Xiao-Jun Ma

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.

Ultrasensitive RNA In Situ Hybridization for Detection of Restricted Clonal Expression of Low-Abundance Immunoglobulin Light Chain mRNA in B-Cell Lymphoproliferative Disorders

OBJECTIVES To assess the feasibility of using a novel ultrasensitive bright-field in situ hybridization approach (BRISH) to evaluate κ and λ immunoglobulin messenger RNA (mRNA) expression in situ in B-cell non-Hodgkin lymphoma (NHL). METHODS A series of 110 semiconsecutive clinical cases evaluated for lymphoma with historic flow cytometric (FCM) results were assessed with BRISH. RESULTS BRISH light chain restriction (LCR) results were concordant with FCM in 108 (99%) of 109 evaluable cases. Additional small B-cell lymphoma cohorts were successfully evaluated. CONCLUSIONS BRISH analysis of κ and λ immunoglobulin mRNA expression is a sensitive tool for establishing LCR in B-cell NHL when FCM results are not available.

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.

Detection of immunoglobulin light-chain restriction in cutaneous B-cell lymphomas by ultrasensitive bright-field mRNA in situ hybridization

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.

Abstract 3953: Visualization of treatment resistant subclones in colorectal cancer by mutation specific RNA in situ hybridization

Introduction: Tumor heterogeneity is a major underlying cause of therapy resistance and disease recurrence. Recent evidence shows that resistant clones are nearly always pre-existing in a cancer, but are present at low frequencies. Moreover, genetic analysis of clonal composition has been largely at the level of ‘bulk’ sequencing, which does not inform of the arrangement of tumor subclones in space. This spatial information contains crucial clues as to the mechanisms of subclone emergence. To address this gap in our knowledge, we have used Basescope™, a novel RNA in situ hybridization (ISH) technology to detect somatic single nucleotide variants (SNVs) within key treatment-resistance genes in sections of archival formalin fixed paraffin embedded (FFPE) colorectal cancer (CRC) and adenoma (CRA) samples. Experimental procedures: Basescope™ probes were designed against common CRC mutations in KRAS, BRAF and PIK3CA, genes that are all implicated in modulating targeted therapy response, and also for the corresponding wildtype genes. Publically available cell lines containing the mutations of interest were used to confirm the sensitivity and specificity of the Basescope™ probes. We then performed RNA-ISH on a series of FFPE colorectal tumors (n=24) using novel Basescope™ technology. The presence of detected mutations was confirmed by DNA sequencing. In additional analyses we multiplexed Basescope™ technology with conventional immunohistochemistry and DNA ISH to study concurrent DNA- and protein-level alterations. Stained sections containing subclonal mutations were digitized and used to create 2D maps depicting the precise location of mutant and wild-type subclones. These were then used to test and refine mathematical models of subclone expansion. Summary of novel results: Our data reports the application of Basescope™ technology for the in situ detection of SNVs in human CRC. We have used custom designed Basescope™ probes to visualize common mutations, and mapped the spatial arrangement of these mutant subclones. We found mutant subclones in 10 samples analyzed, and noted that these subclones appeared generally contiguous in space. Mathematical modeling of these data reveals the patterns of clonal selection and expansion within CRCs. Conclusions: This study is the first to use RNA-ISH sensitive to specific somatic SNVs to visualize the subclonal architecture of CRC, specifically of clones involved in treatment failure. Our results are suggestive of a complex clonal architecture within this common cancer type. Furthermore, we have confidently detected mutant subclones comprising less than 5% of the total lesion - these would not be reliably detected by standard methodology, and would likely be responsible for the failure of targeted therapy. More generally, we envisage that Basescope™ technology will be a useful tool in basic research, and furthermore is readily transferrable to clinical applications. Citation Format: Ann-Marie Baker, Mindy Wang, Xiao-Jun Ma, Courtney Anderson, Weini Huang, Enric Domingo, Annabelle Lewis, John Bridgewater, Marnix Jansen, Nicholas A. Wright, Manuel Rodriguez-Justo, Emily Park, Ian Tomlinson, Trevor A. Graham. Visualization of treatment resistant subclones in colorectal cancer by mutation specific RNA in situ hybridization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2017-3953

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Abstract A24: Evaluation of the expression of immune functional markers in the tumor microenvironment

Recent clinical successes with immune checkpoint blockades have provided promising immune-based therapeutic approaches for controlling malignancy. While therapeutic antibodies against CTLA-4 and PD-1/PD-L1 have resulted in potent and durable clinical responses in many patients, there still remains an urgent need to develop biomarker based assays to identify patients who may benefit from these approaches while avoiding toxicity and without compromising efficacy. While gene and protein expression of immune cell and checkpoint markers can be obtained through traditional techniques, insight regarding spatial and cell-specific expression within the tumor microenvironment is lacking, particularly in regards to secreted proteins with key immune functions. RNAscope® is an advanced in situ hybridization assay that allows for the visualization of single-cell gene expression, targeting mRNA sequences directly in tissues. The probes designed for this approach permit the detection of secreted markers, including cytokines and chemokines. In our investigation of 60 archived formalin-fixed paraffin embedded non-small cell lung cancer biopsies, expression profiles of immune checkpoint markers and immune functional molecules were evaluated in the tissue environment by RNAscope®. Each of the tumors displayed distinct co-expression patterns of immune checkpoint markers as well as varying levels of cytokines (TGFβ, IFNγ, and TNFα) and chemokines, which were measured in respect to their relative location in the tumor or stromal regions. Additionally, the detection of immune cell markers were used to pinpoint the location of infiltrating lymphocytes. The methods and findings in this study provide a valuable glimpse into the understanding of key immune functional markers in the tumor microenvironment to potentially advance treatment opportunities in this era of immuno-oncology therapies. Citation Format: Jeffrey Kim, Na Li, Mingxiao He, Bingqing Zhang, Nan Su, Xiao-Jun Ma, Emily Park. Evaluation of the expression of immune functional markers in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A24.