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Supplementary Table 1: IC20 and IC50 values of ARQ 092 and sorafenib in Hep3B, Huh-7, HepG2, PLC/PRF/5 and HR4 cell lines after 48h of exposure; Supplementary Table 2: List of significantly different phospho-kinases from ARQ 092 treated rats compared to control group.

International audience; No abstract available

Aza-boron-dipyrromethenes (Aza-BODIPYs) are an increasingly studied class of fluorophores. They can be seen as an azadipyrromethene ("aza-DIPY") ligand rigidified by a metalloid, a boron atom. Based on this idea, a series of complexes of group 13 metals (aluminum and gallium) have been synthesized and characterized. The impact of the metal and of the nature of the substituents of aza-DIPY core were investigated. The photophysical and electrochemical properties were determined, and an X-ray structure of an azaGaDIPY was obtained. These data reveal that azaGaDIPY and azaAlDIPY exhibit significant red-shifted fluorescence compared to their analogue aza-BODIPY. Their emission can go up to 800 nm for the maximum emission length and up to NIR-II for the emission tail. This, associated with their electrochemical stability (no metal release whether oxidized or reduced) makes them a promising class of fluorophores for optical medical imaging. Moreover, X-ray structure and molecular modeling studies have shown that this redshift seems to be more due to the geometry around the boron/metal than to the nature of the metal.

Aza-BODIPYs are an increasingly studied class of fluorophores. They can be seen as an "aza-DIPY" ligand rigidified by a metalloid, a boron atom. Based on this idea, a series of complexes of group 13 metals (aluminum and gallium) have been synthesized and characterized. Impact of the metal and of the nature of the substituents of aza-DIPY core were investigated. The photophysical and electrochemical properties were determined and an X-ray structure of an azaGaDIPY was obtained. These data reveal that azaGaDIPY and azaAlDIPY exhibit significant red-shifted fluorescence compared to their analogue aza-BODIPY. Their emission can go up to 800 nm for the maximum emission length and up to NIR-II for the emission tail. This, associated with their electrochemical stability (no metal release whether oxidized or reduced) make them a promising class of fluorophores for optical medical imaging. Moreover, X-ray structure and molecular modeling studies have shown that this red shift seems to be more due to the geometry around the boron/metal than to the nature of the metal.

Following intravenous administration, the interaction of fluorescent exogenous molecules with circulating endogenous transporters can influence their photophysical properties as well as their fate and distribution, and possibly their recognition by different cell types. This type of interaction can be used to optimize the drug delivery but also the imaging properties of a compound of interest. In this study, we investigated the behavior of SWIR-WAZABY-01 fluorophore, a water-soluble aza-BODIPY dye emitting in the NIR-II region, both

Zinc (Zn) is a trace element crucial for oxidative stress, apoptosis, the immune response, and more globally for various processes involved in cellular homeostasis. In some cancers, Zn homeostasis is dysregulated. In this review, the role of Zn in cancer and all the components associated to Zn, the use of Zn and Zn -related proteins as biomarkers and Zn-based strategies for the treatment of tumors will be described. ZIP and ZnT are proteins related to Zn metabolism in normal conditions. In cancer, the level of expression of Zn related proteins is abnormal. These Zn proteins may act as prognostic or diagnostic biomarkers, and may be helpful for detecting early-stage cancers or monitoring the course of the disease. Additionally, Zn and its pathways may also be targeted to treat cancers. Indeed, the use of metals for binding Zn cations allows to regulate the biodistribution of Zn within cells, and will control several downstream signaling pathways. Zinc may also be directly used as a therapeutic substance to improve the prognosis of cancer patients, especially with the supplementation of zinc or the use of Zn oxide nanoparticles.

Multi-elemental imaging of soft tissues using Laser-induced breakdown spectroscopy, also known as LIBS, allows for the direct visualization of the distribution of endogenous or exogenous elements within tissues. LIBS was used to image the kinetics of metal nanoparticles in elimination organs, but also the physiological distribution of biological elements in situ and the topography of chemicals or metals in exposed human tissues. Based on our experience and recent literature in the field of imaging the elemental content of animal and human specimens, this review describes the principle and characteristics of the instrument, technical considerations for setting up experiments with LIBS, its advantages, limitations and possibilities for pre-clinical and medical applications., L’imagerie multi-élémentaire des tissus biologiques grâce à la spectroscopie LIBS (laser-induced breakdown spectroscopy) permet la visualisation directe de la distribution des éléments endogènes ou exogènes. Cette technologie est notamment utilisée pour mettre en image la cinétique des nanoparticules métalliques dans les organes d’élimination, mais également pour analyser la distribution physiologique des éléments biologiques in situ, et pour révéler la topographie des éléments chimiques, comme les métaux, dans des tissus humains qui ont été exposés à des agents extérieurs potentiellement toxiques. Fondée sur notre expérience et les travaux les plus récents dans le domaine de l’imagerie du contenu élémentaire des tissus animaux et humains, cette revue décrit les principes et les caractéristiques de l’instrumentation LIBS, présente certains aspects techniques pour mettre en place des expériences avec cet outil, montre les avantages et les limites de cette technologie, et s’intéresse aux différentes possibilités qu’offre l’imagerie LIBS pour des applications précliniques et médicales.

Biological tissues contain various metals and metalloids ions with central role in the regulation of several pathophysiological functions. In parallel, the development and the evaluation of novel nanocompounds for biomedicine require the monitoring of their biodistribution in tissues of interest. Therefore, researchers need to use reliable and accessible techniques to detect and quantify major and trace elements in space-resolved manner. In this communication, we report how Laser-Induced Breakdown Spectroscopy (LIBS) can be used to image the distribution of chemical elements in brain tissues.

International audience; Boron neutron capture therapy (BNCT) has the potential to specifically destroy tumor cells without damaging the tissues infiltrated by the tumor. BNCT is a binary treatment method based on the combination of two agents that have no effect when applied individually: 10B and thermal neutrons. Exclusively, the combination of both produces an effect, whose extent depends on the amount of 10B in the tumor but also on the organs at risk. It is not yet possible to determine the 10B concentration in a specific tissue using non-invasive methods. At present, it is only possible to measure the 10B concentration in blood and to estimate the boron concentration in tissues based on the assumption that there is a fixed uptake of 10B from the blood into tissues. On this imprecise assumption, BNCT can hardly be developed further. A therapeutic approach, combining the boron carrier for therapeutic purposes with an imaging tool, might allow us to determine the 10B concentration in a specific tissue using a non-invasive method. This review provides an overview of the current clinical protocols and preclinical experiments and results on how innovative drug development for boron delivery systems can also incorporate concurrent imaging. The last section focuses on the importance of proteomics for further optimization of BNCT, a highly precise and personalized therapeutic approach.

Simple Summary Iron is the more abundant metal ion in humans. It is essential for life as it has a role in various cellular processes involved, for instance, in cell metabolism and DNA synthesis. These functions are crucial for cell proliferation, and it is therefore not surprising that iron is accumulated in tumors. In this review, we describe normal and altered iron homeostasis mechanisms. We also provide a vision of iron-related proteins with altered expression in cancers and discuss their potential as diagnostic and/or prognostic biomarkers. Finally, we give an overview of therapeutic strategies acting on iron metabolism to fight against cancers. Abstract Iron (Fe) is a trace element that plays essential roles in various biological processes such as DNA synthesis and repair, as well as cellular energy production and oxygen transport, and it is currently widely recognized that iron homeostasis is dysregulated in many cancers. Indeed, several iron homeostasis proteins may be responsible for malignant tumor initiation, proliferation, and for the metastatic spread of tumors. A large number of studies demonstrated the potential clinical value of utilizing these deregulated proteins as prognostic and/or predictive biomarkers of malignancy and/or response to anticancer treatments. Additionally, the iron present in cancer cells and the importance of iron in ferroptosis cell death signaling pathways prompted the development of therapeutic strategies against advanced stage or resistant cancers. In this review, we select relevant and promising studies in the field of iron metabolism in cancer research and clinical oncology. Besides this, we discuss some co-existing discrepant findings. We also present and discuss the latest lines of research related to targeting iron, or its regulatory pathways, as potential promising anticancer strategies for human therapy. Iron chelators, such as deferoxamine or iron-oxide-based nanoparticles, which are already tested in clinical trials, alone or in combination with chemotherapy, are also reported.

Simple Summary Copper is an essential element for human life. However, its redox activity can be detrimental for the cell that developed highly coordinated pathways to chelate and traffic copper through the cell or the organism. Owing to its important role in functions essential for cell growth and metabolism, copper concentrations are frequently dysregulated in tumors. In this review, we describe normal and cancer-altered copper homeostasis mechanisms. Moreover, on the basis of this knowledge, we expose not only copper-related diagnostic and prognostic markers for oncology but also therapeutic strategies to act on copper homeostasis to fight against cancer. Abstract In the human body, copper (Cu) is a major and essential player in a large number of cellular mechanisms and signaling pathways. The involvement of Cu in oxidation–reduction reactions requires close regulation of copper metabolism in order to avoid toxic effects. In many types of cancer, variations in copper protein levels have been demonstrated. These variations result in increased concentrations of intratumoral Cu and alterations in the systemic distribution of copper. Such alterations in Cu homeostasis may promote tumor growth or invasiveness or may even confer resistance to treatments. Once characterized, the dysregulated Cu metabolism is pinpointing several promising biomarkers for clinical use with prognostic or predictive capabilities. The altered Cu metabolism in cancer cells and the different responses of tumor cells to Cu are strongly supporting the development of treatments to disrupt, deplete, or increase Cu levels in tumors. The metallic nature of Cu as a chemical element is key for the development of anticancer agents via the synthesis of nanoparticles or copper-based complexes with antineoplastic properties for therapy. Finally, some of these new therapeutic strategies such as chelators or ionophores have shown promising results in a preclinical setting, and others are already in the clinic.

Advanced science xx, 2001675 (1-18) (2020). doi:10.1002/advs.202001675, To improve the prognosis of glioblastoma, innovative radiotherapy regimens are required to augment the effect of tolerable radiation doses while sparing surrounding tissues. In this context, nanoscintillators are emerging radiotherapeutics that down‐convert X‐rays into photons with energies ranging from UV to near‐infrared. During radiotherapy, these scintillating properties amplify radiation‐induced damage by UV‐C emission or photodynamic effects. Additionally, nanoscintillators that contain high‐Z elements are likely to induce another, currently unexplored effect: radiation dose‐enhancement. This phenomenon stems from a higher photoelectric absorption of orthovoltage X‐rays by high‐Z elements compared to tissues, resulting in increased production of tissue‐damaging photo‐ and Auger electrons. In this study, Geant4 simulations reveal that rare‐earth composite LaF$_3$:Ce nanoscintillators effectively generate photo‐ and Auger‐electrons upon orthovoltage X‐rays. 3D spatially resolved X‐ray fluorescence microtomography shows that LaF$_3$:Ce highly concentrates in microtumors and enhances radiotherapy in an X‐ray energy‐dependent manner. In an aggressive syngeneic model of orthotopic glioblastoma, intracerebral injection of LaF$_3$:Ce is well tolerated and achieves complete tumor remission in 15% of the subjects receiving monochromatic synchrotron radiotherapy. This study provides unequivocal evidence for radiation dose‐enhancement by nanoscintillators, eliciting a prominent radiotherapeutic effect. Altogether, nanoscintillators have invaluable properties for enhancing the focal damage of radiotherapy in glioblastoma and other radioresistant cancers., Published by Wiley-VCH, Weinheim

Boron neutron capture therapy (BNCT) is a radiotherapeutic modality based on the nuclear capture of slow neutrons by stable 10B atoms followed by charged particle emission that inducing extensive damage on a very localized level (&lt, 10 &mu, m). To be efficient, a sufficient amount of 10B should accumulate in the tumor area while being almost cleared from the normal surroundings. A water-soluble aza-boron-dipyrromethene dyes (BODIPY) fluorophore was reported to strongly accumulate in the tumor area with high and BNCT compatible Tumor/Healthy Tissue ratios. The clinically used 10B-BSH (sodium borocaptate) was coupled to the water-soluble aza-BODIPY platform for enhanced 10B-BSH tumor vectorization. We demonstrated a strong uptake of the compound in tumor cells and determined its biodistribution in mice-bearing tumors. A model of chorioallantoic membrane-bearing glioblastoma xenograft was developed to evidence the BNCT potential of such compound, by subjecting it to slow neutrons. We demonstrated the tumor accumulation of the compound in real-time using optical imaging and ex vivo using elemental imaging based on laser-induced breakdown spectroscopy. The tumor growth was significantly reduced as compared to BNCT with 10B-BSH. Altogether, the fluorescent aza-BODIPY/10B-BSH compound is able to vectorize and image the 10B-BSH in the tumor area, increasing its theranostic potential for efficient approach of BNCT.

International audience; L’étude de la dérégulation des métaux au sein des tissus biologiques intéresse de plus en plus le monde médical car une répartition anormale des métaux est une information importante qui peut contribuer au diagnostic clinique, et orienter le choix du traitement pour de nombreuses pathologies. En tant que méthode d’analyse élémentaire « tout optique », l’imagerie LIBS (laser-induced breakdown spectroscopy) est un candidat idéal pour étudier la composition élémentaire des tissus humains.

International audience; A simple NIR-II emitting water-soluble system has been developed and applied in vitro and in vivo. In vitro, the fluorophore quickly accumulated in 2D and 3D cell cultures and rapidly reached the tumor in rodents, showing high NIR-II contrast for up to 1 week. This very efficient probe possesses all the qualities necessary for translation to the clinic as well as for the development of NIR-II emitting materials.

Objective We synthesized new tamoxifen derivatives as anticancer drug candidates and elaborated on convection-enhanced delivery (CED) as a strategy for delivery. Methods To overcome the issue of their poor solubility, these ferrocenyl-tamoxifen derivatives were esterified and encapsulated into different nanocarriers, that is lipid (LNC) and polymeric nanocapsules (PNL-NC). We describe the chemistry, the encapsulation and the physicochemical characterization of these formulations. Key findings Starting compounds [phthalimido-ferrocidiphenol and succinimido-ferrocidiphenol], esterified prodrugs and their nanocapsules formulations were characterized. These drug candidates displayed a strong in vitro activity against breast and glioblastoma cancer cells. The ester prodrugs were toxic for glioblastoma cells (IC50 = 9.2 × 10−2 μm and 6.7 × 10−2 μm, respectively). The IC50 values for breast cancer cells were higher for these compounds. The encapsulation of the esterified compounds in LNCs (≈50 nm) or PCL-NCs (≈300 nm) did not prevent their efficacy on glioblastoma cells. These anticancer effects were due to both blockade in the S-phase of the cell cycle and apoptosis. Moreover, the tamoxifen derivatives-loaded nanocapsules induced no toxicity for healthy astrocytes and showed no haemolytic properties. Loaded Lipid Nanocapsules (LNCs) presented interesting profiles for the optimal delivery of active compounds. Conclusions Phthalimido- and Succinimido-esters represent an innovative approach to treat cancers with cerebral localizations such as glioblastoma or brain metastases from breast cancers.

International audience; Many Receptor Tyrosine Kinases translocate from the cell surface to the nucleus in normal and pathological conditions, including cancer. Here we report the nuclear expression of insulin-like growth factor-1 receptor (IGF1R) in primary human lung tumours. Using lung cancer cell lines and lung tumour xeno-grafts, we demonstrate that the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) gefitinib induces the nuclear accumulation of IGF1R in mucinous lung adenocarcinoma by a mechanism involving the intracellular re-localization of the growth factor amphiregulin. Amphiregulin allows the binding of IGF1R to importin-b1 and promotes its nuclear transport. The nuclear accumulation of IGF1R by amphiregulin induces cell cycle arrest through p21 WAF1/CIP1 upregulation, and prevents the induction of apoptosis in response to gefitinib. These results identify amphiregulin as the first nuclear localization signal-containing protein that interacts with IGF1R and allows its nuclear translocation. Furthermore they indicate that nuclear expression of IGF1R contributes to EGFR-TKI resistance in lung cancer.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality worldwide. The AKT pathway has been found activated in 50% of HCC cases, making it a promising target. Therefore, we assess efficacy of the allosteric AKT inhibitor ARQ 092 compared with untreated control and standard treatment, sorafenib, in vitro and in vivo. ARQ 092 blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than sorafenib. Similarly, apoptosis and cell migration were strongly reduced by ARQ 092 in vitro. To mimic human advanced HCC, we used a diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that ARQ 092 significantly reduced overall tumor size. Furthermore, number of tumors was decreased by ARQ 092, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the ARQ 092 group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in the surrounding liver of animals treated with ARQ 092. Finally, pAKT/AKT levels in ARQ 092–treated tumors were reduced, followed by downregulation of actors of AKT downstream signaling pathway: pmTOR, pPRAS40, pPLCγ1, and pS6K1. In conclusion, we demonstrated that ARQ 092 blocks AKT phosphorylation in vitro and in vivo. In the HCC-rat model, ARQ 092 was well tolerated, showed antifibrotic effect, and had stronger antitumor effect than sorafenib. Our results confirm the importance of targeting AKT in HCC. Mol Cancer Ther; 16(10); 2157–65. ©2017 AACR.

Celine Bouclier and Matthieu Simon contributed equally ; Amandine Hurbin and May C. Morris contributed equally.; International audience; CDK4/cyclin D kinase constitutes an attractive pharmacological target for development of anticancer therapeutics, in particular in KRAS-mutant lung cancer patients, who have a poor prognosis and no targeted therapy available yet. Although several ATP-competitive inhibitors of CDK4 have been developed for anticancer therapeutics, they suffer from limited specificity and efficacy. Methods: As an alternative to ATP-competitive inhibitors we have designed a stapled peptide to target the main interface between CDK4 and cyclin D, and have characterized its physico-chemical properties and affinity to bind cyclin D1. Results: We have validated a positive correlation between CDK4/cyclin D level and KRAS mutation in lung cancer patients. The stapled peptide enters cells rapidly and efficiently, and inhibits CDK4 kinase activity and proliferation in lung cancer cells. Its intrapulmonary administration in mice enables its retention in orthotopic lung tumours and complete inhibition of their growth when co-administered with Abemaciclib. Conclusion: The stapled peptide targeting the main interface between CDK4 and cyclin D provides promising therapeutic perspectives for patients with lung cancer.

In recent years, important developments have been achieved in the application of laser-induced breakdown spectroscopy (LIBS) for elemental imaging. The aim of this chapter is to report recent instrumental configurations and applications related to LIBS-based imaging. In the first section, different instrumental alternatives for LIBS imaging measurements are presented. The second section reports the wide variety of laboratory applications in geological, industrial, and biomedical fields that have benefited from LIBS mapping techniques.

This review is the second part of the workshop on digital PCR (dPCR) proposed by the working group of the French society of clinical biology. The first part of the paper discusses the advantages and limitations of dPCR for the search of different molecular abnormalities such as point mutations, copy number variants, DNA methylation, RNA analysis and a more innovative application, the single-cell dPCR. This synthesis makes it possible to propose a positioning of the dPCR compared to the other available technologies in a medical laboratory. In a second part, the main current applications of the dPCR will be addressed including the oncology of solid tumors and liquid biopsies, oncohematology and the follow-up of hemopathies treatments by hematopoietic stem cell transplantation. We will also detail non-invasive prenatal diagnosis and diagnosis of mosaic genetic disease, using the example of McCune-Albright syndrome. Several French specialists in the field who have implemented these techniques in their laboratory have written these different examples of applications jointly. In summary, this manuscript offers an up-to-date view of the positioning of dPCR in relation to other existing technologies in order to best meet the expectations of precision medicine.

International audience; Standard chemotherapies that interfere with microtubule dynamics are a chemotherapeutic option used for the patients with advanced malignancies that invariably relapse after targeted therapies. However, major efforts are needed to reduce their toxicity, optimize their efficacy, and reduce cancer chemoresistance to these agents. We previously identified a pyrrolo[2,3d]pyrimidine-based microtubule-depolymerizing agent (PP-13) that binds to the colchicine site of β-tubulin and exhibits anticancer properties in solid human cancer cells, including che-moresistant subtypes. Here, we investigated the therapeutic potential of PP-13 in vitro and in vivo. PP-13 induced a mitotic blockade and apoptosis in several cancer cells cultured in two-dimensions or three-dimensions spheroids, in conjunction with reduced cell proliferation. Capillary-like tube formation assays using HUVECs showed that PP-13 displayed antiangiogenic properties. It also inhibited cancer cell motility and invasion, in in vitro wound-healing and transwell migration assays. Low concentration PP-13 (130 nmol.L −1) treatment significantly reduced the metastatic invasiveness of human cancer cells engrafts on chicken chorioallantoic membrane. In nude mice, 0.5 or 1 mg.kg −1 PP-13 intraperitoneally administered three-times a week reduced the sizes of paclitaxel-refractory orthotopic breast tumors, delayed the progression of metastasis, and decreased the global metastatic load compared to 0.5 mg.kg −1 paclitaxel or vehicle alone. PP-13 did not show any apparent early adverse effect in vivo. These data suggest that PP-13 is a promising alternative to standard chemotherapy in antimitotic drug-refractory tumors, especially through its impact on metastasis.

International audience

Introduction L’exposition professionnelle et/ou environnementale a des poussieres minerales inhalees peut favoriser des pathologies comme la sarcoidose, les pneumopathies infiltratives associees a des connectivites et des fibroses. Ces pathologies sont cependant le plus souvent considerees comme idiopathiques, or parmi elles, un certain nombre sont d’origine environnementale. Par manque de temps et de moyens techniques et humains, l’etiologie de ces pathologies respiratoires idiopathiques n’est pas recherchee. Nous cherchons a caracteriser la nature des (nano)-particules contenues dans divers echantillons pulmonaires de ces patients. Methodes Des patients souffrant de pathologies pulmonaires etiquetees idiopathiques ont ete inclus apres avoir signe un formulaire de consentement eclaire. Les echantillons biopsiques preleves dans le cadre du soin quotidien inclus en paraffine ou FFPE ont ete analyses par imagerie multi-elementaire LIBS ( laser-induced breakdown spectroscopy ) couplee a une analyse mineralogique MEB-EDX (microscopie electronique associee a la microanalyse par energie dispersive de rayons X). Pour chaque echantillon, nous avons recherche la presence d’elements tels Fe, Si, Ti, Cu, Al, Cr, Mn, etc. Resultats Nous avons retrouve des accumulations de metaux exogenes tels Cr, Si, et Ti dans plusieurs echantillons. La presence d’images histologiques realisees sur sections adjacentes permet de localiser precisement ces elements dans des zones granulomateuses, inflammatoires, et meme dans du parenchyme pulmonaire « histologiquement normal ». Ces resultats ont ete confirmes par l’analyse de la composition et de la taille des particules en MEB-EDX. Dans certains cas, la nature des elements exogenes retrouves par les analyses mineralogiques etait en accord avec les expositions professionnelles/environnementales collectees lors des interrogatoires. Conclusion Nous avons developpe une approche technologique innovante qui permet de connaitre la composition elementaire des (nano) particules presentes dans le tissu pulmonaire, ou dans tout autres tissu. L’analyse LIBS permet d’identifier et localiser rapidement la quasi-totalite des elements chimiques au sein des tissus, avec une bonne sensibilite (∼ppm). Les images multi-elementaires LIBS sont superposables avec les images histologiques de microscopie optique utilisee par les anatomo-pathologistes. Il existe une bonne complementarite avec l’analyse mineralogique conventionnelle effectuee par MEB-EDX. L’analyse LIBS couplee a l’analyse MEB-EDX permet d’objectiver les suspicions d’empoussierement professionnel de patients exposes mais aussi de reveler et orienter la recherche d’etiologie de certaines pathologies idiopathiques.

Introduction There is a major health problem due to the occupational related exposures to mineral particles, metals, and dust. Pathologists never report the presence of a possible metal-related aetiology in a specimen by lack of available technology. Therefore, several respiratory diseases are considered idiopathic. We recently developed an all-optical method, fully compatible with standard microscopy systems, for multi-elemental imaging of biological tissues. Methods Our instrument is based on Laser Induced Breakdown Spectroscopy ( LIBS ) and allows the in situ imaging and quantification of the elements of the periodic table within biological tissues. A laser is focused on the sample surface. Elemental images (maps) are obtained by scanning the surface of the specimen. Spectrometers collect the signal of various elements such as Fe-Ca-Na-P-Mg-Zn-Al-Mn-Co-Si-Cr-Ti and Cu in the tissue. Results We identified and mapped the presence of high levels of Si and Al, but also Ti, or Cr in lung and mediastinal lymph nodes biopsies from exposed workers suffering from sarcoidosis or idiopathic pulmonary fibrosis. We found different elements including Lithium in the lymph node of a sarcoidosis patient working in a lithium-battery factory. We were also able to image the presence of Beryllium in the lungs of a patient with Chronic Beryllium Disease. As a matter of fact, Lithium and Beryllium are light-weight elements not detectable by any other elemental imaging technique. We will describe a panel of recent results obtained with LIBS, and confirmed with electron microscopy, focusing on respiratory diseases. Discussion This laser spectrometry technique is versatile because almost any element can be imaged with high sensitivity. These results demonstrate the strong potential of this disruptive technology as a complementary tool for routine classical in-situ pathology examination of human tissues embedded in paraffin , especially for idiopathic respiratory diseases related to occupational or environmental exposure to metals, dust, or particles.

International audience; Biological tissues contain various metal and metalloid ions that play different roles in the structure and function of proteins and are therefore indispensable to several vital biochemical processes. In this review, we discuss the broad capability of laser-induced breakdown spectroscopy (LIBS) for in situ elemental profiling and mapping of metals in biological materials such as plant, animal and human specimens. These biological samples contain or accumulate metal species and metal-containing compounds that can be detected, quantified, and imaged. LIBS enables performing microanalysis, mapping and depth profiling of endogenous and exogenous elements contained in the tissues with a parts-per-million scale sensitivity and microscopic resolution. In addition, this technology generally requires minimal sample preparation. Moreover, its tabletop instrumentation is compatible with optical microscopy and most elements from the periodic table. Specifically, low- and high-atomic-number elements can be detected simultaneously. Recent advances in space-resolved LIBS are reviewed with various examples from vegetable, animal and human specimens. Overall, the performance offered by this new technology along with its ease of operation suggest innumerable applications in biology, such as for the preclinical evaluation of metal-based nanoparticles and in medicine, where it could broaden the horizons of medical diagnostics for all pathologies involving metals.

International audience; Pathologists typically encounter many disparate exogenous materials in clinical specimens during their routine histopathological examinations, especially within the skin, lymph nodes, and lungs. These foreign substances may be free extracellular deposits or induce several clinical abnormalities or histopathological patterns. However, pathologists almost never investigate or report the chemical nature of exogenous metals in clinical specimens due to a lack of convenient and available technologies. In this paper, a novel strategy based on laser-induced breakdown spectroscopy (LIBS) technology is evaluated for in situ multi-elemental tissue imaging. The improved procedures allow visualization of the presence of chemical elements contained within paraffin-embedded specimens of medical interest with elemental images that are stackable with conventional histology images. We selected relevant medical situations for which the associated pathology reports were limited to the presence of lymphohistiocytic and inflammatory cells containing granules (a granuloma and a pseudolymphoma) or to lymph nodes or skin tissues containing pigments or foreign substances. Exogenous elements such as aluminum, titanium, copper, and tungsten were identified and localized within the tissues. The all-optical LIBS elemental imaging instrument that we developed is fully compatible with conventional optical microscopy used for pathology analysis. When combined with routine histopathological analysis, LIBS is a versatile technology that might help pathologists establish or confirm diagnoses for a wide range of medical applications, particularly when the nature of external agents present in tissues needs to be investigated.

International audience; Chemical elements play central roles for physiological homeostasis in human cells, and their dysregulation might lead to a certain number of pathologies. Novel imaging techniques that improve the work of pathologists for tissue analysis and diagnostics are continuously sought. We report the use of Laser-Induced Breakdown Spectroscopy (LIBS) to perform multi-elemental images of human paraffin-embedded skin samples on the entire biopsy scale in a complementary and compatible way with microscope histopathological examination. A specific instrumental configuration is proposed in order to detect most of the elements of medical interest (i.e. P, Al, Mg, Na, Zn, Si, Fe, and Cu). As an example of medical application, we selected and analysed skin biopsies, including healthy skin tissue, cutaneous metastasis of melanoma, Merkel-cell carcinoma and squamous cell carcinoma. Clear distinctions in the distribution of chemical elements are observed from the different samples investigated. This study demonstrates the high complementarity of LIBS elemental imaging with conventional histopathology, opening new opportunities for any medical application involving metals.

Resume Contexte Environ un melanome sur deux presente une mutation sur le codon 600 du gene BRAF. Les patients dont la tumeur est mutee V600 ont une reponse aux traitements par des inhibiteurs de BRAF (vemurafenib ou dabrafenib) le plus souvent rapide et spectaculaire, avec un gain significatif en termes de survie sans progression ou en survie globale. Cependant, s�il existe de rares patients longs repondeurs aux agents anti-BRAF en monotherapie, une resistance au traitement se developpe dans la plupart des cas dans un delai median de 6 mois. Objectifs Il est essentiel de comprendre les mecanismes de resistance aux therapies anti-BRAF afin d�optimiser leur efficacite et d�ameliorer les taux de reponse, ainsi que la duree du benefice clinique. Cette revue de la litterature decrit les voies de signalisation impliquant BRAF. Les principaux mecanismes de resistance a ces traitements identifies a ce jour sont decrits, ainsi que les strategies therapeutiques alternatives potentielles. Methodes Les mots cles (resistance, BRAF, melanoma, targeted therapy, vemurafenib, et dabrafenib) renseignes dans la base de donnees Pubmed/Medline ont permis de selectionner les principales references scientifiques utiles a l�elaboration de ce travail. Cette selection a pris fin le 31 janvier 2014. Discussion Le decryptage et la meilleure comprehension des mecanismes de resistance aux inhibiteurs de BRAF devraient permettre le developpement de nouvelles strategies therapeutiques destinees a contourner ces resistances et ainsi a ameliorer les taux et les durees de reponse. Summary Context In patients with melanoma positive for the BRAF V600 mutation, clinical response to specific BRAF inhibitors is usually rapid and striking, with significant benefits in terms of progression-free survival and overall survival. However, resistance to treatment almost invariably arises, typically within a median timeframe of 6 months. Indeed, very few patients exhibit long-lasting response to these targeted therapies. Aims It is essential to better understand the mechanisms of resistance to targeted anti-BRAF therapies in order to increase both response rates and the duration of clinical response to treatment. This literature review describes the signaling pathways involving BRAF and presents recent data from clinical trials with these molecules. Furthermore, we aim to describe the main resistance mechanisms linked with targeted anti-BRAF therapies. Methods The keywords (resistance, BRAF, melanoma, targeted therapy, vemurafenib, and dabrafenib) were used to extract relevant articles in the Medline/Pubmed database published before 31 January 2014. Discussion Improved knowledge and understanding of the mechanisms of resistance to targeted anti-BRAF therapies should enable the development of new therapeutic strategies in order to overcome such resistance and allow more significant and sustained response rates to be achieved among melanoma patients. Mots cles Mutation; BRAF; Melanome; Therapie ciblee; Vemurafenib; Dabrafenib; Resistance Keywords BRAF; Mutation; Melanoma; Targeted therapy; Vemurafenib; Dabrafenib; Resistance

To select the appropriate patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), it is important to gain a better understanding of the intracellular pathways leading to EGFR-TKI resistance, which is a common problem in patients with lung cancer. We recently reported that mutant KRAS adenocarcinoma is resistant to gefitinib as a result of amphiregulin and insulin-like growth factor-1 receptor overexpression. This resistance leads to inhibition of Ku70 acetylation, thus enhancing the BAX/Ku70 interaction and preventing apoptosis. Here, we determined the intracellular pathways involved in gefitinib resistance in lung cancers and explored the impact of their inhibition. We analyzed the activation of the phosphatidyl inositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) pathway in lung tumors. The activation of AKT was associated with disease progression in tumors with wild-type EGFR from patients treated with gefitinib (phase II clinical trial IFCT0401). The administration of IGF1R-TKI or amphiregulin-directed shRNA decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. The combination of PI3K/AKT inhibition with gefitinib restored apoptosis via Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which decreased the BAX/Ku70 interaction, inhibited AKT signaling and induced gefitinib-dependent apoptosis. The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in lung tumors with KRAS mutation, through the regulation of the BAX/Ku70 interaction. This finding suggests that combined treatments could improve the outcomes for this subset of lung cancer patients, who have a poor prognosis.

Melanoma is a cutaneous cancer with an increasing worldwide prevalence and high mortality due to unresectable or metastatic stages. Mutations inBRAF,NRAS, orKITare present in more than 60% of melanoma cases, but a useful blood-based biomarker for the clinical monitoring of melanoma patients is still lacking. Thus, the analysis of circulating tumor cells (CTCs) and/or cell-free circulating tumor DNA (ctDNA) analysis from blood (liquid biopsies) appears to be a promising noninvasive, repeatable, and systemic sampling tool for detecting and monitoring melanoma. Here, we review the molecular biology-based strategies used for ctDNA quantification in melanoma patients, as well as their main clinical applications. Droplet digital PCR (ddPCR) and next generation sequencing (NGS) technologies appear to be two versatile and complementary strategies to study rare variant mutations for the detection and monitoring of melanoma progression. Among the different clinical uses of ctDNA, we highlight the assessment of molecular heterogeneity and the identification of genetic determinants for targeted therapy as well as the analysis of acquired resistance. Importantly, ctDNA quantification might also be a novel biomarker with a prognostic value for melanoma patients.

International audience; Despite the emergence of targeted therapies and immunotherapy, chemotherapy remains the gold-standard for the treatment of most patients with solid malignancies. Spindle poisons that interfere with microtubule dynamics are commonly used in chemotherapy drug combinations. However, their troublesome side effects and the emergence of chemoresistance highlight the need for identifying alternative agents. We performed a high throughput cell-based screening and selected a pyrrolopyrimidine molecule (named PP-13). In the present study, we evaluated its anticancer properties in vitro and in vivo. We showed that PP-13 exerted cytotoxic effects on various cancer cells, including those resistant to current targeted therapies and chemotherapies. PP-13 induced a transient mitotic blockade by interfering with both mitotic spindle organization and microtubule dynamics and finally led to mitotic slippage, aneuploidy and direct apoptotic death. PP-13 was identified as a microtubule-targeting agent that binds directly to the colchicine site in β-tubulin. Interestingly, PP-13 overcame the multidrug-resistant cancer cell phenotype and significantly reduced tumour growth and metastatic invasiveness without any noticeable toxicity for the chicken embryo in vivo. Overall, PP-13 appears to be a novel synthetic microtubule inhibitor with interesting anticancer properties and could be further investigated as a potent alternative for the management of malignancies including chemoresistant ones. In the last two decades, advances in the understanding of carcinogenesis have revolutionized the management of cancer patients with the development of targeted therapies and immunotherapy. Identification of genetic alterations in subsets of cancers leading to different subcellular signals of tumour growth and progression elicited the clinical use of oncogene-targeted therapies for patients harbouring such anomalies 1-4. In lung cancers with EGFR-activating mutations, anti-EGFR therapies, including gefitinib, erlotinib, and afatinib, have been shown to improve progression-free survival and were approved as first-line options 5. However, despite the initial response, resistance mechanisms almost inevitably ensue and limit the long-term potency of targeted therapies 6, 7. Moreover, efforts are still needed for the management of patients without targetable oncogenic driver

Victor Jeannot,1,2 Benoit Busser,1–3 Laetitia Vanwonterghem,1,2 Sophie Michallet,1,2 Sana Ferroudj,1,2 Murat Cokol,4 Jean-Luc Coll,1,2 Mehmet Ozturk,1,2,5 Amandine Hurbin1,2 1INSERM U1209, Department Cancer Targets and Experimental Therapeutics, Grenoble, France; 2University Grenoble Alpes, Institute for Advanced Biosciences, Grenoble, France; 3Department of Biochemistry, Toxicology and Pharmacology, Grenoble University Hospital, Grenoble, France; 4Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey; 5Faculty of Medicine, Dokuz Eyul University, Izmir Biomedicine and Genome Center, Izmir, Turkey Abstract: Development of drug resistance limits the efficacy of targeted therapies. Alternative approaches using different combinations of therapeutic agents to inhibit several pathways could be a more effective strategy for treating cancer. The effects of the approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (gefitinib) or a multi-targeted kinase inhibitor (sorafenib) in combination with a histone deacetylase inhibitor (vorinostat) on cell proliferation, cell cycle distribution, apoptosis, and signaling pathway activation in human lung adenocarcinoma and hepatocarcinoma cells with wild-type EGFR and mutant KRAS were investigated. The effects of the synergistic drug combinations were also studied in human lung adenocarcinoma and hepatocarcinoma cells in vivo. The combination of gefitinib and vorinostat synergistically reduced cell growth and strongly induced apoptosis through inhibition of the insulin-like growth factor-1 receptor/protein kinase B (IGF-1R/AKT)-dependent signaling pathway. Moreover, the gefitinib and vorinostat combination strongly inhibited tumor growth in mice with lung adenocarcinoma or hepatocarcinoma tumor xenografts. In contrast, the combination of sorafenib and vorinostat did not inhibit cell proliferation compared to a single treatment and induced G2/M cell cycle arrest without apoptosis. The sorafenib and vorinostat combination sustained the IGF-1R-, AKT-, and mitogen-activated protein kinase-dependent signaling pathways. These results showed that there was synergistic cytotoxicity when vorinostat was combined with gefitinib for both lung adenocarcinoma and hepatocarcinoma with mutant KRAS in vitro and in vivo but that the combination of vorinostat with sorafenib did not show any benefit. These findings highlight the important role of the IGF-1R/AKT pathway in the resistance to targeted therapies and support the use of histone deacetylase inhibitors in combination with EGFR-tyrosine kinase inhibitors, especially for treating patients with mutant KRAS resistant to other treatments. Keywords: targeted therapy, combined treatments, non-small cell lung cancer, hepatocarcinoma

Scientific Reports 4: Article number: 6065; 10.1038/srep06065published online: August142014; updated: April222016 In this Article, B. Busser is incorrectly affiliated with ‘Institut Lumiere Matiere, UMR5306 Universite Lyon 1-CNRS, Universite de Lyon 69622 Villeurbanne cedex, France’. The correct affiliation is listed below: Universite Joseph Fourier, Inserm U823, IAB, F-38000 Grenoble, France

Nanomaterials represent a rapidly expanding area of research with huge potential for future medical applications. Nanotechnology indeed promises to revolutionize diagnostics, drug delivery, gene therapy and many other areas of research. For any biological investigation involving nanomaterials, it is crucial to study the behavior of such nano-objects within tissues to evaluate both their efficacy and their toxicity. Here, we provide the first account of 3D label-free nanoparticle imaging at the entire-organ scale. The technology used is known as laser-induced breakdown spectroscopy (LIBS) and possesses several advantages such as speed of operation, ease of use and full compatibility with optical microscopy. We then used two different but complementary approaches to achieve 3D elemental imaging with LIBS: a volume reconstruction of a sliced organ and in-depth analysis. This proof-of-concept study demonstrates the quantitative imaging of both endogenous and exogenous elements within entire organs and paves the way for innumerable applications.

Introduction Des carcinomes epidermoides cutanes (CEC) surviennent chez environ 20 % des patients atteints de melanome (M) sous vemurafenib (V), essentiellement pendant les 3 premiers mois. L’objectif etait de determiner la frequence des CEC sous V chez les patients atteints de tumeurs non melanocytaires dans l’etude pilote de phase II d’acces au V « AcSe-V » et d’etudier leurs caracteristiques cliniques, histologiques et moleculaires. Materiel et methodes Les patients inclus dans l’etude AcSe-V ont un suivi dermatologique organise sous l’egide du Groupe de cancerologie cutanee (GCC). Le schema de suivi comporte une visite de pre-inclusion, puis a M1, puis tous les 3 mois jusqu’a 6 mois apres l’arret du V. Seuls les patients qui ont un suivi ≥ 4 mois et qui n’ont pas d’antecedent de M sont incluables. Les comptes rendus des carcinomes epidermoides et de leurs precurseurs sont revus par un pathologiste. Une revue histologique centralisee et une caracterisation moleculaire des CEC sont en cours. La frequence des CEC etait comparee a celle de l’etude BRIM3. Resultats Parmi 56 patients inclus dans AcSe-V, six (11 % ; 4F, 2H) traites pour un cancer BRAF V600E-mute du poumon, de la thyroide et cerebral ont developpe 10 lesions cutanees qui ont ete analysees histologiquement. La taille mediane des lesions etait de 5,5 mm (5–10 mm). La localisation etait le membre superieur (n = 4) ou inferieur (n = 3), la tete et le cou (n = 2), et le tronc (n = 1). Apres revue des comptes rendus (7/8 disponibles), il y avait 8 CEC, 1 papillome avec une architecture evoquant un keratoacanthome et 1 keratose pre-epitheliomateuse. Au total, cinq patients (9 %) d’âge median 76 ans (23–83 ans) avaient des CE qui etaient multiples dans 2 cas. Il s’agissait de CEC crateriformes (n = 4) ou papilliformes (n = 2), bien differencies (n = 6) ou mal differencie (n = 1). Le delai median de diagnostic du premier CEC etait de 71 jours (29–161 jours) ; 5/6 patients avaient un phototype 3 ; aucun n’avait d’histoire medicale de cancer cutane mais 3 presentaient des keratoses actiniques avant l’initiation du V. Discussion Les CEC induits par le V chez les patients non atteints de M correspondaient le plus souvent a des tumeurs de petite taille bien differenciees diagnostiquees dans les premiers mois de traitement et relevant d’une chirurgie simple. Conclusion Les CEC induits par le V semblent avoir des caracteristiques cliniques et histologiques similaires chez les patients atteints de M et ceux atteints d’autres cancers. La plus faible frequence des CEC chez les patients non atteints de M en comparaison a ceux atteints de M dans l’etude BRIM3 (p = 0,039) pourrait etre due a des differences dans les facteurs de risques. Des facteurs de risques potentiels des CEC induits par le V sont l’âge eleve d’apres une etude australienne recente. Une analyse plus avancee des resultats de cette etude sera presentee. Elle devrait permettre de guider la surveillance dermatologique de ces pts.

Introduction Les metaux et mineraux jouent des roles cruciaux dans de nombreuses pathologies, qu’ils en soient a l’origine, ou qu’ils participent a leur progression. L’identification et la quantification des elements chimiques contenus dans les tissus pourraient permettre de mieux comprendre leurs implications. L’imagerie elementaire demeure un challenge technologique qui reste uniquement accessible par des methodes complexes et onereuses, ce qui limite considerablement les possibilites de faire de la cartographie elementaire sur des tissus. Materiel et methodes Nous proposons une nouvelle technique d’imagerie multi-elementaire basee sur la spectroscopie de plasma induit par laser ou laser induced breakdown spectroscopy (LIBS). Cette technique permet l’analyse d’echantillons inclus en paraffine, a temperature et atmosphere ambiantes. Par ablation laser, une fine quantite de matiere biologique est extraite et excitee sous forme de plasma, permettant son analyse spectrometrique. L’echantillon est scanne afin de realiser une cartographie complete des differents elements chimiques le constituant. Resultats Nous avons analyse plusieurs biopsies cutanees incluses en paraffine, par spectroscopie laser. Nous avons trouve de grandes quantites d’aluminium au sein d’un granulome post-vaccinal, ainsi que dans un pseudo-lymphome cutane. L’aluminium est localise uniquement dans les foyers inflammatoires histiocytaires. De meme, nous avons egalement trouve des elements metalliques tels que le titane ou le tungstene dans une biopsie ganglionnaire presentant une pigmentation a l’analyse histopathologique. Discussion L’apport de la spectroscopie laser dans l’aide au diagnostic pathologique a permis de cartographier et d’identifier avec certitude differents elements dans les biopsies incluses en paraffine. Cette etude preliminaire montre pour la premiere fois la pertinence d’utiliser l’imagerie elementaire sur des echantillons medicaux, en complement de l’histologie conventionnelle. Conclusion L’imagerie elementaire represente un outil innovant et pertinent pour aider les pathologistes dans leur travail de routine diagnostique. Les possibilites d’application en medecine sont immenses, elles incluent notamment les maladies de surcharge, les pathologies professionnelles et environnementales (exposition a des agents exogenes), l’oncologie et la dermatologie.

Introduction L’etude MINASARC est une etude cas-temoins concue pour evaluer l’« exposome » inorganique par un questionnaire professionnel et environnemental en concomitance avec une analyse mineralogique (AM) sur lavage broncho-alveolaire (LBA) et biopsies incluses en paraffine par microscopie electronique. Nous avons applique la technique Laser Induced Breakdown spectroscopy (LIBS) [1] sur les biopsies afin d’imager et quantifier les elements chimiques sur l’ensemble des echantillons. Nous rapportons les resultats obtenus sur deux cas de sarcoidose issus de l’etude MINASARC pour lesquels les techniques de microscopie et de LIBS semblent bien correlees. Methodes Pour chaque patient, le LBA a ete analyse en microscopie electronique a transmission (MET), une coupe histologique (5 μm d’epaisseur) en microscopie electronique a balayage (MEB) et le bloc contenant la biopsie en LIBS. Resultats Les particules de silice observees chez la patiente no 1 pourraient provenir de poudres utilisees lors du polissage des ongles [2] . Avant d’etre employe comme agent dans le domaine de la securite incendie, le patient no 2 a travaille moins d’un an comme plombier et effectue de la soudure sur charpente metallique ( Tableau 1 ). Conclusion Le role d’un facteur d’empoussierement inorganique dans le declenchement de la sarcoidose est discute. Les techniques d’analyses in situ en MEB et en LIBS sur la biopsie incluse en paraffine sont complementaires et doivent etre associees a un interrogatoire professionnel et environnemental du patient pour constituer un faisceau d’indices multiples. La mise en evidence d’un profil mineralogique anormal associe aux donnees de l’interrogatoire peut permettre de rediscuter le diagnostic de sarcoidose vers celui d’une pathologie d’empoussierement mineral.

International audience; An increasing interest has arisen in research focused on metallic and organic ions that play crucial roles in both physiological and pathological metabolic processes. Current methods for the observation of trace elements in biological tissues at microscopic spatial resolution often require equipment with high complexity. We demonstrate a novel approach with an all-optical design and multi-elemental scanning imaging, which is unique among methods of elemental detection because of its full compatibility with standard optical microscopy. This approach is based on laser-induced breakdown spectroscopy (LIBS), which allows the elements in a tissue sample to be directly detected and quantified under atmospheric pressure. We successfully applied this method to murine kidneys with 10 mm resolution and a ppm-level detection limit to analyze the renal clearance of nanoparticles. These results offer new insight into the use of laser spectrometry in biomedical applications in the field of label-free elemental mapping of biological tissues.

Introduction As metastatic pulmonary adenocarcinomas are routinely investigated for EGFR, KRAS, and ALK mutations/rearrangement, adequacy of CT-guided trans-thoracic needle biopsies (TTNB) needs to be evaluated in respect with the 2011 ATS/ERS/IASLC guidelines. Methods Two series of consecutive TTNB with 18-gauge needles performed before and after the publication of the ATS/ERS/IASLC guidelines, were retrospectively compared regarding their adequacy for histological sub-typing and EGFR/KRAS mutations and ALK rearrangement testing; the first series included 43 TTNB collected from January 2010 to February 2011, and the second one 48 TTNB collected from March 2011 to December 2012. Results 28 women and 63 men were included; the 2 groups were comparable in age, in mean size of lesions (32.5 mm), and distance of the lesion from the pleura. By comparing the first to the second series, the number of biopsies increased from 1.6 to 1.85, their mean length increased from 10.9 to 12.5 mm, and the mean number of stainings (TTF1, P63, CK5-6, mucins) per biopsy decreased from 2.6 to 1. Mean tumor cell percentage was 42%, mean total DNA extracted increased from 2.7 to 3.8 μg. In the first series, 85% of TTNB allowed EGFR exons 19 and 21 and KRAS mutations pyrosequencing and 72% additional EGFR exons 18 and 20 mutation analyses, versus 98% and 92% in the second. Conclusions With respect to ATS/ERS/IASLC guidelines, radiologists, biologists and pathologists have improved their practice; accordingly, CT-guided TTNB enable a precise histological sub-typing and provide sufficient DNA amount for genetic analyses.