Introduction

Gastric cancer is the fourth leading cause of cancer-related mortality and the fifth most common malignancy worldwide [1]. Over the past decade, there have been substantial advances in systemic chemotherapy for patients with advanced gastric cancer, especially with targeted and immunotherapeutic agents [2,3,4,5]. In particular, based on the phase 3 CheckMate-649 study [3], the combination of nivolumab, an immune checkpoint inhibitor (ICI) targeting PD-1, with chemotherapy has become the standard first-line treatment option for patients with HER2-negative advanced gastric cancer, establishing the role of ICI-based chemotherapy in the first-line setting. Recently, subsequent phase 3 trials, including RATIONALE-305 [6], ORIENT-16 [7], and KEYNOTE-859 [5], have demonstrated the survival benefits of adding ICI to chemotherapy, which solidifies the role of ICI-based chemotherapy in the first-line setting.

Several predictive biomarkers have been identified in gastric cancer, including PD-L1 combined positive score (CPS), mismatch repair protein (MMR)/microsatellite instability (MSI) status, and Epstein–Barr virus (EBV) positivity [8,9,10]. Among such predictive biomarkers, the MMR/MSI status is a good predictor of a response to ICI-based treatments in gastric cancer. Deficiency of the MMR system (d-MMR) results in the accumulation of DNA mismatches, leading to genomic instability with a high tumor mutation burden in microsatellites, resulting in the production of immunogenic neoantigens [11, 12]. This subsequently results in broad tumor-specific T cell responses, thereby leading to a favorable response to ICIs [13]. These findings were confirmed in phase 3 gastric cancer studies of first-line ICI-based chemotherapy, in which hazard ratios (HRs) for overall survival (OS) for patients with d-MMR/MSI-high tumors were reportedly 0.34–0.38 [3, 5, 14]. These results highlight the MMR/MSI status as a robust predictive biomarker for first-line ICI-based chemotherapy in gastric cancer patients.

While d-MMR/MSI-high tumors generally exhibit favorable clinical outcomes with ICI-based chemotherapy, little is known about the clinicopathologic factors that lead to differential clinical outcomes among patients with d-MMR/MSI-high tumors, possibly due to the rarity of this subset (i.e., a prevalence ranging from 2.7 to 6.6% in a metastatic setting [3, 5, 15, 16]). Indeed, not all patients with MSI-high gastric cancer exhibit excellent outcomes with ICI-based treatments. About 40% of patients with d-MMR/MSI-high tumors develop disease progression within 6 months despite first-line ICI-based chemotherapy [15]. These findings necessitate the identification of additional prognostic biomarkers among patients with d-MMR/MSI-high tumors.

In addition, although d-MMR/MSI-high tumors generally show high PD-L1 expression levels [17], some patients with MSI-high tumors have low expression of PD-L1. In fact, in the CheckMate-649 study, about one-fourth of patients with MSI-high tumors had a low PD-L1 expression level [3]. While little is known about the efficacy of ICI-based chemotherapy among d-MMR/MSI-high patients with low PD-L1 expression, this is an important issue in clinical practice given that nivolumab plus chemotherapy is only approved or reimbursed in some regions for those with a high PD-L1 level. On the other hand, as ICI agents are also available as subsequent lines of treatment, the effect of the use of ICI in different treatment lines in patients with d-MMR/MSI-high remains an unanswered question.

In this study, we aimed to investigate the clinicopathologic factors associated with the efficacy of first-line treatment with nivolumab plus chemotherapy in advanced gastric cancer patients with d-MMR/MSI-high.

Methods

Study patients

We retrospectively reviewed patient clinicopathologic characteristics through electronic medical records of patients with histologically confirmed metastatic, recurrent, or locally advanced un-resectable gastric or gastroesophageal junction adenocarcinoma with d-MMR tumors receiving first-line treatment at Asan Medical Center, Seoul, Korea. Twenty-eight consecutive patients with d-MMR tumors, as determined by the expression of MMR proteins, treated with first-line nivolumab plus chemotherapy between August 2021 and September 2023 were included as the main study population. As a control group, 68 patients with d-MMR tumors who were treated with first-line chemotherapy alone (fluoropyrimidine plus platinum doublet) between February 2015 and June 2023 were included. This study was approved by the Institutional Review Board of Asan Medical Center (IRB No. 2023-1475) and was performed in accordance with the ethical standards of the Institutional Research Committee and the latest Declaration of Helsinki.

Detection of mismatch repair protein expression and microsatellite instability status

The expression of MMR proteins, including MLH1, PMS2, MSH2, and MSH6, in tumor tissues was analyzed by immunohistochemical (IHC) staining. Tissue sections for the IHC assay were prepared from archival formalin-fixed paraffin-embedded tissues and stained for MLH1 (Novo, Newcastle, UK), MSH2 (Cell Marque, CA, USA), MSH6 (Cell Marque, CA, USA), and PMS2 (Cell Marque, CA, USA) using an automated IHC staining device (Benchmark XT, Ventana Medical Systems, AZ, USA). D-MMR tumors were defined as the loss of MLH1, PMS2, MSH 2, or MSH 6 expression. Details regarding the loss of expression of the MMR proteins in each treatment group are summarized in Supplementary Table 1.

To determine the MSI status, a polymerase chain reaction (PCR) method was employed as previously described [18], in which the primers were used to amplify mononucleotide repeats, specifically the microsatellite markers BAT-25 and BAT-26, and the dinucleotide repeats D5S346, D2S123, and D17S250. Tumors were classified as MSI-high if two or more instability markers were detected, as MSI-low if one instability marker was detected, and as microsatellite stable if none of the markers tested for MSI were detected.

PD-L1 immunohistochemistry staining

PD-L1 IHC staining was performed using the PD-L1 28-8 pharmDx kit (Dako, Santa Clara, CA, USA) with the Dako Autostainer Link-48 System, or a diluted PD-L1 22C3 antibody (1:100, Agilent Technologies) on the Ventana Benchmark Ultra using an automated immunostaining system with the OptiView DAB Detection Kit (Ventana Medical Systems). The CPS was calculated as the number of PD-L1-expressing tumor and intra- or peri-tumoral inflammatory cells divided by the number of tumor cells and then multiplied by 100. Among patients treated with nivolumab plus chemotherapy, PD-L1 CPS as assessed with the 28-8 PharmDx assay was available in 21 patients, while that assessed with the 22C3 clone was available in 26 of the study patients.

Panel sequencing and tumor mutation burden

Targeted next-generation sequencing (NGS) was performed using the NextSeq platform (Illumina, San Diego, CA) with OncoPanel AMC version 3 and version 4 as previously described [19]. The tumor mutation burden (TMB) was determined as the sum of the number of single-nucleotide variations and the indel mutations per mega-base (Mb) in the targeted region.

Treatment and response assessment

Patients were administered nivolumab (360 mg every 3 weeks or 240 mg every 2 weeks) plus chemotherapy (CAPOX [capecitabine 1000 mg/m2 twice daily, days 1–14, and oxaliplatin 130 mg/m2, day 1, every 3 weeks] or FOLFOX [leucovorin 400 mg/m2, day 1, fluorouracil 400 mg/m2, day 1 and 1200 mg/m2, days 1–2, and oxaliplatin 85 mg/m2, day 1, every 2 weeks]) or chemotherapy alone [3].

Overall tumor response evaluation was assessed every 6–8 weeks with contrast-enhanced abdominopelvic computed tomography scans. Imaging studies were performed whenever clinically indicated by a suspicion of tumor progression. Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 was used to assess the overall tumor response.

Statistical analysis

Progression-free survival (PFS) was defined as the time from the initiation of first-line treatment (index date) to the date of disease progression according to RECIST version 1.1 criteria or death, whichever occurred first. OS was defined as the time from the index to the date of death from any cause. The Kaplan–Meier method was used to estimate survival outcomes, and the log-rank test was used to compare survival outcomes between the subgroups. The chi-square test or Fisher’s exact test was used to compare categorical variables among the subgroups. A Cox proportional hazard model was used to estimate the HRs and corresponding 95% confidence intervals (CI). The cut-off point for the neutrophil-to-lymphocyte ratio (NLR) as defined by the absolute number of neutrophils divided by the absolute number of lymphocytes was determined based on the median value (3.80). Statistical analysis was performed in R version 4.2.3. A two-sided P value of less than 0.05 was considered statistically significant.

Results

Baseline characteristics of the study patients

Baseline characteristics of the included patients are shown in Table 1. Among 28 patients treated with nivolumab plus chemotherapy (the main study population), the median age was 65 years, 67.9% were male, and 92.9% had an ECOG performance status of 0 or 1. About 60% of these patients had initially metastatic disease at the time of diagnosis. Peritoneal and liver metastases were present in 19 (67.9%) and 6 (21.4%) patients, respectively.

Table 1 Baseline characteristics of the patients
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There were no significant differences in overall baseline characteristics between patients treated with nivolumab plus chemotherapy and those treated with chemotherapy alone, except for the higher proportion of patients who had received prior gastrectomy in the chemotherapy alone group (43.9% vs. 17.9%) (P = 0.030).

Among the study population, 25 patients were tested for the MSI status using the standard PCR assay: 24 had MSI-high tumors, whereas one had a microsatellite stable tumor. Panel NGS was performed in 68 patients, and the median TMB was 79.7/Mb (range, 6.6–196.9).

Overall efficacy outcomes

The median follow-up duration was 16.4 months (interquartile range [IQR] 11.4–20.7) for the nivolumab plus chemotherapy group and 58.4 months (IQR 28.3–74.3) for the chemotherapy alone group. PFS was significantly longer in patients treated with nivolumab plus chemotherapy than those treated with chemotherapy alone, with 12-month PFS rates of 69.4% vs. 20.6%, respectively (HR 0.28, 95% CI 0.13–0.56, P < 0.001) (Fig. 1a). OS tended to be better in patients treated with nivolumab plus chemotherapy, with 12-month OS rates of 80.4% vs. 65.2%, respectively (HR 0.43, 95% CI 0.18–1.03, P = 0.057) (Fig. 1b). Among patients with measurable lesion(s), the nivolumab plus chemotherapy group had a higher objective response rate (ORR) of 86.4% vs. 41.2% (P = 0.002) and disease control rate (DCR) of 100% vs. 70.6% (P = 0.004) compared to the chemotherapy alone group (Table 2).

Fig. 1
figure 1

Kaplan–Meier curves of survival outcomes for the overall study patients treated with nivolumab plus chemotherapy versus chemotherapy alone. a Progression-free survival and b overall survival

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Table 2 Overall response evaluation for the overall study patients treated with nivolumab plus chemotherapy versus chemotherapy alone
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Among patients who received chemotherapy alone, disease progression was documented in 51 patients (75.0%), 33 of whom received ICIs as a subsequent treatment. OS since the date of first-line treatment was not different between patients who were treated with first-line nivolumab plus chemotherapy and those treated with first-line chemotherapy followed by subsequent ICIs (P = 0.182) (Supplementary Fig. 1).

Factors affecting the survival outcomes of patients treated with nivolumab plus chemotherapy

The benefits of PFS and OS with nivolumab plus chemotherapy were consistently noted across the various clinicopathologic subgroups (Fig. 2). The benefits were particularly pronounced in the subgroup of patients with a lower NLR (< 3.80) when stratified by the median NLR of 3.80 (HR 0.10, 95% CI 0.02–0.40, P < 0.001 and HR 0.27, 95% CI 0.06–1.17, P = 0.061 for PFS and OS, respectively) (Fig. 2 and Supplementary Fig. 2a). However, among those with an NLR ≥ 3.80, the benefit of nivolumab plus chemotherapy was not as prominent (HR 0.58, 95% CI 0.24–1.40, P = 0.220 and HR 0.49, 95% CI 0.16–1.48, P = 0.197 for PFS and OS, respectively) (Fig. 2 and Supplementary Fig. 2b).

Fig. 2
figure 2

Forest plot of survival outcomes for the patients treated with nivolumab plus chemotherapy versus chemotherapy alone. a Progression-free survival and b overall survival

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Among patients treated with nivolumab plus chemotherapy, PFS was significantly worse in those with a high NLR (≥ 3.80) compared to those with a low NLR (< 3.80), with 12-month PFS rates of 44.4% vs. 92.3%, respectively (P = 0.025). The OS also tended to be worse for those with a high NLR (≥ 3.80), with 12-month OS rates of 67.3% vs. 92.3%, respectively (P = 0.172) (Fig. 3a). In patients treated with chemotherapy alone, there was no significant difference in PFS between those with a high NLR (≥ 3.80) and those with a low NLR (< 3.80), but OS was a significantly worse in those with a high NLR (≥ 3.80) (P = 0.047) (Supplementary Fig. 3).

Fig. 3
figure 3

Kaplan–Meier curves of progression-free survival and overall survival outcomes. a Patients treated with nivolumab plus chemotherapy stratified according to an NLR of 3.8 (median), b a PD-L1 28-8 assay combined positive score of 5 and c a PD-L1 22C3 assay combined positive score of 1

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Outcomes of patients treated with nivolumab plus chemotherapy according to PD-L1

Among patients treated with nivolumab plus chemotherapy whose PD-L1 CPS as assessed by the 28-8 PharmDx assay were available (n = 21), 12 (57.1%) had a PD-L1 CPS ≥ 5. PFS and OS were similar between those with a PD-L1 CPS (assessed by the 28-8 PharmDx assay) ≥ 5 and < 5 (P = 0.670 and P = 0.706, respectively) (Fig. 3b). Likewise, among 26 patients whose PD-L1 CPS assessed by the 22C3 clone were available, 19 (73.1%) had a PD-L1 CPS ≥ 1. There was no significant difference in PFS and OS between those with CPS ≥ 1 and CPS < 1 (P = 0.801 and P = 0.602, respectively) (Fig. 3c). Similar results were noted when patient subgroups were further stratified by two PD-L1 cut-off values (i.e., CPS of < 1, 1 to 5, and ≥ 5) for both assays (Supplementary Fig. 4).

Discussion

Limited data are currently available on treatment outcomes with ICI-based chemotherapy among patients with d-MMR/MSI-high gastric cancer. In this retrospective study, we investigated the clinicopathologic factors associated with the efficacy of first-line treatment with nivolumab plus chemotherapy in advanced gastric cancer patients with d-MMR tumors. In a real-world setting, nivolumab plus chemotherapy was associated with better efficacy outcomes, which confirms the findings from phase 3 studies [3, 20]. The nivolumab plus chemotherapy group in our current study had an ORR of 86.4% and a DCR of 100%. Of note, we found a differential impact of nivolumab plus chemotherapy in patient subgroups with different NLR levels. In addition, survival outcomes were not inferior in d-MMR patients with a low PD-L1 CPS compared to those with a high PD-L1 CPS. Our results provide practical insights into stratifying clinical outcomes of d-MMR gastric cancer patients, suggesting that additional factors, including NLR, should be taken into account for accurately predicting clinical outcomes with ICI-based treatments for d-MMR/MSI-high gastric cancer patients. To our knowledge, this is the first real-world study reporting factors associated with the efficacy of nivolumab plus chemotherapy specifically among patients with d-MMR advanced gastric cancer.

Although nivolumab plus chemotherapy was overall associated with favorable survival outcomes in patients with d-MMR tumors, a higher NLR was associated with worse survival in patients treated with nivolumab plus chemotherapy. NLR has been shown to have prognostic value in the treatment of advanced gastric cancer with either chemotherapy [21,22,23] or ICI [24, 25]. As NLR values are readily available in clinical practice, this points to the practical aspects of predicting outcomes of d-MMR/MSI-high patients with nivolumab plus chemotherapy.

Mechanistically, neutrophils, the central component of the NLR, can promote a pro-tumor microenvironment by releasing immunosuppressive and angiogenic factors, such as reactive oxygen species, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 9 [24, 26]. Decreased circulating lymphocyte levels may be associated with a reduced presence of tumor-infiltrating lymphocytes, thereby impeding antitumor T cell responses [27]. In addition, cancer patients with a high NLR level may also have elevated levels of myeloid-derived suppressor cells (MDSCs) [28,29,30], which play a critical role in immune evasion [31, 32]. Therefore, the development of novel immunotherapeutic strategies, including myeloid-targeting agents, is warranted to improve the clinical outcomes of patients with a high NLR.

While it is well-known that a high PD-L1 CPS is associated with a greater benefit of adding nivolumab [3], there have been limited data as to its impact on efficacy outcomes in patients with d-MMR/MSI-high gastric cancer. Importantly, while MSI-high gastric cancer is generally associated with high PD-L1 expression, previous studies have shown that 20–25% of MSI-high patients have low PD-L1 expression [3, 33]. However, there was no difference in efficacy outcomes for the nivolumab plus chemotherapy regimen in d-MMR patients regardless of their PD-L1 CPS in our analysis. This supports the concept that MMR/MSI status serves as a positive predictor for nivolumab plus chemotherapy regardless of the PD-L1 expression level. Moreover, our findings may be practically relevant because nivolumab plus chemotherapy has limited availability in some regions, whereas nivolumab plus chemotherapy is approved in the USA and Japan regardless of PD-L1 CPS, the EMA approved nivolumab plus chemotherapy only for those with a PD-L1 CPS ≥ 5, and in Korea, although nivolumab plus chemotherapy is approved regardless of PD-L1 status, reimbursement is limited to cases with PD-L1 CPS ≥ 5. Efforts should be made not to preclude d-MMR/MSI-high patients with a low PD-L1 CPS from benefiting from this efficacious treatment.

In our present analysis, no significant difference in OS was noted between those who were initially treated with nivolumab plus chemotherapy and those who were treated with chemotherapy alone followed by subsequent ICI. These results suggest that ICI treatment either in the first-line setting or in subsequent lines of treatment may be comparably beneficial. However, it should be considered that only 50–60% of patients who receive first-line treatment proceed to second-line treatment, and only 30–40% proceed to third-line treatment [34], suggesting a substantial proportion d-MMR/MSI-high patients may not have a chance to benefit from ICI when treated with first-line chemotherapy alone.

A question that remains with the treatment decision for d-MMR/MSI-high patients is whether chemotherapy should be included in the first-line treatment. Although potential detrimental effects of chemotherapy have been suggested in previous studies in d-MMR patients [35, 36], our previous report found no difference in survival outcomes between patients with d-MMR and proficient MMR [16]. In the post hoc analysis of the KEYNOTE-062 study [15], although there was a numerically lower trend in PFS rates and ORR with pembrolizumab monotherapy compared to pembrolizumab plus chemotherapy in the first-line treatment of patients with d-MMR/MSI-high gastric cancer, the OS rates were comparable between pembrolizumab monotherapy and those who received pembrolizumab plus chemotherapy. Additionally, in the Checkmate-649 study [37], albeit not a direct comparative analysis, the OS of patients with MSI-high gastric cancer who received nivolumab plus ipilimumab was comparable to those who received nivolumab plus chemotherapy. A recent Japanese phase II study demonstrated robust efficacy outcomes with first-line nivolumab plus low-dose ipilimumab for d-MMR/MSI-high patients, with an ORR of 62.1% and a median PFS of 13.8 months [38]. Future studies should prospectively investigate whether chemotherapy-free ICI regimens can replace ICI-based chemotherapy.

In colorectal cancer, it has been suggested that MSI-high tumors often involve locally advanced cases [39, 40]. In our previous report comparing patients with d-MMR and proficient MMR advanced gastric cancer in a palliative setting, the proportion of locally advanced un-resectable cases was not significantly different between these groups (4.2% vs. 6.4%) [16]. In the post hoc analysis of the KEYNOTE-062 study, the proportion of non-metastatic disease was 4.8% in the MSS or MSI non-evaluable group and 8.6% in the MSI-high group, respectively [15]. In our current study population, locally advanced un-resectable disease was observed in 16.7% of patients. Considering the non-significant difference in the locally advanced cases in the previous literatures [15, 16], and the relatively small sample size in our study population, it may be currently difficult to draw any definitive conclusion as to whether the proportion of locally advanced un-resectable cases are high among d-MMR in a palliative setting, suggesting the need for further studies to address this question.

In our present study population, IHC was utilized as the main method to determine the MMR status with MSI testing conducted by PCR in a proportion of patients. We found that 24 of 25 cases with d-MMR tumors had MSI-high status by the PCR assay. This finding is overall in accordance with the previous report of a high concordance between the IHC and PCR methods [41], but also indicates the imperfect aspect of the IHC method to determine the MSI status. On the other hand, as germline MMR gene testing has not previously been a part of routine clinical practice in d-MMR/MSI-high advanced gastric cancer due to the low incidence of Lynch syndrome [42], the germline MMR mutation status was not available in our study population.

There are some limitations to be considered in the current study. Its retrospective nature, the single center-based analysis, and the small number of patients may limit the interpretation and generalizability of our data. It should also be noted that whereas the nivolumab plus chemotherapy group exhibited statistically significant PFS, ORR and DCR benefits over the chemotherapy only group, OS results remained only marginally different possibly due to the effect of subsequent ICI treatment in the chemotherapy alone group and the relatively short follow-up of the nivolumab chemotherapy group. Therefore, our findings warrant further confirmation of the impact of nivolumab chemotherapy on OS with a longer follow-up. Nevertheless, given the low incidence of d-MMR/MSI-high gastric cancer and the fact that this study represents a timely report dealing with factors associated with patients with d-MMR/MSI-high gastric cancer, our results may provide practical insights into regimen selection and prognostication for these patients. Moreover, the use of a control group of patients treated with chemotherapy alone allowed us to investigate the effects of ICI-based treatment compared with chemotherapy alone.

In conclusion, nivolumab plus chemotherapy was associated with better efficacy outcomes among patients with d-MMR gastric cancer, but survival outcomes were poor even with nivolumab plus chemotherapy for those with a high NLR. Survival outcomes were not different according to PD-L1 expression levels among d-MMR gastric cancer patients treated with nivolumab plus chemotherapy. Further studies are warranted to validate our findings.