mRNA In Situ Hybridization (HistoSonda)

Bernet, Laia; Benaclocha, Marcos Martínez; Casterá, Carles; Muñoz, Rafael Cano; Sevilla, Francisco J.; Alba, Javier; Barranco, Juan de Dios; Córdoba, Alicia; García‐Caballero, Tomás; Hardisson, David; Hernandez, Javier Martin de Francisco; Lázaro, J. M.; Polo, Luis; Riu, Francesc; Rezola, Ricardo; Rojo, Federico; Ruiz, Irune; Hernándiz, Ainoha; Heras, Juan María De la Cámara-de las; Coupe, Victoria M.

doi:10.1097/pdm.0b013e3182360b0a

Cited by 11 publications

(1 citation statement)

References 29 publications

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“…However, concordance between IHC and FISH results are lower for IHC 2+ results (~90%–95%) and even lower for IHC 3+ results (~85%) suggesting the potential need for improved methods of evaluating HER2 receptor status [19–21]. New HER2 mRNA quantification methods are emerging that may offer improved specificity or sensitivity than IHC or FISH methods [22]. …”

Section: Discussionmentioning

confidence: 99%

A functional signal profiling test for identifying a subset of HER2-negative breast cancers with abnormally amplified HER2 signaling activity

Huang¹,

Burns²,

Rich³

et al. 2016

Oncotarget

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The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2−, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2− breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%–37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.

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Section: Discussionmentioning

confidence: 99%

A functional signal profiling test for identifying a subset of HER2-negative breast cancers with abnormally amplified HER2 signaling activity

Huang¹,

Burns²,

Rich³

et al. 2016

Oncotarget

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Simultaneous Detection of Protein and mRNA in Jurkat and KG‐1a Cells by Mass Cytometry

Mavropoulos

Allo

et al. 2017

Cytometry Pt A

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Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery. Current techniques allowing the in situ detection of transcripts in single cells are limited to a small number of simultaneous targets and are generally tedious and labor-intensive. In this report, we present the development of a multiplex method for targeted RNA detection by combining the mass cytometry and RNAscope® platforms. This novel assay, called Metal In Situ Hybridization (MISH), includes the hybridization of RNA-specific target probes followed by signal amplification achieved through a cascade of hybridization events, ending with the binding of amplifier-specific detector probes. The detector probes are tagged with isotopically pure metal atoms used for detection by mass cytometry. Proof-of-principle experiments show the simultaneous detection of three mRNA targets in Jurkat cells in suspension cell assay mode. The localization of transcripts was also investigated using the imaging mass cytometry platform in Jurkat and KG-1a cells. In addition, we optimized the antibody staining procedure to allow the co-detection of mRNA and cell surface markers. Our data demonstrate that MISH can be used to complement protein detection by mass cytometry as well as to investigate gene transcription and translation in single cells. © 2017 International Society for Advancement of Cytometry.

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In situ analysis of FGFR2 mRNA and comparison with FGFR2 gene copy number by dual-color in situ hybridization in a large cohort of gastric cancer patients

et al. 2017

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BackgroundFibroblast growth factor receptor (FGFR2) has been proposed as a target in gastric cancer. However, appropriate methods to select patients for anti-FGFR2 therapies have not yet been established.MethodsWe used in situ techniques to investigate FGFR2 mRNA expression and gene amplification in a large cohort of 1036 Japanese gastric cancer patients. FGFR2 mRNA expression was determined by RNAscope. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH).ResultsWe successfully analyzed 578 and 718 samples by DISH and RNAscope, respectively; 2% (12/578) showed strong FGFR2 gene amplification (FGFR2:CEN10 >10); moderate FGFR2 gene amplification (FGFR2:CEN10 <10; ≥2) was detected in 8% (47/578); and high FGFR2 mRNA expression of score 4 (>10 dots/cell and >10% of positive cells with dot clusters under a 20× objective) was seen in 4% (29/718). For 468 samples, both mRNA and DISH data were available. FGFR2 mRNA expression levels were associated with gene amplification; FGFR2 mRNA levels were highest in the highly amplified samples (n = 12). All highly amplified samples showed very strong FGFR2 mRNA expression (dense clusters of the signal visible under a 1× objective). Patients with very strong FGFR2 mRNA expression showed more homogeneous FGFR2 mRNA expression compared to patients with lower FGFGR2 mRNA expression. Gastric cancer patients with tumors that had an FGFR2 mRNA expression score of 4 had shorter RFS compared with score 0–3 patients.ConclusionRNAscope and DISH are suitable methods to evaluate FGFR2 status in gastric cancer. Formalin-fixed paraffin-embedded (FFPE) tissue slides allowed evaluation of the intratumor heterogeneity of these FGFR2 biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1007/s10120-017-0758-x) contains supplementary material, which is available to authorized users.

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mRNA In Situ Hybridization (HistoSonda)

Cited by 11 publications

References 29 publications

A functional signal profiling test for identifying a subset of HER2-negative breast cancers with abnormally amplified HER2 signaling activity

A functional signal profiling test for identifying a subset of HER2-negative breast cancers with abnormally amplified HER2 signaling activity

Simultaneous Detection of Protein and mRNA in Jurkat and KG‐1a Cells by Mass Cytometry

In situ analysis of FGFR2 mRNA and comparison with FGFR2 gene copy number by dual-color in situ hybridization in a large cohort of gastric cancer patients

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