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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 1036-1039

Expression analysis of human epidermal growth factor receptor type 2 transcripts in breast cancer cohort and its association with clinical features


1 Department of Biosciences, COMSATS Institute of Information and Technology, Islamabad, Pakistan
2 Department of Surgery, Pakistan Institute of Medical Sciences, Islamabad, Pakistan

Date of Web Publication25-Jul-2016

Correspondence Address:
Muhammad Faraz Arshad Malik
Department of Biosciences, COMSATS Institute of Information and Technology, Islamabad
Pakistan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.176179

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 > Abstract 

Aim of Study: Increased expression of human epidermal growth factor receptor type 2 (HER2) is significantly associated with poor prognosis in breast cancer patients. However, data on HER2 at transcript levels in Pakistani mammary tumor affected females is still limited. In the current study, HER2 transcripts were explored in breast cancer cohort and correlated with various clinical parameters. Materials and Methods: Freshly excised tumors along with adjacent normal background tissues of 94 patients were collected at the time of surgery and immediately stored in RNAlater ® solution. Clinical data for these samples (disease stage, grade, age, and menopausal status) was also retrieved after a subsequent follow-up. Isolation of RNA and cDNA synthesis was done using an already established protocol. HER2 expression was evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR) technique while β-actin was used as an internal control. Results: In the given cohort, 31 (33%) patients were found positive for HER2. These tumors showed a pronounced increase in HER2 as compared to controls (P = 0.0004). Interestingly, the significant relevance of high HER2 mRNA among moderately differentiated tumor tissues in comparison to controls was also observed (P = 0.02). A significant association of HER2 levels with premenopausal status was also reported. Conclusion: Based on these findings, early screening of HER2 using qRT-PCR should be incorporated for breast cancer patients of Pakistani population diagnosis.

Keywords: Breast cancer, human epidermal growth factor receptor type 2, metastasis


How to cite this article:
Pervez A, Riaz SK, Mehmood A, Rashid R, Arshad Malik MF. Expression analysis of human epidermal growth factor receptor type 2 transcripts in breast cancer cohort and its association with clinical features. J Can Res Ther 2016;12:1036-9

How to cite this URL:
Pervez A, Riaz SK, Mehmood A, Rashid R, Arshad Malik MF. Expression analysis of human epidermal growth factor receptor type 2 transcripts in breast cancer cohort and its association with clinical features. J Can Res Ther [serial online] 2016 [cited 2019 Sep 21];12:1036-9. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/1036/176179




 > Introduction Top


Breast cancer is included among top three most commonly prevailing cancers in women accounting for 14% cancer-related deaths.[1] Interestingly, the prevalence of breast cancer in Pakistan is observed at a relatively young age in contrast to Western women.[2] Patients affected by mammary tumors at a younger age may be influenced by either genetic or nongenetic factors leading to an unfavorable prediction.[3] Human epidermal growth factor receptor type 2 (HER2), a biomarker in breast cancer, is located on chromosome 17q21, encoding transmembrane glycoprotein comprising of 1255 amino acids. Around 20–30% of mammary cancers showed increased HER2 signals detected via immunohistochemistry (IHC).[4] HER2 belongs to four transmembrane tyrosine kinase receptors family that binds growth factor ligand as dimers and facilitate cell growth, differentiation, and survival.[5] Dimerization of HER2 triggers oncogenic potential by increasing cancer cells growth rate.[6] Interestingly, a concordance of HER2 signals among primary and secondary tumor site has also been established.[7] Its activation develops resistant against apoptosis, disrupts organization of epithelial cells and induces uncontrolled proliferation.[8] Up-regulation of HER2 is correlated with several clinical parameters including tumor grade, and hormone receptor status.[9],[10] HER2 overexpression contributes to initiation, progression, dissemination, and metastasis of breast cancer.[11] HER2 is involved in the regulation of normal breast tissue growth and development mediated by tyrosine kinase.[12] HER2 could also be used as a marker for detecting cytotoxic agent's efficiency. Though there is no specific predictor for individual anti-cancer agents, HER2 seems to be the most promising predictor, especially in adjuvant therapeutic approach. Meta-analyses have revealed that the efficacy of anthracycline is confined to HER2 subtype cancers.[13],[14] Based on these findings, an effort to initially assess HER2 positive samples in breast cancer on real-time dataset was designed for the given cohort.


 > Materials and Methods Top


For this study, freshly excised tumor tissues along with adjacent normal background (controls) were collected at the time of surgery from (Pakistan Institute of Medical Sciences). The study was conducted after formal ethical approval of both institution and hospital review committees. These samples were immediately immersed in RNAlater ® stabilization solution (Cat No. AM7020, Thermo Scientific, USA) and shifted to a laboratory for storage at −85°C until further usage.

Isolation of RNA and synthesis of cDNA

RNA isolation was carried out using an already established protocol.[15] cDNA was synthesized using RevertAid First Strand cDNA Kit ( Cat No. K1622, Thermo Scientific, USA) as per manufacturer's guidelines. Briefly, 250 ng of template RNA along with oligo dT and water (12 µl) was incubated at 65°C for 5 min and later shifted on ice for 1 min. Subsequently, a cocktail of the reaction mixture (8 µl) containing (reaction buffer, Ribolock RNase Inhibitor, dNTP, and M-MuLV RT) was added in the same vial for 5 min at room temperature followed by 60 min incubation at 42°C. These tubes were left at 70°C for 5 min to affirm enzyme degradation at the terminal stage.

Optimization of human epidermal growth factor receptor type 2 and quantitative real-time polymerase chain reaction

HER2 and β-actin primers were designed using Primer 3' software and synthesized from IDT. Initially, amplification conditions optimized for both HER2 and β-actin were 94°C for 30 s, 58°C for 1 min, 72°C for 1 min up to 30 cycles using the conventional polymerase chain reaction (PCR) technique. HER2 forward 5'-TTGAGTCCATGCCCAATCC-3' and reverse 5'-GTGTTCCATCCTCTGCTGTC-3' primer set yield 150bp product. β-actin used as internal control yield 580 bp product by using forward 5'-ATGATATCGCCGCGCTCA-3' and reverse 5'-CGCTCGGTGAGGATCTTCA-3'. Amplifications were confirmed using previously mentioned agarose gel electrophoresis. VeriQuest SYBR ® Green qPCR Master Mix (Cat no. 75600, Affymetrix USA) was used to perform quantitative real-time PCR (qRT-PCR). The experiment was carried out with StepOnePlus RT-PCR system (Applied Biosystem, EN60825-1). An initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing at 58°C for 1 min were conducted. Relative expression was assessed using 2ΔΔCt method.

Correlation of human epidermal growth factor receptor type 2 with various parameters

Relative expression of HER2 and β-actin were assessed from data generated by RT-PCR. These values were compared with various clinical parameters including tumor stage, grade, along with age, menopausal status. Both Wilcoxon signed test (paired) and Kruskal–Wallis ANOVA were used from”OriginPro” software (OriginLab Corporation, Northampton, USA) for exploring HER2 relevance. Data were represented as mean of relative expression ± standard error of mean as shown in [Table 1].
Table 1: Association of human epidermal growth factor receptor type 2 with clinicopathological parameters

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 > Results Top


The said cohort (n = 94) contains 48 premenopausal, and 46 postmenopausal breast cancer affected women. In the given cohort, 31 patients were found positive for HER2 expression. Based on Wilcoxon signed test (paired), HER2 overexpression was significantly high among tumors versus their respective controls (P = 0.04) as shown in the [Figure 1].
Figure 1: Human epidermal growth factor receptor type 2 expression between tumor and controls of the cohort

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Effect of human epidermal growth factor receptor type 2 with tumor grade and stage

HER2 positivity among tumors showed significant correlation with early tumor staging. Stage-I patients showed high HER2 for tumor tissues in comparison with their controls (P = 0.01). Data analyzed by Kruskal–Wallis test for tumor grades showed significant HER2 levels among moderately differentiated tumor versus their respective controls (P = 0.02) as shown in [Table 1].

Effect of human epidermal growth factor receptor type 2 with age onset, menopausal status

A positive correlation of HER2 with the menopausal status of affected women was also established. Tumor observed in premenopausal women showed high HER2 expression in comparison to their respective controls (P = 0.03). Women suffering from breast cancer below 45 years of age also showed high HER2 in comparison to their respective controls (P = 0.04) as given in [Table 1].


 > Discussion Top


In Pakistan, breast cancer affected females with an age-adjusted incidence rate of 50.1/100,000/year has been reported.[16] A diversified range of factors responsible for breast cancer in Pakistani women has been observed encompassing both genetic and environmental domains.

Although the mean age of the patients was found to be >45 years while majority of patients belonged to age ≤45 years group. In the current study, age at menarche and menopausal status impart a significant influence toward HER2 regulation in breast cancer affected patients. These results are contradictory to western findings stating breast cancer prevalence higher in old age. Differences may be attributed due to a population-specific genetic signatures prevailing among populations.[17]

In the given cohort, 33% patients were found positive for HER2 expression. Earlier, a quite similar trend of HER2 has been observed in Russian (31.6%) and Brazilian (32.8%) breast cancer affected women.[18],[19] Based on a meta-analysis of 22 studies from Iranian breast cancer affected individuals, data presented 44.5% samples positive for HER2.[20] However, in another study, representing datasets from both European and Asian origin showed that HER2 was not related to the geographic location of the patients with a constitutive positive expression in 21.9% samples of the cohort.[21] Despite these variations (20–30%), based on HER2 prevalence, early screening of breast cancer affected women for HER2 using either IHC or qRT-PCR would be helpful in devising an appropriate therapeutic strategy.

Tumors and along with adjacent background tissues showed a significant variation of HER2 expression according to Wilcoxon signed test (paired). Data sets were analyzed using delta Ct value of qRT-PCR in paired form. Data were also analyzed by using Kruskal–Wallis ANOVA against different parameters such as age, menopausal status, stage, and grade.

Earlier, in few studies conducted on retrospective data, (from 2004 to 2007), no significant correlation of HER2/neu and tumor subtypes (P = 0.980), tumor size (P = 0.455), lymph nodes involved (P = 0.660), and tumor grade (P = 0.062) had been observed.[22],[23] Both these studies used IHC staining technique for data analysis. Interestingly, quite similar findings have also been observed in the given cohort using qRT-PCR approach. Few plausible reasons of lacking significant association are attributed to uneven sample distribution among 4 cancer stages and grading, age at onset of disease and genetic diversity of our population.

Based on these findings, early screening of HER2 will be helpful to devise therapeutic strategies against this disease. Furthermore, apart from conventional IHC screening, qRT-PCR-based approach provides a strong alternate venue for detection of HER2. A concordance of qRT-PCR results with IHC findings using formalin fixed paraffin embedded tissues will be helpful to establish this technique as a diagnostic tool.

HER2 contribute a significant expressional variation among both tumors and control tissues. Based on these findings, early screening of HER2 will be helpful to devise therapeutic strategies against this havoc disease. Furthermore, apart from conventional screening HER2 using IHC, this qPCR-based approach provides a strong alternate venue for both early screening of HER2 positive patients and their response to suggested HER2 inhibitors. A concordance of qPCR results with IHC findings is an interesting area that requires further research and validation in the breast cancer cohort.


 > Conclusion Top


Based on these findings, HER2 screening using qRT-PCR technique should also be incorporated for molecular subtyping of breast cancer. Screening of HER2 along with other biomarkers will impart a significant role towards disease prognosis. A potential concordance of transcript variants with protein profiling of HER2 is an area that requires further research for refining personalized medication.

Acknowledgments

Authors would like to thanks especially HEC (Higher Education Commission of Pakistan) and COMSATS-IT for their valued research funding. We are in immense debt of all participants engaged in this study as patients, clinicians, oncologists, surgeons and medical support staff for their valued patience and needful support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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