Bone Marrow Immunophenotyping Research Articles

Exploring the Relationship between CD66c Expression and Clinico-Haematological Parameters in Pediatric B-Cell Acute Lymphoblastic Leukemia (ALL)

Background: Pediatric B-cell Acute Lymphoblastic Leukemia presents with diverse clinical and hematological manifestations. Understanding these characteristics is crucial for effective management and prognostication.  Methods: This study conducted as prospective observational study at Lady Hardinge Medical College and Kalawati Saran Children Hospital, New Delhi, from November 2017 to March 2019. This study enrolled 30 pediatric patients with B-cell ALL who underwent comprehensive evaluations including routine and specialized hematological tests, bone marrow aspiration (BMA), cytochemistry, and immunophenotyping. Clinical features, hematological parameters, and CD66c expression on lymphoblasts were assessed.  Results: Immunophenotyping revealed that 28 cases (93.3%) were CALLA positive (CD10 positive), while 2 cases (6.7%) were CALLA negative (CD10 negative). The age ranged from 1.5 to 12 years, with a 2:1 male to female ratio. The mean age was 4.8 years.. Common presenting complaints included fever (93.3%) and pallor (60.0%). Hepato-splenomegaly (60.0%) and lymphadenopathy (60.0%) were the most frequent clinical findings. Hematological findings showed moderate to severe anemia (mean hemoglobin 7.19 g/dL), with 56.7% of patients having hemoglobin levels between 4.0-7.9 g/dL. Leukocytosis (>10,000/cumm) was observed in 53.3% of cases, with 20% having total leukocyte counts >50,000/cumm. Thrombocytopenia (platelet count ≤ 25,000/cumm) was noted in 56.7% of patients. Immunophenotyping revealed CD66c expression >20% gated in 46.7% of cases, correlating with more severe clinical features and poorer hematological parameters.  Conclusion: Pediatric B-cell ALL exhibits a spectrum of clinical and hematological features. Elevated CD66c expression correlates with aggressive disease presentation and worse clinical outcomes, suggesting its potential utility as a prognostic marker. Early identification of these parameters can aid in risk stratification and tailored therapeutic approaches.

Spectral Flow Cytometry Methods and Pipelines for Comprehensive Immunoprofiling of Human Peripheral Blood and Bone Marrow.

This study introduces optimized methods and analysis algorithms that enhance capabilities in comprehensive immunophenotyping of human blood and bone marrow using spectral flow cytometry. This approach facilitates detection of rare cell types, enables measurement of cell variations across donors, and provides proof-of-concept in identifying known hematologic malignancies. By unlocking complexities of hematopoietic and immune landscapes at the single-cell level, this advancement holds potential for understanding disease states and therapeutic responses.

Feasibility, accuracy, and usability analysis of MapAML, a first-in-class app for integrated diagnosis in acute myeloid leukemia.

Performing a comprehensive diagnosis of acute myeloid leukemia (AML) is complex and involves the integration of clinical information, bone marrow morphology, immunophenotyping, cytogenetic, and molecular analysis, which can be challenging to the general hematologist. The aim of this study was to evaluate the usability and accuracy of MapAML, a smartphone app for integrated diagnosis in AML, created to aid the hematologist in its clinical practice. App performance was evaluated in dedicated sessions, in which 21 hematologists or fellows in hematology performed an integrated diagnosis of deidentified real-world clinical AML cases, first without and posteriorly with MapAML use. Diagnosis accuracy increased after MapAML utilization, with the average score going from 7.08 without app to 8.88 with app use (on a scale from 0 to 10), representing a significant accuracy improvement (p = .002). Usability evaluation was very favorable, with 81% of users considering the app very or extremely simple to use. There was also a significant increase in confidence to perform a complete and accurate diagnosis in AML after app use, with 61.9% of the participants willing to use the app in their clinical practice. In this study, MapAML increased accuracy with excellent usability for integrated diagnosis in AML.

Diagnostic and Prognostic Value of DNA Repair Gene XRCC1 ARG399GLN Polymorphism in Childhood Acute Lymphoblastic Leukemia: A Single-Center Egyptian Study

Background Repair pathways for DNA damage are essential for repairing various forms of DNA damage. Leukemia risk may be increased by poor DNA repair. This study was conducted to better understand the significance of XRCC1Arg399 Gln polymorphisms in acute lymphoblastic leukemia (ALL) susceptibility and prognosis. Patients and methods Twenty children with ALL participated in the pediatric trial at Tanta University. Thirty healthy controls were of the same age and sex. Complete blood counts, bone marrow aspirates, immunophenotyping, flow cytometry, and XRCC1 genotyping by PCR were performed. Results The allele Gln/Gln was found in one (3.3%) of the controls and two (10%) cases had an odds ratio of 0.13 (0.27–38.1), P=0.34. Eight (40%) patients and six controls [odds ratio of 2.66 (0.75–9.4), P=0.11], had the Arg/Gln allele. Finally, 23 (76.7%) members of the control group and 10 (50%) patients carried this allele (Arg/Arg). The genotype’s prognostic value for alleles: two (20%) of patients with the allele (Arg/Arg) were refractory. Two (25%) patients died during the induction for the allele (Arg/Gln), while four (50%) patients were refractory. Last but not least, no one experienced total remission under the allele (Gln/Gln). Conclusion The XRCC1 polymorphism is not linked to the development of ALL; nevertheless, having mutant alleles was linked to a poorer prognosis.

Clinical Characteristics and Diagnosis of Ph-Positive Mixed Phenotype Acute Leukemia.

The goal was to improve the clinical cognition of Ph-positive mixed phenotype acute leukemia and avoid misdiagnosis or delayed diagnosis. The clinical manifestations and laboratory results (bone marrow cell morphology, multiparameter flow cytometry, and cytogenetics) of a case of Ph-positive mixed phenotype acute leukemia were analyzed, and related literature was reviewed. Blood routine: WBC 386.35 x 109/L, HGB 117.00 g/L, PLT 31 x 109/L; 80% of the original cells can be seen by artificial classification. Morphological examination of bone marrow cells showed that the proliferation of nucleated cells was obviously active, and the original cells accounted for 76%. The size of the original cells was somewhat uniform, most of the cells had less mass, were stained light grayish blue, the cytoplasm particles were not obvious, the nuclei were mostly round or quasi-round, some of them showed distortion and nuclear notch, and the chromatin was coarse. Some of the cells were rich in mass, small azurin granules were seen, the nuclei were regular, most of them were round, the chromatin was fine, the myeloperoxidase and esterase staining were negative, the eosinophils accounted for 2.5%, and the basophils accounted for 0.5%. Flow cytometry immunotyping: Two groups of abnormal cells were seen in the bone marrow. 1. A group included 12.32% of nuclear cells and showed abnormal myeloid primitive cell phenotype. Main expression: CD117, CD34, CD38, HLA-DR, CD33, CD64, CD123, weak expression: CD13, CD19. 2. The other group included 45.61% of the nuclear cells and had a B-lymphoblastic phenotype. Main expression: CD34, CD38, HLA-DR, CD123, CD19, CD10, CD9, cCD79a, TDT, weak expression of CD13, CD22. Mixed phenotype acute leukemia (M/B) immunophenotype was considered. Chromosome: 46,XY,t(9; 22)(q34;q11.2) [20]. BCR-ABL (P210) fusion gene was positive. Mixed phenotype acute leukemia (MPAL) is a rare type of malignant hematologic disease. Its diagnosis is based on the comprehensive evaluation of bone marrow cell morphology, immunophenotype, molecular and cytogenetic features.

Assessing Pancytopenia in Leukemia Patients through flow Cytometry and ELISA to Evaluate the Complete Blood Counts and Cluster of Differentiation Markers

To diagnose cases involving pancytopenia or leukopenia, a comprehensive assessment of various factors is necessary, including blood count, peripheral blood, and bone marrow analysis, immunophenotyping, and cytogenetics. This study aims to examine the complete blood count parameters and CD markers in Sudanese patients with leukemia and pancytopenia, utilizing flow cytometry and ELISA techniques. This study is a laboratory-based addressing the assessment of the target population (acute Leukaemia with pancytopenia) by complete blood count, flow cytometry, and ELISA techniques. The research group was comprised of patients who were diagnosed with acute leukemia and had pancytopenia before undergoing treatment. Another group of patients with acute leukemia but without pancytopenia was also included. In addition, there was a control group consisting of healthy individuals who volunteered for the study. Essentially, the control group was made up of healthy individuals who were not affected by acute leukemia or pancytopenia. In our study, we enrolled a total of 150 participants, comprising three groups: 50 cases of acute leukemia, 50 patients with acute leukemia who subsequently developed pancytopenia, and 50 healthy volunteers. The majority of participants were female, constituting 56% of the sample (84 individuals), while the most prevalent age group represented was individuals aged 65, accounting for 43.3% of the participants. Our analysis revealed a statistically significant correlation between age and both leukemia and leukemia with pancytopenia, with a p-value of 0.00. Furthermore, the presence of either AML or ALL also exhibited a substantial association with the disease, indicated by a p-value of 0.00. Specifically, the use of a flow cytometer allowed us to identify the presence of CD3 with a p-value of 0.00 and CD4 with a slightly higher p-value of 0.04. Improvement of patient management by introducing effective tools for predicting prognosis is the key to success in managing diseases. We recommend that flowcytometry be used routinely to diagnose leukemia and leukemia with cytopenia in patients at all stages of the disease.

Unilateral Orbital mass in a 3-Year-Old Boy of Acute Lymphoblastic Leukemia : A Rare Presentation

Background & objective: We report a case of a 3-year-old boy who presented with left eye swelling, low-grade fever, pallor, and generalized lymphadenopathy. He was diagnosed with a case of B-cell acute lymphoblastic leukemia (ALL) on the basis of morphology and immunophenotyping of bone marrow. Orbital manifestation is rarely seen in ALL; rather it is more common in acute myeloid leukemia (AML). Knowledge regarding the unusual presentation of ALL will help timely diagnosis and treatment which is crucial for preserving the eye as well as the vision. Ibrahim Card Med J 2022; 12 (2): 64-66

A Deep Learning Framework for Classification of Immature White Blood Cells in Acute Myeloid Leukemia Using Convolutional Autoencoder and Transfer-Learning Ensemble

Acute myeloid leukemia (AML) is a type of acute leukemia that affects myeloid cells. It is characterized by uncontrolled proliferation of immature WBCs causing an accumulation of immature WBCs in the bone marrow and peripheral circulation. AML is an aggressive cancer that can be life-threatening if not treated promptly, therefore, early detection is crucial. Identification of immature WBCs and their subtypes is the first step towards AML diagnosis. Microscopic blood smear image analysis is commonly used to diagnose AML as it is less expensive non-invasive diagnostic tool compared to bone marrow biopsy and immunophenotype. Nonetheless, classifying immature WBCs presents challenges due to their high similarity and minimal interclass variation, especially for intermediate stages of myelopoiesis which make is susceptible to misclassification. The current diagnostic methods of AML are based on manual identification of immature WBCs using peripheral blood smear. These methos are time-consuming, prone to errors, and subject to inter-observer variation. Therefore, this study aims to develop a computer-aided diagnostic framework to accurately identify immature WBCs and classify them into various subtypes. The proposed framework consists of two main components: a binary classification system to classify cells into mature versus immature cells and a multiclassification system to further classify immature cells into four subtypes including myeloblast, monoblast, and promyelocytes. The proposed framework was designed to undergo four distinct phases: the first phase involves preprocessing, which includes data augmentation techniques aimed at addressing the imbalance distribution of WBCs. Augmentation encompasses geometric transformation and generation of synthetic images using convolutional Autoencoder (CAE). Feature extraction phase involves using image embedding and transfer-learning. Image embedding representation was obtained using CAE and then utilized by two pre-trained models: VGG19 and Resnet50. Classification phase was carried out using weighted ensemble of the VGG19 and Resnet50 with the optimal weights determined through Grid search. The validation phase evaluated the model performance using overall accuracy, precision, and sensitivity. The area under the ROC curve (AUC) was used to assess model discrimination ability. The proposed framework demonstrated excellent results, achieving an average accuracy of 99.5%, a sensitivity of 97.97%, and a precision of 94.12%. The model exhibited an excellent discriminatory capability, with an AUC of 99.9%. Furthermore, the proposed framework outperformed previous methods, showcasing improved performance. Key word: Acute myeloid leukemia, peripheral blood smear, immature white blood cells, deep learning, feature extraction, computer-aided diagnosis.

Novel, Engineered, Modified Fcγ Receptor I (CD64)-Expressing T Cells (SolidT) Combined with Rituximab Exhibited Profound Anti-Tumor Activity and Memory in a CD20+ Raji Xenograft Model of Lymphoma

Background: CD19 CART cell therapy has been a major advance in the treatment of lymphoma, but is limited due to profound toxicity and relapse, in part due to loss of CD19 expression on malignant cells. Following our discovery of FcγRI (CD64)-expressing T cells in tumors and inflamed tissues (PMID 31449054), we developed novel, engineered cytotoxic T cells (“SolidT”) with increased specificity, reduced exhaustion, and attenuated cytokine secretion compared to other engineered cell therapies (e.g., CAR-T, CAR-NK). SolidT are produced by transducing a retroviral vector encoding an enhanced FcγRI into T cells for use with tumor-targeting monoclonal antibodies (mAbs). SolidT activation correlates with surface tumor antigen density. Unlike CART, where tonic signaling causes premature exhaustion and limited tumor infiltration, SolidT are not exhausted prematurely. They are activated and persist only by mAb bound to tumor-specific antigen at sufficient density, and they take on a T effector memory (T EM) phenotype. We previously showed that SolidT + mAb has anti-tumor activity in vitro and in vivo in several tumor models, including immunodeficient HER2 xenografts and an immunocompetent syngeneic B16-TYRP1 model (PMID 37070661). To further assess the SolidT platform, we examined the effect of SolidT + rituximab (R) in vitro and in vivo in a CD20+ Raji-Luciferase (Luc) xenograft model. Methods: In in vitro Raji-Luc and Daudi-Luc models, SolidT + R was compared to CD19 CART, R + sham transfected T cells (Sham), Sham only, and SolidT only. Cytotoxicity was measured by flow analysis of Annexin V and/or Luc assay. IFNγ and Granzyme B were measured by ELISA. Addition of intravenous immunoglobulin (IVIG; 5mg/ml) to cytotoxicity co-cultures was used to evaluate the effect of non-specific IgGs on the efficacy of the combination treatment. In vitro safety was evaluated in a similar manner using primary cultures of kidney and lung epithelial cells. To assess the anti-tumor activity of SolidT, NSG mice were inoculated intravenously (IV) with 1x10 6 Raji-Luc cells (n=8). SolidT (1x10 7 cells) was given on Day (D) 1. R (250 mg) was given on D1 and then weekly (125 mg). Mice were monitored for weight, clinical signs, and survival until D41 when the experiment was terminated to conduct endpoint analyses including gross pathology, histopathology, and CD3 + immunohistochemistry (IHC). Results: In vitro, SolidT + R showed higher cytotoxicity and attenuated cytokine levels against Raji-Luc compared to CD19 CART cells. Similar results were obtained in a Daudi-Luc model. Addition of IVIG did not activate SolidT cells, and though IFNγ secretion was attenuated, cytotoxicity was not affected. The in vitro co-culture of SolidT cells with primary epithelial cells demonstrated no activation of the effector cells and no death of primary target cells. In vivo, mice treated with SolidT + R had increased survival and reduced tumor burden against Raji-Luc compared to mice treated with R alone (Figure 1). On pathology, 5/8 SolidT + R mice had no tumors detected and 3 had only abdominal tumors. R mice developed tumors in the lungs (2/8), liver (7/8), and abdomen (4/8). Bone marrow immunophenotyping revealed that the presence of T EM phenotype highly correlated to better survival and lower tumor burden. Gross pathology analysis revealed no treatment-related pathology. IHC showed increased inflammation, lymphocyte infiltration, and necrosis in tumors from SolidT + R-treated mice, as well as intense infiltration of CD3 + human cells in the tumors at end point D41. The in vivo Raji-Luc model was performed twice with similar results. Conclusions: Consistent with prior results in solid tumor models targeting HER2 or TYRP1, these data demonstrated that the SolidT platform could be effectively targeted against CD20+ cell lines. A single administration of SolidT cells in combination with R prevented tumor formation and prolonged survival in a CD20+ Raji-Luc model. This experiment demonstrated that marrow-resident SolidT cells developed a T EM phenotype that positively correlated with outcome. These data confirm that the SolidT platform can be targeted against different tumor antigens by using different tumor-targeting mAbs. The SolidT platform could represent a paradigm shift in the approach to engineered cellular therapy for hematological malignancies and solid tumors.

Reduced protein intake and aging affects the sustainment of hematopoiesis by impairing bone marrow mesenchymal stem cells

Protein malnourishment (PM) is common among the elderly, but how aging and PM impact hematopoiesis is not fully understood. This study aimed to assess how aging and PM affect the hematopoietic regulatory function of bone marrow (BM) mesenchymal stem cells (MSCs). Young and aged male C57BL/6J mice were fed with normoproteic or hypoproteic diets and had their nutritional, biochemical, and hematological parameters evaluated. BM MSCs were characterized and had their secretome, gene expression, autophagy, reactive oxygen species production (ROS), and DNA double-stranded breaks evaluated. The modulation of hematopoiesis by MSCs was assayed using in vitro and in vivo models. Lastly, BM invasiveness and mice survival were evaluated after being challenged with leukemic cells of the C1498 cell line. Aging and PM alter biochemical parameters, changing the peripheral blood and BM immunophenotype. MSC autophagy was affected by aging and the frequencies for ROS and DNA double-stranded breaks. Regarding the MSCs’ secretome, PM and aging affected CXCL12, IL-6, and IL-11 production. Aging and PM up-regulated Akt1 and PPAR-γ while down-regulating Cdh2 and Angpt-1 in MSCs. Aged MSCs increased C1498 cell proliferation while reducing their colony-forming potential. PM and aging lowered mice survival, and malnourishment accumulated C1498 cells at the BM. Finally, aged and/or PM MSCs up-regulated Sox2, Nanog, Pou5f1, and Akt1 expression while down-regulating Cdkn1a in C1498 cells. Together, aging and PM can induce cell-intrinsic shifts in BM MSCs, creating an environment that alters the regulation of hematopoietic populations and favoring the development of malignant cells.

Fluorescence In Situ Hybridization (FISH) for Detection of c-MYC Amplifications in AML Patients: A Potential Marker for Risk Stratification

Abstract Background c-MYC is a transcription factor (located on 8q24) which regulates genes involved in proliferation, cell growth, differentiation, and apoptosis. The c-MYC amplification is related to a rapid proliferation of the affected cell clone in patients with myeloid neoplasms. Gene amplification (amp) is one of the basic mechanisms connected with over-expression of oncogenes. c-MYC is routinely analyzed in mature lymphoid neoplasms especially in Burkitt’s Lymphoma while not assessed for acute myeloid leukemia (AML).Despite the fact the c- MYC oncogene is commonly over-expressed in AML it has remained an elusive target. Routine assessment for c- MYC in AML may pave the way for targeting the c-MYC in treatment of AML patients. Several experiments are ongoing investigating both direct and indirect c-MYC targeting therapies and further establishing cMYC as an oncogene of central importance to AML pathogenesis and treatment. Aim of the Work In this study we aimed to define the prognostic value of c-MYC amplification in adult AML patients, and to relate it to other prognostic factors in AML. Patients and Methods The study was a randomized control study, conducted at the Clinical Pathology Department, Ain-Shams University Hospitals during the period from January 2019 till June 2020. It was carried on 50 adult AML patients (30 newly diagnosed and 20 on Therapy) and 10 healthy age-matched volunteers as control. All subjects underwent full history taking, thorough clinical examination, laboratory investigations including; CBC, Examination of Leishman’s stained peripheral blood films, Bone marrow aspiration and examination of Leishman’s stained bone marrow smears, Cytochemical studies using Myeloperoxidase stain, Immunophenotyping of bone marrow or peripheral blood samples and Fluorescence in situ hybridization analysis (FISH). Results we had 9 patients with c-MYC amplification (6 in the newly diagnosed group and 3 in the on therapy group). So the percentage of c-MYC amplification in our patients was 9/50 (18%). To further elucidate the effect of c-MYC amplification on our patients’ clinical, laboratory and prognostic factors, an analytical comparison was done between negative and positive patients for cMYC amplification as regard-studied parameters in each group of patients. Both group of patients showed a statistical significant association between c-MYC amplification and outcome where the majority of patients with positive c-MYC amplification achieved incomplete remission. In addition, c-MYC amplification showed a statically significant association with Trisomy 8 in both patients’ groups and with 17p deletion and hepatosplenomegaly in on therapy group. Univariate logistic regression analysis for the association between c-MYC amplification and outcome showed statistically significant association between c-MYC amplification and incomplete remission in newly diagnosed AML patients with P-value = 0.013 and odds ratio (95% CI) of 14.0. Conclusion Our study has shown that c-MYC amplification is associated with adverse prognosis, poor treatment outcome and it might be involved in the process of AML progression. Targeting cMYC may be of value in the development of new-targeted AML therapeutics.

Biallelic disruption of DDX41 activity is associated with distinct genomic and immunophenotypic hallmarks in acute leukemia.

Inherited DDX41 mutations cause familial predisposition to hematologic malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), with the majority of DDX41 mutated MDS/AMLs described to date harboring germline DDX41 and co-occurring somatic DDX41 variants. DDX41-AMLs were shown to share distinguishing clinical features such as a late AML onset and an indolent disease associated with a favorable outcome. However, genotype-phenotype correlation in DDX41-MDS/AMLs remain poorly understood. Here, we studied the genetic profile, bone marrow morphology and immunophenotype of 51 patients with DDX41 mutations. We further assessed the functional impact of ten previously uncharacterized DDX41 variants of uncertain significance. Our results demonstrate that MDS/AML cases harboring two DDX41 variants share specific clinicopathologic hallmarks that are not seen in other patients with monoallelic DDX41 related hematologic malignancies. We further showed that the features seen in these individuals with two DDX41 variants were concordant with biallelic DDX41 disruption. Here, we expand on previous clinicopathologic findings on DDX41 mutated hematologic malignancies. Functional analyses conducted in this study unraveled previously uncharacterized DDX41 alleles and further illustrate the implication of biallelic disruption in the pathophysiology of this distinct AML entity.

Plasma Cell Leukemia-Clinicopathological Profile from a Tertiary Care Center in Western India.

Poornima ManimaranIntroduction Plasma cell leukemia (PCL) is very uncommon and aggressive neoplasm constituting 2 to 4% of all plasma cell dyscrasias. By definition, clonal plasma cells should make up 20% of peripheral blood or have an absolute plasma cell count of 2 × 10 9 cells/cu.mm. PCL can be primary or secondary. In this study, the clinicohematological features of PCL, and correlation of immunophenotypic profile and conventional therapies with overall survival was analyzed. Materials and Methods This retrospective study involved PCL patients who were diagnosed across a 12-year period, from 2010 to 2021, at a tertiary care center in western India. Clinical, biochemical, peripheral smear, bone marrow aspirate, immunophenotyping, and molecular analysis were performed. Results Total 39 PCL patients were included in the study among which 36 were primary PCL patients. Splenomegaly (10/27), hepatomegaly (6/26), and lymphadenopathy (5/23) were noted. At presentation, all patients had anemia (<11g/dL), thrombocytopenia (33/39), hypercalcemia (>11mg/dl) 10/33 (30.3%) and lytic lesions was noted in 18/26 (69.2%). Immunophenotype of these patients showed CD 38 positivity, CD 138 positivity, CD56 positivity, and CD 117 negativity were 100, 62, 41.6, and 89%, respectively. Overall survival of our patients was 4.1 months and overall survival of patients treated with VTD (bortezomib, thalidomide, dexamethasone) and VCD (bortezomib, cyclophosphamide, dexamethasone) regimen was 3.4 and 4.1 months, respectively, which was not statically significant ( p -value 0.816). CD117 and CD56 markers were also not having any prognostic significance ( p -value 1.000 and 0.873, respectively). Conclusion Because of rarity of the disease, prospective studies are very limited and hence management and outcome of the disease are difficult to analyze. The current treatment protocols have no survival advantage and hence newer therapeutic approach is mandatory to attain better outcome.

Effectiveness of autologous hematopoietic stem cell transplantation in the treatment of high-risk neuroblastoma in children: a single-center clinical study

To investigate the effectiveness of high-dose chemotherapy combined with autologous hematopoietic stem cell transplantation (ASCT) in the treatment of children with high-risk neuroblastoma (NB). A retrospective analysis was performed on 29 children with high-risk NB who were admitted to Shanghai Children's Hospital and were treated with high-dose chemotherapy combined with ASCT from January 2013 to December 2021, and their clinical features and prognosis were analyzed. Among the 29 children treated by high-dose chemotherapy combined with ASCT, there were 18 boys (62%) and 11 girls (38%), with a median age of onset of 36 (27, 59) months. According to the International Neuroblastoma Staging System, 6 children (21%) had stage III NB and 23 children (79%) had stage IV NB, and the common metastatic sites at initial diagnosis were bone in 22 children (76%), bone marrow in 21 children (72%), and intracalvarium in 4 children (14%). All 29 children achieved reconstruction of hematopoietic function after ASCT. After being followed up for a median time of 25 (17, 45) months, 21 children (72%) had continuous complete remission and 8 (28%) experienced recurrence. The 3-year overall survival rate and event-free survival rate were 68.9%±16.1% and 61.4%±14.4%, respectively. Presence of bone marrow metastasis, neuron-specific enolase ≥370 ng/mL and positive bone marrow immunophenotyping might reduce the 3-year event-free survival rate (P<0.05). Children with high-risk NB who have bone marrow metastasis at initial diagnosis tend to have a poor prognosis. ASCT combined with high-dose chemotherapy can effectively improve the prognosis of children with NB with a favorable safety profile.

Isolated meningeal myeloid sarcoma with recurrent MLL-AF6 translocation: a case report

This study aimed to help improve the diagnosis and treatment of isolated myeloid sarcoma. We report the case of a female adolescent patient with isolated meningeal myeloid sarcoma. She was admitted to our department because of vomiting, headache and hearing loss. Positron emission tomography-computed tomography and brain magnetic resonance imaging showed multiple intracranial space-occupying lesions. A complete examination, including morphology, cytology, immunophenotyping, cytogenetics and molecular biology tests of cerebrospinal fluid and bone marrow, was conducted. The diagnosis of primary myeloid sarcoma of the central nervous system with mixed lineage leukemia gene rearrangement with AF6 was established. The patient underwent systemic chemotherapy and intrathecal treatment followed by whole-brain radiotherapy. She achieved complete remission for 84 months and has not developed bone marrow involvement during follow-up. The combination of morphology, cytology, flow cytometry, cytogenetics and molecular analysis can improve the definite diagnosis of isolated myeloid sarcoma.

Clinical Characteristics and Treatment of Blastic Plasmacytoid Dendritic Cell Neoplasm

To explore the clinical manifestations, diagnosis, treatment and prognosis of blastic plasmacytoid dendritic cell neoplasm(BPDCN). The clinical features, bone marrow morphology and immunophenotyping, treatment and prognosis of 4 patients with BPDCN were analyzed retrospectively. 4 patients had bone marrow， spleen and lymph nodes involvement， 2 patients had skin lesions， and 3 patients had central nervous system infiltration． Tailing phenomenon of abnormally cells could be seen in bone marrow. The immunophenotyping showed that CD56, CD4 and CD123 expression was observed in 4 patients， and CD304 in 3 patients． One patient refused chemotherapy and died early. Both patients achieved complete remission after the initial treatment with DA+VP regimen, 1 of them achieved complete remission after recurrence by using the same regimen again． One patient failed to respond to reduced dose of DA+VP chemotherapy, and then achieved complete remission with venetoclax+azacitidine. The malignant cells in BPDCN patients often infiltrate bone marrow, spleen and lymph nodes, and have specical phenotypes, with poor prognosis. The treatment should take into account both myeloid and lymphatic systems. The treatment containing new drugs such as BCL-2 inhibitors combined with demethylation drugs is worth trying.

Characteristics of Bone Marrow Cell Morphology and Immunophenotype in Patients with NHL Leukemia and Evaluation of NHL Bone Marrow Invasion

To observe the evaluation value of characteristics of bone marrow cell morphology and immunophenotype in patients with non-Hodgkin lymphoma (NHL) leukemia on the bone marrow invasion of NHL. The clinical data of 104 patients with NHL treated in the hospital from March 2016 to March 2021 were retrospectively analyzed, the characteristics of bone marrow smear morphology were recorded, and the analysis of bone marrow immunophenotype was performed, the evaluation value of bone marrow cell morphology and immunophenotype on NHL bone marrow invasion was analyzed. One hundred and four patients with NHL leukemia were found to have increased lymphoma cells by examination of bone marrow cell morphology, including 57 cases of B-cell type, 39 cases of T-cell type and 8 cases of NK/T cell type. The characteristics of bone marrow cell morphology were as follows: the cell body was large, irregular or round like in shape, the cytoplasm was much and mostly stained blue, a few cells could see a few granules, the nucleus was round or round like, some were twisted, the chromatin was thick and nucleolus was different, of which the T-cell type lymphoma cells had strong heteromorphism and more obvious nucleolus. B-cell type mainly expressed CD19, HLA-DR and CD20, and the positive rate was ≥70%. T-cell type mainly expressed CD7, HLA-DR and CD38, and the positive rate was ≥40%. NK/T cell types mainly expressed CD56 and CD161, and the positive rates was ≥50%. Compared the proportion of lymphoma cells between bone marrow smear and immunophenotype examination, there was no statistical significant difference (P>0.05). The characteristics of bone marrow cell morphology and immunophenotype have certain application value in the evaluation of bone marrow invasion in patients with NHL leukemia, both can complement each other and provide a feasible mean for the effective evaluation of bone marrow invasion in patients with NHL leukemia.

Detection of Fusion Gene and Prognosis Analysis in Children with Acute Lymphoblastic Leukemia of Different Immunophenotypes

To observe the detection of fusion gene in children with acute lymphoblastic leukemia (ALL) of different immunophenotypes, and analyze the relationship between fusion gene and prognosis. The clinical data of 86 children with ALL treated in the hospital from May 2015 to May 2020 were retrospectively analyzed, the immunophenotypes and the prognosis of children were recorded, the detection of fusion gene in ALL children with different immunophenotypes was compared, the relationship between detection of fusion gene and prognosis was analyzed. The results of bone marrow immunophenotype showed that there were 13 cases of T cell type and 73 cases of B cell type in 86 children with ALL. The detection rate of fusion gene SIL-TAL1 in ALL children with T cell type was significantly higher than that in ALL children with B cell type (P<0.05). The detection rates of fusion genes BCR-ABL1, E2A-PBX1 and TEL-AML1 in ALL children with B cell type were higher than those in ALL children with T cell type, but the differences were not statistically significant (P>0.05). Followed up for 8-12 months, recurrence was taken as the end point, the average follow-up time was (10.14±1.75) months, in 86 children with ALL 15 cases recurred (17.44%). The recurrence curve drawn by Kaplan-Meier method showed that the median recurrence time of 15 children with recurrent ALL was 9 months. The proportions of positive minimal residual disease and extramedullary infiltration in the poor prognosis group were higher than those in the good prognosis group, and the differences were statistically significant (P<0.05). The detection rates of fusion genes BCR-ABL and SIL-TAL1 in the poor prognosis group were higher than those in the good prognosis group, and the differences were statistically significant (all P<0.05). Logistic regression analysis showed that positive minimal residual lesions, extramedullary infiltration, and detection of fusion genes BCR-ABL and SIL-TAL1 were risk factors for poor prognosis in children with ALL (OR>1, P<0.05). The ROC curve analysis showed that the area under the curve (AUC) of combined detection of fusion gene BCR-ABL and SIL-TAL1 for predicting the poor prognosis of ALL children was >0.707, which had a certain predictive value. There are differences in fusion genes among ALL children with different immunophenotypes, minimal residual disease, extramedullary infiltration, and fusion gene are associated with prognosis of ALL children. Fusion gene detection can be used as new method to predict the prognosis of children with ALL.

Expression profile of immunophenotypic marker molecules on B-lymphocytes in patients with chronic lymphocytic leukemia at the stages of immunochemotherapy

Purpose of the study. To study the expression of immunophenotypic marker molecules on B-lymphocytes of patients with chronic lymphocytic leukemia at the stages of immunochemotherapy while monitoring minimal residual disease.Patients and methods. 20 patients with CLL were examined, who in the period 2019–2022 underwent 6 courses of immunochemotherapy (ICT) in the RB/FCR mode at the National Medical Research Centre for Oncology, Rostov-on-Don. Before, after 3, 6 courses of ICT, bone marrow immunophenotyping was performed by flow cytometry. The data is evaluated in Statistica 13.0.Results. Before treatment, 3 groups of patients were identified depending on the expression of prognostic markers (CD38, ZAP‑70, CD11c, CD25, FMC7). I (2 people) – without expression of CD38, ZAP‑70, CD11c, CD25, FMC7 on tumor B-lymphocytes. II (14 people) – with variable expression of CD25, CD38 (0.4–47.6 % and 0.0–57.5 %, respectively), lack of expression of ZAP‑70, CD11c, FMC7. III (4 people)– with high expression of CD38 (57.5–69.2 %), ZAP‑70 (36.6–48.3 %), CD11c (20.0–96.5 %), CD25 (64.9–92.7 %), FMC7 (13.6–88.6 %). After the 3rd course of ICT, the minimum residual disease (MRD): 0 % in group I, 0.48 ± 0.13 % in group II, 33.5 ± 7.84 % in group III. After the 6th course of ICT MRD: 0 % in group I, 0.42 ± 0.09 % in group II, 33.2 ± 8.07 % in group III. The expression of immunophenotypic markers in groups II and III remained unchanged after 3, 6 courses of ICT. According to the criteria for assessing the response to therapy (IWCLL, 2018), patients of groups I, II after the 6th course of ICT have complete remission, 3 patients of group III have partial remission, 1 patient has stabilization of the process. Preliminary data have been obtained indicating that the absence or increased expression of CD38, CD25, ZAP‑70, CD11c, FMC7 on B-lymphocytes of CLL patients before treatment may predetermine the hematological response to therapy according to RB/FCR regimens.Conclusion. Initially, increased expression of all prognostic antigens simultaneously: CD38, CD25, ZAP‑70, CD11c, FMC7 on the tumor population of B-lymphocytes in patients with CLL is associated with an unsatisfactory response to treatment, which seems promising from the point of view of studying the effect of the analyzed marker molecules on achieving a hematological response at the stages of immunochemotherapy.

Immunoregulatory CD71+ Erythroid Cells (CECs) Expand in Multiple Myeloma and Impair Control of L. Monocytogenes Infection

Introduction Although the clinical management of multiple myeloma (MM) patients has significantly improved over recent years, MM remains a largely incurable disease. One of the most common complication of MM, which correlates with the worse clinical outcome, is anemia. Moreover, MM patients are prone to develop life-threatening infections mainly due to the disease-related impairment of the immune system function. Recently, CD71+ erythroid progenitor and precursor cells (CECs) arose as a relevant immunomodulatory population. CECs have been shown to create an immunosuppressive microenvironment via multiple mechanisms including arginase 2 (ARG2), PDL-1, and TGF-beta. It was recently observed in our laboratory that mice bearing late-stage tumors are characterized by the presence of an increased percentage of CECs in their blood and spleens. Moreover, the tumor-associated CECs show elevated levels of arginases. The abovementioned facts may suggest an important role of CECs and their related mechanisms in compromising the immune response in MM. Methods Vk*MYC MM model Recipient C57BL/6 (wild type or ARG2 KO) mice were injected via tail vein with 1 × 106 Vk*MYC cells. M-spike levels were evaluated with serum protein electrophoresis. Five weeks later they were challenged with L. monocytogenes. L.monocytogenes infection and bacterial burden evaluation Mice were injected intraperitoneally with 1 x 103L. monocytogenes cells (strain EGDe). Bacterial burden was analyzed 72h and 7 days post infection. Briefly, spleens and livers of sacrificed mice were isolated, weighed, and homogenized. The homogenates were serially diluted and plated on culture plates. Bacterial burden was calculated using the number of colonies and the dilution factor. Flow cytometry analysis Flow cytometry analyses (immunophenotyping of spleen and bone marrow (BM) cells as well as cytokine beads arrays, Legenplex, BioLegend) were performed using BD Fortessa X-20 flow cytometer operated by BD FACSDiva Software ver. 8.0. The acquired data were re-analyzed using FlowJo™ ver. 10 software. Arginase inhibitor OAT-1746 arginase inhibitor (Molecure SA) was administered intraperitoneally at doses of 5 or 20 mg/kg twice daily, starting from the second or the fifth day before the bacterial challenge, respectively. The treatment was continued until the end of the experiment. Results In Vk*MYC-bearing mice we observed an accumulation of CECs in the blood and spleen in time, correlating with the development of anemia. At the late stage of the disease MM-associated CECs showed less differentiated phenotype and increased expression of arginase-1 (ARG1) and ARG2. MM-associated CECs inhibited T cells activation and proliferation in an ARG and reactive oxygen species-dependent manner. Interestingly, MM development was delayed in ARG2 KO vs WT mice. In the BM aspirates of MM patients we observed increased percentage of CECs expressing high levels of both ARG1 and ARG2 comparing with healthy controls. Moreover, MM impaired control of L. monocytogenes infection as we observed an elevated bacterial burden in the livers and spleens of Vk*MYC-bearing vs control mice. Infected MM-bearing mice had fewer NK cells and activated CD4+ and CD8+ T cells in the spleen than infected control mice. Furthermore, MM altered the dynamics of cytokines involved in the control of intracellular infection. 24h post Listeria challenge we observed their significant decrease followed by an increase at 72h comparing to infected non-MM mice. These results indicate that MM-bearing mice have delayed initial immune response leading to increased bacterial burden followed by excessive cytokines response resulting in sepsis-like condition leading to increased mice mortality. Finally, ARG inhibitor decreased L. monocytogenes burden in MM-bearing mice. Altogether, our results show that accumulation of CECs during MM progression leads to impaired bacterial infection control, at least partially dependent on ARG2 activity. Funding: National Science Centre in Poland [grants No 2017/25/B/NZ6/01139 and 2019/35/B/NZ6/00540]; Polish Ministry of Education and Science [grant No 013/RID/2018/19 (Regional Initiative for Excellence, project budget 12,000,000 PLN)].