ddPCR Analysis Effective in Identifying POLE Mutations in Endometrial Cancer, Study Finds

Investigators recommended that ddPCR testing and immunohistochemical staining be used to detect POLE status and p53/mismatch repair status, respectively, and allow for more rapid classification and risk-stratification.

Droplet digital polymerase chain reaction (ddPCR) could be a useful tool to rapidly identify patients with POLE-mutated endometrial cancer, according to findings from a study published in the Journal of Gynecologic Oncology.

Approximately 1 in 10 patients (10.8%) were found to have POLE mutations based on ddPCR testing. That rate is similar to rates in previous published reports, the authors wrote. Eight of 26 patients who tested positive for POLE mutations via ddPCR testing tested negative with Sanger sequencing. Next-generation sequencing was then conducted in those 8 patients, along with 3 cases in which Sanger sequencing identified a mutation, but ddPCR showed a mutation index (MI) of less than 5%. In 10 of 11 patients, targeted sequencing matched the results from the ddPCR assay.

“The discrepancy was a case in which a very low copy of V411L mutation was observed in ddPCR,” the authors wrote.

Overall, V411L mutations were the most commonly identified POLE mutation (61.54%) followed by P286R (23.07%), S459F (7.69%), S297F (3.85%), and A456P (3.85%). Mutations of S297F and A456P were found in less than 4% of patients. The investigators said the finding that V411L mutations were by far the most common mutation was surprising, since data from the National Institutes of Health’s Cancer Genome Atlas suggests that P286R is more common than V411L. They said the variance could be due to ddPCR’s ability to detect lower-MI mutations, genomic instability associated with the MMR-D subtype, or demographic differences. The authors said larger studies would be needed to fully answer the question.

Theinvestigators explained that patients with POLE mutations generally have an excellent prognosis, whereas patients with p53 mutations have a poor prognosis. The 2 other subtypes included in the study, mismatch repair-deficient (MMR-D) and no specific molecular profile (NSMP) subtype carry an intermediate prognosis. Investigators indicated that classifying patients into subtypes can be challenging. While MMR and p53 status can be detected using immunohistochemical staining, there is no standard method detecting POLE mutations, which are currently identified via NGS or Sanger sequencing.

“Sanger sequencing is useful for evaluating exons 9, 11, 13, and 14 of POLE, where mutation hotspots are concentrated, although it has a low limit of detection and there is a strong possibility of false-negative results for mutations with a low frequency,” the authors wrote.

Investigators theorized that ddPCR testing could be a solution. This quantitative method has the benefit of being able to detect even small levels of genetic mutations, and can be performed using paraffin-embedded specimens in just 1 to 2 days. Investigators evaluated how ddPCR performed against NGS and Sanger sequencing in identifying POLE mutations in patients with endometrial cancer.

Medical records and pathological reports from a total of 240 patients with endometrial cancer were identified via hospital database, assessed via ddPCR assay. Investigators used ddPCR testing to identify 5 previously identified hotspot mutations of the POLE gene: P286R, S297F, V411L, A456P, and S459F. In addition to ddPCR testing, the accuracy of immunohistochemistry in identifying p53 and MMR protein expression was assessed.

In terms of outcomes, the analysis showed that patients with a high FIGO score, deep myometrial invasion, lymphovascular space invasion, advanced stage, and high/advanced risk were more likely to have a p53 mutation and were also more likely to have poor outcomes. Patients in the POLE-mutated group more commonly had lower stages and low/intermediate risk. These patients also had the best outcomes of any subgroup, investigators wrote.

“When only intermediate, high-intermediate, and high-risk groups were analyzed for subgroups, molecular classification still showed differences both in PFS (P = .003) and overall survival (P = .017),” they added.

There are different approaches for when and how to test patients for different molecular subtypes, but the investigators stated that their research has led them to advocate for the use of simultaneous immunohistochemistry and ddPCR testing to evaluate POLE, MMR, and p53 statuses at the same time. Doing so could help prevent misclassification and also shorten the time it takes to classify patients, they concluded.


Kim G, Lee SK, Suh DH, et al. Clinical evaluation of a droplet digital PCR assay for detecting POLE mutations and molecular classification of endometrial cancer. J Gynecol Oncol. 2022;33(2):e15. doi:10.3802/jgo.2022.33.e15