Keywords breast cancer - triple-negative - apocrine - immunohistochemistry - androgen receptor - epidermal growth factor receptor K
i -67
Palavras-chave câncer de mama - triplo-negativo - apócrino - imuno-histoquímica - receptor de androgênio - receptor do fator de crescimento epidérmico - K
i -67
Introduction
Current knowledge: triple-negative breast carcinomas (TNBCs) are, generally, very aggressive neoplasms, more prevalent in young women, with no target for therapy. They proved to be heterogeneous regarding morphology, response to chemotherapy, and genomic changes. There is no doubt that potential targets will work only in subsets of TNBCs. The candidates for these subsets are BRCA-1 mutant, BRCA1-like tumors with underlying defects in homologous recombination-mediated DNA repair, and androgen-receptor positive tumors. In this study, we selected 118 consecutive premenopausal patients with TNBCs to examine the frequency of the apocrine profile defined by the immunohistochemical expression of the androgen receptor (AR), which can be performed in diagnostic routine. Apocrine TNBCs were rare in this population; however, our findings support a more favorable biology of these tumors based on lower proliferative activity and lower tumor grade.
Triple-negative breast carcinomas correspond to a heterogeneous group of neoplasias that usually exhibits a clinically more aggressive phenotype. Triple-negative breast carcinomas commonly exhibit a lack of estrogen and progesterone receptor expression, as well as an absence of overexpression and/or amplification of the epidermal growth factor receptor (EGFR), HER2. Currently there is no targeted therapy for them. Triple-negative breast carcinomas are also more prevalent in young patients.[1 ]
[2 ] While TNBCs are often associated with a basal-like genetic phenotype, 21.4% of them correspond to other molecular types, such as HER2-enriched (7.8%), normal (7.0%), luminal B (4.4%), and luminal A (2.2%).[3 ] Initially, the basal-like phenotype of TNBCs was defined by the immunohistochemical expression of basal cytokeratin ⅚ and/or EGFR.[4 ]
[5 ] Other definitions have been proposed, but none have exhibited sufficient concordance with gene expression profiles, consistent with the heterogeneity of the TNBC phenotype.[6 ] Actually, gene expression-based molecular analyses have identified distinct subgroups of TNBCs, such as claudin-low, immunomodulatory, mesenchymal-like, mesenchymal stem cell-like, androgen luminal, and basal-like types 1 and 2.[7 ] Moreover, the immunohistochemical profiles corresponding to these types are actively being studied.[4 ]
[6 ]
[7 ] Among the currently identified phenotypes, the best characterized, either by genetic and/or immunohistochemical approaches, is the apocrine-based phenotype, which involves the expression of the AR.[7 ]
[8 ]
[9 ] Correspondingly, the AR represents a promising therapeutic target for this carcinoma subtype.[10 ]
The aim of the present study was to analyze the molecular apocrine profile of TNBCs that were biopsied in women 45 years or younger.
Methods
Institutional Approval
This project was approved by the Scientific Committee of the Department of Pathology and by the Ethical Committee for Research Projects of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Brazil (CAPPesq) (protocol n. 311/10). As the study was retrospective, informed patient consent was waived, and any form of patient identification was abolished.
Selection of Cases
Formalin-fixed, paraffin-embedded tissue specimens from 118 patients aged 45 years or younger with triple-negative primary breast carcinomas diagnosed between July 2009 and March 2011 that corresponded with consecutive cases with available paraffin blocks from our larger previous study were selected.[11 ] In the previous study, samples from 5,687 consecutive patients were sent to the Consultoria em Patologia, a private reference surgical pathology laboratory, for routine predictive and prognostic immunohistochemical profiling. Triple-negative breast carcinomas corresponded to 15.7% (894/5,687) of all breast cancer diagnosis. In the subgroup composed by 1,386 patients aged 45 years or younger, the number of TNBCs raised to 19.5% (271/1,386). From the latter, 130 of the more recent cases that had paraffin block specimens available were selected for the present study.
Pathological Study
The same pathologist (FMC) reviewed all of the slides and evaluated their histological subtypes based on World Health Organization criteria.[12 ] Tumor grading was assigned according to the Nottingham criteria.[13 ] The following characteristics were also evaluated: microscopic tumor contour (total, partially circumscribed, or infiltrative); fraction of necrosis (absent, focal, or extensive); in situ component (absent, < 25%, or > 25%); any tubule formation (yes or no); lymphocytic stromal infiltration (absent/slight or moderate/intense); desmoplastic intratumoral reaction (absent/discrete or moderate/intense); vascular embolization; and percentage of Ki -67 expression. A representative area of each tumor was selected for the construction of tissue microarray (TMA) blocks.
TMA Construction
Tissue sections were stained with hematoxylin/eosin, and the corresponding sections in each paraffin donor block were marked. Then, one cylinder of material (2.0 mm in diameter) was taken from each of these regions, and they were mounted into recipient paraffin blocks at 2 mm intervals using a precision microarray instrument (Beecher Instruments, Silver Spring, MD, US). A grid system was established so that each core had an x- and y-coordinate reference for sample identification. The blocks were sealed at 60°C for 10 minutes. Sections (3 µm) from each TMA block were prepared using standard techniques, and were mounted on Starfrost® (Light Labs, Dallas, TX, US) slides. The first histological sections that were cut were stained with hematoxylin/eosin to ensure that the appropriate sections of the tumor had been obtained.
Immunohistochemistry and Scoring
Epitope retrieval methods and the source and dilution of the antibodies used are listed in [Table 1 ]. Bound antibodies were detected with horseradish peroxidase labeled polymers conjugated to goat anti-rabbit or goat anti-mouse immunoglobulins (DAKO EnVisionTM System, US). Peroxidase activity was visualized with diaminobenzidine staining (DAKO).
Table 1
Reagents and methods used for the immunohistochemical analyses performed
Antigen
Clone
Source
Dilution
Epitope retrieval method
AR
F39.44.1
BIOGenex
(Fremont, USA)
1:100
Tris-EDTA buffer (pH 9.0)
20 minutes, 97° C
Ki -67
Mouse, MIB-1
Dako
(Glostrup, Denmark)
1:500
Citrate buffer (pH 6.1)
20 minutes, 97° C
Cytokeratin 5/6
D5/16B4
Dako
(Glostrup, Denmark)
1:4
Tris-EDTA buffer (pH 9.0)
20 minutes, 97° C
EGFR
31G7
Zymed
(South San Francisco)
1:100
Citrate buffer (pH 6.1)
20 minutes, 97° C
Abbreviations: AR, androgen receptor; EGFR, epidermal growth factor receptor.
Androgen receptor expression was evaluated based on the percentage of cells that exhibited positive nuclear staining in a sample ([Fig. 1 ]). Positive EGFR expression was defined based on the presence of complete or moderate/strong membrane staining in ≥ 10% of cells. For cytokeratin ⅚, any cytoplasmic staining of moderate to strong intensity in at least 1% of cells was considered positive. The apocrine subtype was defined by the expression of the AR. All of the cases presented with at least 80% AR-positive cells; therefore, no cut-off was defined for the AR staining. A basal-like phenotype was defined by the expression of cytokeratin ⅚ and/or EGFR.[4 ]
Fig. 1 Representative image of a breast carcinoma stained for AR expression with nuclear immunostaining (original magnification, 200 × ).
Statistical Analyses
Statistical analyses were performed using SPSS software, version 22.0 (SPSS, Chicago, IL, US). The features of the apocrine and non-apocrine subgroups of the TNBCs were described and compared using Fisher's exact test (for categorical variables) or the Mann-Whitney test (for patient age and Ki -67). A p -value lower than 0.05 was considered significant.
Results
The clinicopathological features of all of the examined cases are summarized in [Table 2 ]. An apocrine subset was detected in 6/118 (5.1%) of the TNBC tissues examined. The median patient age for the apocrine samples versus the non-apocrine samples was 41 and 39 years respectively, and this difference was not significant. Apocrine tumors also presented a lower rate of Ki -67 expression (17.5% versus 70.0%, p = 0.02), and a trend toward a lower histological grade (66.7% versus 27.9%, p = 0.06). None of the other studied variables were found to significantly differ between the two groups.
Table 2
Clinicopathological features of TNBCs from 118 patients aged 45 years or younger
Features
Apocrine
Non-apocrine
N
P
Number of cases (%)
6 (5.1%)
112 (94.9%)
118
Mean age ± SD, years
41
39
118
0.2940*
Histological grade
1 / 2
4 (66.7%)
31 (27.9%)
35
0.0646†
3
2 (33.3%)
80 (72.1%)
82
Not assessed
0
1
1
Basal-like phenotype
Yes
5 (95.6%)
82 (73.2%)
87
1.0000†
No
1 (4.4%)
30 (26.8%)
31
EGFR expression
Yes
3 (50%)
59 (52.7%)
62
1.0000†
No
3 (50%)
53 (47.3%)
56
Ki -67, mean ± SD
17.5
70
118
0.0199*
Lymphatic invasion
Yes
3 (60%)
34 (31.7%)
37
0.3295†
No
2 (40%)
73 (68.3%)
75
Not assessed
1
5
6
Tumor necrosis
Yes
3 (50%)
76 (67.8%)
79
0.3953†
No
3 (50%)
36 (32.2%)
39
Abbreviations: EGFR, epidermal growth factor receptor; SD, standard deviation; TNBC, triple-negative breast carcinoma. †Fisher's exact test; *Mann-Whitney test.
Discussion
Triple-negative breast carcinomas represent a very heterogeneous group of breast cancers that have a poor prognosis and no targeted therapy available.[14 ] Most TNBCs exhibit a basal-like molecular subtype, and have been identified at a higher frequency in women carrying BRCA1.[15 ] An important association has been observed between TNBCs, basal-like carcinomas, and BRCA1-associated carcinomas, even though they are not synonymous. In this study, we defined the basal-like phenotype by the immunoexpression of EGFR and/or cytokeratin ⅚, according to the criteria of Nielsen et al.[4 ] Eighty-seven (73.7%) of our 118 cases presented the basal-like phenotype, which was consistent with the rate of cases of previous studies.[16 ]
[17 ] The basal-like phenotype was present in 5 out of 6 (95.6%) cases with apocrine phenotype and in 82 out of 112 (73.2%) cases within the non-apocrine group. However, this difference was not significant, probably because of the small number of apocrine cases. The coexistence of apocrine and basal-like phenotypes was also described by Choi et al.[8 ] These authors studied the immunohistochemical profile of 122 TNBCs, and found the molecular apocrine phenotype in 12 (9.8%) of the cases, the mixed apocrine and basal-like in 5 (4.1%), and the apocrine plus claudin-low in 6 (4.9%) cases. Although their number of apocrine cases was higher than ours, they included older patients (47.5 ± 12.1 years), and the mean age of the apocrine type was even higher (56.9 ± 13.7 years).[8 ] The present set of samples was restricted based on younger patients to include a higher percentage of TNBC cases, albeit with a lower frequency of AR expression[1 ]
[8 ]
[18 ]. For example, AR expression was previously detected in 12% of TNBCs,[10 ] while only 5.1% of the TNBCs examined in the present study were found to express the AR. Another study found AR expression in 17.7% (87/492) of TNBCs, and the mean age of patients with AR-positive was higher than those with AR-negative tumors (53 years versus 47 years, p < 0.001).[18 ] They also observed a significant correlation between AR expression and lower grade tumors. However, they could not explain the worse prognosis they observed in the apocrine group, which differed from the literature.[18 ] The results of our study are consistent with previous observations, despite the limited number of apocrine carcinomas that were examined (n = 6).[8 ]
[19 ]
Efforts to identify subtypes of TNBCs according to predictive variables have been described in recent studies of basal-like carcinomas and/or TNBCs.[7 ]
[20 ] Less than 30% of women with metastatic triple-negative carcinomas survive more than 5 years, and most do not survive regardless of the chemotherapy regimens they undergo.[21 ] However, it has been reported that basal-like carcinomas are more sensitive to platinum-based therapies,[20 ] and drugs acting as poly ADP ribose polymerase (PARP) inhibitors may be effective for the treatment of BRCA1-associated carcinomas once the DNA repair process in these tumors is defective.[22 ] Heretofore, there is a lack of specific and effective therapies for TNBCs. Therefore, a possible role for the AR in defining a distinct subtype of TNBC is of interest.[7 ]
[8 ]
[23 ] The biological and therapeutic applications of the AR have been widely characterized for prostate cancers, but fewer studies have investigated the role of the AR in breast cancer.[24 ] It is also possible that other molecular events may be involved, such as those involving the microenvironment and epigenetic phenomena.
Previously we demonstrated that AR expression in HER2 carcinomas is associated with lower proliferative activity and lower tumor grade, thereby suggesting a less aggressive tumor behavior.[25 ] Mrklić et al[26 ] also observed an inverse correlation between the Ki -67 index and the AR status for TNBC, which suggests a possible antiproliferative effect of androgens.
Other studies have analyzed the relationship between AR immunohistochemistry and tumor clinicopathological features, and an inverse relationship between AR expression and a higher clinical stage, a higher histological grade, and a higher mitotic score was observed.[19 ]
[25 ] These data suggest that AR-positive TNBCs may be less aggressive tumors. In addition, AR expression appears to predict responses to anti-androgen therapies. For example, in the phase II study conducted by Gucalp et al[10 ] with AR-positive, metastatic TNBCs, the clinical benefit rate associated with the antagonist, bicalutamide (150 mg/day), was of 12%.
In spite of the limitations of this study concerning the small number of apocrine cases and the absence of follow-up information, we could confirm the low frequency of the apocrine profile among premenopausal patients and its association with lower grade and lower proliferative activity. Our results reinforce the heterogeneity of TNBCs, a group of different neoplasias that only have in common the absence of estrogen and progesterone receptors, and HER2.
Conclusion
Despite the small number of cases that were analyzed, the present results suggest that the apocrine subgroup of TNBCs is less frequent in premenopausal patients. This subgroup also tends to present as carcinomas of lower grade and lower proliferative activity, which is consistent with a less aggressive biological behavior. These results can be considered in strategies for the development of targeted therapies for TNBCs.
This study was supported by the São Paulo Research Foundation (FAPESP) (Process n. 2014/15472–8).
