Patient material
The patients considered for inclusion in this study were female patients who had been treated with breast surgery in Skåne County and met the criteria for one of the following groups:
Group A (n = 30): breast cancer patients without BRCA1/2 mutation; surgery during the period 1999–2006.
Group B (n = 19): breast cancer patients with BRCA1 mutation; surgery during the period 1984–2009.
Group C (n = 16): breast cancer patients with BRCA2 mutation; surgery during the period 1984–2009.
Group D (n = 13): BRCA1 mutation carriers without breast cancer; prophylactic mastectomy during the period 1996–2010.
Group E (n = 13): BRCA2 mutation carriers without breast cancer; prophylactic mastectomy during the period 1996–2010.
Group F (n = 35): patients without breast cancer or BRCA1/2 mutations; mammoplasty during the period 1993–1994.
Thus a total of 126 patients were reviewed for inclusion in our study. An experienced histopathologist (BLI) examined the original hematoxylin and eosin (H&E) stained microscope slides from each patient without knowledge of the clinical parameters. The tissue blocks containing the largest number of histologically normal TDLUs for each patient were selected for the investigation. Exclusion criteria were any of the following: patient received neoadjuvant therapy; no tissue blocks available in the archives; no tissue block contained ≥10 morphologically benign TDLUs. Based on the mentioned criteria, a total of 106 patients were included in the study (see Table
1).
Table 1
Patient groups in the study
A | Cancer, non-BRCA1/2
| 21 | 50 (31–77) | 4 | NA | NA | 8 | 4 | 9 | 0 | 3 | 1 | 0 |
B | Cancer, BRCA1
| 17 | 43 (23–81) | 17 | 17 | NA | 4 | 10 | 0 | 3 | 4 | 1 | 3 |
C | Cancer, BRCA2
| 11 | 51 (33–74) | 11 | NA | 11 | 0 | 7 | 3 | 1 | 0 | 1 | 2 |
D | Prophylactic mastectomy, BRCA1
| 12 | 37 (23–54) | 12 | 12 | NA | 0 | 6 | 4 | 1 | 0 | 4 | 1 |
E | Prophylactic mastectomy, BRCA2
| 11 | 37 (31–51) | 11 | NA | 11 | 0 | 5 | 5 | 1 | 0 | 0 | 1 |
F | Mammoplasty, non-cancer, non-BRCA
| 34 | 31 (20–68) | 5 | NA | NA | 19 | 7 | 8 | 0 | 0 | 8 | 1 |
Data on the following clinical parameters were available for the majority of the patients: indication for surgery, age at time of surgery, current or previous use and total duration of use of oral contraceptives and or hormone replacement therapy (HRT), age at menarche, number of live childbirths (parity), family history of breast cancer (1st or 2nd degree relative), and
BRCA1/2 mutation status. Table
1 presents information on the age, hormonal exposure, and reproductive status of the women included in the study.
Sixty-seven of the women were pre-menopausal, 35 were post-menopausal, and data were missing for four. The median age at onset of menopause was 49 years (range, 35–55 years). The numbers of live births among the women were as follows: para 0 in 32, para 1 in 11, para 2 in 28, para 3 in 25, and para 4 in four. The median age at menarche was 13 years (range, 10–18 years; data missing for 32 women). Data regarding exogenous hormone treatment status at the time of surgery were as follows: 70 women had no such treatment, 15 used oral contraceptives, seven received HRT, and six had a progestin intrauterine device. Sixty-five women had used oral contraceptives at sometime during their lives (median duration, 7.1 years), and 11 had received HRT at some point (median duration, 5.0 years). Clinical data and breast tissue samples representing 25 of the 28 patients in our previous study [
15] were included in the present analysis.
Histology and immunohistochemistry
If two tissue blocks from the same patient were of similar superior quality, both (from the same or both breasts) were analyzed in the same manner for quality assurance purposes. New sections were obtained from all relevant paraffin tissue blocks, and consecutive sections were H&E stained and used for immunohistochemistry, as described previously in detail [
15]. Briefly, after antigen retrieval, the sections were incubated with mouse monoclonal antibodies directed against human ALDH1 A1 (aa 7–128, N-term, diluted 1:100; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Detection of immunoreactive sites was performed using a system based on horseradish peroxidase (HRP) and di-aminobenzidine (DAPI) labelling of primary antibodies raised in mice (Envision+ System-HRP, Dako, Glostrup, Denmark). Sequential incubations were performed with secondary antibodies and HRP-conjugated antibodies. Following the peroxidase reaction in DAPI and H
2O
2, the sections were rinsed and counterstained with hematoxylin, and then dehydrated and coverslipped in Mountex medium (Histolab Products AB, Göteborg, Sweden). To confirm the specificity of ALDH labelling, slides that were not incubated with the primary antibodies were included in each immunohistochemistry run.
The tissue sections selected for ALDH immunolabeling were assessed regarding the following features: tissue fragment size, epithelial atypia or hyperplasia, invasive carcinoma, and total number of TDLUs. The tissue areas available in each chosen block ranged in size from 48 to 621 mm
2with a median value of 254 mm
2. Areas displaying ductular or lobular hyperplasia, atypia, carcinoma
in situ (four cases), or invasive carcinoma (10 cases) were excluded from further analysis. To evaluate the distribution and number of immunolabelled ALDH+ cells, 50 TDLUs were examined, guided by a grid, at evenly spaced intervals in each tissue section; if fewer than 50 TDLUs were present in a section, all the TDLUs were evaluated. The number of TDLUs found to be available for analysis of ALDH immunoreactivity in each patient ranged from 13 to 50 (mean, 45 TDLUs). Only ductules (i.e., no ducts or stroma) were evaluated. In our previous study [
15], we described ALDH+ cells as being in a basal, luminal, or intermediate location. For each patient in the present study, the number of TDLU cross sections with ductular ALDH+ cells was recorded for each ductular level (luminal, intermediate, or basal) in relation to the total number of TDLU cross sections examined.
ALDH immunoreactive cells that were labelled either strongly or weakly were considered to be positive for ALDH. Furthermore, cells found in luminal and intermediate ductular locations were defined as non-basal (luminal plus intermediate) in some analyses, as indicated in the text. This was done because determination of cell location by ductular level is less accurate when ductules lack microscopically discernible lumina.
Blind testing of ALDH+ cell frequency in duplicate tissue samples from 13 patients yielded correlation coefficients of 0.45 (Pearson) and 0.53 (Spearman), indicating similarity between independent observations (rs = 0.65, p = 0.01). These results confirmed the robustness of the analysis and demonstrated similarity between different areas of breast tissue in the same patient.
Statistical analysis
The number of TDLUs with detectable ALDH+ cells and basal and non-basal localization of those cells were analyzed by linear regression using occurrence of ALDH+ cells in the mammoplasty group as the independent variable. Logistic regression was used to analyze differences between risk factor groups. Correlation between ALDH findings for duplicate samples from the same patient was analyzed using Spearman’s rank test and Pearson’s test. Two-tailed tests were performed to assess the level of statistical significance, and results with p < 0.05 were considered significant. SPSS 18 software (IBM, Armonk, NY, USA) was used for all statistical analyses.