Correlation of P53 Expression with Clinicopathological Parameters, Hormone Receptors and HER 2 Neu Status in Breast Carcinoma

discovered that the HER-2


Introduction
Breast carcinoma is the most prevalent malignancy worldwide in females and is one of the primary reason for mortality in women. It currently accounts for one out of every four malignancies in women [1]. In India, 118000 incident cases were reported in 2016, according to estimates with a 95% confidence level [2].
The tumor's size, type, and grade as well as the condition of its lymph nodes determine the prognosis. Hormone receptor status like estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2/neu) expression are universally employed as biomarkers for breast cancer and are analysed using Immunohistochemistry (IHC) [1]. In 1987, it was discovered that the HER-2/neu proto-oncogene was amplified, overexpressed, and related with a worsening of apjcb.waocp.com Tahmida Ali, et al: Correlation of P53 Expression there are other factors which not only are predictive, but also direct therapies against particular molecular targets [5]. The current therapeutic approaches for breast carcinoma consist of combinations of surgery, postoperative radiation, hormonal treatment, chemotherapy and trastuzumab. The choice between hormonal therapy which has minimal side effects and chemotherapy which has high morbidity and risks is a major responsibility of the clinician. Therefore, an accurate assessment of ER, PR and HER 2 neu status of breast cancers by the pathologist is very essential [6].
Hence, this study aimed to establish a correlation of p53 with ER and PR status and HER 2 neu overexpression, lymph node status, clinical features and tumor histopathology and to effectively use the results to prognosticate and treat invasive breast cancer patients.

Materials and Methods
The study was undertaken at a tertiary care hospital in Bangalore, Karnataka. A total of 86 invasive breast cancer cases which were diagnosed over a period of 23 months between June 2015-May 2017 were studied. Females of all age group were included in the study. Cases where only a trucut biopsy or a limited surgery has been done and in cases where there was extensive tumour necrosis without sufficient viable tumour cells for accurate evaluation of the IHC results were excluded from the study. Detailed history, clinical data and laboratory investigations were collected for each cases. All tissues were fixed in 10% buffered formalin for not more than 24 hours. Representative tumor tissue was submitted for processing and H&E stain for routine histological diagnosis according to modified Bloom Richardson grading system of WHO.
IHC for ER, PR and Her-2/neu were performed on representative blocks of paraffin embedded tissue. For IHC staining 4 μm thick sections were submitted. Sections were taken on slides previously coated with poly L lysine. Antigen retrieval was done by HIER method using Citrate buffer at PH 2.5 for ER/PR and PH 6 for HER 2 neu. The normal epithelial component was served as internal control for ER/PR. IDC with known HER 2 neu over expression was used as external positive control for each lot of staining.
The ER and PR results were screened and interpreted as positive when more than 1% of tumor cells showed positive nuclear staining. For interpretation of HER 2 neu staining in each case, the proportion of positive staining tumour cells (expressed in percentage) and the average intensity of staining (expressed as 0, 1+, 2+ or 3+) was evaluated. The relationship between various parameters such as age, duration of disease presentation, tumor size, tumour extent, histologic grade, lymph node status, size of the largest lymph node and hormone receptors ER, PR, HER2 neu with p53 expression were studied.
The categorical variables were expressed as percentages. Chi-squared test was used for categorical variables for calculating p-value (p-value <0.05 = significant). Statistical analysis was performed using SPSS version 21.

Results
A total of 86 cases of invasive breast carcinoma were studied, out of which most of the women belonged to the age group 41-60 years (54, 62.8%) followed by >60 years (19,22.1%). The duration of symptoms in 36 (41.9%) cases was less than 6 months and in 50 (58.1%) cases was more than 6 months. Family history of breast carcinoma was present in 17 (19.8%) and absent in 69 (80.2%) of 86 cases Table 1.
Out of 86 cases, 54 (62.8%) showed metastasis in the lymph nodes. The tumour size (largest dimension) ranged from 0.5 cm to 4 cm. Of 86 cases, 57 (66.3%) were in the range of 2 -5 cm. The largest lymph node size was less than 2 cm in 58 (67.4%) cases. Scarff Bloom Richardson (SBR) score of 6 -7 was seen in 69 (80.2%) cases and 52 (60.5%) were found to be in Stage II. Of all the age groups studied, most of them had SBR grading of Grade 2 (69, 80.23%) and most of the patients belonged to the age group 40 -60 years (69, 80.23%).
Of the 14 triple positive cases, 6 (42.85%) cases each were seen in the age group of 40 -60 and above 60 years respectively and 2 (14.3%) with age less than 40 years. Of the 25 triple negative cases, 16 (29.63%) were seen in the age group of 40 -60 years, 5 (26.32%) with age more than 60 years and 4 (30.77%) with age less than 40 years as displayed in Table 3.
Of 49 cases with ER and PR positive in 36 (73.47%) have tumour in the size range of 2 -5 cm followed by 11 (22.4%) cases had tumour with size more than 5 cm. Of 37 cases with ER and PR negative, 21 (56.76%) had tumour with size in the range of 2 -5 cm followed by 13 (35.1%) cases with size more than 5 cm ( Figure 1). Cases with ER and PR positive, 41 (83.67%) were in Grade 2 and 4 (8.16%) were in Grade 1 and Grade 3 each, respectively. Table 4.
Of 49 cases with ER and PR positive, metastasis to lymph nodes was present in 32 (65.31%) and absent in 17 (34.7%) patients. P53 expression was positive in 37 (75.5%) cases and negative in 12 (24.5%) cases and it was significant (P-0.047). Of 37 cases with ER/PR negative, p53 expression was positive in 34 (91.9%) cases and negative in 3 (8.1%) cases Table 4. in 60 (69.8%) cases. P53 expression was positive in 71 (82.6%) and negative in 15 (17.4%) of a total of 86 cases as reflected I the Table 1.
Of the 86 patients ER/PR was positive in 49 patients (57 %), ER/PR was negative in 37 patients. ER/PR/Her2 neu was positive in 14 (16.3%) patients and ER/PR/Her2 neu negative in 25 (29.1%) patients. In 35 patients (40.7%) ER/PR was positive with negative HER2 neu and in 12 patients (14%) ER/PR was negative with positive HER2 neu as shown in the Table 2.
Among all the Age groups, most of the patients had tumour size ranging from 2-5 (57, 66.28%) and most people belonged to the age group of 40 -60 years (38, 70.37%) Table 3.    triple positive cases, metastasis to lymph nodes was present in 3 (21.4%) and absent in 11 (78.57%). Of the 25 triple negative cases, metastasis to lymph nodes was present in 12 (48.0%) and absent in 13 (52.0%). (P value 0.000) as shown in Table 5.
Of the 15 p53 negative cases, most of them 10 (66.7%) were in the age group of 40 -60 years.

Discussion
There is evidence that the incidence of breast cancer in India is on the rise. Several risk factors for the development of breast carcinoma have been established. The common denominator for most of the factors is prolonged estrogen stimulation operating on a genetically susceptible background [7].
In our study the age of the patients ranged from 34 to 82 years with a mean age of 59 years. The maximum cases are in the range of 40 to 60 years (62.8%). In various other studies, similar results as our study were obtained. In the study by Ayadi et al 51.6% of cases occurred in less than 50 years of age with median age of 51 years and 46% of cases occurred in less than 45 years [8]. In the study by Raina et al 49.7% of cases occurred in less than 45 years and 48.5% cases in greater than 45 years [9].
Majority of the cases (66.3%) had tumour with size 2-5 cm followed by 27.9% cases with tumour measuring > 5 cm in largest dimension. None of the patients had received any chemo, radio or hormonal therapy. This study results were similar to study by Patnayak et al [7] where tumour size 2-5 cm was seen in most of cases (160, 79.6%) and least cases had < 2cm (19,19.4%) [7].   Of 86 cases, 54 cases (62.8%) showed metastatic disease with the largest lymph node measuring < 2 cm in 67.4% cases similar to studies by Payandeh et al in which 64% had lymph node metastasis [10].
Of the 86 cases in our study, maximum cases (80.2%) were in SBR Grade 2, that is, they have an SBR score of 6 -7. Similarly a study by Reddy SR et al showed 81.61% were in the SBR Grade II [11].
In our study we noted an overall 57.0% of ER positivity and 57.0% of PR positivity (Figure 3a and   3c). The prevalence of hormone receptor positive breast carcinoma in Asian countries has been found to be lower as compared to Western world. Western studies have reported 70 -80% ER and 60 -70% PR expression in cases of invasive ductal carcinoma, respectively [12]. The other study from India has documented lower positivity for the hormone receptors similar to our study. In a study by Dutta et al ER/PR overexpression was found in 33.5% [13].
The percentage of tumours expressing both ER and PR include 57.0%, of which maximum (59.2%) are seen in the age group of 40 -60 years, with a tumour size of 2 -5 cm in 73.5% and 65.3% showing lymphatic invasion.
In this study there was a correlation between metastasis and ER, PR and Her 2 neu (p 0.000). The above results correlated with the study results by Siadati S et al where there was a significant correlation between lymphatic invasion and ER, PR and Her 2 neu expression (p<0.05) [14]. There was also a significant correlation between ER, PR receptors and p53 over-expression. In 91.9% of cases with both ER and PR positivity showed p53 over-expression similar to the study by Bae SY et al (90%) [15].
Triple negative breast cancers (TNBC) have recently attracted a lot of attention, because of their poor prognosis. TNBCs are frequently shown to harbour mutations in Tp53, resulting in loss of the G1 checkpoint and reliance on checkpoint kinase-1 to arrest cell in response to DNA damage. In our study, triple negative cases constituted 29.1% of the total cases, of which 64.0% are in the age group of 40 -60 years, with a tumour size of 2 -5 cm in 60% and 48% showing lymphatic invasion which was similar to study by Rana MJ et al where the mean age of TNBC patients was 53.3+11.6 years with 90% showing invasion [16].
A significant correlation is noted between triple  negative cases and p53 over-expression in our study (Figure 4a, b, c). In 92.0% cases of triple negative show p53 over-expression. A study by Hashmi AA et al also showed correlation between triple negative cases and P 53 expression 49% of cases [17]. Since its initial discovery, HER 2 neu was used as a therapeutic target because of the efficacy of trastuzumab, a recombinant humanized monoclonal antibody that binds with high affinity to HER 2 in advanced breast cancer. Over-expression of HER 2 occurs through either amplification of the gene or mRNA over-expression. HER 2 neu over-expression is seen in 30.2% cases in this study (Figure 5a  One study from north India had shown HER 2 neu oncogene over-expression is higher (46.37%) among Indian patients in comparison to 25 -30% in most western literature [19,20].
However various studies show considerable variation regarding the frequency of HER 2 positivity. There is also evidence from the literature that coexistence of overexpression of HER 2 neu and p53 protein accumulation is a strong prognostic marker in breast cancer [21,22]. However, in our study we did not find a significant correlation between p53 and HER 2 neu over-expression.
In our study the relation of p53 with age, duration of symptoms, tumour size, and lymphatic invasion was not found statistically significant. However, we found significant correlation of p53 over-expression with SBR tumour grade 2 (P-0.019). This finding is similar to that found in study by Deb P et al, where there statistical significance was found between P53 and SBR grade II (P<0.05) [23].
In conclusion, significant positive correlation between p53 over expression and tumour grade was seen in this study. Nodal involvement may be an indicator of metastatic disease, the cause of death in patients with breast cancer. Hence the generally admitted conclusion is that tumour size loses its prognostic role in cases of nodal involvement.
Significant positive correlation of p53 over expression with ER and PR positive tumours and Triple Negative tumours was noted.
Despite improving breast cancer outcomes, distant recurrence remains common and incurable. This study demonstrates the expression of p53 in invasive ductal carcinomas and its correlation with hormone receptors, HER 2 neu and clinico-pathological parameters, thus contributing to an expanding knowledge of prognostic and predictive markers that will allow individualized therapy for invasive breast carcinoma.
To conclude, we suggest that p53 accumulation detected immunohistochemically defines a subgroup of breast carcinoma patients with high tumour aggressiveness and adverse long term prognosis.