From old samples, new breast cancer answers

OHSU scientists have shown how it’s possible to obtain detailed views of breast cancer gene activity from preserved tumor samples more than a decade old.

The researchers say their RNA sequencing process for formalin-fixed, paraffin embedded (FFPE) tissue can be used to explore key problems in the breast cancer field, such as understanding the mechanisms of drug resistance, and discerning life-threatening tumors from those unlikely to spread.

FFPE samples are widely collected and archived for at least 10 years, making them a rich source for studying connections between tumor biology and long-term patient outcomes. But fixation and storage damage RNA and DNA molecules.

The OHSU team developed an RNA sequencing pipeline optimized at every step for FFPE samples. A paper published in BMC Medical Genomics gives the details. The FFPE breast cancer tissues the researchers worked with had been stored at room temperature between 2 and 23 years. The figure above on the left shows RNA yield from different sized sections of FFPE breast tissue. The figure on the right shows the quality of isolated RNA by year of collection as assessed by DV200, the percentage of purified RNA with length greater than or equal to 200 nucleotide bases and thus suitable for RNA sequencing. The optimized sequencing approach achieved high concordance between FFPE gene signatures and those of fresh tissue samples. And it enabled biomarker discovery even though the cohorts of FFPE and fresh tissues were not subject-matched, not prepared by the same library method, and not sequenced by the same facility.

Three scientists share first author position: postdoctoral fellow Nathan Pennock, Ph.D., research assistant professor Sonali Jindal, M.D., and computational biologist Wesley Horton, M.S. The senior co-authors are Pepper Schedin, Ph.D., a professor of cell, developmental and cancer biology, and Zheng Xia, Ph.D., an assistant professor in the Computational Biology Program and the Department of Molecular Microbiology and Immunology. Two OHSU research core directors are co-authors: Christina Harrington, Ph.D., director of the Gene Profiling Shared Resource and DNA Services Core, and Robert Searles, Ph.D., director of the Massively Parallel Sequencing Shared Resource.

While many protocols for extracting RNA from FFPE blocks call for using multiple sections comprising a total of 40 micrometers of tissue thickness, the OHSU team showed it can be done reliably with one-fourth as much tissue. They obtained reliable gene expression information on more than 14,000 genes from a single 10 micrometer section of FFPE breast cancer tissue. RNA sequencing was able to distinguish between breast tumors that are estrogen receptor positive or negative. They confirmed gene expression fidelity by comparing FFPE data to publicly available databases obtained from fresh tissues.

Going further in a proof-of-principle application, Xia’s lab used RNA sequencing data from FFPE samples to explore gene association networks known as regulons. That analysis revealed a possible connection between the transcription factor KDM4B and survival in patients with estrogen receptor positive breast cancers. While needing further confirmation, the authors say it’s the first report of differential expression and activity of KDM4B within ER+ breast cancers correlating to patient survival.

The research is one of six projects selected for testing an enhanced core-coordination process at OHSU, a pilot process that began in February 2017. The aim was to find out to what extent enhanced, more thorough communication and planning among cores and with investigators might contribute to more successful outcomes, improved efficiency and faster turnaround. Despite some challenges, most of the core directors involved concluded that projects requiring support from multiple cores can greatly benefit from a formal coordination process. Schedin said her research team agrees. “Regular meetings with our core staff collaborators resulted in improved research designs and analysis innovations that were essential to the success of this transdisciplinary project.”

Further reading:

RNA-seq from archival FFPE breast cancer samples: molecular pathway fidelity and novel discovery by Nathan D. Pennock, Sonali Jindal, Wesley Horton, Duanchen Sun, Jayasri Narasimhan, Lucia Carbone, Suzanne S. Fei, Robert Searles, Christina A. Harrington, Julja Burchard, Sheila Weinmann, Pepper Schedin and Zheng Xia. BMC Medical Genomics (Dec. 19, 2019)