BRCA1, BRCA2 and PALB2 mRNA Expression as Prognostic Markers in Patients with Early Breast Cancer

Breast cancer (BC) poses a challenge in establishing new treatment strategies and identifying new prognostic and predictive markers due to the extensive genetic heterogeneity of BC. Very few studies have investigated the impact of mRNA expression of these genes on the survival of BC patients. Methods: We examined the impact of the mRNA expression of breast cancer gene type 1 (BRCA1), breast cancer gene type 2 (BRCA2), and partner and localizer of BRCA2 (PALB2) on the metastasis-free survival (MFS) of patients with early BC using microarray gene expression analysis. Results: The study was performed in a cohort of 461 patients with a median age of 62 years at initial diagnosis. The median follow-up time was 147 months. We could show that the lower expression of BRCA1 and BRCA2 is significantly associated with longer MFS (p < 0.050). On the contrary, the lower expression of PALB2 was correlated with a shorter MFS (p = 0.049). Subgroup survival analysis identified the prognostic influence of mRNA expression for BRCA1 among patients with luminal-B-like BC and for BRCA2 and PALB2 in the subset of patients with luminal-A-like BC (p < 0.050). Conclusions: According to our observations, BRCA1, BRCA2, and PALB2 expression might become valuable biomarkers of disease progression.


Introduction
Breast cancer (BC) is, with increasing incidence, the most common cancer among women worldwide [1].Its extensive genetic heterogeneity poses a challenge to establishing new treatment strategies [2].To overcome the obstacles of the prediction of prognosis in terms of heterogeneity, it is crucial to understand how molecular and genetic characteristics affect recurrence and therapy resistance [3].
Multiple studies have explored the specific roles of inherited high-penetrance pathogenic variants of genes such as breast cancer gene type 1 (BRCA1) and breast cancer gene type 2 (BRCA2) and of moderate-penetrance BC risk variants, particularly partner and localizer of BRCA2 (PALB2), checkpoint kinase 2 (CHEK2), and ataxia teleangiectasia mutated (ATM) in the pathogenesis of BC [4].BRCA tumor-suppressor genes have been shown to be associated with an increased risk of breast cancer [5].The two BRCA proteins are physically and functionally linked by PALB2, which is a third tumor-suppressor protein that directly binds BRCA1 and BRCA2 in the homologous recombination (HR) pathway [6].It is noteworthy that the interaction between BRCA genes and PALB2 is essential for cell cycle checkpoint control, genome stability, HR, and tumor suppression [5,6].A better understanding of the role of BRCA genes in the pathogenesis of BC has revolutionized cancer treatment through the development of new targeted therapies.

Patient's Characteristics
In the present study, 461 patients with early breast cancer treated at the Department of Gynecology and Obstetrics at the University Medical Center Mainz between 1986 and 2000 were included.Total RNA samples could be extracted from fresh frozen tumor tissue from each patient.All samples were assayed on Affymetrix Gene Chip in order to determine the mRNA expression values of BRCA1, BRCA2, and PALB2.Clinicopathological data such as age at initial diagnosis, histologic grade of differentiation, tumor size, nodal status, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), Ki-67, treatment details, and follow-up information were obtained from pathology reports and the breast cancer database of our department.
A total of 200 patients with node-negative early BC received no further adjuvant therapy after surgery.Adjuvant therapy with tamoxifen was administered in 165 patients, while 96 patients received chemotherapy in the adjuvant setting.

Gene-Expression Analyses
The following single genes were considered for gene expression analysis: The metagenes of BRCA1 and BRCA2 were calculated for each sample as the average expression of all genes in the gene cluster.
The mRNA expression analyses for PALB2 and for metagenes BRCA1 and BRCA2 were performed with HG-U133A arrays (Affymetrix, Santa Clara, CA, USA), as previously described [18,19].The median mRNA expression of the above-mentioned probe sets was used as the cut-off value.All patients included in this study were subdivided into two groups based on low and high levels of mRNA expression in BRCA1, BRCA2 metagenes, and PALB2, using the median mRNA expression as the cut-off value.

Statistical Analysis
Statistical analyses were performed using SPSS statistical software system version 27.0 (SPSS Inc., Chicago, IL, USA).A two-sided p-value < 0.05 was considered statistically significant.Kaplan-Meier survival analyses and univariable and multivariable Cox regression analyses were performed to examine the prognostic significance of BRCA1, BRCA2, and PALB2 mRNA expression on metastasis-free survival (MFS).Subgroup analyses were performed in order to identify if there were any significant differences among PALB2 and metagenene BRCA1 and BRCA2 levels of mRNA expression in different molecular subtypes.

Subgroup Analyses
The molecular subtypes, also known as intrinsic subtypes, were assessed according to the three-gene model from Haibe-Kains et al., including the estrogen receptor gene (ESR1), HER2, and aurora kinase A (AURKA) [20].
The subgroup analyses were performed taking into consideration the following molecular subtypes:

Patient and Tumor Characteristics
We analyzed 461 patients with a median age of 62 years (range: 30-93) at initial diagnosis.The patient and tumor characteristics are summarized in Table 1.The median follow-up time was 147 months, ranging from 1 to 306 months.The majority of patients presented with ER-positive eBC 252 (73.5%) and PR-positive eBC 204 (59.5%).A total of 39 (8.5%) had an HER2-positive tumor.Among all our patients, 59 (11%) had TNBC.
As shown in Table 1, the level of mRNA expression of BRCA1 was significantly associated with tumor size, axillary nodal status, histological grade of differentiation, Ki-67 expression, and molecular subtypes.The mRNA expression of BRCA2 was significantly associated with the histological grade of differentiation, Ki-67 expression, and molecular subtypes, whereas the mRNA expression of PALB2 was associated with the axillary nodal status, age, ER, PR, and molecular subtypes (Table 1).

Treatment
The surgical treatment of eBC was followed up in 165 patients from adjuvant therapy with tamoxifen and in 96 patients from adjuvant chemotherapy, either cyclophosphamide, methotrexate, fluorouracil, or epirubicin and cyclophosphamide.A total of 200 patients with node-negative early BC, received no further adjuvant therapy after surgery.Since the above-mentioned therapies were performed in an adjuvant setting and the Affymetrix microarray analyses were performed on fresh frozen tumor tissue (fresh frozen), they had no effect on the mRNA expression of BRCA1 and BRCA2 metagenes and also PALB2.

Survival Analyses 3.3.1. Impact of BRCA1 mRNA Expression on Survival
The number of patients with low or high expression levels of BRCA1 was 231 and 230, respectively.The median MFS was 142.0 and 110.5 months in each subgroup, respectively.BRCA1 mRNA expression levels correlated significantly with MFS (HR 1.803; 95% CI 1.269-2.561;p < 0.001).A lower BRCA1 mRNA expression was associated with a longer MFS in the Kaplan-Meier analysis (Figure 1).

Treatment
The surgical treatment of eBC was followed up in 165 patients from adjuvant t with tamoxifen and in 96 patients from adjuvant chemotherapy, cyclophosphamide, methotrexate, fluorouracil, or epirubicin and cyclophospham total of 200 patients with node-negative early BC, received no further adjuvant t after surgery.Since the above-mentioned therapies were performed in an adjuvant and the Affymetrix microarray analyses were performed on fresh frozen tumor (fresh frozen), they had no effect on the mRNA expression of BRCA1 and metagenes and also PALB2.

Impact of BRCA1 mRNA Expression on Survival
The number of patients with low or high expression levels of BRCA1 was 2 230, respectively.The median MFS was 142.0 and 110.5 months in each sub respectively.BRCA1 mRNA expression levels correlated significantly with MF 1.803; 95% CI 1.269-2.561;p < 0.001).A lower BRCA1 mRNA expression was asso with a longer MFS in the Kaplan-Meier analysis (Figure 1).In the multivariable Cox regression analysis adjusted for age, tumor size, histo grade of differentiation, axillary nodal status, and the proliferation marker Ki-67, B mRNA expression showed a significant influence on MFS (HR 1.611; 95% CI 1.047 p = 0.030) (Table 2).Similarly, tumor grading was also identified as an indep prognostic factor in the multivariable Cox regression analysis (HR 2.201; 95% CI 3.480; p < 0.001).In the multivariable Cox regression analysis adjusted for age, tumor size, histological grade of differentiation, axillary nodal status, and the proliferation marker Ki-67, BRCA1 mRNA expression showed a significant influence on MFS (HR 1.611; 95% CI 1.047-2.479;p = 0.030) (Table 2).Similarly, tumor grading was also identified as an independent prognostic factor in the multivariable Cox regression analysis (HR 2.201; 95% CI 1.392-3.480;p < 0.001).BRCA1-breast cancer 1; mRNA-messenger ribonucleic acid; CI-confidence interval; HR-hazard ratio; p-value < 0.05 is considered to be significant.

Impact of BRCA2 mRNA Expression on Survival
The Kaplan-Meier analysis indicated that breast cancer patients with higher mRNA expression of BRCA2 had a shorter MFS (p < 0.001) (Figure 2).In this cohort, the number of patients with low or high expression levels of BRCA2 was 230 and 231, respectively.The median MFS was 151.5 months in the subgroup with lower levels of BRCA2 mRNA expression and 99.0 months in the other group of patients.BRCA1-breast cancer 1; mRNA-messenger ribonucleic acid; CI-confidence interval; HRhazard ratio; p-value < 0.05 is considered to be significant.

Impact of BRCA2 mRNA Expression on Survival
The Kaplan-Meier analysis indicated that breast cancer patients with higher mRN expression of BRCA2 had a shorter MFS (p < 0.001) (Figure 2).In this cohort, the numbe of patients with low or high expression levels of BRCA2 was 230 and 231, respectivel The median MFS was 151.5 months in the subgroup with lower levels of BRCA2 mRN expression and 99.0 months in the other group of patients.Kaplan-Meier analysis of metastasis-free survival in patients with early breast canc according to the levels of BRCA2 metagene mRNA expression.Abbreviations: BRCA2-brea cancer 2; p-value < 0.05 is considered to be significant.
In the univariable analysis, the mRNA expression of BRCA2 correlated significant with MFS (HR 1.881; 95% CI 1.324-2.673;p < 0.001).In contrast, in the multivariable Co regression analysis adjusted for age, tumor size, histological grade of differentiatio axillary nodal status, and the proliferation marker Ki-67, BRCA2 mRNA expression wa not significantly associated with survival (HR 1.315; 95% CI 0.870-1.988;p = 0.194) (Tab 3).Kaplan-Meier analysis of metastasis-free survival in patients with early breast cancer according to the levels of BRCA2 metagene mRNA expression.Abbreviations: BRCA2-breast cancer 2; p-value < 0.05 is considered to be significant.

Impact of PALB2 mRNA Expression on the Survival
The Kaplan-Meier analysis showed that 230 patients with lower levels of PALB2 mRNA expression had a significantly shorter MFS compared to the patients with higher mRNA expression (115 vs. 148 months; p = 0.049) (Figure 3).Abbreviations: BRCA2-breast cancer 2; mRNA-messenger ribonucleic acid; CI-confidence interval; HR-hazard ratio; p-value < 0.05 is considered to be significant.

Impact of PALB2 mRNA Expression on the Survival
The Kaplan-Meier analysis showed that 230 patients with lower levels of PALB2 mRNA expression had a significantly shorter MFS compared to the patients with higher mRNA expression (115 vs. 148 months; p = 0.049) (Figure 3).The impact of PALB2 mRNA expression on MFS could also be verified in the univariable Cox regression analysis (HR 0.710; 95% CI 0.504-1.001;p = 0.050).However, in the multivariable Cox regression analysis, PALB2 mRNA expression failed to show a significant impact on MFS (HR 1.075; 95% CI 0.690-1.676;p = 0.748) (Table 4).As shown in Table 3, when adjusted for age, tumor size, histological grade of differentiation, axillary nodal status, and the proliferation marker Ki-67, only histological grading was found to be a prognostic factor related to MFS (HR 2.244; 95% CI 1.387-3.629;p < 0.001).The impact of PALB2 mRNA expression on MFS could also be verified in the univariable Cox regression analysis (HR 0.710; 95% CI 0.504-1.001;p = 0.050).However, in the multivariable Cox regression analysis, PALB2 mRNA expression failed to show a significant impact on MFS (HR 1.075; 95% CI 0.690-1.676;p = 0.748) (Table 4).As shown in Table 3, when adjusted for age, tumor size, histological grade of differentiation, axillary nodal status, and the proliferation marker Ki-67, only histological grading was found to be a prognostic factor related to MFS (HR 2.244; 95% CI 1.387-3.629;p < 0.001).Abbreviations: PALB2-partner and localizer of BRCA2; BRCA2-breast cancer 2; mRNA-messenger ribonucleic acid; CI-confidence interval; HR-hazard ratio; p-value < 0.05 is considered to be significant.

Impact of BCRA1 mRNA Expression on the Survival among Different Molecular Subtypes of Breast Cancer
The majority of patients (82.9%) with luminal-A-like BC showed a lower expression of BRCA1 mRNA expression.Interestingly, no differences were observed in TNBC (lower vs. higher 25 vs. 26) (Table 5).
Further, subgroup analyses revealed that the MFS rates were significantly increased in patients with luminal-B-like BC (p = 0.003) (Figure 4).Nevertheless, there were no significant differences regarding MFS in other molecular subtypes (all p > 0.050).

Impact of BCRA2 mRNA Expression on the Survival among Different Molecular Subtypes of Breast Cancer
The correlation of BRCA2 mRNA expression and MFS among different molecular subtypes is shown in Table 7. Abbreviations: BRCA2-breast cancer 2; luminal-A-like-estrogen receptor positive, HER2-negative, and low proliferation (AURKA-low); luminal-B-like-estrogen receptor positive, HER2-negative, and high proliferation (AURKA-high).
Subgroup analyses indicated that in patients with luminal-A and HER-2-positive like BC, lower mRNA expression of BRCA2 was associated with higher survival rate (p = 0.039) (Figure 5).There was no significant difference in other molecular subtypes (all p > 0.050).
Further subgroup analyses with univariable and multivariable Cox regression analyses revealed the results shown on Table 8.The level of mRNA expression of BRCA2 correlated significantly with MFS among the patients with luminal A and Her2-positive BC in the univariate analyses.However, in the multivariable model, mRNA expression of BRCA2 could be confirmed as an independent prognostic marker only in the patients with luminal-A-like BC (HR 0.375; 95%-CI 0.209-0.671;p < 0.001) (Table 8).

Impact of PALB2 mRNA Expression on the Survival among Different Molecular Subtypes of Breast Cancer
The correlation of PALB2 mRNA expression and MFS among different molecular subtypes is shown on Table 9.
New  Nevertheless, univariable and multivariable Cox regression analyses were conducted to evaluate the prognostic role of mRNA expression of PALB2 among different molecular subtypes.The results of the Cox regression analyses are shown in Table 10.The level of mRNA expression of PALB2 was identified as an independent prognostic marker related to MFS among the patients with luminal-A-like BC (HR 0.367; 95%-CI 0.216-0.625;p < 0.001) (Table 10).Abbreviations: PALB2-partner and localizer of BRCA2; BRCA2-breast cancer 2; mRNA-messenger ribonucleic acid; luminal-A-like-estrogen receptor positive, HER2-negative, and low proliferation (AURKA-low); luminal-Blike-estrogen receptor positive, HER2-negative, and high proliferation (AURKA-high); CI-confidence interval; HR-hazard ratio; p-value < 0.05 is considered to be significant.

Discussion
The present study investigates the association between the level of mRNA expression of BRCA1, BRCA2, and PALB2 and the MFS in eBC.To our knowledge, this is the first study designed to simultaneously investigate the impact of the mRNA expression of these three genes on the survival of patients with BC.
Given the heterogeneous biology of BC, the different responses to therapy, and the high cost of treatments, there is an increasing interest in identifying the role of prognostic and treatment-predictive factors [21].Inherited monoallelic mutations in PALB2, just like BRCA1/2, cause a high risk of breast cancer and also increase the risks of other cancers [4].However, the role of messenger ribonucleic acid (mRNA) expression of different genes in the prognosis of BC is incompletely understood [22].
In our study, an increased level of mRNA expression of BRCA1 and BRCA2 was associated with several factors that are generally related to poor survival in breast cancer, such as tumor size, axillary nodal status, histological grade of differentiation, Ki-67 expression, and molecular subtypes.However, this observation could not be confirmed in the same analysis for PALB2.
Survival analysis showed a favorable MFS for low BRCA1 mRNA expression in the whole cohort of included patients.Furthermore, a detailed analysis proved that the prognostic influence of BRCA1 mRNA expression was most marked in the subset of patients with luminal-B-like BC.The cause of this finding may be the higher proliferation rate in this molecular subtype, which, together with genetic instability, may increase the need for more DNA damage repair.The observation of Gudas et al., suggesting that the upregulation of BRCA1 expression by steroid hormones is caused indirectly by increasing proliferation of breast cancer cells, could also support our results [23].
Prior studies that have addressed the prognostic significance of BRCA1 mRNA expression have shown conflicting results [12,14,15].Our results are in line with the results of Margeli et al., who showed that low levels of BRCA1 mRNA and a positive progesterone receptor in BC treated with anthracycline-based chemotherapy are associated with significantly longer survival [12].An important difference in both studies is the fact that our patients did not receive any kind of therapy before surgery.These findings are confirmed by the results of a study by Lu et al., proving the impact of BRCA1 expression on survival.The authors assessed the association between the T cell activation status and death risk according to the BRCA1 expression level.An over 50% reduction in the risk of mortality was observed in patients with a low BRCA1 expression level and with an intermediate T cell activation score and in patients with a high T cell activation score compared to patients with a low T cell activation score.Patients in the activation group lived over 8 years longer than those in the exhaustion group if they had a low level of BRCA1 expression.In contrast, no significant association was observed between the T cell activation score and the risk of mortality in patients with a high level of BRCA1 expression [14].Contrary to this report, in another study, a low level of BRCA1 expression has been found to be associated with poor survival in patients with BC [15].These controversial studies imply a compelling need to understand the mechanism of BRCA1 mRNA expression in BC.
Furthermore, the prognostic significance of BRCA2 mRNA expression was explored in our study.Our investigations revealed a significant relation between BRCA2 mRNA expression and MFS, and the survival rates were markedly higher in patients with lower BRCA2 expression.The survival analysis among different molecular subtypes indicated that in luminal-A-like BC, lower mRNA expression is associated with higher survival rates.
Even though BRCA2 plays an important role in DNA repair, the prognostic role of the level of BRCA2 mRNA expression has not been proven in any studies till now.In a cohort of 671 patients without BRCA1/2 germline mutation, it was observed that BRCA2 mRNA expression may not predict responses to anthracycline-based or taxane neoadjuvant chemotherapy, and its level was not associated with survival rates [17].In an experimental study by Wu et al., BRCA2 mRNA levels were significantly down-regulated in breast and ovarian cancer cell lines after exposure to various DNA-damaging agents [24].The authors acknowledged that DNA-damaging agents reduce the rate of BRCA2 mRNA and protein turnover in a p53-dependent manner.
PALB2 is regarded as a BC risk gene and plays a crucial role in the DNA repair process, but the prognostic role of PALB2 expression in BC remains unclear.Few reports have studied the relationship between PALB2 and the survival of patients with cancer, especially BC [25][26][27].In our study, the survival analysis showed that a low mRNA expression of PALB2 correlated with shorter MFS.Similarly, Liu et al. assessed the impact of PALB2 mRNA expression in 563 patients with primary BC and revealed that a low expression of PALB2 mRNA is significantly associated with poor survival (HR = 1.79; 95% CI = 1.21-2.65;p = 0.003 [28].
Interestingly, the prognostic impact of mRNA expression of PALB2 was identified in the subgroup of patients with luminal-A-like BC.Patients with lower levels of PALB2 mRNA expression had a significantly shorter MFS compared to patients with higher mRNA expression.Our observations are consistent with reports in the literature and lend further support to the notion that PALB2 mRNA expression is associated with the survival of patients with BC and has potential clinical applications as a predictive marker of prognosis.
The limitation of the present study is the retrospective design.Another potential weakness of our study is that our analyses were limited to gene expression data, and we do not have information regarding the BRCA1/2 and PALB2 gene germline mutations in the patients included in this study, as this is a historic cohort.
The strength of this study is that we report the prognostic significance of mRNA expression of BRCA1, BRCA2, and PALB2 in a large cohort; the inclusion of patients with a sufficient amount of fresh frozen tissue available for successful mRNA microarray analysis of gene expression; and the long-term follow-up.
In general, the current results support the independent prognostic role of mRNA expression of BRCA1, BRCA2, and PALB2.

Figure 1 .
Figure1.Kaplan-Meier analysis of metastasis-free survival in patients with early breast according to the levels of BRCA1 metagene mRNA expression.BRCA1-breast cancer 1; p-0.05 is considered to be significant.

Figure 1 .
Figure1.Kaplan-Meier analysis of metastasis-free survival in patients with early breast cancer according to the levels of BRCA1 metagene mRNA expression.BRCA1-breast cancer 1; p-value < 0.05 is considered to be significant.

Figure 2 .
Figure 2.Kaplan-Meier analysis of metastasis-free survival in patients with early breast canc according to the levels of BRCA2 metagene mRNA expression.Abbreviations: BRCA2-brea cancer 2; p-value < 0.05 is considered to be significant.

Figure 2 .
Figure2.Kaplan-Meier analysis of metastasis-free survival in patients with early breast cancer according to the levels of BRCA2 metagene mRNA expression.Abbreviations: BRCA2-breast cancer 2; p-value < 0.05 is considered to be significant.

Figure 3 .
Figure 3. Kaplan-Meier analysis of metastasis-free survival in patients with early breast cancer according to the levels of PALB2 mRNA expression.Abbreviations: PALB2-partner and localizer of BRCA2; BRCA2-breast cancer 2, p-value < 0.05 is considered to be significant.

Figure 3 .
Figure 3. Kaplan-Meier analysis of metastasis-free survival in patients with early breast cancer according to the levels of PALB2 mRNA expression.Abbreviations: PALB2-partner and localizer of BRCA2; BRCA2-breast cancer 2, p-value < 0.05 is considered to be significant.

Figure 6 .
Figure 6.Kaplan-Meier analysis of metastasis-free survival in patients with early breast can cording to PALB2 Metagene mRNA expression.

Figure 6 .
Figure 6.Kaplan-Meier analysis of metastasis-free survival in patients with early breast cancer according to PALB2 Metagene mRNA expression.

Table 1 .
Associations between median mRNA expression of BRCA1/2 metagenes and PALB2 and patient and tumor characteristics.

Table 2 .
Multivariable Cox regression analysis of BRCA1 metagene mRNA expression for metastasisfree survival adjusted for age, tumor size, lymph node status, grade of differentiation, and the proliferation marker Ki-67.

Table 2 .
Multivariable Cox regression analysis of BRCA1 metagene mRNA expression f metastasis-free survival adjusted for age, tumor size, lymph node status, grade of differentiatio and the proliferation marker Ki-67.

Table 3 .
Multivariable Cox regression analysis of BRCA2 metagene mRNA expression for metastasisfree survival adjusted for age, tumor size, lymph node status, grade of differentiation, and the proliferation marker Ki-67.

Table 3 .
Multivariable Cox regression analysis of BRCA2 metagene mRNA expression for metastasis-free survival adjusted for age, tumor size, lymph node status, grade of differentiation, and the proliferation marker Ki-67.

Table 4 .
Multivariable Cox regression analysis of PALB2 mRNA expression for metastasis-free survival adjusted for age, tumor size, lymph node status, grade of differentiation, and the proliferation marker Ki-67.

Table 5 .
Correlation of breast cancer subtypes and mRNA expression of BRCA1 metagene.

Table 6 .
Association between BRCA1 mRNA expression and metastasis-free survival using univariable and multivariable Cox regression analysis in different molecular subtypes.

Table 7 .
Correlation of breast cancer subtypes and mRNA expression of BRCA2 metagene.

Table 8 .
Association between BRCA2 mRNA expression and metastasis-free survival using univariable and multivariable Cox regression analysis in different molecular subtypes.

Table 9 .
Correlation of breast cancer subtypes and mRNA expression of PALB2.

Table 10 .
Association between PALB2 mRNA expression and metastasis-free survival using univariable and multivariable Coxregression analysis in different molecular subtypes.