Clinical Significance of Germline Variants in the BRCA2 Gene and Their Association With Prostate Cancer Risk in Polish Men: A Case-Control Study

Abstract

Objectives

Currently, prostate cancer (PC) is the most common medical problem endangering men’s health and life worldwide. We tested the association of detected germline variants in BRCA2 with PC risk and estimated their impact on the clinical course of the disease, including overall survival time, in Polish men with localized PC that qualified for radical prostatectomy (RP).

Materials and Methods

DNA of 97 PC patients from various age groups and with different disease stages was analyzed. Control DNA samples consisted of 100 male volunteers without PC that were age-matched to the study group. Next Generation Sequencing (NGS) and Sanger sequencing were used for variant detection.

Results

Five rare variants of the BRCA2 gene were detected in single PC patients. There were four substitutions (c.8010G>C, c.682-32A>G, c.9257-75G>C, c.516+17G>C) and one deletion (c.6393_6396del). Among the detected variants, one was pathogenic, one was a variant of uncertain significance (VUS), and three were likely benign. The c.8010G>C was a new variant. In the carrier of the c.6393_6396del pathogenic variant, PC was diagnosed at the T3 stage and the patient survived 48 months after PC confirmation (the date of biopsy).

Conclusions

The BRCA2 c.6393_6396del pathogenic variant demonstrates an association with clinical features of the disease (GS and TNM) and shorter survival of patients with localized prostate cancer that qualified for RP. Additionally, our findings suggest that multi-organ cancer aggregation in a family, including prostate cancer aggregation in close relatives, and young age at cancer onset should be taken into consideration by clinicians as an indication to refer patients to molecular testing.

Plain Language Summary

Clinical significance of germline variants in the BRCA2 gene and their association with prostate cancer risk in Polish men: a case-control study.

Keywords

prostate cancer germline variants clinical significance next generation sequencing

Introduction

Prostate cancer (PC) is the world’s second most frequent cancer (after lung cancer) and the fifth leading cause of cancer fatalities (after lung, liver, colorectal and stomach cancer) among men.^1-3 A few risk factors have been identified for PC: well-established advanced age, ethnicity and family history of the disease in close relatives as well as speculative, for example, smoking, excess body weight and nutritional habits.^4-6 The genetic basis of prostate cancer is very complex. Genome-wide association studies have identified common susceptibility loci in more than a dozen independent chromosomal regions associated with an increased risk for PC which can play their role in androgen metabolism, for example, AR, SRD5A2 or CYP17, in DNA repair like BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, NBN, BRIP or ATM and in different human organ development during the embryonic period, for example, HOXB13.^7-13 However, as it turns out, none of the above-mentioned genes is a high-risk for PC development. Detecting high-predisposition gene pathogenic or likely pathogenic germline variants associated with prostate cancer is very important because it can facilitate appropriate treatment or even prevent the disease.

The BRCA2 gene is located on the long arm of chromosome 13 (13q13.1) and consists of 28 exons and codes 3418 amino acids protein.^6,14 The BRCA2 is an autosomal dominant inheritance cancer suppressor gene also performing an essential function in the preservation of genomic control and repair of DNA damage to prevent tumor development. Its N-terminus binds to PALB2, while the C-terminus contains the NLS signal sequence and the S3291 phosphorylation site by cyclin-dependent kinase, which binds to the RAD51 protein.^6,15,16 The BRCA2-RAD51 complex plays a very important role in homologous recombination (HR); BRCA2 recruits RAD51 fibers to where double-stranded DNA has been damaged. Next, the RAD51-BRCA2-DSS1 complex uses sister chromatids as a template for “error-free” homologous recombination, enabling DNA repair.^6,14,17 The gene also participates in cytoplasmic divisions; when damaged, cytoplasmic divisions are disturbed, which increases the incidence of binucleated cells. In addition to changes in the number of cell nuclei, when the BRCA2 gene is damaged or missing, there may also be changes in the number and structure of chromosomes, leading to the formation of aneuploid cancer cells.^14,18 If BRCA2 is missing or damaged, resistance to harmful factors, such as ionizing radiation or poly(ADP-ribose) polymerase (PARP) inhibitors is reduced. This gene also participates in replication and more specifically in stabilizing blocked replication forks and protecting them against degradation.^19,20

The pathogenic or likely pathogenic variants of BRCA2 gene are known as the cause of different cancers in both men and women.¹⁴ These tend to be aggressive and can present at younger age (<50) than non-BRCA–mutated cancers. Mutations in BRCA2 have been detected in 1.2% to 3.2% of patients with prostate cancer in most studies.^1,2,21 The overall PC chance is stated to be up to 8.6-fold for those who are carriers of BRCA2 compared to non-carriers. Additionally, the BRCA2 mutations have been reported to result in reduced survival rates of prostate cancer patients.²¹

Poly (ADP-ribose) polymerases (PARP) are a family of ADP-ribosyl transferase enzymes, of which PARP1 is the most common. PARPs play roles in cellular processes like DNA repair, transcriptional regulation and RNA interference among others.^22,23 The PARP1 is a key component of the base excision repair mechanism (BER). It acts as a DNA damage sensor and a signal transducer. The enzyme detects single-strand breaks, binds to the DNA adjacent to the damage, and synthesizes PAR chains on target proteins (PARylation) leading to the recruitment of additional factors that complete the DNA repair process. In the autoPARylation, PARP1 is released for the site of DNA damage. Poly(ADP-ribose)polymerase inhibitors (PARPi) are a group of drugs that may be used in metastatic castration-resistant prostate cancer (mCRPC) patients with homologous recombination (HR) defects particularly in those with BRCA1/2 changes.^14,24,25

We were looking for rare germline constitutional variants of BRCA2 gene in Polish patients with localized prostate cancer and controls. We genotyped 97 prostate cancer patients and 100 men from the control group to establish whether germline variants contribute to PC development in the study group and to measure their impact on cancer risk and on the clinical characteristics of the disease including survival time.

Materials

The DNA samples were isolated from EDTA anticoagulated peripheral blood of 97 consecutive, newly diagnosed PC patients from all over Poland, regardless of age at prostate cancer onset, family history and histological type of cancer in the Department of Clinical Genetics Collegium Medicum Nicolaus Copernicus University in Bydgoszcz between 2006 and 2007 (79 samples) and 2019 and 2020 (18 samples) (archival material). Patients were hospitalized because of PC at the Department of Urology of the J. Biziel University Hospital in Bydgoszcz. A group of 97 men with prostate cancer was considered representative of the study type.

The age at PC diagnosis ranged from 45 to 76 (the mean age was 60.4 ± 6.3). Family history was analyzed either by the construction of family pedigree or the completion of a standardized questionnaire by patients. All cases of first-and second-degree relatives diagnosed with prostate cancer and other cancer types and their age at the diagnosis were recorded. The estimation of patient families as those with a history suggesting hereditary risk of prostate cancer was performed based on criteria defined by Carter et al²⁶ and Cybulski et al²⁷ Among 97 prostate cancer patients, 21 (21.6%) originated from families suspected of Hereditary Prostate Cancer (HPC). In 62 of 97 (63.9%) families, there was an aggregation of cancers of breast, stomach, colon, ovary, lung, larynx, bladder or kidney as well as melanoma, in addition to prostate cancer. The remaining patients had no family history of cancer.

Information about the history of PC and other cancer aggregation in prostate cancer patients’ families was available for 97 families.

The PSA level before the surgical operation and the information about prostate cancer grade and the tumor stage were available for all 97 patients.

The survival time data were available for 97 patients, who were tracked from the day of the biopsy (confirmation of prostate cancer) until death if applicable, or until the end of the observation. A 5-year survival period was assumed in still-living patients.

The control group consisted of DNA isolated from the peripheral blood of 100 male volunteers, who were healthy at the time of the investigations, that is, without prostate cancer based on PSA concentration and/or digital rectal examination (DRE). These medical examinations were performed as a part of PC prophylaxis. The molecular test was offered to all men in the control group. The age of men from the controls ranged from 46 to 74 (the mean age was 59.9 ± 6.6) and matched to the study group. The purpose of the control group in our study was to estimate with accuracy the frequency of detected BRCA2 germline variants in the Polish population. Men with any cancer diagnosed in a first-degree relative were excluded from controls.

The study protocol was approved by the Ethics Committee of the Collegium Medicum Nicolaus Copernicus University in Bydgoszcz, Poland (approval number: KB 326/2010). All participants provided written informed consent to the analysis and signed the respective form. The personal information of patients in medical materials has been anonymized. Our study was classified as a non-interventional and prognostic clinical study.

Methods

The analysis was performed on genomic DNA (100 ng) (gDNA) extracted from leukocytes with a QIAamp DNA Mini Kit according to the manufacturer’s protocol (Qiagen, Hilden, Germany). The sequencing library preparation was performed using SureSelect XT Reagent Kit (from Agilent) according to the manufacturer’s instructions. The sample was sequenced with NGS technology by Illumina on Miseq sequencer with 2 × 75 bp reads. Demultiplexing of the sequencing reads was performed with Illumina’s bcl2fastq2 v2.19.0. Adapters were trimmed with Skewer version 0.2.9.²⁸ The reads were aligned to GRCh37/hg19 reference sequence using BWA-MEM.²⁹ Read duplicates were removed using Picard 2.18.2 (https://broadinstitute.github.io/picard/). Variant call was performed with GATK v4.0.3.0. HaplotypeCaller^30,31 and FreeBayes (v1.2.0-2-g29c4002).³²

Mutation-positive cases detected by NGS were confirmed by Sanger sequencing analysis using ABI PRISM 3130 (Applied Biosystems). For Sanger sequencing, exons were amplified by PCR (PCR profiles and primer sequences are available upon request). Primers were designed using the Primer3 tool. The sequencing reaction was conducted on PCR product with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s procedure.

The clinical significance of identified variants was evaluated by VarSome or Franklin clinical database. The analysis of BRCA2 detected variants was performed on NCBI reference sequence NG_012772.3. The BRCA2 c.8010G>C new variant was deposited in LOVD database (https://databases.lovd.nl/shared/variants/0001020258#00003479). The reporting of this study conforms to STROBE guidelines.³³

Statistical Analysis

The mean age at PC diagnosis was compared between gene variants carriers (pathogenic, likely pathogenic, and VUS) and non-carriers using Student’s t-test. A Kaplan-Meier survival curve was made from the date of biopsy for the whole group of BRCA2 variant carriers and non-carriers.

Results

The 5 variants of the BRCA2 gene were detected in single PC patients. There were four substitutions (c.8010G>C, c.682-32A>G, c.9257-75G>C, c.516+17G>C) and one deletion (c.6393_6396del). Among detected variants, there was 1 pathogenic, 1 variant of uncertain significance (VUS) and three likely benign. In 92 patients, no variants of tested gene were found. The 4 variants were described previously and have “rs” numbers. The c.8010G>C was a new variant. All variants of BRCA2 gene detected in analyzed patients are presented in Table 1.

Table 1.

Germline Variants in BRCA2 Gene Detected in Prostate Cancer Patients.

Variant	Amino acid change	Exon	Prediction by VarSome or Franklin	Variant ID	Frequency in Europe (%) GnomAD or ExAC	Genotype
c.6393_6396del	p.Lys2131AsnfsTer5	11	Pathogenic	rs397507849	—	het
c.8010G>C	p.Ser2670=	18	VUS	—	—	het
c.682-32A>G	—	—	Likely benign	rs376686897	0.02	het
c.9257-75G>C	—	—	Likely benign	rs276174922	0.02	het
c.516+17G>C	—	—	Likely benign	rs765435155	0.0008	het

VUS, variant of uncertain significance; het, heterozygous carrier.

According to ACMG classification,³⁴ the c.6393_6396del (p.Lys2131AsnfsTer5) is a pathogenic frameshift variant, that is, it meets the criteria of PM2 (is absent in mutation databases such as gnomAD/ExAC), PVS1 (loss of function is a known mechanism of disease), PS4 and PP5 (reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation). The c.8010G>C (p.Ser2670=) is a new synonymous variant of uncertain significance (VUS), fulfilling PM2 (is absent in mutation databases like gnomAD/ExAC) and BP7 (a synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved). The c.682-32A>G, c.9257-75G>C and c.516+17G>C are likely benign variants that meet the ACMG criteria of PM2 (low populational frequency), BP7 (a synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved) and BP6 (reputable source recently reports variant as benign but the evidence is not available to the laboratory to perform an independent evaluation). Our classification of variants done by following under mutation databases is of presumable character.

The frequency, clinical and laboratory characteristics of prostate cancer, and types of cancer diagnosed in relatives of carriers of all detected variants in our study are shown in detail below.

The c.6393_6396del germline variant was detected in 1/97 PC patients (heterozygous carrier) at 64 years of age and in no healthy man. No cancer has been diagnosed in his close relatives. The pedigree of BRCA2 c.6393_6396del carrier family is shown on Figure 1. In the proband, the preoperative PSA level was 6.7 ng/mL, and Gleason score - 9 (4 + 5). PC was diagnosed at T3 stage. The c.6393_6396del carrier survived 48 months after prostate cancer confirmation (the date of biopsy) (Figure 6).

Figure 1.

The Pedigree of BRCA2 c.6393_6396del Germline Variant Carrier. The Proband is Indicated by an Arrow. The Type of Cancer and the Age at Disease Diagnosis are Written Under the Filled Black Square. c.6393_6396del (+) - Presence of the Variant.

The c.8010G>C germline variant was detected in 1/97 men with prostate cancer (heterozygous carrier) at 56 years of age. In the control group, this variant was not detected. According to the Franklin database, it is a variant of uncertain significance (VUS). In addition to PC, other cancer types were diagnosed in close relatives, for example, stomach cancer at 65 in the patient’s mother (died at 65) and at 70 in maternal grandmother (died at 70), colon cancer at 65 in the father (died at the same age) and lung cancer at 72 in the father’s brother (died at the same age). The pedigree of BRCA2 c.8010G>C carrier family is shown on Figure 2. In proband, the preoperative PSA level was 6.71 ng/mL, Gleason score - 5 (2 + 3), and PC was diagnosed at T2c stage. The c.8010G>C carrier survived at least five years from the date of biopsy (prostate cancer confirmation) (Figure 6).

Figure 2.

The Pedigree of BRCA2 c.8010G>C Germline Variant Carrier. The Proband is Indicated by an Arrow. The Type of Cancer, the Age at Disease Diagnosis, and the Age of Death of Family Members are Written Under the Filled Black Square or Circle. c.8010G>C (+) - Presence of the Variant.

The c.682-32A>G, c.9257-75G>C and c.516+17G>C germline variants were found in single PC patients which were classified as likely benign according to Franklin database. None of them was detected in the control group.

In c.682-32A>G carrier the disease was diagnosed at 58 years of age. No other cancers have been diagnosed in carrier’s close relatives. The pedigree of BRCA2 c.682-32A>G carrier family is shown on Figure 3. In proband, the preoperative PSA level was 7.36 ng/mL, Gleason score - 5 (2 + 3), and PC was diagnosed at T2c stage. The c.682-32A>G carrier survived at least five years from the date of biopsy (prostate cancer confirmation) (Figure 6).

Figure 3.

The Pedigree of BRCA2 c.682-32A>G Germline Variant Carrier. The Proband is Indicated by an Arrow. The Type of Cancer, the Age at Disease Diagnosis, and the Age of Death of Family Members are Written Under the Filled Black Square. c.682-32A>G (+) - Presence of the Variant.

In c.9257-75G>C carrier, PC was diagnosed at 61 years of age. The carrier of this likely benign variant originated from a family with HPC. PC was also diagnosed in carrier’s brother at 57 years of age. Additionally, lip cancer was diagnosed in proband’s father at 70 years of age (died at 85), colon cancer in proband’s paternal uncle at 65 (died at 70), nose cancer in proband’s paternal aunt at unknown age (died at unknown age). Besides, leukemia was diagnosed in proband’s mother at 83 (died at the same age), the larynx cancer in proband’s maternal aunt at 67 (died at unknown age) and the pancreas cancer in proband’s maternal grandmother at 70 (died at 70). The pedigree of c.9257-75G>C carrier family is shown on Figure 4. In proband, the preoperative PSA level was 15.06 ng/mL, Gleason score - 6 (3 + 3), and PC was diagnosed at T2 stage. The c.9257-75G>C carrier survived at least five years from the date of biopsy (prostate cancer confirmation) (Figure 6).

Figure 4.

The Pedigree of BRCA2 c.9257-75G>C Germline Variant Carrier. The Proband is Indicated by an Arrow. The Type of Cancer, the Age at Disease Diagnosis, and the Age at Death of the Family Members are Written Under the Filled Black Square or Circle. AOU - Age at Onset Unknown, ADU - Age at Death Unknown, c.9257-75G>C (+) - Presence of the Variant.

In c.516+17G>C likely benign variant carrier, PC was diagnosed at 70 years of age. In proband’s family PC was also diagnosed in his father at 80 years of age (died at 90). Additionally, melanoma was diagnosed in proband’s brother at 55 years of age (died at 60). The pedigree of c.516+17G>C carrier family is shown on Figure 5. In proband, the preoperative PSA level was 5.86 ng/mL, Gleason score - 6 (3 + 3), and PC was diagnosed at T2c stage. The c.516+17G>C carrier survived at least five years from the date of biopsy (prostate cancer confirmation) (Figure 6).

Figure 5.

The Pedigree of BRCA2 c.516+17G>C Germline Variant Carrier. The Proband is Indicated by an Arrow. The Type of Cancer, the Age at Disease Diagnosis, and the Age of Death of the Family Members are Written Under the Filled Black Square. c.516+17G>C (+) - Presence of the Variant.

Figure 6.

The Kaplan-Meier Survival Curve for the Whole Group of BRCA2 Variants Carriers and Non-Carriers. The Five Variant Carriers Were Indicated by an Arrow: 1 - c.6393_6396del, 2 - c.8010G>C, 3 - c.682-32A>G, 4 - c.9257-75G>C, 5 - c.516+17G>C.

Discussion

Prostate cancer is one of the main life-threatening disorders in males. Genetic susceptibility plays an important role in disease development. It was assessed that the overall PC risk is up to 8.6-fold for those who are carriers of BRCA2 pathogenic or likely pathogenic variants compared to non-carriers. Additionally, BRCA2 mutations have been reported to result in an aggressive pattern of the disease with a reduced survival rate. Gene mutations are present in approximately 5% of patients with progressive prostate cancer. In designing strategies for genetic testing, it is important to define the spectrum of germline variants of BRCA2 gene associated with a high risk of prostate cancer development. Genetic testing is critical in PC screening and treatment.

The association between the higher risk of prostate cancer in BRCA2 pathogenic or likely pathogenic variants carriers has been relatively well established.^1,35 However, in the present study, we were looking for BRCA2 rare germline variants in good prognosis of Polish prostate cancer patients with localized disease qualified for radical treatment. We genotyped 97 prostate cancer men and 100 controls to establish whether BRCA2 variants contribute to prostate cancer development and to measure their impact on cancer risk, and on the clinical characteristics of the disease, including survival time.

In the present study, we detected five variants of the BRCA2 gene in single prostate cancer patients out of 97 examined men from Poland. The identified variants included four substitutions (c.8010G>C, c.682-32A>G, c.9257-75G>C, c.516+17G>C) and one deletion (c.6393_6396del). According to Franklin or/and VarSome databases, of these, one variant was classified as pathogenic (c.6393_6396del), one as VUS (c.8010G>C), and three as likely benign (c.8010G>C, c.682-32A>G, c.9257-75G>C).

In the present study, the BRCA2 c.6393_6396del pathogenic variant was detected in a single PC patient above 60 years of age (at 64) and in no healthy men. According to GnomAD/ExAC the populational frequency of this variant is not available. The c.6393_6396del germline variant was also detected in Polish patients, but with ovarian cancer, in another two studies.^36,37 In Koczkowska et al investigation, the c.6393_6396del was found at 62 years of age in patient with the disease diagnosed at III stage³⁶ and in Brozek et al³⁷ study at 76 years old patient with ovarian cancer also diagnosed at III stage.

In our study, at the time of prostate cancer diagnosis, the c.6393_6396del carrier did not know about the presence of prostate or other cancers in his close relatives. Similarly, in Koczkowska et al and in Brozek et al research, no information about other cancers in addition to ovarian cancer diagnosed in c.6393_6396del carriers was available.^36,37

In the present study, the proband’s aggressive disease course was manifested in high Gleason score (9) and an advanced stage (T3) before disease treatment. It contributed to his short survival time (only 48 months) after disease confirmation (the date of biopsy) despite good prognosis. This observation is consistent with the results obtained by other authors.^1,22,35,38 The BRCA2 gene mutations cause a higher risk of prostate cancer, with an estimated relative risk of 2.5-8.6-fold by 65 years of age.^1,2,7,39-47 Gallagher et al¹ showed that men who carried the BRCA2 mutation had a 3 times higher risk of PC than non-carriers (OR = 3.18). Nyberg et al³⁵ found that BRCA2 mutation carriers are at two to five times higher risk of PC compared to men in the general population. Additionally, the association of BRCA2 germline variants with earlier-onset of clinically significant prostate cancer was confirmed by many authors.^22,38 A number of retrospective studies report higher rates of lymph node involvement, distant metastases at diagnosis and higher mortality rates in mutation carriers.^7,39-41 Germline BRCA2 mutation status is reported to be an independent prognostic factor for poorer outcomes.⁷ Gallager et al show that BRCA2 mutation carriers were more than twice as likely to have a Gleason score of 7-10 and were at higher risk of recurrence and death than non-carriers. In their study, the BRCA2 mutation carriers had an increased risk of developing biochemical recurrence (HR = 2.41) and of castrate metastases (HR = 3.01) compared to non-carriers and this translated into a greater risk of death due to prostate cancer (HR = 5.48). Nyberg et al suggest that BRCA2 mutations are associated with a more aggressive PC phenotype; the association was stronger for GS ≥ 7 tumors. Furthermore, the authors observed a significant association between BRCA2 mutations and PC mortality. Associations with high-grade disease and PC mortality are consistent with previous reports for BRCA2 carriers.^43,44,48 Furthermore, tumors of BRCA2 mutation carriers with localized PC have been demonstrated to exhibit genomic instability more typically seen in metastatic castration-resistant PC.⁴²

A key challenge in the management of patients with localized prostate cancer is the identification of men with a high likelihood of progression to an advanced stage. Patients who are carriers of pathogenic germline variants of BRCA2 gene have worse clinical outcomes than noncarriers when treated with conventional methods. Therefore potentially, this group of patients with localized disease may benefit from treatment with PARP inhibitors.

In our study, the new c.8010G>C variant of uncertain clinical significance (VUS) was found in a single prostate cancer patient below 60 years of age (at 56). It was not detected in the control group. This variant frequency is not available in mutation databases such as gnomAD/ExAC. It seems that the c.8010G>C (p.Ser2670=) variant may not have clinical significance because it does not result in a change of amino acid. In addition, in silico analysis do not predict a difference in splicing although, in our study, a family history of various cancers in close relatives (stomach, colon and lung) of c.8010G>C carrier may suggest a potential association of it with broader cancer susceptibility. The presence of c.8010G>C in carrier was not associated with higher Gleason score, higher tumor stage, and shorter overall survival time. However, our observations were made only on one prostate cancer case and his family members and require further examining a larger group of patients to elucidate the final role of it in prostate and other cancer development. In summary, based on the above information, the clinical significance of this variant cannot be determined with certainty at this time.

In the present study, the likely benign variants (c.682-32A>G, c.9257-75G>C, c.516+17G>C) were detected in single PC patients (at 58, 61, 70 years of age respectively) and in no healthy men. According to GnomAD (Aggregated) the populational frequency of c.682-32A>G is very low at 0.02% (it occurred with 0.04% frequency in European-Non Finnish (NFE) population, 0.005% in the African and 0.009% in the American). In GnomAD (Aggregated) the c.9257-75G>C variant also has a very low populational frequency - 0.02% (in European-Non Finnish (NFE) - 0.04%). The frequency of c.516+17G>C was even lower than the two above likely benign variants. According to GnomAD (Exome) it was 0.0008% (0.01% in African population). In our study, the presence of these three likely benign variants is not associated with the age of prostate cancer onset. The c.682-32A>G carrier revealed no other cancers in close relatives. The c.9257-75G>C carrier originated from family with HPC (PC was also diagnosed in his brother at 57) and additionally had close relatives with lip, colon, nose, larynx, pancreas cancers and leukemia. We suppose that in this family, the development of various types of cancer including PC may be associated with other pathogenic germline variants, in BRCA2 or other genes. In c.516+17G>C likely benign variant carrier, prostate cancer was also diagnosed in his father but at an older age (at 80). However, a melanoma was diagnosed in his brother at younger age (55). It is probable that in this family, the development of prostate cancer and melanoma may be associated with other pathogenic variant or variants in BRCA2 or other genes. In our study we did not observe the association between the presence of likely benign variants in PC patients and clinical characteristics of the disease including survival time. Additionally, in silico analysis of c.682-32A>G, c.9257-75G>C, c.516+17G>C predict no impact on the splicing process.

Our study has of course some limitations. The first is an analysis of only one well-known cancer predisposition gene. Currently, NGS technologies allow for genotyping of the whole sequences of many genes to know the individual predisposition to cancer. However, our study aimed was to search for pathogenic or likely pathogenic germline variants of BRCA2 gene to measure their impact on cancer risk and on the clinical characteristics of the disease, including survival time, in the group of good prognosis patients qualified for radical prostatectomy. However, we indicate that in future studies there is a need for multi-gene panels of other DNA damage repair genes, for example, BRCA1, ATM, ATR, CHK1, CHK2, DSS1, RPA1, NBSI, FANCD2, FANCA, CDK12, PALB2, BRIP1, RAD51B, RAD51C, RAD51D, RAD54, MLH1, MSH2, MSH6, PMS2 and multi-centre investigations or whole-genome sequencing (WGS) to understand the broader genetic architecture of prostate cancer. Another limitation may be the number of men included in our study however, the group of 97 men with prostate cancer was considered as representative for the study type. Despite this, we suggest further investigations on larger patient and control groups to finally determine the role of BRCA2 germline variants in prostate cancer development. The limitation of the study may be also the lack of functional analysis for the newly detected c.8010G>C variant. Nevertheless, the functional studies, together with protein modeling, were outside the initial scope of the present investigation and its funding. These will be performed in a follow-up project, and the results will be made available separately.

The identification of germline, constitutional, and pathogenic variants carriers in BRCA2 gene may allow the implementation of individual and family prevention pathways, through the application of validated screening programs and the introduction of risk-reducing strategies. According to the relevant and increasing therapeutic predictive implications, the inclusion of BRCA2 testing in the routine management of patients with prostate cancer represents a key requirement to optimize medical or surgical therapeutic and prevention decision-making and access to specific anticancer therapies. Therefore, accurate patient selection, the use of standardized procedures, and adherence to homogeneous testing criteria are essential to implement BRCA2 testing in clinical practice and into routine prostate cancer screening, especially for individuals with a family history of the disease.

Conclusions

Many years of investigations on prostate cancer pathogenesis indicate the unquestionable role of genetic factors in disease development, both congenital and somatic as well as the high genetic heterogeneity of this cancer. Our results confirm that pathogenic germline constitutional variants of BRCA2 gene are associated with prostate cancer risk as well as the clinical features of the disease. In the present study, the c.6393_6396del pathogenic variant demonstrates the association with shorter survival in carrier (good prognosis prostate cancer patient with localized disease qualified for radical treatment). We also drew attention to prostate cancer aggregation in close relatives together with multi-organ cancer aggregation in a family, and that the young age at cancers onset should be taken under consideration by clinicians as an indication to refer persons to molecular testing. We are convinced that soon genetic testing will be a key factor in the selection of specific prostate cancer patient groups from the whole group of good prognosis patients with localized PC qualified for RP in whom there may be a relapse within the first 5 years after PC diagnosis. Recent years studies indicate that metastatic castration-resistant PC patients (mCRPC) with mutations of DDR genes, including BRCA2, who are treated with PARPi or immunotherapy may achieve positive therapeutic responses. In the era of personalized medicine, genetic tests are of great importance for diagnosis prognostic estimation and cancers treatment.

Footnotes

Authors’ Note

I confirm that this work is original and has not been published elsewhere, nor is it currently under consideration for publication elsewhere.

Acknowledgements

We thank all patients for participating in this study.

ORCID iD

Marta Heise

Statements and Declarations

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the AKRS.480.3.511.2023 grant of the Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, Poland.

Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Gallagher

Gaudet

Pal

, et al. Germline BRCA mutations denote a clinicopathologic subset of prostate cancer. Clin Cancer Res. 2010;16:2115-2121.

Kote-Jarai

Leongamornlert

Saunders

, et al. BRCA2 is a moderate penetrance gene contributing to young-onset prostate cancer: implications for genetic testing in prostate cancer patients. Br J Cancer. 2011;105:1230-1234.

Rawla

. Epidemiology of prostate cancer. World J Oncol. 2019;10:63-89.

Hemminki

Czene

. Age specific and attributable risks of familial prostate carcinoma from the family-cancer database. Cancer. 2002;95:1346-1353.

Bray

Laversanne

Sung

, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74:229-263.

Andreassen

Seo

Wiek

Hanenberg

. Understanding BRCA2 function as a tumor suppressor based on domain-specific activities in DNA damage responses. Genes. 2021;12:1034.

Alkhushaym

Fallatah

, et al. The association of BRCA1 and BRCA2 mutations with prostate cancer risk, frequency, and mortality: a meta-analysis. Prostate. 2019;79:880-895.

Momozawa

Sasai

Usui

, et al. Expansion of cancer risk profile for BRCA1 and BRCA2 pathogenic variants. JAMA Oncol. 2022;8:871-878.

Stanbrough

Bubley

Ross

, et al. Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer. Cancer Res. 2006;66:2815-2825.

10.

Han

Park

Cho

, et al. Characteristics of BRCA2 mutated prostate cancer at presentation. Int J Mol Sci. 2022;23:13426.

11.

Pritchard

Mateo

Walsh

, et al. Inherited DNA-repair gene mutations in men with metastatic prostate cancer. N Engl J Med. 2016;375:443-453.

12.

Pilié

Johnson

Hanson

, et al. Germline genetic variants in men with prostate cancer and one or more additional cancers. Cancer. 2017;123:3925-3932.

13.

Zhen

Syed

Nguyen

, et al. Genetic testing for hereditary prostate cancer: current status and limitations. Cancer. 2018;124:3105-3117.

14.

Xie

Luo

Jiang

Zhong

Shi

. BRCA2 gene mutation in cancer. Medicine. 2022;101:e31705.

15.

Zhang

, et al. PALB2 links BRCA1 and BRCA2 in the DNA damage response. Curr Biol. 2009;19:524-529.

16.

Schlacher

Christ

Siaud

Egashira

Jasin

. Double-strand break repair independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell. 2011;145:529-542.

17.

Pellegrini

Venkitaraman

. Emerging functions of BRCA2 in DNA recombination. Trends Biochem Sci. 2004;29:310-316.

18.

Daniels

Wang

Lee

Venkitaraman

. Abnormal cytokinesis in cells deficient in the breast cancer susceptibility protein BRCA2. Science. 2004;306:876-879.

19.

Mweempwa

Wilson

. Mechanisms of resistance to PARP inhibitors - an evolving challenge in oncology. Cancer Drug Resist. 2019;2:608-617.

20.

Inoue

Sekito

Kageyama

Sugino

Sasaki

. Roles of the PARP inhibitor in BRCA1 and BRCA2 pathogenic mutated metastatic prostate cancer: direct functions and modification of the tumor microenvironment. Gene. 2023;15:2662.

21.

Silvestri

Leslie

, et al. Cancer risks associated with BRCA1 and BRCA2 pathogenic variants. J Clin Oncol. 2022;40:1529-1541.

22.

Junejo

Aikhateeb

. BRCA2 gene mutation and prostate cancer risk. Saudi Med J. 2020;41:9-17.

23.

Robson

Senkus

, et al. Olaparib for metastatic breast cancer in patients with a germline BRCA mutation. N Engl J Med. 2017;377:523-533.

24.

Helleday

. The underlying mechanism for the PARP and BRCA synthetic lethality: clearing up the misunderstandings. Mol Oncol. 2011;5:387-393.

25.

McNevin

Cadoo

Baird

Finn

McDermott

. PARP inhibitors in advanced prostate cancer in tumors with DNA damage signatures. Cancers. 2022;14:4751.

26.

Carter

Beaty

Steinberg

Childs

Walsh

. Mendelian inheritance of familial prostate cancer. Proc Natl Acad Sci USA. 1992;89:3367-3371.

27.

Cybulski

Gliniewicz

Sikorski

, et al. Hereditary prostate cancer: clinical genetics of cancers 2018. In: Lubiński

, ed. Poland: Print Group Sp. Z o.O:179-192.

28.

Jiang

Lei

Ding

Zhu

. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinf. 2014;15:182.

29.

. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. 2013. Preprint at. https://arxiv.org/abs/1303.3997

30.

McKenna

Hanna

Banks

, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297-1303.

31.

Poplin

Ruano-Rubio

DePristo

, et al. Scaling accurate genetic variant discovery to tens of thousands of samples. 2018. Preprint at. https://www.biorxiv.org/content/10.1101/201178v3

32.

Garrison

Marth

. Haplotype-based variant detection from short-read sequencing. 2012. Preprint at. https://arxiv.org/abs/1207.3907

33.

von Elm

Altman

Egger

, et al. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. Ann Intern Med. 2007;147:573-577.

34.

Richards

Aziz

Bale

, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405-424.

35.

Nyberg

Frost

Barrowdale

, et al. Prostate cancer risk by BRCA2 genomic regions. Eur Urol. 2020;78:494-497.

36.

Koczkowska

Zuk

Gorczynski

, et al. Detection of somatic BRCA1/2 mutations in ovarian cancer - next-generation sequencing analysis of 100 cases. Cancer Med. 2016;5:1640-1646.

37.

Brozek

Ochman

Debniak

, et al. High frequency of BRCA1/2 germline mutations in consecutive ovarian cancer patients in Poland. Gynecol Oncol. 2008;108:433-437.

38.

Fettke

Dai

Kwan

, et al. BRCA-deficient metastatic prostate cancer has an adverse prognosis and distinct genomic phenotype. EBioMedicine. 2023;95:104738.

39.

Breast Cancer Linkage Consortium . Cancer risks in BRCA2 mutation carriers. J Natl Cancer Inst. 1999;91:1310-1316.

40.

Kirchhoff

Kauff

Mitra

, et al. BRCA mutations and risk of prostate cancer in Ashkenazi Jews. Clin Cancer Res. 2004;10:2918-2921.

41.

van Asperen

Brohet

Meijers-Heijboer

, et al. Cancer risks in BRCA2 families: estimates for sites other than breast and ovary. J Med Genet. 2005;42:711-719.

42.

Risch

McLaughlin

Cole

DEC

, et al. Population BRCA1 and BRCA2 mutation frequencies and cancer penetrances: a kin-cohort study in Ontario, Canada. Cancer. 2006;98:1694-1706.

43.

Agalliu

Gern

Leanza

Burk

. Associations of high-grade prostate cancer with BRCA1 and BRCA2 founder mutations. Clin Cancer Res. 2009;15:1112-1120.

44.

Moran

O’Hara

Khan

, et al. Risk of cancer other than breast or ovarian in individuals with BRCA1 and BRCA2 mutations. Fam Cancer. 2012;11:235-242.

45.

Akbari

Wallis

CJD

Toi

, et al. The impact of a BRCA2 mutation on mortality from screen-detected prostate cancer. Br J Cancer. 2014;111:1238-1240.

46.

Mersch

Jackson

Park

, et al. Cancers associated with BRCA1 and BRCA2 mutations other than breast and ovarian. Cancer. 2015;121:269-275.

47.

Roed Nielsen

Petersen

Therkildsen

Skytte

Nilbert

. Increased risk of male cancer and identification of a potential prostate cancer cluster region in BRCA2. Acta Oncol. 2016;55:38-44.

48.

Castro

Goh

Leongamornlert

, et al. Effect of BRCA mutations on metastatic relapse and cause-specific survival after radical treatment for localised prostate cancer. Eur Urol. 2015;68:186-193.