Ladanyi, M. | Search bioRxiv

bioRxiv2026

Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.

Genomic, transcriptomic and epigenomic characterization has accelerated the discovery of clinically-relevant alterations in cancer, predominantly using fresh frozen (FF) specimens. However, clinical molecular pathology laboratories prefer formalin-fixed paraffin-embedded (FFPE) methods, known to introduce artifacts at the nucleic acid level, over fresh frozen methods. Extending the multi-platform analysis to FFPE specimens for comprehensive clinical molecular diagnosis requires a thorough understanding of the consequence of formalin-fixation. We present a detailed multi-platform characterization of FFPE preservation using paired FF specimens as the 'gold standard'. DNA and RNA were obtained from 38 patients across 6 cancer types using a FFPE optimized co-isolation. The impact of FFPE on exome sequencing was dependent on filtering, where a minimum coverage or supporting read filter can mitigate FFPE-specific false positives. Copy number alterations, MSI assessment, mutational signatures, and DNA methylation were comparable between FFPE and FF. FFPE biases in RNA expression can be overcome when using biology-relevant genes and we describe a novel consequence of FFPE on miRNA species diversity. Collectively, this data provides a broad view of FFPE artifact and offers best practices for overcome these biases.

bioinformatics↗

bioRxiv2018

YES1 amplification: a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics

In approximately 30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify novel mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1. Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in 5 cases, 4 of which lacked any other known mechanisms of resistance. Pre-inhibitor samples, available for 2 of the 5 patients, lacked YES1 amplification. None of 136 post-inhibitor samples had detectable amplification of other Src family kinases (SRC, FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a novel, recurrent, and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers, and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.\n\nSIGNIFICANCEDespite high response rates to treatment with small molecule inhibitors of EGFR tyrosine kinase activity, patients with EGFR-mutant lung adenocarcinomas eventually develop resistance to these drugs. In many cases, the basis of acquired resistance remains unclear. We have used a transposon mutagenesis screen in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify amplification of YES1 as a novel and targetable mechanism of resistance to EGFR inhibitors in EGFR-mutant lung cancers.

cancer biology↗