Value of combining biological age with assessment of individual frailty to optimize management of cancer treated with targeted therapies: model of chronic myeloid leukemia treated with tyrosine kinase inhibitors (BIO-TIMER trial).

BACKGROUND: In the era of targeted therapies, the influence of aging on cancer management varies from one patient to another. Assessing individual frailty using geriatric tools has its limitations, and is not appropriate for all patients especially the youngest one. Thus, assessing the complementary value of a potential biomarker of individual aging is a promising field of investigation. The chronic myeloid leukemia model allows us to address this question with obvious advantages: longest experience in the use of tyrosine kinase inhibitors, standardization of therapeutic management and response with minimal residual disease and no effect on age-related diseases. Therefore, the aim of the BIO-TIMER study is to assess the biological age of chronic myeloid leukemia or non-malignant cells in patients treated with tyrosine kinase inhibitors and to determine its relevance, in association or not with individual frailty to optimize the personalised management of each patient. METHODS: The BIO-TIMER study is a multi-center, prospective, longitudinal study aiming to evaluate the value of combining biological age determination by DNA methylation profile with individual frailty assessment to personalize the management of chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Blood samples will be collected at diagnosis, 3 months and 12 months after treatment initiation. Individual frailty and quality of life will be assess at diagnosis, 6 months after treatment initiation, and then annually for 3 years. Tolerance to tyrosine kinase inhibitors will also be assessed during the 3-year follow-up. The study plans to recruit 321 patients and recruitment started in November 2023. DISCUSSION: The assessment of individual frailty should make it possible to personalize the treatment and care of patients. The BIO-TIMER study will provide new data on the role of aging in the management of chronic myeloid leukemia patients treated with tyrosine kinase inhibitors, which could influence clinical decision-making. TRIAL REGISTRATION: ClinicalTrials.gov , ID NCT06130787; registered on November 14, 2023.

Receptor for Advanced Glycation End-Products Promotes Activation of Alveolar Macrophages through the NLRP3 Inflammasome/TXNIP Axis in Acute Lung Injury.

The roles of thioredoxin-interacting protein (TXNIP) and receptor for advanced glycation end-products (RAGE)-dependent mechanisms of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-driven macrophage activation during acute lung injury are underinvestigated. Cultured THP-1 macrophages were treated with a RAGE agonist (S100A12), with or without a RAGE antagonist; cytokine release and intracytoplasmic production of reactive oxygen species (ROS) were assessed in response to small interfering RNA knockdowns of TXNIP and NLRP3. Lung expressions of TXNIP and NLRP3 and alveolar levels of IL-1ß and S100A12 were measured in mice after acid-induced lung injury, with or without administration of RAGE inhibitors. Alveolar macrophages from patients with acute respiratory distress syndrome and from mechanically ventilated controls were analyzed using fluorescence-activated cell sorting. In vitro, RAGE promoted cytokine release and ROS production in macrophages and upregulated NLRP3 and TXNIP mRNA expression in response to S100A12. TXNIP inhibition downregulated NLRP3 gene expression and RAGE-mediated release of IL-1ß by macrophages in vitro. In vivo, RAGE, NLRP3 and TXNIP lung expressions were upregulated during experimental acute lung injury, a phenomenon being reversed by RAGE inhibition. The numbers of cells expressing RAGE, NLRP3 and TXNIP among a specific subpopulation of CD16+CD14+CD206- ("pro-inflammatory") alveolar macrophages were higher in patients with lung injury. This study provides a novel proof-of-concept of complex RAGE-TXNIP-NLRP3 interactions during macrophage activation in acute lung injury.

Accuracy of chitotriosidase activity and CCL18 concentration in assessing type I Gaucher disease severity. A systematic review with meta-analysis of individual participant data.

Chitotriosidase activity and CCL18 concentration are interchangeably used for monitoring Gaucher disease (GD) activity, together with clinical assessment. However, comparative studies of these two biomarkers are scarce and of limited sample size. The aim of this systematic review with meta-analysis of individual participant data (IPD) was to compare the accuracy of chitotriosidase activity and CCL18 concentration for assessing type I GD severity. We identified cross-sectional and prospective cohort studies by searching Medline, EMBASE, and CENTRAL from 1995 to June 2017, and by contacting research groups. The primary outcome was a composite of liver volume >1.25 multiple of normal (MN), spleen volume >5 MN, hemoglobin concentration <11 g/dL, and platelet count <100x109/L. Overall, IPD included 1109 observations from 334 patients enrolled in nine primary studies, after excluding 111 patients with undocumented values and 18 patients with deficient chitotriosidase activity. IPD were unavailable for 14 eligible primary studies. The primary outcome was associated with a 5.3-fold (95% confidence interval [CI], 4.2 to 6.6) and 3.0-fold (95% CI, 2.6 to 3.6) increase of the geometric mean for chitotriosidase activity and CCL18 concentration, respectively. The corresponding areas under the receiver operating characteristics curves were 0.82 and 0.84 (summary difference, 0.02, 95% CI, -0.02 to 0.05). The addition of chitotriosidase activity did not improve the accuracy of CCL18 concentration. Estimates remained robust in the sensitivity analysis and consistent across subgroups. Neither chitotriosidase activity nor CCL18 concentration varied significantly according to a recent history of bone events among 97 patients. In conclusion, CCL18 concentration is as accurate as chitotriosidase activity in assessing hematological and visceral parameters of GD severity and can be measured in all GD patients. This meta-analysis supports the use of CCL18 rather than chitotriosidase activity for monitoring GD activity in routine practice.

Contribution of Chronic Myeloid Leukaemia (CML) as a Disease Model to Define and Study Clonal Heterogeneity.

Although tumour cell intra-clonal heterogeneity has been known for many years, its application in the oncology clinical practice (patient management, prognosis, etc.) remains limited. For this, chronic myeloid leukaemia (CML) is a remarkable model. Basic research studies revealed the heterogeneity of the initial clone, and led to the hypothesis of the existence of leukemic stem cells. Nevertheless, the indisputable evidence of the intra-clonal heterogeneity role in the therapeutic response came from the outcomes of the treatment with tyrosine kinase inhibitors (the first targeted therapy in medicine) combined with the early and rigorous clinical and molecular monitoring of these patients. CML management already takes this heterogeneity into account for personalized patient follow-up. The adventure continues with the objectives of better tailoring the treatment and of curing the disease in most of the patients.

Bone marrow hematons: An access point to the human hematopoietic niche.

To understand the complex interactions between hematopoietic stem cells and the bone marrow niche, a human experimental model is needed. Our hypothesis is that hematons are an appropriate ex vivo model of human bone marrow. We analyzed the hierarchical hematopoietic cell content and the tissue organization of single hematons from healthy donors. Most (>90%) hematons contained precursors of all cell lineages, myeloid progenitors, and LTC-ICs without preferential commitment. Approximately, half of the hematons could generate significant levels of lympho-myeloid hematopoiesis after transplantation in an NSG mouse model, despite the low absolute numbers of transplanted CD34+ cells. Mesenchymal STRO-1+ and/or CD271+ cells formed a critical network that preserved hematon cohesion, and STRO-1+ cells colocalized with most hematopoietic CD34+ cells (68%). We observed an influence of age and gender. These structures represent a particularly attractive model for studying the homeostasis of the bone marrow niche and pathological changes that occur during diseases.

Preanalytical, analytical, gestational and pediatric aspects of the S100B immuno-assays.

BACKGROUND: Traumatic brain injury management is a tricky issue in children and pregnant women (due to adverse effects of computer tomography). To facilitate management, we report the main analytical performances and reference ranges for blood tests for the well-established S100B biomarker in under-16 children on a DiaSorin® Liaison XL analyzer and in pregnant women on DiaSorin® Liaison XL and Roche Diagnostics® Cobas e411 analyzers. METHODS: Serum S100B concentrations were determined by chemiluminescent immunoassay on a DiaSorin® analyzer in a population of 409 healthy children aged 0-16 years and on DiaSorin®/Roche Diagnostics® instruments in a population of 50 pregnant women (one blood sample for each trimester). The analytical performances of both instruments and the influence of blood cells and skin pigmentation on the assay were also studied. RESULTS: For children, four age-groups emerged, i.e. 0-3 months (mean: 0.97 µg/L; standard deviation (SD): 0.36; 95th percentile: 1.55), 4-9 months (mean: 0.58 µg/L; SD: 0.30; 95th: 1.18), 10-24 months (mean: 0.31 µg/L; SD: 0.12; 95th: 0.54) and 2-16 years (mean: 0.20 µg/L; SD: 0.07; 95th: 0.32). For pregnant women, serum S100B concentrations were similar to defined ranges for adults and not significantly different between trimesters on DiaSorin® (p=0.652)/Roche Diagnostics® (p=0.877) analyzers. We also found S100B expression (protein, total mRNA) in lymphocytes, an influence of skin pigmentation, and good analytical performances for both instruments. CONCLUSIONS: Data provided here is useful for interpreting serum S100B test results, in terms of preanalytical conditions, analytical performances, pediatric and pregnancy' environment.

Freezing Does Not Alter Sperm Telomere Length despite Increasing DNA Oxidation and Fragmentation.

Correlations were reported between sperm telomere length (STL) and male fertility, sperm DNA fragmentation, and oxidation. Sperm freezing is widely used for assisted reproductive techniques, fertility preservation, and sperm donation. However, its impact on STL remains unknown. For this study, semen surplus from patients who underwent routine semen analysis were used. The impact of slow freezing on STL was analyzed by performing qPCR before and after freezing. Sperm populations with different STL were evaluated using Q-FISH. The relationship between sperm DNA oxidation, DNA fragmentation, and STL was assessed in fresh and frozen sperm samples. No significant impact of slow freezing on STL was observed, neither measured by qPCR nor Q-FISH. However, Q-FISH allowed for the distinguishing of sperm populations with different STLs within individual sperm samples. Slow freezing induced different STL distributions for some of the analyzed sperm samples, but no correlation was found between STL and sperm DNA fragmentation or oxidation. Slow freezing does not alter STL despite increasing sperm DNA oxidation and fragmentation. As STL alterations could be transmitted to offspring, the lack of impact of the slow freezing method on STL ensures the safety of this procedure.

A bicellular fluorescent ductal carcinoma in situ (DCIS)-like tumoroid to study the progression of carcinoma: practical approaches and optimization.

Recently, many types of 3D culture systems have been developed to preserve the physicochemical environment and biological characteristics of the original tumors better than the conventional 2D monolayer culture system. There are various types of models belonging to this culture, such as the culture based on non-adherent and/or scaffold-free matrices to form the tumors. Agarose mold has been widely used to facilitate tissue spheroid assembly, as it is essentially non-biodegradable, bio-inert, biocompatible, low-cost, and low-attachment material that can promote cell spheroidization. As no studies have been carried out on the development of a fluorescent bicellular tumoroid mimicking ductal carcinoma in situ (DCIS) using human cell lines, our objective was to detail the practical approaches developed to generate this model, consisting of a continuous layer of myoepithelial cells (MECs) around a previously formed in situ breast tumor. The practical approaches developed to generate a bi-cellular tumoroid mimicking ductal carcinoma in situ (DCIS), consisting of a continuous layer of myoepithelial cells (MECs) around a previously formed in situ breast tumoroid. Firstly, the optimal steps and conditions of spheroids generation using a non-adherent agarose gel were described, in particular, the appropriate medium, seeding density of each cell type and incubation period. Next, a lentiviral transduction approach to achieve stable fluorescent protein expression (integrative system) was used to characterize the different cell lines and to track tumoroid generation through immunofluorescence, the organization of the two cell types was validated, specific merits and drawbacks were compared to lentiviral transduction. Two lentiviral vectors expressing either EGFP (Enhanced Green Fluorescent Protein) or m-Cherry (Red Fluorescent Protein) were used. Various rates of a multiplicity of infection (MOI) and multiple types of antibodies (anti-p63, anti-CK8, anti-Maspin, anti-Calponin) for immunofluorescence analysis were tested to determine the optimal conditions for each cell line. At MOI 40 (GFP) and MOI 5 (m-Cherry), the signals were almost homogeneously distributed in the cells which could then be used to generate the DCIS-like tumoroids. Images of the tumoroids in agarose molds were captured with a confocal microscope Micro Zeiss Cell Observer Spinning Disk or with IncuCyte® to follow the progress of the generation. Measurement of protumoral cytokines such as IL-6, IL8 and leptin confirmed their secretion in the supernatants, indicating that the properties of our cells were not altered. Finally the advantages and disadvantages of each fluorescent approach were discussed. This model could also be used for other solid malignancies to study the complex relationship between different cells such as tumor and myoepithelial cells in various microenvironments (inflammatory, adipose and tumor, obesity, etc.). Although, this new model is well established to monitor drug screening applications and perform pharmacokinetic and pharmacodynamic analyses.

Measurement of imatinib uptake by flow cytometry.

One of the essential parameters of targeted therapy efficiency in cancer treatment is the amount of drug reaching the therapeutic target area. Imatinib (IM) was the first specifically targeted drug to be developed and has revolutionized the treatment of patients with chronic myeloid leukemia (CML). To evaluate cellular uptake of IM, we developed a method based on the chemical structure of the molecule and using the natural UV fluorescence that we quantified by flow cytometry. In two CML cell lines, we obtained a satisfactory relationship between intracellular IM (ICIM) levels and media concentrations, and we found a strong correlation between ICIM at 1 h and IM efficacy at 24 h, demonstrating that ICIM at 1 h might be a relevant predictive parameter of cell sensitivity. Our method was more sensitive than the standard physicochemical method. We applied our method to primary cells and found cell morphology-dependent IM accumulation. Moreover, in CML cells from patients at diagnosis, IM accumulation was heterogeneous. In all cases, ICIM at the single-cell level was much higher than in culture media arguing in favor of a predominantly active uptake process. We developed a simple method directly applicable to primary cells that has shown two major advantages: only a small number of cells are required, and cell subsets can be identified according to morphological criteria and/or the presence of particular antigenic sites. This method provides a new tool to assess CML cell sensitivity to IM, and ICIM levels in native CML cells could be used to monitor therapeutic response.

Applicability of the long-term uncertainty in measurement (LTUM) method for analytical performance assessment in clinical cytometry laboratories.

BACKGROUND: The estimation of uncertainty in measurement for quantitative analyses is an international obligation of the ISO15189 standard for laboratories. The most widespread method is the Internal Quality Control and External Quality Assessment (IQC + EQA). METHODS: We compared two methods to assess uncertainty in measurement for the quantification of the number of CD34+ stem cells and of the different lymphocyte subpopulations in blood samples: the IQC + EQA method and the Long-Term Uncertainty in Measurement (LTUM) method. RESULTS: We focused on the CD3+/CD4+ T lymphocyte subpopulation for a target value of 350 CD3+/CD4+/µl. The range in terms of uncertainty in the measurement of 350 CD3+/CD4+ cells/µl with the IQC + EQA method was [292.8; 407.2]. With the LTUM method, the uncertainty was 19.1% of the measured value. This represented a range of [283.2; 416.9]. CONCLUSIONS: The relative uncertainty calculated with the LTUM method can be adapted to any level of the measured parameter. IQC and EQA calculate the absolute uncertainty and need a clustering of values at different levels. This clustering can lead to some approximations in the uncertainty in measurement determination, particularly around the cut-off values. Unlike previous reports, uncertainty values were higher when calculated with the LTUM than with the IQC + EQA method. However, LTUM might be more representative of the daily routine practice with patient samples.

The Spliceosome: A New Therapeutic Target in Chronic Myeloid Leukaemia.

RNA splicing factors are frequently altered in cancer and can act as both oncoproteins and tumour suppressors. They have been found mutated or deregulated, justifying the growing interest in the targeting of splicing catalysis, splicing regulatory proteins, and/or specific, key altered splicing events. We recently showed that the DNA methylation alterations of CD34+CD15- chronic myeloid leukaemia (CML) cells affect, among others, alternative splicing genes, suggesting that spliceosome actors might be altered in chronic-phase (CP)-CML. We investigated the expression of 12 spliceosome genes known to be oncogenes or tumour suppressor genes in primary CP-CML CD34+ cells at diagnosis (n = 15). We found that CP-CML CD34+ cells had a distinct splicing signature profile as compared with healthy donor CD34+ cells or whole CP-CML cells, suggesting: (i) a spliceosome deregulation from the diagnosis time and (ii) an intraclonal heterogeneity. We could identify three profile types, but there was no relationship with a patient's characteristics. By incubating cells with TKI and/or a spliceosome-targeted drug (TG003), we showed that CP-CML CD34+ cells are both BCR::ABL and spliceosome dependent, with the combination of the two drugs showing an additive effect while sparing healthy donors cells. Our results suggest that the spliceosome may be a new potential target for the treatment of CML.

DNA Methylation and Intra-Clonal Heterogeneity: The Chronic Myeloid Leukemia Model.

Chronic Myeloid Leukemia (CML) is a model to investigate the impact of tumor intra-clonal heterogeneity in personalized medicine. Indeed, tyrosine kinase inhibitors (TKIs) target the BCR-ABL fusion protein, which is considered the major CML driver. TKI use has highlighted the existence of intra-clonal heterogeneity, as indicated by the persistence of a minority subclone for several years despite the presence of the target fusion protein in all cells. Epigenetic modifications could partly explain this heterogeneity. This review summarizes the results of DNA methylation studies in CML. Next-generation sequencing technologies allowed for moving from single-gene to genome-wide analyses showing that methylation abnormalities are much more widespread in CML cells. These data showed that global hypomethylation is associated with hypermethylation of specific sites already at diagnosis in the early phase of CML. The BCR-ABL-independence of some methylation profile alterations and the recent demonstration of the initial intra-clonal DNA methylation heterogeneity suggests that some DNA methylation alterations may be biomarkers of TKI sensitivity/resistance and of disease progression risk. These results also open perspectives for understanding the epigenetic/genetic background of CML predisposition and for developing new therapeutic strategies.

Beneficial effects of hypotaurine supplementation in preparation and freezing media on human sperm cryo-capacitation and DNA quality.

BACKGROUND: Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal (n = 19) or abnormal (n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with ("H+" arm) or without ("H-" arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. RESULTS: Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p < 0.001), progressive and total motility (+ 39.9% and +  21.6% respectively, p < 0.005) than spermatozoa from the control "H-" arm. Hypotaurine also reduced the non-specific phosphorylation of the capacitation protein markers P110 and P80 (p < 0.01), indicating a decrease in cryo-capacitation. Hypotaurine supplementation reduced chromatin decondensation, measured by chromomycin A3 (- 16.1%, p < 0.05), DNA fragmentation (- 18.7%, p < 0.05) and nuclear vacuolization (- 20.8%, p < 0.05). CONCLUSION: Our study is the first to demonstrate beneficial effects of hypotaurine supplementation in preparation and freezing procedures on human spermatozoa sperm fertilization capacity and nucleus quality. Hypotaurine supplementation limited cryo-capacitation, increased the proportion of live and progressively motile spermatozoa and reduces the percentage of spermatozoa showing chromatin decondensation, DNA fragmentation and nuclear vacuolation. TRIAL REGISTRATION: Clinical Trial, NCT04011813 . Registered 19 May 2019 - Retrospectively registered.

RéSUMé: CONTEXTE: Bien que largement utilisée, la congélation lente modifie considérablement les fonctions des spermatozoïdes humains. La cryoconservation induit des altérations nucléaires du sperme et une cryocapacitation, réduisant les chances de grossesse. L'hypotaurine est. naturellement présente dans les voies génitales masculines et féminines et possède des propriétés capacitantes, osmotiques et anti-oxydantes. Les mesures ont été réalisées sur le reliquat de sperme d'hommes avec des paramètres spermatiques normaux (n = 19) ou anormaux (n = 14). Les spermatozoïdes ont été sélectionnés par centrifugation sur gradient de densité (test de migration survie) avant congélation lente. Pour chaque prélèvement, ces étapes ont été réalisées en parallèle avec des milieux supplémentés en hypotaurine (bras « H+ ¼) ou sans hypotaurine (bras « H- ¼). Après décongélation, nous avons mesuré la mobilité totale et progressive, la vitalité, l'intégrité de l'acrosome, des marqueurs de la voie de signalisation de la capacitation et la qualité nucléaire. Pour cette dernière, nous nous sommes concentrés sur la condensation de la chromatine, la fragmentation de l'ADN et la présence de vacuoles dans le noyau du sperme. RéSULTATS: Post-décongélation, les spermatozoïdes sélectionnés et congelés en présence d'hypotaurine avaient une vitalité plus élevée (+ 16,7%, p < 0,001), une motilité progressive et totale (+ 39,9% et + 21,6% respectivement, p < 0,005) que les spermatozoïdes du bras « H- ¼ sans suplémentation. L'hypotaurine a également réduit la phosphorylation non spécifique des marqueurs protéiques de capacitation P110 et P80 (p < 0,01), indiquant une diminution de la cryocapacitation. La supplémentation en hypotaurine a réduit la décondensation de la chromatine, mesurée par la chromomycine A3 (− 16,1%, p < 0,05), la fragmentation de l'ADN (− 18,7%, p < 0,05) et la vacuolisation nucléaire (− 20,8%, p < 0,05). CONCLUSION: Notre étude est. la première à démontrer les effets bénéfiques de la supplémentation en hypotaurine dans les milieux de préparation et de congélation sur la capacité de fécondation des spermatozoïdes humains et leur qualité nucléaire. La supplémentation en hypotaurine a limité la cryocapacitation, augmenté la proportion de spermatozoïdes vivants et progressivement mobiles et réduit le pourcentage de spermatozoïdes présentant une décondensation de la chromatine, une fragmentation de l'ADN et une vacuolisation nucléaire. ENREGISTREMENT DE L'ESSAI: essai clinique, NCT04011813 . Enregistré le 19 mai 2019 - Enregistré rétrospectivement.

Efficiency of nilotinib to target chronic phase-chronic myeloid leukaemia primary mature CD34- and immature CD34+ cells.

Accumulation in target cells is an essential pharmacokinetic step of targeted therapies. Tyrosine Kinase Inhibitors (TKI) against the BCR-ABL fusion protein in Chronic Phase-Chronic Myeloid Leukaemia (CP-CML) cells constitute a unique model in terms of efficacy, specificity, and in vivo demonstration of response heterogeneity by target cells. The overall therapeutic response to nilotinib is heterogeneous with no satisfactory explanation. To better understand the patients' response heterogeneity, we quantified nilotinib uptake by primary CP-CML cells in standardized conditions using flow cytometry, which allowed also distinguishing mature (polymorphonuclear cells) from immature (CD34+) cells. Nilotinib was undetectable in 13.3% of PMN and 40% of CD34+ cells. Moreover, in CD34+ cells, intracellular nilotinib did not completely abolish BCR-ABL activity (monitored by CrkL phosphorylation inhibition), although nilotinib accumulated well in most CD34+ cell samples. Intracellular nilotinib concentration was inversely correlated with disease burden parameters, Sokal score, and early haematologic response at day 6 ± 1 only in PMN, suggesting an intrinsic ability to limit nilotinib entry in the forms with higher tumor cell burdenat diagnosis. These findings suggest that nilotinib accumulation in CP-CML cells is influenced by individual characteristics and intra-clonal heterogeneity, and might be used for pharmacokinetic studies and to assess the therapeutic response.

Impact of Epicatechin on the Procoagulant Activities of Microparticles.

Microparticles play a role in cardiovascular disease pathology. The flavanol-like epicatechin is increasingly considered due to its cardioprotective effects. The aim of this study was to investigate the impact of epicatechin on microparticle generation, phenotype and procoagulant properties. Plasma samples from 15 healthy subjects were incubated with increasing concentrations of epicatechin (1 to 100 µM). Then, the expression of glycoprotein IIb, phosphatidylserine (PS), glycoprotein Ib (GPIb) and P-selectin was assessed by flow cytometry analysis after (or not) platelet stimulation. Microparticle procoagulant activity was determined using ZymuphenTM MP and ZymuphenTM MP-TF for phospholipid and tissue factor content, and with thrombin generation (TG) assays for procoagulant function. Platelet microparticles that express GPIb (/µL) decreased from 20,743 ± 24,985 (vehicle) to 14,939 ± 14,333 (p = 0.6), 21,366 ± 16,949 (p = 0.9) and 15,425 ± 9953 (p < 0.05) in samples incubated with 1, 10 and 100 µM epicatechin, respectively. Microparticle concentration (nM PS) decreased from 5.6 ± 2.0 (vehicle) to 5.1 ± 2.2 (p = 0.5), 4.5 ± 1.5 (p < 0.05) and 4.7 ± 2.0 (p < 0.05) in samples incubated with 1, 10 and 100µM epicatechin, respectively. Epicatechin had no impact on tissue factor-positive microparticle concentration. Epicatechin decreased TG (endogenous thrombin potential, nM.min) from 586 ± 302 to 509 ± 226 (p = 0.3), 512 ± 270 (p = 0.3) and 445 ± 283 (p < 0.05). These findings indicate that epicatechin affects microparticle release, phenotype and procoagulant properties.

Hormonal Therapy Resistance and Breast Cancer: Involvement of Adipocytes and Leptin.

Obesity, a recognized risk factor for breast cancer in postmenopausal women, is associated with higher mortality rates regardless of menopausal status, which could in part be explained by therapeutic escape. Indeed, adipose microenvironment has been described to influence the efficiency of chemo- and hormonal therapies. Residual cancer stem cells could also have a key role in this process. To understand the mechanisms involved in the reduced efficacy of hormonal therapy on breast cancer cells in the presence of adipose secretome, human adipose stem cells (hMAD cell line) differentiated into mature adipocytes were co-cultured with mammary breast cancer cells and treated with hormonal therapies (tamoxifen, fulvestrant). Proliferation and apoptosis were measured (fluorescence test, impedancemetry, cytometry) and the gene expression profile was evaluated. Cancer stem cells were isolated from mammospheres made from MCF-7. The impact of chemo- and hormonal therapies and leptin was evaluated in this population. hMAD-differentiated mature adipocytes and their secretions were able to increase mammary cancer cell proliferation and to suppress the antiproliferative effect of tamoxifen, confirming previous data and validating our model. Apoptosis and cell cycle did not seem to be involved in this process. The evaluation of gene expression profiles suggested that STAT3 could be a possible target. On the contrary, leptin did not seem to be involved. The study of isolated cancer stem cells revealed that their proliferation was stimulated in the presence of anticancer therapies (tamoxifen, fulvestrant, doxorubicine) and leptin. Our study confirmed the role of adipocytes and their secretome, but above all, the role of communication between adipose and cancer cells in interfering with the efficiency of hormonal therapy. Among the pathophysiological mechanisms involved, leptin does not seem to interfere with the estrogenic pathway but seems to promote the proliferation of cancer stem cells.

Intra-monocyte Pharmacokinetics of Imiglucerase Supports a Possible Personalized Management of Gaucher Disease Type 1.

BACKGROUND AND OBJECTIVES: Intravenous imiglucerase enzyme replacement therapy for Gaucher disease type 1 administered every 2 weeks is at variance with the imiglucerase plasma half-life of a few minutes. We hypothesized that studying the pharmacokinetics of imiglucerase in blood Gaucher disease type 1 monocytes would be more relevant for understanding enzyme replacement therapy responses. METHODS: Glucocerebrosidase intra-monocyte activity was studied by flow cytometry. The pharmacokinetics of imiglucerase was analyzed using a population-pharmacokinetic model from a cohort of 31 patients with Gaucher disease type 1 who either started or were receiving long-term treatment with imiglucerase. RESULTS: A pharmacokinetic analysis of imiglucerase showed a two-compartment model with a high peak followed by a two-phase exponential decay (fast phase half-life: 0.36 days; slow phase half-life: 9.7 days) leading to a median 1.4-fold increase in glucocerebrosidase intra-monocyte activity from the pre-treatment activity (p = 0.04). In patients receiving long-term treatment, for whom the imiglucerase dose per infusion was chosen on the basis of disease aggressiveness/response, imiglucerase clearance correlated with the administered dose. However, the residual glucocerebrosidase intra-monocyte activity value was dose independent, suggesting that the maintenance of imiglucerase residual activity is patient specific. Endogenous pre-treatment glucocerebrosidase intra-monocyte activity was the most informative single parameter for distinguishing patients without (n = 10) and with a clinical indication (n = 17) for starting enzyme replacement therapy (area under the receiver operating characteristic curve: 0.912; 95% confidence interval 0.8-1; p < 0.001), as confirmed also by a factorial analysis of mixed data. CONCLUSION: This study provides novel pharmacokinetic data that support current imiglucerase administration regimens and suggests the existence of a glucocerebrosidase activity threshold related to Gaucher disease type 1 aggressiveness. These findings can potentially improve Gaucher disease type 1 management algorithms and clinical decision making.

DNA methylation profiling reveals a pathological signature that contributes to transcriptional defects of CD34+ CD15- cells in early chronic-phase chronic myeloid leukemia.

Despite the high efficiency of tyrosine kinase inhibitors (TKI), some patients with chronic myeloid leukemia (CML) will display residual disease that can become resistant to treatment, indicating intraclonal heterogeneity in chronic-phase CML (CP-CML). To determine the basis of this heterogeneity, we conducted the first exhaustive characterization of the DNA methylation pattern of sorted CP-CML CD34+ CD15- (immature) and CD34- CD15+ (mature) cells at diagnosis (prior to any treatment) and compared it to that of CD34+ CD15- and CD34- CD15+ cells isolated from healthy donors (HD). In both cell types, we identified several hundreds of differentially methylated regions (DMRs) showing DNA methylation changes between CP-CML and HD samples, with only a subset of them in common between CD34+ CD15- and CD34- CD15+ cells. This suggested DNA methylation variability within the same CML clone. We also identified 70 genes that could be aberrantly repressed upon hypermethylation and 171 genes that could be aberrantly expressed upon hypomethylation of some of these DMRs in CP-CML cells, among which 18 and 81, respectively, were in CP-CML CD34+ CD15- cells only. We then validated the DNA methylation and expression defects of selected candidate genes. Specifically, we identified GAS2, a candidate oncogene, as a new example of gene the hypomethylation of which is associated with robust overexpression in CP-CML cells. Altogether, we demonstrated that DNA methylation abnormalities exist at early stages of CML and can affect the transcriptional landscape of malignant cells. These observations could lead to the development of combination treatments with epigenetic drugs and TKI for CP-CML.