European Journal of Cardiovascular Medicine

Carcinoma Breast is the most common non-skin malignancy in women and has surpassed lung cancer. According to GLOBOCAN cancer statistics 2020, Female breast cancer is the most commonly diagnosed cancer with an estimated 2.3million new cases and 685,00 deaths (1). The most common clinical presentation is painless breast lump followed by axillary lump, retracted nipple, painful lump and nipple discharge (2). The diagnosis of Breast cancer can be achieved by “The Triple Assessment Test” for assessment of breast lump which includes palpation, imaging and percutaneous biopsy (3). All the trucut biopsies are tumor typed and assessed by immunohistochemistry (IHC) for tumor biomarkers status such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2/NEU). Biomarkers status are routinely done in breast carcinoma patients and their expression pattern has been clinically used to guide therapy and predict survival. ER and PR are nuclear hormone receptors while HER2/NEU proteins are cell membrane receptors on breast cells. Based on the hormonal receptor status and HER2/NEU breast cancers are molecularly classified as Luminal type A, Luminal type B, HER2/NEU enriched and Basal like (4). Treatment modalities include surgery, radiation therapy, hormonal therapy, chemotherapy and targeted therapy depending upon the type and extent of disease.

Neoadjuvant chemotherapy [NACT] also known as primary or pre-operative chemotherapy has become a widely used approach to downstage the locally advanced breast carcinoma, making it operable by reducing the vascularity and tumour size. NACT aims to eradicate possible distant micrometastatic disease and to increase breast conservative therapy (5). Most commonly used NACT regimen include four cycles of either Adriamycin and Cyclophosphamide or Epirubicin and Cyclophosphamide followed by four cycles of taxanes. NACT induce various histopathological changes in mastectomy specimen and lymph node (6). However, standard guidelines for pathological evaluation of breast specimen after NACT have not been established. It is proposed in some studies that there may be an alteration in the hormonal receptor status such as Estrogen receptor, Progesterone receptor and human epidermal growth factor receptor-2 following NACT which predicts prognosis, guides clinical management and targeted therapy (5). After NACT patients are categorized on the basis of pathological response as Pathologic complete response (pCR) in which no residual tumour is present in the mastectomy specimen as well as in lymph nodes, pathologic partial response- presence or absence of residual tumour either in the mastectomy specimen or in lymph nodes and pathologic no response (pNR) - no residual tumour in mastectomy specimen and lymph nodes. The purpose of the study was:

1. To evaluate the post- neoadjuvant chemotherapy (NACT) histopathological changes in the mastectomy specimens and lymph nodes.
2. To compare the Immuno-histochemistry (IHC) profiles of hormone receptor status ER, PR and HER2/NEU before and after neoadjuvant chemotherapy.
3. To categorize the patients according to the pathological response.

This was a hospital based prospective study done in the department of Pathology Guru Gobind Singh Medical College and Hospital, Faridkot (Punjab). The study was conducted over a period of one year which includes 50 cases who were diagnosed as carcinoma breast on trucut biopsy and had received neoadjuvant chemotherapy (NACT) while the patients of breast carcinoma who have not received any pre-surgical neoadjuvant chemotherapy and review slides/ blocks were excluded. All the trucut biopsies were tumor typed and then subjected to immunohistochemistry (IHC) for assessment of tumor biomarkers ER, PR and HER2/NEU receptor status. ER and PR are nuclear receptors and their status was assessed by using Allred scoring system which combines both proportion score (PS) and intensity score (IS). Brown nuclei were taken as positive for ER and PR receptors. Score 0-5 is given to cells depending on the proportion of cells which are stained (PS) and score 0-3 is given depending on the intensity of staining (IS). For ER and PR an Allred Score (AS) of 0-2 was considered as negative and Score of 3-8 was considered as positive as shown in table-1[4, 5]. HER2/NEU status was assessed by using HER2/NEU scoring system and depending on the intensity of staining a score of 0-3 is given to cells. Score of 0 and 1+ was considered as negative, Score of 2+ was considered as weakly positive/equivocal while score of 3+ was considered as strongly positive as shown in table- 2 [4, 5].

Then patients received four cycles of NACT (Adriamycin or Epirubicin and Cyclophosphamide). After neoadjuvant chemotherapy (NACT) the Modified radical mastectomy (MRM) specimens were received in 10% buffered formalin. The specimens were adequately fixed and grossed, details like presence or absence of residual and lymph nodes were noted. The specimens taken were processed and paraffin blocks were made. The blocks were cut and sections were stained by Haematoxylin and Eosin (H & E) staining method then mounted by DPX. The slides were examined microscopically for NACT induced histopathological changes.  All the parameters like presence or absence of residual tumor, ductal carcinoma in situ, inflammation, necrosis, fibrosis, calcification, lymphovascular invasion, perineural invasion and lymph nodes metastasis were observed. The residual tumor was graded on the basis of Miller Payne Grading system as shown in table- 3 [7].

After complete evaluation of the histopathological findings in mastectomy and lymph nodes, all the patients were categorized into three categories as follows:

1. Pathologic complete response (pCR): No residual tumour present in the mastectomy specimen and no residual tumor in lymph nodes.
2. Pathologic partial response (pPR): Presence or absence of residual tumour in the mastectomy specimen +/- lymph nodes metastasis.
3. Pathologic no response (pNR): Residual tumour in mastectomy specimen and metastatic deposits in lymph nodes.

Following neoadjuvant chemotherapy (NACT) the specimens with residual carcinoma were again subjected to immunohistochemistry (IHC) tumor biomarkers- ER, PR and HER2/NEU status.

Then comparison of ER, PR and HER2/NEU status was done between pre and post NACT specimens which showed presence of microscopic residual tumor.

Statistics: The data was entered in microsoft excel and analysis was done by using SPSS 23.0 ver. The association between categorical variables was explored using Chi square test and Wilcoxon Signed Rank tests. A p value of <0.05 was considered as statistically significant.

The present study reported that out of 50 patients the mean age of patients was 50 years. On histopathology all 50 cases were diagnosed as invasive carcinoma breast. Pre NACT hormonal receptor- ER, PR and HER2/NEU status in 50 cases were: ER expression was positive in 50% while negative in 50%, PR expression was positive in 44% while negative in 56% and HER2/NEU expression was positive in 16%, equivocal in 12% while negative in 76% as shown in Table 4. Then all the patients received 4 cycles of either Adriamycin and Cyclophosphamide or Epirubicin and Cyclophosphamide followed by four cycles of Taxanes.

Table- 1: Allred scoring system for ER and PR receptors

Table- 2: HER2/NEU scoring system depending on the intensity of staining of cells

Table- 3: The Miller and Payne grading system

Following NACT, Residual tumor grading based on the Miller and payne grading system showed MPG- I in 10%, MPG- II in 58%, MPG- III in 8%, MPG- IV in 6% and MPG- V in 18% as shown in table- 4.

Table- 4: showing results of Pre NACT expression of ER, PR and HER2/NEU status, Miller and Payne grade, Post NACT histopathological changes in mastectomy and lymph nodes, Post NACT expression of ER, PR and HER2/NEU status.

 Out of 50 cases, ductal carcinoma in situ (DCIS) component was seen in 14%, chronic infiltrate (lymphocytic) in 88%, necrosis in 74%, fibrosis in 90%, calcification in 12% and lymphovascular invasion (LVI) in 20% as shown in table- 4. Lymph node with metastatic deposits (MD) were seen in 25 cases (50%) and 25 cases (50%) were free of lymph node metastasis. Among 25 cases with positive lymph node metastasis, post NACT lymph nodes showed NACT related changes- inflammatory infiltrate in 4%, necrosis in 16%, fibrosis in 14%, calcification in 4% and 12% cases were without NACT related changes as shown in table- 4. Out of total 50 cases, 7 cases showed pathological complete response (pCR), 24 cases showed pathological partial response (pPR) and 19 cases showed pathological no response (pNR) as shown in table- 4.  

In our study, among 50 cases of breast carcinoma, 43 cases showed presence of microscopic residual tumor while 7 cases showed no residual tumor. Hence post NACT the hormonal receptor ER, PR and HER2/NEU expression evaluation was done only in 43 cases which showed residual tumor. ER expression was positive in 33% and negative in 67%. PR expression was positive in 23% and negative in 77%. HER2/NEU expression was positive in 16%, equivocal in 12% and negative in 72% as shown in table-4.

Comparison of hormonal receptor ER, PR and HER2/NEU status between pre and post NACT cases was done only in those 43 cases which microscopic residual tumor in post NACT cases.

Out of 43 cases, on immunohistochemical examination prior to NACT ER positive expression was seen in 46.5% and negative in 53.5%. Following NACT ER expression was positive in 33%   and negative in 67%. A statistically significant loss of ER expression was noted when comparison was made in pre and post NACT cases with p value of 0.020 as shown in table-5.

Before NACT PR positive expression was seen in 37% and negative in 63%. Following NACT PR expression was positive in 23% and negative in 77%. A statistically significant increase in PR negative expression was documented when comparison was done in pre and post NACT cases with p value of 0.014 as shown in table-5.

Table- 5: Showing pathological response in post NACT cases.

Prior to NACT HER2/NEU expression was positive in 19%, equivocal in 14% and negative in 67%. Post NACT HER2/NEU expression was positive in 16%, equivocal in 12% and negative in 72%. There was no statistically significant difference observed between pre and post NACT cases with p value of 0.180 as shown in table-5.

Table- 6: Showing results of pre and post NACT comparison of ER, PR and HER2/NEU status which showed residual tumor in post NACT cases.

     P Value: p> 0.05; Not significant, p< 0.05: Significant, p< 0.001 Highly significant

FIGURE LEGENDS:

 Figure:1- Showing comparison of ER, PR and HER2/ NEU status before and after NACT

 in trucut biopsy [pre NACT] and mastectomy specimen [post NACT] (A-H).

A: H&E stained section of trucut biopsy showing invasive carcinoma breast (400X), B: Pre NACT ER showing strong nuclear positivity (score-8) (400X), C: Pre NACT PR showing strong nuclear positivity (score-8) (400X), D: Pre NACT HER2/NEU showing negative membrane staining (score-0) (100X), E: Post NACT mastectomy showing Residual tumor (400X), F: Post NACT ER showing negative nuclear staining (score-0) (400X), G: Post NACT PR showing negative nuclear staining (score-0) (400X), H: Post NACT HER2/NEU showing negative membrane staining (score-0) (400X).

Figure: 2- Showing pathological response after Neoadjuvant chemotherapy (NACT) as Pathological no response (A-C), pathological partial response- micro foci of residual tumor (D-F) and pathological complete response – no residual tumor (G-I)

A: Pre NACT H&E stained section showing invasive carcinoma (100X), B: Gross appearance of mastectomy specimen showing residual tumor (Post NACT), C: Post NACT H&E stained section from mastectomy showing residual tumor (100X), D: Pre NACT H&E stained section showing invasive carcinoma (400X), E: Gross appearance of mastectomy specimen showing residual tumor (Post NACT), F: Post NACT H&E stained section from mastectomy showing micro foci of residual tumor (400X) G: Pre NACT H&E stained section showing invasive carcinoma (100X), H: Gross appearance of mastectomy specimen showing no residual tumor (Post NACT), I: Post NACT H&E stained section from mastectomy showing no residual tumor (100X).

Figure:3- Showing post neoadjuvant chemotherapy (NACT) related histopathlogical changes in mastectomy specimen (A- F) and lymph nodes (G-I)

A: H & E-stained section showing ductal carcinoma in situ (DCIS) component (100X), B: H & E-stained section showing lymphovascular invasion (400X), C: H & E-stained section showing calcification (100X). D: H & E-stained section showing inflammatory cell infiltrate and residual tumor (400X). E: H & E-stained section showing fibrosis (100X). F: H & E-stained section showing foci of necrosis. G: H & E-stained section showing lymph node metastasis (100X). H: H & E-stained section showing lymph node necrosis (100X), I: H&E-stained section from lymph nodes showing fibrosis, calcification and inflammatory cell infiltrate (100X).

In our study the mean age of presentation was 50 years of age. In our study among 50 cases, pre NACT IHC expression of ER expression was positive in 50% cases and 50% cases were negative. Findings of our study were close to study by Kinsella MD et al. (2012) (8) and Shubham S et al. (2017) (9), in which ER positive expression was noted in 45% and 57% respectively Our study reported PR positive expression in 44% cases and negative in 56% cases. The findings of our study were in concordance with study by Kinsella MD et al. (2012) (8) who reported positive expression in 45% cases. Present study reported positive HER2/NEU expression in 16%, equivocal in 12% and negative in 72% cases. Almost similar findings were observed in the study conducted by Nandyal SN et al. (2015) (10) who reported positive HER2/NEU expression in 20% cases.

 According to Miller Payne grading system, out of 50 cases, MPG- I was seen in 10%, MPG- II in 58%, MPG- III in 8%, MPG- IV in 6% and MPG- V in 18% cases. Almost similar findings were reported by Mohapatra M et al. (2020) (11) and Hemavathi N et al. (2021) (12) in which majority of cases were in MPG- II 51% and 50% cases respectively. Our findings were discordant with studies done by Ogston KN et al. (2003) (13) and Corben AD et al. (2013) (14) in which majority of cases were in MPG- III 27% and 29% respectively.

Post NACT spectrum of histopathological changes observed in mastectomy where DCIS component was reported in 14% cases. Our findings were in concordance to Nandyal SN et al. (2015) (10) and Jung YY et al. (2016) (15) who reported DCIS component in 10% and 14% cases respectively.  Chronic inflammatory cell infiltrate (lymphocytic) was observed in majority i.e 88% of cases, similar findings were reported in study conducted by Avci N et al. (2015) (16) in 93% and Figenschau SL et al. (2015) (17) in 87% cases. Necrosis was reported in majority of cases 74%. Our findings were in concordant with studies conducted by Sheereen S et al. (2018), (18), Philipose CS et al. (2019) (19) and Hemavathi N et al. (2021) (12) in which necrosis was observed in 74%, 72% and 60% cases respectively. On the contrary studies conducted by Sethi D et al. (2012) (6), Jung YY et al. (2016) (15) and Chakrabarti S et al. (2016) (20) who reported necrosis in 4%, 40% and 28% cases respectively. In the current study majority of cases showed fibrosis 90%. Our findings were in concordance to study done by Chakrabarti S et al. (2016) (20), Sheereen S et al (2018) (18), Gahine R et al. (2019) (21) and Hemavathi N et al. (2021) (12) who reported fibrosis in 64%, 64%, 96% and 83% cases respectively While a study by Philipose CS et al. (2019) (19) observed fibrosis in 23% cases only. The present study showed calcification only in 12% of cases. The results of our findings were in concordant with the study conducted by Sethi D et al. (2012)  (6) and Gahine R et al. (2019) (21) who reported calcification in 10% and 14% cases respectively. The current study observed LVI in 20% of cases. Similar findings were reported by Vasudevan D et al. (2015) (22) in which 27% of cases showed LVI [22]. Our results were discordant with studies by Ozmen V et al. (2015) (23) and Hemavathi N et al. (2021) (12) who reported LVI in 55% and 50% of cases. In our study out of 50 cases lymph node metastasis was reported in 25 cases (50%). Studies by Ahmad Z et al. (2016) (24) and Mohapatra M et al. (2020) (11) both reported lymph nodes metastasis in 75% of cases. In the current study lymph nodes with chronic inflammatory cell infiltrate was seen in 4% cases, necrosis in 16% cases, fibrosis in 14% cases and calcification in 4% cases. Almost similar findings were reported by Sethi D et al. (2012) (6) who reported lymph node calcification in 4.5% cases. While Chakrabarti S et al. (2016) (20) reported lymph node fibrosis in 30.8% cases.

In our study out of 50 cases, pathological complete response (pCR) was noted in 14% cases, pathological partial response (pPR) in 48% and pathological no response (pNR) in 38% cases. In our study majority of cases documented pathological partial response.  and Concordant findings were reported in the studies conducted by Moon YW et al. (2005) (25), Pu RT et al. (2005) (26), Vasudevan D et al. (2015) (22), Jung YY et al. (2016) (15), Gahine R et al. (2019) (21) and Mohapatra M et al. (2020) (11) who observed pathological partial response in 60%, 84%, 73%, 54%, 84% and 80% cases respectively. While studies by Sethi D et al. (2012) (6) and Sheereen S et al. (2018) (18) reported majority of cases with pathological no response constituted 60% and 67% cases respectively.

Out of 43 cases, post NACT ER expression showed positivity in 30% while 70% were ER negative. Our observations were in concordance with studies conducted by Kinsella MD et al. (2012) (8) who reported majority of cases with negative ER expression 55%. On the contrary studies conducted by Nikura N et al. (2016) (27) and Shubham S et al. (2017) (9) documented ER positive expression in majority of cases i.e 66% and 56% respectively.

Out of 43 cases, post NACT PR expression was positive in 23% and negative in 77%. Our findings were in concordance to the study done by Kinsella MD et al. (2012) (8) and Shubham S et al. (2017) (9) who reported ER positive expression in 21% and 22% respectively. However, on contrary Nikura N et al. (2016) (27) showed ER positivity in 45% cases.

 Out of 43 cases, 16% showed positive HER2/NEU expression, 12% showed equivocal HER/2NEU expression and majority of cases 72% showed negative expression. Almost similar results were noted in Kinsella MD et al. (2012) (8), Nikura N et al. (2016) (27) and Shubham S et al. (2017) (9) which showed positive HER/2NEU expression in 21%, 20% and 22% cases respectively.

Comparison of IHC expression of hormonal receptor- ER, PR and HER2/NEU in pre and post NACT cases was done only in those 43 cases which showed presence of residual tumor in post NACT specimen. A statistically significant loss of ER expression was noted with a p value of 0.020. Our findings were concordant with the study conducted by Taucher S et al. (2003) (28) in which 214 patients were treated with chemotherapy prior to surgery, out of which 14% cases showed a statistically significant loss of expression of ER (p- 0.02) in the post NACT specimen [28]. While the studies conducted by Kinsella MD et al. (2012) (8) and Shubham S et al. (2017) (9) documented no statistically significant change in ER expression following NACT. Also, a statistically significant increase in negative PR expression was noted when comparison was made   between pre and post NACT cases with a p value of 0.014. The results of our findings were concordant with a study by Taucher S et al. (2003) (28), Kasami M et al. (2008) (29), Kinsella M D et al. (2012) (8) and Shubham S et al. (2017) (9) in which a significant negative increase in PR expression was noted. On the contrary studies by Adams AL et al. (2008) (30) did not find any significant change in PR status before and after NACT. There was no statistically significant difference noted in HER2/NEU expression (p- 0.180) between pre and post NACT patients. Similar results were obtained in study conducted by Kinsella M D et al. (2012) (8) and Shubham S et al. (2017) (9) in which no significant difference in expression of HER2/NEU between pre and post NACT noted. While in a study by Adams AL et al. (2008) (30) it was seen that more HER2/NEU positive expression was noted in patients after receiving neoadjuvant chemotherapy with a statistically significant change in HER2/NEU expression (p- 0.027).

NACT can induce significant histomorphological changes in breast cancer and therefore it is important to evaluate the pathological response of resected tumor and lymph nodes by using tumor regression grading system. Characteristic histopathological changes observed after NACT are reduced tumour cellularity, ductal carcinoma in situ (DCIS), lymphocytic infiltration, necrosis, fibrosis, calcification and lymphovascular invasion (LVI). Assessment of pathological response to therapy provides valuable prognostic information to patients and helps in directing the effective hormonal/target treatment. Our study strongly supports the need for evaluation of ER, PR, HER2/NEU status of carcinoma breast patients both before and after neoadjuvant chemotherapy. The post NACT re-evaluation of ER, PR, HER2/NEU documented a significant change in receptor status which provide valuable information regarding change in hormonal/targeted therapy for breast cancer patients.

Acknowledgments: Authors would like to express sincere gratitude to Dr. Sarita nibhoria- Professor department of Pathology, Dr. Vaneet kaur sandhu- Associate Professor, Department of Pathology and Dr Pardeep Garg- Associate Professor, Department of Radiotherapy, GGS Medical College and Hospital, Faridkot,Punjab, India for providing invaluable guidance and support.

Conflicts Of Interest

The authors declare no conflict of interest.

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