Bone Marrow Morphology Research Articles

Morphological Characteristics, Immunophenotyping and Cytogenetics in Acute Myeloid Leukaemia Patients at a Tertiary Care Centre, Gujarat, India

Introduction: Acute Myeloid Leukaemia (AML) is a group of disorders characterized by a spectrum of clinical, morphological, immunophenotypic and associated chromosomal abnormalities. The identification of cytogenetic abnormalities at diagnosis is important for the evaluation of the response to therapy and the identification of an early re-emergence of disease. Aim: To examine the morphological characteristics of AML and emphasize the role of immunophenotyping and cytogenetics in subtyping AML. Materials and Methods: The cross-sectional study was a prospective study that took place from June 2018 to November 2021 at a tertiary-care cancer center, Department of Oncopathology in Gujarat, India. Following the inclusion and exclusion criteria, a total of 232 patients were diagnosed, with 21 being eliminated owing to the unavailability of samples for cytogenetic testing. Diagnosis of AML was based on morphology of Bone Marrow (BM) aspirates and flow cytometric immunophenotyping. Chromosomal analysis was performed on BM and peripheral blood by using standard cytogenetic technique. Results: There were 115 (54.50%) males and 96 (45.49%) females with age group between 1-72 years. The most common subtype was diagnosed as AML M1. Flow cytometry was done on 178 (84.36%) bone marrow and 33 (15.63%) peripheral blood samples. The CD33, CD13, MPO and CD117 were expressed in the vast majority of AML patient, aberrant expression of CD7and CD19 was seen. Total case 121 (57.34%) had normal karyotypes (the majority of cases), 86 (40.75%) cases had anomalous karyotypes consistent with t (8;21), t (15;17), and Inv16, and 4 (1.89%) cases were non informative. Conclusion: The study concluded that flow cytometry and cytogenetics should be performed routinely in all cases of AML. A multimodal diagnostic approach combining cytomorphology, multiparametric flow cytometry accompanied by cytogenetic is needed to arrive at definitive diagnosis of AML.

Risk factors and prognosis of thrombocytopenia in people living with HIV/AIDS.

Thrombocytopenia is a common hematological manifestation in people living with human immunodeficiency virus (HIV) or acquired immune deficiency syndrome (AIDS; PLWHA). Data on the prognostic relationship and associated factors of thrombocytopenia and HIV infection in China are limited. We assessed the prevalence of thrombocytopenia, its association with prognosis, and analyzed the associated risk factors among demographic characteristics, comorbidities, hematological and bone marrow indicators. We collected patients identified as PLWHA in Zhongnan Hospital. The patients were divided into two groups: the thrombocytopenia group and the non-thrombocytopenia group. We analyzed and compared demographic characteristics, comorbidities, peripheral blood cells, lymphocyte subpopulations, infection indicators, bone marrow cytology, and bone marrow morphology of the two groups. Then we analyzed the risk factors for thrombocytopenia and the effect of platelet (PLT) values on the prognosis of patients. Demographic characteristics and laboratory results were obtained from medical records. In contrast to other studies, we included bone marrow cytology and morphology in this study. Data were analyzed with multivariate logistic regression analysis. The Kaplan-Meier method was used to plot 60-month survival curves for the severe, mild, and non-thrombocytopenia groups. The value p < 0.05 was taken as statistically significant. Among 618 identified PLWHA, 510 (82.5%) were male. Overall, thrombocytopenia was found in 37.7% [95% confidence interval (CI): 33.9-41.5%]. Multivariable logistic regression analysis showed that age ⩾40 years [adjusted odds ratio (AOR) 1.869, 95% CI: 1.052-3.320], combined with hepatitis B (AOR 2.004, 95% CI: 1.049-3.826), high procalcitonin (PCT) count (AOR 1.038, 95% CI: 1.000-1.078) were risk factors of thrombocytopenia in PLWHA. An increased percentage of thrombocytogenic megakaryocytes was a protective factor, with an AOR 0.949 (95% CI: 0.930-0.967). Kaplan-Meier survival curve analysis showed that the prognosis was worse in the severe than in the mild (p = 0.002) and non-thrombocytopenia groups (p = 0.008). We discovered a general high pervasiveness of thrombocytopenia in PLWHA in China. Age ⩾40 years, combined with hepatitis B virus infection, high PCT, and decreased percentage of thrombocytogenic megakaryocytes indicated a higher risk for developing thrombocytopenia. A PLT count ⩽50 × 109/liter led to a worse prognosis. Therefore, early diagnosis and treatment of thrombocytopenia in these patients are useful.

Utility of morphologic assessment of bone marrow biopsy in diagnosis of lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are rare disorders and pose a diagnostic challenge for clinicians owing to their generalized symptomatology. In this study, we aim to classify LSDs into two broad categories, namely, Gaucher disease (GD) and Niemann-Pick/Niemann-Pick-like diseases (NP/NP-like diseases) based on the morphology of the storage cells in the bone marrow (BM) aspiration smears and trephine biopsy sections. This retrospective study includes 32 BM specimens morphologically diagnosed as LSDs at our institute, in the last 10 years. Subsequently, they were subclassified into GD and NP/NP-like diseases. Further, we have compared and analyzed the clinical, hematological, and biochemical parameters for the two groups of LSDs. Based on BM morphology, 59.4% (n = 19) cases were diagnosed as NP/NP-like diseases and 40.6% (n = 13) cases as GD. Abdominal distension and failure to thrive were the most common clinical manifestations in both groups of LSDs. Anemia and thrombocytopenia were frequently seen in either of the LSDs. On the assessment of metabolic profile, elevated total/direct bilirubin and liver enzymes were more commonly seen in NP/NP-like diseases when compared with GD. We have classified LSDs into GD and NP/NP-like diseases based on the morphology of the storage cells in the BM specimen. The hallmark findings on BM biopsy annexed with the comparative features of the two proposed categories can aid the clinician in clinching the diagnosis. Formulation of such a methodology will prove instrumental for patient care in an underresourced setting.

A Construction of Object Detection Model for Acute Myeloid Leukemia

The evolution of bone marrow morphology is necessary in Acute Myeloid Leukemia (AML) prediction. It takes an enormous number of times to analyze with the standardization and inter-observer variability. Here, we proposed a novel AML detection model using a Deep Convolutional Neural Network (D-CNN). The proposed Faster R-CNN (Faster Region-Based CNN) models are trained with Morphological Dataset. The proposed Faster R-CNN model is trained using the augmented dataset. For overcoming the Imbalanced Data problem, data augmentation techniques are imposed. The Faster R-CNN performance was compared with existing transfer learning techniques. The results show that the Faster R-CNN performance was significant than other techniques. The number of images in each class is different. For example, the Neutrophil (segmented) class consists of 8,486 images, and Lymphocyte (atypical) class consists of eleven images. The dataset is used to train the CNN for single-cell morphology classification. The proposed work implies the high-class performance server called Nvidia Tesla V100 GPU (Graphics processing unit).

Changes in adipose bone marrow and bone morphology in X-linked hypophosphatemic rickets

IntroductionX-linked hypophosphatemic (XLH) rickets causes significant bone deformities in the lower limbs resulting from a bone mineralization defect. According to Frost's Mechanostat theory, compensatory modeling of the bones takes place during increased mechanical loads. In addition, mechanical stimuli modulate the differentiation of mesenchymal stem cells; common precursors to bone marrow adipocytes and osteoblasts. HypothesisBone deformities of the lower limbs lead to increased femoral bone mass and decreased fatty infiltration of the bone marrow (FIBM) in children with XLH rickets compared to a control group. Patients and methodsEleven children (10.3years [6–17]) with XLH rickets and 22 healthy children (10.2years [5–15.5]) underwent lower limb Magnetic Resonance Imaging. A calculation of FIBM was performed at the mid-femur, as well as a calculation of the total bone cross-sectional area (CSA), the cortical CSA, the anteroposterior and mediolateral axes of the femur, bone marrow and the thickness of the femoral cortices. ResultsTotal bone CSA, total cortical CSA and bone marrow CSA were higher in the XLH group than in the control group (p<0.05). The mid-lateral diameters of the femur and bone marrow were more elongated than those of the control group (p<0.001). Only the anterior cortex was thinned in the XLH group (p=0.001), while there was no difference with the control group for the posterior, medial and lateral cortices. The total percentage of FIBM was 72.81% [±3.95] and 77.4% [±5.52] for the XLH and control groups respectively (p<0.001). DiscussionThe increase in bone mass in the XLH population reflects an adaptation of bone tissue to the bone deformities present in this pathology. The decrease in FIBM indicates a lower risk of osteoporosis in the XLH population and may constitute a new monitoring parameter in this pathology. Level of studyIII; Case-control study.

Novel THPO variant in hereditary thrombocytopenia: A potential candidate variant for predisposition to myeloid neoplasm.

Hereditary thrombocytopenia is a heterogeneous group of congenital disorders with a wide range of symptoms depending on the severity of platelet dysfunction or thrombocytopenia. Because of its clinical phenotypes and the bone marrow morphology associated with this condition, hereditary thrombocytopenia can be misdiagnosed as primary immune thrombocytopenia and myelodysplastic syndrome. Therefore, genetic evidence is necessary for the accurate diagnosis of hereditary thrombocytopenia. Refractory cytopenia of childhood is a subgroup of myelodysplastic syndrome that was added to the World Health Organization classification in 2008. To investigate the germline and somatic variants associated with refractory cytopenia of childhood, we performed targeted multigene sequencing in three patients with refractory cytopenia of childhood. Of the three patients, one progressed from megakaryocytic hypoplasia with thrombocytopenia, and targeted multigene sequencing revealed THPO variants in this patient and his sister. We propose that the monoallelic deletion of THPO is a potential candidate for germline predisposition to myeloid malignancy.

Lymphoplasmacytic Lymphoma with Paraprotein IgG

Lymphoplasmacytic lymphoma (LPL) is a rare indolent mature B-cell lymphoma. LPL secreting immunoglobulins other than IgM are rare. There are very few case series on non-IgM LPL, and little is known about the clinical features and outcomes of patients with this disease. We report the case of a 65-year-old-male who was referred to our hospital for further investigations of persistent chronic anaemia and was diagnosed with IgG-LPL based on the presence of M protein from serum electrophoresis. Bone marrow morphology exhibit a spectrum of B-cell differentiation ranging from small mature lymphocytes to plasma cells. The patient underwent treatment with a combination of bortezomib, dexamethasone and rituximab and showed positive response. LPL with paraprotein IgG is a rare indolent disease and has a heterogeneous clinicopathological presentation with limited literature reviews. Our case report and literature review provide insights and knowledge in the description of the clinicopathological features of IgG LPL.

The Application of RNA-Sequencing in Pediatric B-Cell Acute Lymphoblastic Leukemia

To explore the molecular genetic characteristics of children with B-cell acute lymphoblastic leukemia (B-ALL) and the application value of RNA-sequencing (RNA-seq). The clinical and laboratory examination data of newly diagnosed B-ALL children who were given treatment in the Department of Hematology, Children's Hospital of Chongqing Medical University from May 2015 to April 2020 were collected and analyzed. All children were confirmed by bone marrow morphology, histochemical staining and flow cytometry, and the karyotype analysis, FISH, RT-PCR and RNA-seq detection were conducted. There were 71 males and 58 females with a median age of 50(8-190) months in 129 newly diagnosed children with B-ALL. The fusion gene was positive in 99 children (76.7%). A total of 86 leukemia related or possibly related gene mutations were detected, with a positive rate of 66.7%. There was no significant difference in the detection rates of ETV6-RUNX1, BCR-ABL1, TCF3-PBX1 and KMT2A rearrangements among FISH, RT-PCR and RNA-seq. Rare fusion genes were detected by RNA-seq, including 1 case of KMT2A-USP2, 4 cases of Ph-like related fusion genes, 5 cases of MEF2D rearrangement, 5 cases of PAX5 rearrangement, 3 cases of ZNF384 rearrangement, as well as several fusion genes whose significance were not clear or had not been reported in children with leukemia. Besides, children with ETV6-RUNX1 fusion gene had good response to induction of remission, while children with BCR-ABL1 and ZNF384 rearrangement had poor response, the remission rate of minimal residual disease was statistically significant compared with other types (P<0.05). RNA-seq can not only detect known fusion genes, but also discover new or rare fusion genes and gene mutations. The application of RNA-seq has important guiding significance for risk classification and precise targeted therapy of pediatric B-ALL.

Role of Biomarkers in the Management of Acute Myeloid Leukemia.

Despite many recent advances in treatment options, acute myeloid leukemia (AML) still has a high mortality rate. One important issue in optimizing outcomes for AML patients lies in the limited ability to predict response to specific therapies, duration of response, and likelihood of relapse. With evolving genetic characterization and improving molecular definitions, the ability to predict outcomes and long-term prognosis is slowly improving. The majority of the currently used prognostic assessments relate to molecular and chromosomal abnormalities, as well as response to initial therapy. These risk categories, however, do not account for a large amount of the variability in AML. Laboratory techniques now utilized in the clinic extend beyond bone marrow morphology and single gene sequencing, to next-generation sequencing of large gene panels and multiparameter flow cytometry, among others. Other technologic advances, such as gene expression analysis, have yet to demonstrate enough predictive and prognostic power to be employed in clinical medicine outside of clinical trials, but may be incorporated into the clinic in the future. In this review, we discuss the utility of current biomarkers, and present novel biomarker techniques and strategies that are in development for AML patients. Measurable residual disease (MRD) is a powerful prognostic tool that is increasingly being incorporated into clinical practice, and there are some exciting emerging biomarker technologies that have the potential to improve prognostic power in AML. As AML continues to be a difficult-to-treat disease with poor outcomes in many subtypes, advances in biomarkers that lead to better treatment decisions are greatly needed.

Characterization of Terminal Erythroid Differentiation (TED) and Its Prognosis Impact Inpatients with Myelodysplastic Syndrome (MDS)

INTRODUCTION: Culture assays of normal bone marrow (BM) have shown that TED follows a doubling maturation pattern in each erythroid stage from proerythroblast to orthochromatic erythroblast. For a long time, it has been suggested that TED is profoundly abnormal across all myelodysplastic syndrome (MDS) subtypes. Nevertheless, morphologic quantification of cells undergoing TED in BM both from normal and MDS samples has not been systematically assessed, despite the fact morphological analysis still represents the backbone for the diagnosis. The aims of this study were to describe the pattern of TED by conventional morphology in BM of individuals without myeloid malignancies and in patients with MDS, and to compare clinical characteristics and prognostic features between MDS patients with normal and abnormal TED. METHODS: Three independent cytologists from three different institutions performed the morphological analysis of erythroid cells in BM smears in a 500-cell differential count from 74 patients diagnosed with lymphoma without BM involvement, and from 284 patients with MDS at diagnosis. TED stages were conventionally classified into proerythroblast (PE), basophilic (BA), polychromatic (POLYS), and orthochromatic erythroblasts (ORTHOS), and percentages were calculated based on the total cell population. Furthermore, flow cytometry analysis was performed on 11 further lymphoma patients without BM involvement, by using specific immunofluorescent probe patterns detected through the DxFLEX© cytometer. Patients with MDS were categorized as: TED-Normal, when total TED made up ≥15% of total BM cells and followed the expected doubling pattern; TED-Incomplete, when total TED were ≥15% but did not follow a doubling pattern; and TED-absent, when total TED composed <15%. Statistical analysis was performed by R (4.2.0). RESULTS: Morphologic analysis of normal BM confirmed the doubling growth pattern of TED [median PE of 0.8% (0-1.23); BA of 1.8% (1-2.4; ) POLYS of 8% (6.4-10.89) and POLYS of 8% (6.4-10.89)]. Whereas, immunophenotype analysis showed a linear growth pattern [median PE=0.57% (0.54-0.61), BA=1.35% (1.18-1.75), POLYS=2.64% (2.01-3.01), and ORTHOS=3.91% (3.3-5.37), probably explained by the lysis of the late-stage erythroblast cells by the immunophenotype preparation technique. Among the 284 MDS patients, 211 (75.5%) were TED-Normal, 46 (16.5%) TED-Incomplete, and 22 (8%) TED-Absent. Patients' characteristics are detailed in Table 1. MDS patients with TED-Normal presented significantly higher levels of hemoglobin (99 g/L vs. 89.5 g/L), reticulocytes (56 x109/L vs. 39 x109/L), and a lower percentage of ring sideroblast (0% vs. 26%) compared to TED-Incomplete (Table 1). Survival analysis showed that MDS patients with TED-Normal had a median OS of 39.03 months (95% IC 29.73-49.13), whereas it was of 23.77 months (95% IC 12.17-38.7) for TED-Incomplete, and 13.23 months (95% IC 7.17-NA) for TED-Absent; [HR 2.2; p=0.00132]. Of note, no OS differences were found between TED-Incomplete and TED-Absent [HR=1.41; p= 0.246] [Figure 2]. CONCLUSION: Morphological analysis is a suitable methodology for the study of TED maturation. Remarkably, 24.5% of patients with MDS presented with an abnormal TED (incomplete or absent) at diagnosis, this being associated with poorer clinical features and outcome. Finally, with the advent of newer erythroid maturation agents, analysis of TED might become paramount for treatment selection and response monitoring of patients with MDS in the near future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy and Safety of Ropeginterferon Alfa-2b for Pre-Fibrotic Primary Myelofibrosis and DIPSS Low/Intermediate-1 Risk Myelofibrosis

Most patients with early/pre-fibrotic primary myelofibrosis (pre-PMF) and dynamic international prognostic scoring system (DIPSS) low or intermediate-1 risk myelofibrosis (MF) progress to overt PMF or higher risk MF. There is currently no consensus on the optimal treatment strategies in these patients. Ropeginterferon alfa 2b (P1101) is a next generation monopegylated interferon alfa-2b developed specifically to treat myeloproliferative neoplasms (MPN). The P1101MF study is a multicenter phase 2 study of P1101 for patients with pre-PMF and DIPSS low/intermediate-1 risk MF (NCT04988815). Key eligibility included the need for cytoreduction, morphologically confirmed pre-PMF, overt PMF, post-polycythemia vera MF (PPV-MF) or post-essential thrombocythemia MF (PET-MF), and DIPSS low/intermediate-1 risk category. The primary outcome was the clinicohematologic complete response (CHCR) rate at 24 and 48 weeks. Secondary outcomes included adverse events (AEs), changes in allele burden of driver genes and other non-driver mutations, changes in quality-of-life (QOL), changes in cytokine profiles and changes in bone marrow morphology. Patients received P1101 at a starting dose of 250mcg followed by 350mcg at week 2 and 500mcg every 2 weeks from week 4 onwards. At the data cut-off of 28 July 2022, 56 patients (33 men, 23 women) with a median age of 58 (range: 30-87) were enrolled. Thirty eight patients (68%) had pre-PMF, 5 (9%) had overt PMF, 3 (5%) had PPV-MF and 10 (18%) had PET-MF. Next-generation sequencing (NGS) in 53 patients showed JAK2V617F in 39 patients (74%), CALR mutations in 13 patients (25%) and MPL mutation in 1 patient (2%). On day 1, the mean white blood cell (WBC) count, hemoglobin concentration, platelet count, and lactate dehydrogenase (LDH) level were 9.4 x 109/L (range: 3.3-33.95), 12.5 g/dL (range: 7.1-15.9), 520 x 109/L (range: 113-1038) and 359 IU/L (range: 136-1146) respectively. The median duration of follow-up was 24 (2-28) weeks. Forty-six and 30 patients completed 12 and 24 weeks of treatment respectively. At 12 and 24 weeks the CHCR rate was 74% and 67%, respectively. At 24 weeks, 36 (92%) of 39 JAK2V617F mutated patients had stable or improved JAK2V617F allele burden by droplet digital polymerase reaction (ddPCR). Three patients (8%) had >50% reduction in the JAK2V617F allele burden with one patient achieving undetectable JAK2V617F from week 16 onwards. Disease progression or blastic transformation were not observed during the study. The most common AEs were malaise (N=22, 39%; Grade 1-2, N=21; Grade 3-4, N=1), anemia (N=12, 21%; Grade 1-2, N=8; Grade 3-4, N=4), muscle pain (N=11, 20%; Grade 1-2, N=11; Grade 3-4, N=0) and hair loss (N=10, 18%; Grade 1-2, N=10; Grade 3-4; N=0) . There were no treatment discontinuations, safety signals or deaths related to treatment. In summary, P1101 was well-tolerated, effective in cytoreduction and induced molecular responses. The recruitment of patients is ongoing.

Short-Term Follow-up of Blinatumomab in Children with B-Cell Precursor Acute Lymphoblastic Leukemia：Study of a Single Center from China

Background: Acute lymphoblastic leukemia (ALL) is the most common hematologic disorders in children. The 5 years overall survival (OS) has reached more than 90%, but still 20% of patients relapsed. Positive of microscopic residual disease (MRD+) is the important factors for pediatric ALL. Blinatumomab is a combination of CD3 and CD19 bispecific antibody. Previous studies have shown that it has a significant advantage in improving the efficacy and survival of refractory /relapsed (R/R) ALL and MRD+ patients. Blinatumomab has been approved in China for R/R ALL indications in adults and children respectively since Aug 2021 and Apr 2022. So far, there is a lack of research data on ALL in Chinese children. This study mainly discusses the short-term efficacy and safety of Blinatumomab for R/R ALL and MRD+ of children with B-ALL in China. Methods: We collected the patients' information of children with R/R or MRD+ B-ALL treated with blinatumomab from Aug 2021 to Jun 2022 in our single institute from China. Retrospectively analyzed were carried out for the short-term efficacy and safety. Results: since Aug 2021, blinatumomab has been introduced to China. 13 cases were enrolled in this study, including R/R ALL 6 cases and MRD + ALL 7 cases, the median onset age of 9.5 (range 5-14) years old, male: Female =8:5, in the R/R-ALL group, there was 1 case of sustained no remission (NR), 1 case of very early recurrence(<18 months) and 1 case of late recurrence(≥36 months), and 3 cases of early recurrence, ALL of which were isolated bone marrow recurrence for the first time. 3 cases were treated with reinduction chemotherapy before blinatumomab. The mean proportion of blast cells in bone marrow (BM) prior therapy was 34.3 (range 0-96) %. The mean flow cytometry MRD was 30.26 (range 0.11-78.3) %. 3 patients were with no dose uphill due to low tumor load. 3 patients were given uphill 5ug/m2 qd d1-7, 15ug/m2 d8-28, of which 1 was treated for 14 days and 2 for 21 days. The investigator evaluated the efficacy of the treatment and stopped the drug. After treatment, bone marrow morphology was complete response (CR) in 3 cases, of which 2 cases had MRD negative (MRD-), the Cytokine release syndrome (CRS) incidence was 83.3%, 2 cases had CRS grade 2, 3 cases CRS grade 1, and the main manifestations were fever and hypotension. The levels of cytokines were significantly increased in patients with CRS, especially in IL-6, IL-8 and IL-10. The median level of IL-6 in CRS was 3081.8 (range 12.6-10405.0). IL-8 214.5 (range 23-408.0) and IL-10 377.7 (range 6.7-1398.0) (normal value ≤ 4.91) were significantly higher than those before blinatumomab and after CRS disappeared. In the MRD+ ALL group, there were 2 cases of chemotherapy drug intolerance, 1 case of inadequate treatment, and 4 cases of continuous MRD+ after standard chemotherapy. In MRD+ALL group, the BM of All cases were CR before treatment, the mean flow MRD value was 0.63 (range 0.01-4.00) %, the blinatumomab was 15ug/m2 (weight < 45kg) or 28ug (weight ≥45kg) for 7-28 days. After treatment, the MRD of 7 cases were all turned negative and the incidence of CRS was 71%. All of which were CRS grade I. When CRS occurs, the median IL-6 level is 76.4 (range 4.4-271.0) (normal value ≤ 5.3), IL-8 18.7 (range 8.0-35.5) (normal value ≤ 20.6), IL-10 447.3 (range 8.9-2570.0) (normal value ≤ 4.91) Conclusion: The CR rate of R/R pediatric B-ALL was 50%, the MRD- rate was 67%. The rate of MRD turned negative was 100% in MRD+ B-ALL group. In this study, the tolerance of blinatumomab in children with B-ALL was good, and the incidence of CRS was about grade 1/2, and no neurological complications occurred. The occurrence of CRS was mainly caused by the increase of cytokines, especially IL-6, IL-8 and IL-10. The IL-6 of CRS in R/R ALL group was significantly higher than that in MRD+ ALL (Fig.12.798, P=0.007).

MRD Monitoring of Non-DTA Genes By Digital Droplet PCR in the Early Period after Allogeneic Hematopoietic Stem Cell Transplantation Can Effectively Predict Patients at High Risk of Relapse

Background: Hematological malignancies relapse is the major cause of death after allogeneic hematopoietic stem cell transplantation (HSCT). The detection of measurable residual disease (MRD) after HSCT is the effective and important predictor of relapse. Digital droplet polymerase chain reaction (ddPCR), as the third generation PCR, is a novel technology for absolute quantification of nucleic acids developed in recent years. Compared with real-time quantitative PCR (qPCR, first generation) and next-generation sequencing (NGS), ddPCR has better sensitivity, specificity and repeatability, which provide a new platform for the detection of MRD. The purpose of this study was to evaluate whether early MRD monitoring by ddPCR after HSCT can effectively predict the recurrence of primary disease. Methods: We retrospectively analyzed patients with hematological malignancies who underwent allogeneic HSCT and monitored post-transplant MRD by ddPCR at the BMT Center of the First Affiliated Hospital of Zhejiang University School of Medicine from August 2018 to February 2021. Patients who met the following criteria were included in our study: (1) at least one hematological tumor-associated mutations or fusion genes for MRD monitoring at the time of diagnosis; (2) underwent allogeneic HSCT and the stem cell engraftment was successful; (3) received bone marrow MRD detection by ddPCR at least once in the early period (+30 days to +120 days) after HSCT; (4) bone marrow morphology was in remission status within +30 days to +120 days. Results: 100 patients were included in our study. The median follow-up time of patients was 20 months (range 5-46), and the median age was 41 years (range 14-67). Among them, 59 (59.0%) were male, and the most frequent primary diseases were AML (N=59, 59.0%), ALL (N=21, 21.0%), MDS (N=16, 16.0%), respectively. A total of 43 (43.0%) patients were found to be MRD+ after HSCT by early ddPCR detection. The difference in the 2-year cumulative incidence of relapse (2y CIR, 32.5% vs. 19.6%, P= 0.103), 2-year relapse-free survival (2y RFS, 67.0% vs. 79.8%, P=0.110), and overall survival (OS, 63.0% vs. 84.1%, P=0.566) between the MRD+ and MRD- groups were not statistically significant. 30 patients had at least one mutation related to clonal hematopoiesis gene: DNMT3A, TET2, and ASXL1 (DTA), whereas 97 patients had at least one non-DTA gene. When DTA group and non-DTA group were considered separately, MRD+ were 33.3% in DTA gene group and MRD+ were 36.1% in non-DTA gene group. The 2y CIR (0% vs. 21.5%, P=0.160), 2y RFS (100% vs. 78.5%, P=0.170) and OS (90.0% vs. 85.0%, P=0.737) after HSCT was not statistically different between DTA MRD+ group and DTA MRD- group, suggesting that DTA was not suitable as monitoring genes for relapse after transplantation. However, in the non-DTA group, patients with MRD+ had higher 2y CIR (39.8% vs. 18.4%, P=0.014) and inferior 2y RFS (60.2% vs. 80.8%, P=0.017) compared with MRD- patients. To explore whether ddPCR and multiparameter flow cytometry (MFC) can complement each other and further improve the prediction accuracy of relapse, we conducted a joint analysis on the bone marrow MRD detected simultaneously by non-DTA ddPCR and MFC, and divided into four parts: ①MFC MRD+ and non-DTA MRD+ (F+/M+, n=7); ②MFC MRD- and non-DTA MRD+ (F-/M+, n=26); ③MFC MRD+ and non-DTA MRD- (F+/M-, n=8); ④MFC MRD- and non-DTA MRD- (F-/M-, n=56). The sensitivity of ddPCR was higher than that of MFC. The 2y CIR of the four groups was (F+/M+ 71.4% vs. F-/M+ 34.5% vs. F+/M- 53.1% vs. F-/M- 12.2%, P<0.001), and the 2y RFS was (F+/M+ 28.6% vs. F-/M+ 65.5% vs. F+/M- 46.9% vs. F-/M- 87.3%, P<0.001) and OS was (F+/M+ 31.3% vs. F-/ M+ 61.8% vs. F+/M- 87.5% vs. F-/M- 83.8%, P=0.219). Through multivariate analysis, F+/M+ is an independent risk factor for relapse after HSCT (hazard ratio, 9.531, 95%CI, 2.884 to 31.496). It suggested that F+/M+ detected in the early stage after transplantation had higher CIR and inferior RFS. Conclusion: MRD monitoring for non-DTA gene in the early period after HSCT based on ddPCR has a prognostic effect on the recurrence of primary disease and overall survival after transplantation. When ddPCR is combined with MFC to monitor MRD, it is more accurate in predicting relapse and may help instruct preemptive treatment and then reduce the risk of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Morphologic Spectrum of Myelokathexis in WHIM Syndrome and Germline CXCR4 Variants: New Insights into Cellular Changes in the Bone Marrow and Peripheral Blood

Introduction WHIM syndrome (WHIMs) is a rare disorder caused by germline CXCR4 mutations, classically characterized by warts, hypogammaglobulinemia, infection, and neutrophil retention in the bone marrow (BM), with distinctive morphology termed myelokathexis. Demonstration of myelokathexis by BM evaluation plays a key role in disease recognition and diagnosis. However, the spectrum of BM morphologic features and correlation with specific CXCR4 mutations are not well described. Moreover, diagnostically useful peripheral blood (PB) smear findings and morphologic changes after CXCR4 antagonist therapy are largely unknown. Methods We conducted a multinational, retrospective review of clinicopathologic features of patients with established WHIMs (n=20) or germline CXCR4 variants considered likely pathogenic or of uncertain significance (n=3), including 14 BM specimens and 10 PB smears. Assessments to date were performed by 2 observers in 6 cases and a single observer in the remainder. BM morphology was compared to other neutropenia disorders (Shwachman-Diamond syndrome, n=5; GATA2 deficiency, n=5; severe congenital neutropenia with ELANE variants, n=4) and patients without hematologic disorders (n=10), together "controls." Correlations and contingency analysis (2-tailed Fisher exact test) were performed using Prism v.9.4.0 (GraphPad Software). Results Median age at WHIMs diagnosis was 18.5 years (range, 0.1-53 years), with female:male ratio of 1.1:1. Recurrent infections, warts, and hypogammaglobulinemia were present in 100%, 53%, and 40% of cases, respectively. BM aspirate smears contained neutrophils with long intranuclear chromatin strands and hypermature condensed chromatin ("myelokathectic neutrophils") in all WHIMs cases, making up 32% to 80% (median 66%) of total neutrophils (Table). In comparison, neutrophils with myelokathectic changes were seen in 0% to 4% of control BMs. Myeloid hyperplasia (myeloid:erythroid ratio >4:1) was present in 50% of WHIMs and 4% of controls (P=.0028); vacuolated neutrophils in 92% of WHIMs (≤29% of total neutrophils) and 17% of controls (P<.0001); apoptotic neutrophils in 58% of WHIMs (1%-4% of total neutrophils) and 8% of controls (P=.0025); and nonparatrabecular neutrophil clusters on core biopsy in 3/3 WHIMs and 5% of controls (P=.0017). Vacuolated eosinophils and myeloid shift toward hypermaturity were present in WHIMs. There was no correlation between percentage of BM myelokathectic neutrophils and degree of CXCR4 truncation. BM in 2 patients with CXCR4 p.S341Y showed myeloid hyperplasia, increased neutrophil apoptosis, and infrequent myelokathectic neutrophils (5%-10%). Neutropenia was present in all untreated WHIMs. Myelokathectic neutrophils were present in PB of all patients with known WHIMs and accounted for 8% to 30% (median, 18%) of total neutrophils. Percentage of PB myelokathectic neutrophils showed an inverse correlation with absolute neutrophil count (ANC) (r=-0.783; P=.0074). Neutrophil apoptosis, neutrophil vacuolization, dysmorphic (hypogranular, hyperlobated, vacuolated, or apoptotic) eosinophils, and/or dysmorphic basophils were present in 80%, 30%, 70%, and 60% of WHIMs cases, respectively (Figure). PB in 1 patient with CXCR4p.D84H showed neutrophil hypolobation without myelokathectic morphology. CXCR4 antagonist therapy with plerixafor (n=4) [Dale et al. Blood. 2011] or mavorixafor (n=1) (interval, 4-12 h) resulted in increased white blood cell (WBC) count and ANC, with a similar proportion of myelokathectic neutrophils as before therapy. Conclusions In WHIMs BM specimens, the proportion of myelokathectic neutrophils is variable and may not be prominent; the classic finding of myeloid hyperplasia is only present in a subset of cases, which may result in underrecognition of the disease and delayed diagnosis. Neutrophil vacuolization and apoptosis, eosinophil vacuolization, and prominent neutrophil clusters may also prompt disease recognition. We establish characteristic PB morphologic features which, in a patient with unexplained neutropenia, may suggest a diagnosis of WHIMs, including myelokathectic neutrophils, neutrophil vacuolization or apoptosis, and dysmorphic eosinophils and basophils. CXCR4 antagonist therapy led to an increase of WBC/ANC, while pretreatment morphologic changes persisted after a short interval. Analysis of additional cases is ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Outcome and Treatment-Related Mortality in Patients with Acute Myeloid Leukemia Treated at the Uganda Cancer Institute

Background Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults, accounting for about 80% of cases, and accounts for 20% of childhood leukemias. Treatment of AML is similar in both high-income and low- and middle-income countries (LMICs), and utilizes an anthracycline and cytarabine backbone. Treatment-related mortality (TRM) for AML in high-income countries is significantly lower than in LMICs. Understanding the factors associated with TRM in LMICs is critical to improving AML outcomes. Here we describe the response rate to induction therapy and TRM in patients with AML treated at a tertiary cancer facility in Uganda. Methods A retrospective study of all patients treated for AML at the Uganda Cancer Institute (UCI) in the years 2015-2020 was performed. The diagnosis of AML was based on bone marrow or peripheral blood morphology or flow cytometry. The regimen for remission induction of adult patients with AML at the UCI includes daunorubicin 60mg/m2 given intravenously on days 1-3 and cytarabine 200mg/m2 given intravenously on days 1-7 ("3+7"), while the paediatric regimen (termed "AD") consists of daunorubicin 50mg/m2 given intravenously on days 1, 3, and 5 and cytarabine 100mg/m2 given intravenously twice daily on days 1-10, followed by a second course. Consolidation therapy is given to all patients who achieve complete remission after up to 2 courses of induction. Complete remission was defined as <5% blasts in the marrow identified by morphology or flow cytometry. The study was approved by the UCI Research Ethics Committee (Ref. UCI-2020-9) and the Uganda National Council for Science and Technology (Ref. HS946ES). All patients’ information was anonymized. Results. Medical records were reviewed for 290 patients with a diagnosis of AML, of whom 186 were further analyzed after excluding patients diagnosed with acute promyelocytic leukemia (n=33), those who did not receive induction therapy (n=66), and those who received low dose chemotherapy (n=5). The median age was 20 (IQR, 10-35) years. Patient characteristics are reported in Table 1. One hundred fourteen (61.3%) adult and adolescent patients received a standard "3+7” induction regimen, 65 (34.9%) children were treated with "AD,” and 7 (3.8%) patients received other regimens. Seventy four (39.8%) patients received a second course of induction. Treatment response assessment showed that 86 (46.2%) patients achieved complete remission (CR). Of these, 62 (72%) achieved CR after the first induction, with the remaining 24 (28%) after the second induction. Forty five (24.2 %) patients with evaluable response data at the end of induction did not achieve remission. A total of 55 (29.6%) patients died, all in the first induction course. The overall 30-day induction TRM rate was 31.5% (95% CI, 25.2 - 38.8) with 3-month, 6-month and one-year survival of 52.9% (95% CI, 45.3 - 60), 43.3% (95% CI, 35.8 - 50.6) and 30.8% (95% CI, 23.6 - 30.4) respectively (Figure 1). A pre-treatment HCT - CI score of >2 HR 3.37 (1.084 - 10.461), p=0.04 was associated with increased 30-day TRM. Conclusion This study of 186 patients with AML treated at the UCI with standard induction regimens over a 5-year period demonstrates a CR rate of 46.2% and an overall 30-day induction TRM rate of 31.5%. A pre-treatment HCT-CI score of >2 was associated with increased TRM. Our study demonstrates the urgent need for interventions that can reduce TRM and improve the outcome for patients with AML in LMICs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Assessment of the Frequency of Variants of Undetermined Significance (VUS) in Adult Patients with Chronic Idiopathic Neutropenia Studied for Myeloid Gene Mutations By Next Generation Sequencing

Background: Chronic Idiopathic Neutropenia (CIN) is a neutrophil disorder characterized by the persistent and unexplained reduction in the number of peripheral blood (PB) neutrophils. The diagnosis of CIN is based on exclusion criteria, namely the absence of clinical/laboratory evidence of any underlying disease or use of drugs that might be associated with neutropenia, absence of history of exposure to irradiation or use of chemical compounds, negative antineutrophil antibodies, and normal bone marrow (BM) morphology and karyotype. We have previously performed next generation sequencing (NGS) analysis of genes recurrently mutated in myeloid malignancies in a cohort of CIN patients and we found that 11.35% of the patients display clonal hematopoiesis in term of presence of pathogenic/likely pathogenic variants with VAF higher than 2%. Variants of undetermined significance (VUS) were ruled out from the analysis and their potential significance was not assessed. Aims: In the current study we have assessed the presence, frequency and potential clinical significance of VUS detected in our cohort of CIN patients previously studied for clonal hematopoiesis. Methods: Genomic DNA was extracted from CIN patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted NGS on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies (Tsaknakis G et al. Blood. 2021;138:1249-1257). Results: NGS analysis of myeloid genes has been performed in 139 CIN patients (106 female subjects and 33 male subjects, aged 21 to 89 years with a median age of 59 years). Re-analysis of patients' NGS data and assessment of VUS revealed that 35 out of 139 patients (25.18%) carried one or more VUS (a total of 39 VUS were detected, all with an allelic frequency of 50% and all missense variants). Genetic variants were evaluated according to the ACMG guidelines and classified as VUS. The majority of VUSs were observed in ATM (n=7) and SETBP1 (n=6) genes followed by TET2, NOTCH1 (4 occurrences respectively) and PLCG2 (n=3). Two different patients shared the same variant in TET2 gene whereas two siblings shared the same variant in SETBP1 gene. Pathogenicity score calculation based on the combined evidence from multiple other in-silico predictors identified 3 variants in 3 different genes as having the highest probability to be pathogenic. Namely these were PLCG2K456E,CXCR4R235C and SF3B1T56I. PLCG2 variants have been implicated in familial cold autoinflammatory syndrome 3 (PLCG2 associated antibody deficiency and immune dysregulation, PLAID) and in autoinflammation, antibody deficiency, and immune dysregulation syndrome (APLAID) whereas CXCR4 variants are known to be associated with Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome. SF3B1 mutations have not yet been shown to be involved in hereditary disease. Conclusions: Our study of VUS assessment in a cohort of 139 well characterized adult CIN patients identified 39 VUS, all heterozygous, in a total of 35 patients. Further steps include paired analysis with constitutional reference tissue to determine the germline nature of the mutations. Moreover, these variants should be functionally tested and integrated with functional results and clinical phenotypes to more accurately interpret their potential contribution to disease onset. This study of VUS analysis in CIN patients is anticipated to contribute to the better understanding of the pathogenetic causes of this rare disease.

Lovo-cel (bb1111) Gene Therapy for Sickle Cell Disease: Updated Clinical Results and Investigations into Two Cases of Anemia from Group C of the Phase 1/2 HGB-206 Study

Background: Lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) uses autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an anti-sickling hemoglobin (Hb), HbAT87Q. The ongoing phase 1/2 HGB-206 study (NCT02140554) is the largest clinical trial of GT in SCD to date. We report updated efficacy and safety results for Group C and describe the results of additional investigations in two patients (pts) in Group C with persistent anemia. Methods: Pts (≥12-≤50 yrs of age) with SCD and recurrent severe vaso-occlusive episodes (VOEs) underwent plerixafor mobilization and apheresis followed by myeloablative busulfan conditioning and lovo-cel infusion. Lab evaluations, SCD-related outcomes, and safety were assessed. Data are median (min-max) unless otherwise stated; all timepoints are from lovo-cel infusion. Results: As of July 2021, 35 Group C pts had received a lovo-cel infusion, with a median follow-up of 20.9 months (min-max: 8.5-28.5). Neutrophil and platelet engraftment were achieved on Days 20 (12-35) and 36 (18-136), respectively. The median vector copy number in peripheral blood remained stable in all pts at ≥1 c/dg from 6 months after infusion until the last study visit, and resulted in median HbAT87Q levels of ≥5 g/dL during this period. The median total Hb level increased without transfusions from 8.5 g/dL at baseline to ≥11 g/dL from 6 months through last visit; HbAT87Q contributed at least 40% of total Hb (Fig 1A). Of the 29 pts who could be evaluated, 28 had a complete resolution of severe VOEs after lovo-cel infusion in the 2-yr parent study as of last follow-up, as compared with 3.5 events per yr (1.0-13.5) in the 2 yrs before enrollment (Fig. 1B); no severe VOEs have been reported during the long-term follow up study. Key hemolysis markers approached normal levels. Overall, 15 pts (43%) had ≥1 serious adverse event after lovo-cel infusion; the most frequently reported were abdominal pain, drug withdrawal syndrome (opiate), nausea, vomiting, and dehydration (6% each). No cases of veno-occlusive liver disease, graft failure, replication-competent lentivirus, vector-mediated insertional oncogenesis, or hematologic malignancies were observed. The lovo-cel treatment regimen generally reflected the known side effects of HSPC collection and busulfan conditioning regimen, and/or adverse events commonly seen in the population being evaluated. Further investigations were conducted as of March 2022 in two pts who presented with similar clinical and pathological findings that initially raised suspicion of myelodysplastic syndrome (MDS). Presentation features included neutropenia in one patient; both patients had persistent anemia despite adequate HbAT87Q production, bone marrow morphology showing erythroid specific dysplasia (binucleated or trinucleated cells) consistent with dyserythropoiesis as seen in other hemoglobinopathies, and low level of trisomy 8 (≤7.7%) only observed by fluorescence in situ hybridization analysis. There are no clinical symptoms suggestive of hematologic malignancies, no driver mutations, no clonal process (vector-related or otherwise) as shown by Integration Site Analysis and Next-Generation Sequencing (both subjects had >29,000 Unique Integration Sites with no integration having a relative frequency above 0.425%), and no chromosomal abnormalities on karyotyping among up to 205 metaphases. Both pts have had anemia and one maintained total Hb of 9.8 g/dL at Month 18 without transfusions, and the other required chronic transfusions to maintain 10.3 g/dL at Month 18 with otherwise normal complete blood counts. Both pts experienced a reduction or resolution of severe VOEs after lovo-cel infusion. Of note, these are the only pts in the study to have two α-globin deletions (−α3.7/−α3.7). We propose that α-globin trait may contribute to anemia and dyserythropoiesis after lovo-cel infusion. Summary: In Group C of the HGB-206 study, one-time treatment with lovo-cel resulted in sustained production of HbAT87Qand near-complete resolution of severe VOEs. Investigations to date in both patients with anemia have not confirmed MDS; thus, currently no hematologic malignancies have occurred in Group C. Ongoing long-term follow-up should be continued for all pts receiving GT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A New Genetic Immunodeficiency Syndrome Caused By Autosomal Dominant Mutations in LCP1 Identified By Mosaic Tetraploidy and Severe Neutropenia in Unrelated Pedigrees

The spectrum of genetic abnormalities causing immune deficiencies is still far from complete. We identified independent kindreds in the Netherlands (NL) and the USA in which affected members presented with severe chronic neutropenia (SCN), but lacked the thus far known mutations causing SCN. Bone marrow morphology of the patients showed large hyper-segmented and binucleate neutrophils. In addition to neutropenia, lymphopenia and abnormalities in B cell development were seen, indicating that the hematopoietic defect was not restricted to the neutrophil lineage. An additional remarkable and thus far little-known feature was the presence of cells in the bone marrow with a tetraploid karyotype (Hochberg et al., Pediatr Blood Cancer 2008). Pedigree analysis of the NL and USA families revealed a hereditary pattern consistent with an autosomal dominant inheritance (Figure 1). Comparative whole exome sequencing of both pedigrees identified LCP1, encoding the actin-bundling protein L-plastin, as the single candidate gene, completely segregating affected from unaffected individuals. The LCP1 mutations in the pedigrees are not identical, but give rise to the same altered transcript. The LCPNLmutation is a single nucleotide substitution that disrupts the splice acceptor site at the 5' side of exon 8. In contrast, the mutation LCP1USA causes a deletion of 25 nucleotides at the boundary of intron 7 and exon 8. Theoretically, the LCP1USAmutation could cause a frameshift deletion in exon 8 resulting in a premature stop codon in exon 8. However, because this mutation also disrupts the 5' splice acceptor site of exon 8, it could also lead to alternative RNA splicing, using an alternate cryptic splice acceptor site in exon 8. Sanger sequencing on cDNA derived from mRNA from NL-I unveiled that the splice-site mutation indeed results in an alternative splicing variant with an in-frame loss of 24 nucleotides at the start of exon 8. Next generation RNA sequencing on bone marrow cells from NL-I and USA-III (Figure 1) revealed that the identical alternative splice variant is expressed from LCP1USA and LCP1NL. In both pedigrees, there is approximately a fifty percent drop in reading depth from the start of exon 8 up until the next available splice acceptor site, consistent with a heterozygous 24-nucleotide deletion from the start of exon 8. The resulting in-frame deletion of 8 amino acids affects a highly conserved region between actin binding domain (ABD) 1 and ABD2, thereby predicted to affect the horseshoe-like 3D structure of L-plastin. LCP1USA and LCP1NL will henceforth collectively be termed LCP1ex8mut. Confocal microscopy analysis of HeLa cells expressing LCP1ex8mut showed that mutant L-plastin colocalized with F-actin in excessively formed filopodia, which was not seen in cells expressing wild-type LCP1 (LCP1wt). In addition, binucleate cells were present in cells expressing LCP1ex8mut, but not LCP1wt. A number of cells in G2M phase showed accumulation of F-actin and L-plastin on the contractile ring, a characteristic seen only in cells expressing LCP1ex8mut. This finding suggests that mutant L-plastin interferes with successful completion of cell division. Accumulation of mutated LCP1 on the contractile ring thus could explain the inability of neutrophil progenitors to properly go through cytokinesis, hence giving rise to neutropenia and tetraploidy. Finally, because a recently developed mouse model revealed that phosphorylation of serine 5 (Ser5) of L-plastin is essential for its function in host-pathogen interactions and immune defense in vivo (Anaya et al., J Immunol 2021), we mutated Ser5 to alanine (Ser5Ala) in LCP1ex8mut. This did not affect the function of LCP1ex8mut, suggesting that the mutation confers activation of L-plastin independent of upstream serine/threonine kinase pathways. Altogether, these findings establish that the mutations confer a gain of function rather than haploinsufficiency. In conclusion, by comparing independent pedigrees, we have identified a new immune deficiency syndrome caused by autosomal dominant activating mutations in LCP1 leading to defective neutrophil production and B cell development, which at least in part results from disturbed cytokinesis causing tetraploidy. Figure1. Pedigree information of the NL and USA family. Solid black squares or circles indicate neutropenic males or females, respectively. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Triaging of Bone Marrow for Esoteric Testing – Cost-effective and Patient-centric Approach Utilizing WHO Nomenclature for Hematolymphoid Malignancies

Abstract Introduction/Objective Esoteric testing (ES) plays a major role in bone marrow (BM) evaluation. Providers usually order tests considering differential diagnoses based on clinical history. Hematopathologists (HP) while reviewing the BM consider modifying the standing orders. Knowledge and simple guidelines for triaging BM for ES are limited in the literature. Methods/Case Report Between 2015-2021, a total of 2560 BMs were selected. Initial tests that were ordered by providers were reviewed by HP considering clinical history, medical necessity, and preliminary BM morphology. Average Medicare fee schedule for 2022 was used for cost analysis. Types of ES were evaluated for Flow Cytometry (FC); Cytogenetics (CG); Fluorescence In-Situ Hybridization (FISH) and Molecular Genetics (MG). Immunostains were excluded in this study. Results (if a Case Study enter NA) Cases were evaluated and categorized as cytopenias, cytosis, staging, MPN, MDS, myelomas, treatment follow-up and miscellaneous indications. ES were modified based on medical necessity and blinded with insurance plans or payment status. Initial clinical orders were modified by HP as follows: FC-reduced 15%; CG-reduced 38%; FISH-modified 25%; MG-modified 40% (single assays or NGS). Overall healthcare savings by modifying tests were close to $1.2M. Conclusion Effective triaging of BM is a complex decision and can result in significant cost savings. Factors positively impacting these savings are: 1) expertise training in hematopathology, 2) personalized order of ES considering clinical indication, 3) peer-to- peer interaction with providers, and 4) the types of samples obtained. Collaborative efforts among pathologists and clinicians may lead to appropriate ordering of tests, cost- saving effective patient care with relevant diagnostic, therapeutic and prognostic information obtained as recommended by WHO nomenclature for hematolymphoid malignancies. In addition, established practice guidelines with personalized medicine will reduce the burden for pre-authorization when challenged with time sensitive samples obtained from cancer patients obtained thru invasive procedures.

Carbohydrates and ginsenosides in shenmai injection jointly improve hematopoietic function during chemotherapy-induced myelosuppression in mice

BackgroundShenmai injection (SMI), a traditional Chinese medicine (TCM) injection prepared from Red ginseng and Ophiopogon japonicus, is widely used in clinics to treat chemotherapy-induced myelosuppression. Similar to other TCM injections, SMI contains a high amount of carbohydrates (fructose, sucrose, and maltose) in addition to the bioactive substances, specifically ginsenosides (Rg1, Re, and Rb1). To date, the role of these carbohydrates in the hematopoietic function of SMI remains unclear.PurposeWe aimed to investigate the hematopoietic effects and potential mechanisms of SMI and its components, focusing on the carbohydrates present in SMI.Experimental design/methodsFirst, we evaluated the hematopoietic effect of SMI on 5-fluorouracil (5-FU)-induced myelotoxicity in a tumor-bearing mouse model. Then we prepared mixtures of ginsenosides and carbohydrates according to their proportions in SMI and evaluated their hematopoietic function in mice with 5-FU-induced myelosuppression. Finally, hematopoiesis-related molecular networks were built based on RNA sequencing (RNA-seq) of the bone marrow stromal cells (BMSCs), and the potential mechanisms of carbohydrates and ginsenosides were evaluated.ResultsSMI attenuated 5-FU-induced myelotoxicity in tumor-bearing mice. Both ginsenosides and carbohydrates increased the bone marrow nucleated cell (BMNC) count and improved the bone marrow morphology in myelosuppressive mice; they promoted the proliferation of BMSCs derived from those myelosuppressive mice. Bioinformatics analyses revealed ECM-receptor interaction, Hippo signaling, and Wnt signaling are common pathways regulated by both ginsenosides and carbohydrates; Gstt1, Gstp2, Gsta4 and Oplah in Glutathione metabolism pathway and Cd19, Cd79a, and Cd79b in B cell receptor pathway are uniquely regulated genes related to carbohydrates but not ginsenosides.ConclusionsCarbohydrates may collaborate with ginsenosides and contribute to the hematopoietic function of SMI. Carbohydrates could be considered as a bioactive component in this TCM injection.Graphical