Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become "digitalized", which was deemed important to guide clinicians in treatment decision for better patient care.

Elevated CDKN1A (P21) mediates ß-thalassemia erythroid apoptosis, but its loss does not improve ß-thalassemic erythropoiesis.

ß-thalassemias are common hemoglobinopathies due to mutations in the ß-globin gene that lead to hemolytic anemias. Premature death of ß-thalassemic erythroid precursors results in ineffective erythroid maturation, increased production of erythropoietin (EPO), expansion of erythroid progenitor compartment, extramedullary erythropoiesis, and splenomegaly. However, the molecular mechanism of erythroid apoptosis in ß-thalassemia is not well understood. Using a mouse model of ß-thalassemia (Hbbth3/+), we show that dysregulated expression of the FOXO3 transcription factor is implicated in ß-thalassemia erythroid apoptosis. In Foxo3-/-/Hbbth3/+ mice, erythroid apoptosis is significantly reduced, whereas erythroid cell maturation, and red blood cell and hemoglobin production are substantially improved even with elevated reactive oxygen species in double-mutant erythroblasts. However, persistence of elevated reticulocytes and splenomegaly suggests that ineffective erythropoiesis is not resolved in Foxo3-/-/Hbbth3/+. We found the cell cycle inhibitor Cdkn1a (cyclin-dependent kinase inhibitor p21), a FOXO3 target gene, is markedly upregulated in both mouse and patient-derived ß-thalassemic erythroid precursors. Double-mutant p21/Hbbth3/+ mice exhibited embryonic lethality with only a fraction of mice surviving to weaning. Notably, studies in adult mice displayed greatly reduced apoptosis and circulating Epo in erythroid compartments of surviving p21-/-/Hbbth3/+ mice relative to Hbbth3/+ mice, whereas ineffective erythroid cell maturation, extramedullary erythropoiesis, and splenomegaly were not modified. These combined results suggest that mechanisms that control ß-thalassemic erythroid cell survival and differentiation are uncoupled from ineffective erythropoiesis and involve a molecular network including FOXO3 and P21. Overall, these studies provide a new framework for investigating ineffective erythropoiesis in ß-thalassemia.

Real-World Validation of a Computer-Aided Diagnosis System for Prediction of Polyp Histology in Colonoscopy: A Prospective Multicenter Study.

INTRODUCTION: Computer-aided diagnosis (CADx) of polyp histology could support endoscopists in clinical decision-making. However, this has not been validated in a real-world setting. METHODS: We performed a prospective, multicenter study comparing CADx and endoscopist predictions of polyp histology in real-time colonoscopy. Optical diagnosis based on visual inspection of polyps was made by experienced endoscopists. After this, the automated output from the CADx support tool was recorded. All imaged polyps were resected for histological assessment. Primary outcome was difference in diagnostic performance between CADx and endoscopist prediction of polyp histology. Subgroup analysis was performed for polyp size, bowel preparation, difficulty of location of the polyps, and endoscopist experience. RESULTS: A total of 661 eligible polyps were resected in 320 patients aged ≥40 years between March 2021 and July 2022. CADx had an overall accuracy of 71.6% (95% confidence interval [CI] 68.0-75.0), compared with 75.2% (95% CI 71.7-78.4) for endoscopists ( P = 0.023). The sensitivity of CADx for neoplastic polyps was 61.8% (95% CI 56.9-66.5), compared with 70.3% (95% CI 65.7-74.7) for endoscopists ( P < 0.001). The interobserver agreement between CADx and endoscopist predictions of polyp histology was moderate (83.1% agreement, κ 0.661). When there was concordance between CADx and endoscopist predictions, the accuracy increased to 78.1%. DISCUSSION: The overall diagnostic accuracy and sensitivity for neoplastic polyps was higher in experienced endoscopists compared with CADx predictions, with moderate interobserver agreement. Concordance in predictions increased this diagnostic accuracy. Further research is required to improve the performance of CADx and to establish its role in clinical practice.

An Unusual Case of Ascites.

Cirrhotic portal hypertension is the most common cause of new-onset ascites. The clinician, however, should be cognizant of rarer causes of ascites, especially when clinical and laboratory parameters are not in keeping with cirrhosis. We present a case of urinoma as the cause of an initial presentation of ascites.

Clinical utility of ozone therapy and hyperbaric oxygen therapy in degenerative disc disease.

Ozone can be medically useful concerning healing wounds and relieving pain in various conditions, such as disc disease. The aspects of human blood ozonation have been reviewed, as well as potential complications that may arise. The mechanisms of ozone therapy are discussed in detail. It is imperative to recognize ozone as a useful proxy in oxidative-stress related diseases, consolidating other medical gases recognized for their therapeutic importance. The utility of hyperbaric oxygen therapy is also discussed. Disc herniation is very common, as more than 3 million cases are treated per year. Herein we review the medical, surgical, and gene-based therapies that ozone therapy can provide regarding disc disease.

Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging.

Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here, we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs, which are the most potent subsets, it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol, please refer to Liang et al. (2020) and Qiu et al. (2021).

Characterization of the humoral immune response to the EBV proteome in extranodal NK/T-cell lymphoma.

Extranodal natural killer/T-cell lymphoma (NKTCL) is an aggressive malignancy that has been etiologically linked to Epstein-Barr virus (EBV) infection, with EBV gene transcripts identified in almost all cases. However, the humoral immune response to EBV in NKTCL patients has not been well characterized. We examined the antibody response to EBV in plasma samples from 51 NKTCL cases and 154 controls from Hong Kong and Taiwan who were part of the multi-center, hospital-based AsiaLymph case-control study. The EBV-directed serological response was characterized using a protein microarray that measured IgG and IgA antibodies against 202 protein sequences representing the entire EBV proteome. We analyzed 157 IgG antibodies and 127 IgA antibodies that fulfilled quality control requirements. Associations between EBV serology and NKTCL status were disproportionately observed for IgG rather than IgA antibodies. Nine anti-EBV IgG responses were significantly elevated in NKTCL cases compared with controls and had ORshighest vs. lowest tertile > 6.0 (Bonferroni-corrected P-values < 0.05). Among these nine elevated IgG responses in NKTCL patients, three IgG antibodies (all targeting EBNA3A) are novel and have not been observed for other EBV-associated tumors of B-cell or epithelial origin. IgG antibodies against EBNA1, which have consistently been elevated in other EBV-associated tumors, were not elevated in NKTCL cases. We characterize the antibody response against EBV for patients with NKTCL and identify IgG antibody responses against six distinct EBV proteins. Our findings suggest distinct serologic patterns of this NK/T-cell lymphoma compared with other EBV-associated tumors of B-cell or epithelial origin.

Mitochondrial localization and moderated activity are key to murine erythroid enucleation.

Mammalian red blood cells (RBCs), which primarily contain hemoglobin, exemplify an elaborate maturation process, with the terminal steps of RBC generation involving extensive cellular remodeling. This encompasses alterations of cellular content through distinct stages of erythroblast maturation that result in the expulsion of the nucleus (enucleation) followed by the loss of mitochondria and all other organelles and a transition to anaerobic glycolysis. Whether there is any link between erythroid removal of the nucleus and the function of any other organelle, including mitochondria, remains unknown. Here we demonstrate that mitochondria are key to nuclear clearance. Using live and confocal microscopy and high-throughput single-cell imaging, we show that before nuclear polarization, mitochondria progressively move toward one side of maturing erythroblasts and aggregate near the nucleus as it extrudes from the cell, a prerequisite for enucleation to proceed. Although we found active mitochondrial respiration is required for nuclear expulsion, levels of mitochondrial activity identify distinct functional subpopulations, because terminally maturing erythroblasts with low relative to high mitochondrial membrane potential are at a later stage of maturation, contain greatly condensed nuclei with reduced open chromatin-associated acetylation histone marks, and exhibit higher enucleation rates. Lastly, to our surprise, we found that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation, primarily through pyruvate but independent of in situ glycolysis. These findings demonstrate the critical but unanticipated functions of mitochondria during the erythroblast enucleation process. They are also relevant to the in vitro production of RBCs as well as to disorders of the erythroid lineage.

Applications of genetic-epigenetic tissue mapping for plasma DNA in prenatal testing, transplantation and oncology.

We developed genetic-epigenetic tissue mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung transplant recipients, we showed that, at 72 hr after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles, and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring, and cancer screening.

Psychosocial and Sexual Implications of Repaired Cleft Lip in an Adult Male.

We describe the case of a 32-year-old man with repaired cleft lip complaining of sexual difficulties, which were determined to be multifactorial in origin. A negative body impression, anatomical difficulty in engaging in sexual acts, the indirect impact of secondary complications of cleft lip, and the negative perception of his dysmorphia from potential sexual partners led to his current state. Cleft lip and palate (CLP) are the most common malformations of the craniofacial region. The malformations of anatomical structures involved in CLP can manifest through several variations of clinical features and phenotypes, typically affecting hearing, social integration, speech, and feeding. From birth to the end of growth, a multidisciplinary approach involving orthodontists, speech therapists, psychologists, and social workers is essential for adequate management, even after surgical repair. This case report illustrates the critical importance of the functionality of normal lips essential for sexual function and psychosocial issues encountered in a patient with repaired cleft lip, as this issue likely has a higher prevalence than the literature actually suggests. Adding a psychiatrist on the panel for pediatric reconstructive surgery teams and post-repair rehabilitation would be essential to managing potential sexual and psychological issues into adulthood.

Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.

Following Transcriptome to Uncover FOXO Biological Functions.

Two and half million red blood cells (RBC) are generated every second in a healthy adult. The process of RBC production known as erythropoiesis requires a meticulous synchrony between signaling processes and the activity of many transcription factor complexes. FOXO3 is a transcription factor that is responsive to signaling processes and essential for the erythroid proliferation and maturation, RBC formation, and lifespan. Here, we discuss how using an integrated computational and experimental systems biology approach new and unanticipated FOXO3 functions in terminal erythropoiesis were uncovered. These combinatory approaches identified FOXO3 as a key regulator of terminal erythropoiesis. As a result, a new mode of FOXO3 participation in erythroid transcription complex formation has been proposed.

Stem Cells Seen Through the FOXO Lens: An Evolving Paradigm.

Stem cells self-renew and differentiate to generate all tissues and cells in the body. Stem cell health promotes adaptive responses to tissue damage or loss and is essential for tissue regeneration with age. In the past decade, the evolutionarily conserved transcription factors FOXO with known functions in promoting healthy aging have emerged as key regulators of stem cell homeostasis in various tissues, including blood, neural, and muscle stem cells. Aberrant FOXO functions have been implicated in a variety of disorders including neurodegenerative, blood, cancer, and diabetes some of which are fostered by abnormal stem cell function. As discussed in this chapter, at least in some stem cells FOXO regulatory mechanisms and applied functions follow a complex set of rules distinct from that operating in progenitor cell populations and in cultured cell lines. Elucidating the exact nature of FOXO properties in stem cells will be critical for identifying and targeting aberrant FOXO-mediated mechanisms that promote stem cell-derived disease specifically with age.

Bow-tie signaling in c-di-GMP: Machine learning in a simple biochemical network.

Bacteria of many species rely on a simple molecule, the intracellular secondary messenger c-di-GMP (Bis-(3'-5')-cyclic dimeric guanosine monophosphate), to make a vital choice: whether to stay in one place and form a biofilm, or to leave it in search of better conditions. The c-di-GMP network has a bow-tie shaped architecture that integrates many signals from the outside world-the input stimuli-into intracellular c-di-GMP levels that then regulate genes for biofilm formation or for swarming motility-the output phenotypes. How does the 'uninformed' process of evolution produce a network with the right input/output association and enable bacteria to make the right choice? Inspired by new data from 28 clinical isolates of Pseudomonas aeruginosa and strains evolved in laboratory experiments we propose a mathematical model where the c-di-GMP network is analogous to a machine learning classifier. The analogy immediately suggests a mechanism for learning through evolution: adaptation though incremental changes in c-di-GMP network proteins acquires knowledge from past experiences and enables bacteria to use it to direct future behaviors. Our model clarifies the elusive function of the ubiquitous c-di-GMP network, a key regulator of bacterial social traits associated with virulence. More broadly, the link between evolution and machine learning can help explain how natural selection across fluctuating environments produces networks that enable living organisms to make sophisticated decisions.

Mitochondria and FOXO3 in stem cell homeostasis, a window into hematopoietic stem cell fate determination.

The production of all blood cells from hematopoietic stem cells (HSC) is highly sensitive to reactive oxygen species (ROS). Cumulating evidence suggests that mitochondria are critical for HSC fate determination. FOXO are known regulators of anti-oxidant response and key to the maintenance of HSC. Recent works indicate that FOXO3 is implicated in the control of mitochondrial function beyond regulating levels of ROS in HSC. Here we review these findings and discuss implications for homeostatic blood formation and stem cell fate determination.

FOXO3 Transcription Factor Is Essential for Protecting Hematopoietic Stem and Progenitor Cells from Oxidative DNA Damage.

Accumulation of damaged DNA in hematopoietic stem cells (HSC) is associated with chromosomal abnormalities, genomic instability, and HSC aging and might promote hematological malignancies with age. Despite this, the regulatory pathways implicated in the HSC DNA damage response have not been fully elucidated. One of the sources of DNA damage is reactive oxygen species (ROS) generated by both exogenous and endogenous insults. Balancing ROS levels in HSC requires FOXO3, which is an essential transcription factor for HSC maintenance implicated in HSC aging. Elevated ROS levels result in defective Foxo3-/- HSC cycling, among many other deficiencies. Here, we show that loss of FOXO3 leads to the accumulation of DNA damage in primitive hematopoietic stem and progenitor cells (HSPC), associated specifically with reduced expression of genes implicated in the repair of oxidative DNA damage. We provide further evidence that Foxo3-/- HSPC are defective in DNA damage repair. Specifically, we show that the base excision repair pathway, the main pathway utilized for the repair of oxidative DNA damage, is compromised in Foxo3-/- primitive hematopoietic cells. Treating mice in vivo with N-acetylcysteine reduces ROS levels, rescues HSC cycling defects, and partially mitigates HSPC DNA damage. These results indicate that DNA damage accrued as a result of elevated ROS in Foxo3-/- mutant HSPC is at least partially reversible. Collectively, our findings suggest that FOXO3 serves as a protector of HSC genomic stability and health.

Homoharringtonine (omacetaxine mepesuccinate) as an adjunct for FLT3-ITD acute myeloid leukemia.

An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models. Mechanistically, the effect was mediated by protein synthesis inhibition and reduction of short-lived proteins, including total and phosphorylated forms of FLT3 and its downstream signaling proteins. A phase 2 clinical trial of sorafenib and HHT combination treatment in FLT3-ITD AML patients resulted in complete remission (true or with insufficient hematological recovery) in 20 of 24 patients (83.3%), reduction of ITD allelic burden, and median leukemia-free and overall survivals of 12 and 33 weeks. The regimen has successfully bridged five patients to allogeneic hematopoietic stem cell transplantation and was well tolerated in patients unfit for conventional chemotherapy, including elderly and heavily pretreated patients. This study validated the principle and clinical relevance of in vitro drug testing and identified an improved treatment for FLT3-ITD AML. The results provided the foundation for phase 2/3 clinical trials to ascertain the clinical efficacy of FLT3 inhibitors and HHT in combination.

Characterization of Drug Effect on Leukemia Cells Through Single Cell Assay With Optical Tweezers and Dielectrophoresis.

One of the greatest challenges in acute myeloid leukemia (AML) treatment is preventing relapse. Leukemia cells can hide in bone marrow niche or vascular niche. Hence, many chemical drugs cannot kill these cells. To characterize migration and adhesion properties of leukemia cells in specific niches, CXCR4/SDF- 1α signal pathway has been widely used for investigation. AMD3100 is treated as one of the most common chemical drugs that can inhibit this signal. In the current study, we particularly investigate the effect of AMD3100 on the adhesion property of leukemia cells on stromal cells by using engineering tools, namely, optical tweezers (OT) and dielectrophoresis (DEP), to probe single cell property. AMD3100 not only inhibits the CXCR4/SDF- 1α signal pathway but also reduces gene expression of CXCR4 and VLA-4 on leukemia cells. The drug also softens leukemia cells. This work provides a new way to investigate cell behavior under drug treatment. The use of combined engineering tools will benefit drug discovery and assessment for leukemia treatment.