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. 2016 Jun 30;5(9):e1204507.
doi: 10.1080/2162402X.2016.1204507. eCollection 2016.

Intratumoral expression levels of PD-L1, GZMA, and HLA-A along with oligoclonal T cell expansion associate with response to nivolumab in metastatic melanoma

Hiroyuki Inoue  1 , Jae-Hyun Park  1 , Kazuma Kiyotani  1 , Makda Zewde  1 , Azusa Miyashita  2 , Masatoshi Jinnin  2 , Yukiko Kiniwa  3 , Ryuhei Okuyama  3 , Ryota Tanaka  4 , Yasuhiro Fujisawa  4 , Hiroshi Kato  5 , Akimichi Morita  5 , Jun Asai  6 , Norito Katoh  6 , Kenji Yokota  7 , Masashi Akiyama  7 , Hironobu Ihn  2 , Satoshi Fukushima  2 , Yusuke Nakamura  1
Affiliations

Intratumoral expression levels of PD-L1, GZMA, and HLA-A along with oligoclonal T cell expansion associate with response to nivolumab in metastatic melanoma

Hiroyuki Inoue et al. Oncoimmunology. .

Abstract

Immune checkpoint inhibitors blocking the interaction between programmed death-1 (PD-1) and PD-1 ligand-1 (PD-L1) are revolutionizing the cancer immunotherapies with durable clinical responses. Although high expression of PD-L1 in tumor tissues has been implicated to correlate with the better response to the anti-PD-1 therapies, this association has been controversial. In this study, to characterize immune microenvironment in tumors, we examined mRNA levels of immune-related genes and characterized T cell repertoire in the tumors of 13 melanoma patients before and after nivolumab treatment. We found that, in addition to the PD-L1 (p = 0.03), expression levels of PD-1 ligand-2 (PD-L2), granzyme A (GZMA) and human leukocyte antigen-A (HLA-A) in the pre-treatment tumors were significantly higher (p = 0.04, p = 0.01 and p = 0.006, respectively) in responders (n = 5) than in non-responders (n = 8). With nivolumab treatment, tumors in responders exhibited a substantial increase of CD8, GZMA and perforin 1 (PRF1) expression levels as well as increased ratio of TBX21/GATA3, suggesting dominancy of helper T cell type 1 (Th1) response to type 2 (Th2) response. T cell receptor β (TCR-β) repertoire analysis revealed oligoclonal expansion of tumor-infiltrating T lymphocytes (TILs) in the tumor tissues of the responders. Our findings suggest that melanoma harboring high PD-1 ligands (PD-L1 and PD-L2), GZMA and HLA-A expression may respond preferentially to nivolumab treatment, which can enhance Th1-skewed cellular immunity with oligoclonal expansion of TILs.

Keywords: Metastatic melanoma; PD-1; T-cell receptor sequencing; nivolumab; oligoclonal T cell expansion.

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Figures

Figure 1.
Figure 1.
Comparison of expression levels of multiple immune-related genes in pre-treatment tumors between responders and non-responders. (A–D) Expression levels of PD-L1 (A), PD-L2 (B), GZMA (C), and HLA-A (D) genes in surgically resected pre-treatment tumors in responders and non-responders are shown. The y-axis indicates expression level of each gene relative to that of GAPDH. (E) The expression ratios of CD8/FOXP3, CD8/CD4 and TBX21/GATA3 are presented. (F) Predictive scores for individual patients were calculated based on expression levels of PD-L1, GZMA, and HLA-A which were significantly higher in the tumors of responders compared with those of non-responders. Horizontal lines represent the means. The Mann–Whitney U test was used to examine statistical significance.
Figure 2.
Figure 2.
Nivolumab treatment-induced changes in the intratumoral expression of immune-related genes. Expression levels of CD8 (A), GZMA (B), PRF1 (C), and expression ratio of TBX21/GATA3 (D) in pre-treatment (pre) and post-treatment (post) tumors are presented. The y-axis indicates expression level of each gene relative to that of GAPDH. Red circles and black squares indicate tumor samples from responders and non-responders, respectively. Blue arrows indicate a patient who manifested severe adverse events (SAE). Asterisk indicates a patient whose expression of TBX21 in the tumor of post-treatment was undetectable. The Mann–Whitney U test was used to examine statistical significance.
Figure 3.
Figure 3.
Nivolumab treatment-induced changes of TCR-β repertoires in tumors. Pie charts illustrate distribution of unique CDR3 sequences of TCR-β, detected in paired tumor samples of pre- and post-treatment, from responders (n = 4) (A) or non-responders (n = 6) (B). As each pie chart was separately colored according to CDR3s frequency ranks, the same color between pie charts does not represent an identical CDR3 sequence. Gray color zone indicates combined portion of all clonotypes with the frequencies of less than 0.5%. (C) Fold changes in the diversity index (DI) of TCR-β in tumors are shown according to the patient's response to nivolumab treatment. Horizontal lines represent the means, and the Mann–Whitney U test was used to examine statistical significance.
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