Whole Slide Imaging Versus Microscopy for Primary Diagnosis in Surgical Pathology: A Multicenter Blinded Randomized Noninferiority Study of 1992 Cases (Pivotal Study).

Most prior studies of primary diagnosis in surgical pathology using whole slide imaging (WSI) versus microscopy have focused on specific organ systems or included relatively few cases. The objective of this study was to demonstrate that WSI is noninferior to microscopy for primary diagnosis in surgical pathology. A blinded randomized noninferiority study was conducted across the entire range of surgical pathology cases (biopsies and resections, including hematoxylin and eosin, immunohistochemistry, and special stains) from 4 institutions using the original sign-out diagnosis (baseline diagnosis) as the reference standard. Cases were scanned, converted to WSI and randomized. Sixteen pathologists interpreted cases by microscopy or WSI, followed by a wash-out period of ≥4 weeks, after which cases were read by the same observers using the other modality. Major discordances were identified by an adjudication panel, and the differences between major discordance rates for both microscopy (against the reference standard) and WSI (against the reference standard) were calculated. A total of 1992 cases were included, resulting in 15,925 reads. The major discordance rate with the reference standard diagnosis was 4.9% for WSI and 4.6% for microscopy. The difference between major discordance rates for microscopy and WSI was 0.4% (95% confidence interval, -0.30% to 1.01%). The difference in major discordance rates for WSI and microscopy was highest in endocrine pathology (1.8%), neoplastic kidney pathology (1.5%), urinary bladder pathology (1.3%), and gynecologic pathology (1.2%). Detailed analysis of these cases revealed no instances where interpretation by WSI was consistently inaccurate compared with microscopy for multiple observers. We conclude that WSI is noninferior to microscopy for primary diagnosis in surgical pathology, including biopsies and resections stained with hematoxylin and eosin, immunohistochemistry and special stains. This conclusion is valid across a wide variety of organ systems and specimen types.

High frequency of PTEN mutations in nevi and melanomas from xeroderma pigmentosum patients.

We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) versus 53% (39/73)]. Both had a very high proportion of UV-type mutations (occurring at adjacent pyrimidines) [91% (10/11) versus 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62), and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho-S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus, the clinical and histological appearances and the molecular pathology of these UV-related XP nevi and melanomas were different from nevi and melanomas in the general population.

Epistatic and functional interactions of catechol-o-methyltransferase (COMT) and AKT1 on neuregulin1-ErbB signaling in cell models.

BACKGROUND: Neuregulin1 (NRG1)-ErbB signaling has been implicated in the pathogenesis of cancer and schizophrenia. We have previously reported that NRG1-stimulated migration of B lymphoblasts is PI3K-AKT1dependent and impaired in patients with schizophrenia and significantly linked to the catechol-o-methyltransferase (COMT) Val108/158Met functional polymorphism. METHODOLOGY/PRINCIPAL FINDINGS: We have now examined AKT1 activation in NRG1-stimulated B lymphoblasts and other cell models and explored a functional relationship between COMT and AKT1. NRG1-induced AKT1 phosphorylation was significantly diminished in Val carriers compared to Met carriers in both normal subjects and in patients. Further, there was a significant epistatic interaction between a putatively functional coding SNP in AKT1 (rs1130233) and COMT Val108/158Met genotype on AKT1 phosphorylation. NRG1 induced translocation of AKT1 to the plasma membrane also was impaired in Val carriers, while PIP(3) levels were not decreased. Interestingly, the level of COMT enzyme activity was inversely correlated with the cells' ability to synthesize phosphatidylserine (PS), a factor that attracts the pleckstrin homology domain (PHD) of AKT1 to the cell membrane. Transfection of SH-SY5Y cells with a COMT Val construct increased COMT activity and significantly decreased PS levels as well as NRG1-induced AKT1 phosphorylation and migration. Administration of S-adenosylmethionine (SAM) rescued all of these deficits. These data suggest that AKT1 function is influenced by COMT enzyme activity through competition with PS synthesis for SAM, which in turn dictates AKT1-dependent cellular responses to NRG1-mediated signaling. CONCLUSION/SIGNIFICANCE: Our findings implicate genetic and functional interactions between COMT and AKT1 and may provide novel insights into pathogenesis of schizophrenia and other ErbB-associated human diseases such as cancer.

Aumento de los valores de las proteínas neurregulina 1 y ErbB4 en la corteza prefrontal de pacientes esquizofrénicos / Elevated neuregulin-1 and ErbB4 protein in the prefrontal cortex of schizophrenic patients

La neurregulina 1 (NRG1) y su receptor, ErbB4, se han implicado en la esquizofrenia tanto a nivel genético como de transcripción. En la presente investigación se compararon los niveles de proteína NRG1 y ErbB4 en las fracciones citoplasmática y nuclear de tejido de la corteza prefrontal (CPF) entre individuos sanos, esquizofrénicos, con trastorno bipolar y con depresión mayor procedentes del Stanley Consortium. Utilizamos procedimientos de inmunotransferencia para examinar las posibles bandas inmunorreactivas de NRG1 y ErbB4, pero cuantificamos específicamente las señales inmunorreactivas de NRG1 a 42, 48 y 5kDa y las de ErbB4 a 21, 55, 60 y 180kDa. Se identificó un aumento significativo (alrededor del 20%) de los niveles de proteína NRG1 citoplasmática de 53kDa en CPF de pacientes esquizofrénicos en comparación con los otros grupos de sujetos. También detectamos efectos diagnósticos sobre los niveles de proteína ErbB4 citoplasmática (180kDa) en su forma entera en CPF, y las pruebas post hoc revelaron que estas cantidades eran significativamente mayores (alrededor del 30%) en pacientes esquizofrénicos en comparación con los sanos y lo deprimidos. Además, en fracciones de CPF examinamos los niveles de los posibles productos de la hidrólisis de ErbB4 a 21, 55 y 60kDa en comparación con ErbB4 citoplasmático en su forma entera. Detectamos tendencias de efectos diagnósticos sobre los cocientes citoplasmáticos 21kDa/180kDa y 55kDa/180kDa en CPF y las pruebas post hoc revelaron que estos cocientes eran significativamente más bajos en pacientes esquizofrénicos en comparación con los individuos sanos. La presente investigación sugiere que los aumentos de ARNm de NRG1 y ErbB4 también se producen a nivel proteico y podrían ser específicos de la esquizofrenia. Formulamos la hipótesis de que en esta enfermedad también podría estar alterado el procesamiento proteolítico de ErbB4, lo que depararía una alteración de los cocientes de las formas funcionalmente diferentes de este receptor(AU)

Neuregulin-1 (NRG1) and its receptor, ErbB4, have been implicated in schizophrenia at both gene and transcript levels. The present investigation compared NRG1 and ErbB4 protein levels in prefrontal cortical (PFC) cytoplasmic and nuclear fractions among normal, schizophrenic, bipolar and major depressed subjects from the Stanley Consortium. We used immunoblotting procedures to examine potential NRG1 and ErbB4 immunoreactive bands, but specifically quantified NRG1 immunoreactive signals at 42, 48 and 53kDa and ErbB4 immunoreactive signals at 21, 55, 60 and 180kDa. PFC cytoplasmic 53kDa NRG1 protein levels were significantly increased (¡­20%) in schizophrenic patients relative to each of the other subject groups. We also detected diagnostic effects on PFC cytoplasmic full-length (180kDa) ErbB4 protein levels, and post hoc tests revealed that these quantities were significantly increased (¡­30%) in schizophrenic patients relative to normal and to depressed subjects. In addition, we examined the levels of potential ErbB4 cleavage products at 21, 55 and 60kDa relative to those of full-length ErbB4 in the PFC fractions. We detected trends for diagnostic effects on PFC cytoplasmic 21kDa/180kDa and 55kDa/180kDa ratios, and post hoc tests revealed that these ratios were significantly reduced in schizophrenic patients relative to normal individuals. Our investigation suggests that schizophrenia-associated NRG1 and ErbB4 mRNA elevations also occur at the protein level and may be specific to schizophrenia. We hypothesize that ErbB4 proteolytic processing may also be altered in schizophrenia, yielding altered ratios of functionally distinct forms of ErbB4. Published by Elsevier B.V(AU)

Elevated neuregulin-1 and ErbB4 protein in the prefrontal cortex of schizophrenic patients.

Neuregulin-1 (NRG1) and its receptor, ErbB4, have been implicated in schizophrenia at both gene and transcript levels. The present investigation compared NRG1 and ErbB4 protein levels in prefrontal cortical (PFC) cytoplasmic and nuclear fractions among normal, schizophrenic, bipolar and major depressed subjects from the Stanley Consortium. We used immunoblotting procedures to examine potential NRG1 and ErbB4 immunoreactive bands, but specifically quantified NRG1 immunoreactive signals at 42, 48 and 53 kDa and ErbB4 immunoreactive signals at 21, 55, 60 and 180 kDa. PFC cytoplasmic 53 kDa NRG1 protein levels were significantly increased (approximately 20%) in schizophrenic patients relative to each of the other subject groups. We also detected diagnostic effects on PFC cytoplasmic full-length (180 kDa) ErbB4 protein levels, and post hoc tests revealed that these quantities were significantly increased (approximately 30%) in schizophrenic patients relative to normal and to depressed subjects. In addition, we examined the levels of potential ErbB4 cleavage products at 21, 55 and 60 kDa relative to those of full-length ErbB4 in the PFC fractions. We detected trends for diagnostic effects on PFC cytoplasmic 21 kDa/180 kDa and 55 kDa/180 kDa ratios, and post hoc tests revealed that these ratios were significantly reduced in schizophrenic patients relative to normal individuals. Our investigation suggests that schizophrenia-associated NRG1 and ErbB4 mRNA elevations also occur at the protein level and may be specific to schizophrenia. We hypothesize that ErbB4 proteolytic processing may also be altered in schizophrenia, yielding altered ratios of functionally distinct forms of ErbB4.

Discordant changes in cortical TrkC mRNA and protein during the human lifespan.

Neurotrophin-3 (NT-3) exerts its trophic effects in brain via tyrosine kinase receptor C (trkC) signaling. TrkC splice variants produce receptors with (full-length) and without (truncated) a tyrosine kinase domain. The relative abundance of trkC isoforms and the anatomical localization of trkC in the human prefrontal cortex (PFC) in relationship to development and maturation are currently unknown. We have examined the temporo-spatial expression of trkC protein and mRNA during the development of the human PFC. We have found two major isoforms, a full-length (150 kDa) and a truncated (50 kDa) form of the trkC protein in the human PFC. We report that the full-length form is expressed at low levels throughout development while the truncated form is expressed at moderate levels early in development and increases to reach mature levels by adolescence. In contrast, trkC mRNA levels are uniformly expressed throughout most of postnatal life, but decline in ageing. TrkC protein and mRNA are expressed in both pyramidal and non-pyramidal neurons; additionally, trkC protein is detected in glia and neuropil. Our results suggest that truncated trkC is prevalent in the human PFC and that neurons and glia may be responsive to NT-3 in the PFC throughout life.

A conserved mRNA expression profile of SREB2 (GPR85) in adult human, monkey, and rat forebrain.

SREB is a subfamily of G-protein-coupled receptors, which consists of SREB1 (GPR27), SREB2 (GPR85), and SREB3 (GPR173). Its high evolutionary conservation and predominant expression in the CNS suggest that SREB family members and their undiscovered ligand(s) may have significant functions in the nervous system. SREB2 is the most conserved receptor throughout vertebrate evolution. As a first step in understanding the function of the SREB family, we have determined the anatomical gene expression profile of SREB2 in adult human, monkey, and rat forebrain using in situ hybridization histochemistry. The expression pattern of SREB2 mRNA was well conserved across three mammalian species. SREB2 mRNA was expressed in neurons throughout the brain and the most abundant expression was detected in the hippocampal dentate gyrus in all species examined. The areas expressing high levels of SREB2 mRNA overlap with brain structures known to possess high levels of plasticity, namely, the hippocampal formation, olfactory system, and supraoptic and paraventricular nuclei. Further, the anatomical expression of SREB1 and SREB3 overlapped with that of SREB2 in the adult monkey brain. Together, these data suggest a possible link between SREB family and neural plasticity, which may explain its extremely high conservation throughout vertebrate evolution.

Catechol O-methyltransferase (COMT) mRNA expression in the dorsolateral prefrontal cortex of patients with schizophrenia.

Human prefrontal cortical neurons express catechol O-methyltransferase (COMT), an enzyme that inactivates the neurotransmitter dopamine. A functional polymorphism of COMT, Val(108/158) Met, affects prefrontal function, and the high-activity Val allele has been reported to be a genetic risk factor for schizophrenia. We used in situ hybridization histochemistry to measure mRNA levels of COMT in the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia (N=14) and of normal controls (N=15). While the groups did not differ in terms of mean level of COMT mRNA, there was a significantly different laminar pattern of COMT mRNA expression in pyramidal neurons (F=2.68, df=4,108, P <0.04); patients with schizophrenia had relatively lower levels in the superficial (II/III) layers and higher levels in the intermediate/deep (IV/V) layers (P&<0.01), while in controls, the expression was homogeneous across layers. Neither the mean level nor the laminar distribution of COMT mRNA was related to the Val(108/158) Met genotype, suggesting that the feedback regulation of mRNA level is not a compensation for the functional effect of the COMT polymorphism. The disease-related laminar difference of COMT expression may be involved in dysregulation of dopamine signaling circuits in the DLPFC of patients with schizophrenia.