Breast cancer is the most common cancer among women and is considered as the most important cause of death in women globally. 1 Breast cancer is a multifactorial disease and is under the influences of genes and environment. 1 One out of 10 cases of breast cancer is hereditary. 2 Nearly 2/3 of breast cancers are hormone-dependent. 3 Breast cancer treatment is based on a tumor's genetic and hormonal characteristics, grade, and stage. 4 One of the breast cancer's main genetic prognostic factors is human epidermal growth factor receptor 2 (HER2) gene status. 5 Other prognostic factors include the presence of estrogen and progesterone receptors and Ki-67 index. 6 The expression of estrogen and progesterone receptors are also important predictors of breast cancer prognosis. Estrogen receptors are present in 60 to 70% of breast cancer cases and progesterone receptors are present in 75% of breast cancer cases.

The presence of estrogen receptors is associated with a response to hormonal therapy with estrogen antagonists including tamoxifen. 5 The Ki-67 protein is considered as a cellular proliferation marker. It was previously shown that the Ki-67 concentration is associated with overall and disease-free survival. 7 HER2 gene amplification is seen in about 30% of breast cancer cases and is related to reduced life expectancy and earlier recurrence. 89 On the other hand, HER2 gene amplification is associated with good response to chemotherapy and improved overall survival with medications including Trastuzumab. 10 Therefore, detection of HER2 amplification is important in predicting the survival and quality of life of breast cancer patients. Studies have showed that the expression of hormone receptors has been associated with reduced HER2 gene amplification. 11 Based on the latest guideline by the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP), HER2 status assessment should be performed in all newly diagnosed patients with breast cancer and patients with metastatic breast cancer. 12 The status of HER2 amplification is recommended to be assessed using immunohistochemistry (IHC) or in situ hybridization (ISH). 12 Based on the ASCO/CAP guideline, anti HER2 receptor antibody should be added to chemotherapy regimen in case of documented HER2 amplification (HER2 positive3+). 12 In case of ambiguous HER2 status, the ASCO/CAP recommends repeating HER2 status assessment on another tissue block or performing the assessment using another diagnostic method on the same tissue block. 12 There is a tendency towards performing IHC for detection of HER2 status in breast cancer due to its simplicity and lower cost. The ASCO/CAP has designed a scoring system for the assessment of HER2 status, where scores 0 and 1+ are considered HER2 negative, score 2+ as equivocal and 3+ as HER2 positive. 12 In equivocal cases, assessment using ISH is recommended. 12 It was previously shown that HER2 status was reported to be equivocal in 15 to 25% of breast cancer patients. 13,14 It was also shown that 20 to 30% of the patients with equivocal HER2 status based on IHC were HER2 positive in ISH investigation. 15,16 Regarding the high cost of performing both tests in a large number of breast cancer cases, it is logical to improve the accuracy of IHC assessments in clinics. The primary objective of this study was to identify the prevalence of HER2+ breast cancer cases using chromogenic in situ hybridization (CISH) among tissue samples with equivocal HER2 status in IHC in a referral cancer center from all over the country. This study also aimed to assess the relationship between HER2 amplification and expression of estrogen and progesterone receptors and Ki-67 Ag among cases with equivocal HER status based on IHC.

This cross-sectional study was conducted in the cancer institute of a tertiary hospital in Tehran, Iran. The hospital was chosen due to its high referral rate from all over the country; therefore, the cases represent various geographical regions within the country, and therefore the results might be generalizable to the whole country. The sampling method was non-random stratified probability sampling. The data for the study were collected confidentially from patient records. All breast cancer cases that referred to the hospital from 2015 to 2017 were assessed. The inclusion criteria were presence of breast cancer with equivocal HER2 status based on IHC assessment. Exclusion criteria were lack of data on the IHC assessment of HER2 status, being HER2 positive or HER2 negative based on IHC according to the ASOC/CAP guidelines 13.

Sample size was calculated based on the prevalence of HER2 positive cases based on ISH assessment (26.5%). Considering the minimum significant difference of 0.05, 5% precision and 80% power, the following equation was used.

í µí±‘ 2 Where n is the sample size, d is the minimum significant difference, p is the proportion of HER2 and q is calculated as 1-p. Z is a constant value (1.96 for 80% power). The sample size was calculated to be 280.

The extracted data included the results of HER2 CISH, estrogen (ER) and progesterone (PR) receptors as well as Ki-67 in tissue samples (Figure 1-3).

Data analysis was performed using the statistical package for social sciences (SPSS) software. HER2, ER and PR positivity were presented using frequency and percentage. The percentage of Ki-67 was normally distributed based on Kolmogorov-Smirnov test and was, therefore, presented using mean and standard deviation. The chi-square test was used to assess the relationship between HER2 status based on CISH and ER and PR, while the analysis of variance (ANOVA) was used to assess the relationship between HER2 status and Ki-67. The level of statistical significance was set at P<0.05.

A total of 280 breast cancer cases with equivocal HER2 status based on IHC were assessed in the study. HER2 amplification was evaluated in all samples using the CISH. From 280 cases, 215 (76.8%) were HER2 negative (not amplified) and 60 (21.4%) were HER2 positive (amplified), and 5 (1.8%) were indeterminate. The reason for the indeterminate findings included fixation problems, surgical electrocautery effect, increased cold ischemic time, and exposure of the tissue to acidic materials,high pepsin digestion as well as inappropriate primary tissue processing. The mean and standard deviation for Ki-67 percentage was 26.73 ± 18.56% ranging from 1% to 90% (Figure 4). The results of ER, and PR assessments are presented in Table 1. Among the studied samples, 59 (21.0%) were classified as luminal A, while luminal B, HER2 enriched and triple negative classes were observed in 184 (65.7%), 10 (3.5%) and 27 (9.6%), respectively.

The chi-square test revealed no significant relationship between ER and HER2 status (p=0.373), while a significant relationship was observed between PR positivity and HER2 status (P=0.007) ( Table 2). The chi-square test revealed no significant relationship between HER2 and hormonal receptor status defined as hormone receptor positive (i.e., ER+ and / or PR+) and hormone receptor negative (i.e., ER and PR negative) (P=0.752).

Currently, IHC is one of the most commonly used methods in the assessment of HER2 status in breast cancer cases. 14 The IHC is usually used as the primary method in breast cancer cases. 15 The fluorescent in situ hybridization (FISH) method is considered as the gold standard in detection of HER2 status in breast cancer cases. 16 The CISH procedure has lower cost compared to FISH method. 17 The CISH test has been considered valid and completely concordant in relation to FISH method in the detection of HER2 status in breast cancer in many studies. 1819 Unlike IHC, CISH method is costly and requires specific knowledge and equipment. 17 The CISH method is based on chromogenic detection and has acceptable sensitivity and specificity for the detection of difficult cases. 17 The aim of this study was to count and assess HER2 amplification using CISH method among breast cancer cases with equivocal IHC findings.

The findings of our study revealed that among the 280 equivocal HER2 cases based on IHC method, 21.4% were positive (amplified) based on the CISH method, 76.8% were negative (not amplified) and 1.8% were indeterminate. This finding was in line with the findings of a study by Mohammed 20, that reported 33.3% of HER2 indeterminate breast cancer cases based on IHC were HER2+ while only 0.32% were indeterminate. 20 In a study by 21, 60% of indeterminate HER2 cases based on IHC were HER2-, 30% were HER2+ and 10% were indeterminate in CISH assessment. 21 Meijer et al. (2011) reported 73.6% of indeterminate HER2 cases based on IHC were HER2-in CISH, while 21.3% were HER2+ and 4.9% were indeterminate. 16 Similar results were found in other studies. 222324 These findings indicate that the use of CISH method is useful in indeterminate HER2 status breast cancer cases based on IHC.

The findings of our study revealed that 86.8% and 75.0% of the breast cancer cases were ER+ and PR+, respectively. Previous studies assessed the relationship between HER2 status and ER and PR expression and reported a negative relationship between HER2 positivity and ER and PR expression. 2526[31] Although our study failed to report a significant relationship between HER2 status and ER positivity, a significant negative relationship was observed between HER2 status and PR positivity in our study. The negative relationship between HER2 status and steroid hormone receptors might justify the observation that HER2+ tumors do not respond to hormone receptor specific therapies. 32 On the other hand, the amplification of HER2 in patients with ER+ breast tumors was found to be associated with reduced PR expression. 27 It was previously shown that ER+/PR-breast cancers were mainly found among menopausal women. 28 This finding might be due to the fact that the endogenous estrogen is so low that it may not increase PR and suppress HER2 at menopause. 28 The findings of our study revealed that the mean percentage for Ki-67 protein was 26.73 ± 18.56%, which was lower than what was reported in previous studies. 29303132 Furthermore, our study failed to identify a significant relationship between HER2 status and Ki-67 among HER2 indeterminate breast cancer cases. In contrast a significant relationship was observed between HER2 status and Ki-67 in a previous study. 29 The difference between the findings of our study and the mentioned study might be due to different methodologiesas the later study was conducted on breast cancer cases while our study was conducted on breast cancer cases with indeterminate HER2 based on IHC.

On the other hand, the findings of our study revealed that luminal B (65.7%) was the most common tumor type followed by luminal A (21.0%), triple negative (9.5%) and HER2 enriched (3.5%). This finding was in line with the findings of the studies that followed more recent classification guidelines indicating that luminal B was the most common type with the prevalence of (57.1-68.5%). 3334 A strength of our study was the inclusion of breast cancer cases from different regions and cities of the country, which enables us to generalize the findings to the whole Iranian population. On the other hand, one of the limitations of this study was the exclusion of HER2+ and HER2-cases based on IHC in relationship analysis. As the relationships between HER2 status and ER, PR and Ki-67 were our secondary objective, sampling was based only on HER2 indeterminate cases based on IHC assessment. It is recommended for further researchers to assess the relationship between HER2 status and hormonal receptors and Ki-67 in a population with all types of HER2 status.

This study found that CISH can be of importance in determining the HER2 status of breast cancer tumors that are HER2 equivocal based on IHC assessment. This study also found a significant negative relationship between HER2 status based on CISH and PR among indeterminate HER2 status breast cancer cases based on IHC.

This study was approved by the ethics committee at Cancer Institute, IKHC, TUMS, Tehran.