麻豆传媒

A quantitative genotyping polymerase chain reaction (PCR) assay is a quick, simple, and reliable alternative to DNA sequencing in endometrial cancer, researchers reported.

Molecular classification is essential for prognosis and selection of the most effective treatment for women with endometrial cancer. The introduction of the endometrial cancer molecular classification algorithm, on the basis of four distinct molecular subgroups, has enabled more accurate diagnosis and is used to tailor disease management. Assessment of POLE gene status in endometrial cancer is strongly encouraged, since women with POLEmut disease are effectively cured by surgery alone and are recommended to no or de-escalated adjuvant therapy.

In a new study in , Tjalling Bosse, MD, PhD, of Leiden University Medical Center in the Netherlands, and colleagues described how they developed the QPOLE assay as a quick, low-cost method to identify pathologic variants in endometrial cancer. The following Q&A discusses the details of the study. (The researchers did not respond to requests for comment, and the answers here are from the text of the report.)

What does this study add to the literature?

We developed the QPOLE assay, which is a quick, simple, and reliable alternative for DNA sequencing. In comparison with next-generation sequencing (NGS), QPOLE can easily be implemented in pathology laboratories throughout the world, including those with limited resources.

POLE status is most often assessed by DNA sequencing methods, such as Sanger or NGS in the absence of any immunohistochemical (IHC) markers. These techniques are not widely available in hospitals around the world. In addition, the methods are expensive and time-consuming, since equipment and highly trained personnel are required for correct interpretation, and as such can take up to 1-2 weeks after surgery before results are available.

Consequently, POLE-testing is currently unavailable for most patients with endometrial cancer, both in high-income as well as middle- and low-income countries. As a result, , leading to treatment-related morbidities affecting quality of life and unnecessary costs for radiotherapy and/or chemotherapy.

Lack of access to POLE-testing hampers implementation of the molecular classification of endometrial cancer in routine clinical practice worldwide. Although assessment of mismatch repair deficiency and p53 abnormalities by IHC is increasingly performed, assessment of POLE status is required for correct molecular classification and allocation of adjuvant therapy.

Finally, a cheap, accessible, and rapid assay, which enables assessment of POLE status on tumor material obtained by biopsy or hysterectomy, will aid in counseling and decisions on lymphadenectomy and (neo)adjuvant therapy.

What are the highlights of the study?

We present a quick, simple, and low-cost quantitative genotyping PCR assay for the 11 pathogenic variants in POLE, called QPOLE. Three assays, QPOLE-frequent for the most common mutations and QPOLE-rare-1 and QPOLE-rare-2 for the rare variants, were developed and optimized using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissues.

The simplicity of the design enables POLE status assessment within 4-6 hours after DNA isolation. An interlaboratory external validation study was performed to determine the practical feasibility of this assay.

Cutoffs for POLE wild-type, POLE-mutant, equivocal, and failed results were predefined on the basis of a subset of POLE mutants and POLE wild-types for the internal and external validation. For equivocal cases, additional DNA sequencing is recommended.

Performance in 282 endometrial cancer cases, of which 99 were POLE-mutated, demonstrated an overall accuracy of 98.6%, a sensitivity of 95.2%, and a specificity of 100%. After DNA sequencing of 8.8% equivocal cases, the final sensitivity and specificity were 96.0% and 100%. External validation confirmed the feasibility and accuracy.

What are the advantages of QPOLE over NGS?

Our method ensures that assessment of POLE status can be performed in any laboratory around the world that has a quantitative PCR machine and the capacity to isolate DNA from FFPE tissues.

Quantitative PCR methods are cheap and widely available, have proven clinical reliability, and are used in the first line of diagnostics in many countries today. QPOLE significantly reduces the turnaround time to only 4-6 hours compared with up to 1 or more weeks for conventional NGS.

After establishment of local thresholds for sample calling, QPOLE has shown excellent performance in internal and external validation, and implementation in another laboratory was simple. Even when using DNA from older FFPE blocks, comparing QPOLE with golden standard NGS showed an accuracy of 98.6% and a sensitivity and specificity of 96% and 100%, respectively.

Without taking DNA isolation and preparation into consideration, the simplicity of QPOLE enables POLE status assessment within 4-6 hours. Furthermore, QPOLE does not require interpretation by a molecular biologist, as opposed to NGS. As a result, QPOLE is substantially faster and cheaper in comparison with most current DNA sequencing techniques.

Furthermore, QPOLE results could be used during preoperative counseling on lymphadenectomy.

How cost-effective is QPOLE?

QPOLE will not only enable POLE-testing around the world, but also yield less morbidity through treatment de-escalation and a reduction in healthcare–related costs. Cost-effectiveness of the molecular classification in advanced endometrial cancer has been predicted by a modeling study and is currently under investigation in the .

Additional savings of replacing DNA sequencing with QPOLE will make the assessment of the molecular classification even more cost-effective.

What is your main take-home message for practicing oncologists?

The laboratory-developed QPOLE test is a quick, low-cost, and accurate POLE-hotspot testing alternative for the detection of pathogenic POLE mutations, enabling implementation in clinical practice with consequences for clinical management.

Read the study here.