Table 1.

Summary of studies that disqualify TMB as a biomarker for immunotherapy efficacy in gliomas

Study	Relevant study goal	Method	Relevant result	Conclusion
Select studies relating TMB and neoantigen immunogenicity
Zhang et al. [5]	To identify a subgroup of IDH wild-type GBM patients that will show longest survival and benefit most from immunotherapy	Application of a neoantigen fitness model to 238 IDH wild-type GBM patients	High-quality neoantigen model and quantification of CD8+ T cell infiltration were used to identify a subset of patients that display a higher likelihood of response to immunotherapy, while TMB independently could not	TMB alone is inadequate in determining immunotherapy efficacy in GBMs
Rech et al. [6]	To characterize a new subset of neoantigens that display higher immunogenicity than classically defined neoantigens (CDNs)	Analysis of predicted neoantigens and immune activity in 6324 patients across 27 tumor types from The Cancer Genome Atlas (TCGA)	Characterized alternatively defined neoantigens (ADNs), which display higher affinity to MHC-I and MHC-II molecules than CDNs and can predict immune phenotype and patient survival LGGs and GBMs displayed comparatively lower numbers of mean ADNs (28 and 38, respectively)	LGGs and GBMs tend to harbor lower numbers of immunogenic neoantigens, likely because they produce fewer ADNs per mutation
Select studies relating TMB and neoantigen clonality
Touat et al. [10]	To understand mutational landscape differences between gliomas and other cancers and their impact on immunotherapy response	Analysis of mutational burden and signatures in 10294 gliomas	MMR-deficient gliomas lacked T cell infiltration despite TMB being similar to other hypermutated cancers Hypermutated gliomas demonstrated greater subclonal neoantigens than other hypermutated cancers	GBMs, especially post-TMZ treatment, with high TMB harbor large subclonal neoantigen populations
Kim et al. [9]	To understand the intratumoral clonal composition of GBMs and the impact of therapeutic intervention	Analysis of 252 GBM samples from TCGA and 60 biopsies from 23 pairs of pre- and post-treatment GBMs	Two independently sequenced biopsies from a single post-TMZ hypermutated recurrent glioma revealed 2429 and 5980 mutations, respectively, but only 163 shared mutations Presence of TP53 mutations in both primary and recurrent tumor samples was associated with increased frequency of subclonal neoantigens in samples	Post-TMZ GBMs with high TMB carry large subclonal neoantigen populations TP53-mutated GBMs may show favorable outcomes with combination therapies targeting subclonal neoantigens
Select studies relating TMB and neoantigen expression and presentation
Nejo et al. [11]	To investigate changes in the neoantigen landscape during glioma progression	Exome and RNA-seq analysis of 25 pairs of primary and recurrent grade II–IV gliomas	No difference in the total number of neoantigens between primary and recurrent samples Neoantigen expression ratio decreased significantly in the recurrent tumor samples for neoantigens predicted to be highly immunogenic and clonal	A decrease in neoantigen expression ratio occurs due to immune selective pressure against highly immunogenic neoantigens, resulting in neoantigen ‘invisibility’ Treatments that overcome this immune evasion mechanism are required in addition to immunotherapy
Facoetti et al. [12]	To determine frequency of HLA and APM component abnormalities in malignant brain tumors	Analysis of 88 surgically removed malignant astrocytic tumors	~50% of GBM lesions and ~20% of grade II astrocytoma lesions had HLA-I loss ~70% oftumors showed selective HLA-A downregulation; this is higher than in other malignancies ~20% of GBMs showed downregulation of tapasin, an APM component	Presence of HLA-I and APM defects in malignant brain tumors may explain lack of immunotherapy efficacy in gliomas with high TMB
Yeung et al. [13]	To determine prevalence of LOH in HLA-I and B2M and its impact on survival in GBM patients	Cross-sectional analysis of 60 adult GBM patients	41.4% of cases displayed LOH in HLA-I, which was associated with poorer survival 18.2% ofcases displayed LOH in B2M HLA-I downregulation seen in 22–43% of cases	LOH in HLA-I and B2M is common in GBM patients and may reflect another mechanism of immune escape employed by tumors
Mehling et al. [14]	To investigate impact of APM component defects in astrocytomas with intact HLA-I expression	Analysis of 16 WHO grade I–IV astrocytomas	Downregulation of APM component (LMP2, TAP1, B2M) expression in astrocytomas, in contrast to normal expression of components in non-pathological astrocytes	Peptide-free expression of HLA-I molecules (due to APM impairment) in astrocytomas prevents activation of CD8+ T cells and inhibits NK cells