Introduction

Colorectal cancer (CRC) is the third most prevalent malignant tumor globally and ranks second in terms of mortality rate1, serving as a leading cause of cancer-related deaths. Notably, approximately 25% of CRC patients are diagnosed with metastatic disease, with nearly half of them progressing to metastatic colorectal cancer (mCRC)2. The survival rate after mCRC diagnosis indicates that less than 20% of patients survive for 5 years3. Immune checkpoint blockade (ICB) therapy represents a pivotal area in cancer immunotherapy, focusing on immune checkpoint molecules such as programmed death-1 (PD-1) or programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). These molecules are crucial for regulating immune responses, overcoming tumor-induced immunosuppression, and promoting more effective tumor suppression4,5. The benefits of immune checkpoint inhibitors (ICIs) are particularly pronounced in patients with immunoinflammatory tumors, such as melanoma, non-small cell lung cancer, and renal cell carcinoma, as well as in tumors with an increased neoantigen load and high mutation burden6,7. In contrast, ICIs exhibit limited efficacy in patients with immunologically “cold” tumors and those with a low mutation burden 8. Microsatellite instability-high (MSI-H) CRC was among the first tumors to demonstrate the benefits of ICIs9, and ICIs have since become the standard treatment for MSI-H CRC.

However, the efficacy of ICIs in microsatellite stable (MSS) CRC is limited. Compared to MSI-H tumors, MSS CRC exhibits distinct molecular mechanisms and a tumor microenvironment (TME) characterized by significantly lower tumor mutation burden and more pronounced immunosuppression and immune exhaustion 10. Nevertheless, a series of explorations have been conducted on ICIs in MSS CRC, and combination strategies with tyrosine kinase inhibitors (TKIs), vascular endothelial growth factor inhibitors (anti-VEGF agents), and chemotherapy are being tested in clinical trials. Previous studies have demonstrated that anti-angiogenic therapy can reverse the immunosuppressive tumor microenvironment by normalizing blood vessels and inducing T-cell infiltration and activation11. The phase II CheckMate 9X8 study compared the efficacy of nivolumab combined with mFOLFOX6 plus bevacizumab versus standard treatment (mFOLFOX6 plus bevacizumab) in the first-line treatment of mCRC patients12.Subgroup analysis revealed that patients with RAS-mutant/MSS (CMS3) mCRC could benefit from the addition of nivolumab to mFOLFOX6 plus bevacizumab. The KEYNOTE-651 study evaluated the long-term safety and efficacy of pembrolizumab combined with oxaliplatin, leucovorin, and fluorouracil (mFOLFOX7 regimen) for first-line treatment or pembrolizumab combined with irinotecan, leucovorin, and fluorouracil (FOLFIRI regimen) for second-line treatment of MSS/pMMR mCRC. The results suggested that pembrolizumab combined with chemotherapy is safe and effective for both first-line and second-line treatment of MSS/pMMR mCRC13.

Based on these findings, chemotherapy combined with bevacizumab and anti-PD-1 immunotherapy has a solid theoretical foundation and has demonstrated preliminary efficacy and safety in patients with MSS mCRC. Given the current lack of clinical data on the second-line treatment of advanced colorectal cancer with the combination of immunotherapy, anti-angiogenic drugs, and anti-PD-1 immunotherapy, we conducted a multicenter retrospective cohort clinical study to explore the safety and efficacy of this triplet therapy in the second-line treatment of advanced colorectal cancer patients.

Methods

Study design and participants

This study was a dual-center retrospective cohort study conducted at Peking University First Hospital and Jilin Cancer Hospital.Inclusion Criteria: Histologically or cytologically confirmed unresectable metastatic colorectal cancer (Stage IV according to the AJCC 8th edition) with measurable lesions based on the RECIST criteria. Progression after prior first-line standard two-drug chemotherapy regimen with or without targeted therapy. Proficient mismatch repair (pMMR) or MSS, BRAF wild-type. Patients receiving chemotherapy combined with bevacizumab and anti-PD-1 immunotherapy or chemotherapy combined with bevacizumab. Not having undergone radiotherapy or having completed radiotherapy more than 4 weeks ago. ECOG score ≤ 2. Exclusion Criteria: Patients with deficient mismatch repair (dMMR) or microsatellite MSI-H, or BRAF mutations. Presence of symptomatic brain metastases. Uncontrolled active infection. Dysphagia, intractable vomiting, or known drug absorption disorders. Patients with symptomatic or high-risk obstruction, bleeding, or perforation, or those who have undergone intestinal stent placement to relieve intestinal obstruction. In most cases, the chemotherapy regimen comprised an oxaliplatin-based doublet (FOLFOX: folinic acid, 5-fluorouracil, oxaliplatin, or CAPEOX: capecitabine and oxaliplatin) or a topoisomerase inhibitor (FOLFIRI: folinic acid, 5-fluorouracil, irinotecan). Anti-PD-1 immunotherapy included penpulimab, pembrolizumab, sintilimab, tislelizumab, and toripalimab. The anti-angiogenic agent was bevacizumab.

This study was conducted in compliance with the postulates of the Declaration of Helsinki and approved by the Ethics Committee of Peking University First Hospital and Jilin Cancer Hospital. The requirement for patient approval or informed consent was waived by the Human Ethics Committee of Peking University First Hospital and Jilin Cancer Hospital, owing to the retrospective nature of the study and because the analysis used anonymous clinical data.

Follow-up

Follow-up data were collected through hospital records, telephone interviews, outpatient visits, and rehospitalizations. The data included age, sex, ECOG status, primary tumor location, number of metastatic sites, percentage reduction in tumor volume, PFS, overall survival (OS), follow-up duration, and survival status. The last follow-up date was in December 2024. PFS was the primary outcome, defined as the time from enrollment to the first documented disease progression according to RECIST version 1.1, or death from any cause, whichever occurred first. Secondary outcomes included OS, objective response rate (ORR), disease control rate (DCR), and safety evaluation. OS was calculated from the date of enrollment to the date of death from any cause, with censored cases defined by the last available follow-up. ORR was defined as the proportion of patients with a best objective response of complete response (CR) or partial response (PR) according to RECIST criteria (version 1.1). DCR was defined as the proportion of patients with CR, PR, or stable disease (SD) according to RECIST criteria (version 1.1). Treatment-related adverse events were evaluated according to the Common Terminology Criteria for Adverse Events version 4.0.

Biomarker analysis

Pre- and on-treatment blood samples were collected from all patients to measure neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), systemic immune-inflammation index (SII), and platelet-to-inflammatory index (PIV). Tumor samples, including both fresh and archival biopsies and resections, were obtained at baseline and subjected to multiple immunohistochemical staining, including CD3, CD4_FOXP3, CD8_PD1, CD20, CD56, CD68, PanCK, PD1, PDL1, and CD68_iNOS. Exploratory analysis was conducted to compare these markers in the PFS-long (≥ 16 weeks) group versus the PFS-short group.

Statistical analysis

The log-rank test was used as the primary analysis for comparison between metastasis subgroups. Differences between efficacy response subgroups were analyzed using the (nonparametric) Wilcoxon’s rank sum test (Mann–Whitney U-test), whereas differences between pre-treatment and post-treatment were analyzed using Wilcoxon’s signed-rank test. 95% confidence intervals (CIs) for the response rate were calculated using the Clopper-Pearson method. All reported P values are two-sided, and a P value < 0.05 was considered statistically significant. All statistical analyses in our study were performed using R software (version 4.4.2).

Results

Patients

Between January 2021 and December 2024, 29 patients were enrolled, including 21 males (72.4%) and 15 patients aged over 60 years (51.7%). There were 9 patients with right-sided colon cancer (31%). The vast majority of patients had undergone primary tumor surgery (26/29, 89.7%). Nineteen patients had more than two metastatic lesions (65.5%), among whom 19 had liver metastases (65.5%) and 18 had lung metastases (62.1%). Twenty patients had RAS mutations (69%). Please refer to Table 1 for details.

Table 1 Baseline characteristics of the patients in the cohort.
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Effectiveness

As of December 2024, the median follow-up period was 13.3 months. The median PFS was 7.8 months, and the median OS was 28.8 months (Fig. 1). Based on RECIST version 1.1, the ORR was 17.2%, and the DCR was 79.3%. Among them, 5 patients achieved PR, 18 patients SD, and 6 patients experienced PD. The best percent change in the target lesion diameter from baseline for all 25 patients is shown in Fig. 2A. Six patients were still receiving maintenance therapy at the data cut-off day (Fig. 2B). The median PFS was 6.77 months (95% CI 2.87–10.5) in patients with liver metastasis and 14.7 months (6.9–NA) in patients with non-liver metastasis (HR 3.684, log-rank test P = 0.0118, Fig. 3A). The median PFS was 8.03 months (95% CI 5.7–NA) in patients with lung metastasis and 6.9 months (4.17–NA) in patients with non-lung metastasis (HR 0.6865, log-rank test P = 0.387, Fig. 3B).

Fig. 1
figure 1

(A) KM curve of original cohort (PFS). (B) KM curve of original cohort (OS).

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Fig. 2
figure 2

(A) Waterfall plot of the best percent change in target lesion diameter from baseline. (B) Swimmer plots of patients.

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Fig. 3
figure 3

(A) KM curve of patients with or without liver metastasis. (PFS). (B) KM curve of patients with or without lung metastasis.

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Safety

Treatment was generally well tolerated. Treatment-related adverse events (TRAEs) and immune-related adverse events are summarized in Table 2. At the data cut-off date (December 2024), the most common TRAEs of all grades were anemia (18/29, 62.1%), leukopenia (12/29, 41.4%), and hand-foot syndrome (10/29, 34.5%). The most frequent grade 3 or 4 TRAEs were anemia (2/29, 6.9%) and increased triglycerides (1/29, 3.4%). No grade 5 adverse events occurred during the study.

Table 2 Treatment-emergent adverse events (TEAEs) in the 29 patients of the dataset.
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Biomarker analysis

Dynamic changes in the LMR (p = 0.049), SII (p = 0.0045), and PIV (p = 0.0045) before and after treatment were statistically significant. However, there were no statistically significant differences in the Neutrophil-to-Lymphocyte Ratio (NLR) (p = 0.06) and Platelet-to-Lymphocyte Ratio (PLR) (p = 0.44) between the two groups (Fig. 4A–E).

Fig. 4
figure 4

Kaplan–Meier curves for PFS. Progression-free survival according to the alteration of NLR (A) after 2 cycles of immunotherapy, PLR alteration (B), LMR alteration (C), SII alteration (D) and PIV alteration (E).

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Baseline biopsy or operative specimens were obtained as required from patients for exploratory biomarker assessment. There were no statistically significant differences in CD3, CD8_PD1, CD20, CD56, CD68, PanCK, PD1, PDL1, and CD68_iNOS between the PFS-long (≥ 16 weeks) group and the PFS-short group. However, there were statistical differences in CD4_FOXP3 and CD68_CD163 between the two groups, with the group having better efficacy showing fewer CD4_FOXP3 (p = 0.0033) and CD68_CD163 (p = 0.00007) (Figs. 5, 6).

Fig. 5
figure 5

Association between baseline expression of tumor environment surface proteins and partial response or stable disease for ≥ 16 weeks (Immunohistochemistry results).

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Fig. 6
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Multiplex immunohistochemistry analysis of the tumor immune microenvironment.

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Discussion

Late-stage microsatellite stability (MSS) colorectal cancer accounts for approximately 95% of all colorectal cancers14. Traditional immune checkpoint inhibitors as monotherapy have shown poor efficacy, and in recent years, there have been explorations into various combination therapies. The main research directions for immune-based combination therapies in the later lines of treatment for late-stage MSS colorectal cancer are as follows: 1. Anti-angiogenic agents combined with immunotherapy (e.g., regorafenib + PD-1 inhibitors) have been shown to enhance efficacy, but the benefit is limited to a very small subset of patients. Subgroups such as those without liver metastases, Asian patients, and those with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 tend to benefit more, suggesting that this combination strategy requires precise patient stratification15. 2. Other strategies to enhance the efficacy of PD-1 inhibitors include combining them with other immune checkpoint inhibitors (CTLA-4 antibodies16, LAG-3 antibodies17, epigenetic modulation (HDAC inhibitors to remodel the immune microenvironment)18 and adenosine pathway blockade (dual antagonists of A2aR and A2bR adenosine receptors)19. 3. For patients with BRAF V600E mutations, the combination of targeted therapy against EGFR-BRAF-MEK with PD-1 antibodies has shown promising efficacy and warrants further exploration with larger sample sizes20.

The focal points for optimizing and exploring first-line immunotherapy in late-stage MSS colorectal cancer are as follows:1. High-intensity chemotherapy (e.g., FOLFOXIRI) combined with bevacizumab and PD-1/PD-L1 inhibitors (as in the AtezoTRIBE study) can prolong PFS, but the absolute benefit is small. The question of whether "more chemotherapy drugs are better" remains controversial, and the risks of toxicity and immune cell damage need to be weighed21. 2. Precise patient selection: For example, patients with high T-cell infiltration may benefit more22; POLE/POLD mutations (hypermutated phenotypes), although rare, are sensitive to immunotherapy 23. 3. Building on the "chemotherapy + anti-angiogenic" regimen, adding CTLA-4 antibodies16 (to enhance T-cell priming), CD47 antibodies24,25 (to block the "don’t eat me" signal and activate macrophages), or other immune checkpoint inhibitors (e.g., TIM-3)26 may break through the current efficacy ceiling. Our study shows that chemotherapy plus bevacizumab and anti-PD-1 immunotherapy has demonstrated promising efficacy in the treatment of MSS mCRC, with manageable adverse reactions.

Studies have shown that the LMR is associated with the infiltration of immune cells in the primary tumor microenvironment of gastric cancer27. Li et al.28 demonstrated that neoadjuvant chemotherapy can significantly reduce some inflammatory biomarkers in patients with locally advanced gastric cancer. When combined with factors such as age, gender, tumor location, and clinical stage in different clinical scenarios, high NLR before neoadjuvant chemotherapy, preoperative anemia, and significant changes in LMR often indicate a poor prognosis. Therefore, LMR and its changes before and after chemotherapy are important indicators for evaluating the efficacy and prognosis of neoadjuvant chemotherapy in gastric cancer. Some studies have shown a close association between higher SII levels and poorer prognosis in bladder cancer patients, indicating that SII is an important prognostic predictor for bladder cancer patients29. A retrospective study by Fucà et al. found that the PIV can guide the treatment decision-making process and the development of new first-line treatment strategies for melanoma. Compared with other biomarkers based on complete blood count (CBC), PIV has significant advantages in predicting the prognosis of melanoma patients30. Studies have also found that PIV has irreplaceable value in the prognosis of small cell lung cancer and breast cancer patients, guiding treatment decisions and helping clinicians select advantageous populations for treatment, thereby further improving the individualization and precision of cancer treatment31,32,33. Our study found that dynamic changes in the LMR, SII, and PIV before and after treatment can predict the efficacy of immune combination therapy.

Currently, immunotherapy for MSS colorectal cancer has transitioned from the era of “ineffective monotherapy” to that of "enhanced combination therapy." In the future, it will be necessary to drive innovative combinations through precise subtyping, achieve the transformation of “cold tumors” into “hot tumors” through cross-mechanism synergies (targeted therapy + immunotherapy + microenvironment modulation) and frontier technology-assisted stratification. The role of regulatory T cells (Tregs) in colorectal cancer (CRC) is controversial. In some studies, FOXP3 + T-cell infiltration indicates a poorer prognosis34. Other studies have shown that there are non-Treg cells expressing Foxp3 in the immune microenvironment of CRC, which have stronger infiltration ability than the former, exert pro-inflammatory effects, and do not express the naive Treg cell marker CD45RA, with unstable FOXP3 expression35. High baseline mRNA expression of macrophages (CD68 +, CD163 +) is significantly associated with partial response (PR) or stable disease (SD) at 16 weeks of immune combination therapy in colorectal cancer15. This study shows that patients with low baseline expression of CD4FOXP3 and CD68CD163 before immune combination therapy have a better prognosis.

We acknowledge that our multicenter retrospective cohort study has several limitations.

First, it had a single-arm design without a control group, which may induce some selection bias. Second, this trial had a limited sample size and the results need to be validated in larger studies. The maturity of PFS and OS data in the retrospective analysis was relatively low, and patient management was also relatively poor, introducing certain biases.

In summary

In the second-line treatment of MSS mCRC, chemotherapy combined with bevacizumab and anti-PD-1 immunotherapy demonstrated a high objective response rate (ORR), disease control rate (DCR), and a manageable safety profile in MSS mCRC. This suggests that chemotherapy combined with bevacizumab and anti-PD-1 immunotherapy is a promising combination strategy that is expected to provide more clinical benefits. Exploratory biomarker assessment analysis revealed that patients with reduced LMR, SII, and PIV after immune combination therapy have a better prognosis. Patients with fewer CD4FOXP3 and CD68CD163 in tumor tissue before treatment have a better prognosis.