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Yue-Yu Shi,1 Xing-Lei Chen,1 Qian-Xin Chen,1 Yuan-Zhong Yang,2 Meng Zhou,1 Yue-Xiang Ren,3 Lu-Ying Tang,3 Ze-Fang Ren1 1School of Public Health, Sun Yat-sen University, Guangzhou, People’s Republic of China; 2The Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China; 3The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of ChinaCorrespondence: Ze-Fang Ren, School of Public Health, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, Guangdong, People’s Republic of China, Tel/Fax +86-20-87332577, Email renzef@mail.sysu.edu.cn Lu-Ying Tang, The Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou, Guangdong, People’s Republic of China, Tel +86-20-85253000, Fax +86-20-85253336, Email tangly@mail.sysu.edu.cnPurpose: Enolase-1 (ENO1) plays a key role in malignancies. Previous studies on the association between ENO1 expression and breast cancer prognosis had yielded inconsistent results. In the present study, we assessed the prognostic effect of ENO1 in breast cancer using Guangzhou Breast Cancer Study (GZBCS) cohort with full consideration of the potential confounders and the modification effects. The results were further validated in the TCGA-BRCA cohort and explained by tumor immunity.Methods: ENO1 protein expressions were evaluated by immunohistochemistry in tissue microarrays from 961 patients with primary invasive breast cancer. Chi-square tests were used to assess the association of ENO1 levels with the patient’s characteristics. Cox regression models were applied to assess the prognostic effects. The TCGA-BRCA cohort was utilized to validate the results and explore the potential mechanisms. The immune infiltration was determined using the CIBERSORT and ssGSEA algorithms; the correlation between ENO1 expression and the abundance of tumor-infiltrating immune cells (TIICs) and scores of immune-related functions was evaluated by Wilcoxon signed-rank tests and Spearman’s rank test.Results: ENO1 protein expression exerted a protective effect on OS in stage I/II patients (HR=0.58, 95% CI: 0.35– 0.96) but not in stage III patients (HR=1.42, 95% CI: 0.81– 2.49, P interaction=0.04) in GZBCS; consistent results were obtained at mRNA levels in TCGA cohort. Immune infiltration analyses revealed that ENO1 was positively correlated with multiple antitumor TIICs (including M1 macrophages, B cells, CD8 T cells, T helper 2 cells, and NK cells) only in stage I/II but not stage III patients.Conclusion: A higher expression of ENO1 was associated with a better prognosis only in early-stage breast cancer, which may be related to the different effects of ENO1 on immune infiltration, suggesting that ENO1 may be a promising target for precision immunotherapy in breast cancer.Keywords: ENO1, breast cancer, prognosis, tissue microarray, tumor-infiltrating immune cells

Background: Previous studies have found that acute febrile infection may decrease the risk of breast cancer. Meanwhile, it is well known that interleukin-6 (IL6) played dual roles in the tumor microenvironment. Fever may stimulate IL6 production, and IL6 rs1800796 also influences the expression of IL6. However, the impact of fever and its interaction with IL6 rs1800796 on breast cancer survival remains to be explored. Methods: This was a prospective cohort study of 4,223 breast cancer patients. Exposures were pre-/postdiagnostic infection-induced fever and rs1800796 polymorphism. The endpoints were overall survival (OS) and progression-free survival (PFS). Adjusted hazard ratios were obtained using multivariate Cox proportional hazards regression models. Results: Compared with women without prediagnostic fever, the adjusted hazard ratio (HR) of progression for those with prediagnostic fever was 0.81 (95% CI, 0.66–0.99), particularly for the CC genotype of IL6 rs1800796 (HR, 0.53; 95% CI, 0.36–0.79). OS was also better (HR, 0.59; 95% CI, 0.36–0.99) among women with the CC genotype exposed to prediagnostic fever, accompanied by a significant interaction (P = 0.021). Postdiagnostic fever conferred better PFS for breast cancer (HR, 0.72; 95% CI, 0.52–1.00). Irrespective of the genotype of IL6, lymph node–positive women with postdiagnostic fever (HR, 0.57; 95% CI, 0.37–0.89) had a lower risk of progression than lymph node–negative women (HR, 1.12; 95% CI, 0.70–1.79). Conclusions: Infection-induced fever was beneficial to breast cancer survival, particularly for women who were the CC genotype of IL6 rs1800796 or node positive. Impact: This study provides new insight into the roles of infection-induced fever as a potential prognostic marker and therapy regimen for breast cancer.

Background Cellular experiments revealed that a decreased histone H3 lysine 9 trimethylation (H3K9me3) level was associated with the upregulation of oncogenes in breast cancer cells. Moreover, the role of H3K9me3 in breast cancer was closely associated with estrogen receptor (ER) status. Therefore, we aimed to examine the prognostic value of H3K9me3 on breast cancer by ER status. The level of H3K9me3 in tumors were evaluated with tissue microarrays by immunohistochemistry for 917 women diagnosed with primary invasive breast cancer. Hazard ratios (HRs) and their 95% confidence intervals (CIs) for overall survival (OS) and progression-free survival (PFS) were estimated using Cox regression models. Interaction between H3K9me3 and ER on the prognosis was assessed on multiplicative scale. Results The level of H3K9me3 in tumor tissues was lower than that in adjacent tissues. The high level of H3K9me3 was associated with a better OS (HR = 0.43, 95% CI: 0.21–0.86) and PFS (HR = 0.49, 95% CI: 0.29–0.81) among only ER-positive but not ER-negative tumors. Moreover, the interaction between the level of H3K9me3 and ER status (negative and positive) on the prognosis was significant (Pinteraction = 0.011 for OS; Pinteraction = 0.022 for PFS). Furthermore, the ER-positive tumors were stratified by ER-low and ER-high positive tumors, and the prognostic role of H3K9me3 was significant among only ER-high positive patients (HR = 0.34, 95% CI: 0.13–0.85 for OS; HR = 0.47, 95% CI: 0.26–0.86 for PFS). Conclusions Our study showed that the prognostic value of H3K9me3 on breast cancer was related to ER status and expression level, and the high level of H3K9me3 was associated with a better prognosis among ER-positive tumors, particularly ER-high positive tumors.

Results of previous studies on the associations between Forkhead box A1 (FOXA1) expression in breast cancer tissues and the prognosis varied depending on the follow-up durations. The present study would investigate whether there is a time-varying effect of FOXA1 in breast cancer tissues on the prognosis. FOXA1 expressions were evaluated in 1041 primary invasive breast tumors with tissue microarrays by immunohistochemistry. Cox models with restricted cubic splines and Kaplan–Meier survival analysis were used to examine the associations between FOXA1 and the prognosis. Flexible parametric models were applied to explore the time-varying effect of FOXA1. Overall, the association between FOXA1 expression and the prognosis was not significant but varied on the time of follow-up. Compared to FOXA1 ≤ 270 of H-score, the hazard ratios (HRs) of death for those with 271–285 of FOXA1 expression increased from 0.35 (95% CI 0.14–0.86) at 6 months after diagnosis to 2.88 (95% CI 1.35–6.15) at 120 months with a crossover at around 36 months. Similar patterns were also observed for FOXA1 > 285 of H-score and for progression free survival (PFS). Moreover, when allowed both FOXA1 and estrogen receptor (ER) to change over time in the model (considering that ER had a similar time-varying effect), these time-varying effects remained for FOXA1 on both overall survival (OS) (P

Reproductive factors associated with breast cancer risk may also affect the prognosis. This study aimed to evaluate the associations of multiple reproductive factors with breast cancer prognosis and the modifying effects of menopausal status. We obtained data from 3805 breast cancer patients recruited between October 2008 and June 2016 in Guangzhou. The subjects were followed up until 30 June 2018. The hazard ratios (HRs) and 95% confidence intervals (95% CIs) were calculated using multivariate Cox models to estimate the associations. It was found that there were U‐shaped patterns for the associations of age at first birth and durations from first/last birth to diagnosis with breast cancer prognosis. The adverse effects of old age at first birth [>30 years vs 23‐30 years, HR (95% CI): 1.59 (1.01‐2.50)] and long intervals from first [≥20 years vs 10‐19 years, HR (95% CI): 1.55 (1.07‐2.27)] or last [≥20 years vs 10‐19 years, HR (95% CI): 1.63 (1.08‐2.46)] birth to diagnosis on progression‐free survival (PFS) were significantly more pronounced among premenopausal women. Additionally, long interval (>5 years) between first and second birth was associated with a better PFS [HR (95% CI): 0.64 (0.42‐0.97)]. These results suggested that age at first birth, durations from first/last birth to diagnosis, and intervals between first and second birth should be taken into account when following the patients and assessing the prognosis of breast cancer, particularly for premenopausal patients. These findings would also have implications for further insight into the mechanisms of breast cancer development., This study found U‐shaped patterns for the associations of age at first birth and durations from first/last birth to diagnosis with breast cancer prognosis, particularly for premenopausal women; a relatively long interval between first and second birth may benefit prognosis of breast cancer.

Erythropoietic protoporphyria (EPP) is a rare inherited disease whose morbidity is about 1:75,000 to 1:200,000. It is caused by the deficiency of porphyrin ferrochelatase (FECH). Liver involvement in EPP is even rarer. The diagnosis of EPP with liver involvement mainly relies on clinical manifestations, laboratory examinations, histopathological examinations and genetic testing, which is still a huge challenge for both clinicians and pathologists. Here, 5 cases of EPP with liver injury were collected, and the clinicopathological features of these patients were analyzed. The clinical manifestations and laboratory examinations varied from person to person, whereas the liver biopsies showed that there were dark brown deposits within the hepatocytes, Kupffer cells, bile canaliculi and the lumen of bile ducts, which was a constant finding by histopathological examination. Gene tests were conducted in two of the five cases, and the results confirmed the diagnosis. Fully understanding of the diseases can help us reduce the rate of missed diagnosis and provide proper treatment as early as possible.

Results of the associations between weight change after breast cancer diagnosis and prognosis were inconsistent. The modification effects of menopausal status and endocrine therapy on the associations remain poorly understood.A total of 2016 breast cancer patients were recruited between October 2008 and January 2018 and followed up until December 31, 2019 in Guangzhou. Multivariate Cox models were used to estimate the hazard ratios (HRs) and 95% confidence intervals (95% CIs) for progression-free survival (PFS) in association with weight change after diagnosis.Weight loss at 2 years (HR = 1.34, 95% CI 0.87-2.06) or more than 2 years (HR = 1.95, 95% CI 1.22-3.10) after diagnosis increased risk of breast cancer progression. The adverse effect of weight loss was significantly more pronounced in post-menopausal than pre-menopausal women, particularly for weight loss at 2 years after diagnosis, with the HRs and 95% CIs of 2.41 (1.25-4.63) and 0.90 (0.49-1.64), respectively. Weight gain tended to reduce the risk of disease progression among patients with endocrine therapy but not for those with non-endocrine therapy; the significant interaction between weight gain at 2 years after diagnosis and endocrine therapy was observed (POur finding suggested that weight loss was detrimental to breast cancer prognosis, particularly for post-menopausal women, while weight gain may be a potential beneficial indicator for the patients with endocrine therapy but not for those with non-endocrine therapy.

The antagonistic effect of selenium (Se) against cadmium (Cd)-induced breast carcinogenesis was reported, but underlying mechanisms were unclear. The aim of this study was to identify the epigenetically regulated genes and biological pathways mediating the antagonistic effect. We exposed MCF-7 cells to Cd and Se alone or simultaneously. Cell proliferation was assessed by MTT assay, and differential epigenome (DNA methylation, microRNA, and long non-coding RNA) was obtained by microarrays. We cross-verified the epigenetic markers with differential transcriptome, and the ones modulated by Cd and Se in opposite directions were regarded to mediate the antagonistic effect. The epigenetically regulated genes were validated by using gene expression data in human breast tissues. We further assessed the biological functions of these validated genes. Our results showed that Se alleviated the proliferative effect of Cd on MCF-7 cell. A total of 10 epigenetically regulated genes were regarded to mediate the antagonistic effect, including APBA2, KIAA0895, DHX35, CPEB3, SVIL, MYLK, ZFYVE28, ABLIM2, GRB10, and PCDH9. Biological function analyses suggested that these epigenetically regulated genes were involved in multiple cancer-related pathways, such as focal adhesion and PI3K/Akt pathway. In conclusion, we provided evidence that Se antagonized the Cd-induced breast carcinogenesis via epigenetic modification and revealed the critical pathways.

Glutaminase 1 (GLS) is a therapeutic target for breast cancer; although GLS inhibitors have been developed, only a few subjects responded well to the therapy. Considering that the expression of histone H3 lysine 27 trimethylation (H3K27me3) and menopausal status was closely linked to GLS, we examined the effects of H3K27me3 and menopausal status on GLS to breast cancer prognosis. Data for 962 women diagnosed with primary invasive breast cancer were analyzed. H3K27me3 and GLS expression in tumors were evaluated with tissue microarrays by immunohistochemistry. Hazard ratios (HRs) and their 95% confidence intervals (CIs) for overall survival and progression-free survival were estimated using Cox regression models. Statistical interaction was assessed on multiplicative scale. There was a beneficial prognostic effect of GLS expression on overall survival for those with low H3K27me3 level (HR = 0.50, 95% CI: 0.20–1.28) but an adverse prognostic effect for those with high H3K27me3 level (HR = 3.90, 95% CI: 1.29–11.78) among premenopausal women, and the statistical interaction was significant (Pinteraction = 0.003). Similar pattern was further observed for progression-free survival (HR = 0.44, 95% CI: 0.20–0.95 for low H3K27me3 level, HR = 1.35, 95% CI: 0.74–2.48 for high H3K27me3 level, Pinteraction = 0.024). The statistical interaction did not occur among postmenopausal women. Our study showed that the prognostic effects of GLS on breast cancer correlated to the expression level of H3K27me3 and menopausal status, which would help optimize the medication strategies of GLS inhibitors.

The effect of tea consumption on breast cancer survival remained to be explored. Meanwhile, green tea favorably facilitates lipid metabolisms in breast cancer survivors. This study aimed to examine the effect of tea consumption and the interactions with lipids on breast cancer survival. A total of 1551 breast cancer patients were recruited between April 2008 and March 2012 and followed up until 31 December 2017 in Guangzhou. The endpoint was progression-free survival (PFS). Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using multivariate Cox proportional to estimate the associations. PFS was better among women who regularly drank all teas (mainly green tea) except oolong after cancer diagnosis compared with non-tea drinkers (HR 0.52; 95% CI 0.29 ~ 0.91). This association was more evident among women with normal (HR 0.38; 95% CI 0.18 ~ 0.82) than higher (HR 1.22; 95% CI 0.13 ~ 11.82) total cholesterol, though the interaction was not significant. Moreover, the more they drank (≥ 7 times/week), the better prognosis was (HR 0.30; 95% CI 0.11 ~ 0.84). In contrast, oolong tea was observed to have a potential impaired effect on PFS. Our findings suggested that regularly drinking all teas (mainly green tea) except oolong after diagnosis was beneficial to breast cancer survival, particularly for women with normal lipids, while oolong tea may have an impaired effect.

Purpose This study differentiates patient and care delays of breast cancer and explores the related factors as well as the associations with the prognosis in Guangzhou, a southern city of China. Methods A cohort of female incident breast cancer patients (n=1,551) was recruited from October 2008 to March 2012 and followed up until January 1, 2016 (n=1,374) in the affiliated hospitals of Sun Yat-sen University. The factors associated with patient and care delays were analyzed with multivariable logistic models. Cox proportional hazards regression models were constructed to estimate the impacts of the delays on the prognosis. Results There were 40.4% patient delay (≥3 months) and 15.5% care delay (≥1 month). The patient delay, but not the care delay, was significantly related to the clinical stage and consequently worsened the prognosis of breast cancer (hazard ratio, 1.45; 95% confidence interval, 1.09 to 1.91 for progression-free survival). The factors related to an increased patient delay included premenopausal status, history of benign breast disease, and less physical examination. Conclusion Patient delay was the main type of delay in Guangzhou and resulted in higher clinical stage and poor prognosis of breast cancer. Screening for breast cancer among premenopausal women may be an effective way to reduce this delay.

Objectives Selenium (Se) was a potential anticancer micronutrient with proposed epigenetic effect. However, the Se-induced epigenome in breast cancer cells was yet to be studied. Methods The profiles of DNA methylation, microRNA (miRNA), long non-coding RNA (lncRNA), and message RNA (mRNA) in breast cancer cells treated with sodium selenite were examined by microarrays. We verified the epigenetic modifications by integrating their predicted target genes and differentially expressed mRNAs. The epigenetically regulated genes were further validated in a breast cancer cohort by associating with tumor progression. We conducted a series of bioinformatics analyses to assess the biological function of these validated genes and identified the critical genes. Results The Se-induced epigenome regulated the expression of 959 genes, and 349 of them were further validated in the breast cancer cohort. Biological function analyses suggested that these validated genes were enriched in several cancer-related pathways, such as PI3K/Akt and metabolic pathways. Based on the degrees of expression change, hazard ratio difference, and connectivity, NEDD4L and FMO5 were identified as the critical genes. Conclusions These results confirmed the epigenetic effects of sodium selenite and revealed the epigenetic profiles in breast cancer cells, which would help understand the mechanisms of Se against breast cancer.