Modulation of bone marrow and peripheral blood cytokine levels by age and clonal hematopoiesis in healthy individuals.

Aging is associated with bone marrow (BM) inflammaging and, in some individuals, with the onset of clonal hematopoiesis (CH) of indeterminate potential. In this study conducted on 94 strictly healthy volunteers (18 to 80 yo), we measured BM and peripheral blood (PB) plasma levels of 49 hematopoietic and inflammatory cytokines. With aging, 7 cytokines increased in BM (FLT3L, CXCL9, HGF, FGF-2, CCL27, IL-16, IL-18) and 8 decreased (G-CSF, TNF, IL-2, IL-15, IL-17A, CCL7, IL-4, IL-10). In PB, 10 cytokines increased with age (CXCL9, FLT3L, CCL27, CXCL10, HGF, CCL11, IL-16, IL-6, IL-1 beta, CCL2). CH was associated with higher BM levels of MIF and IL-1 beta, lower BM levels of IL-9 and IL-5 and higher PB levels of IL-15, VEGF-A, IL-2, CXCL8, CXCL1 and G-CSF. These reference values provide a useful tool to investigate anomalies related to inflammaging and potentially leading to the onset of age-related myeloid malignancies or inflammatory conditions.

Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition.

(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

Megaloblastic anemia-related iron overload and erythroid regulators: a case report.

BACKGROUND: In ineffective erythropoiesis, hepcidin synthesis is suppressed by erythroid regulators, namely erythroferrone and growth differentiation factor-15. For the first time, the hypothesis that iron overload in megaloblastic anemia may be related to ineffective erythropoiesis is explored by describing the kinetics of hepcidin, erythroferrone, and growth differentiation factor-15 levels in a patient diagnosed with megaloblastic anemia associated with iron overload. CASE PRESENTATION: An 81-year-old Caucasian male was admitted for fatigue. He had type-2 diabetes previously treated with metformin, ischemic cardiac insufficiency, and stage-3 chronic kidney disease. Vitiligo was observed on both hands. Biological tests revealed normocytic non-regenerative anemia associated with hemolysis, thrombocytopenia, and elevated sideremia, ferritin, and transferrin saturation levels. Megaloblastic anemia was confirmed with undetectable blood vitamin B12 and typical cytological findings like hyper-segmented neutrophils in blood and megaloblasts in bone marrow. The patient received vitamin B12 supplementation. At 3 months, biological parameters reached normal values. Hepcidin kinetics from diagnosis to 3 months inversely correlated with those of erythroferrone and growth differentiation factor-15. CONCLUSIONS: This case suggests that iron-overload mechanisms of dyserythropoietic anemias may apply to megaloblastic anemias.

Automated Early Detection of Myelodysplastic Syndrome within the General Population Using the Research Parameters of Beckman-Coulter DxH 800 Hematology Analyzer.

The incidence of myelodysplastic syndrome increases with aging and the early diagnosis enables optimal care of these diseases. The DxH 800 hematology analyzer measures and calculates 126 cytological parameters, but only 23 are used for routine CBC assessment. The goal of this study was to use the 103 unexploited "research parameters" to develop an algorithm allowing for an early detection of subclinical MDS patients by triggering morphological analysis. Blood sample parameters from 101 MDS patients and 88 healthy volunteers were analyzed to identify the critical "research parameters" with: (i) the most significant differences between MDS patients and healthy volunteers, (ii) the best contributions to principal component analysis (PCA), first axis, and (iii) the best correlations with PCA, first two axes (cos2 > 0.6). Ten critical "research parameters" of white blood cells were identified, allowing for the calculation of an MDS-likelihood score (MDS-LS), based on logistic regression. Automatic calculation of the MDS-LS is easily implementable on the middleware system of the DxH 800 to generate a flag for blood smear review, and possibly early detection of MDS patients in the general population.

Occupational pesticide exposure increases risk of acute myeloid leukemia: a meta-analysis of case-control studies including 3,955 cases and 9,948 controls.

The impact of pesticides on health is a major public health concern. A higher risk to develop chronic lymphoid malignancies has been demonstrated to be associated with occupational pesticide exposure (OPE). By contrast, little is known of the impact of OPE on the occurrence of myeloid malignancies especially acute myeloid leukemia (AML). The purpose of this meta-analysis is to summarize data on the association between OPE and AML. A relevant dataset of case-control studies was extracted. Among 6784 references extracted, 14 were selected, representing 3,955 AML patients and 9,948 control subjects diagnosed between 1976 and 2010. An adverse association was found between OPE and AML (OR = 1.51; 95%CI: 1.10-2.08), not affected by sensitivity analyses. Funnel plot asymmetry suggested a publication bias underestimating OR. Stratified analysis showed the association to be driven by studies with: (1) monocentric AML patients and hospital-based control population, (2) Newcastle-Ottawa scale > 6 and the group of studies identified as with the lowest risk, (3) exposure assessment through peer-to-peer interview, (4) diagnosis in North America and Asia and after 1995, (5) restriction to de novo AML. Moreover, the association between OPE and AML was significant with insecticides. These findings broaden the spectrum of pesticide toxicity to myeloid malignancies.

Involvement of GPx-3 in the Reciprocal Control of Redox Metabolism in the Leukemic Niche.

The bone marrow (BM) microenvironment plays a crucial role in the development and progression of leukemia (AML). Intracellular reactive oxygen species (ROS) are involved in the regulation of the biology of leukemia-initiating cells, where the antioxidant enzyme GPx-3 could be involved as a determinant of cellular self-renewal. Little is known however about the role of the microenvironment in the control of the oxidative metabolism of AML cells. In the present study, a coculture model of BM mesenchymal stromal cells (MSCs) and AML cells (KG1a cell-line and primary BM blasts) was used to explore this metabolic pathway. MSC-contact, rather than culture with MSC-conditioned medium, decreases ROS levels and inhibits the Nrf-2 pathway through overexpression of GPx3 in AML cells. The decrease of ROS levels also inactivates p38MAPK and reduces the proliferation of AML cells. Conversely, contact with AML cells modifies MSCs in that they display an increased oxidative stress and Nrf-2 activation, together with a concomitant lowered expression of GPx-3. Altogether, these experiments suggest that a reciprocal control of oxidative metabolism is initiated by direct cell-cell contact between MSCs and AML cells. GPx-3 expression appears to play a crucial role in this cross-talk and could be involved in the regulation of leukemogenesis.

Reactive oxygen species levels differentiate CD34+ human progenitors based on CD38 expression.

Low reactive oxygen species (ROS) levels are well-established characteristics of mouse hematopoietic stem cells (HSCs). However, little is known about these levels in human HSCs. This study aimed at quantifying ROS levels in human CD34+ CD38low and CD34+ CD38high human progenitors from bone marrow, cord blood and cells mobilized for autologous HSC transplantation. A specifically devised multiparameter flow cytometry method was used to quantify ROS levels (H2 DCFDA staining) in sub-populations of primary cells. Results were confirmed by assessing gene expression level of the ROS scavenger GPX3, a key determinant of HSC self-renewal, in sorted CD34+ CD38low and CD34+ CD38high cells. CD34+ CD38low cells from bone marrow and cord blood displayed significantly lower levels of ROS than CD34+ CD38high cells and other leukocytes. Moreover, the correlation between ROS and GPX3 expression was verified in sorted CD34+ CD38low and CD34+ CD38high cells. These results confirm, in human, data previously reported in mice. Moreover, the flow cytometry assay we developed could allow for a more precise enumeration of repopulating primitive progenitors in the course of HSC transplantation.

High prevalence of clonal hematopoiesis in the blood and bone marrow of healthy volunteers.

Clonal hematopoiesis (CH) of indeterminate potential has been described in blood samples from large series of patients. Its prevalence and consequences are still not well understood because sequencing methods vary and because most studies were performed in cohorts comprising individuals with nonhematologic diseases. Here, we investigated the frequency of CH in 82 paired bone marrow and blood samples from carefully selected healthy adult volunteers. Forty-one genes known to be mutated in myeloid malignancies were sequenced with a 1% threshold of detection. In bone marrow samples, clones were found in almost 40% of healthy volunteers more than 50 years old. The most frequent mutations were found in DNMT3A and TET2, with 1 individual carrying 3 variants. Variant allele frequencies were highly concordant between blood and bone marrow samples. Blood parameters were normal except for those in 2 individuals: 1 had a mild macrocytosis and 1 had a mild thrombocytosis. Furthermore, no morphologic abnormalities or dysplasia were detected when bone marrow smears were carefully evaluated. Individuals with CH differed from others by age (62.8 vs 38.6 years; P < .0001) and platelet count (294 vs 241 ×109/L; P = .0208), the latter being no more significant when removing the 2 individuals who carried the JAK2 p.V617F mutation. These results confirm that CH is a very common condition in healthy adults over 50 years old. Consequently, the detection of driver myeloid mutations should be interpreted with caution in the absence of cytologic abnormalities in the blood and/or the bone marrow.

Hypercalcemia is associated with a poor prognosis in lymphoma a retrospective monocentric matched-control study and extensive review of published reported cases.

The prognostic significance of hypercalcemia in lymphoma has only been studied on small series to date. We conducted a retrospective, monocentric, matched-control study that aimed to compare the outcome of patients diagnosed with any histological subtype of lymphoma associated with hypercalcemia, at diagnosis or relapse, with a group of controls matched for histological and prognostic factors. Sixty-two and 118 comparable patients treated between 2000 and 2016 were included in hypercalcemia and control cohorts, respectively. Hypercalcemia was found mainly at diagnosis (71%) in higher-risk patients (prognosis scores ≥ 3, 76%) and those with diffuse large B cell lymphoma (67.7%), stage III/IV disease (91.9%), and elevated LDH (90.3%). Two-year progression-free survival (PFS) was shorter in the hypercalcemia than control cohort [30.1% (95% confidence interval (95% CI) 18.3-41.9) vs 63.9% (95% CI 5.1-72.7), p < 0.001]. Two-year overall survival (OS) was 40.6% (95% CI 28.1-53.1) and 77.7% (95% CI 70.1-85.3) in the hypercalcemia and control cohorts, respectively (p < 0.001). Hypercalcemia was independently associated with poor PFS [HR = 2.5 (95% CI 1.4-3.5)] and OS [HR = 4.7 (95% CI 2.8-7.8)] in multivariate analysis. Among the 40 patients who received autologous stem cell transplantation (ASCT), hypercalcemia was still associated with shorter OS [2-year OS: 65% (95% CI 40.1-89.9) vs 88.0 (95% CI 75.3-100), p = 0.04]. Hypercalcemia may be associated with chemo-resistance, given its impact on PFS and OS. Hence, these data suggest that alternate strategies for lymphoma patients with hypercalcemia should be developed.

Correction: Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells.

The original version of this Article omitted the following from the Acknowledgements: This research was also supported by grants to KZ (UL and L-CNRS). This has now been corrected in both the PDF and HTML versions of the Article.

Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells.

The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+ progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.

Bone marrow oxidative stress and specific antioxidant signatures in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders with an inherent tendency for transformation in secondary acute myeloid leukemia. This study focused on the redox metabolism of bone marrow (BM) cells from 97 patients compared with 25 healthy controls. The level of reactive oxygen species (ROS) was quantified by flow cytometry in BM cell subsets as well as the expression level of 28 transcripts encoding for major enzymes involved in the antioxidant cellular response. Our results highlight increased ROS levels in BM nonlymphoid cells and especially in primitive CD34posCD38low progenitor cells. Moreover, we identified a specific antioxidant signature, dubbed "antioxidogram," for the different MDS subgroups or secondary acute myeloblastic leukemia (sAML). Our results suggest that progression from MDS toward sAML could be characterized by 3 successive molecular steps: (1) overexpression of enzymes reducing proteic disulfide bonds (MDS with <5% BM blasts [GLRX family]); (2) increased expression of enzymes detoxifying H2O2 (MDS with 5% to 19% BM blasts [PRDX and GPX families]); and finally (3) decreased expression of these enzymes in sAML. The antioxidant score (AO-Score) defined by logistic regression from the expression levels of transcripts made it possible to stage disease progression and, interestingly, this AO-Score was independent of the revised International Scoring System. Altogether, this study demonstrates that MDS and sAML present an important disturbance of redox metabolism, especially in BM stem and progenitor cells and that the specific molecular antioxidant response parameters (antioxidogram, AO-Score) could be considered as useful biomarkers for disease diagnosis and follow-up.

Characteristics, combinations, treatments, and survival of second primary hematological neoplasm: a retrospective single-center cohort of 49 patients (Hemo2study).

The coexistence of dual hematological neoplasms is very rare. Sequential or synchronous neoplasms in hematology are an uncommon and complex clinical situation. The aim of the Hemo2 study was to describe the clinical characteristics and analyze the outcome of these patients. We performed a retrospective review of all patients diagnosed with sequential or synchronous hematological malignancies in the university hospital of Tours, between 2007 and 2018. We identified 49 patients in our study, with a prevalence of 0.89%. Sequential and synchronous combinations were found in 36 (73%) and 13 (27%) patients, respectively. One patient presented three sequential neoplasms. The median cumulative incidence was 6 years (95% CI 3-7). Among all neoplasms diagnosed (n = 99), we found 79 lymphoid neoplasms (LNs) (80%) and 20 myeloid neoplasms (MNs) (20%). Sex ratio was 1.88 with 65% of males and 35% of females. The most common LNs were Hodgkin lymphoma (n = 16; 16%) and multiple myeloma (n = 11; 11%). The most frequent MN was essential thrombocythemia (n = 5; 5%). The most common combination was Hodgkin lymphoma and follicular lymphoma in five (10%) patients. The overall survival from the first diagnosis (OS1) at 5 years was 82.4% (95% CI 72.1-94.3). The median overall survival from the second diagnosis (OS2) was 98 months (95% CI 44-NR) and 5-year OS2 was 58.7% (95% CI 45.5-75.7). Median progression-free survival from the second diagnosis (PFS) was 47 months (95% CI 27-NR) with 5-year PFS of 49% (95% CI 35.9-67). OS and PFS did not statistically differ between synchronous and sequential dual neoplasms. In this cohort, that the death relative risk (RR) was significantly lower if the second neoplasm appeared after more than 4 years following the first diagnosis (OR 0.37 (95% CI 0.16-0.90)). The Hemo2study confirmed the rarity of dual hematological neoplasms. In this cohort, HL and FL were the most frequent combinations. Our results may support that synchronous and sequential dual neoplasms bear the same prognosis. Further studies are needed to better characterize these uncommon clinical situations.

n-3 Polyunsaturated fatty acids induce acute myeloid leukemia cell death associated with mitochondrial glycolytic switch and Nrf2 pathway activation.

Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.

Combined hepatocellular-cholangiocarcinoma in a patient with Abernethy malformation and tetralogy of Fallot: A case report.

Abernethy malformation is a rare congenital anomaly of the portal vein where the portal blood bypasses the liver. We report the first case of a patient with Abernethy malformation and tetralogy of Fallot associated with nodular regenerative hyperplasia and focal nodular hyperplasia (FNH), which finally evolved to a giant hepatocellular-cholangiocarcinoma (HCC-CC) of the liver, successfully resected. (Hepatology 2016;64:1800-1802).

Learning-by-Concordance (LbC): introducing undergraduate students to the complexity and uncertainty of clinical practice.

BACKGROUND: A current challenge in medical education is the steep exposure to the complexity and uncertainty of clinical practice in early clerkship. The gap between pre-clinical courses and the reality of clinical decision-making can be overwhelming for undergraduate students. The Learning-by-Concordance (LbC) approach aims to bridge this gap by embedding complexity and uncertainty by relying on real-life situations and exposure to expert reasoning processes to support learning. LbC provides three forms of support: 1) expert responses that students compare with their own, 2) expert explanations and 3) recognized scholars' key-messages. METHOD: Three different LbC inspired learning tools were used by 900 undergraduate medical students in three courses: Concordance-of-Reasoning in a 1st-year hematology course; Concordance-of-Perception in a 2nd-year pulmonary physio-pathology course, and; Concordance-of-Professional-Judgment with 3rd-year clerkship students. Thematic analysis was conducted on freely volunteered qualitative comments provided by 404 students. RESULTS: Absence of a right answer was challenging for 1st year concordance-of-reasoning group; the 2nd year visual concordance group found radiology images initially difficult and unnerving and the 3rd year concordance-of-judgment group recognized the importance of divergent expert opinion. CONCLUSIONS: Expert panel answers and explanations constitute an example of "cognitive apprenticeship" that could contribute to the development of appropriate professional reasoning processes.

Learning medical professionalism with the online concordance-of-judgment learning tool (CJLT): A pilot study.

CONTEXT: Professionalism development entails learning to make judgments in ambiguous situations. A Concordance of Judgment Learning Tool (CJLT), comprised of 20 vignettes involving professionalism issues, was developed. Students obtained a measure of how concordant their judgments were with a panel of experts and learned from given explanations. METHOD: Twenty clinical vignettes implying professionalism issues were written including, for each, four possible courses of action. Expert panel, nominated by all clerkship students, was made up of attending physicians that best represented professionalism role models. Experts completed CJLT and gave explanations for their answers. All clerks were invited to answer each vignette, and then received automated expert feedback including explanations. RESULTS: Seventy-nine students sat for the activity. The optimized test included 20 cases and 54 questions (Cronbach's alpha coefficient of 0.64). Student - expert concordance scores ranged from 54 to 77 with a mean at 64.6 (standard deviation 5.1). Satisfaction survey results indicated high satisfaction and relevance of tool despite some pitfalls. Post-test focus group data revealed relevant experiential learning on professionalism issues. DISCUSSION: Students' scores and perceptions suggest pedagogic relevance of the CJLT in fostering professionalism development in clerkship. CJLT is user-friendly and shows promise as a situation experiential learning activity.

Heterotopic pancreas presenting as suspicious mass in the gallbladder.

CONTEXT: Heterotopic pancreas is a rare entity. Thirty-three cases in the gallbladder have been reported. We describe the first case of heterotopic pancreas mimicking a gallbladder cancer, identified within a calcified lesion in the thickened posterior wall of the gallbladder. CASE REPORT: A 72-year-old woman with right upper quadrant pain was referred with a suspicion of gallbladder neoplasia. A CT scan demonstrated a 1 cm thickened posterior wall of the gallbladder with a 2 mm punctate calcification. An open cholecystectomy was carried out without complication. The frozen section demonstrated pancreatic tissue. CONCLUSION: Heterotopic pancreas of the gallbladder is highly uncommon. It can mimic a neoplastic process in the gallbladder, particularly in the context of calcification. Its malignant potential in the gallbladder is unknown, in contrast to previously described neoplastic transformation with gastric heterotopic pancreas.