Uncovering Checkpoint Biomarkers

Cancer cells cloak their identity to avoid detection from host defense cells by exploiting genes important to differentiate between self and non-self. Though cells in a tumor mass may go undetected by the immune system, their presence leaves behind molecular clues. Researchers like Lisa Butterfield are sleuthing using archived blood and tumor samples to find biomarkers and capture the presence of these tumor cells.

What is the role of the research laboratory in clinical trials? What is the role of your biomarker core facility in clinical trials?

I direct a research laboratory and a cancer center core facility for the immunologic monitoring and development of cellular products. Initially, we perform translational research and development. Once the clinical trial is rolling, we process and bank both blood and tissue samples in a standardized fashion. Immunological assays are used to elucidate the drug mechanism of action in these clinical research samples and find potential prognostic and predictive biomarkers in the future.

Our core is useful for clinical trials as we have standard operating procedures in place. Competency training is paramount for everyone who touches clinical research samples. This means all samples are processed the same way to minimize variables, reduce error and noise, and to maximize meaningful signal. We also have a large bank of healthy donor serum. While each subject can serve as their own control when we are looking for a change over baseline during time course studies, we also have a big collection of healthy, noncancer donors that are used as a comparison to cancer subjects, giving us an accurate baseline to know what is normal and what is not.

How does a group start the process for biomarker verification?

After discovering a potential biomarker, there is a roadmap to standardization and validation. The Society for Immunotherapy Cancer (SITC) released open-access white papers outlining the analytical and clinical validation of immunotherapy biomarkers. There are 5 white papers and a meeting summary report, all found here on the society’s website: sitcancer.org/research/biomarkers

How did your core facility become involved with the SARC028 clinical trial?

SARC028 is the first clinical study of PD-1 blockade in sarcomas. The SARC cooperative group and their clinical leads, Drs. Hussein Tawbi and Melissa Burgess, were responsible for starting and co-ordinating this clinical trial. Our collaboration formed to standardize handling and banking of blood samples in order to look for mechanisms of action and biomarkers of the immune-tumor response.

Let’s set the stage for the SARC028 clinical trial. Can you give us the background for the study?

In a sense, PD-1 upregulation on lymphocytes is an activation marker. T cells upregulate their expression of PD-1 when exposed to antigen, and that includes tumor antigen. Tumors across a spectrum can upregulate PD-ligand 1 (PD-L1) and express it on the surface of both the tumor cell and myeloid cells in the tumor. PD-1 upregulation and binding to PD-L1 induces T cells to reduce their activity or shut them off. Antibodies that block the PD-1 pathway are designed to interfere with downregulation of T cell activity and allow them to do a better job of eradicating the tumor.

What role did PD-L1 play in the SARC028 trial?

The role of PD-L1 had not yet been investigated in sarcomas. However, it has been hypothesized that patients with higher levels of PD-L1 in tumors are more likely to respond to therapy. The investigators recruited a diverse set of sarcoma patients to test which subset would most likely respond to PD-1 therapy. They collected and analyzed tumor samples for the correlation between PD-L1 expression and positive response to therapy. My group was responsible for testing serum samples to look for generation of tumor antigen–specific immunity and novel prognostic markers to better understand which patients would respond to therapy.

SARC028 found some subgroups of sarcoma patients did not benefit particularly well from the PD-1 pathway blockade. However, there were subsets of patients who did benefit. We found from our biomarker testing that there were novel correlations between soluble PD-1 and PD-L2, and clinical outcomes. Some of our other investigations found possible soluble factors markers, such as IL-15, which correlated with a positive anti-tumor kind of immune milieu in the serum. Our goal is to identify a robust biomarker, then create a panel to test for which patients would benefit from PD-1 blockade.

Can you talk about liquid biopsy and what are the challenges with liquid biopsy?

Finding checkpoint biomarkers from tumors is difficult because the biopsy is an invasive procedure. Ideally, the best tumor samples are taken at points where standard of care biopsies and surgical resections occur. Repeatedly taking a solid tumor sample at different times in the trial is a challenge. Blood samples are more attractive as collection is minimally invasive, can be drawn at any time point and in a reasonable amount. Serum can be aliquoted and easily stored at –80˚C for future testing.

How are the ProcartaPlex panels* for the Luminex platform applied in the SARC028 trial?

Multiplex measurement of soluble forms of the immune checkpoint receptors and ligands is novel. There have been only a few preliminary suggestions that the soluble forms play a role and this is the first evidence that measuring these soluble factors correlates with treatment in these sarcoma patients, and may have clinical utility to potentially predict who might benefit from a given therapy.

Detection of 65 cytokines, chemokines, and growth factors in a single Luminex® assay was also a favorable trait as it combines many analytes into a single assay and with small sample volumes. This diversifies the use of Luminex-based immunoassays for broad biomarker discovery and validation rather than only for testing a specific hypothesis.

What do you think is the future of biomarkers in immuno-oncology research?

An important evolution is taking place. Traditionally, we only had easy access to blood and not the solid tumor. After years of looking in the blood, there were not many robust biomarker signals from those methods. Advancements in biotechnology and pharmaceutical fields provided new technologies and research is to now used to examine both tumor and blood samples for markers. We can now better understand what is happening in the tumor and this can then be applied to finding those signals and biomarkers in the blood.