Mutational burden, immune checkpoint expression, and mismatch repair in glioma: implications for immune checkpoint immunotherapy

Abstract

Background: Despite a multiplicity of clinical trials testing immune checkpoint inhibitors, the frequency of expression of potential predictive biomarkers is unknown in glioma.

Methods: In this study, we profiled the frequency of shared biomarker phenotypes. To clarify the relationships among tumor mutational load (TML), mismatch repair (MMR), and immune checkpoint expression, we profiled patients with glioma (n = 327), including glioblastoma (GBM) (n = 198), whose samples had been submitted for analysis from 2009 to 2016. The calculation algorithm for TML included nonsynonymous mutation counts per tumor, with germline mutations filtered out. Immunohistochemical analysis and next-generation sequencing were used to determine tumor-infiltrating lymphocyte expression positive for programmed cell death protein 1 (PD-1), PD ligand 1 (PD-L1) expression on tumor cells, MMR (MLH1, MSH2, MSH6, and PMS2) protein expression and mutations, and DNA polymerase epsilon (POLE) mutations.

Results: High TML was only found in 3.5% of GBM patients (7 of 198) and was associated with the absence of protein expression of mutL homolog 1 (MLH1) (P = .0345), mutS homolog 2 (MSH2) (P = .0099), MSH6 (P = .0022), and postmeiotic segregation increased 2 (PMS2) (P = .0345) and the presence of DNA MMR mutations. High and moderate TML GBMs did not have an enriched influx of CD8+ T cells, PD-1+ T cells, or tumor-expressed PD-L1. IDH1 mutant gliomas were not enriched for high TML, PD-1+ T cells, or PD-L1 expression.

Conclusions: To clarify the relationships among TML, MMR, and immune checkpoint expression, we profiled the frequency of shared biomarker phenotypes. On the basis of a variety of potential biomarkers of response to immune checkpoints, only small subsets of glioma patients are likely to benefit from monotherapy immune checkpoint inhibition.

Keywords: glioblastoma; glioma; immune checkpoint; mismatch repair; mutational load.

Fig. 1

TML is associated with WHO tumor grade. High TML was defined as more than 20 mutations per 1.4 Mb, moderate TML as 11–20 mutations per 1.4 Mb, and low TML as 10 or fewer mutations per 1.4 Mb. Of the high TML-expressing gliomas, 40% (6/15) were newly diagnosed and 60% (9 of 15) were recurrent. Within the high-TML GBM subset, 57.1% (4 of 7) were newly diagnosed and the remaining were recurrent. °I = grade I glioma, °II = grade II glioma, °III = grade III glioma, °IV = grade IV glioma. Red = newly diagnosed glioma. Blue = recurrent glioma. ns = not significant.

Fig. 2

MMR protein expression corresponds to TML in GBM. MMR protein expression was determined by IHC in GBM cases (n = 30). Only 6.7% (2 of 30) demonstrated loss of MLH1 (A), 10% (3 of 30) loss of MSH2 (B), 13.3% (4 of 30) loss of MSH6 (C), and 6.7% (2 of 30) loss of PMS2 (D). When these GBM cases were analyzed for TML, 6 showed high TML. Of the GBMs with high TML, at least 1 MMR enzyme was found to have defective expression (E). High TML was defined as more than 20 mutations per 1.4 Mb, moderate TML as 11–20 mutations per 1.4 Mb, and low TML as 10 or fewer mutations per 1.4 Mb. ns = not significant.

Fig. 3

MMR mutations do not correspond to TML in glioma. Mutations in MMR and POLE were analyzed by NGS. An association was found between TML and the occurrence of (A) MLH1 mutation, (B) MSH2 mutation, (C) MSH6 mutation, (D) PMS2 mutations, and (E) POLE mutations (P = .109; P = .0144 before FDR adjustment). (F) Of the samples analyzed for mutations in the 4 MMR enzymes, 11 showed high TML; 54.5% (6 of 11) were mutated in at least 1 MMR, and 18% (2 of 11) were mutated in at least 2 MMR. ns = not significant.

Fig. 4

TML is not associated with PD-1 and PD-L1 expression or CD8+ T cell influx. Of all analyzed samples, 38.8% (59 of 152) were PD-1 positive (A) and 7.7 % (24 of 310) were PD-L1 positive (B). Of the GBM patients, 45.7% (43 of 94) were PD-1 positive (C) and 10.1% (19 of 189) were PD-L1 positive (D). No association was found between TML and PD-1/PD-L1 expression. PD-1 and PD-L1 expression levels were determined using IHC staining. (E) CD8+ T-cell influx was determined by IHC in a subset of gliomas with high and moderate TML (n = 9). No association with TML was found.

Fig. 5

Subgroup analysis of IDH1 wild-type GBM. TML was compared between IDH1 wild-type (n = 182) and mutated (n = 16) GBM; 2.7% (5 of 182) of IDH1 wild-type cases and 12.5% (2 of 16) of mutated cases showed high TML (P = .1014) (A). Both PD-1 (B) and PD-L1 expression (C) were predominantly expressed in IDH1 wild-type cases and were not associated with high TML.