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Chenlu Liang, Liuyi Li, Meizhen Zhu, Jiejie Hu, Yang Yu Department of Breast Tumor Surgery, The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang, 310022, People’s Republic of ChinaCorrespondence: Yang YuDepartment of Breast Tumor Surgery, Zhejiang Cancer Hospital, No. 1, Banshan Road, Hangzhou, 310022, Zhejiang Province, People’s Republic of ChinaTel/Fax +86 571 8812 2011Email yuyangkaiyu@163.comPurpose: The results of large randomised trials have changed the treatment strategy of axillary lymph nodes. Axillary lymph node dissection (ALND) can be avoided in some patients with one to two sentinel lymph nodes (SLNs) metastasis based on final paraffin section (FPS) results which called into question the need for intraoperative frozen section (FS). This study aims to assess the guiding value of the number of positive SLN detected via FS for intraoperative ALND.Patients and Methods: This study retrospectively analyzed data from 3303 patients with breast cancer who underwent SLN biopsy between 2015 and 2019. Combined with the FPS results, FS sensitivity, specificity, and false negative rate (FNR) were calculated and the difference in the number of positive SLNs between FS and FPS was analyzed.Results: The overall FNR of FS was 23.21%, which was 76.47% in isolated tumor cells, 62.28% in micrometastasis, and 12.09% in macrometastatic disease. The size of SLN metastasis were significantly associated with a higher FNR (p< 0.001). The accuracy rate of the number of positive SLNs detected via FS was 92.62%. Human epidermal growth factor receptor 2 (HER2) (p< 0.03) and Ki67 (p< 0.02) were significant factors affecting the accuracy rate.Conclusion: FS is a effective method for SLN biopsy, ALND can be avoided in patients with one or two positive SLNs detected via FS.Keywords: breast neoplasms, sentinel lymph node, frozen section, false negative rate, axillary lymph node dissection

BackgroundNeoadjuvant chemotherapy (NACT) is considered a standard treatment strategy for locally advanced triple negative breast cancer (TNBC). TNBC patients who achieve a pathologic complete response (pCR) are predicted to have a better prognosis while unfavorable chemo-sensitivity is still associated with a higher risk of disease relapse. The objective of this study was to construct a nomogram to predict disease-free survival (DFS) for TNBC patients following NACT.MethodsA total of 165 TNBC patients who underwent standard NACT and surgery were retrospectively reviewed, and data on their clinicopathological factors before and after NACT were collected. Independent prognostic factors for DFS were identified by Cox regression based on lower Akaike information criteria (AIC) and Bayesian information criterion (BIC). A nomogram to predict the 2-year and 5-year DFS following NACT for TNBC was constructed based on training cohort (n = 132) and validated by a validation cohort (n = 33).ResultsEither limited or full pCR (breast-only pCR, node-only pCR, or both-pCR) indicated significantly improved DFS and overall survival (OS) (p < 0.001). Lager residual tumor size (hazard ratio [HR] 1.175, p = 0.011) and the presence of lymphatic vessel invasion (LVI) (HR 3.168, p = 0.001) were identified as independent predictors of disease relapse in the training cohort. Five variables, including age, primary tumor size, histological grade, residual tumor size, and LVI were used to establish the nomogram. The C-index of the nomogram was 0.815, and calibration curves showed an acceptable consistency between the actual and nomogram-predicted 2-year and 5-year DFS. The proposed nomogram demonstrated superior predictive performance compared with Residual Cancer Burden (RCB) classification and the 8th American Joint Committee on Cancer Post Neoadjuvant Therapy Classification (AJCC ypTNM) staging system (area under the curve [AUC] for 2-year DFS: 0.870 vs. 0.758 vs. 0.711, respectively; AUC for 5-year DFS: 0.794 vs. 0.731 vs. 0.702, respectively) in the validation cohort.ConclusionsThe nomogram proposed in our study enabled to quantify the risk of disease relapse and demonstrated superior predictive performance than a survival predict instrument. It was an easy-to-use tool for clinicians to guide individualized surveillance of TNBC patients following standard NACT.

Background As the critical regulators for tumorigenesis and progression, long-noncoding RNAs (lncRNAs) are becoming novel prognostic biomarkers for tumor patients. By the levels of lncRNAs expression, the patients with breast carcinoma may be divided into subgroups with different risk scores. Nevertheless, there is limited evidence to evaluate the role of lncRNAs in the prognosis of breast carcinoma. The present study aimed to construct lncRNA signatures for prognostic analysis and assist clinicians in choosing optimal therapies. Methods Abnormal expression profiles of breast cancer-associated lncRNAs were analyzed based on the TCGA datasets. Univariate and multivariate Cox regression analysis was used to build a prognostic risk signature according to the lncRNAs expression. The prognostic ability of this signature was verified in various subgroups. Functional enrichment analysis was employed to reveal the potential roles of these predictive lncRNAs in cancer-related biological processes and pathways. Results Compared with normal breast tissues, the differential analysis demonstrated that 286 lncRNAs were abnormally expressed in breast carcinoma. A four-lncRNA signature (RP1-193H18.2, AL022341.3, WDR86-AS1, LINC00511) was found to be closely related to the prognosis of breast carcinoma. The four-lncRNA signature could also qualify the magnitude of treatment benefits for different breast cancer subtypes. Additionally, it was an independent risk factor out of other clinicopathological parameters based on the multivariate Cox analysis. We also uncovered that the four predictive lncRNAs are involved in multiple cellular progression and pathways of breast cancer. Conclusions The four-lncRNA signature could be an essential reference for prognostic prediction and making therapeutic strategies.

To explore the role of ribosomal protein S15A (RPS15A) in breast cancer. The Oncomine database was used to compare the expression of RPS15A in human breast cancer tissues and normal tissues. RPS15A in breast cancer cell line ZR-75-30 and BT474 was specifically knocked down using lentivirus-mediated short hairpin RNAs (shRNAs). RPS15A knockdown efficiency was validated by quantitative polymerase chain reaction and western blot analysis. Subsequently, the functional effects of RPS15A on proliferation of breast cancer cells were investigated by MTT, colony formation and flow cytometry assays. Functional analysis indicated that RPS15A knockdown could inhibit cell proliferation, induced cell cycle arrest and apoptosis. Mechanism analysis revealed RPS15A mediated apoptosis via activating of caspase-3 and PARP cleavage, upregulating of Bad and BAX and downregulating of Bcl-2. Our preliminary study highlighted the importance of RPS15A in breast cancer growth. The inhibition of RPS15A may be a promising therapeutic target for breast cancer treatment. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10616-018-0221-9) contains supplementary material, which is available to authorized users.

The protein encoded by immature colon carcinoma transcript 1 (ICT1) is a component of the human mitochondrial ribosome, and is reported to be implicated in cell proliferation, viability and apoptosis of HeLa cells. This study was conducted to investigate the role of ICT1 in human breast cancer. Oncomine database was used to investigate ICT1 expression in human breast cancer tissues compared to normal tissues. The results showed that ICT1 was highly overexpressed in various human breast cancer subtypes. Then short hairpin RNA (shRNA)-mediated knockdown of ICT1 was performed in human breast cancer ZR-75-30 and T-47D cells. A series of functional analysis, including MTT, colony formation and flow cytometry assays were conducted after ICT1 knockdown. Our results demonstrated that knockdown of ICT1 significantly suppressed cell viability and proliferation through cell cycle arrest at the G2/M phase and induced apoptosis in breast cancer cells. Furthermore, knockdown of ICT1 altered signaling pathways associated with cell growth and apoptosis, including phospho‑BAD (Ser112), phospho-PRAS40 (Thr246) and induction of phospho‑AMPKα (Thr172). Additionally, it was further confirmed by western blot analysis that ICT1 knockdown altered the expression of apoptosis- or cell cycle‑related proteins such as Bcl-2, caspase-3, CDK1, CDK2 and cyclin B. In conclusion, targeting ICT1 in breast cancer cells may provide a new strategy for breast cancer gene therapy.

Background/Aims: Next-generation sequencing (NGS) has revealed abundant long noncoding RNAs (lncRNAs) that have been characterized as critical components of cancer biology in humans. The present study aims to investigate the role of the lncRNA KCNQ1OT1 in breast cancer (BRCA) as well as the underlying molecular mechanisms and functions of KCNQ1OT1 involved in the progression of BRCA. Methods: The Cancer Genome Atlas (TCGA) and StarBase v2.0 were used to obtain the required gene data. Dual luciferase reporter gene assays were conducted to verify the relevant intermolecular target relationships. QRT-PCR and Western blot were performed to measure the expression levels of different molecules. Cell proliferation was detected by using the MTT and colony formation assays, while cell migration and invasion were examined by transwell assay. Variations in cell apoptosis and cell cycle were determined through flow cytometry. A tumor xenograft model was applied to assess tumor growth in vivo. Results: KCNQ1OT1 was found to be remarkably highly expressed in BRCA tissues and cells. KCNQ1OT1 modulated CCNE2 through sponging miR-145 in BRCA. KCNQ1OT1 promoted tumor growth in vivo by regulating miR-145/CCNE2. Conclusion: The KCNQ1OT1/miR-145/CCNE2 axis plays a critical regulatory role in BRCA, potentially giving rise to BRCA tumorigenesis and progression. These findings provide valuable evidence for improving the diagnosis and treatment of BRCA in the future.

Ethnopharmacological relevance Cimicifuga racemose is previously proved effective on nature menopausal syndrome (MPS). However, its clinical value in treating with MPS induced by luteinizing-hormone releasing hormone analogue (LHRH-a) therapy of pre-/peri-menopausal breast cancer patients is still unknown. Aim of study This perspective randomised-design study is to investigate the effect and safety of cimicifuga racemosa on MPS induced by LHRH-a in breast cancer (clinical trial registered: NCT03339882 ). Materials and methods Breast cancer patients planning for LHRH-a treatment were randomly divided into 2 groups. The control group which was being treated with the standard treatment of LHRH-a. The other group was being treated with Remifemin, the commercialized product of cimicifuga racemose extract, combined with LHRH-a, called Remifemin group. Our main endpoint was Kupperman menopause index (KMI). Hormone levels in peripheral blood and gynecological complications were also evaluated. Results Totally, 85 patients (42 in Remifemin group and 43 in control group) were enrolled in Zhejiang Cancer Hospital. At the 4th, 8th and 12th week after using LHRH-a, the KMI were all significantly lower in Remifemin group than in control group (P 0.05). Conclusions Cimicifuga racemose is effective, oncological safe and reliable for treatment of MPS caused by LHRH-a in breast cancer.

11539 Background: According to Z0011 and AMAROS trials, patients with breast cancer stage cT1~2cN0 and sentinel lymphnode (SLN) 1~2 involvement can avoid axillary lymphnode dissection (ALND). But the risk of non-sentinel lymphnode (nSLN) involvement in those early stage patients is still unclear and it is difficult to predicting the risk before surgery. Our previous study showed CK19 mRNA in peripheral blood had predicative value of nSLN involvement. Also, contrast-enhanced ultrasound (CEUS) is a new effective method exmaming axillary lymph node. We aim to establish a prediction model for nSLN involvement in early breast cancer using CK19 combined with CEUS score. Methods: We identified 119 cases diagnosed early breast cancer (stage cT1~2cN0 and 1~2 SLNs involvement as in Z0011 and AMAROS trials) from Oct 2015 to Nov 2016 in Zhejiang Cancer Hospital. The CK19 mRNA of peripheral blood by RT-PCR and CEUS score of axillary lymph nodes were acquired before surgery. We used logistic regression analysis for filtering out valuable predictive clinical parameters and establishing formulas to calculate the probability of nSLN involvement. Our model was compared with Memorial Sloan Kettering Cancer Center (MSKCC) nomogram, which is one of the most reliable and validated methods for predicting of nSLN. Results: The histological grade, CK19 and CEUS score were screened by logistic regression analysis into the formula to calculate the probability of nSLN involvement. The sensitivity, specificity, total accuracy of this model was 89.13%, 80.82% and 84.03%, respectively. The false negative rate was 10.87%. The model had high quality of consistency (Kappa 0.675, p < 0.01) and goodness of fit (likelihood-ratio test, -2log liklihood = 84.607). The area under curve (AUC) of ROC was significantly higher (P < 0.01) in our model (0.914, 95%CI, 0.863~0.965) than in MSKCC nomogram (0.563, 95%CI, 0.459~0.667). Conclusions: The prediction model based on CK19 and CEUS score has satisfying sensitivity, specificity and accuracy, more effective than MSKCC nomogram. It is a valuable model of evaluating the risk of nSLN involvement in early breast cancer before surgery, picking out the patients who can truly avoid ALND.

Background Male breast cancer (BC) is a kind of rare tumour. There were few researches concerning the effect of chemotherapy for it. The purpose of this study is to estimate the value of chemotherapy on prognosis in male BC. Patients and methods Complete clinical and pathological information of male BC were collected from January 1990 to January 2008 in Zhejiang Cancer Hospital in China. 134 cases of male BC were included for analysis and separated into two groups based on receiving chemotherapy or not receiving chemotherapy. The disease-free survival (DFS) and overall survival (OS) between chemotherapy group and non-chemotherapy group were compared with Kaplan-Meier survival curve. Stratified analysis was used to evaluate the strength of the association between chemotherapy and each risk factor. Multivariate analysis was conducted by using COX proportional hazard regression model. Results There were 58.21% (78/134) cases who underwent chemotherapy and 41.79% (56/134) cases without chemotherapy. There were 20 cases (25.64%) with recurrence/metastasis in patients with chemotherapy and six cases (10.71%) in patients without chemotherapy. The mean DFS time of male BC with chemotherapy and non-chemotherapy is 150.87 and 154.13 months, respectively (χ 2 =3.825, p=0.050). The mean OS time of male BC with chemotherapy and non-chemotherapy is 155.33 and 154.26 months, respectively (χ 2 =2.542, p=0.111). COX proportional hazard regression model showed that the two groups had similar DFS (HR=0.386, p=0.165), while chemotherapy might be a protective fact on OS (HR=0.140, p=0.026). Conclusion The utility of chemotherapy should be considered in the high risk level of recurrence/metastasis in male BC.