| ObjectiveTo compare the concordance between detection of immunohistochemistry (IHC) combine fluorescence in situ hybridization (FISH) and digital droplet Polymerase Chain Reaction (ddPCR) for human epidermal growth factor receptor 2 (HER2) gene amplification in invasive breast cancer patients. To evaluate a less subjective and accurate application of ddPCR for HER2 amplification levels and test the assay performance in clinical form alin-fixed paraffin-embedded(FFPE) breast as well as investigate the correlation between quantitation of HER2 amplification and clinical pathological features, which would offer a useful guidance for clinical diagnosis, treatment and prognosis.Methods1. We collected clinical and pathological data of 120 cases of which patients suffered from breast infiltrating ductal carcinoma, which was confirmed by pathological diagnosis in Guangdong hospital of TCM during July 2012 and December 2013 through CHAS (clinical & health records for analytics & sharing) and Huahai pathological system. Filter out 110 appropriate copies with complete pathologic and immunohistochemical from 120 medical records.2. HER2 were tested by IHC, and IHC 2+ samples were tested by FISH.3. In this study, ddPCR was applied to determinate HER2 amplification in the tumor samples from 110 invasive breast cancer patients which were also tested by IHC scoring as 0,1+,2+,3+.4. Estrogen receptor (ER), progesterone receptor (PR), and nuclear-associated antigen (Ki-67)were tested by IHC5. Use SPSS19.0 to analyze the relations between amplification of HER2 and age, pathological type, as well as tumor size, histological grade, the type of luminal, ER, PR, HER2, Ki-67, the number of lymph nodes metastases, menopausal status, TNM stage, the type of Luminal, TCM Type. The kappa test was used to evaluate the concordance between the two assays. The Spearman’s rank correlation analysis was used to evaluate the correlation between HER2 gene amplification status and clinical pathological features.Use the chi-square test when analyze the relations between TCM Syndrome Types and other prognostic factors.Results1. The IHC results of Her2 showed 53 cases as 0 or 1+,32 cases as 2+(FISH results demonstrated 8 cases as positive; and 24 cases as negative), and 25 cases as 3+.2. There were 31 cases with ddPCR absolute value≥3.2 copies, which means the positive rate is 28.18%(31/100), and 79 cases with ddPCR absolute value ＜3.2, which means the negative rate is 71.82%(79/110)3. The results revealed that the concordance between IHC/FISH positive and ddPCR positive rate was 93.9%(31/33),100%(77/77) concordance between IHC score 0/1+and ddPCR negative. The consistency between IHC& FISH and ddPCR (Kappa=0.96, P＜0.01) is good.4. The IHC results of positive rate of Estrogen receptor (ER), progesterone receptor (PR), and nuclear-associated antigen (Ki-67) were respectively 69.1%,45.5% and 60.0%.5. The results revealed positive significant correlation between HER2 amplification and tumor size, Ki-67, histological grade, and the number of lymph node metastasis, TNM stage, the type of Luminal (P＜0.05) as well as significant inverse correlation between expression of hormone receptors (ER and PR) and HER2 amplification (P＜0.05). However, no correlation was observed between HER2 amplification and age, menopausal status, and histologic types (P＞0.05).6. TCM Syndrome Types was correlated with age(rs=0.219, P＜0.05), ER (X2=39.415, P＜0.01),TNM (x2=32.410, P＜0.05). No correlation was observed between HER2 amplification and PR, Ki-67, and histological grade (P＞0.05).Conclusion1. This study demonstrated that ddPCR could be used as a molecular analysis tool to precisely measure HER2 copy number alterations in breast tumors at DNA level.2. HER2 amplification was correlated with tumor size, ER, PR, Ki-67, histological grade, the number of lymph node metastasis.3. TCM Syndrome Types was correlated with age, ER, TNM.No correlation was observed between HER2 amplification and PR, Ki-67, and histological grade. |
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