Subscribe to RSS
![](/products/assets/desktop/img/oa-logo.png)
DOI: 10.4103/sajc.sajc_344_18
Droplet digital polymerase chain reaction offers an improvisation over conventional immunohistochemistry and fluorescent in situ hybridization for ascertaining Her2 status of breast cancer
Financial support and sponsorship Nil.![](https://www.thieme-connect.de/media/10.1055-s-00049561/201904/lookinside/thumbnails/10-4103_sajc-sajc_344_18_203-1.jpg)
Abstract
Background: Droplet digital polymerase chain reaction (DDPCR) is a recent modality for detecting Her2 expression which is quantitative, cheaper, easier to standardize, and free from interobserver variation. Purpose: The purpose of this study is to incorporate DDPCR in the current diagnostic paradigm with clinical benefit. Materials and Methods: Fifty-four consecutive patients were tested by immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and DDPCR. With FISH result as gold standard, receiver operating characteristic curves for DDPCR ratio were analyzed to label Her2-negative, equivocal, and positive cases as DDPCR score 1, 2, and 3, respectively. Proportion of patients labeled unequivocally as Her2 positive or negative was defined to have “clinically benefitted” from the test. Drawing parallel to inter-relationships between DDPCR, IHC, and FISH in the test cohort, four diagnostic pathways were defined – (1) initial IHC followed by FISH, (2) initial DDPCR followed by FISH, (3) initial IHC followed by DDPCR followed by FISH, and (4) initial DDPCR followed by IHC followed by FISH. Results: Clinical benefit of DDPCR as an initial test in the test cohort was 57%, while it was 65% if used as a second-line test among those with an initial inconclusive IHC result. Sensitivity analysis in the simulation cohort revealed that if DDPCR cost was ≤0.6 times the cost of IHC, then a three-step pathway with DDPCR upfront would near certainly prove most cost beneficial. If DDPCR cost was >0.6 but ≤2 times the cost of IHC, then a three-step pathway with DDPCR as second-line test had a higher probability to prove most cost beneficial. If DDPCR cost was >2 times the cost of IHC, then conventional pathway had a higher probability to prove most cost-effective. Conclusion: Incorporating DDPCR in the current clinical diagnostic paradigm has the potential to improve its cost-effectiveness and benefit.
Key words
Breast cancer - droplet digital polymerase chain reaction - fluorescent in situ hybridization - formalin fixed paraffin embedded - Her2 - immunohistochemistry - RNAPublication History
Article published online:
14 December 2020
© 2019. MedIntel Services Pvt Ltd. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/.)
Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India
-
References
- 1 Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359-86.
- 2 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.
- 3 Dawood S, Broglio K, Buzdar AU, Hortobagyi GN, Giordano SH. Prognosis of women with metastatic breast cancer by HER2 status and trastuzumab treatment: An institutional-based review. J Clin Oncol 2010;28:92-8.
- 4 Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF. Guidelines for human epidermal growth factor receptor 2 testing: Biologic and methodologic considerations. J Clin Oncol 2009;27:1323-33.
- 5 O'Hurley G, Sjöstedt E, Rahman A, Li B, Kampf C, Pontén F, et al. Garbage in, garbage out: A critical evaluation of strategies used for validation of immunohistochemical biomarkers. Mol Oncol 2014;8:783-98.
- 6 Savola S, Nardi F, Scotlandi K, Picci P, Knuutila S. Microdeletions in 9p21.3 induce false negative results in CDKN2A FISH analysis of Ewing sarcoma. Cytogenet Genome Res 2007;119:21-6.
- 7 Gown AM. Current issues in ER and HER2 testing by IHC in breast cancer. Mod Pathol 2008;21 Suppl 2:S8-15.
- 8 Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707-12.
- 9 Park S, Wang HY, Kim S, Ahn S, Lee D, Cho Y, et al. Quantitative RT-PCR assay of HER2 mRNA expression in formalin-fixed and paraffin-embedded breast cancer tissues. Int J Clin Exp Pathol 2014;7:6752-9.
- 10 Nitta H, Kelly BD, Padilla M, Wick N, Brunhoeber P, Bai I, et al. A gene-protein assay for human epidermal growth factor receptor 2 (HER2): Brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections. Diagn Pathol 2012;7:60.
- 11 Perez EA, Cortés J, Gonzalez-Angulo AM, Bartlett JM. HER2 testing: Current status and future directions. Cancer Treat Rev 2014;40:276-84.
- 12 Perez EA, Reinholz MM, Hillman DW, Tenner KS, Schroeder MJ, Davidson NE, et al. HER2 and chromosome 17 effect on patient outcome in the N9831 adjuvant trastuzumab trial. J Clin Oncol 2010;28:4307-15.
- 13 de Cremoux P, Martin EC, Vincent-Salomon A, Dieras V, Barbaroux C, Liva S, et al. Quantitative PCR analysis of c-erb B-2 (HER2/neu) gene amplification and comparison with p185(HER2/neu) protein expression in breast cancer drill biopsies. Int J Cancer 1999;83:157-61.
- 14 Murthy SK, Magliocco AM, Demetrick DJ. Copy number analysis of c-erb-B2 (HER-2/neu) and topoisomerase IIalpha genes in breast carcinoma by quantitative real-time polymerase chain reaction using hybridization probes and fluorescence in situ hybridization. Arch Pathol Lab Med 2005;129:39-46.
- 15 Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 2011;83:8604-10.
- 16 Hayden RT, Gu Z, Ingersoll J, Abdul-Ali D, Shi L, Pounds S, et al. Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus. J Clin Microbiol 2013;51:540-6.
- 17 Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S, et al. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res 2012;40:e82.
- 18 Meehan K, Clynick B, Mirzai B, Maslen P, Harvey JM, Erber WN, et al. HER2 mRNA transcript quantitation in breast cancer. Clin Transl Oncol 2017;19:606-15.
- 19 Suryavanshi M, Mehta A, Jaipuria J, Kumar D, Vishwakarma G, Panigrahi MK, et al. Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients. Breast Cancer 2018;25:416-30.
- 20 Lih CJ, Si H, Das B, Harrington RD, Harper KN, Sims DJ, et al. Certified DNA reference materials to compare HER2 gene amplification measurements using next-generation sequencing methods. J Mol Diagn 2016;18:753-61.
- 21 Doval DC, Sharma A, Sinha R, Kumar K, Dewan AK, Chaturvedi H, et al. Immunohistochemical profile of breast cancer patients at a tertiary care hospital in New Delhi, India. Asian Pac J Cancer Prev 2015;16:4959-64.
- 22 Shah MV, Wiktor AE, Meyer RG, Tenner KS, Ballman KV, Green SJ, et al. Change in pattern of HER2 fluorescent in situ hybridization (FISH) results in breast cancers submitted for FISH testing: Experience of a reference laboratory using US food and drug administration criteria and American Society of Clinical Oncology and College of American Pathologists guidelines. J Clin Oncol 2016;34:3502-10.
- 23 Dendukuri N, Khetani K, McIsaac M, Brophy J. Testing for HER2-positive breast cancer: A systematic review and cost-effectiveness analysis. CMAJ 2007;176:1429-34.
- 24 Müller BM, Kronenwett R, Hennig G, Euting H, Weber K, Bohmann K, et al. Quantitative determination of estrogen receptor, progesterone receptor, and HER2 mRNA in formalin-fixed paraffin-embedded tissue – A new option for predictive biomarker assessment in breast cancer. Diagn Mol Pathol 2011;20:1-10.
- 25 Ginestier C, Charafe-Jauffret E, Penault-Llorca F, Geneix J, Adélaïde J, Chaffanet M, et al. Comparative multi-methodological measurement of ERBB2 status in breast cancer. J Pathol 2004;202:286-98.
- 26 Gjerdrum LM, Sorensen BS, Kjeldsen E, Sorensen FB, Nexo E, Hamilton-Dutoit S, et al. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma: An alternative method for HER-2/neu analysis. J Mol Diagn 2004;6:42-51.
- 27 Kulka J, Tôkés AM, Kaposi-Novák P, Udvarhelyi N, Keller A, Schaff Z, et al. Detection of HER-2/neu gene amplification in breast carcinomas using quantitative real-time PCR – A comparison with immunohistochemical and FISH results. Pathol Oncol Res 2006;12:197-204.
- 28 Nistor A, Watson PH, Pettigrew N, Tabiti K, Dawson A, Myal Y, et al. Real-time PCR complements immunohistochemistry in the determination of HER-2/neu status in breast cancer. BMC Clin Pathol 2006;6:2.
- 29 Bergqvist J, Ohd JF, Smeds J, Klaar S, Isola J, Nordgren H, et al. Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome. Ann Oncol 2007;18:845-50.
- 30 Susini T, Bussani C, Marini G, Nori J, Olivieri S, Molino C, et al. Preoperative assessment of HER-2/neu status in breast carcinoma: The role of quantitative real-time PCR on core-biopsy specimens. Gynecol Oncol 2010;116:234-9.
- 31 Tvrdík D, Staněk L, Skálová H, Dundr P, Velenská Z, Povýšil C, et al. Comparison of the IHC, FISH, SISH and qPCR methods for the molecular diagnosis of breast cancer. Mol Med Rep 2012;6:439-43.
- 32 Olmedillas-López S, García-Arranz M, García-Olmo D. Current and emerging applications of droplet digital PCR in oncology. Mol Diagn Ther 2017;21:493-510.
- 33 Dabbs DJ, Klein ME, Mohsin SK, Tubbs RR, Shuai Y, Bhargava R. High false-negative rate of HER2 quantitative reverse transcription polymerase chain reaction of the oncotype DX test: An independent quality assurance study. J Clin Oncol 2011;29:4279-85.
- 34 Alexander EK, Kennedy GC, Baloch ZW, Cibas ES, Chudova D, Diggans J, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med 2012;367:705-15.
- 35 Wallden B, Storhoff J, Nielsen T, Dowidar N, Schaper C, Ferree S, et al. Development and verification of the PAM50-based prosigna breast cancer gene signature assay. BMC Med Genomics 2015;8:54.
- 36 Schwartz S, Motorin Y. Next-generation sequencing technologies for detection of modified nucleotides in RNAs. RNA Biol 2017;14:1124-37.
- 37 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.
- 38 Garcia-Murillas I, Lambros M, Turner NC. Determination of HER2 amplification status on tumour DNA by digital PCR. PLoS One 2013;8:e83409.
- 39 Belgrader P, Tanner SC, Regan JF, Koehler R, Hindson BJ, Brown AS, et al. Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue. Clin Chem 2013;59:991-4.
- 40 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.
- 41 Otsuji K, Sasaki T, Tanaka A, Kunita A, Ikemura M, Matsusaka K, et al. Use of droplet digital PCR for quantitative and automatic analysis of the HER2 status in breast cancer patients. Breast Cancer Res Treat 2017;162:11-8.
- 42 Wang X, Wu Y, Song X, Sun C, Wu C, Feng H, et al. 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 Cancer Res Ther 2017;13:730-4.
- 43 Wang Y, Tsang JY, Cui Y, Cui J, Lin Y, Zhao S, et al. Robust and accurate digital measurement for HER2 amplification in HER2 equivocal breast cancer diagnosis. Sci Rep 2017;7:6752.
- 44 Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 2012;490:61-70.
- 45 Gyrffy B, Hatzis C, Sanft T, Hofstatter E, Aktas B, Pusztai L, et al. Multigene prognostic tests in breast cancer: Past, present, future. Breast Cancer Res 2015;17:11.
- 46 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.
- 47 Taylor SC, Laperriere G, Germain H. Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: From variable nonsense to publication quality data. Sci Rep 2017;7:2409.
- 48 Baker M. Digital PCR hits its stride. Nat Methods 2012;9:541-4.
- 49 Carlson B. HER2 TESTS: How do we choose? Biotechnol Healthc 2008;5:23-7.