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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 4  |  Page : 730-734

Human epidermal growth factor receptor 2 amplification detection by droplet digital polymerase chain reaction in formalin-fixed paraffin-embedded breast and gastric cancer samples


1 Cancer Center, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, Shandong, China
2 Department of Ophthalmology, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, Shandong, China
3 Clinical Skill Training Center, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, Shandong, China
4 Department of Otorhinolaryngology, Center for Radiation Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, Shandong, China
5 Department of Bone and Joint Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, Shandong, China

Date of Web Publication13-Sep-2017

Correspondence Address:
Hong Feng
Cancer Center, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, 324 Jingwu Road, Jinan, Shandong 250021
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_587_17

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 > Abstract 

Objective: Human epidermal growth factor receptor 2 (HER2) is an important biomarker for the precise individualized treatment including trastuzumab of HER2-positive breast and gastric cancer. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the routine analyses for formalin-fixed paraffin-embedded (FFPE) samples. However, IHC is variable and depends on the evaluator, and FISH is a labor intensive and expensive method. We evaluated the feasibility of droplet digital polymerase chain reaction (ddPCR) as a precise and quantitative method for HER2 amplification test.
Materials and Methods: We used ddPCR to confirm HER2 amplification status in 24 breast cancer and 29 gastric cancer samples to validate the HER2 cutoff value in ddPCR. After setting cutoff value, all the above-mentioned samples were tested by IHC. Afterward, another 51 equivocal IHC 2+ gastric cancer samples were further determined by FISH and ddPCR, respectively, and the concordance between ddPCR and FISH was calculated.
Results: We set the HER2 cutoff value at 1.8. The concordance rate of HER2 status between ddPCR and IHC was 94.4% (17 out of 18) in 24 breast cancer samples. In 29 gastric cancer specimens, the concordance rate of HER2 amplification between ddPCR and IHC was 100% (22 out of 22). At last, compared with FISH determined HER2 status, ddPCR HER2 scores correctly classified 44 of 51 cases with 86.3% concordance in 51 equivocal IHC 2+ gastric cancer samples.
Conclusions: ddPCR was able to identify HER2 amplification status in breast and gastric cancers with precise correlation with IHC and FISH results. This method might become a standard method for testing FFPE samples. However, the technology requires further research.

Keywords: Breast cancer, droplet digital polymerase chain reaction, fluorescence in situ hybridization, gastric cancer, human epidermal growth factor receptor 2, immunohistochemistry


How to cite this article:
Wang X, Wu Y, Song X, Sun C, Wu C, Feng H. Human epidermal growth factor receptor 2 amplification detection by droplet digital polymerase chain reaction in formalin-fixed paraffin-embedded breast and gastric cancer samples. J Can Res Ther 2017;13:730-4

How to cite this URL:
Wang X, Wu Y, Song X, Sun C, Wu C, Feng H. Human epidermal growth factor receptor 2 amplification detection by droplet digital polymerase chain reaction in formalin-fixed paraffin-embedded breast and gastric cancer samples. J Can Res Ther [serial online] 2017 [cited 2018 Aug 16];13:730-4. Available from: http://www.cancerjournal.net/text.asp?2017/13/4/730/214480


 > Introduction Top


Human epidermal growth factor receptor 2 (HER2) overexpression is detected in 15%–25% of breast tumors and associated with higher rate of recurrence and mortality in patients with breast cancer.[1],[2] Moreover, HER2 is overexpressed in 6%–35% of gastric cancer,[3] which is also a key driver of tumorigenesis and a treatment target in patients with gastric cancer.[4],[5],[6] Both breast and advanced metastatic gastric cancer patients with HER2 amplification could benefit from trastuzumab, which is a humanized monoclonal antibody, interfering with the HER2/neu receptor.[2],[7] Thus, the accurate assessment of HER2 plays an important role in the diagnosis and treatment.[8]

Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the recommended standard analyses for formalin-fixed paraffin-embedded (FFPE) samples in breast and gastric cancers.[9],[10] However, IHC and FISH results are based on a subjective determination of the evaluator. de Melo Rêgo et al. reported that IHC is a semiquantitative method due to its subjective measurement of the intensity of the color reaction.[11] In addition, the staining sites in cells are irregular because of intratumoral heterogeneity.[3]

Droplet digital polymerase chain reaction (ddPCR) is an objective nucleic acid quantitative technique with unparalleled sensitivity and absolute quantification.[12] Belmonte et al. reported that ddPCR improves the detection sensitivity due to the reduction of background signal by increasing the signal-to-noise ratio.[13] The aim of this study was to investigate HER2 amplification using ddPCR in patients with breast and gastric carcinomas. In addition, we compared the results of ddPCR with IHC and FISH performed on the same samples. Our results suggested that, as an objective, sensitive, and absolute quantitative assessment, ddPCR might represent a new noninvasive method for HER2 evaluation.


 > Materials and Methods Top


Patients

Biopsy tissues were collected from 24 to 29 consecutive patients with breast and gastric cancer who were treated between April 1, 2011, and August 31, 2013, in Shandong Provincial Hospital affiliated to Shandong University. Of these 24 and 29 subjects, 24 were female and 0 were male in breast cancer patients, and 18 were female and 11 were male in 29 gastric cancer patients, and the median age was 65 and 58 years, respectively (range 51–85 and 50–72). Tissues were formalin-fixed and paraffin-embedded. We carried out our research in two steps. First, we compared ddPCR and IHC in 24 breast cancer and 29 gastric cancer FFPE samples. The aim of the first step is to evaluate whether ddPCR is a feasible technology for testing HER2 amplification and to identify a proper cutoff value for ddPCR. In the second step, we evaluated the concordance between ddPCR and FISH results in another 51 equivocal IHC 2+ gastric cancer FFPE samples. The research procedures were performed with the consent of the patients and ethics committee approval.

Immunohistochemistry and fluorescencein situ hybridization

HER2 IHC and FISH analyses were performed by two senior pathologists of Shandong Provincial Hospital affiliated to Shandong University according to the Recommendations for HER2 Testing in Breast Cancer: ASCO/CAP Guideline Update and HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma.[9],[14] IHC was performed in 24 breast cancer and 29 gastric cancer FFPE samples. The other 51 equivocal IHC 2+ gastric cancer FFPE samples were tested by FISH to identify HER2 status. Scores of IHC 0, IHC 1+, and IHC 2+/FISH negative are negative for HER2, whereas IHC 2+/FISH positive and IHC 3+ are positive for HER2.[3]

DNA isolation

DNA was isolated from breast and gastric cancer FFPE samples using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. DNA was eluted in 50 μL nuclease-free water and stored at −20°C until further use. Purified DNA was quantified with Qubit 2.0 Fluorometer (Life Technologies).

Droplet digital polymerase chain reaction

The copy number variants' analysis was carried out using the HER2 Gene Amplification Kit (QuestGenomics, Nanjing, China) on the Digital PCR System IA-1 (QuestGenomics, Nanjing, China). PCR was run on the TC-EA-4IA PCR System (Bioer) with 96°C for 10 min followed by 39 cycles of 60°C for 2 min, 98°C for 30 s, followed by 2 min at 60°C. Subsequently, the signals were detected by the Digital PCR System IA-1 instrument. RNase P is used as the control gene in ddPCR.

Data analysis

Raw data from the Digital PCR System IA-1 Instrument were transferred to the Digital PCR Analysis Software (Questgenomics, Nanjing, China), and data analysis was performed following the manufacturer's instructions. The reference gene and HER2 gene were identified using VIC and FAM dyes, respectively. The ratio of HER2 gene normalized by reference gene was equally calculated by FAM/VIC. In the ultimate report, we listed the ratio, whereas in the ratio >1.8, the sample was classified as positive.


 > Results Top


Setting a cutoff value for digital polymerase chain reaction

We confirmed HER2 gene amplification status in 24 breast cancer samples and 29 gastric cancer samples by digital PCR to validate the ddPCR assay for FAM/VIC ratio. The median HER2 ratio (FAM/VIC) of the breast cancer samples was 1.32 (range 0.78–11.24), whereas the median HER2 ratio of the gastric cancer samples was 1.37 (range 1.03–11.7). According to the above-mentioned data analysis, 1.8 of FAM/VIC was identified as the HER2 cutoff value, and FAM/VIC >1.8 was considered as HER2 amplified for both types of carcinoma specimens. Zhu et al. set the HER2 cutoff value at 1.66 in both breast and gastric cancer samples,[15] which was in accordance with our results.

The concordance of droplet digital polymerase chain reaction to immunohistochemistry results

To verify the concordance between ddPCR and IHC, we first test 24 breast cancer samples with IHC and ddPCR respectively. Representative images of IHC are shown in [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d. Our results demonstrated that 10 samples were 0/+, 6 were 2+, and 8 were 3+ in IHC, while the ddPCR showed that 9 patients were HER2 positive and 15 patients were HER2 negative. Seven of eight IHC-positive samples also scored positive by ddPCR and 10 of 10 IHC-negative samples also scored negative by ddPCR. HER2 status in breast cancer samples with ddPCR compared with IHC is summarized in [Table 1]. The concordance rate of HER2 status between ddPCR and IHC was 94.4% (17/18) in breast cancer.
Figure 1: Immunohistochemical staining and fluorescence in situ hybridization results of human epidermal growth factor receptor 2 in breast and gastric cancer tissues. Representative images of human epidermal growth factor receptor 2 immunohistochemical staining 0 (a), 1+ (b), 2+ (c), 3+ (d) in breast cancer. Representative images of human epidermal growth factor receptor 2 fluorescence in situ hybridization negative (e) and fluorescence in situ hybridization positive (f) in gastric cancer (a-d: ×200; e and f: ×400)

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Table 1: Human epidermal growth factor receptor 2 status in breast cancer samples with droplet digital polymerase chain reaction compared with immunohistochemistry

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We next evaluated 29 gastric cancer samples with IHC and ddPCR, respectively. HER2 status in gastric cancer samples with ddPCR compared with IHC is shown in [Table 2]. The concordance rate of HER2 amplification between ddPCR and IHC was 100% (22/22) in gastric cancer.
Table 2: Human epidermal growth factor receptor 2 status in gastric cancer samples with droplet digital polymerase chain reaction compared with immunohistochemistry

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The concordance of droplet digital polymerase chain reaction to fluorescencein situ hybridization results

We selected another gastric cancer samples with HER2 2+ of IHC score to investigate the concordance between ddPCR and FISH. Representative images of FISH results are shown in [Figure 1]e and [Figure 1]f. Fifty-one equivocal IHC 2+ gastric cancer samples were further tested with FISH and ddPCR, respectively. HER2 status in IHC 2+ gastric cancer samples with ddPCR compared with FISH is listed in [Table 3]. Compared with FISH results, ddPCR HER2 scores were correctly classified 44 of 51 cases with 86.3% concordance.
Table 3: Human epidermal growth factor receptor 2 status in immunohistochemistry 2+ gastric cancer samples with droplet digital polymerase chain reaction compared with fluorescence in situ hybridisation

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 > Discussion Top


Precise individualized treatment including trastuzumab, monoclonal antibody that interferes with the HER2/neu receptor, is a novel goal for breast and gastric cancer,[16],[17] which depends on the detection of HER2 genetic amplification. The clinical applications of IHC and FISH are limited within the expertise of a seasoned pathologist to make the determination that is prone to significant variation and error between laboratories.[2] The ddPCR could potentially offer a quantitative method for determination of HER2 status that is less sensitive to the judgment of the practitioner and to the laboratory in which it is performed.[18] However, the relationship between ddPCR and IHC/FISH in HER2 detection has not been elucidated.

In this study, we demonstrated that ddPCR is a useful method for evaluating HER2 expression status in FFPE samples of breast and gastric cancers. We calculated the concordance rate between ddPCR and IHC based on 24 breast samples and 29 gastric samples. The concordance rate of HER2 amplification between ddPCR and IHC was 100% and 94.4% in gastric and breast cancer, respectively. In another validation test of 51 IHC 2+ gastric samples, ddPCR was 86.3% concordant with FISH. According to the high concordance with IHC and/or FISH defined HER2 status, ddPCR may be useful as an alternative to IHC/FISH, although this technique requires further research.

The ddPCR is a form of digital PCR with reaction chambers separated by carefully titrated emulsion of oil, water, and stabilizing chemicals,[19] which is capable of detecting rare mutation, and performing absolute quantification of gene copy number and gene expression analysis.[20] The ddPCR has been used in cancer research, mutation detection, and copy number variation studies due to its advantages of high sensitivity, precision, and reproducibility.[21] The method of ddPCR with more accuracy and reproducibility of nucleic acid targets is done based on Poisson algorithm, which needs no endogenous control or standard curve.[22] The other advantages of ddPCR are easy to perform and time-saving compared with FISH which is time-consuming and complicated.[23]

For the intratumoral heterogeneity of FFPE samples, false-negative results are the main drawback of ddPCR. In our study, FISH verified 14 IHC 2+ gastric cancer samples as HER2 positive, while ddPCR tested 13 of 14 as HER2 positive and the rest one was negative. The accuracy of ddPCR results depends on the purity of the DNA extracted from the samples.[24] Hence, microdissection of FFPE samples is necessary. The microdissection techniques have developed from the use of a scalpel blade to the use of micromanipulators and laser microdissection (LMD) within a few years, which could overcome heterogeneity of tissue in their composition.[25] With the development of technology, LMD can be readily used to precisely isolate small numbers of cells from FFPE tissue sections.[26] For those complicated cases, LMD could be used in ddPCR test.

There is not a uniform standard of cutoff value in ddPCR. Gevensleben et al. set the HER2 cutoff value of ddPCR at 1.25 in breast cancer samples.[27] In another clinical research, investigators evaluated HER2 amplification in 25 gastric cancer FFPE samples by ddPCR and set the HER2 cutoff value at 1.2.[28] In a similar study, the authors set the HER2 cutoff value at 1.66 in 145 clinical breast and gastric carcinoma cases who received ddPCR test.[15] In our study, 1.8 of FAM/VIC was identified as the HER2 cutoff value. Among 14 FISH HER2-positive gastric cancer samples, 1 of these 14 samples evaluated by ddPCR was HER2 negative, whose cutoff value is 1.47. Therefore, further researches are required for a proper cutoff value.[29],[30] Our results are in accordance with the study by Zhu et al.,[15] and we further analyzed IHC 2+ with ddPCR as a subgroup to demonstrate that ddPCR is a reasonable test for this obscure IHC 2+ group.


 > Conclusions Top


The ddPCR method was able to identify HER2 amplification status in breast and gastric carcinomas with excellent correlation with IHC and FISH results. Moreover, it might become a standard method for testing FFPE samples.

Acknowledgment

We are grateful to department of pathology in Shandong Provincial Hospital for providing us the assistance with immunostaining and fluorescence in situ hybridisation.

Financial support and sponsorship

This work was supported by grant to Hong Feng from the National Natural Science Foundation of China (81201865) and by grant to Xingwen Wang from the Shandong Science and Technology Development Planning (2012G0021822).

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

1.
Moelans CB, de Weger RA, Van der Wall E, van Diest PJ. Current technologies for HER2 testing in breast cancer. Crit Rev Oncol Hematol 2011;80:380-92.  Back to cited text no. 1
    
2.
Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 2007;25:118-45.  Back to cited text no. 2
    
3.
Rüschoff J, Hanna W, Bilous M, Hofmann M, Osamura RY, Penault-Llorca F, et al. HER2 testing in gastric cancer: A practical approach. Mod Pathol 2012;25:637-50.  Back to cited text no. 3
    
4.
Gravalos C, Jimeno A. HER2 in gastric cancer: A new prognostic factor and a novel therapeutic target. Ann Oncol 2008;19:1523-9.  Back to cited text no. 4
    
5.
Liu X, Xu P, Qiu H, Liu J, Chen S, Xu D, et al. Clinical utility of HER2 assessed by immunohistochemistry in patients undergoing curative resection for gastric cancer. Onco Targets Ther 2016;9:949-58.  Back to cited text no. 5
    
6.
Yuan CL, Li L, Zhou X, Liz H, Han L. Expression of SATB1 and HER2 in gastric cancer and its clinical significance. Eur Rev Med Pharmacol Sci 2016;20:2256-64.  Back to cited text no. 6
    
7.
Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): A phase 3, open-label, randomised controlled trial. Lancet 2010 28;376:687-97.  Back to cited text no. 7
    
8.
Lee S, Yang W, Lan KH, Sellappan S, Klos K, Hortobagyi G, et al. Enhanced sensitization to taxol-induced apoptosis by herceptin pretreatment in ErbB2-overexpressing breast cancer cells. Cancer Res 2002;62:5703-10.  Back to cited text no. 8
    
9.
Bartley AN, Washington MK, Ventura CB, Ismaila N, Colasacco C, Benson AB 3rd, et al. HER2 testing and clinical decision making in gastroesophageal adenocarcinoma: Guideline from the college of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology. Am J Clin Pathol 2016;146:647-69.  Back to cited text no. 9
    
10.
Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med 2014;138:241-56.  Back to cited text no. 10
    
11.
de Melo Rêgo MJ, Cordeiro MF, Cavalcanti Cde L, de Carvalho Junior LB, Beltrão EI. Immunohistochemiluminescence detection: A quantitative tool in breast cancer HER-2 status evaluation. Dis Markers 2013;34:373-7.  Back to cited text no. 11
    
12.
Huggett JF, Whale A. Digital PCR as a novel technology and its potential implications for molecular diagnostics. Clin Chem 2013;59:1691-3.  Back to cited text no. 12
    
13.
Belmonte FR, Martin JL, Frescura K, Damas J, Pereira F, Tarnopolsky MA, et al. Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions. Sci Rep 2016;6:25186.  Back to cited text no. 13
    
14.
Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 2013;31:3997-4013.  Back to cited text no. 14
    
15.
Zhu Y, Lu D, Lira ME, Xu Q, Du Y, Xiong J, et al. Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples. Exp Mol Pathol 2016;100:287-93.  Back to cited text no. 15
    
16.
Fasching PA, Brucker SY, Fehm TN, Overkamp F, Janni W, Wallwiener M, et al. Biomarkers in patients with metastatic breast cancer and the PRAEGNANT Study Network. Geburtshilfe Frauenheilkd 2015;75:41-50.  Back to cited text no. 16
    
17.
Narita Y, Muro K. Challenges in molecular targeted therapy for gastric cancer: Considerations for efficacy and safety. Expert Opin Drug Saf 2017;16:319-27.  Back to cited text no. 17
    
18.
Heredia NJ, Belgrader P, Wang S, Koehler R, Regan J, Cosman AM, et al. Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples. Methods 2013;59:S20-3.  Back to cited text no. 18
    
19.
Manoj P. Droplet digital PCR technology promises new applications and research areas. Mitochondrial DNA A DNA Mapp Seq Anal 2016;27:742-6.  Back to cited text no. 19
    
20.
Ye W, Tang X, Liu C, Wen C, Li W, Lyu J. Accurate quantitation of circulating cell-free mitochondrial DNA in plasma by droplet digital PCR. Anal Bioanal Chem 2017;409:2727-35.  Back to cited text no. 20
    
21.
Roberts CH, Last A, Molina-Gonzalez S, Cassama E, Butcher R, Nabicassa M, et al. Development and evaluation of a next-generation digital PCR diagnostic assay for ocular Chlamydia trachomatis infections. J Clin Microbiol 2013;51:2195-203.  Back to cited text no. 21
    
22.
Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A 1999;96:9236-41.  Back to cited text no. 22
    
23.
Nitta H, Hauss-Wegrzyniak B, Lehrkamp M, Murillo AE, Gaire F, Farrell M, et al. Development of automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence in situ hybridization (FISH). Diagn Pathol 2008;3:41.  Back to cited text no. 23
    
24.
O'Kane SL, Garimella V, Sivarajasingham N, Drew PJ, Cawkwell L. Direct polymerase chain reaction amplification of formalin-fixed, paraffin-wax-embedded tissue after automated sequential laser microdissection and pressure catapulting. J Clin Pathol 2007;60:216-7.  Back to cited text no. 24
    
25.
Okuducu AF, Hahne JC, Von Deimling A, Wernert N. Laser-assisted microdissection, techniques and applications in pathology (review). Int J Mol Med 2005;15:763-9.  Back to cited text no. 25
    
26.
Sho S, Court CM, Kim S, Braxton DR, Hou S, Muthusamy VR, et al. Digital PCR improves mutation analysis in pancreas fine needle aspiration biopsy specimens. PLoS One 2017;12:e0170897.  Back to cited text no. 26
    
27.
Gevensleben H, Garcia-Murillas I, Graeser MK, Schiavon G, Osin P, Parton M, et al. Noninvasive detection of HER2 amplification with plasma DNA digital PCR. Clin Cancer Res 2013;19:3276-84.  Back to cited text no. 27
    
28.
Kinugasa H, Nouso K, Tanaka T, Miyahara K, Morimoto Y, Dohi C, et al. Droplet digital PCR measurement of HER2 in patients with gastric cancer. Br J Cancer 2015;112:1652-5.  Back to cited text no. 28
    
29.
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 Cancer Res Ther 2016;12:1036-9.  Back to cited text no. 29
    
30.
Rohilla M, Bal A, Singh G, Joshi K. Prediction of heterogeneity in breast cancer immunophenotype at ductal carcinoma in situ stage? J Cancer Res Ther 2016;12:1249-56.  Back to cited text no. 30
    


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