Computer-assisted pathological immunohistochemistry scoring is more time-effective than conventional scoring, but provides no analytical advantage

Computer-assisted pathological immunohistochemistry scoring is more time-effective than conventional scoring, but provides no analytical advantage

Immunohistochemistry in the era of personalised medicine

Immunohistochemistry (IHC) plays a central role in the histopathological classification of diseases, including cancer. More recently, the importance of immunohistochemical staining is increasing. IHC usage in diagnostics is invaluable; however, the genetic and therapeutic significance of biomarker immunostaining has become equally relevant.

The prognostic value of the stem-like group in colorectal cancer using a panel of immunohistochemistry markers

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the Western world. It is becoming increasingly clear that CRC is a diverse disease, as exemplified by the identification of subgroups of CRC tumours that are driven by distinct biology. Recently, a number of studies have begun to define panels of diagnostically relevant markers to align patients into individual subgroups in an attempt to give information on prognosis and treatment response. We examined the immunohistochemical expression profile of 18 markers, each representing a putative role in cancer development, in 493 primary colorectal carcinomas using tissue microarrays. Through unsupervised clustering in stage II cancers, we identified two cluster groups that are broadly defined by inflammatory or immune-related factors (CD3, CD8, COX-2 and FOXP3) and stem-like factors (CD44, LGR5, SOX2, OCT4). The expression of the stem-like group markers was associated with a significantly worse prognosis compared to cases with lower expression. In addition, patients classified in the stem-like subgroup displayed a trend towards a benefit from adjuvant treatment. The biologically relevant and poor prognostic stem-like group could also be identified in early stage I cancers, suggesting a potential opportunity for the identification of aggressive tumors at a very early stage of the disease.

PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry

Immunohistochemical staining for phosphatase and tensin homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study, we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC, with 56% of samples on a mixed-tumor tissue microarray (TMA) showing high expression by ISH compared with 42% by IHC. On a prostate TMA, 49% of cases showed high expression by ISH compared with 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumors, clear overexpression of PTEN mRNA on malignant epithelium compared with benign epithelium was frequently observed and quantified. The use of SpotStudio software in the mixed-tumor TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumor samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumors (up to 3-fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumor can be explored with more confidence.

Immunohistochemistry should undergo robust validation equivalent to that of molecular diagnostics

Immunohistochemistry (IHC) is a widely available and highly utilised tool in diagnostic histopathology and is used to guide treatment options as well as provide prognostic information. IHC is subjected to qualitative and subjective assessment, which has been criticised for a lack of stringency, while PCR-based molecular diagnostic validations by comparison are regarded as very rigorous. It is essential that IHC tests are validated through evidence-based procedures. With the move to ISO15189 (2012), not just of the accuracy, specificity and reproducibility of each test need to be determined and managed, but also the degree of uncertainty and the delivery of such tests. The recent update to ISO 15189 (2012) states that it is appropriate to consider the potential uncertainty of measurement of the value obtained in the laboratory and how that may impact on prognostic or predictive thresholds. In order to highlight the problems surrounding IHC validity, we reviewed the measurement of Ki67and p53 in the literature. Both of these biomarkers have been incorporated into clinical care by pathology laboratories worldwide. The variation seen appears excessive even when measuring centrally stained slides from the same cases. We therefore propose in this paper to establish the basis on which IHC laboratories can bring the same level of robust validation seen in the molecular pathology laboratories and the principles applied to all routine IHC tests.

Epidermal growth factor receptor immunohistochemistry: new opportunities in metastatic colorectal cancer

The treatment of cancer is becoming more precise, targeting specific oncogenic drivers with targeted molecular therapies. The epidermal growth factor receptor has been found to be over-expressed in a multitude of solid tumours. Immunohistochemistry is widely used in the fields of diagnostic and personalised medicine to localise and visualise disease specific proteins. To date the clinical utility of epidermal growth factor receptor immunohistochemistry in determining monoclonal antibody efficacy has remained somewhat inconclusive. The lack of an agreed reproducible scoring criteria for epidermal growth factor receptor immunohistochemistry has, in various clinical trials yielded conflicting results as to the use of epidermal growth factor receptor immunohistochemistry assay as a companion diagnostic. This has resulted in this test being removed from the licence for the drug panitumumab and not performed in clinical practice for cetuximab. In this review we explore the reasons behind this with a particular emphasis on colorectal cancer, and to suggest a way of resolving the situation through improving the precision of epidermal growth factor receptor immunohistochemistry with quantitative image analysis of digitised images complemented with companion molecular morphological techniques such as in situ hybridisation and section based gene mutation analysis.

Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia.

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Plasticité intermodale chez le hamster énucléé à la naissance : Études de la distribution des interneurones CaBPir dans les cortex visuel et auditif primaires.

La période postnatale et l’expérience sensorielle sont critiques pour le développement du système visuel. Les interneurones inhibiteurs exprimant l’acide γ-aminobutyrique (GABA) jouent un rôle important dans le contrôle de l’activité neuronale, le raffinement et le traitement de l’information sensorielle qui parvient au cortex cérébral. Durant le développement, lorsque le cortex cérébral est très susceptible aux influences extrinsèques, le GABA agit dans la formation des périodes critiques de sensibilité ainsi que dans la plasticité dépendante de l’expérience. Ainsi, ce système inhibiteur servirait à ajuster le fonctionnement des aires sensorielles primaires selon les conditions spécifiques d’activité en provenance du milieu, des afférences corticales (thalamiques et autres) et de l’expérience sensorielle. Certaines études montrent que des différences dans la densité et la distribution de ces neurones inhibiteurs corticaux reflètent les caractéristiques fonctionnelles distinctes entre les différentes aires corticales. La Parvalbumine (PV), la Calretinine (CR) et la Calbindine (CB) sont des protéines chélatrices du calcium (calcium binding proteins ou CaBPs) localisées dans différentes sous-populations d’interneurones GABAergiques corticaux. Ces protéines tamponnent le calcium intracellulaire de sorte qu’elles peuvent moduler différemment plusieurs fonctions neuronales, notamment l’aspect temporel des potentiels d’action, la transmission synaptique et la potentialisation à long terme. Plusieurs études récentes montrent que les interneurones immunoréactifs (ir) aux CaBPs sont également très sensibles à l’expérience et à l’activité sensorielle durant le développement et chez l’adulte. Ainsi, ces neurones pourraient avoir un rôle crucial à jouer dans le phénomène de compensation ou de plasticité intermodale entre les cortex sensoriels primaires. Chez le hamster (Mesocricetus auratus), l’énucléation à la naissance fait en sorte que le cortex visuel primaire peut être recruté par les autres modalités sensorielles, telles que le toucher et l’audition. Suite à cette privation oculaire, il y a établissement de projections ectopiques permanentes entre les collicules inférieurs (CI) et le corps genouillé latéral (CGL). Ceci a pour effet d’acheminer l’information auditive vers le cortex visuel primaire (V1) durant le développement postnatal. À l’aide de ce modèle, l’objectif général de ce projet de thèse est d’étudier l’influence et le rôle de l’activité sensorielle sur la distribution et l’organisation des interneurones corticaux immunoréactifs aux CaBPs dans les aires sensorielles visuelle et auditive primaires du hamster adulte. Les changements dans l’expression des CaBPs ont été déterminés d’une manière quantitative en évaluant les profils de distribution laminaire de ces neurones révélés par immunohistochimie. Dans une première expérience, nous avons étudié la distribution laminaire des CaBPs dans les aires visuelle (V1) et auditive (A1) primaires chez le hamster normal adulte. Les neurones immunoréactifs à la PV et la CB, mais non à la CR, sont distribués différemment dans ces deux cortex primaires dédiés à une modalité sensorielle différente. Dans une deuxième étude, une comparaison a été effectuée entre des animaux contrôles et des hamsters énucléés à la naissance. Cette étude montre que le cortex visuel primaire de ces animaux adopte une chimioarchitecture en PV similaire à celle du cortex auditif. Nos recherches montrent donc qu’une suppression de l’activité visuelle à la naissance peut influencer l’expression des CaBPs dans l’aire V1 du hamster adulte. Ceci suggère également que le type d’activité des afférences en provenance d’autres modalités sensorielles peut moduler, en partie, une circuiterie corticale en CaBPs qui lui est propre dans le cortex hôte ou recruté. Ainsi, nos travaux appuient l’hypothèse selon laquelle il serait possible que certaines de ces sous-populations d’interneurones GABAergiques jouent un rôle crucial dans le phénomène de la plasticité intermodale.

Immunohistochemistry of synergistic effects of kanamycin and furosemide in the murine model

Adult CBA/CaJ mice were injected with large single dose of both kanamycin and furosemide in order to study hair cell lesion and macrophage recruitment within the cochlea.

The neurochemically diverse intermedius nucleus of the medulla as a source of excitatory and inhibitory synaptic input to the nucleus tractus solitarii

Sensory afferent signals from neck muscles have been postulated to influence central cardiorespiratory control as components of postural reflexes, but neuronal pathways for this action have not been identified. The intermedius nucleus of the medulla (InM) is a target of neck muscle spindle afferents and is ideally located to influence such reflexes but is poorly investigated. To aid identification of the nucleus, we initially produced three-dimensional reconstructions of the InM in both mouse and rat. Neurochemical analysis including transgenic reporter mice expressing green fluorescent protein in GABA-synthesizing neurons, immunohistochemistry, and in situ hybridization revealed that the InM is neurochemically diverse, containing GABAegric and glutamatergic neurons with some degree of colocalization with parvalbumin, neuronal nitric oxide synthase, and calretinin. Projections from the InM to the nucleus tractus solitarius (NTS) were studied electrophysiologically in rat brainstem slices. Electrical stimulation of the NTS resulted in antidromically activated action potentials within InM neurons. In addition, electrical stimulation of the InM resulted in EPSPs that were mediated by excitatory amino acids and IPSPs mediated solely by GABA(A) receptors or by GABA(A) and glycine receptors. Chemical stimulation of the InM resulted in (1) a depolarization of NTS neurons that were blocked by NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonoamide) or kynurenic acid and (2) a hyperpolarization of NTS neurons that were blocked by bicuculline. Thus, the InM contains neurochemically diverse neurons and sends both excitatory and inhibitory projections to the NTS. These data provide a novel pathway that may underlie possible reflex changes in autonomic variables after neck muscle spindle afferent activation.

Immunohistochemistry in diagnostic veterinary pathology: a critical review

A técnica de imuno-histoquímica é usada na rotina diagnóstica e na pesquisa em patologia humana desde 1970, porém seu uso na patologia veterinária é relativamente recente, principalmente com objetivo diagnóstico. A maior dificuldade no uso da imuno-histoquímica na patologia veterinária tem sido a falta de anticorpos específicos para os tecidos animais. Na falta de anticorpos específicos para as espécies domésticas, a patologia veterinária freqüentemente faz uso de anticorpos que apresentam reatividade cruzada entre antígenos humanos e animais. O objetivo deste trabalho foi testar a reatividade cruzada de diversos anticorpos feitos para uso humano em tecido parafinado de algumas espécies animais, utilizando-se dos novos métodos de recuperação antigênica e amplificação da reação imuno-histoquímica. No presente estudo foi possível confirmar a aplicabilidade de que muitos anticorpos produzidos para diagnóstico imuno-histoquímico em patologia humana podem ser utilizados em patologia veterinária. Novos estudos são necessários a fim de se ampliar a lista de aplicabilidade desses anticorpos em diferentes espécies animais, levando sempre em consideração as variações de clones, diluições, métodos de recuperação antigênica e de revelação.

Caracterização de subpopulações de interneurônios imunorreativos para proteínas ligantes de cálcio no córtex pré-frontal do Sagui (Callithrix jacchus): distribuição e morfologia

Cortical interneurons are characterized by their distinct morphological, physiological and biochemical properties, acting as modulators of the excitatory activity by pyramidal neurons, for example. Various studies have revealed differences in both distribution and density of this cell group throughout distinct cortical areas in several species. A particular class of interneuron closely related to cortical modulation is revealed by the immunohistochemistry for calcium binding proteins calbindin (CB), calretinina (CR) and parvalbumin (PV). Despite the growing amount of studies focusing on calcium binding proteins, the prefrontal cortex of primates remains relatively little explored, particularly in what concerns a better understanding of the organization of the inhibitory circuitry across its subdivisions. In the present study we characterized the morphology and distribution of neurons rich in calcium-binding proteins in the medial, orbital and dorsolateral areas of the prefrontal cortex of the marmoset (Callithrix jacchus). Using both morphometric and stereological techniques, we found that CR-reactive neurons (mainly double bouquet and bipolar cells) have a more complex dendritic arborization than CB-reactive (bitufted and basket cells) and PV-reactive neurons (chandelier cells). The neuronal densities of CR- and CB-reactive cells are higher in the supragranular layers (II/III) whilst PV-reactive neurons, conversely, are more concentrated in the infragranular layers (V/VI). CR-reactive neurons were the predominant group in the three regions evaluated, being most prevalent in dorsomedial region. Our findings point out to fundamental differences in the inhibitory circuitry of the different areas of the prefrontal cortex in marmoset