Analytical validation of a 12-gene molecular test for the prediction of distant recurrence in breast cancer.

AIM: To validate the analytical performance of a 12-gene molecular assay that predicts distant recurrence for early-stage ER+/HER2- invasive breast cancer as run within a central reference laboratory. MATERIALS & METHODS: Formalin-fixed paraffin-embedded breast resections were evaluated by quantitative reverse transcription polymerase chain reaction for the expression of eight target genes, three housekeeper genes and one control gene to assess for DNA contamination. RESULTS: The assay results were highly correlated with a validated reference laboratory. The assay had a broad linear range for input RNA, with similar amplicon efficiencies for target and housekeeper genes. The assay test was highly reproducible, with comparable inter- and intrabatch precision to the reference laboratory. CONCLUSION: These studies demonstrate that the 12-gene molecular assay is highly robust and accurate.

Diagnostic Distinction of Malignant Melanoma and Benign Nevi by a Gene Expression Signature and Correlation to Clinical Outcomes.

Background: Histopathologic examination alone can be inadequate for diagnosis of certain melanocytic neoplasms. Recently, a 23-gene expression signature was clinically validated as an ancillary diagnostic test to differentiate benign nevi from melanoma. The current study assessed the performance of this test in an independent cohort of melanocytic lesions against clinically proven outcomes.Methods: Archival tissue from primary cutaneous melanomas and melanocytic nevi was obtained from four independent institutions and tested with the gene signature. Cases were selected according to pre-defined clinical outcome measures. Malignant lesions were defined as stage I-III primary cutaneous melanomas that produced distant metastases (metastatic to sites other than proximal sentinel lymph node(s)) following diagnosis of the primary lesion. Melanomas that were metastatic at the time of diagnosis, all re-excisions, and lesions with <10% tumor volume were excluded. Benign lesions were defined as cutaneous melanocytic lesions with no adverse long-term events reported.Results: Of 239 submitted samples, 182 met inclusion criteria and produced a valid gene expression result. This included 99 primary cutaneous melanomas with proven distant metastases and 83 melanocytic nevi. Median time to melanoma metastasis was 18 months. Median follow-up time for nevi was 74.9 months. The gene expression score differentiated melanoma from nevi with a sensitivity of 93.8% and a specificity of 96.2%.Conclusions: The results of gene expression testing closely correlate with long-term clinical outcomes of patients with melanocytic neoplasms.Impact: Collectively, this provides strong evidence that the gene signature adds valuable adjunctive information to aid in the accurate diagnosis of melanoma. Cancer Epidemiol Biomarkers Prev; 26(7); 1107-13. ©2017 AACR.

An independent validation of a gene expression signature to differentiate malignant melanoma from benign melanocytic nevi.

BACKGROUND: Recently, a 23-gene signature was developed to produce a melanoma diagnostic score capable of differentiating malignant and benign melanocytic lesions. The primary objective of this study was to independently assess the ability of the gene signature to differentiate melanoma from benign nevi in clinically relevant lesions. METHODS: A set of 1400 melanocytic lesions was selected from samples prospectively submitted for gene expression testing at a clinical laboratory. Each sample was tested and subjected to an independent histopathologic evaluation by 3 experienced dermatopathologists. A primary diagnosis (benign or malignant) was assigned to each sample, and diagnostic concordance among the 3 dermatopathologists was required for inclusion in analyses. The sensitivity and specificity of the score in differentiating benign and malignant melanocytic lesions were calculated to assess the association between the score and the pathologic diagnosis. RESULTS: The gene expression signature differentiated benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5%. CONCLUSIONS: These results reflect the performance of the gene signature in a diverse array of samples encountered in routine clinical practice. Cancer 2017;123:617-628. © 2016 American Cancer Society.

Analytical validation of a proliferation-based molecular signature used as a prognostic marker in early stage lung adenocarcinoma.

AIMS: The aim of these studies was to validate the analytical performance of a cell cycle progression (CCP) gene signature that provides prognostic information for early stage lung adenocarcinomas. MATERIALS & METHODS: Formalin-fixed paraffin-embedded (FFPE) lung resections were evaluated by quantitative RT-PCR for the expression of 31 target and 15 housekeeper genes comprising the CCP score. RESULTS: The signature had a standard deviation (SD) of 0.06 score units and a dynamic range spanning CCP scores between -13 and 14. The average amplicon efficiencies for target and housekeeper genes were 107% and 105%, respectively. All but one amplicon had a SD <0.5 CT. CONCLUSION: These studies demonstrate that the gene signature is robust and reproducible, making it suitable for use in a clinical setting.

Analytical validation of a melanoma diagnostic gene signature using formalin-fixed paraffin-embedded melanocytic lesions.

AIM: These studies were to validate the analytical performance of a gene expression signature that differentiates melanoma and nevi, using RNA expression from 14 signature genes and nine normalization genes that generates a melanoma diagnostic score (MDS). MATERIALS & METHODS: Formalin-fixed paraffin-embedded melanocytic lesions were evaluated in these studies. RESULTS: The overall SD of the assay was determined to be 0.69 MDS units. Individual amplicons within the signature had an average amplification efficiency of 92% and a SD less than 0.5 CT. The MDS was reproducible across a 2000-fold dilution range of input RNA. Melanin, an inhibitor of PCR, does not interfere with the signature. CONCLUSION: These studies indicate this signature is robust and reproducible and is analytically validated on formalin-fixed paraffin-embedded melanocytic lesions.

Effects of ADARs on small RNA processing pathways in C. elegans.

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wild-type and ADAR mutant Caenorhabditis elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels. About 40% of siRNAs derived from endogenous genes (endo-siRNAs) also had altered levels in at least one mutant strain, including 63% of Dicer-dependent endo-siRNAs. The 26G class of endo-siRNAs was significantly affected by ADARs, and many altered 26G loci had intronic reads and histone modifications associated with transcriptional silencing. Our data indicate that ADARs, through both direct and indirect mechanisms, are important for maintaining wild-type levels of many small RNAs in C. elegans.

Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer.

MicroRNAs (miRNAs) are small regulatory RNAs that are essential in all studied metazoans. Research has focused on the prediction and identification of novel miRNAs, while little has been done to validate, annotate, and characterize identified miRNAs. Using Illumina sequencing, ∼20 million small RNA sequences were obtained from Caenorhabditis elegans. Of the 175 miRNAs listed on the miRBase database, 106 were validated as deriving from a stem-loop precursor with hallmark characteristics of miRNAs. This result suggests that not all sequences identified as miRNAs belong in this category of small RNAs. Our large data set of validated miRNAs facilitated the determination of general sequence and structural characteristics of miRNAs and miRNA precursors. In contrast to previous observations, we did not observe a preference for the 5' nucleotide of the miRNA to be unpaired compared to the 5' nucleotide of the miRNA*, nor a preference for the miRNA to be on either the 5' or 3' arm of the miRNA precursor stem-loop. We observed that steady-state pools of miRNAs have fairly homogeneous termini, especially at their 5' end. Nearly all mature miRNA-miRNA* duplexes had two nucleotide 3' overhangs, and there was a preference for a uracil in the first and ninth position of the mature miRNA. Finally, we observed that specific nucleotides and structural distortions were overrepresented at certain positions adjacent to Drosha and Dicer cleavage sites. Our study offers a comprehensive data set of C. elegans miRNAs and their precursors that significantly decreases the uncertainty associated with the identity of these molecules in existing databases.

Role of RNA structure in regulating pre-mRNA splicing.

Pre-mRNA splicing involves removing non-coding introns from RNA transcripts. It is carried out by the spliceosome, along with other auxiliary factors. In general, research in splicing has focused on the sequences within the pre-mRNA, without considering the structures that these sequences might form. We propose that the role of RNA structure deserves more consideration when thinking about splicing mechanisms. RNA structures can inhibit or aid binding of spliceosomal components to the pre-mRNA, or can increase splicing efficiency by bringing important sequences into close proximity. Recent reports have identified proteins and small molecules that can regulate splicing by modulating RNA structures, thereby expanding our knowledge of the mechanisms used to regulate splicing.

Pentamidine reverses the splicing defects associated with myotonic dystrophy.

Myotonic dystrophy (DM) is a genetic disorder caused by the expression (as RNA) of expanded CTG or CCTG repeats. The alternative splicing factor MBNL1 is sequestered to the expanded RNA repeats, resulting in missplicing of a subset of pre-mRNAs linked to symptoms found in DM patients. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. We identified the small molecules pentamidine and neomycin B as compounds that disrupt MBNL1 binding to CUG repeats in vitro. We show in cell culture that pentamidine was able to reverse the missplicing of 2 pre-mRNAs affected in DM, whereas neomycin B had no effect. Pentamidine also significantly reduced the formation of ribonuclear foci in tissue culture cells, releasing MBNL1 from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of 2 pre-mRNAs in mice expressing expanded CUG repeats.

Effect of a domain-spanning disulfide on aminoacyl-tRNA synthetase activity.

Enzymes regulated by allostery undergo conformational rearrangement upon binding effector molecules. For modular proteins, a flexible interface may mediate reorientation of the protein domains and transmit binding events to activate catalysis at a distance. Aminoacyl-tRNA synthetases (aaRSs) that use tRNA anticodons as identity elements can be considered allosteric enzymes in which aminoacylation of the tRNA acceptor stem is enhanced upon anticodon binding. We reasoned that anticodon-triggered conformational change might be restricted upon introduction of a disulfide linkage near the core of an aaRS. Here we show that a double cysteine mutation engineered at the Escherichia coli MetRS domain interface spontaneously generates a disulfide linkage. This disulfide clamp has no effect on methionyl adenylate formation but reduces the level of tRNA(Met) aminoacylation approximately 2-fold. Activity is restored upon chemical reduction of the disulfide, demonstrating that E. coli MetRS requires a flexible interface domain for full catalytic efficiency.

The protein factors MBNL1 and U2AF65 bind alternative RNA structures to regulate splicing.

Myotonic dystrophy type 1 (DM1) is a genetic disorder linked to a (CTG)(n) repeat expansion in the 3' untranslated region of the DMPK gene. Upon transcription in the nucleus, the CUG repeats form a stable RNA stem-loop that sequesters the RNA-binding protein MBNL1 from its normal function in the cell. MBNL1 regulates the alternative splicing of many pre-mRNAs, and upon MBNL1's sequestration, the alternative splicing of many genes is mis-regulated, leading to disease symptoms. MBNL1 is known to bind directly to at least 3 of the pre-mRNAs that it regulates, but how MBNL1 binding mechanistically regulates alternative splicing is unclear. Here, we demonstrate that MBNL1 controls the splicing of exon 5 in the cardiac troponin T (cTNT) pre-mRNA by competing directly with the essential splicing factor U2AF65 for binding at the 3' end of intron 4. When U2AF65 is prevented from binding to the pre-mRNA, the U2 snRNP can no longer be recruited and the following exon is skipped. Furthermore, MBNL1 and U2AF65 appear to compete by binding to mutually exclusive RNA structures. When bound by splicing factors, the 3' end of intron 4 can form either a stem-loop or a single-stranded structure. MBNL1 binds a portion of the intron as a stem-loop, whereas U2AF65 binds the same region in a single-strand structure. Mutations that strengthen the stem-loop decrease U2AF65 binding affinity and also repress exon 5 inclusion, independently of MBNL1. Thus, U2AF65 binding can be blocked either by MBNL1 binding or by the stabilization of RNA secondary structure.

Identification of motifs that function in the splicing of non-canonical introns.

BACKGROUND: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment. RESULTS: Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts. CONCLUSION: The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T.

Myotonic dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the RNA splicing factor muscleblind-like (MBNL) is sequestered to the expanded CUG or CCUG RNAs. Mislocalization of MBNL results in missplicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. We demonstrate that MBNL can bind short structured CUG and CCUG repeats with high affinity and specificity. Only 6 base pairs are necessary for MBNL binding: two pyrimidine mismatches and four guanosine-cytosine base pairs in a stem. MBNL also has a preference for pyrimidine mismatches, but many other mismatches are tolerated with decreased affinity. We also demonstrate that MBNL binds the helical region of a stem-loop in the endogenous pre-mRNA target, the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. In vivo splicing results indicate that MBNL-regulated splicing is dependent upon the formation of stem-loops recognized by MBNL. These results suggest that MBNL may bind all of its RNA substrates, both normal and pathogenic, as structured stem-loops containing pyrimidine mismatches.