Cells 6 staining intensity. Using this method, the Autophagy expression of miR-27a or ZBTB10 was evaluated by the SI, scored as 0, 1, 2, 3, 4, 
6, or 9. In cases of disagreement (score discrepancy .1), the slides were reexamined and a consensus was reached by the observers. Cutoff values to define the high- and low-expression of miR-27a or ZBTB10 were chosen by a measurement of heterogeneity with the log-rank test statistic with respect to overall survival. Because the optimal cutoff SIs were identified from the current study as 6, an SI score 6 was taken to define tumors as high expression, and 25033180 SI ,6 defined tumors as low expression of miR-27a and ZBTB10.Patients and Tissue SpecimensParaffin-embedded tumor tissues were obtained from 124 breast cancer patients that were diagnosed and treated at the Sun Yat-sen Memorial Hospital, China, during the period from January 2001 to June 2010 and represented up to 10 years of clinical follow-up information. Of the 124 cases collected, the survival Autophagy information for 102 cases (median age 50, range 23?4) was available. The tissues were acquired from the archival collections of the Department of Pathology, and used for subsequent in situ hybridization and immunohistochemistry. The clinicopathological data are illustrated in Table 1. None of the patients received any chemotherapy or irradiation prior to surgery. Histological diagnosis and scoring of all the cases were performed by 2 independent pathologists according to the WHO Histological Classification. Tumors were staged according to the TNM staging system. The disease-free survival rate of the patients was calculated from the date of resection to the date of local tumor recurrence in the form of either local or distant metastasis, while the actual survival rate was calculated to the date of death.In Situ Hybridization (ISH)This assay was performed according to the manufacturer’s protocol (Exiqon, Vedbaek, Denmark). Briefly, thin sections (4 um thick) of paraffin-embedded specimens were deparaffinized with xylene and rehydrated with graded ethanol dilution. Sections were treated with 0.05 trypsin at room temperature for 15 minutes and re-fixed in 4 paraformaldehyde for 10 minutes. The slides were prehybridized in a hybridization solution at 51uC for 2 hours. Subsequently, 20 nmol/L of aStatistical AnalysisAll statistical analyses were performed using the SPSS 16.0 statistical software package (SPSS Inc., Chicago, IL, USA). The x2 test was used to analyze the relationship between miR-27a or ZBTB10 expression and the clinicopathological characteristics.MiR-27a as a Predictor of Invasive Breast CancerTable 1. Clinicopathological Characteristics of the Patients and the Expression of miR-27a and ZBTB10 in breast cancer.CharacteristicscasesmiR-27a expression level No. of low Expression No. of high expression P 0.ZBTB10 expression level No. of low expression No. of high expression P 0.893 4(40.0) 20(32.8) 11(35.5) 0.967 6(60.0) 41(67.2) 20(64.5) 0.654 12(31.6) 23(35.9) 0.503 26(68.4) 41(64.1) 0.893 10(37.0) 13(31.7) 12(35.3) 0.034 17(63.0) 28(68.3) 22(64.7) 0.020 12(23.1) 15(40.5) 8(61.5) 0.000 40(76.9) 22(59.5) 5(38.5) 0.008 12(26.1) 13(36.1) 3(25.0) 7(87.5) 0.000 34(73.9) 23(63.9) 9(75.0) 1(12.5) 0.000 21(25.0) 14(77.8) 0.072 63(75.0) 4(22.2) 0.993 11(34.4) 24(34.3) 0.527 21(65.6) 46(65.7) 0.641 5(29.4) 30(35.3) 0.411 12(70.6) 55(64.7) 0.997 23(34.3) 12(34.3) 44(65.7) 23(65.7)Age (years) #35 35?5 .55 Menopause Pre-menopausal Post-menopausal Histological grade.Cells 6 staining intensity. Using this method, the expression of miR-27a or ZBTB10 was evaluated by the SI, scored as 0, 1, 2, 3, 4, 6, or 9. In cases of disagreement (score discrepancy .1), the slides were reexamined and a consensus was reached by the observers. Cutoff values to define the high- and low-expression of miR-27a or ZBTB10 were chosen by a measurement of heterogeneity with the log-rank test statistic with respect to overall survival. Because the optimal cutoff SIs were identified from the current study as 6, an SI score 6 was taken to define tumors as high expression, and 25033180 SI ,6 defined tumors as low expression of miR-27a and ZBTB10.Patients and Tissue SpecimensParaffin-embedded tumor tissues were obtained from 124 breast cancer patients that were diagnosed and treated at the Sun Yat-sen Memorial Hospital, China, during the period from January 2001 to June 2010 and represented up to 10 years of clinical follow-up information. Of the 124 cases collected, the survival information for 102 cases (median age 50, range 23?4) was available. The tissues were acquired from the archival collections of the Department of Pathology, and used for subsequent in situ hybridization and immunohistochemistry. The clinicopathological data are illustrated in Table 1. None of the patients received any chemotherapy or irradiation prior to surgery. Histological diagnosis and scoring of all the cases were performed by 2 independent pathologists according to the WHO Histological Classification. Tumors were staged according to the TNM staging system. The disease-free survival rate of the patients was calculated from the date of resection to the date of local tumor recurrence in the form of either local or distant metastasis, while the actual survival rate was calculated to the date of death.In Situ Hybridization (ISH)This assay was performed according to the manufacturer’s protocol (Exiqon, Vedbaek, Denmark). Briefly, thin sections (4 um thick) of paraffin-embedded specimens were deparaffinized with xylene and rehydrated with graded ethanol dilution. Sections were treated with 0.05 trypsin at room temperature for 15 minutes and re-fixed in 4 paraformaldehyde for 10 minutes. The slides were prehybridized in a hybridization solution at 51uC for 2 hours. Subsequently, 20 nmol/L of aStatistical AnalysisAll statistical analyses were performed using the SPSS 16.0 statistical software package (SPSS Inc., Chicago, IL, USA). The x2 test was used to analyze the relationship between miR-27a or ZBTB10 expression and the clinicopathological characteristics.MiR-27a as a Predictor of Invasive Breast CancerTable 1. Clinicopathological Characteristics of the Patients and the Expression of miR-27a and ZBTB10 in breast cancer.CharacteristicscasesmiR-27a expression level No. of low Expression No. of high expression P 0.ZBTB10 expression level No. of low expression No. of high expression P 0.893 4(40.0) 20(32.8) 11(35.5) 0.967 6(60.0) 41(67.2) 20(64.5) 0.654 12(31.6) 23(35.9) 0.503 26(68.4) 41(64.1) 0.893 10(37.0) 13(31.7) 
12(35.3) 0.034 17(63.0) 28(68.3) 22(64.7) 0.020 12(23.1) 15(40.5) 8(61.5) 0.000 40(76.9) 22(59.5) 5(38.5) 0.008 12(26.1) 13(36.1) 3(25.0) 7(87.5) 0.000 34(73.9) 23(63.9) 9(75.0) 1(12.5) 0.000 21(25.0) 14(77.8) 0.072 63(75.0) 4(22.2) 0.993 11(34.4) 24(34.3) 0.527 21(65.6) 46(65.7) 0.641 5(29.4) 30(35.3) 0.411 12(70.6) 55(64.7) 0.997 23(34.3) 12(34.3) 44(65.7) 23(65.7)Age (years) #35 35?5 .55 Menopause Pre-menopausal Post-menopausal Histological grade.
