| Abstract|| |
Introduction: Breast cancer (BC) is the most common cancer and leading cause of death in women. Aim: This study was conducted to study the cyclin D1 expression in BC and its correlation with other clinicopathological parameters such as tumor size, histological grade, lymph node status, estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki67 status. Materials and Methods: Fifty cases of BC diagnosed between 2015 and 2018 were included in the study. A technique of manual tissue microarray was employed for the analysis of expression of immunohistochemical (IHC) markers such as cyclin D1, ER, PR, HER2/neu, and Ki67 in all cases. Results were subjected to statistical analysis. Results: Cyclin D1 positivity was seen in 64% cases of BC cases of which 8% were triple negative BC (TNBC) molecular subtype. Cyclin D1 expression was statistically significantly associated with ER and PR positivity. Maximum cases showing cyclin D1 expression showed negative HER2/neu expression, Ki67 immunopositivity, absent lymphovascular invasion and were of lower grade and stage. 32% cases were TNBC. Cyclin D1 was found positive in 25% TNBC cases. Negative Cyclin D1 expression was seen in TNBC cases of higher grade and higher stage with positive lymph node status, presence of lymphovascular invasion and Ki67 positivity. Conclusion: Cyclin D1 can be potentially used as a prognostic marker and if included in routine IHC workup of BC cases can aid in appropriate patient management with the advent of new targeted therapy that blocks the cyclin D-CDK4/6 axis.
Keywords: Breast carcinoma, cyclin D1, prognosis
|How to cite this article:|
Lengare PV, Sinai Khandeparkar SG, Joshi AR, Gogate BP, Solanke SG, Gore SH. Immunohistochemical expression of cyclin D1 in invasive breast carcinoma and its correlation with clinicopathological parameters. Indian J Pathol Microbiol 2020;63:376-81
|How to cite this URL:|
Lengare PV, Sinai Khandeparkar SG, Joshi AR, Gogate BP, Solanke SG, Gore SH. Immunohistochemical expression of cyclin D1 in invasive breast carcinoma and its correlation with clinicopathological parameters. Indian J Pathol Microbiol [serial online] 2020 [cited 2021 Oct 16];63:376-81. Available from: https://www.ijpmonline.org/text.asp?2020/63/3/376/291657
| Introduction|| |
Breast cancer (BC) is the most common cancer and leading cause of death in women. It is a heterogeneous disease encompassing several pathological and molecular subtypes characterized by different outcomes and responses to a given treatment. In India, almost 100,000 women are diagnosed with BC every year and a rise to 131,000 cases is predicted by 2020. The recent increased knowledge in molecular mechanisms of this cancer and consequent targeted treatments have attempted to improve its outcome.
Cyclin D1, a key cell cycle regulatory protein, is encoded by the gene, CCND1 or PRAD1, positioned on chromosome 11q13. It is necessary for the normal lobulo-alveolar development of the breast. Transgenic mice experiments with targeted deletion of the gene encoding cyclin D1 led to poor mammary gland development and imparted protection from development of BC. Conversely, transgenic mice engineered to overexpress CCND1 in the mammary glands demonstrated abnormal mammary proliferation and, in some instances, developed BC. Cyclin D1 overexpression has been reported in up to 50% of human BC.
Many researchers have shown that cyclin D1 overexpression in BC is related to an unfavorable outcome, but others have yielded different results. Thus, 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 few studies from India that document cyclin D1 expression in BC and their association with other well-established prognostic factors.,
The present study was conducted to evaluate the cyclin D1 expression in BC cases and its correlation with other clinicopathological parameters.
| Materials and Methods|| |
This cross-sectional study was conducted in the department of pathology in a tertiary care hospital. Ethical clearance was obtained from institute's ethical committee. Fifty cases of BC cases operated upon and diagnosed from 2015 to 2018 were included in the study. The available data for all the patients as regards with age, location of tumor, grade, stage, and lymph node status were collected from the records of histopathology section of the department of pathology.
All the slides were evaluated by two senior histopathologists. The modified Bloom-Richardson (MBR) system of cancer grading system was used in this study. TNM classification and staging of the cases was done as per American Joint Committee on Cancer (AJCC) guidelines. The most suitable tissue block of BC cases was selected for immunohistochemical (IHC) evaluation. A technique of manual tissue microarray was employed for the study of cyclin D1, estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki67 in all cases with one tissue core taken from each selected BC block. Antigen retrieval was done using citrate buffer antigen retrieval protocol. Pressure cooker was used as heating source.
The primary antibodies used were anti-human cyclin D1 (Clone EP12, Dako), ER (Clone 6F 11, Novacastra), PR (Clone PGR312, Novacastra), HER2/neu (Clone CB11, Novacastra), and Ki-67 (Clone MM1, Novacastra). Negative control (without adding primary antibody) was included in all batches.
Section from tonsil was used as positive control for cyclin D1 expression. Section from endometrial tissue was used as positive control for ER and PR. Section from BC, which previously showed unequivocal strong immunoreactivity for HER2/neu, was used as positive control for HER2/neu. Section from skin was used as positive control for Ki67. Sections were examined under high power field (HPF) to observe the immunoreactivity.
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.
The intensity of cyclin D1 was scored on a scale from 0 to 3, where 0 = negative staining,
1 = weak staining, 2 = moderate staining, and 3 = strong staining.
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 was considered as positive. HER2 staining was scored according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Moderate to strong complete membrane staining of 10% or more of the tumor cells was considered to be positive (2 + and 3+). Ki67 labeling index (KiLI) of equal to or more than 10% was considered to be positive.
The Primer of Biostatistics 7.0 program was used for calculation of interrelationships between the analyzed cyclin D1 expression and clinicopathological variables by Pearson's Chi-square test. Quantitative data were presented with the help of mean. Qualitative data were presented with the help of frequency and percentage table. The results were considered to be statistically significant when the P value was <0.05 and highly statistically significant when P value was <0.01.
| Results|| |
The various clinicopathological features of BCa are presented in [Table 1]. The age of patients ranged from 33 to 78 years, with a mean value of 52.86 years. The highest number of cases (19) was seen in the age group of 41–50 years (38%).
Cyclin D1 expression in BC and its correlation with various clinicopathological parameters is shown in [Table 2] [Figure 1]a, [Figure 1]b, [Figure 1]c. Cyclin D1 positivity was seen in 32/50 (64%) cases of invasive BC. Cyclin D1 expression was highly statistically significantly associated with ER (χ2 = 25.935, P value <0.01) and PR positivity (χ2 = 21.48, P value <0.01). Maximum cases showing cyclin D1 expression showed negative HER2/neu expression (30/50 cases, 60%), Ki67 immunopositivity (31/50 cases, 62%), absent lymphovascular invasion (23/50 cases, 46%) and were of lower grade (grade I + grade II; 18/50, 36%) and lower stage (stage I + stage II; 17/50, 34%).
|Table 2: Cyclin D1 expression and clinico-pathological parameters of breast carcinoma cases|
Click here to view
|Figure 1: (a) Photomicrograph showing Cyclin D1 negativity (<10% tumor cells are Positive) (IHC, 400×) (b) Photomicrograph showing Cyclin D1 1 + intensity (IHC, 400×). (c) Photomicrograph showing Cyclin D1 1 + intensity (IHC, 400×)|
Click here to view
16/50 (32%) cases were triple negative BC (TNBC) in our study. [Table 3] Cyclin D1 was found negative in 12/16 (75%) TNBC cases. Negative cyclin D1 expression was seen in TNBC cases of higher grade (grade III, 10/16, 62.50%) and higher stage (stage II + III, 11/16, 68.75%) with positive lymph node status (8/16, 50% cases), presence of lymphovascular invasion (8/16, 50%), and Ki67 positivity (12/16, 75%).
|Table 3: Cyclin D1 expression and clinico-pathological parameters of Triple Negative Breast carcinoma cases|
Click here to view
| Discussion|| |
Aberrant cyclin D1 overexpression is known to drive breast carcinogenesis by cell cycle-mediated action. It has been documented that cyclin D1 induction is sufficient for cells arrested in the early Gl phase to complete the cell cycle by binding with cyclin dependent kinase (cdk 4/6) and inactivating the retinoblastoma (Rb) protein in the cell cycle.
Using antibody against cyclin D1 protein, overexpression of the protein can be detected even in the absence of any apparent increase in copy numbers. In the present study, cyclin D1 expression detected by IHC was seen in about 64% of BC cases which is in concordance with that reported in literature of which 8% were TNBC molecular subtype. Many of the studies have documented a positive expression of cyclin D1 in about 60–85% of BC.,,,, [Table 4].
Few studies have shown that over expressed cyclin D1 in BC cases acts in a cdk-independent and Rb-independent fashion by binding directly to the ER and propagate the downstream effects of estrogen.,, Cyclin D1 expression was statistically significantly associated with ER and PR positivity in the present study., These findings confirm the results of some previous studies which is in favor of the effect of cyclin D1 on cell maturation and differentiation. Maximum BC cases showing cyclin D1 expression showed absent HER2/neu expression (60%) consistent with studies done by Peurala et al. and Sarkar et al. The present study had maximum cases of luminal A molecular subtype.
Cyclin D1 is also known to promote other regulatory molecules by cdk-independent mechanisms. It enhances apoptosis by repressing the transcriptional activity of signal transducer and activator of transcriptional 3 (STAT3). Further, cyclin D1 is the intermediary molecule in other cell cycle pathways such as nuclear factor-κB (NFκB), Rac1 and 5′ adenosine monophosphate-activated protein kinase (AMPK) signaling pathways. Decline in Rac1 levels causes inhibition of NFκB signaling and induces downregulation of cyclin D1. Active AMPK leads to loss of cyclin D1 messenger ribonucleic acid and protein.
BCa subtypes included in the present study such as invasive cribriform carcinoma (ICC) (1), mucinous + IDC (NOS) (2), IDC + infiltrating lobular carcinoma (ILC) (2) and IDC + neuroendocrine carcinoma (NEC) (1) expressed cyclin D1. However, BCa subtypes such as metaplastic (4) and mucinous carcinoma (1) did not show cyclin D1 expression. Three cases of medullary carcinoma (MedC) expressed cyclin D1 and 2 cases of MedC showed negative cyclin D1 expression. The type of cyclin D1 expression shown by ICC, ILC, and NEC component of invasive BC was similar to most studies documented in the literature., Cyclin D1 expression in MedC, metaplastic carcinoma, mucinous component of mixed invasive BC, and mucinous carcinoma is variedly mentioned in the literature., Some tumors with defective ER function show low cyclin D1. The importance of loss of PR in BC is not entirely clear, but its positive status indicates properly functioning ER. In case of medullary and metaplastic carcinomas which were triple negative in present study can explain low cyclin D1 expression in these tumors., The rationale for the mucinous carcinoma exhibiting cyclin D1 expression given by one study was that these are well-differentiated tumor types with relatively low nuclear atypicality, low proliferation, positive steroid receptor status, and favorable prognosis in contrast to that seen in our study where single case of mucinous carcinoma as well two cases of mucinous component of mixed invasive BC did not show cyclin D1 expression. Another study has documented 50% of mucinous carcinomas of the breast expressing cyclin D1.
Furthermore, maximum cases showing cyclin D1 expression were seen in BC of lower grade (36%) and lower stage (34%) though not statistically significant in this study. The reverse relationship observed between cyclin D1 overexpression and tumor grade suggests that higher expression of cyclin D1 may directly or indirectly result in maturation of tumor cells. A statistically significant association between cyclin D1 positivity and tumor grade was found in a study done by Paul et al., Peurala et al., and Sarkar et al. No study comparing cyclin D1 expression with stage of tumor was found in the literature for BC cases. Maximum TNBC cases of higher grade (62.5%) and higher stage (68.75%) showed negative cyclin D1 expression. Such correlation of cyclin D1 expression with tumor grade and stage in TNBC cases were not found in the previous references.
Maximum BC cases showing cyclin D1 expression showed Ki67 immunopositivity (62%) though not statistically significant in contrast to studies done by Peurala et al. and Paul et al. Maximum TNBC cases with negative cyclin D1 expression (75%) showed Ki67 positivity. Such study was not found in the literature.
Maximum BC cases showing cyclin D1 expression showed absent lymphovascular invasion (46%), however, this was not statistically significant. Similar finding was seen in the study done by Comut et al. In present study, maximum cases showing negative cyclin D1 expression in TNBC cases showed lymphovascular invasion (50%); however, this was not statistically significant. This is contradictory to study done by Hartel et al. who showed statistically significant correlation between cyclin D1 expression and lymphovascular invasion (P value < 0.005) in TNBC cases.
Tumor cell proliferation in many BC is supported by hyperactivity of the cyclin D–cdk4/6 axis. Potent, selective, orally bioavailable inhibitors of CDK4/6 have only become available as cancer therapeutics in the last decade. In human BC, the subtype for which CDK4/6 inhibition has the strongest rationale is ER positive disease. Moreover, CCND1 is a direct target gene of the ER, and is, thus, often expressed at high levels in ER-positive cancers. Three CDK4/6 inhibitors have now been approved by the FDA for the treatment of ER-positive metastatic BC: Palbociclib, ribociclib, and abemaciclib. The addition of these agents to endocrine therapy has resulted in the longest improvement in progression-free survival seen to date in this subtype of BC. More clinical trial data need to be documented to elucidate the matter further.
| Limitations|| |
TMA technique was used for cyclin D1, ER, PR, Her2, and Ki67. Whole sections were not used for their IHC evaluation. However, utmost care was taken to sample the most representative area from the original whole section blocks for TMA. HER2 was assessed only by IHC. Evaluation by fluorescence in-situ hybridization was not available, especially for the equivocal cases with HER2 expression 2+. Follow-up time for the patients was limited.
| Conclusion|| |
In BC cases, cyclin D1 expression was significantly associated with ER and PR positivity and statistically insignificantly associated with absent LVI, lower tumor grade, and stage and HER2/neu negativity. Thus, increased cyclin D1 expression was associated with mostly well-established good prognostic factors. In TNBC cases, negative cyclin D1 expression was statistically insignificantly associated with higher tumor grade and stage, positive lymph node status, presence of lymphovascular invasion, and Ki67 positivity. Thus, absence of cyclin D1 expression was associated with poor prognostic factors in TNBC. Cyclin D1 can be used as a prognostic marker and should be included in routine IHC workup of BC cases as it can aid in appropriate targeted therapy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Dhande AN, Sinai Khandeparkar SG, Joshi AR, Kulkarni MM, Pandya N, Mohanapure N, et al
. Stromal expression of CD10 in breast carcinoma and its correlation with clinicopathological parameters. South Asian J Cancer 2019;8:18-21.
] [Full text]
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.
] [Full text]
Sarkar S, Kanoi A, Bain J, Gayen R, Das KN. Correlation between cyclin D1 expression and standard clinicopathological variables in invasive breast cancer in Eastern India. South Asian J Cancer 2015;4:155-9.
] [Full text]
Mohammadizadeh F, Hani M, Ranaee M, Bagheri M. Role of cyclin D1 in breast carcinoma. J Res Med Sci 2013;18:1021-5.
Sutherland RL, Musgrove EA. Cyclin D1 and mammary carcinoma: New insights from transgenic mouse models. Breast Cancer Res 2002;4:14-7.
Garrido-Castro AC, Goel S. CDK4/6 inhibition in breast cancer: Mechanisms of response and treatment failure. Curr Breast Cancer Rep 2017;9:26-33.
Lakhani SR, Ellis IO, Schnitt SJ, Tan PH, van de Vijver MJ. World Health Organization Classification of Tumours of the Breast. 4th
ed. Lyon: IARC; 2012. p. 8-108, 152.
Pathak GS, Deshmukh SD, Ashturkar AV. Construction of tissue arrays without prefabricated recipient paraffin block experience of a novel technique in resource poor settings. Indian J Pathol Microbiol 2011;54:654-5.
] [Full text]
Peurala E, Koivunen P, Haapasaari KM, Bloigu R, Jukkola-Vuorinen A. The prognostic significance and value of cyclin D1, CDK4 and p16 in human breast cancer. Breast Cancer Res 2013;15:R5.
Collins LC, Botero ML, Schnitt SJ. Bimodal frequency distribution of estrogen receptor immunohistochemical staining results in breast cancer: An analysis of 825 cases. Am J Clin Pathol 2005;123:16-20.
Gutierrez C, Schiff R. HER2: Biology, detection, and clinical implications. Arch Pathol Lab Med 2011;135:55-62.
Tawfik K, Kimler BF, Davis MK, Fan F, Tawfik O. Ki-67 expression in axillary lymph node metastases in breast cancer is prognostically significant. Hum Pathol 2013;44:39-46.
Li Z, Cui J, Yu Q, Wu X, Pan A, Li L. Evaluation of CCND1 amplification and CyclinD1 expression: Diffuse and strong staining of CyclinD1 could have same predictive roles as CCND1 amplification in ER positive breast cancers. Am J Transl Res 2016;8:142-53.
Gillett C, Fantl V, Smith R, Fisher C, Bartek J, Dickson C, et al
. Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Cancer Res 1994;54:1812-7.
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.
Van Diest PJ, Michalides RJAM, Jannink I, Van der Valk P, Peterse HL, De Jong JS, et al
. Cyclin D1 expression in invasive breast cancer: Correlations and prognostic value. Am J Pathol 1997;150:705-11.
Shanks A, Choi J, Karur V. Dramatic response to cyclin D-dependent kinase 4/6 inhibitor in refractory poorly differentiated neuroendocrine carcinoma of the breast. Proc (Bayl Univ Med Cent) 2018;31:352-4.
Naidu R, Wahab NA, Yadav MM, Kutty MK. Expression and amplification of cyclin D1 in primary breast carcinomas: Relationship with histopathological types and clinico-pathological parameters. Oncol Rep 2002;9:409-16.
Czapiewski P, Wełnicka-Jaśkiewicz M, Seroczyńska B, Skokowski J, Sejda A, Szade J, et al
. CD99 correlates with low cyclin D1, status and triple negative molecular phenotype but isαhigh topoisomerase 2 prognostically irrelevant in breast carcinoma. Pol J Pathol 2015;66:269-75.
Kulkarni MM, Sinai Khandeparkar SG, Joshi AR, Jadhav A, Kamble N, Morale A. Rare breast cancer subtypes- Clinical and immuno-histopathological study with special reference to metaplastic and medullary carcinoma of the breast. Indian J Basic App Med Res 2017;6:2234-44.
Hartel PH, Fleming DR. Cyclin D1 expression in triple-negative breast cancer with new treatment implications. SCIREA J Clin Med 2016;1:49-57.
Pernas S, Tolaney SM, Winer EP, Goel S. CDK4/6 inhibition in breast cancer: Current practice and future directions. Ther Adv Med Oncol 2018;10:1758835918786451.
Siddhi Gaurish Sinai Khandeparkar
E-517, The Island, Wakad, Pune - 411 057, Maharashtra
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]