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Background: Efforts to identify accurate blood biomarkers for neurodegenerative disease hallmarks have been hampered by the lack of a proteomic tool that has the required sensitivity to detect very low concentrations of brain‐derived proteins in plasma or serum and the ability to multiplex many analytes in a single assay. Here we evaluated NULISA™, a recently developed novel immunoassay with attomolar level sensitivity and high multiplex capability1, for its ability to detect serum biomarkers associated with Alzheimer's disease. Method: Serum samples from 31 Alzheimer's disease (AD) patients and 31 non‐AD controls were analyzed using NULISA with a 200plex inflammation panel targeting a broad spectrum of inflammation‐related cytokines, chemokines and other proteins. Five samples failed quality control and were excluded from further analysis, resulting in 28 AD and 29 control samples in the final dataset. Linear model analysis, including age and sex as covariates, was performed for each target for differential expression between AD patients and controls. Result: Two significant proteins were identified. The level of glial fibrillary acidic protein (GFAP) was 1.84‐fold higher in AD patients (adjusted p‐value = 0.00014), whereas the level of S100 calcium‐binding protein A12 (S100A12) was 2‐fold higher in non‐AD controls (adjusted p‐value = 0.031). GFAP serum levels correlated strongly with total tau (Spearman rho = 0.69, p = 1.7e‐09), p‐tau181 (rho = 0.69, p = 1.7e‐09) and amyloid beta 42 (rho = ‐0.71, p = 4.7e‐10) levels in cerebrospinal fluid (CSF). S100A12 levels also correlated significantly with total tau (rho = ‐0.39, p = 0.0031), p‐tau181 (rho = ‐0.36, p = 0.0053) and amyloid beta 42 (rho = 0.38, p = 0.0067) CSF levels. Receiver operating characteristic analysis demonstrated that GFAP strongly discriminated AD from controls with an area under the curve (AUC) of 0.93 (95% CI 0.87‐1.0), and S100A12 also exhibited good discrimination with an AUC of 0.72 (95% CI 0.59‐0.86). Conclusion: This exploratory study identified serum GFAP levels as a strong discriminatory biomarker for AD, consistent with previous studies. S100A12 also demonstrated significant associations with AD and CSF biomarkers and thus warrants further study. NULISA holds great promise for the discovery and validation of blood‐based biomarkers for the early detection and monitoring of neurodegenerative diseases. 1. Feng, W. et al. bioRxiv 2023.04.09.536130 (2023) https://doi.org/10.1101/2023.04.09.536130. [ABSTRACT FROM AUTHOR]

Background: Histopathological image features offer a quantitative measurement of cellular morphology, and probably help for better diagnosis and prognosis in head and neck squamous cell carcinoma (HNSCC). Methods: We first used histopathological image features and machine‐learning algorithms to predict molecular features of 212 HNSCC patients from The Cancer Genome Atlas (TCGA). Next, we divided TCGA‐HNSCC cohort into training set (n = 149) and test set (n = 63), and obtained tissue microarrays as an external validation set (n = 126). We identified the gene expression profile correlated to image features by bioinformatics analysis. Results: Histopathological image features combined with random forest may predict five somatic mutations, transcriptional subtypes, and methylation subtypes, with area under curve (AUC) ranging from 0.828 to 0.968. The prediction model based on image features could predict overall survival, with 5‐year AUC of 0.831, 0.782, and 0.751 in training, test, and validation sets. We next established an integrative prognostic model of image features and gene expressions, which obtained better performance in training set (5‐year AUC = 0.860) and test set (5‐year AUC = 0.826). According to histopathological transcriptomics risk score (HTRS) generated by the model, high‐risk and low‐risk patients had different survival in training set (HR = 4.09, p < 0.001) and test set (HR=3.08, p = 0.019). Multivariate analysis suggested that HTRS was an independent predictor in training set (HR = 5.17, p < 0.001). The nomogram combining HTRS and clinical factors had higher net benefit than conventional clinical evaluation. Conclusions: Histopathological image features provided a promising approach to predict mutations, molecular subtypes, and prognosis of HNSCC. The integration of image features and gene expression data had potential for improving prognosis prediction in HNSCC. [ABSTRACT FROM AUTHOR]

Pathophysiological and atrophic changes in the cerebellum have been well‐documented in schizophrenia. Reduction of gray matter (GM) in the cerebellum was confirmed across cognitive and motor cerebellar modules in schizophrenia. Such abnormalities in the cerebellum could potentially have widespread effects on both sensorimotor and cognitive symptoms. In this study, we investigated how reduction change in the cerebellum affects the static and the dynamic functional connectivity (FC) between the cerebellum and cortical/subcortical networks in schizophrenia. Reduction of GM in the cerebellum was confirmed across the cognitive and motor cerebellar modules in schizophrenic subjects. Results from this study demonstrates that the extent of reduction of GM within cerebellum correlated with increased static FCs between the cerebellum and the cortical/subcortical networks, including frontoparietal network (FPN), and thalamus in patients with schizophrenia. Decreased GM in the cerebellum was also associated with a declined dynamic FC between the cerebellum and the FPN in schizophrenic subjects. The severity of patients' positive symptom was related to these structural‐functional coupling score of cerebellum. These findings identified potential cerebellar driven functional changes associated with positive symptom deficits. A post hoc analysis exploring the effect of changed FC within cerebellum, confirmed that a significant positive relationship, between dynamic FCs of cerebellum–thalamus and intracerebellum existed in patients, but not in controls. The reduction of GM within the cerebellum might be associated with modulation of cerebellum–thalamus, and contributes to the dysfunctional cerebellar‐cortical communication in schizophrenia. Our results provide a new insight into the role of cerebellum in understanding the pathophysiological of schizophrenia. [ABSTRACT FROM AUTHOR]

BACKGROUND Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is a unique form of carcinoma that is important to identify for prognosis and treatment. Immunohistochemistry (IHC) for p16 (also known as cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1) is used as a surrogate marker for transcriptionally active, high-risk HPV. The primary objective of this study was to correlate p16 IHC of cell blocks from fine-needle aspirations (FNAs) with surgical pathology specimens of HPV-related oropharyngeal SCC. METHODS In total, 48 patients who had a diagnosis of oropharyngeal or nonoropharyngeal SCC and also had an FNA that demonstrated metastatic SCC with available cell block material were identified. IHC for p16 was evaluated on both FNA cell blocks and surgical pathology specimens. In situ hybridization for high-risk HPV messenger RNA was performed on 31 of the FNA cell blocks. RESULTS Although partial p16 staining was observed in the majority of cell blocks, there was concordance in 47 of 48 FNAs (98%) with surgical pathology specimens when strong positive p16 staining of at least 15% of tumor cells in FNA cell block material was present. In addition, high-risk HPV RNA in situ hybridization demonstrated a high correlation with p16 staining in surgical pathology specimens (96%) and FNAs (93%). CONCLUSIONS There was excellent correlation between p16 IHC of FNA cell blocks and surgical pathology specimens using a cutoff of at least 15% positive staining in cell blocks. The recommended threshold (70% positive staining) for surgical pathology specimens may yield a high rate of false-negative results if applied to FNA cell blocks. Cancer (Cancer Cytopathol) 2015;123:723-731. © 2015 American Cancer Society. [ABSTRACT FROM AUTHOR]

Background Detection of immunoglobulin light-chain restriction is important in the diagnosis of B-cell non-Hodgkin lymphoma ( B-NHL). Flow-cytometry, commonly used to evaluate light-chain restriction, is impractical to be used in cutaneous specimens. Immunohistochemical and conventional chromogenic in situ hybridization ( CISH) methods on formalin-fixed-paraffin-embedded ( FFPE) tissue lack sufficient sensitivity to detect low-level light-chain expression in B- NHL without plasmacytic differentiation. Ultrasensitive bright-field mRNA-ISH ( BRISH) for in situ light-chain detection in cutaneous B- NHL has been assessed. Design Kappa/lambda mRNA was detected using two-color BRISH (RNAscope 2xPlex, Advanced Cell Diagnostics) on 27 FFPE skin biopsies and excisions from patients with available B-cell PCR clonality studies: 16 clonal B-cell lesions (6 follicle center lymphoma, 5 marginal zone lymphoma, 3 large B-cell lymphoma, and 2 other) and 11 non-clonal B-cell proliferations. Results BRISH was successful in 15/16 clonal B-cell lesions and 11/11 non-clonal proliferations. Light-chain restriction was detected in 15/15 clonal lesions and in 1/11 non-clonal proliferations (96.1% overall concordance with clonality PCR). In 4/5 marginal zone lymphomas, light-chain restriction was detected as strong monotypic mRNA expression in a B-cell subset, consistent with plasmacytic differentiation. Conclusion Ultrasensitive BRISH can successfully detect light-chain restriction in B- NHL from FFPE skin specimens and may be a useful adjunct ancillary tool in cases not resolved by CISH or immunohistochemical methods. [ABSTRACT FROM AUTHOR]

Oropharyngeal squamous cell carcinoma (SCC) is strongly associated with human papillomavirus (HPV) infection, which is distinctively different from most other head and neck cancers. However, a robust quantitative reverse transcription PCR (RT-qPCR) method for comprehensive expression profiling of HPV genes in routinely fixed tissues has not been reported. To address this issue, we have established a new real-time RT-PCR method for the expression profiling of the E6 and E7 oncogenes from 13 high-risk HPV types. This method was validated in cervical cancer and by comparison with another HPV RNA detection method ( in situ hybridization) in oropharyngeal tumors. In addition, the expression profiles of selected HPV-related human genes were also analyzed. HPV E6 and E7 expression profiles were then analyzed in 150 archived oropharyngeal SCC samples and compared with other variables and with patient outcomes. Our study showed that RT-qPCR and RNA in situ hybridization were 100% concordant in determining HPV status. HPV transcriptional activity was found in most oropharyngeal SCC (81.3%), a prevalence that is higher than in previous studies. Besides HPV16, three other HPV types were also detected, including 33, 35 and 18. Furthermore, HPV and p16 had essentially identical expression signatures, and both HPV and p16 were prognostic biomarkers for the prediction of disease outcome. Thus, p16 mRNA or protein expression signature is a sensitive and specific surrogate marker for HPV transcriptional activity (all genotypes combined). [ABSTRACT FROM AUTHOR]

Lewis J S Jr, Ukpo O C, Ma X-J, Flanagan J J, Luo Y, Thorstad W L & Chernock R D (2012) Histopathology 60, 982-991 Transcriptionally-active high-risk human papillomavirus is rare in oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas - a tissue microarray study utilizing E6/E7 mRNA in situ hybridization Aims: Human papillomavirus is well established in oropharyngeal squamous cell carcinoma as both causative and prognostic, but its significance in non-oropharyngeal tumours is unclear. In particular, the significance of finding viral DNA is not known. We sought to evaluate nonoropharyngeal squamous cell carcinomas for transcriptionally-active human papillomavirus and to compare this with the presence of viral DNA. Methods: We evaluated an 87 patient tissue microarray cohort of oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas for high risk human papillomavirus DNA and E6 and E7 mRNA transcripts by in situ hybridization, and for p16 expression by immunohistochemistry. Results: We found only two of the 73 (2.7%) evaluable cases to harbour transcriptionally-active human papillomavirus. Both of these tumours were from the larynx, one was positive for human papillomavirus DNA by in situ hybridization, and both were extensively positive for p16. All oral cavity and hypopharyngeal tumours were negative for human papillomavirus. Conclusions: Transcriptionally-active human papillomavirus appears to be rare in laryngeal, hypopharyngeal, and oral cavity squamous cell carcinomas. As such, it appears unlikely to be a 'driver' or to be clinically significant in most established tumours. [ABSTRACT FROM AUTHOR]

Formalin fixed, paraffin embedded (FFPE) tissue is the most common specimen available for application of diagnostic assays on tissue after microscopic examination. However this approach is not molecular biology friendly, with significant damage to both nucleic acids and proteins by degradation and cross-linking. Extraction of sufficient, quality RNA from FFPE tissue is a barrier to the development of new diagnostic assays. We have optimized a reliable RNA extraction method based on deparaffinization at high temperature coupled with 3-day lysis at 650C. The average total RNA yield is 4.5 to 5.5 pg per 1 µm³ of archival FFPE tissue, and 260/280 ratios are between 1.80 and 1.95. With a new optimized RNA isolation protocol, we are investigated the fundamental factors which influence on RNA integrity within FFPE tissue. Using mouse kidneys as a standardized tissue, we profiled the pattern of RNA degradation based on fixation time, tissue processing time and fixative buffer. Quality was measured by bioanalyzer tracings, RT-PCR and branched DNA assays of specific genes. Fixation time and tissue processing time have significant impact on RNA quality. Our data suggested that the middle region of gene could be a promising target for RNA analysis. Overall, these date of this study provide fundamental knowledge in studies of gene expression analysis using archival FFPE tissues. [ABSTRACT FROM AUTHOR]