Yuling Luo

Quantitative In Situ Measurement of Estrogen Receptor mRNA Predicts Response to Tamoxifen

Purpose Quantification of mRNA has historically been done by reverse transcription polymerase chain reaction (RT-PCR). Recently, a robust method of detection of mRNA utilizing in situ hybridization has been described that is linear and shows high specificity with low background. Here we describe the use of the AQUA method of quantitative immunofluorescence (QIF) for measuring mRNA in situ using ESR1 (the estrogen receptor alpha gene) in breast cancer to determine its predictive value compared to Estrogen Receptor α (ER) protein. Methods Messenger RNA for ER (ESR1) and Ubiquitin C (UbC) were visualized using RNAscope probes and levels were quantified by quantitative in situ hybridization (qISH) on two Yale breast cancer cohorts on tissue microarrays. ESR1 levels were compared to ER protein levels measured by QIF using the SP1 antibody. Results ESR1 mRNA is reproducibly and specifically measurable by qISH on tissue collected from 1993 or later. ESR1 levels were correlated to ER protein levels in a non-linear manner on two Yale cohorts. High levels of ESR1 were found to be predictive of response to tamoxifin. Conclusion Quantification of mRNA using qISH may allow assessment of large cohorts with minimal formalin fixed, paraffin embedded tissue. Exploratory data using this method suggests that measurement of ESR1 mRNA levels may be predictive of response to endocrine therapy in a manner that is different from the predictive value of ER.

HPV E6/E7 RNA In Situ Hybridization Signal Patterns as Biomarkers of Three-Tier Cervical Intraepithelial Neoplasia Grade

Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.

Novel Junction-specific and Quantifiable In Situ Detection of AR-V7 and its Clinical Correlates in Metastatic Castration-resistant Prostate Cancer

BACKGROUNDnAndrogen receptor splice variant 7 (AR-V7) has been implicated in resistance to abiraterone and enzalutamide treatment in men with metastatic castration-resistant prostate cancer (mCRPC). Tissue- or cell-based in situ detection of AR-V7, however, has been limited by lack of specificity.nnnOBJECTIVEnTo address current limitations in precision measurement of AR-V7 by developing a novel junction-specific AR-V7 RNA in situ hybridization (RISH) assay compatible with automated quantification.nnnDESIGN, SETTING, AND PARTICIPANTSnWe designed a RISH method to visualize single splice junctions in cells and tissue. Using the validated assay for junction-specific detection of the full-length AR (AR-FL) and AR-V7, we generated quantitative data, blinded to clinical data, for 63 prostate tumor biopsies.nnnOUTCOME MEASUREMENTS AND STATISTICAL ANALYSISnWe evaluated clinical correlates of AR-FL/AR-V7 measurements, including association with prostate-specific antigen progression-free survival (PSA-PFS) and clinical and radiographic progression-free survival (PFS), in a subset of patients starting treatment with abiraterone or enzalutamide following biopsy.nnnRESULTS AND LIMITATIONSnQuantitative AR-FL/AR-V7 data were generated from 56 of the 63 (88.9%) biopsy specimens examined, of which 44 were mCRPC biopsies. Positive AR-V7 signals were detected in 34.1% (15/44) mCRPC specimens, all of which also co-expressed AR-FL. The median AR-V7/AR-FL ratio was 11.9% (range 2.7-30.3%). Positive detection of AR-V7 was correlated with indicators of high disease burden at baseline. Among the 25 CRPC biopsies collected before treatment with abiraterone or enzalutamide, positive AR-V7 detection, but not higher AR-FL, was significantly associated with shorter PSA-PFS (hazard ratio 2.789, 95% confidence interval 1.12-6.95; p=0.0081).nnnCONCLUSIONSnWe report for the first time a RISH method for highly specific and quantifiable detection of splice junctions, allowing further characterization of AR-V7 and its clinical significance.nnnPATIENT SUMMARYnHigher AR-V7 levels detected and quantified using a novel method were associated with poorer response to abiraterone or enzalutamide in prostate cancer.

Diagnosis of HPV driven oropharyngeal cancers: Comparing p16 based algorithms with the RNAscope HPV-test

BACKGROUNDnAccurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status.nnnMETHODSnTo further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2).nnnRESULTSn105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%. The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07-0.51, p=0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08-0.66, p=0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1-0.65, p=0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13-0.81, p=0.02).nnnCONCLUSIONSnOur findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.

Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues.

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal‐to‐noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA‐box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201–2208, 2016.

Robust RNA-based in situ mutation detection delineates colorectal cancer subclonal evolution

Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth. Current genetic ITH analysis methods do not preserve spatial context and may not detect rare subclones. Here, we address these shortfalls by developing and validating BaseScope—a novel mutation-specific RNA in situ hybridization assay. We target common point mutations in the BRAF, KRAS and PIK3CA oncogenes in archival colorectal cancer samples to precisely map the spatial and morphological context of mutant subclones. Computational modeling suggests that subclones must arise sufficiently early, or carry a considerable fitness advantage, to form large or spatially disparate subclones. Examples of putative treatment-resistant cells isolated in small topographical areas are observed. The BaseScope assay represents a significant technical advance for in situ mutation detection that provides new insight into tumor evolution, and could have ramifications for selecting patients for treatment.Methods that analyze intra-tumor genetic heterogeneity often do not preserve the spatial context of tumor subclones. Here, the authors present BaseScope, a mutation-specific RNA in situ hybridization assay and spatially map colorectal cancer and adenoma KRAS, BRAF and PIK3CA driver gene mutant subclones.

Ribonucleic Acid In Situ Hybridization Is a More Sensitive Method Than Immunohistochemistry in Detection of Thyroid Transcription Factor 1 and Napsin A Expression in Lung Adenocarcinomas

CONTEXTnTTF-1 and napsin A immunomarkers have a crucial role in differentiating lung adenocarcinoma from lung squamous cell carcinoma and in identifying a primary lung adenocarcinoma when working on a tumor of unknown origin.nnnOBJECTIVESnTo investigate the diagnostic sensitivity of ribonucleic acid in situ hybridization (RNAscope) in the detection of expression of these biomarkers in lung adenocarcinomas and to compare RNAscope to immunohistochemical techniques.nnnDESIGNnBoth RNAscope and the immunohistochemical assays for TTF-1 and napsin A were performed on tissue microarray sections containing 80 lung adenocarcinomas and 80 lung squamous cell carcinomas. The RNAscope assay for both TTF-1 and napsin A was also performed on 220 adenocarcinomas from various organs.nnnRESULTSnThe RNAscope assay for TTF-1 gave positive results in 92.5% (74 of 80) of the lung adenocarcinomas; in contrast, immunohistochemistry gave positive results in 82.5% (66 of 80) of those cases. The RNAscope assay for napsin A gave positive results in 90% (72 of 80) of lung adenocarcinomas; immunohistochemistry results were positive in 77.5% (62 of 80) of those cases. Napsin A expression was not seen in lung squamous cell carcinomas by either method. In contrast, TTF-1 expression was seen in 3.8% (3 of 80) (1(+)) and 10% (8 of 80) (1(+)) of the squamous cell carcinomas by immunochemistry and the RNAscope, respectively. All nonpulmonary adenocarcinoma results were negative for TTF-1 by the RNAscope assay.nnnCONCLUSIONSnPreliminary data suggest that RNAscope is superior to immunohistochemistry in detecting TTF-1 and napsin A expression in primary lung adenocarcinomas. Therefore, performing an RNAscope assay may be considered for both TTF-1(-) and napsin A(-) cases with a clinical suspicion of lung adenocarcinoma. The TTF-1 results should be interpreted with caution because a small percentage of squamous cell carcinomas can be focally positive by either assay.

Detection of Albumin Expression by RNA In Situ Hybridization Is a Sensitive and Specific Method for Identification of Hepatocellular Carcinomas and Intrahepatic Cholangiocarcinomas

ObjectivesnInconsistent data on detection of albumin expression by ribonucleic acid (RNA) in situ hybridization have been reported. We investigated the utility of RNAscope (Advanced Cell Diagnostics, Hayward, CA) in detection of albumin in hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (ICCs), and carcinomas from various organs using manual and automated staining.nnnMethodsnRNAscope for albumin detection was performed on 482 cases on tissue microarray sections and on 22 cases of ICC, including 14 surgical resection and eight core biopsy specimens.nnnResultsnThirty-six of 37 (97%) HCCs had detectable mRNA, whereas all non-HCC and non-ICC cases, except one lung adenocarcinoma, were negative for albumin. Fourteen of 22 ICCs (64%) were positive for albumin.nnnConclusionsnRNAscope for albumin is highly sensitive and specific for identifying HCCs and is highly specific and moderately sensitive for detection of ICCs; however, rare carcinomas (non-HCC, non-ICC, and those with no hepatoid histomorphology) can also have aberrant expression of albumin.

Abstract B03: Dissecting molecular pathways in human tumor vs. mouse stromal environment in patient-derived cancer models

The tumor microenvironment plays multiple roles in tumor cell proliferation, differentiation, vascularity, and metastasis through tumor cell-stromal cell interactions. The molecular responses of tumor cells to drug treatment can modify or be modified by the molecular signaling in stromal cells. Therefore, understanding the molecular features of both tumor cells and the tumor microenvironment is crucial for understanding cancer biology and the discovery of targeted therapy. Patient-derived xenograft (PDX) tumor models contain human tumor cells growing in a mouse stromal environment and are widely used models for cancer research and drug discovery. In this study we have applied in situ hybridization to visualize gene expression in human tumor and mouse stroma by developing species-specific probes based on the RNAscope technology. Eight genes were selected for this study based on their roles in a wide range of human cancers: EGFR, ERBB2, FGF19, FGFR1, FGFR4, MET, PECAM1 and TGFB1. We designed human and mouse species-specific probes by targeting the most divergent regions ( In both CRC and liver cancer PDX tumors, human-specific probes detected strong expression of ERBB2, FGFR4, and MET in human tumor cells but no signals in mouse stromal cells. Conversely, the mouse-specific probes only detected expression of Erbb2, Fgfr4, and Met genes in mouse stromal cells. Notably, strong expression of several mouse genes, including Tgfb1, Fgfr1, Egfr, and Pecam1, were detected in the stroma surrounding the human tumor by mouse-specific probes while no human probe signals were detected in the same region. Human TGFB1, FGFR1, EGFR, and PECAM1 probes detected varied levels of gene expression in the human tumor cells from the same sample without cross-detection of the mouse ortholog sequences in the stromal region. We also observed extensive gene expression heterogeneity, both within the same tumor and between tumors. Heterogeneity of FGFR4 and FGF19 expression in the liver cancer model was particularly notable. Our results indicate that RNAscope, a single molecule RNA detection method can robustly detect the expression patterns of FGFR4-FGF19 in PDX tumors and can be utilized for selecting PDX models for mouse trials with therapeutic agents targeting FGFR4 signaling pathway. Overall the findings in this study demonstrate the successful use of RNAscope based in-situ hybridization to examine human tumor specific gene expression in mouse stromal environment in PDX animal models by visualizing gene expression in both the human tumor and mouse stroma with species-specific probes. As human tumor engrafts acquire mouse stromal cells during growth in the murine host, the method presented here will further enable the molecular dissection in PDX tumor models of tumor-host interactions involved in tumor growth, progression, and metastasis as well as responses to cancer drugs and development of drug resistance. Citation Format: Emily Park, Na Li, Katherine Ye, Mingxiao He, Mingxiao He, Zhifu Zhang, Hongzhe Sun, Xin Wang, Courtney Anderson, Yuling Luo, Zhenyu Gu, Xiao-Jun Ma. Dissecting molecular pathways in human tumor vs. mouse stromal environment in patient-derived cancer models. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B03.

Abstract 647: In situ detection of human and mouse species-specific molecules in patient derived xenograft mouse models

Patient derived xenograft (PDX) mouse models are widely used in targeted cancer therapy study and cancer drug development. With human cancer tissues embedded into mouse microenvironment, both human and mouse derived molecules contribute to the proliferation and invasion of the implanted tumor cells. In order to explore the working mechanisms of targeted therapy and evaluate its anti-cancer effects, it is important to uncover the expression and spatial relation of targeted molecules in situ. Although profiling methods such as microarray and RNAseq can address the origin of relevant gene transcripts, they lose tissue spatial and cell morphology information by using tissue lysates. Immunohistochemistry (IHC) can preserve tissue and cell morphology information, however good antibodies are not readily available for every target molecule. In addition, IHC cannot detect long non-coding RNA targets. It is therefore crucial to find a broadly applicable method to separately detect human and mouse derived genes in PDX tissue samples. Species-specific probes targeting on seven cancer related genes, EGFR, ERBB2, FGFR1, FGFR2, FGFR4, PECAM1 and TGFB1, were designed for RNAscope in situ hybridization assays. The assays were performed in colorectal cancer (CRC) PDX sections and liver cancer PDX TMAs. The RNA expression results were categorized into 5 grades according to manufacturer9s scoring guidelines. Probe species-specificity of human probes and mouse probes was tested in human colon cancers and mouse colons. Both tissues passed quality control by hybridizions with probe-Hs-PPIB/probe-Mm-Ppib and probe-dapB. All human species-specific probes produced negative results in mouse colons, whereas mouse species-specific probes generated signals for Egfr, Erbb2, Fgfr2, Pecam1 and Tgfb1 (score 2 or more than 2) in mouse colons. No mouse species-specific probe signals were observed in human colon cancers. In CRC PDX sections, human species-specific probes detected signals of ERBB2 and FGFR4 (score 3) in colorectal tumor cells whereas mouse species-specific probes detected signals for Fgfr1, Pecam1 and Tgfb1 (score 2 or more than 2) in stroma areas. In liver cancer PDX TMAs, human EGFR, ERBB2, FGFR1, FGFR2, FGFR4 and TGFB1 (score 2 or more than 2) were identified by species-specific probes in liver cancer cells; mouse Egfr, Fgfr1, Pecam1 and Tgfb1 signals (score 2 or more than 2) were observed in the stroma regions. RNAscope duplex assays identified both human FGFR1 and mouse Fgfr1 probe signals in the same sections with different colors. RNAscope technology using species-specific probes can detect in situ human and mouse genes, respectively, which provides a powerful method to uncover the location of targeted cancer therapy related genes, their expression levels and the spatial correlation of positive cells with surrounding cells. This method will facilitate targeted therapy studies and cancer drug development in PDX mouse models. Citation Format: Na Li, Xin K. Ye, Ming-Xiao He, Zhifu Zhang, Hongzhe Sun, Xin Wang, Yuling Luo, Xiao-Jun Ma, Zhenyu Gu, Emily Park. In situ detection of human and mouse species-specific molecules in patient derived xenograft mouse models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 647.