ALL Metrics
-
Views
-
Downloads
Get PDF
Get XML
Cite
Export
Track
Research Article

Low prevalence of germline TP53 and PALB2 mutations in unselected cohort of breast cancer patients from Brunei Darussalam

[version 1; peer review: awaiting peer review]
PUBLISHED 30 Nov 2023
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS AWAITING PEER REVIEW

This article is included in the Oncology gateway.

Abstract

Background: Breast cancer is the most frequent malignancy affecting women worldwide. The majority of breast cancer occurs sporadically, with only 5-10% being caused by inheritance of susceptibility genes. In Brunei Darussalam, breast cancer is the leading cause of cancer in women. The prevalence and clinical relevance of breast cancer susceptibility genes in Brunei breast cancer patients is unknown. We investigated the prevalence and clinical relevance of germline TP53 and PALB2 genes, recognised to confer a high and moderate risk respectively, in the development of breast cancer in an unselected cohort of Brunei breast cancer patients.

Methods: Genomic DNA was extracted from peripheral blood samples of 54 unselected Brunei breast cancer patients. The DNA samples were sequenced for germline BRCA1, BRCA2, TP53, and PALB2 variants using targeted panel sequencing on a Hi-Plex NGS platform. Identified variants were analysed for their pathogenicity classification based on clinical/population/mutation databases, in-silico data, and available functional data analysis. Chi-square test was used to determine the association between TP53 codon 72 and response to chemotherapy in Brunei breast cancer patients.

Results: We identified two TP53 and five PALB2 missense variants in our study population. Five of the identified variants were classified as variants of uncertain significance (one in TP53 and four in PALB2) giving a prevalence of TP53 and PALB2 variant of uncertain significance carriers at 1.9% and 9.3%, respectively. No pathogenic TP53 and PALB2 mutation was identified in this study suggesting the rarity of these genes in breast cancer.TP53 codon 72 had no association with Brunei breast cancer patients’ response to chemotherapy supporting the benign characteristic of the variant P72R.

Conclusions: Our current findings suggest that the contribution of germline TP53 and PALB2 genes in unselected Brunei breast cancer patients is rare, and a larger number of participants will be required to confirm this finding.

Keywords

TP53, PALB2, Germline, NGS, Prevalence, Drug Response, Breast Cancer, Brunei population

Introduction

Tumour Protein 53 (TP53) (OMIM #151623) and Partner and Localizer of BRCA2 (PALB2) (OMIM #610355) genes are high- and moderate-penetrance breast cancer susceptibility genes respectively.1,2 TP53 are activated in response to cellular stresses such as expression of activated oncogenes or DNA damage. Following the activation, TP53 undergoes post-translational modifications which activate the protein for DNA binding and activation of downstream genes resulting in suppression of a certain tumour growth, facilitating the tumour suppressor actions of TP53. This includes induction of a specific cellular response such as apoptosis.3,4 Overall, TP53 plays a major role as a transcription factor that is involved in the control of cell growth, DNA damage repair, genomic stability, and in regulating apoptosis.36 PALB2 gene product was initially identified as a BRCA2-interacting protein, localising and accumulating BRCA2 at DNA damage sites.711 It was later shown that PALB2 also interacted with BRCA1 in response to DNA damage,12 linking BRCA1 and BRCA2 to facilitate their function in DNA damage response, specifically for BRCA2-mediated homologous recombination (HR) repair.1214

TP53 was the most common gene mutated in most malignancies. Most of the mutations typically observed occur sporadically and if inherited, the carrier generally suffers from Li-Fraumeni Syndrome (LFS).1,2,5,15,16 Hence, germline TP53 mutations are rare and specific to certain criteria.17,18 A study on early-onset breast cancer Caucasian population revealed that TP53 carriers have a high relative risk (tenfold-increase) of developing cancer.19 Interestingly, TP53 mutations were commonly found to occur in BRCA1- and BRCA2-related breast cancer compared to non-BRCA related breast cancer.5 Mono-allelic pathogenic PALB2 mutations were shown to be associated with an increased risk of developing breast cancer, while bi-allelic pathogenic PALB2 mutations were demonstrated to cause an increase sensitivity to DNA damaging agents and a new subtype of Fanconi anaemia (FA).2022 A study found that 1.1% of BRCA1/2-negative women with familial breast cancer harboured mono-allelic PALB2 mutations which conferred a 2.3-fold high risk.8,13 On the basis of data from 154 families, the risk of developing breast cancer for female PALB2 mutation carriers by the age of 70 were reported to range from 33% for those without considering family history, to 58% for those with two or more first-degree relatives with breast cancer at 50 years of age.22 The findings suggested the risk for PALB2 was higher than other moderate penetrance genes such as CHEK2 and ATM, although the study limitation hindered the certainty of re-categorising PALB2 as a high penetrance risk gene.23These findings were refined in a recent update based on 524 families which revealed the estimated relative risks (RRs) of breast cancer for PALB2 pathogenic variants (PVs) carriers was 7.18 (assumed to be constant with age), and the RRs declined with age.24 Moreover, the study also reported the risk of developing breast cancer for female PALB2 mutation carriers by the age of 70 ranged from 44% for those without considering family history to 68% for those with two or more first-degree relatives with breast cancer at 50 years of age.24 Taken together, this warrants the inclusion of PALB2 in breast cancer gene panels. PALB2 germline mutations were also shown to confer an increased risk of male breast cancer, ovarian cancer, and pancreatic cancer.8,24,25

To date, the reported prevalence of TP53 and PALB2 germline mutations in Asian patients with familial and/or early-onset breast cancers ranges from 0.4% to 6%2629 and 0.3% to 2.6%,21,26,3034 respectively. They are almost similar to the reported prevalence of TP53 and PALB2 germline mutations in familial and/or early-onset breast cancer patients in the Western countries which ranges from 4% to 6.4%35,36and 0.1% to 2.9%,3740 respectively. Notably, the majority of studies in the Western populations have been carried out on selected BRCA1/2 negative familial and/or early-onset breast cancer patients. Meanwhile, a number of studies conducted in unselected Asian breast cancer patients reported a much lower prevalence of TP53 and PALB2 germline mutations which ranges from 0.2% to 0.5%4143and 0.4% to 1.9%,30,41,42,4448 respectively confirming the rarity of germline TP53 and PALB2 mutations.

Brunei Darussalam is a small country located in the South-east Asia (SEA) region, bordering the South China Sea and East Malaysia.49 Breast cancer was the leading cause of cancer among women in Brunei Darussalam with an increasing age-standardized incidence rate [ASIR]) of 45.6-64.8 per 100,000 women during 2011-2020 which is among the highest in countries in the SEA region, but considerably lower compared to the Western Europe (ASIR, 90.7).50,51 The mean age of Brunei breast cancer patients was 52±11.8 years as of 2017,52 approximately 10 years younger than those in Western countries.53 Moreover, 13% of the patients were diagnosed below 40 years old.52 There has not been any formal study conducted on finding the contribution of genetic and non-genetic factors in the rising incidence of breast cancer in Brunei. The contribution of genetics in breast cancer, specifically in the involvement of susceptibility genes, has been continuously researched on in the Western and developed Asian countries, resulting in many reports on the spectrum of variants within the susceptibility genes all over the world. Regionally, only Singapore26and Malaysia21,28,42,47,54 have reported mutation spectrum of TP53 and PALB2 in their breast cancer populations which revealed a low prevalence of TP53 and PALB2 mutations. The advent of next-generation sequencing (NGS) in producing a large number of multigene or targeted panel tests has made the screening of multiple genes simultaneously and at a relatively low cost possible.23,55 In this study, we used targeted panel sequencing through the NGS platform to test our genes of interest; BRCA1, BRCA2, TP53,and PALB2.This article reports on the outcome of screening for mutations in the entire coding sequence of the TP53 and PALB2 genes in an unselected series of Brunei breast cancer patients to determine their prevalence, mutation spectrum, and their clinical implication on drug response.

Methods

Study population

The study population includes unselected incident and prevalent breast cancer patients seen from 2017 till 2018 at The Brunei Cancer Centre (TBCC), Pantai Jerudong Specialist Centre (PJSC), the only cancer referral hospital in Negara Brunei Darussalam. Terminally-ill patients were excluded from the study. Among 218 patients who were seen during this period, 89 patients were approached where only 54 patients consented to participate in the study. All study participants provided written informed consent. Peripheral blood samples, demographic, and detailed family history data were collected from consenting patients. A retrospective review of the recruited study participants’ medical and histopathology reports was performed at TBCC after acquiring access permission from the relevant authorities.

Ethics statement and consent

This study was approved by the Medical and Health Research and Ethics Committee (MHREC) of Brunei Darussalam [MHREC/Edu/2016/4(3)]. Written informed consent for publication of patients’ details was obtained from the patients or through consent by proxy.

Next-generation sequencing (NGS)

Genomic DNA (gDNA) was extracted and purified from the collected peripheral blood sample using Wizard Genomic DNA purification kit (Promega) following manufacturer’s protocol. The DNA samples were then outsourced to Cancer Research Malaysia (CRM) for BRCA1, BRCA2, TP53, and PALB2 targeted panel sequencing.

The HBOC_4_v2 gene panel used was developed by CRM and the University of Melbourne and was used to screen for all coding exons ±2 bp intronic sequence of the BRCA1 (NM_007294.3), BRCA2 (NM_000059.3), PALB2 (NM_024675.3), and TP53 (NM_000546.4) genes. For each sample, 50 ng of gDNA were screened for germline mutations using Hi-PlexNGS platform. The targeted sequencing library was prepared using an amplicon-based approach and sequenced on a MiSeq (Illumina, USA), 2×300bp paired-end sequencing using MiSeq Reagent kit v2 300 cycles. On-target coverage parameter for sequencing was computed using Bedtools (2.17.0). Samples considered as successfully sequenced were those with >95% of amplicons with ≥10 read-pairs (≥20× read depth).

Raw sequencing reads were mapped to the human genome (hg19) using Bowtie2 (2.3.2). Variants were called using ROVER, annotated with ANNOVAR, and according to the Human Genome Variation Society (HGVS) nomenclature using Mutalyzer (2.0.26). Identified variants were classified and assigned to one of the following classes: (1) benign; (2) likely benign; (3) variants of uncertain significance; (4) likely pathogenic; or (5) pathogenic. Class 1 and Class 2 variants were not reported to end-user by CRM. Class 3 variants were reported as equivocal. Class 4 and Class 5 variants were reported as pathogenic mutations. All variants other than neutral polymorphisms were confirmed by Sanger sequencing. Bioinformatic analysis was performed by CRM and reports on the variant results excluding neutral polymorphisms (Class 1 and 2) were delivered upon completion.

Bioinformatics analysis

Although CRM have analysed and validated the results, the data were re-analysed as neutral polymorphisms (Class 1 and Class 2 variants) were not included in the provided reports. Integrative Genomics Viewer (IGV) v2.5.2 (Broad Institute) was used to view the binary files provided by CRM. The data were analysed using the public server at usegalaxy.eu.56 Variants were called using varscan (Galaxy Version 2.4.2) and bcftools call (Galaxy Version 1.9+galaxy 1). The data generated by bcf tools call were filtered using VCF filter. All variants identified were annotated using the SNPeff Eff tool (Galaxy Version 4.3+T.galaxy1).

All variants identified were annotated according to the nomenclature used by HGVS recommendation guidelines, using the A of the ATG translation initiation codon as nucleotide +1. All identified missense variants were analysed in-silico using SIFT,57 PolyPhen-2,58 CADD,59 FATHMM-MKL,60 and DANN61 to predict the effect of amino acid substitution. Each prediction tools scored the variant as damaging or benign/neutral/tolerated. All variants identified in this study were also checked against the NCBI ClinVar, Varsome, and population frequency databases (gnomAD and 1000 genome).

Variant pathogenicity was explored and derived on the basis of the ClinVar database, population data, IARC TP53 database (R20, July 2019),62 UMD TP53 Mutation database (2017_R2), in-silico data, and functional data if available following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines.63 In this study, a variant was considered a pathogenic damaging mutation if it was a protein-truncating mutation caused by deleterious or frameshift mutation, or a missense mutation which has a confirmed association with the disease, or a missense variant which has been classified as likely pathogenic according to the ACMG standards and guidelines. We selectively Sanger sequenced only pathogenic mutations identified using NGS via our own ABI 3500 Genetic Analyzer.

Statistical analysis

Continuous data were presented as median and range. Chi-square or Fisher exact test was used to analyse the association between two independent variables in the population. The statistical analysis was performed using SPSS version 17.0 software for Windows. A p-value <0.05 was considered statistically significant.

Results

This was the first study of its kind in the Brunei breast cancer patients. Of the 54 recruited female breast cancer patients, 40 (74.1%) were Malays, 12 (22.2%) were Chinese, and two(3.7%) were “others”. The median age at diagnosis was 54 years old (range 30-71). Nine (16.7%) were diagnosed with breast cancer ≤40 years old, and three (5.6%) had bilateral breast cancers. A total of 19 (35.2%) had family history of breast and/or ovarian cancers while 23 (42.6%) had no family history of cancers at all. Further assessment was made on patients with family history of breast and/or ovarian cancers on the number of affected family members with breast or ovarian cancer in the first- and second-degree relatives. Nine (16.7%) had one first-degree family member affected with breast cancer, and two (3.7%) had ≥2 first-degree relatives affected with breast cancer. For patients with family history of ovarian cancer, three (5.6%) and two (3.5%) had ≥1 affected member in the first- and second-degree relatives, respectively (Table 1).

Table 1. Demographic characteristics, personal and family history of cancer of study population according to ethnicity.

CharacteristicsMalay (n = 40)Chinese (n = 12)Others (n = 2)Total (n = 54)
n%n%n%n%
Age at first diagnosis (year)
≤3012.5000011.9
31-40615.018.3150.0814.8
41-501127.5433.3150.01629.6
51-601332.5541.7001833.3
≥61922.5216.7001120.4
Bilateral breast cancer
Yes12.518.3150.035.6
No3997.51191.7150.05194.4
Triple negative breast cancer (TNBC)
Yes922.518.3001017.5
No3177.51191.72100.04782.5
Family history of cancer
Breast and/or ovarian cancers1332.5650.0001935.2
Other cancers922.5325.0001222.2
No family history of cancer at all1845.0325.02100.02342.6
No of 1o relatives with breast cancer
03280.0975.02100.04379.6
1717.5216.700916.7
≥212.518.30023.7
No of 2o relatives with breast cancer
03587.51083.32100.04787.0
1410.0216.700611.1
≥212.5000011.9
No of 1o relatives with ovarian cancer
03997.51083.32100.05194.4
≥112.5216.70035.6
No of 2o relatives with ovarian cancer
040100.01083.32100.05296.3
≥100216.70023.7

Tumour characteristics including histological stage and grade, nodal status, receptors status, and molecular subtype were extracted from the histopathology report (Table 2). The majority of the patients were diagnosed with Stage 1 (26.3%) and Grade 3 (78.9) breast cancer. More than half of the patients’ breast tumour were with lymph node metastasis (63.2%), ER-positive (61.4%), PR-negative (54.4%), and Her2-positive (54.4%). In this study, breast cancer is classified into four molecular subtype groups based on the immunohistochemistry (IHC) profile of ER/PR and Her2 and Ki-67 expression.64 Of all patients, 24 (42.1%) were of Luminal-B subtype while the others were distributed equally within the Luminal-A, Her2-enriched, and Triple-negative/Basal-like subtypes.

Table 2. Tumour characteristics of study population.

Tumour characteristicsTotal (n = 57) *
n%
Stage
11526.3
21221.1
358.8
4712.3
Missing1831.6
Grade
135.3
235.3
34578.9
Missing610.5
Lymph node metastasis
Positive3663.2
Negative1729.8
Missing47.0
Estrogen Receptor (ER)
Positive3561.4
Negative2238.6
Progesterone Receptor (PR)
Positive2645.6
Negative3154.4
Her2
Positive3154.4
Negative2645.6
Subtype
Luminal-A1119.3
Luminal-B2442.1
Her2-enriched1119.3
Triple-negative/Basal-like1119.3

* The total number is 57 due to three patients who are affected with bilateral breast cancer.

Prevalence of TP53 and PALB2 germline mutations in unselected breast cancer patients

Although the population included in this study was sequenced for the four genes, this article only reports the prevalence and mutation spectrum of the TP53 and PALB2 genes. The prevalence and mutation spectrum of the BRCA1 and BRCA2 genes for these patients are presented elsewhere (unpublished reports, Siti Nur Idayu Matusin).

Of the 54 unselected breast cancer patients included in this study, two (3.7%) had germline BRCA2 deleterious mutations. A total of seven (7) variants were identified in the study population (Table 3). Two (28.6%) and five (71.4%) were TP53 and PALB2 variants respectively. No deleterious mutations were identified, and all the seven identified missense variants have been reported.

Table 3. TP53 and PALB2 variants identified in the study population.

VariantAA changeTypeFrequency (n =54)EthnicitydbSNP
n%
TP53 (NM_000546.4)
c.79C>TP27SMissense11.9Chinesers922736614
c.215C>GP72RMissense4175.931 Malays, 9 Chinese, 1 Othersrs1042522
PALB2 (NM_024675.3)
c.149A>CK50TMissense11.9Othersrs763598472
c.1676A>GQ559RMissense2037.017 Malays, 2 Chinese, 1 Othersrs152451
c.2289G>CL763FMissense11.9Malayrs373478248
c.2474G>CR825TMissense11.9Chinesers146218439
c.3054G>CE1018DMissense23.71 Malay, 1 Chinesers183489969

Evaluation of variants pathogenicity

Two (2) variants were classified as benign and five (5) as variants of uncertain significance (VUS) (Table 4). Among the VUS variants, four (4) had conflicting evidence for pathogenic and benign criteria, while one (1) did not have enough evidence for its pathogenicity. The one TP53 VUS variant was identified in one patient and the four PALB2 VUS variants were identified in a total of five patients. Thus, the prevalence of TP53 and PALB2 VUS carriers among Brunei breast cancer patients were 1.9% and 9.3%, respectively. All the identified VUS were found to be rare in the general population (<0.05% in gnomAD and/or 1000 Genome).

Table 4. Pathogenicity determination of TP53 and PALB2 variants identified in the study population.

VariantAA changeRefPrediction/Score of functional effect of missense mutationsClassification from database and Population databaseFinal variant classification
SIFTHDIVHVARCADDFATHMM-MKLDANNClinVarVarsomegnomAD AF v2.1.11000 genome AFBrunei BC AFACMG CriteriaACMG Classification
TP53 (NM_000546.4)
c.79C>TP27S65–680.026/D0.097/B0.081/B2.326/D0.864/D0.997/DVUSLP8.01E-06
2 AC only in European
No data1.9E-02PM2, PP3, BS3VUS
c.215C>GP72R720.258/T0.083/B0.045/B0.544/N0.361/N0.570/TDRB6.63E-015.43E-013.8E-01BA1, BP4, BP6B
PALB2 (NM_024675.3)
c.149A>CK50T0.006/D0.989/PrD0.892/PoD3.074/D0.825/D0.998/DCIPVUS3.98E-06No data9.0E-03PM2, PP3VUS
c.1676A>GQ559R701/T0/B0/B-0.522/N0.280/N0.094/TBB1.07E-011.51E-011.94E-01BA1, BS3, BP4, BP6B
c.2289G>CL763F69,700.011/D0.996/PrD0.919/PrD1.638/N0.078/N0.995/DCIPVUS5.30E-05No data9.0E-03PM2, PP3, BS3VUS
c.2474G>CR825T0.043/D0.011/B0.026/B1.158/N0.192/N0.748/TCIPVUS1.63E-041.0E-039.0E-03PM2, BP4VUS
c.3054G>CE1018D710.025/D0.998/PrD0.991/PrD2.907/D0.692/N0.998/DCIPVUS3.85E-041.0E-031.9E-02PM2, PP3, BS3VUS

The two benign variants, TP53 c.215C>G and PALB2 c.1676A>G, were found in 75.9% and 37% of the study population, respectively. Both variants have been reported >10% in the global populations (rs1042522 and rs152451) suggesting the chances of these variants contributing to breast cancer susceptibility to be very low. Furthermore, the clinical significance of these variants has been depicted as benign in the NCBI ClinVar database, and all the in silico tools agreed that they had no impact on the TP53 and PALB2 protein, respectively.

Missense VUS variants

TP53 c.79C>T caused the change of the highly conserved proline at codon 27 to serine which was shown to increase TP53 protein binding affinity to MDM2 due to TP53-P27S being more α-helical compared to the wildtype TP53.65,66 Functional yeast-based assays of this variant have shown that the TP53 protein was able to retain >80% of its transcriptional transactivation activity for the tested promoters, except for WAF (CDKN1A) and AIP where the residual TP53 transcriptional transactivation activity range at 41-50% and 61-70%,67,68 respectively. Four of six in silico tools predicted a damaging effect of the variant on protein function (Table 4). In this study, the variant was found in a 59-year-old patient who exhibited a history of ≥2 familial colon cancer with poor prognosis. Germline TP53 mutations are associated with Li-Fraumeni syndrome and show poor prognosis in several types of cancers, including colon cancer.15,16 The contribution of this variant in the occurrence of colon cancer in this patient’s family members is unknown. Collectively, the evidence provided with this variant was still insufficient to determine its clinical significance.

PALB2 c.149A>C results in a change of Lysine to Threonine at codon 50. All the in silico tools in this study predicted a damaging effect of the variant on protein function (Table 4). To our knowledge, no experimental functional assay demonstrating the impact of this variant on PALB2 protein function has been reported. In our study, the variant was identified in a 35-years old patient who did not have any family history of cancer. Together with the available population data (Table 4) on the variant occurrences in the general population, there is insufficient evidence to determine a definite conclusion on the variant’s significance.

PALB2 c.2289G>C results in a change of leucine at codon 763 to phenylalanine. Four of six in silico tools predict a damaging effect on protein function (Table 4). This variant has been reported in the literature in individuals affected with Hereditary Breast and Ovarian Cancer (HBOC) Syndrome, most of whom were of the East-Asian populations.21,41 However, these studies do not provide clear conclusions on the association of the variant with HBOC syndrome i.e., the variant was classified as VUS. A recent study reported L763F as a moderate-risk somatic missense mutation.69 A cellular homology directed DNA repair (HDR)-based assay showed no damaging effect of this variant on PALB2 protein function.70 Based on the current available evidence, the criteria for benign and pathogenic are contradictory for this variant. In our study, this variant was identified in a 42-year-old patient with a family history of blood and colon cancer.

PALB2 c.2474G>C results in a change of arginine at codon 825 to threonine. Five of six insilico tools predict a benign effect of the variant on protein function (Table 4). To our knowledge, no experimental functional assay demonstrating the impact of this variant on PALB2 protein function has been reported. In our study, this variant is found to co-occur in an early-onset HBOC syndrome patient with a BRCA2 germline pathogenic variant c.5164_5165delAG (S1722Yfs*1725) providing supporting evidence for a benign role of this variant in the HBOC syndrome. However, the available population data (Table 4) on the variant occurrences in the general population are insufficient to allow a definite conclusion on the variant significance.

PALB2 c.3054G>C results in a change of glutamate at codon 1018 to aspartate. Five of six in silico tools predict a damaging effect of the variant on protein function (Table 4). To our knowledge, only one functional study has been conducted on this variant, and it was shown that it did not impair HDR, and also have no impact on the maintenance of IR-induced G2/M checkpoint.71 This variant has been reported in the literature in individuals affected with HBOC Syndrome, most of whom were of the East-Asian populations.21,26,32,33,37,41,42,45 However, these studies do not provide clear conclusions on the association of the variant with HBOC syndrome i.e., the variant was classified as either likely pathogenic based on prediction tools or VUS. The population data, specifically in the East-Asian population, suggested the variant is a benign polymorphism. However, the currently available evidence is still insufficient to fulfil the ACMG criteria for a definite benign or pathogenic classification (Table 4). In our study, this variant was identified in two unrelated patients: a 71-year-old breast cancer patient with a family history of neck and colorectal cancers, and a 45-years old breast cancer patient who did not have any family history of cancers.

TP53 P72R and response to chemotherapy

TP53 polymorphisms have been studied numerously in breast cancer.In one study of a cohort of 40 female patients no significant correlation was found between the studied polymorphisms and risk of premenopausal breast cancer.72 TP53 codon 72 is a hotspot of polymorphisms with rs1042522 (Pro72Arg) being the most studied SNP. The benign Pro72Arg (P72R) was located in the proline-rich region of the TP53, and the substitution directly affects the structure of the SH3-binding domain. TP53-R72 was shown to be more efficient in inducing apoptosis which influenced an increased longevity in humans, whereas TP53-P72 increased the induction of G1 arrest and was better at activating TP53-dependent DNA repair pathway.73 The SNP has been shown to not have any significant association with breast cancer risk.74 Functional analysis of P72R on TP53 protein function, including transcriptional activity, apoptosis, and cell proliferation assays performed both in human cells or in yeast cells showed no defective impact.75 Interestingly, an in vivo study showed the potential mechanisms i.e., chronic inflammation, by which TP53 R72 variant contribute to enhanced breast cancer susceptibility.76

As one of the common SNPs to be identified in different cancers, substantial studies have been conducted to measure the efficacy of drug response in the presence of this variant with other SNPs in different genes in different cancers. Breast cancer patients with P72P variant were shown to be less sensitive to anthracycline-based neoadjuvant treatment compared to heterozygous and homozygous P72R variants77,78 and developed more toxicity.78 The P/P genotype at codon 72 of TP53 was also reported to be associated with poor disease-free survival (DFS), particularly in patients who received adjuvant chemotherapy.79

In the study, 41 of the study population were TP53-P72R carriers (Table 3). Applying the Hardy-Weinberg equilibrium, the genotype distributions of TP53 codon 72 variants in our study showed 15 (27.8%) were R/R, 26 (48.1%) were P/R, and 13 (24.1%) were P/P while the allelic frequencies were 0.519 (n = 56) for R and 0.418 (n = 52) for P. A total of 30 (55.6%) patients received neoadjuvant chemotherapy while the rest received adjuvant chemotherapy. Patients receiving neoadjuvant chemotherapy were assessed for their tumour pathological response based on histopathological criteria.80 A complete response (Grade 3) was defined as no evidence of residual – and invasive if present – disease. A marked response (Grade 2) was defined as ≥50% reduction in tumour size with apparent or a few remaining cancer cells. A slight response (Grade 1) was defined as mild or moderate response to the treatment with marked changes in <50% of cancer cells. An absence of response (Grade 0) was defined as no change in cancer cells after treatment. All patients were assessed for adverse reaction to chemotherapy based on the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 (National Cancer Institute).

Of the 30 patients who received neoadjuvant chemotherapy, 15 (50%) received anthracycline-based (with or without taxane) regimens, 5 (16.7%) received taxane-based regimens, 9 (30%) received taxane/platinum-based (with or without anthracycline) regimens, and 1 (3.3%) patient with missing record. Of the 29 patients, the tumour pathological response of five patients were not available as they had not completed their treatment cycles yet, giving a total of only 24 patients data to be assessed. Two (8.3%) patients achieved complete response, three (12.5%) patients showed no response to the treatment received, while three (12.5%) and sixteen (66.7%) showed slight and marked response, respectively. Due to the small number of patients, the response to neoadjuvant treatment and the TP53 codon 72 status were grouped into two groups to increase the reliability of the statistical analysis. There was no significant association between the TP53 codon 72 status of these patients with their tumour pathological response to neoadjuvant treatment (p = 0.277, Table 5).

Table 5. Association between TP53 codon 72and response to chemotherapy treatment in the study population.

TP53 Codon 72 Statusp-valuea
R/R and P/RP/P
n%n%
Tumour pathological response to neoadjuvant treatment0.277b
Grade 0 – 1633.300
Grade 2 – 31266.76100
Adverse reaction to treatment0.510
No reaction2564.1753.8
Grade 1 – 31435.9646.2

a Chi-square test for independence.

b Fisher’s exact test.

Records for 2/54 patients with P72R variant on any development of adverse reaction to treatment were not available for analysis giving a total of only 52 patients records to be assessed. A total of 20 (38.5%) patients presented any grade of adverse reaction to chemotherapy, with one (5%) of them experiencing a severe Grade 3 reaction to treatment. There was no significant association between the TP53 codon 72 status with the development of adverse reactions to treatment in these patients (p = 0.510, Table 5).

Discussion

In this study, we analysed the prevalence of germline TP53 and PALB2 mutations in an unselected cohort of 54 breast cancer patients, two of whom have pathogenic BRCA2 mutation. Our study showed that none of the study population were TP53 and PALB2 carriers. Our findings support the rarity of germline TP53 and PALB2 mutations in the unselected cohort of breast cancer which has been reported at a prevalence of 0.2-0.5% and 0.4-1.9%, respectively.30,4148 Moreover, analysis of TP53 and PALB2 mutations in other populations that reported prevalence of both genes in their study cohorts were shown to be selective in their recruitment criteria such as only recruiting: (1) breast cancer patients with personal and family history of breast and/or pancreatic cancer25; (2) BRCA1/2-negative familial breast cancer patients21,25; and (3) early-onset BRCA1/2-negative breast cancer patients.28 It is acknowledged that the small number of recruited patients in this study possibly resulted in the low prevalence and mutation spectrum reported. Notably, we did not conduct large rearrangement analysis which potentially could lead to the possibility of missed carriers in our population.

Our study identified two TP53 and five PALB2 variants, all of which have been reported (Table 3). Of the seven variants, five were classified as VUS (Table 4). As agreed by other studies, elucidating the clinical implication of VUS remains a great challenge especially when disclosing the results to patients and family members as the implication of VUS in the risk of developing breast cancer and disease prevention strategies are still not well understood.41 This could be attributed to the higher frequency of VUS compared to known pathogenic mutations. Moreover, sequencing more genes using high-throughput NGS has vastly increased the number of VUS identified in the genome.

In line with the idea of analysing the association of TP53 mutations with drug response,78 it can be concluded in this study that regardless of the genotype, TP53 codon 72 is not associated with the outcome of neoadjuvant chemotherapy on the patients’ tumour and the development of adverse reaction to the drugs used. Because of the absence of pathogenic mutation and low prevalence of VUS in our study, we decided not to investigate the association between the study population’ characteristics and mutation-status. Furthermore, we did not investigate the association of TP53 codon 72 with the study population’ DFS, as most of our recruited participants were still undergoing treatment at the time of the study.

Conclusions

This is the first report on germline TP53 and PALB2 mutations in Brunei Darussalam. Our data showed two TP53 and five PALB2 missense variants were identified in the Brunei breast cancer patients. The absence of germline pathogenic TP53 and PALB2 mutations among Brunei breast cancer population in our data supports the rarity in the involvement of these genes in all breast cancer cases unless specific clinical presentations have been observed. Further studies consisting of a larger number of patients and controls are needed to shed more light on the significance of TP53 and PALB2 mutations in Brunei breast cancer patients.

Comments on this article Comments (0)

Version 1
VERSION 1 PUBLISHED 30 Nov 2023
Comment
Author details Author details
Competing interests
Grant information
Copyright
Download
 
Export To
metrics
Views Downloads
F1000Research - -
PubMed Central
Data from PMC are received and updated monthly.
- -
Citations
CITE
how to cite this article
Matusin SNI, Lu ZH and Haji Abdul Hamid MRW. Low prevalence of germline TP53 and PALB2 mutations in unselected cohort of breast cancer patients from Brunei Darussalam [version 1; peer review: awaiting peer review]. F1000Research 2023, 12:1537 (https://doi.org/10.12688/f1000research.136362.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
track
receive updates on this article
Track an article to receive email alerts on any updates to this article.

Open Peer Review

Current Reviewer Status:
AWAITING PEER REVIEW
AWAITING PEER REVIEW
?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Comments on this article Comments (0)

Version 1
VERSION 1 PUBLISHED 30 Nov 2023
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
Sign In
If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password.

The email address should be the one you originally registered with F1000.

Email address not valid, please try again

You registered with F1000 via Google, so we cannot reset your password.

To sign in, please click here.

If you still need help with your Google account password, please click here.

You registered with F1000 via Facebook, so we cannot reset your password.

To sign in, please click here.

If you still need help with your Facebook account password, please click here.

Code not correct, please try again
Email us for further assistance.
Server error, please try again.