581 Background: In a previous study, the patient-derived tumor-like cell clusters (PTC) were confirmed to be an accurate medicine effect-predicting model in vitro for clinical practice (1). We designed this prospective randomized phase II trial to research if this PTC-guiding strategy could improve the effect of neoadjuvant therapy in human epidermal growth factor receptor 2 (HER2) positive breast cancer (NCT05103293). Methods: We enrolled HER2-positive early breast cancer patients planning for neoadjuvant therapy to accept the PTC-guiding regimen as an experimental group or regimens of the physician's choice as a control group randomly. All eligible patients in the experimental group would receive medicine-sensitive testing by PTC then accepted the regimen with the highest PTC killing rate (30% as the lowest cut-off value) from the three candidate regimens (Cyclophosphamide combined with epirubicin -- Docetaxel combined with trastuzumab and pertuzumab, EC-THP, Docetaxel and carboplatin with trastuzumab and pertuzumab, TCbHP and Docetaxel with trastuzumab and pyrotinib, THPy). The primary endpoint is pathological complete response (pCR) in two groups using superiority odd ratio Fisher's exact test (one-sided alpha =0.05, power of 80%, and superiority margin at 0.1). The secondary endpoints are Miller-Payne (MP) grade 4~5 proportion, objective response rate (ORR), disease-free survival (DFS), and overall survival (OS). Results: Finally, 86 patients were enrolled in this study, 44 in the experimental and 42 in the control groups. The experimental group had 65.9% of pCR, significantly higher than 42.9% in the control group (Hazard ratio=0.388, P=0.033). The PTC-guiding treatment also considerably increased MP 4~5 proportion (79.5% vs. 59.4%, Hazard ratio=0.283, P=0.009) and objective response rate (90.9% vs 71.4%, Hazard ratio=0.250, P=0.027). After a two-year medium follow-up, the disease-free survival of the PTC-guiding treatment group was 84.1% higher than 71.4% of the control group without a significant difference (Log Rank, P=0.106). The two groups had similar overall survival (Log Rank, P=0.958). Furthermore, the PTC-guiding strategy significantly impacted pCR in univariate and multivariate regression analyses but not survival. Conclusions: In this trial, we concluded that the PTC-guiding neoadjuvant therapy could improve the effect, including pCR, MP score, and ORR in HER2-positive breast cancer. 1. Yin et al., Sci.Transl. 2020. Clinical trial information: NCT05103293 .

Human bone marrow mesenchymal stem cells (BMSCs) are efficient mass producers of exosomes that can potentially be utilized for delivery of miRNAs in cancer therapy. The current study aimed to assess the role of MSC-exosomal miR-99b-5p during the development of colorectal cancer (CRC). The potential value of using plasma levels of exosomal miR-99b-5p for predicting the liver metastasis of colorectal cancer was also assessed. In this study, we found that overexpression of fibroblast growth factor receptor 3 (FGFR3) was associated with tumor progression in CRC and FGFR3 was the target gene of miR-99b-5p, which was down-regulated in CRC tissues. Furthermore, we observed that elevated miR-99b-5p inhibited CRC cell proliferation, invasion and migration, while reduced levels had the opposite effect on CRC cells. Moreover, exosomal miR-99b-5p delivered by BMSCs was able to limit the proliferation, invasion and migration of CRC cells in vitro, as well as suppressing tumor growth in vivo. Collectively, these findings revealed that MSC-derived exosomal miR-99b-5p can be transferred into CRC cells and which can suppress tumor progression by targeting FGFR3. This highlights the potential of using exosomal miR-99b-5p as a novel diagnostic marker for CRC, while providing a therapeutic target to combat CRC.

Aim: The role of plasma heat shock protein 90 alpha (HSP90α) in colorectal cancer patients remains unclear. This study aimed to evaluate the relationship between HSP90α and the occurrence and development of colorectal cancer through diagnosis and prognosis value.Methods: 635 colorectal cancer patients and 295 healthy controls were recruited. The HSP90α was measured by using the ELISA kit in all objects and the immune cells and common biomarkers as CEA, AFP, CA125, CA153 and CA199 were measured in all colorectal cancer patients. The relationship between plasma HSP90α with clinical features, common tumor markers and immune cells were also conducted. The survival analysis endpoint was progression-free survival (PFS).Results: The levels of plasma HSP90α were significantly higher in colorectal cancer patients compared to healthy controls [51.4 (ng/ml) vs. 43.7 (ng/ml), p < 0.001]. In additional, the levels of plasma HSP90α were associated with gender and disease progress as stage, lymphatic and distant metastasis. Furthermore, plasma HSP90α was closed correlation with CEA, CA125, CA199 and percentage of B cells. However, the initial expression level of plasma HSP90α failed to show a prognostic value for progression-free survival in colorectal cancer.Conclusion: The plasma Hsp90α was remarkable higher in colorectal cancer and correlated with common tumor biomarkers and immune cells. Plasma Hsp90α levels were associated with disease progress but a poor diagnosis performance and also failed to show a prognostic value in colorectal cancer.

Objective To evaluate a model for axillary lymph node involvement combining CK19 mRNA with contrast enhanced ultrasound sonography (CEUS) score in operable breast cancer.Methods Operable breast cancer patients planned for sentinel lymph node (SLN) biopsy were enrolled.Preoperative CK19mRNA expressions in peripheral blood and CEUS score of axillary lymph nodes were tested before surgery.In the training set,postoperative sentinel lymph node (SLN) and non-sentinel lymph node (nSLN) pathological results were taken as the gold standard,effective modeling variables were screened,logistic regression was used to establish the prediction model.Parallel control studies were conducted between the validation set and the MSKCC model to evaluate the prediction accuracy and prediction efficiency.Results From Oct 2015 to Nov 2016,359 cases (training set) were enrolled and mathematical formulas for predicting SLN and nSLN were established,respectively.The sensitivity,specificity and AUC of predicting SLN involvement were 91.36％,94.92％ and 0.979 respectively.The sensitivity,specificity and AUC of predicting nSLN metastasis were 91.04％,90.53％ and 0.932 respectively.From Dec 2016 to Jul 2017,219 cases (verification set) were included.The sensitivity of SLN metastasis predicted by the model was 91.84％,the specificity was 96.69％,and the AUC was 0.979,significantly superior to the MSKCC model (0.739).The sensitivity,specificity and AUC of predicting nSLN metastasis were 95.35％,92.73％ and 0.945 respectively,significantly superior to the MSKCC model (0.873).Concolusions Combined with peripheral blood CK19 mRNA and CEUS score,the prediction model for axillary lymph node involvement for operable breast cancer,SLN/nSLN involvement probability can be calculated and qualitative judgment can be made.The overall accuracy and AUC of this model are better than the prediction model of MSKCC.

Background Early breast cancer with one or two sentinel lymph nodes (SLNs) may omit axillary lymph node dissection (ALND) if followed by radiotherapy. However, only less than one-third of the patients have positive non-SLNs and can truly benefit from radiotherapy. Before any regional treatment decision, the risk of non-SLN metastasis must be identified. The authors previously developed a predictive model for non-SLN involvement using CK19 mRNA and contrast-enhanced ultrasound (CEUS) score in a training set. They designed a further study to evaluate the predictive effect using the model prospectively in a validation set of one or two involved SLNs. Methods This study identified early breast cancer patients at Zhejiang Cancer Hospital from July 2017 to June 2018. The CK19 mRNA tested by quantitative real-time polymerase chain reaction and CEUS scores were collected before surgery. Patients with one or two involved SLNs were enrolled and underwent ALND. The estimated percentage of non-SLN involvement was calculated by the authors' model formula and the Memorial Sloan Kettering Cancer Center (MSKCC) nomogram. The false-negative rates, predictive accuracy, and area under curve (AUC) were compared between two predictive models. Results The study enrolled 235 patients, and 35.36% (83/235) of them had non-SLN involvement. The authors' model had a false-negative rate of 6% and an accuracy of 94.9%. The AUC was 0.952 (95% confidence interval [CI] 0.922-0.982), which was significantly higher than that of the MSKCC model at all three cutoff value levels. Conclusion The authors' model, using CK19 mRNA and the CEUS score, showed the potential predictive value of non-SLNs before surgery for early breast cancer patients. Clinicaltrials Registry NCT02992067, NCT03280134.

e12059 Background: Axillary lymph node (LN) involvement is significantly related with high recurrent risk and influenced by tumor size, histologic grade and subtype in early breast cancer. For hormone receptor positive (HR+) and human epidermal growth factor receptor-2 negative (HER2-) breast cancer without LN involvement, 21-gene recurrence score (RS) provides an effective method evaluating the risk of recurrence and metastasis. But the correlation between RS and LN involvement is not clear currently. To investigate the effect of RS on LN involvement, we designed a retrospective cohort study based on the data of tumor size-match LN+/- HR+HER2- breast cancer. Methods: The luminal type (ER+, PR+ and HER2-) early breast cancer (cT1-2) patients with 1~3 LN+ were enrolled and were tumor size-match paired (1:3) with those LN- during the same period. We compared the 21-gene RS between the LN+ and LN- group by univariate and multivariate analyses. Results: From April 2016 to May 2018, there were 65 LN+ cases undergoing 21-gene test included and 195 LN- cases were paired. With tumor size matched, the LN+ group had significantly younger age, more positive family history, higher histologic grade and higher RS than LN- group. All those factors, excepted histologic grade, were further confirmed in multivariate analysis (Table). Conclusions: After adjusted by tumor size, RS may have a significantly independent correlation with LN involvement in HR+HER2- early breast cancer. Total HR+HER2- cases’ tumor size-match paired (1:3) clinic pathologic and RS data. Clinical trial information: NCT03638648. [Table: see text]

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.

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.

In recent decades, the incidence rate of breast cancer has increased dramatically worldwide and has become one of the most prevalent cancers in women. According to the global data provided by the WHO, there were more than 1.7 million new cases of breast cancer in 2012, accounting for 25% of all reported cancer cases and 15% of all reported deaths among females. To most young patients, especially those younger than 40 years of age, chemotherapy should be taken into consideration as a treatment modality, even if the tumor size is small or without lymphatic metastasis. A significant portion of the patients will suffer from the side effects that result from long-term chemotherapy regimens, such as myelosuppression, heart failure, and development of a second primary malignancy. Ovarian suppression and premature ovarian failure (POF) are the most common side effects reported as they have notable clinical symptoms; the incidence rates have been reported to be in a range of 10-90%. Gonadotropin-releasing hormone agonists (GnRHa), also termed luteinizing hormone release hormone agonists (LHRH-a), may very well offer a solution to treating these side effects. This article aims to summarize advanced studies concerning protective effects of GnRH-a on ovarian functions in the setting of breast cancer under chemotherapy.

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.

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.

Reverse-transcription polymerase chain reaction (RT-PCR) is used to detect CK19 mRNA in sentinel lymph node biopsy (SLNB) tissues from breast cancer patients. We examined whether CK19 mRNA in peripheral blood is predictive of non-sentinel lymph node (nSLN) metastasis. Breast cancer cases diagnosed with clinical stage cT1-3cN0 and registered in our medical biobank were identified retrospectively. This study then included 120 breast cancer cases treated at Zhejiang Cancer Hospital from Aug 2014 to Aug 2015, including 60 SLN-positive and 60 SLN-negative cases. CK19 mRNA levels in peripheral blood samples were assessed using RT-PCR prior to tumor removal. During surgery, if SLNB tissue showed evidence of metastasis, axillary lymph node dissection (ALND) was performed. No ALND was performed if SLNB and nSLN tissues were both negative for metastasis. CK19 expression was higher in nSLN-positive patients than in nSLN-negative patients (p < 0.05). Logistic regression indicated that lymphatic vessel invasion and CK19 levels were predictive of nSLN status (p < 0.05). The area under the ROC curve for CK19 was 0.878 (p < 0.05). We conclude that high CK19 levels in peripheral blood may independently predict nSLN metastasis in breast cancer patients.

562 Background: In our previous study, goserelin has been reported downregulating the estradiol level, breast density and endometrial thickness in Chinese pre-/peri-menopausal hormone positive (HR+) breast cancer. Here, the prognositic results of this study will be reported: disease-free survival (DFS) and overall survival (OS) of goserelin plus tamoxifen (TAM) versus TAM alone in pre-/peri-menopausal HR+ early-stage breast cancer in China (NCT00827307). Methods: Pre-/peri-menopausal patients with HR+ early-stage breast cancer (after finishing surgery, chemotherapy, radiotherapy if applicable) were randomized to receive either goserelin 3.6mg ih. every 28 days for 1.5 years plus TAM 10 mg po. twice a day for 5 years (Group A) or TAM alone for 5 years (Group B) as adjuvant endocrine therapy. The patients also were classified according to recurrence-risk grade for stratified analysis. The DFS and OS in total patients and in stratified risk grade subgroups were analyzed. Results: From 22 June 2008 to 31 December 2009, 110 eligible patients were recruited. The medium follow-up time is 72 months. Because of 19 cases dropped out, there were 47 patients of group A and 44 patients of group B included in for final analysis. Patients in group A had better DFS and OS than those in group B although not statistically significant due to small sample size (DFS: HR = 0.452, P = 0.261, OS: HR = 0.301, P = 0.299). In high-risk (1~4 LN+ and HER2+/ ≥ 4 LN+) subgroup, there were no patients with recurrent/metastasis (0/10) in group A while three patients with recurrent/metastasis (3/8) in group B, goserelin combined with TAM has better DFS than TAM alone (Log-rank test, χ2= 4.324, P = 0.038). In total patients, COX proportional hazard regression model (forward conditional method) showed that the number of LN+ significantly impacts the DFS (P = 0.011). Meanwhile, both the age and the tumor size have significant impacts on OS (P = 0.028 and P = 0.030, respectively). Conclusions: Goserelin combined with TAM was suggested to improve DFS in HR+ pre-/peri-menopausal breast cancer with high-recurrence risk grade. Clinical trial information: NCT00827307.

Paired box 6 (PAX6) plays a significant role in the development of human neuroectodermal epithelial tissues. Previous studies have suggested that the PAX6 promoter is hypermethylated in breast cancer and that it is involved in breast cancer cell proliferation. The present study aimed to investigate the expression of PAX6 in invasive breast cancer tissues, and to evaluate its prognostic significance. Immunohistochemistry (IHC) was used to detect PAX6 expression on a breast cancer tissue microarray containing tissues from 111 patients. Associations of PAX6 expression with staging and prognosis were analyzed. PAX6 was mainly expressed in the nucleus. The PAX6 staining intensity was not associated with age, histological grade, lymph node status, tumor size, or progesterone receptor and human epidermal growth factor receptor 2 expression (all P > 0.05). A high level of PAX6 staining was more frequent in estrogen receptor (ER) -negative cases compared with ER-positive cases (43.9 vs. 25.7%; P = 0.049). After a median follow-up time of 110 months, the patients with low PAX6 expression exhibited an improved survival rate compared with the patients with high PAX6 expression (P < 0.001). Cox analysis showed a worse survival rate in the patients with high PAX6 staining (hazard ratio, 3.458; 95% confidence interval, 1.575-7.593; P = 0.002). In conclusion, high tumor PAX6 staining intensity by IHC was associated with a poor prognosis in breast cancer patients.