Targeted Next-Generation Sequencing Validates the Use of Diagnostic Biopsies as a Suitable Alternative to Resection Material for Mutation Screening in Colorectal Cancer

  • Hersh A. Ham-Karim
  • Henry Okuchukwu EbiliEmail author
  • Kirsty Manger
  • Wakkas Fadhil
  • Narmeen S. Ahmad
  • Susan D. Richman
  • Mohammad Ilyas
Original Research Article



Mutation testing in the context of neoadjuvant therapy must be performed on biopsy samples. Given the issue of tumour heterogeneity, this raises the question of whether the biopsies are representative of the whole tumour. Here we have compared the mutation profiles of colorectal biopsies with their matched resection specimens.


We performed next-generation sequencing (NGS) analysis on 25 paired formalin-fixed, paraffin-embedded colorectal cancer biopsy and primary resection samples. DNA was extracted and analysed using the TruSight tumour kit, allowing the interrogation of 26 cancer driver genes. Samples were run on an Illumina MiSeq. Mutations were validated using quick-multiplex-consensus (QMC)-polymerase chain reaction (PCR) in conjunction with high resolution melting (HRM). The paired biopsy and resection tumour samples were assessed for presence or absence of mutations, mutant allele frequency ratios, and allelic imbalance status.


A total of 81 mutations were detected, in ten of the 26 genes in the TruSight kit. Two of the 25 paired cases were wild-type across all genes. The mutational profiles, allelic imbalance status, and mutant allele frequency ratios of the paired biopsy and resection samples were highly concordant (88.75–98.85%), with all but three (3.7%) of the mutations identified in the resection specimens also being present in the biopsy specimens. All 81 mutations were confirmed by QMC-PCR and HRM analysis, although four low-level mutations required a co-amplification at lower denaturation temperature (COLD)-PCR protocol to enrich for the mutant alleles.


Diagnostic biopsies are adequate and reliable materials for molecular testing by NGS. The use of biopsies for molecular screening will enhance targeted neoadjuvant therapy.



The authors would like to thank Gareth Cross for enabling the process of NGS data generation and analyses.

Compliance with Ethical Standards


This work was funded by Universities of Nottingham (for MI) and Leeds (for SDR).

Conflict of interest

All the authors (HH-K, HOE, KM, WF, NSA, SDR and MI) declare that they have no conflicts of interest in publishing this manuscript.

Ethical approval and informed consent

Access to tissues and ethics approval were granted by Nottingham Health Sciences Biobank, which has approval as an IRB from North West–Greater Manchester Central Research Ethics Committee (REC reference: 15/NW/0685).

Supplementary material

40291_2019_388_MOESM1_ESM.tif (5.9 mb)
Supplementary material 1 (TIFF 6040 kb) Online Resource Figure 1: Validation of NGS-detected mutations by HRM analysis. Difference plots obtained for (A) TP53 and (B) KRAS, by HRM analysis. The samples shown were identified by NGS as harbouring mutations and were confirmed by HRM analysis
40291_2019_388_MOESM2_ESM.tif (3.8 mb)
Supplementary material 2 (TIFF 3908 kb) Online Resource Figure 2: HRM Analysis Difference plots showing enrichment of mutant allele by COLD-PCR. (A) A PIK3CA (c.331_333delAAG) mutation was detected by NGS in this sample. Plot 1 represents PCR products obtained by QMC-PCR, whilst plot 2 denotes PCR products obtained by COLD-PCR. (B) A SMAD4 (c.1082G>A) mutation detected by NGS. Plot 1 is PCR products obtained by QMC-PCR, whereas plot 2 is PCR products obtained by COLD-PCR. * denotes baseline normal DNA
40291_2019_388_MOESM3_ESM.tif (886 kb)
Supplementary material 3 (TIFF 885 kb) Online Resource Figure 3: A grid chart showing the agreement status between Bx and Rx using the ‘mutation-present-or-absent’ test. The coloured boxes denote presence of mutations, whilst the white boxes denote absence of mutations. The coloured boxes without numbers denote that there is only one mutation type between Rx/Bx pair; the numbers in some of the boxes denote the number of mutations for each gene found in the sample pair, whilst the * denotes that the matched Bx lacked the mutation that was found in the Rx. C = concordance, D = discordance
40291_2019_388_MOESM4_ESM.doc (196 kb)
Supplementary material 4 (DOC 196 kb)
40291_2019_388_MOESM5_ESM.xlsx (112 kb)
Supplementary material 5 (XLSX 111 kb)


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Hersh A. Ham-Karim
    • 1
    • 2
  • Henry Okuchukwu Ebili
    • 1
    • 3
    Email author
  • Kirsty Manger
    • 4
  • Wakkas Fadhil
    • 1
  • Narmeen S. Ahmad
    • 5
    • 6
  • Susan D. Richman
    • 7
  • Mohammad Ilyas
    • 1
  1. 1.Division of Pathology, School of Medicine, Queen’s Medical CentreUniversity of NottinghamNottinghamUK
  2. 2.Department of Medical Laboratory Sciences, College of Health SciencesKomar University of Science and TechnologySulaimaniIraq
  3. 3.Department of Morbid Anatomy and HistopathologyOlabisi Onabanjo UniversityAgo-IwoyeNigeria
  4. 4.Centre for Medical GeneticsNottingham University Hospitals NHS Trust, City Hospital CampusNottinghamUK
  5. 5.Clinical OncologyUniversity of Nottingham, City Hospital CampusNottinghamUK
  6. 6.Kurdistan Institution for Strategic Studies and Scientific ResearchSulaimaniIraq
  7. 7.Department of Pathology and Tumour Biology, Leeds Institute of Cancer and Pathology, Wellcome Trust Brenner BuildingSt James University HospitalLeedsUK

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