High tumor mutational burden and T-cell activation are associated with long-term response to anti-PD1 therapy in Lynch syndrome recurrent glioblastoma patient

Abstract

Background: Glioblastomas (GBMs) in patients harboring somatic or germinal mutations of mismatch-repair (MMR) genes exhibit a hypermutable phenotype. Here, we describe a GBM patient with increased tumor mutational burden and germline MMR mutations, treated using anti-PD1 therapy.

Methods: A woman with newly diagnosed GBM (nGBM) was treated by surgery, radiotherapy, and temozolomide. The tumor recurred after 13 months leading to a second surgery and treatment with nivolumab. Whole-exome sequencing was performed on the nGBM, recurrent GBM (rGBM), and blood. Immune infiltration was investigated by immunohistochemistry and the immune response in the blood during treatment was analyzed by flow cytometry.

Results: High density of infiltrating CD163 + cells was found in both GBM specimens. Large numbers of CD3 + and CD8 + T cells were homogeneously distributed in the nGBM. The infiltration of CD4 + T cells and a different CD8 + T cell density were observed in the rGBM. Both GBM shared 12,431 somatic mutations, with 113 substitutions specific to the nGBM and 1,683 specific to the rGBM. Germline variants included pathogenic mutation in the MSH2 (R359S) gene, suggesting the diagnosis of Lynch syndrome. Systemic immunophenotyping revealed the generation of CD8 + T memory cells and persistent activation of CD4 + T cells. The patient is still receiving nivolumab 68 months after the second surgery.

Conclusions: Our observations indicate that the hypermutator phenotype associated with germinal mutations of MMR genes and abundant T-cell infiltration contributes to a durable clinical benefit sustained by a persistent and robust immune response during anti-PD1 therapy.

Keywords: Glioblastoma (GBM); Lynch syndrome (LS); Mismatch repair (MMR); Nivolumab/anti-PD1 therapy; Tumor infiltration lymphocytes (TILs); Tumor mutational burden (TMB).

Fig. 1

Patient brain neuroimaging. a July 2013 (Time 0): pre-surgery brain computed tomography (CT) showing an intra-axial frontal expansive lesion with edema and mass effect. b–f Brain MRI, top T2 and bottom T1 with contrast agent weighted imaging, respectively; b April 2014 (Time 9): MRI after gross total resection (GTR) of the lesion, slight gliosis without mass effect; c, d August–October 2014 (Time 13–Time 15): recurrence with T1 enhancing lesion with increasing size, edema and mass effect; e November 2014 (Time 16): early brain MRI post-second surgery and GTR of the rGBM, showing persisting edema and mass effect; f June 2020 (Time 84): brain MRI shows gliosis and ex-vacuum right lateral ventricle enlargement, with no signs of recurrence

Fig. 2

Expression and distribution of PD-L1 and CD163 in the nGBM and rGBM specimens. a, b Entire sections (magnification 0 ×), and representative areas (black rectangles) of nGBM (a) and rGBM (b) show that PD-L1 is only expressed after recurrence, and positive cells are mainly distributed at the margin of the specimen (scale bar 200 μm). c, d Entire section and representative marginal and central areas display that CD163 + infiltrating cells are found around the vessels and scattered in the tumor center of the specimens (scale bar 200 μm). e An automated quantification analysis revealed that the percentage of CD163 + cells, calculated with respect to the total nucleated cells, was higher in the margin compared to the tumor center of nGBM (43.2 ± 4.0% vs. 30.7 ± 9.7%, respectively; P = 0.04), as well as rGBM specimen (51.9 ± 9.0% vs. 31.4 ± 11.5%, respectively; P = 0.0008)

Fig. 3

CD8 + cell infiltration are abundant in both nGBM and rGBM specimens. a, b Representative adjacent sections of the nGBM specimen showing a high distribution of CD3 + and CD8 + and total absence of CD4 + TILs. High density of CD3 + and CD8 + TILs was observed in both tumor center (a) and tumor margins (b) of the nGBM specimen. c, d Changes in CD3 + and CD8 + T cell distribution and increased of CD4 + TILs were found in the rGBM. A manual count performed in the center area and tumor margins separately confirmed a significant higher density of CD3 + CD8 + TILs at the tumor margins (Scale Bar 300 μm)

Fig. 4

a Phylogenetic tree of the patient. Branch lengths are proportional to the number of mutations detected. b Mutation signature analysis. c Stacked bar plot indicating the contribution of each mutational signature in the nGBM and rGBM specimens

Fig. 5

Immune monitoring of the peripheral immune response activated by the anti-PD1 therapy. a Representative dot plots showing the CD8 + T-cell positivity for IFN-γ at two different time points (Time 51 and Time 60) in anti-PD1-treated patient. b Kinetics of the frequency of CD3 + CD8 + T cells (white square) and CD8 + T cells expressing IFN-γ (black square) assessed by flow cytometry. The dotted and dashed lines represent the median of the CD8 + T-cell activation evaluated by IFN-γ expression in V-DENDR2 OS > 9 and OS ≤ 9, respectively. c Representative dot plots showing the memory CD8 + T-cell subsets at two different time points. CM central memory, EM effector memory, N naïve. d Time course of the frequency of CD8 + central (CM) and effector memory (EM). e Representative dot plots showing the double positive CD38 and HLA-DR cells at two different time points. f Kinetics of the frequency of CD38⁺/HLA-DR⁺-activated cells evaluated in CD45/CD3/CD4⁺ T cells. The dotted and dashed lines represent the median of the CD8 + T-cell activation evaluated by IFN-γ expression in V-DENDR2 OS > 9 and OS ≤ 9, respectively. g Kinetics of the frequency of CD4 + T cells expressing CD62L as low and high in anti-PD1-treated patient. h Flow cytometry histogram showing the distribution of the CD62L levels in anti-PD1-treated patient compared to control patients (DC OS ≤ 9 and DC OS > 9)