Solving the FFPE Problem: Scientists Coax High-Quality Results from Low-Quality Samples
NGS expert Brian Walker is helping to bring personalized treatment to cancer patients. Along the way, he built a pipeline using library prep kits from Kapa Biosystems to generate high-quality sequencing results from even the most degraded FFPE samples.
For scientists trying to usher genomics into routine clinical use, one of the biggest hurdles is finding enough samples necessary for testing workflows, discovering biomarkers, and evaluating treatments. Formalin-fixed, paraffin-embedded (FFPE) tissue samples represent a possible solution to this issue — indeed, by some estimates there are more than a billion FFPE samples stored around the world, representing countless phenotypes — but unlocking their value has been a significant challenge. FFPE samples often yield degraded, highly fragmented DNA that usually leads to poor sequencing results.
Brian Walker wants to help his fellow translational scientists tap these gold mines of patient data. As a senior research scientist in the Center for Molecular Pathology at the Royal Marsden Hospital in London, he led an optimization effort that developed a robust pipeline for processing and sequencing FFPE samples, achieving high-quality results in a clinically useful time frame. Critical to that pipeline is the single-tube DNA fragmentation and library prep provided by KAPA HyperPlus Kits from Kapa Biosystems.
At the Royal Marsden Hospital, Walker’s goal is to improve molecular diagnostic protocols and develop new tools for better diagnosis and treatment of patients with cancer. The hospital and its academic partner, the Institute of Cancer Research, serve as the largest comprehensive cancer center in Europe, treating more than 50,000 patients each year.
Walker’s base is the Translational Research Centre, a cutting-edge facility designed to speed the deployment of these molecular diagnostic techniques to clinical utility. With a background in immunology and myeloma genetics, Walker now focuses on the implementation of next-generation sequencing technologies in clinical labs; he has sequenced hundreds of patients for the hospital. “My main responsibilities are to evaluate new molecular diagnostic techniques, implement standard operating procedures and wet lab protocols, and eventually, push them through to our molecular diagnostics laboratory,” he says. “I am responsible for ensuring these techniques pass all of our rigorous validation processes and meet the Clinical Pathology Accreditation standards before they are transferred over.”
Walker is part of a team that has developed NGS panels to assess relevant mutations in cancers of the breast, lung, blood, and gastrointestinal tract, as well as pediatric cancers. “NGS provides us with a sensitive and faster method to test many genes at once, leading to quicker turnaround times, a more cost-efficient process, and more effective treatment of patients,” he says. The panels must be able to detect single nucleotide changes, indels, copy number abnormalities, wild type status, and more. In some cases, the team has taken panels previously designed for PCR and reinvented them for NGS workflows. In one such example, Walker and his colleagues worked with the EuroClonality Consortium on an NGS capture protocol that enabled better detection of rearrangements and translocations in B cells and T cells.
Two of the team’s panels — breast and gastrointestinal cancers — are currently being used in clinical trials. For the GI study, which has been going on for about a year and aims to recruit 250 patients, Walker and his team are using a panel to look at 46 genes in tumor and matched normal samples from each participant to recommend more personalized treatment courses. “It’s so much easier to do analysis when you have a matched blood sample from the same patient,” he says, noting that this process allows for the straightforward removal of germline SNPs from analysis, focusing interpretation efforts on the most likely candidates. “It makes reporting the data in the clinic much easier and more robust,” Walker adds.
The GI study shows just how far the team has come in a short time. When it first launched, turnaround time for results clocked in at 5.5 months — a delay too long to assist in treatment decisions for these patients. Most of the lag could be attributed to long wait times to get samples from other hospitals and to the sequencing workflow, Walker says. But in the last six months, technology and other improvements have slashed three full months from the process, leading to delivery of results in nine or 10 weeks.
Some of that optimization work led to increased panel uniformity and excellent metrics for sensitivity, specificity, and reproducibility of results. The overall process has been validated for use in clinical labs, Walker says.
Finding Value in FFPE Samples
Prior to his molecular diagnostics work, Walker most frequently used fresh-frozen DNA samples that are known for their excellent quality in NGS and other pipelines. The shift to a hospital setting introduced him to the frustrations of FFPE samples. “The DNA has been degraded and is often low quality,” he says. “The differences in DNA sample qualities have been shocking.”
But those FFPE samples hold so much potential value — for biomarker discovery, translational research, and diagnostics — that Walker knew he had to come up with some way to get better results from them. His team tackled the challenge head-on. “We’ve done a lot of work in the last year and a half to try and improve how we can use FFPE DNA for NGS technology,” he says. They quickly realized that there were quality nuances among FFPE samples as well. “We often see that samples coming from different hospitals behave in a different way. This usually depends on how the samples were fixed in the first place,” Walker says.
What they needed was a robust sample preparation approach that would be effective across all FFPE samples. Walker tested out KAPA Hyper Prep Kits and immediately found that median sequencing coverage increased significantly. The team has now switched to KAPA HyperPlus Kits, which incorporate enzymatic fragmentation for a more streamlined workflow. The kits generate higher yields while minimizing amplification bias, producing superior sequencing results even from highly fragmented or degraded samples.
“We now have a lot more diversity, our coverage is up, and our libraries have fewer duplicates,” Walker says. “The performance of the KAPA HyperPlus Kit essentially turns my FFPE samples into regular samples.”
For Research Use Only. Not for use in diagnostic procedures.
Brian Walker is now at the University of Arkansas for Medical Sciences (UAMS), 4301 W. Markham, #816, Little Rock, Arkansas 72205.
There is no conflict of interest.
Written by Meredith Salisbury of Bioscribe, a public relations consultancy dedicated to supporting the communications goals of organizations that are advancing biotechnology, genomics, bioinformatics, molecular diagnostics, and molecular and synthetic biology.