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Mitochondrial DNA (mtDNA) variations affect the efficiency of the electron transport chain and production of reactive oxygen species, contributing to carcinogenesis. The D-loop region of mtDNA has emerged as a variation hotspot region in human neoplasia; however, the potential contribution of these variations in breast cancer risk prediction remains unknown. We investigated the relationship between germline single nucleotide polymorphisms (SNPs) in the entire D-loop region and breast cancer risk in Chinese women. Peripheral blood-isolated mtDNA from 2329 patients with breast cancer and 2328 cancer-free controls was examined for SNPs. In the combined cohort, we used traditional risk factors, susceptibility germline polymorphisms, and logistic regression analysis to evaluate the predictive value of susceptibility variants for breast cancer risk. We calculated the area under the receiver operating characteristic curve (AUC) as a measure. We also measured the content of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Individual polymorphisms SNP573 were significantly associated with breast cancer risk in both the discovery and validation cohorts. In the combined cohort, the AUC of the traditional risk factors was 64.3%; after adding susceptibility variants, the AUC was 64.9% (DeLong test, p = 0.007). 8-OHdG levels were significantly higher in patients with breast cancer than in controls and higher in individuals with SNP573 than in those negative for this variation. Overall, oxidative stress might be associated with the risk of breast cancer, and SNP573 might be associated with oxidative stress. Our results indicate the risk potential of polymorphisms in the D-loop region in breast cancer in Southern China., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

To assess the predictive and prognostic value of a subtyping method based on immunohistochemistry in patients with triple-negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NAC). This study included patients with TNBC treated with anthracycline- and taxane-based NAC and curative surgery. Immunohistochemical (IHC) subtyping was performed using core needle biopsy specimens before NAC (pre-NAC) and residual tumors after NAC (post-NAC). Logistic regression was performed to identify predictive biomarkers of pathological complete response (pCR). Invasive disease-free survival (iDFS), distant disease-free survival (DDFS), and overall survival (OS) were assessed using the log-rank test and Cox proportional hazards regression. A total of 230 patients were followed up for a median of 59 months. Clinical lymph node status and the pre-NAC subtype were independent predictors of pCR (P=0.006 and 0.005, respectively). The pre-NAC subtype was an independent prognostic factor for long-term survival (iDFS: P < 0.001, DDFS: P=0.010, and OS: P=0.044). Among patients with residual disease (RD) after NAC, approximately 45% of tumors changed their IHC subtype. Furthermore, the post-NAC subtype, but not the pre-NAC subtype, was strongly associated with the survival of patients with RD (iDFS: P < 0.001, DDFS: P=0.005, and OS: P=0.006). The IHC subtype predicted response to NAC and long-term survival in patients with early TNBC. In patients with RD, almost 45% of the tumors changed subtype after NAC. The IHC subtype should be considered when planning additional therapies pre- and post-NAC., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Radiation pneumonitis (RP) is one of the common side effects after adjuvant radiotherapy in breast cancer. Irradiation dose to normal lung was related to RP. We aimed to propose an organ features based on deep learning (DL) model and to evaluate the correlation between normal lung dose and organ features., Methods: Patients with pathology-confirmed invasive breast cancer treated with adjuvant radiotherapy following breast-conserving surgery in four centers were included. From 2019 to 2020, a total of 230 patients from four nationwide centers in China were screened, of whom 208 were enrolled for DL modeling, and 22 patients from another three centers formed the external testing cohort. The subset of the internal testing cohort (n = 42) formed the internal correlation testing cohort for correlation analysis. The outline of the ipsilateral breast was marked with a lead wire before the scanning. Then, a DL model based on the High-Resolution Net was developed to detect the lead wire marker in each slice of the CT images automatically, and an in-house model was applied to segment the ipsilateral lung region. The mean and standard deviation of the distance error, the average precision, and average recall were used to measure the performance of the lead wire marker detection model. Based on these DL model results, we proposed an organ feature, and the Pearson correlation coefficient was calculated between the proposed organ feature and ipsilateral lung volume receiving 20 Gray (Gy) or more (V20)., Results: For the lead wire marker detection model, the mean and standard deviation of the distance error, AP (5 mm) and AR (5 mm) reached 3.415 ± 4.529, 0.860, 0.883, and 4.189 ± 8.390, 0.848, 0.830 in the internal testing cohort and external testing cohort, respectively. The proposed organ feature calculated from the detected marker correlated with ipsilateral lung V20 (Pearson correlation coefficient, 0.542 with p < 0.001 in the internal correlation testing cohort and 0.554 with p = 0.008 in the external testing cohort)., Conclusions: The proposed artificial Intelligence-based CT organ feature was correlated with normal lung dose in adjuvant radiotherapy following breast-conserving surgery in patients with invasive breast cancer., Trial Registration: NCT05609058 (08/11/2022)., (© 2023. The Author(s).)

While overweight/obesity has become a major public health issue worldwide, any association between body mass index (BMI) and therapeutic response in neoadjuvant targeted therapy treated HER2 positive breast cancer patients remain unclear. The information from a total of four-hundred and ninety-one neoadjuvant targeted therapy treated HER2 positive breast cancer patients from four institutions were retrospectively collected. Univariate and multivariate logistic analysis was developed to determine the association between BMI and therapeutic response. A meta-analysis of published literature was then conducted to confirm the effect of overweight/obesity on pCR for patients treated with neoadjuvant targeted therapy. Restricted cubic spline (RCS) adjusted for confounding factors demonstrated a decrease pCR with increasing BMI (OR = 0.937, P = 0.045). Patients were then categorized into under/normal weight (n = 299) and overweight/obesity (n = 192). Overweight/obese patients were independently associated with a poor therapeutic response. In the subgroup analysis, a significant negative impact of overweight/obesity on pCR can be observed both in single-targeted (OR = 0.556; P = 0.02) and dual-targeted (OR = 0.392; P = 0.021) populations. Six eligible studies involving 984 neoadjuvant targeted therapy treated HER2 positive breast cancer patients were included in the meta-analysis. The meta-analysis also demonstrated that overweight/obesity was significantly associated with a poor response to neoadjuvant anti-HER2 therapy (OR = 0.68; P = 0.007). Our result show that overweight and obese HER2 positive breast cancer patients are less likely to achieve pCR after neoadjuvant targeted therapy., (© 2023. The Author(s).)

Background: Compelling evidence has indicated a significant association between leukocyte mitochondrial DNA copy number (mtDNAcn) and prognosis of several malignancies in a cancer-specific manner. However, whether leukocyte mtDNAcn can predict the clinical outcome of breast cancer (BC) patients has not been well investigated., Methods: The mtDNA copy number of peripheral blood leukocytes from 661 BC patients was measured using a Multiplex AccuCopy™Kit based on a multiplex fluorescence competitive PCR principle. Kaplan-Meier curves and Cox proportional hazards regression model were applied to investigate the association of mtDNAcn with invasive disease-free survival (iDFS), distant disease-free survival (DDFS), breast cancer special survival (BCSS), and overall survival (OS) of patients. The possible mtDNAcn-environment interactions were also evaluated by the Cox proportional hazard regression models., Results: BC patients with higher leukocyte mtDNA-CN exhibited a significantly worse iDFS than those with lower leukocyte mtDNAcn (5-year iDFS: fully-adjusted model: HR = 1.433[95%CI 1.038-1.978], P = 0.028). Interaction analyses showed that mtDNAcn was significantly associated with hormone receptor status (adjusted p for interaction: 5-year BCSS: 0.028, 5-year OS: 0.022), so further analysis was mainly in the HR subgroup. Multivariate Cox regression analysis demonstrated that mtDNAcn was an independent prognostic factor for both BCSS and OS in HR-positive patients (HR+: 5-year BCSS: adjusted HR (aHR) = 2.340[95% CI 1.163-4.708], P = 0.017 and 5-year OS: aHR = 2.446 [95% CI 1.218-4.913], P = 0.011)., Conclusions: For the first time, our study demonstrated that leukocyte mtDNA copy number might influence the outcome of early-stage breast cancer patients depending on intrinsic tumor subtypes in Chinese women., (© 2023. The Author(s).)

Background: Bilateral breast cancer (BBC), as well as ovarian cancer, are significantly associated with germline deleterious variants in BRCA1/2, while BRCA1/2 germline deleterious variants carriers can exquisitely benefit from poly (ADP-ribose) polymerase (PARP) inhibitors. However, formal genetic testing could not be carried out for all patients due to extensive use of healthcare resources, which in turn results in high medical costs. To date, existing BRCA1/2 deleterious variants prediction models have been developed in women of European or other descent who are quite genetically different from Asian population. Therefore, there is an urgent clinical need for tools to predict the frequency of BRCA1/2 deleterious variants in Asian BBC patients balancing the increased demand for and cost of cancer genetics services., Methods: The entire coding region of BRCA1/2 was screened for the presence of germline deleterious variants by the next generation sequencing in 123 Chinese BBC patients. Chi-square test, univariate and multivariate logistic regression were used to assess the relationship between BRCA1/2 germline deleterious variants and clinicopathological characteristics. The R software was utilized to develop artificial neural network (ANN) and nomogram modeling for BRCA1/2 germline deleterious variants prediction., Results: Among 123 BBC patients, we identified a total of 20 deleterious variants in BRCA1 (8; 6.5%) and BRCA2 (12; 9.8%). c.5485del in BRCA1 is novel frameshift deleterious variant. Deleterious variants carriers were younger at first diagnosis (P = 0.0003), with longer interval between two tumors (P = 0.015), at least one medullary carcinoma (P = 0.001), and more likely to be hormone receptor negative (P = 0.006) and HER2 negative (P = 0.001). Area under the receiver operating characteristic curve was 0.903 in ANN and 0.828 in nomogram modeling individually (P = 0.02)., Conclusion: This study shows the spectrum of the BRCA1/2 germline deleterious variants in Chinese BBC patients and indicates that the ANN can accurately predict BRCA deleterious variants than conventional statistical linear approach, which confirms the BRCA1/2 deleterious variants carriers at the lowest costs without adding any additional examinations., (© 2022. The Author(s).)

Purpose: Current trials support the application of sentinel lymph node biopsy (SLNB) in node-positive breast cancer treated with neoadjuvant chemotherapy (NAC) with a lower false-negative rate (FNR) if dual-tracer (radioisotope and blue-dye) is used. However, radioisotopes are not available in many areas of the world. In this study, we evaluated the feasibility and accuracy of SLNB mapped with methylene-blue-dye alone., Methods: This study enrolled 132 patients with biopsy-proven node-positive breast cancer with a clip placed in the positive node who then received NAC. After chemotherapy and before operation, all patients underwent axillary ultrasound (AUS) assessment and were classified as either negative (AUS-) or positive (AUS +) according to the axillary status. All patients underwent both SLNB and axillary lymph node dissection (ALND). SLNB was mapped with methylene-blue-dye alone. FNRs were evaluated on factors potentially affecting false-negative SLN finding., Results: Using methylene-blue-dye alone, the FNR of SLNB was 9.9%. Post-NAC AUS assessment (p = 0.009) and the number of SLNs retrieved (p = 0.029) showed association with FNRs in multivariate analysis. In AUS- group, FNR was as low as 2.5%. In AUS + group, retrieving ≥ 4 SLNs including the clipped node improved FNR from 17.1% to 4.8%. A flowchart was designed with the combination of post-NAC AUS assessment, retrieved SLN number, and the retrieved of clipped node further improve overall FNR to 3.3%., Conclusion: In biopsy-proven node-positive breast cancer treated with NAC, using a flowchart to optimize patient selection reduces the FNR of single-tracer (methylene-blue-dye) guided SLNB., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Peripherally inserted central catheters have been extensively applied in clinical practices. However, they are associated with an increased risk of thrombosis. To improve patient care, it is critical to timely identify patients at risk of developing peripherally inserted central catheter-related thrombosis. Artificial neural networks have been successfully used in many areas of clinical events prediction and affected clinical decisions and practice., Objective: To develop and validate a novel clinical model based on artificial neural network for predicting peripherally inserted central catheter-related thrombosis in breast cancer patients who underwent chemotherapy and determine whether it may improve the prediction performance compared with the logistic regression model., Design: A prospective cohort study., Setting: A large general hospital in Fujian Province, China., Participants: One thousand eight hundred and forty-four breast cancer patients with peripherally inserted central catheters placement for chemotherapy were eligible for the study., Methods: The dataset was divided into a training set (N = 1497) and an independent validation set (N = 347). The synthetic minority oversampling technique (SMOTE) was used to handle the effect of imbalance class. Both the artificial neural network and logistic regression models were then developed on the training set with and without SMOTE, respectively. The performance of each model was evaluated on the validation set using accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve (AUC)., Results: Of the 1844 enrolled patients, 256 (13.9%) were diagnosed with peripherally inserted central catheter-related thrombosis. Predictive models were constructed in the training set and assessed in the validation set. Eight factors were selected as input variables to develop the artificial neural network model. Without SMOTE, the artificial neural network model (AUC = 0.725) outperformed the logistic regression model (AUC = 0.670, p = 0.039). SMOTE improved the performance of both two models based on AUC. With the SMOTE sampling, the artificial neural network model performed the best across all evaluated models, the AUC value remained statistically better than that of the logistic regression model (0.742 vs. 0.675, p = 0.004)., Conclusion: Artificial neural network model can effectively predict peripherally inserted central catheter-related thrombosis in breast cancer patients receiving chemotherapy. Identifying high-risk groups with peripherally inserted central catheter-related thrombosis can provide close monitoring and an opportune time for intervention., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Triple-negative breast cancer (TNBC) is characterized by a more aggressive biological behavior and unfavorable outcome. Circulating and histological expression of THBS2 has been demonstrated to be a novel diagnostic and prognostic biomarker in patients with various types of tumors. However, few studies have evaluated the predictive and prognostic value of THBS2 in TNBC specifically. Methods: In total, 185 triple-negative breast cancer patients (TNBC) with preoperative neoadjuvant chemotherapy were enrolled in this study. Serum THBS2 (sTHBS2) level was measured both prior to the start of NAC and at surgery by enzyme-linked immunosorbent assay (ELISA). Histological THBS2 (hTHBS2) expression in patients with residual tumors was evaluated by immunohistochemistry (IHC) staining method. Correlations between variables and treatment response were studied. Kaplan-Meier plots and Cox proportional hazard regression model were applied for survival analysis. Functional activities of THBS2 in TNBC cells were determined by CCK-8 assay, colony formation, wound healing, and transwell assay. Results: Of the 185 patients, 48 (25.9%) achieved pathological complete response (pCR) after completion of NAC. Elevated pCR rates were observed in patients with a lower level of sTHBS2 at surgery and higher level of sTHBS2 change (OR = 0.88, 95%CI: 0.79-0.98, p = 0.020 and OR = 1.12, 95%CI: 1.02-1.23, p = 0.015, respectively). In survival analysis, hTHBS2 expression in residual tumor was of independent prognostic value for both disease-free survival (HR = 2.21, 95%CI = 1.24-3.94, p = 0.007) and overall survival (HR = 2.07, 95%CI = 1.09-3.92, p = 0.026). For functional studies, THBS2 was indicated to inhibit proliferation, migration, and invasion abilities of TNBC cells in vitro . Conclusion: Our findings confirmed the value of serum THBS2 level to predict pCR for TNBC patients and the prognostic performance of histological THBS2 expression in non-pCR responders after NAC. THBS2 might serve as a promising functional biomarker for patients with triple-negative breast cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lin, Lin, Li, Chen, Chen, Huang, Guo, Chen, Wu, Zhang, Zhang, Jin, Zhang, Fu and Wang.)

Biomarkers are indispensable for precision medicine. However, focused single-biomarker development using human tissue has been complicated by sample spatial heterogeneity. To address this challenge, we tested a representation of primary tumor that synergistically integrated multiple in situ biomarkers of extracellular matrix from multiple sampling regions into an intratumor graph neural network. Surprisingly, the differential prognostic value of this computational model over its conventional non-graph counterpart approximated that of combined routine prognostic biomarkers (tumor size, nodal status, histologic grade, molecular subtype, etc.) for 995 breast cancer patients under a retrospective study. This large prognostic value, originated from implicit but interpretable regional interactions among the graphically integrated in situ biomarkers, would otherwise be lost if they were separately developed into single conventional (spatially homogenized) biomarkers. Our study demonstrates an alternative route to cancer prognosis by taping the regional interactions among existing biomarkers rather than developing novel biomarkers., (© 2022. The Author(s).)

Background: Cytidine nucleotide triphosphate synthase 1 (CTPS1) is a CTP synthase which play critical roles in DNA synthesis. However, its biological regulation and mechanism in triple-negative breast cancer (TNBC) has not been reported yet., Methods: The expression of CTPS1 in TNBC tissues was determined by GEO, TCGA databases and immunohistochemistry (IHC). The effect of CTPS1 on TNBC cell proliferation, migration, invasion, apoptosis and tumorigenesis were explored in vivo and in vitro. In addition, the transcription factor Y-box binding protein 1 (YBX1) was identified by bioinformatics methods, dual luciferase reporter and chromatin immunoprecipitation (CHIP) assays. Pearson correlation analysis was utilized to assess the association between YBX1 and CTPS1 expression., Results: CTPS1 expression was significantly upregulated in TNBC tissues and cell lines. Higher CTPS1 expression was correlated with a poorer disease-free survival (DFS) and overall survival (OS) in TNBC patients. Silencing of CTPS1 dramatically inhibited the proliferation, migration, invasion ability and induced apoptosis of MDA-MB-231 and HCC1937 cells. Xenograft tumor model also indicated that CTPS1 knockdown remarkably reduced tumor growth in mice. Mechanically, YBX1 could bind to the promoter of CTPS1 to promote its transcription. Furthermore, the expression of YBX1 was positively correlated with CTPS1 in TNBC tissues. Rescue experiments confirmed that the enhanced cell proliferation and invasion ability induced by YBX1 overexpression could be reversed by CTPS1 knockdown., Conclusion: Our data demonstrate that YBX1/CTPS1 axis plays an important role in the progression of TNBC. CTPS1 might be a promising prognosis biomarker and potential therapeutic target for patients with triple-negative breast cancer., (© 2022. The Author(s).)

Background: The YTH domain family protein 3 (YTHDF3) is an important N6-methyladenosine (m 6 A) reader which is involved in multiple cancers. However, the biological role and mechanisms of action for YTHDF3 in triple-negative breast cancer (TNBC) remains to be elucidated., Methods: The expression of YTHDF3 in TNBC tissues was evaluated using The Cancer Genome Atlas (TCGA) database, BC-GenExMiner, and immunohistochemistry (IHC) staining. Cell migration, invasion, and epithelial-mesenchymal transition (EMT) were validated by wound healing assays, transwell assays, and Western blot (WB) analyses. The association between YTHDF3 and zinc finger E-box-binding homeobox 1 (ZEB1) was confirmed by Pearson correlation analysis. RNA-binding protein immunoprecipitation (RIP) assays and mRNA actinomycin stability analyses were applied to confirm whether YTHDF3 could interact with ZEB1in an m 6 A-dependent manner., Results: The expression of YTHDF3 was correlated with poorer disease-free survival (DFS) and overall survival (OS) in TNBC patients. Functional experiments indicated that YTHDF3 positively regulated cell migration, invasion, and EMT in TNBC cells. Moreover, ZEB1 was identified as a key downstream target for YTHDF3 and YTHDF3 could enhance ZEB1 mRNA stability in an m 6 A-dependent manner. Inhibition of YTHDF3 reduced migration, invasion, and EMT, all of which were reversed by rescue experiments overexpressing ZEB1., Conclusions: The findings herein confirmed that the YTHDF3/ZEB1 axis plays an important role in the progression and metastasis of TNBC. YTHDF3 is a promising prognosis biomarker and potential therapeutic target for patients with TNBC., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-21-6857/coif). The authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

Purpose: We investigate the prognostic value of tumour-infiltrating lymphocytes (TILs) based on the evaluation of the present frequency in patients with breast cancer rather than that of the density proposed in previous research., Methods: Multiphoton microscopy (MPM) was introduced to label-freely obtain TIL images from a total of 564 patients, and then TILs were redefined as TILs-1 to TILs-3 from MPM images according to the relative positions between TILs, tumour cells and collagen fibres. More seminally, a new method, which was based on the present frequency of TILs-1 to TILs-3, was presented for assessing the predictive ability of TILs, and then a tumour-infiltrating lymphocytes score (TILs-score) for each patient was obtained by ridge regression analysis., Results: Data results from Cox proportional hazards regression analysis showed that the TILs-score was an independent prognostic factor for both disease-free survival (DFS) and overall survival (OS) in the complete cohort (n = 564), oestrogen receptor (ER)-positive subgroup (n = 352) and ER-negative subgroup (n = 212), but was more suitable for the ER-positive subgroup. Furthermore, the nomogram model combining the TILs-score with independent clinical factors further improved the predictive ability for the ER-positive subgroup: area under the curve (AUC) at 5-year DFS (OS) and hazard ratio (HR) for DFS (OS) in the training cohort increase from 0.735 (0.785) to 0.814 (0.830) and from 3.156 (5.845) to 4.643 (7.006), respectively, and in the validation cohort from 0.749 (0.748) to 0.804 (0.830) and from 3.104 (3.701) to 3.729 (5.132), respectively., Conclusion: The TILs-score is an independent prognostic factor and displays a strong prognostic value for ER-positive breast cancer. To our knowledge, this is the first time to use MPM for studying the prognostic value of TILs in breast cancer., Competing Interests: Conflict of interest statement The authors declare that they have no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: To study the prognostic parameters in patients with breast cancer of size smaller than or equal to 2 cm which are useful for treatment and follow-up., Methods: Four hundred and seventy-five patients with breast cancer of size smaller than or equal to 2 cm diagnosed and treated in Affiliated Union Hospital, Fujian Medical University, China during the period from January, 2002 to December, 2008 were enrolled into the study. The clinical features, pathologic findings and follow-up data were analyzed. Kaplan-Meier curve method and Cox proportional hazard regression model were used to study factors which influenced disease-free survival and overall survival of the patients., Results: Onset below 40 years of age (P=0.000), presence of axillary lymph node metastasis (P=0.000) and histologic grade 3 (P=0.013) negatively correlated with 5-year disease-free survival. Onset below 40 years of age (P=0.000), presence of axillary lymph node metastasis (P=0.000), histologic grade 3 (P=0.012) and negative estrogen receptor status (P=0.035) negatively correlated with 5-year overall survival. Multivariate analysis indicated that onset below 40 years of age (HR=3.249, 95% CI: 1.514-6.974, P=0.002) and presence of axillary lymph node metastasis (HR=3.177, 95% CI: 1.695-5.953, P=0.000) were independent predictors of 5-year disease-free survival. Onset below 40 years of age (HR=5.006, 95% CI: 2.013-12.449, P=0.001), presence of axillary lymph node metastasis (HR=4.461, 95% CI: 1.948-10.218, P=0.000) and negative estrogen receptor status (HR=2.612, 95% CI: 1.092-6.246, P=0.031) were independent predictors of 5-year overall survival., Conclusions: Onset below 40 years of age, presence of axillary lymph node metastasis and negative estrogen receptor status are prognostic indicators in patients with breast cancer of size less than or equal to 2 cm. Assessment of these prognostic parameters would be helpful in treatment and follow-up of this group of breast cancer patients.

Mutation analysis in breast cancer has failed to explain the inactivation of RhoBTB2, a candidate breast cancer tumor suppressor gene on chromosome 8p. Some breast cancer-related genes in this region become inactivated by hypermethylation, and hypermethylation of RhoBTB2 abrogates its expression in bladder cancers. The aim of the present study was to determine whether RhoBTB2 was silenced by methylation in breast cancer. Nested methylation-specific PCR (nMSP) and quantitative reverse transcription PCR were used to analyze the methylation status and mRNA levels of RhoBTB2 in 50 paired breast cancer and normal tissues and the results were correlated with clinicopathological characteristics. Promoter methylation and the downregulation of RhoBTB2 mRNA was observed in tumor tissues (P<0.001). mRNA levels were decreased in samples with methylation (χ² = 15.751, P<0.001). RhoBTB2 methylation was observed preferentially in progesterone receptor (PR)-negative samples (P<0.05). The results demonstrated that aberrant methylation of RhoBTB2 may be responsible for the suppression of RhoBTB2 mRNA expression in breast cancer, a significant event during the genesis of breast cancer that correlated with PR status.