Medical Research Journal:
Study of Her-2/neu and TOP2A expression in familial versus sporadic breast carcinoma
El-Gerzawy, Asaad M.S.a; Al Ansary, Mervat M.S.b; Hussein, Iman L.c; Mohamed, Amal M.a; El-Kasem, Fatma M. Aboud; Hammad, Sayeda A.a; Abd-Allah, Sally G.a
aDepartment of Human Cytogenetics, Division of Human Genetics and Genome Research, National Research Centre
bDepartment of Clinical and Chemical Pathology, Faculty of Medicine
Departments of cPathology
dMedical Oncology, National Cancer Institute, Cairo University, Cairo, Egypt
Correspondence to Asaad M.S. El-Gerzawy, MD, Department of Human Cytogenetics, Division of Human Genetics and Genome Research, National Research Centre, El-Bohouth Street, Dokki, 12311 Cairo, Egypt Tel: +20 0100 5017045; fax: +20 233 70931; e-mails: firstname.lastname@example.org, email@example.com
Received March 19, 2013
Accepted April 18, 2013
Introduction: Genetic predisposition is one of the important risk factors that increase the incidence of breast cancer. The Her-2/neu gene is considered the most frequently amplified oncogene and is accompanied with either amplification or deletion of the TOP2A gene with an equal frequency.
Aim of the study: The aim of the study was to compare patients with familial breast cancer (FBC) with those having sporadic breast cancer (SBC) and also to evaluate the combined study on the hormonal status, immunostaining for Her-2/neu, and Her-2/neu and TOP2A copy number alterations by fluorescence in-situ hybridization (FISH).
Patients and methods: A total of 22 patients with invasive breast carcinomas were involved: 12 positive for the criteria that define FBC and 10 positive for SBC. Representative sections of formalin-fixed paraffin-embedded blocks were subjected to immunostaining for Her-2/neu (Hercep test), estrogen receptors, and progesterone receptors and also to FISH in order to evaluate TOP2A and Her-2/neu copy number alterations.
Results: FBC tumors were more aggressive than SBC tumors, as assessed by pathological parameters and immunohistochemical markers (lower estrogen receptor and progesterone receptor expression). However, the frequency of Her-2/neu gene amplification and protein overexpression was equal between the two groups. TOP2A gene amplification was observed in 25% of FBC patients compared with 20% of SBC patients. Coamplification of both Her-2/neu and TOP2A genes was observed in 8.3% of FBC patients and in 20% of SBC patients. In contrast, Her-2/neu amplification alone was observed in 25% of FBC patients compared with 10% of SBC patients. Importantly, TOP2A amplification without Her-2/neu gene amplification was observed in 16.7% of FBC patients but was not observed among SBC patients.
Conclusion: FBC tumors were more aggressive than SBC tumors. This study suggests that TOP2A gene amplification occurs both in FBC and in SBC patients but more frequently among FBC patients. Moreover, TOP2A gene amplification may occur independent of Her-2/neu amplification. We can conclude that this parameter should be determined on a routine basis in breast cancer patients, especially when FBC is suspected. In addition, a combined approach using immunohistochemical analysis and FISH can optimize Her-2/neu testing for breast carcinoma patients, especially among those with immunohistochemistry-equivocal tumors.
Breast cancer is ranked as the most common malignancy affecting women, contributing up to 22% of all cancers worldwide 1. In its latest registry, breast cancer constitutes about 17.5% of all malignant tumors presented to the National Cancer Institute, Cairo University 2.
Genetic predisposition is considered to be one of the important risk factors that increase the incidence of breast cancer 3, as about 5–10% of all cases are believed to be predominantly hereditary in nature 4. Several specific genes are involved in hereditary breast cancer; among them are the BRCA1 gene, which is mapped to chromosome 17 (17q12–21), and the BRCA2 gene, which is mapped to chromosome 13 (13q12–13). Both these genes are considered as tumor suppressor genes 5. Mutations in these two genes account for 2–3% of all breast cancer cases and around 40–50% of all familial cases 6.
Other important genes include the Her-2/neu gene, which is located on chromosome 17 (17q11.2–12) 7 and is considered one of the most frequently amplified oncogenes in breast cancer 8. Her-2/neu gene amplification is accompanied with either amplification or deletion, with equal frequency, of the TOP2A gene located on chromosome 17 (17q12–21) 9. Other relevant genes include P53, Rb1, Myc, and CCNd1 10.
Well-characterized biomarkers for the assessment of breast cancer exist. The hormonal receptor status [estrogen (ER) and progesterone (PR) receptors] is used for patients candidate for hormonal therapy 11, the Her-2/neu status is important for herceptin therapy 12, and TOP2A is a therapeutic target for anthracycline treatment 13.
The aim of this study was to evaluate patients with familial breast cancer (FBC) and compare them with sporadic breast cancer (SBC) and also to evaluate the combined study on the hormonal status, immunostaining for Her-2/neu, and Her-2/neu and TOP2A copy number alterations by fluorescence in-situ hybridization (FISH) in the management of breast cancer.
Patients and methods
A total of 22 patients (18 female and four male) with duct carcinoma of the breast were selected retrospectively by retrieval of formalin-fixed paraffin-embedded tissue sections from archival blocks of breast invasive duct carcinoma collected from the Department of Pathology, National Cancer Institute, Cairo University.
Clinical status variables included the age at diagnosis, the menopausal status in case of women, and the presence of family history of breast cancer, according to the criteria of Lynch et al. 14, which included the following:
Having three or more first-degree relatives who had been affected by breast cancer.
Having two first-degree relatives who had been affected by breast cancer and at least one of them was younger than 40 years and/or had had been affected by bilateral breast cancers.
Patients who fulfilled the above-mentioned criteria were included into the first group. However, in the absence of the above criteria, the patients were considered as having sporadic tumors and were included into the second group.
Histological diagnosis was based on the recent WHO classification 15. Pathological variables such as tumor size, tumor type, multifocality, and lymph node status were evaluated. Histological grading was evaluated according to the Nottingham combined histological grade (Elston–Ellis modification of the Scarff–Bloom–Richardson grading system) 16.
Hematoxylin and eosin-stained slides were prepared for each patient from each paraffin block for proper evaluation of tumor type and grade. Tumor size and lymph node status were categorized according to the guidelines of the American Joint Committee on Cancer (AJCC) 17.
Three positively charged slides were prepared from representative tumor blocks of each specimen and stained with primary monoclonal antibodies against estrogen receptors (mouse monoclonal, clone 1D5, ready to use; Dako, Agilent Technologies Company, Denmark), PRs (mouse monoclonal, clone PgR 636, ready to use; Dako), and Her-2/neu (rabbit polyclonal, dilution 1 : 250; Dako) using an avidin–biotin-based detection method.
The hormonal receptor status, ER and PR status, were evaluated according to the Allred scoring system 18. The monoclonal antibodies used for immunocharacterization of breast carcinoma are listed in Table 1.
Her-2/neu immunostaining results were estimated according to the Her-2/neu scoring system (Hercep test) 19, including the following scores: 0 (no detectable staining); 1+ (<10% positive cells with partial membrane staining); 2+ (>10% positive cells with concentric and weak staining); and 3+ (>10% positive cells with concentric and intense membrane staining).
Evaluation of fluorescence in-situ hybridization
The formalin-embedded paraffin blocks were subjected to FISH using positively charged slides – two slides for every patient. Deparaffinization was carried out using xylene. Pretreatment of the slides was carried out using a Vysis Paraffin Pretreatment Reagent Kit (Abbot laboratories, Illinois, USA). Locus-specific probes for Her-2/neu (spectrum red) with a control probe for the centromere of chromosome 17 (spectrum green) were added to one slide (LSI Her-2/CEP 17 dual-color probe; Q-biogene, MP Biomedicals, Irvine, California, USA); in addition, probes for TOP2A genes (spectrum red) with a control probe for the centromere of chromosome 17 (spectrum green) (LSI TOP2A/CEP 17 dual-color probe; Vysis) were added to the second slide, according to the method described by Pinkel et al. 20 and according to manufacturer’s instructions. The sections were examined at a ×100 magnification and photographed using an Olympus BX51 (Olympus, Tokyo, Japan) fluorescent microscope. The analysis was carried out using the Cytovision Software (Applied Imaging, Newcastle Upon Tyne, UK). Signals were counted in 60 cell nuclei (from two different areas of the histological section) limited to regions of invasive duct carcinoma cells. The results were expressed as a ratio of the number of signals for Her-2/neu and TOP2A (red) to those for the centromere of chromosome 17 (green), with a ratio of 2 or more being considered as an amplification of both genes, a ratio of less than 1.8 considered as a deletion of the Her-2/neu gene, and a ratio of less than 0.8 considered as a deletion of the TOP2A gene 21,22.
Data were analyzed using the SPSS win statistical package version 17 (SPSS Inc., Chicago, Illinois, USA). The χ2-test was used to examine the relation between qualitative variables, whereas the Mann–Whitney U-test was used for quantitative variables. The agreement between the markers was calculated using the κ-test. A P-value of less than 0.05 was required to reject the null hypothesis (a statistically significant level).
Twenty-two patients of breast duct carcinoma were recruited from the Department of Pathology, National Cancer Institute, Cairo University; their ages ranged from 24 to 81 years (18 females and four males with a ratio of 4.5 : 1). The patients were classified into two groups: group Ι (12 patients) comprised patients with FBC (54.5%) and group ΙΙ (10 patients) comprised patients with no family history of the disease (SBC; 45.5%). Comparisons between FBC patients and SBC patients as regards clinical and pathological parameters are shown in Table 2.
The age distribution of patients with SBC and those with FBC did not show any difference (P=0.539). There was a marked difference between the two groups with regard to the menopausal status, which was statistically significant: 75% of women with FBC were premenopausal in contrast to 25% of those with SBC (P=0.01). As regards the tumor size, a higher frequency of large-sized tumors was recorded among FBC patients (50% of tumors were in the T4 category) compared with SBC patients (10%), with a high statistical difference (P=0.019).
The histological grade was significantly higher among FBC patients compared with SBC patients (indicating a more aggressive phenotype in FBC; P=0.005), whereas lymph node involvement was higher among FBC patients (83.4%) compared with SBC patients (60%) but did not reach statistical significance (P=0.494).
ER staining evaluation showed a marked difference between the two groups: 40% of SBC patients were positive compared with 16.7% of FBC patients (P=0.001). As regards PR staining, 50% of SBC patients were positive compared with 25% of FBC patients (P=0.02). Carcinomas with a strongly positive or moderately positive reaction for ER are shown in Figs 1 and 2, respectively. Figure 3 shows a carcinoma with a weak positive (PR) reaction.
The incidence of Her-2/neu protein expression in FBC patients (33.3%) compared with (20%) that in SBC patients showed no statistical difference (P=1.00). The results are shown in Table 3. A carcinoma with a strong positive staining reaction for Her-2/neu gene expression (score 3+) is shown in Fig. 4, whereas a carcinoma negative for the staining reaction (score 0) is shown in Fig. 5.
Fluorescence in-situ hybridization results
The incidence of Her-2/neu gene amplification in FBC patients (33.3%) and SBC patients (30%) was equal (P=1.00). In contrast, the frequency of TOP2A gene amplification was slightly higher among FBC patients (25%) than in SBC patients (20%) but did not reach a statistically significant level (P=1.00), as shown in Table 4.
Coamplification of Her-2/neu and TOP2A genes was observed in one FBC patient (8.3%) and in two SBC patients (20%). In contrast, Her-2/neu amplification alone was observed in three FBC patients (25%) and in one SBC patient (10%). TOP2A gene amplification with normal Her-2/neu gene expression was observed in two FBC patients (16.7%) but was not observed among SBC patients (P=0.274, κ=0.320). The results are shown in Table 5.
Carcinomas with normal Her-2/neu gene copy numbers or gene amplification are shown in Figs 6 and 7, respectively. Carcinomas with normal TOP2A gene copy numbers or gene amplification are shown in Figs 8 and 9, respectively.
All samples from patients (13) that showed negative staining for Her-2/neu by immunohistochemical (IHC) (score 0 or 1+) also showed an absence of gene amplification by FISH, and all samples from patients (6) that showed positive staining by IHC (score 3+) also showed gene amplification by FISH. However, among the three patients with IHC-equivocal/inconclusive (score 2+) tumors, one patient (33.3%) was found to be positive for gene amplification by FISH.
Twenty-two patients (18 female and four male) with duct carcinoma of the breast were selected from the Department of Pathology, National Cancer Institute, Cairo University. The diagnosis was based on the recent WHO classification. Some of the well-characterized biomarkers used for the assessment of breast cancer were used: the hormonal receptor status (ER and PR status), which is used for patients candidate for hormonal therapy; the Her-2/neu status, which is important for herceptin therapy; and TOP2A, which is a therapeutic target for anthracycline treatment.
The mean age of SBC patients was 52.5 years, which is comparable to that in other reports 23,24; however, this figure is slightly lower than that report in other studies from Western countries 25,26. The mean age of FBC patients was 49.5 years, which is slightly lower than that in other reports 27,28; this could be explained by considering breast cancer in Egyptian patients (both SBC and FBC) as a biologically more aggressive disease compared with what has been encountered in western studies 24. The frequency of premenopausal women was higher among FBC patients than in SBC patients, whereas the frequency of postmenopausal women was higher among SBC patients than in FBC patients. These results are similar to those reported in other studies 27–29 but are in contrast to those reported by D’Eredita et al. 28, who reported no difference between FBC and SBC patients.
There was a statistically significant difference as regards tumor size between the two groups, with a larger tumor size observed among FBC patients, which is in accordance with the results of some studies 28,29 but is in contrast with those of others 27–30 who found no significant difference between the two groups. A highly significant difference was observed between the two groups as regards tumor grade, with a higher grade among FBC patients, which confirms the presence of more aggressive tumors among patients with a genetic predisposition. These results are in accordance with those of some studies 29,30 but are in contrast with those of another study 28 in which no significant difference between the two groups was found. There was no significant difference between FBC and SBC patients as regards the lymph node status, which is in agreement with the results of other studies 28–30 but is in contrast with the study by Veronesi et al. 29, who reported that FBC patients tended to have positive lymph node affection.
In the present study, we compared the results of ER, PR, and Her-2/neu statuses as important biological IHC markers in both FBC and SBC groups.
There was a highly significant difference among FBC and SBC patients, with a lower ER expression in the FBC group (P=0.001). Other studies have also shown low levels of ER expression in FBC patients 29–31. It is known that ER expression is inversely correlated with tumor grade 32: FBC patients who showed a higher tumor grade than SBC patients would therefore be predicted to be more ER negative. There was a significant difference between FBC and SBC patients (P=0.02) as regards the expression of PR, with a lower expression among FBC patients compared with SBC patients (25 vs. 50%); these results are in accordance with those of other studies 29,30–33. As regards Her-2/neu protein expression, the incidence of its expression in FBC patients (33.3%) compared with SBC patients (30%) showed no statistical difference (P=1.00), which is in agreement with the results of other studies 31–33.
Her-2/neu gene amplification by FISH was observed in 31.8% of patients, which is similar to the results of other studies 34,35. The Her-2/neu gene was equally amplified in both FBC and SBC patients (33.3 vs. 30%; P=1.00), which is in accordance with the results of other studies 30–37. In contrast, another study 21 recorded infrequent Her-2/neu gene amplification among FBC patients, whereas other studies 27,28 reported more frequent Her-2/neu gene amplification among FBC patients compared with SBC patients.
Amplification of the TOP2A gene was detected by FISH in 22.7% of patients, whereas 77.3% of patients showed a normal gene copy number. The amplification was more frequent among FBC patients (25%) compared with SBC patients (20%). The frequency of amplification of TOP2A among SBC patients is in accordance with what has been observed in other studies 38,39. However, in another study 21, the frequency of TOP2A gene amplification in FBC patients was 15%, which is lower than that observed in our results.
The TOP2A gene was amplified in 42.9% of Her-2/neu-amplified tumors and in 13.3% of nonamplified tumors. Therefore, a fair concordance between Her-2/neu and TOP2A amplification was observed (P=0.274, κ=0.320), which is in agreement with the results of other studies 40,41.
Some studies 42,43 suggest that Her-2/neu and TOP2A were coamplified as a result of amplification of a large segment of chromosome 17 – the 17q12–21 amplicon – as Her-2/neu gene amplification is the initial genetic aberration that takes place, followed by additional chromosomal rearrangement, which could result in TOP2A gene amplification or deletion. Other recent studies 44–46 report a difference in the copy number of Her-2/neu and TOP2A in coamplified tumor samples and concluded that the chromosome region 17q11.2–22 contained at least two distinct amplicons, which explains the difference in gene copy numbers and why Her-2/neu and TOP2A are not amplified together in some patients. In our study, coamplification of Her-2/neu and TOP2A genes was observed in one FBC patient (8.3%) and in two SBC patients (20%). In contrast, Her-2/neu gene amplification alone was observed in three (25%) patients of the FBC group and in one (10%) patient of the SBC group. TOP2A gene amplification with a normal Her-2/neu gene status was observed in two FBC patients (16.7%) but was not observed in any SBC patient (0%). This result was supported by other studies 21–47, in which it was reported that TOP2A gene amplification without amplification of the Her-2/neu gene was a rare event in SBC patients.
Her-2/neu-amplified tumors are treated with anthracyclines at higher doses 48. Other studies 38–49 have suggested the association between Her-2/neu amplification and anthracycline sensitivity to be due to coamplification of TOP2A. In our study, we demonstrated that some FBC patients may harbor TOP2A gene amplification without Her-2/neu gene amplification, and this is why TOP2A measurement should be included into the routine study, especially for FBC patients.
All patients (13) who showed negative staining for Her-2/neu by IHC (score 0 or 1+) showed an absence of gene amplification by FISH, whereas all patients (6) with positive staining by IHC (score 3+) showed gene amplification by FISH. There was a concordance among the results of all patients. However, among the three patients with IHC-equivocal/inconclusive (score 2+) tumors, one patient (33.3%) was found to be positive for gene amplification by FISH. FISH has been shown to be a superior technique to IHC, as DNA is a more stable target than the Her-2/neu protein and is less susceptible to variation caused by tissue handling. The Her-2/neu gene status is important in selecting candidates for herceptin therapy 12, whereas identification of the TOP2A gene status in patients is important for anthracycline treatment selection 13–51. Inactivation of a tumor suppressor gene called Nischarin is among the recently identified mechanisms by which the Her-2/neu gene promotes breast cancer progression and spread; this finding provides a new therapeutic target to block this function of Her-2/neu 52.
Finally, we conclude that FBC tumors seem to be more aggressive compared with SBC tumors. Our study suggests that TOP2A gene amplification seems to occur in both FBCs and SBCs, however with a higher frequency in FBCs, together with the finding that TOP2A gene amplification may occur independent of Her-2/neu amplification. Thus, we suggest this parameter be determined on a routine basis in patients with breast cancer in general, and especially when FBC is suspected. Moreover, a combined approach using IHC and FISH can optimize Her-2/neu testing for breast carcinoma patients, especially in patients with IHC-equivocal tumors.
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Conflicts of interest
There are no conflicts of interest.
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