Hersh A Ham-Karim1,2, Henry Okuchukwu Ebili3,4, Kirsty Manger5, Wakkas Fadhil1, Narmeen S Ahmad6,7, Susan D Richman8, Mohammad Ilyas1. 1. Division of Pathology, School of Medicine, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK. 2. Department of Medical Laboratory Sciences, College of Health Sciences, Komar University of Science and Technology, Chaq-Chaq-Qualaraisi, Sulaimani, Iraq. 3. Division of Pathology, School of Medicine, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK. Henry.Ebili@nottingham.ac.uk. 4. Department of Morbid Anatomy and Histopathology, Olabisi Onabanjo University, Ago-Iwoye, Nigeria. Henry.Ebili@nottingham.ac.uk. 5. Centre for Medical Genetics, Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham, UK. 6. Clinical Oncology, University of Nottingham, City Hospital Campus, Nottingham, UK. 7. Kurdistan Institution for Strategic Studies and Scientific Research, Qirga, Sulaimani, KRG, Iraq. 8. Department of Pathology and Tumour Biology, Leeds Institute of Cancer and Pathology, Wellcome Trust Brenner Building, St James University Hospital, Leeds, UK.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Diagnostic biopsies are adequate and reliable materials for molecular testing by NGS. The use of biopsies for molecular screening will enhance targeted neoadjuvant therapy.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Diagnostic biopsies are adequate and reliable materials for molecular testing by NGS. The use of biopsies for molecular screening will enhance targeted neoadjuvant therapy.
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