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  Title	Anti-PD1 based precision induction therapy in unresectable stage III non-small cell lung cancer: a phase II umbrella clinical trial
  ID	7242422
  External ID	7576542538
  Author	Unknown
  Published Date	Feb. 24, 2025, 12:08 p.m.
  Summary	Study design This open-label, single-center, phase II clinical umbrella trial was conducted at Tongji University affiliated Shanghai Pulmonary Hospital, which was registered at https://www.chictr.org
  Tags	None
  URL	https://www.nature.com/articles/s41467-025-57184-z
  Sentiment	Negative: 0.05031    Neutral: 0.92194    Positive: 0.02775
  Relevance	0.00370

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  Content ID	4547668
  Key	168a6871-0a17-4e83-b8a2-a3de3edd4a4a
  Content	Study design This open-label, single-center, phase II clinical umbrella trial was conducted at Tongji University affiliated Shanghai Pulmonary Hospital, which was registered at https://www.chictr.org.cn/index.html (registration number: ChiCTR2000035367; Fig. S1A). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the independent ethic committee in Tongji University affiliated Shanghai Pulmonary Hospital (L20-335-2). All participants provided written informed consent prior to their inclusion in the study. Informed consent included permission to publish aggregated demographic information, including age, sex distribution, and the medical center involved in the study. To ensure patient confidentiality, any identifiable information has been anonymized in accordance with ethical guidelines and privacy regulations. The demographic data presented reflects the overall distribution of participants, without disclosing any personal identifiers. Patient eligibility Between January 21st, 2021, and March 1st, 2023, this study enrolled treatment-naïve participants aged over 18 years with initially unresectable stage III NSCLC (including N2 and N3) according to the 8th edition of the American Joint Committee on Cancer staging manual. Their Eastern Cooperative Oncology Group (ECOG) performance status was 0 or 1. The pathological diagnosis, driver gene mutation status, and PD-L1 expression were confirmed by endobronchial ultrasound (EBUS) or percutaneous lung biopsy. PD-L1 status was determined by immunohistochemical (IHC) staining of PD-L1 protein (clone 22C3) in the submitted tumor samples and categorized as follows: PD-L1 positive was defined as ≥ 1% tumor cell membrane staining positive in a minimum of 100 evaluable tumor cells; PD-L1 negative was defined as <1% tumor cell membrane staining positive in a minimum of 100 evaluable tumor cells and PD-L1 not evaluable/indeterminate was defined as participants with insufficient sample quantity or quality to stain for PD-L1 status or those participants in whose samples PD-L1 status could not be determined despite appropriate amounts of tissue sample. Clinical staging was confirmed through positron emission tomography/computed tomography (PET/CT), chest computed tomography (CT), and brain magnetic resonance imaging (MRI). Eligible patients had to exhibit normal hematological indexes as well as qualified hepatic, renal, and pulmonary functions to ensure tolerance for induction therapy followed by radical resection. Initially unresectable disease was evaluated by MDT, and was defined as (1) pathological confirmed N3 disease; (2) tumor invading vital structures, such as large blood vessels, the trachea, or primary bronchus, with potential for curative resection after tumor downgrading by induction therapy, as judged by a preoperative assessment; (3) clinically confirmed metastatic lymph nodes with multi-station N2 disease or bulky N2 disease, and patients could tolerate hilar and mediastinal lymph node dissection after tumor downgrading by induction therapy as determined by a preoperative evaluation; (4) Possibility of non-R0 resection, even through procedures such as pneumonectomy, specifically right pneumonectomy. The MDT consisted of experts in surgery, radiology, pathology, and oncology. The thoracic surgery specialists had extensive experience, each performing an average of 1,100 thoracic surgeries annually, with over 40 cases involving post-neoadjuvant immunotherapy. All surgeries in this trial were performed by the thoracic surgeons from this MDT team. Patients with contraindication to IBI305 was defined as per the provided instructions of IBI305, including (1) History of hemoptysis within 3 months prior to screening, with a volume of blood greater than 2.5 mL each time; (2) Symptomatic brain metastases with evidence of hemorrhage; (3) History of myocardial infarction or stroke within 6 months prior to enrollment; (4) History of abdominal fistula, gastrointestinal perforation, or intra-abdominal abscess; (5) Patients who have arterial thrombotic events, esophageal varices, peptic ulcers, wounds, or bone fractures; (6) Radiographic evidence of tumor invasion in great vessels or tumor cavitation. The exclusion criteria included a history of other malignancies, previous antitumor therapy, severe comorbidities, active autoimmune disease, prior non-infectious pneumonitis, interstitial lung disease, pregnancy or breastfeeding, and EGFR or ALK mutation positive. The detailed inclusion and exclusion criteria are listed in the trial protocol (Supplemental Text 1). Procedures Patients who consented to enroll in the trial were assigned to one of these treatment arms based on the expression level of PD-L1 in the primary tumor of patients and whether patients have contraindications to IBI305 (a biosimilar of Bevacizumab). The Treatment subgroups included: (A) immuno-monotherapy for patients with PD-L1 expression level ≥ 50% but with contraindications to IBI305; (B) immunotherapy plus anti-angiogenesis therapy for patients with PD-L1 expression level ≥ 1% and without contraindications to IBI305 (Chemo-free subgroup: A + B); (C) chemoimmunotherapy for patients with PD-L1 expression level ranging from 1% to 49% but with contraindications to IBI305; (D) chemoimmunotherapy for patients with negative or unknown expression level of PD-L1 (Chemo subgroup: C + D). Sintilimab (200 mg) on day 1 for all participants; IBI305 (15 mg/kg) on day 1 for participants in Treatment-subgroup B; carboplatin (area under curve 5) plus pemetrexed (500 mg/m2) for participants with adenocarcinoma and carboplatin (area under curve 5) plus nab-paclitaxel (260 mg/m2) for participants with other pathological subtypes on day 1 in the Chemo subgroup. All participants were scheduled to receive two to four 21-day cycles of induction therapy. Chest CT was performed at the 6th and/or 12th weeks after induction treatment. All CT scans of participants were assessed by the same radiologist and 3 experienced thoracic surgeons via Response Evaluation Criteria in Solid Tumors measurement criteria (RECIST, version 1.1)53. After completing two cycles, radiographic assessments and lymph node pathological evaluations (restricted to N3) were conducted. Participants were deemed unresectable if radiographic evaluations (PET/CT, chest CT, or cranial MRI) revealed distant metastasis or tumor invasion into critical structures such as the root of the main vessels, trachea, or other unresectable organs, impacting indispensability. Alternatively, if lymph node pathologic evaluation remained positive in the N3 node, additional treatment cycles were contemplated. For assessing N3 status after induction treatment, patients initially presenting with N3 status underwent reassessment using EBUS or supraclavicular lymph node biopsy. However, sampling challenges, such as difficulty in evaluating stations 5 and 6 lymph nodes, meant that PET/CT results were primarily relied upon. For patients without N3 status prior to induction therapy, PET/CT imaging was primarily employed for assessment. If PET/CT indicated a high suspicion of N3 lymph node involvement, further confirmatory assessments were performed using EBUS or supraclavicular lymph node biopsy to verify potential metastasis. In cases where complete resection was unattainable after four cycles of induction treatment, participants were considered for withdrawal from the study, and their management would transition to an MDT approach. If CR, PR or SD participants after induction could be resected completely, surgery could be performed within 4 ~ 12 weeks after the induction treatment discontinuation. Adjuvant therapy would be administered 4 to 6 weeks postoperatively, unless the participants were assessed to be unable to tolerate adjuvant therapy or refuses to accept adjuvant treatment. For patients who received chemoimmunotherapy induction therapy, we supplemented the number of chemoimmunotherapy cycles based on the number of cycles they received during induction therapy, ensuring a total of 4 cycles of chemoimmunotherapy. This was followed by one year of maintenance therapy with 17 cycles mono-immunotherapy. For patients who received chemo-free induction therapy, we recommended 4 cycles of chemoimmunotherapy during the adjuvant therapy period. This was also followed by one year of maintenance therapy with 17 cycles mono-immunotherapy, contingent upon the patient’s tolerance to chemotherapy. Efficacy and safety assessment The primary endpoint was major pathological response (MPR: ≤ 10% residual viable tumor cells in the primary tumor) rate according to independent pathological review in each arm. The secondary endpoints were pathological response rate (pCR rate, proportion of patients without residual viable tumor cell in the primary tumor and sampled lymph nodes)54, the objective response rate (ORR), the conversion rate of surgery, event-free survival (EFS, time from initial treatment to any progression of disease [precluding surgery], progression or recurrence of disease after surgery, progression of disease in the absence of surgery, or death from any cause), overall survival (OS, time from treatment assignment to death from any cause), safety and tolerability. Adverse events (AEs) were assessed in all treated patients, with both the assessment and grading of AEs and serious adverse events (SAEs) conducted in accordance with the NCI-CTCAE v5.0 guidelines. Sample size estimation The primary endpoint of this research was MPR, which was taken to calculate the sample size. Previous randomized clinical trial reported that the MPR of neoadjuvant chemotherapy for NSCLC at stage IIIA/IIIB was 20% (17/84, P0) in the patients with R0 resection55. The null hypothesis (H0) posited that the MPR of anti-PD1-based precision induction therapy would be no greater than 20%, equivalent to the MPR observed with standard neoadjuvant chemotherapy. The MPR for neoadjuvant immune checkpoint inhibitors (ICIs) monotherapy from CheckMate-159 was 45% (9/20)56, and the MPR of neoadjuvant ICIs plus chemotherapy (LungMate-001) at stage IIIA was 43% (13/30)36. Another phase 2 clinical trial revealed the MPR for neoadjuvant ICIs plus anti-VEGFR was 57% (37/65)18. Given that the small sample size of these trials may lead to overestimation of the MPR rate, the MPR rate was expected to be 45% (P1) in this study. It was calculated with α = 0.05 (two-tailed) and the power of test (1-β) = 95%. According to the sample calculation of the one-sample rate test, when P0 = 20%, and P1 = 45%, the sample size will be 42 as calculated for patients with R0 resection. Based on experience from LungMate-001 trial of our hospital (60%, 30 / 50)36, R0 resection is expected to be achieved in 45% of patients considering the relatively more advanced tumor and lymph node status in this trial. Therefore, 93 patients need to be recruited. Assuming that 5% would drop out early or be lost to follow-up, at least 98 patients will be recruited. According to the study on the proportion of PD-L1 expression level in Chinese population, the expression level of PD-L1 ≥ 50% accounted for 11.4%, 1-49% accounted for 22.4%, and <1% accounted for 66.3%57. Because the determination of PD-L1 expression depends on the amount of tissue punctured, the tumor tissue obtained by puncture in some patients is too small to be determined. The experience of thoracic surgery department in our hospital accounts for about 40% (98*40% = 39 patients)16,36. Therefore, it is expected that at least 20 patients (7 [98*60%*11.4%] + 13 [98*60%*22.4%]) will be enrolled in Chemo-free subgroup, and at least 78 (39 [98*60%*66.3%] + 39) will be enrolled in Chemo subgroup. Follow-up Follow-up was acquired by telephone contact or outpatient visit. For surgical treated participants, chest CT were performed every 3 months for the first year postoperatively, every 6 months for 2 to 5 years, and annually from then on. MRI of the brain, ultrasonography of abdominal regions, bone scans were performed annually. Local recurrence was defined as the recurrence in the primary site or mediastinal lymph nodes, while distant recurrence was defined as recurrence in other sites. The follow-up was conducted until death. Exploratory analysis The distribution of PD-L1 expression in the intention-to-treat analysis were examined. Additionally, other potential predictive biomarkers, such as gene expression profiling, tumor mutational burden, and infiltration of immune cells into archived and/or fresh tumor tissue, were evaluated either prior to or after surgery during the course of the study treatment. Furthermore, their correlation with disease status and/or therapeutic efficacy was assessed. These analyses were prespecified in the approved study protocol. RNA sequencing and data processing Total RNA from fresh frozen tissues was extracted with TRIzol (Invitrogen, 15596026). Sequencing libraries were generated using a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7770), and index codes were added to attribute sequences to each sample. The libraries were pooled, and paired-end sequencing (2×150 bp reads) was performed using an Illumina NovaSeq 6000. After RNAseq sequencing, raw fastq files were trimmed via fastp (v0.20.1)58 and aligned to GRCh38 reference genome by STAR (v2.7.6a)59 with default settings. After obtaining the BAM files, read counts were summarized by featureCounts (v2.0.1)60. TPMs (Trans Per Million) were generated using Salmon (v0.6.0)61. Batch effects were adjusted using “combat” function in sva package62. Differentially expressed gene analysis We used DESeq2 to calculate differential gene expression between sample groups63. The DESeq2 profiles genes according to model gene count expression data and calculates |Log2Fold Change | , which estimates the effect size and represents gene changes between comparison groups. The two-sided Wald-test statistics are computed to exam the differential expression across the comparison groups. Genes with |Log2Fold Change | > 1 and Wald test P < 0.05 were defined as differentially expressed genes. We used volcano plots to visualize the differential gene expression results. Gene set enrichment analysis For gene set enrichment analysis (GSEA) analysis, results for all protein coding genes were ranked by Log2fold change and evaluated with the “GSEA” algorithm64. “Hallmark” and “KEGG” gene sets were acquired from MSigDb. We filtered GSEA result based on the criteria that the P-value <0.05 and visualized the pathways as candidates based on the normalized enrichment score. Tumor micro-environment estimation The immune scores of each sample were calculated using the “ESTIMATE” R package65. The infiltration of immune cells was evaluated by “mcpcounter” (v1.2.0)66. TCGA data analysis The correlation between expression of BST1 and tumor microenvironment immune infiltration was conducted by TIMER2.0 (http://timer.comp-genomics.org/timer/)67. Additionally, the correlation between the expression of BST1 in lung cancer (LUAD plus LUSC) was analyzed by GEPIA68. DNA sequencing and data processing In this study, 35 baseline samples were collected and successfully conducted for DNA sequencing. According to the manufacturer’s protocol, genomic DNA was extracted from tumors using QIAamp Fast DNA tissue kit (QIAGEN). Total DNA was quantified by the Qubit 2.0 Flurometer (Life Technologies) and NanoDrop 2000 (Thermo Fisher Scientific) and the integrity was assessed by TapeStation (Agilent Technologies). For Illumina sequencing library construction, the genomic DNA was fragmented to an average size of 180-280 bp using a Covaris focused-ultrasonicator. Then, WES libraries were prepared and captured using the Agilent SureSelect Human All Exon V6 kit (Agilent Technologies, 5190-8865) following the manufacturer’s instructions. The DNA library with 150 bp paired-end reads was sequenced on the Illumina Novaseq 6000 platform. To detect single nucleotide variant (SNV) and small insertion/deletion (INDEL), the GATK (v4.0.6.0) best practice guideline was followed69. After excluding low-quality reads, qualified paired-end WES sequencing reads were aligned to human reference genome (hg38) with BWA MEM (v0.7.15)70. The resulting BAM files were further processed with Picard tools to remove PCR duplicates. SNVs and INDELs were detected using MuTect2 in tumor-only mode with panel-of-normals71. Then we filtered out short tandem repeat regions downloaded from UCSC table browser, and finally annotated using Funcotator from GATK. Oncoplot was plotted using “Maftools” R package72. Immunohistochemistry analysis Tumor tissues were fixed in 10% neutral buffered formalin, embedded into paraffin blocks, cut into 4 - 5 μm slides. The tumor samples were provided by the Pathology Department of Shanghai Pulmonary Hospital. The slides were stained with Anti-BST1 antibody (Abcam: ab137718, dilusion:1/100) and nuclei were stained with hematoxylin dye. At last the sections were scanned using the Pannoramic 250/ MIDI. The immunohistochemical (IHC) score were determined in five randomly selected areas. The staining results were semi-quantitatively evaluated by the multiply of staining intensity and the percentage of positive staining cells73. The percentage of positive cells was given into four grades by ImageJ: 0 for <5%; 1 for 6%–25%; 2 for 26%–50%; 3 for 51%–75% and 4 for >75%. Staining intensity was assessed by four degrees: 0, negative; 1, weak; 2, moderate; and 3, strong. The intensity was assessed by IHC Profiler74. Single-cell RNA sequencing analysis To explore the association between BST1 and immune cells, we used the single-cell RNAseq data of immunochemotherapy from our previous study23. The data processing, cell clustering, annotation and tumor cell identification have been described in previous study. The mean value of BST1 in tumor cells was calculated for each sample. Then, we calculated the cellular fraction for each immune lineage in all CD45+ cells. Notably, to avoid the bias caused by inaccurate estimations, samples with less than 10 cells were excluded for calculating cellular factions and average gene expression. Spearman correlation test was performed to test correlation between BST1 expression and the cellular fraction. Statistical analysis & reproducibility Considering that our study population consists of patients with stage III initially unresectable NSCLC, including patients who did not convert to resectable status and received surgery in the effectiveness analysis may introduce selection bias. The pathological effectiveness of the treatment was evaluated based on patients who had comprehensive pathological assessment reports from our hospital. Patients who were deemed resectable but discontinued the trial for reasons other than disease progression were excluded from the primary analysis. Additionally, to better demonstrate the real-world impact of precise stratification of induction therapy in patients with stage III initially unresectable NSCLC, further analyses of ORR, survival, and safety were conducted using the ITT population who had received at least one dose of induction therapy. The 95% CIs of MPR rate, pCR rate and ORR were estimated by means of the Clopper-Pearson method. Time-to-event analyses were estimated with the Kaplan–Meier method, with median survival time presented with 95% CIs based on the Brookmeyer-Crowley method and survival rates presented along with 95% CIs based on the complementary log-log transformation method. The median follow-up time was determined using the reverse Kaplan-Meier method. TRAEs were summarized with frequencies (percentages). Additionally, EFS and OS stratified by surgery were explored by post-hoc analyses. Wilcoxon test was applied to compare the expression differences between sample groups. To separate patients into low- or high-BST1 expression groups, the cutoff was generated based on the association between gene expression and survival data using the survminer package. All P values were two-sided, with P < 0.05 considered statistically significant. Statistical analyses were conducted using R programming (version 4.3.1). Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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  Publisher URL	https://www.nature.com/
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  Citation Flow Score	58

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