Association Between Preanalytical Factors and Tumor Mutational Burden Estimated by Next-Generation Sequencing-Based Multiplex Gene Panel Assay

Abstract

Background Tumor mutational burden (TMB) measured via next‐generation sequencing (NGS)‐based gene panel is a promising biomarker for response to immune checkpoint inhibitors (ICIs) in solid tumors. However, little is known about the preanalytical factors that can affect the TMB score. Materials and Methods Data of 199 patients with solid tumors who underwent multiplex NGS gene panel (OncoPrime), which was commercially provided by a Clinical Laboratory Improvement Amendments‐licensed laboratory and covered 0.78 megabase (Mb) of capture size relevant to the TMB calculation, were reviewed. Associations between the TMB score and preanalytical factors, including sample DNA quality, sample type, sampling site, and storage period, were analyzed. Clinical outcomes of patients with a high TMB score (≥10 mutations per megabase) who received anti‐programmed cell death protein 1 antibodies (n = 22) were also analyzed. Results Low DNA library concentration (<5 nM), formalin‐fixed paraffin‐embedded tissue (FFPE), and the prolonged sample storage period (range, 0.9–58.1 months) correlated with a higher TMB score. After excluding low DNA library samples from the analysis, FFPE samples, but not the sample storage period, exhibited a marked correlation with a high TMB score. Of 22 patients with a high TMB score, we observed the partial response in 2 patients (9.1%). Conclusion Our results indicate that the TMB score estimated via NGS‐based gene panel could be affected by the DNA library concentration and sample type. These factors could potentially increase the false‐positive and/or artifactual variant calls. As each gene panel has its own pipeline for variant calling, it is unknown whether these factors have a significant effect in other platforms. Implications for Practice A high tumor mutational burden score, as estimated via next‐generation sequencing‐based gene panel testing, should be carefully interpreted as it could be affected by the DNA library concentration and sample type.

Qualification of tumour mutational burden by targeted next‐generation sequencing as a biomarker in hepatocellular carcinoma

“…Firstly, we have to take into account inter‐sample differences given the groups of untreated, UDG‐treated FFPE and FF DNA samples were not extracted from the same tumours. In addition, DNA integrity is inversely proportional to the duration of tissue fixation 33 . Because our study included archival material processed during routine care, we cannot confidently reconstruct the influence of fixation on DNA quality.…”

Tumor Mutational Burden as a Potential Biomarker for Immunotherapy in Pancreatic Cancer: Systematic Review and Still-Open Questions

“…However, this method is challenging to adopt in daily clinical practice, due to high costs and long turnaround times. As a consequence, an increasing number of studies has tried to demonstrate whether targeted NGS gene panels might determine TMB with reliable precision [15,[18][19][20]. While initial studies have shown that targeted NGS gene panels offer reliable estimates of TMB, substantial concerns regarding the stochastic error associated to limited gene panel sequencing have been raised [15,[21][22][23][24][25][26].…”

“…Some of these factors can impact TMB reproducibility, even when using the same assay [8]. Another significant issue is represented by the lack of standardization of the genes of interest, which can vary greatly among commercial and custom panels [3,11,12,18,19].…”

“…Extensive investigations on tumors of different origins highlighted how high-TMB may be observed in almost all cancer types [3]. Recent evidence has clarified that several factors may determine a high-TMB in different neoplasms, including polymerase-epsilon (POLE) mutations [28], environment-based etiologies such as tobacco smoke, polycyclic aromatic hydrocarbons and UV exposure [19,27,29,30], or the presence of MSI [4,6]. Currently, TMB-based approved immunotherapy approaches include non-small cell lung cancers [31], bladder cancers [32], and malignant melanomas [33] with TMB cut-off of 10 mutations per Mb [5,30].…”

Inter-assay variability of next-generation sequencing-based gene panels

“…The minimum input DNA quantity required for creating libraries was 150 ng per sample, while the median depth of coverage was more than 3000. Variant calling software VarPROWL (r20278) was used for variant calling [ 13 ].…”