Molecular profiling of breast carcinoma with IHC surrogates in a tertiary care centre in South India

Introduction and Aim: Breast cancer is the most common malignancy in females worldwide. Almost 1.4 million new cases have been diagnosed with breast cancer every year. This aims to study the clinicopathological profile and molecular subtypes of invasive breast carcinoma in resected mastectomy specimens over a period of 5 years. Materials and Methods: A retrospective study of 90 mastectomy and wide local resection specimens received during the period of January 2012 to June 2017 were analyzed. The clinical data of patients including age, gender, and stage of the disease were obtained from the medical records section. Immunohistochemical staining for Estrogen Receptor [ER], Progesterone Receptor [PR] and Human Epidermal Growth Factor Receptor 2 HER2neu were done. The cases were classified according to the molecular classification based on the ER, PR and HER2 receptor status. Results: The peak incidence of breast carcinoma was in the age group 50 to 60 years. Invasive ductal carcinoma, Not otherwise specified [NOS] accounted for the most common histologic type. There was higher incidence of pT2 tumors in our study. The most common molecular subtype was luminal A, followed by triple negative tumors. These molecular subtypes associated well with Tumor grade and HGDCIS with a statistically significant p value of 0.001 and 0.015 respectively. An increased proportion of Grade 3 tumors were Triple Negative tumors. Conclusion: In breast carcinomas the routine histopathological features provide inexpensive method for understanding tumour biology and prognosis. It`s essential in areas with poor resources. ER, PR and HER2 assessment helps in identifying hormonal status and enables for hormone therapy and anti HER2 therapy.


INTRODUCTION
reast cancer is the most common malignancy in females worldwide. Almost 1.4 million new cases have been diagnosed with breast cancer every year. About half of the cases occur in economically developing countries (1). The incidence of breast cancer has grown rapidly during the last decade in many developing countries and accounts for major cause of mortality. The molecular classification of invasive breast carcinoma using global gene profiling is not economical in a poor resource setting. The expression of clinically significant immunohistochemistry (IHC)surrogates has been analysed in many studies. The present study aims at identifying the molecular subtypes using IHC surrogates in our population and analyzing the relation of the molecular subtypes with the other various clinicopathological features of invasive breast carcinoma. The objective was to study the clinicopathological profile and molecular subtypes of invasive breast carcinoma.

MATERIALS AND METHODS
This is a retrospective study on paraffin blocks of 90 cases of Invasive Breast Carcinoma specimens Exclusion criteria: Breast malignancies other than carcinoma. Trucut core biopsy Specimens The clinical data of patients including age, gender, and stage were obtained from the medical records section.
The histopathological data were collected from the pathological case files. Paraffin blocks which contained the tumor with adjacent tissue were collected for the study. Five micron sections were cut and stained with hematoxylin and eosin. Tumors were type specified and stage based on WHO guidelines. Invasive breast carcinomas were graded based on the Nottingham combined histologic grade (Elston-Ellis modification of Scarf-Bloom-B Richardson grading system). Immunostaining for ER was done using monoclonal antibody to estrogen receptor prediluted antibody procured from Biogenex laboratories. Immunostaining for PR was done using Mouse monoclonal antibody to progesterone receptor (Clone: PR88), procured from Biogenex Laboratories. Immunostaining for HER2neu was done using monoclonal antibody to c-erbB-2 Protein (HER2) prediluted antibody procured from Biogenex Laboratories. ER/PR and HER2 staining were reported as per the American Joint Committee on cancer protocol guidelines. A cutoff of a minimum of 1% of tumour cells showing nuclear positivity for ER/PR was considered positive (ASCO guidelines, 2010). Reporting Immunohistochemical results of Her2neu was done in the following manner (Table 1). No immunoreactivity or immunoreactivity in < or equal to 10% of tumour cells. 1+ (Negative) Faint weak immunoreactivity in >10% of tumour cells but only a portion of the membrane is positive.
Weak to moderate complete membrane immunoreactivity in >10% of tumour cells or circumferential intense membrane staining in < or equal to 30% of cells.
More than 30% of the tumour cells must show circumferential intense and uniform membrane staining. A homogeneous (chicken wire) pattern should be present.
The cases were classified according to the molecular classification based on the ER, PR and HER2 receptor status. (Table 2).

RESULTS
The study parameters include age, laterality, size of the tumor, clinical staging, histopathological grade, lymph node status and molecular subtyping.
Age: The age of the patients ranged from 20 to 80 years and above. The mean age was 54.5years. Highest incidence was noted in the 51-60year age group accounting for 28% of the cases. The second highest incidence was seen in the 41-50 years age group accounting for 19% of cases followed by the age group of 61-70 years (15%; Graph 1).

Tumor Laterality:
The incidence of right breast carcinoma 53% which is higher when compared to the incidence of left breast carcinoma 37% (Graph 2). Among the left sided breast carcinoma 19 cases showed nodal positivity, 16 cases showed nodal positivity on the right side (Graph 3).

Molecular Classification
The most common molecular subtype was Luminal A accounting for43.3% of cases, closely followed by Triple Negative subtype with 25.6% % of cases. Luminal B and Her 2 subtypes were 16.7% and 13.3% respectively (Graph 9).
There was statistically insignificant association seen between tumour staging and molecular classification (p value=0.054

DISCUSSION
The various prognostic factors that determine patient therapy and outcome include age, tumor burden, histological type, grade, lymph node status and hormone receptor status. According to a study done by Leong et al., the peak age for breast cancer is between 40 and 50 years in the Asian countries, whereas the peak age in the Western countries is between 60 and 70 years. In our study, the patient's age ranged from 51-60 years with mean age of 54.5. Studies by Zeeneldin et al., and Weiss et al showed increased preponderance for left breast carcinoma than right (2,3). In a study done by Fatima et al, it was mentioned that right sided breast carcinoma tend to occur at a younger age with higher incidence of nodal metastasis (4). This study showed an increased incidence of right breast carcinoma than left breast carcinoma. Nodal metastasis is seen more in the left breast carcinoma (Graph 3).
Li et al., collected all invasive breast cancer cases for a period of 15 years which showed invasive ductal carcinoma (80.2%) was the most common type, followed by lobular (11.8%) and mucinous type (2.4%). Saxena et al studied various morphological variants in 569 invasive breast cancers out of which 502 cases (88.2%) were invasive ductal carcinoma with no special type (NST). In our study, invasive ductal carcinoma was the most common type followed by mixed and papillary carcinoma, mucinous and metaplastic carcinoma in the decreasing order of frequency. Rakha et al., studied around 2,608 cases out of which the author found that Grade 3 tumors (45.6%) were the highest in the study population (5). A study done in Mumbai, India on 1022 breast cancer patients reported a higher proportion (70%) of patients were found to have grade 3 tumours (6). In contrast, we found that the incidence of Grade 2 tumours were high.
According to Leonard et al., patients with high grade ductal carcinoma in situ are at highest risk of developing recurrence (7) . Similar results were found in our study with 41 cases of high-grade ductal carcinoma in situ. Breast cancer staging significantly influences the overall survival rate. Diagnosis of breast cancer at an early tumor stage remains vital. According to a study done by Abbas et al., where they studied 60 samples of cases of breast cancer, showed pT2 was the most common group contributing to 56% (8). Similar results were also seen in our study where 62% of cases fell under the group pT2. Metastasis to the regional lymph node or internal mammary lymph node has an overall poor survival rate. In our study, 44 cases fell under the category of pN0 status which was seen similar to study by Talvensaari et al., (9).
, he found significant differences between the subtypes and grade (p=0.000). Both basal like and Her2 over expressing subtypes were associated with a higher grade, which is also seen in our study.

CONCLUSION
Breast cancer is heterogeneous neoplasm with many factors affecting its prognosis. The routine histopathological features provide an inexpensive method for understanding the tumor biology and prognosis. It's essential in areas with poor resources. ER, PR and HER2 assessment helps in identifying hormonal status and enables to start the patient on hormone therapy and anti HER2 therapy. The molecular classification can be used an adjunct to the histopathological findings.
The most commonly used IHC surrogates are oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), dividing invasive mammary carcinoma into luminal, HER2, and triple-negative subtypes. More genetic features will be available in the future which may throw light into the finer subtypes and ultimately help in accurate prognostication and treatment.