Mechanisms of primary and acquired resistance to immune checkpoint inhibitors in advanced non-small cell lung cancer: A multiplex immunohistochemistry-based single-cell analysis.

Objective: Immune checkpoint inhibitors (ICIs) have become a key therapeutic modality for advanced non-small cell lung cancer (NSCLC), but most patients experience primary or acquired resistance to these drugs. We here explored the mechanisms underlying both types of ICI resistance by analysis of the tumor immune microenvironment (TME).
Materials and Methods: Four patients who experienced a long-term response to ICI treatment (progression-free survival [PFS] of ≥12 months) followed by disease progression, after which a rebiopsy was immediately performed (cohort-A), as well as four patients who experienced early tumor progression during ICI treatment (PFS of <9 weeks, cohort-B) were enrolled in this retrospective study. The pretreatment TME was evaluated by 16- or 17-color multiplex immunohistochemistry (mIHC)-based spatial profiling at the single-cell level for both cohorts. In cohort-A, changes in the TME after disease progression during ICI treatment were also investigated by mIHC analysis and transcriptomic analysis.
Results: Pretreatment tumor tissue from cohort-B manifested poor infiltration of tumor-reactive CD8 ⁺ T cells characterized by CD39 and CD103 expression or by programmed cell death-1 expression, implicating insufficient recognition of tumor cells by CD8 ⁺ T cells as a mechanism of primary ICI resistance. Analysis of the paired tumor specimens from cohort-A revealed various changes in the TME associated with acquired ICI resistance, including substantial infiltration of myeloid-derived suppressor cells and M2-type tumor-associated macrophages without a marked decline in the number of tumor-reactive CD8 ⁺ T cells; a decrease in the number of tumor-reactive CD8 ⁺ T cells; and an apparent decrease in neoantigen presentation by tumor cells.
Conclusion: The presence of intratumoral tumor-reactive CD8 ⁺ T cells may be a prerequisite for a long-term response to ICI treatment in advanced NSCLC, but it is not sufficient for cancer cell eradication. Various TME profiles are associated with acquired ICI resistance, suggesting that patient-specific strategies to overcome such resistance may be necessary.
Competing Interests: Declaration of Competing Interest KH reports research funding regarding the submitted work from KANAE Foundation for the Promotion of Medical Science, SGH Foundation, and YOKOYAMA Foundation for Clinical Pharmacology; other research funding from AstraZeneca K.K. and MSD K.K.; honoraria from AS ONE Corporation, Bristol-Myers Squibb Company, MSD K.K., and Ono Pharmaceutical Co., Ltd., outside the submitted work; TT reports research funding from Ono Pharmaceutical Co., Ltd.; honoraria from Ono Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., Merck Sharp & Dohme Corp, and Bristol Myers Squibb Co., Ltd., outside the submitted work; HK reports grants from Eisai Co., Ltd., Kobayashi Pharmaceutical. Co., Ltd., and Taiho Pharmaceutical Co., Ltd.; consulting fees from Daiichi-Sankyo Co., Ltd.; honoraria from Bristol-Myers Squibb Company, Eli Lilly Japan K.K., Ono Pharmaceutical Co., Ltd, Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd, Otsuka Pharmaceutical Co., Ltd., Bayer Yakuhin Ltd., MSD K.K., Chugai Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., Yakult Pharmaceutical Industry, and Taiho Pharmaceutical Co., Ltd., outside the submitted work; MT has received honoraria from Chugai Pharmaceutical Co., Ltd., AstraZeneca K.K., Bristol-Myers Squibb Company, Novartis Pharma K.K., Ono Pharmaceutical Co., Ltd., Boehringer Ingelheim Japan Inc., and Takeda Pharmaceutical Co., Ltd., outside the submitted work; KY reports research funding from Daiichi Sankyo Co., Ltd.; honoraria from AstraZeneca K.K., Chugai Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Takeda Pharmaceutical Co., Ltd., Merck Sharp & Dohme K.K., and Nippon Kayaku; patents US20200115467 and US20200061031 pending, outside the submitted work; KT has received honoraria from AstraZeneca K.K., Eisai Co., Ltd., Ono Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., Bristol-Myers Squibb Company, MSD K.K., Takeda Pharmaceutical Co., Ltd., and Kyowa Hakko Kirin Co., Ltd., outside the submitted work; HH reports grants from AstraZeneca K.K., Astellas Pharma Inc., MSD K.K., Ono Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., Novartis Pharma K.K., Pfizer Japan Inc., Bristol Myers Squibb Co., Ltd., Eli Lilly Japan K.K., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Merck Serono Co., Ltd. / Merck Biopharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Limited., AbbVie Inc, inVentiv Health Japan, ICON Japan K.K., GRITSONE ONCOLOGY.INC, PAREXEL International Corp., Kissei Pharmaceutical Co., Ltd., EPS Corporation., Syneos Health., Pfizer R&D Japan G.K., A2 Healthcare Corp., Quintiles Inc. / IQVIA Services JAPAN K.K., EP-CRSU CO., LTD., Linical Co., Ltd., Eisai Co., Ltd., CMIC Shift Zero K.K., Kyowa Hakko Kirin Co., Ltd, Bayer Yakuhin, Ltd, EPS International Co., Ltd., and Otsuka Pharmaceutical Co., Ltd.; honoraria from Amgen K.K., AstraZeneca K.K., Boehringer Ingelheim Japan Inc., Bristol-Myers Squibb Company, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan K.K., Guardant healthcare, Kyorin Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., MSD K.K., Novartis Pharmaceuticals K.K., Ono Pharmaceutical Co., Ltd., Shanghai Haihe Biopharm, Taiho Pharmaceutical Co., Ltd., Pfizer Japan Inc., and Takeda Pharmaceutical Co., Ltd., outside the submitted work; KN reports grants from Boehringer Ingelheim Japan Inc., West Japan Oncology Group, Thoracic Oncology Research Group, North East Japan Study Group, Clinical Research Support Center Kyushu, Nichirei Biosciences Inc., and Eli Lilly Japan K.K.; consulting fees from SymBio Pharmaceuticals Ltd., Solasia Pharma, Eli Lilly Japan K.K., and Otsuka Pharmaceutical Co., Ltd.; honoraria from Boehringer Ingelheim Japan Inc., AstraZeneca K.K., Chugai Pharmaceutical Co., Ltd., Novartis Pharma K.K., Eisai Co., Ltd., MSD K.K., Bristol-Myers Squibb Company, Ono Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Guardant Health, Inc., Eli Lilly Japan K.K., Amgen K.K., Merck Biopharma Co., Ltd., Roche Diagnostics K.K., Yakult Honsha Co., Ltd., Takeda Pharmaceutical Co., Ltd., Janssen Pharmaceutical K.K., FUJIREBIO Inc., outside the submitted work; KN reports grants from MSD K.K., ICON Japan K.K., Takeda Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Bristol-Myers Squibb Company, Taiho Pharmaceutical Co., Ltd., PAREXEL International Corp., Ono Pharmaceutical Co., Ltd., Sysmex Corporation, A2 Healthcare Corp., AbbVie Inc., Chugai Pharmaceutical Co., Ltd., Boehringer Ingelheim Japan Inc., SymBio Pharmaceuticals Limited., AstraZeneca K.K., EPS International Co., Ltd., CMIC Shift Zero K.K., Eisai Co., Ltd., Mochida Pharmaceutical Co., Ltd., Kissei Pharmaceutical Co., Ltd., GlaxoSmithKline K.K., Kyowa Hakko Kirin Co., Ltd., EPS Corporation Co., Ltd., Daiichi Sankyo Co., Ltd., Quintiles Inc. / IQVIA Services JAPAN K.K., Pfizer Japan Inc., Bayer Yakuhin Ltd., Otsuka Pharmaceutical Co., Ltd., PRA HEALTHSCIENCES, Merck Biopharma Co., Ltd., Covance Japan Inc., Medical Research Support, Sanofi K.K., Syneos Health., Pfizer R&D Japan G.K., PPD-SNBL K.K., and Japan Clinical Research Operations; consulting fees from Astellas Pharma Inc., Takeda Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Pfizer Japan Inc., KYORIN Pharmaceutical Co., Ltd., and Ono Pharmaceutical Co., Ltd.; honoraria from Astellas Pharma Inc., AstraZeneca K.K., MSD K.K., Ono Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Taiho Pharmaceutical Co., Ltd., Bristol Myers Squibb Co., Ltd., Medical Review Co., Ltd., Thermo Fisher Scientific K.K., KYORIN Pharmaceutical Co., Ltd., Nikkei Business Publications, Inc., Takeda Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Merck Biopharma Co., Ltd., Novartis Pharma K.K., Pfizer Japan Inc., CareNet, Inc, YODOSHA CO., LTD., Hisamitsu Pharmaceutical Co., Inc., MEDICUS SHUPPAN Publishers Co., Ltd., YOMIURI TELECASTING CORPORATION., NANZANDO Co., Ltd., Roche Diagnostics K.K., Nippon Kayaku Co., Ltd., Bayer Yakuhin, Co., Ltd., Kyowa Hakko Kirin Co., Ltd., AbbVie Inc., Amgen K.K., 3H Clinical Trial Inc., Nichi-Iko Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., and Medical Mobile Communications Co., Ltd., outside the submitted work. The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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