Yang et al. Anti-PD-1/CTLA-4 Dual Immunotherapy for LARC

According to the latest statistics from the National Cancer Center of China, the incidence and mortality of colorectal cancer rank 2nd and 4th among all malignant tumors, respectively. Locally advanced rectal cancer (LARC) accounts for more than 60% of all colorectal cancer cases, predominantly presenting as mid-low and locally advanced stages[1]. The standard therapeutic paradigm for LARC centered on "neoadjuvant chemoradiotherapy (NCRT) + total mesorectal excision + adjuvant chemotherapy" has significantly improved local tumor control, reducing local recurrence rate to below 5% and achieving pathological complete response (pCR) in a subset of patients[2]. Total neoadjuvant therapy has also been widely applied in the treatment of high-risk rectal cancer, with notable short-term efficacy, yet its long-term benefit remains controversial. The distant metastasis rate of rectal cancer reaches 25%-30%, representing a critical determinant of prognosis.

To further improve therapeutic efficacy, reduce distant metastasis risk, and enhance long-term prognosis, the oncological efficacy of neoadjuvant immunotherapy combined with chemoradiotherapy has been validated by an increasing number of multicenter randomized controlled trials. The 2025 Chinese Society of Clinical Oncology (CSCO) Guidelines for the Diagnosis and Treatment of Colorectal Cancer has listed neoadjuvant immunotherapy combined with chemoradiotherapy as a Grade II recommended regimen for pMMR/MSS rectal cancer. Multiple randomized controlled trials have demonstrated that neoadjuvant immunotherapy combined with chemoradiotherapy can elevate the pCR rate from 15%-20% to 30%-50%, allowing patients with clinical complete response (cCR) to adopt the "wait-and-watch" strategy for organ and function preservation[2-4]. Despite remarkable therapeutic outcomes achieved by neoadjuvant immunotherapy combined with chemoradiotherapy, several clinical research priorities and challenges remain to be addressed through further investigation, including radiotherapy field selection, optimal timing of immunotherapy initiation, clinical efficacy of different radiotherapy fractionation regimens, prediction of neoadjuvant immunotherapy response, and precise screening of eligible candidates for the "wait-and-watch" strategy.

Distant metastasis remains the primary challenge affecting long-term prognosis of LARC patients after neoadjuvant therapy[2]. In recent years, the synergistic effect between immune checkpoint inhibitors and radiotherapy has provided a novel approach to overcome this dilemma[1-4]. Existing studies have shown that radiotherapy induces immunogenic cell death, releases tumor antigens, activates the STING/cGAS pathway, promotes type I interferon secretion, and upregulates MHC-I and PD-L1 expression, thereby enhancing CD8⁺ T cell infiltration, inhibiting immunosuppressive cell populations (e.g., Treg cells, M2 macrophages), and improving the immune microenvironment[5]. Other studies indicate that hypofractionated radiotherapy (e.g., short-course radiotherapy) exerts stronger DNA damage and danger signal release effects, which not only promotes dendritic cell maturation and antigen cross-presentation but also causes less damage to peripheral immune cells, facilitating the transformation of "cold tumors" to "hot tumors"[6].

1.1 Exploration and Limitations of Long-Course Chemoradiotherapy Combined with Immunotherapy Research on neoadjuvant long-course chemoradiotherapy combined with immunotherapy was initiated earlier, mainly comprising three modes: sequential, concurrent, and induction immunotherapy, yet its overall efficacy is inferior to short-course radiotherapy combined regimens. Regarding sequential immunotherapy: The Japanese VOLTAGE-A study first confirmed that sequential nivolumab after long-course radiotherapy increased the pCR rate to 30% in MSS LARC patients, with a grade 3 adverse event rate of 11.9%[7]. The Chinese NECTAR study adopted long-course radiotherapy followed by sequential tislelizumab for LARC, achieving a 40% pCR rate in 50 pMMR patients with a 4% grade 3 adverse event rate, demonstrating the potential of drug optimization[2]. Regarding concurrent immunotherapy: The Italian AVANA study investigated concurrent avelumab administration during long-course radiotherapy, reporting a 23% pCR rate in 96 patients, suggesting that direct killing of immune cells by radiotherapy may attenuate therapeutic efficacy[3]. Regarding induction immunotherapy: The US NRG-GI002 study employed a total neoadjuvant therapy modality, consisting of FOLFOX induction chemotherapy followed by sequential long-course radiotherapy plus pembrolizumab, which yielded a 30.9% pCR rate with no significant difference compared with the control group. It was hypothesized that the immunosuppressive effect of induction chemotherapy may offset the combined efficacy[4]. Overall, the pCR rate of long-course chemoradiotherapy combined with immunotherapy generally ranges from 23% to 40%, with limitations including long study duration, high time cost, potential attenuation of overall efficacy due to radiotherapy effects on lymphoid tissues, and possible suppression of peripheral blood lymphocytes by chemotherapy.

1.2 Breakthrough Progress of Short-Course Radiotherapy Combined with Immunotherapy The UNION study adopted the regimen of "short-course radiotherapy + CAPEOX chemotherapy + camrelizumab" for mid-low LARC, achieving a pCR rate of 39.8%, which was significantly superior to the 15.3% rate of traditional long-course chemoradiotherapy, establishing the standard therapeutic status of this regimen. The TORCH study employed the total neoadjuvant therapy modality of "short-course radiotherapy + CAPOX + toripalimab", reporting an overall CR rate of 55.4% in 121 LARC patients, with approximately 25% of patients achieving cCR and safely adopting the "wait-and-watch" strategy, while the pCR rate reached 50% in patients who underwent surgery[8]. The PRECAM study innovatively applied "sequential CAPEOX + envafolimab after short-course radiotherapy", achieving a pCR rate as high as 66.7% (12/18). Short-course radiotherapy enhances tumor immunogenicity through hypofractionated dose (5×5Gy), resulting in significant chemoradioimmunotherapy efficacy. However, acute toxicity within two weeks after short-course radiotherapy is more pronounced compared with long-course radiotherapy, and its biologically equivalent dose is lower than that of long-course radiotherapy[9]. The aforementioned studies have established the role of short-course radiotherapy combined with chemotherapy and immunotherapy in the perioperative treatment of LARC, and this modality demonstrates great potential for continuous optimization to further improve efficacy and reduce toxicity.

1.3 Clinical Research Progress of Dual Immunotherapy in LARC pMMR/MSS rectal cancer exhibits low response to single-agent immunotherapy. Dual immunotherapy has become a research focus, with its core mechanism lying in the amplification of anti-tumor immune response through multi-target and multi-pathway synergistic effects, thereby improving oncological efficacy[10,11]. Theoretically, the PD-1 pathway primarily acts in the tumor microenvironment, relieving effector T cell functional suppression by blocking PD-1/PD-L1 binding[12], while the CTLA-4 pathway mainly functions during the initial activation phase of T lymphocytes in lymph nodes, inhibiting excessive immune responses[13]. The combination of these two agents enhances the breadth and depth of immune responses: CTLA-4 blockade expands the "repertoire" of immune responses, whereas PD-1 blockade enhances the killing efficacy of effector cells against tumors[14,15].

The NeoCaCRT study adopted the regimen of "sequential paromlimab and Tuvonralimab combined with mFOLFOX6 after short-course radiotherapy", achieving a 37% pCR rate and 55.6% major pathological response rate in pMMR/MSS rectal cancer. This indicates that neoadjuvant dual immunotherapy combined with chemoradiotherapy can provide clinical benefit even in pMMR/MSS rectal cancer[16]. Compared with single-agent immunotherapy or immunotherapy combined with chemotherapy, the theoretical advantage of dual immunotherapy lies in activating effector T cells while simultaneously reversing the immunosuppressive microenvironment, which is particularly critical for pMMR/MSS rectal cancer.

Despite promising signals from preclinical and some phase II studies, the application of dual immunotherapy in LARC still faces multiple challenges. First, the issues of efficacy and population adaptability remain unresolved: current clinical benefits are mainly derived from a small number of MSI-H/dMMR patients, while the actual response rate and long-term benefit of dual immunotherapy in MSS/pMMR patients lack confirmatory evidence. Second, toxicity management is more challenging: the incidence of immune-related adverse events (irAEs) is significantly higher with the combination of PD-1 and CTLA-4 monoclonal antibodies compared with single-agent therapy[6,17]. Third, the optimal combination modality and timing remain undefined: different studies show substantial variations in whether to combine radiotherapy and chemotherapy, selection of short-course or long-course radiotherapy, and timing of dual immunotherapy administration (concurrent or sequential), with no unified standard available[18]. Overall, dual immunotherapy simultaneously targeting PD-1 and CTLA-4 represents a novel approach in neoadjuvant immunotherapy for LARC, demonstrating positive trends in organ function preservation and pCR. However, larger-scale clinical trials with longer follow-up periods are required to confirm its actual value, and precise molecular typing and immunological characterization should be applied to screen eligible populations, ultimately promoting its development into a mature regimen in the comprehensive treatment of rectal cancer.

1.4 Core Controversies Regarding Dose, Field, and Timing of Immunotherapy Combined with Chemoradiotherapy Based on the above analysis, although neoadjuvant immunotherapy combined with chemoradiotherapy exhibits significant advantages in improving local tumor control, several critical issues remain to be addressed through targeted research.

• Selection strategy for short-course vs. long-course radiotherapy combined with immunotherapy: Eligible populations, optimal timing of immunotherapy initiation, and dose adjustment strategies for different radiotherapy modalities (short-course vs. long-course) combined with immunotherapy require further clarification.
• Optimal timing and duration of immunotherapy: Based on our team's three-arm randomized controlled study published in Nature Medicine in 2025, sequential immunotherapy is superior to concurrent immunotherapy, yet differences in oncological outcomes and safety between different combination modalities remain controversial, requiring identification of the optimal sequence of immunotherapy and chemoradiotherapy.
• Clinical application prospects of dual immunotherapy: Dual immunotherapy has demonstrated favorable oncological efficacy in single-arm studies, but with small sample sizes and low level of evidence, requiring further validation through clinical research.
• Optimization of anus-preservation strategy and screening of "wait-and-watch" candidates: Neoadjuvant immunotherapy combined with chemoradiotherapy significantly increases the cCR rate, enabling the "wait-and-watch" strategy[5]. How to precisely screen patients suitable for the "wait-and-watch" strategy through imaging and endoscopic evaluation to avoid overtreatment or delayed surgery remains a challenge in clinical practice.

This study focuses on the key challenges in the field of neoadjuvant immunotherapy for LARC. Through a prospective study comparing the efficacy and safety of different radiotherapy fractionation modalities, immunotherapy initiation timings, and efficacy prediction models, this study aims to define the optimal treatment sequence and duration, optimize the anus-preservation strategy and screening criteria for "wait-and-watch" populations, enhance local tumor control, reduce distant metastasis rate, improve long-term prognosis, provide individualized, highly effective, and low-toxicity treatment regimens for patients, and achieve the precise diagnosis and treatment goal of organ and function preservation.

Inclusion criteria

• Age between 18 and 80 years; ECOG performance status 0-1;
• Histopathologically confirmed rectal adenocarcinoma via colonoscopy; pMMR or MSS phenotype;
• Rectal MRI stage II/III (excluding T4b); distal tumor margin ≤ 12 cm from the anal verge;
• Willingness to comply with study procedures; consent to use tissue and blood samples for medical research purposes;
• No prior history of radiotherapy, chemotherapy, or immunotherapy;
• No immune system diseases (e.g., systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, hyperthyroidism/hypothyroidism, ulcerative colitis, autoimmune hemolytic anemia, HIV infection, etc.);
• No severe cardiac, pulmonary, hepatic, or renal dysfunction; no jaundice or gastrointestinal obstruction;
• No concurrent acute infection;
• Baseline laboratory evaluations completed as required, with results obtained within 14 days before randomization, and laboratory values meeting the following criteria (per CTCAE 5.0):

• White blood cell count ≥ 2000/μL;
• Neutrophil count ≥ 1500/μL;
• Platelet count ≥ 100×10³/μL;
• Hemoglobin ≥ 9.0 g/dL;
• Serum creatinine ≤ 1.5×upper limit of normal (ULN) or creatinine clearance > 50 mL/min (female: creatinine clearance = [140 - age (years)] × body weight (kg) × 0.85 / (72 × serum creatinine (mg/dL)); male: creatinine clearance = [140 - age (years)] × body weight (kg) × 1.00 / (72 × serum creatinine (mg/dL)));
• AST ≤ 3×ULN, ALT ≤ 3×ULN, total bilirubin ≤ 1.5×ULN;
• No psychiatric/psychological disorders affecting social function;
• Negative serum pregnancy test (blood HCG) within 1 week before randomization for women of childbearing potential;
• Women of childbearing potential must agree to use effective contraception during the study period and for 5 months after the last dose of study drug;
• Male subjects who are sexually active with women of childbearing potential must agree to use effective contraception during the study period and for 7 months after the last dose of study drug, and must refrain from sperm donation during this period.

Exclusion criteria

• Multiple primary cancers or concurrent other malignant tumors;
• Patients requiring emergency surgery due to intestinal obstruction, intestinal perforation, gastrointestinal bleeding, etc.;
• Factors affecting oral drug absorption (e.g., inability to swallow, nausea/vomiting, diarrhea, intestinal obstruction, etc.);
• Any uncontrolled, severe concomitant diseases;
• Hypersensitivity to any component of the study drugs;
• Expected survival < 5 years for any reason;
• Planned or previous organ/bone marrow transplantation;
• Treatment with immunosuppressants or corticosteroids within 1 month before enrollment;
• Central nervous system disorders that may impair ability to provide informed consent or comply with study procedures, as determined by the investigator;
• Other conditions that may prevent completion of study treatment (e.g., alcoholism, drug addiction, etc.);
• Pregnant or breastfeeding women.