Diversity of peripheral CD8+PD-1+ T cells is a novel predictive biomarker for response to anti-PD-1 antibody treatment in lung cancer patients.
Seiji Matsumoto1) , Takaji Matsutani2), Yoshiko Fujita1), Kazutaka Kitaura2), Toru Nakamichi1), Akifumi Nakamura1), Ayumi Kuroda1), Masaki Hashimoto1), Nobuyuki Kondo1), Ryuji Suzuki2), Seiki Hasegawa1)
1) Department of Thoracic Surgery, Hyogo College of Medicine, Hyogo, Japan

2) Repertoire Genesis Incorporation, Osaka, JapanBackgroundPFS

Anti-PD-1 antibodies (nivolumab) are effective in thetreatment of many cancers, including malignant
melanoma, non-small cell lung cancer (NSCLC), renal
cell cancer, and head and neck squamous cell carcinoma.
Immune checkpoint inhibitors (ICIs) are only effective
in around 20% of patients, which saw a demand for thedevelopment of biomarkers that predict a therapeutic
response before administering treatment. As thebiomarkers intratumoral PD-L1 expression and tumor
mutation burden (TMB) both require tumor tissue
biopsy, there is now a demand for safer and lessinvasive biomarkers.
Peripheral and tumor CD8+PD-1+ T cells shareneoantigen-specific T-cell receptors (TCRs), and arepresumed to act as effector T cells with an antitumor
effect at the tumor site. We analyzed the diversity interms of TCR α and β repertoires on peripheral
CD8+PD-1+ T cells and examined therelationship between this diversity andtherapeutic effect of nivolumab.

Fig.3 Representative 3D plots of TRB repertoire in Responder (left) and Non-responder (right) of lungcancer patient
Percentage usage of combinations of TRBV and TRBJ were represented by 3D plots. Responder showed
diverse TCR repertoire while Non-responder had higher clonality of TCR repertoire.

OSP=0.01

20.6

P < 0.05

403020100Responder Non-Responder

0.004

P < 0.05

0.4
0.2
0.0

Responder Non-Responder

DE50 (TCR)

0.003
0.002

Fig. 6 PFS and OS
Kaplan-Meier curves of PFS and OS comparing Responder withNon-responder.

0.001
0.000

Responder Non-ResponderP < 0.01

Responder Non-Responder

Fig.4 Comparison of diversity between Nivolumab responder and Non-responder (TCR α diversity )TCR α diversity was significantly higher among responders (n=6) than non-responders (n=6) based on Shannon
index, Simpson index and DE50 (P < 0.05, P < 0.05, P < 0.01, respectively).
6P < 0.01

420Responder Non-Responder

Inverse Simpson (TCR)Normalized Shannon (TCR )

600.6

P < 0.01

40200Responder Non-Responder

0.0025

P < 0.01

0.40.20.0

DE50 (TCR)Diversity Index

Shannon (TCR)Fig.2 Comparison of PD-1 positive cell between Nivolumab
responder and Non-responder
Six of 12 patients responded to treatment. Upon comparing these
responders (CR, PR) with non-responders (SD, PD), there were
no differences in the proportion of PD-1+ in CD8+ T cells andthe proportion of CD8+PD-1+ T cells in gated lymphocytes.

50

Diversity Index

Results

Normalized Shannon (TCR)Diversity Index40

Fig1. Study design and flow of biomarker test with NGSbased TCR repertoire analysis

P < 0.05

Diversity Index

Diversity Index

6Inverse Simpson (TCR )

Diversity Index

Shannon (TCR)Diversity Index

This study used patients administered nivolumab after
exhibiting no response to chemotherapy for recurrence
following l surgery. Peripheral blood mononuclear cells
were collected from patients before administration ofnivolumab.
CD8+PD-1+ T cells were subjected to FACS sorting,
NGS-based TCR repertoire analysis was performed byRepertoire Genesis Inc., and TCR diversity wasevaluated statistically. CT scan was performed during
week 12 of treatment and used to determine response tonivolumab. This study was approved by the EthicalCommittee of Hyogo College of Medicine.

P=0.01

Diversity IndexMethods

Non-responder

Responder0.0020
0.00150.00100.00050.0000

Responder Non-ResponderP < 0.01

Responder Non-Responder

Fig.5 Comparison of diversity between Nivolumab responder and Non-responder (TCR β diversity )TCR β diversity was also significantly higher among responders (n=6) than non-responders (n=6) based on
Shannon index, Simpson index and DE50 (all P < 0.01).

Conclusions
TCR repertoire analysis was performed on CD8+PD-1+
T cells in easily-obtainable peripheral blood before
nivolumab treatment in patients with NSCLC, andnivolumab was observed to be effective in patients withhigh TCR diversity. This result indicates the TCR
diversity of peripheral CD8+PD-1+ T cells is effective asa predictive biomarker for response to ICI therapy. In thefuture, we intend to analyze TMB and neoepitopes using
surgical specimens of these patients and determine theefficacy of this biomarker for cancers other than NSCLC.
Corresponding author: Seiji Matsumoto
smatumo@hyo-med.ac.jp
AACR Annual Meeting 2019
April, 2019 Atlanta, GA, USA

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