|Year : 2020 | Volume
| Issue : 2 | Page : 190-197
Study of ER, PR, HER2/neu, p53, and Ki67 expression in primary breast carcinomas and synchronous metastatic axillary lymph nodes
Tejal Abasaheb Khande, Avinash R Joshi, Siddhi Gaurish Sinai Khandeparkar, Maithili M Kulkarni, Bageshri P Gogate, Aniket R Kakade, Piyush D Sahu, Chaitnya D Khillare
Department of Pathology, Shrimati Kashibai Navale Medical College and General Hospital, Pune, Maharashtra, India
|Date of Submission||15-Sep-2018|
|Date of Decision||25-Dec-2018|
|Date of Acceptance||12-Jan-2019|
|Date of Web Publication||17-May-2020|
Siddhi Gaurish Sinai Khandeparkar
Department of Pathology, Shrimati Kashibai Navale Medical College and General Hospital, Pune, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Breast cancer (BCA) is the second most common cancer among women in India and accounts for 7% of global burden of BCA. The axillary lymph node status is an independent prognostic factor. The combined estrogen receptor (ER), progesterone receptor (PR), and HER2/neu biomarker expression is a predictor of BCA status for therapeutic guidance. Studies have demonstrated that these biomarkers are unstable throughout their tumor progression. Varying concordance and discordance rates in the biomarker expression between primary breast carcinoma (PBC) and metastatic axillary lymph node (MALN) status are reported.
Aim: This study was conducted for studying and comparing the expression of immunohistochemistry (IHC) markers, i.e., ER, PR, HER2/neu, p53, and Ki67 between PBC and their corresponding MALN for prognostication and therapeutic purpose. Methods: Sixty cases of PBC with metastasis to axillary lymph nodes diagnosed between years 2008 and 2014 were included in the study. A technique of manual tissue array was employed for cases subjected to IHC. Analyses of the expression of IHC markers were attempted between the PBC and their corresponding synchronous MALN and classified as concordant or discordant. Results were subjected to statistical analysis.
Results: Substantial agreement was observed for biomarker ER, PR, HER2/neu, p53, and Ki67 expression between PBC and MALN with k-value 0.79, 0.75, 0.89, 0.7, and 0.6, respectively.
Conclusion: There was high concordance for the IHC markers: ER, PR, HER2/neu, p53, and Ki67 expression in matched pairs of PBC and corresponding synchronous MALN. However, the discordance noted in small subgroups cannot be overlooked. Thus, there is a need to perform ER, PR, HER2/neu, p53, and Ki67 IHC studies routinely in both PBC and MALN to help design therapies that are tailored to target the specific tumor clones and render maximum benefit to patients.
Keywords: Breast carcinoma, estrogen receptor, HER2/neu, Ki67, metastatic axillary lymph node, p53, progesterone receptor
|How to cite this article:|
Khande TA, Joshi AR, Khandeparkar SG, Kulkarni MM, Gogate BP, Kakade AR, Sahu PD, Khillare CD. Study of ER, PR, HER2/neu, p53, and Ki67 expression in primary breast carcinomas and synchronous metastatic axillary lymph nodes. Indian J Cancer 2020;57:190-7
|How to cite this URL:|
Khande TA, Joshi AR, Khandeparkar SG, Kulkarni MM, Gogate BP, Kakade AR, Sahu PD, Khillare CD. Study of ER, PR, HER2/neu, p53, and Ki67 expression in primary breast carcinomas and synchronous metastatic axillary lymph nodes. Indian J Cancer [serial online] 2020 [cited 2020 May 28];57:190-7. Available from: http://www.indianjcancer.com/text.asp?2020/57/2/190/284481
| » Introduction|| |
Breast cancer (BC), a major health burden both in the developed and developing countries, is the foremost cause of death in women worldwide with more than one million cases occurring annually. The axillary lymph node-status is an inde-pendent prognostic factor for disease free and overall survival of BCA. Estrogen receptor (PR) and progesterone receptor (PR) were the first predictive molecular markers utilized for BCA. Patients with positive status of these steroid hormone receptors generally have high sensitivity to hormone therapy. The next molecular marker that was included in clinical practice was HER2/neu, the positive status of which in BCA correlates with high sensitivity to targeted therapy with trastuzumab. However, despite recognition of these prognostic and predictive factors and prompt utilization of the effective therapies, 20%–30% of BCA patients present with relapse. Yet, treatment decisions are solely based on the biological features of the primary breast carcinoma (PBC).
Studies have demonstrated that these biomarkers are unstable throughout tumor progression. Varying concordance and discordance rates in the biomarker expression between PBC and metastatic axillary lymph node (MALN) are reported., The explanations proposed for this alteration are intratumoral heterogeneity, inherent host and tumor biological factors, and adjuvant therapy that may have been administered to the PBC patients. In such circumstances, therapy provided to the patients, if is solely based on PBC biomarker expression profile, may not be the optimum treatment.
High Ki-67 is a sign of poor prognosis associated with a good chance of clinical response to chemotherapy. Studies show that p53 mutations are present in up to 50% of invasive breast carcinomas with poor clinical outcome. There are studies which document concordance and discordance in the expression of these proliferative markers, i.e., p53 and Ki67 between PBC and their corresponding MALN.,,,
This study was conducted for studying and comparing the expression of ER, PR, HER2/neu, p53, and Ki67 between PBC and their corresponding MALN for prognostication and therapeutic purpose.
| » Materials and Methods|| |
This is a cross-sectional/descriptive study. Ethical clearance was obtained from institute's “Ethical Clearance Committee. ” Sixty cases of PBC with MALN and diagnosed from operated upon 2008 to 2014 were included in the study. The available data for all the patients as regard age, location of tumor, menopausal status, and stage were collected from the records.
Only those cases of PBC were included in the study which showed MALN at the time of presentation of the disease (synchronous MALN). Cases in which records/slides/blocks were not available, stage I and stage IV PBC cases with MALN and distant metastasis were excluded. Patients who had received any hormonal therapy or HER2 targeted therapy with Herceptin or any other chemotherapy prior to the modified radical mastectomy (MRM) surgery were not included in this study.
The MRM specimens of the test population were evaluated histopathologically. All the slides were evaluated by two senior histopathologists. The modified Bloom–Richardson system of cancer grading system was used in this study. TNM classification and staging of the cases were done as per AJCC guidelines.
The most suitable tissue block of PBC and paired MALN was selected for immunohistochemistry (IHC) evaluation. A technique of manual tissue array was employed for all the cases subjected for IHC. The primary antibodies used were ER (Clone 6F11; Novacastra), PR (Clone PGR312; Novacastra), HER2/neu (clone CB11; Novacastra), p53 (clone DO-7; Dako), and Ki67(clone MM-1; Novacastra). Negative control (without adding primary antibody) was included in all batches. Section from endometrial tissue was used as positive control for ER and PR. Section from BCA, which previously showed unequivocal strong immunoreactivity for HER2/neu, was used as positive control for HER2/neu. Section from prostate and skin was used as positive control for p53 and Ki67, respectively. Sections were examined under high-power field to observe the immunoreactivity.
Allred score was used to evaluate the ER and PR, and a score of 3–8 was considered positive. Immunoreactivity for ER and PR was assessed by estimating the percentage of tumor cells showing nuclear staining. More than 10% of the tumor cells showing immunoreactivity were considered as positive. HER2 staining was scored according to the American Society of Clinical Oncology/College of American Pathologists guidelines. Moderate to strong complete membrane staining of 10% or more of the tumor cells was considered to be positive (2+ and 3+) p53 expression in ≥10% of the tumor cell nuclei was labeled as positive. Ki-67 Labeling index (Ki67LI) was recorded as percentage of positively stained tumor nuclei in 1,000 tumor cells in the hot spot of tumor. The Ki67LI of >10% was labeled as positive.
The statistical analysis was done using Primer software and Epi Info Version 7.0 (manufactured by McGraw-Hill). Data analysis was done with the help of Chi-square test. Kappa statistics was used to see the agreement (concordance) and disagreement (discordance). Quantitative data were presented with the help of mean. Qualitative data were presented with the help of frequency and percentage table. P values of ≤0.05 were considered statistically significant.
| » Results|| |
This study comprised of 60 cases of PBC with synchronous MALN. The various clinicopathological features of PBC and MALN are mentioned in [Table 1]. IDC included one case of IDC with ductal carcinoma in situ and two cases of IDC with Paget disease of nipple. In all the 60 cases studied, it was observed that minimum of four lymph nodes (LNs) were dissected in each case. In one case, a maximum of 34 LNs were dissected. The mean number of LN dissected was 16.2. In 14 cases, the total number of LN dissected ranged from 4 to 9. In the remaining 46 cases, ≥10 LN were dissected in each case. In the 60 cases studied, there were 7 cases showing 1 MALN and 1 case in which the tumor metastasis was seen in 31 ALN. The mean value for MALN was 7.6. The PBC were divided based on molecular classification and their clinicopathological features are given in [Table 2].
|Table 2: Clinicopathological characteristics of molecularly classified 60 cases|
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Hormonal receptor status was compared between PBC and MALN [Table 1] and [Figure 1]. ER positivity was observed in 45% cases of PBC, which reduced to 41.7% cases in the MALN (P = 0.854). PR positivity was observed in 30% cases of PBC, which reduced to 23.3% cases in MALN (P = 0.536). Her-2/neu protein positivity was noted in 40% cases of PBC, which reduced to 35% cases in the MALN (P = 0.706). p53 positivity was observed in 53.3% cases of PBC, which increased to 56.7% cases in the MALN (P = 0.854). KiLI was ≥10% in 63.3% cases in the PBC, which increased to 66.7% cases in the MALN (P = 0.848). Thus, there was no statistically significant difference between expression of biomarkers in PBC and MALN.
|Figure 1: Photomicrograph showing (a) strong nuclear estrogen receptor (ER), (b) strong nuclear progesterone receptor (PR), and (c) uniform intense membrane HER2/neu immunoreactivity in invasive ductal carcinoma (NST) of breast. Photomicrograph showing (d) strong nuclear ER, (e) strong nuclear PR, and (f) uniform intense membrane HER2/neu immunoreactivity in tumor cells of NST of breast metastasized to the ALN (IHC, ×400)|
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In [Table 3] substantial agreement was observed for biomarker ER, PR, HER2/neu, p53, and Ki67 expression between PBC and MALN with k-value 0.79, 0.75, 0.89, 0.7, and 0.6, respectively. Concordance of biomarker (ER, PR, HER2/neu, P53, and Ki67) expression between PBC and synchronous MALN was high.
|Table 3: Concordance and discordance of IHC marker (ER, PR, HER2/neu, p53, and Ki67) expression between PBC and MALN|
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| » Discussion|| |
All the treatment predictive IHC biomarkers (ER, PR, HER2, Ki67, and p53) are routinely analyzed in PBC and are crucial for choosing the most appropriate adjuvant therapy after BCA surgery. Administration of tamoxifen in ER, PR positive, and trastuzumab in HER2-positive PBC is standard practice. It is observed that tamoxifen reduces the risk of BCA recurrence by 50%, after 5 years of treatment in women with early BCA. However, despite the prompt tamoxifen adjuvant therapy, 15%–20% of these women presented with relapse. In another study, it was observed that 60% of hormone receptor-positive PBC patients derived no benefit from endocrine therapies. Only 30%–40% patients receiving trastuzumab derived benefit.
There are important explanations for this tumor resistance to the adjuvant therapy. PBC demonstrate intratumoral heterogeneity with various metastatic capacities. Inherent host and tumor biological factors along with microenvironment in the blood and other organs are involved in the clonal expansion and metastatic spread of cells with specific characteristics. This micrometastatic spread of disease is not targeted by adjuvant therapy, as the selection of treatment at the time of diagnosis is based solely on expression profile of biomarkers in the PBC. In this study, 10% cases showed ER and PR and 5% cases showed HER2/neu expression discordance between PBC and MALN.
Lack of stability is noted in clinically used markers throughout tumor progression. One in three patients with BCA experiences alteration of hormone receptor status, and 15% of patients experience a change in HER2/neu status during tumor progression. The importance of changed receptor status in primary and local or distant metastases is that it is associated with a statistically significant differential survival in women harboring BCA. Importantly, patients with ER-positive disease at relapse have better prognosis than ER-negative patients, independent of the ER status of the primary tumor. In contrast, PR status for the primary tumor rather than PR status at relapse seems to have an impact on patient survival. Adjuvant therapies administered may also influence and be a cause for the variability in the biomarker expression throughout tumor progression. Since tumor instability is seen not only between the primary and relapse setting but throughout tumor progression, this dynamic alteration renders clinical decisions more difficult and emphasizes the potential need for taking biopsies in a consecutive manner in the advanced setting to optimize treatment for the patient.
Various studies have reported varying expression of these tumor markers in PBC and their MALN,,,,, [Table 4], [Table 5], [Table 6], [Table 7]. In our study, overall ER and PR immunoexpression was less in the MALN as compared with that in PBC as seen in study done by Azam et al. However, in the study done by Atasevena et al., overall ER expression was more and PR expression was less in the MALN as compared with the PBC. In our study, the overall positivity of HER2/neu expression in PBC was more as compared with MALN as observed in a study done by Santinelli et al. A few other studies show slight amplification of HER2/neu expression in the MALN compared with PBC., Overall, p53 expression was more in the MALN compared with the PBC observed in the study conducted by Cardoso et al. However, in the study by Arun et al., overall p53 expression was less in the MALN as compared with its expression in PBC. Overall Ki67 expression increased from 63.3% in PBC to 66.7% in the MALN in our study. Similar findings were noted in studies done by Falck et al. and Buxant et al.,
|Table 4: Comparison of results of concordance and discordance of ER, PR, and HER2/neu IHC expression between PBC and MALN in this study with other studies|
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|Table 5: Comparison of results of concordance and discordance of p53 expression between PBC and MALN in this study with other studies|
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|Table 6: Comparison of results of concordance and discordance of Ki67 expression between PBC and MALN in this study and other studies|
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|Table 7: Various studies with their results (concordance/discordance) on comparison of IHC markers between matched pairs of PBC and MALN|
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In this study, out of the six discordant cases, in four cases, ER was positively expressed in PBC, but its expression was negative in MALN. Out of the six discordant cases; in five cases, PR was positively expressed in PBC, but its expression was negative in MALN. Similar type of change in the discordant cases, i.e., ER-positive PBC to ER-negative- MALN, and PR-positive PBC to PR-negative MALN, was observed in 6% and 9% cases, respectively, in the study done by Azam et al. and in 18% and 14.6% cases, respectively, in study done by Aitken et al.,
We know that a proportion of ER-positive and PR-positive patients fail to respond to tamoxifen and have a poor outcome. In one study, it was argued that patients with hormone receptor-positive PBC, but hormone receptor-negative MALNs were inappropriately treated with endocrine therapy, which was solely based on the hormone receptor status of the PBC and that they could be spared of unnecessary therapy. In our study, negative ER expression in PBC changed to positive expression in MALN in two cases (3.3%). Similarly, negative PR expression in PBC changed to positive in MALN in one case (1.7%). Thus, initially if the patient was not receiving any hormonal therapy, there could be benefit with administration of targeted therapy, as metastatic site is hormone receptor positive.
In this study, in 5% cases, loss of HER2/neu expression was noted in the MALN as compared with PBC as reported by few other authors., Herceptin is administered in patients in whom the breast tumor overexpresses HER2/neu. However, if the HER2 status at the metastatic site is different than what it is at the PBC, then therapy would have to be revised. The study also supported the finding that patients who have discordance in HER2/neu status have poor prognosis compared with those who have concordance. It is essential that biopsy at the metastatic site be performed.
In this study, p53 was discordant in 8/60 (13.3%) cases. Among the discordant cases, in five cases, i.e., 8.3% cases, the expression changed from negative in PBC to positive in MALN. Similarly in studies done by many workers, varying proportions of differences were noted in the p53 expression in matched pairs of PBC and MALN.,, Particularly, p53 being a proliferation marker, its overexpression in MALN as compared with its expression in PBC is a sign of aggressive nature of the tumor. Thus, as complete concordance is not observed in p53 expression at PBC and MALN, it is important to study the difference in immunoexpression at the two sites for making effective treatment decisions.
In this study, discordance in Ki67 expression was in 6/60 (10%) cases, where Ki67 expression changed from negative in PBC to positive in MALN. This implies that in these cases, the KiLI in PBC was <10% but increased to ≥10% in MALN. In a study done by Park et al., discordance was observed in 28.4% cases. In another study done by Tawfiq et al., it was observed that, Ki67 expression showed 81.3% concordance in PBC and MALN. These patients had a favorable outcome. However, a small subgroup, i.e., 6.7% cases, were noted to have a nodal Ki67 of ≥10% as compared with <10% in PBC and these patients were reported to have worse survival. The group of patients in which, KiLI decreased to <10% in MALN as compared with PBC, had better overall survival. This implies that there are prognostically significant biological differences between primary and metastatic tumor cells and that identifying subpopulations of metastatic tumor cells with high proliferative activities may provide critical prognostic information and assist in the selection of therapeutic options.
It is, thus, evident that some studies show substantial concordance, whereas some others demonstrate significant amount of discordance between matched pairs of PBC and MALN,,,,,,,, [Table 6]. In our study, concordance between biomarker expression in PBC and synchronous MALN was high when analyzed for IHC markers: ER, PR, HER2/neu, p53, and Ki67. However, the discordance noted in small subgroup of patients cannot be overlooked. Subtle, dynamic changes happen all the time in the tumor, due to its crosstalk with surrounding environment and ongoing carcinogenesis. Inherent host and tumor biological factors are also involved in the clonal expansion and metastatic spread of cells with specific characteristics. Patients with BCA, thus, may experience altered hormone receptor status throughout tumor progression. Thus, there is a need to perform ER, PR, HER2/neu, p53, and Ki67 IHC studies routinely in both PBC and MALN to help design therapies that are tailored to target the specific tumor clones and render maximum benefit to the patients.,
| » Summary and Conclusion|| |
There was high concordance for the IHC markers, ER, PR, HER2/neu, p53, and Ki67, expression in matched pairs of PBC and corresponding synchronous MALN. However, the discordance noted in small subgroup of patients cannot be overlooked. Patients with BCA thus may experience altered hormone receptor status throughout tumor progression. Thus, there is a need to perform ER, PR, HER2/neu, p53, and Ki67 IHC studies routinely in both PBC and MALN to help design therapies that are tailored to target the specific tumor clones and render maximum benefit to the patients.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]