To detect the molecular changes of malignancy in histologically normal breast (HNB) tissues, we recently developed a novel 117-gene-malignancy-signature. Here we report validation of our leading malignancy-risk-genes, topoisomerase-2-alpha (TOP2A), minichromosome-maintenance-protein-2 (MCM2) and “budding-uninhibited-by-benzimidazoles-1-homolog-beta” (BUB1B) at the protein level. Using our 117-gene malignancy-signature, we classified 18 fresh-frozen HNB tissues from 18 adult female breast cancer patients into HNB-tissues with low-grade (HNB-LGMA;
Despite recent advances in biomarker discovery, no clinically proven biomarkers of increased breast cancer risk have been identified and validated in histologically normal breast. However, there is increasing evidence in the current literature for the presence of specific genetic abnormalities in histologically normal breast tissue in patients with and without breast cancer [
In order to elucidate the molecular changes of malignancy in HNB tissues, we used the Affymetrix platform to profile a large prospective series of fresh-frozen HNB tissues and invasive ductal breast carcinomas (IDCs). Based on the differential expression of a number of IDC-specific genes in HNB tissues, we developed a novel 117-gene malignancy signature for molecular classification of HNB tissues into two subsets that we designated as those with high-grade and low-grade molecular abnormalities. We subsequently validated our microarray data on HNB tissues using real-time PCR (qPCR) [
Many of the genes in our 117-gene signature were “proliferation genes.” Some of these “proliferation genes” (TOP2A, MCM2, and BUB1B) are also important targets for breast cancer therapy. Here we report cross-platform validation of these 3 genes as our leading candidate malignancy genes at the protein level. We used immunohistochemistry on a new set of archival sections of HNB tissues from 18 breast cancer/DCIS/prophylactic mastectomy patients whose breast tissues (cancer and normal) were used to derive our 117-gene malignancy signature in the original microarray experiment. Since the cells lining the terminal duct lobular units (TDLUs) are thought to be the precursor cells of breast cancer [
This study was carried out under approval by the Institutional Review Board (IRB) at the University of South Florida, Tampa, FL. It included 24 adult female patients who underwent mastectomy for their breast cancers at Moffitt Cancer Center (MCC), Tampa, FL between 2002 and 2005. Eighteen of these patients had fresh-frozen histologically normal breast (HNB) tissues previously analyzed using Affymetrix Plus 2.0 Gene chip to develop a 117 gene signature to be used for molecular classification of histologically normal breast tissues. Based on the expression levels of 117-genes in our malignancy signature (Figure
Clinicopathologic summary of IDC, molecularly high-risk and molecularly low-risk, histologically normal breast tissue specimens.
Outside institution | Moffitt Cancer Center (MCC) | ||||||||||||||
Case no. | Tiisue core ID (frozen specimens for microarray) | Patient age (Years) | Side of breast sampled | Date of first-tissue diagnosis of BRCA | Surgical procedure at first-tissue diagnosis of BRCA; INA = Information not available | Pathologic diagnosis of BRCA | Date of surgical procedure at MCC | Side of breast sampled | Surgical procedure | Axillary LN dissection = AX; Sentinel LN dissection, axillary = SLN-AX; Not done = ND | Final pathologic diagnosis | Histologic grade (Invasive carcinoma) | Tumor size (cm) | DCIS | Other histopathologic findings (resected specimen) |
1 | X | 41 | Left | 2001.04.10 | Core biopsy | IDC, DCIS, high grade, comedo type | 2005.08.04 | Left | Lumpectomy | Intramammary lymph nodes | IDC | 3 | 5 | Present | X |
1 | X | 41 | Right | X | X | X | 2005.08.04 | Right | Lumpectomy | ND | IDC | 3 | 4 | Present | Skeletal muscle invasion, multifocal |
2 | X | 38 | Left | 2005.07.01 | Core biopsy | IDC | 2005.08.03 | Left | Modified radical mastectomy | SLN-AX | Papillary CA with a focus of IDC | 3 | 7.5 | Present | IDC focus 0.8 cm |
3 | X | 68 | Right | 2005.07.20 | INA | IDC | 2005.08.09 | Right | Total mastectomy | SLN-AX | IDC | 3 | 2.2 | Present | X |
4 | X | 47 | Left | 2005.11.28 | INA | IDC | 2006.01.09 | Left | Mastectomy | SLN-AX | IDC | 3 | 2 | Present | Focal ductal hyperplasia, radial scar, sclerosing adenosis, microcalcifications |
4 | X | 47 | Right | X | X | X | 2006.01.09 | Right | Mastectomy | SLN-AX | Fibroadenomatoid hyperplasia, florid ductal hyperplasia, sclerosing adenosis, microcalcifications | NA | NA | Absent | X |
5 | X | 53 | Right | 2006.05.09 | Core biopsy | IDC | 2006.07.31 | Right | Mastectomy | AX | Residual IDC | 3 | 2.8 | NS | involve dermis of skin/nipple |
6 | X | 71 | Left | 2006.04.12 | Core biopsy | Ductal & Lobular CA | 2006.05.23 | Left | Modified radical mastectomy | SLN-AX | IDC w/lobular features | 3 | 4 | Present | Tumor involves dermal angiolymphatic space in areola |
7 | 1480 | 61 | Right | 2006.05.09 | INA | IDC | 2006.08.01 | Right | Mastectomy | SLN-AX | Benign breast | NA | NA | Absent | ALH, proliferative fibrocystic changes |
7 | X | 61 | Left | X | X | X | 2006.08.01 | Left | Mastectomy | SLN-AX | IDC | 2 | 2.6 | Absent | |
8 | 1481 | 86 | Right | 2006.07.31 | Core biopsy | IDC | 2006.08.29 | Right | Total mastectomy | SLN-AX | IDC | 3 | 8.5 | Present | involve dermis of skin/nipple |
9 | 1482 | 52 | Left | 2006.02.22 | Core biopsy | IDC | 2006.03.30 | Left | Total mastectomy, skin-sparing | SLN-AX | IDC | 2 | 1.5 | Present | X |
10 | 1483 | 80 | Left | 2005.06.13 | Excisional biopsy | Mucinous Carcinoma | 2005.08.16 | Left | Total mastectomy | Not done | No residual carcinoma | NA | NA | Absent | X |
11 | 1484 | 42 | Left | 2005.07.06 | Core biopsy | DCIS solid type | 2005.08.15 | Left | Mastectomy | SLN-AX | Residual DCIS | NA | 0.4 | Present | DCIS, multifocal |
11 | X | 42 | Right | X | X | X | 2005.08.15 | Right | Mastectomy | SLN-AX | Benign breast | NA | NA | Absent | X |
12 | 1485 | 52 | Left | 2006.03.31 | Core biopsy | IDC | 2006.05.30 | Left | Total mastectomy | ND | No residual carcinoma | NA | NA | Absent | X |
12 | X | 52 | Right | X | X | X | 2006.05.30 | Right | Rt modified radical prophylactic mastectomy | AX | Benign breast | NA | NA | Absent | X |
13 | 1486 | 67 | Right | 2006.05.02 | Core biopsy | DCIS | 2006.07.12 | Right | Mastectomy | SLN-AX | DCIS | NA | 1.2 | Present | X |
13 | X | 67 | Left | X | X | X | 2006.07.12 | Left | prophylactic mastectomy | SLN-AX | Benign breast | NA | NA | Absent | X |
14 | 1487 | 67 | Left | 2006.06.12 | Core biopsy | DCIS | 2006.07.31 | Left | Mastectomy | SLN-AX | No residual DCIS | NA | NA | Absent | |
14 | X | 67 | Right | X | X | X | 2006.07.31 | Right | prophylactic mastectomy | SLN-AX | Benign breast | NA | NA | Absent | microcalcifications |
15 | 1488 | 49 | Left | 2005.05.23 | Excisional biopsy | DCIS | 2005.08.02 | Left | Total Mastectomy | SLN-AX | Residual DCIS | NA | X | Present | X |
16 | 1489 | 49 | Left | 2005.05.27 | Excisional biopsy, subareolar | Adenoid cystic CA | 2005.06.14 | Left | Total Mastectomy | SLN-AX | No residual carcinoma | NA | NA | Absent | ADH |
16 | X | 49 | Right | X | X | X | 2005.06.14 | Right | prophylactic mastectomy | SLN-AX | Benign breast | NA | NA | Absent | X |
17 | 1490 | 39 | Right | 2005.04.26 | Excisional biopsy | DCIS solid type | 2005.06.29 | Right | Mastectomy | SLN-AX | DCIS, multifocal | NA | 2.2 | Present | X |
18 | 1491 | 85 | Left | 2005.06.30 | Excisional biopsy | Intracystic CA | 2005.07.28 | Left | Mastectomy | SLN-AX | No residual carcinoma | NA | NA | Absent | ADH |
19 | 1492 | 47 | Left | 2005.11.11 | Needle-loc excisional biopsy | IDC | 2006.05.02 | Left | Skin-sparing total mastectomy | SLN-AX | IDC focal micropapillary features | 3 | 2.5 | Present | X |
19 | X | 47 | Right | 2006.02.27 | Needle core biopsy | Fibrocystic changes | 2006.05.02 | Right | Needle-loc excisional biopsy | SLN-AX | Adenoma w/adenomyoepitheliomatous features and focal atypia | NA | NA | Absent | Sclerosing adenosis, cystic/apocrine changes |
20 | 1493 | 55 | Right | 2006.01.03 | Cytology: Adenocarcinoma | IDC | 2006.02.20 | Right | Mastectomy | SLN-AX | IDC | 3 | 2 | NS | X |
20 | X | 55 | Left | X | X | X | 2006.02.20 | Left | Mastectomy | SLN-AX | ALH; No invasive carcinoma | NA | X | X | ALH involving lactiferous duct |
21 | 1494 | 50 | Right | 2006.03.15 | Core biopsy | IDC | 2006.05.22 | Right | Bilateral nipple sparing mastectomy | SLN-AX | No residual carcinoma | NA | NA | Absent | X |
21 | X | 50 | Left | X | X | X | 2006.05.22 | Left | As above | SLN-AX | Benign breast | NA | NA | Absent | X |
22 | 1495 | 18 | Right | NA | NA | No clinical evidence of invasive carcinoma | 2006.06.19 | Right | prophylactic mastectomy* | SLN-AX | Benign breast | NA | NA | Absent | X |
22 | X | 18 | Left | X | X | X | 2006.06.19 | Left | prophylactic mastectomy* | SLN-AX | Benign breast | NA | NA | Absent | X |
23 | 1496 | 56 | Right | 2006.04.04 | Unknown | DCIS | 2006.06.15 | Right | Bilateral mastectomy | SLN-AX | No residual DCIS | NA | NA | Absent | Ductal hyperplasia, microcalcifications |
23 | X | 56 | Left | X | X | X | 2006.06.15 | Left | Bilateral mastectomy | SLN-AX | Benign breast | NA | NA | Absent | Fat necrosis, microcalcifications |
24 | 1497 | 48 | Right | 2006.06.27 | Lumpectomy | IDC, ILC | 2006.07.28 | Right | Modified radical mastectomy | SLN-AX | No residual invasive carcinoma; LCIS involves nipple duct | NA | NA | Absent | X |
24 | X | 48 | Left | X | X | X | 2006.07.28 | Left | prophylactic mastectomy | ND | Benign breast | NA | NA | Absent | Focal secretory change |
*Strong family history of breast cancer and patient tested positive for BRCA1 gene.
Molecular markers of malignancy in histologically normal breast tissues. Context and evolution of our prospective experimental plan.
All available formalin fixed, paraffin-embedded (FFPE) sections from the mastectomies of the study patients
Patient age distribution for IDC, molecularly high-risk and low-risk, histologically normal breast tissue groups.
IDC patients | Patients with histologically normal breast tissues with low-grade molecular abnormality on microarray confirmed by IHC | Patients with histologically normal breast tissues with high-grade molecular abnormality on microarray confirmed by IHC | |
---|---|---|---|
Mean age | 63 | 55 | 50 |
Standard deviation | 14.3 | 15.16 | 17.48 |
Total no. of cases | 6 | 9 | 9 |
Five-micron thick serial FFPE sections from each selected IDC (
The mouse monoclonal antibody that reacts with human TOP2A protein (#MS-1819-SO, Neomarkers) was used at a 1 : 50 concentration in Dako antibody diluent and incubated for 60 min. The mouse monoclonal antibody that reacts with human MCM2 protein (#MS1726PO, Neomarkers) was used at a 1 : 100 concentration in Dako antibody diluent and incubated for 4 hours. The BUB1B staining required a 4-minute treatment with Ventana Protease 1 prior to a 60-minute incubation with the BUB1B antibody (diluted 1 : 100, Abcam, #AB54894). The Ventana Omni Anti-Mouse HRP Secondary Antibody (prediluted) was used for 16 min. The detection system used was the Ventana Omni UltraMap kit, and slides were then counterstained with hematoxylin. Slides were dehydrated and cover-slipped as per standard tissue core laboratory protocol.
Positive control tissues that were used for optimization of the above IHC protocols included tonsillar lymphoid tissue for TOP2A and MCM2 and spleen for BUB1B protein, per manufacturer’s recommendations. For negative controls, the respective primary antibodies were replaced by commercially available nonimmunized normal serum. Both types of controls showed satisfactory results.
The stained slides were evaluated by the breast pathologist on the study with extensive experience in immunohistochemistry (AN). Immunohistochemical staining for TOP2A and MCM2 was localized to the nuclei of the tumor cells and the normal breast epithelium, while the expression of BUB1B protein was localized to the cytoplasm of the tumor and normal breast epithelial cells. In order to calculate TOP2A and MCM2 nuclear staining indices in IDC tissue sections, up to 2000 tumor cells and in the case of histologically normal breast tissues (HNB-HGMA and HNB-LGMA) tissue sections up to 500 nonneoplastic breast epithelial cells were evaluated by absolute counting of positive (stained) and negative (unstained) cells in each section. TOP2A and MCM2 indices were recorded as per cent positive nuclei as previously described [
Serial archival sections representative of an IDC stained for H&E, TOP2A, MCM2 and BUB1B proteins. (a) Primary invasive ductal carcinoma (IDC) of the breast, grade 2, featuring focal tubular differentiation. (b, c, d) Distinct nuclear immunoreactivity for TOP2A marking the presence of cycling cells in about 15% of the infiltrating tumor cells, and for MCM2 marking the “licensed” population in about 1/3rd of the infiltrating tumor cells and diffuse cytoplasmic immunoreactivity (2+) with focal cell membrane accentuation for BUB1B protein (Immunoperoxidase staining (IMPOX staining); original magnifications 200x).
Serial archival sections representative of histologically normal breast tissues with high-grade molecular abnormality stained for H&E, TOP2A, MCM2 and BUB1B proteins. (a) Portion of a TDLU from a histologically normal breast tissue with high-grade molecular abnormality (Case 22, specimen 1495). Serial sections showing the same TDLU as in (a) with distinct nuclear immunoreactivity for TOP2A (b) and MCM2 (c) in the epithelial cell nuclei, and diffuse cytoplasmic immunoreactivity (2+) for BUB1B protein (d) in the mammary epithelial cells. (IMPOX staining; original magnifications 400x).
Serial archival sections representative of a histologically normal breast tissues with low-grade molecular abnormality stained for H&E, TOP2A, MCM2 and BUB1B proteins. (a) Portion of a TDLU from a molecularly low-risk, histologically normal breast tissue (Case 8, specimen 1481). Serial sections showing the same TDLU as in (a) without any expression of TOP2A (b) and MCM2 (c) in the epithelial cell nuclei. There is a focal cytoplasmic immunoreactivity (1+ to 2+) for BUB1B protein (d) in some of the mammary epithelial cells in this field. (IMPOX staining; original magnifications 200x).
In the IDCs, both TOP2A- and MCM2-positive tumor cells were often more frequent at the peripheral/advancing edge of the tumor mass (Figures
Since the intensity of cytoplasmic staining and the percentage of epithelial cells stained for BUB1B protein was variable from case to case and from lobule to lobule within the same case, a comprehensive immunohistochemical scoring method (
Mean TOP2A and MCM2 indices and BUB1B protein expression scores in IDCs and molecularly high-risk and low-risk, histologically normal breast tissues.
Archival specimen type | Average no. of TDLUs evaluated/specimen (Range) | Mean TOP2A index (%) by IHC | Mean MCM2 index (%) by IHC | Mean BUB1B protein expression score ( |
---|---|---|---|---|
IDCs ( | Not applicable | 27 (15–35) | 47 (30–80) | 149 (80–200) |
Histologically normal breast tissues with high-grade molecular abnormality ( | 31 (6–39) | 11 (2–30) | 20 (8–35) | 68 (33–113) |
Histologically normal breast tissues with low-grade molecular abnormality ( | 24 (17–35) | 2 (1–3) | 4 (1–12) | 17 (10–22) |
.18 | <.005 | <.05 | <.005 |
Whisker plot showing BUB1B protein expression scores for each individual histologically normal breast tissue with high-grade (
Apart from cross-platform validation of 3 of our leading malignancy genes in archival HNB tissue samples, we further demonstrated the differential expression of TOP2A protein on independent test sets of Histologically normal breast tissues, including reduction mammoplasty samples, benign breast tissue from patients with and without synchronous breast cancer, and a set of DCIS and invasive breast carcinomas in a custom-designed breast TMA (Figure
Analysis of variance was used to test the differences among the three sample groups (IDC, HNB-HGMA, and HNB-LGMA tissues) with the Tukey method to adjust for
The 18 histologically normal breast tissues with low-grade (
Mean age for the patient groups with IDCs, HNB-HGMA, and HNB-LGMA tissues was 63, 50, and 55 years, respectively (Table
TOP2A and MCM2 immunostaining was localized to the nuclei of the tumor cells (Figures
Expression of TOP2A was nuclear both in the tumor cells (Figure
TOP2A, MCM2, and BUB1B protein expression scores in IDCs, molecularly low-risk and molecularly high risk, histologically normal breast tissues.
Case no. | Breast tissue specimen category (based on gene expression profiling) | Histologic tumor type on initial biopsy/lumpectomy | Final pathologic diagnosis on mastectomy | Histopathologic findings on archival tissue sections selected for IHC validation | Topoisomerase II-alpha (TOP2A) index (%) | MCM2-index (%) | BUB1B protein expression score ( |
---|---|---|---|---|---|---|---|
1 | Carcinoma | IDC | IDC, DCIS | Invasive Cancer | 25 | 30 | 170 |
2 | Carcinoma | IDC | Invasive papillary CA with a focus of IDC | Invasive Cancer | 30 | 30 | 135 |
3 | Carcinoma | IDC | IDC | Invasive cancer | 15 | 80 | 145 |
4 | Carcinoma | IDC | IDC | Invasive Cancer | 35 | 60 | 80 |
5 | Carcinoma | IDC | IDC | Invasive Cancer | 25 | 50 | 165 |
6 | Carcinoma | IDC, ILC | IDC w/ lobular features | Invasive cancer | 30 | 30 | 200 |
HNB tissues with low-grade molecular abnormality (HNB-LGMA) | |||||||
7 | HNB-LGMA 1 | IDC | IDC | Benign breast tissue | 2.5 | 2 | 21 |
8 | HNB-LGMA 2 | IDC | IDC | Benign breast tissue | 2.5 | 6 | 20 |
9 | HNB-LGMA 3 | IDC | IDC | Benign breast tissue | 2.5 | 12 | 22 |
10 | HNB-LGMA 4 | Mucinous carcinoma | No residual mucinous carcinoma | Benign breast tissue | 1 | 2 | 15 |
11 | HNB-LGMA 5 | DCIS | Residual DCIS, multifocal | Benign breast tissue | 1 | 1 | 10 |
12 | HNB-LGMA 6 | IDC | No residual IDC | Benign breast tissue | 1 | 1 | 18 |
13 | HNB-LGMA 7 | DCIS | Residual DCIS | Benign breast tissue | 2 | 2 | 15 |
14 | HNB-LGMA 8 | DCIS | No residual DCIS | Benign breast tissue | 3 | 8 | 11 |
15 | HNB-LGMA 9 | DCIS | Residual DCIS | Benign breast tissue | 1 | 1 | 17 |
HNB tissues with high-grade molecular abnormality (HNB-HGMA) | |||||||
16 | HNB-HGMA 1 | Adenoid cystic carcinoma | No residual adenoid cystic carcinoma | Benign breast tissue | 8 | 12 | 33 |
17 | HNB-HGMA 2 | DCIS | DCIS, multifocal | Benign breast tissue | 12.5 | 20 | 64 |
18 | HNB-HGMA 3 | Intracystic carcinoma | No residual intracystic carcinoma | Benign breast tissue | 12.5 | 8 | 35 |
19 | HNB-HGMA 4 | IDC | IDC focal papillary features | Benign breast tissue | 9 | 30 | 113 |
20 | HNB-HGMA 5 | IDC | IDC | Benign breast tissue | 2 | 12 | 46 |
21 | HNB-HGMA 6 | IDC | No residual IDC | Benign breast tissue | 15 | 30 | 46 |
22 | HNB-HGMA 7 | No prior biosy performed | Benign breast tissue-patient BRCA1+, strong family history of BC | Benign breast tissue | 10 | 25 | 106 |
23 | HNB-HGMA 8 | DCIS | No residual DCIS | Benign breast tissue | 4 | 12 | 43 |
24 | HNB-HGMA 9 | IDC, ILC | No residual invasive carcinoma | Benign breast tissue | 30 | 35 | 94 |
Immunohistochemical expresssion of TOP2A protein in HNB tissues with low-grade and high-grade molecular abnormalities and in IDCs. There is an obvious trend toward increasing expression from HNB tissues with low-grade molecular abnormality (white bars) to those with high-grade molecular abnormality (gray bars), and the IDCs (black bars), thus providing evidence for cross-platform validation of our original expression profiling data for TOP2A at the protein level. (a) Is the specimen-wise distribution of immunohistochemical expression of
MCM2 protein expression in IDCs and histologically normal breast tissues with high-grade and low-grade molecular abnormality on microarray Expression of MCM2 was nuclear both in the IDC cells (Figure
Immunohistochemical expression of BUB1B protein in HNB tissues with low-grade and high-grade molecular abnormalities and in IDCs. There is an obvious trend toward increasing expression from HNB tissues with low-grade molecular abnormality (white bars) to those with high-grade molecular abnormality (gray bars), and the IDCs (black bars), thus providing evidence for cross-platform validation of our original expression profiling data for BUB1B at the protein level. (a) Is the specimen-wise distribution of immuno-histochemical expression of
Mean BUB1B protein cytoplasmic staining scores for IDCs and histologically normal breast tissues with high-grade and low-grade molecular abnormality on microarray were 149, 68, and 17, respectively (Table
The immunohistochemical expression scores for TOP2A, MCM2, and BUB1B protein in the HNB-HGMA tissues were in the intermediate range between the higher scores (expression) for the IDCs and the lower scores (expression) for the HNB-LGMA tissues (Tables
Immunohistochemical expresssion of MCM2 protein in HNB tissues with low-grade and high-grade molecular abnormalities and in IDCs. There is an obvious trend toward increasing expression from HNB tissues with low-grade molecular abnormality (white bars) to those with high-grade molecular abnormality (gray bars), and the IDCs (black bars), thus providing evidence for cross-platform validation of our original expression profiling data for MCM2 at the protein level. (a) Is the specimen-wise distribution of immunohistochemical expression of MCM2 for the HNB tissues with low-grade and high-grade molecular abnormality and IDC groups. (b) Is the pairwise comparison of MCM2 immunostaining among the three groups. The adjusted
In order to further validate the differential expression of TOP2A protein in various benign, atypical, premalignant, and cancerous breast tissues, we immunostained a breast TMA for TOP2A, using the same IHC protocol as outlined above. The various groups of breast lesions represented on this TMA were as follows.
In this group seven adult females had undergone unilateral or bilateral reduction mammoplasty (RM). Others underwent diagnostic breast tissue sampling. Final pathologic evaluation showed histologically normal breast tissues with areas of benign breast disease (BBD) (
All specimens in this group showed BBD with foci of ADH. In addition, six (66%) cases showed columnar cell change and four (44%) had atypical lobular hyperplasia. There was one case with pseudoangiomatous stromal hyperplasia (PASH) and one case with an intraductal papilloma.
All of these cases showed ADH. In addition, three cases showed areas of invasive ductal carcinoma (IDC) while 5 cases had ductal carcinoma in situ (DCIS), 2 cases showed focal columnar cell change, and one of them also had an intraductal papilloma with atypia, a radial scar, and a fibroadenoma.
Of the fifteen specimens in this group, 14 (93%) were intermediate to high-nuclear grade DCIS and one low-nuclear grade DCIS. Among these two specimens had areas of adenosis, focal ductal hyperplasia, PASH, and a fibroadenoma in addition.
These were histologically confirmed IDCs, of which 2 cases also had focal DCIS, intermediate to high nuclear grade. One IDC showed focal mucinous differentiation.
We found a striking trend toward increasing expression of TOP2A protein in this independent test set of histologically normal and benign breast tissues, ADH with or without synchronous invasive breast carcinoma, DCIS and invasive ductal breast carcinoma tissues, represented on the breast TMA. These results provide further validation of increasing expression of TOP2A protein along the histologic continuum of various breast lesions from benign to premalignant to invasive breast carcinomas it’s (Figures
Immunohistochemical expression of TOP2A protein. (a) Histologically normal breast tissue from a reduction mammoplasty (RM) case featuring lack of nuclear expression of TOP2A in the epithelial cells lining a normal TDLU. (b) Histologically normal breast tissue from a patient with synchronous breast cancer showing positive nuclear staining in 4-5% of the mammary epithelial cells-higher TOP2A expression than the HNB tissues from a reduction mammoplasty case illustrated in (a). (c-d) A larger proportion of epithelial cells are immunoreactive for nuclear TOP2A protein in ductal carcinoma in situ (DCIS) and in the invasive ductal carcinoma (IDC) infiltrating the mammary fat. These cases illustrate an obvious increase in TOP2A protein expression from the lowest risk specimen from a reduction mammoplasty case (a), to the higher-risk specimens (c) and (d) (IMPOX staining for TOP2A; original magnifications 200x).
Mean TOP2A index in independent test sets of histologically normal breast (including reduction mammoplasty tissues), histologically normal and benign breast tissues from patients without and with synchronous cancer, DCIS and invasive breast carcinoma tissues. There is an obvious trend toward increasing TOP2A expression from benign to malignant breast tissues.
There is increasing evidence to support the hypothesis that histologically normal breast tissues contain genetic and epigenetic abnormalities that render them more susceptible to neoplastic transformation and that they might be detected through molecular analyses. In patients with sporadic breast cancer, abnormalities of breast cancer susceptibility genes, including TP53, BRCA1, and BRCA2, have been identified in tumor tissue, and also in histologically normal TDLUs adjacent to carcinoma [
Using specific epigenetic biomarkers, we have previously mapped a number of DNA methylation changes in histologically normal breast tissues as a potential explanation as to why histologically normal breast tissues are at risk for local recurrence after surgical therapy for breast cancer [
Here we present the results of cross-platform immunohistochemical validation of these candidate malignancy gene products (TOP2A, MCM2, and BUB1B proteins) on archival histological normal breast tissue sections from the mastectomies of the two patient groups in the original microarray experiment (those with HNB tissues with high-grade and low-grade molecular abnormalities). These candidate biomarkers were selected for validation based on the gene expression data and the availability of commercially available antibodies and to further investigate their usefulness as biomarkers of molecular abnormalities in histologically normal and benign breast tissues. We further confirmed the increasing expression of one of our malignancy-risk gene products in the present analysis on independent test sets of histologically normal breast tissues including reduction mammoplasty samples, which mostly represent the specimens with lowest risk of breast malignancy, histologically normal/benign breast tissues from patients with and without synchronous breast cancer and a set of DCIS and invasive breast carcinomas (IDCs) using a custom-designed breast TMA (Figure
One of our leading malignancy risk genes identified on microarray analysis of the histologically normal breast tissues was topoisomerase II alpha (TOP2A). TOP2A is a key enzyme in regulating various chromosomal events during tumor cell replication. It is one of the markers of cell proliferation in human breast cancer [
We identified TOP2A as a part of the malignancy-risk signature on our microarray experiment and, in this study, validated its expression at protein level as a potential biomarker of risk of malignancy in histologically normal breast tissues. In the archival sections from histologically normal breast tissues with high-grade and low-grade molecular abnormalities, we evaluated a large number of “morphologically normal TDLUs” and found that the level of expression of TOP2A protein in HNB tissues with high-grade molecular abnormality on microarray was intermediate between the expression levels in the HNB tissues with low-grade molecular abnormality on microarray and invasive ductal breast carcinoma tissues analyzed. Furthermore, the differences in the TOP2A expression levels between the two subsets of molecularly abnormal histologically normal breast tissues and IDCs were statistically significant. Since amplification of TOP2A gene leads to the overexpression of the TOP2A protein and better response to anthracycline therapy [
Since TOP2A amplified tumor cells tend to be sensitive to topo-II inhibitor therapy while TOP2A deleted tumor cells tend to be resistant to anthracycline chemotherapy [
MCM family of proteins are a novel class of proliferation markers, of which MCM2 is part of the prereplicative complex (pre-RC) that is assembled at the site of future DNA replication during the G1 phase to allow genome replication in the subsequent S phase. High-MCM2 index has been shown to correlate with high Ki-67 labeling [
In our malignancy-gene signature, MCM2 was one of the leading malignancy-associated genes in a set of histologically normal breast tissues from peri-menopausal beast cancer patients. In this study, using the standard immunohistochemical approach, we have observed that the MCM2 index in HNB tissues with high-grade molecular abnormality was in the intermediate range between IDCs and HNB tissues with low-grade molecular abnormality, thus validating the overexpression of MCM2 protein in the set of HNB tissues that were showed high-grade molecular abnormality on our original microarray data analysis.
In this study, we found expression of MCM2 protein in all of our cases of histologically normal breast tissues. Considering all of our normal breast samples together, the observed MCM2 index ranged from 1% to 35%. This wider variation is a reflection of an inherent case selection bias in our study, since we selected the 2 subsets of histologically normal breast tissues (with high- and low-grade molecular abnormality) based on differential expression of our malignancy- (proliferation-) associated genes. In a set of normal breast tissues from reduction mammoplasties, Shetty et al. found a median MCM2 expression of 35% [
In another recent study of benign breast tissues from 30 patients who underwent lumpectomy for fibrocystic changes, ductal hyperplasia, and fibroadenomas, the overall MCM2 labeling index was from 0% to 12% [
BUB1B protein is a mitotic checkpoint kinase required for cell mitotic divisions following severe cell damage or mutation [
In this study, we have validated overexpression of BUB1B protein in histologically normal breast tissues that were found to be molecularly abnormal on microarray, thus validating our prior microarray and real-time PCR results. Our study suggests that BUB1B overexpression may be a new immunohistochemical biomarker of malignancy in histologically normal breast tissues. It will also be interesting to investigate the role of BUB1B overexpression as a potential therapeutic target for microtubule inhibitors and an immunohistochemical biomarker of predictive of chemosensitivity of atypical and pre-malignant breast lesions to these antimitotic agents.
Expression of hormone receptors is an established predictor of response of breast cancer to hormonal therapy in breast cancer, but markers predictive of chemosensitivity of breast cancer are less well defined [
To our knowledge, this is the first IHC-based analysis focusing on the pattern of coexpression of newer proliferation-associated proteins (TOP2A, MCM2, and BUB1B) in histologically normal breast tissues. In continuation of our prior transcriptional validation using qPCR, in this immunohistochemical validation study, we have demonstrated significantly higher expression of TOP2A, MCM2, and BUB1B proteins in a set of histologically normal breast tissues that were found to have high-grade molecular abnormality on microarray, based on our novel 117-gene malignancy signature. Taken together, these data further validate our leading candidate malignancy-risk genes (TOP2A, MCM2, and BUB1B) at the protein level. In addition, we have shown incremental expression of TOP2A protein on independent test sets of histologically normal breast tissues (including reduction mammoplasty samples), histologically normal/benign breast tissue from patients with and without synchronous breast cancer, and a set of DCIS and invasive breast carcinomas using a custom breast TMA. This study reveals new information about the coexpression of TOP2A, MCM2, and BUB1B proteins in histologically normal breast tissues and provide preliminary evidence to support further analyses of these proteins on larger series of histologically normal, benign, pre-malignant, and malignant breast tissues. Specifically, determination of TOP2A, MCM2, and BUB1B protein expression status may provide an objective tool to evaluate of the molecular signature of malignancy in histologically normal and benign breast tissues.
The immunohistochemical approach used here offered some distinct technical advantages over other techniques like qPCR or microarray: (1) combined assessment of the degree of expression (high versus low), microanatomical distribution (diffuse versus patchy), tissue (epithelial versus stromal), and subcellular (nuclear versus cytoplasmic) localization of the biomarker proteins in a given sample; (2) comparative evaluation of the relative expression of these marker proteins in histologically normal TDLUs and various incidental benign and pre-malignant breast lesions present in the same archival breast tissue sections. We do recognize one of the limitations of our study—the fewer numbers of histologically normal breast tissue analyzed. However, since we have successfully validated the expression of TOP2A, MCM2, and BUB1B proteins in HNB tissues with various grades of molecular abnormalities, we are in the process of now expanding our investigation to larger sample size and a wider range of benign pre-malignant and malignant breast tissues.
The data presented in this technical validation study of a novel set of molecular biomarkers (TOP2A, MCM2, and BUB1B proteins) in histologically normal breast tissues confirms our microarray data at the protein level. We have also unraveled a preliminary association between the expression of these marker proteins and different stages of mammary carcinogenesis (histologically normal to benign to pre-malignant and fully invasive malignant breast tissues). Additional studies on larger selection of histologically normal, benign, and pre-malignant breast tissues are needed to fully explore the clinical utility of these biomarkers in the stratification of histologically normal breast and benign and premalignant breast lesions into those with various levels of molecular abnormalities. Such classification may potentially be predictive of response of various benign, atypical, and pre-malignant to targeted chemopreventive approaches.
Topoisomerase 2 alpha
Minichromosome maintenance protein 2
Benzimidazoles 1 homolog beta’
Histologically normal breast
Terminal duct lobular units
Benign breast
Hyperplastic enlarged lobular units
Epithelial hyperplasia
Focal ductal hyperplasia
Atypical ductal hyperplasia
Ductal carcinoma in situ
Breast cancer
Invasive ductal carcinoma
Tissue microarray
Real-time PCR
Histologically normal breast with high-grade molecular abnormality
Histologically normal breast with low-grade molecular abnormality
Formalin fixed, paraffin-embedded
Sentinel lymph node dissection, AXILLARY
Standard deviation.
National Cancer Institute RO1grant, CA098522 “Screening for Breast Cancer Using Molecular Signatures” (PI: TJY).
In the future, the authors would like to evaluate the expression of TOP2A, MCM2, and BUB1B on larger clinical validation sample sets of breast tissues, in order to correlate the immunohistochemical expression of these biomarkers with future risk of development of malignancy and subsequent recurrence.
The authors have no conflict of interests to disclose.
The authors, would like to thank Mary Willis, Jean Stern, and Debbie Bir in organization of the study material, Tissue Core Histology Laboratory for optimization experiments and services on new antibodies, and Magaly Mendez and Dane Gregor in the preparation of the paper.