Introduction
Ovarian cancer is the leading cause of death in women from gynecological malignant diseases. The lifetime probability of ovarian cancer in the general population is approximately 1.6 %. This risk increases to 5 % for women with one first-degree relative with ovarian cancer and to 7.2 % for women with two or three relatives with ovarian cancer [
1].
BRCA1 and
BRCA2 genes are high-penetrating with important roles during tumorigenesis. Both genes encode proteins that interact with a machinery of recombination of DNA or DNA repair pathways [
2]. Twenty percent of breast cancer has familial basis and approximately 5 % to 10 % of breast cancer is hereditary. Two-thirds of these hereditary cancers occur in carriers with mutations of
BRCA1 or
BRCA2 which are germline mutations [
3]. Some preclinical studies have shown that
BRCA1 appears to be an important responding factor to both DNA-damaging (platimun-compounds) and taxane-based chemotherapy [
4]. Molecularly, these anticancer agents are to be as crucial modulators of
BRCA-dependent pathways, independently of detected
BRCA1/2 mutations defined germline or sporadic [
5].
The
BRCAness phenotype may be a result of defective homologous recombination related to several mechanisms, including epigenetic hypermethylation of the
BRCA1 promoter, somatic mutation of
BRCA1/2, or loss of function mutations in other homologous recombination orchestrating molecules. The complex profile of the
BRCAness phenotype correlates with responsiveness to platinum and poly-ADP ribose polymerase (PARP) inhibitors [
6]. On the other hand, there have been known at least, two molecular mechanisms responsible for chemotherapy resistance and recurrence of ovarian cancer such as 1.secondary mutations restoring
BRCA1/2, and 2.high levels of PARP, Fanconi anemia proteins and P53 [
7,
8]. Determination of the
BRCA1/2 status may be a relevant clinical biomarker both for survival prognosis and prediction both for response or resistance to chemotherapy in sporadic ovarian cancer [
9,
10].
BRCA mutations may have impact on better survival of patients with ovarian cancer when compared with those without mutations. Several studies have investigated the possible effects of
BRCA1 mutation on clinical and pathologic characteristics defined by earlier age of onset in any mutation carriers with the possible better response to platinum-based chemotherapy, but results of these studies were inconclusive. Some of these studies have demonstrated longer survival in epithelial invasive ovarian cancer patients [
11,
12] who are
BRCA1 mutation carriers in comparison with noncarriers, but other investigators did not find a survival benefit in
BRCA1 mutation carriers [
13‐
15]. Recently reported results have revealed that after 3 years since the ovarian cancer diagnosis, the presence of a
BRCA1 or
BRCA2 mutation was associated with a clinically better prognosis (HR 0.68; 95 % CI: 0.48–0.98,
p = 0.03), which has not been sustained 10 years after the diagnosis of ovarian cancer (HR 1.00; 95 % CI: 0.83–1.22,
p = 0.90) [
16,
17].
The aim of our study was to assess clinical features and treatment outcomes in ovarian cancer patients, having a founder mutation in BRCA1 gene in comparison with clinical results of sporadic ovarian cancer patients.
Patients and methods
Patients
A consecutive series of 125 patients with ovarian cancer diagnosed and treated at the Military Institute of Medicine in Warsaw, Poland, between 2002–2008 was studied. All patients underwent surgery defined as radical, optimal tumor debulking with residual disease < 1 cm, or suboptimal as residual disease > 1 cm. The pathology reports were classified as epithelial ovarian cancer serous, endometrioid, mucinous, clear cell, mixed, or unspecified. Chemotherapy was used in all patients. Mutation analysis was performed in all enrolled patients. The study protocol was approved by the local Ethics Committee (The Resolution of The Bioethics Committee of Military Institute of Medicine at Warsaw, No 48/WIM/2008 data November, 19th 2008), and written informed consent was obtained from all participants of the study.
Chemotherapy regimen
The first line chemotherapy consisted of 6 courses. The first version of chemotherapy regimen consisted of 135 mg/m2 of intravenous infusion paclitaxel over 24-h on day 1 followed by 75 mg/m2 of intravenous infusion cisplatin on day 2. The second version of chemotherapy regimen consisted of 175 mg/m2 of intravenous infusion paclitaxel over 3-h on day 1 followed by AUC6 according to Calvert formula of intravenous 30-min infusion carboplatin on day 2. Standard premedication (dexamethasone 20 mg, ranitidinum 50 mg, clemastinum 2 mg) was given intravenously to prevent hypersensitivity reaction to paclitaxel. Treatments were administered every 3 weeks. The third version of chemotherapy was based upon carboplatin AUC5 according to Calvert formula of intravenous 30-min infusion. The fourth version of treatment was given as neoadjuvant triple chemotherapy regimen based upon paclitaxel (175 mg/m2 over 3-h intravenously), carboplatin (AUC5 intravenously), and caelyx (20 mg/m2 intravenously) or epirubicin (50 mg/m2 intravenously) which was administered for three courses before debulking surgery with further continuation of standard chemotherapy with paclitaxel and carboplatin in mentioned doses up to total number of six courses. The fifth version of chemotherapy was a regimen based upon combination of gemcitabine (1000 mg/m2 intravenously on days 1,8 every 21 days) and cisplatin (75 mg/m2 intravenously on day 1 every 21 days) given as the primary chemotherapy as well.
Mutational analysis
High-molecular-weight DNA was isolated from peripheral blood leukocytes by nonenzymatic and rapid method described by Lahiri and Nurnberger [
18]. Mutation analysis was performed for three common in Poland founder mutations in
BRCA1 (5382insC – exon 20; 4153delA – exon 11.17) by a multiplex allele-specific polymerase chain reaction (PCR) assay. 300 T > G – exon 5 mutation generates a novel restriction enzyme site. This mutation can be detected after digesting amplified DNA with Ava II. To show the different
BRCA1 alleles, the PCR products were subjected to horizontal electrophoresis in a 2.0 % agarose gel and stained with ethidium bromide. We analyzed samples of all enrolled patients [
19].
Statistical analysis
Demographic data are shown as median or mean with standard deviation (SD) and 95 % confidence interval (CI). Relationships between categorical variables were assessed using the Chi-square test, Yates-corrected Chi-square test or Mann–Whitney U test. Progression-free survival (PFS) was calculated from the start date of chemotherapy to the first evidence of treatment failure. Overall survival (OS) was defined as the time interval between the date of starting chemotherapy to death of any cause. PFS and OS were estimated using the Kaplan-Meier methods and differences in survival were compared by using log-rank test. On univariate comparisons of survival between groups, statistical significance was assessed using the Cox-Mantel test. Multivariate regression analyses were performed using the Cox proportional hazards model. A p value of <0.05 was considered statistically significant. For calculations we used STATISTICA for Windows Version 7.0 software.
Discussion
Our results found the total rate of
BRCA1 mutations was 13.6 % in 125 ovarian cancer patients. This result is in accordance with other previously published studies in the Polish population with germline mutations in
BRCA1 and
BRCA2 in 13.5 % and 13.9 % of patients, respectively [
20,
21]. We also show that
BRCA1 germline mutations are associated with good prognosis for patients with ovarian cancer and OS was better for carriers of
BRCA1 mutations (HR 0.14, 95 % CI: 0.032–0.650,
p = 0.012) than for noncarriers. This observation is in accordance with results of other authors, for example, Rubin et al. [
22] for the first time determined that ovarian cancer associated with
BRCA1 mutations had a more favorable clinical course, and it was confirmed by other studies in which this advantage in survival of mutation carriers was reported to be an independent protective factor [
23].
Here we report that PFS assessed both on univariate and on multivariate analyses (
BRCA1 mutations vs
BRCA1 wild type, median 21.5 months vs 14.6 months,
p = 0.58, respectively) show a slight favor with predictive value for patients with mutated
BRCA1 but without statistical significance. We present that FIGO stage, serous histologic type, primary debulking surgery and chemotherapy based upon combination of platinum compound with paclitaxel, are typical clinical variables that correlate with PFS. Among 71 Jewish patients with ovarian cancer, 22 had
BRCA1 three germline mutations and one truncating mutation, and 12 had
BRCA2 truncating mutations vs 37 patients with sporadic ovarian cancer. Overall survival was improved in patients with mutated forms of
BRCA in comparison with sporadic ovarian cancer patients (91 months vs 54 months, respectively;
p = 0.046). In accordance with our results, the authors showed that patients with
BRCA mutations had slightly longer disease free survival but not statistically significant (49 months vs 19 months, respectively;
p = 0.16) [
24]. Tan et al. [
25] coined the term of a clinical syndrome of
BRCAness which relies on better prognosis for response rate after first- line chemotherapy and at subsequent recurrences, overall survival, serous histology (but not necessary), and TFI (treatment-free interval defined as the time between each line of treatment calculated from the date of the last course of the previous chemotherapy to the date of the first cycle of the next chemotherapy). In ovarian cancer patients with
BRCA mutations the median TFI for three lines of chemotherapy was significantly longer than in sporadic ovarian cancer patients (first-line,
p < 0.001; second-line,
p < 0.015; third-line,
p = 0.002, respectively). The median overall survival for
BRCA-positive ovarian cancer patients was better than in controls (from the time of diagnosis 8.4 years vs 2.9 years, respectively;
p < 0.002) [
25,
26]. Gallagher et al. [
27] reported in a group of 110 patients (36 with three germline mutations of
BRCA genes (20
BRCA1 mutations, 16
BRCA2 mutations) vs 74 controls) that ovarian cancer patients with
BRCA mutations had better survival (median not reached vs 67.8 months for controls, respectively;
p = 0.02). The multivariate analysis sustained crucial role of
BRCA mutations in overall survival prognosis (HR 0.36; 95 % CI 0.14–0.93) but disease-free survival was not significantly different between
BRCA mutations and control ovarian cancer patients (median 26.9 months vs 24.0 months, respectively;
p = 0.30).
In our study, the age at the onset of ovarian cancer for
BRCA1 wild type was almost 10 years postponed than in carriers of
BRCA1 mutations (median 56 years vs 47 years, respectively,
p = 0.004). Thigpen et al. [
28] decided to determine major prognostic factors in 2123 ovarian cancer patients who were studied in the six GOG (Gynecologic Oncology Group) clinical trials. It turned out that only three factors had impact on prognosis for overall survival of the entire investigated population, i.e., age, volume of residual disease, and performance status. Based upon the results of studied patients, elder patients with ovarian cancer (>69 years) had poorer survival. Therefore, age seems to have indirect roles in better survival of patients with mutated
BRCA1 through better response to chemotherapy, better tolerance of such a therapy without other health problems which typically are associated with elder patients. But on the other hand, the second-line chemotherapy due to relapsed ovarian cancer could be used in elder patients after detailed assessment of performance status rather than making-decision based primarily on the age [
29].
Malignant diseases are the final results of incorrect interactions between immune-surveillance in a healthy organism and unrestricted proliferation of a small portion of cells that constitute a solid tumor or leukemia. The fundamental process that is primarily responsible for tumorigenesis embraces changes in the DNA sequences of the genomes of malignant cells. Recently accumulating knowledge clearly shows that detection cancer genome structural changes at the levels of DNA sequences (mainly somatic mutations) and epigenetic alterations lead to improper functions in cells that constantly become malignant. This dynamic process defines cancer as an evolutionary entity determining highly probabilistic events providing finally advanced malignant disease [
30]. Naturally occurring during malignant progression, genome changes and epigenetic alterations seem to have impact on detection of molecular abnormalities with clinical usefulness at a bed in oncology ward. TCGA (The Cancer Genome Atlas) project is a well-know initiative to establish a series of somatic mutations and epigenetic alterations in human cancer samples in relation to clinical data. Analyses performed in 489 ovarian cancer samples (mainly high-grade serous ovarian adenocarcinoma) revealed changes in expression of mRNA, noncoding RNA, methylation of promoter of many genes, and changed DNA copy numbers. The most important gene mutations were ascribed to P53 detected in 96 % of investigated samples. Other low prevalent with statistical relevance aberrations in the form of somatic mutations were noted in such genes as
NF1,
BRCA1,
BRCA2,
RB1 and
CDK12. In 168 genes, the authors detected promoter methylation alterations and prognosis for survival was performed in patients with
BRCA mutations and
CCNE1 aberrations [
31].
Based on a cohort of 316 ovarian cancer patients taken from TCGA, 77.8 % had
BRCA1/2 wild type, 8.5 % had germline mutations, 3.1 % had somatic mutations, and 10.4 % had
BRCA1 hypermethylation. Detailed analyses showed that the only prognostic factor for OS in ovarian cancer was mutated
BRCA2 (HR 0.33; 95 % CI: 0.16–0.69,
p = 0.003). Taking into account platinum-based chemotherapy efficacy, mutated
BRCA2 gene had significantly longer PFS (HR 0.40; 95 % CI: 0.22–0.74,
p = 0.004) [
32]. Also, Hyman et al. [
33] reported that better survival was seen in patients carrying
BRCA2 mutations [HR 0.20; 95 % CI 0.06–0.65,
p = 0.007] compared with either
BRCA1 carriers [HR 0.70; 95 % CI 0.36–1.38,
p = 0.31] or noncarriers. The presented results are surprising, especially in the context of many published studies showing prognostic role of germline mutations of
BRCA1 gene. It seems that patient selection through a specific definition of primary response to adjuvant chemotherapy or variety in stage distribution will play a crucial role in the process of analysis and interpretation of collected results, but further studies should be planned as randomized and controlled with statistically well-founded number of participating patients. Some commentaries on the potential role of
BRCA2 mutations as a real prognostic factor in ovarian cancer patients show such a particular role of patient selection and differences in surgical and oncologic managements [
34].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AS conceived and designed the study, carried out molecular genetic studies, analysed, interpreted data, and drafted the manuscript. GW conceived and designed the study, analysed, interpreted data, and drafted the manuscript. LB participated in data collection and performed statistical analyses. BG designed molecular genetic studies. JS carried out molecular genetic studies. KSW participated in clinical data collection. CS participated in coordination and helped to draft the manuscript. All authors read and approved the final manuscript.