|Year : 2017 | Volume
| Issue : 4 | Page : 475-480
|Assessment of topoisomerase II-alpha gene status by dual color chromogenic in situ hybridization in a set of Iraqi patients with invasive breast carcinoma
Rasha Abd Alraouf Neama1, Manal A Habib2, Sahira A Ali2, Ali H Al-Khafaji3, Mohammed F Alqanbar1
1 Department of Pathology, College of Medicine, Karbala University, Karbala, Iraq
2 Department of Pathology, College of Medicine, University of Baghdad, Baghdad, Iraq
3 Department of histopathology, Central Public Health Laboratory, Ministry of Health, Baghdad, Iraq
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|Date of Web Publication||12-Jan-2018|
| Abstract|| |
Background: The human epidermal growth factor receptor 2(HER2) proto-oncogene is overexpressed or amplified in approximately 15%–25% of invasive breast cancers. Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene encoding an enzyme that is a major target of anthracyclines. Hence, the determination of genetic alteration (amplification or deletion) of both genes is considered as an important predictive factor that determines the response of breast cancer patients to treatment. The aims of this study are to determinate TOP2A status gene amplification in a set of Iraqi patients with breast cancer that have had an equivocal (2+) and positive HER2/neu by immunohistochemistry (IHC) and to compare the results with estrogen receptor (ER) and progesterone receptor (PR) and HER2/neu status. Patients and Methods: A cross-sectional prospective study done on 53 patients with invasive breast carcinoma. Twenty-six out of total 53 cases were positive HER2/neu (3+), the remaining 27 equivocal HER2-IHC (2+) cases reanalyzed using dual-color chromogenic in situ hybridization (ZytoVision) probe kit for further identification of HER2/neu gene amplification. Using chromogenic in situ hybridization (CISH), TOP2A gene status determination was done for all cases. Results: There is a direct significant correlation between TOP2A gene amplification and HER2/neu positivity, P < 0.05 in that 15 (39.4%) out of 38 positive HER2/neu cases were associated with topoisomerase gene amplification. Regarding relation of topoisomerase gene to hormone receptor status (ER and PR), there was a significant negative relationship between the gene and ER receptor status. The higher level of gene amplification was noticed in ER and PR negative cases in about 13 (43.3%) and 14 (48.2%) for ER and PR, respectively. Conclusion: TOP2A gene status has a significantly positive correlation with HER2/neu status while it has a significantly negative correlation with hormone receptor status.
Keywords: Breast cancer, dual-color chromogenic in situ hybridization, human epidermal growth factor receptor 2/neu, topoisomerase II-alpha gene
|How to cite this article:|
Neama RA, Habib MA, Ali SA, Al-Khafaji AH, Alqanbar MF. Assessment of topoisomerase II-alpha gene status by dual color chromogenic in situ hybridization in a set of Iraqi patients with invasive breast carcinoma. Indian J Pathol Microbiol 2017;60:475-80
|How to cite this URL:|
Neama RA, Habib MA, Ali SA, Al-Khafaji AH, Alqanbar MF. Assessment of topoisomerase II-alpha gene status by dual color chromogenic in situ hybridization in a set of Iraqi patients with invasive breast carcinoma. Indian J Pathol Microbiol [serial online] 2017 [cited 2020 Aug 13];60:475-80. Available from: http://www.ijpmonline.org/text.asp?2017/60/4/475/222984
| Introduction|| |
Human epidermal growth factor receptor 2 (HER2/neu) is one of the most important prognostic and predictive factors of breast cancer. It is frequently involved in a lot of studies as an important participating biomarker in the determination and evaluation of responsiveness to chemotherapeutic agents  and it is amplified in approximately 20%–30% of all breast cancers. Amplification of this gene is associated with the rapid advancement of the disease, increased rate of metastasis, increased resistance to tamoxifen therapy, and better response to anthracycline-based chemotherapy although the treatment is costly and may accompany with significant side effects such as cardiac toxicity with trastuzumab. Hence, it is important to select the group of patients who will benefit from this therapy.
Topoisomerase II-alpha (TOP2A) gene is located on long arm of chromosome 17 and telomeric to HER2/neu gene. The protein coded by the TOP2A gene helps in situ unbinding of the DNA double strands and it releases twisting stress throughout cellular replication and the action is accomplished by creation of temporally double helical breaks with further separation, thus facilitating rapid cellular turnover and proliferation. As an important participant in the proliferative process, TOP2A gene amplification may associate with abnormal genetic events that associated with malignancy. TOP2A/HER2 coamplification was considered as a beneficial predictive indicator of improving the response to anthracycline-based chemotherapy, so more accurate and easy method for evaluation of this gene becomes crucial.
| Patients and Methods|| |
A cross-sectional prospective study was done, from July 2014 to February 2015, in Cancer Research Center in Baghdad Medical Complex in Iraq. This center received blocks belonging to patients newly diagnosed to have invasive breast carcinoma and immunohistochemical (IHC) study performed for estrogen receptor (ER) and progesterone receptor (PR), HER2/neu, and Ki67. All cases that reached to the center in that period were included and according to the criteria that mentioned below. The geographical distribution of cases was mainly from Baghdad, middle and south of Iraq.
The cancer research center is authorized by the Ministry of Health and Ethical Committee, but we do not know whether these regulations meet the standard once around the world.
After receiving paraffin-embedded blocks of tissue, an hematoxylin and eosin slide was taken to confirm the diagnosis and to determine the proper area in the tumor to perform IHC study. ER, PR, HER2/neu, and Ki67 IHC markers were performed in all the cases reaching the center. All the slides were usually examined by two pathologists and the results usually adapted with consent.
A total number of 75 cases were included according to the inclusion criteria, but just 53 were successfully hybridized applying HER2/neu or TOP2A genes while the remaining samples could not be evaluated because of technical errors, insufficient tumor tissue, and/or very weak signals. Twenty-six out of total 53 cases were positive HER2/neu (3+), the remaining 27 equivocal HER2-IHC (2+) cases reanalyzed using dual-color chromogenic in situ hybridization (DC-CISH) (ZytoVision) probe kit for further identification of HER2/neu gene amplification. Using chromogenic in situ hybridization (CISH), TOP2A gene status determination was done for all cases.
Only patients with HER2/neu score 2 and 3 were included in the study.
Patients with HER2/neu score 0 and 1 are excluded from the study.
Preparation of tissue sections
Twenty-six out of total 53 representative paraffin-embedded tissues were scored as equivocal cases (score 2+) for HER2/neu receptor protein by IHC according to the scoring criteria of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP), 2007. These cases were dealt with to determine HER-2 gene status according to HER-2 gene/CEN17 ratio by in situ hybridization (ISH). All cases were processed by the same steps for the detection of TOP2A gene/CEN17 ratio.
The status of both HER-2 and TOP2A genes was determined by the DC-CISH technology of ZytoVision.
Controls for HER2/neu and TOP2A were taken for ISH:
- HER-2 gene/CEN17
- Positive control: A case of breast carcinoma, with score 3+ IHC HER-2 protein expression, showing gene amplification by DC-CISH was used as a positive control
- Negative control: Tumor sections in which HER-2 probe was not used, but phosphate-buffered saline (PBS) was considered as negative controls.
- TOP2A gene/CEN17
- Positive control: A case of breast carcinoma, with score 3+ IHC HER-2 protein expression, showing TOP2A gene amplification by DC-CISH was used as a positive control
- Negative control: A case of breast carcinoma in which TOP2A probe was not used, but PBS was considered as negative controls.
Staining protocol for human epidermal growth factor receptor Her2/neu and topoisomerase II-alpha by dual-color chromogenic in situ hybridization
The procedure was performed according to manufacturer's instructions and along 2 days.
Denaturation and hybridization (day 1)
After incubation at 65°C overnight, the slides were deparaffinized in xylene and graded ethanols. Heat pretreatment was done with pretreatment buffer at 98°C–100°C for 15 min and adding pepsin for 2–3 min at room temperature. For each probe (HER2/neu and TOP2A), shake and pipette 10 μl on each sample. The slides were coverslipped and edges sealed with rubber cement. Slides were incubation overnight at 37°C using a moisturized chamber.
Posthybridization and detection (day2)
After removing the rubber cement from the slides, they were washed in wash buffer SSC (WB1) at 75°C–80°C for 5 min in water bath. The slides were washed in the buffer tris-buffered-saline (TBS) and drained off. The slides were put in humidity chamber and inside the oven we applied Anti-DIG/DNP-Mix (AB14) dropwise (3–4 drops/slide) to the slides and incubated for 15 min at 37°C. Washing in buffer TBS was done for 2 × 2 min and drained off. HRP/AP-Polymer-Mix (AB13) was applied dropwise (3–4 drops/slide) to the slides; the slides were incubated for 15 min at 37°C in humidity chamber without exposing to strong direct light. After washing with TBS in two jars, slides were put in humidity chamber, then applied AP-red solution dropwise (3–4 drops/slide) and incubated for 10 min at room temperature (protected from strong direct light by covering the humidity chamber). Then, the slides were washed for 2 min in distilled water, blotting, then applied HRP-green solution dropwise (3–4 drops/slide) and incubated for 10 min at room temperature (protected from strong direct). The slides were washed for 2 min in distilled water; counterstain was added to the tissue samples for 2 min with nuclear blue solution (CS2).
The slides washed for 2 min in running tap water, then dehydration in 100% ethanol and incubated in xylene. Slides were coverslipped using mounting solution (alcoholic) (MT4) and air dried for approximately 30 min. Evaluation of the sample material was carried out by light microscopy.
Evaluation of tumor marker receptors was done depending on scoring systems. That for ER, PR was done depending on Allred scoring system. In concern to HER2/neu receptor protein, scoring system involves the evaluation of the intensity and pattern of membranous staining determination of immunoreactivity and this was done using new recommendations of the ASCO/CAP  as bellow:
- Score 0: No reactivity or membranous reactivity in <10% of the tumor cell
- Score 1+: Faint or barely perceptible incomplete membranous reactivity in >10% of tumor cells
- Score 2+: Weak to moderate reactivity of the entire membrane in >10% of the tumor cells
- Score of 3+: Strong reactivity of the entire membrane in >30% of the tumor cells.
Depending on ASCO/CAP protocol published in 2007, the evaluation of HER2/neu gene amplification by ISH (CISH) was used as such:
- Negative: HER2/neu gene copy number <4/nucleus or HER2/neu gene/CEN17 <1.8
- Equivocal (suspect positive): HER2/neu gene copy number = 4–6, HER2/neu gene/CEN17 = 1.8–2.2
- Positive: HER2/neu gene copy number >6 or HER2/neu/CEN17 >2.2 in 20 tumor cells in two different fields.
At least 30 tumor cells from each specimen were counted. The final results were documented after counting 100 nuclei per sample and after repeating the ISH for equivocal cases.
The scoring system for TOP2A gene alteration by ISH is the same as that for HER2/neu which was done depending on company instruction leaf let  as well as other studies.
Statistical Package for the Social Sciences SPSS version 21 (SPSS Inc., Chicago, IL, USA) and Microsoft Office Excel 2010 used in analysis of these data; Chi-square test was used to study association between any two nominal variables. P ≤ 0.05 was considered statistically significant.
| Results|| |
The age of the patients ranged between 29 and 76 years with mean age ± standard deviation of 47.47 ± 10.89 years. About 30 (56.6%) out of total patients were younger than 50 years (premenopausal age).
The number of cases in different grades and stages of invasive breast cancer in this study are illustrated in [Figure 1] and [Table 1].
The immunohistochemical markers' (ER, PR, and HER2/neu) distribution of all cases was shown in [Table 2]. HER2/neu cases were analyzed by CISH and HER2/neu was divided as positive 38 cases (12/CISH and 26 by IHC) and 15 negative cases as shown in [Table 2] and [Figure 2],[Figure 3].
|Figure 2: Invasive ductal mammary carcinoma showing positive membranous staining for human epidermal growth factor receptor 2/neu (score +2) (human epidermal growth factor receptor 2/neu IHC, ×100)|
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|Figure 3: Chromogenic in situ hybridization of a tissue section with breast cancer. There are greenish signals of multiple dense clusters of intranuclear human epidermal growth factor receptor 2/neu gene. This is a case of highly amplified human epidermal growth factor receptor 2/neu gene. Human epidermal growth factor receptor 2/neu/CEN17 ratio was > 5 (oil immersion, ×1000)|
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|Table 2: Immunohistochemical profile of hormone receptors and human epidermal growth factor receptor 2/neu with chromogenic in situ hybridization study performed for equivocal human epidermal growth factor receptor 2/neu cases of breast cancer|
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CISH of TOP2A gene was done to all patients that included in the study [Figure 4]. Depending on the scoring system of TOP2A gene, gene amplification was detected when ratio of Topo gene/chromosomal 17 centromere was ≥2.3 (TOPO2A/CEN17 ≥2.3). We found that Topo gene was amplified in 19 (36%) of total cases of patients. Low-level amplification (LA-TOPO2A/CEN17, 2.3–4.9) was seen in 8 (15%) and high-level amplification (HA-TOPO2A/CEN17, ≥5) in 11 (21%) of cases [Figure 5]. 34 (64%) of cases had have nonamplified gene [Figure 6], while no gene deletion detected in all cases (0%).
|Figure 4: Topoisomerase II-alpha gene amplification in 53 cases with breast cancer|
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|Figure 5: Chromogenic in situ hybridization of a tissue section of breast cancer. The picture shows multiple dense clusters of greenish signals of intranuclear topoisomerase II-alpha gene, red signals for chromosome 17 centromere. This is a highly amplified topoisomerase II-alpha gene, Topo/CEN17 ratio was >5 (oil immersion, ×1000)|
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|Figure 6: Chromogenic in situ hybridization of a tissue section of breast cancer showing normal diploid signals for intranuclear topoisomerase II-alpha gene/CEN17 (no amplification), green signal for Topo gene, and red signal for chromosome 17 centromere (oil immersion, ×1000)|
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Correlation of topoisomerase II-alpha gene status with HER2/neu/neu and immunohistochemical markers
Correlation between topoisomerase II-alpha gene status and human epidermal growth factor receptor 2/neu
The correlation between TOP2A gene status and HER2/neu status was significantly positive (P< 0.05) as about 40% of HER2/neu positive cases have amplified TOP2A gene with higher amplification accounts for 28.9%. In negative HER2/neu, 75% of cases had no gene amplification as seen in [Table 3].
|Table 3: Association between topoisomerase II-alpha gene status and human epidermal growth factor receptor 2/neu expression in patients with breast cancer|
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Correlation between topoisomerase II-alpha gene status and estrogen receptor
The correlation between TOP2A gene status and ER expression that detected by IHC was significantly negative (P< 0.05) as about 74% of ER positive cases have nonamplified TOP2A gene. While 35% of highly amplified TOP2A gene have negative ER as seen in [Table 4].
|Table 4: Correlation between topoisomerase II-alpha gene status and estrogen receptor expression in patients with breast cancer|
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Correlation between topoisomerase II-alpha gene status and progesterone receptors
The correlation between TOP2A gene status and PR expression that detected by IHC was significantly negative (P< 0.05) as 79% of PR positive cases have nonamplified TOP2A gene. While again 35% of highly amplified TOP2A gene had have negative PR [Table 5].
|Table 5: Correlations between topoisomerase II-alpha gene status and progesterone receptor expression in patients with breast cancer|
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| Discussion|| |
It is highly recognized that breast cancer is a heterogeneous disease with special clinical and molecular characteristics. ER and PR are important prognostic factors. ER-positive tumors usually have a higher survival rate when compared with ER-negative tumors in that the rate can reach to 10% better than ER negative. This discrepancy then starts to be diminished with time. Cancers which are ER and/or PR positive are more responsive to hormonal therapy and experience a longer disease-free interval.
The most active chemotherapeutic agents for breast cancer patients are anthracyclines and taxanes. A limited number of patients, may be <5%, are long-time survivors in spite of attaining complete remission. The restricted influence of chemotherapy is due to intrinsic or acquired resistance to the drugs in use. Although several researches were done to study the sensitivity of different tumors to chemotherapeutic agents, but till now it is difficult to be assessed precisely. The significance of a factor that can give an expectation about the efficacy of chemotherapy in both the metastatic and adjuvant setting is clear and important. The nuclear enzymes TOP2A and proto-oncogene c-erbB-2 (HER2) are two factors that have shown predictive potential with regard to anthracycline-based chemotherapy in breast cancer. This study in Iraq conducted to detect such a relation between TOP2A and HER2, and it may pave the way for subsequent studies that try to confirm such a connection between these factors. Hence, such a fact will be a highly beneficial to the oncologist to ensure a higher response to anthracyclins chemotherapy in patients with breast cancer.
In this study, the relation of TOP2A gene to ER and PR status was significant negative relationship, which agree to those documented by other studies.,, Whereas the study conducted by Engstrøm et al. stated that there is a strong direct relationship between TOP2A gene changes and hormonal receptors status.
The result of this study can be interpreted according to results of other studies which state that TOPO2A gene amplification can have the advantage from the usage of anthracyclines containing treatment which targeting TOP2A in hormone receptors (ER and PR) negative breast cancer. So accordingly, hormonal receptor negativity is associated inversely with topo-gene status.
According to the results of this study, there was a significant relationship between TOP2A gene and HER2/neu positive cases and such result was compatible to other studies.,, An interpretation is that high HER2-positive tumors have higher levels of overall genomic instability than HER2-negative tumors and topo2α gene is near her2/neu gene thus can be involved in small region of amplification of HER2/neu gene which in turn may lead to that most cases of HER2/neu gene-amplified breast cancer cases to be associated with coamplified of TOP2A gene.
| Conclusion|| |
DC-CISH technique is a simple applicable method for the revealing of TOP2A gene alterations and determination of HER2/neu gene status whether amplified or not in equivocal (2+) cases detected by IHC with invasive breast cancer. Positive significant correlation of TOP2A gene with HER2/neu gene and its negative correlation with steroid hormones receptor may indicate that it could be considered as a bad prognostic factor. However, this needs further studies in the future for best understanding and confirmation of the role of this gene and its relation with the anthracyclines' chemotherapeutic agents used in treatment of patients with breast cancer.
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Conflicts of interest
There are no conflicts of interest.
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Dr. Rasha Abd Alraouf Neama
Department of Pathology, College of Medicine, Karbala University, Karbala
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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