Tumor Biol. DOI 10.1007/s13277-015-3390-6
RESEARCH ARTICLE
Clinical significance of topoisomerase 2A expression and gene change in operable invasive breast cancer Jiang-Hua Qiao 1 & De-Chuang Jiao 1 & Zhen-Duo Lu 1 & Sen Yang 2 & Zhen-Zhen Liu 1
Received: 28 February 2015 / Accepted: 24 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract This study aims to investigate clinical significance of topoisomerase 2A (TOP2A) expression and TOP2A gene change in operable invasive breast cancer. This is a retrospective analysis, which includes 256 patients diagnosed as operable invasive breast cancer. All postoperational waxed specimens were subjected to resectioning for staining. Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), KI-67, TOP2A expression, and TOP2A gene changes were detected by immunohistochemistry (IHC) and fluorescent in situ hybridization technique (FISH), respectively. Correlation between TOP2A expression and clinicopathological characteristics was also investigated. Effects of TOP2A protein or gene changes on survival rate were detected. Results indicated that 165 were TOP2A positive (64.5 %), and 31 were gene amplification positive (12.1 %). Positive rate of TOP2A expression showed significant correlations with ER, KI-67, and HER-2. The difference of 5-year overall survival (OS) between TOP2Apositive and TOP2A-negative groups did not reach statistical significance (OS: P=0.321, 85.9 vs. 79.6 %; disease-free survival [DFS]: P=0.247, 83.3 vs. 75.3 %). Five-year OS in TOP2A amplification group was 68.8 %, which is lower than deficiency and control group (P>0.05). Subgroup analysis showed no significant differences of OS and DFS either * Zhen-Zhen Liu [email protected] 1
Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, No. 127 Dongming Road, Zhengzhou 450008, People’s Republic of China
2
China-US (Henan) Hormel Cancer Institute, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450008, People’s Republic of China
between TOP2A-positive and TOP2A-negative groups or between TOP2A amplification and control group in population of patients with HER-2 amplification, triple negative breast cancer, or hormone-positive breast cancer. In conclusion, positive rate of TOP2A expression correlates significantly with ER, KI-67, and HER-2. However, prognostic significance of either TOP2A expression or TOP2A gene changes in breast cancer and its various subtypes is limited. Keywords Operable invasive breast cancer . Topoisomerase . Immunohistochemistry . Situ hybridization technique . Clinical significance
Introduction Topoisomerase 2A (TOP2A), the key enzyme required for generating transient double-stranded breaks in DNA topological structure, played a significant role in DNA replication and transcription and the formation, enrichment, and separation of chromosome structure [1]. TOP2A gene located on chromosome 17 is adjacent to and co-expressed with the gene encoding human epidermal growth factor receptor 2 (HER-2) [2]. It has been widely acknowledged that HER-2 can be used as an independent indicator of prognosis in breast cancer. Moreover, TOP2A has been linked with cell proliferation. In the previous studies, TOP2A has been investigated in a few cancer, such as breast cancer, ovarian cancer [3], prostate cancer [4], gastric cancer [5], and electrochemotherapy (ECT). However, the prognostic effects of the TOP2A on the operable invasive breast cancer have not been investigated in the previous studies. Meanwhile, the previous biomarkers for the operable invasive breast cancer prognosis, such as ULK1 [6], KI67 [7], and HER2 [8], always indicate lower sensitivity. Therefore, this study aims to explore the clinical significance
Tumor Biol.
of topoisomerase 2A protein expression and gene change in operable invasive breast cancer.
Materials and methods Clinical materials A retrospective analysis of 256 patients with T1–T3 stage, operable invasive breast cancer, who were enrolled between June 2009 and December 2009, was conducted. The patients’ age ranged from 27 to 83 years, with the median age of 48 years. The study individuals comprised 216 invasive ductal carcinoma, 21 invasive lobular carcinoma, and 19 other types of carcinomas such as mixed carcinoma and medullary carcinoma. All patients did not receive any preoperative therapies and instead receive postoperative treatments, including six breast-conserving surgery and 250 modified radical operation. Postoperative systematic chemotherapy was given. More specifically, 124 individuals chose chemotherapy based on antharcycline, 23 taxanes, 79 combining of both, and 30 other regimens. Radiotherapy was given to patients who had accepted the breast-conserving surgery or with lymphatic metastasis or tumor diameter being about 5 cm. Endocrinotherapies were given to patients with hormone receptor-positive tumors. Targeted therapy was not given to patients with HER-2 amplification due to the constrained treatment conditions. The follow-up continued until November 30, 2014, with a median time of 58 months (6–65 months). During the follow-up, 16 were lost, 53 suffered with adverse events such as recurrences and metastases, and 34 were dead. This study has been approved by the Ethics Committee of Affiliated Cancer Hospital of Zhengzhou University. All of the patients participated in this study have approved and gave their consent for this study. Immunohistochemical staining assay Estrogen receptor (ER), progesterone receptor (PR), HER-2, KI-67 indexes, and TOP2A protein expression were detected via immunohistochemical (IHC) staining assay. The waxed species of 256 patients were collected and resliced, and further reevaluated by two skilled pathologists. The evaluation criteria were as follows: ER- or PR-positive staining was defined as the condition in which >1 % cancer cells were stained. HER-2 protein expression was ranked between 0 and 3+. Specifically, 0 were the specimens with no HER-2 protein amplification, while 3+ were assigned for the specimens with the presence of HER-2 protein amplification. 36 were 2+ via IHC detection, without further confirmation of the HER-2 status. The KI-67 evaluation criteria were as follows. KI-67-positive staining was nucleus-targeted, and cytoplasm- and membrane-
positive cancer cells were not counted in. Ten high-powered field in each light microscope slices were randomly selected, in which the amount of all cancer cells were counted. The average proportion of positive cell of all cancer cells was treated as quantitative index, which is further subcategorized into low expression (≤14 %) and high expression (>14 %) [9]. There is currently no agreed conclusion concerning the cutoff value of TOP2A. In our study, we defined it as follows: the specimens were ranked as positive (>10 % positive cells) and negative (≤10 % positive cells). Molecular typing was classified according to the St Gallen International Expert Consensus into luminal A, luminal B, basal-like, and HER-2 amplification [10]. Luminal A is defined as ER (+), PR (+), HER-2 (−), and KI-67≤14 %, while luminal B is defined as ER (+), HER2 (−), and KI-67>14 %. Triple negative breast cancer is defined as ER (−), PR (−), and HER-2 (−), whereas HER-2 amplification was defined as ER (−), PR (−), and HER-2 (+). Fluorescence in situ hybridization analysis TOP2A status changes were detected via fluorescence in situ hybridization (FISH) methods. All waxed specimens were subjected to a 2- to 3-cm-thick serial sectioning before fixed in 75 °C oven for 2–3 h. After 10-min xylene dewaxing twice at room temperature, the slides were dehydrated using anhydrous alcohol for 5 min at room temperature, after which the slides were respectively immersed in 100 and 80 % alcohol, and deionized water 2 min for each (gradient rehydration). Afterwards, the splices were placed in the deionized water high–pressure cooker for 3 min, then immersed in the two times SSC buffer solution for 3 min. The water surrounding the species were then blotted, and the tissues were then digested with 80 μl pepsin and incubated at 37 °C for 10– 20 min when the extent of absorption was closely monitored. After washing in two times SSC solution for 5 min and subsequent dehydration with 80 and 100 % ethanol for 2 min, the species were taken out for natural drying, after which tissue region was circled by diamond pen, probe was added dropwise, and coverslip was lidded and sealed. They were denatured in hybridization oven for 5 min and were hybridized overnight at 37 °C for 16 h. After removing the seals, they were immersed in two times SSC at 37 °C, after which the coverslip was removed and the species were waggled for 1– 3 s in 37 °C, 0.1 % NP-40 immersion for 5 min, dehydration for 2 min in 80 % alcohol and natural drying, 10 μl 4′,6diamidino-2-phenylindole (DAPI) counterstain added dropwise to the hybrid area, with coverslip covered. After 20 min, they were observed by fluorescence microscope. Image acquisition and processing were conducted via FISH 3.0 imaging system. The red signal is representative of TOP2A gene, while the green signal is chromosome 17 centromere probe. Firstly, the areas of invasive carcinoma on FISH
Tumor Biol.
section same as that on HE-dyed section were searched under ten times objective lens, the criteria required at least two areas. Second, the FISH results of cancerous cell nuclei were observed through specific single channel filter under 100× objective lens, after which the red and green signal counting and ratio calculation were conducted. The ratio > 2.0 means TOP2A gene amplification, the ratio50 years old
65.1 % (95/146) 63.6 % (70/110)
Tumor size T1–T2 T3 Lymph node Negative Positive Pathological type Invasive ductal carcinoma Invasive lobular carcinoma Other types Histological grade 1–2 grade 3 grade ER Positive Negative PR Positive Negative HER-2 Amplification Negative KI-67 Low expression (
RESEARCH ARTICLE
Clinical significance of topoisomerase 2A expression and gene change in operable invasive breast cancer Jiang-Hua Qiao 1 & De-Chuang Jiao 1 & Zhen-Duo Lu 1 & Sen Yang 2 & Zhen-Zhen Liu 1
Received: 28 February 2015 / Accepted: 24 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract This study aims to investigate clinical significance of topoisomerase 2A (TOP2A) expression and TOP2A gene change in operable invasive breast cancer. This is a retrospective analysis, which includes 256 patients diagnosed as operable invasive breast cancer. All postoperational waxed specimens were subjected to resectioning for staining. Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), KI-67, TOP2A expression, and TOP2A gene changes were detected by immunohistochemistry (IHC) and fluorescent in situ hybridization technique (FISH), respectively. Correlation between TOP2A expression and clinicopathological characteristics was also investigated. Effects of TOP2A protein or gene changes on survival rate were detected. Results indicated that 165 were TOP2A positive (64.5 %), and 31 were gene amplification positive (12.1 %). Positive rate of TOP2A expression showed significant correlations with ER, KI-67, and HER-2. The difference of 5-year overall survival (OS) between TOP2Apositive and TOP2A-negative groups did not reach statistical significance (OS: P=0.321, 85.9 vs. 79.6 %; disease-free survival [DFS]: P=0.247, 83.3 vs. 75.3 %). Five-year OS in TOP2A amplification group was 68.8 %, which is lower than deficiency and control group (P>0.05). Subgroup analysis showed no significant differences of OS and DFS either * Zhen-Zhen Liu [email protected] 1
Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, No. 127 Dongming Road, Zhengzhou 450008, People’s Republic of China
2
China-US (Henan) Hormel Cancer Institute, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450008, People’s Republic of China
between TOP2A-positive and TOP2A-negative groups or between TOP2A amplification and control group in population of patients with HER-2 amplification, triple negative breast cancer, or hormone-positive breast cancer. In conclusion, positive rate of TOP2A expression correlates significantly with ER, KI-67, and HER-2. However, prognostic significance of either TOP2A expression or TOP2A gene changes in breast cancer and its various subtypes is limited. Keywords Operable invasive breast cancer . Topoisomerase . Immunohistochemistry . Situ hybridization technique . Clinical significance
Introduction Topoisomerase 2A (TOP2A), the key enzyme required for generating transient double-stranded breaks in DNA topological structure, played a significant role in DNA replication and transcription and the formation, enrichment, and separation of chromosome structure [1]. TOP2A gene located on chromosome 17 is adjacent to and co-expressed with the gene encoding human epidermal growth factor receptor 2 (HER-2) [2]. It has been widely acknowledged that HER-2 can be used as an independent indicator of prognosis in breast cancer. Moreover, TOP2A has been linked with cell proliferation. In the previous studies, TOP2A has been investigated in a few cancer, such as breast cancer, ovarian cancer [3], prostate cancer [4], gastric cancer [5], and electrochemotherapy (ECT). However, the prognostic effects of the TOP2A on the operable invasive breast cancer have not been investigated in the previous studies. Meanwhile, the previous biomarkers for the operable invasive breast cancer prognosis, such as ULK1 [6], KI67 [7], and HER2 [8], always indicate lower sensitivity. Therefore, this study aims to explore the clinical significance
Tumor Biol.
of topoisomerase 2A protein expression and gene change in operable invasive breast cancer.
Materials and methods Clinical materials A retrospective analysis of 256 patients with T1–T3 stage, operable invasive breast cancer, who were enrolled between June 2009 and December 2009, was conducted. The patients’ age ranged from 27 to 83 years, with the median age of 48 years. The study individuals comprised 216 invasive ductal carcinoma, 21 invasive lobular carcinoma, and 19 other types of carcinomas such as mixed carcinoma and medullary carcinoma. All patients did not receive any preoperative therapies and instead receive postoperative treatments, including six breast-conserving surgery and 250 modified radical operation. Postoperative systematic chemotherapy was given. More specifically, 124 individuals chose chemotherapy based on antharcycline, 23 taxanes, 79 combining of both, and 30 other regimens. Radiotherapy was given to patients who had accepted the breast-conserving surgery or with lymphatic metastasis or tumor diameter being about 5 cm. Endocrinotherapies were given to patients with hormone receptor-positive tumors. Targeted therapy was not given to patients with HER-2 amplification due to the constrained treatment conditions. The follow-up continued until November 30, 2014, with a median time of 58 months (6–65 months). During the follow-up, 16 were lost, 53 suffered with adverse events such as recurrences and metastases, and 34 were dead. This study has been approved by the Ethics Committee of Affiliated Cancer Hospital of Zhengzhou University. All of the patients participated in this study have approved and gave their consent for this study. Immunohistochemical staining assay Estrogen receptor (ER), progesterone receptor (PR), HER-2, KI-67 indexes, and TOP2A protein expression were detected via immunohistochemical (IHC) staining assay. The waxed species of 256 patients were collected and resliced, and further reevaluated by two skilled pathologists. The evaluation criteria were as follows: ER- or PR-positive staining was defined as the condition in which >1 % cancer cells were stained. HER-2 protein expression was ranked between 0 and 3+. Specifically, 0 were the specimens with no HER-2 protein amplification, while 3+ were assigned for the specimens with the presence of HER-2 protein amplification. 36 were 2+ via IHC detection, without further confirmation of the HER-2 status. The KI-67 evaluation criteria were as follows. KI-67-positive staining was nucleus-targeted, and cytoplasm- and membrane-
positive cancer cells were not counted in. Ten high-powered field in each light microscope slices were randomly selected, in which the amount of all cancer cells were counted. The average proportion of positive cell of all cancer cells was treated as quantitative index, which is further subcategorized into low expression (≤14 %) and high expression (>14 %) [9]. There is currently no agreed conclusion concerning the cutoff value of TOP2A. In our study, we defined it as follows: the specimens were ranked as positive (>10 % positive cells) and negative (≤10 % positive cells). Molecular typing was classified according to the St Gallen International Expert Consensus into luminal A, luminal B, basal-like, and HER-2 amplification [10]. Luminal A is defined as ER (+), PR (+), HER-2 (−), and KI-67≤14 %, while luminal B is defined as ER (+), HER2 (−), and KI-67>14 %. Triple negative breast cancer is defined as ER (−), PR (−), and HER-2 (−), whereas HER-2 amplification was defined as ER (−), PR (−), and HER-2 (+). Fluorescence in situ hybridization analysis TOP2A status changes were detected via fluorescence in situ hybridization (FISH) methods. All waxed specimens were subjected to a 2- to 3-cm-thick serial sectioning before fixed in 75 °C oven for 2–3 h. After 10-min xylene dewaxing twice at room temperature, the slides were dehydrated using anhydrous alcohol for 5 min at room temperature, after which the slides were respectively immersed in 100 and 80 % alcohol, and deionized water 2 min for each (gradient rehydration). Afterwards, the splices were placed in the deionized water high–pressure cooker for 3 min, then immersed in the two times SSC buffer solution for 3 min. The water surrounding the species were then blotted, and the tissues were then digested with 80 μl pepsin and incubated at 37 °C for 10– 20 min when the extent of absorption was closely monitored. After washing in two times SSC solution for 5 min and subsequent dehydration with 80 and 100 % ethanol for 2 min, the species were taken out for natural drying, after which tissue region was circled by diamond pen, probe was added dropwise, and coverslip was lidded and sealed. They were denatured in hybridization oven for 5 min and were hybridized overnight at 37 °C for 16 h. After removing the seals, they were immersed in two times SSC at 37 °C, after which the coverslip was removed and the species were waggled for 1– 3 s in 37 °C, 0.1 % NP-40 immersion for 5 min, dehydration for 2 min in 80 % alcohol and natural drying, 10 μl 4′,6diamidino-2-phenylindole (DAPI) counterstain added dropwise to the hybrid area, with coverslip covered. After 20 min, they were observed by fluorescence microscope. Image acquisition and processing were conducted via FISH 3.0 imaging system. The red signal is representative of TOP2A gene, while the green signal is chromosome 17 centromere probe. Firstly, the areas of invasive carcinoma on FISH
Tumor Biol.
section same as that on HE-dyed section were searched under ten times objective lens, the criteria required at least two areas. Second, the FISH results of cancerous cell nuclei were observed through specific single channel filter under 100× objective lens, after which the red and green signal counting and ratio calculation were conducted. The ratio > 2.0 means TOP2A gene amplification, the ratio50 years old
65.1 % (95/146) 63.6 % (70/110)
Tumor size T1–T2 T3 Lymph node Negative Positive Pathological type Invasive ductal carcinoma Invasive lobular carcinoma Other types Histological grade 1–2 grade 3 grade ER Positive Negative PR Positive Negative HER-2 Amplification Negative KI-67 Low expression (
