Clinical Implications of a Targeted RNA-Sequencing Panel in the Detection of Gene Fusions in Solid Tumors.

The detection of recurrent gene fusions can help confirm diagnoses in solid tumors, particularly when the morphology and staining are unusual or nonspecific, and can guide therapeutic decisions. Although fluorescence in situ hybridization and PCR are often used to identify fusions, the rearrangement must be suspected, with only a few prioritized probes run. It was hypothesized that the Illumina TruSight RNA Fusion Panel, which detects fusions of 507 genes and their partners, would uncover fusions with greater sensitivity than other approaches, leading to changes in diagnosis, prognosis, or therapy. Targeted RNA sequencing was performed on formalin-fixed, paraffin-embedded sarcoma and carcinoma cases in which fluorescence in situ hybridization, RT-PCR, or DNA-based sequencing was conducted during the diagnostic workup. Of the 153 cases, 138 (90%) were sequenced with adequate quality control metrics. A total of 101 of 138 (73%) cases were concordant by RNA sequencing and the prior test method. RNA sequencing identified an additional 30 cases (22%) with fusions that were not detected by conventional methods. In seven cases (5%), the additional fusion information provided by RNA sequencing would have altered diagnosis and management. A total of 19 novel fusion pairs (not previously described in the literature) were discovered (14%). Overall, the findings show that a targeted RNA-sequencing method can detect gene fusions in formalin-fixed, paraffin-embedded specimens with high sensitivity.

Impact of Reducing DNA Input on Next-Generation Sequencing Library Complexity and Variant Detection.

PCR amplification, a key step in next-generation sequencing (NGS) library construction, can generate an unlimited amount of product from limited input; however, it cannot create more information than was present in the original template. Thus, NGS libraries can be made from very little DNA, but reducing the input may compromise assay sensitivity in ways that are difficult to ascertain unless library complexity (ie, the number of unique DNA molecules represented in the library) and depth of coverage with unique sequence reads (those derived from input DNA molecules) versus duplicate sequence reads (those resulting from overamplification of particular molecules) are discretely measured. A series of experiments was performed to explore the impact of low DNA input on an amplicon-based NGS assay using unique molecular identifiers to track unique versus duplicate reads. At high sequencing depths, unique and total (unique plus duplicate) read coverage are not well correlated, so increasing the number of sequenced reads does not necessarily improve sensitivity. Unique coverage depth tends to improve with more input, but improvements are not consistent. Fluctuations in library complexity complicated variant detection using both standardized and clinical specimens, often resulting in technical replicates with vastly different estimates of variant allelic fraction. In conclusion, depth of coverage with unique reads must be tracked in clinical NGS to ensure that sensitivity and accuracy are maintained.

Requirement for MUC5AC in KRAS-dependent lung carcinogenesis.

With more than 150,000 deaths per year in the US alone, lung cancer has the highest number of deaths for any cancer. These poor outcomes reflect a lack of treatment for the most common form of lung cancer, non-small cell lung carcinoma (NSCLC). Lung adenocarcinoma (ADC) is the most prevalent subtype of NSCLC, with the main oncogenic drivers being KRAS and epidermal growth factor receptor (EGFR). Whereas EGFR blockade has led to some success in lung ADC, effective KRAS inhibition is lacking. KRAS-mutant ADCs are characterized by high levels of gel-forming mucin expression, with the highest mucin levels corresponding to worse prognoses. Despite these well-recognized associations, little is known about roles for individual gel-forming mucins in ADC development causatively. We hypothesized that MUC5AC/Muc5ac, a mucin gene known to be commonly expressed in NSCLC, is crucial in KRAS/Kras-driven lung ADC. We found that MUC5AC was a significant determinant of poor prognosis, especially in patients with KRAS-mutant tumors. In addition, by using mice with lung ADC induced chemically with urethane or transgenically by mutant-Kras expression, we observed significantly reduced tumor development in animals lacking Muc5ac compared with controls. Collectively, these results provide strong support for MUC5AC as a potential therapeutic target for lung ADC, a disease with few effective treatments.