| Abstract|| |
Context: It is well established that breast cancer subtypes differ in their outcome and treatment response. Aim: To observe tumor characteristics of different molecular subgroup and patients with postoperative (PO) raised cancer antigen 15.3 (CA 15.3) group and variation of tumor nature between pre- and post-menopausal breast cancer patients. Materials and Methods: Blood samples and tumor blocks were collected from 95 nonmetastatic female breast cancer patients. Immunohistochemical stains for estrogen receptors (ER), progesterone receptor (PR), and HER2/Neu were used to classify molecular subtypes. CA 15.3 level was detected by ELISA. Significance levels were ascertained by Pearson Chi-square test. Results: Prevalence of luminal A tumor with grade 3 was high. Triple negative and ER positive (ER+) types showed tumors with high grade and high lymph node (LN) metastasis. More nodal involvement was noticed in patients with PO raised CA 15.3. In addition, premenopausal patients with triple-negative and ER+ subtypes exhibited more aggressive tumors which were characterized by high grade and large numbers of LN metastasis. Conclusion: Clinicopathological characteristics of certain molecular subtypes and influence of menopausal status on it can predict disease recurrence or overall survival of breast cancer patients.
Keywords: Breast cancer, cancer antigen 15.3, menopausal status, molecular subtypes, traditional prognostic indexes
|How to cite this article:|
Saha S, Ganguly S, Sarkar DK, Hazra A. Clinicopathological characteristics of patients of certain molecular subtypes and elevated postoperative cancer antigen 15.3 levels and its correlation with menopausal status. Indian J Pathol Microbiol 2016;59:172-6
|How to cite this URL:|
Saha S, Ganguly S, Sarkar DK, Hazra A. Clinicopathological characteristics of patients of certain molecular subtypes and elevated postoperative cancer antigen 15.3 levels and its correlation with menopausal status. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Dec 15];59:172-6. Available from: http://www.ijpmonline.org/text.asp?2016/59/2/172/182029
| Introduction|| |
Breast cancer is the most commonly diagnosed cancer among women worldwide. The major risk factors for this disease are female gender, early menarche, or late menopause. Within menopausal groups, premenopausal females are at much higher risk, although only 30% of all patients with breast cancer are premenopausal. Null parity, older age at first birth, family history of breast cancer, personal history of proliferative benign breast disease, and history of radiation exposure are other factors associated with the origin of breast cancer.
Female breast cancer at young age can be considered as a more aggressive subtype, such as triple negative or HER2-positive breast cancer. It is more likely to present at an advanced stage in young women, either because of its biological aggressive subtype or because of delayed diagnosis. As a result, more loco-regional recurrences and distant metastases may occur in these patients, which contributes to the poorer outcome.
Study reveals that breast cancer is characterized by its molecular and clinical heterogeneity. According to gene expression profiling studies, it is classified into four major categories: luminal A, luminal B, HER2 (+), basal-like, and normal breast-like., Both luminal A and B types are estrogen receptor positive (ER+), but the A type has progesterone receptor (PR+) and no HER2/neu receptor, whereas the B type is either triple positive or PR−. The HER2 type over expresses only HER2/neu receptor. Basal-like tumor is more commonly negative for all three receptors. On the other hand, approximately, 73% of triple negative tumors are basal-like., Triple-negative subtype shows a unique molecular profile and most clinically aggressive behavior.
Diagnosis and treatment of this disease at young age remains a major challenge because of its strong clinical, morphological, and genetical differences. There are some clinical parameters such as tumor size, lymph node (LN) involvement, histological grade, and circulating marker such as cancer antigen 15.3 (CA 15.3) that have been used as predictive marker as well as for diagnosis and treatment of patients., Many reports have suggested that CA 15.3 can be used as an independent prognostic marker in breast cancer., However, still understanding breast cancer in molecular level is an enigma to the researchers. Furthermore, differences in the etiology of the respective breast cancer subtypes are not fully understood.
In the present study, we stratified breast cancer patients according to menopausal status and studied the distribution of the various molecular subtypes in these groups. The aim of our study is to observe: (a) The nature of tumor of certain molecular subgroups and patients with postoperative (PO) elevated CA 15.3 group by relating it to traditional predictive factors, that is, tumor size, grade, LN metastasis, and (b) whether it varies within pre- and post-menopausal patients
| Materials and Methods|| |
A total of 95 female breast cancer patients who underwent surgery were considered for inclusion in this study. All patients were aged between 20 and 75 years and had no metastasis at the time of primary diagnosis. Patients with any other malignancy from their previous history or undergoing chemotherapy or radiotherapy were excluded from the study. Relevant clinical data such as menopausal status and age were collected. All patients gave written informed consent. Patients were treated with either modified radical mastectomy (MRM) or breast conservation therapy (BCS). After completion of surgery, radiotherapy and appropriate adjuvant chemotherapy or hormone therapy were administered as per the international guidelines.
Collection of sample
Blood samples were collected in clotting vials from all patients and serum was separated by centrifugation. Serum was used to test CA 15.3 level. Paraffin embedded tissue blocks were collected. Histopathological characteristics of tumor such as tumor size, grade, and involvement of LN were taken after the biopsy.
CA 15.3 level was measured on the preoperative day by Sandwiched method of ELISA. The test was repeated on certain selected PO days to compare the marker level. Patients who showed marker level above the cut off 25 U/ml  on PO follow-up were considered as PO raised CA 15.3. Assay was performed according to Calbiotech ELISA kit (Ref CA153T; LOT RN-50051). Optical density was read by ELISA plate analyzer (Robonik Readwell Touch) at 450 nm within 15 min.
2–7 µm section of tissues were sliced by microtome and layered on poly-L-lysine coated glass slides. Slides were put in the incubator at 65°C for 30–40 min, dipped in xylene-I, and then xylene-II. The slides were dipped in different concentrations of alcohol for dehydration. All the slides were then immersed in hot citrate buffer for antigen retrieval, cooled and washed with Tris buffer. The slides were allowed for peroxide blocking for 5–8 min and then for protein blocking for 8–10 min. The slides were wiped clearly. Primary antibodies for ER, PR, and HER2/neu were applied for 45 min and washed. With postprimary block, the slides were incubated for 30 min. Novolink polymer was added for 30 min. All the slides were washed with Tris Buffer. DAB chromogen was applied for 3–5 min. After rinsing properly, hematoxylin was applied for 5 min. Dehydration was done again as mentioned before. Finally, all the slides were mounted before examination (Novolink Min Polymer Detection System-Leica, Ref RE7290-K, LOT 6030030. Anti-ER: Rabit monoclonal, Anti-PR: Rabit monoclonal antibody, Anti-Her2/Neu: Mouse monoclonal antibody; Cell Marque, USA. All antibodies are in ready to use form [diluted in TBS, pH: 7.3–7.7 with carrier protein and preservatives]).
ELISA for CA 15.3 was performed at a regular interval for up to 2 years.
Before commencement of the project, Human Ethical Committee clearance was obtained for carrying out the project work. Written informed consent form in three local languages (English, Hindi, and Bengali) was obtained from each of the patients prior to their recruitment in the study.
The collected data was statistically analyzed and the significance levels were ascertained. Statistical analysis was performed with Statistica version 6 (Tulsa, Oklahoma: StatSoft Inc., 2001). Descriptive statistical analysis was performed for numerical variables to calculate mean, median, and corresponding standard deviations. Pearson Chi-square test and Fisher' exact test (two-tailed) were used to compare the categorical variables. Significance level was set at P < 0.05.
| Results|| |
All the patients were distributed according to menopausal status [Table 1]. The mean menopausal age of total 95 breast cancer patients was measured 45.3 years. 37.8% of total studied breast cancer patients were premenopausal, whereas 62.2% were postmenopausal.
|Table 1: Distribution of breast cancer patients according to menopausal status|
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Prevalence of patients with luminal subtypes (i.e., Luminal A, luminal B, Her2/neu positive, and triple negative), ER+ type (irrespective of other two receptors), and PO high CA 15.3 type were observed among pre- and post-menopausal breast cancer patients [Table 2]. The difference in the prevalence of luminal A and triple-negative breast cancer between premenopausal and postmenopausal cases was statistically significant (P = 0.012, P = 0.032). 55.9% of patients were accounting for ER+ type from the postmenopausal group, but the difference of distribution between two menopausal groups was found not significant. Similarly, higher percentage (55.5%) of premenopausal patients showed PO raised CA 15.3 level, but no significant difference (P = 0.527) was detected in marker levels between premenopausal and postmenopausal groups. The other two subtypes - luminal B and HER2/neu were observed in very low frequency among patients; hence, they were not included in the further investigation.
|Table 2: Distribution of patients of different molecular subgroups and PO raised CA 15.3 group based on menopausal status|
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Tumor characteristics of certain molecular group, that is, luminal A, ER+, triple negative, and patients with PO raised CA 15.3 group were summarized in [Table 3]. These groups differed significantly either by tumor size, grade, and the number of LN metastasis. It was observed that luminal A, ER+, and triple-negative type breast tumors showed aggressive nature as they were mostly of grade 3 type and had large numbers of LN metastasis. Patients with postoperatively elevated CA 15.3 had significantly high number of node metastasis (P = 0.023).
|Table 3: Tumor characteristics of molecular subgroups and PO raised CA 15.3 group|
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It was further investigated whether the tumor aggressiveness of different groups was related to menopausal status of patients. The prevalence of grade 3 luminal A type tumors (P = 0.016) was significantly high in postmenopausal patients only. ER + grade 3 tumors were predominated in both menopausal groups (P = 0.037, 0.033). Only significant numbers of premenopausal patients showed ER + tumors with high LN metastasis (P = 0.011). In case of triple negative subtype, most of the premenopausal patients exhibited more aggressive tumors in terms of high grade (P = 0.017) and large number of node metastasis (P = 0.001) than postmenopausal group. Menopausal status had not showed any significant impact on the postoperatively elevated CA 15.3 level and any of the traditional predictive parameters [Table 4].
|Table 4: Tumor aggressiveness of different groups based on menopausal status|
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| Discussion|| |
Breast cancer in young aged women comprised a small proportion of the total incidence, but its burden on older counterpart is very high. Risk factors that have been proved to be associated with the breast cancer do not have the same impact on young and older patients. This can be explained by hormonal condition which may be an important part in breast carcinogenesis and differentiation.
In the present study, we found that occurrence of luminal A type tumors was higher in postmenopausal breast cancer patients. On contrary to our finding, Lin et al. reported that young Taiwanese patients with breast cancer had a higher prevalence of luminal A subtype. Our result was in agreement with several prior studies. Highest prevalence of luminal A was evident among postmenopausal white women. Carey et al. also reported that luminal A subtype was more frequent among postmenopausal African-American (59%) or non-African-American (54%) women compared with premenopausal African-American women (36%). ER+ tumors were found to be higher among postmenopausal women than that of premenopausal women which was supported by a study conducted on Turkish breast cancer patients. Other reports suggested conversely that ER/PR negative, HER2 positive, and high-grade type tumors were highly predominant in young breast cancer patients., Yamamoto et al. explained the reason behind ER + tumors in postmenopausal patients. Increase of ER+/PR− tumors in patients older than 50 years could be due to the decrease in circulating estrogen after menopause. We observed that triple negative cases were greater in percentage in premenopausal patients. The result was consistent with previous findings where Carvalho et al. reported that triple negative tumors were over represented in breast cancer patients of 35 years old or younger age. CA 15.3 level was monitored in connection with menopausal state. Menstrual cycle-dependent variability of serum tumor markers was being studied in healthy women previously. Erbagci et al. published that CA 15.3 level was found to be significantly elevated in the midluteal phase of the menstrual cycle compared to midfollicular phase. We observed that high percentage of premenopausal patients showed elevated marker level in PO period compared to the postmenopausal group. However, we found no significant differences of PO elevated marker level between pre- and post-menopausal groups.
Nature of aggressiveness of luminal A, ER+, triple negative groups, and PO elevated marker group was analyzed by observing their tumor characteristics. Tumor size 5 cm or more, grade 3, and LN metastasis 4 or more was incorporated as parameters to investigate disease aggressiveness of above group. In general, the luminal A tumors were tend to be smaller in size, lower histologic grade, and less likely to have nodal involvement than the other molecular subtypes. Another report by Zaha et al. showed that most of luminal A tumors were of medium grade. However, the present study found that significant numbers of luminal A tumors were from high-grade category. The association between ER+ breast tumor and LN metastasis has been studied previously. ER expression level varies greatly between primary and metastatic breast tumor. In addition, if the primary tumors are ER+, then the probability of retaining the receptors is more than 80% in case of LN metastases and 65–70% in case of distant metastases. Our studied subject did not have any sign of metastasis during primary diagnosis, though we found that significant numbers of ER+ cases possessed high grade and higher node metastasis. Estrogen level might play a role in attributing the spread of tumor cells from the primary site or their deposition and growth at metastatic sites. The exact mechanism of hormone responsiveness in LN microenvironment was not clear. Triple negative groups were more aggressive since significant number of cases were represented with high grade and had large number of LN metastasis. This was consistent with several prior investigations., Another two studies showed that patients with basal-like subtypes were associated with a higher prevalence of grade 3 tumors., Significant numbers of patients from PO elevated CA 15.3 group were characterized by high LN metastasis in our study. No significance was obtained in the prevalence of PO elevated patients with high tumor size and high grade. This result was supported by similar other studies. CA 15.3 was reported to be elevated with the increase of LN positivity. Mahindocht Keyhani et al. found a significant correlation of PO CA 15.3 elevation when node metastasis was ≥4. They did not observe any significant correlation between marker elevation and patient's tumor size. Higher preoperative CA 15.3 concentrations are generally associated with large tumor size and increasing nodal burden. After surgical removal of tumor, PO elevation of CA 15.3 level with nodal involvement might be an indicator of the metastatic diffusion of breast cancer.
The present study also monitored the influence of menopausal status with respect to the aggressive tumor features of few molecular subtypes. The prevalence of patients with large tumor size and any of the subtype did not differ by menopausal status. Evidence suggests that luminal A tumors in premenopausal breast cancer patients are likely to be grade 1 and 2. In our study, luminal A subtype was aggressive in postmenopausal group than premenopausal one by correlating with grade 3 tumors. ER + tumors were aggressive in both menopausal groups when characterized by high grade and only in premenopausal patients by showing intense nodal involvement. Premenopausal patients with ER positivity and high LN metastasis were significantly noticeable. Triple-negative patients only at premenopausal age showed aggressive tumor in terms of high grade and node metastasis. There were no supporting findings on the variation of clinicopathological tumor characteristics of molecular subtypes by menopausal status. Plausible explanation for more aggressive breast tumors in premenopausal triple-negative and premenopausal ER+ patients than postmenopausal ones might be due to the variation of certain cycle related factor between pre- and post-menopausal groups and that could affect recurrence or overall survival.
| Conclusion|| |
A study of 95 breast cancer patients from Eastern India showed breast cancer is still more common in the elderly and the postmenopausal patients such as the developed countries. Prevalence of luminal A and ER+ tumors was significantly higher in postmenopausal patients. Triple-negative and PO raised CA 15.3 cases dominated the population of premenopausal breast cancer patients. Luminal A type tumors were grade 3 type. Triple negative and ER+ tumors were mostly of high grade and had high nodal metastasis. Patients with PO raised CA 15.3 showed intense nodal involvement. Furthermore, premenopausal breast tumor either with ER+ or triple negative for receptor were aggressive in nature by exhibiting high grade and large numbers of LN metastasis. Though our result is indicating that menopausal status can give greater accuracy in predicting the aggressiveness of tumor of few molecular subtypes when correlated with grade and nodal metastasis, our small sample size is insignificant to draw a direct extrapolation to the general population; it gives scope for similar studies to establish Indian statistics.
Authors are indebted to the Director and the H.O.D. of Surgery of IPGMER, Kolkata, for providing the infrastructural facilities. We must express our obligation and gratitude to the Ethical Committee of IPGMER, Kolkata, for their kind permission to carry out this study in this institution. We are also grateful to the funding body, Government of West Bengal, Department of Science and Technology. We also thank our patients for their continuing co-operation.
Financial support and sponsorship
Department of Science and Technology, West Bengal.
Conflicts of interest
There are no conflicts of interest.
| References|| |
World Health Organization. Breast Cancer: Prevention and Control. Europe: World Health Organization [Online]; 2012.
Finek J, Holubec L Jr, Topolcan O, Elgrova L, Skalova A, Pecen L. The importance of prognostic factors in premenopausal women with breast cancer. Anticancer Res 2007;27:1893-6.
Assi HA, Khoury KE, Dbouk H, Khalil LE, Mouhieddine TH, El Saghir NS. Epidemiology and prognosis of breast cancer in young women. J Thorac Dis 2013;5 Suppl 1:S2-8.
Tamimi RM, Colditz GA, Hazra A, Baer HJ, Hankinson SE, Rosner B, et al.
Traditional breast cancer risk factors in relation to molecular subtypes of breast cancer. Breast Cancer Res Treat 2012;131:159-67.
Turkoz FP, Solak M, Petekkaya I, Keskin O, Kertmen N, Sarici F, et al.
Association between common risk factors and molecular subtypes in breast cancer patients. Breast 2013;22:344-50.
Phipps AI, Malone KE, Porter PL, Daling JR, Li CI. Body size and risk of luminal, HER2-overexpressing, and triple-negative breast cancer in postmenopausal women. Cancer Epidemiol Biomarkers Prev 2008;17:2078-86.
Adamo B, Anders CK. Stratifying triple-negative breast cancer: which definition (s) to use? Breast Cancer Res 2011;13:105.
Anders C, Carey LA. Understanding and treating triple-negative breast cancer. Oncology (Williston Park) 2008;22:1233-9.
Islam T, Matsuo K, Ito H, Hosono S, Watanabe M, Iwata H, et al.
Reproductive and hormonal risk factors for luminal, HER2-overexpressing, and triple-negative breast cancer in Japanese women. Ann Oncol 2012;23:2435-41.
Martín A, Corte MD, Alvarez AM, Rodriguez JC, Andicoechea A, Bongera M, et al.
Prognostic value of pre-operative serum CA 15.3 levels in breast cancer. Anticancer Res 2006;26:3965-71.
Eroles P, Bosch A, Pérez-Fidalgo JA, Lluch A. Molecular biology in breast cancer: Intrinsic subtypes and signaling pathways. Cancer Treat Rev 2012;38:698-707.
Duffy MJ, Shering S, Sherry F, McDermott E, O'Higgins N. CA 15-3: A prognostic marker in breast cancer. Int J Biol Markers 2000;15:330-3.
Park BW, Oh JW, Kim JH, Park SH, Kim KS, Kim JH, et al.
Preoperative CA 15-3 and CEA serum levels as predictor for breast cancer outcomes. Ann Oncol 2008;19:675-81.
Saha S, Singh V, Ganguli S, Das S, Mandal S, Sarkar D. Serial estimation of CA 15.3 can predict the outcome in patients with breast cancer – A prospective study. Int J Pharma Biosci 2014;5:570-6.
Lin CH, Liau JY, Lu YS, Huang CS, Lee WC, Kuo KT, et al.
Molecular subtypes of breast cancer emerging in young women in Taiwan: Evidence for more than just westernization as a reason for the disease in Asia. Cancer Epidemiol Biomarkers Prev 2009;18:1807-14.
Millikan RC, Newman B, Tse CK, Moorman PG, Conway K, Dressler LG, et al.
Epidemiology of basal-like breast cancer. Breast Cancer Res Treat 2008;109:123-39.
Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, et al.
Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 2006;295:2492-502.
Mutlu H, Ozdogan M, Colak T, Akca Z, Buyukcelik A. Variation in prognostic factors and molecular phenotype with menopausal status in Turkish patients with breast cancer. Int J Hematol Oncol 2013;23:109-16.
Anders CK, Hsu DS, Broadwater G, Acharya CR, Foekens JA, Zhang Y, et al.
Young age at diagnosis correlates with worse prognosis and defines a subset of breast cancers with shared patterns of gene expression. J Clin Oncol 2008;26:3324-30.
Collins LC, Marotti JD, Gelber S, Cole K, Ruddy K, Kereakoglow S, et al.
Pathologic features and molecular phenotype by patient age in a large cohort of young women with breast cancer. Breast Cancer Res Treat 2012;131:1061-6.
Yamamoto Y, Yamamoto-Ibusuki M, Iwase H. Menopausal status should be taken into consideration for patients with luminal a breast cancer in terms of the effect of differential biology on prognosis. J Clin Oncol 2013;31:2516.
Carvalho FM, Bacchi LM, Santos PP, Bacchi CE. Triple-negative breast carcinomas are a heterogeneous entity that differs between young and old patients. Clinics (Sao Paulo) 2010;65:1033-6.
Erbagci AB, Yilmaz N, Kutlar I. Menstrual cycle dependent variability for serum tumor markers CEA, AFP, CA 19-9, CA 125 and CA 15-3 in healthy women. Dis Markers 1999;15:259-67.
Zaha DC, Lazar E, Lazureanu C. Clinicopathologic features and five years survival analysis in molecular subtypes of breast cancer. Rom J Morphol Embryol 2010;51:85-9.
Zheng WQ, Lu J, Zheng JM, Hu FX, Ni CR. Variation of ER status between primary and metastatic breast cancer and relationship to p53 expression. Steroids 2001;66:905-10.
Harrell JC, Dye WW, Allred DC, Jedlicka P, Spoelstra NS, Sartorius CA, et al.
Estrogen receptor positive breast cancer metastasis: Altered hormonal sensitivity and tumor aggressiveness in lymphatic vessels and lymph nodes. Cancer Res 2006;66:9308-15.
Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, et al.
Triple-negative breast cancer: Clinical features and patterns of recurrence. Clin Cancer Res 2007;13:4429-34.
Ramirez MT, Vazquez-Lopez MI, Vela BE, Flores D, Uribe MD, Vazquez MI, et al
. Correlation of CA15-3 elevation with number and site of metastasis in breast cancer patients. J Clin Oncol (Meeting Abstracts) 2004;22:875.
Nasizadeh S. Serum CA15-3 measurement in breast cancer patients before and after mastectomy. Arch Iran Med 2005;8:263-6.
Ehinger A. The Role of Histological Grade in Discrimination between Luminal A-like and Luminal B-like HER-2 Normal Subtypes in a Series of Premenopausal Breast Cancer Patients. 26th
European Congress of Pathology, London; 2014.
Senior Research Scholar, Department of General Surgery, IPGMER, 244, A.J.C. Bose Road, Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]