Turkish Journal of Pathology

Türk Patoloji Dergisi

Turkish Journal of Pathology

Turkish Journal of Pathology

2011, Vol 27, Num, 3     (Pages: 196-203)

Correlation of HER 2/neu Gene Amplification with Immunohistochemistry and Other Prognostic Factors in Breast Carcinoma

Saime SEZGİN RAMADAN 1, Özlem YAPICIER 1, Selçuk KİHTİR 2, Ayhan ERDEMİR 2, Tuğser Hakan DOĞAN 3, İsmail Necdet Hakkı ÜSKENT 4, Haluk ONAT 4, Metin ÇAKMAKÇI 2

1 Department of Pathology, Anadolu Medical Center, KOCAELİ, TURKEY
2 Department of General Surgery, Anadolu Medical Center, KOCAELİ, TURKEY
3 Department of Radiology, Anadolu Medical Center, KOCAELİ, TURKEY
4 Department of Medical Oncology, Anadolu Medical Center, KOCAELİ, TURKEY

DOI: 10.5146/tjpath.2011.01075
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The purpose of this study was to determine relationship between HER-2/neu status and estrogen receptor, progesterone receptor, grade and age by comparing fluorescence in situ hybridization and immunohistochemistry. One hundred invasive breast carcinomas were reviewed and fluorescence in situ hybridization analysis was performed in all cases. Immunohistochemical scores showed a perfect concordance with fluorescence in situ hybridization amplification ratios (p<0.0001). The results indicated a significant correlation between HER-2/neu and grade, but an inverse relationship between HER-2/neu and hormone receptors. In women aged ≤45 years, an inverse relationship between HER-2/neu and progesterone receptor was found and no association was noted between HER-2/neu and other factors. In women aged >45 years, the results indicated a significant correlation between HER-2/neu and grade, and there was an inverse relationship between HER-2/neu and hormone receptors.


Breast cancer is the most common life-threatening malignant neoplasm in women. Clinical outcome is affected by a number of established prognostic factors, including age, tumour grade, estrogen receptor (ER) and progesterone receptor (PR) status[1-4]. One of the most common genetic alterations associated with human breast cancer is the HER-2/neu amplification. Molecular alterations in breast cancer are being incorporated into the development of new treatment strategies.

The HER-2/neu (C-erbB-2) gene is localised to chromosome 17q and encodes a transmembrane tyrosine kinase receptor protein that is a member of the epidermal growth factor receptor family. Slamon et. al. were the first authors to find a strong and highly statistically significant correlation between the degree of gene amplification and both time to disease relapse and survival[5]. Previous studies have shown the prognostic and predictive value of HER-2/neu overexpression in node positive breast cancer[6,7,8] and for metastatic disease[9,10].

Trastuzumab is a humanized antibody against the external domain of HER-2/ neu that presents antitumoral activity[11]. Trastuzumab improves response rates, time to progression and even survival when used alone or added to chemotherapy in metastatic breast cancer[12]. It is also active as a single agent and reduces the risk of recurrence and mortality in patients with early-stage breast cancer[13-15]. The interesting point of such a specific treatment is that only those tumors that overexpress HER-2/neu benefit from such therapy. The determination of HER-2/ neu in breast cancer is very important as it has significant benefits and some side effects like cardiotoxicity[16]. Overexpression of HER-2/neu is most commonly caused by amplification of the HER-2/neu gene, which results in increased HER-2/neu mRNA levels and concomitant overexpression of the HER-2/neu receptor on the tumor cell surface. There is no “gold standard” for HER-2/neu testing but immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the most commonly used methods. In addition, chromogenic in situ hybridization and silver in situ hybridization has recently been validated as alternatives to FISH.

Early studies suggested that as many as 30% of breast cancers have HER-2/neu overexpression[17]. However, recent studies showed lower results around 20%[18-20]. The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) convened an expert panel and recommended a new HER-2/neu testing guideline in 2007 to decrease discordant results[12].

HER-2/neu overexpression has been correlated with several poor prognostic paramaters. They are more likely to be hormone-receptor negative and have higher tumour grade[3,7,21]. The inverse association between HER-2/neu and hormone receptors has been linked with the fact that estrogens suppress HER-2/neu through the ER[22]. One recent report showed that this inverse association differs in different age groups[23]. Tumor grade, as an important predictor for HER-2/neu overexpression shows an association with hormone receptor expression that differs between young and older women and this may be reflected with an age-related association between hormone receptors and HER-2/neu[24-25].

Taken together, the aim of our study was to better understand the relationship between HER-2/neu status and other prognostic factors like age, tumour grade, ER and PR receptor status by comparing the results of immunohistochemistry and FISH in invasive female breast carcinomas at our institution.


Tissue Samples
The study material comprised 100 cases of invasive mammary carcinoma diagnosed at or referred to Anadolu Health Center, Kocaeli, between February 2005 and June 2008. The cases were included in the study only if paraffin blocks were available for FISH. Some of the cases which were sent to our Department for second look had only H&E slides and were excluded from the study. FISH was applied to all cases with IHC scores 0 to 3+. Tumours were graded according to the Bloom Richardson grading system. Among the 100 cases were 86 ductal carcinomas, 3 tubular carcinomas, 3 mucinous, 3 mixed, 2 medullary, 1 papillary, 1 lobular and 1 basal-like carcinoma. Only medullary carcinomas were not graded according to the American Joint Committee on Cancer 2002[26].

From all cases a set of 4 micrometer-thick paraffin sections were cut and stained for ER (Novocastra-Clone 6 F 11), PR (Neomarkers- Clone SP 2) and HER-2/neu (Novocastra- Clone 10 A 7) antibodies by using microwave for antigen retrieval. The staining for ER and PR was classified as positive if more than 10% of the tumor cells exhibited nuclear staining. The HER-2/neu stained slides were scored on a scale of 0 to 3+ according to the new ASCO/CAP guideline recommendations. Scoring was done on a 0-3 scale. Strongly positive (3+) was defined as strong complete membranous staining in more than 30% of the tumor cell population. Weakly positive (2+) was defined as moderate membranous staining in more than 10% of tumor cells and strong complete membranous staining in less than 30% of tumor cells. 1+ was defined as either weak or barely perceptible membranous staining in more than 10% of the tumor cells. 0 was completely negative staining. Scores of 0 and 1+ were considered as negative for HER2/neu expression, 3+ as immunopositive, while 2+ was weakly or borderline positive.

FISH analysis was performed to all cases. Amplification of HER-2/neu was evaluated using the Path-Vysion DNA Probe Kit (nodul 35-161060; Vysis) which uses a dual-color probe for determining the number of copies of both HER-2/ neu (orange) and the chromosome 17 centromeres (green). The kit was used following the manufacturers instructions with a few minor modifications. Slides containing 4 micrometerthick paraffin–embedded tissue sections were placed on a slide warmer overnight at 62°C followed by deparaffinization in Hemo-De, dehydration in 100% ethanol, and drying on a slide warmer at 45 to 50°C. Slides were then pretreated with 0, 2N hydrochloric acid for 22 minutes, followed by washes in purified water and immersion in Vysis wash buffer. They were subsequently immersed in Vysis protease solution at 37°C for 40 minutes, washed in Vysis wash buffer, and dried on the slide warmer and 10 ml of probe was applied. Slides are then put in Hybride for 5 minutes at 72°C. They were then coverslipped, sealed with rubber conent, and placed in a prewarmed humid incubation chamber at 37°C for 18 to 22 hours. This was followed by immersion in prewarmed postwash solution at 73°C for 4 minutes. The slides were air–dried, and a 4;6 – diamidino -2- phenylindole (DAPI) counterstain was applied.

The scoring system used is described in detail in the manufacturers instructions. In brief, a minimum of 60 nuclei were scored by each of 2 observers using on Olympus BX41 fluorescent microscope with a Chroma filter set (DAPI / spectrum orange / spectrum green triple bandpass) Areas scored were limited to regions of invasive disease as compared with a companion hematoxylin and eosinstained section. The ratio of HER-2/ neu signals (orange) to chromosome 17 centromere signals (green) was determined.

FISH results were than compared to previously stained and evaluated IHC scores to determine how well protein overexpression correlates with gene amplification.

Statistical Analysis
All statistical analyses were performed with the Statistical Package for the NCSS 2007 version for Windows. The chi-square test and odds ratio were used to examine categorical variables and the association between HER-2/ neu overexpression and other clinicopathological factors in univariate analysis. Logistic regression analysis was used to evaluate HER-2/neu FISH scores. All statistical tests were two–sided and p<0.05 was considered significant.


The age, nuclear and histologic grade, immunostaining results for ER, PR and HER-2/neu, and FISH results of 100 invasive carcinomas are shown in Table I. Comparison of HER-2/neu immunostaining scores with FISH amplification is shown in Table II. Immunohistochemistry scores showed a good correlation with FISH amplification (p<0.0001). None of the cases with immune scores at 0 or 1 showed amplification. Four cases (25%) within 16 cases of score 2+ (Figure 1) showed amplification, 13 cases (86.7%) with score 3+ (Figure 2) were amplified. The association between HER-2/neu amplification and different clinicopathological factors in all patients are presented in Table III. The results indicated a significant correlation between HER-2/neu and grade, but inverse relationship between HER-2/neu and hormone receptors. There is no significant difference in HER-2/neu amplification between women age ≤45 and >45 years. The results from the univariate analysis for the association between HER-2/neu amplification and different clinicopathological factors in women aged ≤45 years and >45 years are given in Table IV and V, respectively.

Table I: Clinicopathological features of 100 invasive mammary carcinomas

Table II: Comparison of HER-2/neu immune scores with FISH amplification (p<0.0001)

Figure 1: Score 3+ staining (HER-2/neu, x400).

Figure 2: Score 3+ staining (HER-2/neu, x100).

Table III: The association between HER-2/neu amplification and clinicopathological features in all age groups

Table IV: Univariate analysis of clinicopathological factors predicting HER- 2/neu amplification in women aged ≤45

Table V: Univariate analysis of clinicopathologic factors predicting HER- 2/neu amplification in women aged >45

In women aged ≤45 years, we found an inverse relationship between HER-2/neu overexpression and progesterone receptor, but we found no association within other factors. However, in women aged >45 years, results revealed that ER status, PR status and tumor grade were significantly associated within HER-2/neu amplification.


Since the first report by Slamon et al[5] showing that HER- 2/neu amplification in breast carcinoma correlates with poor prognosis, accurate detection of the HER-2/neu gene alteration has become increasingly important. Moreover the selection of patients for trastuzumab therapy relies on the presence of this alteration. Given trastuzumab’s toxicity[27] accurate determination of HER-2/neu status is crucial.

IHC and FISH are the two FDA-approved methodologies. IHC is by far the most popular and accessible testing modality. It directly detects HER-2/neu protein overexpression and provides very accurate results with advantages including relative ease of performance, rapid turn-around time, and relatively low cost. FISH methodology is quantitatively more precise but also time consuming, technically demanding and more expensive.

FISH methodology theoretically may not be completely concordant with IHC results as it measures HER-2/neu gene amplification and not protein overexpression. HER- 2/neu gene amplification occurs in about 15-25% of breast cancers, where as the interstudy range at HER-2/neu protein overexpression is higher (10-50%)[2831]. The range of HER-2/neu protein overexpression falls to 15-20% when gene amplification is also present.

In our study, we found good correlation between the IHC and FISH methods (p= 0.0001). None of the cases with score 0 or 1 showed amplification. Twenty five percent of the cases with immune score 2+ showed amplification. Eighty six percent of the cases with immune score 3+ were amplified but 2 cases with immune score 3+ did not show amplification. In the literature, concordance rates between IHC and FISH range from 79% to 100% for 3+ cases[32,33] and between 12% and 36% for 2+ cases[34,35]. Although there is good correlation between HER-2/neu gene amplification and protein overexpression, approximately 5-10%[36] of breast carcinomas overexpress HER-2/neu without amplification and a small undetermined percentage amplify HER-2/neu without overexpression[37]. In the study of Rossi et al, the percentage of cases with HER-2/ neu protein overexpression (3+) and FISH negativity rose to 22%[38]. In the literature it is proposed that these 3+ cases could be due to single copy overexpression of the HER-2/neu gene[39]. These cases are considered clinically similar to the immunohistochemically HER-2/neu negative patients[40].

HER-2/neu amplification in different histological types has been a subject of interest. While some prior studies have reported the absence of a significant association between tumor type and HER-2/neu status[40], other studies have shown that HER-2/neu amplification or overexpression was significantly more likely in infiltrating ductal carcinomas than infiltrating lobular carcinomas. But Hoff et.al.[41] demonstrated that infiltrating ductal carcinomas were significantly more likely to show HER-2/neu amplification than infiltrating lobular carcinomas. In this study, 15 of the 17 amplified cases were of the infiltrating ductal carcinoma type and 2 cases were medullary carcinomas. None of the other types was amplified.

Previous studies have shown a strong correlation between HER-2/neu protein overexpression/amplification and steroid receptor negativity and high tumour grade[1,4,25,27,42]. We also found similar results through all age groups. The relationship between the age of patients and HER-2/neu status and other prognostic factors is another subject of interest. We did not find a significant relationship between HER-2/neu gene amplification and patient age as previous studies[1]. Although Huang et.al. also did not find a statistically significant difference in expression of HER-2/ neu by age ≤45 and > 45 years, they suggested an age related association between ER, PR, tumour grade and HER-2/neu overexpression[22]. Their results suggested that the inverse association between the hormone receptor and HER-2/ neu status only appeared in women aged beyond 45 but not under 45. In this study, we found inverse relationship between PR and HER-2/neu amplification in all age groups. However, we found a relationship between HER-2/neu status with ER negativity and high tumor grade only in women aged beyond 45. Konecny et.al. also found that the relatively low levels of HER-2/neu amplification/overexpression were associated with more marked decreases of PR than of ER[43], because PR expression is linked to a biologically active and functional ER[42-44]. It has been shown that the activation of growth factor receptors such as HER-2/neu can result in direct phosphorylation and activation of ER in an estrogen–independent manner, which may itself be an important mechanism for tamoxifen resistance in addition to the subsequent reduction in hormone receptor levels[45]. However, it appears likely that this reduced expression is not the only mechanism for endocrine resistance in patients with HER-2/neu and that steroid receptors are most likely determined by multiple complex mechanisms, such as phosphorylation of the ER via HER-2/neu activation[46], overexpression of the steroid receptor cofactorA1B1 in HER-2/neu positive tumors[47] and competition between ER and other coregulatory receptor proteins resulting in altered HER-2/neu expression[48].

In our institution, we perform immunohistochemistry for detecting HER-2/neu and hormonal status. In this study, we compared our IHC scores with FISH method and found a significant correlation. In conclusion, we suggest performing FISH only in cases with an IHC score of 2+. Although we have a limited number of patients, we found that mainly PR negativity predicts HER-2/neu overexpression through all age groups. In women beyond the age of 45, there is also a strong correlation between HER-2/neu, ER negativity and tumor grade. A combination of HER-2/neu and histological prognostic factors can help to determine subgroups for more specific treatment protocols.


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Keywords : Breast, Carcinoma, HER-2/neu, Immunohistochemistry, Fluorescent in situ hybridization