Pap-ctDNA Yields High Sensitivity in Endometrial Cancer

Patients with Lynch Syndrome (LS) face a significantly elevated risk for endometrial cancer (EC) and are advised to undergo risk-reducing hysterectomy with bilateral salpingo-oophorectomy (RR-Hys/BSO) due to the absence of effective screening methods. Given the limited sensitivity of plasma-derived circulating tumor DNA (plasma-ctDNA) in detecting EC, researchers aimed to investigate the potential of Pap-derived ctDNA (Pap-ctDNA) as a novel noninvasive tool for EC detection.

The results will be presented at the American Society for Clinical Oncology 2025 Annual Meeting, from May 30 to June 3, 2025.

Genomic alterations identified in paired-tumor and normal-tissue sequencing were compared with alterations detected in plasma- and Pap-derived ctDNA. Plasma-ctDNA and Pap-ctDNA samples were collected from a total of 19 patients with EC undergoing surgery (n=16) and from individuals with LS undergoing RR-Hys/BSO (n=3). Among EC cases, 56% were of low-grade endometrioid histology, and 81% were early stage.

Somatic variants found in plasma and Pap samples were validated using matched tumor tissue, and variant allele frequency was calculated. Tumors were evaluated for microsatellite instability (MSI) and/or mismatch repair deficiency (MMRD) using MSISensor or immunohistochemistry (IHC). In LS patients, pathology was reviewed for evidence of precursor lesions.

Mismatch repair deficiency (MMRD) due to MLH1 hypermethylation was identified in 12.5% of tumors. The median DNA yield was significantly higher from Pap-ctDNA than from plasma-ctDNA (91 ng vs. 19.5 ng; P=0.0004). Mutations within the targeted regions of both assays were identified in 94% of EC tumors, with mutations detected by Pap-ctDNA in 93% of cases compared with only 33% with plasma-ctDNA.

In the five patients with detectable mutations in both sample types, median variant allele frequency was consistently higher in Pap-ctDNA than in plasma-ctDNA (P=0.012). Among the 13 patients with early-stage EC, 92% tested positive using Pap-ctDNA versus 23% using plasma-ctDNA.

Of the three patients with LS, one had MMRD atypical hyperplasia consistent with germline MMR mutation; however, both plasma- and Pap-ctDNA were negative.

“Pap-ctDNA from EC [patients] results in higher ctDNA yield than plasma-based testing. Tumor mutations were detected in more than 90% of Pap-ctDNA samples, even in early-stage EC, versus only 23% of plasma-ctDNA samples. Pap-ctDNA for early-detection of EC is feasible and is a promising tool for average and high-risk individuals with potential applicability in other neoplasms,” the researchers concluded.