In Situ Quantitative Measurement of HER2mRNA Predicts Benefit from Trastuzumab-Containing Chemotherapy in a Cohort of Metastatic Breast Cancer Patients

PLoS One. 2014 Jun 26;9(6):e99131.

Vassilakopoulou M, Togun T, Dafni U, Cheng H1, Bordeaux J, Neumeister VM, Bobos M, Pentheroudakis G, Skarlos DV, Pectasides D, Kotoula V, Fountzilas G, Rimm DL, Psyrri A.
PMID: 24968015 | DOI: 10.1371/journal.pone.0099131.eCollection2014.

BACKGROUND: We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients. METHODS: A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan - Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates. RESULTS: HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson's Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman's Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31-0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22-0.70, p = 0.002). CONCLUSIONS: The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment.

Quantitative, in situ analysis of mRNAs and proteins with subcellular resolution

Sci Rep.

2017 Nov 28

Kwon S, Chin K, Nederlof M, Gray JW.
PMID: 29184166 | DOI: 10.1038/s41598-017-16492-1

We describe here a method, termed immunoFISH, for simultaneous in situ analysis of the composition and distribution of proteins and individual RNA transcripts in single cells. Individual RNA molecules are labeled by hybridization and target proteins are concurrently stained using immunofluorescence. Multicolor fluorescence images are acquired and analyzed to determine the abundance, composition, and distribution of hybridized probes and immunofluorescence. We assessed the ability of immunoFISH to simultaneous quantify protein and transcript levels and distribution in cultured HER2 positive breast cancer cells and human breast tumor samples. We demonstrated the utility of this assay in several applications including demonstration of the existence of a layer of normal myoepithelial KRT14 expressing cells that separate HER2+ cancer cells from the stromal and immune microenvironment in HER2+ invasive breast cancer. Our studies show that immunoFISH provides quantitative information about the spatial heterogeneity in transcriptional and proteomic features that exist between and within cells.

Simultaneous Multiplexed Imaging of mRNA and Proteins with Subcellular Resolution in Breast Cancer Tissue Samples by Mass Cytometry.

Cell Syst.

2017 Dec 26

Schulz D, Zanotelli VRT, Fischer JR, Schapiro D, Engler S, Lun XK, Jackson HW, Bodenmiller B.
PMID: 29289569 | DOI: 10.1016/j.cels.2017.12.001

To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.

In situ Tumor PD-L1 mRNA expression is associated with increased TILs and better outcome in breast carcinomas.

Clin Cancer Res. Mar 19.

Schalper KA, Velcheti V, Carvajal D, Wimberly H, Brown J, Pusztai L, Rimm DL (2014)
PMID: 24647569 | DOI: 10.1158/1078-0432.CCR-13-2702.

PURPOSE: Blockade of the PD-1/PD-L1 axis emerged as a promising new therapeutic option for cancer that has resulted in lasting responses in metastatic renal, lung carcinomas and melanomas. Tumor PD-L1 protein expression may predict response to drugs targeting this pathway. Measurement of PD-L1 protein is limited by the lack of standardized immunohistochemistry methods and variable performance of antibodies. Our goal was to correlate PD-L1 mRNA expression with clinical variables in primary breast carcinomas. EXPERIMENTAL DESIGN: The fluorescent RNAscope paired-primer assay was used to quantify in situ PD-L1 mRNA levels in 636 stage I-III breast carcinomas on two sets of tissue microarrays (YTMA128 [n=238)] and YTMA201 [n=398]). Tumor infiltrating lymphocytes (TILs) were assessed by hematoxylin-eosin stain and quantitative fluorescence. RESULTS: On YTMA128 and YTMA201, 55.7% and 59.5% of cases showed PD-L1 mRNA expression, respectively. Higher PD-L1 mRNA expression was significantly associated with increased TILs (P=0.04) but not with other clinical variables. Elevated TILs (scores 2&3+) occurred in 16.5% on YTMA128 and 14.8% on YTMA201 and was associated with estrogen receptor-negative status (P=0.01 on YTMA128 and 0.0001 on YTMA201). PD-L1 mRNA expression was associated with longer recurrence free survival (log-rank P=0.01) which remained significant in multivariate analysis including age, tumor size, histological grade, nodal metastasis, hormone receptor, HER2 status and extent of TILs (HR=0.268 [CI=0.099-0.721], P=0.009). CONCLUSIONS: PD-L1 mRNA expression is identified in nearly 60% of breast tumors and it is associated with increased TILs and improved recurrence free survival. These observations support the evaluation of PD-1/PD-L1 targeted therapies in breast cancer

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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ACD’s data images for HER2 gene.

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