Introduction

Cervical cancer is one of the main female reproductive malignant tumors worldwide, especially in developing countries [1]. Radiotherapy may be selected as the primary non-fertility-sparing treatment for all cervical cancer patients. Particularly for locally advanced cervical cancer (LACC, IIB to IVA), concurrent chemoradiotherapy (CCRT) followed by brachytherapy has become the standard choice on the basis of 5 randomized clinical studies, which reported that CCRT reduced the risk of death by 30–50% [2]. However, approximately one-third of patients with LACC still experience persistent or recurrent disease despite appropriate multidisciplinary management [3]. Therefore, more effective therapeutic strategies are needed to improve the outcomes of these patients. Nimotuzumab is an anti-EGFR targeted therapy and a classic anti-tumor drug. Some researchers reported that this drug increased the 2-year PFS of LACC patients to 83.08% [4].

Approximately 99.7% of all cervical cancers are associated with persistent genital high-risk human papillomavirus (HPV) infection [5]. The relationship between HPV infection and inflammation has been researched, and a recent study revealed that the HPV-positive status was associated with the immune microenvironment and an improved response to anti-programmed death 1 (PD-1) therapy in head and neck carcinoma [6]. Indeed, a high level of PD-L1 expression of 96% [7] has been reported in cervical cancer, which strongly supports the use of anti-PD-L1 therapy in cervical cancer patients.

Immune checkpoint blockade has been studied in metastatic and persistent cervical cancer patients and has shown promising outcomes [8,9,10,11,12]. The overall objective response rate (ORR) ranged between 12.2 and 55.6%. Several studies confirmed that immune checkpoint inhibitors (ICIs) combined with radiotherapy had synergistic anti-tumor effects on local and distant tumors [13, 14]. However, there is a lack of prospective clinical data on the addition of ICIs as radiosensitizers to definitive CCRT in LACC patients.

Toripalimab is a humanized immunoglobulin G4 monoclonal antibody against PD-1 that has shown promising anti-tumor efficacy in multiple solid tumors [15, 16]. The addition of toripalimab to chemotherapy as a first-line treatment for patients with advanced nasopharyngeal carcinoma provided superior PFS (1-year PFS rate from 27.9 to 47.4%) [17]. In the current clinical trial (TRACE), we evaluated the efficacy and safety of toripalimab combined with definitive CCRT for newly diagnosed patients with locally advanced cervical squamous cell carcinoma and identified the population most likely to benefit from ICI treatment.

Patients and methods

Patients

Eligible patients were 18–75-year-old females with newly diagnosed and previously untreated locally advanced squamous cell carcinoma of the uterine cervix. LACC was referred to as International Federation of Gynecology and Obstetrics (FIGO) 2018 stage IB3 to IVA, with no evidence of distant metastasis. All patients were enrolled regardless of their PD-L1 expression status. Patients had an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1 and adequate organ function. Patients who previously suffered from immunodeficiency disorders or had any condition that researchers believed to be associated with an increased risk of treatment were excluded. The Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, approved this study. The study is registered with the Chinese Clinical Trial Registry (ChiCTR2000032879).

Study design and treatment regimens

This study was a single-center, prospective, open-label, single-arm study of toripalimab combined with definitive CCRT for LACC. Before enrollment, the medical history and clinical physical examination findings were documented and screened. The initial laboratory and radiological evaluations included a complete blood count (CBC), liver and renal function measurement, contrast computed tomography or whole-body 18F-fluoro-2-deoxy-2-D-glucose positron emission tomography/computed tomography (18F-FDG PET-CT) to exclude the presence of distant metastasis and pelvic magnetic resonance imaging (MRI) to evaluate the extent of infiltration.

Written informed consent was obtained from all of the candidates before they were enrolled in the study. External beam radiotherapy (EBRT) of 45–50.4 Gy was delivered via the intensity-modulated radiation therapy (IMRT) technique to the pelvic ± para-aortic fields in 25–28 fractions 5 days per week, followed by brachytherapy of 24–30 Gy in 3–5 fractions once weekly. The target volume and normal tissue were contoured in accordance with the consensus guidelines [18]. The patients received HDR (high-dose rate) brachytherapy via an iridium-192 source, and the total dose achieved was 80–90 Gy (EQD2, biologically equivalent dose of 2 Gy per fraction) to D90 of the high-risk clinical target volume. The high-risk clinical target volume (HR-CTV) in brachytherapy was contoured according to the GEC-ESTRO recommendations [19]. Concurrent chemotherapy with cisplatin at a dose of 40 mg/m2 via intravenous infusion was administered once weekly for 5 weeks during EBRT. Toripalimab (240 mg via intravenous infusion) was administered on days 1, 22 and 43 (Fig. 1A).

Fig. 1
figure 1

Trial profile (A) and patient enrollment (B)

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Assessments and study endpoints

All patients were assessed weekly during the treatment course and subsequently every 3 months for the first 2 years to evaluate toxicity and disease status for study purposes and then every 6 months pursuant to our institutional practice. Adverse events were graded and recorded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0. Radiological responses were assessed independently by an experienced radiologist (YM) according to the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 [20], using MRI, CT or PET/CT. A complete response (CR) was defined as the disappearance of all target lesions, and a partial response (PR) was defined as a greater than 30% decrease in the sum of the diameters of the target lesions. The objective response rate (ORR) was defined as the proportion of CR and PR.

The dual primary endpoints were the incidence of dose-limiting toxicities (DLTs) during or within 28 days of treatment completion and 2-year progression-free survival (PFS). DLTs were defined as grade ≥ 3 immune-related adverse events and radiotherapy delay greater than 2 weeks related to treatment toxicity, and PFS was measured from the date of treatment to the date of disease progression or death from any cause in the absence of progression. The secondary endpoints included (1) 2-year local control (LC), defined as the absence of disease in the cervix (uterus), upper vagina or parametria on clinical examination, imaging and biopsy, (2) 2-year local regional control and (3) 2-year overall survival (OS). OS was defined as the time from the start of treatment until the date of death from any cause.

Biomarker analysis and immunofluorescences

The peripheral complete blood count (CBC), with differential measurements of the percentages of each type of leukocyte present, and peripheral T cell subsets were obtained at baseline, 1 month and every 3 months after treatment. The peripheral T cell subsets were evaluated using flow cytometry.

Tumor tissue samples were obtained via biopsy when patients were enrolled. Formalin-fixed, paraffin-embedded (FFPE) sections 4 mm in thickness from biopsy samples were assayed for the expression of CD3, CD8 and CD28 using immunofluorescence.

PD-L1 status was assessed using immunohistochemical staining via the 22C3 assay (Dako North America, Carpinteria, CA, USA). PD-L1 expression was reported as a combined positive score (CPS), defined as the number of PD-L1-positive cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells and multiplied by 100.

Statistical analysis

Based on a two-sided type 1 error of 0.05 and a power of 80%, we estimated that a total sample size of 19 patients would be needed to show an improvement in 2-year PFS from 60 to 90%. Assuming a 5% drop out rate, the final sample size was estimated to be 20 patients. Continuous variables are presented as medians with ranges or means with standard deviations (SDs). Categorical variables are presented as frequencies with percentages. Continuous variables were compared using nonparametric tests. P values were two-sided, with a significance level of 0.05 for all analyses. The Statistical Package for Social Sciences software, version 22.0 (IBM Corporation, Chicago, IL, USA), and GraphPad Prism software, version 9, were used for statistical analyses and plotting.

Results

Patients and treatment

From June 2020 to June 2021, we evaluated 37 candidates with LACC for potential participation in this study. Due to the unsatisfactory enrollment rate (18/35, 51.4%) and the risk of increased dropout rate associated with the COVID-19 pandemic, four patients were screened simultaneously and enrolled in the study. Therefore, a total of 22 patients were ultimately analyzed in this study (Fig. 1B).

The baseline clinical characteristics of the patients are summarized in Table 1. The median age was 55 years (range 42–72 years), with a median longest tumor diameter of 56.5 mm (range, 30–85 mm). Stage IIIC (FIGO, 2018) LACC accounted for 68.2% of these patients (15/22), whereas five patients (22.7%) had stage IVA disease. Sixteen patients were HPV-positive, and 20 had elevated squamous cell carcinoma antigen (SCCAg) levels.

Table 1 Demographic and disease characteristics of the patients
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All of the participants showed good compliance with the planned treatment. The median overall treatment time was 56 days (range 49–65 days). During the treatment period, all of the patients completed CCRT with at least four cycles of weekly cisplatin and three cycles of toripalimab, according to protocol-defined criteria.

Safety

The early adverse events (AEs) of the participants during or within 28 days of treatment completion are listed in Table 2. There were no treatment-related deaths, and none of the patients experienced DLT. Most AEs were grade 1 or 2, and no treatment discontinuation occurred. The most common AEs were leukopenia (20/22, 90.9%), neutropenia (17/22, 77.3%), nausea (17/22, 77.3%) and thrombocytopenia (12/22, 54.5%). Among 11 of the 22 patients (50%), grade 3 or higher AEs were observed, the most common of which was leukopenia (8/22, 36.4%), and all of these AEs were considered related to CCRT. The most common immune-mediated AE was hypothyroidism (grade 2, 2/22, 9.1%).

Table 2 Treatment-emergent adverse events reported in patients
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Efficacy

All patients underwent pelvic evaluation via magnetic resonance imaging (MRI) every 3 months after treatment. CR was achieved in 20 patients (20/22, 90.9%), PR was achieved in 2 patients (2/22, 9.1%), and the ORR was 100%.

At the data cutoff (June 30, 2023), the median follow-up was 31.8 months (9.5 to 37.8 months). The 2-year PFS rate was 81.8%. The 2-year LC and local regional control rates were both 95.5%, and the 2-year OS rate was 90.9%.

Four patients developed disease progression, and 2 died during follow-up. Among them, one developed multiple metastases (bones, lungs, liver, left adrenal gland, soft tissue, etc.) and local relapse of her uterine cervix 6 months after of enrollment (Patient #3 in Fig. 2B, Fig. 3A and B) and died of multiple organ failure 9.53 months later. One of these patients developed isolated pulmonary metastasis 9 months after enrollment, which was proven by pulmonary lobectomy. One patient developed isolated inguinal lymph node metastasis 14 months after enrollment and received palliative radiotherapy. Another patient developed pelvic recurrence 27 months after treatment, but she underwent surgery and continued chemotherapy. However, one patient with FIGO stage IVA (with bladder and rectal infiltration) disease developed rectovesicovaginal fistula 10 months after treatment completion. She required ostomy surgery after an event of gastrointestinal perforation but passed away with poor nutritional status despite all of the surgical interventions (Patient #7 in Fig. 2A).

Fig. 2
figure 2

Clinical features and efficacy after toripalimab and CCRT and selective pathological and radiological images. A Clinical features are presented for each patient, including baseline ALC count, PD-L1 CPS, radiological best response (based on RECIST, P = PR, C = CR) and disease control status. B Radiological and pathological images of Patient 3 before treatment, showed a stage IIIC1r patient achieving radiological PR 1 month after treatment completion. However, this patient developed multiple metastases as well as local relapse of her uterine cervix later, with a PFS of 6.07 months. C Radiological and pathological images of Patient 6 before treatment, showed numerous lymphocytic infiltrations in biopsy pathology and the patient achieved radiological CR after treatment until the last visit

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Fig. 3
figure 3

Representative multiplex immunohistochemical staining of the tumor microenvironment (TME) for CD8 + (red), CD3 + (green) and CD28 + (pink) cells. Representative images (A) and statistical analysis (C) of tumor-infiltrating CD8 + lymphocytes (CD8 + TILs) in the CR and non-CR groups. Representative images (B) and statistical analysis (D) of CD3 + CD8 + CD28 + triple-positive cells in the CR and non-CR groups. The Mann‒Whitney U test was used to analyze the P value. The source data are provided in the Source Data File

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Results of biomarker analysis

PD-L1 expression was evaluable in 22 patients, and 11 (50%) patients had a CPS of 1 or greater. The associations of clinical outcomes with PD-L1 expression were analyzed. The CR rate was the same (90.9%, 10 of 11) in patients with a CPS ≥ 1 and in patients with a CPS < 1. Moreover, OS and PFS did not differ significantly between these two groups (Fig. 4B1 and B2). While we chose a PD-L1 expression CPS cutoff value of 5, the CR rate was 100% (7 of 7) in patients with a PD-L1 CPS ≥ 5 versus 86.7% (13 of 15) in patients with a PD-L1 CPS < 5, but no significant difference was observed (P = 0·46). Consistent with the results based on the cutoff value of 1, no significant differences were observed in PFS or OS in patients with a CPS ≥ 5 and a CPS <5 (shown in Fig. 4C1 and C2).

Fig. 4
figure 4

Progression-free survival and overall survival for the whole group (A), PD-L1 CPS < 1 vs. ≥ 1 (B) and PD-L1 CPS < 5 vs. ≥ 5 (C)

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Tumor microenvironment analysis

We stained formalin-fixed, paraffin-embedded (FFPE) sections of 22 biopsy samples for CD3, CD8 and CD28 using immunofluorescence and processed the results via analysis pipelines from CellProfiler. The immunofluorescence of select samples is shown in Fig. 3. Compared with the non-CR group, the number of CD8 + T cells tended to increase in the CR group (P = 0.7, Fig. 3C). In addition, the percentage of CD3 + CD8 + CD28 + cells among CD3 + CD8 + cells was significantly greater in the CR group than in the non-CR group (P = 0.009, Fig. 3D).

Peripheral T lymphocyte subset analysis

Compared to patients with a baseline absolute lymphocyte count (ALC) > 1.255 × 10^9/L, patients with an ALC ≤ 1.255 × 10^9/L were more likely to progress (57.1%, 4/7 vs. 0%, 0/15, P = 0.001). In addition, the median PFS of patients with a baseline absolute lymphocyte count (ALC) > 1.255 × 10^9/L was significantly longer than patients who did not (not reached and 26.97 months, P = 0.0067). Figure 2 shows the clinical features of each patient, including baseline ALC counts, PD-L1 CPS and efficacy after toripalimab and CCRT, and selective pathological and radiological images.

Compared to the baseline, the total peripheral CD3 + T lymphocyte absolute counts and the relative percentages of CD3 + and CD4 + T lymphocytes significantly decreased 1 month after treatment completion (P < 0.001). However, the percentage of CD3 + CD8 + T lymphocytes increased, and the CD4 + /CD8 + ratio decreased significantly in all participants (P < 0.001). In addition, the percentages of CD4 + CD25 + CD127low T lymphocytes (regulatory T cells, Tregs) and CD4 + CD25 + T lymphocytes decreased 1 month after treatment completion (P = 0.088). Additionally, the percentages of CD4 + CD28 + T lymphocytes and CD8 + CD28 + T lymphocytes decreased 1 month after treatment completion (P < 0.001 and P = 0.001). Table 3 shows the peripheral T lymphocyte subset counts at baseline and during early follow-up. All of the peripheral T lymphocyte subset counts gradually returned to baseline levels 3 months after treatment completion.

Table 3 Peripheral T lymphocyte subset counts at baseline and during early follow-up (mean ± SD)
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Discussion

In this exploratory trial (TRACE) investigating the administration of ICIs combined with CCRT for LACC patients, we found that the addition of the anti-PD-1 agent toripalimab demonstrated manageable toxicity and presented an ORR of 100%. All enrolled patients completed CCRT and three cycles of toripalimab without delay. The most common grade 3 and higher AE in our study was leukopenia, with no grade 5 AEs, which was similar to the reported treatment-related AEs of CCRT alone [21, 22]. These results validated the safety and feasibility of combined treatment in LACC patients.

Several other prospective studies with small sample sizes are being performed to explore ICIs prior to, concurrent with or following CCRT for LACC (NCT02635360, NCT03833479, NCT03298893 [23] and NCT03738228 [24]). PD-L1 expression on tumor cells was not required for inclusion in any of these trials, which is consistent with our study. Although PD-L1 expression is recommended for guiding the clinical management of recurrent and metastatic cervical cancer [9, 12], its role in LACC patients receiving ICIs, especially ICIs combined with CCRT, is obscure.

In all the above-mentioned studies, the duration of anti-PD-1 or anti-PD-L1 therapy varied depending on its purpose. To evaluate the safety and effectiveness of immunotherapy in combination with chemoradiotherapy, anti-PD-1 agents were administered for 3 months or up to 6 months in combination with CCRT (NCT02635360, NCT03298893 [23]). To evaluate the efficacy of ICIs as consolidation therapy following CCRT, an anti-PD-1 agent was administered for up to 24 months after CCRT (NCT03833479). To investigate how well ICIs affect immune system activation in these patients, atezolizumab was administered for just 3 cycles before or with CCRT (NCT03738228).

To understand the rationale of CCRT-ICI combination treatment in LACC patients, including whether the ICI worked as a radiosensitizer and immune activator in combination treatment, we assessed feasibility and tolerability in this exploratory study and were designed to administer 3 cycles of toripalimab as a radiosensitizer concurrently with CCRT. In addition, cervical adenocarcinoma is associated with a poor prognosis [25]. We focused on squamous cell LACC in this study to eliminate confounding factors associated with different histological types.

Compared to the above studies, the patients in our study had more advanced disease. More than 90% of the patients enrolled had stage IIIC-IVA disease (FIGO 2018) and represented a subgroup of LACC patients with a poor prognosis [26]. Sturzda et al. [27] reported the outcomes of a retroEMBRACE study including 731 women treated with image-guided brachytherapy (IGBT) following EBRT ± chemotherapy. The LC rate for the whole group was 90.6%, with a 3/5 year LC rate of 71%-76% in the III-IVA stage (FIGO 2009). Reiko et al. retrospectively analyzed 137 LACC patients who received definitive RT and reported a CR rate of 88% and a 5-year PFS of 60%. Non-CR patients significantly correlated with advanced-stage disease, and all patients die from disease [28]. In addition, the CALLA study [24, 29], which was a randomized trial evaluating the benefit of the PD-L1 inhibitor durvalumab, added to CCRT in LACC patients, which did not reach the primary endpoint. Another single-arm phase I trial, Nicol (NCT03298893) [23], explored the PD-1 inhibitor nivolumab plus CCRT for LACC, with 12.5% adenocarcinoma and reported a 2-year PFS of 75%. The Keynote-A18 trial is the largest phase III randomized clinical trial to compare the PD-1 inhibitor pembrolizumab with placebo in combination with CCRT for the treatment of LACC with 16% non-squamous cell carcinoma and reported a 2-year PFS of 68% [30]. Notably, there are differences in immunological targets and intracellular signaling between PD-L1 and PD-1 inhibitors [31]. In our study, the ORR of all 22 LACC patients was 100%, with an excellent CR rate of 90.9%. In terms of survival outcomes, our 2-year PFS rate of 81.8% is promising compared to the 2-year PFS reported in the Nicol and Keynote-A18 trials, both of which reported the outcome of a PD-1 inhibitor combined with CCRT for LACC [23] and previous studies investigating CCRT alone for LACC [32, 33], although it did not reach our estimated 2-year PFS of 90%. All of the enrolled patients in our study were diagnosed with squamous cell carcinoma, which eliminated the impact of tumor cell heterogeneity on outcomes, and the results demonstrated a potential benefit of immunotherapy for patients with squamous cell carcinoma. Moreover, we should consider the shorter follow-up and the limited number of patients in this trial. Therefore, our study revealed a 2-year PFS of 81.8%, with a total of 90.9% for stage IIIC-IVA patients, which indicated that our innovative combination treatment strategy may provide more opportunities for LACC patients, especially patients with more aggressive disease (IIIC-IVA), to achieve a higher rate of clinical response and survival. Therefore, another single-arm phase I trial exploring the efficacy of concurrent and maintained toripalimab during CCRT in stage III-IVA cervical cancer patients is ongoing (NCT05084677) [34], which focuses on more advanced LACC patients and provides more experience in the combination of ICIs with CCRT for these patients.

It is important to identify biomarkers to predict which patients could benefit most from immunotherapy. The Keynote 158 and Keynote 826 trials consistently reported positive associations between the efficacy of pembrolizumab plus chemotherapy and PD-L1 expression in patients with metastatic cervical cancer, with PD-L1 CPS cutoff values of 1 and 10, respectively [9, 12]. There have been contradictory results of the predictive value of PD-L1 status for patients with locally advanced cancer. We found that a PD-L1 expression CPS ≥ 5 may be associated with a higher CR rate after CCRT and PD-1 inhibition. However, our study did not demonstrate a statistically significant survival benefit in these patients. Considering that more than 90% of the patients included in our study had FIGO 2018 stage III-IVA, the above conclusion was similar to the conclusions of the subgroup analysis of the Keynote-A18 trial [30]. The Keynote-A18 trial suggested that FIGO 2014 stage III-IVA patients could benefit from immunotherapy combined with CCRT regardless of their PD-L1 expression status [30]. However, the prevalence rate of PD-L1 expression in cervical cancer patients remains inconclusive, with estimated rates in the literature ranging from 19 [35] to 88.8% [12]. Additionally, we found that only 50% of the 22 enrolled patients in our study were PD-L1 expression positive at baseline (CPS > 1), which was lower than metastatic patients reported in Keynote 158 and Keynote 826 (83.7% [9] and 88.8% [12], respectively). This difference may be related to the relatively low HPV-positive rate of 72.7% for SCC in our study. Meng Y. et al. [36] reported that there were direct correlations between the expression of PD-L1 and the rate of HPV positivity in patients with cervical cancer. Therefore, considering the small sample size and the biases in the enrolled population, the results must be treated with caution, and long-term follow-up is needed.

Radiotherapy-induced lymphopenia has received increasing attention recently [37], but the exact mechanism is not yet fully understood. The decreased total CD3 + lymphocyte count after treatment in our study may be associated with chemoradiotherapy. Theoretically, lymphopenia reflects impaired cell-mediated immunity [38, 39]. This finding illustrates the potential of combination therapeutic strategies, such as concurrent radiotherapy and ICIs, to improve clinical efficacy via immunoregulatory pathways. Researchers showed that a high baseline absolute lymphocyte count was associated with a better response to anti-PD-1 or anti-PD-L1 therapy in several solid tumors [40,41,42]. In our study, a lower baseline ALC was associated with a significantly greater rate of disease progression (57.1 vs. 0%, P = 0.001).

Cytotoxic CD8 + T cells are the most powerful effectors of the anticancer immune response and constitute the backbone of anticancer immunotherapy. In contrast, T regulatory cells (Tregs) are one of the most difficult obstacles in anticancer immunotherapy. In our study, the percentage of CD3 + CD8 + lymphocytes significantly increased after the combined treatment, whereas the absolute CD4 + CD25 + CD127low and relative CD4 + CD25 + counts (Tregs) decreased. These results suggest that anti-PD-1 treatment may reverse radiotherapy-induced immune suppression, such as cytotoxic CD8 + T cell depletion [43]. Meir et al. [44] reported that conventional radiotherapy significantly decreased the absolute number of CD8 + T cells and their capacity to respond to antigenic stimulation, whereas blockade of PD-1 successfully increased T cell reactivity in vitro. We also investigated the immunofluorescence of CD8 + T cells for their potential role in anti-tumor immunity. The presence of CD8 + T cells was previously recognized as an indicator of response [45]. However, the dependence of immunotherapy on CD28 + CD8 + T cells was recently well characterized, which showed that CD28 expression on T cells is PD-1-mediated inhibition, and CD28-B7 interaction is required for the rejuvenation of CD8 + T cells depleted after PD-1 blockade [46,47,48]. In our study, the percentage of CD8 + CD28 lymphocyte significantly increased after the combined treatment. Our results also revealed an increase in the percentage of CD3 + CD8 + CD28 + T cells in samples from CR patients, but the percentage of CD8 + T cells in the CR group was not significantly greater than the non-CR group.

Unfortunately, one patient with an initial diagnosis of stage IIIC cervical cancer experienced rapid tumor progression just 3 months after her treatment completion. The patient’s medical history, physical examination and laboratory and imaging results were not unusual before treatment started. A previous study reported that hyperprogressive disease was associated with inferior overall survival [49]. However, the mechanism underlying hyperprogressive disease induced by PD-1/PD-L1 inhibitors is not clear [50]. Research [51, 52] has focused on identifying possible predictors of hyperprogression, including mutated genes and tumor microenvironment biomarkers. These predictors require further validation. Further efforts are needed to identify more accurate predictors of hyperprogression prior to immunotherapy as well as the potential underlying mechanisms.

The primary limitation of the trial was the small sample size and single-center design, which may restrict the generalizability of the conclusions. Second, a comparator group is needed to explore the role of toripalimab, and our exploratory research on biomarkers should be interpreted with caution.

In conclusion, our study demonstrated that toripalimab combined with CCRT achieved good tolerance and promising anti-tumor effects. Further phase III studies and outcomes from longer durations of follow-up are warranted.