Predicting response to immune checkpoint blockade in NSCLC with tumour-only RNA-seq

Targeted RNA sequencing (RNA-seq) from FFPE specimens is used clinically in cancer for its ability to estimate gene expression and to detect fusions. Using a cohort of NSCLC patients, researchers at the Peter MacCallum Cancer Centre sought to determine whether targeted RNA-seq could be used to measure tumour mutational burden (TMB) and the expression of immune-cell-restricted genes from FFPE specimens and whether these could predict response to immune checkpoint blockade.

Using The Cancer Genome Atlas LUAD dataset, the researchers developed a method for determining TMB from tumour-only RNA-seq and showed a correlation with DNA sequencing derived TMB calculated from tumour/normal sample pairs (Spearman correlation = 0.79, 95% CI [0.73, 0.83]. They applied this method to targeted sequencing data from our patient cohort and validated these results against TMB estimates obtained using an orthogonal assay (Spearman correlation = 0.49, 95% CI [0.24, 0.68]). The researchers observed that the RNA measure of TMB was significantly higher in responders to immune blockade treatment (P = 0.028) and that it was predictive of response (AUC = 0.640 with 95% CI [0.493, 0.786]). By contrast, the expression of immune-cell-restricted genes was uncorrelated with patient outcome.

Filtering variants from tumour-only RNA-seq to compute RNA TMB

a Flowchart illustrating the sequence of filtering steps used to remove unwanted variants. b All variants in the cohort are aggregated into a table containing loci as rows and samples as columns. This enables the identification of loci-containing multiple variants. These commonly occurring variants (with loci marked by a red cross in the figure) are unlikely to be due to randomly distributed mutations and hence can be removed from TMB calculations based on their frequency within the cohort. c Variants are accepted or rejected based on their allele frequency in each sample. VAFs close to 0 or 1 are removed as are those in a range around 0.5. d RNA TMB versus DNA TMB for the TCGA-LUAD dataset with a linear regression fitted through the origin.