Center in Medical City Directorate

Evaluation of Nurses Knowledge about Diagnostic Bone Marrow Aspiration and Biopsy at Blood Diseases Center in Medical City Directorate

Effectiveness of an Educational Program on Oncology Nurses Knowledge about Diagnostic Bone Marrow Aspiration and Biopsy at Blood Diseases Center in Medical City Directorate

Background: Bone marrow aspiration and biopsy is a procedure which done for patients may be have a blood disorder or a suspected or previously diagnosed blood malignancy. Bone marrow examination is done during the initial diagnosis and/or staging and used to determine treatment response in patients with leukemia, lymphoma, and other blood disorders. Aims: To evaluate the effect of an educational program on nurse’s knowledge related to about Diagnostic Bone Marrow Aspiration and Biopsy at blood diseases center in medical city directorate. Methodology: A pre experimental study design (one group Pretest-Posttest study design) on 20 nurses who work in bone marrow examination unit, it is carried out through the application of pre-test and post-test approach for the study group, from the period 13th June to 13th of December 2021. Three sessions were submitted to the nurses that participate in an educational program. Results: The results of current study indicated that that there were significances differences between pre and post educational program related to nurses knowledge. Conclusion: Based on the study findings that the program applied has an observational enhanced nurses’ knowledge relating to the bone marrow aspiration and biopsy management. Keywords Effectiveness, Educational Program, Oncology Nurses, Knowledge, Bone Marrow Aspiration,

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Laboratory quality assessment of candidate gene panel testing for acute myeloid leukaemia: a joint ALLG / RCPAQAP initiative

Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories. This study compared the results obtained from MPS testing of ten diagnostic AML bone marrow aspirate samples sent to eight participating laboratories across Australasia. A reassuringly high concordance of 94% was observed with regard to variant detection and characterisation of pathogenicity. The level of discordance observed, although low, demonstrates the need for ongoing assessment of concordance between diagnostic testing laboratories through quality assurance programs.

After Treatment Decrease of Bone Marrow Tregs and Outcome in Younger Patients with Newly Diagnosed Acute Myeloid Leukemia.

An emerging body of evidence demonstrates that defects in antileukemic effector cells in patients with acute myeloid leukemia (AML) can contribute to the development and/or persistence of the disease. In particular, immune suppressive regulatory T cells (Tregs) may contribute to this defective antileukemic immune response, being recruited by bone marrow leukemic cells to evade immune surveillance. We evaluated Tregs (CD4+/CD45RA-/CD25high/CD127low), performing multiparametric flow cytometry on freshly collected bone marrow aspirate (BMA), in addition to the usual molecular and cytogenetic work-up in newly diagnosed AML patients to look for any correlation between Tregs and the overall response rate (ORR). We studied 39 AML younger patients (<65 years), all treated with standard induction chemotherapy. ORR (complete remission (CR)+CR with incomplete hematologic recovery (CRi)) was documented in 21 out of 39 patients (54%); two partial responder patients were also recorded. Apart from the expected impact of the molecular-cytogenetic group (p = 0.03) and the NPM mutation (p = 0.05), diagnostic BMA Tregs did not show any correlation with ORR. However, although BMA Tregs did not differ in the study population after treatment, their counts significantly decreased in responder patients (p = 0.039), while no difference was documented in nonresponder ones. This suggested that the removal of Treg cells is able to evoke and enhance anti-AML immune response. However, the role of BMA Tregs in mediating immune system-AML interactions in the diagnostic and posttreatment phase should be confirmed in a greater number of patients.

RAS Mutations Are Independently Associated with Decreased Overall Survival and Event-Free Survival in Patients with AML Receiving Induction Chemotherapy

Cai: Imago Biosciences, Inc.: Consultancy. Viny:Hematology News: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Other: Sponsored travel. Goldberg:American Society of Clinical Oncology: Research Funding; Abbvie: Research Funding; ADC Therapeutics: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Celgene: Consultancy. Tallman:BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stein:Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

Transcriptome Alterations of Isolated Primary Bone Marrow Endothelial Cells Provide Insigt to MGUS and Multiple Myeloma Pathobiology

Multiple myeloma (MM) is characterized by an abnormal clonal expansion of plasma cells in the bone marrow, production of monoclonal immunoglobulins and finally organ damage (CRAB). The premalignant precursor of MM is Monoclonal gammopathy of undetermined significance (MGUS) and one percent of all MGUS patients progress to MM yearly. The bone marrow microenvironment is thought to play an important role in plasma cell growth, migration, and survival mainly via cytokine secretion and cell-cell interactions. Endothelial cells (ECs) are a major component in the bone marrow microenvironment, they regulate trafficking and homing of hematopoietic progenitor and stem cells. In MM increased bone marrow angiogenesis and recruitment of endothelial progenitors to the bone marrow niche has been reported. However, the specific EC contribution to myelomagenesis is not yet known. This study therefore aimed to investigate transcriptome alterations in prospectively isolated bone marrow ECs from MGUS and MM patients to identify possible disease-stage related changes. We isolated primary ECs from MGUS and MM patients undergoing diagnostic bone marrow aspirations and age-matched healthy donors by FACS. RNA from Lin- CD45- CD71- CD235a- CD271- CD31+ cells of MGUS (n=4) and MM (n=7) patients and healthy donors (n=6) was extracted. Sequencing was done using the Illumina® NextSeq 500/550 High Output Kit v2.5 (300 cycles). Gene expression analysis was performed in R. Differential gene expression analysis (DEseq2) identified 1,507 genes with p adjusted values below 1e-2 that were significantly differentially expressed between the three groups. Hierarchical clustering was done following Ward's method (ward.D2). Unsupervised clustering on the data showed that one MGUS-EC sample clustered with the healthy controls, and that one healthy control sample clustered with the MGUS samples. We therefore decided to restrict the analysis to those samples that clearly clustered separately, to be able to better depict the MGUS-, MM- and healthy EC specific profiles. Further clustering of differential expressed genes into 8 clusters revealed two especially interesting expression patterns. One cluster (#4) contained 102 genes that where higher expressed in the healthy controls with lower expression in MGUS and lowest expression in MM Samples. These genes thus reflect the downregulation during progression from a healthy bone marrow microenvironment to a reduced expression MGUS and further downregulation in MM. Another cluster (#6) showed the opposite pattern, with 105 genes being low or not expressed in healthy controls while the expression was higher in MGUS and highest in MM. Gene sets where further analyzed in the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Cluster 4 showed a high number of downregulated transmembrane genes. Six genes of the major histocompatibility complex conserved site where identified might indicate a possible immunomodulating effect in disease progression. Furthermore, within cluster 4 we identified a cluster of genes involved in cell adhesion and receptor binding. Cluster 6 most strikingly showed a group of 6 genes of the melanoma-associated antigen (MAGE) gene family that were upregulated with disease progression. MAGE genes which belong to the cancer-testis group of germline genes have previously been reported in MM, as being involved in tumorigenesis, and plasma cell MAGE expression has been associated with chemotherapy resistance. Furthermore, cluster 6 contained a high number of extracellular matrix genes, and genes for proteins having an extracellular region, respectively, hinting towards a differential microenvironment composition upon MM development. Taken together RNA sequencing analysis of prospectively isolated bone marrow endothelial cells identified genes that were specifically upregulated/suppressed in MM-ECs compared to MGUS-ECs and healthy donor-ECs. These genes thus represent potential gene candidates involved in the disruption of normal microenvironment function, thus leading to disease development and progression. Accordingly, studies are underway to investigate selected transcriptional deregulation EC-MM microenvironmental functions in the context of the disease. Disclosures No relevant conflicts of interest to declare.

SUN-583 Immune Thrombocytopenic Purpura Associated with Stiff Person Syndrome and Polyglandular Autoimmune Syndrome Type 2

Background Idiopathic thrombocytopenic purpura (ITP) is an acquired autoimmune disease caused by antibodies against platelet glycoproteins. Stiff person syndrome (SPS) is a progressive neurological disorder characterized by axial muscle rigidity and involuntary spasms associated with anti-glutamic acid decarboxylase (GAD) antibodies. Polyglandular autoimmune syndrome type 2 (APS-2) is defined by the occurrence of Addison disease with thyroid autoimmune disease and/or Type 1 diabetes mellitus (T1DM). SPS has been associated with APS-2 as well as to T1DM and autoimmune thyroid disease alone. ITP has been linked T1DM in rare cases as well as autoimmune thyroid disease, yet SPS and ITP have not previously been associated. We present the first reported case to our knowledge of a patient with ITP subsequent to Graves disease, SPS and T1DM. Clinical Case The patient is a 35-year-old female evaluated in endocrinology clinic for follow up of Graves disease treated with RAI ablation 5 years prior. She presented with fluctuating muscle rigidity and spasms involving the axial muscles leading to gait disturbance and frequent falls. She displayed the classic “wooden-man” appearance when ambulating first described by Moersch and Wotmann which lead to the name of SPS. She was treated with diazepam due to clinical suspicion for SPS. Neurology was consulted, both the referring physician and neurologist agreed with the diagnosis of SPS. Her symptoms gradually improved yet they have proven to be relapsing and remitting in nature requiring modification of dosing depending on response. 7 years later she was diagnosed with T1DM, presenting to the ED with generalized weakness and fatigue. Initial laboratory work was consistent with DKA. During her hospitalization routine laboratory work showed incidental thrombocytopenia with a platelet count of 15,000. Over the next few days her platelet count dropped to 5,000 prompting diagnostic bone marrow aspiration and biopsy which revealed a hypercellular marrow and adequate megakaryopoiesis. ITP was the working diagnosis and patient was discharged with prednisone. A favorable response to therapy was noted and prednisone was tapered accordingly. 2 years later she presented with a relapse of ITP with a platelet count of 6,000; treatment was initiated with intermittent prednisone and IVIG. At this time T1DM and thyroid disease were well controlled. Serum GAD-antibodies tested after presentation of ITP were >250 IU/ml (0-5.0 IU/ml). Conclusion To our knowledge ITP has not previously been documented in a patient with SPS. Our goal is to create physician awareness regarding the association between these disorders as well as the need for regular monitoring of patients with an endocrine autoimmune disease. SPS has been linked to APS-2; yet is it possible that SPS can independently be associated with ITP?

Human Acute Myeloid Leukemia Inhibits Normal Erythroid Differentiation through the Paracrine Effects of IL-6

Abstract Acute Myeloid Leukemia (AML) is an aggressive cancer resulting in severe cytopenias related to bone marrow (BM) failure. The common assumption for AML-induced BM failure is overcrowding due to clonal expansion of immature myeloid blasts, leading to failure of normal hematopoiesis. However, in a cohort of 293 AML patients, we found that disease burden (% of blasts determined on diagnostic BM aspirate) did not predict severity of cytopenia (hemoglobin rs=-0.053; p=0.49; WBC rs=-0.030, p=0.70; platelet rs=0.091, p=0.026), strongly arguing against simple crowding as the main mechanism underlying AML-induced BM failure. Thus, the goal of our study is to identify novel mechanism(s) associated with AML-induced BM failure, potentially enabling development of new therapies to improve AML management and reverse morbidity. Conventional xenograft models of human AML do not typically exhibit cytopenias, making them unsuitable to study AML-induced BM failure. We speculated that in the mouse, increased splenic extramedullary hematopoiesis compensated for failed intramedullary hematopoiesis due to AML. To test this hypothesis, we performed surgical splenectomy on NSG mice prior to their transplantation with human AML. Strikingly, splenectomized NSG mice engrafted with primary human AML at a 30-70% disease burden (n=8 for primary AML samples, n=5-10 for each group of splenectomized NSG mice) developed leukopenia and severe anemia compared to sham-operated AML-engrafted controls. AML-engrafted splenectomized NSG mice showed early mortality compared to AML-engrafted NSG mice with intact spleens (10.1 weeks vs. 34.2 weeks p&lt;0.0001). Thus, our splenectomized murine xenograft model uniquely recapitulates human disease and allows for novel characterization of AML-induced BM failure. Utilizing our model, splenectomized NSG mice engrafted with human AML demonstrated depletion of normal hematopoietic progenitors (HSPCs) including HSCs (11.1 fold p&lt;0.0001), MPPs (8.90 fold, p&lt;0.001)), CMPs (11.4 fold p&lt;0.0001), MEPs (1.76 fold p=0.0005) and GMPs (5.07 fold p&lt;0.0001). Focusing on anemia, AML also depleted erythroid progenitors including proerythroblasts (1.39 fold p=0.04), normoblasts (4.96 fold p&lt;0.0001), late normoblasts (10.0 fold p&lt;0.0001), and reticulocytes (4.34 fold p=0.007). The relative preservation of proerythroblasts and depletion of normoblasts indicated that AML blasts impart a specific in vivo differentiation blockade in erythroid differentiation. To explore mechanisms by which AML blasts inhibit normal HSPCs, we generated conditioned media (CM) from patient-derived AML blasts, and found that AML-CM suppressed BFU-E colony formation from normal HSPCs (3.1-5.1 fold). Furthermore, in a direct co-culture system with AML blasts and CD34+ HSPCs, AML blasts inhibited erythroid differentiation from the CFU-E to normoblast stage by 81%. Removing CD34+ cells from the AML co-culture allowed cells to resume differentiation to the proerythroblast stage. These experiments demonstrate that AML imparts a differentiation blockade along the MEP-proerythroblast axis in a cell non-autonomous, reversible fashion. Using cytokine array analysis, we identified elevated IL-6 levels in AML sample-derived CM (n=10, 2841±766.4 pg/ml, 7.80 fold increase, p=0.03) compared to CD34+-derived CM (n=5, 364±36.0 pg/ml). Increased IL-6 was also found in BM aspirates from human AML engrafted splenectomized NSG mice (715±125pg/ml, p=0.001) compared to mice engrafted with normal CD34+ HSPCs (undetectable). Thus, we hypothesized that IL-6 produced by AML blasts acts as a paracrine factor to suppress erythropoiesis. Consistent with this hypothesis, an IL-6 neutralizing antibody reversed the inhibition of BFU-E formation imparted by AML-CM. Furthermore, the addition of recombinant IL-6 to liquid cultures of erythroid differentiation resulted in a 23% reduction in proerythroblasts. Together, our data suggests that (1) overcrowding is not the primary mechanism resulting in BM failure in AML; (2) AML blasts play a previously unrecognized role in imparting a differentiation blockade along the MEP-proerythroblast axis, resulting in progressive anemia; and (3) this differentiation blockade is at least partially attributable to IL-6 secreted from AML blasts as a paracrine factor. Disclosures No relevant conflicts of interest to declare.

The Common Causes Leading to Pancytopenia at Tertiary Care Hospital Hossain

Pancytopenia is a triad of simultaneous presence of anemia, leucopenia and thrombocytopenia. It is result from a number of disease processes. Both hematopoietic and nonhematopoietic conditions manifest with features of pancytopenia.Methods: This was a cross-sectional study carried out in the Department of Medicine, Dhaka Medical College Hospital between December 2016 to July 2017. We included adult patients of both sexes having age 16 years and above. Criteria for inclusion were persistent pancytopenia on peripheral blood film of more than one week duration. All patients underwent a detailed medical history and full physical examination followed by blood sampling for the investigations. After taking all the aseptic measures and with standard technique the diagnostic bone marrow aspiration and trephine biopsy were done.Results: There were 36 patients with pancytopenia which were included in this study. There were 20 (56%) males and 16 (44%) females with a 1.25:1 male to female ratio and a mean age 47.30 years ± 15.01 SD. The most common complaints were bleeding manifestation (29/36), followed by generalized weakness (27/36) and fever (25/36). Anaemia was the most common (100%) clinical feature followed by jaundice and splenomegaly (6/36). Aplastic anemia was the commonest cause that was observed in 27.78% (10/36) cases followed by Megaloblastic anemia 16.67% (6/36) and erythroid hyperplasia 13.88% (5/36).Conclusion: Aplastic anaemia is leading cause of pancytopenia in this study followed by megaloblastic anaemia and hypersplenism being second and third common causes respectively. Pancytopenia should be suspected on clinical grounds when a patient presents with unexplained anemia, prolonged fever and tendency to bleed.J Dhaka Medical College, Vol. 26, No.2, October, 2017, Page 157-161

Impact of Bone Marrow Aspirate Tregs on the Response Rate of Younger Newly Diagnosed Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is widely considered a distinct clinical entity with a well-defined molecular and genetics-based prognosis. Particularly in a younger patient, the therapeutic approach depends largely on diagnostic risk stratification, which has an impact on the outcome after therapy. We added Treg evaluation to the usual molecular and cytogenetics profile in the AML younger patients' diagnostic bone marrow aspirate (dBMA) in order to search for any correlation between Tregs and overall response (OR) as well as survival (OS) rates. We studied 23 AML young patients, all treated with standard induction chemotherapy: OR (complete remission (CR) + CR incomplete (CRi)) was documented in 10 of 23 patients (44%); there were two partial responder patients. The optimal dBMA Treg cut-off value for predicting response to treatment (≥21/μL) was obtained by ROC curve analysis. However, in multivariate analysis, apart from the expected impact of the molecular/cytogenetic risk (p = 0.049) and NPM mutation (p = 0.001), dBMA Tregs ≥ 21/μL was not correlated with OR. Actually, higher dBMA Tregs were associated with the good intermediate molecular/cytogenetic risk group (p = 0.02), whose median OS was confirmed to be better as compared with that of the poor risk group (18 versus 5 months, p = 0.05) and equal to the dBMA Tregs ≥ 21/μL group (5 versus 5 months, p = 0.902), respectively. The possible prognostic value of such an immunological player as BMA Tregs in the diagnostic and successive phases of AML needs to be confirmed in larger patient numbers.

Association of mutations with morphological dysplasia in de novo acute myeloid leukemia without 2016 WHO Classification-defined cytogenetic abnormalities.

Despite improvements in our understanding of the molecular basis of acute myeloid leukemia (AML), the association between genetic mutations with morphological dysplasia remains unclear. In this study, we evaluated and scored dysplasia in bone marrow (BM) specimens from 168 patients with de novo AML; none of these patients had cytogenetic abnormalities according to the 2016 World Health Organization Classification. We then performed targeted sequencing of diagnostic BM aspirates for recurrent mutations associated with myeloid malignancies. We found that cohesin pathway mutations [q (FDR-adjusted P)=0.046] were associated with a higher degree of megakaryocytic dysplasia and STAG2 mutations were marginally associated with greater myeloid lineage dysplasia (q=0.052). Frequent megakaryocytes with separated nuclear lobes were more commonly seen among cases with cohesin pathway mutations (q=0.010) and specifically in those with STAG2 mutations (q=0.010), as well as NPM1 mutations (q=0.022 when considering the presence of any vs. no megakaryocytes with separated nuclear lobes). RAS pathway mutations (q=0.006) and FLT3-ITD (q=0.006) were significantly more frequent in cases without evaluable erythroid cells. In univariate analysis of the 153 patients treated with induction chemotherapy, NPM1 mutations were associated with longer event-free survival (EFS) (P=0.042), while RUNX1 (P=0.042), NF1 (P=0.040), frequent micromegakaryocytes (P=0.018) and presence of a subclone (P=0.002) were associated with shorter EFS. In multivariable modeling, NPM1 was associated with longer EFS, while presence of a subclone and frequent micromegakaryocytes remained significantly associated with shorter EFS.

Presumed primary immune‐mediated neutropenia in 35 dogs: a retrospective study

To describe, in a cohort of dogs with presumed primary immune-mediated neutropenia, the presenting clinical characteristics, haematology results, bone marrow characteristics, therapies used (drugs and doses), clinical response to treatment, relapse and outcome at six months and one year. Multi-institutional recruited retrospective descriptive case series with voluntary submissions. Presumed immune-mediated neutropenia was diagnosed based on a neutrophil concentration <1·5×109 cells/L on a minimum of two complete blood counts, exclusion of other causes of neutropenia based on a diagnostic bone marrow aspirate or biopsy, and exclusion of secondary immune-mediated neutropenia. Dogs meeting these diagnostic criteria between 2006 and 2013, and that had a haematocrit of ≥29% and minimum of two complete blood clounts performed after initiation of therapy, were included. Information on 35 dogs was included. Neutropenia was less than 0·5×109 cells/L in most cases (21 dogs), 0·5 to ·99×109 cells/L in 11, and 1.0 to 1·49×109 cells/L in three. Eight dogs had thrombocytopenia, which was severe (<49·9×109 cells/L) in three. [Correction added on 23 May 2017, after first online publication: the cell numbers were incorrect due to errors in the conversion of cell measurements to international units. The numbers have been corrected throughout the article and Table 2.] Twenty-three dogs had myeloid hyperplasia, 10 dogs had myeloid hypoplasia and two dogs had normal myelopoiesis. Neutropenia resolved in 32 of 33 dogs within two weeks of starting corticosteroid therapy and in all dogs within one month. Relapse of neutropenia occurred in 12 cases within one year. Initial response of presumed primary immune-mediated neutropenia cases to corticosteroid therapy can be excellent. Long-term monitoring for relapse is warranted because 34% of cases relapsed during or after taper of immunosuppressive medications.

Non-tumour bone marrow lymphocytes correlate with improved overall survival in childhood acute lymphoblastic leukaemia.

Composition of tumour immune cell infiltrates correlates with response to treatment and overall survival (OS) in several cancer settings. We retrospectively examined immune cells present in diagnostic bone marrow aspirates from paediatric patients with B‐cell acute lymphoblastic leukaemia. Our analysis identified a sub‐group (∼30% of patients) with >2.37% CD20 and >6.05% CD7 expression, which had 100% OS, and a sub‐group (∼30% of patients) with ≤2.37% CD20 and ≤6.05% CD7 expression at increased risk of treatment failure (66.7% OS, P < 0.05). Immune cell infiltrate at diagnosis may predict treatment response and could provide a means to enhance immediate treatment risk stratification.