Immunohistochemical profile of infiltrating ductal carcinoma breast-prognostic and therapeutic use.
Breast carcinoma is the most common malignant tumour and the leading cause of deaths due to carcinoma in women.  In India, breast cancer is the second most common cancer (after cervical cancer) & second most common cause of cancerrelated deaths among women.  There are so many types of breast carcinomas, but infiltrating ductal carcinoma is most common histological type of breast cancer.  The infiltrating ductal carcinoma term is used for all breast carcinomas that cannot be subclassified into one of the specialised types.
Carcinomas of "no special type" or "not otherwise specified"(NOS) are synonyms for ductal carcinomas. Traditional morphological prognostic factors include tumour size, tumour grade, axillary lymph node metastasis, etc. Now a days, more importance is given to biological molecular prognostic factors, because a significant number of patients with early stage breast cancer harbour microscopic metastasis at the time of diagnosis.
Prognostic and predictive factors are used in the management of breast cancer. Prognostic factors are those which evaluate patient's overall outcome such as chances of recurrence after treatment. These factors help in selection of patients for a specific treatment.  Predictive factors evaluate the likelihood of benefit from a specific treatment. ER, PR & HER2/neu are prognostic as well as predictive factors. 
The oestrogen receptor plays a very important role in the pathophysiology of breast cancer. Oestrogen receptor is of two types-ER[alpha] and ER[beta]. Receptor ER[alpha] is a well-established prognostic and predictive factor in breast cancer. The prognostic significance of ER[beta] is not well defined. [5,6] The majority of ER-positive breast cancers contain both ER[alpha] and ER[beta] subtypes, although some cancers have only ER[beta] expression. This may lead to distinct clinical behaviours and responses. It is observed that in contrast to ER[alpha], ER[beta] expression declines during breast carcinogenesis. [7,8]
Progesterone receptor is of two types: PR-A & PR-B. Progesterone acts as a modulator of oestrogen function. [9,10] PR expression increases, as the atypia in breast tissue, increases. It is observed that ER-positive breast cancers which lack PR expression, are less responsive to hormonal treatment than those that are PR positive. It is also seen that ER & PR are not stable phenotypes. These can change the natural history of the disease or as consequence of treatment.  HER2/neu (c-erbB-2).
It is a member of four member family of closely related growth factor receptors, including EGFR or HER1, HER2, HER3, HER4. HER2/neu amplification or over expression is involved in oncogenic transformation and tumorigenesis in breast cancer. Inappropriately increased signalling occurs as a result of receptor over-expression. It may lead to increased & uncontrolled cell proliferation, decreased apoptosis, increased cancer cell motility and angiogenesis and hence worse prognosis. 
The present study was conducted to correlate the expression of ER, PR & HER2/neu with each other & to various clinicopathological parameters.
MATERIAL AND METHODS
This study was conducted on 100 Patients, of infiltrating ductal carcinoma breast (NOS) to analyse the expression of ER, PR, and HER2/neu by immunohistochemistry.
Histopathologically diagnosed cases of IDC-NOS.
1. Histopathological subtypes other than IDC-NOS.
2. Patients who received neo-adjuvant chemotherapy.
3. Inflammatory breast lesions.
4. Post-traumatic breast lesions.
5. Benign breast diseases.
From histologically confirmed cases of infiltrating ductal carcinoma breast (nos.), paraffin blocks containing cancer tissue were selected. Slides were prepared from these paraffin blocks. Standard procedure was followed for immunohistochemical staining of slides for ER, PR, and HER2/neu. 
Preparation of Paraffin Slides
Paraffin sections were cut at 5 micrometres, mounted on silanized slides, and melted at 65oC in an oven for 2 hours. Slides were dipped into xylene (3 times) for 5 minutes each to remove the paraffin. Tissues were dehydrated by dipping the slides into absolute ethanol (100%), then 95% ethanol, and finally 70% ethanol. Slides were washed with distilled water for 5 min. & then dipped into a fresh aqueous solution of 3% peroxide for 3 min. and then rinsed with Tris buffer for 3 minutes.
Antigen Retrieval (In Decloaking Chamber)
Heat retrieval was done with citrate buffer in Decloaking chamber for forty minutes at 950C & then brought to room temperature after removing from Decloaking chamber and by placing the slides in Tris-Saline buffer.
Detection of Antigens in Paraffin Sections
1% Mouse serum (Diluted in antibody dilution buffer) was added to the tissue section for 20 minutes, to block non-specific immunostaining.
The primary antibody was added and the sections are exposed to the primary antibody, usually for one hour & primary antibody was washed from slide with Tris buffer. Sections were soaked in Tris buffer for 10 minutes (2X 5 min. washes).
Secondary Detection of the Primary Antibody
Sections were then incubated with biotinylated mouse anti-species antibody for 10 minutes. Sections were rinsed for 10 min (2X 5 min. washes) in Tris buffer. A solution of chromogen, 3,3'-diaminobenzidine (DAB) at 1 mg/mL in Tris buffer with 0.016% fresh [H.sub.2][O.sub.2] was made & added to the slides and incubated for approximately 8 minutes. DAB from the slides was washed with tap water.
Slides were dipped in a solution of haematoxylin that is diluted 1:1 in distilled water and stained for one minute to produce a very light nuclear counterstaining. Washed for 1 min. in distilled water & dehydrated by dipping in 95% ethanol for 1 min., then 100% ethanol for 1 min. Washed 3 times in xylene and cover slip applied for viewing & reporting.
ER/PR scoring system and criteria as per Allred scoring system : Figure 1.
0--No cells are ER +ve.
1--[less than or equal to] 1% of cells are ER +ve.
2--1-10% of cells are ER +ve.
3--11-33% of cells are ER +ve.
4--34-66% of cells are ER +ve.
5--67-100% of cells are ER +ve.
Total (proportion score + intensity score) 0-2=Negative; 3-8 = Positive.
HER2/neu Scoring System and Criteria According to ASCO-CAP Guidelines 
0 = No staining or incomplete faint and barely perceptible in <10% of tumour cells. 1+= Incomplete membrane staining which is faint and barely perceptible and within>10% of tumour cells.
2+ = Circumferential membrane staining that is incomplete and/or weak/moderate and within >10% of the invasive tumour cells; or complete and circumferential membrane staining that is intense and within <10% of the invasive tumour cells.
3+ = Circumferential, complete and intense staining and within >10% of tumour cells.
FISH is required for equivocal HER2/neu positivity. So, HER2/neu 2+ was taken as negative along with HER2/neu 0 and 1+. Only 3+ on IHC was taken as positive.
Chi-square test was used to determine the statistical significance between ER/PR status and HER2/neu status along with their correlation with various clinicopathological parameters like patient's age, axillary lymph node status, tumour size and tumour grade with respect to IDC-NOS. A p value of < 0.05 was considered statistically significant.
In our study, the mean age was 55.28 years. The majority of ER-positive (96.82%) cases were of age >40 years and 44.44% were of age >60 years. The majority of PR-positive (96.55%) cases were of age > 40 years and 43.10% were of age >60 years. The majority of HER2/neu positive (71.43%) were of age <40 years i.e. younger age group (Table-1).
Using chi-square statistics, it was observed that various values for ER expression were [x.sup.2] = 25.305; df = 3; p = 0.000, for PR expression were [x.sup.2] =18.051; df = 3; p = 0.000; & for HER2/neu expression [x.sup.2] = 19.363; df = 3; p = 0.000 & it was concluded that ER, PR& HER2/neu expression compared to age distribution was statistically highly significant.
Axillary lymph Node Status
38 patients had axillary lymph nodes positive for metastasis. Out of 63 ER-positive cases, 25 had positive axillary lymph nodes, whereas out of 58 PR-positive cases 23 had positive axillary lymph nodes, and out of 7 HER2/neu-positive cases, 2 had positive axillary lymph nodes. (Table 2).
Using chi-square statistics, various values for ER expression were [x.sup.2] = 0.205; df = 1; p = 0.651; for PR expression were [x.sup.2] = 0.161; df = 1; p = 0.689 & HER2/neu expression were [x.sup.2] = 0.284; df = 1; p = 0.594; It was concluded that axillary lymph node status with ER, PR and HER2/neu was not statistically significant.
Tumour size was highly variable ranging from 0.1 cm to 12 cm. The average tumour size was 4.3 cm. The majority of ER/PR positive (46-47%) tumours were of size between 2 to 5 cm & majority of HER2/neu positive (71.43%) tumours were of size <2 cm.(Table-3).
Using chi-square statistics, expression of ER compared to tumour size was [x.sup.2] = 3.098; df = 2; p = 0.001; expression of PR was [x.sup.2] = 8.587; df = 2; p = 0.014; & expression of HER2/neu was [x.sup.2] = 7.144; df = 2; p = 0.028. So it was concluded that expression of ER, PR& HER2/neu compared to tumour size was statistically significant.
In our study, according to Nottingham Modified BloomRichardson system (MBR) score, majority tumours were in grade II (43%), followed by grade III (31%) and then grade I (26%). The majority of ER/PR positive (48-49%) tumours were of grade II & majority of HER2/neu positive (57.14%) tumours were of grade III.
Using chi-square statistics, it was seen that expression of ER compared to tumour grade was [x.sup.2] = 11.534; df = 2; p = 0.003; expression of PR was [x.sup.2] = 6.976; df = 2; p = 0.031 & expression of HER2/neu was [x.sup.2] = 2.421; df = 2; p = 0.298. It was concluded that expression of ER/PR compared to tumour grade was statistically significant & HER2/neu was not significant.
Oestrogen Receptor Status
Majority (63%) of tumours were ER positive and 37% were ER negative. ER-positive tumours showed weak, moderate to strong nuclear positivity in >1% of tumour cells.
Progesterone Receptor Status
58 tumours were PR Positive and 42 were PR negative. PRpositive cases showed weak, moderate to strong nuclear positivity in >1% of tumour cells.
Kappa value = 0.854; ASE = 0.053; p value = 0. 000 Out of 100 cases, 58 cases were ER & PR positive, 37 cases negative & 5 cases showed different expressions of ER & PR. (Table 5). On statistical analysis using kappa as measure of agreement, it is concluded that expressions of ER & PR agree significantly to each other.
Only 7 were HER2/neu positive and 93 were HER2/neu negative.
Kappa value = -0.079; ASE = 0.048; p value = 0.058 [ER compared to HER2/neu].
Only 2 cases were ER, PR as well as HER2/neu positive. 32 cases were both ER & HER2/neu negative. Majority i.e. 66 cases showed different expressions of ER & HER2/neu (Table 6). On statistical analysis using kappa as a measure of agreement, it is concluded that expressions of ER & HER2/neu do not agree to each other.
Breast cancer is the most common malignancy in women. It is highly curable, if diagnosed at early stage, because a significant number of patients with early stage breast cancer harbour microscopic metastasis at the time of diagnosis. Prognostic and predictive factors are used in the management of breast cancer. ER, PR & HER2/neu are prognostic as well as predictive factors. Correlation of the expression of ER, PR & HER2/neu with each other & to various clinicopathological parameters is significant.
Patients were in age group between 24 and 80 years. The mean age was 55.28 years. Studies conducted by Kaul R et al  showed similar results. In the present study, majority of ER-positive (44.44%) and PR positive (43.10%) cases were of age >60 years. Studies conducted by Onitilo AA et al  Alzaman AS et al  showed similar results. The majority of HER2/neu positive (71.43%) were of age <40 years in the present study. Studies conducted by Alzaman AS et al  showed similar results.
There was significant correlation seen between the age of the patient and ER (p = 0.000) & PR (p = 0.000) expression in present study. Studies by Dodiya H et al  and Ghosh S et al  showed similar results. Significant correlation was also observed between age of the patient and HER2/neu expression (p = 0.000), similar to studies conducted by Ramic S et al  and Ganesan M et al. 
Axillary Lymph Node Status
38% patients had axillary lymph nodes positive for metastasis. 39.68% of ER-positive cases had positive axillary lymph nodes for metastasis. 39.65% of PR-positive cases had positive axillary lymph nodes. 28.57% of HER2/neu-positive cases had positive axillary lymph nodes for metastasis. A study conducted by Ali EM et al  showed similar results. There was no significant correlation observed between axillary lymph node status with ER (p = 0.651), PR (p = 0.689) and HER2/neu (p=0.594) expression in present study, similar to studies conducted by Azizun Nisa et al. 
Tumour size was 0.1 cm to 12 cm, with average size 4.3 cm. The majority of cases (43%) had size 2 to 5 cm, similar to Siadati S et al study. 47.61% of ER-positive & 46.55% of PR-positive tumours were of size 2 to 5 cm. 71.43% of HER2/neu tumours were of size <2 cm. A study conducted by Prasad HLK et al  showed similar results.
There was significant correlation seen between tumour size and ER (p=0.001), PR (p=0.014) expression in present study. Studies by Prasad HLK et al  showed similar results. Similar to Almasri NM et al  study, significant correlation was seen between tumour size and HER2/neu expression (p = 0.028).
In the present study, majority of tumours were in grade II (43%) followed by grade III (31%) and then grade I (26%). The majority of ER-positive (49.21%) and majority of PRpositive (31.03%) tumours were of grade II, but majority of HER2/neu-positive (57.14%) tumours were of grade III, similar to study conducted by Siadati S et al.  There was significant correlation of tumour grade with ER (p=0.003) & PR (p=0.031), similar to study done by Onitilo AA et al  and Dodiya H et al.  As shown by Dodiya H et al,  no association was seen between tumour grade and HER2/neu expression (p=0.298).
Majority (63%) of tumours were ER positive and 37% were ER-negative. 57% tumours were PR Positive and 43% were PR negative. Only 7% were HER2/neu positive and 93% were HER2/neu negative.
There was wide variation seen in the ER, PR & HER2/neu expression in breast carcinoma in different studies, possibly due to variations in different populations. In the present study, ER positivity was 63%, closely matched the results of the study conducted by Idirisinghe PK et al  & PR-positivity was 58%, closely matched the results of the study conducted by Engstrom MJ et al.  HER2/neu positivity was only 7%, much lower as compared to other studies. The possible explanation for this is due to variations in different populations. Also, HER2/neu assay results are influenced by multiple biologic, technical and performance factors. Since many aspects of HER2/neu assays have not been standardised, the effects of these disparate influences could not be isolated.
ER and PR correlated with each other (p = 0.000), whereas expression of HER2/neu was inversely related to ER (p = 0.058) & PR expression (p = 0.102). Similar results were found in studies conducted by Siadati S et al,  Maha A et al,  etc.
Invasive ductal carcinomas of breast were seen in age of 24 to 80 years, with mean age of 55.28 years. The maximum number of cases were seen in the age of >50 years (66%). The majority of tumours were ER & PR positive and HER2/neu negative. The majority of ER & PR positive tumours were of grade II; whereas majority of HER2/neu-positive tumours were of grade III. The present study confirmed that ER & PR correlated significantly with age, tumour size and tumour grade; whereas HER2/neu correlated significantly with age & tumour size only. No association was seen with axillary lymph node metastasis. ER and PR expression correlated with each other, but none was correlated with HER2/neu.
Assessment of hormone receptors for clinical management of breast cancer patients is strongly recommended to provide prognostic information and therapeutic options.
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Prem Chand (1), Ashok Kumar (2), Vandana Singla (3), Goldendeep Singh (4), Ramandeep (5)
(1) Assistant Professor, Department of Surgery, GMC, Patiala.
(2) Associate Professor, Department of Surgery, GMC, Patiala.
(3) Lecturer, Department of Pathology, GMC, Patiala.
(4) Senior Resident, Department of Surgery, GMC, Patiala.
(5) Junior Resident, Department of Pathology, GMC, Patiala.
Financial or Other, Competing Interest: None.
Submission 02-02-2017, Peer Review 27-02-2017, Acceptance 04-03-2016, Published 13-03-2017.
Dr. Prem Chand, #108-Hem Bagh, Near Shiv Mandir, Kheri Gujjran Road, Patiala-147001, Punjab.
Caption: Figure 1. Allred Scoring System
Table 1. ER & PR Expression compared to Age Age Group ER PR (Years) Positive Negative Total Positive Negative Total <40 02 13 15 02 13 15 41-50 09 10 19 09 10 19 51-60 24 08 32 22 10 32 >60 28 06 34 25 09 34 Total 63 37 100 58 42 100 Age Group HER2/neu (Years) Positive Negative Total <40 05 10 15 41-50 01 18 19 51-60 01 31 32 >60 00 34 34 Total 07 93 100 Table 2. Expression of ER, PR & HER2/neu compared to Axillary Lymph Node Status Axillary ER PR Lymph Positive Negative Total Positive Negative Total Node Status Positive 25 13 38 23 15 38 Negative 38 24 62 35 27 62 Total 63 37 100 58 42 100 Axillary HER2/neu Lymph Positive Negative Total Node Status Positive 02 36 38 Negative 05 57 62 Total 07 93 100 Table 3. ER, PR & HER2/neu Expression compared to Tumour Size Tumour ER PR Size Positive Negative Total Positive Negative Total (cm) <2 23 06 29 21 08 29 2-5 30 13 43 27 16 43 >5 10 18 28 10 18 28 Total 63 37 100 58 42 100 Tumour HER2/neu Size Positive Negative Total (cm) <2 05 24 29 2-5 02 41 43 >5 00 28 28 Total 07 93 100 Table 4. ER, PR& HER2/neu Expression compared to Tumour Grade Tumour ER PR Grade Positive Negative Total Positive Negative Total Grade I 20 06 26 18 08 26 Grade II 31 12 43 28 15 43 Grade III 12 19 31 12 19 31 Total 63 37 100 58 42 100 Tumour HER2/neu Grade Positive Negative Total Grade I 01 25 26 Grade II 02 41 43 Grade III 04 27 31 Total 07 93 100 Table 5. ER Expression compared to PR Expression ER/PR PR Positive PR Negative Total ER Positive 58 5 63 ER Negative 0 37 37 Total 58 42 100 Table 6. ER & PR Expression compared to HER2/neu Expression ER/ HER2/neu HER2/neu PR/ HER2/neu HER2/neu Positive Negative Total HER2/neu Positive ER Positive 02 61 63 PR Positive 02 ER Negative 05 32 37 PR Negative 05 Total 07 93 100 Total 07 ER/ HER2/neu Total HER2/neu Negative ER Positive 56 58 ER Negative 37 42 Total 93 100
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|Title Annotation:||Original Research Article|
|Author:||Chand, Prem; Kumar, Ashok; Singla, Vandana; Singh, Goldendeep; Ramandeep|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Mar 13, 2017|
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