Impact of Regorafenib on Endothelial Transdifferentiation of Glioblastoma Stem-like Cells.

Glioblastomas (GBM) are aggressive brain tumours with a poor prognosis despite heavy therapy that combines surgical resection and radio-chemotherapy. The presence of a subpopulation of GBM stem cells (GSC) contributes to tumour aggressiveness, resistance and recurrence. Moreover, GBM are characterised by abnormal, abundant vascularisation. Previous studies have shown that GSC are directly involved in new vessel formation via their transdifferentiation into tumour-derived endothelial cells (TDEC) and that irradiation (IR) potentiates the pro-angiogenic capacity of TDEC via the Tie2 signalling pathway. We therefore investigated the impact of regorafenib, a multikinase inhibitor with anti-angiogenic and anti-tumourigenic activity, on GSC and TDEC obtained from irradiated GSC (TDEC IR+) or non-irradiated GSC (TDEC). Regorafenib significantly decreases GSC neurosphere formation in vitro and inhibits tumour formation in the orthotopic xenograft model. Regorafenib also inhibits transdifferentiation by decreasing CD31 expression, CD31+ cell count, pseudotube formation in vitro and the formation of functional blood vessels in vivo of TDEC and TDEC IR+. All of these results confirm that regorafenib clearly impacts GSC tumour formation and transdifferentiation and may therefore be a promising therapeutic option in combination with chemo/radiotherapy for the treatment of highly aggressive brain tumours.

Molecular diagnosis of T-cell lymphoma: a correlative study of PCR-based T-cell clonality assessment and targeted NGS.

Immunomorphological diagnosis of T-cell lymphoma (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays detect only 80% of TCL, and clonal lymphocyte populations may also appear in nonneoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques' performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 nonneoplastic T-cell infiltrates were divided into 2 cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7%-45.5%), whereas no differences were observed in terms of sensitivity (95.1%-97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.

ALK-positive histiocytosis associated with chronic lymphocytic leukaemia/small lymphocytic lymphoma: a multitarget response under ibrutinib.

ALK-positive histiocytosis is a recently described entity with few reported cases in literature. Herein, we report an unusual case of ALK-positive histiocytosis showing an Erdheim-Chester disease (ECD)-like presentation, occurring in a 37-year-old woman with a 2-year history of chronic lymphocytic leukaemia (CLL). Our CLL patient relapsed 6 months after the end of fludarabine, cyclophosphamide and rituximab frontline therapy and complained of lower limb pains. A bone marrow biopsy was performed and showed concomitant CLL/small lymphocytic lymphoma and ALK-positive histiocytosis with an identical immunoglobulin heavy-chain gene rearrangement in both neoplasms, suggesting clonal relationship. After 4 years under ibrutinib therapy, our patient remains free of both diseases. This report extends the spectrum of composite hematolymphoid neoplasms and shows that ALK rearrangement should be considered in all histiocytosis subtypes. Moreover, both tumours eradication under ibrutinib suggests that BTK inhibitors may also be effective in histiocytic neoplasms.

Which Method for Diagnosing Small Fiber Neuropathy?

Introduction: Small fiber neuropathies (SFN) induce pain and/or autonomic symptoms. The diagnosis of SFN poses a challenge because the role of skin biopsy as a reference method and of each neurophysiological test remain to be discussed. This study compares six methods evaluating small sensory and autonomic nerve fibers: skin biopsy, Quantitative Sensory Testing (QST), quantitative sweat measurement system (Q-Sweat), Laser Evoked Potentials (LEP), Electrochemical Skin Conductance (ESC) measurement and Autonomic CardioVascular Tests (ACVT). Methods: This is a single center, retrospective study including patients tested for symptoms compatible with SFN between 2013 and 2016 using the afore-mentioned tests. Patients were ultimately classified according to the results and clinical features as "definite SFN," "possible SFN" or "no SFN." The sensitivity (Se) and specificity (Sp) of each test were calculated based on the final diagnosis and the best diagnostic strategy was then evaluated. Results: Two hundred and forty-five patients were enrolled (164 females (66.9%), age: 50.4 ± 15 years). The results are as follows: skin biopsy: Se = 58%, Sp = 91%; QST: Se = 72%, Sp = 39%; Q-Sweat: Se = 53%, Sp = 69%; LEP: Se = 66%, Sp = 89%; ESC: Se = 60%, Sp = 89%; Cardiovascular tests: Se = 15%, Sp = 99%. The combination of skin biopsy, LEP, QST and ESC has a Se of 90% and a Sp of 87%. Conclusion: Our study outlines the benefits of combining skin biopsy, ESC, LEP and QST in the diagnosis of SFN.

A 4-Year Retrospective Analysis of Salivary Gland Cytopathology Using the Milan System for Reporting Salivary Gland Cytology and Ancillary Studies.

The cytopathology of salivary glands presents major challenges due to the heterogeneity of benign and malignant neoplasms, which is reflected in the large range of WHO 2017 Classifications. Fine needle aspiration (FNA) of salivary gland tumours is still the favoured initial approach as it results in good sensitivity and specificity. The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) was published in 2018 and comprises seven categories. We report results from a 4-year retrospective analysis of 328 salivary gland FNAs which were reviewed and classified according to the MSRSGC. We assess the risk of neoplasm, the risk of malignancy and the contribution of ancillary studies to the diagnosis. Benign neoplasms were the most frequent diagnosis (44.2%). Malignant and suspicious for malignancy were identified in 11.3% and 4.9% of diagnosed cases, respectively. Histopathological analysis after surgery was available for 216 (65.8%) of the cases. All malignant cases were confirmed post-surgery, and 68.8% of suspicious for malignancy were confirmed as malignant tumours. Immunocytochemistry was informative in 72.3% of cases. Immunocytochemistry and FISH provided the definitive diagnosis in 23.7% and 33% of cases, respectively. In conclusion, the MSRSGC is more effective when specific features of neoplasms can be identified. Ancillary studies help to further characterise salivary gland tumours and thereby increase the accuracy of MSRSGC.

MIAmS: microsatellite instability detection on NGS amplicons data.

MOTIVATION: Microsatellite instability (MSI) is a molecular marker of DNA mismatch repair deficiency frequently at play in oncogenesis. MSI testing is used for diagnostic, prognostic and therapeutic purposes in several cancers. The current gold standard analysis for microsatellite status is based on length distribution analysis of multiplex-PCR generated DNA fragments from tumor samples which is a laborious and time consuming method. Next generation sequencing (NGS) using amplicon panels is an easy, accurate and scalable technique to determine both the microsatellite status and tumor genome mutations at the same time. Here, we describe MIAmS, an application designed to tag microsatellite status from amplicon NGS of tumor samples. Interestingly, this tool does not require paired normal tissue for comparison. In addition, this scalable application provides a user-friendly report for the interpretation of the results by biologists and exhibits a strong accuracy and robustness for determination of the MSI status. AVAILABILITY: https://github.com/bialimed/miams. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online, evaluation data are available at http://www.ebi.ac.uk/ena/data/view/PRJEB31725.

Ionizing radiation induces endothelial transdifferentiation of glioblastoma stem-like cells through the Tie2 signaling pathway.

Glioblastomas (GBM) are brain tumors with a poor prognosis despite treatment that combines surgical resection and radio-chemotherapy. These tumors are characterized by abundant vascularization and significant cellular heterogeneity including GBM stem-like cells (GSC) which contribute to tumor aggressiveness, resistance, and recurrence. Recent data has demonstrated that GSC are directly involved in the formation of new vessels via their transdifferentiation into Tumor Derived Endothelial Cells (TDEC). We postulate that cellular stress such as ionizing radiation (IR) could enhance the transdifferentiation of GSC into TDEC. GSC neurospheres isolated from 3 different patients were irradiated or not and were then transdifferentiated into TDEC. In fact, TDEC obtained from irradiated GSC (TDEC IR+) migrate more towards VEGF, form more pseudotubes in MatrigelTM in vitro and develop more functional blood vessels in MatrigelTM plugs implanted in Nude mice than TDEC obtained from non-irradiated GSC. Transcriptomic analysis allows us to highlight an overexpression of Tie2 in TDEC IR+. All IR-induced effects on TDEC were abolished by using a Tie2 kinase inhibitor, which confirms the role of the Tie2 signaling pathway in this process. Finally, by analyzing Tie2 expression in patient GBMs by immunohistochemistry, we demonstrated that the number of Tie2+ vessels increases in recurrent GBM compared with matched untreated tumors. In conclusion, we demonstrate that IR potentiates proangiogenic features of TDEC through the Tie2 signaling pathway, which indicates a new pathway of treatment-induced tumor adaptation. New therapeutic strategies that associate standard treatment and a Tie2 signaling pathway inhibitor should be considered for future trials.

Multicenter Evaluation of the Fully Automated PCR-Based Idylla EGFR Mutation Assay on Formalin-Fixed, Paraffin-Embedded Tissue of Human Lung Cancer.

Before initiating treatment of advanced non-small-cell lung cancer with tyrosine kinase inhibitors (eg, erlotinib, gefitinib, osimertinib, and afatinib), which inhibit the catalytic activity of epidermal growth factor receptor (EGFR), clinical guidelines require determining the EGFR mutational status for activating (EGFR exons 18, 19, 20, or 21) and resistance (EGFR exon 20) mutations. The EGFR resistance mutation T790M should be monitored at cancer progression. The Idylla EGFR Mutation Assay, performed on the Idylla molecular diagnostics platform, is a fully automated (<2.5 hours turnaround time) sample-to-result molecular test to qualitatively detect 51 EGFR oncogene point mutations, deletions, or insertions. In a 15-center evaluation, Idylla results on 449 archived formalin-fixed, paraffin-embedded tissue sections, originating from non-small-cell lung cancer biopsies and resection specimens, were compared with data obtained earlier with routine reference methods, including next-generation sequencing, Sanger sequencing, pyrosequencing, mass spectrometry, and PCR-based assays. When results were discordant, a third method of analysis was performed, when possible, to confirm test results. After confirmation testing and excluding invalids/errors and discordant results by design, a concordance of 97.6% was obtained between Idylla and routine test results. Even with <10 mm2 of tissue area, a valid Idylla result was obtained in 98.9% of the cases. The Idylla EGFR Mutation Assay enables sensitive detection of most relevant EGFR mutations in concordance with current guidelines, with minimal molecular expertise or infrastructure.

Rapid and Efficient Production of Human Functional Mast Cells through a Three-Dimensional Culture of Adipose Tissue-Derived Stromal Vascular Cells.

Mast cells (MC) are innate immune cells involved in many physiological and pathological processes. However, studies of MC function and biology are hampered by the difficulties to obtain human primary MC. To solve this problem, we established a new method to produce easily and rapidly high numbers of MC for in vitro studies using human adipose tissue, which is an abundant and easy access tissue. Stromal vascular fraction of adipose tissue, obtained from human abdominal dermolipectomy, was cultured as spheroids in serum free medium supplemented in stem cell factor. Using this method, we generated, within 3 wk, a highly pure population of connective tissue-type MC expressing MC typical peptidases (tryptase, chymase, and carboxypeptidase-A3) with a yield increasing over time. Stem cell factor was required for this culture, but unlike MC derived from CD34+ cells, this culture did not depend on IL-3 and -6. MC obtained with this method degranulated following FcεRI cross-linking or stimulation by C5a, compound 48/80, and substance P. Interestingly, activation by anti-IgE of both white adipose tissue-MC and MC obtained from peripheral blood-derived CD34+ pluripotent progenitor cells induced the production of PGs as well as proinflammatory cytokines (TNF-α, Il-6, and GM-CSF). In conclusion, we developed a new time saving and reproducible method to produce highly pure and functional human MC in 3 wk from human adipose tissue.

One year of experience using the Paris System for Reporting Urinary Cytology.

BACKGROUND: The Paris System for Reporting Urinary Cytology (TPS) was published in November 2015. It focuses on the diagnosis of high-grade urothelial carcinoma (HGUC) and provides criteria with which to define the category of atypical urothelial cells (AUC). The objective of the current study was to compare two 1-year consecutive periods before and after use of TPS. METHODS: A total of 1634 and 1814 urine cytology cases, respectively, were analyzed before and after use of TPS. Histological diagnosis within 6 months was available for 330 and 299 cases, respectively. RESULTS: After use of TPS, the authors reported significantly fewer low-grade urothelial neoplasms (0.94% vs 1.84%; P<.05) and more cases that were suspicious for HGUC (2.09% vs 0.73%; P<.01) compared with before use of TPS. For the AUC category, there was no significant change in frequency noted for before versus after TPS (6.12% vs 5.18%), whereas the rate of detection of HGUC on histology significantly increased after TPS when compared with before TPS (49.02% vs 28.17%; P<.02). For the HGUC category, neither the frequency (4.69% vs 4.47%) nor the risk of malignancy (89.39% vs 91.04% with HGUC on histology) were found to be significantly different when comparing before and after use of TPS. CONCLUSIONS: In the authors' practice, TPS helped to better characterize the categories of AUC, low-grade urothelial neoplasm, and suspicious for HGUC, which were associated with a higher risk of HGUC compared with the authors' previous classification. Cancer Cytopathol 2018;126:430-6. © 2018 American Cancer Society.

Endothelial to mesenchymal transition is common in atherosclerotic lesions and is associated with plaque instability.

Endothelial to mesenchymal transition (EndMT) plays a major role during development, and also contributes to several adult cardiovascular diseases. Importantly, mesenchymal cells including fibroblasts are prominent in atherosclerosis, with key functions including regulation of: inflammation, matrix and collagen production, and plaque structural integrity. However, little is known about the origins of atherosclerosis-associated fibroblasts. Here we show using endothelial-specific lineage-tracking that EndMT-derived fibroblast-like cells are common in atherosclerotic lesions, with EndMT-derived cells expressing a range of fibroblast-specific markers. In vitro modelling confirms that EndMT is driven by TGF-ß signalling, oxidative stress and hypoxia; all hallmarks of atherosclerosis. 'Transitioning' cells are readily detected in human plaques co-expressing endothelial and fibroblast/mesenchymal proteins, indicative of EndMT. The extent of EndMT correlates with an unstable plaque phenotype, which appears driven by altered collagen-MMP production in EndMT-derived cells. We conclude that EndMT contributes to atherosclerotic patho-biology and is associated with complex plaques that may be related to clinical events.

Targeting VEGFR1 on endothelial progenitors modulates their differentiation potential.

OBJECTIVES: We studied whether plasma levels of angiogenic factors VEGF and placental growth factor (PlGF) in coronary artery disease patients or undergoing cardiac surgery are modified, and whether those factors modulate endothelial progenitor's angiogenic potential. METHODS AND RESULTS: A total of 143 patients' plasmas from two different studies were analyzed (30 coronary artery disease patients, 30 patients with stable angina, coupled with 30 age and sex-matched controls; 53 patients underwent cardiac surgery). Among factors screened, only PlGF was found significantly increased in these pathological populations. PlGF-1 and PlGF-2 were then tested on human endothelial-colony-forming cells (ECFCs). We found that PlGF-1 and PlGF-2 induce VEGFR1 phosphorylation and potentiate ECFCs tubulogenesis in vitro. ECFCs VEGFR1 was further inhibited using a specific small interfering RNA (siRNA) and the chemical compound 4321. We then observed that the VEGFR1-siRNA and the compound 4321 decrease ECFCs tubulogenesis potential in vitro. Finally, we tested the compound 4321 in the preclinical Matrigel(®)-plug model with C57Bl/6J mice as well as in the murine hindlimb ischemia model. We found that 4321 inhibited the plug vascularization, attested by the hemoglobin content and the VE-Cadherin expression level and that 4321 inhibited the post-ischemic revascularization. CONCLUSION: PlGF plasma levels were found increased in cardiovascular patients. Disrupting PlGF/VEGFR1 pathway could modulate ECFC-induced tubulogenesis, the cell type responsible for newly formed vessels in vivo.

Coronary artery calcification is inversely related to body morphology in patients with significant coronary artery disease: a three-dimensional intravascular ultrasound study.

AIMS: Emerging data have indicated unexpected complexity in the regulation of vascular and bone calcification. In particular, several recent studies have challenged the concept of a universally positive relationship between body morphology [weight, height, body mass index (BMI), body surface area (BSA)] and the extent of vascular calcification. We sought to clarify these discrepancies and investigated the relationship between index lesion coronary artery calcification (CAC) and body morphology in patients undergoing percutaneous coronary intervention (PCI) using three-dimensional intravascular ultrasound (IVUS). METHODS AND RESULTS: We analysed CAC in patients who underwent PCI with pre-intervention IVUS imaging. The main outcome measure was the calcium index (CalcIndex); a three-dimensional IVUS-derived measure of total calcification per obstructive coronary lesion. A total of 346 patients (65.3 ± 10.6 years; 29.5% females) underwent PCI with IVUS-based CAC assessment. CalcIndex was categorized as zero-low (0-0.1399; n = 152) or intermediate-high (0.1400-1.2541; n = 194). All measures of body morphology were lower in patients with intermediate-high CalcIndex (height, P = 0.024; weight, P = 0.008; BMI, P = 0.064; BSA, P = 0.005). In adjusted multivariable models, weight and BSA were independent inverse predictors of intermediate-high CalcIndex [weight: odds ratio (OR) 0.986, P = 0.017; BSA: OR 0.323, P = 0.012] while CalcIndex also trended towards an inverse association with both height (P = 0.068) and BMI (P = 0.064). These independent inverse associations were consistent across multiple clinical subgroups, including stratification by age, race, gender, diabetes, and renal impairment. CONCLUSION: Using three-dimensional IVUS to assess vascular calcification, these data confirm an independent, inverse relationship between body size and index lesion CAC in patients with obstructive coronary artery disease.

Inverse relationship between body mass index and coronary artery calcification in patients with clinically significant coronary lesions.

AIMS: Mounting data support a 'calcification paradox', whereby reduced bone mineral density is associated with increased vascular calcification. Furthermore, reduced bone mineral density is prevalent in older persons with lower body mass index (BMI). Therefore, although BMI and coronary artery calcification (CAC) exhibit a positive relationship in younger persons, it is predicted that in older persons and/or those at risk for osteoporosis, an inverse relationship between BMI and CAC may apply. We sought to explore this hypothesis in a large group of patients with coronary artery disease undergoing percutaneous coronary intervention (PCI). METHODS AND RESULTS: We accessed our single-center registry for 07/01/1999 to 06/30/2009, extracting data on all patients that underwent PCI. To minimize bias we excluded those at the extremes of age or BMI and non-Black/Hispanic/Caucasians, leaving 9993 study subjects (age 66.6±9.9 years). Index lesion calcification (ILC) was analyzed with respect to BMI. Comparing index lesions with no angiographic calcification to those with the most severe, mean BMI decreased by 1.11 kgm(-2); a reduction of 3.9% (P<0.0001). By multivariable modeling, BMI was an independent inverse predictor of moderate-severe ILC (m-sILC; odds ratio [OR] 0.967, 95% CI 0.953-0.980, P<0.0001). Additional fully adjusted models identified that, compared to those with normal BMI, obese patients had an OR of 0.702 for m-sILC (95% CI 0.596-0.827, P<0.0001). CONCLUSIONS: In a large group of PCI patients, we identified an inverse correlation between BMI and index lesion calcification. These associations are consistent with established paradigms and suggest a complex interrelationship between BMI, body size and vascular calcification.