Researchers from Princess Margaret Cancer Centre (PM) have developed an approach to predict outcomes in patients treated with pembrolizumab—an immunotherapy drug used to treat various cancers such as head and neck, breast, and skin cancer.
Analysis for this study was performed using blood samples collected from patients with different cancer types treated with pembrolizumab in the INSPIRE trial, an investigator-initiated study that was designed and carried out at PM.
Pembrolizumab is a type of antibody that attaches to a protein known as programmed death 1 (PD-1), hindering its activity. PD-1 acts as a checkpoint for the immune system, preventing it from becoming overactive and attacking healthy cells. But in the case of cancer, PD-1 can also act as a brake to prevent the immune system from attacking cancer cells. Therefore, inhibiting PD-1 releases the brakes on the immune system and allows it to recognize and attack cancer cells more effectively.
“Although pembrolizumab is used to target many cancers, some patients may develop resistance to the drug,” says Dr. Stutheit-Zhao, Postdoctoral Researcher at PM and co-first author of the study. “Biomarkers like circulating tumour DNA (ctDNA) can provide insights into the effectiveness of treatments without the need for invasive procedures. However, many current ctDNA analyses are so-called tumour-informed, thus requiring molecular sequencing of patients' tumour tissue to identify alterations that can be tracked in the blood.”
ctDNA are pieces of tumour DNA that circulate in the bloodstream. Previous studies—including a clinical trial from this team—have shown that changes in levels of ctDNA using a tumour-informed assay early on during the treatment course can predict pembrolizumab treatment success and patient survival. However, as this assay also requires tumour samples, it may not be feasible for some patients as tumours may be in challenging locations to biopsy, or biopsy specimens may contain insufficient tumour cells for genetic analysis.
“We wanted to investigate a way to predict treatment outcome using ctDNA alone without needing to analyze a tumour biopsy,” says Dr. Enrique Sanz-Garcia, Clinician Investigator at PM and co-first author of the study. “DNA from tumours is known to be enriched for a type of modification, called methylation. Therefore, we analyzed methylated cell-free DNA from 204 plasma samples from 87 patients before and during treatment with pembrolizumab from the INSPIRE trial. This particular assay does not require access to patients’ tumour samples.”
Previous studies have demonstrated the ability of a technique, first introduced by scientists at UHN, that isolates and analyzes this methylated DNA, called cell-free methylated DNA immunoprecipitation and sequencing (cfMeDIP-seq), to estimate ctDNA levels. This technique can also quantify DNA fragment lengths, which are known to be shorter in tumour tissue compared to DNA derived from normal tissue.
The team therefore retrospectively tested whether methylation and fragmentation status of cell-free DNA using cfMeDIP-seq analysis can monitor the response to pembrolizumab in the INSPIRE study.
“Our findings indicated a strong correlation between the ctDNA analysis of methylation patterns and DNA fragmentation in blood samples alone with tumour-informed ctDNA analysis,” says Dr. Lilllian Siu, Senior Scientist at PM and senior author of the study. “Importantly, methylation analysis predicted overall survival and progression-free survival, and fragment length analysis was able to predict overall survival in patients treated with pembrolizumab.”
Using statistical analysis, the team found that a decrease in cancer-specific methylation over the first six weeks of pembrolizumab treatment was associated with a 60% lower risk of death and a 65% lower risk of disease progression compared to the reference group. For fragmentation analysis, a decrease in fragment length score over time was significantly associated with a 60% lower risk of death.
This suggests that analysis of circulating DNA assayed by cfMeDIP-seq yields promising tumour-naïve (i.e. not requiring access to tumour samples) predictive biomarkers for response to pembrolizumab.
“This is the first reported examination of methylated cell-free DNA in advanced cancer patients during treatment with pembrolizumab and one of the first direct comparisons between ctDNA analysis with and without tumour samples,” adds Dr. Trevor Pugh, Senior Scientist at PM and co-senior author of the study.
This study, published in Cancer Discovery, the lead journal of the American Association for Cancer Research, reveals the potential of a minimally invasive blood test (i.e., a liquid biopsy) for predicting response to cancer treatments. This could enable earlier response assessment by doctors and lead to prompt redirection to next-line treatment options in non-responders before radiological assessments.
Circulating tumour DNA (ctDNA) can be analyzed using blood tests as part of a liquid biopsy. Liquid biopsies are blood tests used to detect cancer cells or other markers of disease.
This work was supported by The Princess Margaret Cancer Foundation, the Ontario Institute for Cancer Research, the Terry Fox Research Institute, the Princess Margaret Cancer Centre Global Oncology Program, and the Cancer Research Institute.
Dr. Enrique Sanz-Garcia is an Assistant Professor at the University of Toronto (U of T). Dr. Lilian Siu is a Professor of Medicine at U of T and holds the BMO Chair in Precision Cancer Genomics. Dr. Pugh is a Professor in the Department of Medical Biophysics at U of T and is a tier 2 Canada Research Chair in Biological Physics.
Dr. Sanz Garcia reported research funding from Novartis. Dr. Siu reported either a leadership role, financial interest in, or a consulting role with Treadwell Therapeutics, Agios, Merck, AstraZeneca/MedImmune, Roche, Voronoi Health Analytics, Oncorus, GlaxoSmithKline, Seattle Genetics, Arvinas, Navire, Janpix, Relay Therapeutics, Daiichi Sankyo/UCB Japan, Janssen, Hookipa Pharma, InterRNA, Tessa Therapeutics, Sanofi, Amgen, and research funding from Bristol Myers Squibb (Inst),Genentech/Roche (Inst), GlaxoSmithKline (Inst), Merck (Inst), Novartis (Inst), Pfizer (Inst), AstraZeneca (Inst), Boehringer Ingelheim (Inst), Bayer (Inst), Amgen (Inst), Astellas Pharma (Inst), Shattuck Labs (Inst), Symphogen (Inst), AVID Radiopharmaceuticals (Inst), Mirati Therapeutics (Inst), Intensity Therapeutics (Inst), Karyopharm Therapeutics (Inst). Dr. Pugh has provided consultation for AstraZeneca, Chrysalis Biomedical Advisors, Merck, and SAGA Diagnostics (compensated); and receives research support (institutional) from Roche/Genentech.
For a full list of competing interests, see manuscript.
Stutheit-Zhao EY*, Sanz-Garcia E*, Liu ZA, Wong D, Marsh K, Abdul Razak AR, Spreafico A, Bedard PL, Hansen AR, Lheureux S, Torti D, Lam B, Yang SYC, Burgener J, Luo P, Zeng Y, Cheng N, Awadalla P, Bratman SV, Ohashi PS, Pugh TJ, Siu LL. Early changes in tumor-naive cell-free methylomes and fragmentomes predict outcomes in pembrolizumab-treated solid tumors. Cancer Discov. 2024 Feb 23. doi: 10.1158/2159-8290.CD-23-1060. Epub ahead of print. PMID: 38393391.
*authors contributed equally to this article.