Identification of the tissue of origin of a tumor is vital to its management. Previous studies showed tissue-specific expression patterns of microRNA and suggested that microRNA profiling would be ...useful in addressing this diagnostic challenge. MicroRNAs are well preserved in formalin-fixed, paraffin-embedded (FFPE) samples, further supporting this approach. To develop a standardized assay for identification of the tissue origin of FFPE tumor samples, we used microarray data from 504 tumor samples to select a shortlist of 104 microRNA biomarker candidates. These 104 microRNAs were profiled by proprietary quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) on 356 FFPE tumor samples. A total of 48 microRNAs were chosen from this list of candidates and used to train a classifier. We developed a clinical test for the identification of the tumor tissue of origin based on a standardized protocol and defined the classification criteria. The test measures expression levels of 48 microRNAs by qRT-PCR, and predicts the tissue of origin among 25 possible classes, corresponding to 17 distinct tissues and organs. The biologically motivated classifier combines the predictions generated by a binary decision tree and K-nearest neighbors (KNN). The classifier was validated on an independent, blinded set of 204 FFPE tumor samples, including nearly 100 metastatic tumor samples. The test predictions correctly identified the reference diagnosis in 85% of the cases. In 66% of the cases the two algorithm predictions (tree and KNN) agreed on a single-tissue origin, which was identical to the reference diagnosis in 90% of cases. Thus, a qRT-PCR test based on the expression profile of 48 tissue-specific microRNAs allows accurate identification of the tumor tissue of origin.
For patients with primary lung cancer, accurate determination of the tumor type significantly influences treatment decisions. However, techniques and methods for lung cancer typing lack ...standardization. In particular, owing to limited tumor sample amounts and the poor quality of some samples, the classification of primary lung cancers using small preoperative biopsy specimens presents a diagnostic challenge using current tools. We previously described a microRNA-based assay (miRview squamous; Rosetta Genomics Ltd., Rehovot, Israel) that accurately differentiates between squamous and nonsquamous non–small cell lung cancer. Herein, we describe the development and validation of an assay that differentiates between the four main types of lung cancer: squamous cell carcinoma, nonsquamous non–small cell lung cancer, carcinoid, and small cell carcinoma. The assay, miRview lung (Rosetta Genomics Ltd.), is based on the expression levels of eight microRNAs, measured using a sensitive quantitative RT-PCR platform. It was validated on an independent set of 451 samples, more than half of which were preoperative cytologic samples (fine-needle aspiration and bronchial brushing and washing). The assay returned a result for more than 90% of the samples with overall accuracy of 94% (95% CI, 91% to 96%), with similar performance observed in pathologic and cytologic samples. Thus, miRview lung is a simple and reliable diagnostic assay that offers an accurate and standardized classification tool for primary lung cancer using pathologic and cytologic samples.
Subtypes of renal tumors have different genetic backgrounds, prognoses, and responses to surgical and medical treatment, and their differential diagnosis is a frequent challenge for pathologists. New ...biomarkers can help improve the diagnosis and hence the management of renal cancer patients. We extracted RNA from 71 formalin-fixed paraffin-embedded (FFPE) renal tumor samples and measured expression of more than 900 microRNAs using custom microarrays. Clustering revealed similarity in microRNA expression between oncocytoma and chromophobe subtypes as well as between conventional (clear-cell) and papillary tumors. By basing a classification algorithm on this structure, we followed inherent biological correlations and could achieve accurate classification using few microRNAs markers. We defined a two-step decision-tree classifier that uses expression levels of six microRNAs: the first step uses expression levels of hsa-miR-210 and hsa-miR-221 to distinguish between the two pairs of subtypes; the second step uses either hsa-miR-200c with hsa-miR-139-5p to identify oncocytoma from chromophobe, or hsa-miR-31 with hsa-miR-126 to identify conventional from papillary tumors. The classifier was tested on an independent set of FFPE tumor samples from 54 additional patients, and identified correctly 93% of the cases. Validation on qRT-PCR platform demonstrated high correlation with microarray results and accurate classification. MicroRNA expression profiling is a very effective molecular bioassay for classification of renal tumors and can offer a quantitative standardized complement to current methods of tumor classification.
We describe a novel activity of the SV40 large T-ag helicase, the unwinding of four stranded DNA structures linked by stacked G-quartets, namely stacked groups of four guanine bases bound by ...Hoogsteen hydrogen bonds. The structures unwound by the helicase were of two types: (i) quadruplexes comprising four parallel strands that were generated by annealing oligonucleotides including clustered G residues in a buffer containing Na+ ions. Each parallel quadruplex consisted of four oligonucleotide molecules. (ii) Complexes comprising two parallel and two antiparallel strands that were generated by annealing the above oligonucleotides in a buffer containing K+ ions. Each antiparallel complex consisted of two folded oligonucleotide molecules. Unwinding of these unusual DNA structures by the T-ag was monitored by gel electrophoresis. The unwinding process required ATP and at least one single stranded 3′-tail extending beyond the four stranded region. These data indicated that the T-ag first binds the 3′-tail and moves in a 3′→5′ direction, using energy provided by ATP hydrolysis; then it unwinds the four stranded DNA into single strands. This helicase activity may affect processes such as recombination and telomere extension, in which four stranded DNA could play a role.
High-grade gliomas defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active ...chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.
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•H3K27M mutant gliomas exhibit an enhancer landscape reflecting lineage of origin•Elevated H3K27 acetylation is pervasively distributed across the epigenome•Repeat element expression is de-repressed by H3K27M mutation•H3K27M cells are more vulnerable to DNA demethylation triggering viral mimicry
Krug et al. reveal increased global H3K27ac deposition across the genome without creation of de novo active enhancers or promoters in high-grade glioma (HGG) with H3K27M mutations. H3K27ac enrichment at repeat elements in H3K27M HGG increases their expression, conferring sensitivity to epigenetic therapies.
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