| Breast cancer is a heterogeneous tumor composed of molecular and biological heterogeneity.There is no standardized therapy for breast cancer heterogeneity.Tumor biomarkers including ER, PR, HER2 and Ki-67 are routinely tested in breast cancer and their status guides clinical management, predicts prognosis and analyze the reactivity of tumor to chemotherapy. In this study we take advantage of a relatively large cohort and aim to study the effect of neoadjuvant chemotherapy (NAC) on biomarker expression and explore the impact of tumor size and lymph node involvement on biomarker status changes.In addition we studied cases with HER2 status changes after NAC treatments in detail and emphasized the nature of tumor heterogeneity.Methods:1.107 patients with breast invasive ductal carcinomas diagnosed from 2011 to 2013 at Shandong University Qilu hospital were enrolled into this study. Each patient received at least three cycles of NAC. For each case, core biopsy before NAC and excisional tumor sample following NAC were collected.2.Immunohistochemistry was used to detect the ER, PR, HER2 and Ki-67 expression in specimens before and after NAC.Fluorescent in situ hybridization (FISH) was used todetermine the HER2 genestatus before and after NAC treatments.3. ER and PR status was assessed following both H-score scoring method and the guidelines from American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP). HER2 scoring was performed according to HercepTest criteria.The cut-off point of 14% for Ki67-positive cells was used to distinguish low proliferation and high proliferation tumors.4. For cases with positive tumor heterogeneity, at least 3 tissue spots were counted for the heterogeneity assessment by two pathologists in a blind manner. Each spot was counted for at least 20 nuclei according to ASCO/CAP guideline 2013. A tumor was considered homogeneous if all counted tissue spots showed identical results, while others were considered HER2 gene heterogeneous.5. All the tumors were classified according to the biomarker status before NAC: luminal A (ER+/PR+/HER2-/Ki67< 14%), luminal B (ER+/PR+/HER2-/Ki67≥ 14%), HER2amplified (ER-/PR-/HER2+), triple negative (ER-/PR-/HER2-), and luminal-HER2 (ER+/PR+/HER2+).Results:1. Among 107 NAC-treated cases, changes of ER, PR, and HER2status were observed in 14%(15/107),24.3%(26/107), and 4.7%(5/107) cases respectively.A significant reduction of PR (P= 0.013) and Ki-67 (P= 0.000) expressionwas found inpost-NAC tumors when compared to the pre-NAC core specimens.2. No receptor status alternation was observed in the triple negative group. The luminal B as well as Luminal-HER2 tumors showed the most frequently altered biomarker status after NAC treatment.3. A significant alteration was found in tumors greater than 2cm and tumors with positive lymph node involvement (P<0.05) after NAC. We proposed that as tumor grows and metastasizes to lymph nodes, microenvironment change and enhance the tumor heterogeneity, which lead to the alteration of biomarker after NAC.4. The heterogeneous tumor may be composed of different subtypes.The tumor cells of different molecular cloning is associated with different clinical responsiveness to chemotherapy.Conclusions:1. In the study of biomarker profiles after neoadjuvant chemotherapy in breast cancer, tumor heterogeneity should be taken into consideration.2. Luminal B and luminal-HER2 subgroups showed the most frequently altered biomarker status after NAC treatment. We believe those types of tumor might be more heterogeneous.3. The tumor cells of molecular heterogeneity is associated with different clinical responsivenesstochemotherapy.The heterogeneity of HER2 gene cluster amplification should be defined in the pathology report.Chkl (checkPoint kinasel) plays an essential role in regulate cell cycle arrest at the G2 checkpoint and DNA damage response. It’s expressed highly in breast cancer, especially in the triple negative breast carncer. Chk1 can weaken the anti -tumor efficacy of the chemotherapy drugs. Our previous study demonstrated that CHK1 duplication or amplification was rare in breast cancer.We hypothesized that regulating the post-transcription of CHK1gene plays a key role in the expression of Chk1 protein by the bioinformatic analysis. In the present study, we use MDA-MB-231cell line to show for the first time that miR-424 could increase cell apoptosis and release cell cycle arrest in G2/M, suppress the process of invasion and metastasis by regulating it’s direct target gene CHK1.Finally,Chk1 may be an effective chemoresistance target in tumor. In this regard, miR-424 could be used to enhance the chemotherapy efficacy in breast cancer.Methods:1. The detection of luciferase assay was further used to identify that CHK1 is the direct target gene of miR-424.2. RT-qPCR was used to determine the Chk1 mRNA expression and Western blot was performed to detect the Chk1 protein expression in breast cancer after tansfection.3. RT-qPCR was used to determine the levels of miR-424 in 50 cases of human breast cancer tissues and 15 cases of adjacent normal tissues.4. MiR-424 mimic, miR-424 inhibitor, as well as negative control were instantaneously transfected in breast cancer cell line MDA-MB-231.Then Flow cytometry was used to explore the cell cycle and cell apoptosis.Transwell assay was used to detect the ability of migration and invasion after transfection.Results:1. A significant reduction of luciferase activity was observed in MDA-MB-231 cells with miR-424 mimics and the plasmid of CHK1 transfection,compared to the negative control group (P<0.05).2. RT-qPCR showed that miR-424 couldn’t regulate the level of Chkl mRNA.Western Blot showed that miR-424 downregulated the expression of Chkl protein.3. The expression level of miR-424 in tumor tissues was 0.5-fold lower than that of adjacent normal tissues by RT-qPCR, which showed significant differences (P<0.05)4..Flow cytometry showed that miR-424 release cell cycle arrest in G2/M phase in breast cancer cell. By transfection with miR-424 mimic and negative control in cell line MDA-MB-231, The proportion of G2/M phase in the group transfected with miRNA mimics was 13.97% ± 0.81%, decreased apperantly compared with the control group(20.33% ± 1.99%), which showed significant differences (P< 0.05); Flow cytometry showed that miR-424 induced cell apoptosis in breast cancer cell significantly. The proportion of apoptosis cells in the group transfected with miRNA mimic was 18.23±1.29%, compared to the control group (8.57±0.52%) (P< 0.05); Transwell assay proved a significant suppressive effect of miR-424 on the ability of migration and invasion compared with negative control in breast cancer cell (P<0.05).Conclusions:1. CHK1 is the direct target gene of miR-424.2. miR-424 could increase breast cancer cell apoptosis, release cell cycle arrest in G2/M, and suppress cell invasion. |
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