What if science had the ability to dip into past genes to investigate a current ailment?
The results of a recent study, a collaboration between researchers at the Notre Dame genomics & bioinformatics core facility (GBCF) and the Mayo Clinic, mean that science might be headed in that 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 Sept. 16 and has 18 contributors from the two organizations.
Humble beginnings
The years-long project started with a cohort of patient samples from Amy Degnim, a specialized breast cancer surgeon and researcher at the Mayo Clinic. Degnim, a graduate of Notre Dame herself, had old benign samples from a subset of her patients that later developed breast cancer.
“Our interest is in looking back at these benign biopsies [the women] had years before they ever developed the cancer and comparing the biopsies of the women who did get cancer to those who didn’t,” she explained. “What are the differences, the molecular differences in the tissue that would give us some clues?”
Degnim added that having samples from the same person at two different time periods can give insights into possible somatic mutations, instead of hereditary, germline mutations that are commonly studied such as the BRCA 1 and 2 in breast cancer.
“Most women who get breast cancer don’t have a BRCA mutation or another genetic mutation. Most women who get breast cancer have, we think, somatic mutations — cumulative errors in genome that then translate into errors in cell proliferation genes and cause certain cells that have these mutations to proliferate out of control.”
Degnim said she realized that she had a very unique opportunity to almost literally look back in time to a period before the cancer developed; however, the samples were formalin-fixed and paraffin-embedded, a kind of preservation technique that allowed for the degradation of the DNA and RNA molecules needed for sequencing.
Degnim brought her problem to a colleague, who connected her to the Notre Dame GBCF. There, director Michael Pfrender, assistant director Melissa Stephens and technical scientist Brent Harker were up to the challenge.
“They were willing to take this project to push the limits of how successfully we can do RNA sequencing on these really, really old tissues,” Degnim said.
The legwork
According to Stephens, the partnership started in 2017, took a small break during the COVID-19 pandemic and the work for the paper was finally finished in 2021. She also noted that the GBCF worked closely with Derek Radisky, another collaborator on the paper and a cancer biology researcher at the clinic.
While Degnim, Radisky and others dealt with larger brainstorming and clinical applications, the collaborators at the GBCF did a majority of the 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 differences — what’s turned on, what’s turned off in these genes — and get information about the function of the genes,” she said. “[RNAseq] allows you to better understand the underlying biology of, in this case, a particular disease.”
The issue, Stephens said, was not with the archived preserved (FFPE) samples themselves but with the method of RNA extraction and the manner of enriching, or marking, the strands of interest.
“You have to use these other methods to try and get the coding transcriptome out [of the samples] without using the traditional approaches,” she said.
Stephens outlined two main methods that she and Harker used in the paper: the ribosomal depletion method or the exome sequence capture approach. The conclusion drawn in the paper was that the exome capture approach yielded better enrichment results than the depletion method.
At the end of a long trial and error period to figure out the best method, Pfrender explained that there was a need to figure out which samples’ data was reliable or “trustworthy.”
“In a whole range of samples, some were in pretty good shape, and some were in really rough shape. So, the big challenge for us was to try to figure out how to quantify that so we know which ones were safe to move forward,” he said. “The statisticians really took a harsh look at the data to try to figure out ‘what’s the roadmap? where are those cut offs?’”
The paper’s conclusion consisted of method recommendations on how to get usable data and guidance of how to judge good and bad samples.
Pfrender also took the opportunity to talk up the work ethic and patience of the center’s researchers.
“It’s really quite an accomplishment, and it’s completely up to their expertise and infinite patience trying to work through this project. I think most facilities would have just given up,” he said. “You lose a lot of potentially very important information … these kinds of samples are quite rare and really precious.”
Although the samples were localized to breast tissue and the study about breast cancer, Stephens said she believed 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 into the past and beyond.
Into the future
Moving forward, Degnim told The Observer that they are currently in the process of analyzing the results of the RNAseq.
“The first step was just, ‘Can we trust this data?’” she explained. “Now we’re analyzing the data in those samples that pass the quality metrics to find out what the differences are in gene expression in women who develop cancer in the future and the women who did not.”
Degnim said she hopes to identify some biomarker, causative of the cancer or otherwise, that she can incorporate into future research endeavors and more studies.
“[This research] is a discovery effort to try to find new factors that either may predispose to breast cancer or will tell us that if a woman is expressing these changes in their RNA, they will be at an increased risk to get a breast cancer,” Degnim mentioned.
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 very thrilling and very endearing for me to be able to circle back now and have this collaboration with my alma mater,” she said.
Contact Bella Laufenberg at ilaufenb@nd.edu
