Bone Marrow Immunophenotyping Research Articles

A simple score derived from bone marrow immunophenotyping is important for prognostic evaluation in myelodysplastic syndromes

Immunophenotyping of bone marrow (BM) precursors has been used as an ancillary diagnostic tool in myelodysplastic syndromes (MDS), but there is no general agreement about which variables are the most relevant for prognosis. We developed a parsimonious prognostic model based on BM cell populations well-defined by phenotype. We analyzed 95 consecutive patients with primary MDS diagnosed at our Institution between 2005 and 2012 where BM immunophenotyping had been performed at diagnosis. Median follow-up: 42 months (4–199). Median age: 67 years (33–79). According to IPSS-R, 71 cases were low or intermediate risk. Flow variables significant in the univariate Cox analysis: “%monocytes/TNCs”, “% CD16+ monocytes/TNCs”, “total alterations in monocytes”, “% myeloid CD34+ cells”, “number of abnormal expressions in myeloblasts” and “% of B-cell progenitors”. In the multivariate model remained independent: “% myeloid CD34+ cells”, B-cell progenitors” and “% CD16+ monocytes/TNCs”. These variables were categorized by the extreme quartile risk ratio strategy in order to build the score: % myeloid CD34+ cells” (≥ 2.0% = 1 point), B-cell progenitors” (< 0.05% 1 point) and “CD16+ monocytes/TNCs” (≥ 1.0% 1 point). This score could separate patients with a different survival. There was a weak correlation between the score and IPSS-R. Both had independent prognostic values and so, the flow score adds value for the prognostic evaluation in MDS.

First Description of Upfront Target Therapy Treating AML and Bcll Presented Simultaneously As First Line

INTRODUCTION: The occurrence of acute myeloid leukemia (AML) and chronic lymphoid leukemia (B-CLL) simultaneously is rarely described. We describe a case report of AML and B-CLL, diagnosed simultaneously, without any previous treatment for any of the hematological neoplasms [1]. The patient received low-dose cytarabine (ARA-C) and Venetoclax, which is medically indicated on label for both hematological neoplasms. CASE REPORT: A 75-year-old male, presented with edema and joint pain one month before hospital admission, showing pancytopenia on a complete blood count and presence of blasts in peripheral blood. The patient was then referred to the hematology service. The morphological analysis of the bone marrow aspirate showed 67.7% of myeloblasts, compatible with AML. Bone marrow immunophenotyping was performed, which identified 34.10% of myeloblasts, compatible with AML and 50.48% of monoclonal B lymphocytes (chronic B-cell lymphoproliferative disease). In flow cytometry there were two distinct populations of myeloblasts. Type 1 myeloblasts labeling CD7 +, CD13 +, CD34 ++, CD38 ++, CD45 ++, CD56 ++, CD117 ++, CD123 +, HLA-DR +++ and MPO + / ++. The second population marked CD13 + / ++, CD34 + / ++, CD38 ++, CD45 +, CD117 ++, CD123 +, HLA-DR ++ / +++ and MPO + (30%). Monoclonal lymphocytes showed CD11c + / ++ (70%), CD19 ++, CD20 + / ++ (84%), CD22 + (39%), CD23 + / ++, CD25 +, CD31 +/-, CD43 ++, CD45 ++ / +++, CD81 + (38%), CD200 + (85%) and Lambda +. The molecular study was negative for genetic abnormalities: FLT3, KIT and NPM, configuring the patient as an intermediate risk for AML. In the cytogenetic analysis there was no growth of metaphases. Patient received simultaneous diagnosis of AML and B-CLL. As he was ineligle to intensive chemotherapy (IC), we started original protocol Subcutaneous Cytarabin+venetoclax(VIALE C). The patient had grade 2-3 AE(neutropenia managed with GCSF) ending the fourth cycle in July 2020. The evolution of hematimetric parameters and diseases are described in graphics. DISCUSSION: This is the first described case in our knowledge treated upfront with bcl2-inh target therapy for two absolutely different hematological neoplasms: AML and BCLL. Nowadays we are experiencing a new therapeutic model in oncohematology, in which the targeted therapy is gaining ground in relation to IC with excellent results. In this way, the importance of comprehension of the pathophysiological mechanism of the neoplasms and the way we can stop the disease proliferation is progressively guiding the new protocols. Elderly patients are more likely to have early treatment-related death and exhibit therapeutic resistance, limiting alternatives. We decided to start first-line treatment with ARA-C and Venetoclax [2]. Venetoclax associated with ARA-C has a manageable safety profile, producing quick and durable remissions in elderly people with AML ineligible for IC, as well as in B CLL, being the best therapeutic alternative for the case, in our opinion. Venetoclax belongs to a group of drugs called Bcl-2 inhibitors, an anti-apoptotic protein, which works by blocking this protein in the body, causing apoptosis of both neoplastic cells. The high rate of remission and low early mortality, combined with fast and durable remission, make Venetoclax and ARA-C a new and attractive treatment for the elderly [2]. In our case, the intention of the product in the first line was not B-CLL, but it would certainly be a good option for this profile of elderly patients. CONCLUSION: We report the first description of simultaneous diagnosis of AML and B CLL treated with a Bcl-2 inhibitor, demonstrating that antitumor mechanisms can be extremely effective in completely different diseases. We have a long way to go in the search for full knowledge of oncohematological diseases and targeted therapies. However, this case report shows that we are on the right track.

Immunophenotyping of Baseline Bone Marrow Reveals a Specific Pattern of Immune Cells Associated with Greater Depth and Sustained Response in Newly Diagnosed Patients Randomized to Krd or Ktd Followed By Carfilzomib Maintenance or Control (AGMT MM 02 Study)

Immunophenotyping of Baseline Bone Marrow Reveals a Specific Pattern of Immune Cells Associated with Greater Depth and Sustained Response in Newly Diagnosed Patients Randomized to Krd or Ktd Followed By Carfilzomib Maintenance or Control (AGMT MM 02 Study)

Construction of a Novel CD34+ Hematopoietic Stem Cell Derived Xenograft Model of Human Hyperleukocytic Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an abnormal clonal proliferative disease of hematopoietic progenitors in the bone marrow, which is the most common malignant disease in the blood system. Hyperleukocytic leukemia (HLL) is one of the high-risk types and commonly defined as peripheral blood leukocyte counts exceeding 50-100×109/l. The patients with hyperleukocytic acute myeloid leukemia (HL-AML) usually suffer from poor prognosis due to low responsiveness to initial chemotherapeutic agents, so these patients have higher mortality with life-threatening complication than those without hyperleukocytosis. However, there is no available patient derived xenograft model (PDX) in HL-AML, especially the CD34+ hematopoietic stem cell derived xenograft model of human HL-AML. In this study, firstly, the primary HL-AML cells were collected and separated from the patients who underwent leukapheresis by using Fresenius COM.TEC machine, and the CD34+ hematopoietic stem cells were isolated from primary HL-AML cells using magnetic beads. Then, the NOD-SCID-IL2rγnull (B-NSG) mice were treated with 1.5 Gy χ-ray for total body irradiation at day 0, and intravenously injected with isolated 1.5 × 107 HLL cells or 1.5 × 106 CD34+ cells mixed with G-CSF within 24 h of irradiation. After inoculation, the blood smear, bone marrow smear, immunophenotyping and histopathology were used to test whether the model was successfully constructed. For construction of hematopoietic stem cell derived xenograft model in HL-AML, at day 9, the immunophenotypes of hCD45+ and hCD34+ cells were found and had higher ratios in CD34+ group than HLL group in peripheral blood, which suggested the model was successfully established. The body weight of mice in CD34+ group were decreased faster than the HLL group due to severe tumor load and the leukocyte counts were increased higher than the correspondent HLL group after cell inoculation. In CD34+ group, the blood routine examination had changed, especially the WBC counts and PLT counts. On the 25th day, leukemic cells were found by the blood smears analysis and detected by flow cytometry, and which were more obviously found in bone marrow, and the immunophenotypes ratio of hCD45+ and hCD34+ were increased in CD34+ group. A lot of CD34+ cells were infiltrated into bone marrow, liver and spleen tissues on the 25th day that detected by histopathological analysis, which indicated that the more obviously model construction using hematopoietic stem cell derived leukemic cell. In a conclusion, we successfully constructed a novel CD34+ hematopoietic stem cells derived animal model of human hyperleukocytic AML, which is available for mechanism research and drug treatment. Disclosures No relevant conflicts of interest to declare.

Clinical manifestations and diagnosis of acute myeloid leukemia in a patient with a medical history of pulmonary tuberculosis (a case report)

The aim is to familiarize practitioners with the initial clinical manifestations of acute myeloid leukemia and its diagnosis in a patient with a past medical history of pulmonary tuberculosis.Materials and methods. The article presents a clinical case of acute myeloid leukemia clinical manifestations based on our own clinical experience and its diagnosis in the patient with previous pulmonary tuberculosis. The patient was followed-up and diagnosed with acute myeloid leukemia in the Municipal Institution “Zaporizhzhia Regional Tuberculosis Clinical Dispensary”.Results. The patient presented in this study was diagnosed with pulmonary tuberculosis and received antimycobacterial treatment including isoniazid and rifampicin, which may have initially contributed to the hematopoietic mechanisms impairment. Perhaps the stressful situation triggered the development of acute myeloid leukemia due to underlying impaired mechanisms of hematopoiesis that had occurred. In this case, the initial clinical manifestations were weakness, loss of appetite and weight loss. A clinical blood analysis with leukogram and the bone marrow biopsy with aspirate examination became priority methods in diagnosing acute myeloid leukemia. The clinical case presented primarily demonstrates patient’s irresponsibility for their own health, who having symptoms such as weakness, loss of appetite, and weight loss for 5 months as well as past medical history of tuberculosis, did not seek medical advice timely.Conclusions. In a patient with a past medical history of pulmonary tuberculosis, priority task is to confirm or rule out reactivation tuberculosis. The initial clinical manifestations of acute myeloid leukemia are: unprovoked weakness for several months; increasing exertional dyspnea; anemia, leukopenia, neutropenia, ESR acceleration. The primary methods to diagnose acute myeloid leukemia are: clinical blood test with leukogram, biochemical blood tests to determine the levels of erythropoietin, ferritin and vitamin B12; bone marrow biopsy with aspirate examination (myelogram); cytochemical examination of bone marrow (myeloperoxidase); bone marrow immunophenotyping (expression of myeloid-associated antigens).

Morphological and immunofenotypic features of the monoclonal population of B-lymphocytes in chronic lymphocytic leukemia

Purpose of the study. To evaluate the features of morphological and immunophenotypic characteristics of the lymphoid population with different restriction of light chains of immunoglobulins in patients with chronic lymphocytic leukemia (CLL).Materials and methods. The study included 30 CLL patients aged 47–79 years (20 men and 10 women). All patients underwent a General clinical blood test (SysmexXE 2100, Japan), morphological examination of the bone marrow (BioVision; Micros, Austria), immunophenotyping of bone marrow and peripheral blood by flow cytofluorometry (Navios10/3, Beckman Coulter, USA). B-cell clonality established by detection of restriction of light chains of surface immunoglobulins kappa or lambda. Morphological analysis of lymphocytes that differ in the expression of light chains of surface immunoglobulins: kappa (k) — group I (22 people — 73,3%), lambda (λ) — group II (8 people — 26,7%).Results. Determination of cell types by values of direct (FSC) and lateral (SSC) light scattering during immunophenotyping of peripheral blood and bone marrow samples showed that in patients of group I (CD19k+/CD5+/CD23+) on the light scattering diagram, the lymphoid population had low parameters: on the FSC scale — from 200 to 400, on the SSC — from 10 to 160 units, which indicates morphological uniformity of cells. In group II (CD19λ+/CD5+/CD23+), on the contrary, on the light scattering sketogram, the lymphoid zone was heterogeneous and stretched: on the FSC scale — from 200 to 1000, on the SSC — from 10 to 400 units, which indicates morphological polymorphism of cells. There were also differences in the expression of the common leukocyte antigen CD45. In group I, the expression is higher: the population of B-lymphocytes in terms of fluorescence intensity is on the dot graph on the CD45 scale in the second half of the third decade and in the fourth decade — to the right, than in group II, in which B-lymphocytes lie in the third decade. The data indicate that the CD19k+/CD5+/CD23+ population is represented by Mature cells, while the Cd19k+/CD5+/CD23+ population is represented by less Mature and / or intermediate forms. Significant morphological differences in lymphocyte populations were also observed in microscopic studies of blood and bone marrow preparations.Conclusion. The established immunophenotypic and morphological differences in lymphoid populations expressing either kappa — or lambda-light chains of immunoglobulins may be important for identifying risk groups among patients with biologically heterogeneous variants of chronic lymphocytic leukemia.

Acute Renal Failure: A Rare Initial Presentation of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia has several presentations associated with bone marrow and extramedullary involvement. The unusual presentation may be due to the infiltration of leukemic cells in any organ. An 11-year-old girl presented with fever and vomiting since one day before admission after starfish biting during swimming. Her vital signs were: blood pressure 150/100 mmHg, pulse 98 beats per minute, respiration 18 breathes per minute, and temperature 37.2 °C (99 F). Laboratory work-up showed blood urea nitrogen 38 mg/dl and creatinine 2.8 mg/dl. In peripheral blood smear, few atypical cells, mild anemia (Hb: 9.2 g/dl), and mild 2 thrombocytopenia (Platelet: 109,000/μL) were detected. Bone marrow aspiration and immunophenotyping were in favor of acute precursor B cell type lymphoblastic leukemia. The patient had a favorable response to treatment after initiating high-risk chemotherapy. Therefore, acute renal failure can be a rare initial presentation of acute lymphoblastic leukemia, and azotemia will improve with an early chemotherapy treatment.

Immunophenotyping of Peripheral Blood, Lymph Node, and Bone Marrow T Lymphocytes During Canine Leishmaniosis and the Impact of Antileishmanial Chemotherapy.

Dogs are a major reservoir of Leishmania infantum, etiological agent of canine leishmaniosis (CanL) a zoonotic visceral disease of worldwide concern. Therapeutic protocols based on antileishmanial drugs are commonly used to treat sick dogs and improve their clinical condition. To better understand the impact of Leishmania infection and antileishmanial drugs on the dog's immune response, this study investigates the profile of CD4+ and CD8+ T cell subsets in peripheral blood, lymph node, and bone marrow of sick dogs and after two different CanL treatments. Two CanL groups of six dogs each were treated with either miltefosine or meglumine antimoniate combined with allopurinol. Another group of 10 clinically healthy dogs was used as control. Upon diagnosis and during the following 3 months of treatment, peripheral blood, popliteal lymph node, and bone marrow mononuclear cells were collected, labeled for surface markers CD45, CD3, CD4, CD8, CD25, and intracellular nuclear factor FoxP3, and T lymphocyte subpopulations were immunophenotyped by flow cytometry. CanL dogs presented an overall increased frequency of CD8+ and CD4+CD8+ double-positive T cells in all tissues and a decreased frequency of CD4+ T cells in the blood. Furthermore, there was a higher frequency of CD8+ T cells expressing CD25+FoxP3+ in the blood and bone marrow. During treatment, these subsets recovered to levels similar to those of healthy dogs. Nevertheless, antileishmanial therapy caused an increase of CD4+CD25+FoxP3+ T cells in all tissues, associated with the decrease of CD8+CD25−FoxP3− T cell percentages. These findings may support previous studies that indicate that L. infantum manipulates the dog's immune system to avoid the development of a protective response, ensuring the parasite's survival and the conditions that allow the completion of Leishmania life cycle. Both treatments used appear to have an effect on the dog's immune response, proving to be effective in promoting the normalization of T cell subsets.

Frequency of Discrepancy between Bone Marrow Morphology and Immunophenotype in Acute Leukaemia

Background: Acute leukaemia (AL) are a heterogenous group of haematological malignancy characterized by uncontrolled clonal proliferation of haematopoietic progenitor cells. Objectives: The study was conducted to have a detailed understanding of immunophenotyping profile, the frequency of discrepancy between bone marrow morphology and immunophenotyping and importance of immunophenotyping in diagnosis of acute leukaemia. Methods: This prospective type of observational study was carried out with an aim to correlate the immunophenotype with bone&#x0D; marrow morphology and to see the discrepancy between this two in acute leukaemia. A total of 38 untreated acute leukemia patients attending in the Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Dhaka, during the period from October 2016 to September 2017were included in this study. At first the morphological diagnosis was done. Then the immunophenotypic profile was compared. Result: Around eighty-two cases of acute leukaemia did find similarity with immunophenotyping and remaining 18.4% shows discrepancy. Diagnosis in this 18.4% changes after immunophenotyping. Aberrant phenotypes were detected in 20 (52.6%) samples among them 13 (34.21%) cases were AML, 3 (7.8%) cases were B-ALL and 4 (10.52%) cases were T-ALL. Significant relation was not found between aberrant marker and FAB subtypes. Conclusion: In acute leukaemia morphological appearance of bone marrow does not always match with immunophenotyping. It is therefore imperative and absolutely essential to ascertain the lineage of leukaemia by immunophenotyping before starting treatment.

Feasibility of evidence-based treatment of childhood acute myeloid leukemia in a Sub-Sahara Africa center.

e22508 Background: The proportion of Acute Myeloid Leukemia (AML) among childhood leukemias is putatively higher in Sub-Sahara Africa (SSA) compared to Western experiences. However, the treatment of childhood AML is challenging in SSA settings because of inadequate facilities for supportive care to deliver intensive AML regimens and, hence, high treatment-related mortality (TRM). Many pediatric oncology centers in SSA treat childhood AML with a palliative intent, with or without chemotherapy. At the Mulago National Referral Hospital (MNRH)/Global HOPE Center in Uganda, we treat AML with a curative intent. We distinguish Acute Promyelocytic Leukemia (APL) and AML with Down Syndrome (AML-DS) from other subtypes of AML. We treat APL with Children’s Oncology Group (COG) AAML1331 and AML-DS and other AML with modified reduced intensity regimens. We hereby share our experience of anti-cancer and supportive treatment, TRM, remission rates, and short-term overall survival (OS). Methods: We performed a retrospective cohort analysis of children diagnosed with AML between March 2019 and February 2020. The diagnosis of AML was established by bone marrow or peripheral blood morphology and immunophenotyping by flow cytometry according to WHO criteria. All cases of APL were confirmed by cytogenetics for t(15;17). Children without DS or APL received 2 cycles of Cytarabine 100mg/m2 12 hourly Days 1-10, Daunorubicin 50mg/m2 Days 1,3,5 and a single triple intrathecal chemotherapy for induction and 2 cycles of high dose Cytarabine 3000mg/m2 for consolidation. Supportive care comprised warm saline oral rinses, antiseptic baths, antimicrobial prophylaxis with cotrimoxazole, ciprofloxacin and itraconazole, and blood product support. Results: Of the 74 children diagnosed with leukemia 23 (31%) were AML; 4/23 (17%) of AML were APL and 3/23 (13%) were AML-DS. The mean age at diagnosis of AML was 6.1 (SD 4.3) years and 16/23 (70%) were males. For the 4 cases of APL, TRM 1/4 (25%) due to differentiation syndrome, remission rate 2/4 (50%), 1-year OS 75%; 95% CI 32.5-100. For the 3 cases of AML-DS, TRM 1/3 due to severe sepsis with respiratory failure, remission rate 1/3, and 1 was referred to another treatment center. For the other AML, TRM 2/16 (12.5%) due to severe sepsis, remission rate 9/16 (56%) and 1-year OS 78.6%; 95% CI 57.1-100. Conclusions: It is feasible to treat, manage TRM, and achieve remission in childhood AML in the SSA setting. Although there were several TRMs, a remission rate of 56% in the non-APL non-AML-DS is a promising outcome.

High CD38 expression in childhood T-cell acute lymphoblastic leukemia is not associated with prognosis.

Prognostic factors are not well exploited in childhood T-cell acute lymphoblastic leukemia (T-ALL). The aim of this study was to analyze the prognostic role of CD38 as well as minimal residual disease (MRD) and other biological factors in T-ALL. Immunophenotyping of bone marrow (BM) at diagnosis and MRD levels were determined using a standard panel of antibodies by 4-colour flow cytometry. A total of 96 children with T-ALL were enrolled. The results showed that 97.9% of T-ALL patients were positive for CD38 with a median level of 85.3%. CD38-high group had a worse early treatment response than the CD38-low group. However, CD38 levels were not associated with prognosis, albeit CD38-high group had a worse 5-year event free survival rate (55.1% vs. 66.6%, P> 0.05) and a higher 5-year cumulative incidence of relapse (35.6% vs. 19.8%, P> 0.05). Very high MRD levels (> 10%) were related to the worse survival. Neither flow cytometry based minimal residual disease (MRD) levels nor CD38 expression levels showed significant relation to the hazard of relapse (P> 0.05). We conclude that T-ALL has a high level of CD38 expression which is not associated with prognosis. Very high MRD level (> 10%) is related to the worse survival, however, FCM based MRD detection does not convey a significant prognostic value.

Leukemia cutis: A study from a tertiary care hospital in North India

Background/Objectives: Only anecdotal case reports or small case series have attempted to study the histomorphology patterns of leukemic infiltration. This study explored the possible association of leukemia cutis with, temporal course of disease, subtypes of leukemia, blast count, total leukocyte count, lactate dehydrogenase (LDH) levels, and histopathological patterns. Materials and Methods: Clinical and laboratory data were retrieved from the hospital information system. Selected cases were reviewed by two pathologists. Data in terms of demographics, clinical presentation, bone marrow findings, immunophenotype, and temporal course were recorded where available. Results: Twenty-three diagnosed cases of leukemia cutis were reported during the study period. There was no gender predilection with a wide age range (8–82 years). No particular trend was noted in relation to total leukocyte or bone marrow blast count. Common skin manifestations included multiple erythematous papular eruptions, frequently involving the skin of the limbs. All cases showed a consistently high serum LDH level. 11/23 (48%) showed cutaneous manifestation of undiagnosed leukemia or indicated worsening in a diagnosed case. The histological patterns varied from perivascular to diffuse dermal interstitial infiltration. In addition, subtle changes were seen in the form of fibrosis, thrombosis, and epidermotropism in 12%–50% of cases. The judicious use of a panel of immunohistochemistry was vital to establish a diagnosis. Conclusions: Leukemia cutis is a diagnostic challenge on histopathology due to lack of any specific pattern in absence of a clinical suspicion. Its diagnosis may be an indicator of undiagnosed leukemia or worsening in an already known case of leukemia.

B Cell Specific Knockdown of the Erythropoietin (EPO) Receptor Attenuates EPO-Induced Bone Loss in Mice

Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production, commonly used in clinical practice to treat certain forms of anemia. Our studies and those of others have demonstrated that EPO administration induces substantial trabecular bone loss. We proposed that EPO-induced bone loss is partially mediated by subsets of bone marrow (BM) B cells that express EPO-R. Mechanistically, EPO upregulates the surface expression of RANKL by BM B cells and augments B cell-derived osteoclastogenesis in vitro. We showed that the latter is likely mediated by pro-B cells expressing the MCS-F receptor (CD115) and capable of transdifferentiation to osteoclasts (Abstract # 1007, EHA 2017). Here we address the role of B cell-specific EPO-R in EPO-induced bone loss (i.e. at supra-physiological EPO levels). Moreover, we demonstrate, for the first time, the occurrence of B cell-derived osteoclastogenesis in vivo, a finding of critical importance in the field of osteohematology. Methods: In order to trace the B cell lineage from its earliest precursors, we used the MB1-Cre mouse line combined with either the R26R-EYFP or the EPO-Rfl/fl mice for lineage tracing and B cell-specific EPO-R knockdown, respectively. Sequential fluorescence and light microscopy were used for the demonstration of B cell-derived osteoclastogenesis in vivo. Human recombinant EPO was administered in vivo at a dose of 180IU thrice weekly for two weeks. Immunophenotyping of BM B cell populations was assessed by multi-color flow cytometry. Results: Using female MB1-Cre; EPO-Rfl/fl (cKD) mice, we found that B cell-specific EPO-R knockdown attenuated the profound EPO-induced trabecular bone loss in the proximal part of the femoral distal metaphysis (proximal BV/TV 0.034±0.012% vs 0.007±0.003% in the cKD vs control mice, p&amp;lt;0.05, Figure 1). Remarkably, this effect was observed despite the fact that cKD mice attained higher hemoglobin levels following EPO treatment (21.1±0.1 mg/dL vs 20.4±0.2 mg/dL in the cKD vs control mice, p&amp;lt;0.05). An EPO-induced increase in CD115+ Pro-B cells was observed in EPO-treated control mice but was absent in the cKD mice. The latter finding correlates with the observed bone loss and indicates that the increased number of MCSF-R-expressing pro-B cells is dependent on B cell EPO-R. Supporting the osteoclastic potential of this specific B cell subpopulation is the fact that most of the CD115+ Pro-B cells also express β3 integrin (CD61) which is essential for osteoclast differentiation and function. Using the MB1-Cre;R26R-EYFP murine model for B cell lineage tracing, we could demonstrate that some of the TRAP+/ β3 integrin+ bone lining cells were also positive for EYFP (Figure 2). This demonstrates the B cell origin of some of the osteoclasts in vivo. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling that regulates bone homeostasis and might also indirectly affect EPO-stimulated erythropoietic response. The relevance and the mechanisms of the latter phenomenon merits further investigation. Importantly, we present here, for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo, thus opening novel research avenues. DN and YG Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and by the Israel Science Foundation (ISF) Grant No. 343/17 to DN. Disclosures Mittelman: Novartis: Honoraria, Research Funding, Speakers Bureau.

The Relationship between Baseline Biomarkers and Efficacy of Isatuximab in Combination with Pomalidomide and Dexamethasone in RRMM: Insights from Phase 1 and Phase 3 Studies

The Relationship between Baseline Biomarkers and Efficacy of Isatuximab in Combination with Pomalidomide and Dexamethasone in RRMM: Insights from Phase 1 and Phase 3 Studies

Depletion of the Long Non-Coding RNA MALAT1 primes Chronic Myelomonocytic Leukemia (CMML) for Differentiation Therapy with All-Trans retinoic Acid (ATRA) through the Transcription Factor CREB

Improvements in antisense technology have now enabled clinically relevant therapeutic credentialing of the noncoding genome. MALAT1 is a long non-coding RNA that, among other functions, is thought to serve as a nuclear scaffold for splicing and transcription factors. MALAT1 expression is associated with inferior prognosis across solid tumors and its depletion impairs proliferation and metastasis in preclinical solid tumor models. We found that elevated MALAT1 levels are independently associated with inferior overall survival in patients with CMML. Further, RNA-sequencing of primary CMML monocytes identified MALAT1 as the fourth most over-expressed transcript compared to controls. Therefore, we explored the biologic relevance and therapeutic candidacy of MALAT1 across several human and murine models of CMML. First, we crossed NRASQ61R/+Mx1-cre driven mice, which display a CMML-like phenotype, to MALAT1KO/KOmice. Although MALAT1KO/KOmice did not have abnormalities in complete blood counts, immunophenotyping of the hematopoietic stem cell (HSC) compartment identified statistically significantly lower numbers of HSC compared to wild type (WT) controls and a non-significant decrease in NRASQ61R/+/MALAT1KO/KOcompared to NRASQ61R/+alone. This decrease in HSC was not a result of impaired self-renewal as no differences were observed in these models after in vivo competitive transplant experiments. Therefore, we reasoned that MALAT1 expression may be controlling HSC differentiation. To test this, we transformed bone marrow cells from these models with an estrogen-regulated (ER) Hoxb8 construct enabling cells to maintain an HSC state until ER is withdrawn and myeloid differentiation is induced. ER-Hoxb8NRASQ61R/+/MALAT1KO/KOcells had increased basal levels of Gr-1 compared to ER-Hoxb8 transformed NRASQ61R/+alone that was dramatically enhanced upon ER withdrawal suggesting that MALAT1 depletion regulates myeloid differentiation. These findings were validated by assessment of morphology, transcriptome, and in vivo immunophenotyping of bone marrow and spleen cells. Further, moribund NRASQ61R/+/MALAT1KO/KOmice displayed a reduction in organomegaly typically associated with leukemic burden. We validated this in human monocytic leukemia by generating MALAT1 depleted THP-1 isogeneic cell lines using the CRISPR/Cas9 system. MALAT1 depleted THP-1 cells (MKO) had greater terminal differentiation according to immunophenotypic markers and morphology that was greatly enhanced when treated with phorbol myristate acetate. Last, MKO orthotopic xenografts demonstrated inferior human leukemia engraftment and decreased spleen and liver weights, and heterotopic xenografts exhibited reduced tumor volume, collectively suggesting diminished leukemic burden. Because ATRA has been clinically tested in CMML with minimal effects, we next explored whether MALAT1 depletion could potentiate ATRA differentiation in CMML. First, we treated MKO cells with ATRA and observed a large induction of myeloid differentiation by marker expression and morphologic assessment compared to isogenic controls. This was validated by NRASQ61R/+/MALAT1KO/KOmice demonstrating that ATRA more robustly induced differentiation compared to vehicle which was not seen in NRASQ61R/+/MALAT1+/+mice. Next, we tested MALAT1 antisense oligonucleotides (ASOs) currently under clinical development in THP-1 cells +/- ATRA and demonstrated both an increase in myeloid differentiation and apoptosis compared to ATRA alone. To test this therapeutic strategy in primary CMML specimens, we generated CMML patient-derived xenografts (n=30 mice) and treated each with ASO, ATRA, the combination, or controls and identified a more robust reduction in human HSC engraftment with the combination. To explore the mechanistic basis for these findings, we performed RNA-sequencing of MALAT1-depleted or control cells and identified that CREB target genes were differentially expressed. Basal protein levels of p-CREB were also decreased in MKO cells and were further reduced in the nucleus of MKO by western and microscopy. Lastly, overexpression of WT or constitutively active CREB but not its dominant negative rescued the differentiation effect seen in ATRA treated MKO cells. Taken together, MALAT1 is a novel, CREB-dependent regulator of myeloid differentiation and its depletion potentiates ATRA therapy. Disclosures Cluzeau: Menarini: Consultancy; Jazz Pharma: Consultancy; Abbvie: Consultancy. Komrokji:celgene: Consultancy; pfizer: Consultancy; DSI: Consultancy; JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy. MacLeod:Ionis Pharmaceuticals: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Epling-Burnette:Forma Therapeutics: Research Funding; Celgene Corporation: Patents &amp; Royalties, Research Funding; Incyte Corporation: Research Funding.

A case of acute monocytic leukemia complicated with multiple myeloma

A case of acute monocytic leukemia complicated with multiple myeloma

Myelodysplastic syndromes combined with mature plasmacytoid dendritic cells proliferation: report of one case and review of literature

Objective To investigate the clinical features, diagnosis, treatment and prognosis of myelodysplastic syndromes (MDS) with mature plasmacytoid dendritic cells (PDC) proliferation. Methods The clinical data of one case of MDS with excess blasts (EB)-1 with mature PDC proliferation in Air Force Medical Center was retrospectively analyzed, and the literature was reviewed. Results The patient's physical examination revealed anemia and thrombocytopenia. Bone marrow smears showed 0.064 of myeloblasts and 0.152 of dendritic cells. Immunophenotyping showed two groups of abnormal proliferation cells, namely, myeloblasts and mature PDC. Decitabine treatment was given, and the red blood cells and platelets were infused intermittently. The condition of patient was basically stable. Conclusions MDS with mature PDC proliferation is extremely rare. No special clinical manifestations are found, and the diagnosis is based on bone marrow cytology and immunophenotyping. There is no standard regimen for treatment of MDS with mature PDC proliferation, and the prognosis depends on the progression of MDS. Key words: Myelodysplastic syndromes; Plasmacytoid dendritic cells; Diagnosis; Treatment

Acute Myeloid Leukemia After Treatment of Early Breast Cancer: Case Report and Literature Review

Therapy-related acute myeloid leukemia has been widely accepted for decades as one of the most serious long-term complications of treatment for early breast cancer. We report the case of a patient with therapy-related acute myeloid leukemia that may have been caused by regional radiation, chemotherapy, or endocrine therapy after surgery. A 35-year-old female was diagnosed 7 years ago as having stage IIA left breast cancer; she was treated by conservative surgery, followed by chemotherapy, radiotherapy, and endocrine therapy for 5 years. After 1 year of active follow-up, she presented with complaints of mild fever, weakness, loss of appetite, weight loss, and multiple bruises all over the body. Complete blood count revealed a hemoglobin level of 4 g/dl and total leukocyte count of 40,000 cells/mm3, with multiple blast cells in peripheral smear. Bone marrow biopsy and immunophenotyping established the diagnosis as AML. Before starting the treatment, she died following multiple organ failure.

Abstract 1340: High NK cell number predicts poor overall survival in de novo treatment naive acute myeloid leukemia

Abstract Introduction Acute myeloid leukemia (AML) is a deadly disease associated with high mortality and morbidity. Previous studies report defects in NK cell maturation and function in AML patients leading to innate immune evasion. Considering that the number of NK cells may be an important indicator of innate immune regulation of AML, our group sought to understand the prognostic significance of NK cell number in de novo AML patients. Methods Patient samples were obtained from the Oregon Health and Science University (OHSU) Pathology Department between September 2010 and March 2016. This cohort study includes 82 newly diagnosed adult AML patients who had a bone marrow biopsy that was subjected to flow cytometry and a targeted-NGS panel within 30 days prior to induction treatment. Patients were excluded if they had antecedent hematological diseases or therapy-related AML. Bone marrow immunophenotyping was performed using the Ion Torrent PGM platform. Absolute numbers of NK cells were estimated from flow cytometric analysis of the bone marrow samples whereby lymphocytes were identified based on CD45 staining and light side scatter and NK cells were defined as CD3-/CD56+. Results This retrospective cohort of 82 newly diagnosed AML patients were 53.7% male with a median age of 61 years (range 18 - 83). Using 2017 ELN risk stratification guidelines, 36.6% of patients were classified as favorable, 26.8% as intermediate, and 36.6% as adverse. Absolute NK cell numbers in the bone marrow ranged from 1 to 896 cells/μl with a median of 98. When stratifying NK cells by staining intensity to identify subpopulations, the number of CD56 bright NK cells ranged from 0 to 69 cells/μl (median 3) and the number of CD56 dim NK cells ranged from 1 to 585 cells/μl (median 77). Overall survival (OS) was measured from diagnosis to date of death or last contact and varied from 1 day to 5.7 years (median 20.5 months). There were 3 deaths in the first week after diagnosis. Separate Cox proportional hazards regression models were applied to each NK cell group in both the univariable and multivariable settings, with the latter models accounting for patient age and ELN risk. Higher absolute numbers of NK cells (CD3-/CD56+) were significantly correlated with worse OS in both model settings, with a multivariable p-value of 0.001. The number of bright NK cells was a significant predictor of OS in the univariable model but lost significance when controlling for other variables. However, similar to the number of all NK cells and contrary to previous reports, higher counts of dim NK cells were significantly associated with shorter survival times, with a multivariable p-value of 0.001. Conclusions In this study, we show that higher NK cell numbers at diagnosis are associated with worse OS and thus play an important role in prognostication for AML patients. This association with survival is most evident in patients at least 60 years old or those with intermediate or adverse ELN risk. Citation Format: Weiwei Wang, Guang Fan, Andy Kaempf, evan Lind, Tomi Mori, Byung Park, Jennifer Saultz. High NK cell number predicts poor overall survival in de novo treatment naive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1340.

Abstract 3100: Blinatumomab is safe and effective in relapsed and MRD positive B-ALL CD19+ patients: The bologna compassionate program experience

Abstract Adult B-ALL patients still have a dismal prognosis, due to a high incidence of relapse even after allogenic SCT. Safety and efficacy of Blinatumomab, an anti CD3-CD19 Bite antibody, has been demonstrated both in MRD positive patients and in relapsed/refractory (R/R) setting. To evaluate safety profile and efficacy of Blinatumomab, obtained through a compassionate use, in a cohort of 18 adult patients affected by MRD+ or R/R B-ALL treated at Bologna University. From March 2015 to December 2017, 18 patients received Blinatumomab at the standard dosage (9 mcg/d x 7 days, 28 mcg/d x 21 days) in 28-days courses. All the patients were hospitalized to receive the first course of therapy. The following courses, based on the good safety profile of the compound, were administered in outpatient setting. 18 patients (M/F = 10/8; median age 43, range 18-73) have been treated. Philadelphia (Ph) chromosome was detected in 8/18 patients. 10 patients were MRD+ (5 Ph pos and 5 Ph neg); E2A-PBX1 and MLL-AF4 rearrangements were found in two patients. 8 patients had a R/R disease (3 Ph pos and 5 Ph neg). Median WBC count before starting therapy with Blinatumomab was 5400/mmc (range 500-76500). All the patients had previously received many lines of therapy (median 4, range 1-7). In 4 cases an alloTMO was already performed, and two patients had received two transplants. 12/18 patients were referred to us by other Italian Institutions. All the patients received at least one course of Blinatumomab. In one case three courses were administered; an elderly patient is actually receiving the fifth course. Globally, 32 courses of therapy have been administered (median 2, range 1-5). Bone marrow evaluations, including cytogenetics, molecular biology and immunophenotyping analysis were performed at the beginning of every course of therapy in order to assess patients' disease status. MRD evaluation was assessed through BCR-ABL fusion transcript quantitative analysis in Ph pos ALL patients and Ig rearrangment in Ph negative patients. Monitoring of adverse events was periodically performed. 16/18 patients are evaluable for response, at least to one cycle (one patient died during the first course, one patient is still receiving the first course). 9/16 (56%) patients obtained a CR (7/9 MRD+ and 2/7 R/R). In 7/9 (78%) responders patients a molecular CR was reached, (in 6 patients after the first course, in one case after the second one). 5 responders proceeded to alloBMT and are actually alive in CR (median follow-up after transplant 240 days). In terms of toxicity, one patient developed a grade IV neurological event (mental confusion, tremor), which completely resolved after a transient drug withdrawal. Our results confirm the high rate of response and to Blinatumomab in a poor patients' population, and the good management profile of the compound. Note: This abstract was not presented at the meeting. Citation Format: Giovanni Martinelli, Stefania Paolini, Claudio Cerchione, Alessandra Santoro, Valentina Robustelli, Simona Soverini, Caterina De Benedittis, Enrica Imbrogno, Andrea Ghelli Luserna Di Rora, Sarah Parisi, Chiara Sartor, Giovanni Marconi, Silvia Lo Monaco, Maria Chiara Abbenante, Maria Chiara Fontana, Antonella Padella, Anna Ferrari, Giorgia Simonetti, Elena Tenti, Federica Frabetti, Francesca Volpato, Samantha Bruno, Fabiana Mammoli, Maria Teresa Bocchicchio, Carmen Baldazzi, Cristina Papayannidis. Blinatumomab is safe and effective in relapsed and MRD positive B-ALL CD19+ patients: The bologna compassionate program experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3100.