Introduction
Triple-negative breast cancer represents a distinct subset of breast cancer that exhibits a more aggressive course as compared to other biological subtypes of cancer [
1‐
7]. The aggressiveness of the disease is best illustrated by the fact that the peak risk of recurrence of the disease is between the first and third year following diagnosis, and that survival after recurrence is significantly shorter than that observed in patients with non-triple-negative controls [
4,
6,
8‐
10]. Surprisingly, patients who did not have a recurrence of the disease within the first 8 years after the diagnosis did not subsequently relapse. However, in other subtypes of breast cancer, the risk of recurrence keeps rising as time progresses [
4,
11]. The recurrence pattern of triple-negative breast cancer also differs from other biological subtypes of cancer. In addition, the most characteristic sites of metastases include the brain and lungs [
3,
6,
12].
From a biological point of view, triple-negative breast cancer remains a heterogeneous group with difficult-to-define subtypes. Gene expression studies have shown that ER-negative and HER2-negative tumors are clustered into at least three distinct molecular classes: basal-like, normal-like and claudin-negative breast cancer [
1,
11,
13‐
19]. Most of triple-negative tumors show basal-like phenotype which can be subdivided into pure variant and myoepithelial variant [
20]. Many “immunohistochemical (IHC) signatures” have been described as a surrogate of microarray, but the panel proposed by Nielsen et al. [
21] seems to be the best example of the classification to date. In this classification, basal-like cancers are defined as those lacking ER and HER2 expression and expressing cytokeratin 5/6 (CK 5/6) and/or Epidermal Growth Factor Receptor (EGFR or HER1). This panel has 100% specificity and 76% sensitivity for identification of basal-like cancers.
Patients with triple-negative breast cancer and brain metastases have the poorest prognosis out of all biological subtypes of breast cancer after dissemination to the brain [
3]. It is not known whether such a short survival depends on clinical features (performance status, dissemination of the disease to other organs), or the affiliation to the specific biological subset, which can be selected by molecular markers, for example basal cytokeratins.
The aim of the present study was to analyze clinical features and survival of triple-negative breast cancer patients with brain metastases and to compare basal-like and non-basal-like subtypes in order to establish the clinical value of basal biological markers differentiating these subtypes.
Materials and methods
Between 1 January 2003 and 31 December 2009, 111 patients with triple-negative (ER-negative, PgR-negative, HER2-negative) breast cancer and brain metastases were treated in the Breast Cancer and Reconstructive Surgery Department at The Maria Sklodowska-Curie Memorial Cancer Center, Warsaw, Poland. The observation of the patients started at the time of the detection of brain metastases and all data were collected prospectively in our database. In each case, treatment options were approved by a team of medical oncologists, radiation oncologist, neurologist and neurosurgeon, and were performed after patients had signed written consent. Immunohistochemistry (IHC) was carried out in order to evaluate levels of ER, PgR and HER2 expression in primary breast tumors. Staining was performed using primary antibodies against ER (Clone 6F11, Novocastra, dilution scale 1:100); against PR (Clone 16, Novocastra, dilution scale 1:200) and against HER2 (Polyclonal Hercep Test, DAKO). For the evaluation of ER and PR expression, all cases with 10% or higher stained cancer nuclei were classified as positive. Fluorescence in situ hybridization (FISH) was used for all HER2 2+ tumors using the HER2 DNA Probe Kit Abbott (Vysis). HER2-positive staining was defined as IHC 3+ or, in the case of IHC 2+, FISH-positive. HER2-negativity was defined as IHC 0, 1+ or 2+, along with negative FISH results.
Immunohistochemistry for cytokeratin 5/6 (Clone D5/16B4, DAKO, dilution scale 1:100), HER1 (EGFR pharm Dx, Clone 2-18C9, DAKO, RTU) and c-KIT (CD117, Policlonal, DAKO, dilution scale 1:500), as a surrogate of cDNA microarray, was performed in 75 patients in whom paraffin blocs with primary tumor samples were available in the pathology archives. These assays were performed after the detection of brain metastases. Based on the definition by Nielsen et al. [
21], tumors were divided into basal-like and non-basal-like biological subsets.
In 36 out of 111 patients, the analysis of basal markers was impossible to perform for the following reasons: firstly, there was an insufficient amount of biological material to perform detailed analysis. In some of those patients, only core biopsy or fine needle biopsy was performed to confirm the initial diagnosis of breast cancer and, after detection of brain metastases, we did not have enough biological material to perform IHC analysis. Secondly, some patients were treated in different hospitals before the dissemination of the disease, and after the detection of brain metastases, it was impossible to gain access to paraffin blocks from their primary tumors. Thirdly, according to information provided by the remaining patients, the IHC was impossible to perform due to technical difficulties that occurred during pathological processing.
Statistical analysis
Descriptive statistics were used to determine patient demographics and clinical characteristics. In order to compare categorical tumor features in the basal-like and non-basal-like subsets, the chi-square test was used. For those categorical variables in which the chi-square test was inappropriate because of small sample size, the Fisher exact test was used. A univariate analysis and Cox proportional hazards model were developed to identify factors influencing survival after brain metastasis in patients with triple-negative breast cancer. The following factors were analyzed in a Cox model: KPS (<70 vs. ≥70), age at detection of brain metastases (<50 vs. ≥50), extracranial disease (absent vs. present), extracranial disease (controlled vs. uncontrolled), lung metastases (absent vs. present), brain metastases as the first or the only metastatic site (yes vs. no), neurosurgery (yes vs. no), systemic treatment after brain metastases (yes vs. no), basal subtype vs. non-basal subtype. Disease-free survivals (DFS), overall survivals (OS, from initial diagnosis of breast cancer), and survivals from the detection of brain metastases in the entire group and in biological subgroups were estimated using the Kaplan–Meier method and compared using the log-rank test.
Results
Clinical characteristics of the entire group are presented in Table
1. In the group of 111 patients, histopathology and metastatic spread that is typical for triple-negative breast cancer was revealed. In most of the patients, ductal cancer with histological grade 3 was detected, although medullary and metaplastic cancers were also found. Lungs and brain were the most relevant sites of distant metastases. Among 75 patients in whom basal markers were assessed, 48 (64%) tumors expressed CK5/6, 26 (36%) tumors expressed HER1 and 13 (17%) expressed c-KIT. In 6 tumors (8%), expression of three basal biological markers was detected and 13 tumors (17%) did not express any of them. Based on the definition by Nielsen et al. [
21], basal-like tumors were identified based on panel of four antibodies (ER, HER2, HER1 and cytokeratin 5/6). There were 57 patients (76%) with basal-like and 18 (24%) with non-basal-like biological subtypes. The comparison between basal-like and non-basal-like subsets is presented in Table
2. There were no significant differences between both groups in relation to patients’ age at initial diagnosis, clinical stage and tumor histologic grade; however, medullar and metaplastic cancers were observed only in the basal-like subset. Bone metastases, liver metastases and brain metastases as a first site of dissemination were all equally distributed in both groups, but lung metastases were more common in the non-basal subtype.
Table 1
Characteristics of 111 patients with triple-negative breast cancer patients with brain metastases
Initial TNM stage |
I | 18 | 16 |
II | 45 | 41 |
III | 39 | 35 |
IV | 9 | 8 |
Histological type |
Ductal carcinoma | 78 | 70 |
Lobular carcinoma | 2 | 1 |
Medullar carcinoma | 3 | 3 |
Papillar carcinoma | 1 | 1 |
Mucinous carcinoma | 1 | 1 |
Metaplastic carcinoma | 1 | 1 |
Neuroendocrine carcinoma | 1 | 1 |
Cancer cells or invasive cancer after |
Chemotherapy | 24 | 22 |
Number of brain metastases |
1 | 27 | 24 |
2 | 7 | 6.5 |
3 | 6 | 5.5 |
Multiple | 71 | 64 |
Localization of metastases |
Brain as the first or only site | 36 | 32 |
Liver | 19 | 17 |
Lung | 54 | 49 |
Bone | 27 | 24 |
Locoregional recurrence | 34 | 31 |
Neurosurgery | 20 | 18 |
Systemic therapy after WBRTa,b
| 59 | 53 |
Table 2
Differences in clinical features between patients with basal-like (ER/PgR/HER2-negative, CK5/6-positive and/or HER1-positive) and non-basal-like (ER/PgR/HER2-negative, CK5/6-negative and HER1-negative) biological subtypes of triple-negative breast cancer (75 patients)
Number of patients | 57 (76%) | 18 (24%) | |
Age at initial diagnosis (years) | 50 | 47 | 0.343 |
Age at brain metastases (years) | 54 | 50 | 0.495 |
KPS |
<70 | 28 (49%) | 6 (33%) | |
≥70 | 29 (51%) | 12 (67%) | 0.184 |
Initial TNM stage |
I | 8 (14%) | 0 | |
II | 20 (35%) | 10 (56%) | |
III | 23 (40%) | 7 (39%) | |
IV | 6 (11%) | 1 (5%) | 0.062 |
Histological type and grade |
Ductal carcinoma Grade 3 | 30 (55%) | 10 (59%) | |
Other | 25 (45%) | 7 (41%) | 0.49 |
Number of brain metastases |
1 | 13 (23%) | 4 (22%) | |
2 | 3 (5%) | 1 (6%) | |
3 | 3 (5%) | 0 | |
Multiple | 38 (67%) | 13 (72%) | 1.0 |
Brain as the first/only site | 20 (35%) | 4 (22%) | 0.308 |
Extracranial metastases | 37 (65%) | 15 (83%) | 0.116 |
Lung metastases | 22 (39%) | 13 (72%) | 0.013 |
Liver metastases | 9 (16%) | 2 (11%) | 0.447 |
Bone metastases | 15 (26%) | 3 (17%) | 0.310 |
Loca/locoregional recurrence | 14 (25%) | 7 (39%) | 0.200 |
Neurosurgery of brain metastasis | 12 (21%) | 1 (6%) | 0.120 |
Systemic treatment after WBRT | 29 (51%) | 11 (61%) | 0.314 |
c-KIT | 9 (16%) | 4 (22%) | 0.575 |
DFS, OS and survival from brain metastases in triple-negative, basal-like and non-basal-like subsets are presented in Table
3. In the entire group of 111 triple-negative breast cancer patients with brain metastases, median DFS, OS and survival from brain metastases were 20, 29 and 4 months, respectively. In 75 patients with known basal markers, median DFS, OS and survival from brain metastases were 18, 26 and 3.2 months, respectively. In the basal-like subtype, they were 15, 26 and 3 months, respectively, and in the non-basal-like subtype they were 20, 30 and 2.8 months, respectively. No statistically significant differences in DFS, OS and survival from brain metastases were detected between basal-like and non-basal-like biological subtypes.
Table 3
Median survival of patients with triple-negative breast cancer (75 patients), basal-like subtype (57 patients) and non-basal-like subtype (18 patients)
Disease-free survival |
Triple-negative | 75 | 18 | 13.728; 21.600 | |
Basal-like | 57 | 15 | 10.764; 19.308 | |
Non-basal-like | 18 | 20 | 8.892; 31.560 | 0.284 |
Overall survival |
Triple-negative | 75 | 26 | 17.172; 34.248 | |
Basal-like | 57 | 24 | 17.292; 31.428 | |
Non-basal-like | 18 | 30 | 19.188; 40.764 | 0.227 |
Survival from brain metastases |
Triple-negative | 75 | 3.2 | 2.532; 3.900 | |
Basal-like | 57 | 3.2 | 2.904; 3.528 | |
Non-basal-like | 18 | 2.8 | 0.000; 5.892 | 0.880 |
The analysis of factors influencing survival from brain metastases in triple-negative breast cancer patients with brain metastases, including clinical and biological factors was assessed in univariate and multivariate analysis. The results of univariate analysis of the group of 111 patients are presented in Table
4. The results of Cox multivariate analysis (final model) is presented in Table
5. Multivariate analysis revealed that clinical, and not biological factors influenced survival from brain metastases. They included KPS and the status of extracranial metastases. Patients with good performance status and those with controlled extracranial disease lived longer. Age at the detection of brain metastases was a factor of borderline significance; younger patients lived longer. None of three analyzed basal markers (CK 5/6, HER1, c-KIT) had any influence on survival from brain metastases.
Table 4
Factors influencing survival from brain metastases in patients with triple-negative breast cancer: univariate analysis (111 patients)
KPS |
<70 | 2.2 | |
≥70 | 6.3 | <0.0001 |
Age at initial diagnosis |
<50 | 3.3 | |
50–65 | 3.9 | |
>65 | 3.6 | 0.692 |
Age at the detection of brain metastases |
<50 | 6.3 | |
50–65 | 3.0 | |
>65 | 3.6 | 0.024 |
Initial TNM stage |
I + II | 3.3 | |
III + IV | 4.8 | 0.158 |
Extracranial disease |
Present | 3.2 | |
Absent | 6.3 | 0.063 |
Extracranial disease |
Controlled (stable/responsive) | 8.2 | |
Uncontrolled (progressive) | 2.8 | <0.0001 |
Locoregional recurrence |
Present | 4.2 | |
Absent | 2.8 | 0.304 |
Lung metastases |
Present | 3.1 | |
Absent | 6.3 | 0.045 |
Neurosurgery |
Yes | 12 | |
No | 3.2 | 0.002 |
Systemic treatment after WBRT |
Yes | 4.9 | |
No | 2.6 | 0.148 |
Type of chemotherapy after WBRT |
With capecitabine | 3.1 | |
With vinorelbine | 5.9 | |
With taxanes | 5.4 | |
With antracyclines | 9.8 | |
With platinum salts or etoposide | 3.9 | |
Without chemotherapy | 2.6 | 0.496 |
Table 5
Cox multivariate analysis of factors influencing survival from brain metastases, final model
Karnofsky performance status KPS ≥ 70 | 0.319 | <0.0001 | 0.181; 0.563 |
Age at brain metastases >50 | 1.681 | 0.059 | 0.981; 2.881 |
Controlled extracranial disease | 0.383 | 0.001 | 0.222; 0.660 |