Demographics and clinical features
Thirteen tissue specimens from newly diagnosed GBM, 11 from recurrence and 2 benign control specimens from patients with epilepsy were analyzed in this study. Among them, there were 10 paired samples from 5 patients at both initial diagnosis and at recurrence. The demographic and clinical features of all patients (n=26) are summarized in Table 1. The median age of all GBM patients at diagnosis was 59.25 (range 30.56-84.36). Among them, 57.7% were females, and all the patients in this study were Caucasian. No statistically significant difference in gender and median age was found between patients at initial diagnosis and patients at recurrence. Gross total resection (GTR) was performed in 53.8% at initial diagnosis and 81.8% at recurrence. Radiotherapy was administered in 84.6% at initial diagnosis and in 91% at recurrence, both as standard adjuvant treatment after initial diagnosis with 6000 cGy as the median dose. Seventy-seven percent of patients at initial diagnosis and 100% of recurrent patients received standard chemotherapy (TMZ). The median OS after the initial diagnosis was 19.7 months, and 96.2% of patients died during follow-up. The median length of survival after date of surgery for recurrence (SR) was 12.77 months (Table 1).
The impact of clinical features in the prognosis of newly diagnosed and recurrent GBM were evaluated using univariate survival analysis. KPS ≤ 70 is an adverse prognostic factor for OS with HR (Hazard Ration) of 9.6 (95% CI 1.065-86.874) when compared to KPS of > 70. GTR showed a trend towards improved OS compared with biopsy only. Gender and age had no significant association with OS (Table 2). None of these were associated with survival after recurrence (Table 3).
Immune cell infiltration is greater at recurrence than in newly diagnosed GBM
To explore the immune cell infiltration features in GBM patients, we analyzed TILs and macrophages as well as PD-L1 expression in tumor tissues from newly diagnosed and recurrent GBM patients. Additionally, we used two brain tissue samples from patients undergoing non-lesional epilepsy surgery as control. Compared to GBM tissue at primary diagnosis, the recurrent tumors showed significantly higher levels of infiltration of CD4+T cells, CD8+ T cells, and CD68+ macrophages, whereas there was no significant difference in the CD8 to CD4 ratio observed between the two groups (Fig. 1A, B, C, D). Moreover, we found the expression level of PD-L1 greatly increased in recurrent GBM compared with tumors at initial diagnosis (Fig. 1E). The density of infiltrating TILs in epilepsy patients is numerically similar compared to recurrent GBM and higher than GBM at initial diagnosis but GBMs have much more CD68+ macrophages than epilepsy specimen. On average, about one third of all cells in GBM tissue are CD68+ macrophage in recurrent GBM specimen (Fig. 1D). However, no statistically significant difference was found, likely due to the insufficient number of patients in the epilepsy group. There was no significant correlation between OS and the infiltration of CD4+ T cells, CD8+ T cells, CD68+ macrophage, as well as the CD8/CD4 ratio and PD-L1 expression level for patients at initial diagnosis (Fig. 2A, B, C, D, E).
Intra-tumoral heterogeneity is one of the most prominent features of GBM, which is responsible for the different responses to treatment as well as the tumor relapse after the treatment . Tumor samples from different patients may carry even greater heterogeneity caused by the phenotypic and genetic differences of tumor cells between individual patients. Therefore, we identified and selected five patients who had tumor samples from both the initial diagnosis and recurrence (paired samples) and studied the changes of infiltrating immune cells between the paired samples.
Consistent with the unmatched patients, the number of CD4+, CD8+ TIL as well as CD68+ macrophage were robustly higher at recurrence. The expression of PD-L1 also increased in recurrent GBM compared with matched samples at initial diagnosis (Fig. 3). No significant differences were observed in the CD8/CD4 ratio between the two groups. Notably, one patient with a remarkably increased CD8/CD4 ratio in the recurrent tumor had the highest overall survival (2411 days) among all GBM patients included in this study. This patient, a 73-year-old man, was first diagnosed with GBM in February 2008, with two recurrences in August 2011 and July 2013, and passed away in September 2014. The CD8/CD4 ratios of the tumor tissue samples collected at his first diagnosis and twice recurrence were 0.65, 2.64 and 2.31, respectively, with quite drastic increase at recurrence.
Increased densities of infiltrating immune cells in recurrent GBM with CD4+ TIL predominant in the perivascular region
To learn more about the distribution patterns of tumor-infiltrating immune cells in GBM, we further looked at their infiltrations at perivascular and intratumoral regions separately. There were significantly increased densities of CD4+ TIL, CD8+ TIL, macrophage, and PD-L1 positive cells in recurrent GBM compared with GBM samples at initial diagnosis in intratumoral regions (Fig. 4B). Interestingly, the densities of CD4+ TIL and CD8+ TIL infiltration in the perivascular regions were also robustly increased in recurrent GBM tumor tissues than in initially diagnosed GBM, while there was no difference in the distributions of macrophage and PD-L1+ cells in perivascular spaces between initially diagnosed and recurrent specimens (Fig. 4B). The infiltration of CD4 TIL was almost twice denser than CD8 TIL in initially diagnosed GBM tumors across the entire tumor area. The predominance of CD4+ over CD8+ TIL was also seen in recurrent GBM, with the difference being more significant in the perivascular region (Fig. 4B).
In order to compare the immune cell infiltration levels between the intratumoral region and perivascular region, we used a 0-3 scoring system to evaluate the cells infiltration pattern (Fig. 4C). There were significant higher number of TIL in the intratumoral regions than in perivascular areas at both initial diagnosis and recurrence. The distribution of macrophage and PD-L1+ cells were also dramatically higher in the intratumoral regions than perivascular areas at either initial diagnosis or recurrence (Fig. 4A, C).
We further look into the pattern of TILs in recurrent GBM parenchymal tissues and their correlations with the prognosis after tumor recurrence, ie, the event-free survival (EFS) which is defined as the time interval from the recurrence when the tissues were obtained to the next recurrence or death, whichever came first. Interestingly, both the infiltrating CD4 and CD8 T cell densities predicted worse EFS with trend toward significance in recurrent specimens and significantly when all specimens were combined (Table 4). No significant correlations to EFS of CD8 to CD4 ratio (CD8/CD4), macrophage infiltration (CD68) or PD-L1 expression levels in recurrent only or combined tumor tissues were identified.