Original Article
Cyclin D1 expression in ductal carcinoma of the breast and its correlation with other prognostic parameters Purpose: Cyclin D1 is a cell cycle regulatory gene emerging as a potentially significant oncogene in invasive breast cancers. In this study, we attempted to see the expression of Cyclin D1 in invasive ductal carcinomas of the breast in our population and correlate its expression with other known prognostic parameters.
Gayatri Ravikumar, Anuradha Ananthamurthy
Materials and Methods: A total of 39 cases were selected from our case files from January 2011. Immunohistochemistry for Cyclin D1 was performed and interpreted as positive when >10% of the tumor cells expressed the marker with a moderate to strong intensity of staining. Clinicopathological parameters such as laterality, focality, tumor size, grade, ductal carcinoma in situ (DCIS), axillary lymph node (ALN) metastasis, hormone receptor status and human epidermal growth factor receptor 2 status were analyzed and correlated with Cyclin D1 expression.
Department of Pathology, St. John’s Medical College, Koramangala, Bengaluru, Karnataka, India
ABSTRACT
Results: The patients’ age ranged from 30 to 76 years (mean = 53.18). The tumors were unilateral and unifocal in 38 cases; one patient had bilateral synchronous tumors. The majority were grade2 (67.5%) and tumor size T2 (57.5%). Nearly 35% were associated with DCIS and 57.5% had ALN metastasis. Estrogen receptors (ER) and progesterone receptor (PgR) positivity was seen in 65% of the cases and 25% was triple negative. Cyclin D1 expression was seen in 67.5% of the cases in our study. Among the ER, PgR positive and Her‑2 negative tumors, Cyclin D1 expression was seen in the majority of cases (92%) cases, whereas none of the triple negative tumors showed Cyclin D1 expression. The other prognostic parameters such as tumor size, grade and lymph node status did not show any association with Cyclin D 1 positivity. Conclusions: Cyclin D1 expression was seen in 67.5% of ductal carcinoma and it showed a significant correlation with ER, PgR expression (92% in this study), which is in concordance with other similar studies in literature.
For correspondence: Dr. Gayatri Ravikumar, Department of Pathology, St. John’s Medical College, Sarjapur Road, Koramangala, Bengaluru ‑ 560 034, Karnataka, India. E‑mail: gayatri. [email protected]
KEY WORDS: Breast, cyclin D1, ductal carcinoma, immunohistochemistry, prognostic factors
INTRODUCTION Human breast cancer is a heterogeneous disease with varied genetic alterations implicated in its pathogenesis. It is one of the most common cancers in women and a leading cause of cancer related deaths. Various prognostic parameters are described and validated, but the search for newer prognostic factors continues as the existing parameters do not provide sufficient information for accurate risk assessment and treatment planning.[1,2] Among the various types of breast cancers, invasive ductal carcinomas (IDC), not otherwise specified (NOS) is the most common type accounting for about 60‑70% of the primary breast cancers.[3] Tumor size, grade, axillary lymph node (ALN) metastasis, mitotic rate, hormone receptor status and human epidermal growth factor receptor 2 (Her‑2/neu) status have traditionally been regarded as prognostic makers in patients with breast cancer. The interaction of these known
prognostic markers with newer molecular markers is a subject of intense investigation in predicting outcome in breast cancers. Cyclin D1 is a known cell cycle regulator which acts by binding with cyclin dependent kinase (cdk 4/6) in the cell cycle and inactivating the retinoblastoma (Rb) protein, helping in the progression of the cell cycle. The gene encoding Cyclin D1 is located on chromosome 11q13, which is commonly amplified in many human cancers including breast cancers. This aberrant over expression of Cyclin D1 is known to drive breast carcinogenesis by cell cycle mediated action.
Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.138135 PMID: ***
On the other hand, some of the studies have shown that over expressed Cyclin D1 in breast cancer patients acts by binding directly to the estrogen receptors (ERs) and propagate the downstream effects of estrogen in a cdk independent and Rb independent fashion.[4] There is strong evidence
Journal of Cancer Research and Therapeutics - July-September 2014 - Volume 10 - Issue 3
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Ravikumar and Ananthamurthy: Cyclin D1 in ductal carcinoma breast
that over expression of Cyclin D1 is a prognostic factor for better outcome in ER positive invasive breast cancer. However, other studies have shown that over expression and amplification of Cyclin D1 is a predictor of poor response to anti‑estrogen treatment.[5] It is evident that studies on Cyclin D1 have shown inconsistent and conflicting results with regard to its role in pathogenesis and also prognosis. There are hardly any studies from India which document the expression of Cyclin D1 in invasive breast cancers and its relation to other biomarkers. The current study was done to evaluate Cyclin D1 expression in IDC, NOS of the breast and to correlate it with other prognostic factors, in the Indian setting. MATERIAL AND METHODS
were performed using Fischer’s exact test and P value obtained. RESULTS The clinicopathological features and immunohistochemical findings of the 39 cases included in the study are summarized in Table 1. The mean age of presentation was 53.18 years, with an age range of 30‑76 years. In 38 cases, the tumors were unilateral and unifocal. One patient had bilateral synchronous tumors (considered as two separate tumors). In the majority of cases, the tumors were of MBR grade 2 (67.5%), tumor size pT2 (57.5%) and had a positive lymph node status (57.5%). In 8 patients, there was associated extra nodal extension of the tumor. Ductal carcinoma in situ (DCIS) was seen in 14 tumors (35%).
A total of 39 consecutive cases of IDC, NOS in female patients were collected from the archives of department of pathology, St. John’s medical college, between January 2011 and June 2012. All these patients had a complete staging workup performed as part of clinical assessment. Immunohistochemical data for hormone receptor status was available in all these cases. The relevant clinical and pathological details like age, laterality, focality, modified Bloom Richardson (MBR) grade, tumor size (pT), ALN status, ER, PgR and Her-2/neu status were recorded from the pathology reports. Patients who had undergone diagnostic core biopsies, lumpectomy without axillary clearance and neoadjuvant chemotherapy were excluded from the study.
OnIHC, 25 tumors (62.5%) were positive for ER, PgR and negative for Her‑2/neu and one tumor was positive for all three. Cyclin D1 staining showed positive expression in 27 out of the 40 tumors which accounts for 67.5% of the tumors in this study. Of the 26 tumors (inclusive of one triple positive tumor) which were ER, PgR positive, 92% tumors (24 cases) were also Cyclin D1 positive. None of the triple negative tumors showed Cyclin D1 positivity. In the case with bilateral tumors, both tumors were positive for ER and PgR and negative for Her‑2/neu. Cyclin D1 expression was also seen in both the tumors and also in the in situ component. 11 out of the
The histopathology slides were reviewed and a suitable block was chosen to perform immunohistochemistry (IHC) for Cyclin D1. IHC was performed using polymer technique on tissue sections of 4‑5 µm thickness, floated on sialinised slides and incubated overnight at 60 C. The slides were deparaffinized with xylene and rehydrated with ethanol. The slides were then placed in hydrogen peroxide solution. Antigen retrieval was done by steam treatment in Tris ethylenediaminetetraacetic acid buffer. The slides were coated and incubated at room temperature for 30 min with primary rabbit monoclonal antibody to Cyclin D1, clone EP12 from Dako. Subsequently, the slides were incubated with secondary antibody. Reactivity was detected using diaminobenzidine as chromogen and was counterstained with Harris’ hematoxylin.
Age distribution 30‑76 years Clinicopathological characteristics Laterality Right Left Bilateral MBR grade Grade 1 Grade 2 Grade 3 Tumor size (pT) pT1 pT2 pT3 pT4 ALN metastasis (N) N0 N1 N2 N3 DCIS High and low grade Immunohistochemistry ER, PgR positive and Her‑2/neu negative ER, PgR negative and Her‑2/neu positive Triple negative Triple positive Cyclin D1 positive
The staining for Cyclin D1 was interpreted as positive when at least 10% or more of the tumor cells showed nuclear expression of the marker with a moderate to strong intensity of staining.[6] The clinicopathological parameters like age, MBR grade, tumor size (pT), ALN metastasis, ER, PgR and Her‑2/neu status were compared and correlated with Cyclin D1 expression. Statistical analysis for correlation of Cyclin D1 expression with the other recorded clinicopathological parameters 672
Table 1: Clinicopathological and immunohistochemical characteristics of the 39 patients Mean 53.18 Number of tumors 20 18 1 2 27 11 12 23 3 2 17 13 3 7 14 25 4 10 1 27
DCIS=Ductal carcinoma in situ, MBR=Modified Bloom Richardson, ALN=Axillary lymph node, ER=Estrogen receptors, PgR=Progesterone receptor
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Ravikumar and Ananthamurthy: Cyclin D1 in ductal carcinoma breast
14 tumors (79%) with DCIS showed Cyclin D1 positivity. This was an additional significant finding in this study. The positive and negative staining for Cyclin D1 is shown in Figures 1 and 2 respectively. The Cyclin D1 positivity was correlated with the tumor grade, tumor size and ALN metastasis and hormone receptor status. Of the 27 tumors that were positive for Cyclin D1, 23 were ER/PgR positive and one was triple positive (ER, PgR and Her‑2/neu) and 3 were ER/PgR negative and Her 2/neu positive. Of the 13 cases that were Cyclin D 1 negative, 10 were triple negative, 2 were ER/PgR positive and 1 was ER/PgR negative and Her 2/neu positive. Only ER/PgR status was correlated with Cyclin D1 positivity as the numbers with Her 2/neu positivity was low (only 5 cases). The results of the correlation of Cyclin D1 positivity with other parameters such as grade, stage, ALN status and hormone receptor status are summarized in Table 2. A statistically significant correlation of Cyclin D1 expression with ER/PgR expression is noted and no significant correlation is seen with tumor size, grade and ALN metastasis. DISCUSSION The breast cancers are a group of heterogeneous diseases with multiple genetic alterations contributing to the pathogenesis. A number of genetic alterations are being implicated in the pathogenesis, which may provide a clue to the varied clinical behavior and prognosis in patients with morphologically similar tumors. Some of these genetic alterations may be inherent to the development of the tumor and others may help in the progression of the same. One such basic genetic alteration in breast carcinogenesis is the amplification of Cyclin D1 leading to overexpression.[4]
breast as transgenic mice experiments with targeted deletion of the gene encoding Cyclin D1 leads to poor mammary gland development and also protects against development of breast carcinoma.[7,8] CCND1 genetic locus is commonly amplified in many human tumors including those of the breast.[4] It is therefore considered as one of the basic genetic alterations in the development of breast carcinomas. Many of the studies in western literature have documented a positive expression of Cyclin D1 detected by IHC in about 65‑70% of breast carcinomas.[9‑12] In the present study, Cyclin D1 expression was seen in about 67.5% of IDC, NOS which is in concordance with that reported in western studies. It is now known that Cyclin D1 is necessary for the normal development of the breast and dysregulated expression stimulates aberrant mammary epithelial proliferation. Alle et al. studied the expression of Cyclin D1 in the histological model of progression of normal breast epithelium to proliferative breast disease with atypia, atypical ductal hyperplasia, DCIS and invasive carcinomas, to evolve a paradigm for the course of genetic alterations in the development of breast carcinomas. They found that the overexpression occurs in the early stages of breast oncogenesis and plays a crucial role in the further progression of the tumor.[13] Interestingly, we found Cyclin D1 expression in 75% of the associated DCIS loci in the current study indicating that Cyclin D1 overexpression is already present in the pre invasive stages of breast cancer.
Cyclin D1 is a cell cycle regulatory protein encoded by the CCND1 gene located on chromosome 11q13. Cyclin D1 is necessary for the normal lobulo alveolar development of the
The histopathological parameters like tumor size, MBR grade and ALN metastasis did not show a significant correlation with Cyclin D1 expression in the present study. A similar study by Lee et al. showed a high expression of Cyclin D1 with smaller tumor size although the other factors failed to show a positive correlation.[1] The study by Reis‑Filho et al. showed no significant association between these factors and Cyclin D1 expression, which was a finding similar to the present study.[14]
Figure 1: Immunohistochemistry showing nuclear positivity for Cyclin D1 in the neoplastic cells of infiltrating ductal carcinoma (×200) higher magnification shown in the inset (×400)
Figure 2: Immunohistochemistry showing negative staining for Cyclin D1 in the neoplastic cells of infiltrating ductal carcinoma (×200) higher magnification shown in the inset (×400)
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Ravikumar and Ananthamurthy: Cyclin D1 in ductal carcinoma breast
Table 2: Correlation of cyclin D1 immunostaining with tumor grade, size, axillary lymphnode metastasis and hormone receptor expression Parameters (no. of cases) Grade Grade 1 (2) Grade 1 (27) Grade 1 (11) Tumor size (pT) pT1 (12) pT2 (23) pT3 (3) pT4 (2) ALN metastasis (N) N0 (17) N1 (13) N2 (3) N3 (7) ER, PgR expression Positive (26) Negative (14)
Cyclin D1 positive
Cyclin D1 negative
P value
2 20 5
0 7 6
0.1402
8 16 1 2
4 7 2 0
0.4557
13 6 2 6
4 7 1 1
0.2232
24 3
2 11
0.000*(significant)
REFERENCES
ALN=Axillary lymph node, ER=Estrogen receptor, PgR=Progesterone receptor
Breast cancer cells are known for their expression of hormone receptors. The role that these receptors play as important therapeutic targets and prognostic parameters is well‑established. ER content of breast carcinomas is important as a prognostic and predictive biomarker and the evaluation of ER status is part of the routine assessment of breast neoplasms.[15‑17] PgR status is also an independent predictive factor for benefit from adjuvant endocrine therapy with tamoxifen.[18] Most of the well‑differentiated tumors are known to show a positive expression of these receptors. In the present study the ER, PgR positivity was seen in 65%. Triple negative tumors formed the next majority 25% in our cohort. Studies on Asian women with breast cancer have quoted a higher incidence of triple negative phenotype. Kim et al. in their study on 776 consecutive cases of breast carcinoma have found an incidence of triple negative phenotype to be 30%[19,20] which is in near concordance with the present study. Cyclin D1 expression in ER/PgR positive tumors was 92%, while 78.57% of the hormone receptor negative tumors were negative for Cyclin D1. This finding was statistically significant in the present study. In addition, none of the triple negative tumors showed Cyclin D1 expression in this study. This finding is comparable to Reis‑Filho’s study on 245 invasive breast cancers where they found a positive correlation of ER/PgR expression with Cyclin D1 expression and an inverse correlation between expression of basal like markers (comparable to triple negative phenotype) and Cyclin D1.[14] A similar finding was noted in the Lee et al. study.[1] In summary, 40 tumors (39 cases) of IDC analyzed in the present study, a significant positive association was found between the expression of the hormone receptors ER/PgR and the cell cycle regulator protein Cyclin D1 with a strong inverse relationship between the triple negative tumors and 674
Cyclin D1 expression. This is one of the first study from India attempting to analyze Cyclin D1 expression in breast cancers. Although the small number of cases was a limitation of the present study, this significant association may throw light on the proposed mechanism of action of Cyclin D1 in breast cancer and its relation to hormone receptors. Further larger studies are warranted to explore Cyclin D1 as a potential biomarker and prognostic indicator in breast cancer.
1. Lee A, Park WC, Yim HW, Lee MA, Park G, Lee KY. Expression of c‑erbB2, cyclin D1 and estrogen receptor and their clinical implications in the invasive ductal carcinoma of the breast. Jpn J Clin Oncol 2007;37:708‑14. 2. Hicks DG, Dawson A, Mattingly S, Crowe JP. The unmet clinical need for new molecular genetic markers in the prognosis and therapeutic management of breast cancer. Arch Pathol Lab Med 2005;129:1372‑4. 3. Ellis IO, Schnitt SJ, Sastre‑Garau X, Bussolati G, Tavassoli FA, Eusebi V, et al. Invasive breast carcinoma. In: Tavassoli FA, Devilee P, editors. World Health Organization Classification of Tumours Pathology and Genetics of Tumours of the Breast and Female Genital Organs. Lyon: IARC Press; 2003. p. 20. 4. Arnold A, Papanikolaou A. Cyclin D1 in breast cancer pathogenesis. J Clin Oncol 2005;23:4215‑24. 5. Kenny FS, Hui R, Musgrove EA, Gee JM, Blamey RW, Nicholson RI, et al. Overexpression of cyclin D1 messenger RNA predicts for poor prognosis in estrogen receptor‑positive breast cancer. Clin Cancer Res 1999;5:2069‑76. 6. Joe AK, Memeo L, Mckoy J, Mansukhani M, Liu H, Avila‑Bront A, et al. Cyclin D1 overexpression is associated with estrogen receptor expression in Caucasian but not African‑American breast cancer. Anticancer Res 2005;25:273‑81. 7. Yu Q, Geng Y, Sicinski P. Specific protection against breast cancers by cyclin D1 ablation. Nature 2001;411:1017‑21. 8. Sutherland RL, Musgrove EA. Cyclin D1 and mammary carcinoma: New insights from transgenic mouse models. Breast Cancer Res 2002;4:14‑7. 9. Joe AK, Arber N, Zhang Y, Heitjan T, Han E, Sgambato A, et al. Over expression of Cyclin D1 in breast cancer, clinical and pathologic correlations. ASCO Annual Meeting. 1998. 10. Hwang TS, Han HS, Hong YC, Lee HJ, Paik NS. Prognostic value of combined analysis of cyclin D1 and estrogen receptor status in breast cancer patients. Pathol Int 2003;53:74‑80. 11. Zukerberg LR, Yang WI, Gadd M, Thor AD, Koerner FC, Schmidt EV, et al. Cyclin D1 (PRAD1) protein expression in breast cancer: Approximately one‑third of infiltrating mammary carcinomas show overexpression of the cyclin D1 oncogene. Mod Pathol 1995;8:560‑7. 12. Zhang SY, Caamano J, Cooper F, Guo X, Klein‑Szanto AJ. Immunohistochemistry of cyclin D1 in human breast cancer. Am J Clin Pathol 1994;102:695‑8. 13. Alle KM, Henshall SM, Field AS, Sutherland RL. Cyclin D1 protein is overexpressed in hyperplasia and intraductal carcinoma of the breast. Clin Cancer Res 1998;4:847‑54. 14. Reis‑Filho JS, Savage K, Lambros MB, James M, Steele D, Jones RL, et al. Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: An immunohistochemical and chromogenic in situ hybridisation analysis. Mod Pathol 2006;19:999‑1009. 15. McGuire WL. Breast cancer prognostic factors: Evaluation guidelines. J Natl Cancer Inst 1991;83:154‑5. 16. Clark GM. Prognostic and predictive factors. In: Harris JR, Lippman ME, Morrow M, OsborneCK.(eds):Diseases of the Breast. Philadelphia, PA: Lippincott‑Raven; 1996. p. 461‑85. 17. Clinical practice guidelines for the use of tumor markers in breast
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and colorectal cancer. Adopted on May 17, 1996 by the American Society of Clinical Oncology. J Clin Oncol 1996;14:2843‑77. 18. Bardou VJ, Arpino G, Elledge RM, Osborne CK, Clark GM. Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases. J Clin Oncol 2003;21:1973‑9. 19. Patil VW, Singhai R, Patil AV, Gurav PD. Triple‑negative (ER, PgR, HER‑2/neu) breast cancer in Indian women. Breast Cancer: Targets and Therapy 2011;3:9‑19.
20. Kim MJ, Ro JY, Ahn SH, Kim HH, Kim SB, Gong G. Clinicopathologic significance of the basal‑like subtype of breast cancer: A comparison with hormone receptor and Her2/neu‑overexpressing phenotypes. Hum Pathol 2006;37:1217‑26. Cite this article as: Ravikumar G, Ananthamurthy A. Cyclin D1 expression in ductal carcinoma of the breast and its correlation with other prognostic parameters. J Can Res Ther 2014;10:671-5. Source of Support: Nil, Conflict of Interest: None declared.
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Cyclin D1 expression in ductal carcinoma of the breast and its correlation with other prognostic parameters Purpose: Cyclin D1 is a cell cycle regulatory gene emerging as a potentially significant oncogene in invasive breast cancers. In this study, we attempted to see the expression of Cyclin D1 in invasive ductal carcinomas of the breast in our population and correlate its expression with other known prognostic parameters.
Gayatri Ravikumar, Anuradha Ananthamurthy
Materials and Methods: A total of 39 cases were selected from our case files from January 2011. Immunohistochemistry for Cyclin D1 was performed and interpreted as positive when >10% of the tumor cells expressed the marker with a moderate to strong intensity of staining. Clinicopathological parameters such as laterality, focality, tumor size, grade, ductal carcinoma in situ (DCIS), axillary lymph node (ALN) metastasis, hormone receptor status and human epidermal growth factor receptor 2 status were analyzed and correlated with Cyclin D1 expression.
Department of Pathology, St. John’s Medical College, Koramangala, Bengaluru, Karnataka, India
ABSTRACT
Results: The patients’ age ranged from 30 to 76 years (mean = 53.18). The tumors were unilateral and unifocal in 38 cases; one patient had bilateral synchronous tumors. The majority were grade2 (67.5%) and tumor size T2 (57.5%). Nearly 35% were associated with DCIS and 57.5% had ALN metastasis. Estrogen receptors (ER) and progesterone receptor (PgR) positivity was seen in 65% of the cases and 25% was triple negative. Cyclin D1 expression was seen in 67.5% of the cases in our study. Among the ER, PgR positive and Her‑2 negative tumors, Cyclin D1 expression was seen in the majority of cases (92%) cases, whereas none of the triple negative tumors showed Cyclin D1 expression. The other prognostic parameters such as tumor size, grade and lymph node status did not show any association with Cyclin D 1 positivity. Conclusions: Cyclin D1 expression was seen in 67.5% of ductal carcinoma and it showed a significant correlation with ER, PgR expression (92% in this study), which is in concordance with other similar studies in literature.
For correspondence: Dr. Gayatri Ravikumar, Department of Pathology, St. John’s Medical College, Sarjapur Road, Koramangala, Bengaluru ‑ 560 034, Karnataka, India. E‑mail: gayatri. [email protected]
KEY WORDS: Breast, cyclin D1, ductal carcinoma, immunohistochemistry, prognostic factors
INTRODUCTION Human breast cancer is a heterogeneous disease with varied genetic alterations implicated in its pathogenesis. It is one of the most common cancers in women and a leading cause of cancer related deaths. Various prognostic parameters are described and validated, but the search for newer prognostic factors continues as the existing parameters do not provide sufficient information for accurate risk assessment and treatment planning.[1,2] Among the various types of breast cancers, invasive ductal carcinomas (IDC), not otherwise specified (NOS) is the most common type accounting for about 60‑70% of the primary breast cancers.[3] Tumor size, grade, axillary lymph node (ALN) metastasis, mitotic rate, hormone receptor status and human epidermal growth factor receptor 2 (Her‑2/neu) status have traditionally been regarded as prognostic makers in patients with breast cancer. The interaction of these known
prognostic markers with newer molecular markers is a subject of intense investigation in predicting outcome in breast cancers. Cyclin D1 is a known cell cycle regulator which acts by binding with cyclin dependent kinase (cdk 4/6) in the cell cycle and inactivating the retinoblastoma (Rb) protein, helping in the progression of the cell cycle. The gene encoding Cyclin D1 is located on chromosome 11q13, which is commonly amplified in many human cancers including breast cancers. This aberrant over expression of Cyclin D1 is known to drive breast carcinogenesis by cell cycle mediated action.
Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.138135 PMID: ***
On the other hand, some of the studies have shown that over expressed Cyclin D1 in breast cancer patients acts by binding directly to the estrogen receptors (ERs) and propagate the downstream effects of estrogen in a cdk independent and Rb independent fashion.[4] There is strong evidence
Journal of Cancer Research and Therapeutics - July-September 2014 - Volume 10 - Issue 3
Quick Response Code:
671
Ravikumar and Ananthamurthy: Cyclin D1 in ductal carcinoma breast
that over expression of Cyclin D1 is a prognostic factor for better outcome in ER positive invasive breast cancer. However, other studies have shown that over expression and amplification of Cyclin D1 is a predictor of poor response to anti‑estrogen treatment.[5] It is evident that studies on Cyclin D1 have shown inconsistent and conflicting results with regard to its role in pathogenesis and also prognosis. There are hardly any studies from India which document the expression of Cyclin D1 in invasive breast cancers and its relation to other biomarkers. The current study was done to evaluate Cyclin D1 expression in IDC, NOS of the breast and to correlate it with other prognostic factors, in the Indian setting. MATERIAL AND METHODS
were performed using Fischer’s exact test and P value obtained. RESULTS The clinicopathological features and immunohistochemical findings of the 39 cases included in the study are summarized in Table 1. The mean age of presentation was 53.18 years, with an age range of 30‑76 years. In 38 cases, the tumors were unilateral and unifocal. One patient had bilateral synchronous tumors (considered as two separate tumors). In the majority of cases, the tumors were of MBR grade 2 (67.5%), tumor size pT2 (57.5%) and had a positive lymph node status (57.5%). In 8 patients, there was associated extra nodal extension of the tumor. Ductal carcinoma in situ (DCIS) was seen in 14 tumors (35%).
A total of 39 consecutive cases of IDC, NOS in female patients were collected from the archives of department of pathology, St. John’s medical college, between January 2011 and June 2012. All these patients had a complete staging workup performed as part of clinical assessment. Immunohistochemical data for hormone receptor status was available in all these cases. The relevant clinical and pathological details like age, laterality, focality, modified Bloom Richardson (MBR) grade, tumor size (pT), ALN status, ER, PgR and Her-2/neu status were recorded from the pathology reports. Patients who had undergone diagnostic core biopsies, lumpectomy without axillary clearance and neoadjuvant chemotherapy were excluded from the study.
OnIHC, 25 tumors (62.5%) were positive for ER, PgR and negative for Her‑2/neu and one tumor was positive for all three. Cyclin D1 staining showed positive expression in 27 out of the 40 tumors which accounts for 67.5% of the tumors in this study. Of the 26 tumors (inclusive of one triple positive tumor) which were ER, PgR positive, 92% tumors (24 cases) were also Cyclin D1 positive. None of the triple negative tumors showed Cyclin D1 positivity. In the case with bilateral tumors, both tumors were positive for ER and PgR and negative for Her‑2/neu. Cyclin D1 expression was also seen in both the tumors and also in the in situ component. 11 out of the
The histopathology slides were reviewed and a suitable block was chosen to perform immunohistochemistry (IHC) for Cyclin D1. IHC was performed using polymer technique on tissue sections of 4‑5 µm thickness, floated on sialinised slides and incubated overnight at 60 C. The slides were deparaffinized with xylene and rehydrated with ethanol. The slides were then placed in hydrogen peroxide solution. Antigen retrieval was done by steam treatment in Tris ethylenediaminetetraacetic acid buffer. The slides were coated and incubated at room temperature for 30 min with primary rabbit monoclonal antibody to Cyclin D1, clone EP12 from Dako. Subsequently, the slides were incubated with secondary antibody. Reactivity was detected using diaminobenzidine as chromogen and was counterstained with Harris’ hematoxylin.
Age distribution 30‑76 years Clinicopathological characteristics Laterality Right Left Bilateral MBR grade Grade 1 Grade 2 Grade 3 Tumor size (pT) pT1 pT2 pT3 pT4 ALN metastasis (N) N0 N1 N2 N3 DCIS High and low grade Immunohistochemistry ER, PgR positive and Her‑2/neu negative ER, PgR negative and Her‑2/neu positive Triple negative Triple positive Cyclin D1 positive
The staining for Cyclin D1 was interpreted as positive when at least 10% or more of the tumor cells showed nuclear expression of the marker with a moderate to strong intensity of staining.[6] The clinicopathological parameters like age, MBR grade, tumor size (pT), ALN metastasis, ER, PgR and Her‑2/neu status were compared and correlated with Cyclin D1 expression. Statistical analysis for correlation of Cyclin D1 expression with the other recorded clinicopathological parameters 672
Table 1: Clinicopathological and immunohistochemical characteristics of the 39 patients Mean 53.18 Number of tumors 20 18 1 2 27 11 12 23 3 2 17 13 3 7 14 25 4 10 1 27
DCIS=Ductal carcinoma in situ, MBR=Modified Bloom Richardson, ALN=Axillary lymph node, ER=Estrogen receptors, PgR=Progesterone receptor
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Ravikumar and Ananthamurthy: Cyclin D1 in ductal carcinoma breast
14 tumors (79%) with DCIS showed Cyclin D1 positivity. This was an additional significant finding in this study. The positive and negative staining for Cyclin D1 is shown in Figures 1 and 2 respectively. The Cyclin D1 positivity was correlated with the tumor grade, tumor size and ALN metastasis and hormone receptor status. Of the 27 tumors that were positive for Cyclin D1, 23 were ER/PgR positive and one was triple positive (ER, PgR and Her‑2/neu) and 3 were ER/PgR negative and Her 2/neu positive. Of the 13 cases that were Cyclin D 1 negative, 10 were triple negative, 2 were ER/PgR positive and 1 was ER/PgR negative and Her 2/neu positive. Only ER/PgR status was correlated with Cyclin D1 positivity as the numbers with Her 2/neu positivity was low (only 5 cases). The results of the correlation of Cyclin D1 positivity with other parameters such as grade, stage, ALN status and hormone receptor status are summarized in Table 2. A statistically significant correlation of Cyclin D1 expression with ER/PgR expression is noted and no significant correlation is seen with tumor size, grade and ALN metastasis. DISCUSSION The breast cancers are a group of heterogeneous diseases with multiple genetic alterations contributing to the pathogenesis. A number of genetic alterations are being implicated in the pathogenesis, which may provide a clue to the varied clinical behavior and prognosis in patients with morphologically similar tumors. Some of these genetic alterations may be inherent to the development of the tumor and others may help in the progression of the same. One such basic genetic alteration in breast carcinogenesis is the amplification of Cyclin D1 leading to overexpression.[4]
breast as transgenic mice experiments with targeted deletion of the gene encoding Cyclin D1 leads to poor mammary gland development and also protects against development of breast carcinoma.[7,8] CCND1 genetic locus is commonly amplified in many human tumors including those of the breast.[4] It is therefore considered as one of the basic genetic alterations in the development of breast carcinomas. Many of the studies in western literature have documented a positive expression of Cyclin D1 detected by IHC in about 65‑70% of breast carcinomas.[9‑12] In the present study, Cyclin D1 expression was seen in about 67.5% of IDC, NOS which is in concordance with that reported in western studies. It is now known that Cyclin D1 is necessary for the normal development of the breast and dysregulated expression stimulates aberrant mammary epithelial proliferation. Alle et al. studied the expression of Cyclin D1 in the histological model of progression of normal breast epithelium to proliferative breast disease with atypia, atypical ductal hyperplasia, DCIS and invasive carcinomas, to evolve a paradigm for the course of genetic alterations in the development of breast carcinomas. They found that the overexpression occurs in the early stages of breast oncogenesis and plays a crucial role in the further progression of the tumor.[13] Interestingly, we found Cyclin D1 expression in 75% of the associated DCIS loci in the current study indicating that Cyclin D1 overexpression is already present in the pre invasive stages of breast cancer.
Cyclin D1 is a cell cycle regulatory protein encoded by the CCND1 gene located on chromosome 11q13. Cyclin D1 is necessary for the normal lobulo alveolar development of the
The histopathological parameters like tumor size, MBR grade and ALN metastasis did not show a significant correlation with Cyclin D1 expression in the present study. A similar study by Lee et al. showed a high expression of Cyclin D1 with smaller tumor size although the other factors failed to show a positive correlation.[1] The study by Reis‑Filho et al. showed no significant association between these factors and Cyclin D1 expression, which was a finding similar to the present study.[14]
Figure 1: Immunohistochemistry showing nuclear positivity for Cyclin D1 in the neoplastic cells of infiltrating ductal carcinoma (×200) higher magnification shown in the inset (×400)
Figure 2: Immunohistochemistry showing negative staining for Cyclin D1 in the neoplastic cells of infiltrating ductal carcinoma (×200) higher magnification shown in the inset (×400)
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Ravikumar and Ananthamurthy: Cyclin D1 in ductal carcinoma breast
Table 2: Correlation of cyclin D1 immunostaining with tumor grade, size, axillary lymphnode metastasis and hormone receptor expression Parameters (no. of cases) Grade Grade 1 (2) Grade 1 (27) Grade 1 (11) Tumor size (pT) pT1 (12) pT2 (23) pT3 (3) pT4 (2) ALN metastasis (N) N0 (17) N1 (13) N2 (3) N3 (7) ER, PgR expression Positive (26) Negative (14)
Cyclin D1 positive
Cyclin D1 negative
P value
2 20 5
0 7 6
0.1402
8 16 1 2
4 7 2 0
0.4557
13 6 2 6
4 7 1 1
0.2232
24 3
2 11
0.000*(significant)
REFERENCES
ALN=Axillary lymph node, ER=Estrogen receptor, PgR=Progesterone receptor
Breast cancer cells are known for their expression of hormone receptors. The role that these receptors play as important therapeutic targets and prognostic parameters is well‑established. ER content of breast carcinomas is important as a prognostic and predictive biomarker and the evaluation of ER status is part of the routine assessment of breast neoplasms.[15‑17] PgR status is also an independent predictive factor for benefit from adjuvant endocrine therapy with tamoxifen.[18] Most of the well‑differentiated tumors are known to show a positive expression of these receptors. In the present study the ER, PgR positivity was seen in 65%. Triple negative tumors formed the next majority 25% in our cohort. Studies on Asian women with breast cancer have quoted a higher incidence of triple negative phenotype. Kim et al. in their study on 776 consecutive cases of breast carcinoma have found an incidence of triple negative phenotype to be 30%[19,20] which is in near concordance with the present study. Cyclin D1 expression in ER/PgR positive tumors was 92%, while 78.57% of the hormone receptor negative tumors were negative for Cyclin D1. This finding was statistically significant in the present study. In addition, none of the triple negative tumors showed Cyclin D1 expression in this study. This finding is comparable to Reis‑Filho’s study on 245 invasive breast cancers where they found a positive correlation of ER/PgR expression with Cyclin D1 expression and an inverse correlation between expression of basal like markers (comparable to triple negative phenotype) and Cyclin D1.[14] A similar finding was noted in the Lee et al. study.[1] In summary, 40 tumors (39 cases) of IDC analyzed in the present study, a significant positive association was found between the expression of the hormone receptors ER/PgR and the cell cycle regulator protein Cyclin D1 with a strong inverse relationship between the triple negative tumors and 674
Cyclin D1 expression. This is one of the first study from India attempting to analyze Cyclin D1 expression in breast cancers. Although the small number of cases was a limitation of the present study, this significant association may throw light on the proposed mechanism of action of Cyclin D1 in breast cancer and its relation to hormone receptors. Further larger studies are warranted to explore Cyclin D1 as a potential biomarker and prognostic indicator in breast cancer.
1. Lee A, Park WC, Yim HW, Lee MA, Park G, Lee KY. Expression of c‑erbB2, cyclin D1 and estrogen receptor and their clinical implications in the invasive ductal carcinoma of the breast. Jpn J Clin Oncol 2007;37:708‑14. 2. Hicks DG, Dawson A, Mattingly S, Crowe JP. The unmet clinical need for new molecular genetic markers in the prognosis and therapeutic management of breast cancer. Arch Pathol Lab Med 2005;129:1372‑4. 3. Ellis IO, Schnitt SJ, Sastre‑Garau X, Bussolati G, Tavassoli FA, Eusebi V, et al. Invasive breast carcinoma. In: Tavassoli FA, Devilee P, editors. World Health Organization Classification of Tumours Pathology and Genetics of Tumours of the Breast and Female Genital Organs. Lyon: IARC Press; 2003. p. 20. 4. Arnold A, Papanikolaou A. Cyclin D1 in breast cancer pathogenesis. J Clin Oncol 2005;23:4215‑24. 5. Kenny FS, Hui R, Musgrove EA, Gee JM, Blamey RW, Nicholson RI, et al. Overexpression of cyclin D1 messenger RNA predicts for poor prognosis in estrogen receptor‑positive breast cancer. Clin Cancer Res 1999;5:2069‑76. 6. Joe AK, Memeo L, Mckoy J, Mansukhani M, Liu H, Avila‑Bront A, et al. Cyclin D1 overexpression is associated with estrogen receptor expression in Caucasian but not African‑American breast cancer. Anticancer Res 2005;25:273‑81. 7. Yu Q, Geng Y, Sicinski P. Specific protection against breast cancers by cyclin D1 ablation. Nature 2001;411:1017‑21. 8. Sutherland RL, Musgrove EA. Cyclin D1 and mammary carcinoma: New insights from transgenic mouse models. Breast Cancer Res 2002;4:14‑7. 9. Joe AK, Arber N, Zhang Y, Heitjan T, Han E, Sgambato A, et al. Over expression of Cyclin D1 in breast cancer, clinical and pathologic correlations. ASCO Annual Meeting. 1998. 10. Hwang TS, Han HS, Hong YC, Lee HJ, Paik NS. Prognostic value of combined analysis of cyclin D1 and estrogen receptor status in breast cancer patients. Pathol Int 2003;53:74‑80. 11. Zukerberg LR, Yang WI, Gadd M, Thor AD, Koerner FC, Schmidt EV, et al. Cyclin D1 (PRAD1) protein expression in breast cancer: Approximately one‑third of infiltrating mammary carcinomas show overexpression of the cyclin D1 oncogene. Mod Pathol 1995;8:560‑7. 12. Zhang SY, Caamano J, Cooper F, Guo X, Klein‑Szanto AJ. Immunohistochemistry of cyclin D1 in human breast cancer. Am J Clin Pathol 1994;102:695‑8. 13. Alle KM, Henshall SM, Field AS, Sutherland RL. Cyclin D1 protein is overexpressed in hyperplasia and intraductal carcinoma of the breast. Clin Cancer Res 1998;4:847‑54. 14. Reis‑Filho JS, Savage K, Lambros MB, James M, Steele D, Jones RL, et al. Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: An immunohistochemical and chromogenic in situ hybridisation analysis. Mod Pathol 2006;19:999‑1009. 15. McGuire WL. Breast cancer prognostic factors: Evaluation guidelines. J Natl Cancer Inst 1991;83:154‑5. 16. Clark GM. Prognostic and predictive factors. In: Harris JR, Lippman ME, Morrow M, OsborneCK.(eds):Diseases of the Breast. Philadelphia, PA: Lippincott‑Raven; 1996. p. 461‑85. 17. Clinical practice guidelines for the use of tumor markers in breast
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Ravikumar and Ananthamurthy: Cyclin D1 in ductal carcinoma breast
and colorectal cancer. Adopted on May 17, 1996 by the American Society of Clinical Oncology. J Clin Oncol 1996;14:2843‑77. 18. Bardou VJ, Arpino G, Elledge RM, Osborne CK, Clark GM. Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases. J Clin Oncol 2003;21:1973‑9. 19. Patil VW, Singhai R, Patil AV, Gurav PD. Triple‑negative (ER, PgR, HER‑2/neu) breast cancer in Indian women. Breast Cancer: Targets and Therapy 2011;3:9‑19.
20. Kim MJ, Ro JY, Ahn SH, Kim HH, Kim SB, Gong G. Clinicopathologic significance of the basal‑like subtype of breast cancer: A comparison with hormone receptor and Her2/neu‑overexpressing phenotypes. Hum Pathol 2006;37:1217‑26. Cite this article as: Ravikumar G, Ananthamurthy A. Cyclin D1 expression in ductal carcinoma of the breast and its correlation with other prognostic parameters. J Can Res Ther 2014;10:671-5. Source of Support: Nil, Conflict of Interest: None declared.
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