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The contribution of Ca 2+ in TGF-β-induced EMT is poorly understood. We aimed to confirm the effect of TGF-β on the gene expression of intracellular calcium-handling proteins and to investigate the potential underlying mechanisms in TGF-β-induced EMT. T47D and MCF-7 cells were cultured in vitro and treated with TGF-β. The mRNA expression of EMT marker genes and intracellular calcium-handling proteins were quantified by qRT-PCR. qRT-PCR and Western blot analysis results verified the changes of EMT marker gene expression. Furthermore, we found that TGF-β induced cell morphological changes significantly with an increase of cell surface area and cell length. These results indicated that TGF-β induced EMT. The mRNA expression levels of SPCA1, SPCA2 and MCU were not influenced by TGF-β treatment, while NCX1 expression was decreased in T47D cells. In addition, the mRNA levels of SERCAs and IP 3 Rs were significantly changed due to TGF-β-induced EMT. The TGF-β-treated T47D cells exhibited markedly greater response to ATP than the control cells, and the descent velocity of cytosolic calcium concentration was faster in TGF-β-treated cells than in control cells. This is the first report to demonstrate that TGF-β-induced EMT in human breast cancer cells is associated with alterations in endoplasmic reticulum calcium homeostasis. [ABSTRACT FROM AUTHOR]

Anti-GP-1D8 monoclonal antibody was produced in our laboratory. Immunoaffinity purification of GP-1D8 glycoprotein from human breast carcinoma tissues, on column with the monospecific antibody, is developed. The procedure permits purification of GP-1D8 to a highly purified state. It appeared as a single band in sodium dodcyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of GP-1D8 was determined to be 66 kDa by mass spectrometry. Its antigenicity was stable between 0 and 70°C. It was breast cancer specific, as determined by immunohistochemistry and immunocytochemistry. The preparation of anti-GP-1D8 monoclonal antibody will facilitate further detection of, and functional study on, GP-1D8. The study also provides a simple, economical, and efficient method for affinity purification of target proteins from human or animal tissues. [ABSTRACT FROM AUTHOR]

Background: The status of axillary lymph nodes (ALNs) is one of the important factors in decision‐making for breast cancer treatment. Physical examination (PE) has long been the main, or even the only, means of clinical staging for ALNs in breast cancer. However, the sensitivity and accuracy of PE remains unsatisfactory. The results from this study suggest that axillary ultrasonography (US) should replace PE as a standard method for the clinical staging of ALNs in breast cancer. Methods: Consecutive and nonselective breast cancer patients treated between September 2018 and November 2018 in our center were enrolled in the study. Comparisons of ALN results between PE/US and pathological results were conducted and the difference in sensitivity, specificity and accuracy between PE and US were tested by McNemar chi‐square test. Results: A total of 123 patients were enrolled into the study. Their ages ranged from 28 to 76 years with a median age of 53 ± 10. There were 83 ALN positive cases and 40 ALN negative cases confirmed pathologically. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of PE and US were 54.2%, 90.0%, 65.9%, 91.8%, 48.7% versus 86.8%, 72.5%, 82.1%, 86.8%, 72.5%, respectively. The sensitivity and accuracy of US was significantly higher than that of PE (P = 0.004 and P = 0.002). Conclusion: The results of this study demonstrated that US is superior in evaluating ALNs when compared with PE and that US should replace PE as the standard method for the clinical staging of ALNs in breast cancer. [ABSTRACT FROM AUTHOR]

Purpose: The role of long-acting hematopoietic growth factor in supporting dose-dense chemotherapy and minimizing hematologic toxicity has not been established. We investigated the efficacy and safety of once-per-cycle pegfilgrastim in breast cancer patients receiving neoadjuvant dose-dense epirubicin and cyclophosphamide (ddEC).Methods: Newly diagnosed stage I to III breast cancer patients received four cycles of ddEC (E, 100 mg/m2 and C, 600 mg/m2 every 2 weeks) and 6 mg of subcutaneous pegfilgrastim on day 2 of each cycle. The primary endpoint was to evaluate the incidence of chemotherapy delay. Secondary endpoints include the incidences of febrile neutropenia (FN) and grade 3/4 neutropenia during the four ddEC cycles.Results: A total of 240 patients were enrolled and 913 ddEC cycles were administered in the study. Chemotherapy delay occurred in 15 patients (6.3% of patients, 95% CI 3.2-9.4%) for 17 cycles (1.9% of cycles, 95% CI 1.0-2.8%). The most frequent cause of chemotherapy delay was transaminase elevation (10 patients, 12 cycles). A total of 12 patients (5.0%, 95% CI 2.2-7.8%) developed 13 episodes of FN. Of the 221 patients that completed four ddEC cycles with pegfilgrastim support, 209 patients (94.6%, 95% CI 91.6-97.6%) had a 100% relative dose intensity (RDI). A RDI ≥ 85% was achieved in 217 of 221 patients (98.2%, 95% CI 96.5-99.9%). Bone pain of any grade was recorded in 85 of 220 evaluable patients (38.6%, 95% CI 32.2-45.0%).Conclusions: Pegfilgrastim is effective and safe in facilitating four cycles of neoadjuvant ddEC, with low incidences of chemotherapy delay and febrile neutropenia. [ABSTRACT FROM AUTHOR]

Purpose: The prevalence and clinical relevance of TP53 and PIK3CA mutations in pretreatment breast cancer have been previously reported. However, little is known regarding these mutations in residual tumor tissues after neoadjuvant chemotherapy. Here, we investigated the association between TP53 and PIK3CA mutations in residual disease and survival of breast cancers. Methods: TP53 and PIK3CA somatic mutations were examined in 353 post-neoadjuvant chemotherapy residual tumor tissues by Sanger sequencing. Survival curves of patients with TP53 and PIK3CA mutations were compared using the Kaplan–Meier method. Results: Fifty-six (15.9%) of the 353 patients carried a TP53 somatic mutation and 79 patients (22.4%) carried a PIK3CA somatic mutation. A total of 18 patients carried co-mutation of TP53 and PIK3CA. Patients with somatic co-mutation were more likely to have high-grade tumors (35.3% vs. 10.6%, P = 0.010), estrogen receptor-negative tumors (55.6% vs. 26.7%, P = 0.009), progesterone receptor-negative tumors (61.1% vs. 30.5%, P = 0.008) and triple-negative tumors (35.3% vs. 13.3%, P = 0.025) compared with non-carriers. More importantly, co-mutation of TP53 and PIK3CA carriers had a significantly worse disease-free survival (DFS) and distant disease-free survival (DDFS) than non-carriers (5-year DFS: 58.0% vs. 83.2%, P < 0.001; 5-year DDFS: 70.3% vs. 86.4%, P = 0.024). Furthermore, in multivariate regression analysis, TP53 and PIK3CA co-mutation carriers showed a significantly worse DFS (adjusted hazard ratio = 3.70; 95% confidence interval, 1.79–7.63; P < 0.001). Conclusions: Patients with somatic co-mutation of TP53 and PIK3CA were associated with unfavorable survival compared with non-carriers. Co-mutation of TP53 and PIK3CA could be used as a potential prognosis marker in post-neoadjuvant chemotherapy breast cancer patients. [ABSTRACT FROM AUTHOR]

PURPOSE: To investigate the association between the HER2 germline mutation Ala270Ser (A270S), located in HER2 extracellular domain, and survival in breast cancer patients. METHODS: HER2 germline mutation A270S was identified in 5395 consecutive patients with operable primary breast cancer using direct Sanger sequencing analysis. Survival curves for patients with HER2 A270S mutation were compared using the Kaplan-Meier method with log-rank test. RESULTS: We identified that 31 cases carried HER2 germline mutation A270S in 5395 patients (0.6%, 31/5395). The HER2 A270S mutation was significantly associated with recurrence-free survival (RFS) and distant recurrence-free survival (DRFS) in the entire cohort of 5395 patients (RFS, unadjusted hazard ratio [HR] = 2.23; 95% confidence interval [CI] = 1.00–5.00; P = 0.045; DRFS, unadjusted HR = 2.80; 95% CI = 1.25–6.28; P = 0.009). Among the HER2-negative patients (n = 3825), those with the HER2 A270S mutation had a significantly worse RFS (unadjusted HR = 3.19; 95% CI = 1.42–7.16; P = 0.003) and DRFS (unadjusted HR = 3.98; 95% CI = 1.77–8.96; P < 0.001) than did those with wild type. Moreover, the A270S mutation remained an independent unfavorable factor for RFS and DRFS in the HER2-negative patients (RFS, HR = 3.30; 95% CI = 1.34–8.10; P = 0.009; DRFS, HR = 4.26; 95% CI = 1.73–10.47; P = 0.002). CONCLUSIONS: Breast cancer patients with the HER2 germline mutation A270S had a worse survival, especially in HER2-negative patients. Therefore, HER2-negative patients with a HER2 germline mutation A270S might be potential candidates for HER2-targeted therapy. [ABSTRACT FROM AUTHOR]

Abstract: Knowledge of the pathology of axillary lymph nodes (ALN) in breast cancer patients is critical for determining their treatment. Ultrasound is the best noninvasive evaluation for the ALN status. However, the correlation between negative ultrasound results and the sentinel lymph nodes (SLN) pathology remains unknown. To test the hypothesis that negative ultrasound results of ALN predict the negative pathology results of SLN in breast cancer patients, we assessed the association between ALN ultrasonography‐negative results and the SLN pathology in 3115 patients with breast cancer recruited between October 2010 and April 2016 from a single cancer center, prospective database. Of these patients who met the inclusion criteria, 2317 (74.4%) had no SLN pathological metastasis. In the univariate analysis, other 798 patient with positive SLN tended to be under age 40 and premenopausal, having large tumor sizes (>2 cm), higher histological grade of primary tumor, positive hormone receptors, and negative HER‐2 status (P < .05 for all). In the multivariate analysis, menstrual status, tumor size, ER status and histological types of primary tumor remained to be independent predictors for SLN pathological metastasis. The area under curve (AUC) was 0.658 (95% CI = 0.637‐0.679), P > .05. In conclusion, only a 74.4% consistency between ALN ultrasonography‐negative results and negative pathological SLN results, although menstrual status, tumor size, histologic subtypes of primary tumor and ER status were found to be statistically independent predictors of positive SLN among patients negative for ALN ultrasonography. Therefore, the present study suggests that negative ultrasound results of ALN do not adequately predict the negative pathology results of SLN in breast cancer patients. [ABSTRACT FROM AUTHOR]

Background: RECQL is a number of the RecQ DNA helicase family and plays an important role in maintaining genome stability. Although several studies have reported that RECQL mutations were correlated with the susceptibility to breast cancer, the effect on prognosis in breast cancer was not yet clarified. Here, we explored the association between RECQL expression level and survival in patients with breast cancer.Methods: In the first cohort, the RECQL mRNA expression level was evaluated in 774 primary breast cancer patients using a quantitative real-time PCR assay. Then, in the second independent cohort, the level of RECQL protein expression was detected in 322 patients with breast cancer using immunohistochemistry assay. Survival curves of patients with RECQL expression were compared using the Kaplan-Meier method with log-rank test.Results: In the first cohort of 774 breast cancer patients, the low expression level of RECQL mRNA was significantly correlated with aggressive clinicopathological characteristics, including the positive lymph node status (P = 0.026), HER2 overexpression (P < 0.001), ER negative status (P = 0.047) and high tumor grade (P = 0.041). Moreover, the low expression level of RECQL mRNA was significantly associated with poor distant recurrence-free survival (DRFS, unadjusted hazard ratio (HR): 2.77, 95% confidence interval (CI): 1.88-4.09, P < 0.001) and disease-specific survival (DSS, unadjusted HR: 3.10, 95% CI: 1.84-5.20,P < 0.001), and it remained an independent unfavorable factor for DRFS and DSS (DRFS: adjusted HR: 3.04, 95% CI: 1.89-4.87, P < 0.001; DSS: adjusted HR: 4.25, 95% CI: 2.12-8.46, P < 0.001). In the second cohort of 322 breast cancer patients, low expression of RECQL protein was also subject to poor survival in breast cancer, and it was an independent prognosis factor of poor DRFS by multivariate analysis (DRFS: adjusted HR: 2.12, 95% CI: 1.16-3.88, P = 0.015).Conclusions: Breast cancer patients with low RECQL expression had a worse survival. The expression level of RECQL may be a potential prognosis factor for breast cancer. [ABSTRACT FROM AUTHOR]

The genetic cause for approximately 80% of familial breast cancer patients is unknown. Here, by sequencing the entire exomes of nine early-onset familial breast cancer patients without BRCA1/2 mutations (diagnosed with breast cancer at or before the age of 35) we found that two index cases carried a potentially deleterious mutation in the RECQL gene (RecQ helicase-like; chr12p12). Recent studies suggested that RECQL is involved in DNA double-strand break repair and it plays an important role in the maintenance of genomic stability. Therefore, we further screened the RECQL gene in an additional 439 unrelated familial breast cancer patients. In total, we found three nonsense mutations leading to a truncated protein of RECQL (p.L128X, p.W172X, and p.Q266X), one mutation affecting mRNA splicing (c.395-2A>G), and five missense mutations disrupting the helicase activity of RECQL (p.A195S, p.R215Q, p.R455C, p.M458K, and p.T562I), as evaluated through an in vitro helicase assay. Taken together, 9 out of 448 BRCA-negative familial breast cancer patients carried a pathogenic mutation of the RECQL gene compared with one of the 1,588 controls (P = 9.14×10-6). Our findings suggest that RECQL is a potential breast cancer susceptibility gene and that mutations in this gene contribute to familial breast cancer development. [ABSTRACT FROM AUTHOR]

A relatively little is known of whether CYP2D6 *10 (188 C to T) polymorphism mediates susceptibility to breast cancer. In this study the CYP2D6 *10 polymorphism was detected in Chinese women (286 breast cancer patients and 305 healthy women) by a PCR-RFLP assay. We found that women with the 188T/T genotype displayed a slightly increased risk for breast cancer as compared with those with the 188C/C genotype (OR 1.36, CI 0.89–2.1), the association of the 188T/T genotype with breast cancer risk was more pronounced among postmenopausal women (OR 1.49, CI 0.8–2.76), but the association did not reach statistical significance. Furthermore, we found that patients carrying the 188T/T or T/C genotype were more likely to be a positive lymph node status than those with the 188C/C genotype (OR 2.12, CI 1.08–4.18, P = 0.019). Our results suggest that CYP2D6 *10 mutant 188T/T genotype displays a non-significant increased risk for breast cancer. Moreover, patients carrying 188T/T or T/C genotype might exhibit a more aggressive phenotype than those carrying 188C/C genotype, as the observation association of genotype with clinical outcome may be due to chance, therefore, further studies are required to confirm our present findings. [ABSTRACT FROM AUTHOR]