PD-L1 (CD274) copy number gain, expression, and immune cell infiltration as candidate predictors for response to immune checkpoint inhibitors in soft-tissue sarcoma

Abstract

Soft-tissue sarcomas (STS) are rare malignancies that account for 1% of adult cancers and comprise more than 50 entities. Current therapeutic options for advanced-stage STS are limited. Immune checkpoint inhibitors targeting the PD-1/PD-L1 signaling axis are being explored as new treatment modality in STS; however, the determinants of response to these agents are largely unknown. Using the sarcoma data set of The Cancer Genome Altas (TCGA) and an independent cohort of untreated high-grade STS, we analyzed DNA copy number status and mRNA expression of PD-L1 in a total of 335 STS cases. Copy number gains (CNG) were detected in 54 TCGA cases (21.1%), of which 21 (8.2%) harbored focal PD-L1 CNG and that were most prevalent in myxofibrosarcoma (35%) and undifferentiated pleomorphic sarcoma (34%). In the untreated high-grade STS cohort, we detected CNG in six cases (7.6%). Analysis of co-amplified genes identified a 5.6-Mb core region comprising 27 genes, including JAK2. Patients with PD-L1 CNG had higher PD-L1 expression compared with STS without CNG (fold change, 1.8; p = 0.02), an effect that was most pronounced in the setting of focal PD-L1 CNG (fold change, 3.0; p = 0.0027). STS with PD-L1 CNG showed a significantly higher mutational load compared with tumors with a diploid PD-L1 locus (median number of mutated genes; 58 vs. 40; p = 3.6E-06), and PD-L1 CNG were associated with inferior survival (HR = 1.82; p = 0.025). In contrast, T-cell infiltrates quantified by mRNA expression of CD3Z were associated with improved survival (HR = 0.88; p = 0.024) and consequently influenced the prognostic power of PD-L1 CNG, with low CD3Z levels conferring poor survival in cases with PD-L1 CNG (HR = 1.8; p = 0.049). These data demonstrate that PD-L1 GNG and elevated expression of PD-L1 occur in a substantial proportion of STS, have prognostic impact that is modulated by T-cell infiltrates, and thus warrant investigation as response predictors for immune checkpoint inhibition.

Keywords: Amplification; CD274; PD-L1; immune checkpoint inhibition; soft-tissue sarcoma.

Figure 1.

PD-L1 CNG in STS. (A) Percentages of focal gains, 9p gains, and chromosome 9 gains in different STS subtypes in the TCGA cohort. (B) Percentages of PD-L1 CNG in the independent high-grade sarcoma cohort. (C) Overview of PD-L1 co-amplified genes on chromosome 9p. A core region including 27 genes (length, 5.6 Mb; inset) was co-amplified in more than 80% of STS with focal PD-L1 CNG (TCGA cohort).

Figure 2.

Immunohistochemical and FISH analysis of PD-L1 in 48 cases of the high-grade STS cohort. (A) Immunhistochemical staining and FISH for PD-L1. (1) PD-L1 staining of lung adenocarcinoma serving as a positive control. Note the membranous and cytoplasmic staining of tumor cells as well as membranous staining of inflammatory cells. (2) PD-L1 staining of tonsillar tissue illustrates membranous staining of lymphocytes and antigen presenting cells. PD-L1 CNG and protein expression in STS cases without (3) and with (5, and 7) CNG as detected by aCGH. Higher magnifications are presented aside (4, 6, and 8). A case of UPS (3, and 4) with balanced PD-L1 copy number on aCGH analysis showing absent PD-L1 protein expression. Representative pictures showing membranous staining of PD-L1 in a sample of DDLS (5, and 6) and UPS (7, and 8). Insets illustrates results of FISH analyses demonstrating PD-L1 copy number status (PD-L1/CEN9 dual-color FISH probe; green, PD-L1; red, CEN9; blue, DNA). Scale bars, 50 µm. (B) Pie chart showing the association of PD-L1 CNG, PD-L1 staining of tumors cells and PD-L1 staining of immune cells.

Figure 3.

PD-L1 mRNA expression in the TCGA cohort (A) and in the independent cohort of high-grade STS (B). In the TCGA cohort, sarcomas with PD-L1 CNG showed higher PD-L1 expression than STS without PD-L1 CNG (fold change, 1.8; p = 0.02). PD-L1 expression was significantly higher in MFS compared with DDLS (fold change, 2.2; p = 0.036) and SS (fold change, 12.2; p = 0.00012). Furthermore, PD-L1 expression in LMS was significantly higher than in DDLS (fold change, 1.5; p = 0.039) and SS (fold change, 8.6; p = 0.00047). In the high-grade STS cohort, LMS and UPS cases showed highest PD-L1 expression levels, whereas PD-L1 expression was significantly lower (p < 0.05) in MLS compared with all other subtypes except MPNST.

Figure 4.

Analysis of mutational load in STS (TCGA cohort). (A) The median number of mutated genes was highest in MFS, followed by MPNST, UPS, LMS, DDLS, and SS. (B) Sarcomas with PD-L1 CNG and PD-L1 copy number losses showed a significantly higher mutational load compared with tumors without PD-L1 CNG.

Figure 5.

Prognostic impact of PD-L1 CNG in STS (TCGA cohort). PD-L1 CNG were associated with shortened overall survival in (A) the entire TCGA cohort (162 patients, 56 events) and (B) in the LMS subcohort 63 patients, 22 events). Chromosome 9p gains were associated with shortened overall survival (C) in the entire TCGA cohort (162 patients, 56 events) and (D) in the LMS subcohort (63 patients, 22 events).

Figure 6.

Expression and prognostic impact of immune genes in STS (TCGA cohort). (A) Analysis of co-expression patterns and correlation of mRNA expression with overall survival in all STS. (B) Analysis of co-expression patterns and correlation of mRNA expression with overall survival in LMS. (C) Inferior prognosis of CD3Z low/PD-L1 CNG sarcomas. (D) Inferior prognosis of CD3Z low/PD-L1 CNG LMS.