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Tumor Discovery Somatic mutations in POLE and POLD1 in colorectal cancer

4 Division of Cancer and Stem Cell, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, United Kingdom
(This article belongs to the Special Issue: Colorectal Cancer: Best Tools for Diagnosis to Management Strategies)

1. Introduction The aim of the present study is to investigate the
rate of somatic mutations in the exonuclease domains
Colorectal cancer (CRC) is a complex disease driven by of both POLE and POLD1 genes in a well-characterized
various genetic alterations that influence its initiation cohort of CRC and to define the clinicopathological and
and progression. It ranks as the fourth most common molecular characteristics of POLE and POLD1-mutated
1
cancer in both men and women. The majority of CRC cancers.
cases – approximately 70 – 80% – are sporadic. In contrast,
about 20 – 30% have a hereditary component, resulting 2. Materials and methods
from either rare, high-risk genetic tumor syndromes such
as Lynch syndrome (3 – 4%) and familial adenomatous 2.1. Sample population
polyposis (about 1%) or from more common but lower-risk One hundred formalin-fixed and paraffin-embedded
alleles identified through genome-wide association studies. (FFPE) primary CRC samples had been previously
Only 1 – 2% of CRC cases are associated with inflammatory retrieved from the archives of the pathology department of
bowel diseases. Among these alterations, impairments in the Nottingham University Hospitals NHS Trust. The CRC
2
DNA replication fidelity, particularly due to mutations in tissue samples were accompanied by 40 cases of matched
the proofreading domains of DNA polymerases ε (POLE) normal mucosa, each carefully located away from the
and δ (POLD1), have garnered significant attention. Wang tumor site and processed in separate blocks.
3
et al. revealed that somatic mutations in POLE and POLD1 All patients had undergone surgery between 2003
4
occurred at rates of 2.79% and 1.37%, respectively.
and 2010 at Queen’s Medical Center, Nottingham,
Both the POLE and POLD1 play crucial roles in United Kingdom (UK). The cases were selected based on
ensuring accurate DNA replication and repair, making the availability of clinicopathological data and the presence
them integral components of cellular genomic integrity. of at least 50% of tumor tissue in the biopsy specimens and
Germline mutations in these genes have been associated tumor block (Table 1).
with an increased risk of CRC development, implicating The ploidy and microsatellite status of all samples
their involvement in hereditary CRC syndromes. For were previously determined by Fadhil et al. In brief,
5,6
15
example, mutations such as POLE P286R and POLD1 ploidy status was examined using flow cytometry, whereas
S478N have been identified in familial CRC cases, leading the microsatellite status of the cases was determined
to an elevated mutation burden and distinct mutational by polymerase chain reaction (PCR) followed by high-
signatures. Furthermore, somatic mutations in POLE resolution melting (HRM) evaluation of instability at
7,8
and POLD1 have been observed in sporadic CRC cases, mononucleotide repeat sequences using a panel of 6 markers
contributing to tumor heterogeneity and progression. (BAT25, BAT26, NR21, NR22, NR24, and B-CAT25).
These somatic mutations are often associated with
microsatellite stability (MSS) in CRC tumors, influencing 2.2. DNA extraction
their mutational landscape. 9-11 Six 10 mm sections were cut from each block, and DNA
The functional consequences of POLE and POLD1 was extracted using the QIAamp DNA FFPE tissue kit
mutations extend beyond increased mutational burden. (Qiagen, UK) following the manufacturer’s protocol, as
For instance, POLE V411L and POLD1 C319Y have previously described. 16
been linked to a hypermutator phenotype, resulting in
an increased neoantigen load and potential implications 2.3. Primer design and optimization
for immunotherapy response. Understanding the The POLE and POLD1 nested primers were designed for
3,12
implications of both germline and somatic mutations use in quick-multiplex consensus (QMC)-PCR assays,
in POLE and POLD1 is crucial for advancing our as previously described. The genomic sequences of the
17
knowledge of CRC pathogenesis and guiding exonuclease domains of POLE (codons 268 – 471) and
personalized therapeutic approaches. These mutations POLD1 (codons 304 – 517) were obtained from the National
13
not only provide insights into CRC etiology but also hold Center for Biotechnology Information (NCBI) (http://www.
promise as biomarkers for risk assessment and treatment ncbi.nlm.nih.gov/pubmed/). Primer3 software (http://
stratification. 14 biotools.umassmed.edu/bioapps/primer3_www.cgi) was

Volume 3 Issue 3 (2024) 2 doi: 10.36922/td.3659