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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 5  |  Page : 981-988

Assessment of human epidermal growth factor receptor 2/neu gene amplification and expression as a biomarker for radiotherapy and hormonal-treated breast cancer patients in upper Egypt


1 Department of Radiobiology, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority, Cairo, Egypt
2 Department of Radiotherapy, South Egypt Cancer Institute, Assiut University, Asyut, Egypt
3 Department of Clinical Pathology, South Egypt Cancer Institute, Assiut University, Asyut, Egypt
4 Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt

Date of Web Publication4-Oct-2019

Correspondence Address:
Tarek K El-Maghraby
Department of Radiobiology, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority, Naser City, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_42_17

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 > Abstract 


Background: Breast cancer plays major public health in Egyptian women. In upper Egypt, There is an increase in the incidence of breast cancer compared to other Egyptian areas without know the reasons. In this study, we aimed to evaluate the potential of HER-2/neu status as one of the important markers to classify the women suffering from breast cancer in upper Egypt and monitoring the responsiveness to different therapies.
Settings and Design: The present study was performed on 67 female breast cancer patients in the South Egypt Cancer Institute to evaluate HER-2/neu gene amplification and expression.
Patients and Methods: Tissue samples were used for immunohistological analysis of endocrine receptors, HER-2/neu, and HER-2/neu gene amplification. In addition, the blood samples were also used to determine HER-2/neu gene expression.
Statistical Analysis: All statistical analyses were performed using Chi-square test. The statistical difference is considered statistically significant at P < 0.05.
Results: There was a statistically significant association between HER-2/neu gene expression and the age of patients. There is decrease in the level of HER-2/neu mRNA expression in group treated with chemotherapy and group treated with chemotherapy and radiotherapy compared to each group baseline level of HER-2/neu mRNA expression before treatment. On the contrary, the group treated with chemotherapy, radiotherapy, and hormonal therapy revealed increase on the level of HER-2/neu mRNA expression when compared with their baseline for the same patients before treatment.
Conclusion: We need further studies on the large group of upper Egypt breast cancer patients to confirm that the level of HER-2/neu mRNA expression can be used as a marker for classified them and their response to different treatment.

Keywords: Breast cancer, gene expression, human epidermal growth factor receptor 2/neu gene, upper Egypt


How to cite this article:
Essmat MK, Abdelwanis MA, Mosad EZ, El-Maghraby TK, Othman AE. Assessment of human epidermal growth factor receptor 2/neu gene amplification and expression as a biomarker for radiotherapy and hormonal-treated breast cancer patients in upper Egypt. J Can Res Ther 2019;15:981-8

How to cite this URL:
Essmat MK, Abdelwanis MA, Mosad EZ, El-Maghraby TK, Othman AE. Assessment of human epidermal growth factor receptor 2/neu gene amplification and expression as a biomarker for radiotherapy and hormonal-treated breast cancer patients in upper Egypt. J Can Res Ther [serial online] 2019 [cited 2019 Oct 22];15:981-8. Available from: http://www.cancerjournal.net/text.asp?2019/15/5/981/244215




 > Introduction Top


Breast carcinoma is the more prevalence tumor cause of cancer death in women, annually about 1.7 million cases occurring worldwide.[1]

In Egypt, breast cancer is the most common type of cancer. The proportion of incident breast cancer was highest in upper Egypt (38.7%) and next in Middle Egypt (33.8%) and lowest in the lower Egypt (26.8%).[2]

Numerous studies have been worried about its pathogenesis, biological behavior, and it has been recorded that breast malignancy is a heterogeneous infection with variable biological and clinical feature in view of its diverse hereditary modification.[3]

Protooncogenes and tumor silencer qualities are two types of genes that assume a focal part in the regulation of cell development. Hence, any modification in one or a greater amount of these genes seems to assume an imperative part in the pathogenesis of most human malignancies.[4]

Human epidermal growth factor receptor 2 (HER-2)/neu (otherwise called C-erbB2) proto-oncogene amplification as well as overexpression is a standout among the most critical adjustments experienced in breast malignancy. HER-2/neu is an individual form epidermal development variable receptor family HER-1, HER-3, and HER-4.[5] It encodes a transmembrane glycoprotein with tyrosine kinas activity and structural homology to the human epidermal development component (EGFR; ERBBI). HER-2/neu is expressed in numerous cell systems, triggers a network of signaling pathways, and assumes an essential part in ordinary development and advancement.[6]

HER-2/neu proto-oncogene is amplified as well as overexpressed in around in 20%–30% of obtrusive primary breast cancers.[7] Such change of this gene has been connected with poor prognosis.[8]

Currently, no single assay is globally accepted as the gold standard for HER-2 determination in breast cancer. Immunohistochemistry (IHC) is used for measurement protein overexpression and the detection of gene amplification by fluorescence in situ hybridization (FISH), chromogenic in situ hybridization, or polymerase chain reaction (PCR)-based methods including quantitative reverse transcription-PCR (RT-PCR) and more advanced, quantitative real-time PCR method for RNA, and DNA analysis.[9]

Indeed, IHC is a reliable, easy to perform, and accessible technique, which is far less expensive and less time-consuming than FISH.[10] while, real-time PCR is an easy, rapid, sensitive, accurate and reliable method to quantify amplification and expression of HER-2/neu.[11]

Real-time RT-PCR has the feasibility of quantitation of circulating HER-2/neu as a sensitive marker for the detection of micrometastases in the blood of breast cancer patients. Therefore, HER-2/neu is helpful for varied clinical applications. These include recognizable proof and observing of ladies with metastatic breast cancer to help in patient administration; expectation of the reaction to hormonal treatment; and the chose patients for trastuzumab treatment.[12]

Diverse diseases react contrastingly to radiation treatment. The reaction of a tumor to radiation is depicted by its radiosensitivity. In addition, the measure of radiation utilized as a part of radiation treatment changes relying on the type and stage of malignancy being dealt with. In the breast, radiotherapy is a very focused on, exceedingly compelling approach to decimate tumor cells that may stick around after surgery. Radiation can decrease the danger of bosom growth repeat by around 70%.[13] Radiation will be taken alone or in mix with chemotherapy and/or hormone treatment, to decrease the danger of tumor redeveloping in the breast.[13] There have been a various trials demonstrating that HER-2/neu overexpression give hormone resistance.[14]


 > Patients and Methods Top


The present study was performed on 87 participants, 67 diagnosed female breast cancer patients beside, and 20 blood samples were collected from healthy volunteers as control during period from 2011 to 2013. The ages of the patients and healthy volunteers ranged from 27 to 65 years with a mean of 49.64 ± 1.432. They underwent surgery either in the form of conservative wide local excision and axillary lymph node dissection or modified radical mastectomy.

Sampling

Blood samples

Two blood samples were collected from each patient, before and after 0–3 and 6 months after treatment for mRNA extraction to determine HER-2/neu gene expression.

Tissue samples

Surgically removed tissue samples, collected from all the patients enrolled in this study were used for IHC analysis for HER-2/neu. In addition, the same tissue was also used for DNA extraction from paraffin-embedded tissues to determine HER-2/neu gene amplification. Where more investigation showed that HER-2/neu gene is duplicated (amplified) and copy numbers are increased.

Methods

Immunostaining for human epidermal growth factor receptor 2/neu gene

IHC measures the level of HER-2 receptor overexpression. IHC analysis of the HER-2 protein expression was carried out in histologic sections of breast cancer specimens using the Hercep Test (DAKO, CA, USA) according to manufacture's guidelines and instructions.

Assessment of human epidermal growth factor receptor 2/neu Gene amplification using real time-polymerase chain reaction

Real-time PCR was performed in the Light Cycler 2 (Roche, Penzberg, Germany) and the “Light Cycler-HER-2/neu DNA quantification kit” (Roche, Penzberg, Germany) was used for HER-2 gene copy quantification according to the manufacturer's instruction.[15]

Quantification of RNA corresponding to human epidermal growth factor receptor 2/neu gene

RNA was extracted then from blood samples using the extraction kitRNA extraction kit (Real Genomic, Real Biotech Corporation, Taiwan) was used to extract samples according to the manufacturer's protocol based on [16] methods, with quantification of RNA concentrations with nanodropper. cDNA was synthesized then according to the instructions of cDNA synthesis kit (Fermentus, Thermo Fisher Scientific Inc, UK). PCR amplifications for cDNAs were accomplished in (Rotor Gene 2000 real-time fluorescence thermal cycler [Corbett Ltd., Australia]) with a heated lid (105°C) based on the PCR programs. PCR conditions were 15s at 95°C (denaturation step), 20s at 55°C (annealing step), and 20s at 72°C (extension step) for 40 cycles. Each cDNA fragment was amplified in duplicate for HER-2/neu gene as well as Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH). All primers were purchased from (Jena Bioscience, Germany) primers were designed according to Cuadros et al., 2010.[17]

Statistical analysis

All statistical analyses were performed using Chi-square test. The statistical difference is considered significant at P < 0.05.


 > Results Top


All samples include in the present study were obtained from 67 female patients during the period from 2011 to 2013. The age of patients with breast ranged from 27 to 75 with a mean of 46.045 ± 1.432. There were three patients (4.5%) with Grade I and 55 patients (82.09%) with Grade II and 9 patients (13.43) with Grade III. Twenty-three out of 67 cases (34.33%) were premenopausal while 44 out of 67 (65.67%) were postmenopausal. Thirty patients out of 67 cases (44.78%) had right breast cancers while 37 (55.22%) had left breast cancers. Radiotherapy was delivered to 26 patients (39%). Chemotherapy was administered to 52 patients (77.6%). Hormonal therapy was administered to 15 patients (22.4%).

IHC analysis was performed on breast cancer samples. HER-2/neu were positive in 24 patients (35.29%) and 43 patients (64.18%) were HER-2/neu negative [Image 1].



Correlation between human epidermal growth factor receptor 2/neu by immunohistochemistry and clinicopathological parameters

According to age, 18/24 cases (75%) of positive immunoreactivity for HER-2/neu cases were 45 years or above. Regarding tumor grade, cases were classified into Grade I, II, and III.

The relationship between clinicopathologic variables (age, histological, and grade) and HER-2/neu gene by IHC is shown in [Table 1]. No significant association was detected between the HER-2/neu gene histological grade. There was a statistically significant association detected between HER-2/neu gene and age of patients [Table 1].
Table 1: Correlation between human epidermal growth factor receptor 2/neu and clinicopathological parameters

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Correlation between human epidermal growth factor receptor 2/neu expression by IHC and human epidermal growth factor receptor 2/neu amplification by real time-polymerase chain reaction

[Table 2] shows the correlation between HER-2/neu amplification by RT-PCR versus HER-2/neu expression by IHC. No significant association was detected between HER-2/neu amplification by RT-PCR and HER-2/neu by IHC.
Table 2: Correlation between human epidermal growth factor receptor 2/neu immunohistochemistry and human epidermal growth factor receptor 2/neu DNA amplification by real time-polymerase chain reaction

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The comparison between the levels of human epidermal growth factor receptor 2/neu cDNAs in different treated groups

[Figure 1] shows the comparison between the effect of different types of treatments on the level of positive HER-2/neu mRNA expression after 3 or 6 months. Our results demonstrated that all treatment groups revealed decrease in the level of HER-2/neu mRNA expression compared to nontreated groups. In the same direction, there is decrease in the level of HER-2/neu mRNA expression in group treated with chemotherapy and group treated with chemotherapy and radiotherapy compared for each group to the baseline level of HER-2/neu mRNA expression before treatment. Moreover, the highest decrease in the level of HER-2/neu mRNA expression was shown in the group treated with chemotherapy and radiotherapy. On the contrary, the group treated with chemotherapy, radiotherapy, and hormonal therapy revealed increase on the level of HER-2/neu mRNA expression when compared with their baseline for the same patients before treatment. There is no difference in the direction of the results between, 3 and 6 months, while the decrease and the increase on the level HER-2/neu mRNA expression are more clearly in the 6 months from treatments [Figure 1].
Figure 1: The comparison between positive human epidermal growth factor receptor 2/neu mRNA expression in the baseline of treatment and after 3 months of different treatments

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[Figure 2] showed amplification plots with good baseline for real time-PCR HER-2/neu cDNAs clarify the difference between plots in Ct values. Moreover, [Figure 3] demonstrated sharp melting curves for the PCR products of HER-2/neu and GAPDH cDNA, these melting curves indicating the homogeneity of these products without primer dimer.
Figure 2: Amplification curves for the quantitative of real-time polymerase chain reaction human epidermal growth factor receptor 2/neu cDNAs

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Figure 3: Single sharp melting curves for the polymerase chain reaction products of human epidermal growth factor receptor 2/neu cDNA and reference gene Glyceraldehyde 3-phosphate dehydrogenase cDNA

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 > Discussion Top


In Egypt, as in numerous different parts of the world, breast cancer is the most well-known type of cancer: it represents around 38% of reported malignancies among Egyptian females.[18]

In perspective of past facts, the present study addresses the possibility using real-time RT-PCR-based quantization of circulating HER-2/neu as a sensitive marker for detection of micrometastases to obtain effective treatment for breast cancer that reduce HER-2/neu gene expression and to prevent recurrence or reduce relapse of breast cancer and using it to measure amplification (DNA level) in tissue and compares the latest results obtained with clinical prognostic indices and those obtained using IHC.[19]

Our results demonstrated that, by IHC 24/67 (35.8%) patients were positive, 43/67 (64%). This finding was in concordance with the previous studies which have identified HER-2/neu overexpression in 9%–40% of human breast carcinoma with the great majority reporting incidence around 20% of tumors.[20]

However, lower incidence of HER-2/neu gene expression were reported by Fabi et al.,[21] who reported that, HER-2/neu expression was 18.24%18.24%. Rydén et al.,[22] showed that over-expression or Her-2 gene amplification is identified in 10-30% of primary mammary carcinomas.

Previous studies have shown HER-2/neu-positive tumors to have a higher association with estrogen receptor (ER) and progesterone-receptor (PR) negativity.[23]

It had been shown that 50%–70% of breast cancer patients with positive ER and PR. regressed with hormone therapy.[24]

However, other investigators reported that there was a relation between hormone-receptor status and HER-2/neu overexpression.[25] The reason for this discrepancy may be the different assay methods of detecting HER-2/neu overexpression and hormone-receptor status or may be due to the different cutoff.

The analysis of conjoint expression of ER and PR seen in the previous paper.[26] The same authors reveals no association of ER and PR with HER-2/neu expression. Although ER and PR expression were significantly decreased in HER-2/neu positive tumors, a substantial proportion of ER +ve and PR +ve cases also expressed HER-2/neu, which makes it mandatory to perform hormone receptors and HER-2/neu in all the cases, as one cannot predict expression in any particular patients. Moreover, there are a significant number of cases which did not show expression of ER, PR, or HER-2/neu whereas the percent of ER -ve and PR -ve and positive HER-2/neu is small (25%). These data concluding no correlation between HER-2/neu and hormone receptors.

An increment in the quantity of copies of cell oncogenes brought about by DNA amplification frequently prompts deregulated protein expression and is connected with neoplastic change. It has been accounted for that c-erbB2/HER-2/neu gene amplification from 2 to >20 fold is found in 30% of human breast tumors additionally [27],[28] demonstrated the same result. The level of amplification is related with a poor anticipation for a scope of tumors including those of breast tissue. High sensitivity is required on the grounds that tissue biopsies are regularly of constrained weight and it might be important to get material by microdissection.[29]

Our results demonstrated that, on paraffin-embedded tissue samples, 24 (35.8%) out of 67 specimens tested positive for HER-2/neu in the IHC, while 25 (37.31%) out of 67 specimens tested positive in real-time PCR. Along these lines, the examination of protein overexpression by IHC demonstrated a little number of positive cases than were identified by real-time PCR on DNA level. This could imply that the amplified region does exclude the promoter, or it may be the case that amplification of HER-2/neu is an early occasion and thus may continue now and again distinguishable overexpression of the protein.[30] All cases with allelic uneven characters indicated protein overexpression, and every one of these cases was recognized by real-time PCR.

These results are in agreement with other studies.[31],[32]

In addition, Bánkfalvi et al.[30] and his associates, who found that the percentage of HER-2/neu overexpression by IHC was less than identified by real-time PCR. This implies real-time PCR could distinguish HER-2/neu in patients known not HER-2 negative in tissue tests. They disclosed their discoveries to be because of the way that amplification of HER-2/neu is an early occasion and along these lines, may go before protein expression; another conceivable clarification ascribed that real-time PCR can distinguish and measure little measures of particular nucleic acid sequences most specifically, sensitively, and reproducibly.[33]

In the present study, no amplification of HER-2/neu had been detected in blood samples at the healthy studied control group. Our findings are in accordance to the results of Wasserman et al.[34] Approximately 0%–38% of patients with primary breast cancer and 23%–80% of patients with metastatic breast cancer have increased levels of the HER-2/neu extracellular domain (ECD, P185) in serum.[35] where the ECD of HER-2/neu is shed into the circulation by metalloproteases protein [36] and it can be detected by real-time-PCR. Overwhelming evidence from numerous studies indicates that a significant correlation of circulating HER-2/neu protein levels with disease recurrence, metastasis, or shortened survival.[37]

What's more positive HER-2 status predicts a reaction to treatment with trastuzumab (Hercep tin), which is an adapted monoclonal antibody acting agent coordinated against the external domain of the HER-2 protein which has been appeared to be powerful in delaying survival in patients with HER-2 receptor-positive metastatic breast carcinoma.[38],[39]

In our study, we observed that serum HER-2 ECD levels varied during chemotherapy, regardless of the type of response and the regimens utilized. Moreover, the decrease in HER-2 ECD levels during the first 3 months after initiation of therapy appeared to be more accurate than baseline HER-2 mRNA expression levels before treatment.

This was in agreement with the results reported by other studies on females cancer patients, as those described by Kumaki et al.[40] even so, conflicting findings have also been reported, for example Tinari et al.,[41] reported in their experimental study, no significant change was observed for the expression C-erbB-2 after neoadjuvant chemotherapy, Neubauer et al.,[42] found HER-2/neu expression rarely changes after chemotherapy.

In our results, we found that the highest decrease in the level of HER-2/neu mRNA expression in group treated with chemotherapy and radiotherapy after 3 or 6 months compared to the baseline level of HER-2/neu mRNA expression before treatment.

This was in agreement with results reported by Lee et al.,[43] who reported that, Patients who were treated with surgery + CT + RT showed significantly (P < 0.05) reduced serum HER-2/neu levels, showing good response to treatment.

In addition, McArdle et al.[44] found that the mix of radiotherapy and chemotherapy was connected with diminished recurrence and a pattern toward increased malignancy-specific survival; and that disease-related survival was for the most part subject to the quantity of positive nodes. Zhang et al.[45] reported that it the patients who got radiotherapy alongside chemotherapy and/or hormonal treatment had a lower danger of developing second cancers compared with those patients got radiotherapy alone.

As shown in our results, there is an increase on the level of HER-2/neu mRNA expression in Patients who were treated with hormonal therapy in addition to chemotherapy and radiotherapy.

This was in agreement with results reported by Lee et al.,[43] who reported that patients who were treated with endocrine therapy in addition to surgery + CT + RT regimen did not show any significant reduction in serum HER-2/neu levels showing resistance to treatment. The overexpression of HER-2 and dimerisation with HER family members triggers several downstream pathways as a consequence of phosphorylation of kinases, resulting in anti-apoptotic encouragement, and enhancement of cell proliferation or even cell migration.[46] PI3K/Akt or MAPK might be involved in the activation of these responses; however, they can be also implicated in the activation of ER or even its coregulators. This way ER is able to develop its cellular outcomes, which means higher stimulation on tumor development and endocrine treatment resistance in those cells showing high expression of HER-2 and ER.[47]

For instance, it is found that there is an association between overexpression of proto-oncogene HER-2/neu and resistance to tamoxifen in ER-positive, primary, and metastatic breast cancer has been suggested. HR+/HER-2/neu+ patients have a poor response to endocrine therapy than ER+/HER-2/neu-patients. This explained by cross talking between the ER/HER family are involved in mechanisms of resistance in patients with breast carcinoma when submitted to endocrine treatments.[48]


 > Conclusion Top


We concluded that there was an increase in the level of HER-2/neu mRNA expression in patients who were treated with hormonal therapy in addition to chemotherapy and radiotherapy. The highest decrease in the level of HER-2/neu mRNA expression in group treated with chemotherapy and radiotherapy after 3 or 6 months compared to the baseline level of HER-2/neu mRNA expression before treatment.

Financial support and sponsorship

This study was financially supported by the Egyptian Academy of Scientific Research (ASRT).

Conflicts of interest

There are no conflicts of interest.



 
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