Abstract
Background/Aim: Knowing the molecular footprint of tumors is a precondition for personalized medicine. For breast cancer, targeted therapies are frequently based on the molecular status of the tissue gained from the primary tumor operation. However, it is unclear whether metastases in different organs maintain the same status. Patients and Methods: We compared the estrogen- (ER), progesterone- (PgR) and HER2/neu receptor status of the primary tumor with brain metastases in a series of 24 consecutive breast cancer patients. Results: 62.5-75% of patients exhibited a constant receptor status between the primary tumor and the brain metastasis, whereas discordance rates of 25-37.5% were found, depending on the receptor. The rate of ER and PgR expression was each 41.6% in the primary tumors and decreased to 12.5% and 16.6% in the brain metastases. In contrast, the rate for Her2+ tumors increased from 41.6% in primary breast cancer to 65.2% in the respective brain metastases. The Ki-67 proliferation index increased significantly from a mean of 21% at the primary tumor site to 60% in brain metastases (p<0.001). All anti-estrogen treated breast tumors lost the estrogen receptor expression in the brain metastases, whereas no Her2/neu conversions occurred after treatment with trastuzumab. Conclusion: In summary, receptor conversion is frequent during disease progression. Therefore, the receptor status of the primary tumor is invalid for planning a therapy targeted against brain metastases, especially after hormone-therapy. In these cases, new tissue collection by biopsy or resection is mandatory for the selection of adequate therapeutic targets and accurate decision-making for systemic therapies.
Breast cancer is the most common cancer type and the second leading cause of death in women with 14.5 cancer deaths per 100,000 women within the European Union in 2014 (1, 2). The most prevalent cause of mortality is the early metastatic spread to different organs like lung, bone and brain (1). Approximately 6% of all breast cancer patients have distant metastases at the time of first diagnosis and 20-50% may develop metastases during the course of the disease, with brain metastases accounting for 10-16% of cases (3, 4). The development of brain metastases is associated with about 80% mortality within 1 year of diagnosis (3). In recent years, the incidence of brain metastases is increasing due to new agents in breast cancer therapy allowing prolonged survival (5). Therefore, better therapy options against brain metastases are needed, besides conventional surgical resection and radiation (3).
Elevated expression of different receptor types, such as hormone receptors, i.e. estrogen-receptor (ER), progesterone-receptor (PgR) and the human epidermal growth factor receptor 2 (Her2/neu) are common in breast cancer and represent targets for personalized therapies. For example, the competitive ER-inhibitor Tamoxifen and the monoclonal antibody against the growth factor receptor Her2/neu, Trastuzumab (Herceptin®), are drugs used in the personalized treatment of breast cancer patients (6, 7). It has been reported, that patients with HR-negative and Her2/neu-positive tumors have a higher proportion of brain metastases (8).
Analysis of these receptors is routinely done by only using the primary tumor tissue. Consequently, metastatic lesions are treated according to this receptor phenotype. Since several studies indicate a change in receptor expression between the primary tumor and subsequent metastasis in different organs (9-12), one could expect a significant risk that targeted therapies would only hit the primary tumor and not the subsequent metastasis, if changes in the expression pattern occur. There is little data about conversion rates of brain metastases (12) which often are the most limiting ones for the patient's prognosis.
Therefore, it was the aim of our study to evaluate the expression status of ER, PgR and HER2/neu in primary breast cancer and their corresponding brain metastasis in the same patient in order to define potential discordance rates.
Materials and Methods
Patients. Informed consent of the patients to use tissue samples for scientific investigations was obtained according to the Helsinki declaration of ethical requirements and the local ethical committee (Application No. 03-170). Medical records and the pathological results were analyzed retrospectively. The tumor tissue bank of the Dept. of Neurosurgery of the University Hospital of Cologne harbored 114 specimens of patients who underwent excision of breast cancer brain metastasis from 2001 to 2012. These data were cross-checked with the tissue bank of the Dept. of Pathology containing specimens of primary breast cancer.
In 24 patients, samples from the primary tumor and the corresponding brain metastasis could be identified. These patients were included in our study and characteristics recorded (for clinical parameters see Table I). Data regarding treatment prior to surgical excision of the brain metastasis were collected (Table II). Clinical data on metastases other than brain metastases are displayed in Table III.
Immunohistochemistry and FISH analysis. Patient data as well as histological and immunohistochemical results from the pathological analysis of the primary tumor and the brain metastasis were collected and compared. Both, the primary breast carcinomas and the brain metastases were analyzed by immunohistochemistry in the Departments of Pathology and the Division of Neuropathology, University of Cologne. The expression level of the estrogen and progesterone receptors were measured by the immunoreactive score (IRS) score and the Her2/neu expression using the DAKO score. Fluorescence in situ hybridization (FISH) as described before was used (13, 14). Histological features such as differentiation grade, Ki-67 and tumor type were also collected (Table I).
The Her2/neu receptor conversion rate was analysed by IHC and FISH. These results were validated when comparing the different scores (IRS, DAKO). An overview of all receptor changes within the 24 patient pairs is depicted in Table IV. No correlation was found between the changes of hormone receptors and HER2/neu (p>0.05) (15-17).
Statistical analysis. The status of the three receptors of the primary tumor was compared to the matched brain metastasis. The proportions between the paired groups were compared using the χ2 test. Mann Whitney U and Kruskal-Wallis test where applied when comparing different groups. All statistical analysis was performed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). The level of significance was set at ≤0.05. All results are depicted with SEM (standard error of the mean).
Systematic review of the literature: Analysis of concordance and conversion rates of ER, PgR and Her2/neu in brain metastatic breast cancer in the literature. A database research in MEDLINE (http://www.ncbi.nlm.nih.gov/pubmed) was performed. We included studies with accessible data on concordance and/or conversion rates in subsequent brain metastases of breast cancer patients. Inclusion criteria were matched tissue pairs of the same patient and performance of immunohistochemistry as minimal histopathological requirement. After collection of all studies, the data were summed up with our own results and overall concordance and conversion rates were calculated. The results are displayed in Table V.
Results
Patient data. Twenty-four patients were included in this study. 86% of the patients had an invasive ductal carcinoma whereas 10% of the breast cancer patients showed an invasive lobular carcinoma (Table I). The histological subtype of one patient remained unclear. 63% of the patients received surgical resection of the primary breast tumor. 32% of these patients underwent axillary dissection and breast preservative surgery, 26% breast preservative surgery, 21% ablation, 37% axillary dissection, 11% axillary dissection and ablation. Furthermore, 74% of patients received radiation with an average dose of 51.7 Gy. 5% of patients got neoadjuvant radiation therapy whereas 47% were treated postoperatively. 11% of the patients underwent multiple radiation therapies and 26% received radiation during the history of the disease. 89% of the patients were treated by chemotherapy at least once (Table II).
Neoadjuvant chemotherapy was applied before breast surgery in 42% of cases, and adjuvant in 37%. A combined preoperative and postoperative chemotherapy was used in 11% of the patients. Taxanes were the most commonly used agents (41%), whereas the EC-scheme (Epirubicin and Cyclophosphamide) was the most favorable combined therapy (29%). Hormonal therapy was used in 46% of cases (Table II).
Most brain metastases occurred metachronously, whereas in single patients the brain metastases were found simultaneously with the primary tumor. 84% of the patients harboured a metastasis elsewhere in the body prior to the brain metastasis (Table III): 37% exhibited metastases in the lung, 32% in liver, 21% in bone, 16% in thorax, 42% in more distant lymph nodes, and 5% in soft tissue.
The average age at first diagnosis was 55.88 years (+/− 2.15), the average age at neurosurgical treatment was 60.00 years (+/−2.22). The time interval between the first diagnosis of breast cancer and the diagnosis of the brain metastasis averaged 33.47 months (+/− 5.56). Brain metastases were found fronto-temporally in 43.5% of the cases and in 39% within the cerebellum (Table I).
Patient characteristics.
Histology and receptor expression. At initial diagnosis, most breast cancers leading to a brain metastasis were moderately differentiated (G2), whereas the brain metastases were regularly poorly differentiated (G4). The average Ki-67 increased from 21% in the primary breast cancer to 60% in the brain metastasis (p<0.001). Ten out of 24 tumor samples were Her2/neu positive at first diagnosis and 5 out of 24 were triple negative (21%).
The conversion of receptor status is shown in Figure 1. The ER remained stable in 15 cases (62.5%), while in 8 cases expression was lost (33.3%). A single case of gained expression was observed. The PgR was stable in 18 cases (75%) and its expression was lost in 6 cases (25%). No gained expression was observed. On the contrary, Her2/neu remained stable in 16 cases (69.6%), expression was lost in just one case but gained in 6 cases (26.1%). In 4 out of 9 cases, in which expression of either ER or PgR was lost in the brain metastasis, Her2/neu gained expression. The cases of conversion in ER and PgR overlapped, a joint loss was seen in 55.6% (5/9). The individual receptor status changes are shown in Table IV, the concordance and conversion rates are depicted in Figure 1.
Seven ER positive patients were treated with Tamoxifen, aromatase inhibitors or both. In all of these seven cases, the estrogen receptor status changed and the brain metastasis was tested negative. Five out of these seven patients also exhibited a negative conversion of the progesterone receptor. In contrast, in patients without prior anti-estrogen treatment, only one patient out of 10 had an estrogen conversion. No change in progesterone receptor expression was seen in these ten patients (Table VI). Four patients received Trastuzumab, none of these patients had a change in the Her2/neu status comparing the primary tumor with the brain metastasis (Table VI). The influence of potential changes of receptor status on the time interval between initial diagnosis and brain metastasis is shown in Figure 2. The average time interval was longest in case of gain of Her2/neu and loss of hormone receptors status (>7 years), followed by patients with a gain in Her2/neu only (5.93 years). Short intervals were seen in patients with constant receptor status (2.71 years) or with a loss in the progesterone receptor (3.1 years) (Figure 2). As the development of brain metastases is associated with about 80% mortality within 1 year of diagnosis (3), this time interval indicates a prognosis depending on the receptor status.
Therapies performed before brain surgery.
Patients with metastatic disease (n=24).
Conversion of estrogen-, progesterone- and Her2/neu receptor expression in primary breast carcinoma compared to brain metastasis within the same patients. Estrogen-receptor: Loss of expression in 8 cases. Progesteron-receptor: loss of expression in 6 cases; Her2/neu: loss of expression in 1 case, gain of expression in 6 cases.
Review of the literature. The data of 7 published studies (8, 10, 14, 18-21) and our own findings built the basis for computation of concordance rates (CR), positive conversion rates (PCR) and negative conversion rates (NCR) with a high number of patients (300 to 345). The review and analysis of the published data demonstrate the heterogeneity in different studies. Taking all data together, the concordance rate is 77.7% in ER, 75.6% in PgR, and 86.7% in Her2/neu. The highest conversion rate was found in PgR (positive conversion: 10.1%, negative: 49.4%), followed by ER (pos.: 15.1, neg.: 36.0%), and Her2/neu (pos.: 16.6%, neg.: 10.1%). ER and PgR expression decreased in breast cancer brain metastasis, while Her2/neu and Ki-67 increased (Table V).
Discussion
In our study, we found a high discordance rate of PgR, ER and HER2/neu expression between the primary lesion and the brain metastasis. In a considerable number of cases, PgR and ER were lost in brain metastases. Compared to published data, ER loss was more common in our series (NCR 80% vs. 36%), especially as only one of the ER negative patients gained ER expression (Table V). The data on PgR conversion in the literature are varying widely from 1 conversion in 41 matched pairs (19) to a 25% conversion rate (8) that is very similar to our study. Former studies have shown Her2/neu expression to be more stable than the ER or PgR (22, 23). As shown in Table V, the Her2/neu status only changed in 32 out of 259 (=12.4%) patients during metastatic spread to the brain. The pooled discordance rate for Her2/neu is estimated at 5.5%; in metachronous and distant metastases this rate is expected to be higher (23, 24). In our patients, CR was only 69.6% for Her2/neu. Broom et al. investigated conversion rates of Her2/neu in non-brain metastases. Interestingly, they found that the expression of Her2/neu does not significantly change during metastatic spread to different organs (10). Comparing this result with our finding of 46% PCR for Her2/neu, one could assume that changes of Her2/neu status might be more common in brain metastases than in metastases of other organs. This could be explained by a potential selection towards HER2/neu positive metastatic cells due to their higher affinity to brain tissue, immunogenic selection or after chemotherapy, as no gain of Her2/neu was seen without chemotherapy (25-27)
Individual change of receptor status (n=24).
Differences of the time to brain metastasis in breast cancer patients and the changes of HR and Her2/neu-expression. The longest time to metastasis have the combined loss of HR+ and gain of Her2/neu while constant receptor status and gain of HR demonstrate a much shorter time from primary tumor to the metastasis. HR: Hormone receptor.
There are inconsistent data about the role of conversion on prognosis. A recent study comparing matched tissue from primary breast cancer and different metastases (only 3% brain metastases) showed the highest discordance in PgR and lowest in Her2/neu. A significantly worse clinical outcome was shown in patients who either lost their positive hormone-receptor status or converted to triple-negative in the metastasis (12). Some studies have shown a significant increase of brain metastasis-free interval for patients with positive ER, while others see no influence (13, 14). ER discordance was found to be an independent prognostic factor for progression-free survival (25). When comparing our conversion rates to the interval from primary breast cancer to the occurrence of brain metastases, a Her2/neu receptor gain and an estrogen receptor loss seem to have a favorable effect.
Chemotherapy influences hormone receptor expression and may change HR or Her2/neu status (25, 26). Our results also show receptor changes in most patients, who received chemotherapy (8 out of 13). Only a single change occurred in a patient without prior chemotherapy in our study and one (ER), respective two (PgR) changes were reported in Ustaalioglu et al. (2014) compared to 27 changes after chemotherapy (25). Our study shows that hormone-therapy even has a stronger impact than chemotherapy. A total of 16 out of 21 receptors changed after hormone-therapy compared to 2 changes out of 30 without prior hormone-therapy. In contrast, radiation did not significantly influence receptor status changes (data not shown).
Conclusion
The high conversion rates in our series show that the expression of receptors varies widely between primary breast carcinomas and brain metastases. Obviously, chemotherapy and especially hormone therapy influence receptor expression significantly. This finding is of utmost importance for the potential selection of targeted therapies. For these reasons, biopsies of brain metastases have to be performed before starting targeted therapies for breast cancer with brain metastases.
Changes in the estrogen-, progesterone- and Her2/neu receptor expression in brain metastatic breast cancer shown in literature.
Correlation between therapy and hormone status change (n=17).
Footnotes
↵* These Authors contributed equally to this study.
- Received May 27, 2017.
- Revision received July 15, 2017.
- Accepted July 17, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
