The complexity of screening PMS2 in DNA isolated from formalin-fixed paraffin-embedded material

Area of Science:

• Genetics
• Molecular Biology
• Oncology

Background:

• Germline variants in the DNA mismatch repair (MMR) gene PMS2 are implicated in 1-14% of Lynch Syndrome cancers.
• Accurate PMS2 variant analysis is complicated by pseudogenes, gene conversion, and fragmented DNA in formalin-fixed paraffin-embedded (FFPE) tissues.

Purpose of the Study:

• To develop and validate a reliable approach for detecting true PMS2 variants in fragmented DNA from FFPE samples.
• To improve the accuracy of genetic testing for Lynch Syndrome by addressing PMS2 analysis complexities.

Main Methods:

• A custom next-generation sequencing (NGS) panel was designed targeting four MMR genes, POLE, and POLD1, with specific amplicon design for PMS2 based on paralogous sequence variants (PSVs).
• The method was validated using 125 MMR-deficient tumors, assessing PMS2 variants and protein expression loss.

Main Results:

• The custom NGS panel successfully identified true PMS2 variants in fragmented DNA, distinguishing them from pseudogenes.
• In six unexplained MMR-deficient tumors with solitary PMS2 loss, two unclassified (class 3) and five likely pathogenic (class 4/5) PMS2 variants were detected.
• One microsatellite unstable tumor with intact MMR protein expression harbored a pathogenic frameshift PMS2 variant (class 5).

Conclusions:

• This custom NGS panel provides a reliable method for accurate PMS2 variant detection in FFPE tissues, crucial for Lynch Syndrome diagnosis.
• The findings highlight the importance of comprehensive PMS2 analysis, even in tumors with seemingly normal MMR protein expression patterns.