T-cell invigoration to cell-free DNA ratio associated with anti-PD-1 response in gastric or gastro-esophageal junction adenocarcinoma: VOYAGER trial.

Introduction
Gastric cancer is the sixth most common neoplasm and the third highest cause of cancer deaths (GLOBOCAN 2020 data).1
Nivolumab is a fully human immunoglobulin G4 (IgG4) monoclonal anti-human programmed cell death protein 1 (PD-1) antibody that inhibits tumor growth by blocking the binding of PD-1 to its ligands programmed death-ligand 1 (PD-L1) and PD-L2, thereby enhancing proliferation, activation, and cytotoxic activity of cancer antigen-specific T cells.2 The ATTRACTION-2 trial reported that nivolumab significantly improved the prognosis of gastric cancer patients compared with placebo [median overall survival (OS) 5.32 months for nivolumab versus 4.14 months for placebo; hazard ratio (HR) 0.63, 95% confidence interval (CI) 0.50-0.78, P < 0.0001]3 as third-line or later treatment. However, only 11% of patients in the nivolumab group responded, and the 6-month progression-free survival (PFS) rate was ∼20%. Therefore, increasing research has been conducted to identify and develop predictive biomarkers for nivolumab efficacy. A subgroup analysis of the ATTRACTION-2 trial demonstrated that PD-L1 expression, blood neutrophil-to-lymphocyte ratio, and serum sodium levels could serve as predictive biomarkers for the efficacy of nivolumab monotherapy.4 Identifying biomarkers associated with other mechanisms may further deepen the understanding of immune checkpoint inhibitor (ICI) treatment efficacy and improve therapeutic outcomes.
Anti-PD-1 therapy restores and activates nonresponsive cancer antigen-specific T cells, resulting in an antitumor effect. In patients with melanoma, correlation of T-cell reinvigoration with tumor burden and impact of reinvigoration/tumor burden ratio on clinical outcome for pembrolizumab therapy have been reported.5 This suggests that both host immune status and tumor burden are important in predicting the efficacy of anti-PD-1 therapy, which may be more useful for patients with low tumor burden and who have T cells that easily recover from exhaustion.
Therefore, we conducted the phase II, multicenter, single-arm VOYAGER study (jRCTs071190025) with the hypothesis that the ratio of activated lymphocytes/tumor volume is also associated with nivolumab efficacy in patients with gastric or gastro-esophageal junction adenocarcinoma (GGEJA). Since the ratio would be higher in patients with low tumor volume, we conducted early introduction of nivolumab before progressive disease (PD) according to RECIST version 1.1 in patients with GGEJA who had achieved complete (CR)/partial response (PR) or stable disease (SD) during paclitaxel ± ramucirumab therapy. In addition, blood samples were collected to analyze the number of activated lymphocytes before and at 2, 4, and 6 weeks after treatment to nivolumab. Furthermore, we investigated cell-free DNA (cfDNA) as a surrogate marker of tumor burden before nivolumab administration.

Patients and methods

Study design
This was a multicenter, single-arm, phase II study of early induction of nivolumab monotherapy in GGEJA patients after demonstrating tumor response or SD according to RECIST version 1.1 during prior chemotherapy and before tumor progression. This clinical trial protocol was approved by the Kyushu University-certified institutional review board for clinical trials and approved by the director of each participating site. The names of all participating facilities are presented in the Supplementary Material, available at https://doi.org/10.1016/j.esmogo.2025.100148. The study was registered in the Japan Registry of Clinical Trials (jRCTs071190025) and conducted in accordance with the Declaration of Helsinki, the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects, and the Clinical Trial Acts in Japan. The first patient was enrolled on 12 September 2019 and the last patient on 10 February 2021. All patients provided written informed consent before enrollment. Simplified inclusion and exclusion criteria are shown as follows. The full version is provided in the Supplementary Material, available at https://doi.org/10.1016/j.esmogo.2025.100148.
Patients were eligible if they: (i) were aged ≥20 years when providing consent; (ii) had histologically confirmed GGEJA; (iii) had presence of measurable lesions according to RECIST version 1.1; (iv) had prior chemotherapy with fluoropyrimidine (±platinum or trastuzumab, if applicable) and taxane for ≥28 days; (v) best response according to imaging assessment before treatment would be CR/PR or SD according to RECIST version 1.1; (vi) met any of the following conditions during pretreatment or within 12 weeks of the date of final administration of taxane: (1) imaging assessment reveals tumor enlargement, (2) poorer laboratory results (e.g. tumor marker levels), (3) worsening clinical signs, such as worsening symptoms (as determined by the patient’s primary physician), (4) pretreatment is discontinued because the patient is unable to tolerate medication (in any event, a patient who is deemed to have PD in pretreatment, based on imaging assessment according to RECIST version 1.1, will not be enrolled); (vii) had no prior exposure to ICIs and had an Eastern Cooperative Oncology Group performance status of 0-2; (viii) had adequate organ function and were likely to survive ≥3 months post-enrollment.
Participants were excluded if they: (i) had severe comorbidities such as uncontrolled infections, cardiac conditions, or autoimmune diseases requiring systemic immunosuppressants; (ii) had active brain metastases, severe pulmonary conditions, or significant pleural effusion/ascites requiring treatment; (iii) had received transfusions, hematopoietic growth factors, or radiotherapy within 14 days before enrollment; (iv) were pregnant, nursing, or had active multiple cancers; (v) were deemed unsuitable for the study by the investigator.
The primary endpoint was the rate of PFS at 6 months. Secondary endpoints included safety, OS, response rate (RR), time to treatment failure, and duration of response.
The PFS rate at 6 months was defined as the percentage of patients without RECIST-assessed PD or death from any cause at 24 weeks (168 days) from the date of enrollment. OS was defined as the length of the date of enrollment to death. PFS was defined as the length of the date of enrollment to disease progression (including death). Time to treatment failure was defined as the period from the date of enrollment to the earliest date of protocol treatment discontinuation, disease progression, or death. The duration of response was defined as the time from the onset of response to disease progression or death in patients who achieve CR or PR. Safety was evaluated by the worst grade of adverse event observed during the entire course according to the National Cancer Institute’s (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0.

Treatment plan
Patients received nivolumab 240 mg/body every 2 weeks with no dose modification implemented. Adverse events and causal relationship to protocol treatment were assessed by the investigator for each treatment cycle. Tumors were evaluated by RECIST version 1.1 using computed tomography or magnetic resonance imaging at a frequency of every 8 weeks and confirmed by the investigator at each participating site.

Sample collection
Peripheral blood was obtained from 42 patients, in EDTA-2K tubes before treatment and before each nivolumab infusion every 2 weeks for 6 weeks. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll gradient and stored using standard protocols (−80°C). Peripheral blood was obtained from 39 patients before nivolumab introduction, with a volume of 8 ml per patient, using whole blood collection tubes (Cell-Free DNA BCT, Streck, La Vista, NE) for cfDNA extraction. cfDNA concentration was measured with a TapeStation 4200 (Agilent, Santa Clara, CA) and a NanoDrop (ThermoFisher, Waltham, MA). cfDNA concentration values from the TapeStation measurements were used for all statistical analyses.

Measurement of activated lymphocytes in peripheral blood (PBMC)
Activated lymphocytes were defined as Ki67+ PD-1+ CD8+ T cells.4 Since Ki67+ is considered to be human leukocyte antigen (HLA)-DR+ CD38+, HLA-DR+ CD38+ PD-1+ CD8+ T cells were defined as activated lymphocytes.5,6 Regarding the measurement of PD-1, after administration of nivolumab, nivolumab strongly binds to PD-1 on the surface of lymphocytes so that the binding of other PD-1 antibodies is inhibited and cannot be detected by flow cytometry. Therefore, measurement of PD-1 requires indirect detection of PD-1 using an antibody that binds to nivolumab bound to PD-1.7 Nivolumab is of isotype IgG4, and PD-1 is measured indirectly through measuring nivolumab bound to PD-1 using an anti-IgG4 antibody. HLA-DR, CD38 and CD8 are measured directly using flow cytometry. The proportion of activated lymphocytes was defined as the percentage of HLA-DR+ CD38+ PD-1+ CD8+ T cells among the PD-1+ CD8+ T cells.
For PD-1 measurement, it was necessary to saturate PD-1 of lymphocytes with nivolumab first. According to the previous report,7 addition of nivolumab at 20 μg/ml can saturate PD-1 of lymphocytes. In this study, nivolumab 50 μl (40 μg/ml) was added to 50 μl of a cell suspension (at a cell concentration of 105/ml) to saturate the suspension.

Flow cytometry
Cryopreserved PBMC samples from pretreatment and cycles 2-4 (weeks 2-6) were thawed and stained with master mix of antibodies including fluorescein isothiocyanate anti-human HLA-DR antibody (L243, BioLegend, San Diego, CA, catalog no.: 307604), CD38 APC (HB7, Biosciences, catalog no.: 345807), and APC-Cy™7 Mouse Anti-Human CD8 (SK1, Biosciences, Franklin Lakes, NJ, catalog no.: 557834). PD-1 on specimens was detected using Mouse anti-Human IgG4 Fc Secondary Antibody (Biotin, Invitrogen, Waltham, MA, catalog no.: MH1542) and PE Streptavidin (Biosciences, catalog no.: 349023). PBMC samples were pretreated with nivolumab 40 μg/ml in vitro for 30 min at room temperature, washed twice, and stained with standard antibody mix. Cells were resuspended in phosphate-buffered saline buffer until acquisition on flow cytometry (Sony Biotechnology, Tokyo, Japan) and analyzed using FlowJo software (BD, Franklin Lakes, NJ).

Statistical analysis
Based on the results of the ATTRACTION-2 trial, which showed a PFS at 6 months of 20.2% (95% CI 15.7% to 25.1%) in the nivolumab arm, the threshold for PFS at 6 months was set at 16%. Although there are no clear data on the expected value of PFS at 6 months, we set it at 30% because it would be clinically meaningful to obtain an additional PFS of 14%. Based on the above, assuming a one-sided significance level of 10%, a power of 80%, an enrollment period of 12 months, and a follow-up period of 6 months, the number of patients required using Southwest Oncology Group’s One Arm Survival (non-parametric) was calculated to be 39 patients. Considering dropouts, the expected number of enrolled patients was set at 42 patients. Median survival was estimated using the Kaplan–Meier method, and the 95% CIs were calculated based on the Brookmeyer and Crowley method. Response was calculated based on RECIST version 1.1. All data were stored by the data center of SRL Medisearch Inc. Patients who completed at least one treatment course were included in all safety and efficacy analyses. All statistical analyses were carried out using SAS version 9.4 (SAS Institute, Cary, NC).
In biomarker analyses, associations were assessed using the Mann–Whitney U test for continuous variables, such as the proportion of activated lymphocytes and cfDNA concentration. The chi-square test was applied to analyze associations for categorical variables. OS and PFS were estimated using the Kaplan–Meier method, and survival curves were compared using the log-rank test, which is widely used for time-to-event data. The cut-off values for the proportion of activated lymphocytes and cfDNA concentration were determined using the minimum P value approach, a comprehensive method for identifying the optimal cut-off point to separate risk in continuous variables for survival analysis.8 A two-sided P < 0.05 was considered significant. Data analyses were carried out as described,9 using JMP Pro 15 software (SAS Institute) and R software version 3.3.2 (The R Foundation, Vienna, Austria).

Results

Patient characteristics
Forty-two cases were enrolled from September 2019 to February 2021, with data cut-off on 3 December 2021. The median observation period was 10.3 months. Figure 1 shows the flow of patients in this study. Since there were no ineligible patients, the full analysis population and safety analysis population were 42 patients. As of the analysis cut-off date, 34 patients had discontinued treatment, of whom 30 patients had PD, 3 had discontinued treatment due to adverse events, and 1 had discontinued protocol treatment due to surgery. Eight patients were continuing treatment at the cut-off date.
Patient characteristics are reported in Table 1. Gender was male in 28 (66.7%) patients and female in 14 (33.3%) patients, median age was 71 years (range 39-87 years), and 21 (50.0%) patients, 19 (45.2%) patients, and 2 (4.8%) patients had a performance status (PS) 0, 1, and 2, respectively. Only one patient with microsatellite instability-high was included. Fourteen (33.3%) patients had liver metastases, 25 (59.5%) had peritoneal metastases, and 16 (38.1%) had ascites on computed tomography.

Toxicity analysis
The number and percentage of adverse events during the entire treatment course are shown in Table 2. Grade ≥3 serious adverse events were mainly anorexia in four (9.5%) patients, hypokalemia in three (7.1%) patients, hyponatremia in three (7.1%) patients, and lymphocyte count decreased in three (7.1%) patients. No treatment-related deaths were observed.

Efficacy analysis
The PFS at 6 months was 35.7% (80% CI 26.4% to 45.1%), which means that this trial met the primary endpoint. The median PFS and OS were 4.0 (95% CI 2.3-5.7) months (Figure 2A) and 10.9 (95% CI 9.9-16.0) months (Figure 2B), respectively. Of 34 patients with baseline target legions, RR was 17.5% (95% CI 6.8% to 34.5%) (all confirmed PR) and the rate of SD was 41.2%. Time to treatment failure and duration of response were 3.6 (95% CI 2.2-5.1) months and 14.3 (95% CI 3.9-14.3) months, respectively.

Measurement of activated lymphocytes before and after treatment and the amount of change
In the VOYAGER study, a total of 42 patients were enrolled. PBMCs were extracted from the participants before nivolumab introduction and 2, 4, and 6 weeks after nivolumab treatment (Figure 3A). The quantification of activated lymphocytes was carried out according to the methods described in the study. Activated lymphocytes in PBMCs were defined as Ki67+ PD-1+ CD8+ T cells4 and HLA-DR+ CD38+ was considered Ki67+.5 As shown in Supplementary Figure S1A, available at https://doi.org/10.1016/j.esmogo.2025.100148, the population of activated lymphocytes exhibited an increase during the initial 4 weeks of treatment, reaching a peak, and subsequently declining by week 6 of treatment (Supplementary Figure S1B, available at https://doi.org/10.1016/j.esmogo.2025.100148). Furthermore, at the start of week 4, the percentage of Ki67+ cells within the PD1+ CD8+ T-cell subset was significantly higher compared with the PD-1− CD8+ T-cell subset (Supplementary Figure S1C, available at https://doi.org/10.1016/j.esmogo.2025.100148). These findings were consistent with a previous study conducted in melanoma patients,4 suggesting that ICI therapy can modify the local immune environment in gastric cancer. Notably, the median increase in activated lymphocytes in gastric cancer patients treated with nivolumab was 1.32-fold (Supplementary Table S1, available at https://doi.org/10.1016/j.esmogo.2025.100148). However, the previous study in melanoma reported an increase of threefold or greater in activated lymphocytes following ICI administration. Therefore, modification of immune environment induced by ICI therapy might be lower in gastric cancer than in melanoma.

Change in activated lymphocytes before and after nivolumab treatment and its association with efficacy and prognosis
In accordance with the previous melanoma study,4 our initial focus revolved around assessing the potential correlation between the escalation of activated lymphocytes induced by ICI treatment and the therapeutic efficacy of nivolumab in gastric cancer. The maximum fold change in activated lymphocytes, observed following nivolumab administration in comparison to the baseline measurements (before nivolumab introduction), was calculated for individual cases. Our findings did not reveal any significant associations between the maximum fold change and the RR (Supplementary Figure S2A, available at https://doi.org/10.1016/j.esmogo.2025.100148), the 6-month PFS (Supplementary Figure S2B, available at https://doi.org/10.1016/j.esmogo.2025.100148), or the 12-month OS (Supplementary Figure S2C, available at https://doi.org/10.1016/j.esmogo.2025.100148) among the patients who received nivolumab treatment.

Association of activated lymphocytes before and after nivolumab treatment with efficacy and prognosis
We investigated the correlation between the proportion of activated lymphocytes before and after nivolumab treatment and the efficacy and prognosis of nivolumab treatment. Notably, patients who achieved a CR or PR exhibited a significantly higher percentage of activated lymphocytes before nivolumab introduction compared with those with SD or PD (Figure 3B). Additionally, patients with 6-month PFS, which is the primary endpoint of this study, also showed a substantially higher proportion of activated lymphocytes before nivolumab introduction than patients with PD (Figure 3C). Similarly, patients with a 12-month OS displayed a significantly higher proportion of activated lymphocytes before nivolumab introduction than patients who died within 12 months (Figure 3D). Furthermore, we found that a high proportion of activated lymphocytes before nivolumab introduction were significantly associated with improved PFS (Supplementary Figure S3A, available at https://doi.org/10.1016/j.esmogo.2025.100148). However, the proportion of activated lymphocytes was not associated with OS (Supplementary Figure S3B, available at https://doi.org/10.1016/j.esmogo.2025.100148). Additionally, no significant associations were observed between the proportion of activated lymphocytes after nivolumab treatment (2, 4, 6 weeks) and the response to nivolumab treatment (Supplementary Figure S4A-C, available at https://doi.org/10.1016/j.esmogo.2025.100148).

Association of cfDNA before nivolumab treatment with efficacy and prognosis
cfDNA was extracted from peripheral blood samples before the introduction of nivolumab, and its concentration was measured. The median concentration of cfDNA in the peripheral blood was 20.32 (range 5.31-60.80) ng/ml (Supplementary Table S2, available at https://doi.org/10.1016/j.esmogo.2025.100148). The cfDNA concentration did not correlate with the response to nivolumab treatment (Figure 4A), the 6-month PFS (Figure 4B), or the 12-month OS (Figure 4C). However, it is noteworthy that patients with notably higher levels of cfDNA demonstrated significantly poorer PFS compared with other patients (Figure 4D). This observation suggests that patients with substantially elevated cfDNA levels experienced a poorer prognosis, possibly due to a larger tumor burden reflected by cfDNA.

Association of activated lymphocyte/cfDNA index before nivolumab with efficacy and prognosis
The index of activated lymphocyte/cfDNA concentration was calculated for each case to determine its association with nivolumab treatment response and prognosis. The index was defined as the percentage of activated lymphocytes before nivolumab introduction multiplied by 100 and divided by the concentration of cfDNA. The index of activated lymphocytes/cfDNA concentration was measured in 39 patients, with a median value of 2.1 (range 0.6-10.1) (Supplementary Table S3, available at https://doi.org/10.1016/j.esmogo.2025.100148). The median index was used as the cut-off value to divide the patients into a higher index and a lower index group. The higher index group had a significantly higher nivolumab responder rate, 6-month PFS, and 12-month OS than the group with a lower index (Figure 5A). Furthermore, the higher index group had a significantly better PFS than the group with a lower index (Figure 5B). Univariate and multivariate analyses revealed that a higher index tended to be independently associated with improved PFS (Supplementary Table S4, available at https://doi.org/10.1016/j.esmogo.2025.100148), although the association did not reach statistical significance due to the small number of events. Areas under the curve for the model that predicts cases of CR or PR, 6-month PFS, and 12-month OS, based on the activated lymphocyte/cfDNA index, were determined to be 0.74, 0.78, and 0.67, respectively (Supplementary Figure S5, available at https://doi.org/10.1016/j.esmogo.2025.100148). Taken together, activated lymphocyte/cfDNA index calculated before nivolumab introduction may be a predictive biomarker of nivolumab efficacy.

Discussion
This study was conducted to explore and evaluate the utility of the ICI nivolumab in the third-line treatment of GGEJA when administered at an earlier stage (with less tumor burden) than when the patient is clearly refractory to prior therapy so as to maximize the efficacy of nivolumab. We also sought to explore whether the host’s immune status and tumor burden affect the therapeutic effect of nivolumab. The results showed that the primary endpoint, 6-month PFS rate, had a point estimate of 35.7% (80% CI 26.4% to 45.1%), which exceeded the pre-specified lower threshold of 16% for a two-sided 80% CI, and thus nivolumab, administered according to the study concept, was judged to be a promising treatment option. The study also demonstrated, for the first time, that the pretreatment activated lymphocyte/cfDNA ratio may predict the prognosis of GGEJA patients and the efficacy of nivolumab. The secondary endpoints of median PFS and OS were 4.0 months and 10.9 months, respectively, the RR was 17.6%, and the median duration of response was 14.3 months. These results compare favorably with those of the ATTRACTION-2 trial, which demonstrated a median PFS of 1.61 (95% CI 1.54-2.30) months, a median OS of 5.26 (95% CI 4.60-6.37) months, an RR of 11.2% (95% CI 7.7% to 15.6%), and a median duration of response of 9.53 (95% CI 6.14-9.82) months,3 and the early induction of nivolumab treatment appears promising based on secondary evaluations. Furthermore, no adverse events exceeding 10% were observed especially in immune-related adverse events, and the only adverse events exceeding 5% were anorexia (9.5%), hypokalemia (7.1%), hyponatremia (7.1%), and lymphocyte count decreased (7.1%). There was no increase or occurrence of new adverse events compared with previously reported data.
These results demonstrate that the concept of early induction of nivolumab has the potential to improve therapeutic efficacy without increasing the risk of adverse events. The anticipated phase III trial is difficult to conduct because nivolumab is already indicated for first-line therapy (CheckMate 649).10 However, the early induction concept presented in these results does not only mean that nivolumab should be used for first-line therapy, but also indicates that host immunocompetence and tumor volume may affect the efficacy of nivolumab.
Activated lymphocytes are defined as Ki67+ PD-1+ CD8+ T cells, representing effector cells that exhibit responsiveness to ICI treatment.5 Previous research has shown that ICI treatment significantly enhances the population of Ki67+ PD-1+ CD8+ T cells in both melanoma and lung cancer, and the extent of this increase is associated with therapeutic efficacy.5,11 However, the similar association was not observed in gastric cancer. As is well known, immunosuppressive cells, such as regulatory T cells, are abundant in the tumor microenvironment of gastric cancer.12 In this study, gastric cancers exhibited a weak response to ICI treatment, with a lower enhancement of activated lymphocytes in this study. This may explain why an increase in activated lymphocytes following nivolumab administration did not correlate with the therapeutic effect of nivolumab.
Upon the administration of anti-PD-1 antibodies, the release of immunosuppression triggers activated lymphocytes to target tumor cells, thereby resulting in a powerful antitumor response.13 Therefore, the level of activated lymphocytes before nivolumab introduction was associated with therapeutic efficacy. Tumor burden was involved in the efficacy of ICI treatment in several cancers.14 In melanoma, larger tumor sizes are linked to lower treatment efficacy.5 However, tumor size cannot accurately reflect tumor burden in gastric cancer, due to the morphological features and the presence of distant metastases. According to recent reports, plasma circulating tumor DNA (ctDNA) is a surrogate biomarker for tumor burden in metastatic melanoma.15 Although ctDNA can more precisely reflect tumor burden, quantifying ctDNA is cost-effective and expeditious. It was reported that the concentration of cfDNA in peripheral blood partially reflects ctDNA and correlates with tumor progression.16 Thus, this study measured cfDNA concentration to assess its correlation with the therapeutic efficacy of ICI. Unfortunately, our findings did not show a significant correlation between cfDNA concentrations and the RR to nivolumab in gastric cancer. This may be attributed to the majority of cfDNA in peripheral blood being of normal cell origin. Interestingly, the seven patients with the highest cfDNA concentrations exhibited significantly shorter PFS compared with others, likely due to a higher proportion of ctDNA, reflecting an elevated tumor burden. Taken together, baseline values before nivolumab introduction were likely the most predictive because they capture the pretreatment immune and tumor burden status without the confounding effects of treatment.
In melanoma, the change in activated lymphocytes divided by tumor diameter has demonstrated an association with treatment response.5 In this study, we formulated a hypothesis that the combination of pretreatment activated lymphocytes and cfDNA concentration would yield a more robust predictive capacity for treatment response and prognosis. Our data showed that patients with elevated pretreatment activated lymphocytes exhibited improved efficacy of ICI treatment, while those with higher pretreatment cfDNA concentrations in peripheral blood displayed a poorer prognosis. Hence, the activated lymphocyte percentage was multiplied by 100 and then divided by the cfDNA concentration to generate a predictive index for treatment efficacy of nivolumab. Surprisingly, the group with a higher activated lymphocyte/cfDNA index exhibited a higher RR and more favorable prognosis. Thus, the activated lymphocyte/cfDNA index is considered an ideal biomarker for predicting nivolumab response and prognosis in gastric cancer, due to its cost-effectiveness and easy availability in clinical practice.
There were three limitations in the biomarker exploratory analyses. Firstly, it was challenging to precisely evaluate tumor burden since ctDNA was not utilized. However, ctDNA measurement requires expensive and time-consuming next-generation sequencing, making it impractical as a frequently utilized biomarker in daily clinical practice. A recent study suggested that the proportion of short fragments in cfDNA may enhance tumor signal detection. Therefore, assessing tumor burden using short fragment cfDNA could be a promising alternative strategy for future studies.17 Secondly, the biomarker for predicting the efficacy of nivolumab therapy, administered as a first-line regimen, has not been explored. Presently, combination of nivolumab and chemotherapy is the standard first-line regimen in gastric cancer.10 However, when this study was designed, nivolumab was employed as a late-line regimen for gastric cancer.3 Thirdly, the utility of this novel biomarker was not compared with conventional biomarkers, such as the combined positive score (CPS) of PD-L1. In the future, the relationship between the activated lymphocyte/cfDNA index combined with CPS and the therapeutic efficacy of nivolumab as a first-line regimen should be thoroughly evaluated.
In conclusion, our analysis provides valuable insights into responses to PD-1-targeted therapies among patients with gastric cancer. Moreover, activated lymphocyte/cfDNA index may be a predictive biomarker for nivolumab efficacy in gastric cancer.