Abstract
Purpose
Squamous non-small cell lung cancer (sq-NSCLC) is a distinct subtype of NSCLC. This exploratory, phase II study investigated the feasibility and efficacy of a four-cycle perioperative regimen combining serplulimab with a taxane (paclitaxel or nab-paclitaxel) and carboplatin in patients with resectable stage II-IIIA sq-NSCLC.
Methods
This investigator-initiated, single-arm, phase II exploratory trial (NCT05775796) enrolled patients with histologically confirmed, resectable clinical stage II-IIIA squamous NSCLC. Patients received two to three cycles of neoadjuvant serplulimab plus taxane-carboplatin, followed by curative-intent surgery and one to two cycles of adjuvant treatment. The primary endpoint was major pathological response (MPR). Secondary endpoints included pathological complete response (pCR), R0 resection rate, overall response rate (ORR), safety, event-free survival (EFS), and overall survival (OS).
Results
A total of 30 patients without actionable driver alterations were enrolled and 29 underwent surgery. The median age was 65 years, and most were male smokers (n=28, 93.33%). Surgery was performed in 29 patients, and R0 resection was achieved in 96.55% (28/29) of the surgically resected patients. Among all enrolled patients, MPR and pCR rates were 76.67% and 50.00%, respectively. Based on radiological assessments during the neoadjuvant phase, the ORR was 73.33% (95% CI 54.11% to 87.72%). Grade ≥3 treatment-related adverse events were predominantly hematologic and were generally manageable. Long-term EFS and OS data are not yet mature. Additionally, exploratory minimal residual disease analysis using circulating tumor DNA (ctDNA) in 27 patients showed a strong correlation between ctDNA clearance and pCR (p=0.004), suggesting ctDNA as a promising biomarker for immunochemotherapy response.
Conclusions
A four-cycle perioperative regimen of serplulimab combined with taxane-carboplatin demonstrated promising MPR and pCR rates with an acceptable safety profile in patients with resectable sq-NSCLC. Long-term follow-up and future phase III trials are warranted to confirm these exploratory findings.
Trial registration number
Keywords: Lung Cancer, Immune Checkpoint Inhibitor, Neoadjuvant, Minimal residual disease - MRD
WHAT IS ALREADY KNOWN ON THIS TOPIC
Perioperative immunochemotherapy improves outcomes in resectable non-small cell lung cancer (NSCLC), and the standard regimen consists of four neoadjuvant cycles of chemoimmunotherapy followed by up to a year of adjuvant immunotherapy. Squamous NSCLC (sq-NSCLC) is a distinct histological subtype, but few studies explored simplified perioperative regimens specifically for this population.
WHAT THIS STUDY ADDS
This phase II trial demonstrated that a simplified, four-cycle perioperative regimen (serplulimab plus chemotherapy) was feasible in resectable stage II-IIIA sq-NSCLC, achieving a major pathological response rate of 76.7% and a pathological complete response (pCR) rate of 50.0%. Additionally, exploratory analysis revealed that circulating tumor DNA (ctDNA) clearance correlated strongly with pCR, outperforming radiological assessment and programmed death-ligand-1 expression in predicting pCR.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
These findings offer preliminary evidence supporting a simplified, four-cycle perioperative serplulimab-based regimen for resectable sq-NSCLC. Furthermore, this study underscores the potential of ctDNA as a non-invasive biomarker for predicting pCR. Future large-scale studies are warranted to validate these results.
Introduction
Lung cancer is one leading cause of cancer incidence and mortality in the USA.1 Over the past two decades, remarkable advances have been made in the diagnosis and treatment of non-small cell lung cancer (NSCLC), which accounts for over 80% of all lung cancers. The identification of actionable driver gene alterations has accelerated the development of targeted therapies, while the discovery of immune checkpoints has revolutionized NSCLC treatment. Squamous NSCLC (sq-NSCLC), constituting approximately 25–30% of NSCLC cases, is a distinct histological subtype characterized by low frequencies of actionable driver genomic alterations such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and c-ros oncogene 1 (ROS1), leading to limited targeted therapeutic options.2 3 Immunotherapy targeting programmed cell death protein-1 (PD-1), or programmed death-ligand-1 (PD-L1) represents a promising antitumor strategy for sq-NSCLC. The randomized phase III KEYNOTE-407 trial demonstrated that pembrolizumab-based combination therapy significantly improved outcomes in patients with metastatic sq-NSCLC across PD-L1 expression levels, with greater benefits observed in patients with higher PD-L1 expression.4
Early-stage (stage I-IIIA) NSCLC, representing approximately 40% of cases, is primarily treated with radical surgery.5 However, recurrence or metastasis after surgery remains common. While adjuvant chemotherapy improves prognosis, the absolute increase in 5-year survival rate is only about 5%.6 7 Recent landmark studies have highlighted the potential benefits of perioperative immunochemotherapy. In the CheckMate-816 trial, neoadjuvant nivolumab (an anti-PD-1 monoclonal antibody (mAb)) combined with chemotherapy resulted in a major pathological response (MPR) of 36.9% and pathological complete response (pCR) of 24.0% in patients with resectable stage IB-IIIA NSCLC, with comparable safety to chemotherapy alone.8 The subsequent KEYNOTE-671 trial further confirmed improved survival following perioperative pembrolizumab-based treatment.9 Notably, subgroup analyses suggested enhanced benefit among patients with squamous histology, although dedicated studies specifically targeting sq-NSCLC in the perioperative setting remain limited.
Serplulimab, a fully humanized anti-PD-1 mAb, has demonstrated higher affinity for the PD-1 receptor and greater antitumor activity compared with nivolumab and pembrolizumab. Its efficacy in lung cancer has been validated by the ASTRUM-005 and ASTRUM-004 trials, showing improved outcomes with serplulimab-based therapy in extensive-stage small-cell lung cancer and advanced PD-L1-positive sq-NSCLC, respectively.3 10 Building on these findings, we have initiated a single-center, single-arm, exploratory phase II clinical trial to evaluate the feasibility and preliminary efficacy of a simplified, four-cycle perioperative regimen combining serplulimab with chemotherapy in patients with resectable stage II-IIIA sq-NSCLC. Additionally, we planned to explore circulating tumor DNA (ctDNA)-based minimal residual disease (MRD) as a potential predictive biomarker for treatment response.
Methods
Study design and participants
This investigator-initiated, single-center, single-arm phase II exploratory clinical trial (NCT05775796) planned to enroll 30 patients with histologically or cytologically confirmed, resectable clinical stage II-IIIA sq-NSCLC. It aimed to evaluate the feasibility of a perioperative regimen consisting of four cycles of serplulimab combined with taxane and platinum-based chemotherapy (online supplemental figure 1). Key eligibility criteria are summarized in online supplemental table 1, with full eligibility details provided in the study protocol. Patients were excluded if they had actionable driver alterations, which included, but were not limited to: EGFR sensitizing mutations (eg, exon 19 deletions, L858R), ALK fusions, ROS1 fusions, BRAF V600E mutations, RET fusions, MET exon 14 skipping mutations, etc.
Eligible participants received a total of four cycles of perioperative serplulimab-based treatment, comprising two to three neoadjuvant cycles and one to two postoperative adjuvant cycles. After treatment completion, patients were followed-up every 3 months until death. The primary endpoint was MPR. Secondary endpoints included pCR, R0 resection rate, overall response rate (ORR), event-free survival (EFS), overall survival (OS), and safety.
This study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice Guidelines (online supplemental file 3).
Treatment and procedures
All enrolled patients in the study received perioperative serplulimab (fixed dose, 300 mg intravenously every 3 week), combined with paclitaxel (175 mg/m²) or nab-paclitaxel (260 mg/m²) and carboplatin (area under the curve=5). Treatment consisted of two to three cycles administered preoperatively (neoadjuvant phase), followed by curative-intent surgery and one to two postoperative cycles (adjuvant phase). The exact number of cycles was adjusted based on imaging-based tumor response, clinical tolerability, and investigator judgment. Specifically, patients achieving substantial tumor regression or complete response (CR) may proceed directly to surgery. If the investigator determined that poor physical tolerance might compromise the patient’s ability to withstand surgery following further neoadjuvant treatment, the additional cycle was not recommended to ensure surgical eligibility. Detailed treatment regimens and procedures are described in the study protocol. The time window between the last neoadjuvant cycle and surgery was 4–6 weeks. Patients were followed-up every 3 months post-treatment.
Study endpoints and assessment
The primary endpoint was the postoperative MPR rate, defined as the proportion of patients with ≤10% viable tumor cells remaining in the primary tumor bed after neoadjuvant therapy, regardless of residual tumor cells in lymph nodes. Key secondary endpoints included the R0 resection rate and the pCR rate. An R0 resection required fulfillment of specific criteria established by the International Association for the Study of Lung Cancer Staging and Prognostic Factors Committee, including microscopically negative resection margins, a systematic lymph node dissection covering at least six lymph node stations (minimum three intrapulmonary/hilar and three mediastinal stations), no extracapsular nodal involvement, and no involvement of the highest resected lymph node.11 The pCR rate was defined as the proportion of patients with 0% residual viable tumor cells in both the primary tumor bed and resected lymph nodes after neoadjuvant therapy. Pathological response was assessed by two experienced senior pulmonary pathologists from the Department of Pathology at Fudan University Shanghai Cancer Center who were blinded to the treatment assignment.
Secondary endpoints also included tumor response during the neoadjuvant phase, EFS, OS, and the safety profile. Tumor response was assessed by investigators according to Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1, with ORR defined as the proportion of patients achieving a partial response (PR) or CR after neoadjuvant therapy. EFS was defined as the time from enrollment to disease progression precluding surgery, local or distant recurrence, or death from any cause. OS was defined as the time from enrollment to death from any cause. Safety assessments were conducted according to the National Cancer Institute Common Terminology Criteria for Adverse Events V.5.0. Adverse events were recorded throughout the treatment and follow-up periods. This study also planned to explore the efficacy differences between clinical stage II and III patients to further evaluate the feasibility of the perioperative regimen.
DNA sequencing and MRD detection
Additionally, pretreatment tumor tissue samples were collected for whole exome sequencing (WES) to identify gene mutations in sq-NSCLC. Specifically, only samples containing ≥30% tumor cells were included in the analysis. The MagPure FFPE DNA/RNA LQ kit (Magen, Guangzhou, China) was used to extract genomic DNA (gDNA) from formalin-fixed, paraffin-embedded (FFPE) tumor tissues. Peripheral blood gDNA was extracted with the MagPure Universal DNA Kit (Magen, Guangzhou, China). DNA samples meeting quality criteria were subjected to WES library preparation, exome capture, and quantification. Sequencing was performed on a NovaSeq 6000 platform (Illumina, San Diego, California, USA) with paired-end reads, targeting an average coverage of approximately 500× for tumor samples and 150× for matched normal samples.12
The tumor-informed MRD detection was based on the PROPHET assay (Burning Rock Biotech, China),12 13 which tracks up to 50 patient-specific variants. The analytical limit of detection (LOD) was established via serial dilutions of cancer cell lines and reference standards, yielding 0.0036% at 20 ng DNA input and 0.0018% at 60 ng. Consequently, the validated assay LOD was set at 0.004% with a specificity exceeding 99%. Peripheral blood samples were collected at predefined time points using Cell-Free DNA BCT tubes (Streck, La Vista, Nebraska, USA). Cell-free DNA (cfDNA) was isolated using the QIAamp Circulating Nucleic Acid Kit (Qiagen) and analyzed using the PROPHET assay to detect MRD.12 High-depth sequencing, using unique molecular identifier-based technology, was conducted on the Illumina NovaSeq 6000 platform (San Diego, California, USA) with paired-end reads, targeting a raw sequencing depth of 100,000× for personalized panels.
After adapter trimming and quality control, single nucleotide variants and insertions/deletions with at least five reads and a variant allele frequency (VAF) of ≥3% were identified. Germline mutations were filtered by comparing the fold change in VAF between tumor and matched normal samples, excluding variants with a fold change <3 or both VAFs >10%. Variant annotation was conducted using ANNOVAR and SnpEff V.3.6. Up to 50 high-priority variants with a VAF ≥3.0% were selected, excluding those in repetitive, high guanine and cytosine (GC) (>75%), or homologous regions. A personalized biotinylated capture probe pool was created, and ctDNA was sequenced using this custom panel. Mutations with p<0.05 were considered significant. MRD-positive status was defined by the presence of at least two significant mutations with p<0.005, and ctDNA clearance was defined as conversion from MRD-positive status at baseline to MRD-negative status after treatment.
Statistical analysis
This single-arm study employed a statistical hypothesis based on historical data. According to previous research, the MPR rate for neoadjuvant chemotherapy was approximately 9%.8 Assuming an anticipated MPR rate of 27%, with a two-sided alpha of 0.05, a power of 80%, and a dropout rate of 10%, the calculated sample size was 30 patients. Three analysis sets were defined for statistical analysis: the full analysis set (FAS), including all patients who received at least one dose of study treatment and had at least one post-treatment assessment; the per-protocol set (PPS), a subset of FAS excluding patients with major protocol deviations; and the safety set, including all patients who received at least one dose of study treatment with available safety data.
Statistical analyses were performed using R software (V.4.3.2). Analyses comprised: (1) a summary of patient enrollment, exclusions, withdrawals, and completion status; (2) descriptive summaries of baseline demographic, clinical, and treatment characteristics; (3) assessment of treatment efficacy for primary (MPR) and secondary endpoints (pCR, R0 rate, ORR, EFS, OS); and (4) safety evaluations, including treatment-related adverse events (TRAEs). Continuous variables were described using means, SDs, medians, and ranges. Categorical variables were summarized using counts and percentages. The 95% CIs for ORR were calculated using the Clopper-Pearson method. EFS and OS were estimated using the Kaplan-Meier method. To compare differences between subgroups, the independent samples t-test or paired samples t-test was used for normally distributed continuous variables, the Mann-Whitney U test or Kruskal-Wallis H test was used for non-normally distributed continuous variables, and the Fisher’s exact test was used for categorical variables. Additionally, logistic regression analysis was conducted in this study based on the outcome variable to evaluate the associations between potential influencing factors and the outcome. All statistical tests were two-sided, and a p value of <0.05 was considered statistically significant.
Results
Study overview, baseline characteristics, and treatment
Between April 2023 and February 2024, 30 patients with stage II-IIIA sq-NSCLC were enrolled and received at least one dose of the study treatment. All 30 patients were Asians and included in the FAS (figure 1). Two patients received only one cycle of neoadjuvant therapy. Among the remaining 28 patients who completed the protocol-specified neoadjuvant treatment, 19 received two cycles and 9 received three cycles. There were no significant differences of baselines between patients receiving two or three cycles (online supplemental table 2). 29 patients underwent curative-intent surgery, of whom 6 declined subsequent adjuvant consolidation therapy, while the remaining 23 completed the full perioperative regimen as planned.
Figure 1. Trial profile. CR, complete response; FAS, full analysis set; PPS, per-protocol set; PR, partial response; SD, stable disease; SS, safety set.
Baseline and treatment characteristics are summarized in table 1. The median age was 65 years (range: 35–75), and most patients were male smokers (n=28, 93.33%). 14 patients (46.67%) had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0. Nearly half of the patients presented with stage II disease (2 with stage IIA and 12 with stage IIB), while the remaining 16 patients had stage IIIA disease; 8 patients (26.67%) had N2 lymph node involvement. Positive PD-L1 expression (tumor proportion scores (TPS) ≥1%) was detected in 24 patients (80.00%), and 12 patients (40.00%) demonstrated high PD-L1 expression (TPS ≥50%). 22 patients (73.33%) received nab-paclitaxel plus carboplatin, and the remaining 8 patients (26.67%) received paclitaxel plus carboplatin. Among the 29 patients who completed surgery, the median interval from the last neoadjuvant dose to surgery was 35 days (table 2). Lobectomy was the most common surgical approach (n=17, 58.62%). All patients underwent complete mediastinal lymph node dissection, with a mean of 19.79 lymph nodes removed (table 2).
Table 1. Baseline characteristics.
| Characteristics | All patients (n=30) |
|---|---|
| Age, years | |
| Median (range) | 65 (35–75) |
| <65, n (%) | 17 (56.67) |
| ≥65, n (%) | 13 (43.33) |
| Sex, n (%) | |
| Male | 28 (93.33) |
| Female | 2 (6.67) |
| BMI, kg/m² | |
| Mean±SD | 23.47±3.10 |
| Smoking, n (%) | |
| Yes | 28 (93.33) |
| Former | 12 (40.00) |
| Current | 16 (53.33) |
| No | 2 (6.67) |
| ECOG PS, n (%) | |
| 0 | 14 (46.67) |
| 1 | 16 (53.33) |
| PD-L1 status, n (%) | |
| Positive (TPS ≥1%) | 24 (80.00) |
| TPS 1%–50% | 12 (40.00) |
| TPS >50% | 12 (40.00) |
| Negative (TPS <1%) | 5 (16.67) |
| Unknown | 1 (3.33) |
| Clinical T stage, n (%) | |
| cT1 | 4 (13.33) |
| cT2 | 11 (36.67) |
| cT3 | 10 (33.33) |
| cT4 | 5 (16.67) |
| Clinical N stage, n (%) | |
| N0 | 13 (43.33) |
| N+ | 17 (56.67) |
| N1 | 9 (30.00) |
| N2 | 8 (26.67) |
| Clinical stage, n (%) | |
| Stage II | 14 (46.67) |
| IIA | 2 (6.67) |
| IIB | 12 (40.00) |
| Stage IIIA | 16 (53.33) |
| Chemotherapy regimens, n (%) | |
| Paclitaxel+carboplatin | 8 (26.67) |
| Nab-paclitaxel+carboplatin | 22 (73.33) |
BMI, body mass index; ECOG PS, Eastern Cooperative Oncology Group Performance Status; PD-L1, programmed cell death protein ligand 1; TPS, tumor positive score.
Table 2. Surgery characteristics.
| Characteristics | Surgical patients (n=29) |
|---|---|
| Time from the last dose of neoadjuvant therapy to surgery, days | |
| Median | 35 |
| Surgical approach, n (%) | |
| Muscle-sparing thoracotomy | 29 (100.00) |
| Lobectomy | 17 (58.62) |
| Bi-lobectomy | 5 (17.24) |
| Sleeve resection | 6 (20.69) |
| Pneumonectomy | 1 (3.45) |
| Blood loss, mL | |
| Median (range) | 100 (50–550) |
| Number of lymph nodes dissected | |
| Mean±SD | 19.79±11.01 |
By the data cut-off in November 2024, all patients had completed the study treatment, with a median follow-up of 13.6 months. Four patients experienced EFS events, including two with recurrence and two deaths. Survival data remain immature, with median EFS and OS not yet reached (online supplemental figures 2 and 3).
Antitumor activity of serplulimab-based neoadjuvant treatment
In the intention-to-treat (ITT) population (n=30), the ORR during neoadjuvant treatment was 73.33% (22/30, 95% CI 54.11% to 87.72%) with no progressive disease (table 3). The 29 patients who underwent curative-intent surgery were included in the PPS as the primary efficacy analysis population. 28 achieved R0 resection (28/29, R0 resection rate: 96.55%) in patients receiving surgery, while one patient had R2 resection. Postoperative pathological evaluation revealed that 23 patients achieved MPR, and 15 patients achieved pCR, resulting in MPR and pCR rates of 76.67% (95% CI 57.72% to 90.07%) and 50.00% (95% CI 31.30% to 68.70%) in the ITT population (79.31% and 51.72% in the PPS, respectively). Notably, 11 patients with PR and 2 with stable disease as their best radiological responses achieved pCR post-surgery (figure 2). Additionally, postoperative pathology showed negative lymph node involvement in 23 patients (79.31%), including 5 with baseline N2 disease. Subgroup analysis demonstrated no significant differences in MPR or pCR rates based on clinical stage (II vs III) (table 4). Further analysis indicated pCR rates were 52.6% and 55.6% for two-cycle and three-cycle neoadjuvant therapy, respectively, while MPR rates were 84.2% and 77.8%. Logistic regression analysis also showed no significant associations between pCR and patients’ characteristics (online supplemental table 3). The findings in the ITT population were consistent with those in the PPS (online supplemental tables 4 and 5).
Table 3. Tumor response of patients in the intention-to-treat population.
| All patients (n=30) | Stage IIA/IIB (n=14) | Stage IIIA (n=16) | |
|---|---|---|---|
| Radiographic response according to RECIST V.1.1, n (%) | |||
| CR | 2 (6.67) | 1 (7.14) | 1 (6.25) |
| PR | 20 (66.67) | 9 (64.29) | 11 (68.75) |
| SD | 8 (26.67) | 4 (28.57) | 4 (25.00) |
| PD | 0 (0.00) | 0 (0.00) | 0 (0.00) |
| ORR, n (%) | 22 (73.33) | 10 (71.43) | 12 (75.00) |
| 95% CI | 54.11 to 87.72 | 41.9 to 91.61 | 47.62 to 92.73 |
| Pathological response, n (%) | |||
| MPR | 23 (76.67) | 11 (78.57) | 12 (75.00) |
| pCR | 15 (50.00) | 8 (57.14) | 7 (43.75) |
CR, complete response; MPR, major pathological response; ORR, overall response rate; pCR, pathological complete response; PD, progressive disease; PR, partial response; RECIST 1.1, Response Evaluation Criteria in Solid Tumors V.1.1; SD, stable disease.
Figure 2. Tumor response. *Patient who achieved SD after one cycle of neoadjuvant therapy and did not undergo the planned surgery. CR, complete response; ECOG PS, Eastern Cooperative Oncology Group Performance Status; MPR, major pathological response; pCR, pathological complete response; PD-L1, programmed cell death protein ligand 1; PR, partial response; RECIST V.1.1, Response Evaluation Criteria in Solid Tumors V.1.1; SD, stable disease; TPS, tumor positive score.
Table 4. Subgroup analysis of pCR and MPR in the intention-to-treat population.
| Subgroup | All patients (n=30) | |||||
|---|---|---|---|---|---|---|
| pCR | MPR | |||||
| Yes (n=15) | No (n=15) | P value | Yes (n=23) | No (n=7) | P value | |
| Sex, n (%) | 1.000 | 1.000 | ||||
| Male | 14 (93.33) | 14 (93.33) | 21 (91.30) | 7 (100.00) | ||
| Female | 1 (6.67) | 1 (6.67) | 2 (8.70) | 0 (0.00) | ||
| Age, years | 1.000 | 0.666 | ||||
| <65 | 8 (53.33) | 9 (60.00) | 14 (60.87) | 3 (42.86) | ||
| ≥65 | 7 (46.67) | 6 (40.00) | 9 (39.13) | 4 (57.14) | ||
| Smoking | 1.000 | 1.000 | ||||
| No | 1 (6.67) | 1 (6.67) | 2 (8.70) | 0 (0.00) | ||
| Yes | 14 (93.33) | 14 (93.33) | 21 (91.30) | 7 (100.00) | ||
| ECOG PS | 1.000 | 1.000 | ||||
| 0 | 7 (46.67) | 7 (46.67) | 11 (47.83) | 3 (42.86) | ||
| 1 | 8 (53.33) | 8 (53.33) | 12 (52.17) | 4 (57.14) | ||
| PD-L1 status* | 1.000 | 0.569 | ||||
| Negative | 2 (13.33) | 3 (20.00) | 3 (13.04) | 2 (28.57) | ||
| Positive | 12 (80.00) | 12 (80.00) | 19 (82.61) | 5 (71.43) | ||
| Number of neoadjuvant cycles | 0.536 | 0.062 | ||||
| 1 | 0 (0.00) | 2 (13.33) | 0 (0.00) | 2 (28.57) | ||
| 2 | 10 (66.67) | 9 (60.00) | 16 (69.57) | 3 (42.86) | ||
| 3 | 5 (33.33) | 4 (26.67) | 7 (30.43) | 2 (28.57) | ||
| Clinical T stage | 1.000 | 0.390 | ||||
| T1/2 | 7 (46.67) | 8 (53.33) | 10 (43.48) | 5 (71.43) | ||
| T3/4 | 8 (53.33) | 7 (46.67) | 13 (56.52) | 2 (28.57) | ||
| Clinical N stage | 1.000 | 0.104 | ||||
| N0 | 7 (46.67) | 6 (40.00) | 12 (52.17) | 1 (14.29) | ||
| N+ | 8 (53.33) | 9 (60.00) | 11 (47.83) | 6 (85.71) | ||
| Clinical stage | 0.715 | 1.000 | ||||
| IIA/IIB | 8 (53.33) | 6 (40.00) | 11 (47.83) | 3 (42.86) | ||
| IIIA | 7 (46.67) | 9 (60.00) | 12 (52.17) | 4 (57.14) | ||
PD-L1 expression status was not available for one patient.
ECOG PS, Eastern Cooperative Oncology Group performance status; MPR, major pathological response; pCR, pathological complete response; PD-L1, programmed cell death protein ligand 1.
A post hoc analysis assessed changes in tumor biomarkers before and after neoadjuvant therapy, including serum concentrations of carcinoembryonic antigen, squamous cell carcinoma antigen, neuron-specific enolase, cancer antigen 125, and cytokeratin-19 fragments. All biomarkers showed a significant decrease following neoadjuvant therapy (all p value<0.05) (online supplemental figure 4, online supplemental table 6).
Safety profile
Generally, the TRAEs from this trial were manageable. All patients experienced at least one TRAE, and the incidence of grade ≥3 TRAEs was 26.66%. The most common TRAE was hematologic, including anemia and neutropenia. Neutropenia was the most common grade ≥3 TRAE, accounting for 23.33% of patients (online supplemental table 7).
Predicting pCR after immunochemotherapy using MRD
It is widely recognized that pCR serves as a surrogate marker of improved clinical outcomes and is frequently used as a key endpoint in clinical trials.14 Consequently, accurately predicting pCR status following immunochemotherapy is of great importance. In our study, tumor-informed MRD assays were successfully performed in 27 patients. Blood samples were collected at multiple time points: before the neoadjuvant phase (time point (TP)-0), prior to surgical resection (TP-1), at the start of the adjuvant phase (TP-2), and during follow-up (TP-3, TP-4, etc) (figure 3A). By the data cut-off, a total of 97 blood samples had been analyzed for MRD. Baseline WES revealed that TP53 mutation was the most common, detected in 93% of patients (figure 3B). At baseline (TP-0), all patients were ctDNA-positive (figure 3C). Following immunochemotherapy, MRD negativity was achieved in 52% of patients, while 48% remained MRD-positive (figure 3C). Most patients showed a decline in ctDNA ratios after immunochemotherapy, though a subset displayed increased ctDNA levels during follow-up (figure 3D).
Figure 3. The association between ctDNA levels and pCR. (A) Schematic representation of the study design, including treatment timeline and blood sample collection points. (B) Heatmap showing the top 30 most frequently altered genes in the cohort. (C) Bar plots illustrating the percentage of patients with positive ctDNA at various time points. (D) Graphs depicting the dynamic changes in ctDNA ratios across different time points. (E) Performance of ctDNA clearance after treatment in predicting pathological response. (F) Comparison of ctDNA ratios between non-pCR and pCR patients. (G) Bar plots revealing the percentage of patients with positive ctDNA in patients with non-pCR and pCR. (H) Representative case (LC01002) demonstrating dynamic changes in ctDNA ratio alongside radiological findings. (I) Comparative C-index values of different predictive models for pCR in patients with sq-NSCLC undergoing immunochemotherapy. CR, complete response; ctDNA, circulating tumor DNA; ECOG, Eastern Cooperative Oncology Group; MPR, major pathological response; pCR, pathological complete response; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand-1; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; sq-NSCLC, squamous non-small cell lung cancer; TP, time point.
Further analyses showed that ctDNA clearance could accurately predict pCR status with a sensitivity of 78.6% and a specificity of 76.9% (figure 3E). Patients who achieved pCR had lower mean ctDNA ratios compared with those in the non-pCR group (figure 3F). Of the non-pCR patients, 77% remained MRD-positive, whereas only 21% of pCR patients exhibited positive MRD status (figure 3G). Interestingly, one patient (LC01002) achieved pCR after neoadjuvant immunochemotherapy but retained a positive MRD status (figure 3H). 6 months post-surgery, this patient developed brain metastasis, suggesting that ctDNA clearance may serve as a more sensitive indicator of residual tumor burden than pCR alone. We next compared the predictive capabilities of baseline characteristics, RECIST response, PD-L1 expression, ctDNA clearance, and a combined model for determining pCR status. While ctDNA clearance alone achieved a C-index of 0.776, outperforming both RECIST response (C-index: 0.655) and PD-L1 expression (C-index: 0.667), the integrated model incorporating all variables achieved the highest C-index (figure 3I). Taking together, these findings underscore the potential of ctDNA clearance as a robust predictor of pCR in resectable sq-NSCLC, which may need future validation.
Discussion
Several landmark trials have established the benefits of perioperative immunochemotherapy in resectable NSCLC. The CheckMate-816 trial demonstrated that neoadjuvant nivolumab combined with chemotherapy significantly improved the pCR rates and survival outcomes.15 Subsequently, the KEYNOTE-671 trial advanced the perioperative immunotherapy approach by combining neoadjuvant pembrolizumab and chemotherapy with mandatory adjuvant pembrolizumab, resulting in significantly increased pCR rate and extended the median EFS.9 Building on the standard of care established by these landmark trials, our study explores a potential refinement of the treatment strategy by evaluating the feasibility of a simplified perioperative immunochemotherapy regimen in resectable stage II-IIIA sq-NSCLC, using serplulimab combined with chemotherapy. Our study employs a perioperative approach that differs from several major phase III trials, such as CheckMate 77T, KEYNOTE-671, and AEGEAN (online supplemental table 8).16,18 While the standard perioperative regimen typically comprises 4 cycles of neoadjuvant chemoimmunotherapy followed by 12–13 cycles of adjuvant immunotherapy maintenance, we compressed the perioperative regimen to only 4 cycles in total, either 2 or 3 cycles of neoadjuvant therapy, followed by 1 or 2 postoperative cycles. This flexibility may significantly maximize patient compliance and reduce treatment burden. However, the single-arm design and the small sample size preclude definitive conclusions regarding the efficacy of this regimen. Our results should be considered exploratory and hypothesis-generating.
We enrolled 30 patients without actionable driver alterations, of whom 29 underwent surgery (96.67%). The R0 resection rate reached 96.55% among patients receiving surgery. MPR and pCR rates were 76.67% and 50.00% in all enrolled patients, respectively, surpassing our predefined endpoints. While our study demonstrated encouraging pathological response rates, suggesting potential efficacy of a shorter perioperative regimen,19 20 the limited small sample size and lack of a contemporaneous control group preclude definitive conclusions regarding long-term clinical benefits such as survival outcomes.
Compared with lung adenocarcinoma, sq-NSCLC is more strongly associated with smoking and predominantly affects males,21 22 as observed in our cohort, where only 2 participants were non-smoking females, while the remaining 28 were male smokers. Furthermore, common driver mutations frequently found in adenocarcinomas (such as EGFR alterations) are rarely identified in sq-NSCLC. Even when driver mutations are present, responses to targeted therapies and survival outcomes in sq-NSCLC remain inferior. For instance, the biomarker-driven phase II LUNG-MAP S1400 study reported an ORR of only 7% for targeted therapies in previously treated patients with sq-NSCLC.2123,25 High tumor mutational burden, prevalent in sq-NSCLC, is associated with a greater abundance of neoantigens and potentially enhanced antitumor immune responses.26 In addition, sq-NSCLC tumors commonly exhibit higher PD-L1 expression and more extensive immune infiltration, both predictive of favorable immunotherapy outcomes.27 Although several perioperative chemoimmunotherapy trials included patients with sq-NSCLC, dedicated prospective trials specifically designed to evaluate perioperative immunotherapy exclusively in resectable sq-NSCLC remain relatively limited. Recent evidence indicates that sq-NSCLC may uniquely benefit from immunotherapy,28 29 indicating the need for subtype-specific strategies. The encouraging MPR and pCR rates observed in our study further substantiate the efficacy of immunochemotherapy in early-stage sq-NSCLC. These findings support the notion that sq-NSCLC should be recognized as a distinct subgroup within NSCLC, potentially benefiting from more tailored, histology-specific treatment strategies.
Notably, subgroup analyses for MPR and pCR did not show any significant differences based on baseline characteristics. These findings suggest that serplulimab combined with paclitaxel or nab-paclitaxel and carboplatin is broadly effective in patients with resectable sq-NSCLC regardless of PD-L1 expression, consistent with the ASTRUM-004 trial.3 Our exploratory subgroup analysis indicated no significant difference in pathological responses between patients receiving two versus three neoadjuvant cycles. However, due to limited sample size and statistical power, this observation requires validation in larger studies. Typically, standard neoadjuvant regimens for early-stage NSCLC involve three to four treatment cycles. In this study, we prospectively explored a shorter, imaging-guided neoadjuvant regimen of two to three cycles, which aligns more closely with the primary goals of neoadjuvant therapy: achieving rapid tumor shrinkage, facilitating R0 resection, and minimizing surgical morbidity. Notably, nearly all patients (96.7%) successfully underwent definitive surgery, contrasting with previous studies like the phase II NADIM trial, where approximately 10.9% of patients did not proceed to surgery after three cycles of nivolumab plus chemotherapy.30 In other trials, about 20% of patients also failed to reach surgery,8 9 16 18 31 32 underscoring the advantages of a shorter regimen in reducing preoperative treatment burden. Furthermore, our innovative approach limited adjuvant treatment to one or two cycles, significantly shortening treatment duration compared with the 9–12 months of adjuvant therapy. We hypothesize that this shorter perioperative strategy could potentially enhance patient adherence and reduce treatment burden. Nevertheless, these presumed benefits need prospective validation through patient-reported outcomes and cost-effectiveness analyses in larger randomized studies. Collectively, our findings support the clinical feasibility and efficacy of a personalized perioperative regimen with fewer cycles for resectable sq-NSCLC, encouraging further validation in phase III studies.
Additionally, we explored the predictive role of ctDNA clearance in identifying pCR. Previous studies have shown that ctDNA levels can predict clinical outcomes in early-stage treatment-naïve NSCLC33 and advanced NSCLC undergoing immunotherapy.34 Our exploratory analysis indicated that ctDNA clearance correlated with pCR status, showing preliminary superiority over baseline characteristics, RECIST response, and PD-L1 expression. Our study demonstrated ctDNA clearance tracked closely with pathological tumor regression, and this biological consistency supports the biological plausibility of using ctDNA as a promising non-invasive surrogate for residual disease burden. However, given the limited sample size, these findings should be validated in larger trials before clinical implementation.
This study has several limitations. First, as a small-sample, single-arm, exploratory phase II trial without a contemporaneous control group, our results must be cautiously interpreted and viewed as hypothesis-generating. The variability in neoadjuvant treatment cycles introduces heterogeneity that limits interpretability and generalizability. Second, although not by design, all enrolled patients in this study were of Asian ethnicity. Considering the epidemiological characteristics of sq-NSCLC, our small sample size of only 30 patients inevitably resulted in a predominance of male smokers. Future studies are warranted to determine the generalizability of these results to broader patient populations. Third, while MPR is associated with improved prognosis, the absence of mature long-term survival data currently precludes definitive conclusions regarding OS benefit. Lastly, there is a potential risk of overfitting in the ctDNA analyses. Consequently, the findings regarding the association between ctDNA and pCR should be considered exploratory. The combined prediction model is not intended for direct clinical application at this stage, but rather aims to provide insights that warrant subsequent prospective validation.
In conclusion, our study provides preliminary evidence suggesting promising efficacy and a manageable safety profile of the simplified four-cycle perioperative serplulimab-based regimen in resectable sq-NSCLC. However, these findings require confirmation in larger randomized controlled trials with extended follow-up before clinical application.
Supplementary material
Footnotes
Funding: This work was supported by the National Natural Science Foundation of People’s Republic of China (82430099 and 82573547), the Clinical Research Special Project of Shanghai Municipal Health Commission (202440070), the Medical Research Special Project for Shanghai Science and Technology Innovation Action Plan (24Y12800400), and the National Key R&D Program of China (2022YFA1103900). The study was funded by Shanghai Henlius Biotech and Burning Rock Biotech.
Provenance and peer review: Not commissioned; externally peer reviewed.
Patient consent for publication: Not applicable.
Ethics approval: This study involves human participants and was approved by the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center (Approval No: 2211264-18). Participants gave informed consent to participate in the study before taking part.
Data availability free text: All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Data availability statement
Data are available upon reasonable request.
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Associated Data
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Supplementary Materials
Data Availability Statement
Data are available upon reasonable request.