The advent of immune checkpoint inhibitors (ICIs) has driven the progress of tumor therapy, transformed the treatment landscape of multiple tumor types and provided clinicians with new therapeutic strategies (1, 2). However, since the overall response rate of ICI therapy is around 20%, only a small proportion of patients benefit from this treatment (3). Recent findings demonstrate that some chemotherapeutics given using specific administration schedules display positive immunological effects that contribute to tumor eradication (4–6). Therefore, there is a growing interest in combining ICIs with chemotherapy to enhance the efficacy of immunotherapy.

In established tumor models, gemcitabine induces tumor cell apoptosis and thereby elicits antitumor immunity by increasing the amount of antigen cross-presentation (7–10). Gemcitabine also enhances CD8⁺ T cell and natural killer (NK) cell-mediated anti-tumor immunity through depletion of myeloid-derived suppressor cells and regulatory T cells (11). Some preclinical data provide a rationale for combining gemcitabine with ICIs (12, 13); however, the clinical efficacy of gemcitabine combined with ICIs for treating solid tumors is not as expected. For example, in the first-line treatment of advanced non-small-cell lung cancer, progression-free survival (PFS) and overall survival (OS) in patients treated with gemcitabine combined with nivolumab did not increase compared with single drug use (14). In pancreatic ductal adenocarcinoma, gemcitabine plus ipilimumab achieves a similar objective response rate (ORR) to gemcitabine monotherapy (15). The same results were reported in metastatic disease or locoregional nasopharyngeal carcinoma patients treated with camrelizumab (SHR-1210) along with gemcitabine (16).

Multiple factors may contribute to the lack of a significant increase in the efficacy of combination therapy. Among them, the decrease in the number and quality of T cells caused by standard-dose gemcitabine may be an important factor (17). Anti-PD-1 monotherapy efficacy is limited by the number and specificity of tumor-directed T cells, and defects in either will lead to an inability to reach a critical threshold to elicit immune infiltration, especially for tumors with low mutational burdens (18). To alleviate these adverse effects on T cells, researchers have begun exploring the effect of low-dose gemcitabine in anti-tumor immunity, and investigations into the combination of low-dose gemcitabine and PD-1 for treating tumors are underway. There is some evidence that tumors exposed to low-dose gemcitabine secrete interferons that can help mature dendritic cells, which ultimately enhance T-cell responses (19). Additionally, low-dose gemcitabine selectively inhibits tumor-associated myeloid-derived suppressor cells in mice bearing 4T1 mammary carcinomas, which is beneficial to the amplification of tumor-targeting T cells (20). Moreover, a systematic review revealed that, compared with standard-dose infusion for gemcitabine, prolonged low-dose infusion is an effective and well-tolerated regimen for multiple solid tumors (21).

These data provide a strong rationale for the use of a regimen including low-dose gemcitabine along with inhibitors of the PD-1 pathway. Thus, we hypothesized that the combination of low-dose gemcitabine with PD-1 inhibitors would improve the response rate to ICI therapy and reduce the incidence of adverse reactions.

Our study demonstrates that the use of low-dose gemcitabine along with a PD-1 inhibitor is safe and feasible in patients with advanced solid malignancies and results in an impressive 29.5% ORR and 62.3% DCR. The median PFS was 4.3 months and the median OS was 15.0 months.

Parikh et al. (22) suggested pembrolizumab along with standard dose gemcitabine as a second or third-line therapy in patients with advanced or metastatic UC had an ORR of 33%, DCR of 50%, and median PFS of 3.7 months. In the IMvigor130 study, patients with metastatic UC received first-line atezolizumab plus platinum chemotherapy (1,000 mg/m²), gemcitabine plus carboplatin or cisplatin, and obtained a median PFS of 8.2 months and a median OS of 16.0 months (23). In our study, ORR and DCR in patients with urothelial carcinoma were 33 and 100%, respectively, and the median PFS and OS were 24.0 and 32.9 months, respectively. These data suggest that a higher response rate may be achieved with low-dose, rather than standard-dose, gemcitabine plus a PD1 inhibitor. For cervical cancer, the phase II NCT02257528/NRG-GY002 trial evaluated the efficacy and safety of nivolumab in 26 persistent or recurrent patients. In that trial, the ORR was only 4%, and the estimated PFS and OS at 6.0 months were 16 and 78.4%, respectively (24). In our study, ORR in patients with cervical cancer was 60%; median PFS and OS were 6.8 and 14.7 months, respectively; these results suggest that the efficacy of ICIs combined with chemotherapy is much better than that of single-agent ICIs.

Different doses of gemcitabine have different effects on peripheral lymphocytes. In a mouse model, gemcitabine at high doses resulted in complete reductions in CD4⁺, CD8⁺, and B220⁺ cell numbers (25), whereas low doses selectively reduced myeloid-derived suppressor cell levels without affecting lymphocyte counts (20). In a clinical setting, gemcitabine caused statistically significant decreases in the absolute numbers of CD3 and CD20 lymphocytes when administered in routine doses (26). Unlike in other studies, the chemotherapy drug gemcitabine used in our study was administered at a lower dose; the results showed that this dose did not affect the absolute numbers of T, B, and NK cells (Supplementary Figure 3). The regimen proposed here does not reduce the number of lymphocytes; ensuring sufficient immune cells may be an important reason why the combined regimen can exert a superior therapeutic effect.

In our previous study, we found that elevated B-cell levels in the peripheral blood were associated with poor prognosis of anti-PD-1-based immunotherapy, which indicates that B cells may affect the efficacy of immunotherapy with PD-1 inhibitors (27). To evaluate whether there is a correlation between B cells and the efficacy of combination therapy, we detected pre-treatment levels of various lymphocytes, including B cells, in patients within the PR, SD, and PR groups; however, no significant differences were observed (Supplementary Figure 2), suggesting that in addition to B cells, other factors can also affect the efficacy of immunotherapy.

Our study has several limitations: first, the retrospective design may have permitted selection bias; second, the non-interventional design may have led to heterogeneity in patient management and poor data quality; third, the heterogeneity of tumor types and small numbers for each type of tumor limits the conclusions that can be drawn for a given tumor type (but, the larger sample size was valuable for stratified analysis to identify the efficiency of combination therapy, especially for cervical and urologic cancers); fourth, data related to the positive predictive biomarkers for PD-1 inhibitor therapy were not presented because of incomplete baseline data. To address these limitations, we are currently working on a prospective clinical trial on a fixed tumor (NCT04331626) to confirm these findings.

Low-dose gemcitabine combined with PD-1 inhibitors offers a novel option for treatment in patients with advanced malignancies and appears particularly promising for post-operative patients. Our results suggest that patients with urological and cervical cancers benefit significantly and that this specific combination therapy is well tolerated. Future studies with larger sample sizes will help further verify these results.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by Medical Ethics Committee of Henan caner hospital (2019090507). The patients/participants provided their written informed consent to participate in this study.

HH, LP, and BZ performed experiments, analyzed data, and wrote the manuscript. BZ and BT edited the manuscript. HH, YY, XZ, LZ, XF, TL, LH, and PQ were responsible for patient management. LC, XY, YL, WW, ZY, LR, and LG analyzed data. LH, QG, and ZW designed and supervised the study. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (Grant No. 81972690) and the Medical Science and Technology Research Project of Health Commission of Henan Province (YXKC2021007).The funding bodies played no role in the design of the study and collection, analysis, or in the interpretation of data and writing the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.