Ching Ngar Wong1, Petros Fessas1, Kathy Dominy2, Francesco A Mauri1, Takahiro Kaneko1,3, Persephone Du Parcq2, Jamshid Khorashad2, Pierluigi Toniutto4, Robert D Goldin5, Claudio Avellini6, David J Pinato1. 1. Department of Surgery & Cancer, Imperial College London, Hammersmith Hospital, London, UK. 2. Molecular Pathology Laboratory, Hammersmith Hospital, London, UK. 3. Tokyo Medical and Dental University, Tokyo, Japan. 4. Hepatology and Liver Transplantation Unit, Department of Medical Area (DAME), University of Udine, Udine, Italy. 5. Centre for Pathology, Imperial College London, London, UK. 6. Azienda Ospedaliero-Universitaria "Santa Maria della Misericordia", Institute of Histopathology, Udine, Italy.
Abstract
BACKGROUND & AIMS: Tumour mutational burden (TMB) predicts improved response and survival to immunotherapy. In this pilot study, we optimized targeted next-generation sequencing (tNGS) to estimate TMB in hepatocellular carcinoma (HCC). METHODS: We sequenced 48 non-paired samples (21 fresh-frozen [FF] and 27 paraffin-embedded [FFPE]), among which 11 FFPE samples were pretreated with uracil-DNA glycosylase (UDG). Thirty samples satisfied post-sequencing quality control. High/low TMB was defined by median number of mutations/Mb (Mut/Mb), across different minimum allele frequency (MAF) thresholds (≥0.05, ≥0.1 and ≥0.2). RESULTS: Eligible patients (n = 29) were cirrhotic (84%) with TNM stage I-II HCC (75%). FFPE samples had higher TMB (median 958.39 vs 2.51 Mut/Mb, P < .0001), estimated deamination counts (median 1335.50 vs 0, P < .0001) and C > T transitions at CpG sites (median 60.3% vs 9.1%, P = .002) compared to FF. UDG-treated samples had lower TMB (median 4019.92 vs 353 Mut/Mb, P = .041) and deamination counts (median 6393.5 vs 328.5, P = .041) vs untreated FFPE. At 0.2 MAF threshold with UDG treatment, median TMB was 5.48 (range 1.68-16.07) and did not correlate with salient pathologic features of HCC, including survival. CONCLUSION: While tNGS on fresh HCC samples appears to be the optimal source of tumour DNA, the low median TMB values observed may limit the role of TMB as a predictor of response to immunotherapy in HCC.
BACKGROUND & AIMS:Tumour mutational burden (TMB) predicts improved response and survival to immunotherapy. In this pilot study, we optimized targeted next-generation sequencing (tNGS) to estimate TMB in hepatocellular carcinoma (HCC). METHODS: We sequenced 48 non-paired samples (21 fresh-frozen [FF] and 27 paraffin-embedded [FFPE]), among which 11 FFPE samples were pretreated with uracil-DNA glycosylase (UDG). Thirty samples satisfied post-sequencing quality control. High/low TMB was defined by median number of mutations/Mb (Mut/Mb), across different minimum allele frequency (MAF) thresholds (≥0.05, ≥0.1 and ≥0.2). RESULTS: Eligible patients (n = 29) were cirrhotic (84%) with TNM stage I-II HCC (75%). FFPE samples had higher TMB (median 958.39 vs 2.51 Mut/Mb, P < .0001), estimated deamination counts (median 1335.50 vs 0, P < .0001) and C > T transitions at CpG sites (median 60.3% vs 9.1%, P = .002) compared to FF. UDG-treated samples had lower TMB (median 4019.92 vs 353 Mut/Mb, P = .041) and deamination counts (median 6393.5 vs 328.5, P = .041) vs untreated FFPE. At 0.2 MAF threshold with UDG treatment, median TMB was 5.48 (range 1.68-16.07) and did not correlate with salient pathologic features of HCC, including survival. CONCLUSION: While tNGS on fresh HCC samples appears to be the optimal source of tumour DNA, the low median TMB values observed may limit the role of TMB as a predictor of response to immunotherapy in HCC.
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