Immunohistochemical localisation of androgen receptor in apocrine metaplasia and apocrine adenosis of the breast: relation to oestrogen and progesterone receptors

AIM: To investigate the receptor status of the sex steroid hormones in apocrine metaplasia of the breast. METHODS: 82 cases of apocrine metaplasia, including 18 of the rare lesion apocrine adenosis, were studied immunohistochemically for the expression of androgen receptor, oestrogen receptor, and progesterone receptor proteins on formalin fixed, paraffin embedded tissue sections. The standard avidin biotin complex (ABC) technique was followed and appropriate positive and negative controls were used. RESULTS: All the studied cases (82/82) were positive for androgen receptor, but were negative for oestrogen receptor and progesterone receptor. CONCLUSIONS: Apocrine metaplastic epithelium, unlike the normal breast epithelium, is responsive to androgens, through androgen receptors, rather than to the female sex hormones. This may have clinical implications.     

Androgen and oestrogen receptors as potential prognostic markers for patients with ductal carcinoma in situ treated with surgery and radiotherapy

Ductal carcinoma in situ (DCIS) is a heterogeneous disease that has been investigated less extensively than invasive breast cancer. Women with DCIS are mainly treated with conservative surgery almost exclusively followed by radiotherapy. However, as radiation treatment is not always effective, the search for biomarkers capable of identifying DCIS lesions that could progress to invasive cancer is ongoing. Although conventional biomarkers have been thoroughly studied in invasive tumours, little is known about the role played by androgen receptor (AR), widely expressed in DCIS. A series of 42 DCIS patients treated with quadrantectomy and radiotherapy were followed for a period of up to 95 months. Of these, 11 had recurrent DCIS or progressed to invasive cancer. All tumours were analysed for clinical pathological features. Conventional biomarkers and androgen receptor expression were determined by immunohistochemistry. Our results showed that AR was higher in tumours of relapsed patients than non‐relapsed patients (P value: 0.0005). Conversely, oestrogen receptor (ER) was higher, albeit not significantly, in non‐relapsed patients than in relapsed patients. AR/ER ratio was considerably different in the two subgroups (P value: 0.0033). Area under the curve (AUC) values were 0.85 for AR and 0.80 for the AR/ER ratio. These preliminary results highlight the potentially important role of both AR and the AR/ER ratio as prognostic markers in DCIS.     

Imprint cytology in immunocytochemical analysis of oestrogen and progesterone receptors of breast carcinoma.

Cytological imprint material from 26 mammary carcinomas was stained with monoclonal antibodies to oestrogen and progesterone receptors in an immunoperoxidase procedure. The staining result was compared with that of parallel stainings of frozen tissue sections of the same tumours. The peroxidase reactions in both techniques were semiquantitatively assessed (histoscore). In both sets of stainings the results agreed in 25 of 26 cases (oestrogen receptor: 19 positive, six negative; progesterone receptor: 14 positive, 11 negative). The histoscores of imprint preparations and cryostat sections showed a significant correlation in linear regression analysis (oestrogen receptor: r = 0.755, p less than 0.001; progesterone receptor: r = 0.740, p less than 0.001). Imprint cytology is simple, does not require expensive instruments, and no separate specimen has to be sequestered. It is especially suitable for immunocytochemical steroid receptor analysis of small breast carcinomas.     

CD24 expression in ductal carcinoma in situ and invasive ductal carcinoma of breast: an immunohistochemistry-based pilot study

CD24 is a small, heavily glycosylated cell surface protein, that is expressed in a large variety of solid tumors. It is considered to play an important role in tumor progression and metastasis. We aimed to evaluate CD24 expression in invasive ductal carcinomas (IDCa), ductal carcinoma in situ (DCIS) and non-tumorous breast tissues, and to investigate the relationship between histopathological parameters, estrogen and progesterone receptors, and c-erbB2 expressions. The study included 34 IDCa, 25 DCIS, and 13 non-tumorous breast tissues. All cases were reevaluated histopathologically, and immunohistochemistry was performed with monoclonal CD24 antibody. The results clearly demonstrated that CD24 expression, including membranous and cytoplasmic staining, was significantly higher in DCIS and IDCa than in the non-tumorous breast (p=0.001, p=0.000, and p=0.035, p=0.000, respectively). Cytoplasmic staining was detected predominantly in neoplastic tissues and was significantly increased in high grade DCIS (p=0.013). In invasive carcinomas, although the level of membranous staining was significantly positively correlated with tumor grade (p=0.040), there was no such an association with the cytoplasmic level. However, it showed a trend towards pT (p=0.089). In conclusion, our results suggest that higher CD24 expression may be associated with malignant transformation and progression in breast cancer biology. Furthermore, higher membranous expression and, in particular, cytoplasmic staining seem to predict malignant transformation, and different patterns of CD24 expression may be associated with different pathological features in breast tumors.     

Genetic relation of lobular carcinoma in situ, ductal carcinoma in situ, and associated invasive carcinoma of the breast.

AIMS: The mutual relation of lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS) of the breast, as accepted precursor lesions of invasive breast cancer, is controversial. Because they display genetic heterogeneity, it is not clear how genetically advanced these entities are and what causes the transition to an invasive carcinoma. METHODS: Six cases of LCIS, four of them with associated lobular invasive carcinoma, four cases of intermediately differentiated DCIS with an associated invasive lobular carcinoma, and nine cases of intermediately and poorly differentiated DCIS with associated ductal invasive carcinoma were investigated by means of comparative genomic hybridisation (CGH) after microdissection and immunohistochemical staining of E-cadherin. RESULTS: LCIS was characterised by a low average rate of copy number changes, no evidence of amplifications, and a high rate of gains and losses of chromosomal material at 1q and 16q, respectively. A high degree of genetic homology with well differentiated DCIS was obvious, as reported previously. The cases of intermediately differentiated DCIS with associated lobular invasive components and lobular differentiation revealed striking homologies, and a significant difference of E-cadherin expression. The comparison of preinvasive and invasive breast lesions, irrespective of differentiation within the same patient, revealed no specific alteration that might be associated with invasion. Genetic alterations seen in invasive carcinoma were not necessarily seen in the adjacent precursor lesions. CONCLUSIONS: These results provide strong evidence that invasive breast cancer is a disease with multiple cytogenetic subclones already present in preinvasive lesions. Moreover, specific CGH alterations associated with invasion were not observed. Furthermore, the close genetic association between well differentiated and a subgroup of intermediately differentiated DCIS and LCIS led to the hypothesis that LCIS and a subgroup of DCIS are different phenotypic forms of a common genotype.     

Immunolocalization of oestrogen and progesterone receptors in the human decidua in relation to prolactin production

The distribution of oestrogen and progesterone receptors withinthe decidualized stroma of the uterus was examined in earlyand term human pregnancy and the results related to the effectof oestradiol and progesterone on prolactin production by deciduain vitro. In early pregnancy progesterone receptors were presentin the nucleus of decidualized cells of both the capsularisand parietalis but not in glandular cells. In contrast at termprogesterone receptors were located within the cytoplasm ofdecidual cells. Oestrogen receptors were detected only in thenucleus and were present in greater amounts in decidua capsularisthan parietalis in early pregnancy, but were not detectablein term decidua. Both oestrogen and progesterone receptors werepresent in the nuclei of cells of arterioles within the decidua.In early pregnancy prolactin production decreased during in-vitroculture of decidua parietalis but was maintained in deciduacapsularis, associated with an increase in progesterone productionby the decidua capsularis. In term decidua, prolactin productionin vitro was only stimulated by a combination of oestradioland progesterone. These results suggest, firstly, that maintaineddecidualization and prolactin production by decidua capsularisduring treatment of women in early pregnancy with the anti-progestinmifepristone is not due to an absence of progesterone receptor;secondly, there is a shift in immunoreactive progesterone receptorin decidual cells from the nucleus in early pregnancy to thecytoplasm in term pregnancy. This may indicate an alterationin the action of progesterone around the time of parturition;and thirdly, in term decidua, progesterone, apparently actingthrough the cytoplasmic receptor, is active in increasing prolactinproduction in vitro only when combined with oestradiol.     

Prognostic role of oestrogen and progesterone receptors in patients with breast cancer: relation to age and lymph node status.

AIMS: To consider the prognostic role of oestrogen receptor and progesterone receptor status in relation to the age at surgery, length of follow up and lymph node status. METHODS: The study population comprised 977 patients with histologically confirmed breast carcinoma, with a median follow up of nine years. The actuarial life table method was used to test for survival differences. The Cox proportional hazard model was used to test for interaction effects between each hormone receptor and age, lymph node status and length of follow up. As the analysis involved multiple subgroups, significance was set at the 1% level (p < 0.01). RESULTS: When the patients were subdivided into groups according to lymph node status and age, progesterone and oestrogen receptor status predicted prognosis in middle aged (46-60 years) patients with lymph node positive breast cancer. Their prognostic effect in this subgroup, however, was restricted to the first five years after surgery. Progesterone receptor status was the strongest predictor of outcome. CONCLUSION: The prognostic power of oestrogen and progesterone receptor status varies depending on age, lymph node status and length of follow up after surgery.     

Thymidine phosphorylase expression and stromal vascularity in ductal carcinoma in situ of the breast

AIMS: Periductal angiogenesis in ductal carcinoma in situ is associated with an increased risk of subsequently developing a recurrence. This study aimed to (1) identify the relation between periductal and stromal vascularity and recurrence and (2) determine whether thymidine phosphorylase (TP) is associated with angiogenesis or recurrence in ductal carcinoma in situ (DCIS). METHODS: Twenty cases of DCIS that did not subsequently recur, 20 that developed a subsequent in situ recurrence, and 12 that developed a subsequent invasive recurrence were investigated. Periductal and stromal (hotspot) microvessel density were determined quantitatively using antibodies to CD34 and von Willebrandt factor (vWF). TP expression by DCIS was assessed semiquantitatively using the H score method. RESULTS: Stromal and periductal microvessel density assessed by anti-vWF gave similar mean values, and showed a strong positive correlation. When angiogenesis was assessed with anti-CD34 this association was lost. Not only were the mean values for both types of microvessel density higher than those obtained with anti-vWF, but the periductal microvessel density was significantly greater than the stromal microvessel density. TP expression was associated with stromal microvessel density assessed with anti-vWF, but not with anti-CD34. TP expression was not related to recurrence. No significant difference was identified in TP expression or stromal vascularity in DCIS between cases that recurred as DCIS and those that recurred as invasive carcinoma. CONCLUSIONS: Recurrent in situ or invasive disease after excision of DCIS does not appear to be related to stromal microvessel density or to TP expression by DCIS cells.     

Topoisomerase IIalpha expression in ductal carcinoma in situ of the breast: a preliminary study

Ductal carcinoma in situ (DCIS) of the breast, a precursor lesion of invasive breast cancer, is a heterogeneous disease in terms of histomorphologic features and biologic behavior. Our aim was to assess the proliferative activity, expressed as topoisomerase IIalpha (Topo IIalpha) immunoreactivity and c-erbB-2 expression in relation to morphologic features and architectural pattern of DCIS. The study included 26 DCIS, which were reclassified according to the recommendations of Consensus Conference. Topo-IIalpha and c-erbB-2 immunoreactivity were detected on paraffin sections. Topo IIalpha was consistently negative in normal ductal epithelium. Topo IIalpha-labeling index (Topo IIalpha-LI) was 0.7+/-0.6% for grade I, 4.3+/-3.9% for grade II, and 13.4+/-8.9 for grade III lesions (P<.01). For mixed nuclear grade DCIS, Topo IIalpha-LI was 6.8+/-4.8. There was no difference in Topo IIalpha-LI between different architectural patterns in low- and intermediate-grade lesions. In high nuclear grade DCIS, there was a progressive increase in Topo IIalpha-LI from solid toward cribriform and comedo-type DCIS. Positive c-erbB-2 immunoreactivity was found in 46% of DCIS, being highest in DCIS with high nuclear grade (78%) and in lesions with extensive necrosis. Topo IIalpha-LI was significantly higher in c-erbB-2-positive lesions (Topo IIalpha-LI- 12.4+/-8.5) as compared with negative lesions (Topo IIalpha-LI- 3.9+/-4.5, P<. 0001). Overexpression of c-erbB-2 and Topo IIalpha is associated with poorly differentiated lesions. Proliferative activity in individual ducts of DCIS depended primarily on the nuclear grade and was independent of architectural patterns of individual ducts in architecturally heterogeneous lesions.     

Immunohistochemical localization of androgen receptor in the human ovary throughout the menstrual cycle in relation to oestrogen and progesterone receptor expression.

In order to elucidate the role of androgen receptors (AR) in human ovaries, we examined their immunohistochemical localization, in comparison with oestrogen receptors (ER) and progesterone receptors (PR), at various stages of the menstrual cycle and follicular development. Primordial and primary follicles did not express AR. In granulosa and thecal cells of secondary follicles there was weak nuclear staining for AR. Granulosa cells of dominant follicles showed moderate nuclear staining for AR, which was stronger than that in thecal cells. In the luteal phase, the staining intensity for AR was strongest in the early luteal phase just after ovulation and declined gradually thereafter. Thecal cells of atretic follicles showed moderate nuclear staining for AR, which was a little stronger than that in dominant follicles. There was weak nuclear staining for AR in stromal cells surrounding follicles. Though there was variation in the staining intensity, AR were present at almost all stages of the menstrual cycle. There is a possibility that androgens, mediated by AR, may play an essential role in follicular growth and maturation, atresia and luteinization as autocrine or paracrine agents.     

Immunolocalization of oestrogen and progesterone receptors in prostatic hyperplasia and carcinoma

Oestrogen receptors and progesterone receptors were immunolocalized in 19 patients with benign prostatic hyperplasia and in 26 patients with prostatic carcinoma. Immunohistochemistry was performed on tissue that had been fixed in 8% paraformaldehyde and then paraffin-embedded, using microwave irradiation for antigen retrieval. Oestrogen receptor expression was observed exclusively in the stromal cells of six out of 26 (23%) patients with prostatic carcinoma, but in none of the cells of patients with benign prostatic hyperplasia. Progesterone receptor expression was detected in 16 of 19 (84%) and 17 of 19 (89%) of the epithelial cells and stromal cells of patients with benign prostatic hyperplasia, respectively. In patients with prostatic carcinoma, progesterone receptor immunoreactivity was observed in 12 of 20 (46%) and 20 of 26 (77%) of the carcinoma and stromal cells of prostatic carcinoma, respectively. The ratio of epithelial cells with progesterone receptor immunoreactivity corresponded well with that of stromal cells with immunoreactivity in patients with benign prostatic hyperplasia. However, the ratio of stromal cells with progesterone receptor immunoreactivity was much higher than that in carcinoma cells in patients with prostatic carcinoma. Immunolocalization patterns or the ratio of the cells with progesterone receptor immunoreactivity did not significantly correlate with histological differentiation or patient's age in carcinoma cases. However, patients with advanced surgical stages of disease demonstrated a significantly smaller number of carcinoma and stromal cells with progesterone immunoreactivity in patients with prostatic carcinoma. These results suggest that oestrogens do not have a direct effect on the biological behaviour of benign prostatic hyperplasia and prostatic carcinoma, but that progesterone appears to play a role in the pathogenesis of benign prostatic hyperplasia and prostatic carcinoma.     

P-cadherin expression is associated with high-grade ductal carcinoma in situ of the breast

Cadherins are calcium-dependent cell-cell adhesion glycoproteins, separated into several subclasses with distinct adhesive specificities and tissue distribution, which play an important role in many cellular events. We analyse the expression of E-, N- and P-cadherin in a series of ductal carcinoma in situ (DCIS) of the breast, since this disease represents a heterogeneous group, with different risks of progression to invasive breast carcinoma. We also studied the correlation between cadherin expression and DCIS classification systems, namely the Van Nuys and the Holland et al. classification, this latter based on cytonuclear differentiation and cell polarity. Our results showed that, regardless the classification applied, P-cadherin expression is strongly associated with high histological grade of DCIS (P=0.0047) and lack of estrogen receptors (P=0.0008). The use of Holland et al. classification showed a significant correlation between P-cadherin expression and decreased cell polarity (P=0.01). In conclusion, P-cadherin expression seems to be more relevant in DCIS pathogenesis than the altered expression of any other cadherin, including the decrease of E-cadherin expression.     

Apocrine ductal carcinoma in situ of the breast: histologic classification and expression of biologic markers

Apocrine ductal carcinoma in situ (ADCIS) has been called a special type of ductal carcinoma in situ (DCIS) because the histologic grading is considered difficult using the classification schemes that have been proposed for common DCIS. However, ADCIS encompasses a spectrum of lesions with different morphologic aspects ranging from minimally atypical to overtly malignant. To define a classification scheme for ADCIS, 35 cases (22 pure and 13 associated with invasive carcinoma) were selected on the basis of conventional morphology on hematoxylin and eosin (H&E)-stained sections. Each case was assigned to 1 of 3 histologic grades (low, intermediate, and high) based on nuclear morphology and the presence of necrosis. In addition, the expression of hormone receptors p53, bcl-2, c-erbB-2, and Ki-67 was evaluated by immunohistochemistry, and the DNA ploidy was determined by image cytometry. Fifteen cases were classified as high histologic grade, 10 as low histologic grade, and the other 10 as intermediate grade. All but 4 cases, irrespective of grade, had the same hormonal immunophenotype: androgen receptor positivity (97.1%) and estrogen receptor and progesterone receptor negativity (94.3% and 97.1% respectively). Twenty-one cases (61.8%) showed p53 expression, and 47.1% of the cases were positive for c-erbB-2. The median positivity for Ki-67 was 5.2%. ADCIS has a unique morphologic and hormonal profile, distinct from common DCIS, deserving a specific classification. The proposed classification scheme allows for categorization of ADCIS according to the most important morphologic features already seen in common DCIS, ie, nuclear grade and necrosis. The expression of biologic markers other than hormonal receptors and bcl2 in ADCIS seems in general to be similar to that in common DCIS. Ki-67 and c-erbB-2 are expressed more frequently in intermediate and high histologic grade ADCIS.     

Immunohistochemical and biochemical analysis of the oestrogen regulated protein pS2, and its relation with oestrogen receptor and progesterone receptor in breast cancer.

AIMS--(i) To assess the validity of an immunocytochemical technique for detecting pS2 protein in paraffin wax embedded tissue; (ii) to provide further data on the relation between pS2 protein and oestrogen receptor (ER) and progesterone receptor (PgR). METHODS--Breast cancer excision biopsy specimens were obtained from 35 previously untreated patients. An immunoradiometric assay was compared with an immunohistochemical method for measuring pS2 protein. ER and PgR were measured in cytosol fractions by enzyme immunoassay and the relation between the presence of these receptors and pS2 protein was assessed before and after subdivision of the women into groups of over or under 50 years of age. RESULTS--A good correlation was seen between the immunoradiometric and immunohistochemical methods for pS2 protein measurement (r = 0.84; p = 0.0001). Log-transformed data showed a significant correlation between increasing values of ER and pS2 protein (r = 0.45; p = 0.006) and to a lesser extent between pS2 protein and PgR (r = 0.38; p = 0.03). Correlations were also shown between pS2+ and PgR+ status (p = 0.01), and between ER and PgR positivity (p = 0.05; Fisher's exact test). pS2+ protein status was only associated with ER+ status in patients aged 50 years or less. CONCLUSIONS--The two methods for pS2 analysis are virtually interchangeable. This provides strong support for using immunohistochemistry for pS2 in paraffin wax embedded tissue. The association with ER positivity and pS2+ protein status only in the premenopausal patients may be due to the higher levels of oestrogenic stimuli in that group.     

KPNA2 protein expression in invasive breast carcinoma and matched peritumoral ductal carcinoma in situ

The aim of this study was to evaluate protein expression of Karyopherin alpha 2 (KPNA2) in invasive breast cancer and matched ductal carcinoma in situ (DCIS) and to correlate it with clinicopathological data, including patient survival. KPNA2 protein expression was assessed by immunohistochemistry in breast tissue samples, containing invasive carcinomas, DCIS, and adjacent histologically benign breast tissues. A polyclonal goat KPNA2 antibody was used for immunostaining of 83 clinicopathologically characterized cases. For statistical analysis, staining of at least 10% of nuclei was considered KPNA2 positive. Immunohistochemical detection of KPNA2 in invasive carcinoma showed a significant correlation with higher tumor stage, positive lymph node status, higher tumor grade, and negative ER status. Concordantly, KPNA2-positive tumors (31.3%) showed significantly shorter disease-free survival times (69 months vs 118 months; p = 0.007). KPNA2 protein expression was also detected in DCIS (21.3%) adjacent to invasive tumor and correlated with nuclear grade (p = 0.013). Expression of KPNA2 in invasive breast cancer correlates with conventional prognostic parameters and shorter disease-free survival. Additionally, KPNA2 is overexpressed in DCIS, particularly high grade lesions, which emphasizes its potential role in carcinogenesis of invasive breast carcinomas.     

Immunohistochemical evaluation of c-erbB-2 oncogene expression in ductal carcinoma in situ and atypical ductal hyperplasia of the breast

Cases of ductal carcinoma in situ (DCIS) and atypical ductal hyperplasia (ADH) of the breast were examined for expression of the protein product of the c-erbB-2 (neu, HER-2) oncogene using two different polyclonal antibodies via an avidin-biotin immunoperoxidase method on formalin- or Bouin'-fixed, paraffin-embedded tissue. Fifty-five percent (18/33) of DCIS and 10% (2/21) of ADH were positive. Significant c-erbB-2 expression in DCIS was generally divided on histologic grounds: ten of ten comedocarcinomas showed strong membrane staining, while only one of 14 small cell DCIS cases (micropapillary or cribiform patterns) showed immunostaining (which was weak and basilar in this single case). DCIS cases of mixed histology were strongly positive in areas of comedocarcinoma. In two of three cases of associated Paget's disease strong membrane staining was seen. The two c-erbB-2-positive ADH cases showed weak basilar staining akin to the small cell DCIS cases. Five cases of lobular neoplasia (atypical lobular hyperplasia or lobular carcinoma in situ) associated with DCIS or ADH were negative for c-erbB-2 expression. We conclude that comedocarcinoma in situ and Paget's disease frequently express the c-erbB-2 protein and are both histologically and biochemically distinct from ADH and small cell patterns of DCIS. We advocate precise subclassification of DCIS on histopathologic reports, particularly in view of reports that overexpression of the c-erbB-2 oncogene in infiltrating breast carcinomas may be associated with a poor prognosis.     

Lysyl oxidase gene expression in the stromal reaction to in situ and invasive ductal breast carcinoma.

Lysyl oxidase is involved in the main pathway of collagen and elastin cross-linking: it has a role in the maturation of fibrillar matrix proteins in fibrosing processes and dictates their stability against metalloproteases. The stromal reaction patterns in ductal breast carcinoma are known to be morphologically varied. This has raised the hypothesis that there might be a differential expression of the lysyl oxidase gene as a function of stromal reaction pattern. The present study investigates this potential correlation and the role of matrix protein cross-linking in stromal differentiation. Lysyl oxidase was detected by immunohistochemistry and lysyl oxidase gene expression by in situ hybridization. Maximal expression was observed in myofibroblasts and myoepithelial cells around in situ tumors and in the reactive fibrosis facing the invasion front of infiltrating tumors. The lysyl oxidase substrates were observed in parallel, resulting in the stabilization of a scar-like peritumor barrier. In contrast, a lack of lysyl oxidase was associated with the loose or scirrhous stroma accompanying invading tumors; here, in situ hybridization revealed type I collagen synthesis, resulting in the deposition of non-cross-linked matrix proteins susceptible to degradation. The early development of a cross-linked matrix around ductal breast carcinoma suggests a possible bost defense mechanism, whereas the synchronous or late stromal reaction lacking lysyl oxidase favors tumor dispersion.