Hormone receptor status, Her 2- neu amplification and their correlation with prognostic indices in patients with invasive ductal carcinoma of breast in Jaffna

Knowing the tumour biology can help to stratify patients for prognostic and therapeutic purposes. Expression of estrogen and progesterone receptors (ER, PR), amplification of human epidermal growth factor receptor 2 (Her 2 neu), size and histological grading of tumour , axillary lymph node state and NPI ( Nottingham Prognostic Index) are well established parameters that have influence on prognosis and treatment of breast carcinoma


Introduction
Carcinoma of breast accounts for 12.6% of all cancers and 27% of female cancers in Sri Lanka. It is the most common cancer among females in our country (1). The incidence of breast cancer in females in Sri Lanka has increased from 17.3 per 100,000 in 2001 to 24.7 per 100,000 in 2010. Highest incidence was noticed among female patients in the 60 to 64-year age group. Among the different histological types of breast cancer, 79.3 % are the invasive ductal carcinoma in Sri Lanka (2).
Breast cancer has diverse characteristics in o n c o l o g i c a l b e h a v i o u r a n d t r e a t m e n t responsiveness. Apart from early identification of breast cancer it is also important to distinguish those breast cancers that are likely to cause morbidity and mortality. So stratifying the patients based on outcome prediction and tumour biology could aid to institute more personalized and responsive treatment (3,4). Analyzing prognostic indices on one particular histological type of breast carcinoma could nullify these variations.
The aim of this study is to assess the expression of hormone receptors (ER/PR) and amplification of Her 2-neu receptors in the infiltrating ductal carcinoma of breast and to analyse the correlation between these receptor status and prognostic indices among a cohort of female patients primarily treated by mastectomy in Jaffna.

Methodology
It is an institutional based retrospective cross sectional analytic study. Approval for the study was obtained from Ethics Review Committee, Faculty of Medicine, and University of Jaffna. Female patients who underwent primary mastectomy and axillary clearance for invasive ductal carcinoma of breast in Teaching Hospital Jaffna from 2014 to 2018 were included in this study. Patients who received neo-adjuvant therapy were excluded from this study. The expression of ER & PR and Her 2-neu amplification were identified by immunohistochemistry based on American Society of Clinical Oncology guidelines. Nuclear stain appearing in more than 1% of tumour cells would be considered positive for ER & PR. Her 2-neu amplification was scored from 0 to 3+ based on the pattern of staining. 0 and 1+ would be negative for Her 2-neu amplification. 3+ would considered Her 2-neu +. There were five patients with equivocal Her 2 neu expression (2+) and they were excluded from this study. Informed written consent was obtained from patients. Patient's age at diagnosis, histopathological features (nodal status, tumour size and histological grade) and immunohistochemical marker expression (ER/PR/ Her 2) were recorded.
Statistical analysis was carried out using SPSS 21.0(SPSS Inc). Correlation between the receptor status and prognostic indices were calculated using Spearman's correlation, chi-square and Kruskal-Wallis tests. The p value < 0.05 was considered as being significant.   Table   (2)]. There is statistically significant inverse correlation between the Her 2-neu amplification and expression of either one or both of ER and PR (chi square=12.93, p value < 0.0001).

Discussion
Hormone receptor status (ER/PR) in invasive breast cancer has more predictive strength than prognostic strength. Expression of these hormone receptors can predict benefit from adjuvant endocrine therapy. Over expression of Her 2 neu is associated with increased risk of recurrence and poor prognosis and it is considered as a prognostic factor. Her 2 neu expression can predict treatment responsiveness to anthracyline based chemotherapy and not to endocrine therapy. So it is a strong predictive factor as well (4,7,8).
Predictive factors are complementary to prognostic factors. Prognostic indices when used in combination with predictive factors could help to correctly identify patients with high risk and low risk after primary surgery. This will guide to tailor adjuvant therapy in order to improve patient outcome (9, 10). When compared with similar studies carried out in different provinces in Sri Lanka, the prevalence of invasive ductal carcinoma of breast with HER 2-neu amplification (HER 2-neu +) is relatively high in Jaffna while the prevalence of tumours with expression of ER ( ER +) status is relatively low. Other histological types of breast carcinoma were also taken into account in studies from different provinces and could have contributed to the differences observed.
A positive correlation between ER and PR expression was observed in this study population as well as in other studies in Sri Lanka by Ratnatunga et al and Bimalka et al (11,7). The negative correlation between Her 2-neu and ER/PR was observed in this study as well as in other studies from Sri Lanka and India (7,11,12).
Correlation between various demographic factors were analysed and the existence of a statistically significant relationship between ER expression and age of patients has been reported by Ratnatunga et al from Kandy, Sri Lanka (11). Faheem et al from Pakistan have identified higher incidence of ER and PR expression among post-menopausal patients with breast cancer (15). ER + tumours tend to occur in slightly older patients in Jaffna. But this correlation was not statistically significant (p =0.853) No statistically significant correlation between the Her 2-neu amplification and age of breast cancer patients was observed in studies from Sri Lanka and India (11,16,12). Faheem et al from Pakistan found that Her 2-neu amplification was significantly higher in breast carcinomas occurring in premenopausal patients (p < 0.001) (15).
The Nottingham Prognostic Index (NPI) is a numerical value calculated from histopathological parameters such as the nodal status, tumour size and histological grade. Thus, NPI stratify the breast cancers based on their metastatic potential, rate of growth and extent of genetic instability. NPI distinguishes good, moderate, and poor prognostic groups (NPI 1, 2 & 3 respectively) in breast cancer. It is a well recognized method to predict survival of operable breast cancer (17). The 10 year survival rates of these prognostic groups, namely NPI 1, NPI 2 and NPI 3, are 83%, 52% and 13 % respectively (18).
ER expression is more frequent in NPI types of favourable prognosis. This correlation was statistically significant (p = 0.047). There was no statistically significant correlation observed between NPI groups and expression of PR, Her 2neu amplification or the age of patient at diagnosis of invasive breast cancer in Jaffna (p values 0.081, 0.908 and 0.106 respectively). Peiris et al from Galle found that more NPI 1 tumours occurring with increasing age and the correlation between age of patients at presentation and NPI was statistically significant ( p =0.011) (16). Kurshumliu et al has demonstrated that the NPI 1 and NPI 2 groups had tumours expressing more ER and PR (p < 0.01) whereas the NPI 3 group had tumours expressing more Her 2-neu (p =0.01) (19).
When ER/PR status and Her 2 neu status are used together in invasive breast cancer they have more prognostic and therapeutic yield. For example, triple negative (ER -, PR -, Her 2 neu -) invasive breast cancers have the worst overall and disease free survival (14).
21% of patients with invasive ductal carcinoma of breast in Jaffna had triple negative tumour [Table (5)]. Triple negative invasive breast carcinoma was found in 28.23% patients in Sri Lanka and 30% of patients India (11,12). This included all other histological types of breast carcinomas as well. Bimalka et al demonstrated that in breast carcinomas with histological types other than ducal and lobular carcinomas, 33.3% had triple negative tumours (7). Considering these facts, the prevalence of triple negative breast cancer in Jaffna is comparable to other regions in Sri Lanka.
Interesting finding in this study is that relatively more ( 11%) of triple negative invasive ductal carcinomas were found to be in NPI 1 and less (2.8%) of triple negative invasive ductal carcinomas were found in NPI 3 groups [ The correlation between the Her 2 -neu amplification and the axillary lymph node involvement was statistically significant (p =0.0001). There was no statistically significant correlation between expression of ER or PR and associated axillary lymph node involvement in this study. Bimalka et al from Colombo found axillary lymph node involvement in 31.52% of patients and there was no significant correlation existed between tumours with Her 2-neu amplification and associated axillary lymph node involvement (7). Peiris et al from Galle identified axillary lymph node involvement in 55.2% of patients and the frequency of lymph node involvement was reducing with increasing age (p =0.012). They have also described that lymph node staging was the most important independent predictor of survival in breast cancer irrespective of the age of patient at diagnosis (16).

Conclusion
There is positive correlation between the expression of ER and PR and an inverse correlation between the expression of hormone receptors and the Her 2 -neu amplification among invasive ductal carcinoma patients in Jaffna. The expression of ER is significantly associated with favourable NPI groups and also with low grade tumours. The prevalence of invasive ductal carcinoma with Her 2-neu amplification is relatively high in Jaffna and the tumours with Her 2-nue amplification are significantly associated with axillary lymph node involvement.