Probes for CDKN2A

Genomic DNA anomalies such as copy number variations (gene duplication, amplification, deletion) and gene rearrangements are important biomarkers and drug targets in many cancer types. DNA in-situ hybridization (ISH) is the gold standard method to directly visualize these molecular alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissues at single-cell resolution within a histological section. However, currently available fluorescent ISH (FISH) assays provide limited morphological detail due to the use of fluorescent nuclear staining compared to chromogenic staining. Furthermore, FISH techniques rely on expensive fluorescence microscopes, risk loss of fluorescent signal over time and involve tedious imaging at high magnifications (100X). There is thus an unmet need for a sensitive and robust chromogenic DNA-ISH assay that can enable high-resolution detection of genomic DNA targets with the ease of bright-field microscopy. We present here DNAscope - a novel chromogenic DNA-ISH assay - for detecting and visualizing genomic DNA targets under a standard light microscope. DNAscope is based on the widely used RNAscope double-Z probe design and signal amplification technology and provides unparalleled sensitivity and specificity with large signal dots readily visualized at 40X magnification and with full morphological context. Furthermore, DNAscope ensures specific DNA detection without interference from RNA due to the use of a novel RNA removal method. Using a duplex chromogenic detection assay in red and blue, we demonstrate highly specific and efficient detection of gene rearrangements (ALK, ROS1, RET and NTRK1), gene amplification (ERBB2, EGFR, MET) and deletion (TP53 and CDKN2A). The DNAscope assay has been carefully optimized for probe signal size and color contrast to enable easy interpretation of signal patterns under conventional light microscopy or digital pathology. Compared to conventional FISH assays, DNAscope probes are standard oligos that are designed in silico to be free of any repetitive sequences and can be rapidly synthesized for any DNA target. In conclusion, the DNAscope assay provides a powerful and convenient alternative to commonly used FISH assays in many cancer research applications.

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is an age-related progressive and fatal lung disease with limited therapeutic options. IPF exhibits several pathological features of epithelial reprogramming, including cellular senescence. Moreover, distal airway remodeling and bronchiolization can result in honeycomb cysts. The molecular and cellular mechanisms that lead to this prominent phenotype, however, still remain poorly characterized. Here, we aimed to decipher the IPF distal bronchiole and alveolar cell subtypes and their potential contribution to IPF development and progression. METHODS: EpCAM+ cells were isolated and enriched from human lung tissue (IPF/age-matched donors, each n=3) followed by single-cell RNA sequencing on 10x Genomics platform. Single cell RNA-sequencing (scRNA-Seq) data was pre-processed on Scanpy pipeline, including filtered, ambient gene correction, batch correction, clustering and annotation. RNA scope and fluorescent immunolabeling were used to confirm the gene expression and further localization of proteins to specific cell types. Air-liquid interface cultures (ALI) were used to generate a functional in vitro 3D airway model and to conduct functional studies. RESULTS: We generated a dataset of 46199 cells and found distinct cell clusters, including rare cell types, such as suprabasal cells, recently reported in the healthy lung. We identified features of distal alveolar epithelial cells enriched in healthy donor while airway epithelial cells were enriched in IPF patients. We further analyzed our data set for marker that are enriched in aberrant basal cells in IPF and identified Gprotein coupled receptor (GPR) 87 as a novel surface marker of distal Keratin (KRT)5 + basal cells. Correlation analysis showed GPR87 expression was highly correlated with the expression of CDKN2A (ρ= 0.84), a senescence marker. KEGG pathway analysis further revealed genes positive correlated with GPR87 expression were enriched in P53 signaling pathway further corroborating a link with cellular senescence. GPR87 expression was localized to distal bronchioles and honeycomb cysts in IPF in situ by RNA Scope and immunolabeling. ALI cultures stimulated with TGF- β, a fibrotic inducer, led to increased GPR87 expression. Furthermore, modulation of GPR87 in primary human bronchial epithelial cell cultures resulted in impaired airway cell maturation and ciliogenesis. CONCLUSION: GPR87 is a novel surface marker of KRT5 + basal progenitor cells likely contributing to senescence, bronchiole remodeling and honeycomb cyst development in IPF. Further studies are ongoing to elucidate whether GPR87 is a potential druggable target of fibrosis and senescence.