Exome-Based ctDNA Positivity Predicts Worse DFS But Falls Short for Adjuvant Pembrolizumab Selection in High-Risk ccRCC

Exome-based circulating tumor DNA (ctDNA) positivity at baseline post-surgery was associated with significantly worse disease-free survival (DFS) with both adjuvant pembrolizumab (Keytruda) and placebo in patients with high-risk resected clear cell renal cell carcinoma (ccRCC), but the low positivity rate and low sensitivity of the assay do not support routine use of current exome-based ctDNA technology to select patients for adjuvant pembrolizumab, according to data from an analysis of the phase 3 KEYNOTE-564 trial (NCT03142334) presented during the 2026 ASCO Annual Meeting.¹

Among 736 ctDNA-evaluable patients across both treatment arms, the baseline ctDNA positivity rate was 5.4% using the 16-plex exome-based assay and 8.2% using the 64-plex assay. Specificity of baseline ctDNA for disease recurrence ranged from 96% to 99% across assays and treatment arms, but sensitivity was only 10% to 15%.

Adjuvant pembrolizumab improved DFS vs placebo regardless of baseline ctDNA status according to the 64-plex assay; the 24-month DFS rates were 79.9% and 38.8% in the pembrolizumab arm among patients who were ctDNA negative (n = 344) and positive (n = 30), respectively (P < .0001). These respective rates were 72.9% and 18.3% in the placebo arm among patients who were ctDNA negative (n = 332) and positive (n = 30; P < .0001). Similarly, among patients evaluated with the 16-plex assay, the 24-month DFS rates were 79.5% and 28.6% in ctDNA-negative (n = 353) and -positive (n = 21) patients in the pembrolizumab arm (P < .0001). In the placebo arm, the 24-month DFS rates were 71.9% and 5.3% among patients who were ctDNA negative (n = 343) and ctDNA positive (n = 19), respectively (P < .0001).

“These data highlight the limitations of current testing with exome-based ctDNA, not ctDNA in general, in adjuvant ccRCC,” -Toni Choueiri, MD, the director of the Lank Center for Genitourinary Oncology and the medical director of International Strategic Initiatives at Dana-Farber Cancer Institute, as well as the Jerome and Nancy Kohlberg Professor of Medicine at Harvard Medical School, both in Boston, Massachusetts.

What were the notable prior data from KEYNOTE-564?

Findings from KEYNOTE-564 presented during the 26th Annual Meeting of the Society of Urologic Oncology demonstrated that adjuvant pembrolizumab led to a significant DFS benefit compared with placebo (HR, 0.71; 95% CI, 0.59-0.86).² Pembrolizumab also produced a significant overall survival benefit compared with placebo (HR, 0.66; 95% CI, 0.48-0.90).

Adjuvant pembrolizumab was previously approved by the FDA for the treatment of patients with RCC who are at intermediate-high or high risk of recurrence following nephrectomy or following nephrectomy and resection of metastatic lesions in November 2021.³ The regulatory decision was supported by prior data from KEYNOTE-564.

What were the design and baseline characteristics of the KEYNOTE-564 ctDNA analysis?

KEYNOTE-564 enrolled patients with histologically confirmed ccRCC, no prior systemic therapy for advanced disease, and surgery within 12 weeks before random assignment.¹ Risk criteria included post nephrectomy intermediate-high risk (pT2 grade 4 or sarcomatoid N0; pT3 any grade N0), high risk (pT4 any grade N0; any pT any grade N+), or M1 no evidence of disease (NED) after complete resection of metastasis.

KEYNOTE-564 randomly assigned 994 patients 1:1 to receive pembrolizumab at 200 mg intravenously every 3 weeks for approximately 1 year (up to 17 cycles) or placebo. Patients were stratified by M stage (M0 vs M1 NED) and, within M0, by ECOG performance status and geographic region.

The ctDNA analysis population comprised treated participants with tumor tissue, whole blood, and plasma samples available. Plasma ctDNA was assessed using a modified version of Natera's Signatera personalized, tumor-informed, tissue-exome-based circulating tumor DNA assay; the 64-plex version was used as the primary assay, with 16-plex results simulated from the 64-plex data. The data cutoff was September 25, 2024, representing a minimum 5-year follow-up.

At baseline, the median age among ctDNA-evaluable patients at baseline was 61 years among patients in the pembrolizumab arm (n = 374) and 60 years in the placebo arm (n = 362). Most patients in both arms were male (69.3% vs 71.8%), had an ECOG performance status of 0 (84.0% vs 83.4%), had a PD-L1 combined positive score of at least 1 (76.7% vs 79.6%), and had M0 intermediate-high risk disease (86.4% vs 88.1%).

What did baseline ctDNA status show for DFS?

Pembrolizumab improved DFS vs placebo regardless of baseline ctDNA status. In the intention-to-treat population, the DFS HR was 0.71 (95% CI, 0.59-0.86). Among ctDNA-negative patients, the DFS HRs were 0.78 (95% CI, 0.61-1.00) with the 64-plex assay and 0.78 (95% CI, 0.61-0.98) with the 16-plex assay. Among ctDNA-positive patients, the DFS HRs were 0.68 (95% CI, 0.38-1.21) with the 64-plex assay and 0.59 (95% CI, 0.29-1.19) with the 16-plex assay.

What were the specificity, sensitivity, and ctDNA clearance findings?

The diagnostic performance of exome-based ctDNA testing at baseline was characterized by high specificity but low sensitivity. For the 64-plex assay in the pembrolizumab arm, specificity was 98%, sensitivity was 15%, positive predictive value (PPV) was 73%, and negative predictive value (NPV) was 64%. In the placebo arm, specificity was 98%, sensitivity was 15%, PPV was 87%, and NPV was 56%. For the 16-plex assay, specificity was 99% and 99% in pembrolizumab and placebo arms, respectively, while sensitivity was 10% and 10%.

ctDNA clearance at cycle 5 day 1 was higher in the pembrolizumab arm than in the placebo arm: 60.0% (n = 6 of 10) vs 21.4% (n = 3 of 14) with the 16-plex assay and 55.6% (n = 10 of 18) vs 36.0% (n = 9 of 25) with the 64-plex assay.

References

1. Choueiri TK, Tomczak P, Haas NB, et al. ctDNA analysis in participants with renal cell carcinoma treated with adjuvant pembrolizumab or placebo in the KEYNOTE-564 trial. J Clin Oncol. 2026;44(suppl16):4502. doi:10.1200/JCO.2026.44.16_suppl.4502
2. Haas NB, Powles T, Tomczak P, et al. Five-year follow-up results from the phase 3 KEYNOTE-564 study of adjuvant pembrolizumab for the treatment of clear cell renal cell carcinoma. Presented at: Society of Urologic Oncology Annual Meeting; December 2-5, 2025; Phoenix, Arizona. Poster 158.
3. FDA approves pembrolizumab for adjuvant treatment of renal cell carcinoma. News release. FDA; November 17, 2021. Accessed June 11, 2026. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-pembrolizumab-adjuvant-treatment-renal-cell-carcinoma