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Neoadjuvant nivolumab with or without relatlimab in resectable non-small-cell lung cancer: a randomized phase 2 trial

Martin Schuler 1,2,3 , Kristof Cuppens 4,5 , Till Plönes2,6,18, Marcel Wiesweg 1,2,3, Bert Du Pont7, Balazs Hegedus2,6, Johannes Köster 2,3,8, Fabian Mairinger2,9, Kaid Darwiche 2,3,10, Annette Paschen 2,11, Brigitte Maes 12, Michel Vanbockrijck13, David Lähnemann 1,2,8, Fang Zhao 2,11, Hubertus Hautzel 2,3,14, Dirk Theegarten2,9, Koen Hartemink15, Henning Reis2,9,16, Paul Baas 17, Alexander Schramm 1,2,20 & Clemens Aigner 2,6,19,20

Nature Medicine APRIL 30 2024

Abstract: Antibodies targeting the immune checkpoint molecules PD-1, PD-L1 and CTLA-4, administered alone or in combination with chemotherapy, are the standard of care in most patients with metastatic non-small-cell lung cancers. When given before curative surgery, tumor responses and improved event-free survival are achieved. New antibody combinations may be more efficacious and tolerable. In an ongoing, open-label phase 2 study, 60 biomarker-unselected, treatment-naive patients with resectable non-small-cell lung cancer were randomized to receive two preoperative doses of nivolumab (anti-PD-1) with or without relatlimab (anti-LAG-3) antibody therapy. The primary study endpoint was the feasibility of surgery within 43 days, which was met by all patients. Curative resection was achieved in 95% of patients. Secondary endpoints included pathological and radiographic response rates, pathologically complete resection rates, disease-free and overall survival rates, and safety. Major pathological (≤10% viable tumor cells) and objective radiographic responses were achieved in 27% and 10% (nivolumab) and in 30% and 27% (nivolumab and relatlimab) of patients, respectively. In 100% (nivolumab) and 90% (nivolumab and relatlimab) of patients, tumors and lymph nodes were pathologically completely resected. With 12 months median duration of follow-up, disease-free survival and overall survival rates at 12 months were 89% and 93% (nivolumab), and 93% and 100% (nivolumab and relatlimab). Both treatments were safe with grade ≥3 treatment-emergent adverse events reported in 10% and 13% of patients per study arm. Exploratory analyses provided insights into biological processes triggered by preoperative immunotherapy. This study establishes the feasibility and safety of dual targeting of PD-1 and LAG-3 before lung cancer surgery.

摘要：针对免疫检查点分子PD-1、PD-L1和CTLA-4的抗体，单独使用或与化疗联合使用，是大多数转移性非小细胞肺癌患者的标准护理。如果在根治性手术前给予，可改善肿瘤反应和无事件生存。新的抗体组合可能更有效和更容易耐受。本研究是一项正在进行的开放标签，II期临床研究，60名未经过生物标记物选择、未接受治疗的可切除非小细胞肺癌患者被随机分成两组，在接受或不接受relatlimab(抗LAG-3)抗体治疗的情况下，在手术前接受两剂nivolumab(抗PD-1)治疗。主要的研究终点是在43 天内手术的可行性，所有患者均满足此条件。95%的患者获得了根治性切除。次要终点包括病理和影像学反应率、病理完全切除率、无病存活率和总存活率，以及安全性。主要病理反应(≤10%活肿瘤细胞)和客观影像学反应分别为27%和10%(尼伏单抗)和30%和27%(尼伏单抗和瑞拉莫单抗)。在100%(Nivolumab)和90%(nivolumab和relatlimab)患者中，肿瘤和淋巴结病理完全切除。随访12个 月的中位无瘤生存率和总生存率分别为89%和93%(尼伏单抗)和93%和100%(尼伏单抗和瑞拉莫单抗)。12 月的中位无瘤生存率和总生存率分别为89%和93%。这两种治疗都是安全的，≥3级治疗--每个研究组分别有10%和13%的患者报告了紧急不良事件。探索性分析提供了对术前免疫治疗触发的生物过程提供了新的见解。本研究确定了肺癌手术前应用PD-1和LAG-3双靶向治疗的可行性和安全性。

目录

1. INTRODUCTION (Fig. 1a and Supplementary information)

2. Materials and Methods 

    2.1 Clinical study

    2.2 Metabolic hybrid imaging

    2.3 Phenotyping of peripheral blood T cells (Supplementary Fig. 1)

    2.4 Phenotyping of immune cell subsets in resected tumors

    2.5 Gene expression analyses

    2.6 Genome sequencing

    2.7 Inference of subclonal diversity

    2.8 Data availability

    2.9 Code availability

3. Results

    3.1 Study design and patient disposition (Fig. 1a,b)(Table 1)

    3.2 Primary outcome (Fig. 1a)

    3.3 Secondary outcomes (Fig. 1 c,d)(Fig. 2)

    3.4 Safety (Table 2)

    3.5 Exploratory outcomes

    3.5.1 Metabolic responses (Supplementary Fig. 1)(Fig. 1e)(Extended Data Fig. 1)

    3.5.2 Immune cell phenotyping (Fig. 3; Supplementary Fig. 3 and Extended Data Fig. 2b)

    3.5.3 Expression of immune- and cancer pathway-related genes (Fig. 3c)

    3.5.4 Shaping of cancer genomes by immunotherapy  (Fig. 4 and Supplementary Fig. 3)

4. DISCUSSION

— 图表汇总—

    2.3 Phenotyping of peripheral blood T cells

Supplementary Fig. 1 Metabolic response assessment per FDG-PET/CT. 

Deidentified imaging data from exemplary patients with lung adenocarcinomas stage with partial metabolic response (001-R-037) and metabolically progressive disease (001-R-006) following neoadjuvant immunotherapy. Both patients have consented publication. Representative images (computed tomography – left panels, positron emission tomography – left center panels, fusion images – right center panels, topograms – right panels) taken during screening/staging (upper lines) and following study therapy prior to surgery (lower lines) are shown. Preoperative clinical stages, metabolic response per PERCIST, postoperative histopathological stages, and pathological response to study therapy are shown. In patient 001-R-006 ipsilateral (N2) and contralateral (N3) lymph nodes were sampled preoperatively by EBUS-TBNA and intraoperatively (in total 35 lymph nodes were retrieved during surgery including sampling of FDG-avid contralateral hilar and paratracheal lymph nodes). Contralateral lymph node metastases were ruled out by both modalities. 

    2.4 Phenotyping of immune cell subsets in resected tumors

a, Primary antibodies and gating strategy for detection of T lymphocyte subsets in the peripheral blood. 

b, Primary antibodies and gating strategy for detection of T lymphocyte subsets in single cell suspensions generated from resected tumors. 

c, Primary antibodies and gating strategy for detection of myeloid cell subsets in single cell suspensions generated from resected tumors. 

    2.6 Genome sequencing

Left: Waterfall plot of pathologic response (% regression of viable tumor cells) in tumors and lymph nodes from patients resected following neoadjuvant study treatment (arm A light blue, arm B dark blue). The category of PD-L1 expression by tumors cells (Tumor Proportional Score, TPS <1% light blue, TPS 1 to 49% medium blue color, TPS 50 to 100% dark blue) for each patient is represented above the oncograms. The lower panel is an oncogram of cancer genes with increased variant allele frequencies following neoadjuvant immunotherapy per patient. Boxes represent pathogenic genomic aberrations in the respective gene.

Right: List and details of mutated cancer genes per patient, which exhibited increased variant allele frequencies following neoadjuvant immunotherapy as compared to diagnostic pretherapeutic biopsies. 

3. Results

    3.1 Study design and patient disposition

Fig. 1 Study design, patient deposition and secondary endpoints.  

a, Graphical representation of clinical study design including key inclusion criteria.

b, Patient deposition during the phases of the clinical study including screening, preoperative immunotherapy and curative resection. Reasons for screening failure and outcomes of surgery are summarized (*including one patient with single bone metastasis).

Table 1. Patient demographics and characteristics

    3.2 Primary outcome

    3.3 Secondary outcomes

Fig. 1

c, Fraction of patients (n = 60) with microscopically complete (R0, green), microscopically incomplete (R1, purple) and macroscopically incomplete (pleural carcinosis, M1a (PLE), orange) resection of primary lung cancers and, if present, lymph node metastases per study arm.  

d, Fraction of patients (n = 60) with complete (none), partial response (PR, green), stable (SD, yellow) and progressive disease (PD, red) per RECIST evaluation of CT scans per study arm.

Fig. 2 Pathological responses, biomarkers and survival outcomes. 

a, Waterfall plots of pathologic tumor regression (percentage reduction of viable tumor cells) in resected tumors and lymph nodes following neoadjuvant treatment with nivolumab (arm A, blue) or nivolumab and relatlimab (arm B, red). The color intensity encodes the category of PD-L1 expression by tumor cells (TPS <1% light color, TPS 1–49% medium dark color, TPS 50–100% dark color). The lower panel depicts the oncogram of each tumor using next-generation DNA sequencing of 500 cancer-related genes. Boxes represent pathogenic genomic aberrations in the respective gene. Genes with pathogenic aberrations in at least two study patients are listed. 

b, Kaplan–Meier plots for OS (left) and DFS (right) per study arm (arm A nivolumab, blue; arm B nivolumab and relatlimab, red).  

c, Kaplan–Meier plot for DFS in patients achieving a MPR (≤10% viable tumor cells (green)), and not achieving a MPR (>10% viable tumor cells (orange)). Statistical comparisons by log-rank test, vertical lines indicate censored patients. Two patients (both arm A) had died from noncancer causes. Six patients (four in arm A and two in arm B) have recurred or died. No patient with MPR has recurred, one patient with MPR had died from a noncancer cause. 

d, Fraction and number of patients with complete pathological response (pCR, upper) and MPR (lower) in study arms A (nivolumab (blue)) and B (nivolumab and relatlimab (red)).

    3.4 Safety

Table 2. Summary of adverse events

    3.5 Exploratory outcomes

    3.5.1 Metabolic responses

Supplementary Fig. 1 Metabolic response assessment per FDG-PET/CT. 

Deidentified imaging data from exemplary patients with lung adenocarcinomas stage with partial metabolic response (001-R-037) and metabolically progressive disease (001-R-006) following neoadjuvant immunotherapy. Both patients have consented publication. Representative images (computed tomography – left panels, positron emission tomography – left center panels, fusion images – right center panels, topograms – right panels) taken during screening/staging (upper lines) and following study therapy prior to surgery (lower lines) are shown. Preoperative clinical stages, metabolic response per PERCIST, postoperative histopathological stages, and pathological response to study therapy are shown. In patient 001-R-006 ipsilateral (N2) and contralateral (N3) lymph nodes were sampled preoperatively by EBUS-TBNA and intraoperatively (in total 35 lymph nodes were retrieved during surgery including sampling of FDG-avid contralateral hilar and paratracheal lymph nodes). Contralateral lymph node metastases were ruled out by both modalities. 

Fig. 1e Fraction of patients (n = 31) with complete (none), partial metabolic response (PMR, green), metabolically stable (SMD, yellow) and metabolically progressive disease (PMD, red) per PERCIST evaluation of positron emission tomography scans per study arm. SoC, standard of care.

Extended Data Fig. 1 Response assessment per FDG-PET/CT and CT in relation to pathological response and nodal upstaging.

Metabolic responses (PERCIST), radiographic responses (RECIST), nodal upstaging (yes, no), pathological response category (Viable tumor cells), and treatment arm (A – nivolumab, B – nivolumab/relatlimab) of 30 patients from study site Essen, who underwent FDG-PET/CT scanning following preoperative immunotherapy. One patient was excluded because surgery was aborted due to pleural carcinosis.

    3.5.2 Immune cell phenotyping

Fig. 3 Immune cell subsets and gene expression in peripheral blood and resected tumors.

a, Fraction of total CD8+ T cells (left), CD8+GrzB+ effector T cells (center) and CD8+GrzB− T cells (right) in the peripheral blood of responding (≤50% viable tumor cells in resected tumors and lymph nodes) and nonresponding patients (>50% viable tumor cells). Each dot represents an individual patient: baseline values are in black and values at day 28 are in red. Whiskers and boxes represent the minimum, first, second and third quartiles and the maximum. Wilcoxon matched pairs signed-rank test was applied for statistical comparison. All P values are two-sided, no adjustments were made for multiple comparisons. 

b, Fraction of CD16+ neutrophil granulocytes (left), CD14+ monocytes (center) and CD4+CD25+ regulatory T cells (Treg, right) in single-cell suspensions from resected tumors. Each dot or box represents a single patient (black, nivolumab; red, nivolumab and relatlimab; MPR, ≤10% viable tumor cells in resected tumors and lymph nodes; no MPR, >10% viable tumor cells). Horizontal lines indicate the mean and s.e.m. 

c, Differential expression of immune-related and cancer pathway-related genes in response to treatment with nivolumab (left) and nivolumab and relatlimab (right) are presented as volcano plots. Significantly (FDR ≤ 0.05) upregulated (right of 0 line on x axes) and downregulated (left of 0 line on x axes) genes are depicted as blue closed circles. Selected significantly regulated genes are indicated. P values on the y axes were calculated using the two-sided quasi-likelihood F-test approach of EdgeR. 

d, Differential expression of immune-related genes and cancer pathway-related genes in resected tumors with MPR following treatment with nivolumab (left) and nivolumab and relatlimab (right) compared with resected tumors without MPR. Significantly (FDR ≤ 0.05) upregulated (right of 0 line on x axes) and downregulated (left of 0 line on x axes) genes in tumors with MPR are depicted as blue closed circles. Selected significantly regulated genes are indicated. P values on the y axes were calculated using the two-sided quasi-likelihood F-test approach of EdgeR. There was no significant interaction with MPR following nivolumab treatment.

Left: Waterfall plot of pathologic response (% regression of viable tumor cells) in tumors and lymph nodes from patients resected following neoadjuvant study treatment (arm A light blue, arm B dark blue). The category of PD-L1 expression by tumors cells (Tumor Proportional Score, TPS <1% light blue, TPS 1 to 49% medium blue color, TPS 50 to 100% dark blue) for each patient is represented above the oncograms. The lower panel is an oncogram of cancer genes with increased variant allele frequencies following neoadjuvant immunotherapy per patient. Boxes represent pathogenic genomic aberrations in the respective gene.

Right: List and details of mutated cancer genes per patient, which exhibited increased variant allele frequencies following neoadjuvant immunotherapy as compared to diagnostic pretherapeutic biopsies. 

Extended Data Fig. 2 Immune cell subsets in peripheral blood and resected tumors.

a, Induction of CD8+GrzB+ effector T cells in the peripheral blood in response to neoadjuvant nivolumab or nivolumab and relatlimab treatment. Each dot represents an individual patient, base line values in black, values at day 28 (after neoadjuvant immunotherapy) in red. Responders are defined by ≤ 50% viable tumor cells in resected tumors and lymph nodes, non-responders by >50% viable tumor cells. The Wilcoxon matched pairs signed-rank test was applied for statistical comparison. All p-values are two-sided, no adjustment was made for multiple comparisons. 

b, Characterization of infiltrating T lymphocytes in resected tumors in relation to study treatment (arm A: nivolumab, arm B: nivolumab and relatlimab) and achieving a major pathological response (MPR, ≤ 10% viable tumor cells in resected tumors and lymph nodes) or not achieving a MPR (no MPR). Each symbol represents an individual patient. Tregs – regulatory T lymphocytes. Horizontal lines indicate the mean value and standard error of the mean.

    3.5.3 Expression of immune- and cancer pathway-related genes

Fig. 3 Immune cell subsets and gene expression in peripheral blood and resected tumors.

c, Differential expression of immune-related and cancer pathway-related genes in response to treatment with nivolumab (left) and nivolumab and relatlimab (right) are presented as volcano plots. Significantly (FDR ≤ 0.05) upregulated (right of 0 line on x axes) and downregulated (left of 0 line on x axes) genes are depicted as blue closed circles. Selected significantly regulated genes are indicated. P values on the y axes were calculated using the two-sided quasi-likelihood F-test approach of EdgeR. 

    3.5.4 Shaping of cancer genomes by immunotherapy

Fig. 4 Dynamic changes in the mutational spectra in response to immunotherapy.

a, Prevalence of mutations per megabase (Mb, y axes) of 500 cancer-related genes in pretherapeutic diagnostic biopsies (left) and resected tumors (right) of two exemplary patients without (001-R-010) and with response (002-R-052) to study therapy. The specific mutations (nucleotide exchanges from C to A (C>A), G (C>G) or T (C>T), from T to A (T>A), C (T>C) or G (T>G), complex nucleotide replacements (complex) or multiple nucleotide variants (MNV)) are color-coded from dark blue to yellow. The minimal VAFs are depicted on the x axes. 

b, Subclonal dynamics between pretherapeutic biopsies and resected tumors of 14 patients; each line depicts an individual patient. Left, pathological regression (percentage reduction of viable tumor cells) following immunotherapy. Center, estimated total number of subclones in the resected tumor. Right, fraction of subclones enriched (‘fraction gained’) and depleted (‘fraction lost’) in the resected tumors. Fractions are visualized by color (with yellow for high, purple for low), and bubble size (large for high, small for low, no bubble for zero). 

c, Selection of genomically encoded putative resistance mechanisms in one of 43 patients with pretreatment and posttreatment tumor specimens for genomic analyses. Left, representative microphotographs of the pretherapeutic diagnostic tumor biopsy stained with H&E and with an anti-PD-L1 primary antibody. DNA sequencing of the tumor biopsy revealed pathogenic mutations of KRAS and TP53 and amplification of the CD274 (PD-L1)-encoding gene. Center, low magnification image of a H&E-stained section of the resected tumor showing massive necrosis, but a residual region of vital tumor cells on the left-hand margin. Right, high magnification photomicrographs representing the transition zone from necrotic tumor to residual viable tumor cells stained with H&E and with an anti-CD8 primary antibody demonstrating tumor-infiltrating T lymphocytes. DNA sequencing of the resected tumor confirmed the presence of pathogenic mutations of KRAS and TP53 and amplification of the CD274 (PD-L1)-encoding gene. In addition, copy number gain of MYC and a pathogenic IDH1 mutation were newly detected. A complete list of patients with enrichment of genomically encoded putative resistance mechanisms in resected tumors is presented in Supplementary Fig. 3.

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