Updated Efficacy Outcomes of Anti-PD-1 Antibodies plus Multikinase Inhibitors for Patients with Advanced Gastric Cancer with or without Liver Metastases in Clinical Trials

Recently, immune checkpoint inhibitors (ICI) such as anti–programmed cell death-1 (PD-1) or programmed cell death ligand-1 (PD-L1) mAbs have become one of the standards of care for various types of cancers including advanced gastric cancer (AGC; refs. 1–8). In AGC, nivolumab, monoclonal anti-PD-1 antibody, improved survival outcomes in pivotal phase III trials such as the Asian ATTRACTION-2 study in the third-line or subsequent treatment and the global CheckMate-649 study in the first-line treatment combined with standard cytotoxic agents (8, 9). Most recently, adding pembrolizumab to trastuzumab and chemotherapy improved the response rate in the first-line treatment for patients with HER2-positive AGC in the phase III KEYNOTE-811 study (10). However, a substantial number of patients with AGC showed resistance to ICIs, highlighting the importance of the development of further combined immunotherapy.

In previous reports, inhibition of the VEGF pathway could suppress tumor growth together with the inhibition of immune-suppressive cell infiltration such as tumor-associated macrophages, regulatory T cells, and myeloid-derived suppressor cells, while increasing the mature dendritic cell fraction (11, 12). In an in vivo model, multikinase inhibitors of VEGF receptors and other receptor tyrosine kinases substantially decreased immune-suppressive cells with the enhancement of antitumor activity of PD-1 inhibitors (13–15). Indeed, we previously reported the promising antitumor activity of anti-PD-1 antibodies plus multikinase inhibitors [regorafenib plus nivolumab (REGONIVO) or lenvatinib plus pembrolizumab (LENPEM)] for AGC in early clinical trials (16, 17). Meanwhile, several preclinical and clinical studies demonstrated that liver metastases were less responsive to ICIs, presumably due to enriched immune-suppressive cells in liver metastases (18–22). We hypothesized that targeting immune-suppressive cells with multikinase inhibitors could overcome the resistance to ICIs in patients with AGC with liver metastases.

Here, we investigated updated efficacy outcomes of REGONIVO and LENPEM for AGC with or without liver metastases in these clinical trials. The impact of the presence of liver metastases on the efficacy of anti-PD-1 monotherapies in AGC was also investigated. Furthermore, comparison of the immune microenvironments between gastric primary tumors and liver metastases was conducted by multiplex IHC.

The current study examined combined efficacy outcomes with a longer follow-up in a phase Ib trial of REGONIVO (NCT 03406871) and a phase II trial of LENPEM (NCT 03609359) in patients with AGC (16, 17). The detailed methods of these trials were reported previously (16, 17). We also retrospectively reviewed the medical records of patients with AGC treated with anti-PD-1 monotherapy at the National Cancer Center Hospital East. The eligibility criteria for the anti-PD-1 monotherapy cohort were as follows: (i) an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1; (ii) histologically proven, unresectable, locally advanced or metastatic gastric adenocarcinoma; (iii) adequate bone marrow, hepatic, and renal function; and (iv) history of previous treatment with one or more regimens and at least one treatment with nivolumab or pembrolizumab from September 2017 to September 2019. Efficacy outcomes were compared between patients with liver metastases and those without liver metastases in the REGONIVO plus LENPEM cohort and the anti-PD-1 monotherapy cohort. We also additionally analyzed survival outcomes according to the presence of peritoneum metastases.

All the patients provided written informed consent prior to chemotherapy. Furthermore, patients who underwent biomarker analysis provided written informed consent for the analysis. The study protocol was approved by the Institutional Review Board at the National Cancer Center Japan. This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki.

As reported previously, molecular characteristics, such as the status of HER2, PD-L1, and mismatch repair deficiency (MMR) were analyzed with formalin-fixed paraffin-embedded (FFPE) tissue specimens from archival tissue samples if available (23, 24). IHC using a monoclonal anti-HER2 antibody [PATHWAY HER2 (4B5)] and FISH using the PathVysion HER-2 probe kit (Abbott Laboratories) were performed to assess HER2 status, and HER2 positive was defined as IHC 3 + or IHC 2 + and FISH positive. PD-L1 IHC was performed using an anti-PD-L1 mAb (Clone 28-8, 22C3, SP142, or SP263) and measured using a combined positive score (CPS), defined as the number of PD-L1–positive cells (tumor cells, lymphocytes, and macrophages) as a proportion of the total number of tumor cells multiplied by 100. MMR status was assessed by IHC using mAbs for anti-mutL homolog 1 (MLH1, ES05), anti-mutS homolog 2 (MSH2, FE11), anti-postmeiotic segregation increased 2 (PMS2, EP51), and anti-mutS homolog 6 (MSH6, EP49; Agilent Technologies), and tumors lacking either MLH1, MSH2, PMS2, or MSH6 expression were considered MMR deficient, whereas tumors that maintained expression of MLH1, MSH2, PMS2, and MSH6 were considered MMR proficient. All specimens in this study were reviewed by a pathologist (T. Kuwata).

We assessed the objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) for each cohort. Tumor response was assessed in patients with measurable lesions according to the guidelines of the RECIST version 1.1. The ORR was defined as the proportion of patients with the best overall response of complete response (CR) or partial response (PR). The DCR was defined as the proportion of patients with the best overall response of CR, PR, or stable disease (SD). The PFS was defined as the time from the patient enrollment (REGONIVO or LENPEM) or the initiation of nivolumab or pembrolizumab (anti-PD-1 monotherapy cohort) until the date of disease progression or the date of death from any cause. The OS was defined as the time from the patient enrollment (REGONIVO or LENPEM) or the initiation of anti-PD-1 monotherapy until the date of death from any cause. Statistical comparison of the ORR and DCR according to the presence of liver metastases was performed using Fisher exact test. The PFS and OS were estimated by the Kaplan–Meier method, compared according to the presence of liver metastases using Cox proportional hazards models, and presented as HRs with 95% confidence intervals (CI). PFS and OS in anti-PD-1 monotherapy cohort were analyzed using multivariate Cox regression analyses. Confounders in multivariate analysis included age (≥65 vs. <65), sex (male vs. female), ECOG PS (0 vs. 1), histology (intestinal vs. diffuse), numbers of previous chemotherapy (2 vs. ≥3), metastatic site (lymph node, liver, lung, and peritoneum), HER2 status (positive vs. negative), MMR (deficient vs. proficient), and CPS (≥10 vs. <10). Statistical analyses were done using SAS software (version 9.4).

Next, we performed multiplex IHC (mIHC) to compare the immune microenvironments between gastric primary tumors and liver metastases using biopsy or surgical tumor samples in patients with AGC. Gastric primary tumor and liver metastases were obtained from the same patients without prior chemotherapy at the National Cancer Center Hospital East, from January 2009 to May 2019. The FFPE blocks of tumor samples were sliced into 4-μm-thick sections onto adhesion microscope slides (Matsunami). The tissue slides were deparaffinized and rehydrated for mIHC staining. Antigen retrieval and staining followed the protocol of Opal 7-Color IHC Kits (AKOYA Biosciences) provided by the manufacturer. Images were acquired using a Vectra 3 System (PerkinElmer). The protein expression levels of CD4 (Clone 4B12), CD8α (Clone C8/144B), CD206 (Clone CL0387), CD11b (Clone D6×1N), FOXP3 (Clone 236A/E7), and cytokeratin (Clone AE1/AE3) were assessed. Cell phenotyping was identified by inForm Tissue Analysis Software (AKOYA Biosciences), and cell density was calculated from the average density of at least three regions of interest (682 μm × 510 μm/region). The density of the indicated immune cells was plotted using a heatmap and was normalized by Z-score transformation. The feature of infiltrating immune cells was analyzed by principal component analysis, and the differences between organs were analyzed by an analysis of similarities (ANOSIM).

The data generated in this study are available upon request from the corresponding authors.

Baseline patient characteristics for each cohort are shown in Table 1. Each characteristic of REGONIVO and LENPEM is also available in Supplementary Table S1. Most patients had an ECOG PS of 0 in the REGONIVO plus LENPEM cohort, while about half of the patients had an ECOG PS of 0 in the anti-PD-1 monotherapy cohort. Liver metastases were observed in 28 (52%) and 46 (34%) patients in the REGONIVO plus LENPEM cohort and anti-PD-1 monotherapy cohort, respectively. Peritoneum metastases were frequently observed in the anti-PD-1 monotherapy cohort compared with the REGONIVO plus LENPEM cohort: 85 (63%) and 15 (28%) patients, respectively.

The data cutoff for the updated efficacy analysis was December 15, 2020, with a median follow-up of 14.0 months (range, 2.0–31.3 months). All patients (n = 54) had measurable lesions. The ORR and DCR were 57% (31/54 patients) and 94% (51/54 patients) in the overall population (Table 2). The ORR was 46% in patients with liver metastases and 69% in patients without liver metastases (P = 0.0938; Table 2; Fig. 1A). The median PFS was 7.0 months (95% CI, 5.4–9.7) in the overall population (Fig. 1B). The median PFS was 7.8 months (95% CI, 4.3–13.7) with liver metastases and 6.9 months (95% CI, 4.6–9.8) without liver metastases [HR: 0.817 (95% CI, 0.462–1.444), P = 0.4813; Fig. 1C]. The median OS was 15.6 months (95% CI, 10.6–24.5) in the overall population, with 35 patients (65%) being already deceased (Fig. 1D). The median OS was 15.6 months (95% CI, 9.8–not reached) with liver metastases and 15.5 months (95% CI, 7.2–22.2) without liver metastases [HR: 0.723 (95% CI, 0.371–1.411), P = 0.3398; Fig. 1E]. Patients with peritoneum metastases had shorter PFS and OS compared to those without (Supplementary Fig. S1A and S1B). Clinical outcomes according to HER2, MMR, and CPS were available in Supplementary Table S2. Each efficacy of REGONIVO and LENPEM is also available in Supplementary Table S3 and Supplementary Fig. S2 and S3.

The data cutoff was September 30, 2020, with a median follow-up of 27.6 months (range, 0.7–57.3 months). Of 136 patients, 109 patients (80%) had measurable lesions. The ORR and DCR were 17% (18/109 patients) and 37% (40/109 patients) in the overall population (Table 2). The ORR tended to be lower in patients with liver metastases than in those without liver metastases (9% vs. 22%, P = 0.1144; Table 2; Fig. 2A). The median PFS was 1.9 months (95% CI, 1.6–2.3) in the overall population (Fig. 2B). The PFS was significantly shorter in patients with liver metastases than in those without liver metastases [median 1.4 months (95% CI, 0.9–1.8) vs. 2.3 months (95% CI, 1.9–3.7), HR: 1.856 (95% CI, 1.276–2.698), P = 0.0009; Fig. 2C]. The median OS was 8.7 months (95% CI, 6.0–10.3) in the overall population, with 102 patients (75%) being already deceased (Fig. 2D). The median OS was 6.4 months (95% CI, 3.8–10.9) with liver metastases and 9.0 months (95% CI, 6.7–10.9) without liver metastases [HR: 1.253 (95% CI, 0.836–1.878), P = 0.2724; Fig. 2E]. Multivariate analysis showed that patients with liver metastases had a significantly shorter PFS (HR, 2.015; 95% CI, 1.253–3.241; P = 0.0039) and tended to have a shorter OS (HR, 1.662; 95% CI, 0.961–2.872; P = 0.069) compared with those without liver metastases. Patients with peritoneum metastases had shorter PFS and OS compared with those without (Supplementary Fig. S1C and S1D).

Ten paired specimens of gastric primary tumors and liver metastases were analyzed by mIHC (Fig. 3A and B). The characteristics of the tumor-infiltrating immune cells showed a clear difference between gastric primary tumors and liver metastases (Fig. 3C). Enrichment of CD206⁺CD11b⁺ cells, which are regarded as tumor-associated macrophages, was observed in liver metastases. Principal component analysis summarized the features of the infiltrating immune cells, and an ANOSIM showed a significant difference between gastric primary tumors and liver metastases (Fig. 3D). The density of CD206⁺CD11b⁺ cells was significantly higher in liver metastases than in gastric primary tumors, while the density of CD8⁺ T cells tended to be lower in liver metastases than in gastric primary tumors (Fig. 3E). The density of FOXP3⁺CD4⁺ cells, which are regarded as regulatory T cells, was numerically higher in liver metastases compared with gastric primary tumors, although the difference was not statistically significant. The CD206⁺CD11b⁺/CD8⁺ and FOXP3⁺CD4⁺/CD8⁺ ratios were significantly higher in liver metastases than in gastric primary tumors.

In this study, we investigated the updated clinical activity of REGONIVO or LENPEM for patients with AGC in clinical trials. To the best of our knowledge, this is the first report to provide information on the efficacy of anti-PD-1 antibodies plus multikinase inhibitors with a longer follow-up for patients with AGC with or without liver metastases. Also, multiplex fluorescent IHC demonstrated the immune-suppressive microenvironment in liver metastases compared with gastric primary tumors.

In REGONIVO, with a median follow-up of 14.0 months, the median PFS and OS were 5.6 and 12.3 months in the third- or later-line setting. In LENPEM, with a median follow-up of 17.8 months, the median PFS and OS were 7.1 and 24.5 months in the first- or second-line setting. These survival outcomes seem to be better than those of standard chemotherapy in comparable treatment lines, although cross-trial comparison require careful interpretation because these are early clinical trials with a select patient population (8, 9, 25, 26). Meanwhile, REGONIVO for colorectal cancer was not as effective in North American population as in Japanese patients in REGONIVO, especially in patients with liver metastases (16, 27). Furthermore, a subgroup analysis of the phase III KEYNOTE-062 trial showed that the hazard ratio for OS (pembrolizumab to chemotherapy) was better in an Asian population than in a non-Asian population (28). Therefore, a further study would be needed to investigate the efficacy of REGONIVO or LENPEM for non-Japanese AGC patients. Currently, a phase III trial (INTEGRATEIIb; NCT 04879368) of REGONIVO compared with standard chemotherapy for AGC in the third- or later-line setting and a phase III trial (LEAP-015; NCT 04662710) of LENPEM plus chemotherapy followed by LENPEM versus chemotherapy for AGC in the first-line setting is being investigated.

In this study, both REGONIVO and LENPEM showed promising clinical activity irrespective of liver metastases status. In patients with liver metastases, the ORR was 40% in REGONIVO and 54% in LENPEM with favorable survival outcomes. Meanwhile, in the anti-PD-1 monotherapy cohort, the ORR (9% vs. 22%) and PFS (1.4 vs. 2.3 months) were worse in patients with liver metastases than those without liver metastases, which is in line with previous reports showing that ICIs are less effective in patients with melanoma or non–small cell lung cancer with liver metastases (20, 21). Although the survival benefit of nivolumab was observed in the ATTRACTION-2 trial (nivolumab vs. placebo) or the ATTRACTION-4 trial (nivolumab plus chemotherapy vs. placebo plus chemotherapy) regardless of liver metastases, retrospective studies suggest that the presence of liver metastases in AGC is associated with rapid disease progression or a lower response rate compared with other metastases (8, 29–31). Recently, it was reported that liver metastases create a systemic immune desert through an interaction between T cells and macrophages in preclinical models (19). Another preclinical study also demonstrated that antigen-specific immune suppression by activation of regulatory T cells and CD11b⁺ monocytes in liver metastases led to the systemic suppression of antitumor immunity in mouse models (18). In our study, liver metastases were associated with an abundance of immune-suppressive cells, such as tumor-associated macrophages and regulatory T cells with fewer CD8⁺ T cells, compared with gastric primary tumors. These findings from our study and previous reports suggest that targeting immune-suppressive cells by multikinase inhibitors could relieve the systemic immunosuppressive effect led by liver metastases. Also, other agents targeting immune-suppressive cells such as PGE2-receptor EP4 antagonists, TGFβ inhibitors, FLOUNT (a regulator of CCR2 and CCR5 signaling) inhibitors, and local therapies including photodynamic therapy are expected to overcome resistance to anti-PD-1 antibodies in patients with liver metastases in future studies. Meanwhile, patients with peritoneum metastases had shorter PFS and OS compared with those without in both REGONIVO plus LENPEM cohort and anti-PD-1 monotherapy cohort, in line with a previous report (32). Tumor immune microenvironment in peritoneum should also be investigated in future study.

The major limitation to the current study was the small sample size, which comprised a select population as early clinical trials. Thus, any efficacy analysis is preliminary in nature. Also, efficacy in the anti-PD-1 monotherapy cohort was investigated with a retrospective manner at a single institution. Finally, comparison of the immune microenvironments between gastric primary tumors and liver metastases by multiple immunofluorescence IHC was performed in a limited number of tumor samples.

In conclusion, the combination of anti-PD-1 antibodies plus multikinase inhibitors (REGONIVO or LENPEM) for patients with AGC showed promising antitumor activity with a longer follow-up in these clinical trials, irrespective of liver metastases. Also, an immune-suppressive microenvironment was observed by multiple immunofluorescence IHC in liver metastases compared with gastric primary tumors. These results suggest that targeting immune-suppressive cells by multikinase inhibitors could overcome the resistance to ICIs in patients with AGC with liver metastases, which should also be investigated in ongoing pivotal phase III trials of these combination immunotherapies.

A. Kawazoe reports personal fees from Daiichi Sankyo, Lilly, Ono, Taiho, Bristol Myers Squibb, Merck Serono Biopharma, Sumitomo Dainippon, and AstraZeneca outside the submitted work. H. Hara reports grants from Astellas, AstraZeneca, Amgen, Beigene, Janssen, and Bristol Myers Squibb; grants and personal fees from Bayer, Boehringer Ingelheim, Chugai, Dainippon Sumitomo, Daiichi-Sankyo, Merck Biopharma, MSD, and Ono; and personal fees from Sanofi, Taiho, Takeda, Yakult, Asahi Kasei, and Lilly outside the submitted work. T. Kojima reports grants from BeiGene Ltd., EPS Corporation, MSD K.K., Amgen Inc., Shionogi & Co., Ltd., Ono Pharmaceutical, Ltd., Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., Parexel International, Bristol Myers Squibb, and Merck Biopharma Co., Ltd, as well as personal fees from Bristol Myers Squibb, Ono Pharmaceutical Co., Ltd., Covidien Japan, Inc., MSD K.K., Taiho Pharmaceutical Co., Ltd., and Merck Biopharma Co., Ltd. outside the submitted work. M. Asayama reports personal fees from Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Merck Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., and Eli Lilly Pharmaceutical Co., Ltd. outside the submitted work. H. Bando reports personal fees from Eli Lilly Japan, Taiho Pharmaceutical, and Ono Pharmaceutical, as well as grants from Ono Pharmaceutical outside the submitted work. D. Kotani reports personal fees from Takeda, Chugai, Lilly, Taiho, Daiichi-Sankyo, Bristol Myers Squibb, Merck Biopharma, and Pfizer; grants and personal fees from Ono and MSD; and grants from Novartis, IQVIA, CMIC Shift Zero, Janssen, Syneos Health, and Servier outside the submitted work. Y. Nakamura reports grants from Taiho, Genomedia, Guardant Health, Daiichi-Sankyo, Seagen, and Roche Diagnostics; grants and personal fees from Chugai Pharmaceutical; and personal fees from Merck Biopharma and Guardant Health AMEA outside the submitted work. Y. Kuboki reports grants and personal fees from Taiho, Lilly, Takeda, Boehringer Ingelheim, and Amgen; grants from Astellas, Daiichi-Sankyo, AstraZeneca, Chugai, Genmab, GlaxoSmithKline, Incyte, and Abbie; and personal fees from Bristol Myers Squibb outside the submitted work. S. Mishima reports personal fees from Merck Serono Biopharma and grants from Roche Diagnostics outside the submitted work. M. Wakabayashi reports personal fees from Nihon Medi-Physics Co., Ltd. outside the submitted work. T. Kuwata reports personal fees from AstraZeneca, Astellas Pharma, Bristol Myers Squibb Japan, Celltrion, and MSD; grants and personal fees from Ono Pharmaceutical; and grants from Daiichi Sankyo and Roche Diagnostics Japan outside the submitted work. M. Goto reports personal fees from Daiichi-Sankyo Co., Ltd., Ono Pharmaceutical Co., Ltd., MSD K.K., Takeda Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Yakult Pharmaceutical Industry Co., Ltd., and Eli Lilly Japan K.K.; grants and personal fees from Taiho Pharmaceutical; and grants from Chugai Pharma and Nippon Kayaku outside the submitted work. T. Yoshino reports grants from Taiho, Daiichi Sankyo, Amgen, Sanofi, Pfizer, Genomedia, Sysmex, and Nippon Boehringer Ingelheim; grants and personal fees from Ono, Chugai, and MSD; and personal fees from Merck Biopharma and Bayer Yakuhin outside the submitted work. T. Doi reports grants from Lilly, MSD, Novartis, Merck Biopharma, Janssen Pharma, Boehringer Ingelheim, Pfizer, Eisai, and IQVIA; grants and personal fees from Daiichi Sankyo, Sumitomo Dainippon, Taiho, BMS, AbbVie, and Chugai Pharma; and personal fees from Takeda, Bayer, Rakuten Medical, Otsuka Pharma, Kaken Pharma, Kyowa Kirin, Shionogi, PRA Health Science, Ono Pharma, and AstraZeneca outside the submitted work. H. Nishikawa reports grants and other support from Ono Pharmaceutical, Bristol Myers Squibb, MSD, and Chugai Pharmaceutical, as well as grants from Taiho Pharmaceutical, Daiichi-Sankyo, Zenyaku Kogyo, Kyowa Kirin, Oncolys BioPharma, Debiopharma, BD Japan, Asahi Kasei, Fujifilm, Sysmex, Astellas Pharmaceutical, and Sumitomo Dainippon Pharma outside the submitted work. K. Shitara reports grants from Astellas Pharma, Daiichi Sankyo, Chugai Pharma, Medi Science, and Eisai; personal fees from Eli Lilly and Company, Bristol Myers Squibb, Takeda Pharmaceuticals, Pfizer Inc, Novartis, AbbVie Inc, GlaxoSmithKline, Boehringer Ingelheim, and Janssen; and grants and personal fees from Ono Pharmaceutical, Taiho Pharmaceutical, Merck Pharmaceutical, and Amgen outside the submitted work. No disclosures were reported by the other authors.

H. Yukami: Investigation, writing–original draft. A. Kawazoe: Conceptualization, supervision, writing–review and editing. Y.-T. Lin: Investigation, writing–review and editing. S. Koyama: Investigation, writing–review and editing. S. Fukuoka: Writing–review and editing. H. Hara: Writing–review and editing. N. Takahashi: Writing–review and editing. T. Kojima: Writing–review and editing. M. Asayama: Writing–review and editing. T. Yoshii: Writing–review and editing. H. Bando: Writing–review and editing. D. Kotani: Writing–review and editing. Y. Nakamura: Writing–review and editing. Y. Kuboki: Writing–review and editing. S. Mishima: Writing–review and editing. M. Wakabayashi: Investigation, writing–review and editing. T. Kuwata: Writing–review and editing. M. Goto: Writing–review and editing. K. Higuchi: Writing–review and editing. T. Yoshino: Writing–review and editing. T. Doi: Writing–review and editing. H. Nishikawa: Writing–review and editing. K. Shitara: Supervision, writing–review and editing.

REGONIVO was supported by Bayer HealthCare Pharmaceuticals Inc and Ono Pharmaceuticals. LENPEM was supported by Merck Sharp & Dohme. The funders of the study had no role in study design; data collection, analysis, interpretation; or writing of the report.

We thank the patients and their families, the nurses, and the investigators who participated in this study.

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