Breast Cancer DOI 10.1007/s12282-014-0528-0

ORIGINAL ARTICLE

A meta-analysis on concordance between immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) to detect HER2 gene overexpression in breast cancer Fatemeh Bahreini • Ali Reza Soltanian Parvin Mehdipour

•

Received: 27 August 2013 / Accepted: 17 March 2014 Ó The Japanese Breast Cancer Society 2014

Abstract Background We performed this meta-analysis study to evaluate the concordance and discordance between immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in detecting HER2 alteration in human breast cancer. Methods As a meta-analysis, the present study evaluated the available data from previous studies on the HER2 gene detected by IHC and FISH. To indicate the meta-analysis results, a forest plot was used. Results We identified 172 citations, for which our inclusion criteria were met by 18 articles, representing 6629 cases. The overall concordance and discordance rate between IHC staining with score 0/1? and FISH for detection failure of HER2 expression was 96 and 4 %, respectively. The present study showed that the overall proportion of FISH positive and negative rate for IHC score 2? for detection of HER2 expression was 36 and 64 %, respectively; and 91 and 9 % for 3? IHC scores. Conclusion The results of this study show that IHC score 0/1? and 3? cannot be completely considered as negative

F. Bahreini  P. Mehdipour (&) Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Pour Sina Avenue, 14176-13151 Tehran, Iran e-mail: [email protected] F. Bahreini e-mail: [email protected]; [email protected] A. R. Soltanian Department of Biostatistics and Epidemiology, Modeling of Noncommunicable Diseases Research Center, School of Public Health, Hamadan University of Medical Sciences, Shahid Fahmideh Street, P.O.Box 4171, 65155 Hamadan, Iran e-mail: [email protected]; [email protected]

and positive breast cancer test, respectively. Therefore, we suggest a valid and complementary test, the same as FISH, to explore HER2 expression. Keywords HER2  Immunohistochemistry  Fluorescence in situ hybridization  Breast cancer  Meta-analysis

Introduction The HER2 protein is a transmembrane tyrosine kinase growth factor receptor and is found in normal and malignant epithelial cells. The need for accurate detection of the Her2 alteration has now become even more important, because therapeutic decisions for patients are increasingly dependent on this information [1]. Previous studies showed that HER2 alteration may be predictive of response to certain types of chemotherapy [2, 3]. For example, the HER2 gene product, P185HER2, represents a target for specific therapy with the monoclonal antibody trastuzumab [1]. The HER2 gene is amplified and overexpressed in 15–20 % of breast cancers [4, 5]. In 90 % of these carcinomas, HER2 protein overexpression is attributable to gene amplification [5]. The expression of HER2 protein was detected by immunohistochemistry (IHC) in routine practice because of ease of performance and low costs. IHC is widely used for measuring HER2 protein expression on formalin-fixed, paraffin-embedded samples of breast tumors, and IHC is a preferred method for screening and determining cases which need to be genetically evaluated [1, 4, 6, 7]. In addition, HER2 gene amplification as detected by fluorescence in situ hybridization (FISH) may be a reliable predictor of clinical response to treatment of breast cancer.

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Tissue-based detection of the HER2 alteration by FISH and/or IHC offers clear advantages, which suffer from dilutional artifacts resulting from a mixture of normal and abnormal cell populations within the tissue [1, 5]. Both FISH and IHC facilitate the specific assessing of HER2 alteration in individual cells. As a result, FISH and IHC techniques have emerged as the preferred methods to detect HER2 alteration. FISH has already important applications in clinical hematology as well as medical genetics, but it is more time consuming and expensive than IHC. Therefore, IHC may be considered as appropriate to detect the alteration of HER2 expression in cells instead of FISH [1, 6]. However, there is a question whether IHC is accurate for detecting HER2 alteration in all breast cancer stages. So far, detection of HER2 protein by IHC instead of FISH, especially in the early stages of breast cancer, provides no consensus among researchers [6, 7]. Many studies have examined agreement between IHC and FISH [1, 6–8]. But, still no overall result is available. Some of the studies show that IHC staining with score of 3? is mostly in agreement with FISH in detecting HER2 protein, compared to IHC staining score 2? , 1? and 0 [6–8]. Thus, we performed this meta-analysis study to evaluate the efficacy of IHC compared to FISH in detecting HER2 alteration in human breast cancer.

Materials and methods Search strategy All studies evaluating the concordance and discordance between IHC and FISH were searched using ‘‘immunohistochemical (IHC)’’, ‘‘fluorescence in Situ Hybridization (FISH)’’, ‘‘HER2’’ and ‘‘breast cancer’’. In the study, seven electronic search engines were used for searching: PubMed, ISI Web of Knowledge, ScienceDirect, Medline, Embase, Scopus and Scholar-Google. All research results were evaluated independently by two experts in medical genetics. In cases where difference existed between them, the judgment of a third browser was taken to reject or accepts articles. In this study, we searched papers published with any language from January 2001 to December 2011. After extraction of documents and information, article characteristics were imported to Excel software and repeated cases were excluded. In the next step, articles were reviewed, and if they were irrelevant to our aim they were eliminated [9–81, 83–99]. Then with regard to the article abstracts/full text among the remaining studies, we ensured the relevance of the available publications to the aim of our study (Fig. 1).

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Quality assessment and data extraction The quality of the studies was assessed based on their aim (using IHC and FISH in human community) and measuring tools (how did IHC and FISH methods score). The number of HER2 gene amplification was determined by FISH using a dual-color probe FDA-approved PathVysionTM HER2/ neu DNA Probe Kit and Paraffin Pretreatment Kit. It contains a mixture of spectrum orange-labeled HER-2/neu gene probe and spectrum green-labeled centromere control for chromosome 17. The HER2/CEN-17 ratio was calculated by dividing the total number of HER2 signals by the total number of CEN-17 signals. Ratio of HER2/CEN-17 signal\2 was considered as non-amplified (\2 or FISH-), and at least 2 was considered as amplified (C2 or FISH ?). The full text of the articles was reviewed and their quality was finally assessed. Most articles compiled in this study had a good quality score. Statistical methods For the meta-analysis, all data related to the results of IHC (IHC staining with score 0, 1?, 2? and 3?) and FISH (the number of amplifications and non-amplifications) were extracted. Then, the proportion of positive results (confirmed HER2 expression in breast cancer patients) in the FISH method compared to the IHC method was calculated. In FISH by determining concordance and discordance rates between FISH and IHC, binomial distribution was considered to be in agreement between FISH and IHC methods, and also, Jeffrey method was applied to determine variance and confidence intervals in all the reviewed studies. Since in some studies the agreement rate between FISH and IHC was zero, Jeffrey correction was used following confidence interval determination [82]. Consequently, based on the variances, each study was weighed according to the fixed effect model and the inverse standard error. After obtaining the weight of each study, the proportions were obtained using within and between homogeneity techniques, and the agreement proportion between FISH and IHC in HER2 detection was estimated. Cochran test showed that there was significant heterogeneity between the results in different studies. Therefore, the random effects model was used for final analysis. To assess the factor/or factors causing heterogeneity in the results, sensitivity analysis and meta-regression model were used. To achieve this, the type of antibody used in the studies, years of study, location of study, publisher of the paper and quality of studies (as possible factors causing heterogeneity) were extracted from existing documents, and the metaregression model was adjusted. To indicate the meta-analysis results, forest plot was used. Possible publication bias was sought by a funnel plot and Egger’s test. Finally,

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Fig. 1 Selection of publication studies from the last decade, 2001–2011

analysis was performed using STATA statistical software (version 11).

Results We identified 172 citations, of which 124 remained after omission of duplicate articles (Fig. 1). Overall, there were 26 potentially relevant concordance/discordance studies between IHC and FISH for the detection of HER2 gene. Six were further excluded because either the scoring system in IHC/or FISH method was not clear or mentioned. Our inclusion criteria were met by 24 articles, representing 6629 cases (Fig. 1). Table 1 provides the characteristics of the studies to evaluate agreement between IHC and FISH for the detection of HER2 alteration. To compare concordance, false-positive and false-negative IHC with FISH results in detecting HER2 alteration, we divided the dataset into three categories: first, IHC staining with score 0/1? ; second, IHC staining scored 2?; third, IHC staining scored 3? . Then, we evaluated the

concordance of IHC staining score 3? with FISH for the detection of HER2 expression (representing 1434 patients), false-negative rate of IHC score 0/1? with FISH (representing 3958 patients), and also the proportion of FISH positive rate for IHC score 2? (representing 1346 patients). In other words, 1325 (19.99 %), 1346 (20.30 %), and 3958 (59.71 %) patients had IHC score 3? (positive), 2? (equivalent), and 0/1? (negative), respectively. Discordance between IHC and FISH results is defined as: IHC negative (0/1?) and FISH positive; or IHC positive (3?) and FISH negative. Discordance rates of approximately 4 % in 0/1? , and 9 % in 3? IHC scores were observed (Table 2). Among the 64 % of cases with IHC staining score 2?, FISH was negative. Part 1: patients with IHC staining with score 0/1? In the present study, the total of 3958 cases yielded a high concordance between IHC and FISH in the results for detection of HER2/-neu protein. The range of concordance

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Breast Cancer Table 1 Characteristics of 36 subsets of 24 publication studies to evaluate concordance between IHC and FISH for detection of HER2 protein Studies

Year

Country

Antibody

IHC score 0/1? compared to FISH No. of cases

Concordance (%)

S.E.a

IHC score 2? compared to FISH No. of cases

Proportion of FISH positive rate(%)b

S.E.a

IHC score 3? compared to FISH No. of cases

Concordance (%)

S.E.a

Lebeau et al. [83]

2001

Germany

TAB 250

–

–

–

3

0.666

0.187

18

1

0.061

Lebeau et al. [83]

2001

Germany

GSF

–

–

–

6

0.500

0.158

15

1

0.070

Lebeau et al. [83]

2001

Germany

A8010

–

–

–

9

0.222

0.128

18

1

0.061

Lebeau et al. [83]

2001

Germany

A0485

–

–

–

15

0.133

0.094

18

1

0.061

Lebeau et al. [83]

2001

Germany

CB11

–

–

–

11

0.818

0.114

11

1

0.088

Lebeau et al. [83]

2001

Germany

HerceptTest

–

–

–

14

0.143

0.098

18

Perez et al. [7]

2002

USA

HerceptTest

244

0.963

0.013

88

0.239

0.045

197

McCormick et al. [84]

2002

USA

HerceptTest

112

0.938

0.025

45

0.422

0.071

41

1

0.061

0.893

0.022

1

0.956

Dowsett et al. [6]

2003

UK

HerceptTest

270

0.993

0.007

54

0.481

0.066

102

0.941

0.027

Ellis et al. [85]

2005

UK

HerceptTest

71

0.944

0.031

21

0.333

0.096

20

0.900

0.076

Ainsworth et al. [86]

2005

UK

HerceptTest

140

0.914

0.025

13

0.077

0.092

12

0.917

0.098

Loring et al. [42]

2005

Ireland

HerceptTest

62

1

0.021

17

0

0.064

31

0.871

0.064

Dybdal et al. [87]

2005

USA

CTA

244

0.963

0.013

88

0.239

0.045

197

0.893

0.022

Sapino et al. [88]

2006

Italy

HerceptTest

176

0.994

0.009

36

0.278

0.072

25

0.920

0.064

Bergqvist et al. [89]

2006

Sweden

HerceptTest

–

–

–

11

0.454

0.129

20

0.950

0.067

91

0.912

0.031

131

0.931

0.023

–

–

Powell et al. [90]

2007

USA

4B5

Drev et al. [91]

2007

Slovenia

HerceptTest

Powell et al. [90]

2007

USA

CB11

58

0.828

0.050

–

903

0.977

0.005

85

85

0.906

0.033

–

–

–

0.259

0.047

–

–

–

Umemura [92]

2008

Japan

HerceptTest

73

1

0.018

45

0.444

0.071

62

1

0.021

Gilbert et al. [93]

2008

USA

HerceptTest

12

0.917

0.098

7

0.286

0.145

16

0.313

0.107

Vegt et al. [94]

2009

Netherlands

4B5

408

0.953

0.011

15

0.800

0.101

43

1

0.029

Vegt et al. [94]

2009

Netherland

CB11

394

0.969

0.009

40

0.575

0.075

39

1

0.032

Vogel et al. [99]

2010

Germany

HerceptTest

5

0.800

0.157

–

–

–

8

1

0.108

Rhodes et al. [95]

2010

UK

A0485

1

0.018

6

0.167

0.145

12

1

0.083

0.231

0.110

11

1

0.1

73

Rhodes et al. [95]

2010

UK

CB11

68

1

0.019

13

Rhodes et al. [95]

2010

UK

SP713

81

0.975

0.023

3

0.983

0.008

205

0.229

0.029

94

0.862

0.036

1

0.018

8

0.250

0.136

11

1

0.088

Grimm et al. [96]

2010

USA

HerceptTest

Rhodes et al. [95]

2010

UK

4B5

364 73

a

S.E. measures denote standard errors and were calculated based on Jeffery method [84]

b

The column shows the proportions of FISH positive rate in IHC score 2?

8

0.909

0.103

1

0.108

Table 2 Common discordance rate based on meta-analysis between IHC and FISH results for Her2/neu in breast cancer Immunohistochemistry (IHC)

Fluorescence in situ hybridization (FISH) Positive

Discordance rate (%)

135

3823

2? (equivalent)

393

953

953/1346 (70.80)a

135/3958 (3.41)

1299

26

26/1325 (1.96)

a

The proportion of FISH negative rate in IHC 2? (equivalent cases)

b

The proportion of FISH positive rate in IHC 2? score

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Concordance FISH positive rate (%)

Negative

0/1? (negative) 3? (positive)

Adjusted discordance/FISH positive rate (%)

4 64a 9

3823/3958 (96.59) 393/1346 (29.19)b 1299/1325 (98.04)

Adjusted concordance rate (%) 96 36b 91

Breast Cancer Fig. 2 Concordance measures (95 % CI) in published studies in the recent decade, from 2001 to 2011, between IHC staining with score 0/1? and FISH for detection of HER2 expression

between FISH and IHC staining score 0/1? among 21 subsets based on 16 studies was 67–99 %. A funnel plot and Cochran test showed noticeable asymmetry in the dataset (PQtest ¼ 0:001) and there was publication bias (Egger’s test P = 0.002). The smallest and the largest sample sizes were 5 and 903, respectively. Random effects meta-analysis of 21 subsets showed that the overall concordance between IHC (when IHC staining score was 0/1?) and FISH for detection failure of HER2 expression was 96 % (95 % CI 95–97 %); Fig. 2). Meta-regression on concordance rate shows that factors such as study location, publication date, and sample size were not significant for causing heterogeneity (P [ 0.05). The heterogeneity may be due to the type of antibody, including monoclonal and polyclonal antibodies (P = 0.001). Cumulative meta-analysis shows that there is no trend in the changes over time and there seems to be a uniform trend in the last decade. Figure 3 shows that overall false-negative rate of IHC staining with score 0/1? is 4 %. The lowest false negative was reported by Dowsett [6, 7]. The largest false negative was reported by Vogel [91, 99].

showed publication bias (P = 0.017). In this case, the smallest and the largest sample sizes were 3 and 205 cases, respectively. An initial random effect meta-analysis comprising 1346 cases yielded a low concordance between IHC and FISH for detecting HER2 expression in human breast cancer. Random effects meta-analysis of 25 subsets based on 16 published studies showed that using FISH, the detection of HER2 alteration in IHC 2? (equivalent) was 36 % (95 % CI 30–43 %; Fig. 4). Meta-regression on concordance rate showed that factors such as study location, publication date, and antibody type did not lead to heterogeneity (P \ 0.05). After meta-regression on concordance measures and publication date was considered, with type of antibody and studies’ sample size as covariates, I2 index decreased from 62.7 % (P \ 0.001) to 38.1 % (P = 0.002), yet there was publication bias. In this case, as in the previous case (part1), only the type of antibody was a significant cause of heterogeneity (P = 0.021).

Part 2: patients with IHC staining with score 2?

A meta-analysis including 1434 cases yielded a high concordance between IHC and FISH in the detection of HER2 protein. Concordance measure variations between IHC and FISH in 27 subsets based on 18 studies were 31–97 %. A

A funnel plot and Cochran test showed noticeable asymmetry in the dataset (PQtest ¼ 0:013), and also Egger’s test

Part 3: patients with IHC staining with score 3?

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Breast Cancer Fig. 3 False-negative measures (95 % CI) in published studies in the recent decade, from 2001 to 2011, between IHC staining with score 0/1? and FISH for detection of HER2 expression

Fig. 4 The proportions of FISH positive rate in IHC score 2? (95 % CI) in published studies from 2001 to 2011 for detecting HER2 expression

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Breast Cancer Fig. 5 Concordance measures (95 % CI) in published studies in the recent decade, from 2001 to 2011, between IHC staining with score 3? and FISH for detection of HER2 expression

funnel plot and Cochran test showed noticeable asymmetry in the dataset (PQtest ¼ 0:003) and there was no publication bias (Egger’s test P = 0.47). The smallest and the largest sample sizes were 8 and 197, respectively. Random effects meta-analysis of 27 subsets shows that the overall concordance between IHC (when IHC score is 3?) and FISH for the detection of HER2 expression was 91 % (95 % CI 89–93 %); Fig. 5). Overall false-positive rate in our study was 9 % in 3? IHC score. Meta-regression on concordance rate shows that factors such as study location, publication date, and antibody type did not produce heterogeneity (P \ 0.05).

Discussion Precise and accurate detection of HER2 gene expression is crucial in breast cancer to determine the future course of treatment. Before starting trastuzumab treatment, physicians must be sure of the accuracy of the test results that show HER2 overexpansion [36, 39, 84, 85]. Overall, we evaluated IHC results compared to FISH for the detection of HER2 gene expression among 6629 patients (Fig. 1). There have been conflicting results for the putative concordance between IHC and FISH. The results of previous studies on the agreement between IHC and FISH are still

controversial [6, 7, 42, 83–85, 87–99]. We have tried to find an overall concordance between them, especially for IHC staining with score 3?. True-positive results may be confounded by underlying population substructure or stage of breast cancer within studies and patients, respectively, especially when IHC staining score is 2?. Also, heterogeneity, in the manner by which a particular antibody variant or small sample size increases in discordance between both methods, may also diminish our ability to detect HER2 alteration. Even after minimizing some above discussed confounding factors including antibody, publication date, etc., our metaanalysis contains residual heterogeneity. To address this issue, we performed a meta-regression by considering sample size and antibody as covariates. Our data show that discordance is smaller in studies conducted with large sample size and CB11 or 4B5 antibody in IHC scoring 0/1? and 3?. The true positive of IHC may be more improved if a second immunohistochemical reaction such as 4B5 or HercepTest is performed with CB11, and genelevel extraction could be excluded only when both IHC reactions are positive. However, after adjustment for control antibody and sample size, unexplained heterogeneity remains, but the concordance between IHC and FISH is no longer significant when IHC staining scores are 0/1? and 3?.

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In this study, although for 1325 patients with IHC staining score 3? the overall concordance between both methods was 0.91, there does not appear to be a reasonable concordance. In other words, 9 percent of patients were false positive in IHC 3? score and might have been receiving expensive treatments. Most studies that have used the CB11 antibody in the IHC method have reported approximately 100 % agreement between IHC and FISH results for detection of HER2 alteration when IHC staining score is 3? (Table 1), while researchers who have applied other antibodies such as HerceptTest and 4B5 had \95 % concordance between HER2 alteration and IHC staining score 3? (Table 1). Although concordant IHC 3? and FISH results were satisfactory, we showed that IHC could provide false-positive (9 %) and false-negative (4 %) results as well, due to technical inconsistencies. In addition, the results in Table 1 show that when CB11 antibody was used in IHC, agreement between IHC and FISH was more than when using other antibodies for the detection of HER2 alteration. Such results have been reported in some previous publications [6, 86, 87]. In addition, Ainsworth et al. [86] showed that CBE356 provides a more accurate predictor of HER2 overexpression than HerceptTest. Undoubtedly, inaccurate diagnostics with IHC compared to FISH takes the form of false positives. False-positive diagnosis incorrectly ascribes a poorer prognosis and a failure to benefit from administered trastuzumab, which results in patient anxiety and disappointment as well as wasted resources. Given the level of adjusted discordance rates between FISH and IHC, 0/1? and 3? score were 4 and 9 %, respectively. In addition, the proportions of FISH negative rate in IHC score 2? (equivalent cases) was 64 %. Therefore, we propose that according to the current protocol used in the UK and some other countries, before prescribing trastuzumab, the application of FISH for detection of HER2 expression is recommended [47]. In FISH we can maneuver the quality and quantity of HER2 which is not possible by IHC due to two reasons [1, 7]: first, the copy number is not accessible by IHC and qualitative value of protein is under a remarkable reason due to exposure of protein to formaldehyde during process; second, copy number intensity of signal is reflective of the quality of HER2 protein. If we have more than 4 signals as polysomy, it does not necessarily mean that all additional gene copy numbers act similarly, but amplification has an influential rule on decision making for Herceptin therapy. Therefore, such problems will lead to an inappropriate therapy by Herceptin, providing target cells an overdose or underdose during the treatment. These are all related to the precise diagnosis of signal copy number as a quantitative target and amplification as a qualitative value, which are

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closely correlated to Herceptin therapy. This determines whether the technique is trustable and/or reliable. Conflicting results in previous studies may be due to the disease duration before diagnosis [6, 7, 42, 83–85, 87–99]. The causes of discrepancy in studies such as by Vogel [99] may be due to small sample size. In addition, an update of ASCO/CAP guidelines may be another reason for the discrepancies in studies during times. However, the use of diagnostic tests depends on the researches’ goal. If the goal is to reduce false-positive and false-negative rates to a very low level (\1 %), then IHC techniques alone are not to be utilized as the first-line test. In this case, we suggest using either CB11 with HerceptTest to detect HER2 overexpression or FISH test alone as the first-line test. If the researchers’ goal is breast cancer screening in the community or to obtain its prevalence, the IHC method will be suitable using antibody such as CB11. It should be noted that the IHC method is reliable for score staining 0/1? and 3? to detect HER2 overexpression. On the other hand, if clinicians have decided to recommend trastuzumab therapy, FISH is a valid and supplement method to HER2 overexpression rather than IHC. In the study we have found that CB11 and HerceptTest are incomplete to explore HER2 overexpression. Therefore, we suggest the following algorithm to explore HER2 overexpression among breast cancer cases. First, all suspicious cases in clinical must go through a conventional IHC staining with the CB11 antibody to provide a preliminary result. Second, immunohistochemical reaction, HerceptTest, and FISH are performed on cases’ slides. If in both, IHC is concordant with FISH, we have our final results. If one or both IHC reactions did not show concordance with FISH, large slide FISH is performed, and the results are considered as final. Finally, the concordance or discordance of results between two complementary tests (FISH, IHC) depends on the following points: 1. 2.

Adequate number of cells in the analysis in different groups of cell populations in the same slide. As a matter of fact, IHC reflects the expression of tissue and FISH reflects the copy number, intensity, and amplification of HER2.

Conclusion The results of this study show that IHC scores 0/1? and 3? cannot be completely considered as negative and positive breast cancer test, respectively. Immunohistochemical tests to explore HER2 overexpression may have false-positive and false-negative results. Our data show that concordance is greater in studies conducted with CB11 or HerceptTest antibody. Therefore, sensitivity/specificity of

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IHC may be more improved if a second immunohistochemical reaction such as HerceptTest is performed. Complementary test for IHC may provide negative or positive results, then an approval test is needed. Acknowledgments We thank the editorial boards and reviewers for editorial assistance with this manuscript. Conflict of interest P. Mehdipour, F. Bahreini, and A. R. Soltanian have nothing to disclose.

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