What if science had the ability to tap into the genes of the past to investigate a current disease?
The results of a recent study, a collaboration between researchers from the Notre Dame Genomics and Bioinformatics Platform (GBCF) and the Mayo Clinic, mean that science could be heading in this direction for breast cancer and other diseases.
The paper, titled “Quality Control Recommendations for RNASeq Using FFPE Samples Based on Pre-sequencing Lab Metrics and Post-sequencing Bioinformatics Metrics,” was published in BMC Medical Genomics Online on September 16 and has 18 contributors from both organizations.
The multi-year project began with a cohort of patient samples from Amy Degnim, a breast cancer surgeon and researcher at the Mayo Clinic. Degnim, herself a Notre Dame graduate, had old benign samples from a subset of her patients who later developed breast cancer.
“Our interest is to come back to these benign biopsies [the women] had years before developing cancer and comparing biopsies from women who had cancer to those who did not,” she explained. “What are the differences, the molecular differences in the tissues that would give us clues?”
Degnim added that having samples from the same person at two different time periods can provide insight into possible somatic mutations, instead of inherited germline mutations that are commonly studied such as BRCA 1 and 2 in breast cancer.
“Most women who have breast cancer do not have a BRCA mutation or another genetic mutation. We believe that most women who have breast cancer have somatic mutations – cumulative errors in the genome that then translate into errors in cell proliferation genes and cause certain cells carrying these mutations to grow out of control.
Degnim said she realized she had a once-in-a-lifetime opportunity to almost literally rewind time to a period before the cancer developed; however, samples were formalin-fixed and paraffin-embedded, a sort of preservation technique that allowed the degradation of DNA and RNA molecules needed for sequencing.
Degnim shared her problem with a colleague, who put her in touch with the Notre Dame GBCF. The director Michael Pfrenderassistant director Melissa Stephens and technical scientist Brent Harker took on the challenge.
“They were willing to take on this project to push the boundaries of successful RNA sequencing on these really, really old tissues,” Degnim said.
The leg work
According to Stephens, the partnership began in 2017, went on a short hiatus during the COVID-19 pandemic, and work for the newspaper was finally completed in 2021. She also noted that GBCF was working closely with Derek Radiskyanother contributor to the article and a cancer biology researcher at the clinic.
While Degnim, Radisky and others handled brainstorming and broader clinical applications, GBCF collaborators did the majority of legwork on the project, including RNAseq, a specialty of the center.
Stephens explained that RNAseq is a technique used to quantify the amount of RNA transcribed using next-generation sequencing.
“Using RNAseq allows you to look at the differences – what’s on, what’s off in these genes – and get insights into gene function,” she said. “[RNAseq] helps to better understand the underlying biology, in this case, of a particular disease.
The problem, Stephens said, was not with the archived preserved samples (FFPE) themselves, but with the method of RNA extraction and how to enrich or label strands of interest.
“You have to use these other methods to try to get the coding transcriptome [of the samples] without using traditional approaches,” she said.
Stephens described two main methods that she and Harker used in the article: the ribosomal depletion method or the exome sequence capture approach. The conclusion drawn in the paper was that the exome capture approach gave better enrichment results than the depletion method.
At the end of a long period of trial and error to find the best method, Pfrender explained that it was necessary to determine which sample data was reliable or “trustworthy”.
“Across a whole range of samples, some were in pretty good shape, and some were in really bad shape. So the big challenge for us was trying to figure out how to quantify that so we knew which ones were safe to go from. forward,” he said. “The statisticians really looked at the data closely to try to figure out ‘what’s the roadmap? where are these cuts?’
The article’s conclusion consisted of methodological recommendations on how to obtain usable data and advice on how to judge good and bad samples.
Pfrender also took the opportunity to talk about the work ethic and patience of the center’s researchers.
“It’s truly quite an accomplishment, and it’s entirely up to their expertise and infinite patience to try to bring this project to fruition. I think most facilities would have just given up,” he said. “You lose a lot of potentially very important information…these types of samples are quite rare and really valuable.”
Although the samples were located on breast tissue and the breast cancer study, Stephens said she believes the recommendations they created are viable for all FFPE samples.
Pfrender added in his belief that the standards and methods developed could be used to go back and study FFPE samples from 30 years in the past and beyond.
In the future
Moving forward, Degnim told The Observer that they are currently analyzing the RNAseq results.
“The first step was just, ‘Can we trust this data?'” she explained. “Now we’re analyzing the data from those samples that pass the quality measures to find out what the differences are in gene expression in women who will develop cancer in the future and women who don’t. “
Degnim said she hopes to identify a biomarker, cancer causative or otherwise, that she can incorporate into future research efforts and other studies.
“[This research] is a discovery effort to try to find new factors that can either predispose to breast cancer or tell us that if a woman expresses these changes in her RNA, she will be at increased risk of developing breast cancer,” said Denim.
A graduate of Notre Dame herself, Degnim noted that she was really excited to work with the University on this project.
“It’s been really, really exciting and really endearing for me to be able to go back now and have this collaboration with my alma mater,” she said.
Contact Bella Laufenberg at email@example.com