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BMC Cancer
Published in: BMC Cancer 1/2017

Open Access 01-12-2017 | Research article

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients

Authors: Joost H. van Ginkel, Manon M. H. Huibers, Robert J. J. van Es, Remco de Bree, Stefan M. Willems

Published in: BMC Cancer | Issue 1/2017

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Abstract

Background

During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR).

Methods

TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data.

Results

In all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2–422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667–156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%.

Conclusions

Our results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.
Appendix
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Literature
1.
Ferlito A, Partridge M, Brennan J, Hamakawa H. Lymph node micrometastases in head and neck cancer: a review. Acta Otolaryngol. 2001;121:660–5.CrossRefPubMed
2.
de Bree R, van der Putten L, Brouwer J, Castelijns JA, Hoekstra OS, Leemans CR. Detection of locoregional recurrent head and neck cancer after (chemo)radiotherapy using modern imaging. Oral Oncol. 2009;45:386–93.CrossRefPubMed
3.
Muller J, Hullner M, Strobel K, Huber GF, Burger IA, Haerle SK. The value of (18) F-FDG-PET/CT imaging in oral cavity cancer patients following surgical reconstruction. Laryngoscope. 2015;125:1861–8.CrossRefPubMed
4.
Gleber-Netto FO, Braakhuis BJ, Triantafyllou A, Takes RP, Kelner N, Rodrigo JP, et al. Molecular events in relapsed oral squamous cell carcinoma: Recurrence vs. secondary primary tumor. Oral Oncol. 2015;51:738–44.CrossRefPubMed
5.
Yom SS, Machtay M, Biel MA, Sinard RJ, El-Naggar AK, Weber RS, et al. Survival impact of planned restaging and early surgical salvage following definitive chemoradiation for locally advanced squamous cell carcinomas of the oropharynx and hypopharynx. American Journal of Clinical Oncology-Cancer Clinical Trials. 2005;28:385–92.
6.
Kalia M. Biomarkers for personalized oncology: recent advances and future challenges. Metabolism. 2015;64:S16–21.CrossRefPubMed
7.
Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients: Quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001;61:1659–65.PubMed
8.
9.
Nemunaitis J, Clayman G, Agarwala SS, Hrushesky W, Wells JR, Moore C, et al. Biomarkers predict p53 gene therapy efficacy in recurrent squamous cell carcinoma of the head and neck. Clin Cancer Res. 2009;15:7719–25.CrossRefPubMed
10.
Lui VW, Hedberg ML, Li H, Vangara BS, Pendleton K, Zeng Y, et al. Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers. Cancer Discov. 2013;3:761–9.CrossRefPubMedPubMedCentral
11.
Ndiaye C, Mena M, Alemany L, Arbyn M, Castellsague X, Laporte L, et al. HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis. Lancet Oncol. 2014;15:1319–31.CrossRefPubMed
12.
Koole K, Brunen D, van Kempen PM, Noorlag R, de Bree R, Lieftink C, et al. FGFR1 Is a Potential prognostic biomarker and therapeutic target in head and neck squamous cell carcinoma. Clin Cancer Res. 2016;22:3884–93.CrossRefPubMed
13.
Arantes LMRB, de Carvalho AC, Melendez ME, Carvalho AL, Goloni-Bertollo EM. Methylation as a biomarker for head and neck cancer. Oral Oncol. 2014;50:587–92.CrossRefPubMed
14.
Noorlag R, van Kempen PMW, Moelans CB, de Jong R, Blok LER, Koole R, et al. Promoter hypermethylation using 24-gene array in early head and neck cancer Better outcome in oral than in oropharyngeal cancer. Epigenetics. 2014;9:1220–7.CrossRefPubMedPubMedCentral
15.
Zhang M, Zhao LJ, Liang WQ, Mao ZP. Identification of microRNAs as diagnostic biomarkers in screening of head and neck cancer: a meta-analysis. Genet Mol Res. 2015;14:16562–76.CrossRefPubMed
16.
Boyle JO, Hakim J, Koch W, van der Riet P, Hruban RH, Roa RA, et al. The incidence of p53 mutations increases with progression of head and neck cancer. Cancer Res. 1993;53:4477–80.PubMed
17.
Nees M, Homann N, Discher H, Andl T, Enders C, Herold-Mende C, et al. Expression of mutated p53 occurs in tumor-distant epithelia of head and neck cancer patients: a possible molecular basis for the development of multiple tumors. Cancer Res. 1993;53:4189–96.PubMed
18.
Hedberg ML, Goh G, Chiosea SI, Bauman JE, Freilino ML, Zeng Y, et al. Genetic landscape of metastatic and recurrent head and neck squamous cell carcinoma. J Clin Invest. 2016;126:169–80.CrossRefPubMed
19.
Morris LG, Chandramohan R, West L, Zehir A, Chakravarty D, Pfister DG, et al. The Molecular landscape of recurrent and metastatic head and neck cancers: insights from a precision oncology sequencing platform. JAMA Oncol. 2016;
20.
Hiley C, de Bruin EC, McGranahan N, Swanton C. Deciphering intratumor heterogeneity and temporal acquisition of driver events to refine precision medicine. Genome Biol. 2014;15:453.CrossRefPubMedPubMedCentral
21.
van Ginkel JH, de Leng WW, de Bree R, van Es RJ, Willems SM. Targeted sequencing reveals TP53 as a potential diagnostic biomarker in the post-treatment surveillance of head and neck cancer. Oncotarget. 2016;
22.
Seiwert TY, Zuo ZX, Keck MK, Khattri A, Pedamallu CS, Stricker T, et al. Integrative and comparative genomic analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas. Clin Cancer Res. 2015;21:632–41.CrossRefPubMed
23.
Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, et al. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14:985–90.CrossRefPubMed
24.
Dawson SJ, Tsui DWY, Murtaza M, Biggs H, Rueda OM, Chin SF, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. 2013;368:1199–209.CrossRefPubMed
25.
Gray ES, Rizos H, Reid AL, Boyd SC, Pereira MR, Lo J, et al. Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma. Oncotarget. 2015;6:42008–18.PubMedPubMedCentral
26.
Reinert T, Scholer LV, Thomsen R, Tobiasen H, Vang S, Nordentoft I, et al. Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery. Gut. 2015;
27.
Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods. 2013;10:1003–5.CrossRefPubMedPubMedCentral
28.
de Leng WW, Gadellaa-van Hooijdonk CG, Barendregt-Smouter FA, Koudijs MJ, Nijman I, Hinrichs JW, et al. Targeted Next generation sequencing as a reliable diagnostic assay for the detection of somatic mutations in tumours using minimal DNA amounts from formalin fixed paraffin embedded material. PLoS One. 2016;11:e0149405.CrossRefPubMedPubMedCentral
29.
Hoogstraat M, Hinrichs JW, Besselink NJ, Radersma-van Loon JH, de Voijs CM, Peeters T, et al. Simultaneous detection of clinically relevant mutations and amplifications for routine cancer pathology. J Mol Diagn. 2015;17:10–8.CrossRefPubMed
30.
Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, et al. The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem. 2013;59:892–902.CrossRefPubMed
31.
32.
Fleischhacker M, Schmidt B. Circulating nucleic acids (CNAs) and cancer--a survey. Biochim Biophys Acta. 2007;1775:181–232.PubMed
33.
Rothwell DG, Smith N, Morris D, Leong HS, Li YY, Hollebecque A, et al. Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample. Mol Oncol. 2016;10:566–74.CrossRefPubMedPubMedCentral
34.
El Messaoudi S, Rolet F, Mouliere F, Thierry AR. Circulating cell free DNA: preanalytical considerations. Clin Chim Acta. 2013;424:222–30.CrossRefPubMed
35.
Swarup V, Rajeswari MR. Circulating (cell-free) nucleic acids - a promising, non-invasive tool for early detection of several human diseases. FEBS Lett. 2007;581:795–9.CrossRefPubMed
36.
Jung M, Klotzek S, Lewandowski M, Fleischhacker M, Jung K. Changes in concentration of DNA in serum and plasma during storage of blood samples. Clin Chem. 2003;49:1028–9.CrossRefPubMed
37.
Wang P, Jing F, Li G, Wu Z, Cheng Z, Zhang J, et al. Absolute quantification of lung cancer related microRNA by droplet digital PCR. Biosens Bioelectron. 2015;74:836–42.CrossRefPubMed
38.
Tang H, Cai Q, Li H, Hu P. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA. Biosci Biotechnol Biochem. 2016:1–6.
39.
Ruelle J, Yfantis V, Duquenne A, Goubau P. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification. J Int AIDS Soc. 2014;17:19675.CrossRefPubMedPubMedCentral
40.
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.CrossRefPubMedPubMedCentral
41.
Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2012;84:1003–11.CrossRefPubMed
42.
Wang Y, Springer S, Mulvey CL, Silliman N, Schaefer J, Sausen M, et al. Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas. Sci Transl Med. 2015;7:293ra104.CrossRefPubMedPubMedCentral
43.
Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014;6:224ra224.CrossRef
44.
Ignatiadis M, Lee M, Jeffrey SS. Circulating Tumor Cells and Circulating Tumor DNA: Challenges and Opportunities on the Path to Clinical Utility. Clin Cancer Res. 2015;21:4786–800.CrossRefPubMed
45.
Sozzi G, Roz L, Conte D, Mariani L, Andriani F, Verderio P, et al. Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays. J Natl Cancer Inst. 2005;97:1848–50.CrossRefPubMed
46.
Trypsteen W, Vynck M, De Neve J, Bonczkowski P, Kiselinova M, Malatinkova E, et al. ddpcRquant: threshold determination for single channel droplet digital PCR experiments. Anal Bioanal Chem. 2015;407:5827–34.CrossRefPubMed
47.
Devonshire AS, Whale AS, Gutteridge A, Jones G, Cowen S, Foy CA, et al. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem. 2014;406:6499–512.CrossRefPubMedPubMedCentral
48.
Nygaard AD, Holdgaard PC, Spindler KLG, Pallisgaard N, Jakobsen A. The correlation between cell-free DNA and tumour burden was estimated by PET/CT in patients with advanced NSCLC. Br J Cancer. 2014;110:363–8.CrossRefPubMed
49.
Coulet F, Blons H, Cabelguenne A, Lecomte T, Lacourreye O, Brasnu D, et al. Detection of plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis. Cancer Res. 2000;60:707–11.PubMed
50.
Heitzer E, Ulz P, Geigl JB. Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem. 2015;61:112–23.CrossRefPubMed
51.
Burrell RA, McGranahan N, Bartek J, Swanton C. The causes and consequences of genetic heterogeneity in cancer evolution. Nature. 2013;501:338–45.CrossRefPubMed
52.
Netherlands. Medical research involving human subjects act. Bull Med Ethics. 1999;No. 152:13–8.
53.
van Diest PJ. For and against - No consent should be needed for using leftover body material for scientific purposes - For. Br Med J. 2002;325:648–9.CrossRef
Metadata
Title
Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients
Authors
Joost H. van Ginkel
Manon M. H. Huibers
Robert J. J. van Es
Remco de Bree
Stefan M. Willems
Publication date
01-12-2017
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2017
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/s12885-017-3424-0

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