TY - JOUR

T1 - Tumor mutational burden and purity adjustment before and after treatment with temozolomide in 27 paired samples of glioblastoma

T2 - a prospective study

AU - Nørøxe, Dorte Schou

AU - Flynn, Aidan

AU - Yde, Christina Westmose

AU - Østrup, Olga

AU - Cilius Nielsen, Finn

AU - Skjøth-Rasmussen, Jane

AU - Brennum, Jannick

AU - Hamerlik, Petra

AU - Weischenfeldt, Joachim

AU - Skovgaard Poulsen, Hans

AU - Lassen, Ulrik

N1 - Publisher Copyright: © 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

PY - 2022

Y1 - 2022

N2 - Treatment of glioblastoma (GBM) remains a challenging task, with limited treatment options, none offering a cure. Immune therapy has proven effective across different cancers with remarkable response rates. Tumor mutational burden (TMB) is a marker of response, but technical and methodological differences in TMB estimates have made a proper assessment and comparison challenging. Here, we analyzed a prospective collection of paired samples from 35 patients with newly diagnosed GBM, all of whom were wild-type (WT) for isocitrate dehydrogenase, before and after treatment with radiotherapy and temozolomide. Seven patients (20%) had O6-methylguanine-DNA methyltransferase-methylated tumors. Six patients (17%) had two relapse surgeries, and tissue from all three surgeries was collected. We found that accurate evaluation of TMB was confounded by high variability in the cancer cell fraction of relapse samples. To ameliorate this, we developed a model to adjust for tumor purity based on the relative density distribution of variant allele frequencies in each primary–relapse pair. Additionally, we examined the mutation spectra of shared and private mutations. After tumor purity adjustment, we found TMB comparison reliable in tumors with tumor purity between 15% and 40%, resulting in 27/35 patients (77.1%). TMB remained unchanged from 0.65 mutations per megabase (Mb) to 0.67/Mb before and after treatment, respectively. Examination of the mutation spectra revealed a dominance of C > T transitions at CpG sites in both shared and relapse-private mutations, consistent with cytosine deamination and the clock-like mutational signature 1. We present and apply a cellularity correction approach that enables more accurate assessment of TMB in paired tumor samples. We did not find a significant increase in TMB after correcting for cancer cell fraction. Our study raises significant concerns when determining TMB. Although a small sample size, corrected TMB can have a clinical significance when stratifying patients to experimental treatment, for example, immune checkpoint therapy.

AB - Treatment of glioblastoma (GBM) remains a challenging task, with limited treatment options, none offering a cure. Immune therapy has proven effective across different cancers with remarkable response rates. Tumor mutational burden (TMB) is a marker of response, but technical and methodological differences in TMB estimates have made a proper assessment and comparison challenging. Here, we analyzed a prospective collection of paired samples from 35 patients with newly diagnosed GBM, all of whom were wild-type (WT) for isocitrate dehydrogenase, before and after treatment with radiotherapy and temozolomide. Seven patients (20%) had O6-methylguanine-DNA methyltransferase-methylated tumors. Six patients (17%) had two relapse surgeries, and tissue from all three surgeries was collected. We found that accurate evaluation of TMB was confounded by high variability in the cancer cell fraction of relapse samples. To ameliorate this, we developed a model to adjust for tumor purity based on the relative density distribution of variant allele frequencies in each primary–relapse pair. Additionally, we examined the mutation spectra of shared and private mutations. After tumor purity adjustment, we found TMB comparison reliable in tumors with tumor purity between 15% and 40%, resulting in 27/35 patients (77.1%). TMB remained unchanged from 0.65 mutations per megabase (Mb) to 0.67/Mb before and after treatment, respectively. Examination of the mutation spectra revealed a dominance of C > T transitions at CpG sites in both shared and relapse-private mutations, consistent with cytosine deamination and the clock-like mutational signature 1. We present and apply a cellularity correction approach that enables more accurate assessment of TMB in paired tumor samples. We did not find a significant increase in TMB after correcting for cancer cell fraction. Our study raises significant concerns when determining TMB. Although a small sample size, corrected TMB can have a clinical significance when stratifying patients to experimental treatment, for example, immune checkpoint therapy.

KW - glioblastoma

KW - immune therapy

KW - paired samples

KW - temozolomide

KW - tumor mutational burden

KW - tumor purity

U2 - 10.1002/1878-0261.13015

DO - 10.1002/1878-0261.13015

M3 - Journal article

C2 - 34018316

AN - SCOPUS:85108840009

VL - 16

SP - 206

EP - 218

JO - Molecular Oncology

JF - Molecular Oncology

SN - 1574-7891

IS - 1

ER -