June 13, 2017

Story by Rebecca Hood

The Paper of the Month for May 2017 is "Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis," published in Cell Reports. 

This highly collaborative paper features research from multiple departments at the OHSU School of Medicine, including Cell, Developmental and Cancer Biology, Biomedical Engineering, Otolaryngology/Head Neck Surgery and Dermatology as well as the school's Computational Biology Program and the School of Public Health. 

Takahiro Tsujikawa, M.D., Ph.D., Sushil Kumar, Ph.D., Rie Kawashima, D.D.S., Ph.D., Gina Choe, B.S. and Lisa M. Coussens, Ph.D., chair and professor, all of whom are in the Department of Cell, Developmental and Cancer Biology, OHSU School of Medicine, coordinated and oversaw the project while developing the experimental platform supporting in the research.

A closer look at immune therapies

While scientists have known that the body's own immune system can be harnessed to attack cancer cells, they have also come to realize that patients will respond to immune therapy with varying degrees of success. Some patients may respond well initially but then develop a resistance to the therapy. And, as an additional challenge, some patients may have tumors that do not express many of the markers used as signals for the immune system to target the cells. 

To understand why immune therapies have such varying levels of success, Drs. Tsujikawa and Coussens set out to develop experimental tools. Their goal was to identify tumor biomarkers with which to stratify cancer patients in order to improve the odds that they will respond to immune therapy. In addition, they were looking for new ways to prospectively monitor patient responses, like remission and resistance, over time.

To subclassify tumors into types that might be more or less responsive to immune therapy, the multi-disciplinary team designed panels of antibodies recognizing lineage-specific markers on immune cells (leukocytes) that infiltrate solid tumors. 

While this research is promising, there is a bottleneck: getting samples to study. "A major obstacle for deployment of such a strategy is based on limited quantities of precious tumor-derived specimens for monitoring," said Dr. Tsujikawa. So not only must researchers find a way to use tumor leukocyte biomarkers to reliably predict patient outcomes, they must develop experimental methods using limited amounts of precious tissue.

Utilizing IHC

In order to tackle this problem, Drs. Tsujikawa and Coussens and team implemented a commonly used method called immunohistochemistry (IHC). IHC allows researchers to use an antibody to specifically identify a protein of interest in a slice of tissue and visualize where the protein is located. Computer programs can quantify how much of the protein is present. 

Dr. Tsujikawa was able to develop a method where IHC is done sequentially, allowing up to 12 different proteins to be analyzed in a step-wise manner from a single piece of tissue. The team obtained tissue with patient permission from Johns Hopkins University and the OHSU School of Medicine Departments of Otolaryngology/Head Neck Surgery and Dermatology.

Once tumor sections were sequentially stained with the panels of antibodies, they were digitally scanned. To quantitatively interpret results from the IHC, software-enabling visualization and image analysis was developed in collaboration with Rohan Borkar and Vahid Azimi – current OHSU medical students – and Guillaume Thibault, Ph.D., Young Hwan Chang, Ph.D., and Joe Gray, Ph.D., all of the Department of Biomedical Engineering, and Ariel Balter, Ph.D., Patrick Leyshock, Ph.D. and Adam Margolin, Ph.D., in the Computational Biology Program. Once the data were collected, statistical analysis was performed by Motomi Mori, Ph.D., a biostatistician in the School of Public Health. 

The multi-disciplinary team used three separate panels of twelve biomarkers each to investigate tumor leukocyte complexity. One panel contained biomarkers specific to lymphoid cells; one panel consisted of biomarkers specific to myeloid cells; and a third panel was specific for cytotoxic T cells. Lymphoid and myeloid cell types play important roles in immune response to pathogens, but in tumor tissue, they can either facilitate tumor growth, or instead eradicate tumor cells by direct killing. 

Validation

To initially validate the IHC methodology, the Coussens Lab used oropharyngeal head and neck squamous cell carcinomas. Some of the samples had been infected with human papilloma virus (HPV), which creates specific and well-known effects on the immune system. 

The team was able to separate the samples by HPV status using their technique and revealed that tumors containing higher percentages of myeloid immune cells correlated with reduced patient survival. Moreover, using pancreatic carcinomas from patients that had received a prior immune therapy, e.g., a vaccine designed to stimulate cytotoxic T cells, the team was able to stratify patients based on their response to immune therapy and identified a specific immune complexity within the pancreatic tumors that predicted that response, all using only three tissue sections.

Although the team focused on just two specific types of cancers in the present manuscript, Dr. Tsujikawa explained, "The method can be expanded to include antibody panels reflecting a diversity of biomarkers illuminating a wide variety of biological features in tissue and tumors." 

Importantly, the method can be readily adopted without significant expense. "Since basic procedures in this method are shared with traditional IHC, it is highly practical in terms of equivalent cost and instrument requirements to standard IHC," said Dr. Tsujikawa. 

According to Dr. Mori, "This [paper] will facilitate biomarker studies of early detection and progression, as well as those aimed at understanding complexities of the tumor microenvironment."

Power of collaboration

Mary Heinricher, Ph.D., associate dean for basic research, OHSU School of Medicine, describes what caught her interest about this study. "This paper truly exemplifies the power of partnerships and collaboration," she said. 

Dr. Mori elaborates. "It demonstrates that we can collaborate and work together for a common goal." 

Thanks to this highly collaborative project, researchers now have a powerful new tool to study tissues and tumors.

This project was supported by a 2016 OCTRI Catalyst Award, in addition to other funding to the Coussens laboratory.

Resources

• Read the paper
• OHSU Cancer Translated article

Citation

Quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor-immune complexity associated with poor prognosis Takahiro Tsujikawa, Sushil Kumar, Rohan N. Borkar, Vahid Azimi, Guillaume Thibault, Young Hwan Chang, Ariel Balter, Rie Kawashima, Gina Choe, David Sauer, Edward El Rassi, Daniel R. Clayburgh, Molly F. Kulesz-Martin, Eric R. Lutz, Lei Zheng, Elizabeth M. Jaffee, Patrick Leyshock, Adam A. Margolin, Motomi Mori, Joe W. Gray, Paul W. Flint, and Lisa M. Coussens. Cell Reports. April 4, 2017.

More Published Papers

• Previous School of Medicine Papers of the Month
• Recent OHSU published papers

Pictured above: Members of the Coussens Lab.