Tislelizumab combined with radiotherapy for unresectable locally advanced esophageal squamous cell carcinoma: A prospective, single-arm, phase II study (TREC)

Tislelizumab combined with radiotherapy for unresectable locally advanced esophageal squamous cell carcinoma: A prospective, single-arm, phase II study (TREC) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tislelizumab combined with radiotherapy for unresectable locally advanced esophageal squamous cell carcinoma: A prospective, single-arm, phase II study (TREC) Changmin Liu, Yue Wang, Bichun Xu, Zeshun Yu, Jieyong Tian, Lijun Tian, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7857613/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Treatment options for unresectable locally advanced esophageal squamous cell carcinoma (ESCC) that cannot tolerate concurrent chemoradiotherapy (CCRT) are exceedingly limited. This study aimed to evaluate the efficacy and safety of tislelizumab, a programmed cell death protein 1 (PD-1) inhibitor, in combination with definitive radiotherapy as a novel chemotherapy-free first-line treatment for patients with unresectable, locally advanced ESCC who are ineligible for standard CCRT. Methods: This prospective, single-arm, phase II clinical trial (ChiCTR2100053182) recruited treatment-naïve patients diagnosed with unresectable, locally advanced ESCC at Binzhou Medical University Hospital. Eligibility was limited to patients considered ineligible for standard CCRT due to medical contraindications or patient preference. The investigational regimen consisted of tislelizumab (200 mg intravenously) administered on a three-week cycle, initiated concurrently with the first fraction of radiotherapy. The primary endpoint was progression-free survival (PFS). Additionally, the protocol included a prespecified exploratory analysis of tumor immune microenvironment markers using high-throughput multiplex immunofluorescence. Results:From May 2021 to October 2022, 32 eligible patients were enrolled. The mPFS was 23.5 months (95% CI 18.2–not reached), the 1-, 2-, and 3-year PFS rates were 75.0%, 57.1% and 42.9% respectively. The median overall survival (mOS) was not reached, with a 3-year OS rate of 53.6%. The regimen demonstrated substantial efficacy, achieving an objective response rate (ORR) of 82.1% and a pathological complete response (pCR) rate of 64.3%. Treatment was well-tolerated; the most frequent grade ≥3 treatment-related adverse event (TRAE) was lymphopenia (53.6%). Exploratory biomarker analysis revealed that high densities of T-cell exhaustion markers (e.g., intratumoral PD-1+ cells) were significantly associated with poorer PFS and OS, whereas high expression of stromal CD68+PD-L1+ cells was a strong favorable prognostic factor. Conclusion: This phase II study demonstrates that the combination of tislelizumab and definitive radiotherapy yields promising efficacy and manageable toxicity in patients with unresectable, locally advanced ESCC who are ineligible for standard CCRT. Moreover, the observed associations between immune cell signatures and treatment outcomes highlight the potential for biomarker-driven patient selection in future trials. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Esophageal cancer is the sixth-leading cause of cancer-related mortality worldwide [1]. For patients with unresectable, locally advanced esophageal squamous cell carcinoma (ESCC), definitive concurrent chemoradiotherapy (CCRT) is the established standard of care, based on landmark trials including RTOG 8501, RTOG 9405, and RTOG 0436[2–4]. Although CCRT offers superior efficacy to single-modality therapy, its clinical application is constrained by a significant toxicity bottleneck. The regimen's considerable toxicity profile, which includes acute high-grade esophagitis and hematologic events as well as the risk of late, life-threatening cardiopulmonary complications, renders it unsuitable for many patients. This ineligibility, particularly among the elderly or those with significant comorbidities, compromises their prognosis. Consequently, there is a critical unmet need to develop more effective and tolerable therapeutic strategies to improve survival outcomes for this specific patient population. The advent of immune checkpoint inhibitors (ICIs) has fundamentally reshaped the treatment landscape for esophageal cancer, with their clinical application successfully extending to the entire course of disease management. In advanced or metastatic ESCC, ICIs combined with chemotherapy have become the standard of care for first-line treatment. In the adjuvant setting, following neoadjuvant chemoradiotherapy, the use of nivolumab has also been established as a standard of care based on the landmark CheckMate 577 trial [5]. Furthermore, studies such as TD-NICE have preliminarily confirmed the positive effects of combining immunotherapy with CCRT in the neoadjuvant setting [6]. For patients with unresectable ESCC, numerous studies are exploring the integration of ICIs with CCRT, offering new hope for this challenging population [7, 8]. Radiotherapy can initiate a cascade of anti-tumor immune responses by inducing immunogenic cell death (ICD), stimulating the release of inflammatory cytokines, and modulating the tumor microenvironment. These actions collectively upregulate PD-L1 expression on tumor cells, providing a robust theoretical basis for its combination with ICIs. This synergy is conceptually framed as an "in situ vaccine" effect, whereby radiotherapy can transform immunologically "cold" (immunosuppressive) tumors into "hot" (immune-infiltrated) tumors, thereby enhancing their susceptibility to immune checkpoint blockade [9]. Radiotherapy promotes immunogenic cell death, leading to the release of damage-associated molecular patterns (DAMPs) such as ATP and high-mobility group box-1 (HMGB1), and surface exposure of calreticulin. This process enhances antigen presentation by dendritic cells. Furthermore, radiation-induced DNA damage can activate the cytosolic cGAS-STING pathway, triggering a type I interferon response that is critical for priming antitumor CD8 + T cells [10]. Tislelizumab is a humanized IgG4 monoclonal antibody (mAb) that targets PD-1 to exert its anti-tumor effects and has been approved for several malignancies, including ESCC [11–12]. Early evidence from the RATIONALE 205 study showed significant clinical activity for tislelizumab combined with chemotherapy in ESCC, achieving an objective response rate (ORR) of 46.7%, a disease control rate (DCR) of 80%, a median duration of response (DOR) of 12.8 months, and a median progression-free survival (PFS) of 10.4 months [13]. These promising findings were subsequently validated in the pivotal Phase III RATIONALE-306 study. In this trial, first-line tislelizumab plus chemotherapy demonstrated a significant survival benefit in advanced/metastatic ESCC, extending the median overall survival (OS) to 17.2 months compared to 10.6 months with chemotherapy alone (HR = 0.66), thereby establishing its potent activity [14]. Moreover, the ETNT study explored its role in an earlier disease stage, showing that for patients with locally advanced ESCC without a complete clinical response (cCR) after neoadjuvant chemoradiotherapy, sequential treatment with tislelizumab plus chemotherapy provided a favorable safety and efficacy profile [15]. For unresectable ESCC patients who cannot tolerate standard CCRT, our TREC study pioneers a chemotherapy-free regimen of tislelizumab plus radiotherapy. By leveraging immunogenic synergy to achieve high pathological complete response (pCR) rates and durable efficacy, this approach may establish a new standard of care and challenges the traditional necessity of chemotherapy in immunoradiotherapy. MATERIALS AND METHODS Study design and population The TREC study was a single-center, single-arm, open-label, phase II study used to evaluate the efficacy and safety of tislelizumab combined with radiotherapy in patients with unresectable ESCC. The trial was conducted at Binzhou Medical University Hospital in China, with patient enrollment occurring between May 2021 and October 2022 (Registration: ChiCTR2100053182). This trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. Eligible participants were patients aged 18 to 90 years with histologically confirmed, unresectable, locally advanced ESCC (stage II–IVA, AJCC 8th edition), who were deemed unable or unwilling to receive CCRT. Eligible participants were required to have baseline imaging confirming the absence of distant metastasis and no prior anti-tumor therapy, including chemoradiotherapy, targeted therapy, or immunotherapy. Participants were also expected to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and adequate hepatic, renal, bone marrow, and cardiopulmonary function. Patients with a history of autoimmune disease, a second primary malignancy, or those who were pregnant or lactating were excluded. Written informed consent was obtained from all participants prior to enrollment. Treatment and assessments Participants received tislelizumab at a dose of 200 mg intravenously every 3 weeks (Q3W), with the first dose administered on Day 1 of radiotherapy. Treatment continued until documented disease progression, the occurrence of unacceptable adverse events (AEs), or withdrawal of patient consent. Tumor assessments via imaging were performed at baseline and subsequently every 6 weeks. Treatment response and disease progression were evaluated according to the Immune-Related Response Evaluation Criteria in Solid Tumors (irRECIST). Intensity-modulated radiotherapy (IMRT) was used for treatment, targeting both the primary esophageal lesions and regional lymph nodes. Lymph node metastases were irradiated to a total dose of 60 Gy in 30 fractions. For esophageal lesions, if pathological examination by continuous deep biopsy or multi-point sampling via gastroscopy confirmed complete remission(CR), a total dose of 50 Gy in 25 fractions was delivered; otherwise, a total dose of 60 Gy in 30 fractions was administered. The trial utilized a response-adapted radiotherapy strategy to individualize treatment. Patients who achieved an early pCR had their radiation dose de-escalated to 50 Gy to minimize severe toxicities, while those with residual disease received the standard 60 Gy curative dose to ensure robust local tumor control. Endpoints The primary endpoint was investigator-assessed PFS, defined as the time from enrollment to the first documented disease progression or death from any cause, whichever occurred first. Secondary endpoints included OS, defined as the time from enrollment to death from any cause; major pathological response (MPR), defined as ≤ 10% residual viable tumor in the primary tumor bed following treatment, irrespective of lymph node status; ORR, the proportion of patients achieving a CR or partial response (PR); and DCR, the proportion of patients achieving a CR, PR, or stable disease (SD). Safety was assessed by monitoring and grading AEs according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE), version 4.0. Differential protein expression and tumor immune microenvironment analysis Prior studies have suggested that the expression of immune markers may exhibit significant heterogeneity between tumor parenchyma and stroma, potentially leading to differential prognostic implications. To address this and identify potential prognostic biomarkers in our cohort, we analyzed pre-treatment tumor specimens using multiplex immunofluorescence (mIF) staining for key markers including PD-L1, PD-1, CD8, CD68, and Pan-Cytokeratin (PanCK) .Subsequently, we employed high-throughput fluorescence analysis to quantify a comprehensive set of cellular populations and scoring systems across the whole tissue, tumor parenchyma, and stromal compartments. These parameters included PANCK + PD-L1 + cells, CD68 + PD-L1 + cells, PANCK + cells, CD68 + cells, CD8A + PD-1 + cells, CD8A + cells, the combined positive score (CPS), PD-1 + cells, PD-L1 + cells, and the tumor proportion score (TPS). Finally, the prognostic significance of these markers within each compartment was assessed using the Kaplan-Meier method. Statistical analyses The sample size was calculated to detect a hypothesized 20% absolute improvement in the 2-year OS rate, from a baseline of 10% with radiotherapy alone to 30% with the addition of immunotherapy. The trial was designed to have 80% power at a two-sided significance level (α) of 0.05, based on a one-sample test for proportions. Accounting for a potential 10% loss to follow-up, the required sample size was determined to be 27 patients using PASS software (version 25.0). Efficacy was analyzed in the modified intention-to-treat (mITT) population, defined as all enrolled patients who received at least one dose of tislelizumab and had a baseline tumor assessment. The primary endpoint of PFS and all secondary efficacy endpoints, including OS and ORR, were evaluated in this mITT population. Safety was analyzed in the safety analysis set (SAS), which comprised all patients who received at least one treatment dose. For statistical analysis, continuous variables were presented as mean and standard deviation (SD), while categorical variables were presented as frequencies and percentages. Comparisons for categorical data were performed using the chi-square test or Fisher’s exact test. The Wilcoxon test was used to compare non-parametric data. PFS and OS were estimated using the Kaplan-Meier method. All statistical analyses were conducted using SPSS software (version 25.0, IBM Corp., Armonk, NY, USA), and a two-sided p-value of less than 0.05 was considered statistically significant. Results Study Design and Baseline Characteristics This prospective phase II trial enrolled patients between May 2021 and October 2022. The patient screening and enrollment process is detailed in the CONSORT diagram (Fig. 1). In brief, 32 consecutive patients were screened for eligibility, of whom 29 met the protocol-specified inclusion criteria. Three subjects were excluded for not meeting these criteria, and one additional patient withdrew consent before the initial treatment. This resulted in a modified intention-to-treat (mITT) population of 28 patients who received at least one dose of therapy and completed baseline radiographic evaluations. Patient status was assessed as of the data cutoff date on June 30, 2024. At this time, 3 of the 28 patients (10.7%) remained on active treatment. Among the other 25 patients, 15 (53.6%) had discontinued treatment due to radiologically confirmed disease progression or death, and 10 (35.7%) had discontinued after completing the planned 24 months of therapy. Two additional patients (7.1%) were lost to follow-up. Overall, nine deaths were recorded; six (66.7%) were attributed to tumor-related causes, while three (33.3%) were non-cancer deaths (one grade 5 pulmonary embolism, one acute myocardial infarction, and one grade 5 pneumonia). The baseline demographic and clinical characteristics of the mITT population (N = 28) are presented in detail in Table 1. The cohort had a median age of 67 years (range 55–80), with a predominance of male patients (57.1%, 16/28). For performance status, 39.3% (11/28) of patients had an ECOG score of 0, while 60.7% (17/28) had a score of 1. According to the AJCC 8th edition staging, the patient distribution was Stage II (14.3%, 4/28), Stage III (67.9%, 19/28), and Stage IV (17.9%, 5/28). The five Stage IV cases included two with IVA and three with IVB disease, the latter presenting with isolated supraclavicular metastases. The primary tumor epicenter was most commonly located in the middle thoracic esophagus (50.0%, 14/28), followed by the lower (35.7%, 10/28), upper (10.7%, 3/28), and cervical esophagus (3.6%, 1/28). Efficacy Outcomes and Pathological Correlation In the mITT population (n = 28), treatment response assessments per RECIST v1.1 criteria demonstrated substantial antitumor efficacy (Fig. 2a). The ORR reached 82.1% (23/28; 95% CI 64.5%–92.1%), including 3 CR (10.7%; 95% CI: 2.8%–27.3%) and 20 PR (71.4%; 95% CI: 52.2%–85.9%). DCR was 100% (28/28; 95% CI: 87.7%–100.0%), with 5 patients (17.9%; 95% CI: 6.7%–35.9%) achieving SD and no instances of progressive disease observed during the treatment period (Table 2 & Fig. 2a). To evaluate the depth of response, post-radiotherapy deep-cup endoscopic biopsies were performed for histopathological validation. A pCR was achieved in 64.3% (18/28) of patients, while 35.7% (10/28) had residual tumor (non-pCR) (Fig. 2a). Notably, a significant discordance was observed between radiographic and pathological outcomes (Fisher's exact test, p = 0.047; Cramer's V = 0.41, Fig. 2b). The pCR rate increased with better radiographic response categories: 40% (2/5) in patients with SD, 65% (13/20) in those with a PR, and 100% (3/3) in those with a CR. While a pairwise comparison revealed a significant difference in pCR rates between the SD and CR groups (p = 0.049), this difference did not remain significant for other inter-group comparisons after Bonferroni correction (all p > 0.05). Radiographic CR demonstrated a 100% positive predictive value (PPV) for achieving pCR, though its sensitivity was limited to 16.7%. The mPFS was 23.5 months (95% CI 18.2–not reached), with 1-, 2-, and 3-year PFS rates of 75.0% (62.5–85.7), 57.1% (42.9–71.4), and 42.9% (28.6–57.1), respectively (Fig. 2c). A Kaplan-Meier analysis revealed an initial decline in survival probability within the first year, followed by a period of sustained disease control beyond 24 months. For OS, the median survival was not reached at the data cutoff (median follow-up: 24.7 months; range 4–34.5 months). The 1-, 2-, and 3-year OS rates were 82.1% (71.4–90.5), 64.3% (50.0–77.8), and 53.6% (38.1–67.9), respectively (Fig. 2d). A plateau emerged in the OS curve after 24 months (log-rank p = 0.032 vs. historical controls), suggesting durable survival in a clinically relevant subset of patients (n = 9/28, 32.1% of patients remaining progression-free at 2 years). Safety The safety profile was analyzed in all 28 patients who received treatment. The regimen was generally manageable, with a detailed summary of AEs provided in Table 3. All patients experienced at least one AE, the majority of which were Grade 1–2 in severity. The most frequent AEs of any grade included lymphopenia (22 patients, 78.6%), esophagitis (20, 71.4%), and hyponatremia (14, 50.0%). The most common Grade 2 event was esophagitis, occurring in 15 patients (53.6%). Among Grade 3 or higher AEs, the most frequent was Grade 3 lymphopenia, observed in 15 patients (53.6%). Other notable Grade 3 events included lung infection (2, 7.1%), esophageal fistula (1, 3.6%), and immune-related hepatotoxicity (1, 3.6%). Immune-related adverse events (irAEs) were common but predominantly low-grade. The most frequent irAE was hypothyroidism, occurring in 4 patients (14.3%, all Grade 2). Additionally, three cases of Grade 2 checkpoint inhibitor pneumonitis (10.7%), one case of Grade 2 myocarditis (3.6%), and one case of Grade 3 immune-related hepatotoxicity (3.6%) were reported. Three Grade 5 (fatal) AEs were reported, with one case each attributed to lung infection, myocardial infarction, and a vascular access complication. Biomarker To identify potential prognostic biomarkers, we performed multiplex immunofluorescence staining on tumor specimens. A total of 25 specimens were subsequently analyzed, targeting PD-L1, PD-1, CD8, CD68, and PANCK (Fig. 3a & 3b). Quantitative analysis across all patient samples revealed that parenchymal (PANCK_P) and stromal (PANCK_S) tumor cells were the most abundant populations overall (Fig. 3c). Further stratification by tissue compartment highlighted distinct microenvironments: the tumor parenchyma was characterized by a predominance of PANCK + tumor cells and CD68 + macrophages (Fig. 3d), whereas the stromal compartment was primarily composed of CD68 + macrophages and PD-L1 + cells, indicating a different immune infiltrate (Fig. 3e). High densities of markers associated with T-cell exhaustion, such as PD-1 + and CD8A + PD-1 + cells, were generally associated with unfavorable outcomes. In the whole-tissue analysis, increased PD-1 + cell abundance correlated with shorter OS (Fig. 4) and PFS (Fig. 5). This effect was primarily driven by their presence within the tumor parenchyma; compartmental analysis revealed that a high density of intratumoral PD-1 + cells was a strong factor for poor prognosis, significantly associating with both shorter OS (p = 0.024) and PFS (p = 0.038) (Fig. 6&7). In contrast, stromal PD-1 + cells only showed a non-significant trend toward worse PFS (p = 0.089) (Fig. 6&7). Similarly, a high abundance of CD8A + PD-1 + cells in the whole tissue was associated with shorter PFS (Fig. 5). This negative effect was also localized to the parenchyma, where a high density of these cells significantly correlated with shortened PFS (p = 0.022) but not OS (p = 0.170) (Fig. 6&7). No significant associations were found for stromal CD8A + PD-1 + cells (Fig. 6&7). Conversely, markers related to PD-L1 expression generally indicated a more favorable prognosis, with their predictive value being highly dependent on the cell type and spatial compartment. In the whole-tissue analysis, a higher proportion of PANCK + PD-L1 + cells was associated with improved PFS (Fig. 5), and along with a positive TPS, exhibited protective effects on OS (Fig. 4).Compartmental analysis provided further insights. High expression of stromal CD68 + PD-L1 + cells was a strong favorable prognostic factor, significantly associated with improved OS (p = 0.017) and PFS (p = 0.013) (Fig. 6&7). While a high density of these cells in the parenchyma also correlated with better OS (p = 0.040), it had no significant impact on PFS (p = 0.400) (Fig. 6&7). For total PD-L1 + cells, a high density within the tumor parenchyma was significantly associated with improved OS (p = 0.032) and PFS (p = 0.038), while a high density in the stroma was also significantly associated with improved PFS (p = 0.043) and showed a trend toward improved OS (p = 0.056) (Fig. 6&7). A high density of total PD-L1 + cells in the whole tissue also showed a borderline trend toward improved PFS (P = 0.054) (Fig. 5). In contrast to TPS, the CPSshowed no significant impact on survival outcomes in this cohort (Figs. 4 & 5). DISCUSSION CCRT stands as the standard treatment modality for inoperable locally advanced ESCC [2–4]. Meta-analysis of multicenter randomized controlled trials shows that, compared to radiotherapy alone, CCRT significantly improves patient prognosis, reducing the risk of death by 27% (HR = 0.73, 95% CI 0.64–0.84) and increasing the absolute survival rates by 9% and 4% at 1 and 2 years, respectively [4]. This therapeutic gain, however, is offset by a substantially higher toxicity profile. CCRT elevates the risk of grade 3–4 acute toxicity more than five-fold (OR = 5.16, 95% CI 2.83–9.38), with severe hematologic toxicity observed in approximately 30% of patients, compared to just 2% in the radiotherapy-alone cohort [4]. Consequently, patients with advanced age or comorbidities are often ineligible for CCRT, leaving them with suboptimal options like radiotherapy or chemotherapy alone [16–17]. This highlights a pressing clinical need for more effective and better-tolerated therapeutic strategies for this specific population. Recent regulatory and guideline advancements underscore the clinical utility of tislelizumab, a humanized anti-PD-1 antibody, in ESCC. In March 2024, it secured U.S. FDA approval as a monotherapy for unresectable/metastatic ESCC post-chemotherapy. This is mirrored by its designation as a Category 1 preferred agent for second-line and subsequent therapy in the NCCN Guidelines (v2.2024). Building upon this validation, evaluating its efficacy in the first-line setting, especially combined with radiotherapy, is a logical and compelling next step in clinical investigation. While the combination of radiotherapy and immunotherapy is an active area of investigation, a consensus on the optimal timing for initiating immunotherapy is lacking [18]. Preclinical evidence indicates superior efficacy when the two modalities are administered concurrently. This is corroborated by early-phase clinical trials, which show greater benefit when immunotherapy is given with or shortly after radiotherapy, as opposed to sequentially. Sequential strategies are often hampered by low rates of immunotherapy initiation after the completion of chemoradiotherapy [19, 20]. Therefore, our study protocol involved the concurrent administration of tislelizumab with radiotherapy, followed by a maintenance phase of tislelizumab monotherapy. In this phase II trial, we demonstrated that a chemotherapy-free regimen of tislelizumab plus radiotherapy yielded remarkable clinical outcomes for unresectable locally advanced patients ineligible for standard CCRT. The ORR of 82.1% and mPFS of 23.5 months represent a substantial improvement over historical outcomes with radiotherapy alone [16, 17]. Furthermore, the high pathological complete response rate of 64.3% underscores the potent synergy between immunotherapy and radiotherapy. Notably, the efficacy observed in our study compares favorably with recent trials that combined immunotherapy with a full CCRT regimen. For instance, studies adding camrelizumab or toripalimab to CCRT reported 2-year OS rates of 70% and 1-year OS rates of 78%, respectively [21, 22]. That our chemotherapy-sparing approach achieved comparable survival outcomes (a 2-year OS rate of 64.3%) is a key finding. It suggests that for patients who cannot tolerate concurrent chemotherapy, the combination of a PD-1 inhibitor with radiotherapy may offer a highly effective and better-tolerated alternative, positioning this strategy strongly within the current therapeutic landscape. A particularly noteworthy finding of our study was the significant discordance between radiographic and pathological responses. While radiographic CR demonstrated a 100% positive predictive value for pCR, its low sensitivity suggests that imaging alone is insufficient for accurately assessing treatment efficacy. Post-radiotherapy effects, such as inflammation and fibrosis, can often mimic residual disease on imaging, leading to an underestimation of the true pathological response. This has profound clinical implications: it suggests that even patients with stable disease or partial response on imaging may have already achieved pCR. Therefore, our findings strongly advocate for the routine use of post-treatment endoscopic biopsy to confirm pathological response, which is critical for guiding subsequent clinical decisions, such as the duration of immunotherapy or the consideration of consolidative surgery. Furthermore, this discordance underscores the limitations of imaging-based endpoints and supports the establishment of pCR as a more robust and objective surrogate endpoint for survival in future clinical trials of immunoradiotherapy for ESCC. Evaluating the safety of this combination therapy was critical, particularly given the theoretical risk of overlapping toxicities[23, 24] and our inclusion of a vulnerable cohort intolerant to standard CCRT, which included a significant proportion of elderly patients (32.1% ≥75 years). Overall, the regimen was well-tolerated, with most TRAEs being Grade 1–2. Two adverse events of special concern in this setting are esophageal fistula and pneumonitis. The incidence of esophageal fistula was notably low at 3.57% (one Grade 3 event), which compares favorably to the 10–14% rates reported in prior studies combining immunotherapy with CCRT [21, 22]. Pneumonitis is another critical concern, as it accounts for a significant portion of deaths during anti-PD-1/PD-L1 therapy [25] and its risk may be elevated when radiotherapy is combined with immunotherapy [26]. In our study, the toxicity from pneumonitis was manageable. Both checkpoint inhibitor-related and radiation-induced pneumonitis occurred at a rate of 10.7% each, and importantly, all cases were confined to Grade 2 severity. The most frequent high-grade toxicity was Grade 3 lymphopenia, occurring in 53.6% of patients. While clinically manageable, this high incidence warrants careful monitoring due to its potential to impair long-term anti-tumor immunity. Three Grade 5 (fatal) AEs were reported: one lung infection, one myocardial infarction, and one vascular access complication. These events were considered by investigators to be primarily related to severe patient comorbidities during the COVID-19 pandemic rather than direct treatment toxicity. However, indirect therapeutic contributions cannot be fully excluded, as the high incidence of Grade 3 lymphopenia may have increased infection susceptibility, and a systemic inflammatory response could have factored into the cardiovascular event. These outcomes underscore the fragility of this patient population, necessitating exceptionally vigilant monitoring and supportive care when administering this potent regimen. Our study's biomarker analysis validates the predictive value of PD-L1 expression in ESCC and provides deeper insights into the complex, context-dependent role of the tumor immune microenvironment. Landmark trials such as KEYNOTE-590 and ESCORT have consistently demonstrated that higher PD-L1 expression, whether measured by Combined Positive Score (CPS ≥ 10) or Tumor Proportion Score (TPS ≥ 10%), correlates with significantly greater survival benefits for patients receiving immunotherapy combinations [27, 28]. Aligning with these findings, our study also identified a link between PD-L1 expression and favorable outcomes. Specifically, an increased proportion of PD-L1 on tumor cells (PANCK + PD-L1+) was significantly associated with improved PFS (p < 0.05), while a high density of PD-L1 + cells showed a trend toward improved OS (P = 0.054). Considering that region-specific expression patterns of immune biomarkers may critically impact immunotherapy efficacy, we also examined the expression of PD-L1 within the cancer nest and tumor stroma. A key finding emerged in the tumor stroma, where a high abundance of CD68 + PD-L1 + cells was a significant favorable prognostic factor, associated with both longer PFS (p = 0.013) and improved OS (p = 0.017). High expression of general PD-L1 + cells was also a positive prognostic indicator; its expression in both the cancer nest and stroma was significantly associated with better PFS and OS (p < 0.05), and showed a notable trend toward improved OS specifically in the stroma (p = 0.056). The expression level of PD-1 on T cells and its role in tumor immunotherapy have been widely studied. As a key immune checkpoint molecule, its expression level is closely related to T cell functional status and directly affects the efficacy of anti-PD-1 therapy[29, 30]. High expression of PD-1 is often associated with T cell exhaustion, characterized by decreased T cell proliferation ability and impaired effector function, which might explain why some patients are resistant to immunotherapy[30, 31]. Consistent with this biological mechanism, our study identified PD-1 expression as a key negative prognostic factor.We found that a high abundance of T-cell exhaustion markers, including both CD8A + PD-1 + and general PD-1 + cells, was significantly associated with shorter PFS (p < 0.05). A high abundance of PD-1 + cells also correlated with shorter OS (p < 0.05). This negative prognostic impact was particularly pronounced within the tumor parenchyma, where high PD-1 expression was significantly associated with both inferior PFS and OS (p < 0.05). The findings of this study should be interpreted in the context of several limitations. First, the single-arm, non-randomized design is susceptible to selection bias and precludes a direct comparison against a control group, potentially influencing the interpretation of therapeutic effectiveness. Furthermore, the small sample size of 28 patients limits the statistical power for definitive conclusions and may restrict the generalizability of our promising efficacy and safety outcomes. Second, the reliance on radiographic assessments may not fully capture treatment response complexity, a point underscored by the observed discordance between radiographic and pathological outcomes. From a translational perspective, our biomarker analysis also has certain constraints. Our investigation was based solely on pre-treatment specimens, providing a static snapshot of the immune microenvironment without capturing the dynamic immunological changes induced by the immunoradiotherapy regimen. While we identified significant correlations between specific immune cell signatures and clinical outcomes, these associations are correlative and do not establish causality. Further mechanistic studies are required to elucidate the functional roles these biomarkers play in treatment response and resistance. Collectively, these factors highlight the necessity of a larger, randomized controlled trial to validate these findings and enhance the robustness of our conclusions. Future research should prospectively validate our prognostic biomarkers (e.g., stromal CD68 + PD-L1 + and PD-1 + cells) within a randomized controlled trial (RCT) to develop a predictive signature for this chemo-free regimen. Furthermore, the success of this chemo-sparing paradigm in ESCC warrants its investigation in other malignancies with high chemoradiotherapy-related toxicity, such as head and neck or cervical cancers. A confirmatory phase III RCT is required to establish this regimen as a new standard of care. Conclusion In conclusion, this Phase II trial provides compelling evidence that the chemotherapy-free regimen of tislelizumab combined with radiotherapy demonstrates substantial antitumor activity and a manageable safety profile in patients with unresectable, locally advanced ESCC who are ineligible for standard CCRT. Furthermore, our identification of prognostic immune signatures within the tumor microenvironment provides a strong rationale for developing predictive biomarkers to guide patient selection. While acknowledging the study's limitations, these findings represent a significant advance for this vulnerable patient population and strongly support the need for a definitive randomized controlled trial. Abbreviations ChiCTR Chinese Clinical Trial Registry ESCC Esophageal Squamous Cell Carcinoma CCRT Concurrent Chemoradiotherapy PD-1 Programmed cell Death protein 1 PFS Progression-Free Survival mPFS median Progression-Free Survival CI Confidence Interval mOS median Overall Survival OS Overall Survival ORR Objective Response Rate pCR pathological complete response TRAE Treatment-Related Adverse Event RTOG Radiation Therapy Oncology Group ICIs Immune Checkpoint Inhibitors ICD Immunogenic Cell Death DAMPs Damage-Associated Molecular Patterns HMGB1 High-Mobility Group Box-1 IgG4 Immunoglobulin G4 mAb monoclonal Antibody DCR Disease Control Rate DOR Duration of Response cCR complete Clinical Response AJCC American Joint Committee on Cancer ECOG Eastern Cooperative Oncology Group AEs Adverse Events irRECIST Immune-Related Response Evaluation Criteria in Solid Tumors IMRT Intensity-modulated Radiotherapy CR Complete Remission MPR Major Pathological Response PR Partial Response SD Stable Disease NCI-CTCAE National Cancer Institute's Common Terminology Criteria for Adverse Events mIF multiplex Immunofluorescence PanCK Pan-Cytokeratin CPS Combined Positive Score TPS Tumor Proportion Score mITT modified Intention-to-Treat SAS Safety Analysis Set RECIST Response Evaluation Criteria in Solid Tumors PPV Positive Predictive Value irAEs Immune-related adverse events FDA Food and Drug Administration NCCN National Comprehensive Cancer Network RCT randomized controlled trial Declarations Funding acknowledgment The authors received no specific funding for this work. Authors’ contributions Changmin Liu, Yue Wang, and Bichun Xu contributed equally to this work. Changmin Liu and Yue Wang designed the study, collected the data, and performed the statistical analysis. Bichun Xu was responsible for model development, validation, and drafting the manuscript. Zeshun Yu, Jieyong Tian, and Lijun Tian provided technical support and assisted with data interpretation. Judong Luo, Shuanghu Yuan, and Fangling Ning conceived of and supervised the study, provided funding, and critically revised the manuscript. All authors reviewed and approved the final version. Date Availability The datasets generated and analyzed for this study are securely stored in a controlled-access repository at Binzhou Medical University Hospital and are not publicly available to protect patient confidentiality. Anonymized data may be made available by the corresponding author upon reasonable request. Ethics approval and consent to participate This prospective study was conducted in accordance with the principles of the Declaration of Helsinki and received full approval from the Ethics Committee of Binzhou Medical University Hospital. Written informed consent was obtained from all participants prior to their enrollment in the trial. Before providing consent, each patient received a comprehensive explanation from their oncologist regarding the potential risks, benefits, and procedures associated with the radiotherapy and immunotherapy treatment. Consent for publication Not applicable. Declaration of interest The authors declare that they have no competing interests. References Morgan E, Soerjomataram I, Rumgay H, Coleman HG, Thrift AP, Vignat J, et al. The Global Landscape of Esophageal Squamous Cell Carcinoma and Esophageal Adenocarcinoma Incidence and Mortality in 2020 and Projections to 2040: New Estimates From GLOBOCAN 2020. Gastroenterology. 2022;163(3):649–58.e2. 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Yan X, Duan H, Li G, Zhang Y, Li Z, Liu J, et al. Tislelizumab combined with chemotherapy as neoadjuvant therapy for surgically resectable esophageal cancer: A prospective, single-arm, phase II study (TD-NICE). Int J Surg. 2022;103:106680. Kakeji Y, Oshikiri T, Takiguchi G, Kanaji S, Matsuda T, Nakamura T, et al. Multimodality approaches to control esophageal cancer: development of chemoradiotherapy, chemotherapy, and immunotherapy. Esophagus. 2021;18(1):25–32. Yu R, Wang W, Li T, Li J, Zhao K, Wang W, et al. RATIONALE 311: tislelizumab plus concurrent chemoradiotherapy for localized esophageal squamous cell carcinoma. Future Oncol. 2021;17(31):4081–9. Zhang Y, Sriramaneni RN, Clark PA, Jagodinsky JC, Ye M, Jin W, et al. Multifunctional nanoparticle potentiates the in situ vaccination effect of radiation therapy and enhances response to immune checkpoint blockade. Nat Commun. 2022;13(1):4948. Vanpouille-Box C, Alard A, Aryankalayil MJ, Sarfraz Y, Diamond JM, Schneider RJ, et al. 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Tislelizumab plus chemotherapy versus placebo plus chemotherapy as first-line treatment for advanced or metastatic oesophageal squamous cell carcinoma (RATIONALE-306): a global, randomised, placebo-controlled, phase 3 study. Lancet Oncol. 2023;24(5):483–95. He W, Wang C, Wu L, Wan G, Li B, Han Y, et al. Tislelizumab Plus Chemotherapy Sequential Neoadjuvant Therapy for Non-cCR Patients After Neoadjuvant Chemoradiotherapy in Locally Advanced Esophageal Squamous Cell Carcinoma (ETNT): An Exploratory Study. Front Immunol. 2022;13:853922. Chen F, Luo H, Xing L, Liang N, Xie J, Zhang J. Feasibility and efficiency of concurrent chemoradiotherapy with capecitabine and cisplatin versus radiotherapy alone for elderly patients with locally advanced esophageal squamous cell carcinoma: Experience of two centers. Thorac Cancer. 2018;9(1):59–65. Gockel I, Kneist W, Junginger T. Incurable esophageal cancer: patterns of tumor spread and therapeutic consequences. World J Surg. 2006;30(2):183–90. Dovedi SJ, Adlard AL, Lipowska-Bhalla G, McKenna C, Jones S, Cheadle EJ, et al. Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res. 2014;74(19):5458–68. Jiang M, Hu Y, Lin G, Chen C, Li H. Radiotherapy combined with immune checkpoint inhibitors in locally advanced/metastatic esophageal squamous cell carcinoma: clinical trials, efficacy and future directions. Front Immunol. 2023;14:1177085. Chen Y, Yu R, Liu Y. Combine radiotherapy and immunotherapy in esophageal squamous cell carcinoma. Crit Rev Oncol Hematol. 2023;190:104115. Zhang W, Yan C, Zhang T, Chen X, Dong J, Zhao J, et al. Addition of camrelizumab to docetaxel, cisplatin, and radiation therapy in patients with locally advanced esophageal squamous cell carcinoma: a phase 1b study. Oncoimmunology. 2021;10(1):1971418. Zhu Y, Wen J, Li Q, Chen B, Zhao L, Liu S, et al. Toripalimab combined with definitive chemoradiotherapy in locally advanced oesophageal squamous cell carcinoma (EC-CRT-001): a single-arm, phase 2 trial. Lancet Oncol. 2023;24(4):371–82. Herrera FG, Bourhis J, Coukos G. Radiotherapy combination opportunities leveraging immunity for the next oncology practice. CA Cancer J Clin. 2017;67(1):65–85. Vanpouille-Box C, Alard A, Aryankalayil MJ, Sarfraz Y, Diamond JM, Schneider RJ, et al. DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity. Nat Commun. 2017;8:15618. Wang DY, Salem JE, Cohen JV, Chandra S, Menzer C, Ye F, et al. Fatal Toxic Effects Associated With Immune Checkpoint Inhibitors: A Systematic Review and Meta-analysis. JAMA Oncol. 2018;4(12):1721–8. Li M, Gan L, Song A, Xue J, Lu Y. Rethinking pulmonary toxicity in advanced non-small cell lung cancer in the era of combining anti-PD-1/PD-L1 therapy with thoracic radiotherapy. Biochim Biophys Acta Rev Cancer. 2019;1871(2):323–30. Sun JM, Shen L, Shah MA, Enzinger P, Adenis A, Doi T, et al. Pembrolizumab plus chemotherapy versus chemotherapy alone for first-line treatment of advanced oesophageal cancer (KEYNOTE-590): a randomised, placebo-controlled, phase 3 study. Lancet. 2021;398(10302):759–71. Huang J, Xu J, Chen Y, Zhuang W, Zhang Y, Chen Z, et al. Camrelizumab versus investigator's choice of chemotherapy as second-line therapy for advanced or metastatic oesophageal squamous cell carcinoma (ESCORT): a multicentre, randomised, open-label, phase 3 study. Lancet Oncol. 2020;21(6):832–42. Liu J, Chen Z, Li Y, Zhao W, Wu J, Zhang Z. PD-1/PD-L1 Checkpoint Inhibitors in Tumor Immunotherapy. Front Pharmacol. 2021;12:731798. Zuazo M, Gato-Cañas M, Llorente N, Ibañez-Vea M, Arasanz H, Kochan G, et al. Molecular mechanisms of programmed cell death-1 dependent T cell suppression: relevance for immunotherapy. Ann Transl Med. 2017;5(19):385. Lei Q, Wang D, Sun K, Wang L, Zhang Y. Resistance Mechanisms of Anti-PD1/PDL1 Therapy in Solid Tumors. Front Cell Dev Biol. 2020;8:672. Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files table1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":43931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow chart of the clinical trial\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/61e062df3b74b620933a8943.jpg"},{"id":96979265,"identity":"cb5ba7e1-f2bf-4651-8aa5-b508d18d9196","added_by":"auto","created_at":"2025-11-28 08:54:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":84350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-tumor activity and survival outcomes. (a) Scatter plot demonstrating the association between best percentage change in target lesion size (RECIST 1.1 criteria) and pathological complete response (pCR) status in the efficacy-evaluable population (n=28). Dashed line indicates 30% threshold for partial response. (b) Stacked bar graph comparing pCR rates across radiographic response categories: stable disease (SD, n=5), partial response (PR, n=20), and complete response (CR, n=3). (c) Kaplan–Meier curve for PFS.(d) Kaplan–Meier curve for OS\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/b4b3a924ea66b35d97c3dd91.jpg"},{"id":96979252,"identity":"9dbb15b2-7c89-4def-939e-75ac048187b1","added_by":"auto","created_at":"2025-11-28 08:54:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial Heterogeneity and Quantitative Profiling of Immune Cell Subpopulations in the Tumor Microenvironment. (a) Representative 6-channel immunofluorescence staining of tumor tissue sections showing. (b) Comparative analysis of cellular subpopulations across 25tumor specimens. (c) Tissue-compartment specific cellular densities across all samples. (d) Tumor core subpopulation stratification. (e) Stromal compartment cellular dynamics.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/7b5f704727f84bbcfa852486.jpg"},{"id":96979263,"identity":"ab4f75a1-5850-4696-8846-79283e4dfa6a","added_by":"auto","created_at":"2025-11-28 08:54:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall survival stratified by immune markers in whole tissue sections.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/d3f3c17baf910aab736c1cfb.jpg"},{"id":96979256,"identity":"3c3b8985-2134-475f-bf5e-94e85921c91e","added_by":"auto","created_at":"2025-11-28 08:54:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":103969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePFS stratified by immune markers in whole tissue sections.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/de6460f5ec87bd55203e96f2.jpg"},{"id":96979239,"identity":"551a1927-d8e5-4837-a983-759ffbf7db41","added_by":"auto","created_at":"2025-11-28 08:54:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor parenchyma-specific biomarker profiles.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/e5dbc7b6ebfc7e1bca1670a0.jpg"},{"id":96979270,"identity":"f1aef921-8344-4b51-8fc2-9d09e503e23a","added_by":"auto","created_at":"2025-11-28 08:54:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":114872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStromal compartment analysis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/e2592c1f0e08a3c3e9d37dd3.jpg"},{"id":98424557,"identity":"3cc82820-cbb9-4aa6-a4dc-23ac97ebe69a","added_by":"auto","created_at":"2025-12-17 16:33:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1854839,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/d4594e1f-b2c8-4d00-9245-9de56948f3a6.pdf"},{"id":96979266,"identity":"553588aa-b11c-459b-8167-e74c07871d40","added_by":"auto","created_at":"2025-11-28 08:54:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":231284,"visible":true,"origin":"","legend":"","description":"","filename":"table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7857613/v1/ce72eb07881145ffb3b79443.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tislelizumab combined with radiotherapy for unresectable locally advanced esophageal squamous cell carcinoma: A prospective, single-arm, phase II study (TREC)","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEsophageal cancer is the sixth-leading cause of cancer-related mortality worldwide [1]. For patients with unresectable, locally advanced esophageal squamous cell carcinoma (ESCC), definitive concurrent chemoradiotherapy (CCRT) is the established standard of care, based on landmark trials including RTOG 8501, RTOG 9405, and RTOG 0436[2\u0026ndash;4]. Although CCRT offers superior efficacy to single-modality therapy, its clinical application is constrained by a significant toxicity bottleneck. The regimen's considerable toxicity profile, which includes acute high-grade esophagitis and hematologic events as well as the risk of late, life-threatening cardiopulmonary complications, renders it unsuitable for many patients. This ineligibility, particularly among the elderly or those with significant comorbidities, compromises their prognosis. Consequently, there is a critical unmet need to develop more effective and tolerable therapeutic strategies to improve survival outcomes for this specific patient population.\u003c/p\u003e\u003cp\u003eThe advent of immune checkpoint inhibitors (ICIs) has fundamentally reshaped the treatment landscape for esophageal cancer, with their clinical application successfully extending to the entire course of disease management. In advanced or metastatic ESCC, ICIs combined with chemotherapy have become the standard of care for first-line treatment. In the adjuvant setting, following neoadjuvant chemoradiotherapy, the use of nivolumab has also been established as a standard of care based on the landmark CheckMate 577 trial [5]. Furthermore, studies such as TD-NICE have preliminarily confirmed the positive effects of combining immunotherapy with CCRT in the neoadjuvant setting [6]. For patients with unresectable ESCC, numerous studies are exploring the integration of ICIs with CCRT, offering new hope for this challenging population [7, 8]. Radiotherapy can initiate a cascade of anti-tumor immune responses by inducing immunogenic cell death (ICD), stimulating the release of inflammatory cytokines, and modulating the tumor microenvironment. These actions collectively upregulate PD-L1 expression on tumor cells, providing a robust theoretical basis for its combination with ICIs. This synergy is conceptually framed as an \"in situ vaccine\" effect, whereby radiotherapy can transform immunologically \"cold\" (immunosuppressive) tumors into \"hot\" (immune-infiltrated) tumors, thereby enhancing their susceptibility to immune checkpoint blockade [9].\u003c/p\u003e\u003cp\u003eRadiotherapy promotes immunogenic cell death, leading to the release of damage-associated molecular patterns (DAMPs) such as ATP and high-mobility group box-1 (HMGB1), and surface exposure of calreticulin. This process enhances antigen presentation by dendritic cells. Furthermore, radiation-induced DNA damage can activate the cytosolic cGAS-STING pathway, triggering a type I interferon response that is critical for priming antitumor CD8\u0026thinsp;+\u0026thinsp;T cells [10].\u003c/p\u003e\u003cp\u003eTislelizumab is a humanized IgG4 monoclonal antibody (mAb) that targets PD-1 to exert its anti-tumor effects and has been approved for several malignancies, including ESCC [11\u0026ndash;12]. Early evidence from the RATIONALE 205 study showed significant clinical activity for tislelizumab combined with chemotherapy in ESCC, achieving an objective response rate (ORR) of 46.7%, a disease control rate (DCR) of 80%, a median duration of response (DOR) of 12.8 months, and a median progression-free survival (PFS) of 10.4 months [13]. These promising findings were subsequently validated in the pivotal Phase III RATIONALE-306 study. In this trial, first-line tislelizumab plus chemotherapy demonstrated a significant survival benefit in advanced/metastatic ESCC, extending the median overall survival (OS) to 17.2 months compared to 10.6 months with chemotherapy alone (HR\u0026thinsp;=\u0026thinsp;0.66), thereby establishing its potent activity [14]. Moreover, the ETNT study explored its role in an earlier disease stage, showing that for patients with locally advanced ESCC without a complete clinical response (cCR) after neoadjuvant chemoradiotherapy, sequential treatment with tislelizumab plus chemotherapy provided a favorable safety and efficacy profile [15].\u003c/p\u003e\u003cp\u003eFor unresectable ESCC patients who cannot tolerate standard CCRT, our TREC study pioneers a chemotherapy-free regimen of tislelizumab plus radiotherapy. By leveraging immunogenic synergy to achieve high pathological complete response (pCR) rates and durable efficacy, this approach may establish a new standard of care and challenges the traditional necessity of chemotherapy in immunoradiotherapy.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy design and population\u003c/h2\u003e\u003cp\u003eThe TREC study was a single-center, single-arm, open-label, phase II study used to evaluate the efficacy and safety of tislelizumab combined with radiotherapy in patients with unresectable ESCC. The trial was conducted at Binzhou Medical University Hospital in China, with patient enrollment occurring between May 2021 and October 2022 (Registration: ChiCTR2100053182). This trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines.\u003c/p\u003e\u003cp\u003eEligible participants were patients aged 18 to 90 years with histologically confirmed, unresectable, locally advanced ESCC (stage II\u0026ndash;IVA, AJCC 8th edition), who were deemed unable or unwilling to receive CCRT. Eligible participants were required to have baseline imaging confirming the absence of distant metastasis and no prior anti-tumor therapy, including chemoradiotherapy, targeted therapy, or immunotherapy. Participants were also expected to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and adequate hepatic, renal, bone marrow, and cardiopulmonary function. Patients with a history of autoimmune disease, a second primary malignancy, or those who were pregnant or lactating were excluded. Written informed consent was obtained from all participants prior to enrollment.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTreatment and assessments\u003c/h3\u003e\n\u003cp\u003eParticipants received tislelizumab at a dose of 200 mg intravenously every 3 weeks (Q3W), with the first dose administered on Day 1 of radiotherapy. Treatment continued until documented disease progression, the occurrence of unacceptable adverse events (AEs), or withdrawal of patient consent. Tumor assessments via imaging were performed at baseline and subsequently every 6 weeks. Treatment response and disease progression were evaluated according to the Immune-Related Response Evaluation Criteria in Solid Tumors (irRECIST). Intensity-modulated radiotherapy (IMRT) was used for treatment, targeting both the primary esophageal lesions and regional lymph nodes. Lymph node metastases were irradiated to a total dose of 60 Gy in 30 fractions. For esophageal lesions, if pathological examination by continuous deep biopsy or multi-point sampling via gastroscopy confirmed complete remission(CR), a total dose of 50 Gy in 25 fractions was delivered; otherwise, a total dose of 60 Gy in 30 fractions was administered. The trial utilized a response-adapted radiotherapy strategy to individualize treatment. Patients who achieved an early pCR had their radiation dose de-escalated to 50 Gy to minimize severe toxicities, while those with residual disease received the standard 60 Gy curative dose to ensure robust local tumor control.\u003c/p\u003e\n\u003ch3\u003eEndpoints\u003c/h3\u003e\n\u003cp\u003eThe primary endpoint was investigator-assessed PFS, defined as the time from enrollment to the first documented disease progression or death from any cause, whichever occurred first. Secondary endpoints included OS, defined as the time from enrollment to death from any cause; major pathological response (MPR), defined as \u0026le;\u0026thinsp;10% residual viable tumor in the primary tumor bed following treatment, irrespective of lymph node status; ORR, the proportion of patients achieving a CR or partial response (PR); and DCR, the proportion of patients achieving a CR, PR, or stable disease (SD). Safety was assessed by monitoring and grading AEs according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE), version 4.0.\u003c/p\u003e\n\u003ch3\u003eDifferential protein expression and tumor immune microenvironment analysis\u003c/h3\u003e\n\u003cp\u003ePrior studies have suggested that the expression of immune markers may exhibit significant heterogeneity between tumor parenchyma and stroma, potentially leading to differential prognostic implications. To address this and identify potential prognostic biomarkers in our cohort, we analyzed pre-treatment tumor specimens using multiplex immunofluorescence (mIF) staining for key markers including PD-L1, PD-1, CD8, CD68, and Pan-Cytokeratin (PanCK) .Subsequently, we employed high-throughput fluorescence analysis to quantify a comprehensive set of cellular populations and scoring systems across the whole tissue, tumor parenchyma, and stromal compartments. These parameters included PANCK\u0026thinsp;+\u0026thinsp;PD-L1\u0026thinsp;+\u0026thinsp;cells, CD68\u0026thinsp;+\u0026thinsp;PD-L1\u0026thinsp;+\u0026thinsp;cells, PANCK\u0026thinsp;+\u0026thinsp;cells, CD68\u0026thinsp;+\u0026thinsp;cells, CD8A\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;cells, CD8A\u0026thinsp;+\u0026thinsp;cells, the combined positive score (CPS), PD-1\u0026thinsp;+\u0026thinsp;cells, PD-L1\u0026thinsp;+\u0026thinsp;cells, and the tumor proportion score (TPS). Finally, the prognostic significance of these markers within each compartment was assessed using the Kaplan-Meier method.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eThe sample size was calculated to detect a hypothesized 20% absolute improvement in the 2-year OS rate, from a baseline of 10% with radiotherapy alone to 30% with the addition of immunotherapy. The trial was designed to have 80% power at a two-sided significance level (α) of 0.05, based on a one-sample test for proportions. Accounting for a potential 10% loss to follow-up, the required sample size was determined to be 27 patients using PASS software (version 25.0).\u003c/p\u003e\u003cp\u003eEfficacy was analyzed in the modified intention-to-treat (mITT) population, defined as all enrolled patients who received at least one dose of tislelizumab and had a baseline tumor assessment. The primary endpoint of PFS and all secondary efficacy endpoints, including OS and ORR, were evaluated in this mITT population. Safety was analyzed in the safety analysis set (SAS), which comprised all patients who received at least one treatment dose.\u003c/p\u003e\u003cp\u003eFor statistical analysis, continuous variables were presented as mean and standard deviation (SD), while categorical variables were presented as frequencies and percentages. Comparisons for categorical data were performed using the chi-square test or Fisher\u0026rsquo;s exact test. The Wilcoxon test was used to compare non-parametric data. PFS and OS were estimated using the Kaplan-Meier method. All statistical analyses were conducted using SPSS software (version 25.0, IBM Corp., Armonk, NY, USA), and a two-sided p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design and Baseline Characteristics\u003c/h2\u003e\u003cp\u003eThis prospective phase II trial enrolled patients between May 2021 and October 2022. The patient screening and enrollment process is detailed in the CONSORT diagram (Fig.\u0026nbsp;1). In brief, 32 consecutive patients were screened for eligibility, of whom 29 met the protocol-specified inclusion criteria. Three subjects were excluded for not meeting these criteria, and one additional patient withdrew consent before the initial treatment. This resulted in a modified intention-to-treat (mITT) population of 28 patients who received at least one dose of therapy and completed baseline radiographic evaluations.\u003c/p\u003e\u003cp\u003ePatient status was assessed as of the data cutoff date on June 30, 2024. At this time, 3 of the 28 patients (10.7%) remained on active treatment. Among the other 25 patients, 15 (53.6%) had discontinued treatment due to radiologically confirmed disease progression or death, and 10 (35.7%) had discontinued after completing the planned 24 months of therapy. Two additional patients (7.1%) were lost to follow-up. Overall, nine deaths were recorded; six (66.7%) were attributed to tumor-related causes, while three (33.3%) were non-cancer deaths (one grade 5 pulmonary embolism, one acute myocardial infarction, and one grade 5 pneumonia).\u003c/p\u003e\u003cp\u003eThe baseline demographic and clinical characteristics of the mITT population (N\u0026thinsp;=\u0026thinsp;28) are presented in detail in Table\u0026nbsp;1. The cohort had a median age of 67 years (range 55\u0026ndash;80), with a predominance of male patients (57.1%, 16/28). For performance status, 39.3% (11/28) of patients had an ECOG score of 0, while 60.7% (17/28) had a score of 1. According to the AJCC 8th edition staging, the patient distribution was Stage II (14.3%, 4/28), Stage III (67.9%, 19/28), and Stage IV (17.9%, 5/28). The five Stage IV cases included two with IVA and three with IVB disease, the latter presenting with isolated supraclavicular metastases. The primary tumor epicenter was most commonly located in the middle thoracic esophagus (50.0%, 14/28), followed by the lower (35.7%, 10/28), upper (10.7%, 3/28), and cervical esophagus (3.6%, 1/28).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEfficacy Outcomes and Pathological Correlation\u003c/h3\u003e\n\u003cp\u003eIn the mITT population (n\u0026thinsp;=\u0026thinsp;28), treatment response assessments per RECIST v1.1 criteria demonstrated substantial antitumor efficacy (Fig.\u0026nbsp;2a). The ORR reached 82.1% (23/28; 95% CI 64.5%\u0026ndash;92.1%), including 3 CR (10.7%; 95% CI: 2.8%\u0026ndash;27.3%) and 20 PR (71.4%; 95% CI: 52.2%\u0026ndash;85.9%). DCR was 100% (28/28; 95% CI: 87.7%\u0026ndash;100.0%), with 5 patients (17.9%; 95% CI: 6.7%\u0026ndash;35.9%) achieving SD and no instances of progressive disease observed during the treatment period (Table\u0026nbsp;2 \u0026amp; Fig.\u0026nbsp;2a).\u003c/p\u003e\u003cp\u003eTo evaluate the depth of response, post-radiotherapy deep-cup endoscopic biopsies were performed for histopathological validation. A pCR was achieved in 64.3% (18/28) of patients, while 35.7% (10/28) had residual tumor (non-pCR) (Fig.\u0026nbsp;2a). Notably, a significant discordance was observed between radiographic and pathological outcomes (Fisher's exact test, p\u0026thinsp;=\u0026thinsp;0.047; Cramer's V\u0026thinsp;=\u0026thinsp;0.41, Fig.\u0026nbsp;2b). The pCR rate increased with better radiographic response categories: 40% (2/5) in patients with SD, 65% (13/20) in those with a PR, and 100% (3/3) in those with a CR. While a pairwise comparison revealed a significant difference in pCR rates between the SD and CR groups (p\u0026thinsp;=\u0026thinsp;0.049), this difference did not remain significant for other inter-group comparisons after Bonferroni correction (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Radiographic CR demonstrated a 100% positive predictive value (PPV) for achieving pCR, though its sensitivity was limited to 16.7%.\u003c/p\u003e\u003cp\u003eThe mPFS was 23.5 months (95% CI 18.2\u0026ndash;not reached), with 1-, 2-, and 3-year PFS rates of 75.0% (62.5\u0026ndash;85.7), 57.1% (42.9\u0026ndash;71.4), and 42.9% (28.6\u0026ndash;57.1), respectively (Fig.\u0026nbsp;2c). A Kaplan-Meier analysis revealed an initial decline in survival probability within the first year, followed by a period of sustained disease control beyond 24 months. For OS, the median survival was not reached at the data cutoff (median follow-up: 24.7 months; range 4\u0026ndash;34.5 months). The 1-, 2-, and 3-year OS rates were 82.1% (71.4\u0026ndash;90.5), 64.3% (50.0\u0026ndash;77.8), and 53.6% (38.1\u0026ndash;67.9), respectively (Fig.\u0026nbsp;2d). A plateau emerged in the OS curve after 24 months (log-rank p\u0026thinsp;=\u0026thinsp;0.032 vs. historical controls), suggesting durable survival in a clinically relevant subset of patients (n\u0026thinsp;=\u0026thinsp;9/28, 32.1% of patients remaining progression-free at 2 years).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSafety\u003c/h2\u003e\u003cp\u003eThe safety profile was analyzed in all 28 patients who received treatment. The regimen was generally manageable, with a detailed summary of AEs provided in Table\u0026nbsp;3. All patients experienced at least one AE, the majority of which were Grade 1\u0026ndash;2 in severity. The most frequent AEs of any grade included lymphopenia (22 patients, 78.6%), esophagitis (20, 71.4%), and hyponatremia (14, 50.0%). The most common Grade 2 event was esophagitis, occurring in 15 patients (53.6%). Among Grade 3 or higher AEs, the most frequent was Grade 3 lymphopenia, observed in 15 patients (53.6%). Other notable Grade 3 events included lung infection (2, 7.1%), esophageal fistula (1, 3.6%), and immune-related hepatotoxicity (1, 3.6%). Immune-related adverse events (irAEs) were common but predominantly low-grade. The most frequent irAE was hypothyroidism, occurring in 4 patients (14.3%, all Grade 2). Additionally, three cases of Grade 2 checkpoint inhibitor pneumonitis (10.7%), one case of Grade 2 myocarditis (3.6%), and one case of Grade 3 immune-related hepatotoxicity (3.6%) were reported. Three Grade 5 (fatal) AEs were reported, with one case each attributed to lung infection, myocardial infarction, and a vascular access complication.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBiomarker\u003c/h2\u003e\u003cp\u003eTo identify potential prognostic biomarkers, we performed multiplex immunofluorescence staining on tumor specimens. A total of 25 specimens were subsequently analyzed, targeting PD-L1, PD-1, CD8, CD68, and PANCK (Fig.\u0026nbsp;3a \u0026amp; 3b). Quantitative analysis across all patient samples revealed that parenchymal (PANCK_P) and stromal (PANCK_S) tumor cells were the most abundant populations overall (Fig.\u0026nbsp;3c). Further stratification by tissue compartment highlighted distinct microenvironments: the tumor parenchyma was characterized by a predominance of PANCK\u0026thinsp;+\u0026thinsp;tumor cells and CD68\u0026thinsp;+\u0026thinsp;macrophages (Fig.\u0026nbsp;3d), whereas the stromal compartment was primarily composed of CD68\u0026thinsp;+\u0026thinsp;macrophages and PD-L1\u0026thinsp;+\u0026thinsp;cells, indicating a different immune infiltrate (Fig.\u0026nbsp;3e).\u003c/p\u003e\u003cp\u003eHigh densities of markers associated with T-cell exhaustion, such as PD-1\u0026thinsp;+\u0026thinsp;and CD8A\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;cells, were generally associated with unfavorable outcomes. In the whole-tissue analysis, increased PD-1\u0026thinsp;+\u0026thinsp;cell abundance correlated with shorter OS (Fig.\u0026nbsp;4) and PFS (Fig.\u0026nbsp;5). This effect was primarily driven by their presence within the tumor parenchyma; compartmental analysis revealed that a high density of intratumoral PD-1\u0026thinsp;+\u0026thinsp;cells was a strong factor for poor prognosis, significantly associating with both shorter OS (p\u0026thinsp;=\u0026thinsp;0.024) and PFS (p\u0026thinsp;=\u0026thinsp;0.038) (Fig.\u0026nbsp;6\u0026amp;7). In contrast, stromal PD-1\u0026thinsp;+\u0026thinsp;cells only showed a non-significant trend toward worse PFS (p\u0026thinsp;=\u0026thinsp;0.089) (Fig.\u0026nbsp;6\u0026amp;7). Similarly, a high abundance of CD8A\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;cells in the whole tissue was associated with shorter PFS (Fig.\u0026nbsp;5). This negative effect was also localized to the parenchyma, where a high density of these cells significantly correlated with shortened PFS (p\u0026thinsp;=\u0026thinsp;0.022) but not OS (p\u0026thinsp;=\u0026thinsp;0.170) (Fig.\u0026nbsp;6\u0026amp;7). No significant associations were found for stromal CD8A\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;cells (Fig.\u0026nbsp;6\u0026amp;7).\u003c/p\u003e\u003cp\u003eConversely, markers related to PD-L1 expression generally indicated a more favorable prognosis, with their predictive value being highly dependent on the cell type and spatial compartment. In the whole-tissue analysis, a higher proportion of PANCK\u0026thinsp;+\u0026thinsp;PD-L1\u0026thinsp;+\u0026thinsp;cells was associated with improved PFS (Fig.\u0026nbsp;5), and along with a positive TPS, exhibited protective effects on OS (Fig.\u0026nbsp;4).Compartmental analysis provided further insights. High expression of stromal CD68\u0026thinsp;+\u0026thinsp;PD-L1\u0026thinsp;+\u0026thinsp;cells was a strong favorable prognostic factor, significantly associated with improved OS (p\u0026thinsp;=\u0026thinsp;0.017) and PFS (p\u0026thinsp;=\u0026thinsp;0.013) (Fig.\u0026nbsp;6\u0026amp;7). While a high density of these cells in the parenchyma also correlated with better OS (p\u0026thinsp;=\u0026thinsp;0.040), it had no significant impact on PFS (p\u0026thinsp;=\u0026thinsp;0.400) (Fig.\u0026nbsp;6\u0026amp;7). For total PD-L1\u0026thinsp;+\u0026thinsp;cells, a high density within the tumor parenchyma was significantly associated with improved OS (p\u0026thinsp;=\u0026thinsp;0.032) and PFS (p\u0026thinsp;=\u0026thinsp;0.038), while a high density in the stroma was also significantly associated with improved PFS (p\u0026thinsp;=\u0026thinsp;0.043) and showed a trend toward improved OS (p\u0026thinsp;=\u0026thinsp;0.056) (Fig.\u0026nbsp;6\u0026amp;7). A high density of total PD-L1\u0026thinsp;+\u0026thinsp;cells in the whole tissue also showed a borderline trend toward improved PFS (P\u0026thinsp;=\u0026thinsp;0.054) (Fig.\u0026nbsp;5). In contrast to TPS, the CPSshowed no significant impact on survival outcomes in this cohort (Figs.\u0026nbsp;4 \u0026amp; 5).\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCCRT stands as the standard treatment modality for inoperable locally advanced ESCC [2\u0026ndash;4]. Meta-analysis of multicenter randomized controlled trials shows that, compared to radiotherapy alone, CCRT significantly improves patient prognosis, reducing the risk of death by 27% (HR\u0026thinsp;=\u0026thinsp;0.73, 95% CI 0.64\u0026ndash;0.84) and increasing the absolute survival rates by 9% and 4% at 1 and 2 years, respectively [4]. This therapeutic gain, however, is offset by a substantially higher toxicity profile. CCRT elevates the risk of grade 3\u0026ndash;4 acute toxicity more than five-fold (OR\u0026thinsp;=\u0026thinsp;5.16, 95% CI 2.83\u0026ndash;9.38), with severe hematologic toxicity observed in approximately 30% of patients, compared to just 2% in the radiotherapy-alone cohort [4]. Consequently, patients with advanced age or comorbidities are often ineligible for CCRT, leaving them with suboptimal options like radiotherapy or chemotherapy alone [16\u0026ndash;17]. This highlights a pressing clinical need for more effective and better-tolerated therapeutic strategies for this specific population.\u003c/p\u003e\u003cp\u003e Recent regulatory and guideline advancements underscore the clinical utility of tislelizumab, a humanized anti-PD-1 antibody, in ESCC. In March 2024, it secured U.S. FDA approval as a monotherapy for unresectable/metastatic ESCC post-chemotherapy. This is mirrored by its designation as a Category 1 preferred agent for second-line and subsequent therapy in the NCCN Guidelines (v2.2024). Building upon this validation, evaluating its efficacy in the first-line setting, especially combined with radiotherapy, is a logical and compelling next step in clinical investigation.\u003c/p\u003e\u003cp\u003eWhile the combination of radiotherapy and immunotherapy is an active area of investigation, a consensus on the optimal timing for initiating immunotherapy is lacking [18]. Preclinical evidence indicates superior efficacy when the two modalities are administered concurrently. This is corroborated by early-phase clinical trials, which show greater benefit when immunotherapy is given with or shortly after radiotherapy, as opposed to sequentially. Sequential strategies are often hampered by low rates of immunotherapy initiation after the completion of chemoradiotherapy [19, 20]. Therefore, our study protocol involved the concurrent administration of tislelizumab with radiotherapy, followed by a maintenance phase of tislelizumab monotherapy.\u003c/p\u003e\u003cp\u003eIn this phase II trial, we demonstrated that a chemotherapy-free regimen of tislelizumab plus radiotherapy yielded remarkable clinical outcomes for unresectable locally advanced patients ineligible for standard CCRT. The ORR of 82.1% and mPFS of 23.5 months represent a substantial improvement over historical outcomes with radiotherapy alone [16, 17]. Furthermore, the high pathological complete response rate of 64.3% underscores the potent synergy between immunotherapy and radiotherapy. Notably, the efficacy observed in our study compares favorably with recent trials that combined immunotherapy with a full CCRT regimen. For instance, studies adding camrelizumab or toripalimab to CCRT reported 2-year OS rates of 70% and 1-year OS rates of 78%, respectively [21, 22]. That our chemotherapy-sparing approach achieved comparable survival outcomes (a 2-year OS rate of 64.3%) is a key finding. It suggests that for patients who cannot tolerate concurrent chemotherapy, the combination of a PD-1 inhibitor with radiotherapy may offer a highly effective and better-tolerated alternative, positioning this strategy strongly within the current therapeutic landscape.\u003c/p\u003e\u003cp\u003eA particularly noteworthy finding of our study was the significant discordance between radiographic and pathological responses. While radiographic CR demonstrated a 100% positive predictive value for pCR, its low sensitivity suggests that imaging alone is insufficient for accurately assessing treatment efficacy. Post-radiotherapy effects, such as inflammation and fibrosis, can often mimic residual disease on imaging, leading to an underestimation of the true pathological response. This has profound clinical implications: it suggests that even patients with stable disease or partial response on imaging may have already achieved pCR. Therefore, our findings strongly advocate for the routine use of post-treatment endoscopic biopsy to confirm pathological response, which is critical for guiding subsequent clinical decisions, such as the duration of immunotherapy or the consideration of consolidative surgery. Furthermore, this discordance underscores the limitations of imaging-based endpoints and supports the establishment of pCR as a more robust and objective surrogate endpoint for survival in future clinical trials of immunoradiotherapy for ESCC.\u003c/p\u003e\u003cp\u003eEvaluating the safety of this combination therapy was critical, particularly given the theoretical risk of overlapping toxicities[23, 24] and our inclusion of a vulnerable cohort intolerant to standard CCRT, which included a significant proportion of elderly patients (32.1% \u0026ge;75 years). Overall, the regimen was well-tolerated, with most TRAEs being Grade 1\u0026ndash;2. Two adverse events of special concern in this setting are esophageal fistula and pneumonitis. The incidence of esophageal fistula was notably low at 3.57% (one Grade 3 event), which compares favorably to the 10\u0026ndash;14% rates reported in prior studies combining immunotherapy with CCRT [21, 22]. Pneumonitis is another critical concern, as it accounts for a significant portion of deaths during anti-PD-1/PD-L1 therapy [25] and its risk may be elevated when radiotherapy is combined with immunotherapy [26]. In our study, the toxicity from pneumonitis was manageable. Both checkpoint inhibitor-related and radiation-induced pneumonitis occurred at a rate of 10.7% each, and importantly, all cases were confined to Grade 2 severity. The most frequent high-grade toxicity was Grade 3 lymphopenia, occurring in 53.6% of patients. While clinically manageable, this high incidence warrants careful monitoring due to its potential to impair long-term anti-tumor immunity. Three Grade 5 (fatal) AEs were reported: one lung infection, one myocardial infarction, and one vascular access complication. These events were considered by investigators to be primarily related to severe patient comorbidities during the COVID-19 pandemic rather than direct treatment toxicity. However, indirect therapeutic contributions cannot be fully excluded, as the high incidence of Grade 3 lymphopenia may have increased infection susceptibility, and a systemic inflammatory response could have factored into the cardiovascular event. These outcomes underscore the fragility of this patient population, necessitating exceptionally vigilant monitoring and supportive care when administering this potent regimen.\u003c/p\u003e\u003cp\u003eOur study's biomarker analysis validates the predictive value of PD-L1 expression in ESCC and provides deeper insights into the complex, context-dependent role of the tumor immune microenvironment. Landmark trials such as KEYNOTE-590 and ESCORT have consistently demonstrated that higher PD-L1 expression, whether measured by Combined Positive Score (CPS\u0026thinsp;\u0026ge;\u0026thinsp;10) or Tumor Proportion Score (TPS\u0026thinsp;\u0026ge;\u0026thinsp;10%), correlates with significantly greater survival benefits for patients receiving immunotherapy combinations [27, 28]. Aligning with these findings, our study also identified a link between PD-L1 expression and favorable outcomes. Specifically, an increased proportion of PD-L1 on tumor cells (PANCK\u0026thinsp;+\u0026thinsp;PD-L1+) was significantly associated with improved PFS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while a high density of PD-L1\u0026thinsp;+\u0026thinsp;cells showed a trend toward improved OS (P\u0026thinsp;=\u0026thinsp;0.054). Considering that region-specific expression patterns of immune biomarkers may critically impact immunotherapy efficacy, we also examined the expression of PD-L1 within the cancer nest and tumor stroma. A key finding emerged in the tumor stroma, where a high abundance of CD68\u0026thinsp;+\u0026thinsp;PD-L1\u0026thinsp;+\u0026thinsp;cells was a significant favorable prognostic factor, associated with both longer PFS (p\u0026thinsp;=\u0026thinsp;0.013) and improved OS (p\u0026thinsp;=\u0026thinsp;0.017). High expression of general PD-L1\u0026thinsp;+\u0026thinsp;cells was also a positive prognostic indicator; its expression in both the cancer nest and stroma was significantly associated with better PFS and OS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and showed a notable trend toward improved OS specifically in the stroma (p\u0026thinsp;=\u0026thinsp;0.056).\u003c/p\u003e\u003cp\u003eThe expression level of PD-1 on T cells and its role in tumor immunotherapy have been widely studied. As a key immune checkpoint molecule, its expression level is closely related to T cell functional status and directly affects the efficacy of anti-PD-1 therapy[29, 30]. High expression of PD-1 is often associated with T cell exhaustion, characterized by decreased T cell proliferation ability and impaired effector function, which might explain why some patients are resistant to immunotherapy[30, 31]. Consistent with this biological mechanism, our study identified PD-1 expression as a key negative prognostic factor.We found that a high abundance of T-cell exhaustion markers, including both CD8A\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;and general PD-1\u0026thinsp;+\u0026thinsp;cells, was significantly associated with shorter PFS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A high abundance of PD-1\u0026thinsp;+\u0026thinsp;cells also correlated with shorter OS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This negative prognostic impact was particularly pronounced within the tumor parenchyma, where high PD-1 expression was significantly associated with both inferior PFS and OS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eThe findings of this study should be interpreted in the context of several limitations. First, the single-arm, non-randomized design is susceptible to selection bias and precludes a direct comparison against a control group, potentially influencing the interpretation of therapeutic effectiveness. Furthermore, the small sample size of 28 patients limits the statistical power for definitive conclusions and may restrict the generalizability of our promising efficacy and safety outcomes. Second, the reliance on radiographic assessments may not fully capture treatment response complexity, a point underscored by the observed discordance between radiographic and pathological outcomes. From a translational perspective, our biomarker analysis also has certain constraints. Our investigation was based solely on pre-treatment specimens, providing a static snapshot of the immune microenvironment without capturing the dynamic immunological changes induced by the immunoradiotherapy regimen. While we identified significant correlations between specific immune cell signatures and clinical outcomes, these associations are correlative and do not establish causality. Further mechanistic studies are required to elucidate the functional roles these biomarkers play in treatment response and resistance. Collectively, these factors highlight the necessity of a larger, randomized controlled trial to validate these findings and enhance the robustness of our conclusions. Future research should prospectively validate our prognostic biomarkers (e.g., stromal CD68\u0026thinsp;+\u0026thinsp;PD-L1\u0026thinsp;+\u0026thinsp;and PD-1\u0026thinsp;+\u0026thinsp;cells) within a randomized controlled trial (RCT) to develop a predictive signature for this chemo-free regimen. Furthermore, the success of this chemo-sparing paradigm in ESCC warrants its investigation in other malignancies with high chemoradiotherapy-related toxicity, such as head and neck or cervical cancers. A confirmatory phase III RCT is required to establish this regimen as a new standard of care.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this Phase II trial provides compelling evidence that the chemotherapy-free regimen of tislelizumab combined with radiotherapy demonstrates substantial antitumor activity and a manageable safety profile in patients with unresectable, locally advanced ESCC who are ineligible for standard CCRT. Furthermore, our identification of prognostic immune signatures within the tumor microenvironment provides a strong rationale for developing predictive biomarkers to guide patient selection. While acknowledging the study's limitations, these findings represent a significant advance for this vulnerable patient population and strongly support the need for a definitive randomized controlled trial.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eChiCTR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChinese Clinical Trial Registry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eESCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEsophageal Squamous Cell Carcinoma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCCRT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eConcurrent Chemoradiotherapy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProgrammed cell Death protein 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProgression-Free Survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emPFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emedian Progression-Free Survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eConfidence Interval\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emedian Overall Survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOverall Survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eORR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eObjective Response Rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003epCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epathological complete response\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTRAE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTreatment-Related Adverse Event\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRTOG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRadiation Therapy Oncology Group\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eICIs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmune Checkpoint Inhibitors\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eICD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmunogenic Cell Death\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAMPs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDamage-Associated Molecular Patterns\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHMGB1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHigh-Mobility Group Box-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIgG4\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmunoglobulin G4\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emAb\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emonoclonal Antibody\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDisease Control Rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDOR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDuration of Response\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecomplete Clinical Response\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAJCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAmerican Joint Committee on Cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eECOG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEastern Cooperative Oncology Group\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAEs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAdverse Events\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eirRECIST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmune-Related Response Evaluation Criteria in Solid Tumors\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIMRT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIntensity-modulated Radiotherapy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eComplete Remission\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMPR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMajor Pathological Response\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePartial Response\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStable Disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNCI-CTCAE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNational Cancer Institute's Common Terminology Criteria for Adverse Events\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emIF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emultiplex Immunofluorescence\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePanCK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePan-Cytokeratin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCPS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCombined Positive Score\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTPS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumor Proportion Score\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emITT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emodified Intention-to-Treat\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSafety Analysis Set\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRECIST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eResponse Evaluation Criteria in Solid Tumors\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePPV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePositive Predictive Value\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eirAEs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmune-related adverse events\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFood and Drug Administration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNCCN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNational Comprehensive Cancer Network\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRCT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003erandomized controlled trial\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding acknowledgment\u003c/p\u003e\n\u003cp\u003eThe authors received no specific funding for this work.\u003c/p\u003e\n\u003cp\u003eAuthors’\u0026nbsp;contributions\u003c/p\u003e\n\u003cp\u003eChangmin Liu, Yue Wang, and Bichun Xu contributed equally to this work. Changmin Liu and Yue Wang designed the study, collected the data, and performed the statistical analysis. Bichun Xu was responsible for model development, validation, and drafting the manuscript. Zeshun Yu, Jieyong Tian, and Lijun Tian provided technical support and assisted with data interpretation. Judong Luo, Shuanghu Yuan, and Fangling Ning conceived of and supervised the study, provided funding, and critically revised the manuscript. All authors reviewed and approved the final version.\u003c/p\u003e\n\u003cp\u003eDate Availability\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed for this study are securely stored in a controlled-access repository at Binzhou Medical University Hospital and are not publicly available to protect patient confidentiality. Anonymized data may be made available by the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis prospective study was conducted in accordance with the principles of the Declaration of Helsinki and received full approval from the Ethics Committee of Binzhou Medical University Hospital. Written informed consent was obtained from all participants prior to their enrollment in the trial. Before providing consent, each patient received a comprehensive explanation from their oncologist regarding the potential risks, benefits, and procedures associated with the radiotherapy and immunotherapy treatment.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eDeclaration of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMorgan E, Soerjomataram I, Rumgay H, Coleman HG, Thrift AP, Vignat J, et al. The Global Landscape of Esophageal Squamous Cell Carcinoma and Esophageal Adenocarcinoma Incidence and Mortality in 2020 and Projections to 2040: New Estimates From GLOBOCAN 2020. 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World J Surg. 2006;30(2):183\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eDovedi SJ, Adlard AL, Lipowska-Bhalla G, McKenna C, Jones S, Cheadle EJ, et al. Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res. 2014;74(19):5458\u0026ndash;68.\u003c/li\u003e\n\u003cli\u003eJiang M, Hu Y, Lin G, Chen C, Li H. Radiotherapy combined with immune checkpoint inhibitors in locally advanced/metastatic esophageal squamous cell carcinoma: clinical trials, efficacy and future directions. Front Immunol. 2023;14:1177085.\u003c/li\u003e\n\u003cli\u003eChen Y, Yu R, Liu Y. Combine radiotherapy and immunotherapy in esophageal squamous cell carcinoma. Crit Rev Oncol Hematol. 2023;190:104115.\u003c/li\u003e\n\u003cli\u003eZhang W, Yan C, Zhang T, Chen X, Dong J, Zhao J, et al. Addition of camrelizumab to docetaxel, cisplatin, and radiation therapy in patients with locally advanced esophageal squamous cell carcinoma: a phase 1b study. Oncoimmunology. 2021;10(1):1971418.\u003c/li\u003e\n\u003cli\u003eZhu Y, Wen J, Li Q, Chen B, Zhao L, Liu S, et al. Toripalimab combined with definitive chemoradiotherapy in locally advanced oesophageal squamous cell carcinoma (EC-CRT-001): a single-arm, phase 2 trial. Lancet Oncol. 2023;24(4):371\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eHerrera FG, Bourhis J, Coukos G. Radiotherapy combination opportunities leveraging immunity for the next oncology practice. CA Cancer J Clin. 2017;67(1):65\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eVanpouille-Box C, Alard A, Aryankalayil MJ, Sarfraz Y, Diamond JM, Schneider RJ, et al. DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity. Nat Commun. 2017;8:15618.\u003c/li\u003e\n\u003cli\u003eWang DY, Salem JE, Cohen JV, Chandra S, Menzer C, Ye F, et al. Fatal Toxic Effects Associated With Immune Checkpoint Inhibitors: A Systematic Review and Meta-analysis. JAMA Oncol. 2018;4(12):1721\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eLi M, Gan L, Song A, Xue J, Lu Y. Rethinking pulmonary toxicity in advanced non-small cell lung cancer in the era of combining anti-PD-1/PD-L1 therapy with thoracic radiotherapy. Biochim Biophys Acta Rev Cancer. 2019;1871(2):323\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eSun JM, Shen L, Shah MA, Enzinger P, Adenis A, Doi T, et al. Pembrolizumab plus chemotherapy versus chemotherapy alone for first-line treatment of advanced oesophageal cancer (KEYNOTE-590): a randomised, placebo-controlled, phase 3 study. Lancet. 2021;398(10302):759\u0026ndash;71.\u003c/li\u003e\n\u003cli\u003eHuang J, Xu J, Chen Y, Zhuang W, Zhang Y, Chen Z, et al. Camrelizumab versus investigator's choice of chemotherapy as second-line therapy for advanced or metastatic oesophageal squamous cell carcinoma (ESCORT): a multicentre, randomised, open-label, phase 3 study. Lancet Oncol. 2020;21(6):832\u0026ndash;42.\u003c/li\u003e\n\u003cli\u003eLiu J, Chen Z, Li Y, Zhao W, Wu J, Zhang Z. PD-1/PD-L1 Checkpoint Inhibitors in Tumor Immunotherapy. Front Pharmacol. 2021;12:731798.\u003c/li\u003e\n\u003cli\u003eZuazo M, Gato-Ca\u0026ntilde;as M, Llorente N, Iba\u0026ntilde;ez-Vea M, Arasanz H, Kochan G, et al. Molecular mechanisms of programmed cell death-1 dependent T cell suppression: relevance for immunotherapy. Ann Transl Med. 2017;5(19):385.\u003c/li\u003e\n\u003cli\u003eLei Q, Wang D, Sun K, Wang L, Zhang Y. Resistance Mechanisms of Anti-PD1/PDL1 Therapy in Solid Tumors. Front Cell Dev Biol. 2020;8:672.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7857613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7857613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Treatment options for unresectable locally advanced esophageal squamous cell carcinoma (ESCC) that cannot tolerate concurrent chemoradiotherapy (CCRT) are exceedingly limited. This study aimed to evaluate the efficacy and safety of tislelizumab, a programmed cell death protein 1 (PD-1) inhibitor, in combination with definitive radiotherapy as a novel chemotherapy-free first-line treatment for patients with unresectable, locally advanced ESCC who are ineligible for standard CCRT.\u003c/p\u003e\n\u003cp\u003eMethods: This prospective, single-arm, phase II clinical trial (ChiCTR2100053182) recruited treatment-naïve patients diagnosed with unresectable, locally advanced ESCC at Binzhou Medical University Hospital. Eligibility was limited to patients considered ineligible for standard CCRT due to medical contraindications or patient preference. The investigational regimen consisted of tislelizumab (200 mg intravenously) administered on a three-week cycle, initiated concurrently with the first fraction of radiotherapy. The primary endpoint was progression-free survival (PFS). Additionally, the protocol included a prespecified exploratory analysis of tumor immune microenvironment markers using high-throughput multiplex immunofluorescence.\u003c/p\u003e\n\u003cp\u003eResults:From May 2021 to October 2022, 32 eligible patients were enrolled. The mPFS was 23.5 months (95% CI 18.2–not reached), the 1-, 2-, and 3-year PFS rates were 75.0%, 57.1% and 42.9% respectively. The median overall survival (mOS) was not reached, with a 3-year OS rate of 53.6%. The regimen demonstrated substantial efficacy, achieving an objective response rate (ORR) of 82.1% and a pathological complete response (pCR) rate of 64.3%. Treatment was well-tolerated; the most frequent grade ≥3 treatment-related adverse event (TRAE) was lymphopenia (53.6%). Exploratory biomarker analysis revealed that high densities of T-cell exhaustion markers (e.g., intratumoral PD-1+ cells) were significantly associated with poorer PFS and OS, whereas high expression of stromal CD68+PD-L1+ cells was a strong favorable prognostic factor.\u003c/p\u003e\n\u003cp\u003eConclusion: This phase II study demonstrates that the combination of tislelizumab and definitive radiotherapy yields promising efficacy and manageable toxicity in patients with unresectable, locally advanced ESCC who are ineligible for standard CCRT. Moreover, the observed associations between immune cell signatures and treatment outcomes highlight the potential for biomarker-driven patient selection in future trials.\u003c/p\u003e","manuscriptTitle":"Tislelizumab combined with radiotherapy for unresectable locally advanced esophageal squamous cell carcinoma: A prospective, single-arm, phase II study (TREC)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 08:53:32","doi":"10.21203/rs.3.rs-7857613/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf0462e3-2150-4595-8ea7-9d18c3bbe360","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-11T11:24:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-28 08:53:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7857613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7857613","identity":"rs-7857613","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}