Clonal Disease Research Articles

Clinical Utility of a Novel Triplex Digital PCR Assay for Clone Monitoring in Sequential and Relapsed Pediatric B-Cell Acute Lymphoblastic Leukemia Patients.

Digital polymerase chain reaction (PCR) studies for clonal disease monitoring in B-acute lymphoblastic leukemia patients are currently limited due to the heterogeneous nature of mutations, which limit cost-effective assay designs. In this study, 70 samples (14 relapse and 56 sequential therapy samples) were tested for 13 recurrent mutations identified on deep sequencing in our published cohort (KRAS, NRAS, NT5C2, PMS2, UHRF1, KMT2D, and TP53 genes) via a novel triplex digital PCR assay. A total of seven major clones of NRAS [five] and NT5C2 [two] were noted in six out of 14 (43%) relapse patients, accounting for 44% of early relapses. In addition, 10 minor clones (PMS2 [two], NRAS [four], NT5C2 [three], and TP53 [one]) were noted in five out of 14 (36%) patients. In the 56 sequential therapy samples, six major clones were noted (NRAS [five], KRAS [one]) in four out of 14 (28.5%) patients, with two increasing in size in maintenance samples, leading to subsequent relapse in both cases. In addition, therapy-acquired minor clones in NT5C2 [four] and PMS2 [one] were seen to emerge in maintenance samples in four out of 14 (28.5%) patients, with concordant detection of such major and minor clones in unpaired relapse samples, indicating the need for their active surveillance during therapy. Overall, digital PCR validated NRAS and NT5C2 major clones in one-third (10 out of 27; 37%) of cases, driving nearly half of early relapses.

Emerging Role of CAR-T Therapy in Acute Myeloid Leukemia

Acute myeloid leukemia (AML), a malignant clonal disease originating from hematopoietic stem cells. In the past few decades, AML treatment has been dominated by a "3+7" regimen (anthracyclines + cytarabine), and the survival curve has stagnated. However, these procedures are associated with serious adverse effects. The development of chimeric antigen receptor (CAR)-T cell treatment provides a viable alternative, with decreased non-relapse death rates. Despite its potential, CAR-T cell therapy for AML confronts significant obstacles due to the disease's heterogeneity and accompanying toxicity. Genetic alteration strategies are being investigated to improve its safety and efficacy. Engineering CAR-T cells to express inhibitory receptors or suicide genes can help regulate T cell activity and prevent excessive immune activation. In summary, while CAR-T cell treatment has promise for AML, overcoming its hurdles through genetic modification and target refining is crucial. This review focuses on the existing landscape and future directions of CAR-T cell treatment for AML.

Monitoring clonal burden as an alternative to blast count for myelodysplastic neoplasm treatment response.

Accurate assessment of therapy response in myelodysplastic neoplasm (MDS) has been challenging. Directly monitoring mutational disease burden may be useful, but is not currently included in MDS response criteria, and the correlation of mutational burden and traditional response endpoints is not completely understood. Here, we used genome-wide and targeted next-generation sequencing (NGS) to monitor clonal and subclonal molecular disease burden in 452 samples from 32 patients prospectively treated in a clinical trial. Molecular responses were compared with International Working Group (IWG) 2006-defined response assessments. We found that myeloblast percentage consistently underestimates MDS molecular disease burden and that mutational clearance patterns for marrow complete remission (mCR), which depends on myeloblast assessment, was not different than stable disease or bone marrow aplasia, underscoring a major limitation of using mCR. In contrast, achieving a complete remission (CR) was associated with the highest level of mutation clearance and lowest residual mutational burden in higher-risk MDS patients. A targeted gene panel approach was inferior to genome-wide sequencing in defining subclones and their molecular responses but may be adequate for monitoring molecular disease burden when a targeted gene is present in the founding clone. Our work supports incorporating serialNGS-based monitoring into prospective MDS clinical trials.

A-284 VEXAS: a new clonal disease with specific diagnostic clues

A-284 VEXAS: a new clonal disease with specific diagnostic clues

Prognostic Impact of CD200 and CD56 Expression in Patients with Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) is a clonal malignant disease of hematopoietic tissue which results from a block of normal differentiation of hematopoietic progenitor cells along with uncontrolled proliferation of cells of myeloid origin with maturation arrest leading to infiltration of bone marrow and other tissues by myeloblasts. It escapes from immunosurveillance by induction of immunosuppression through expression of specific cell surface molecules with immune modulatory function. Novel immune-directed therapeutic approaches form a major focus of current and clinical research. Objective to assess the expression level of CD200 and CD56 in de-novo acute myeloid leukemia patients and evaluate the prognostic value of their positive expressions in patients with AML. Methods This Cohort study was conducted at Ain Shams University Hospitals, on 51 newly diagnosed adult AML patients attending the Hematology Oncology Unit of Ain Shams University Hospitals from February, 2022 until June, 2023. Results CD200+ expression was reported in 74.5% of patients while 9.8% of patients showed CD56+ expression. M1-M2 were found to be the most common FAB subtypes. CD200+ was higher among female patients (p = 0.045). On the other hand, CD56+ patients were younger in comparison to CD56- patients (p = 0.002). Total death was higher among CD200+ patients than CD200- patients (p = 0.037). Conclusion Our study reveals that CD200 expression is associated with higher incidence of mortality which suggests a negative impact of CD200 expression on survival rate while CD56 is not associated with a significant mortality.

CBL mutations in chronic myelomonocytic leukemia often occur in the RING domain with multiple subclones per patient: Implications for targeting

Chronic myelomonocytic leukemia (CMML) is a rare blood cancer of older adults (3 in every 1,000,000 persons) characterized by poor survival and lacking effective mutation-specific therapy. Mutations in the ubiquitin ligase Cbl occur frequently in CMML and share biological and molecular features with a clonal disease occurring in children, juvenile myelomonocytic leukemia (JMML). Here we analyzed the clinical presentations, molecular features and immunophenotype of CMML patients with CBL mutations enrolled in a prospective Phase II clinical trial stratified according to molecular markers. Clinically, CBL mutations were associated with increased bone marrow blasts at diagnosis, leukocytosis and splenomegaly, similar to patients harboring NRAS or KRAS mutations. Interestingly, 64% of patients presented with more than one CBL variant implying a complex subclonal architecture, often with co-occurrence of TET2 mutations. We found CBL mutations in CMML frequently clustered in the RING domain in contrast to JMML, where mutations frequently involve the linker helix region (P<0.0001). According to our comparative alignment of available X-ray structures, mutations in the linker helix region such as Y371E give rise to conformational differences that could be exploited by targeted therapy approaches. Furthermore, we noted an increased percentage of CMML CD34+ stem and progenitor cells expressing CD116 and CD131 in all CBL mutant cases and increased CD116 receptor density compared to healthy controls, similar to CMML overall. In summary, our data demonstrate that CBL mutations are associated with distinct molecular and clinical features in CMML and are potentially targetable with CD116-directed immunotherapy.

Clinical efficacy and immune response of BCL-2 inhibitors combined with hypomethylating agents in the treatment of acute myeloid leukemia

ObjectiveAcute myeloid leukemia (AML) is a malignant clonal proliferative disease with a high mortality rate. The combination therapy of BCL-2 inhibitor Venetoclax (VEN) and hypomethylating agents (HMAs) has significant anti-leukemia activity.MethodsWe analyzed the efficacy, safety and immune response characteristics of AML patients who were unfit for high-dose chemotherapy and accepted the medication of VEN + HMAs.ResultsAfter VEN + HMAs treatment, 31 newly diagnosed AML patients had the morphologic leukemia-free state rate (MLFS%) of 80.6% (25/31), complete response rate (CR%) of 54.8% (17/31), the minimal residual disease negative rate (MRD-%) of 51.6% (16/31), and the median progression-free survival (PFS) of 14 months. After treatment, the proportion of bone marrow primitive cells, the MRD level, white blood cell (WBC) count, fibrinogen (FIB) level and the proportion of B cells were significantly decreased. The red blood cell (RBC) count, hemoglobin (HGB) level, platelet count (PLT) count, activated partial thromboplastin time (APTT), the proportion of total T cells, CD8 + T cells and the IFN-γ level were significantly increased. After VEN + HMAs treatment, 12 relapsed AML patients had a MLFS% of 50% (6/12), CR% of 33.3% (4/12), MRD-% of 25% (3/12), and a median PFS of 7 months. After treatment, the proportion of bone marrow primitive cells and MRD level were slightly decreased, the proportions of CD8 + T cells and NK cells were significantly increased, the proportion of B cells and IL-10 level were significantly decreased. 12 AML patients who receive microtransplantation (MST) treatment using VEN + HMAs as a pretreatment regimen had a PFS of 20.5 months, which was much greater than VEN + HMAs group alone. Hematological recovery was better in the MST group with significantly increased RBC count, HGB level and PLT count. The most common adverse events were myelosuppression, agranulocytosis, infection and cardiovascular toxicity. No fatal adverse events were reported.ConclusionThe combination of BCL-2 inhibitors and HMAs had good efficacy and safety in AML patients who were unfit for high-dose chemotherapy, which may improve the immune microenvironment and enhance anti-leukemia immune response.

Factors impeding organ recovery despite a deep haematological response in patients with systemic AL amyloidosis.

Patients with AL amyloidosis can have persistent organ dysfunction despite achieving a haematological complete response (hemCR). We aimed to identify factors for organ non-response among 143 patients who achieved hemCR for ≥6 months. Kidney, heart and liver non-response were observed in 40/117 (34%), 19/68 (28%) and 3/17 (18%) patients respectively. Predisposing factors varied by organ system. Kidney non-responders had more advanced organ dysfunction at diagnosis, whereas heart non-responders had disproportionately more lambda-typic amyloidogenic light chains. Most patients without an apparent reason for organ non-response had detectable residual clonal disease. The interplay of factors impeding organ recovery in AL amyloidosis is complex.

Divergent Evolution in Bilateral Prostate Cancer: a Case Study.

Multifocal prostate cancer is a prevalent phenomenon, with most cases remaining uncharacterized from a genomic perspective. A patient presented with bilateral prostate cancer. On systematic biopsy, two indistinguishable clinicopathologic lesions were detected. Whole-genome sequencing displayed somatically unrelated tumours with distinct driver CNA regions, suggesting independent origins of the two tumors. We demonstrated that similar clinicopathologic multifocal tumours, which might be interpreted as clonal disease, can in fact represent independent cancers. Genetic prognostics can prevent mischaracterization of multifocal disease to enable optimal patient management.

Peculiarities of the influence of recombinant cytokines on the functional activity of hematopoietic progenitor cells in myelodysplastic syndrome in vitro

Populations of hematopoietic progenitor cells are the closest descendants of stem cells. It is at their level that the processes of proliferation and differentiation occur, since they are sensitive, unlike stem cells, to the action of cytokines, which are released in the event of a shortage of blood cells in the periphery. However, for a long time the role of hematopoietic progenitor cells in the implementation of the pathological process in disorders of hematopoiesis was underestimated, while now it has turned out to be more significant than previously thought. This is especially true of myelodysplastic syndrome, which, despite its name, is a clonal disease that precedes acute leukemia. The purpose of the study was to determine the peculiarities of the functioning of hematopoietic progenitor cells in normal and impaired hematopoiesis at the initial stages of the malignant process of MDS (MDS-IB) under the conditions of exposure to different concentrations of cytokines to assess the hematopoietic potential of these cells. Their colony-forming activity (CFU) was studied in two groups of patients — control (10 people) and experimental (20 people) in culture in vitro. It was found that CFU increases with increasing concentration of cytokines and requires twice as much stimulus when culturing hematopoietic cells from patients with MDS-IB. The optimal concentration in the control for G-CSF, GM-CSF, IL-3 was 20 ng/ml, and for cells from patients with MDS-IB — 40 ng/ml. It has been proven that in the case of using a complex of cytokines (GM-CSF, G-CSF, IL-3), the colonyforming ability of progenitor cells from patients with MDS-IB increases significantly, compared to such indicators for cytokines acting alone (28.7±3.2 to 18.3±1.8, 12.1±1.5 and 24.5±2.1, respectively). The paper reveals the latent potential for cell proliferation from patients with MDS-IB, which can be used both in experimental studies and in the creation of protocols for the treatment of patients with MDS in the initial stage of the disease.

Risk Factors for Severe Sting Reactions and Side Effects During Venom Immunotherapy

Understanding the risk factors leading to severe systemic sting reactions (SSR) is crucial for initiating venom immunotherapy (VIT) and for educating affected individuals and their families. Some of these risk factors are well-established, some are no longer considered risk factors, and some remain controversial. Well-established risk factors for severe SSR include clonal mast cell disease, high baseline serum tryptase, and advanced age. The absence of skin symptoms and the rapid onset of symptoms are indicators of severe SSR. Recent publications indicate that antihypertensive treatment and stings in the head and neck area are not risk factors for severe SSR. VIT is the only available treatment that can potentially prevent further anaphylactic reactions. Although rare and generally manageable, individuals undergoing VIT may experience systemic adverse events (sAE). More sAE are expected in patients undergoing bee VIT compared to vespid VIT. The role of elevated baseline serum tryptase as a risk factor for sAE remains debated, but if it is a factor, the risk is increased by only about 1.5-fold. Rapid up-dosing protocols, depending on the specific regimen, can also be associated with more sAE. Severe initial SSR, antihypertensive medication, high skin test reactivity, and high specific IgE levels are not risk factors for sAE.

Mast Cell Disorders and Hymenoptera Venom-Triggered Anaphylaxis: Evaluation and Management

Patients with Hymenoptera venom allergy (HVA), especially those with severe anaphylaxis, frequently have concomitant clonal mast cell disease (MCD) in the form of systemic mastocytosis or monoclonal mast cell activation syndrome. Detection of clonal MCD is important since it will have significant consequences for management of HVA. Therefore, we recommend patients with HVA be systematically screened for clonal MCD. The pre-test probability of clonal MCD can be assessed in a stepwise fashion starting with examination of the skin for typical monomorphic maculopapular cutaneous mastocytosis (MPCM) lesions; measurement of the baseline serum tryptase (BST) and tryptase genotyping for patients with BST>11 ng/mL; followed by the REMA score which is calculated by utilizing anaphylaxis clinical features, BST, and patient sex. A bone marrow biopsy should be performed in patients with monomorphic MPCM, a REMA score ≥2, or an elevated BST based upon tryptase genotype. Patients with HVA and a clonal MCD should be treated with immunotherapy directed against the Hymenoptera venom for which they are sensitized. For this high-risk subgroup of HVA patients, it is recommended to continue immunotherapy for more than 5 years or indefinitely and to carry at least three epinephrine autoinjectors. Future studies should determine whether KIT D816V-selective tyrosine kinase inhibitors are effective at preventing or reducing severity of Hymenoptera-venom triggered anaphylaxis in patients with clonal MCD.

Decoding Clonal Hematopoiesis: Emerging Themes and Novel Mechanistic Insights.

Clonal hematopoiesis (CH), the relative expansion of mutant clones, is derived from hematopoietic stem cells (HSCs) with acquired somatic or cytogenetic alterations that improve cellular fitness. Individuals with CH have a higher risk for hematological and non-hematological diseases, such as cardiovascular disease, and have an overall higher mortality rate. Originally thought to be restricted to a small fraction of elderly people, recent advances in single-cell sequencing and bioinformatics have revealed that CH with multiple expanded mutant clones is universal in the elderly population. Just a few years ago, phylogenetic reconstruction across the human lifespan and novel sensitive sequencing techniques showed that CH can start earlier in life, decades before it was thought possible. These studies also suggest that environmental factors acting through aberrant inflammation might be a common theme promoting clonal expansion and disease progression. However, numerous aspects of this phenomenon remain to be elucidated and the precise mechanisms, context-specific drivers, and pathways of clonal expansion remain to be established. Here, we review our current understanding of the cellular mechanisms driving CH and specifically focus on how pro-inflammatory factors affect normal and mutant HSC fates to promote clonal selection.

Mimicry of inherited red cell disorders: the result of somatic mutations in a clonal myeloid disease.

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Advancements in the impact of human microbiota and probiotics on leukemia.

The human gut microbiota is a complex ecosystem that plays a crucial role in promoting the interaction between the body and its environment. It has been increasingly recognized that the gut microbiota has diverse physiological functions. Recent studies have shown a close association between the gut microbiota and the development of certain tumors, including leukemia. Leukemia is a malignant clonal disease characterized by the uncontrolled growth of one or more types of blood cells, which is the most common cancer in children. The imbalance of gut microbiota is linked to the pathological mechanisms of leukemia. Probiotics, which are beneficial microorganisms that help maintain the balance of the host microbiome, play a role in regulating gut microbiota. Probiotics have the potential to assist in the treatment of leukemia and improve the clinical prognosis of leukemia patients. This study reviews the relationship between gut microbiota, probiotics, and the progression of leukemia based on current research. In addition, utilizing zebrafish leukemia models in future studies might reveal the specific mechanisms of their interactions, thereby providing new insights into the clinical treatment of leukemia. In conclusion, further investigation is still needed to fully understand the accurate role of microbes in leukemia.

Cladribine in the Treatment of Multifocal Multisystem Langerhans Cell Histiocytosis in a Patient with a Poor Prognosis. A Case Report

Langerhans cell histiocytosis is a hematologic clonal disease. The treatment of localized histiocytosis includes local methods such as radiotherapy or surgery. In cases of disseminated lesions with multiple system damage, chemotherapy is administered, which provides recovery in some patients. The present paper is a case report of multifocal multisystem Langerhans cell histiocytosis with bone, liver, spleen, abdominal lymph node and pituitary lesions in a 40-year-old patient. The diagnosis was verified by histology and immunohistochemistry of bone biopsy specimens. Polymerase chain reaction revealed no V600E mutation in the BRAF gene in tumor tissue. Persistent antitumor response was achieved after 6 chemotherapy cycles with cladribine monoregimen. This drug was well tolerated by the patient. By the time of this publication, the patient had preserved the persistent response for 38 months.

Hesperidin/Salinomycin Combination; a Natural Product for Deactivation of the PI3K/Akt Signaling Pathway and Anti-Apoptotic Factors in KG1a Cells.

AML is a highly aggressive malignant clonal disease of hematopoietic origin. Hesperidin as a polyphenol glycoside, Activates the apoptotic pathway and salinomycin as a k + selective ionophore. We examined how hesperidin and salinomycin induce pro-apoptotic effects in KG1a cells. Cells were divided into four groups; 1) control cells (CRTL), 2) cells treated with hesperidin 85μM, 3) cells treated with 2μM salinomycin, 4) cells treated with combination of salinomycin and hesperidin. The MTT assay was implemented to determine the IC50 of hesperidin and salinomycin in KG1a cell lines. Propidium iodide staining and flow cytometry were used to analyze the distribution of the cell cycle. The level of ROS was evaluated by fluorescent microscopy and spectrophotometry. Additionally, Akt, XIAP, Bad, and FOXO1 gene expression was analyzed by real-time PCR. Hesperidin/Salinomycin decreased the viability of KG1a leukemic cells more than Hesperidin and Salinomycin separately. Changes in the shape of apoptotic cells and rise in ROS levels were detected after Hesperidin/Salinomycin treatment. Our findings showed that following Hesperidin/Salinomycin treatment, the expression of PI3K/AKT signaling pathway related genes (AKT, PTEN and FOXO1), were in line with the destruction of KG-1a cells. Furthermore, XIAP and BAD mRNA were regulated to trigger apoptosis in cancer cells. The study discovered that hesperidin and salinomycin, could effectively hinder the PI3K/Akt signaling pathway in leukemia cancer cells. Also, the combination of hesperidin and salinomycin has the potential to be a treatment option for acute myeloid leukemia.

Anti-tumor activity of niclosamide-mediated oxidative stress against acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a heterogeneous clonal disease originated from B- or T-cell lymphoid precursor cells. ALL is often refractory or relapses after treatment. Novel treatments are anxiously needed in order to achieve a better response and prolonged overall survival in ALL patients. In the present study, we aimed at examining the anti-tumor effect of niclosamide on ALL. We investigated the effects of niclosamide on the proliferation and apoptosis in vitro, the growth of ALL cells in xenografted NCG mice. The results showed that niclosamide treatment potently inhibited the growth of ALL cells and induced apoptosis via elevating the levels of reactive oxygen species (ROS) and activating TP53. These findings suggest that niclosamide may be a promisingly potential agent for ALL therapy.

Unveiling mitochondrial and ribosomal gene deregulation and tumor microenvironment dynamics in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant clonal hematopoietic disease with a poor prognosis. Understanding the interaction between leukemic cells and the tumor microenvironment (TME) can help predict the prognosis of leukemia and guide its treatment. Re-analyzing the scRNA-seq data from the CSC and G20 cohorts, using a Python-based pipeline including machine-learning-based scVI-tools, recapitulated the distinct hierarchical structure within the samples of AML patients. Weighted correlation network analysis (WGCNA) was conducted to construct a weighted gene co-expression network and to identify gene modules primarily focusing on hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and natural killer (NK) cells. The analysis revealed significant deregulation in gene modules associated with aerobic respiration and ribosomal/cytoplasmic translation. Cell-cell communications were elucidated by the CellChat package, revealing an imbalance of activating and inhibitory immune signaling pathways. Interception of genes upregulated in leukemic HSCs & MPPs as well as in NKG2A-high NK cells was used to construct prognostic models. Normal Cox and artificial neural network models based on 10 genes were developed. The study reveals the deregulation of mitochondrial and ribosomal genes in AML patients and suggests the co-occurrence of stimulatory and inhibitory factors in the AML TME.

Advances in research on renal injury in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disease. Clinical manifestations include intravascular hemolysis, renal dysfunction, fatigue, jaundice, pulmonary hypertension, and so on. Renal injury, as a clinical feature of PNH, is difficult to diagnose and is one of the causes of death in patients with PNH. This article reviews the progress in research on PNH combined with renal injury to improve clinicians' understanding of renal injury in PNH patients, define and judge staging in a timely and accurate manner, enable patients to receive timely and appropriate treatment and reduce mortality.