[Evaluation of an algorithm based on quantitative assays of glomerular parameters (albuminuria and urinary IgG) for proteinuria typing]. / Évaluation d'un algorithme basé sur des dosages quantitatifs de paramètres glomérulaires (albuminurie et IgG urinaires) afin de typer une protéinurie.

The first orientation test for proteinuria typing is electrophoresis. However, this technique has several drawbacks, such as delayed turnaround time and subjective readings. Some laboratories therefore use quantitative assays of glomerular markers combined with tubular markers. However, the cost of reagents and the instability of certain markers are significant drawbacks for some peripheral laboratories. The aim of this study is to evaluate the implementation of an algorithm based on parameters that can be used by all laboratories for proteinuria typing within a timeframe compatible with the urgency of the situation. Albuminuria and urinary IgG were determined on 161 urines. ROC curves were produced, using urine electrophoresis read by an expert center as the reference method. The decision thresholds used are: glomerular proteinuria is defined by a Albumin+IgGproteinsratio greater than 75.4% (100% specificity), and tubular or overload proteinuria is defined by by a Albuminproteinsratio less than 37.3% (100% sensitivity). Agreement between the results of the algorithm selected and the reference method used in our study was 88 %, with a kappa value of 0.807 (95% CI [0.729 to 0.885]). The algorithm's performance suggests that it can find its place in the diagnostic strategy for clinically significant proteinuria, despite its limited indications. It is up to each biologist to assess the value of this algorithm in relation to the recruitment, habits and needs of clinicians.

The TRIPLEX study: use of patient-derived tumor organoids as an innovative tool for precision medicine in triple-negative breast cancer.

BACKGROUND: Triple negative breast cancers (TNBC) account for approximately 15% of all breast cancers and are associated with a shorter median survival mainly due to locally advanced tumor and high risk of metastasis. The current neoadjuvant treatment for TNBC consists of a regimen of immune checkpoint blocker and chemotherapy (chemo-ICB). Despite the frequent use of this combination for TNBC treatment, moderate results are observed and its clinical benefit in TNBC remains difficult to predict. Patient-derived tumor organoids (PDTO) are 3D in vitro cellular structures obtained from patient's tumor samples. More and more evidence suggest that these models could predict the response of the tumor from which they are derived. PDTO may thus be used as a tool to predict chemo-ICB efficacy in TNBC patients. METHOD: The TRIPLEX study is a single-center observational study conducted to investigate the feasibility of generating PDTO from TNBC and to evaluate their ability to predict clinical response. PDTO will be obtained after the dissociation of biopsies and embedding into extra cellular matrix. PDTO will be cultured in a medium supplemented with growth factors and signal pathway inhibitors. Molecular and histological analyses will be performed on established PDTO lines to validate their phenotypic proximity with the original tumor. Response of PDTO to chemo-ICB will be assessed using co-cultures with autologous immune cells collected from patient blood samples. PDTO response will finally be compared with the response of the patient to evaluate the predictive potential of the model. DISCUSSION: This study will allow to assess the feasibility of using PDTO as predictive tools for the evaluation of the response of TNBC patients to treatments. In the event that PDTO could faithfully predict patient response in clinically relevant time frames, a prospective clinical trial could be designed to use PDTO to guide clinical decision. This study will also permit the establishment of a living biobank of TNBC PDTO usable for future innovative strategies evaluation. TRIAL REGISTRATION: The clinical trial (version 1.2) has been validated by local research ethic committee on December 30th 2021 and registered at ClinicalTrials.gov with the identifier NCT05404321 on June 3rd 2022, version 1.2.

Shortening identification times: comparative observational study of three early blood culture testing protocols.

Background: While early appropriate antibiotic therapy is a proven means of limiting the progression of infections, especially bacteremia, empirical antibiotic therapy in sepsis is ineffective up to 30%. The aim of this study was to compare early blood culture testing protocols in terms of their ability to shorten the delay between blood sampling and appropriate antibiotic therapy. Methods: In this french observational study, we compared three blood culture testing protocols. Positive blood cultures were tested using either GenMark ePlex panels (multiplex PCR period), a combination of MRSA/SA PCR, ß-Lacta and oxidase tests (multitest period), or conventional identification and susceptibility tests only (reference period). Conventional identification and susceptibility tests were performed in parallel for all samples, as the gold standard. Results: Among the 270 patients with positive blood cultures included, early and conventional results were in good agreement, especially for the multitest period. The delay between a blood culture positivity and initial results was 3.8 (2.9-6.9) h in the multiplex PCR period, 2.6 (1.3-4.5) h in the multitest period and 3.7 (1.8-8.2) h in the reference period (p<0.01). Antibiotic therapy was initiated or adjusted in 68 patients based on early analysis results. The proportion of patients receiving appropriate antibiotic therapy within 48 h of blood sampling was higher in the multiplex PCR and multitest periods, (respectively 90% and 88%) than in the reference period (71%). Conclusion: These results suggest rapid bacterial identification and antibiotic resistance tests are feasible, efficient and can expedite appropriate antibiotic therapy.

ORGAVADS: establishment of tumor organoids from head and neck squamous cell carcinoma to assess their response to innovative therapies.

BACKGROUND: Radiotherapy is one of the cornerstones of the treatment of Head and Neck Squamous Cell Carcinomas (HNSCC). However, radioresistance is associated with a high risk of recurrence. To propose strategies (such as combinations with drugs) that could over intrinsic radioresistance, it is crucial to predict the response to treatment. Patient-Derived Tumor Organoids (PDTO) are in vitro tridimensional microtumors obtained from patient' own cancer samples. They have been shown to serve as reliable surrogates of the tumor response in patients. METHODS: The ORGAVADS study is a multicenter observational trial conducted to investigate the feasibility of generating and testing PDTO derived from HNSCC for the evaluation of sensitivity to treatments. PDTO are obtained after dissociation of resected tumors remaining from tissues necessary for the diagnosis. Embedding of tumor cells is then performed in extracellular matrix and culture in medium supplemented with growth factors and inhibitors. Histological and immunohistochemical characterizations are performed to validate the resemblance between PDTO and their original tumor. Response of PDTO to chemotherapy, radiotherapy and innovating combinations are assessed, as well as response to immunotherapy using co-cultures of PDTO with autologous immune cells collected from patient blood samples. Transcriptomic and genetic analyses of PDTO allow validation of the models compared to patients' own tumor and identification of potential predictive biomarkers. DISCUSSION: This study is designed to develop PDTO models from HNSCC. It will allow comparing the response of PDTO to treatment and the clinical response of the patients from whom they are derived. Our aim is to study the PDTO ability to predict the clinical response to treatment for each patient in view of a personalized medicine as well as to establish a collection of HNSCC models that will be useful for future innovative strategies evaluation. TRIAL REGISTRATION: NCT04261192, registered February 7, 2020, last amendment v4 accepted on June, 2021.

Multiomic analysis of malignant pleural mesothelioma identifies molecular axes and specialized tumor profiles driving intertumor heterogeneity.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with rising incidence and challenging clinical management. Through a large series of whole-genome sequencing data, integrated with transcriptomic and epigenomic data using multiomics factor analysis, we demonstrate that the current World Health Organization classification only accounts for up to 10% of interpatient molecular differences. Instead, the MESOMICS project paves the way for a morphomolecular classification of MPM based on four dimensions: ploidy, tumor cell morphology, adaptive immune response and CpG island methylator profile. We show that these four dimensions are complementary, capture major interpatient molecular differences and are delimited by extreme phenotypes that-in the case of the interdependent tumor cell morphology and adapted immune response-reflect tumor specialization. These findings unearth the interplay between MPM functional biology and its genomic history, and provide insights into the variations observed in the clinical behavior of patients with MPM.

[Evaluation of the performance of Cepheid® GeneXpert MRSA/SA Blood Culture assay and results' extrapolation to coagulase-negative staphylococci]. / Évaluation des performances du test GeneXpert MRSA/SA Blood Culture de Cepheid® et extrapolation des résultats aux staphylocoques à coagulase négative.

In hospitalized patients, staphylococcal blood infection is common and mortality is high. Rapid diagnosis using molecular assay aims to identify the presence of Staphylococcus aureus and its resistance to methicillin as soon as the blood culture is positive. We evaluate performance of GeneXpert MRSA/SA Blood Culture assay (Cepheid®) before and after interpretation of the positivity levels of the various probes estimated by the Cycle threshold (Ct), as well as its contribution to the characterization of coagulase-negative staphylococci blood cultures not offered by the supplier. The study involved 145 samples with gram-positive cocci bacteremias. Ct analysis of the different probes revealed a few positive results with very high Ct values distants from the mean. The reclassification of these results as negative improves the specificity of the probes (spa: 100% vs. 96,8% and mec 100% vs. 91,9%) without degrading the sensitivity (spa: 98,1% vs. 100% and mec 98,6% vs. 98,6%). Then, based on an algorithm integrating the amplification results of each target, we extrapolated the results to the coagulase-negative staphylococci. In the end, reclassifying probes with extreme Ct values as negative and using the algorithm for coagulase-negative staphylococci resulted in a 97,9% (142/145) agreement between the molecular assay conclusion and conventional culture.

A rapid and inexpensive protocol to screen for third generation cephalosporin-resistant and non-fermenting Gram-negative rods directly in positive blood cultures.

Third generation cephalosporins are frequently used in the first-line treatment of Gram-negative rod (GNR) bacteremia but are unsuitable in the case of extended-spectrum-beta-lactamase-producing Enterobacterales (ESBL-E) or non-fermenting GNR infections. The aim of this study was to develop and evaluate a simple and rapid two-test protocol involving oxidase and ß-Lacta tests performed directly on positive blood culture broth as a preliminary screen for non-fermenting or third generation cephalosporins-resistant GNR. The diagnostic performance of this approach was evaluated on 294 bottles for the oxidase test and 267 bottles for the ß-Lacta Test. The sensitivity and specificity of the oxidase test were respectively 93.1% and 100%, and the sensitivity of the ß-Lacta Test for ESBL-E was 100% and the specificity 99.5%. This simple protocol, which can be implemented in all laboratories and performed in only 20 min, may be a valuable tool to optimize first-line antibiotic therapy for bacteremia.

Hypophosphatasia: a genetic-based nosology and new insights in genotype-phenotype correlation.

Hypophosphatasia (HPP) is caused by pathogenic variants in the ALPL gene. There is a large continuum in the severity, ranging from a lethal perinatal form to dental issues. We analyzed a cohort of 424 HPP patients from European geographic origin or ancestry. Using 3D modeling and results of functional tests we classified ALPL pathogenic variants according to their dominant negative effect (DNE) and their severity. The cohort was described by the genotypes resulting from alleles s (severe recessive), Sd (severe dominant), and m (moderate). Many recurrent variants showed a regional anchor pointing out founder effects rather than multiple mutational events. Homozygosity was an aggravating factor of the severity and moderate alleles were rare both in number and frequency. Pathogenic variants with DNE were found in both recessive and dominant HPP. Sixty percent of the adults tested were heterozygous for a variant showing no DNE, suggesting another mechanism of dominance like haploinsufficiency. Adults with dominant HPP without DNE were found statistically less severely affected than adults with DNE variants. Adults with dominant HPP without DNE represent a new clinical entity mostly diagnosed from 2010s, characterized by nonspecific signs of HPP and low alkaline phosphatase, and for which a high prevalence is expected. In conclusion, the genetic composition of our cohort suggests a nosology with 3 clinical forms: severe HPP is recessive and rare, moderate HPP is recessive or dominant and more common, and mild HPP, characterized by low alkaline phosphatase and unspecific clinical signs, is dominantly inherited and very common.

Redefining malignant pleural mesothelioma types as a continuum uncovers immune-vascular interactions.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive disease related to asbestos exposure, with no effective therapeutic options. METHODS: We undertook unsupervised analyses of RNA-sequencing data of 284 MPMs, with no assumption of discreteness. Using immunohistochemistry, we performed an orthogonal validation on a subset of 103 samples and a biological replication in an independent series of 77 samples. FINDINGS: A continuum of molecular profiles explained the prognosis of the disease better than any discrete model. The immune and vascular pathways were the major sources of molecular variation, with strong differences in the expression of immune checkpoints and pro-angiogenic genes; the extrema of this continuum had specific molecular profiles: a "hot" bad-prognosis profile, with high lymphocyte infiltration and high expression of immune checkpoints and pro-angiogenic genes; a "cold" bad-prognosis profile, with low lymphocyte infiltration and high expression of pro-angiogenic genes; and a "VEGFR2+/VISTA+" better-prognosis profile, with high expression of immune checkpoint VISTA and pro-angiogenic gene VEGFR2. We validated the gene expression levels at the protein level for a subset of five selected genes belonging to the immune and vascular pathways (CD8A, PDL1, VEGFR3, VEGFR2, and VISTA), in the validation series, and replicated the molecular profiles as well as their prognostic value in the replication series. INTERPRETATION: The prognosis of MPM is best explained by a continuous model, which extremes show specific expression patterns of genes involved in angiogenesis and immune response.

Functional Analysis of Somatic Mutations Affecting Receptor Tyrosine Kinase Family in Metastatic Colorectal Cancer.

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

Auricular Clyster, Otenchytes, and Pyoulcos: Precursors of the Ear Syringe.

OBJECTIVES/HYPOTHESIS: In Western medicine, the long history of the ear syringe dates back at least to the end of the 1st millennium BCE; but the corresponding Ancient Greek word surinx designates another tool. Other Greek and Latin words and phrases, in particular auricular clyster, otenchytes, and pyoulcos, were known as names of the ear syringe until modern times. The aim of this article is to study the Greek and Latin words and phrases referred to as names of the ear syringe up until modern times before syringe became the standard word. METHOD: Historical and philological review of ancient Greek and Latin medical literature dealing with the subject. RESULTS: Careful reading of ancient medical texts mentioning these tools shows a variety of shapes and uses: beside the piston-driven syringe, the system of a bladder attached to a catheter remained in use throughout Antiquity; the otenchytes, being a piston-driven syringe, obviously was not used to squirt the liquid when the remedy put inside was warmed by a flame; the piston-driven pyoulcos is most likely of greater size, and never linked with ear care in Antiquity. CONCLUSION: Latin auricular clyster and Greek otenchytes and pyoulcos, in the few ancient texts in which they occur, designate tools of a large variety of shapes and uses, significantly different from Heron's description of piston-driven pyoulcos.

What is the acceptable hemolysis index for the measurements of plasma potassium, LDH and AST?

Hemolysis is a cause of variability in test results for plasma potassium, LDH and AST and is a non-negligible part of measurement uncertainty. However, allowable levels of hemolysis provided by reagent suppliers take neither analytical variability (trueness and precision) nor the measurand into account. Using a calibration range of hemolysis, we measured the plasma concentrations of potassium, LDH and AST, and hemolysis indices with a Cobas C501 analyzer (Roche Diagnostics(®), Meylan, France). Based on the allowable total error (according to Ricós et al.) and the expanded measurement uncertainty equation we calculated the maximum allowable bias for two concentrations of each measurand. Finally, we determined the allowable hemolysis indices for all three measurands. We observed a linear relationship between the observed increases of concentration and hemolysis indices. The LDH measurement was the most sensitive to hemolysis, followed by AST and potassium measurements. The determination of the allowable hemolysis index depends on the targeted measurand, its concentration and the chosen level of requirement of allowable total error.

Erythropoietin Combined with Liposomal Amphotericin B Improves Outcome during Disseminated Aspergillosis in Mice.

Disseminated aspergillosis is responsible for a high mortality rate, despite the use of antifungal drugs. Adjuvant therapies are urgently needed to improve the outcome. The aim of this study was to demonstrate that the cytoprotective effect of erythropoietin (EPO) combined with amphotericin B can reduce the mortality rate in a murine model of disseminated aspergillosis. After infection with Aspergillus fumigatus, neutropenic mice were randomized to receive vehicle or 7.5 mg/kg liposomal amphotericin B (LAmB) or 7.5 mg/kg LAmB combined with 1000 IU/kg EPO (16 mice per group). Aspergillus galactomannan and organ cultures were performed to evaluate fungal burden at day 5. Cumulative long-term survival was analyzed at day 12 post-infection according to the Kaplan-Meier method. At day 5, fungal burden was similar between non-treated and treated groups. At day 12, mortality rates were 75, 62.5, and 31% in control group, LAmB group, and EPO/LAmB group, respectively. We observed a significant decrease in mortality using EPO/LAmB combination compared to control group (p < 0.01). LAmB single treatment did not improve the survival rate compared to control group (p = 0.155). Our results provide the first evidence that EPO improved the outcome of mice presenting with disseminated aspergillosis when combined with amphotericin B.

Establishment of an operating room committee and a training program to improve aseptic techniques for rodent and large animal surgery.

Investigators of our research facility generally accept the concept of asepsis as an important component of adequate surgical care for animals. However, they experience difficulties putting it into practice, especially in the case of rodents. The reasons for this are inconvenience, cost, and lack of training. To better assist investigators in the implementation of aseptic surgical techniques in their laboratories, we have created an Operating Room (OR) Committee modeled after OR committees found in human hospitals. A reconstructive surgeon, a veterinarian, a research scientist, a nurse involved in the training of OR personnel, interns, graduate students, and an animal health technician were chosen as committee members in light of their OR and animal care expertise. The first task of the OR Committee was to establish institutional guidelines for aseptic surgery, taking into account the costs imposed on research budgets by these procedures. The OR Committee also supports a complete training program in aseptic surgery techniques, which consists of lectures, a training manual, videos, and a practical course. Furthermore, when experimental procedures require specialized equipment, the OR Committee collaborates with researchers to develop strategies to achieve asepsis. This OR Committee and the training program proved to be important tools to promote and improve the quality of animal care during surgery.