It has long been recognized that some human breast cancers are hormone dependent in that their growth depends on a continued supply of female sex hormones. ¹ Estrogen is important in regulating the differentiation and proliferation of breast epithelial cells and interacts with estrogen receptor in the nucleus. Prolonged exposure has been shown to be an important risk factor for cancer. PR expression in normal breast epithelium is regulated by ER. Presence of ER and PR in invasive carcinoma correlates positively with survival and is an important prognostic factor^{2, 3}.The determination of ER and PR levels in breast cancer has become part of the standard work-up by the pathologist.

There are mainly 2 types of hormone assays: the biochemical assay and the immunohistochemical assay. Immunohistochemical assay – Monoclonal antibodies are used to localize the presence of steroid hormone receptors on frozen and paraffin sections. Both methods have been shown to have high concordance rates,^{4, 5} although some studies indicate that the immunohistochemical assay is more predictive for prognosis than the biochemical assay⁵.

Some workers feel that the ER is best used in combination with other tumor characteristics such as nuclear grade, in which case the predictive power together is greater than that of either alone⁶. ^{King, Fischer and Lesser et al have shown that ER and PR positivity correlates significantly with low grade tumors 7, 8, 9.}

Parl et al suggest that ER and PR status is a better prognostic indicator when used along with tumor grading¹⁰. In concordance with their findings, Berger et al¹¹ have found that prognosis associated with poorly differentiated ER negative tumors is worse than that of ER positive tumors of equal histopathologic grade.

Pertshuk et al¹² have shown ER positivity to correlate well with well differentiated, grade I tumors. No such association was studied for progesterone receptors. In addition, they suggest that PR status is a better indicator of survival and disease-free interval than ER status for women at the same stages of disease. Molino et al¹³ have found no correlation between receptor status and grade and suggest that the receptor status is indicative of prognosis only in patients with 4 or more positive nodes and in the stage T1 and premenopausal groups. Nixon et al¹⁴ have demonstrated a positive correlation between patients with higher grade tumors and ER negativity.

Initially considered to be genetic material from a retrovirus that results in malignant transformation when incorporated into normal cells, the majority of HER-2 oncogenes are now thought to be activated homologues of protooncogenes that have persisted in normal cell¹⁵. HER-2/neu, also known as c-erbB-2 is a member of the erbB oncogene family, and is related, but distinct from, epidermal growth factors¹⁶.

The expression of this protein has been associated with a poor histologic grade,^{15 ,17} the spread to axillary nodes, and the number of nodes involved;¹⁶ a negative association between c-erbB-2 expression and a ER and PR has been noted.¹⁸ In axillary node-positive patients, there is a significant correlation between amplification of HER-2 protein and shorter disease –free and overall survival.¹⁹ HER-2 oncogene amplification correlates with the absence of estrogen and progesterone receptors.¹⁸ Amplification of c-erbB-2 strongly correlates with each element (architectural pattern, nuclear atypia, and number of mitotic figures) of histologic grade.²⁰ Even if a primary tumor does not express c-erbB-2 protein, subsequent metastases may express the protein; in contrast, if a primary tumor expresses c-erbB-2 protein, this capacity is retained in all subsequent tumor metastases. Amplification of c-erbB-2 has been noted among in situ carcinomas as well. Numerous subsequent studies found that either HER2 gene amplification or protein expression predicted poor prognosis¹⁵. There is also some evidence of a relationship between HER2 and response of cancers to chemotherapy and endocrine therapies¹⁷.

Following the development of a humanized monoclonal antibody against HER2 (transtuzumab), the reasons for establishing the HER2 status of breast cancers changed, since it is a prerequisite for transtuzumab’s clinical use. Transtuzumab was originally licensed for the treatment for the treatment of patients with metastatic disease who had HER-2 positive cancers. More recently several prospective randomized trials have shown that adjuvant transtuzumab reduces the risk of recurrence and mortality in patients with HER2 positive early stage breast cancer^{17, 21}.

The principal testing methods used are immunohistochemistry and/or in situ hybridization using either fluorescence (FISH) or a chromagen. Immunohistochemistry-monoclonal antibodies/polyclonal anti-sera are used to analyze HER-2. In situ hybridization-fluorescently labeled probes complementary to centromeres of either HER2 gene or chromosome 17 are used.¹⁷

Expression of these immunohistochemical receptors is associated with a characteristic clinical outcome and in the present day, the breast cancers are classified and prognosticated by its molecular/immunohistochemical expression. The treatment regimen too is customized for the patient according to expression of molecular markers.

Hence the aim of the study is to compare between the initial reporting and reviewed reporting of estrogen receptor, progesterone receptor and Her2-neu receptor status

Subjects with invasive primary breast cancer were extracted from the medical records department, Kasturba hospital, Manipal using ICD-O-3 codes with the date of diagnosis between January 1, 2006 and December 31, 2007. The registry identified 217 breast cancer subjects of which 65 without complete ER/PR and Her2 data were excluded.

The following information regarding cancer characteristics (morphology, grade, size, ER/PR and Her2 expression), were obtained from case records. Information regarding ER/PR and Her2 expression of cases with available stored slides was reviewed and analyzed concurrently by 2 pathologists.

Four micrometer sections attached on poly-lysine coated and incubated overnight at 37̇̇̇·C slides were dewaxed in xylene, rehydrated in graded ethanol, distilled water and covered with 10mM citrate buffer (pH 6). They were then incubated for 30 min with primary monoclonal anti-bodies against Her2 (DAKO, clone 250, 1:100, antigen retrieval: 2 min pressure cooker), ER (DAKO, clone SP1, 1:50, antigen retrieval: 2 min pressure cooker) and PR (DAKO, clone PgR636, 1:50, antigen retrieval: 2 min pressure cooker), followed by incubation with biotin-labeled secondary antibodies. The streptavidin-peroxidase complex was visualized using di-aminobenzidine as a chromogenic substrate. For each run of staining, a positive control slide was prepared from breast carcinoma known to be positive for the proteins studied.

The best-preserved and best-stained areas of the sections were assessed. Nuclear staining was assessed for ER and PR. Quick score was determined by adding the scores for the proportion of cells stained and the intensity of staining, which ranged from 0 to 8.

Figure 1

Table 1: Quick score for ER/PR

For the purpose of this study, even a single cell with weak staining intensity was considered positive and no single cell with no staining was considered negative.

Figure 2

Fig 1: Hormone receptor - QUICK scoring

The best-preserved and best-stained areas of the sections were assessed. Only membrane staining was assessed for Her2-neu.

Figure 3

Table 2: Her2 protein assessment

For the current study, a Her2-neu result of 2+ is considered negative result unless verified by fluorescent in-situ hybridization (FISH).

Figure 4

Fig 2: HER2- neu receptor status

The retrieved slides were assessed for the ER, PR, Her2-neu status according to above mentioned criteria concurrently by 2 pathologists.

Statistics- determination of level of significance by Fischer test using SPSS 16.0 software.