Acute promyelocytic leukemia (APL) is considered a hematologic emergency due to high risk of bleeding and fatal hemorrhages being a major cause of death. Despite lower death rates reported from ...clinical trials, patient registry data suggest an early death rate of 20%, especially for elderly and frail patients. Therefore, reliable diagnosis is required as treatment with differentiation-inducing agents leads to cure in the majority of patients. However, diagnosis commonly relies on cytomorphology and genetic confirmation of the pathognomonic t(15;17). Yet, the latter is more time consuming and in some regions unavailable.
In recent years, deep learning (DL) has been evaluated for medical image recognition showing outstanding capabilities in analyzing large amounts of image data and provides reliable classification results. We developed a multi-stage DL platform that automatically reads images of bone marrow smears, accurately segments cells, and subsequently predicts APL using image data only. We retrospectively identified 51 APL patients from previous multicenter trials and compared them to 1048 non-APL acute myeloid leukemia (AML) patients and 236 healthy bone marrow donor samples, respectively.
Our DL platform segments bone marrow cells with a mean average precision and a mean average recall of both 0.97. Further, it achieves high accuracy in detecting APL by distinguishing between APL and non-APL AML as well as APL and healthy donors with an area under the receiver operating characteristic of 0.8575 and 0.9585, respectively, using visual image data only.
Our study underlines not only the feasibility of DL to detect distinct morphologies that accompany a cytogenetic aberration like t(15;17) in APL, but also shows the capability of DL to abstract information from a small medical data set, i. e. 51 APL patients, and infer correct predictions. This demonstrates the suitability of DL to assist in the diagnosis of rare cancer entities. As our DL platform predicts APL from bone marrow smear images alone, this may be used to diagnose APL in regions were molecular or cytogenetic subtyping is not routinely available and raise attention to suspected cases of APL for expert evaluation.
Patients with acute promyelocytic leukemia (APL) often present with potentially life-threatening hemorrhagic diathesis. The underlying pathomechanisms of APL-associated coagulopathy are complex. ...However, two pathways considered to be APL-specific had been identified: 1) annexin A2 (ANXA2)-associated hyperfibrinolysis and 2) podoplanin (PDPN)-mediated platelet activation and aggregation. In contrast, since disseminated intravascular coagulation (DIC) is far less frequent in patients with non-APL acute myeloid leukemia (AML), the pathophysiology of AML-associated hemorrhagic disorders is not well understood. Furthermore, the potential threat of coagulopathy in non-APL AML patients may be underestimated. Herein, we report a patient with non-APL AML presenting with severe coagulopathy with hyperfibrinolysis. Since his clinical course resembled a prototypical APL-associated hemorrhagic disorder, we hypothesized pathophysiological similarities. Performing multiparametric flow cytometry (MFC) and immunofluorescence imaging (IF) studies, we found the patient's bone-marrow mononuclear cells (BM-MNC) to express ANXA2 - a biomarker previously thought to be APL-specific. In addition, whole-exome sequencing (WES) on sorted BM-MNC (leukemia-associated immunophenotype (LAIP)1: ANXA
, LAIP2: ANXA
) demonstrated high intra-tumor heterogeneity. Since ANXA2 regulation is not well understood, further research to determine the coagulopathy-initiating events in AML and APL is indicated. Moreover, ANXA2 and PDPN MFC assessment as a tool to determine the risk of life-threatening DIC in AML and APL patients should be evaluated.
Although extramedullary manifestations (EMs) are frequent in patients with acute myeloid leukemia (AML), they are often not detected during clinical workup and neither imaging- nor molecularly based ...diagnostic strategies are established to reveal their existence. Still, the detection of EM is essential for therapeutic decision-making, as EM present with aggressive and resistant disease and since mutational profiling might render patients within a different risk category, requiring personalized therapeutic strategies. Here, we report the case of an AML patient presenting with AML bone marrow (BM) infiltration and molecularly distinct EM at time of diagnosis followed by multiple EM relapses while undergoing several intensive chemotherapies including allogeneic hematopoietic cell transplantations (alloHCTs). 18Fluorodesoxy-glucose positron emission tomography (18FDG-PET)-imaging revealed EM sites in the mediastinum, duodenum, skin, and in retroperitoneal tissue, whereas recurrent BM biopsies showed continuous cytomorphologic and cytogenetic remission after alloHCT. To investigate the molecular background of the aggressive character of extramedullary disease and its differential treatment response, we performed amplicon-based next generation sequencing. An exon 4 (c.497_498insGA) frameshift RUNX1 mutation was exclusively found in all of the patient’s EM sites, but not in the BM or in peripheral blood samples at time of EM reoccurrence. In addition, we detected an exon 13 (c.3306G>T) ASXL1 point mutation only in the retroperitoneal tumor tissue at the time of the fourth relapse. In contrast to the patient’s intermediate-risk BM AML at diagnosis according to ELN2017, EM sites showed molecular adverse-risk features implicating intensified strategies like cellular therapies. Notably, disease relapse could only be detected by imaging throughout the course of disease. This case demonstrates both the necessity of continuous molecular profiling of EM to reveal differential molecular composition of EM and BM-derived AML, supposedly leading to divergent susceptibility to established therapies, as well as recurrent 18FDG-PET-imaging for detecting residual disease and assessment of treatment response in case of EM AML.
Lysyl oxidase (LOX) has been described as necessary for premetastatic niche formation in epithelium-derived malignancies and its expression level therefore correlates with risk of metastatic disease ...and overall survival. However, its role in acute myeloid leukemia (AML) has not been sufficiently analyzed.
We investigated LOX plasma expression in 683 AML patients (age 17-60 years) treated within the prospective AML2003 trial (NCT00180102). The optimal cut-off LOX value was determined using a minimal-
-value method dichotomizing patients into a LOX-high group (> 109 ng/mL,
= 272, 40%) and a LOX-low group (≤ 109 ng/mL,
= 411, 60%).
Higher LOX expression was associated with lower peripheral white blood cells, lower serum LDH, and a lower frequency of
-ITD and
mutations at diagnosis. Higher LOX expression was found significantly more frequently in patients with secondary AML and therapy-related AML, in patients with French-American-British M5 subtypes, and in patients with adverse-risk cytogenetics. Comparing patients in the LOX-high group and the LOX-low group revealed a 3-year overall survival (OS) of 47 and 53% (
= 0.022) and 3-year event-free survival (EFS) of 27 and 35% (
= 0.005), respectively. In the LOX-high group significantly more patients had extramedullary AML compared to the LOX-low group (
= 0.037). Combining extramedullary AML and LOX as interacting factors in a multivariate analysis resulted in an independent impact on survival for the LOX-high-extramedullary interaction for OS (HR = 2.25,
= 0.025) and EFS (HR = 2.48,
= 0.008). Furthermore, in patients with extramedullary disease (
= 59) the LOX level predicted survival. Patients within the LOX-low group had an OS of 43% and EFS of 36% as compared to the LOX-high group with an OS of 13% and EFS of 6% (
= 0.002 and
= 0.008, respectively).
We hypothesize LOX expression to be a new potential biomarker to predict outcome in AML, specifically in AML subgroups such as the prognostic heterogeneous group of AML patients with extramedullary disease.
This retrospective study was performed with patient samples registered within the prospective AML2003 trial (NCT00180102). Patients were enrolled between December 2003 and November 2009.
The mammalian neocortex shows a conserved six-layered structure that differs between species in the total number of cortical neurons produced owing to differences in the relative abundance of ...distinct progenitor populations. Recent studies have identified a new class of proliferative neurogenic cells in the outer subventricular zone (OSVZ) in gyrencephalic species such as primates and ferrets. Lissencephalic brains of mice possess fewer OSVZ-like progenitor cells and these do not constitute a distinct layer. Most in vitro and in vivo studies have shown that oxygen regulates the maintenance, proliferation and differentiation of neural progenitor cells. Here we dissect the effects of fetal brain oxygen tension on neural progenitor cell activity using a novel mouse model that allows oxygen tension to be controlled within the hypoxic microenvironment in the neurogenic niche of the fetal brain in vivo. Indeed, maternal oxygen treatment of 10%, 21% and 75% atmospheric oxygen tension for 48 h translates into robust changes in fetal brain oxygenation. Increased oxygen tension in fetal mouse forebrain in vivo leads to a marked expansion of a distinct proliferative cell population, basal to the SVZ. These cells constitute a novel neurogenic cell layer, similar to the OSVZ, and contribute to corticogenesis by heading for deeper cortical layers as a part of the cortical plate.
Reduced oxygen levels (1–5% O2, named herein ‘physioxia’) are beneficial for stem cell cultures leading to enhanced proliferation, better survival and higher differentiation potential, but the ...underlying molecular mechanisms remain elusive. A potential link between physioxia and the canonical Wnt pathway was found recently, but the differential involvement of this signalling pathway for the various stem cell properties such as proliferation, stem cell maintenance, and differentiation capacity remains enigmatic. We here demonstrate increased Wnt target gene transcription and stabilised active β-catenin upon physioxic cell culture in primary tissue-specific foetal mouse neural stem cells. Knock-out of the main oxygen sensing molecule, hypoxia-inducible factor-1α (Hif-1α), had no impact on Wnt activation assuming that physioxia induces the Wnt pathway independently of Hif-1α. To determine the physiological relevance of physioxia-induced Wnt/β-catenin signalling, we examined proliferation, cell cycle kinetics, survival and stem cell maintenance upon Wnt activation and inhibition. Whereas survival and stem cell maintenance seem to be independent of the Wnt pathway, our studies provide first evidence that Wnt/β-catenin signalling positively stimulates proliferation of physioxic cells by affecting cell cycle regulation. Together, our results provide mechanistic insight into oxygen-mediated regulation of the self-renewal activity of neural stem cells.
•Wnt/β-catenin signalling is induced by low O2 (physioxia) in neural stem cells.•Physioxia-induced Wnt signalling is independent of Hif-1α.•Wnt/β-catenin signalling regulates the proliferation of neural stem cells in physioxia.•Physioxia-induced Wnt signalling is a therapeutic target for regenerative medicine.
Ex vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different ...sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient.
We generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state.
Morphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5–7 days.
Our results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future.
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The limited proliferative capacity of neuroprogenitor cells (NPCs) within the periventricular germinal niches (PGNs) located caudal of the subventricular zone (SVZ) of the lateral ventricles together ...with their high proliferation capacity after isolation strongly implicates cell‐extrinsic humoral factors restricting NPC proliferation in the hypothalamic and midbrain PGNs. We comparatively examined the effects of norepinephrine (NE) as an endogenous candidate regulator of PGN neurogenesis in the SVZ as well as the periventricular hypothalamus and the periaqueductal midbrain. Histological and neurochemical analyses revealed that the pattern of NE innervation of the adult PGNs is inversely associated with their in vivo NPC proliferation capacity with low NE levels coupled to high NPC proliferation in the SVZ but high NE levels coupled to low NPC proliferation in hypothalamic and midbrain PGNs. Intraventricular infusion of NE decreased NPC proliferation and neurogenesis in the SVZ‐olfactory bulb system, while pharmacological NE inhibition increased NPC proliferation and early neurogenesis events in the caudal PGNs. Neurotoxic ablation of NE neurons using the Dsp4‐fluoxetine protocol confirmed its inhibitory effects on NPC proliferation. Contrarily, NE depletion largely impairs NPC proliferation within the hippocampus in the same animals. Our data indicate that norepinephrine has opposite effects on the two fundamental neurogenic niches of the adult brain with norepinephrine being a negative regulator of adult periventricular neurogenesis. This knowledge might ultimately lead to new therapeutic approaches to influence neurogenesis in hypothalamus‐related metabolic diseases or to stimulate endogenous regenerative potential in neurodegenerative processes such as Parkinson's disease.
Intracerebroventricular (i.c.v.) infusion of norepinephrine (NE) decreases neuroprogenitor cell (NPC) proliferation (arrows) within the subventricular zone (SVZ) of the lateral ventricles (LV) and neurogenesis in the SVZ‐olfactory bulb system without changing cells type composition of the SVZ. In contrast, pharmacological NE inhibition increases the proliferation of various NPC types and early neurogenesis events in the caudal periventricular germinal niche (PGN) of the third ventricle (3V). Neurotoxic ablation of norepinephrine neurons within the Locus coeruleus (LC) using Dsp4 confirms the inhibitory effects of NE on NPC proliferation within the 3V‐PGN. This effect is reversed by NE i.c.v. infusion. Contrarily, NE depletion largely impairs NPC proliferation within the hippocampus in the same animals. Our data indicate that NE has opposite effects on the two fundamental neurogenic niches of the adult brain with norepinephrine being a negative regulator of adult periventricular neurogenesis.
Oxygen tension is an important factor controlling stem cell proliferation and maintenance in various stem cell populations with a particular relevance in midbrain dopaminergic progenitors. Further ...studies have shown that the oxygen-dependent transcription factor hypoxia-inducible factor 1α (HIF-1α) is involved in these processes. However, all available studies on oxygen effects in dopaminergic neuroprogenitors were performed in vitro and thus it remains unclear whether tissue oxygen tension in the embryonic midbrain is also relevant for the regulation of dopaminergic neurogenesis in vivo. We thus dissect here the effects of oxygen tension in combination with HIF-1α conditional knockout on dopaminergic neurogenesis by using a novel experimental design allowing for the control of oxygen tension within the microenvironment of the neurogenic niche of the murine fetal midbrain in vivo. The microenvironment of the midbrain dopaminergic neurogenic niche was detected as hypoxic with oxygen tensions below 1.1%. Maternal oxygen treatment of 10%, 21%, and 75% atmospheric oxygen tension for 48 h translates into robust changes in fetal midbrain oxygenation. Fetal midbrain hypoxia hampered the generation of dopaminergic neurons and is accompanied with restricted fetal midbrain development. In contrast, induced hyperoxia stimulated proliferation and differentiation of dopaminergic progenitors during early and late embryogenesis. Oxygen effects were not directly mediated through HIF-1α signaling. These data--in agreement with in vitro data-indicate that oxygen is a crucial regulator of developmental dopaminergic neurogenesis. Our study provides the initial framework for future studies on molecular mechanisms mediating oxygen regulation of dopaminergic neurogenesis within the fetal midbrain as its natural environment.
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