RESUMEN
Precision medicine promises to enhance patient treatment through the use of emerging molecular technologies, including genomics, transcriptomics, and proteomics. However, current tools in surgical pathology lack the capability to efficiently isolate specific cell populations in complex tissues/tumors, which can confound molecular results. Expression microdissection (xMD) is an immuno-based cell/subcellular isolation tool that procures targets of interest from a cytological or histological specimen. In this study, we demonstrate the accuracy and precision of xMD by rapidly isolating immunostained targets, including cytokeratin AE1/AE3, p53, and estrogen receptor (ER) positive cells and nuclei from tissue sections. Other targets procured included green fluorescent protein (GFP) expressing fibroblasts, in situ hybridization positive Epstein-Barr virus nuclei, and silver stained fungi. In order to assess the effect on molecular data, xMD was utilized to isolate specific targets from a mixed population of cells where the targets constituted only 5% of the sample. Target enrichment from this admixed cell population prior to next-generation sequencing (NGS) produced a minimum 13-fold increase in mutation allele frequency detection. These data suggest a role for xMD in a wide range of molecular pathology studies, as well as in the clinical workflow for samples where tumor cell enrichment is needed, or for those with a relative paucity of target cells.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Microdisección/métodos , Animales , Núcleo Celular/metabolismo , Epitelio/metabolismo , Humanos , Ratones , Células 3T3 NIH , Coloración y EtiquetadoRESUMEN
BACKGROUND: We conducted a validation study of digital pathology for the quantitative assessment of tissue-based biomarkers with immunohistochemistry. OBJECTIVE: To examine observer agreement as a function of viewing modality (digital versus optical microscopy), whole slide versus tissue microarray (TMA) review, biomarker type (HER2 incorporating membranous staining and Ki-67 with nuclear staining), and data type (continuous and categorical). METHODS: Eight pathologists reviewed 50 breast cancer whole slides (25 stained with HER2 and 25 with Ki-67) and 2 TMAs (1 stained with HER2, 1 with Ki-67, each containing 97 cores), using digital and optical microscopy. RESULTS: Results showed relatively high overall interobserver and intermodality agreement, with different patterns specific to biomarker type. For HER2, there was better interobserver agreement for optical compared to digital microscopy for whole slides as well as better interobserver and intermodality agreement for TMAs. For Ki-67, those patterns were not observed. CONCLUSIONS: The differences in agreement patterns when examining different biomarkers and different scoring methods and reviewing whole slides compared to TMA stress the need for validation studies focused on specific pathology tasks to eliminate sources of variability that might dilute findings. The statistical uncertainty observed in our analyses calls for adequate sampling for each individual task rather than pooling cases.
Asunto(s)
Biomarcadores de Tumor/análisis , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Patología Clínica/métodos , Patología Clínica/normas , Neoplasias de la Mama/diagnóstico , Femenino , Humanos , Inmunohistoquímica , Variaciones Dependientes del Observador , Análisis de Matrices TisularesRESUMEN
BACKGROUND: With the emerging role of digital imaging in pathology and the application of automated image-based algorithms to a number of quantitative tasks, there is a need to examine factors that may affect the reproducibility of results. These factors include the imaging properties of whole slide imaging (WSI) systems and their effect on the performance of quantitative tools. This manuscript examines inter-scanner and inter-algorithm variability in the assessment of the commonly used HER2/neu tissue-based biomarker for breast cancer with emphasis on the effect of algorithm training. MATERIALS AND METHODS: A total of 241 regions of interest from 64 breast cancer tissue glass slides were scanned using three different whole-slide images and were analyzed using two different automated image analysis algorithms, one with preset parameters and another incorporating a procedure for objective parameter optimization. Ground truth from a panel of seven pathologists was available from a previous study. Agreement analysis was used to compare the resulting HER2/neu scores. RESULTS: The results of our study showed that inter-scanner agreement in the assessment of HER2/neu for breast cancer in selected fields of view when analyzed with any of the two algorithms examined in this study was equal or better than the inter-observer agreement previously reported on the same set of data. Results also showed that discrepancies observed between algorithm results on data from different scanners were significantly reduced when the alternative algorithm that incorporated an objective re-training procedure was used, compared to the commercial algorithm with preset parameters. CONCLUSION: Our study supports the use of objective procedures for algorithm training to account for differences in image properties between WSI systems.