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Original article
Diagnostic and prognostic relevance of Cullin1 expression in invasive ductal carcinoma of the breast
  1. Kyueng-Whan Min1,
  2. Dong-Hoon Kim2,
  3. Sung-Im Do2,
  4. Jin Hee Sohn2,
  5. Seoung Wan Chae2,
  6. Jung-Soo Pyo2,
  7. Chan Heun Park3,
  8. Young-Ha Oh1,
  9. Ki-Seok Jang1,
  10. Hack-Lyoung Kim4,
  11. Min Kim5
  1. 1Department of Pathology, College of Medicine, Hanyang University, Seoul, South Korea
  2. 2Department of Pathology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea
  3. 3Department of Surgery, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea
  4. 4Department of Internal Medicine, Seoul National University Hospital, Seoul, South Korea
  5. 5Department of Ophthalmology, Severance Eye and ENT Hospital, Yonsei University College of Medicine, Seoul, South Korea
  1. Correspondence to Professor Dong-Hoon Kim, Department of Pathology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, 78 Saemunan-gil, Jongno-Gu, Seoul 110-746, South Korea; idavid.kim{at}samsung.com

Abstract

Background Cullin1 (Cul1) is a matrix degrading enzyme known to be involved in the remodelling of extracellular matrix proteins. This enzyme has recently been reported to play a key role in tumour progression and its presence is associated with poor clinical outcome for several different types of tumours.

Methods 159 patients diagnosed with invasive ductal carcinoma between 2000 and 2005 were studied. Cul1 expression was analysed by immunohistochemical staining on a tissue microarray. The relationship between Cul1 expression and clinicopathological parameters was evaluated.

Results Tumour expression of Cul1 was correlated with prognostic factors such as high histological grade and p53 expression, and was also linked to negative ER and positive HER2 as therapeutic markers (all p<0.05). There was a significant association between poor overall survival and high Cul1 expression in both univariate and multivariate analyses (all p<0.05).

Conclusions Cul1 expression was significantly associated with high-grade tumours and poor prognosis, suggesting that it may play a role in breast tumour progression. Cul1 expression may therefore be crucial for the prediction of disease outcome in breast cancer patients.

  • Invasive ductal carcinoma
  • Cdk-associated Cullin1
  • human
  • prognosis
  • breast

https://doi.org/10.1136/jclinpath-2012-200847

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    Introduction

    Breast cancer is the most commonly diagnosed neoplasm and the leading cause of cancer death among women.1 Histopathological characteristics and/or grade, tumour size, hormone receptor status, lymph node metastasis, age and other associated diseases correlate with breast cancer prognosis.2 In addition, variation in transcriptional programmes accounts for much of the biological diversity among breast cancers.3 Gene-expression analyses have supported various molecular classifications for breast cancers, which are highly correlated with patient prognosis.4–6 Recently, the development of molecular techniques has provided a framework for new molecular classifications of breast cancers.7 Gene expression profiling has identified five majority patterns in the no special type group (NST; invasive ductal carcinoma): luminal A, luminal B, normal, basal-like and HER2 positive.

    The ubiquitin-proteasome system regulates the expression of various proteins involved in cell-cycle control, such as p53, β-catenin, p21, p27 and cyclins.8 ,9 An aberrant ubiquitin-proteasome pathway has been implicated in the pathogenesis of numerous human diseases and contributes to deregulated cell-cycle control, which is a fundamental aspect of cancer. As the major class of E3 ubiquitin ligase, the Skp1/cullin1/F-box protein (SCF) complex is involved in the proteolysis of core components of the cell-cycle machinery.10 Cullin1 (Cul1) serves as a rigid scaffold in SCF complex assembly, and aberrant expression of Cul1 results in the dysfunction of SCF E3 ligases.11 SCF E3 ligase-mediated demethylase degradation was recently found to block JMJD2A-dependent faster S phase progression in a Cul1-dependent manner.12 Another study showed that Cul1 may function as a tumour suppressor by regulating PLK4 protein levels.13 In contrast, c-Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression. Furthermore, regulation of Cul1 expression in immunological tissues may affect susceptibility to rheumatoid arthritis by altering lymphocyte signal transduction.14 However, less is known about differential Cul1 expression in tumours, although Bai et al recently described the relationship between Cul1 expression and poor prognosis in gastric cancer.15

    The aim of this study was to investigate Cul1 expression in 159 patients with invasive ductal carcinoma (IDC) of the breast and assess any statistical correlations with clinicopathological variables, molecular subtypes and patient survival.

    Methods

    Patient selection

    One hundred fifty-nine patients diagnosed with IDC at Kangbuk Samsung Hospital in Korea between 2000 and 2005 were reviewed. H&E-stained slides were reviewed by at least two pathologists for each case (K-WM, D-HK, S-ID, SWC, J-SP, Y-HO and K-SJ). Tumours were initially characterised based on clinicopathological features—mainly patient age and cancer characteristics such as tumour size, margins, number of tumours (single or multiple), Paget's disease, histological grade, invasion, necrosis and central tumour fibrosis. Histological grading was based on tubular formation, nuclear pleomorphism and mitotic counts using the modified Bloom–Richardson–Elston grading system.16 The predominant histological grade was decided by consensus. In addition, tumours were staged according to the tumour, lymph node and metastasis (TNM) classification of the American Joint Committee on Cancer (AJCC).

    Tissue microarray construction

    The most morphologically representative and non-necrotic area was carefully selected and marked on the H&E-stained slide. The tissue microarray (TMA) specimens were assembled using a tissue-array instrument (AccuMac Arrayer; ISU ABXIS Co., Seoul, Korea) consisting of thin-walled stainless steel punches and stylets for emptying and transferring the needle content. The assembly was held in an X-Y position guide equipped with semiautomatic micrometers, with a 1-mm interval between the individual samples and a 4-mm punch depth stop device. The instrument was briefly used to create holes in a recipient block with defined array cores. The fitted needle was used to transfer the tissue cores into the recipient block. Taking into account the limitations of the representative areas of the tumour, we used duplicate 3-mm-diameter tissue cores from each donor block. Additionally, two sample cores were representative for the whole tumour in 95% of the cases.

    Immunohistochemistry

    Serial 4-μm sections were cut from the array blocks and deparaffinised by routine techniques. For immunohistochemical (IHC) staining, the TMA slides were deparaffinised by heating at 55°C for 30 min and three 5-min washes with xylene. The sections were rehydrated with a series of 5-min washes in 100%, 90% and 70% ethanol. Antigens were retrieved by microwaving the samples for 4 min 20 s in 250 ml of 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase for 20 min. Immunostaining for progesterone receptor (PR), oestrogen receptor (ER), p53, human epidermal growth factor receptor 2 (HER2) and CK5/6 was performed using a Dako Autostainer with a Universal Staining System (DakoCytomation, Carpinteria, California, USA) and a ChemMate DAKO EnVision Detection Kit. Primary antibodies were raised against PR (1:200, Dako, Glostrup, Denmark), ER (1:200, Lab Vision Corporation, Fremont, California, USA), p53 (1:5000, Cell Marque, Hot Spring, Arkansas, USA), HER2 (1:200, Dako) and CK5/6 (1:200, Dako). For Cul1, affinity-purified rabbit anti-human-cul1 polyclonal antibody (1:400, Santa Cruz Biotechnology, San Francisco, California, USA) was used, and detection was performed with the Ultra Tech HRP Streptavidin-Biotin Detection System and an automatic staining machine (Bond Intense Detection Kit, Leica Biosystems, Newcastle, UK). Diaminobenzidine (DAB) was the chromogen. Cul1 IHC staining was performed in normal breast tissue as a negative control.

    Immunohistochemical evaluation

    IHC results were used to classify the tumours according to the published grading system17 into different molecular breast cancer subtypes: luminal A (ER- and/or PR-positive, HER2-negative), luminal B (ER- and/or PR-positive, HER2-positive), HER2 (ER- and PR-negative, HER2-positive), and basal-like (ER-, PR- and HER2-negative, CK5/6-positive).

    For this study, positive Cul1 immunostaining was defined as only cytoplasmic with or without nuclear staining, and staining was graded according to both the intensity and percentage of positively-stained tumour cells.15 Cul1 staining intensity was scored on a scale of 0 to 3 (0=negative; 1=weak; 2=moderate; 3=strong) (figure 1). The percentage of Cul1 positive cells was also scored into one of four categories: 1 (0–25%), 2 (26–50%), 3 (51–75%) or 4 (76–100%). When a discrepancy occurred between duplicate cores, the high score from the two tissue cores was used as the final score. The level of Cul1 staining was analysed as an immunoreactive score (IRS), which was calculated by multiplying the score of staining intensity and the percentage of positive cells.18

    Figure 1
    Figure 1

    Representative microphotographs showing Cullin1 expression of negative (A), weak (B), moderate (C) and strong intensity (D) by immunohistochemical staining in invasive ductal carcinoma (original magnification ×400).

    For determination of the optimal cut-off values of Cul1, receiver operating characteristic (ROC) curves plotting sensitivity versus 1 − specificity were used. The cut-off value calculated by the ROC was used to evaluate the relationship between overall survival rate and Cul1 expression. ROC values exhibited good discriminatory power for Cul1 expression considering the overall survival (area under the ROC = 0.668) (figure 2). Cul1 expression was divided into low expression (IRS ≤7) and high expression groups (IRS >7), according to the ROC curve.

    Figure 2
    Figure 2

    ROC curve for determination of the optimal cut-off value for Cullin1 expression according to patient survival rate in invasive ductal carcinoma of the breast (area under the ROC: 0.668 in tumour cells).

    Statistical analysis

    Comparisons between clinicopathological parameters and Cul1 expression were made using the χ2 test and Fisher's exact test. Overall survival curves were generated using the life table method by applying the cut-off values calculated using the ROC, and were compared by the log-rank test. Multivariate analysis was performed to identify independent prognostic markers for overall survival using a Cox multistep regression model. A two-tailed p value <0.05 was considered statistically significant. All data were analysed using SPSS for Windows V.13.0.

    Results

    Patient clinicopathological characteristics

    The mean age of the patients was 48.7 years (range 25–79 years). The distribution of AJCC stages was as follows: stage I, 24 (15.1%); stage II, 76 (47.8%); stage III, 27 (17%); and stage IV, 32 (20.1%). The T category distribution was as follows: T1, 52 (32.7%); T2, 97 (61%); T3, 10 (6.3%); and T4, 0 (0%). The N category distribution was as follows: N0, 65 (40.9%); N1, 56 (35.2%); N2, 21 (13.2%); and N3, 17 (10.7%). Metastasis was detected in 32 cases (20.1%), and the average tumour size was 2.7 cm (range 0.2–8 cm). The tumours were mostly classified as single lesions (151 cases; 95%). The tumours in 122 samples (76.7%) had ill-defined borders, and those in four patients (2.5%) were associated with Paget's disease (table 1).

    View this table:
    Table 1

    Correlation between clinical characteristics and Cullin1 expression in 159 patients with invasive ductal carcinoma

    Regarding the histological grade, the tumours were grade 1 and 2 in 99 patients (62.3%), whereas 60 (37.7%) of the tumours were classified as grade 3. Tubular formation was <10% in 10 patients, 10–75% in 27 patients and >75% in 122 patients. Nuclear pleomorphisms were mild in 10 patients, moderate in 108 and marked in 41. The mitotic counts were <10 mitoses/10 high power fields (HPF) in 44 patients, 10–19 mitoses/10 HPF in 56 patients and >19 mitoses/10 HPF in 59 patients. Lymphatic, vascular and perineural invasion was found in 81 (50.9%), 6 (3.8%) and 22 (13.8%) instances, respectively.

    Histologically, the tumours were associated with necrosis in 66 patients (41.5%), central tumour fibrosis in 24 patients (15.1%) and extensive intraductal components in 26 patients (16.4%) (table 2). The treatment comprised modified radical mastectomy (n=150), quadratectomy (n=6) and excision (n=3). During the follow-up period (mean 80.3 months, range 5–138 months), 20 out of 159 patients died (overall survival rate 87.4%).

    View this table:
    Table 2

    Correlations between histopathological findings and Cullin1 expression in 159 patients with invasive ductal carcinoma

    Correlations of tumour characteristics and Cul1 expression with clinicopathological parameters

    High Cul1 expression was statistically correlated with high histological grade, poor tubular formation and high mitotic count (OR 2.065, 95% CI 1.076 to 3.962, p=0.028; OR 2.57, 95% CI 1.196 to 5.524, p=0.014; OR 2.448, 95% CI 1.266 to 4.735, p=0.007, respectively). Cul1 expression was associated with marked nuclear pleomorphism, but the association was not statistically significant (p=0.091). In addition, high expression was more frequently observed in patients with ER negativity, HER2 positivity and p53 expression (OR 2.283, 95% CI 1.145 to 4.545, p=0.018; OR 2.588, 95% CI 1.246 to 5.374, p=0.01; OR 2.245, 95% CI 1.047 to 4.815, p=0.035, respectively) (table 3). Table 4 shows that high expression of Cul1 was frequently observed in the HER2-positive subtype, compared to the luminal A, luminal B and basal-like subtypes (p=0.004). However, we did not observe any significant relationship between Cul1 expression and other clinicopathological variables.

    View this table:
    Table 3

    Correlations between Cullin1 expression and the expression of ER, PR, HER2 and p53 in 159 patients with invasive ductal carcinoma

    View this table:
    Table 4

    Expression of Cullin1 in tumour cells according to breast cancer molecular subtype

    Comparison between Cul1 expression and patient survival

    Histological grade, AJCC stage and vascular invasion were associated with worse overall survival on univariate analysis (HR 2.716, 95% CI 1.11 to 6.646, p=0.022; HR 35.677, 95% CI 4.775 to 266.557, p=0.001; HR 7.787, 95% CI 2.257 to 26.861, p=0.001, respectively) (table 5), but parameters other than AJCC stage and vascular invasion (HR 39.392, 95% CI 4.979 to 311.631, p=0.001; HR 5.119, 95% CI 1.15 to 22.779, p=0.032, respectively) were not correlated with survival on multivariate analysis (table 6). High Cul1 expression was associated with poor overall survival (HR 2.819, 95% CI 1.083 to 7.388, p=0.026) (figure 3). After adjustment for confounding factors such as age, histological grade, AJCC stage and vascular, lymphatic and perineural invasion, high Cul1 expression was an independent prognostic factor for overall survival (HR 3.123, 95% CI 1.142 to 8.541, p=0.026).

    View this table:
    Table 5

    Overall survival analysis according to Cullin1 expression in 159 patients with invasive ductal carcinoma; univariate analysis

    View this table:
    Table 6

    Overall survival analysis according to Cullin1 expression in 159 patients with invasive ductal carcinoma; multivariate analysis

    Figure 3
    Figure 3

    Overall survival curves for the life table model according to tumour expression of Cullin1 in invasive ductal carcinoma in 159 patients (log-rank test, p=0.026).

    Discussion

    In this study, we showed that Cul1 expression correlated with several clinicopathological factors such as histological grade, ER, HER2, p53 and overall survival in 159 patients with IDC. Therefore, Cul1 could be considered to play an important role in promoting tumour progression, and the identification of this protein may be helpful in predicting outcome and improving prognostic models.

    The SCF complex plays well established roles in cell growth control pathways.19 Cul1 as a scaffold protein of the SCF complex plays a key role in the ubiquitin-dependent degradation pathway regulating the expression of cyclins (cyclin D1 and cyclin E) and Cdk inhibitors (p27 and p21). Indeed, contrary functions such as oncoprotein and tumour suppressor can occur, according to variable components (F-box proteins) of the SCF complex.10

    Many recent studies have demonstrated that Cul1 overexpression is associated with various malignant tumours. One study suggested that high expression of Cul1 is associated with lymph node metastasis and poor survival in gastric cancer.15 Chen et al20 reported that Cul1 expression is increased in early stage malignant melanoma, but it is not correlated with patient survival. Another study demonstrated that increased expression of Cul1 is frequently found in advanced melanoma.21 Thus, controversy still exists regarding the relationship between Cul1 and clinical outcomes in various malignancies.

    In this study, high Cul1 expression was significantly correlated with high histological grade, ER negativity, HER2 positivity, p53 expression and worse overall survival. In addition, we analysed the linkage between Cul1 nuclear expression (low group: 31 patients vs high group: 128 patients) and clinicopathological findings with reference to a previous study design, but there was no statistical significance (supplementary tables 1–6).22 Also, the Cul1 cytoplasmic expression was divided into negative (11 patients) and positive (148 patients) groups, the results were statistically correlated to other clinicopathological parameters, except for mitotic count and lymphatic invasion (supplementary tables 7–12) (all p<0.05). The discrepancy in the correlations between Cul1 expression and clinical outcomes among different studies can be explained by several factors, including study design, ethnic factors, types of malignancy and the number of cases. Moreover, the bivalent functions of the SCF complex, especially its previously described roles in oncogenic and tumour suppressor functions, can be assessed based on its expression, but many aspects of its functions remain unknown.

    We note that our study has some limitations. First, due to its retrospective and cross-sectional design, our study did not show a continuous relationship over time compared to longitudinal studies, making it difficult to draw concrete conclusions. Further longitudinal studies with step-by-step reliability monitoring are needed to clarify a cause–effect relationship. Second, representative tumour areas may not have been evaluated, because Cul1 evaluation was carried out on only two cut surfaces of each tumour specimen without IHC analysis of whole-tumour section. Third, these results have various limitations associated with IHC staining such as the possible lack of specificity of the antibody or inappropriate antigen retrieval.

    In summary, we found that high Cul1 expression was significantly associated with high histological grade and p53 expression as well as poor prognosis in patients with IDC of the breast. High Cul1 expression was also correlated with ER negativity and HER2 positivity, which are important in determining therapeutic plans. Consequently, Cul1 expression could affect overall survival with tumour progression and thus represents a potential target for the treatment of breast cancer.

    Take-home messages

    • High Cul1 expression was significantly correlated with aggressive clinicopathological parameters such as high histological grade and p53 expression.

    • Cul1 expression was frequently observed in patients with ER negativity and HER2 positivity, and may be an important factor for therapeutic planning.

    • There was a significant correlation between worse overall survival and high Cul1 expression.

    Acknowledgments

    We are grateful to English teacher Joung Woo Hong, for editing this manuscript.

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    Footnotes

    • Additional tables are published online only. To view these files please visit the journal online (http://dx.doi.org/10.1136/jclinpath-2012-200847).

    • Competing interests None.

    • Ethics approval The study was performed according to the Declaration of Helsinki and approved by the Ethics Committee of the Kangbuk Samsung Hospital (KBSMC11105).

    • Provenance and peer review Not commissioned; externally peer reviewed.