Bone Marrow Neoplasms Research Articles

Bithionol eliminates acute myeloid leukaemia stem-like cells by suppressing NF-κB signalling and inducing oxidative stress, leading to apoptosis and ferroptosis

Acute myeloid leukaemia (AML) is a lethal bone marrow neoplasm caused by genetic alterations in blood cell progenitors. Leukaemic stem cells (LSCs) are responsible for the development of AML, drug resistance and relapse. Bithionol is an old anthelmintic drug with potential antibacterial, antiviral, antifungal, anti-Alzheimer, and antitumour properties. In this work, we focused on the anti-AML LSC properties of bithionol. This compound inhibited the viability of both solid and haematological cancer cells, suppressed AML stem-like cells, and inhibited AML growth in NSG mice at a dosage of 50 mg/kg, with tolerable systemic toxicity. Bithionol significantly reduced the levels of phospho-NF-κB p65 (Ser529) and phospho-NF-κB p65 (Ser536) and nuclear NF-κB p65 translocation in AML cells, indicating that this molecule can suppress NF-κB signalling. DNA fragmentation, nuclear condensation, cell shrinkage, phosphatidylserine externalisation, loss of transmembrane mitochondrial potential, caspase-3 activation and PARP-(Asp 214) cleavage were detected in bithionol-treated AML cells, indicating the induction of apoptosis. Furthermore, this compound increased mitochondrial superoxide levels, and bithionol-induced cell death was partially prevented by cotreatment with the selective ferroptosis inhibitor ferrostatin-1, indicating the induction of ferroptosis. In addition, bithionol synergised with venetoclax in AML cells, indicating the translational potential of bithionol to enhance the effects of venetoclax in patients with AML. Taken together, these data indicate that bithionol is a potential new anti-AML drug.

Role of the STINGpathway in myeloid neoplasms: a prospero-registered systematic review of principal hurdles of STINGon the road to the clinical practice.

Myeloid neoplasms are a group of bone marrow diseases distinguished by disruptions in the molecular pathways that regulate the balance between hematopoietic stem cell (HSC) self-renewal and the generation of specialized cells. Cytokines and chemokines, two important components of the inflammatory process, also influence hematological differentiation. In this scenario, immunological dysregulation plays a pivotal role in the pathogenesis of bone marrow neoplasms. The STING pathway recognizes DNA fragments in the cell cytoplasm and triggers an immune response by type I interferons. The role of STING in cancer has not yet been established; however, both actions, as an oncogene or tumor suppressor, have been documented in other types of cancer. Therefore, we performed a systematic review (registered in PROSPERO database #CRD42023407512) to discuss the role of STING pathway in the advancement of pathogenesis and/or prognosis for different myeloid neoplasms. In brief, scientific evidence supports investigations that primarily use cell lines from myeloid neoplasms, such as leukemia. More high-quality research and clinical trials are needed to understand the role of the STING pathway in the pathology of hematological malignancies. Finally, the STING pathway suggests being a promising therapeutic molecular target, particularly when combined with current drug therapies.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Phase 3b Study Design for a Comparison of Treatment Preference between Oral Decitabine/Cedazuridine and Azacitidine in Adult Patients with IPSS-R Intermediate Myelodysplastic Syndrome, IPSS Intermediate-2 or High-Risk Myelodysplastic Syndrome, Low-Blast Acute Myeloid Leukaemia, or Chronic Myelomonocytic Leukaemia

Myelodysplastic syndromes (MDS) are heterogeneous clonal bone marrow neoplasms characterised by marrow dysplasia, low blood counts and risk of progression to acute myeloid leukaemia (AML). Hypomethylating agents (HMAs) decitabine and azacitidine (AZA) are effective treatments for hematologic cancers including MDS. AZA is indicated to treat International Prognostic Scoring System (IPPS) defined Intermediate-2 (Int-2) and high-risk (HR) MDS, low blast AML (LB-AML; 20-30% blasts) with multi-lineage dysplasia, and chronic myelomonocytic leukaemia (CMML) with 10-29% blasts without myeloproliferative disorder. Oral decitabine/cedazuridine is currently the only oral hypomethylating agent indicated to treat IPSS Int-1, Int-2 or HR MDS and CMML, in the United States, Canada and Australia. AZA administration requires daily visits to a hospital or clinic for 7 days every 28 days, for either intravenous infusions, or large-volume subcutaneous (SC) injections for a minimum of 6 cycles.This places a burden on this patient population, resulting in low treatment adherence, early discontinuation, and subsequently low treatment uptake. Oral decitabine/cedazuridine is administered as one tablet daily for the first 5 days of a 28-day cycle for a minimum of 4 cycles. This allows patients to take their medication at a location of their choice. Alongside previously reported preference for oral therapy, patients may favour oral decitabine/cedazuridine over SC AZA due to improved treatment convenience and reduced discomfort. However, this is untested in a controlled setting. This study aims to evaluate preference between oral decitabine/cedazuridine and SC AZA treatment for adult patients with Revised International Prognostic Scoring System (IPSS-R) intermediate, IPSS Int-2 or HR MDS, LB-AML or CMML. This phase 3b, open-label, multi-centre study (NCT05883956) has sites planned in Australia (4 sites) and New Zealand (5 sites), to compare preference for oral decitabine/cedazuridine (Treatment A) and SC AZA (Treatment B). The study design includes 28 days of screening, four continuous 28-day cycles of study treatment, and a follow-up period with two 28-day cycles of continued therapy. Patients will be randomised to two balanced treatment sequences: ABBA or BAAB (Figure 1). Patients will express a preference twice in the study; after completing Cycle 1 and 2, and after completing Cycle 3 and 4. This cross-over design is twice the length of a typical 2-period AB vs BA design and allows patients to act as their own controls. The study design allows estimation of the treatment effect while mitigating factors that may influence preference. These include the order of treatments, and fluctuations in the patient's physical and disease state over the course of the trial. All available preferences expressed throughout the trial will be used for primary analysis. A total of 42 patients are planned for this study. The primary objective of this study is to compare patients' treatment preference for oral decitabine/cedazuridine and SC AZA for the treatment of MDS, LB-AML or CMML. Secondary objectives include evaluation of carers' and clinicians' treatment preference between oral decitabine/cedazuridine and SC AZA for the treatment and continued treatment of adult patients with MDS, LB-AML or CMML. The primary objective will be assessed by a treatment preference in myelodysplasia questionnaire (pTPMQ), which instructs patients to indicate a preference for injection, oral/tablets or no preference. The secondary objectives will be evaluated by a carer treatment preference in myelodysplasia questionnaire (cTPMQ) and a medical clinician treatment preference in myelodysplasia questionnaire (mTPMQ). Treatment discontinuation rates, quality of life and safety of SC AZA and oral decitabine/cedazuridine will also be compared. The primary analysis will be conducted with a multilevel logistic regression model analysing all available preferences. Descriptive statistics including frequencies, proportions, mean, standard deviation, median, and interquartile range will be used to summarize the study data. When complete, this study will address an evidence gap in the comparison of patient preference for oral decitabine/cedazuridine and SC AZA, and subsequently inform the value of oral decitabine/cedazuridine for the treatment of MDS, LB-AML and CMML.

ß-catenin expression in myelodysplastic syndromes and myeloproliferative neoplasms in bone marrow, in relation to CD34 and CD117 status

Aims: The activation of the Wnt/ ß-catenin signaling pathway has been demonstrated to play a crucial role in the development of myeloid neoplasms. In addition to CD34, which has been used until now in the diagnosis and staging of clonal hematopoietic diseases, Myelodysplastic Syndromes (MDS) and Myeloproliferative Neoplasms (MPN), CD117, which has found its place in hematopoietic diseases, also provides significant benefits in these respects. In this study, we evaluated the immunohistochemical presence and utility of ß-catenin in blasts relative to other markers, as the inhibition of ß-catenin activity may be an attractive therapeutic approach Methods: By retrospectively analyzing bone marrow samples with ß-catenin immune marker, we determined the staining rates, intensities, and patterns of 30 MDS, 29 MPN cases and 30 normal bone marrow controls, in comparison to the efficacy of the the well-known CD34 and CD117. We statistically interpreted the correlation between them. Results: Based on the findings, ß-catenin, which has recently been used in hematopoietic diseases and is said to have a high efficacy in acute myeloid leukemia (AML) cases, was not immunohistochemically detectable in our study. As expected, CD34 and CD117 immun markers exhibited significant blast staining. MPN cases were more prone to staining with CD117. Conclusion: CD34 continues to be the most reliable marker for identifying blasts for diagnosing and grading bone marrow neoplasms while CD117 may have a supportive role in this process. Further investigation is required to ascertain the true effectiveness of ß-catenin, a molecule that has demonstrated encouraging potential in the context of AML.

Fibrinogen-to-albumin ratios are related to mortality in patients with secondary malignant neoplasm of bone and bone marrow

BackgroundSystemic inflammation has long been considered to be one of the mechanisms in the development and progression of secondary malignant neoplasm of bone and bone marrow (BM). Fibrinogen-to-albumin ratio (FAR) has been demonstrated to be vital to the poor prognosis of lots of disease such as tumor and coronary heart disease. This study aimed to determine whether FAR could be used as an independent risk prognostic factor in patients with BM. MethodsFirstly, the baseline data of patients with BM, who met our inclusion/exclusion criteria, were extracted and analyzed from MIMIC-III database. The association between FAR and endpoints was analyzed using a Cox proportional hazards regression model and a propensity score matched analysis. ResultsThe univariate analysis revealed that for the 30-day mortality, the HR (95% CI) in the FAR ≥99.5group was 1.47 (1.15,1.91). After adjusting various factors, FAR ≥99.5 was found to be an independent significant risk factor for death in patients with BM (HR = 1.11, 95% CI: 1.01–1.42). Similarly, for the one-year mortality, HR (95% CI) in the FAR ≥99.5 group was 1.51 (1.11, 2.06). ConclusionIt has been found that FAR provides independent prognostic information for mortality among patients with secondary malignant neoplasms of the bone and bone marrow.

Does the SORG Machine-learning Algorithm for Extremity Metastases Generalize to a Contemporary Cohort of Patients? Temporal Validation From 2016 to 2020.

The ability to predict survival accurately in patients with osseous metastatic disease of the extremities is vital for patient counseling and guiding surgical intervention. We, the Skeletal Oncology Research Group (SORG), previously developed a machine-learning algorithm (MLA) based on data from 1999 to 2016 to predict 90-day and 1-year survival of surgically treated patients with extremity bone metastasis. As treatment regimens for oncology patients continue to evolve, this SORG MLA-driven probability calculator requires temporal reassessment of its accuracy. Does the SORG-MLA accurately predict 90-day and 1-year survival in patients who receive surgical treatment for a metastatic long-bone lesion in a more recent cohort of patients treated between 2016 and 2020? Between 2017 and 2021, we identified 674 patients 18 years and older through the ICD codes for secondary malignant neoplasm of bone and bone marrow and CPT codes for completed pathologic fractures or prophylactic treatment of an impending fracture. We excluded 40% (268 of 674) of patients, including 18% (118) who did not receive surgery; 11% (72) who had metastases in places other than the long bones of the extremities; 3% (23) who received treatment other than intramedullary nailing, endoprosthetic reconstruction, or dynamic hip screw; 3% (23) who underwent revision surgery, 3% (17) in whom there was no tumor, and 2% (15) who were lost to follow-up within 1 year. Temporal validation was performed using data on 406 patients treated surgically for bony metastatic disease of the extremities from 2016 to 2020 at the same two institutions where the MLA was developed. Variables used to predict survival in the SORG algorithm included perioperative laboratory values, tumor characteristics, and general demographics. To assess the models' discrimination, we computed the c-statistic, commonly referred to as the area under the receiver operating characteristic (AUC) curve for binary classification. This value ranged from 0.5 (representing chance-level performance) to 1.0 (indicating excellent discrimination) Generally, an AUC of 0.75 is considered high enough for use in clinical practice. To evaluate the agreement between predicted and observed outcomes, a calibration plot was used, and the calibration slope and intercept were calculated. Perfect calibration would result in a slope of 1 and intercept of 0. For overall performance, the Brier score and null-model Brier score were determined. The Brier score can range from 0 (representing perfect prediction) to 1 (indicating the poorest prediction). Proper interpretation of the Brier score necessitates a comparison with the null-model Brier score, which represents the score for an algorithm that predicts a probability equal to the population prevalence of the outcome for each patient. Finally, a decision curve analysis was conducted to compare the potential net benefit of the algorithm with other decision-support methods, such as treating all or none of the patients. Overall, 90-day and 1-year mortality were lower in the temporal validation cohort than in the development cohort (90 day: 23% versus 28%; p < 0.001, and 1 year: 51% versus 59%; p<0.001). Overall survival of the patients in the validation cohort improved from 28% mortality at the 90-day timepoint in the cohort on which the model was trained to 23%, and 59% mortality at the 1-year timepoint to 51%. The AUC was 0.78 (95% CI 0.72 to 0.82) for 90-day survival and 0.75 (95% CI 0.70 to 0.79) for 1-year survival, indicating the model could distinguish the two outcomes reasonably. For the 90-day model, the calibration slope was 0.71 (95% CI 0.53 to 0.89), and the intercept was -0.66 (95% CI -0.94 to -0.39), suggesting the predicted risks were overly extreme, and that in general, the risk of the observed outcome was overestimated. For the 1-year model, the calibration slope was 0.73 (95% CI 0.56 to 0.91) and the intercept was -0.67 (95% CI -0.90 to -0.43). With respect to overall performance, the model's Brier scores for the 90-day and 1-year models were 0.16 and 0.22. These scores were higher than the Brier scores of internal validation of the development study (0.13 and 0.14) models, indicating the models' performance has declined over time. The SORG MLA to predict survival after surgical treatment of extremity metastatic disease showed decreased performance on temporal validation. Moreover, in patients undergoing innovative immunotherapy, the possibility of mortality risk was overestimated in varying severity. Clinicians should be aware of this overestimation and discount the prediction of the SORG MLA according to their own experience with this patient population. Generally, these results show that temporal reassessment of these MLA-driven probability calculators is of paramount importance because the predictive performance may decline over time as treatment regimens evolve. The SORG-MLA is available as a freely accessible internet application at https://sorg-apps.shinyapps.io/extremitymetssurvival/ .Level of Evidence Level III, prognostic study.

Preclinical pharmacokinetics of a promising antineoplastic prototype piperazine-containing compound (LQFM018) in rats by a new LC-MS/MS bioanalytical method

LQFM018 is a novel antineoplastic prototype, showing an expressive drug-triggered K562 leukemic cells death mechanism, through necroptotic signaling. Due to its promising effect, this study aimed to evaluate the pharmacokinetics of LQFM018 in rats, using a new validated bioanalytical LC-MS/MS-based method. Chromatographic column was an ACE® C18 (100 mm × 4.6 mm, 5 µm) eluted by a mobile phase composed of ammonium acetate 2 mM and formic acid 0.025%:methanol (50:50, v/v), under flow of 1.2 mL/min and injection volume of 3.0 µL. LQFM018 was extracted from rat plasma by a simple liquid–liquid method, using MTBE solvent. Rats were administered intraperitoneally at LQFM018 100 mg/kg dose and blood samples were collect at times of 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 h. Bioanalytical-LC-MS/MS-based method was rapid, high throughput and sensitive with a good linearity ranging from 10 (LLOQ) to 15000 ng/mL, besides precise and accurate, ranging of 0.8–7.3% and 96.8–107.6%, respectively. The prototype LQFM018 was rapid and well absorbed, and highly distributed, apparently due to its high lipid solubility. These features are primordial for an anticancer agent in the treatment of deep tumors, such as bone marrow neoplasms, in which the drug might permeate easily tissue barriers. Also, LQFM018 has demonstrated a high clearance, according to a low t1/2in rats, indicating a relative fast elimination phase related to a possible intense hepatic biotransformation. These information support further studies to establish new understands on pharmacokinetics of promising antineoplastic prototype LQFM018 from preclinical and clinical evaluations.

Abstract 7 Reduced Intensity Conditioning Umbilical Cord Blood Transplantation in Combination with Splenic Irradiation in a Patient with Secondary Myelofibrosis and Massive Splenomegaly

IntroductionMyelofibrosis (MF) is a bone marrow neoplasm characterized by marrow fibrosis/failure and splenomegaly, causing pain, early satiety, and cytopenia. The only curative option is allogeneic hematopoietic stem cell transplant (allo-HSCT); however, its use is limited due to donor availability and patient age, functional status, and comorbidities. Umbilical cord blood (UCB) has been explored in this population for this reason with variable success, partly related to splenomegaly, which might influence engraftment in MF patients.ObjectiveThe objective was to report the successful use of UCB transplantation in conjunction with splenic irradiation to treat a patient with advanced MF and massive splenomegaly.MethodsA 71-year-old male with post-essential thrombocythemia MF was treated with ruxolitinib for 2 years but developed worsening cytopenia and transfusion dependence. Splenomegaly (19.1×7.1×20.8 cm) was identified. Allo-HSCT was recommended on the basis of high-risk disease (DIPSS Plus score: 4) and few alternative options. Although suitable HLA-matched donors were unattainable, two cord blood units were available. A reduced intensity conditioning regimen of fludarabine, cyclophosphamide, and TBI 300 was chosen. Given the primary resistance to ruxolitinib, the spleen was irradiated with 450 cGy prior to transplant.ResultsNeutrophil and platelet engraftment occurred on days +30 and +49, respectively. The patient developed engraftment syndrome and acute graft versus host disease (GVHD) that resolved with methylprednisolone and early introduction of ruxolitinib. His immunosuppressive medications were discontinued by 6-months without evidence of chronic GVHD thereafter. He is now 1 year post-transplant with 100% donor chimerism and no clinical evidence of MF or GVHD.DiscussionWe present a novel therapeutic approach combining the use of splenic irradiation and a double unit UCB HSCT in a patient with MF and massive splenomegaly. UCB transplant should be considered in patients with MF who do not have an HLA-matched donor with the additional benefit of less chronic GvHD. Massive splenomegaly can negatively impact transplant outcomes due to entrapment of donor stem cells, delayed engraftment, and prolonged cytopenia. Splenic irradiation before UCB transplant can potentially reduce these complications, as it did in this case, and in turn decrease transplant-related mortality. Our future aim is to develop a clinical trial to study this approach.

A Novel Nomogram for Predicting Morbidity Risk in Patients with Secondary Malignant Neoplasm of Bone and Bone Marrow: An Analysis Based on the Large MIMIC-III Clinical Database.

ObjectiveBone and bone marrow are the third most frequent sites of metastases from many cancers and are associated with low survival and high morbidity rates. Currently, there are no effective bedside tools to predict the morbidity risk of these patients in general intensive care units (ICUs). The main objective of this study was to establish and validate a nomogram to predict the morbidity risk of patients with bone and bone marrow metastases.MethodsData on patients with bone and bone marrow metastases were extracted from the Medical Information Mart for Intensive Care III (MIMIC-III) database. The patients were divided into training and validation cohorts. The data were analyzed using univariate and multivariate Cox regression methods. Factors significantly and independently prognostic of survival were used to construct a nomogram predicting 30-day morbidity. The nomogram was validated by various methods, including Harrell’s concordance index (C-index), area under the receiver operating characteristic curve (AUC), calibration curve, integrated discrimination improvement (IDI), net reclassification index (NRI), and decision curve analysis (DCA).ResultsThe study included 610 patients in the training cohort and 262 in the validation cohort. Multivariate Cox regression analysis showed that temperature, SpO2, Sequential Organ Failure Assessment (SOFA) score, Oxford Acute Severity of Illness Score (OASIS), comorbidities with coagulopathy, white blood cell count, heart rate, and respiratory rate were independent predictors of patient survival. The resulting nomogram had good discriminative ability, as shown by high AUCs, and was well calibrated, as demonstrated by calibration curves. Improvements in NRI and IDI values suggested that the nomogram was superior to the SOFA scoring system. DCA curves revealed that the nomogram showed good value in clinical applications.ConclusionThis prognostic nomogram, based on demographic and laboratory parameters, was predictive of the 30-day morbidity rate in patients with secondary malignant neoplasms of the bone and bone marrow, suggesting its applicability in clinical practice.

Multiple Myeloma Involving Left Hemicranium

AbstractMultiple myeloma (MM) is a malignant neoplasm of bone marrow affecting plasma cells. It is commonly seen as multiple punched-out lesions in the skull bone as a characteristic feature. Its presentation as hemicranial involvement with intracranial extension is rare. A 46-year-old male presented with left side scalp swelling, prominent over parietal region. X-ray showed multiple punched out lesions involving left hemicranium. CT and MRI brain showed intracranial extension of lesion without brain parenchyma invasion. He was treated with biopsy of lesion followed by chemotherapy.

Multiparameter Flowcytometry for Diagnosis and Subtyping of Mature Lymphocytic Neoplasms in Peripheral Blood and Bone Marrow: A Prospective Observational Study

Introduction: Multiparameter Flowcytometry (MFC) is a high throughput, quick, and practical technique for diagnoses of Chronic Lymphoproliferative Disorders (CLPDs). Indian CLPDs cases have distinct distribution and presentation than the developed world. Moreover, limited studies have confirmed the diagnostic utility of MFC in Indian CLPDs cases. Aim: To evaluate the diagnostic utility of MFC in peripheral blood and bone marrow aspirate of CLPDs cases. Materials and Methods: This was a single centre, prospective, observational study involving clinico-morphologically suspected or diagnosed 85 CLPDs cases. It was carried out in the Department of Pathology, Government Medical College and Hospital, Nagpur, Maharashtra, India, from January 2016 to November 2019. The patients were followed up for peripheral smear (PS) and Bone Marrow(BM) MFC and staging in nodal or extranodal Non-Hodgkin’s Lymphoma was done. Results: Clinico-morphological examination led to the diagnosis of 74 CLPD cases, while remaining 11 cases were strongly suspected. MFC immunophenotyping was contributory in diagnosing 74 CLPD cases which on further subtyping consisted of B-cell CLPD (N=70), and T-cell CLPD (N=3), while one case of B-NHL could not be subtyped. The most common B-cell CLPD included multiple myeloma (n=27), chronic lymphocytic leukemia (n=25), diffuse large B-cell lymphoma (n=7). T-cell CLPD included hepatosplenic gamma delta T-cell lymphoma (N=2) and adult T-cell lymphoma, follicular lymphoma (n=3), burkitt’s lymphoma (n=2), mantle cell lymphoma (n=2), prolymphocytic leukemia (n=2), splenic marginal zonal lymphoma (n=2), B Cell Non-Hodgkin’s Lymphoma (n=1). Finally, 11 suspected cases mostly comprised of reactive lymphocytosis (81.8%). Conclusion: MFC immunophenotyping led to diagnoses and determination of CLPD sub-class. It also resulted in rapid diagnoses of reactive hyperplasia and non-hematolymphoid malignancy which may mimic CLPD on morphology and hence, difficult to diagnose based on morphology alone.

Immunogenicity of Covid-19 Vaccination in Subjects with Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) represent a spectrum of clonal bone marrow neoplasms from low risk disease through to those transforming into acute myeloid leukaemia. The COVID-19 pandemic has presented a great risk to those with hematological malignancies who are at higher risk of severe disease and death than the general population. Previous studies looking at the immune response to influenza vaccination in those with MDS had shown promising results, with immune responses not differing from those of healthy family members. Whilst some data exist to reassure the MDS community that majority of patients show seroconversion following Covid-19 vaccination, little data exists on their neutralizing capacity or post vaccination T-cell responses in this cohort. In addition, the majority of patients in these studies received BNT162b2 and there is little published data on vaccine response to the ChAdOx1 nCoV-19 vaccine. We have investigated the humoral and T-cell response of 39 patients with MDS two to four weeks following Covid-19 booster vaccination with BNT162b2 or ChAdOx1 nCoV-19 through the SOAP study (Sars-cov-2 fOr cAncer Patients, IRAS project ID:282337).Plasma and PBMCs from MDS cases and healthy controls have been collected, and are being assessed for both humoral and cellular responses to SARS_CoV_2, the alpha (B.1.1.7) and delta (B.1.617.2) variants. Humoral responses will be assessed using ELISA (peptide binding) and functional viral neutralization assays. Cellular responses will be assessed using IFNy ELISPOT and flow cytometry (CD25 and CD69 expression) after 24h peptide stimulation. All data at time point 1 (2 - 4 weeks following booster vaccination) have been collected and will subsequently be collected at 6 months and 12 months post-vaccination. We also report on the safety data for these vaccines within this patient population.Of this cohort 64% were male with a median age of 65 years (range 21-84). 54% received vaccination with ChAdOx1 nCoV-19 and 44% received BNT162b2 (2% unrecorded). The vaccines were well tolerated with no serious adverse events to date. The mean interval between doses was 70.7 days (range 50 - 90 days). 71% of the cohort were receiving no disease modifying therapy at the time of vaccination, half of whom were receiving supportive therapy and the other half no intervention for their MDS. Of those receiving disease modifying therapy; 5 were receiving azacitidine, (1 in conjunction with low-dose cytarabine) and 3 ciclosporin.We will report the largest study of the humoral and T-cell mediated response to the Covid-19 vaccine in MDS patients to date. This will include cellular response to the delta variant and immunogenicity of both the BNT162b2 and ChAdOx1 nCoV-19 vaccines. Given the vulnerability of these patients to severe disease, investigating the immune response to the vaccines begins to build an evidence base for advising MDS patients on their ongoing risk of infection during the pandemic and going forward. The SOAP study will reassess the immune response at 6 and 12 months post-vaccination to continue to investigate post-vaccine immunity in this cohort. DisclosuresKulasekararaj: F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy; Akari: Consultancy, Honoraria, Speakers Bureau; Biocryst: Consultancy, Honoraria, Speakers Bureau; Achilleon: Consultancy, Honoraria, Speakers Bureau; Alexion: Consultancy, Honoraria, Speakers Bureau; Ra Pharma: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Alexion, AstraZeneca Rare Disease Inc.: Consultancy, Honoraria, Other: Travel support. Patten: JANSSEN: Honoraria; NOVARTIS: Honoraria; GILEAD SCIENCES: Honoraria, Research Funding; ROCHE: Research Funding; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.

Abstract 316: Effect of schweinfurthins on malignant plasma cells

Abstract Multiple myeloma (MM), a bone marrow neoplasm is currently incurable and effective alternative therapies are needed. Schweinfurthin-3114 (S-3114) purified from a plant extract, inhibits growth of established MM cell lines. The mechanism(s) for this effect are not fully characterized. To advance understanding, MM cell lines RPMI-8226 and MM.1s were incubated with increasing concentrations of S-3114 for up to 48 hours. S-3114 decreased cell viability (trypan blue assay) and mitochondrial activity (MTT assay) in a concentration and time-dependent manner (p≤0.05). RPMI-8226 cells were more sensitive (EC50 2.36 nM) than MM.1s (EC50 11.1 nM) at 48 hours. Flow cytometry analyses revealed increased apoptosis in a concentration and time-dependent manner in both cell lines (64.0% in RPMI-8226 with 20 nM S-3114; 86.3% in MM.1s with 270 nM S-3114, both at 48 hours). S-3114 also induced cell cycle arrest at G2/M. In RPMI-8226, the percent of cells arrested in G2/M increased from 45.9 to 66.1 with 3.5 nM S-3114 at 24 and 48 hours, respectively. In RPMI-8226, 20 nM S-3114 for 24 and 48 hours caused a 100% G2/M arrest. In both cell lines, S-3114 reduced protein kinase B (Akt) activation (Western blot) in a concentration and time-dependent manner. In MM.1s the apoptotic extrinsic pathway (caspase 8), and not the intrinsic pathway (caspase 9), is highly involved. In fact, S-3114 (20 nM for 48 hours, and 270 nM for 24 and 48 hours) caused a major cleavage for both 43 and 18 KDa caspase 8 fragments. Also cleaved Poly ADP-Ribose Polymerase 1 (PARP-1) increased at S-3114 concentrations of 20 and 270 nM at 24 and 48 hours. S-3114 influence on Golgi-mediated secretion was evaluated by measuring the soluble chemokine Macrophage Inflammatory Protein-1 alpha (MIP-1α), which is secreted by the Golgi and it is involved in the progression of MM disease. S-3114 treatment for 48 hours stimulated the MIP-1α negative cell line (RPMI-8226) to significantly increase MIP-1α RNA levels and to secrete MIP-1α at low concentrations. On the contrary, despite that prolonged S-3114 treatment significantly increased MIP-1α RNA levels, its secretion is decreased in the MIP-1α positive cell line (MM.1s). These in vitro studies demonstrate that S-3114 impairs the Phosphoinositide 3-Kinases (PI3K)/Akt signaling pathway resulting in cell death and/or cell cycle arrest. Data also shows an increased gene expression of the pro-survival MIP-1α by S-3114. Our ongoing studies are focused on schweinfurthin effects on MIP-1 α and Golgi secretion. We postulate that the cytotoxic impact of S-3114 requires disruption of MIP-1α signaling to regulate cell cycle, survival, and impairment of Golgi architecture and secretion in malignant plasma cells. In aggregate, these results reveal mechanism(s) for S-3114 effects and support further work advancing S-3114 as a novel therapy for MM. Citation Format: Barbara Manfredi, Raymond J. Hohl. Effect of schweinfurthins on malignant plasma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 316.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

Symptom load assessment of Ph chromosome/BCR-ABL fusion gene negative myeloproliferative tumor patients at different efficacy evaluation periods

Objective To observe the disease type and the changes of symptom load during treatment of patients with Ph chromosome/BCR-ABL fusion gene negative myeloproliferative neoplasm (MPN). Methods A total of 84 patients with MPN diagnosed from May 2017 to January 2019 in People′s Hospital of Longhua District of Shenzhen were selected, and were divided into polycythemia vera (PV) group, essential thrombocyhemia (ET) group, and myelofibrosis (PMF) group according to their subtypes, with 28 cases in each group. The scores of MPN-SAF-TSS were compared among the three groups. Besides, the scores of the scale (myeloproliferative neoplasm symptom assessment form total symptom score, MPN-SAF-TSS) in different treatment periods (at the time of the visit, when the disease progressed, when the disease was stable, when the clinical improvement was made, when the partial remission was completed, at the time of remission and recurrence) were also compared. Results At the time of initial diagnosis, there were significant differences in the incidences of symptom burdens among the three groups of MPN patients with abdominal fullness (χ2=6.095, P=0.047), abdominal discomfort (χ2=7.342, P=0.025), poor mobility (χ2=13.029, P=0.001), inattention (χ2=6.099, P=0.047), pruritus (χ2=6.956, P=0.031), bone pain (χ2=7.807, P=0.020), fever (χ2=8.000, P=0.018) and weight loss (χ2=27.340, P<0.001). The incidences of poor mobility (85.71%, 24/28), inattention (67.86%, 19/28) and weight loss (82.14%, 23/28) in PMF group were significantly higher than those in PV group [42.86% (12/28), 39.29% (11/28), 35.71% (10/28)] and ET group [46.43% (13/28), 39.29% (11/28), 14.29% (4/28)] (all P<0.05). The incidences of abdominal discomfort (75.00%, 21/28) and bone pain (60.71%, 17/28) in PMF group were higher than those in PV group [39.29% (11/28), 25.00% (7/28)] (both P<0.05). The incidences of abdominal fullness (89.29%, 25/28) and fever (42.86%, 12/28) in PMF group were higher than those in ET group [60.71% (17/28), 10.71% (3/28)] (both P<0.05). The incidence of pruritus in PV group (71.43%, 20/28) was higher than that in ET group (42.86%, 12/28) and PMF group (39.29%, 11/28) (both P<0.05). Symptom load scores of patients with fatigue (χ2=368.594, P<0.001), abdominal fullness (χ2=261.312, P<0.001), abdominal discomfort (χ2=195.629, P<0.001), poor mobility (χ2=217.862, P<0.001), lack of concentration (χ2=280.664, P<0.001), night sweats (χ2=239.650, P<0.001), pruritus (χ2=254.418, P<0.001), bone pain (χ2=180.291, P<0.001), fever (χ2=231.613, P<0.001) and weight loss (χ2=227.831, P<0.001) were significantly different during different therapeutic periods. The fatigue symptom load score was higher when the disease progressed than that at the time of the visit (P<0.05), and the symptom score of abdominal fullness was lower than that at the time of visit (P<0.05). Symptom load scores of weakness and pruritus when the condition was stable was lower than those when the disease progressed (both P<0.05). When the clinical improvement was made, symptom load scores of weakness, abdominal discomfort, inattention, night sweats, weight loss were lower than those when the disease was stable (all P<0.05). Symptom load scores of abdominal fullness, poor mobility, inattention, night sweats and pruritus in partial remission period decreased compared to temporary improvement period (all P<0.05). Compared to the partial remission period, the symptom load scores of weakness, abdominal fullness, night sweats, pruritus, bone pain and weight loss in complete remission period were lower (all P<0.05). At last, symptom load scores of weakness, abdominal fullness, abdominal discomfort, poor mobility, inattention, night sweats, pruritus, bone pain, fever and weight loss in recurrence period were higher than those in complete remission period (all P<0.05). Conclusion There are several differences in the main clinical symptoms among patients with different MPN subtypes, and there are significant changes in the main clinical symptoms as the disease progresses or turns around. Key words: Bone marrow neoplasms; Myeloproliferative disorders; Polycythemia vera; Thrombocythemia, essential; Primary myelofibrosis; Myeloproliferative neoplasm symptom assessment form total symptom score

PB1959 THE ROLE OF FLOW CYTOMETRY IN DIFFERENTIAL DIAGNOSIS OF CMML AND REACTIVE MONOCYTOSIS. THE LIPID SIGNAL PATHWAY CHARACTERISTICS IN CLONAL MONOCYTOSIS

Background:Myelodysplastic syndrome (MDS) is a heterogeneous group of diseases characterized by ineffective erythropoiesis and dysplasia. MDS and its subtype CMML is highly associated with chronic inflammatory and autoimmune diseases; however the combination of MDS with other bone marrow neoplasms is not a common entity. Only few cases of MDS combined with other blood malignancies have been described. The pathogenesis and mechanisms of two different clones evolution in MDS is an unclear multistep process involving molecular genetic instability, epigenetic factors and innate immune response deregulation. The role of plasma lipid signaling pathway have been also defined in recent years.Aims:Is to investigate the role of immunophenotyping in differential diagnosis of CMML and reactive monocytosis and clarify the possible significance of phosphoinositides in diagnosis of CMML.Methods:Blood samples of patient with CMML combined with plasma cell disorder, patient with CMML and blood samples of 2 patients with monocytosis were examined in our laboratory. Peripheral blood CBC, bone marrow smear cytological examination, pathohistology, flow cytometry and PCR were performed for the diagnostic purposes. In parallel with classic diagnostic work‐up the chromatograph method was used to investigate the cytoplasmatic mono and di‐phosphoinositides in patients with CMML.Results:CBC reveal monocytosis &gt;1000 less than 1 month duration, PCR analysis were negative for BCR ABL in all 4 cases. Case N = 1 histology examination Results: &gt;30% of plasma cells express CD56 and CD138, atypical cells express MPO and CD14 and epithelioid granulomas. In case N = 1 Immuntophenotype results were the following: (CD64+)‐52%, (CD16+)‐44%, (CD14‐). In case N = 2 the dysplasia, megakaryocytes and monocytes were observed by histology examination. Immunophenotype Results: (CD14+/CD16)‐31.1%, (CD14+/‐CD16+)‐49.3%, (CD14‐/CD16‐)‐4.3%. There were monocytosis and secondary changes of hematopoietic tissue in histology examination of cases N = 3 and N = 4. Immunophenotype showed: (CD14‐/16‐)‐13%, (CD64+)15%, (CD56‐)‐3%. High level of mono and di‐phosphoinositides were registered in first 2 cases in compare with cases N3 and N4. The flow cytometry revealed monocyte specific markers in all 4 cases with certain variations.Summary/Conclusion:The exact immunophenotype markers for clonal monocytosis must be defined. Lipid signaling pathway components, such as cytoplasmatic mono and di‐phosphoinositides must be investigated in the context of clonal monocytosis and diagnostic purposes.

Methylation and PTEN activation in dental pulp mesenchymal stem cells promotes osteogenesis and reduces oncogenesis

Lineage commitment and tumorigenesis, traits distinguishing stem cells, have not been well characterized and compared in mesenchymal stem cells derived from human dental pulp (DP-MSCs) and bone marrow (BM-MSCs). Here, we report DP-MSCs exhibit increased osteogenic potential, possess decreased adipogenic potential, form dentin pulp-like complexes, and are resistant to oncogenic transformation when compared to BM-MSCs. Genome-wide RNA-seq and differential expression analysis reveal differences in adipocyte and osteoblast differentiation pathways, bone marrow neoplasm pathway, and PTEN/PI3K/AKT pathway. Higher PTEN expression in DP-MSCs than in BM-MSCs is responsible for the lineage commitment and tumorigenesis differences in both cells. Additionally, the PTEN promoter in BM-MSCs exhibits higher DNA methylation levels and repressive mark H3K9Me2 enrichment when compared to DP-MSCs, which is mediated by increased DNMT3B and G9a expression, respectively. The study demonstrates how several epigenetic factors broadly affect lineage commitment and tumorigenesis, which should be considered when developing therapeutic uses of stem cells.

Epigenetic Modifiers in Myeloid Malignancies: The Role of Histone Deacetylase Inhibitors.

Myeloid hematological malignancies are clonal bone marrow neoplasms, comprising of acute myeloid leukemia (AML), the myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), the myeloproliferative neoplasms (MPN) and systemic mastocytosis (SM). The field of epigenetic regulation of normal and malignant hematopoiesis is rapidly growing. In recent years, heterozygous somatic mutations in genes encoding epigenetic regulators have been found in all subtypes of myeloid malignancies, supporting the rationale for treatment with epigenetic modifiers. Histone deacetylase inhibitors (HDACi) are epigenetic modifiers that, in vitro, have been shown to induce growth arrest, apoptotic or autophagic cell death, and terminal differentiation of myeloid tumor cells. These effects were observed both at the bulk tumor level and in the most immature CD34+38− cell compartments containing the leukemic stem cells. Thus, there is a strong rationale supporting HDACi therapy in myeloid malignancies. However, despite initial promising results in phase I trials, HDACi in monotherapy as well as in combination with other drugs, have failed to improve responses or survival. This review provides an overview of the rationale for HDACi in myeloid malignancies, clinical results and speculations on why clinical trials have thus far not met the expectations, and how this may be improved in the future.