Ultra-high-frequency ultrasound imaging of sural nerve: A comparative study with nerve biopsy in progressive neuropathies.

INTRODUCTION: Nerve ultrasound has been used increasingly in clinical practice as a complementary test for diagnostic assessment of neuropathies, but nerve biopsy remains invaluable in certain cases. The aim of this study was to compare ultra-high-frequency ultrasound (UHF-US) to histologic findings in progressive polyneuropathies. METHODS: Ten patients with severe, progressive neuropathies underwent ultrasound evaluation of the sural nerve before nerve biopsy. Ultrasound data were compared with histologic results in a retrospective manner. RESULTS: Sural nerves were easily identified on UHF-US. Nerve hyperechogenicity correlated with inflammatory infiltrates on biopsy. Nerve fascicles could be identified and measured on ultrasound in the majority of patients. DISCUSSION: Hyperechogenicity on UHF-US may be a marker of nerve inflammation in neuropathies. Furthermore, the UHF-US probe allows for evaluation of sensory nerves in spite of their small size, providing valuable information on their size and on their internal structure.

Motor axonal neuropathy associated with GNE mutations.

BACKGROUND: Mutations in the GNE gene have been so far described as predominantly associated with distal lower-limb myopathies. Recent reports describe mutations in this gene in patients with peripheral neuropathy and motor neuron disease. METHODS: We describe three patients displaying motor neuropathy in association with GNE mutations. Clinical, electrophysiological, imaging, pathological, and genetic data are presented in a retrospective manner. RESULTS: The three patients had different phenotypes, ranging from mildly progressive lower limb weakness to a rapidly progressive 4-limb weakness. Genetic testing revealed GNE gene mutations in all patients; of those mutations, p.(His186Arg) has not been previously reported. All patients showed evidence of axonal motor nerve involvement on electrodiagnostic examination and/or muscle biopsy. CONCLUSIONS: Nerve involvement associated with GNE gene mutations may be an underdiagnosed pathology and may influence clinical presentation and disease progression.

[PD1/PD-L1 immunohistochemistry in thoracic oncology: Where are we?] / Immunohistochimie PD-1/PD-L1 en oncologie thoracique : où en sommes-nous ?

The assays for the assessment of the PD-L1 status by immunohistochemistry are available in clinical studies in thoracic oncology to predict response to immunotherapies targeting the PD-1/PD-L1 pathway. With the arrival of this new class of molecules in second line and very soon in first line of treatment for patients with advanced or metastatic non-small cell lung cancer, these tests will certainly be required in routine once these new drugs will be granted marketing authorization. The rapid introduction of these "companion" or "complementary" tests seems essential to select patients to benefit from these effective but also expensive and sometimes toxic therapies. Although challenged by some oncologists (as some patients not expressing PD-L1 may sometimes respond to PD-1/PD-L1 blockade), the anti-PD-L1 immunohistochemically approach seems inevitable in 2017. This new activity developed in the pathology laboratories raises several questions: which anti-PD-L1 clone should be used? On which device? What threshold of positivity should be considered? Should PD-L1 expression be assessed on tumor cells as well as on the immune cells? What controls should be used? Comparative studies are underway or have been already implemented in order to answer some of these questions. This review addresses the different evaluation criteria for immunohistochemistry using the main anti-PD-L1 antibodies used to date as well the recently published studies using these antibodies in thoracic oncology.

PD-L1 expression in basaloid squamous cell lung carcinoma: Relationship to PD-1+ and CD8+ tumor-infiltrating T cells and outcome.

PD-1/PD-L1 inhibitors demonstrated durable clinical responses in patients with lung squamous cell carcinoma. However, the expression pattern of PD-L1 and the presence of CD8+ and PD-1+ tumor-infiltrating T cells in the basaloid variant of squamous cell carcinoma remain unknown. immunohistochemistry analysis of PD-L1 expression, with three recently validated monoclonal antibodies used in clinical trials (clones SP142, SP263, and 28-8), and detection of CD8+ and PD-1+ tumor-infiltrating T cells was performed on whole-tissue sections from 56 patients following surgery for basaloid squamous cell carcinoma. Data were correlated to clinicopathological parameters and outcome. Fair to poor concordance was observed between the SP142 vs SP263 clones, and SP142 vs 28-8 (κ range, 0.018-0.412), while the 28-8 and SP263 demonstrated a strong correlation in both the tumor cell and immune cell compartments (κ=0.883, and κ=0.721). Expression of PD-L1 correlated with a high content of CD8+ and PD-1+ tumor-infiltrating T cells when using SP142 (P=0.012; P=0.022), but not with SP263 or 28-8 (P=0.314; P=0.611). In the multivariate analysis, we found significantly better disease-free and overall survival rates for high PD-L1 expression with SP142, CD8+ and PD-1+ tumor-infiltrating T cells (P=0.003; P=0.007). No significant prognosis value was observed for SP263 and 28-8 clones, except a correlation between improved overall survival and SP263 in the univariate analysis (P=0.039), not confirmed in the multivariate model. In conclusion, we report that the expression of PD-L1 and the content of CD8+ and PD-1+ tumor-infiltrating T cells is an independent indicator of better outcome in basaloid squamous cell carcinoma patients, although the observed effect is dependent on the PD-L1 immunohistochemistry assay.

Immunohistochemistry as a potential tool for routine detection of the NRAS Q61R mutation in patients with metastatic melanoma.

BACKGROUND: It can be useful to assess the NRAS mutation status in patients with metastatic melanoma because NRAS-activating mutations confer resistance to RAF inhibitors, and NRAS-mutated patients appear to be sensitive to mitogen-activated protein kinase (MEK) inhibitors. OBJECTIVE: We aimed to assess the diagnostic accuracy of an immunohistochemistry (IHC) approach using a novel anti-NRAS (Q61R) monoclonal antibody on formalin-fixed paraffin-embedded tissue samples from patients with metastatic melanoma. METHODS: We conducted a retrospective multicenter cohort study on 170 patients with metastatic melanoma. The automated IHC assay was performed using the SP174 clone, and compared with results of the molecular testing. RESULTS: Evaluation of a test cohort with knowledge of the mutation status established a specific IHC pattern for the mutation. In the independent blinded analysis of the remaining cases, the anti-NRAS (Q61R) antibody accurately identified all NRAS Q61R-mutated tumors, and demonstrated 100% sensitivity and specificity. LIMITATIONS: Limitations include retrospective design and lack of multicenter interobserver reproducibility. CONCLUSION: The NRAS (Q61R) IHC assay is reliable and specific for the evaluation of the Q61R mutation status in metastatic melanoma and may be an alternative to molecular biology in evaluation of metastatic melanoma in routine practice.

[Setting up indicators in biobanking: why and how?]. / Mise en place d'indicateurs de suivi au sein d'une tumorothèque et/ou d'un centre de ressources biologiques : pourquoi et comment ?

The biobanking area is highly complex, and its complexity is increasing along with its growth and demand. Due to the advancements in genetic research, stem cell research and regenerative medicine, biobanking has become ever more important and plays a key role in biomedical research. The robustness and the reproducibility of research results depend greatly on the quality and on the number of the samples used, and thus on the expertise of biobanks having supplied these samples. Undoubtedly, the recognition of a research biobank depends on the impact of the research projects conducted with samples obtained from tumour bank(s), but also on many other criteria. It thus seems important to determine a number of indicators within a biobank to estimate objective criteria for the performance of these structures. These indicators can allow to make some strategic decisions knowing that biobanks are expensive structures to maintain in the present hospital context. The use of these indicators could also contribute to the elaboration of an "biobank impact factor of" or so called "bioresource research impact factor" (BRIF). We describe here four major categories of indicators (quality, activity, scientific production, visibility), which seem to be useful for the evaluation of a biobank by making a proposition of allocation of coefficients for the various considered items.

Specific infiltration of langerin-positive dendritic cells in EBV-infected tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma.

We report here the existence of a novel subset of langerin (CD207)-positive, immature dendritic cells (DCs) (CD83(neg) ) abundantly infiltrating Epstein Barr virus (EBV)-infected areas in tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma. These CD207(+) DCs differ from conventional epidermal Langerhans cells in their lack of CD1a and CCR6 and their unusual tissue localization. CD207(+) DC infiltration strongly correlates with EBV infection because it was neither detected in EBV negative specimens nor in tissues infected with other human viruses. These immature DCs might represent good candidates for induction of the EBV-specific immune response.

Isolation of a highly myogenic CD34-negative subset of human skeletal muscle cells free of adipogenic potential.

The differentiation of multipotent cells into undesirable lineages is a significant risk factor when performing cell therapy. In muscular diseases, myofiber loss can be associated with progressive fat accumulation that is one of the primary factors leading to decline of muscular strength. Therefore, to avoid any contribution of injected multipotent cells to fat deposition, we have searched for a highly myogenic but nonadipogenic muscle-derived cell population. We show that the myogenic marker CD56, which is the gold standard for myoblast-based therapy, was unable to separate muscle cells into myogenic and adipogenic fractions. Conversely, using the stem cell marker CD34, we were able to sort two distinct populations, CD34(+) and CD34(-), which have been thoroughly characterized in vitro and in vivo using an immunodeficient Rag2(-/-)gamma(c) (-/-) mouse model of muscle regeneration with or without adipose deposition. Our results demonstrate that both populations have equivalent capacities for in vitro amplification. The CD34(+) cells and CD34(-) cells exhibit equivalent myogenic potential, but only the CD34(-) population fails to differentiate into adipocytes in vitro and in vivo after transplantation into regenerative fat muscle. These data indicate that the muscle-derived cells constitute a heterogeneous population of cells with various differentiation potentials. The simple CD34 sorting allows isolation of myogenic cells with no adipogenic potential and therefore could be of high interest for cell therapy when fat is accumulated in diseased muscle.

Mouse model of skeletal muscle adiposity: a glycerol treatment approach.

Fat cell accumulation in skeletal muscle is a major characteristic of various disorders, such as obesity, sarcopenia and dystrophies. Moreover, these fat cells could be involved in muscle homeostasis regulation as previously described for adipocytes in bone marrow. Despite recent advances on the topic, no clearly characterized mouse model is currently available to study fat accumulation within skeletal muscle. Here, we report a detailed characterization of a mouse model of skeletal muscle fat cell accumulation after degeneration induced by intra-muscular injection of glycerol. Information is provided on the kinetics of degeneration/fat deposition, including the quantity of fat deposited based on various parameters such as glycerol concentration, age, sex and strain of mice. Finally, these fat cells are characterized as true white adipocytes morphologically and molecularly. Our study shows that the mouse adipocyte accumulation within skeletal muscle after glycerol degeneration is a reproducible, transposable and easy model to use. This mouse model should allow a more comprehensive understanding of the impact of adipocyte accumulation in skeletal muscle pathophysiology.

[What is new in 2010 for electron microscopy in surgical pathology?]. / Que reste t-il de la microscopie électronique pour le diagnostic anatomopathologique en 2010 ?

In the last decades, several ancillary methods, such as immunohistochemistry and molecular biology techniques, have increased the possibilities for the diagnosis and to evaluate the prognosis of lesions observed in a laboratory of pathology. Conversely, the impact of another method largely used a couple of years ago in a laboratory of pathology, the electron microscopy (EM), is currently limited. EM is a difficult, quite expensive and long method, which requires technicians with a high qualification. Therefore, EM is currently rarely available at the hospital in a laboratory of pathology and is essentially established in research centers. However, EM is still an essential tool for the surgical pathologist. This method allows in some circumstances to confirm or, more rarely, to make the diagnosis of a couple of tissular and cellular lesions observed in human pathology. EM is also an interesting method to better understand the etiopathogenesis of emerging human diseases, in particular of emerging infectious diseases. In this review, we report the main indication of EM in human pathology, we lay special emphasize in certain infectious diseases and neoplasia.

[Role of the surgical pathology laboratory in the pre-analytical approach of molecular biology techniques]. / Rôle du laboratoire d'anatomie pathologique dans l'approche pré-analytique des examens de biologie moléculaire réalisés en pathologie tumorale.

The advent of the targeted cancer therapies administered to patients, according to the results of molecular biology techniques (in particular, in situ hybridization, "polymerase chain reaction" amplification and sequencing), has modified the practice of the surgical pathology laboratories. The necessity to answer to the needs of physicians for optimizing the medical care for patients who develop cancer has led to a policy of national debate, spurred by the National Institute of Cancer (INCa), in order to implement new procedures in the pathology laboratories. Thus, in addition to the structuring of molecular biology platforms and their labeling by INCa, the upstream control of the steps present between resection of tumor samples and molecular analysis has proved to be crucial. Indeed, the quality of this upstream time, called "pre-analytical" phase, determines the reliability of the molecular biology results and therefore the therapeutic strategy. We describe here the main steps to be checked in the pre-analytical phase. The optimization of this pre-analytical phase within the surgical pathology laboratory aims to reduce or render insignificant the risk of errors of molecular biology tests. These errors can indeed lead to false negative or false positive results whose therapeutic consequences can be particularly harmful to patients with cancer.

[The Nice CHU biobank experience to collect patients' informed consent for research context (2004-2009)]. / L'expérience de la tumorothèque du CHU de Nice pour le recueil des consentements éclairés dans le cadre de la recherche (2004-2009).

Over the last 10 years, significant financial support from the French National Institute of Cancer (INCa), the Ministry of Health (DGOS), and the Health and Research National Institute (Inserm) helped biobanks--of which tumour banks represent a prominent example of hospital-based infrastructures--to improve their operations, and in some instances to adopt the rules of Biological Ressource Centers as defined by OECD. Nowadays, the use of biological samples of human origin is strictly subordinated to regulations that integrate bioethical principles. However, in spite of the establishment of these regulations, requirement to obtain an authorisation and/or to register the biological collections with the Ministry of Research, many uncertainties persist. While French regulations mandate that samples can be used for research as long as patients did not oppose to such use, many biobank curators face practical and theoretical issues when establishing a Material Transfer Agreement with scientists, due to the lack of harmonization between national regulations--particularly due to a different perception of privacy and free will in anglo-american and other countries--and different demands on the side of private industry or editorial boards of scientific journals. The goal of this article is (1) to describe the procedure followed to collect patients' informed consent at the Biobank of CHU de Nice and (2) to assess the number of obtained consents in comparison to the number of collected samples between 01/09/2004 and 31/12/2009, the number of consents obtained before or after collecting the samples, and the number of patients' refusal to collect their biological resources. This balance-sheet is settled for the three major collections (thoracic, thyroid and head and neck tissues) from the Biobank of CHU de Nice. Results show that 88 % of consents were obtained during this period (82 % in a prospective manner and 6 % in a retrospective manner). Refusal was notified by writing in nine cases only. The percentage of consents varies slightly according to the collection involved and is stable from 2004 to 2009. Overall, our procedure is quite efficient at obtaining informed consents from a majority of patients for whom the tumour bank stores biological samples. This situation provides optimal conditions for the use of collected samples in the context of national and international research projects.

[Pathologic findings and main aetiologies of rhinosinusal infections]. / Caractéristiques morphologiques et principales étiologies des infections rhino-sinusiennes.

A large variety of infectious diseases may involve the rhinosinusal tract. They include bacterial, viral, fungal, and parasite infections. These infections can induce an acute and/or a chronic inflammatory reaction. They can develop both in immunocompetent or in immunodeficient patients. Clinically, the consequences of these infections are variable, but a few of them have to be rapidly diagnosed for an immediate specific treatment. This article describes the pathologic features of a variety of infectious diseases that surgical pathologists may encounter in analysis of biopsy specimens taken from the rhinosinusal tract.

Pericardial effusion as primary manifestation of metastatic cutaneous adenoid cystic carcinoma: diagnostic cytopathology from an exfoliative sample.

Adenoid cystic carcinoma (ACC) occurs not only as a tumor of salivary glands, but also in very unusual locations, such as in the skin. Only very few cases of primary cutaneous of ACC have metastasized to the lymph nodes and lungs. We present a 53-year-old man with metastasis of the pericardium from a primary cutaneous ACC (PCACC) of the scalp, which had been surgically treated 14 years ago. Exfoliative cytologic findings from pericardial effusion included small clusters of basaloid cells with occasional cystlike spaces containing mucoid material. To our knowledge, this is the first case of pericardial metastasis from a PCACC.

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (≥1%, ≥50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

Any Place for Immunohistochemistry within the Predictive Biomarkers of Treatment in Lung Cancer Patients?

The identification of certain genomic alterations (EGFR, ALK, ROS1, BRAF) or immunological markers (PD-L1) in tissues or cells has led to targeted treatment for patients presenting with late stage or metastatic lung cancer. These biomarkers can be detected by immunohistochemistry (IHC) and/or by molecular biology (MB) techniques. These approaches are often complementary but depending on, the quantity and quality of the biological material, the urgency to get the results, the access to technological platforms, the financial resources and the expertise of the team, the choice of the approach can be questioned. The possibility of detecting simultaneously several molecular targets, and of analyzing the degree of tumor mutation burden and of the micro-satellite instability, as well as the recent requirement to quantify the expression of PD-L1 in tumor cells, has led to case by case development of algorithms and international recommendations, which depend on the quality and quantity of biological samples. This review will highlight the different predictive biomarkers detected by IHC for treatment of lung cancer as well as the present advantages and limitations of this approach. A number of perspectives will be considered.

New variant of necklace fibres display peculiar lysosomal structures and mitophagy.

Here, we describe a new variant of necklace fibres with specific myopathological features that have not been described thus far. They were observed in two patients, from two independent families with identical DNM2 (dynamin 2) mutation (c.1106 G > A (p.Arg369Gln)), displaying mildly heterogeneous clinical phenotypes. The variant is characterized by lysosomal inclusions, arranged in a necklace pattern, containing homogenous material, devoid of myonuclei. The so-called necklace region has a certain characteristic distance to the sarcolemma. Electron microscopy, including three dimensional reconstructions of serial section images highlights their ultrastructural properties and relation to neighbouring organelles. This new pattern is compared to the previously reported patterns in muscle biopsies containing necklace fibres associated with MTM1- and DNM2-mutations.

Chromogenic Multiplex Immunohistochemistry Reveals Modulation of the Immune Microenvironment Associated with Survival in Elderly Patients with Lung Adenocarcinoma.

With underrepresentation of elderly patients with lung adenocarcinoma (LADC) in anti-PD-1/PD-L1 clinical trials, better understanding of the interplay of PD-L1 and tumor-associated immune cells (TAICs) could assist clinicians in stratifying these patients for immunotherapy. One hundred and one patients with LADCs, stratified by age, were included for analysis of PD-L1 expression and density of TAICs expressing CD4, CD8, and CD33, by using multiplex chromogenic immunohistochemistry (IHC) assays and automated digital quantification. The CD4⁺/CD8⁺ ratio was significantly higher in elderly patients. In patients <75 years, the density of CD4⁺, CD8⁺, and PD-L1 in TAICs showed a positive significant correlation with PD-L1 expression in tumor cells (TCs), while a lower correlation was observed in the elderly population. In the latter, a high CD4⁺/CD8⁺ ratio, and combined PD-L1 expression ≥1% TCs with a low CD8⁺ density, low CD33⁺ density, and a high CD4⁺ density correlated to worse overall survival. We identified differences according to age in the CD4⁺/CD8⁺ ratio and in correlation between PD-L1 expression and the density of TAICs in LADC patients. Distinct groups of tumor microenvironments had an impact on the OS of elderly patients with LADC.