Point mutation P174L of the penA gene endowing ceftazidime resistance to Burkholderia pseudomallei in China.

In a clinical isolate of Burkholderia pseudomallei from Hainan, the association between the emergence of ceftazidime resistance and a novel PenA P174L allele was identified for the first time, providing an understanding of one mechanism by which ceftazidime resistance arises in B. pseudomallei.

A novel lytic phage potentially effective for phage therapy against Burkholderia pseudomallei in the tropics.

BACKGROUND: Burkholderia pseudomallei is a tropical pathogen that causes melioidosis. Its intrinsic drug-resistance is a leading cause of treatment failure, and the few available antibiotics require prolonged use to be effective. This study aimed to assess the clinical potential of B. pseudomallei phages isolated from Hainan, China. METHODS: Burkholderia pseudomallei strain (HNBP001) was used as the isolation host, and phages were recovered from domestic environmental sources, which were submitted to the host range determination, lytic property assays, and stability tests. The best candidate was examined via the transmission electron microscope for classification. With its genome sequenced and analyzed, its protective efficacy against B. pseudomallei infection in A549 cells and Caenorhabditis elegans was evaluated, in which cell viability and survival rates were compared using the one-way ANOVA method and the log-rank test. RESULTS: A phage able to lyse 24/25 clinical isolates was recovered. It was classified in the Podoviridae family and was found to be amenable to propagation. Under the optimal multiplicity of infection (MOI) of 0.1, an eclipse period of around 20 min and a high titer (1012 PFU/ml) produced within 1 h were demonstrated. This phage was found stabile at a wide range of temperatures (24, 37, 40, 50, and 60 °C) and pH values (3-12). After being designated as vB_BpP_HN01, it was fully sequenced, and the 71,398 bp linear genome, containing 93 open reading frames and a tRNA-Asn, displayed a low sequence similarity with known viruses. Additionally, protective effects of applications of vB_BpP_HN01 (MOI = 0.1 and MOI = 1) alone or in combination with antibiotics were found to improve viability of infected cells (70.6 ± 6.8%, 85.8 ± 5.7%, 91.9 ± 1.8%, and 96.8 ± 1.8%, respectively). A significantly reduced mortality (10%) and a decreased pathogen load were demonstrated in infected C. elegans following the addition of this phage. CONCLUSIONS: As the first B. pseudomallei phage was isolated in Hainan, China, phage vB_BpP_HN01 was characterized by promising lytic property, stability, and efficiency of bacterial elimination during the in vitro/vivo experiments. Therefore, we can conclude that it is a potential alternative agent for combating melioidosis.

Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hainan, China: from 2014 to 2019.

BACKGROUND: The emergence of antimicrobial resistance against Mycobacterium tuberculosis (M. tuberculosis) has become the major concern in global tuberculosis control due to its limited therapy options and high mortality. However, the clinical and molecular characteristics of drug-resistant strains vary in different geographical areas. Hainan Island located in southern China, is a high drug-resistant tuberculosis burden area. This study aimed to determine the dynamic changes of drug-resistance patterns and drug-related gene mutation types of M. tuberculosis in Hainan from 2014 to 2019. RESULTS: A total of 1484 culture-confirmed M. tuberculosis were included in this study. It was found that the proportions of drug resistance to isoniazid and rifampin were 31.3 and 31.1% respectively. Overall the proportion of multidrug resistant M. tuberculosis was 24.9%. Multivariate logistic regression analysis showed that age and the treatment history were independent influencing factors of drug resistant tuberculosis. The proportions of drug-resistant tuberculosis in retreatment patients were considerably higher than those in new patients. The most common mutation types of isoniazid were Ser315 → Thr (66.3%), and the most common mutation types of rifampin were Ser531 → Leu (41.5%). CONCLUSIONS: Our data suggests that the prevalence of drug resistant TB remains high in Hainan, and the risks for developing drug resistance with diversified mutation types increased significantly in retreatment patients. These results contribute to the knowledge of the prevalence of drug resistance in Hainan Province and expand the molecular characteristics of drug resistance in China simultaneously.

Small molecular fluorescence dyes for immuno cell analysis.

Many diseases, including cancers, AIDS, diabetes, asthma, Parkinson's, and lymphoma, are associated with the immune cell responses of patients suffering from them. Identifying the underlying immune response in such diseases is critical to correctly diagnose their root cause and determine the correct medications to target that root cause for personal therapy and immunotherapy. This work focuses on small molecular CF dyes to conjugate with antibodies, such as CD4 and CD19, for their application in flow cytometry. The CF dyes enable the expansion of flow cytometry reagent panels to support high dimensional flow cytometry analysis of the resulting emissions of 30-40 fluorescent colors, a record in flow cytometry. The CF dyes can be used along with existing flow cytometry dyes to provide a quick, accurate, and cost-effective method for the diagnosis and immunology treatment of diseases such as minimal residual disease (MRD) after cancer therapy. The CF dyes will also be an effective tool for the clinical studies of immune response to SARS-CoV-2 and the related vaccine development.

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 ß 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.

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

BACKGROUND: Androgen 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. OBJECTIVE: To 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. DESIGN, SETTING, AND PARTICIPANTS: We 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. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We 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. RESULTS AND LIMITATIONS: Quantitative 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). CONCLUSIONS: We 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. PATIENT SUMMARY: Higher AR-V7 levels detected and quantified using a novel method were associated with poorer response to abiraterone or enzalutamide in prostate cancer.

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.

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. © 2016 Wiley Periodicals, Inc.

Correlation of p16 immunohistochemistry in FNA biopsies with corresponding tissue specimens in HPV-related squamous cell carcinomas of the oropharynx.

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.

Dual-color ultrasensitive bright-field RNA in situ hybridization with RNAscope.

In situ hybridization (ISH) techniques have been important to the study of gene expression signatures in cells and tissues. The ability to detect multiple targets simultaneously is especially valuable, since it allows dissecting gene expression of distinct cell types with precise cellular and subcellular resolution within morphological context. Recently, we have reported using a novel dual-color ultrasensitive bright-field RNA in situ hybridization for detection of clonally restricted immunoglobulin light chain mRNA expression in B cell lymphomas. Here, we present detailed protocols of RNAscope 2-Plex assays for FFPE tissue sections. The protocols describe the tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis, as well as emphasize the critical steps for ensuring successful staining.

Quantitative ultrasensitive bright-field RNA in situ hybridization with RNAscope.

RNA in situ hybridization (ISH) can provide valuable morphological context for molecular markers on one hand and enable morphological analysis in molecular context on the other hand. It has become increasingly important, thanks to increasing interest in new biomarkers and noncoding RNAs in both research and clinical applications. We have developed an ultrasensitive RNA ISH technology, RNAscope, employing a unique probe design strategy that allows target-specific signal amplification while suppressing background noise. This approach enables single RNA molecule detection in formalin-fixed paraffin-embedded (FFPE) specimens under standard bright-field microscopy and is capable of multiplex detection at the single cell level. After staining, target-specific signals appear as punctate dots present in individual cells in well-preserved tissue morphological context, which facilitates both semiquantitative manual scoring and software-assisted quantitative analysis. Here, we present detailed protocols of RNAscope for FFPE tissue sections. The step-by-step protocols describe tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis. We also highlight the critical steps for ensuring successful staining.

RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma.

The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

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.

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.

RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.

In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays.

Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots.

Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small molecules, can specifically and effectively label molecular targets at a subcellular level. Here we have used QDs linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection.