Year : 2009 | Volume
: 52 | Issue : 2 | Page : 171--174
Hormone receptors over the last 8 years in a cancer referral center in India: What was and what is?
Tanuja Shet1, Atin Agrawal1, Mandar Nadkarni2, Mahendra Palkar1, Rohini Havaldar3, Vani Parmar2, Rajendra Badwe2, RF Chinoy1,
1 Department of Pathology, Tata Memorial Hospital, Parel, Mumbai - 400 012, Maharashtra, India
2 Department of Surgical Oncology, Tata Memorial Hospital, Parel, Mumbai - 400 012, Maharashtra, India
3 Department of Central Research Secretariat, Tata Memorial Hospital, Parel, Mumbai - 400 012, Maharashtra, India
Department of Pathology, Tata Memorial Hospital, Parel, Mumbai - 400 012
This study was carried out to observe the trend in hormone receptors over the last 8 years in a tertiary cancer center in India. A total of 11,780 tumors analyzed for hormone receptors over the last 7 years were compared with the results of hormone receptor expression in a prior published study on
798 cases of breast cancer from the same institute. The patient«SQ»s ages ranged from 18 to 102 years, Sixty percent of the patients were in the age group of 31-50 years. Seventy percent of the tumors were grade III tumors. The percentage of hormone receptor expression in breast cancer in the last 8 years varied from 52 to 57%. The overall receptor expression in the last 8 years shifted within a 5% range, confirming that the hormone receptor expression in Indian patients with breast cancer is low. However, there was redistribution within the pattern of estrogen receptor (ER) and progesterone receptor (PR) expression among tumors showing hormone receptor expression. Breast cancers showing only PR expression reduced dramatically from 21% in the year 1999 to in the year 2006, with a parallel increase in breast cancers showing combined ER and PR positivity (from 25 to 41.8%) and only ER expression (from 7.4 to 10.6%). The hormone receptor expression in breast cancers in India is and continues to be low but the high incidence of only PR-positive tumors in our population reported earlier was misrepresented.
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Shet T, Agrawal A, Nadkarni M, Palkar M, Havaldar R, Parmar V, Badwe R, Chinoy R F. Hormone receptors over the last 8 years in a cancer referral center in India: What was and what is?.Indian J Pathol Microbiol 2009;52:171-174
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Shet T, Agrawal A, Nadkarni M, Palkar M, Havaldar R, Parmar V, Badwe R, Chinoy R F. Hormone receptors over the last 8 years in a cancer referral center in India: What was and what is?. Indian J Pathol Microbiol [serial online] 2009 [cited 2019 Nov 20 ];52:171-174
Available from: http://www.ijpmonline.org/text.asp?2009/52/2/171/48909
Hormone receptor estimation in breast cancer has shifted from the biochemistry laboratory to the immunohistochemistry (IHC) laboratory and from there on to a surgical pathologist preview in the last decade. Immunohistochemical demonstration of the hormone receptors in breast cancer is here to stay given its convenience, cost effectiveness, applicability to paraffin-processed material and the fact that hormone receptors by this method are specifically assessed within the actual tumor.  It was observed that in patients receiving endocrine treatment, estrogen receptor (ER) positivity by IHC was associated with an improved disease-free survival (DFS) and overall survival, while no such significance was seen with the ligand binding assay/dextran-coated charcoal assay results. 
The main concern in the demonstration of hormone receptors by IHC is the high degree of interlaboratory variance. ,, In a quality assurance program by the United Kingdom National External Quality Assurance Scheme (UKNEQAS), where 200 labs from 26 countries participated, only one-third of the laboratories scored the low-expressing tumors correctly while one-third of the tumors were labeled as false negative. 
In a publication from our institute 5 years ago we documented a total hormone receptor expression of only 53.5% breast cancers in Indian patients as opposed to 75-80% reported in the western literature. , The percentage of tumors expressing only progesterone receptor (PR) was also high (21%) in our previous study. 
Was this low hormone receptor positivity and high percentage of only PR expression in breast tumors due to the lack of quality control or did it represent an unusual biology of tumors in this part of the continent?
Subsequent to our initial published work, we have implemented several changes in the processing of the tissues of breast cancer and in the IHC technique. We undertook this analysis of tumors at our hospital to observe whether the trend in receptors has changed following these measures over the last 7 years.
Materials and Methods
All the cases of breast cancer accessioned at our institute from the year 2000 to the year 2006 were retrieved to document the hormone receptor status. The previous data published in year 1999 was used for comparative purposes, making this an 8-year study.  A total of 11,780 cases were studied for hormone receptors during the 7-year study period while 798 additional cases had been evaluated in the previous study.  In the latter 7 years, we included only in-house cases where the processing was performed at our institute. All referral material was excluded from this analysis. Fixation of tissues till January 2001 was in 10% formalin and after that time it was in 10% neutral buffered formalin (NBF), with the pH monitored to 7.2.
IHC for the hormone receptors was performed using the Avidin Biotin complex method and the Vecta stain ABC kit, Vector laboratories, UK. Primary antibody used for the demonstration of the ER was monoclonal mouse antibody anti-human ER clone 1D5 and for the PR was monoclonal mouse antibody anti-human PR clone PGR636. Both the antibodies were from the Immunotech Company, Marseille, France till December 2000 and subsequently antibodies from the DAKO Company, Glostrup, Denmark were used. Antigen retrieval was performed by immersing the sections into preheated sodium citrate, pH 6, and either microwaving for 30min or pressure cooking for 5-10min (till 1 whistle blows). Samples before May 2001 underwent microwave-based antigen retrieval after which we shifted to the pressure cooking method for antigen retrieval. The rest of the steps were similar to the usual IHC method. Any tumor with >5% of cells showing nuclear staining was considered as positive.
Hormone receptors were positive in 6349/11,780 (53.9%) breast cancers tested for the hormone receptors over the last 8 years. All patients were females except 1.6%, who were males. Patients age ranged from 18 to 102 years; however, 60% of the patients were in the age group of 31-50 years of age. The tumor grade as per the modified Scarff-Richardson Bloom scoring system was Grade I = 2%, Grade II = 28% and Grade III = 70%.
The ER and PR expression of tumors over the last 8 years is given in [Table 1]. As seen from the Table, the percentage of hormone receptor expression in the last 8 years varied from 52 to 57%. An interesting drift in the pattern of ER and PR expression was observed. Comparing the pattern of hormone receptor expression in 1999 and 2006, the percentage of tumors showing only PR expression reduced remarkably from 21 to 3.4%, with a parallel increase in the number of tumors expressing both ER and PR (25-41.8%) and only the ER antigen (7.4-10.6%).
The hormone receptor status was significantly associated with the age of patients, as depicted in [Table 2]. The hormone receptor expression significantly correlated with tumor grade ( P -value P -value = 0.004), wherein infiltrating lobular carcinoma, mucinous carcinoma and variants of papillary carcinoma expressed hormone receptors at a significantly higher level than infiltrating duct carcinoma (IDC), not otherwise specified. When the results of ER positivity in specific histological types in the year 2005 were compared with our previous study in 1999, there were a few differences.
The percent ER positivity in IDC improved from 29 to 39% while it reduced from 63 to 49% in invasive lobular carcinoma (ILC). The PR expression on the other hand reduced from 44.8 to 34% invasive duct carcinoma and from 69.7 to 48% in ILC. The ER expression in mucinous cancers remained the same in both these years whereas the PR was reduced by half. This proves the redistribution within the ER and PR groups even in the special histological types of breast cancer. Hormone receptor estimation during the study period was performed only in patients with intraduct carcinoma showing foci of microinvasion. Of these, 5% showed ER and PR expression. One-third of the patients (39%) presented with tumors >5cm or locally advanced breast cancers. These patients however did not show any different hormone receptor expression than the operable breast cancers.
The Early Breast Cancer Trialists Collaborative Group has confirmed that the amount of benefit from adjuvant endocrine therapy in breast cancer is proportional to the amount of ER present in the primary tumor.  The option of hormonal therapy is now offered to every patient of breast cancer irrespective of age based on the tumor receptor status.  Hence, ER/PR estimation needs to be carried out in every patient diagnosed with breast cancer. The tremendous clinical importance of this test has improved its commercial value and coerced many laboratories to offer ER and PR testing in breast cancer. Consequently, there is a need for strict quality assurance in the immunohistochemical demonstration of hormone receptors in breast cancer.  With this in mind, many quality assurance programs across various countries have been started. , These have confirmed that there is a great degree of variability in the methodology of IHC demonstration of hormone receptors.
Recommendations for improving hormone receptor estimation include participation in an external quality assurance scheme for immunocytochemistry, including an audit of the percentage-positive tumors in a laboratory against the accepted norms annually.  For the last issue, it is important to record the standard hormone receptor expression in a given population. It is clear that there are some racial differences in hormone receptor expression in breast cancer.  This study was an attempt to audit the hormone receptor status of tumors at our institute in the last 8 years with a view to observe the trend and to analyze the actual status of hormone receptor expression in breast cancer at our institute.
The hormone receptor expression in breast cancers in India is low. , A study from the same city as ours attributed the incidence of ER negativity and PR positivity (ER-/PR+) to the use of suboptimal manual assays rather than true genetic differences.  The authors repeated 37 ER-/PR+ cases using automated IHC and a different set of ER antibodies. On repeat IHC, only 24.3% of the tumors remained ER negative while 28 (75.6%) tumors retested as ER positive.  We agree that high PR+ tumors reported in our earlier paper  were overrepresented due to underreporting of ER.
Nevertheless, the current study enforces a fact documented in a previous study from our institute  that the overall hormone positivity in India is low. The patient demographic features in this study are nearly similar to previous studies , from India indicating that the cause for the low receptor expression in Indian patients may be due to younger age and higher grade of breast cancers. The ER expression in our patient population in the best possible situation was 50.5% and the PR expression was 42% as opposed to the 75% ER and 58% PR positivity reported in the literature. 
The chief cause for false negativity in hormone receptor demonstration by IHC is inefficient antigen retrieval.  Different combinations of heating devices and antigen retrieval solutions are available. Ethylene Diammine Tetraacetic acid (EDTA) is thought to be a superior antigen retrieval solution irrespective of the heating method used.  Pressure cooking is found to be the most convenient heating method on practical grounds as it allows for the simultaneous handling of a large number of slides and saves time.  In this study, a stark jump in the percentage of positivity was observed in the year 2001 after implementing use of NBF for fixation, changing the antibody and using pressure cooking for antigen retrieval. We have introduced the pressure method for antigen retrieval in 2001 and this was associated with an increased detection of ER-positive tumors with a reduction in PR only expressing tumors. Hence, we agree that pressure cooking is a more accessible and effective method for antigen retrieval in hormone receptor estimation than the microwave-based methods in developing nations, although we continue to use sodium citrate rather than EDTA as the antigen retrieval solution for economical reasons.
The UKNEQAS documented that the hormone receptor estimation results are not compromised when histological material from different sources is processed by a common protocol of antigen retrieval and analyzed with automated IHC systems.  Most laboratories in Europe use alcoholic formalin for fixation of tissues from breast cancer for better demonstration of ER and PR.  In our experience, many laboratories in our country do not follow strict fixation policies and may not even use buffered formalin for fixation, resulting in suboptimal processing and some degree of irreversible damage.
Lowering the cut-off points for reporting hormone receptors will help in reducing the false negatives for hormone receptors in breast cancers. A consensus development panel of the US National Institutes of Health recommended in 2001 that any ER staining in breast cancers should be sufficient to consider a tumor ER positive and the patient a suitable candidate for endocrine therapy.  In a recent paper, the all or none method of scoring hormone receptors for scoring ER appeared valid as a prognostic indicator of survival in patients with positive lymph nodes.  We stuck to 5% positivity to ensure uniformity for this analysis although our practice is to report any degree of hormone receptors in our routine histology report.
There has been controversy about the importance of PR receptor estimation in breast cancer, with a strong opinion that PR estimation should be stopped.  But, several studies have shown that patients with PR+ tumors benefit with endocrine therapy although PR is a weaker predictor of response to endocrine therapy than ER. ,, Preliminary data suggest that there is no difference in DFS between tamoxifen and anastrozole in the subgroup of patients with ER- and PR+ tumors, while anastrozole was found to be significantly superior to tamoxifen in the subgroup of ER+ and PR− patients. 
The results of our study indicate that a high degree of only PR+ tumors in a laboratory calls for a need for quality check on the ER antigen retrieval methods. In this study, patients with only PR+ tumors received endocrine therapy, which would have not been the case if the PR testing had not been performed. In our opinion, PR serves as a check of the ever so frail ER testing and helps in quality control of the ER antigen.
To conclude, the high incidence of only PR+ tumors in our population is what was but hormone receptor expression in breast cancers in India is and continues to be low. This can be attributed to the younger age of patients and the increased incidence of Grade III cancers. The hormone receptor results appear to have now stabilized at our institute but there is scope for additional quality checks from laboratories abroad. We have started using the polymer technology for demonstration of hormone receptors in breast cancer recently and it will be some time before these results emerge. Lessons from this exercise were useful to other states in our country and may help others facing similar problems.
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