The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low–copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2′-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA–based probes and the optimized ISH assay enable simple and fast detection of low–copy number miRNA targets, such as miR-130a in mouse brain.
MicroRNA-21 (miR-21) is upregulated in many cancers including colon cancers and is a prognostic indicator of recurrence and poor prognosis. In colon cancers, miR-21 is highly expressed in stromal fibroblastic cells and more weakly in a subset of cancer cells, particularly budding cancer cells. Exploration of the expression of inflammatory markers in colon cancers revealed tumor necrosis factor alpha (TNF-α) mRNA expression at the invasive front of colon cancers. Surprisingly, a majority of the TNF-α mRNA expressing cells were found to be cancer cells and not inflammatory cells. Because miR-21 is positively involved in cell survival and TNF-α promotes necrosis, we found it interesting to analyze the presence of miR-21 in areas of TNF-α mRNA expression at the invasive front of colon cancers. For this purpose, we developed an automated procedure for the co-staining of miR-21, TNF-α mRNA and the cancer cell marker cytokeratin based on analysis of frozen colon cancer tissue samples (n = 4) with evident cancer cell budding. In all four cases, TNF-α mRNA was seen in a small subset of cancer cells at the invasive front. Evaluation of miR-21 and TNF-α mRNA expression was performed on digital slides obtained by confocal slide scanning microscopy. Both co-expression and lack of co-expression with miR-21 in the budding cancer cells was noted, suggesting non-correlated expression. miR-21 was more often seen in cancer cells than TNF-α mRNA. In conclusion, we report that miR-21 is not linked to expression of the pro-inflammatory cytokine TNF-α mRNA, but that miR-21 and TNF-α both take part in the cancer expansion at the invasive front of colon cancers. We hypothesize that miR-21 may protect both fibroblasts and cancer cells from cell death directed by TNF-α paracrine and autocrine activity.
Trastuzumab is established as standard care for patients with HER2-positive breast cancer both in the adjuvant and metastatic setting. However, 50% of the patients do not respond to the trastuzumab therapy, and therefore new predictive biomarkers are highly warranted. MicroRNAs (miRs) constitute a new group of biomarkers and their cellular expression can be determined in tumor samples by in situ hybridization (ISH) analysis. miR-21 is highly prevalent and up-regulated in breast cancer and has been linked to drug resistance in clinical and in vitro settings. To determine expression patterns of miR-21 in high-grade breast cancers, we examined miR-21 expression in 22 HER2-positive tumors and 15 HER2-negative high-grade tumors by ISH. The histological examination indicated that patient samples could be divided into three major expression patterns: miR-21 predominantly in tumor stroma, predominantly in cancer cells, or in both stromal and cancer cells. There was no obvious difference between the HER2-positive and HER2-negative tumors in terms of the miR-21 expression patterns and intensities. To explore the possibility that miR-21 expression levels and/or cellular localization could predict resistance to adjuvant trastuzumab in HER2-positive breast cancer patients, we analyzed additional 16 HER2-positive tumors from patients who were treated with trastuzumab in the adjuvant setting. Eight of the 16 patients showed clinical recurrence and were considered resistant. Examination of the miR-21 expression patterns and intensities revealed no association between the miR-21 scores in the cancer cell population (p = 0.69) or the stromal cells population (p = 0.13) and recurrent disease after adjuvant trastuzumab. Thus, our findings show that elevated miR-21 expression does not predict resistance to adjuvant trastuzumab.
Specific chromogen- and fluorescence-based detection of microRNA by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded (FFPE) tissue sections has been facilitated by locked nucleic acid (LNA)-based probe technology and can be performed within a single working day. In the current method paper, we present a similar simple 1-day ISH method developed for cryostat sections obtained from clinical cryo-embedded tissue samples. The presented chromogen-based ISH method does not involve proteolytic pretreatment, which is mandatory for FFPE sections, but still retains a sensitivity level similar to that obtained in FFPE sections. The LNA-based ISH method is not only applicable in situations where only access to cryo-embedded material is possible, but it also has a potential use if combining microRNA ISH with immunohistochemistry in double fluorescence staining with antibodies not being compatible with proteolytic predigestion.
The chromosomes of a lymphoblastic sarcoma were investigated in 2 biopsies from the malignant lymph nodes. Both biopsies had the same highly stable stemline karyotype, analyzed both by the classical orcein technique, including tritium autoradiography and measurements of all chromosomes of 10 cells, and by the new fluorescence technique. In relation to the normal human karyotype, the stemline had undergone changes in the proportions of the normal chromosome types and had acquired several marker chromosomes. With the aid of fluorescence technique some of the marker chromosomes were identified as parts of normal chromosomes, whereas others were not recognized. The latter included 3 medium‐sized metacentrics with completely median centromere and the same fluorescence pattern in both arms. They were similar in every respect to marker chromosomes recently found in 4 other malignant lymphomas. The possible significance of these findings is discussed.
MicroRNAs (miRNAs) are small noncoding RNAs that constitute a novel group of biomarkers with exciting functions in cell differentiation, proliferation and apoptosis by mediating degradation or destabilization of target mRNAs. Expression profiling provides identification of disease-related miRNAs; however, questions towards molecular interactions in vivo remain un-answered. In situ hybridization (ISH) allows visualization the miRNAs in histological sections and thereby allows determination of specific cellular origin, which is an essential step for understanding the miRNAs role in any biological setting. We have developed fast double fluorescence and quantitative ISH technologies that facilitates interpretation of roles for miRNA in cancer progression. On a semi-automated platform, HistoFlex, the double fluorescence assay can be completed within 2 hours without loss of sensitivity and specificity. In order to characterize the miR-21 positive cells in the breast cancer stroma, we combined miR-21 ISH with smooth muscle α-actin in a double fluorescence assay, and found that most, but not all miR-21 positive cells, are smooth muscle α-actin positive and include both myofibroblasts and myoepithelial cells in the neoplastic tissue. Subsequent combination of miR-21 ISH and programmed cell death-4 (PDCD4) immunofluorescence showed differential expression in many but not all miR-21 positive cells. The findings suggest that PDCD4 (mRNA), as a predicted miR-21 target according to sequence alignment, may also take place in vivo. Thus, a role of miR-21 in cell survival and apoptosis is likely. miRNA ISH also allows quantitative estimation of miRNA expression levels in archived formalin-fixed and paraffin embedded tissue samples. Using chromogenic detection of miRNA and subsequent image analysis, semi-quantitative expression estimates can be obtained, which can be used for correlation with clinical parameters, such as drug response, diagnosis, prognosis and other molecular profiles. We have established an automated 1-day assay that provides quantitative estimates with a precision of app. 15%. We also show that a quantitative estimate obtained in one paraffin section with tumor tissue is representative for the whole tumor. For clinical applications, we present data showing that the miRNA ISH signal can be quantified in routinely processed paraffin samples from colon and breast cancer, and that this approach can provide novel diagnostic possibilities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-480. doi:1538-7445.AM2012-LB-480
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