Abstract
Background/Aim: Abnormalities in the cyclin D1–CDK4/6 complex have been implicated in breast cancer proliferation and resistance to treatment. Recently, new drugs have been developed to target CDK4/6. Meanwhile, liquid biopsy has received great interest in oncology. In this study, we analyzed cyclin D1 gene (CCND1) copy number variation (CNV) in circulating tumor DNA (ctDNA) from luminal B breast cancer patients. Patients and Methods: This study included 31 patients with luminal B breast cancer who underwent resection. We analyzed CCND1 CNV in ctDNA by digital droplet PCR. Results: Of the 31 luminal B breast cancers, CCND1 CNV was positive in 5 cases. Patients with CCND1 CNV positivity had significantly shorter recurrence-free survival than patients with negative CCND1 CNV. Conclusion: CCND1 CNV in ctDNA was associated with poor prognosis in patients with luminal B breast cancer. This biomarker could be a useful prognostic factor.
Breast cancer is broadly classified into subtypes according to the presence or absence of hormone receptors and HER2. Hormone receptor-positive breast cancers are further categorized into luminal A (low Ki67) and luminal B (high Ki67) according to their proliferative potential. Luminal B breast cancer has a poor prognosis without adjuvant therapy (1). Abnormalities in the cyclin D1–CDK4/6 complex, which regulates the G1 to S phase of the cell cycle, have been implicated in the growth of breast cancer and its resistance to treatment. In particular, luminal B breast cancer has been reported to have amplified cyclin D1 gene (CCND1) compared with other subtypes (2), but a means to easily assess cyclin D1 expression in a clinical setting has not yet been established. The U.S. Food and Drug Administration officially approved palbociclib CDK4/6 inhibitor in 2017 for hormone receptor-positive and HER2-negative unresectable and recurrent breast cancer, and in combination with aromatase inhibitor (letrozole), it was shown to significantly improved progression-free survival (PFS) and prolong overall survival (OS) (3, 4). CDK4/6 inhibitors represent an option for patients with unresectable or recurrent luminal B breast cancer who have a poor prognosis, and their therapeutic efficacy is expected to improve in clinical practice. However, a marker targeting the cyclin D1–CDK4/6 complex for effective administration of the drug is yet to be identified.
Liquid biopsy is a peripheral blood-based molecular diagnosis approach that avoids the need for an invasive tumor biopsy, in which the genomic profiling provides information on driver mutations, therapeutic resistance, and treatment response, and has gained increasing attention in oncology (5). In clinical practice, predictive markers for therapeutic response have been established for many cancer types, and in the case of breast cancer, Carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 15-3 are used. Several reports indicated that the concentration of tumor-derived circulating tumor DNA (ctDNA) is more reflective of disease status than these existing markers (6, 7).
CCND1 is known as one of the most frequently altered genes and is associated with hyperactive PI3K/mTOR pathway, which was shown to lead to hormonal therapy resistance in the BOLERO-2 next generation research subgroup and The Cancer Genome Atlas network (8). By confirming the relationship between CCND1 in ctDNA and CCND1 in tumor tissue, we aimed to validate whether CCND1 in ctDNA can be used as a therapeutic or prognostic marker. Several studies focused on the mutation and concentration of ESR1 and PIK3CA in breast cancer (9, 10), but there are no reports on CCND1 copy number variation (CNV) in both tissue and ctDNA in primary breast cancer. In this study, we first evaluated the association between CCND1 CNV in ctDNA and the prognosis of patients with luminal B breast cancer (Cohort 1). Next, we assessed whether CCND1 CNV in ctDNA reflected the biological characteristics of the lesion (Cohort 2).
Patients and Methods
Patients. We first evaluated the relationship between the CNV of CCND1 in ctDNA and prognosis (Cohort 1) in 31 patients with luminal B breast cancer who underwent resection at Kyushu University Hospital (Fukuoka, Japan) between September 2006 and March 2009.
Next, we assessed whether CCND1 CNV in ctDNA from peripheral blood reflected the biological characteristics of the tumor tissues (Cohort 2) in 15 patients with luminal B breast cancer who underwent resection at Kyushu University Hospital between January and June 2017. Tumor subtypes were identified using immunohistochemical (IHC) staining on tissue acquired by surgical resection. Estrogen receptor (ER)-positive or progesterone receptor (PR)-positive tissues were defined as tumors with ≥1% of tumour cells staining positive for ER or PR, respectively, as previously reported (11). Tumor specimens were defined as HER2-positive when HER2 IHC staining was scored as 3+ according to standard criteria (12) or when HER2 gene amplification was detected using fluorescence in situ hybridization. Cancer specimens were defined as luminal B when the Ki67 status was high (>20%) or the PR status was low (<20%) in ER-positive disease. The clinicopathological information of patients was collected retrospectively from the electronic medical records, including patient outcome and OS. The patients were treated according to the National Comprehensive Cancer Network Guidelines for the treatment of breast cancer (13), and the Clinical Practice Guidelines for Breast Cancer of the Japanese Breast Cancer Society (14).
The study conformed to the principles of the Declaration of Helsinki and was approved by the institutional review board of Kyushu University Hospital (no. 2020-591). Before surgery, patients provided comprehensive written consent, which stated that their medical information could be used for research purposes.
Samples. Patient blood samples (5 ml) were collected immediately before undergoing resection. In Cohort 1, blood samples were centrifuged at 3,000×g for 5 min to separate the serum, and the supernatant was collected and stored at –80°C until further use. In Cohort 2, blood samples were centrifuged at 1,600×g for 20 min. The supernatant was then centrifuged at 12,000×g for 10 min to separate the plasma, and the supernatant was collected and stored at –80°C until further use. In addition, we recruited one healthy volunteer who had no underlying disease and undertook venous puncture for collection of a blood sample. Formalin-fixed paraffin-embedded (FFPE) tumor tissues and frozen tissues were also collected after surgery.
DNA preparation from tissue samples and blood samples. DNA was extracted from each frozen tissue sample using an AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). The DNA concentration was determined by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and DNA samples were confirmed to be >20 ng/μl in concentration. ctDNA was extracted using a QIAamp Circulating Nucleic Acid Kit (Qiagen) from plasma.
Digital droplet PCR for CCND1 copy number variation analysis. Digital droplet PCR (ddPCR) was performed to measure CCND1 CNV in the DNA extracted from tissues and plasma using a QX200 Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. CN determination was performed as follows: From the results of ddPCR, the proximal curve to the input was drawn, and the theoretical value at CN=2 was calculated from the proximal curve. If the minimum value of the measured value was greater than the maximum value of the theoretical value, it was judged to be positive (Table I).
Analysis of CCND1 CNV in ctDNA of 31 patients in cohort 1 and one healthy volunteer.
IHC staining. Anti-cyclin D1 antibody (monoclonal rabbit, SP4; Nichirei Bioscience, Tokyo, Japan) was used according to the manufacturer’s protocol. Cyclin D1 positivity was defined as cyclin D1 expression in ≥10% of tumour cells (15). Furthermore, ≥50% cyclin D1 expression was considered to be strongly positive.
Statistical analysis. Logistic regression was used to compare continuous variables and χ2 tests were performed to compare categorical variables between CCND1-positive and -negative groups. The survival endpoint was recurrence-free survival (RFS), which was defined as the time from surgery to recurrence, including both local relapse and metastatic disease. A survival curve was generated using the Kaplan-Meier method and compared with the log-rank test. A p-value of <0.05 was considered statistically significant. Statistical analysis was performed using JMP 16 (SAS Institute, Cary, NC, USA).
Results
The clinicopathological characteristics of Cohort 1, including age at diagnosis, pathological stage, histological characteristics, and outcomes, are listed in Table II. The mean age of the patients was 55 years (range=27-79 years) and one man (3%) was included. Most patients had clinical stage I (35.5%) or II (58.0%) disease and all had no vascular invasion. Of 31 patients, 27 (87%) were recurrence-free and four (13%) had local or distant recurrence. Five (16%) patients were CCND1 CNV-positive. Three (11%) of the 27 recurrence-free cases were CNV-positive. In addition, two (50%) of four recurrent cases showed CNV positivity. A prognostic analysis with the endpoint of RFS showed that RFS was significantly lower in CCND1 CNV-positive cases compared with negative cases (p=0.0359) (Figure 1).
Clinicopathological characteristics of patients in Cohort 1.
Prognostic value of CCND1 CNV expression in ctDNA. Kaplan-Meier curves showing estimated RFS for CCND1 CNV expression in ctDNA. p-Value indicates the significance of the difference between the two groups.
The clinicopathological characteristics of patients in Cohort 2, including age at diagnosis, pathological stage, and histological characteristics, are listed in Table III. The mean age of the patients was 58.3 years (range=38-84 years) and all were female. Most patients had clinical stage I (33.3%) or II (46.7%) disease. The relationship between the CNV of CCND1 in plasma ctDNA, CNV of CCND1 in tissue, and tissue IHC are shown in Figure 2. There were three positive cases. Of the 15 luminal B breast cancer patients, 11 (73%) were cyclin D1-positive by IHC (Figure 3). Cases that were positive for CCND1 CNV in ctDNA tended to be strongly positive for cyclin D1 protein by IHC and had high expression of Ki67. However, there was no match for CCND1 CNV in ctDNA and tissue.
Clinicopathological characteristics of 15 patients in Cohort 2.
Relationship between CCND1 CNV in plasma ctDNA, CCND1 CNV in tissue, and tissue IHC. Cases that were positive for CCND1 CNV in ctDNA (*) tended to be strongly positive for cyclin D1 protein and had high expression of Ki67.
Cyclin D1 expression in breast carcinomas analyzed by immunohistochemistry (magnification, ×200; bar, 100μm). (a–c) Haematoxylin-eosin staining shows tumors. (d) Cyclin D1 expression was strongly positive. (e) Cyclin D1 expression was moderately positive. (f) Cyclin D1 was not expressed.
Discussion
To the best of our knowledge, this is the first report to show that it is possible to measure CCND1 CNV in ctDNA and blood from patients with luminal B breast cancer. Analysis of CCND1 CNV in ctDNA and the nature of the lesions was also performed but did not match the tissue CCND1 CNV.
ctDNA in blood samples can offer essential advantages over tissue biopsies, including minimal invasiveness, access to tissue, use of preserved samples, and tumor heterogeneity, and as a result, is used to guide treatment decisions more quickly. ctDNA detection research is of great significance to the early diagnosis, prognostic evaluation, disease monitoring, and individualized treatment of breast cancer (7). Cyclin D1 over-expression occurs in half of all breast cancers (8, 16-18). In particular, it has been reported that CCND1 is amplified in luminal B breast cancer (2). The prognostic utility of associated proteins has great potential, with over-expression associated with favorable prognosis (19, 20) and gene amplification signifying poor prognosis (21). This study showed that RFS was significantly lower in cases that were positive for CCND1 CNV compared with those that were negative. The threshold for determining the increase or decrease in copy number is difficult because it varies depending on various factors such as sample quality and quantity, and the combination of target and reference probes. In the present study, we generated an approximate curve from the ddPCR results and calculated theoretical values from the curve, and then used them as the basis for determining the increase or decrease in copy number.
When verified at the mRNA level using the Kaplan-Meier plotter, high CCND1 mRNA expression was significantly correlated with shorter RFS among luminal B breast cancer patients (Figure 4). In the BOLERO-2 study, genetic alternations in the PI3K/mTOR pathway and cell-cycle control genes (CCND1, CDK4, CDK6, and CDKN2A) and amplification of CCND1 had minimal benefit on the progression-free survival gain with mTOR inhibitor (everolimus) (8), which supported our study data.
Prognostic value of CCND1 mRNA expression in the Kaplan-Meier plotter (kmplot.com). Kaplan-Meier curve showing estimated RFS for CCND1 mRNA expression in luminal B breast cancer patients (n=904).
The concordance trend of ctDNA and tissue CCND1 CNV in this study is shown in Figure 2. However, in a study without breast cancer by Nakamura et al. (GOZILA study), ctDNA-based testing demonstrated 97%-100% concordance with tissue-based testing for identifying actionable mutations (22). In a study of breast cancer by Turner et al. (plasmaMATCH trial), there was 98%-100% concordance (23). Although the difference between primary and metastatic lesions may affect the result, further studies on ctDNA for primary breast cancer are needed.
This study has some limitations. First the sample size was small because we focused on a limited population. Second, it included only retrospectively collected cases. Increasingly, however, disease-tracking markers targeting the cyclin D1–CDK4/6 complex will be required for effective drug administration. This study suggests that the use of ctDNA may enable minimally invasive and real-time monitoring. In conclusion, we were able to detect CCND1 CNV in ctDNA from patients with luminal B breast cancer. Positive CCND1 CNV may be associated with worse survival.
Acknowledgements
The Authors thank Emiko Manabe, Shoko Sadatomi, Tamaki Nakagaki, Nobuhiro Torata, Midori Kojyo, and Emi Nagao for their technical assistance. We are thankful to Jun Furukawa and Akihiro Tsuyada of Riken Genesis Co., Ltd. for performing the analysis of CNV in ctDNA and tissue. The Authors would also like to thank H. Nikki March, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This work was supported by Japan Society for the Promotion of Science KAKENHI Grant Number 18K08577.
Footnotes
↵* These Authors contributed equally to this work.
Authors’ Contributions
AS and M Kubo wrote the manuscript; M Kubo, M Kai and K Kurata designed the study; AS, K Kurata, UT, SH, YH, HK, K Kaneshiro, and MY collected samples; AS and K Kurata performed the experiments and analysed data; MN provided intellectual input. All Authors reviewed the manuscript draft, revised it critically regarding the intellectual content, and approved the final version of the manuscript to be published.
Conflicts of Interest
There are no conflicts of interest regarding this study.
- Received May 20, 2022.
- Revision received June 6, 2022.
- Accepted June 7, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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