For years, oncologists have prescribed targeted cancer therapies based on the DNA sequences of individual patient tumors. But more recently, studies focused on data from RNA analysis also provide critical information to more efficiently address cancer with great effect. Now, new research from a European team confirms that sequencing these molecules in parallel to DNA can help clinicians make cancer diagnoses faster, cheaper and more accurate.
The retrospective study, which is the largest of its kind to-date, analyzed both the DNA and RNA from tumors of 3,000 patients diagnosed with late-stage non-small cell lung cancer (NSCLC) between July 2014 and April 2018. The integrated, next-generation sequencing (NGS) approach requires less tissue and is faster and more cost-effective than traditional single-gene testing, said Albrecht Stenzinger, M.D., head of the Center for Molecular Pathology at the Institute of Pathology (IPH), Heidelberg University Hospital. Stenzinger spearheaded the study, which was recently published in the International Journal of Cancer.
Stenzinger, working with a multi-disciplinary clinical research team from several contributing German institutions, showed that the results from simultaneous, targeted NGS of DNA and RNA exceeded current international guidelines for clinical diagnosis on several fronts. And the specially designed assay they used in the study helped cast a wider net to identify a broader set of therapeutic options.
The team leveraged its Ion AmpliSeq-based assay for solid tumor analysis and the Ion Torrent sequencing platform to reduce turnaround time to six working days on average (international guidelines currently recommend 10 working days). Dropout rates, which occur when there is an insufficient quantity of tumor material, were also reduced to 3.4 percent from 6.5 percent with average sample sizes of 10ng of DNA and 20ng of RNA.
In total, the combined benefits of the integrated NGS approach contributed to the clinical research team’s goal of obtaining suitable diagnostic information for as many patients as possible. They also highlight the practicability of applying the method in a routine clinical setting to support individual decisions in patient care and clinical and translational research.
“What we are demonstrating is a one-stop shop approach, where we extract DNA and RNA from small biopsies in parallel and consequently sequence them to give us the opportunity not only to have a look at mutations from the DNA level, but also on gene fusions on the transcript level,” Stenzinger said. “So, as an example, we can state that it’s not only an ALK-translocated tumor, but that the tumor also harbors an in-framed gene fusion of ELM 4 that is actively transcribed.”
While current guidelines recommend RNA analysis to be performed by fluorescence in situ hybridization (FISH), Stenzinger’s analysis also found that RNA sequencing offers a superior method to identify gene fusion variants known to have prognostic and predictive significance in the treatment of NSCLC. Gene fusions occur when part of the DNA from one chromosome moves to another chromosome; however, only the fusions that are transcribed into RNA have the potential to be expressed and potentially cause cancer. The result of the team’s findings could help physicians more effectively identify candidates for aggressive therapeutic and surveillance strategies, Stenzinger said.
“The beauty of this workflow is that it’s not sequential. You immediately know which genetic event is there and the genetic microenvironment that actually plays a role. We know that TP53 mutations shape the outcome of ALK fusion positive non-small cell lung cancers. So, for example, a variant 3 of ALK gene fusions conveys very poor survival,” Stenzinger said. “So with this parallel sequencing approach, we can provide prognostic information that goes way beyond what is currently being reported,” he said.
“Targeted therapies currently confer the longest overall survival for patients with non-small cell lung cancer, but determining who is likely to benefit from these novel treatments relies on the detection of specific biomarkers in tumor biopsies,” Stenzinger explained. “Combining targeted DNA and RNA sequencing offers clinicians and their patients the information needed to expand therapeutic options.”
The study is an ongoing effort, and the team expects to add 1,000 samples each year to the existing cohort. Stenzinger said the current approach can also be scaled and adapted for other applications, including clinical research in tumor mutation burden.
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