Journal of Cancer Research and Clinical Oncology

, Volume 143, Issue 7, pp 1199–1207 | Cite as

Non-reproducible sequence artifacts in FFPE tissue: an experience report

  • Richard Ofner
  • Cathrin Ritter
  • Selma Ugurel
  • Lorenzo Cerroni
  • Mathias Stiller
  • Thomas Bogenrieder
  • Flavio Solca
  • David Schrama
  • Jürgen C. Becker
Original Article – Cancer Research



Recent advances in sequencing technologies supported the development of molecularly targeted therapy in cancer patients. Thus, genomic analyses are becoming a routine part in clinical practice and accurate detection of actionable mutations is essential to assist diagnosis and therapy choice. However, this is often challenging due to major problems associated with DNA from formalin-fixed paraffin-embedded tissue which is usually the primary source for genetic testing.


Here we want to share our experience regarding major problems associated with FFPE DNA used for PCR-based sequencing as illustrated by the mutational analysis of ERBB4 in melanoma. We want to focus on two major problems including extensive DNA fragmentation and hydrolytic deamination as source of non-reproducible sequence artifacts. Further, we provide potential explanations and possible strategies to minimize these difficulties and improve the detection of targetable mutations.


Genomic DNA from formalin-fixed paraffin-embedded tumor samples was isolated followed by PCR amplification, Sanger sequencing and statistical analysis.


Analysis of Sanger sequencing data revealed a total of 46 ERBB4 mutations in 27 of 96 samples including the identification of 11 mutations at three previously unknown mutational hotspots. Unfortunately, we were not able to confirm any assumed hotspot mutation within repeated sequencing of relevant amplicons suggesting the detection of sequence artifacts most likely caused by DNA lesions associated with FFPE tissues.


Since DNA from FFPE tissue is usually the primary source for mutational analyses, appropriate measures must be implemented in the workflow to assess DNA damage in formalin-fixed tissue to ensure accurate detection of actionable mutations and minimize the occurrence of sequence artifacts.


Melanoma ERBB4 Sequencing artifacts Sanger sequencing FFPE 



We thank Isabella Fried for supporting this study, and Gerlinde Mayer and Ulrike Schmidbauer for excellent technical assistance (all Department of General Dermatology, Medical University Graz, Graz, Austria).

Compliance with ethical standards

Conflict of interest

The authors RO, CR, MS, LC, and DS state no conflict of interest. Author SU has received advisory board honorariums from Roche, BMS and Novartis. The authors TB and FS are employees of Boehringer Ingelheim. Author JCB has received speaker honorariums from Amgen, MerckSerono, and Pfizer, advisory board honorariums from Amgen, CureVac, Lytex, MerckSerono, Novartis, Rigontec, and Takeda as well as research funding from Boehringer Ingelheim, BMS and MerckSerono; none of these activities are related to the submitted report.

Ethics approval

The study was approved by the institutional review board of the Medical University of Graz (ethics votum 24-397 ex 11/12).

Informed consent

Informed consent was obtained from all individual participants included in the study.


This study was funded in part by a Research Grant of Boehringer Ingelheim RCV, Vienna, Austria.

Supplementary material

432_2017_2399_MOESM1_ESM.docx (20 kb)
Supplementary material 1 (DOCX 20 KB)


  1. Akbani R, Akdemir KC, Aksoy BA, Albert M, Ally A, Amin SB, Zou L (2015) Genomic classification of cutaneous melanoma. Cell 161(7):1681–1696. doi: 10.1016/j.cell.2015.05.044 CrossRefGoogle Scholar
  2. Akbari M, Doré Hansen M, Halgunset J, Skorpen F, Krokan HE (2005) Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. J Mol Diagn†¯ 7(1):36–39. Retrieved from
  3. Astolfi A, Urbini M, Indio V, Nannini M, Genovese CG, Santini D, Pantaleo MA (2015) Whole exome sequencing (WES) on formalin-fixed, paraffin-embedded (FFPE) tumor tissue in gastrointestinal stromal tumors (GIST). BMC Genom 16(1):892. doi: 10.1186/s12864-015-1982-6 CrossRefGoogle Scholar
  4. Berger MF, Hodis E, Heffernan TP, Deribe YL, Lawrence MS, Protopopov A, Garraway LA (2012) Melanoma genome sequencing reveals frequent PREX2 mutations. Nature 485(7399):502–506. doi: 10.1038/nature11071 PubMedPubMedCentralGoogle Scholar
  5. Betge J, Kerr G, Miersch T, Leible S, Erdmann G, Galata CL, Boutros M (2015) Amplicon sequencing of colorectal cancer: variant calling in frozen and formalin-fixed samples. PLoS One 10(5):e0127146. doi: 10.1371/journal.pone.0127146 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cai L, Yuan W, Zhang Z, He L, Chou K-C (2016). In-depth comparison of somatic point mutation callers based on different tumor next-generation sequencing depth data. Sci Rep 6:36540. doi: 10.1038/srep36540 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Carrick DM, Mehaffey MG, Sachs MC, Altekruse S, Camalier C, Chuaqui R, Schully SD (2015) Robustness of next generation sequencing on older formalin-fixed paraffin-embedded tissue. PLoS One 10(7):e0127353. doi: 10.1371/journal.pone.0127353 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen G, Mosier S, Gocke CD, Lin M-T, Eshleman JR (2014). Cytosine deamination is a major cause of baseline noise in next-generation sequencing. Mol Diagn Ther 18(5):587–593. doi: 10.1007/s40291-014-0115-2 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Do H, Dobrovic A (2012). Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase. Oncotarget 3(5):546–558. doi: 10.18632/oncotarget.503 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Do H, Dobrovic A (2015). Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem 61(1):64–71. doi: 10.1373/clinchem.2014.223040 CrossRefPubMedGoogle Scholar
  11. Dutton-Regester K, Hayward NK (2012) Reviewing the somatic genetics of melanoma: from current to future analytical approaches. Pigment Cell Melanoma Res 25(2):144–154. doi: 10.1111/j.1755-148X.2012.00975.x CrossRefPubMedGoogle Scholar
  12. Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Boutselakis H, Campbell PJ (2015) COSMIC: exploring the world’s knowledge of somatic mutations in human cancer. Nucleic Acids Res 43(D1):D805–D811. doi: 10.1093/nar/gku1075 CrossRefPubMedGoogle Scholar
  13. Gagan J, Van Allen EM (2015) Next-generation sequencing to guide cancer therapy. Genome Med 7(1):80. doi: 10.1186/s13073-015-0203-x CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gallegos Ruiz MI, Floor K, Rijmen F, Grünberg K, Rodriguez JA, Giaccone G (2007) EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens. Cell Oncol†¯ 29(3):257–64. Retrieved from
  15. Gao J, Wu H, Wang L, Zhang H, Duan H, Lu J, Liang Z (2016) Validation of targeted next-generation sequencing for RAS mutation detection in FFPE colorectal cancer tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR. BMJ Open 6(1):e009532. doi: 10.1136/bmjopen-2015-009532 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hamilton A, Tétreault M, Dyment DA, Zou R, Kernohan K, Geraghty MT, Boycott KM (2016) Concordance between whole-exome sequencing and clinical Sanger sequencing: implications for patient care. Mol Genet Genom Med 4(5):504–512. doi: 10.1002/mgg3.223 CrossRefGoogle Scholar
  17. Haracska L, Prakash L, Prakash S (2002) Role of human DNA polymerase kappa as an extender in translesion synthesis. Proc Natl Acad Sci USA 99(25):16000–16005. doi: 10.1073/pnas.252524999 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hedegaard J, Thorsen K, Lund MK, Hein A-MK, Hamilton-Dutoit SJ, Vang S, Dyrskjøt L (2014) Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS One 9(5):e98187. doi: 10.1371/journal.pone.0098187 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat J-P, Chin L (2012) A landscape of driver mutations in melanoma. Cell 150(2):251–263. doi: 10.1016/j.cell.2012.06.024 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kerick M, Isau M, Timmermann B, Sültmann H, Herwig R, Krobitsch S, Schweiger MR (2011) Targeted high throughput sequencing in clinical cancer settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity. BMC Med Genom 4(1):68. doi: 10.1186/1755-8794-4-68 CrossRefGoogle Scholar
  21. Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M, Halmos B (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352(8):786–792. doi: 10.1056/NEJMoa044238 CrossRefPubMedGoogle Scholar
  22. Krauthammer M, Kong Y, Ha BH, Evans P, Bacchiocchi A, McCusker JP, Halaban R (2012) Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat Genet 44(9):1006–1014. doi: 10.1038/ng.2359 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lin J, Kennedy SH, Svarovsky T, Rogers J, Kemnitz JW, Xu A, Zondervan KT (2009) High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil. Anal Biochem 395(2):265–267. doi: 10.1016/j.ab.2009.08.016 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Ludyga N, Grünwald B, Azimzadeh O, Englert S, Höfler H, Tapio S, Aubele M (2012). Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Virchows Arch 460(2):131–140. doi: 10.1007/s00428-011-1184-9 CrossRefPubMedGoogle Scholar
  25. Oh E, Choi Y-L, Kwon MJ, Kim RN, Kim YJ, Song J-Y, Shin YK (2015) Comparison of accuracy of whole-exome sequencing with formalin-fixed paraffin-embedded and fresh frozen tissue samples. PLoS One 10(12):e0144162. doi: 10.1371/journal.pone.0144162 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Pabinger S, Dander A, Fischer M, Snajder R, Sperk M, Efremova M, Trajanoski Z (2014) A survey of tools for variant analysis of next-generation genome sequencing data. Brief Bioinform 15(2):256–278. doi: 10.1093/bib/bbs086 CrossRefPubMedGoogle Scholar
  27. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Varmus H (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2(3):e73. doi: 10.1371/journal.pmed.0020073 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray SJ, Greenman CD, Stratton MR (2010) A comprehensive catalogue of somatic mutations from a human cancer genome. Nature 463(7278):191–196. doi: 10.1038/nature08658 CrossRefPubMedGoogle Scholar
  29. Pokholok DK, Le JM, Steemers FJ, Ronaghi M, Gunderson KL (2014) Abstract LB-134: analysis of restored FFPE samples on high-density SNP arrays. Cancer Res 70(8 Supplement):LB-134-LB-134. doi: 10.1158/1538-7445.AM10-LB-134 Google Scholar
  30. Prickett TD, Agrawal NS, Wei X, Yates KE, Lin JC, Wunderlich JR, Samuels Y (2009) Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4. Nat Genet 41(10):1127–1132. doi: 10.1038/ng.438 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Quach N, Goodman MF, Shibata D (2004) In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BMC Clin Pathol 4:1. doi: 10.1186/1472-6890-4-1 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Sah S, Chen L, Houghton J, Kemppainen J, Marko AC, Zeigler R, Latham GJ (2013). Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies. Genome Med 5(8):77. doi: 10.1186/gm481 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Schormann N, Ricciardi R, Chattopadhyay D (2014) Uracil-DNA glycosylases-structural and functional perspectives on an essential family of DNA repair enzymes. Protein Sci†¯ Publ Protein Soc 23(12):1667–1685. doi: 10.1002/pro.2554 CrossRefGoogle Scholar
  34. Schweiger MR, Kerick M, Timmermann B, Albrecht MW, Borodina T, Parkhomchuk D, Lehrach H (2009) Genome-wide massively parallel sequencing of formaldehyde fixed-paraffin embedded (FFPE) tumor tissues for copy-number- and mutation-analysis. PLoS One 4(5):e5548. doi: 10.1371/journal.pone.0005548 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sedlackova T, Repiska G, Celec P, Szemes T, Minarik G (2013) Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods. Biol Proced Online 15(1):5. doi: 10.1186/1480-9222-15-5 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Solassol J, Ramos J, Crapez E, Saifi M, Mangé A, Vianès E, Maudelonde T (2011) KRAS mutation detection in paired frozen and formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Int J Mol Sci 12(5):3191–3204. doi: 10.3390/ijms12053191 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Spencer DH, Sehn JK, Abel HJ, Watson MA, Pfeifer JD, Duncavage EJ (2013) Comparison of clinical targeted next-generation sequence data from formalin-fixed and fresh-frozen tissue specimens. J Mol Diagn†¯ 15(5):623–633. doi: 10.1016/j.jmoldx.2013.05.004 CrossRefGoogle Scholar
  38. Stiller M, Sucker A, Griewank K, Aust D, Baretton GB, Schadendorf D, Horn S (2016). Single-strand DNA library preparation improves sequencing of formalin-fixed and paraffin-embedded (FFPE) cancer DNA. Oncotarget. doi: 10.18632/oncotarget.10827 PubMedPubMedCentralGoogle Scholar
  39. Sturm RA, Fox C, McClenahan P, Jagirdar K, Ibarrola-Villava M, Banan P, Soyer HP (2014) Phenotypic characterization of nevus and tumor patterns in MITF E318K mutation carrier melanoma patients. J Invest Dermatol 134(1):141–149. doi: 10.1038/jid.2013.272 CrossRefPubMedGoogle Scholar
  40. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35(suppl 2):W71–W74. doi: 10.1093/nar/gkm306 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Wong SQ, Li J, Tan A Y-C, Vedururu R, Pang J-MB, Do H, Dobrovic A (2014) Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing. BMC Med Genom 7:23. doi: 10.1186/1755-8794-7-23 CrossRefGoogle Scholar
  42. Ye X, Zhu Z-Z, Zhong L, Lu Y, Sun Y, Yin X, Ji Q (2013) High T790M detection rate in TKI-naive NSCLC with EGFR sensitive mutation: truth or artifact? J Thorac Oncol†¯ 8(9):1118–1120. doi: 10.1097/JTO.0b013e31829f691f CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of General DermatologyMedical University GrazGrazAustria
  2. 2.Translational Skin Cancer Research-TSCR, German Cancer Consortium (DKTK), Partner Site Essen/Düsseldorf, German Cancer Research Center (DKFZ)University Hospital EssenEssenGermany
  3. 3.Department of DermatologyUniversity Hospital of EssenEssenGermany
  4. 4.Department of DermatologyUniversity Hospital of WürzburgWürzburgGermany
  5. 5.Boehringer Ingelheim RCVViennaAustria

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