NEW: POLE
ORDER CODE: LABPOL
SUMMARY
POLE by SNaPshot is now available at Stanford Clinical Laboratory. This assay is designed to detect 15 somatic mutation sites within exons 9, 11, 13, and 14 of the proofreading exonuclease domain of POLE.
The DNA replicase Pol ℇ is essential for DNA replication fidelity and the maintenance of genomic stability. Specifically, POLE encodes one of the major catalytic subunits of the DNA Polymerase Epsilon complex and plays a critical role in DNA replication, including proofreading newly synthesized DNA strands and catalyzing 3’-5’ exonuclease activity in the event of any base misincorporations. In addition, POLE has also been implicated in base excision repair, nucleotide excision repair, MMR, double strand break repair, cell checkpoint regulation and propagation of chromatin modification states (Rayer et al. 2016).
Somatic mutations in the proofreading exonuclease domain of the POLE gene have been identified in multiple tumor types and cause an ‘ultramutation’ phenotype secondary to dysfunctional proofreading and a high rate of base substitution mutation from replicative error. Deleterious mutations in the proofreading exonuclease domain of POLE occur in approximately 7-12% of endometrial carcinomas, 1-2% of colorectal carcinomas and a small number of other cancer types including ovarian, pancreas, stomach, brain, and breast cancers. Of note, several germline POLE mutations have also been identified and are associated with an increased risk of several cancer types (Rayer et al. 2016).
Identifying pathogenic mutations in the POLE gene is clinically important as such mutations are associated with a more favorable prognosis and can impact oncologic management (Church et al. 2015, TCGA 2013). Importantly, POLE mutated tumors may also be eligible for immunotherapy (e.g. check-point inhibitors) given the strong correlation between these mutations and high tumor mutation burden (Mehnert et al. 2016). In addition, several POLE-mutant cancers display increased tumor infiltrating lymphocyte density, a phenotype which also portends better tumor immune response to immunotherapy.
This assay is designed to detect 15 somatic mutation sites within exons 9, 11, 13, and 14 of the proofreading exonuclease domain of POLE. Mutation sites covered in this assay were selected based on evidence of functional impact, association with high tumor mutation burden, and/or mutations reported in clinical outcome studies for POLEmutations (Campbell et al. 2017, Church et al. 2014, TCGA 2013). The most common POLE variants, p.P286R and p.V411L, are detectable by this assay and these two mutations alone account for approximately 70-90% of the reported, pathogenic POLE mutations in endometrial and colorectal carcinoma across different studies (Church et al. 2015, Cambell et al. 2017). The other variant sites covered by this assay were selected to include those particularly enriched or studied in endometrial and colorectal carcinomas, and account for the majority of additional pathogenic POLE mutations in these cancer types. Notably, the majority of somatic mutations in POLE are actually passenger mutations, with unknown effect or no effect on POLE function (Campbell et al. 2018, Domingo et al 2017). As such, this assay focuses on variants with evidence to support clinical utility, but will not identify all potential pathogenic variants. There continues to be ongoing discovery and characterization of novel deleterious mutations in POLE, which may be considered for inclusion in future assays.
The POLE SNaPshot assay requires DNA extraction from paraffin-embedded tissue, DNA purification with the Qiagen kit, and amplification reactions for each of the above mentioned exons and detection of the mutations by 12 “SNaPshot” reactions. The chemistry of the SNaPshot Multiplex kit is based on the dideoxy single-base extension of an unlabeled oligonucleotide extension primer. Each primer binds to a complementary template in the presence of fluorescently labeled ddNTPs and DNA polymerase. The polymerase extends the primer by one nucleotide, adding a single labeled ddNTP to its 3’ end. SNaPshot products are collected on 3500xL genetic analyzer and the data collected is analyzed with Genemapper software