Neoadjuvant chemotherapy plus nivolumab with or without ipilimumab in operable non-small cell lung cancer: the phase 2 platform NEOSTAR trial

Neoadjuvant ipilimumab + nivolumab (Ipi+Nivo) and nivolumab + chemotherapy (Nivo+CT) induce greater pathologic response rates than CT alone in patients with operable non-small cell lung cancer (NSCLC). The impact of adding ipilimumab to neoadjuvant Nivo+CT is unknown. Here we report the results and correlates of two arms of the phase 2 platform NEOSTAR trial testing neoadjuvant Nivo+CT and Ipi+Nivo+CT with major pathologic response (MPR) as the primary endpoint. MPR rates were 32.1% (7/22, 80% confidence interval (CI) 18.7–43.1%) in the Nivo+CT arm and 50% (11/22, 80% CI 34.6–61.1%) in the Ipi+Nivo+CT arm; the primary endpoint was met in both arms. In patients without known tumor EGFR/ALK alterations, MPR rates were 41.2% (7/17) and 62.5% (10/16) in the Nivo+CT and Ipi+Nivo+CT groups, respectively. No new safety signals were observed in either arm. Single-cell sequencing and multi-platform immune profiling (exploratory endpoints) underscored immune cell populations and phenotypes, including effector memory CD8+ T, B and myeloid cells and markers of tertiary lymphoid structures, that were preferentially increased in the Ipi+Nivo+CT cohort. Baseline fecal microbiota in patients with MPR were enriched with beneficial taxa, such as Akkermansia, and displayed reduced abundance of pro-inflammatory and pathogenic microbes. Neoadjuvant Ipi+Nivo+CT enhances pathologic responses and warrants further study in operable NSCLC. (ClinicalTrials.gov registration: NCT03158129.)

lmmunohistochemistry analysis: PD-L1 stained slides were scored by standard microscopy following the recommendations of the International Association for the Study of Lung Cancer guidelines (PMID: 29800747). The results were plotted using GraphPad Prism v.9.00. Flow Cytometry analysis: Data were analyzed using FlowJo Software v.10.5.3 (Tree Star, Inc.). Dead cells were stained using LIVE/DEAD Fixable Yellow Dead Cell Stain dye (catalog no. L-34968, Life Technologies) and excluded from the analysis. Analyzed data were plotted using GraphPad prism v. 9.00. Multiplex immunofluorescence analysis: ROIs were analyzed by a pathologist using InForm v.2.8.2 image analysis software (Akoya Biosciences). All the data were consolidated using the R studio v.3.5.3 (Phenopter v.0.2.2 packet, Akoya Biosciences/PerkinElmer) and SAS v.7.1 Enterprise. The data were plotted using GraphPad Prism v.9.00. NanoString analysis: nCounter Digital Analyzer was used to tabulate the counts of the reporter probes and for further analysis raw data output was imported into nSolver analysis software (v4.0.70) (http://www.nanostring.com/products/nSolver). Normalization, cell type and differential gene expression analyses were performed using the nSolver Advanced data analysis package (v2.0.134). The data were plotted using GraphPad prism v. 9.0.0.
Gut microbiome analysis: The V4 region of the bacterial 16S rRNA gene was amplified and sequenced on the Illumina MiSeq (Illumina, Inc.) platform using the 2x250 bp paired-end protocol yielding paired-end reads with near-complete overlap. Raw FASTQ files were processed using DADA2 (1.18) to generate amplicon sequence variants (ASVs) and taxonomies assigned with SILVA database v138 (https://www.arbsilva.de). The resulting ASV table and taxonomies were used to compute alpha and beta diversity metrics as well as taxonomic relative abundances. The sequencing depths ranged from 19,310 to 159,961 with a mean of 59,057 reads per sample. Alpha diversity was calculated using Shannon Index. Bray-Curtis dissimilarity were used to calculate the pairwise dissimilarities and perform principal coordinate analysis (PCoA) between samples. PERMANOVA analyses (with 999 permutations) and beta-dispersion tests were used to compare microbiota diversity and dispersion between the two trials. Differentially abundant taxa were identified in each of the trials using the statistical method implemented in the R package DESeq2. The results were plotted in R (R Core Team 2020; https://www.R-project.org) using ggplot2 package (https://ggplot2.tidyverse.org).
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Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy De-identified single-cell RNA-sequencing raw data reported in this manuscript have been deposited in the European Genome-phenome Archive with accession number EGAS00001006728. Access to this dataset is controlled by the institutional Data Access Committee in compliance with the NIH policy for Data Management and Sharing and in accordance with an alliance agreement between MD Anderson Cancer Center and Bristol Myers Squibb. Access to this dataset will be granted upon review and acceptance of academic requests. Further information about EGA can be found at https://egaarchive.org. The raw reads were aligned to human reference genome GRCh38 (hg38). The 16S fecal microbiome sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive under the SRA BioProject ID PRJNA665109 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA665109). Taxonomies were assigned with SILVA database v138 (https://www.arb-silva.de). Source data for Figure  Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Simon's minimax two-stage design was applied to test the major pathologic response rate for each one of the two treatment arms. We assumed the 15% major pathologic response rate under the null hypothesis versus the 40% major pathologic response rate under the alternative hypothesis. For each treatment arm, 15 patients were enrolled in the first stage. If only two or less of the 15 patients have major pathologic response, enrollment to that treatment arm would be terminated and the treatment is considered inefficacious. Otherwise, with at least three major pathologic response, additional 6 patients would be enrolled to reach a total of 21 patients. At the end of trial, if we observe 6 or more patients have major pathologic response, the treatment is considered efficacious and inefficacious otherwise. The trial would have 90% power when the major pathologic response rate is 40%. When the major pathologic response rate is 15%, the probability of early termination is 0.60 with an average sample size of 17.4 and one-sided 10% type I error rate.
Data exclusions Clinical analyses: All eligible patients enrolled into the study were included in the analyses.
Correlative analyses: All samples available and considered appropriate based on QC for correlative studies at time of analyses were included.
Flow cytometry analysis: Available samples were excluded from analysis if they did not pass the respective QC for a given assay as detailed in Methods and Figure

Replication
Replication was not applicable to this study as this was a clinical study with unique patient samples. All techniques and reagents used for the correlative analyses of this study had been previously optimized and validated.
Randomization Patient were enrolled to the nivolumab plus chemotherapy arm followed by ipilimumab plus nivolumab plus chemotherapy arm of the NEOSTAR platform study. Randomization was not performed in these two arms. Treatment allocation was not relevant in this multi-arm platform trial with two independent single-arm. These two arms were expected to be analyzed and reported separately with the goal to expedite the investigation of novel immunotherapy-based strategies in the neoadjuvant setting.

Blinding
The trial was not a blinded study. Blinding was not practical in this multi-arm platform phase 2 trial of single studies performed in sequence as experimental treatments were administered intravenously with different doses and schedules. However, after initial clinical reporting, the primary endpoint of the study was reviewed in a blinded manner by two pathologists experienced in the evaluation of tumor response after neoadjuvant therapy. Furthermore, the study was designed to compare the primary endpoint to historical controls of neoadjuvant chemotherapy and both experimental arms were novel strategies added to standard-of-care approach.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. -antibody internally titrated and validated with respect to differential staining patterns on CD8 T cells from expanded tumorinfiltrating lymphocytes as a positive control and normal donor PBMCs as a negative control; Vendor validation and technical information can be found at https://www.thermofisher.com/antibody/product/CD279-PD-1-Antibody-clone-MIH4-Monoclonal/64-9969-42; https://www.biolegend.com/en-us/products/percp-cyanine5-5-anti-human-cd279-pd-

Human research participants
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Population characteristics
Male and female patients who met inclusion criteria for the study were 18 years of age and older and had stage IB (equal to or greater than 4 cm) to IIIA NSCLC according to American Joint Commission on Cancer (AJCC) 7th edition staging system. Only single mediastinal ipsilateral N2 station was allowed for the enrollment. All patients had to have surgically resectable disease and Eastern Cooperative Group performance status 0-1, adequate organ function, and cardiopulmonary status. Patients were excluded from the study if they had autoimmune disease, immunodeficiency, or previously received immunotherapy for other disease, if they had active infectious disease requiring ongoing treatment or cancer within the last two years. A complete list of inclusion and exclusion criteria is included in the Methods of the manuscript. Patient characteristics, including self-reported sex, are reported in Table 1. Sex and/or gender was not considered in the trial design. Twelve females and ten males were recruited in the Nivo+CT arm; seven females and fifteen males were recruited in the Ipi +Nivo+CT arm. Ten and thirteen patients were less than 65 years of age in the Nivo+CT and in the Ipi+Nivo+CT arms, respectively; twelve and nine patients were more than 65 years of age in the Nivo+CT and in the Ipi+Nivo+CT arms, respectively. The participants were not compensated for their participation on the studies. Potential biases applicable to the study were the relatively subjective operability/resectability of the disease prior to enrollment and the self-selection bias, which could derive from patient health literacy about the study (clinicaltrial.gov). The impact of both biases on the results of our study were minimized by presenting and discussing eligible patients at multidisciplinary tumor board conference before enrollment and by the objective evaluation of all study endpoints.

Ethics oversight
Written informed consent was provided by all study participants or their legal representatives. The study was approved by the University of Texas MD Anderson Cancer Center's Institutional Review Board.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Outcomes
The primary endpoint of the trial was major pathologic response (MPR), defined as less than or equal to 10% viable tumor cells in the original resected tumor bed following neoadjuvant therapy on trial and assessed by the pathologists involved in the study as detailed in the methods of the manuscript. Select secondary endpoints included treatment toxicity, perioperative morbidity and mortality, quantification of CD8+ TILs in resected tissues, ORR, pCR, completeness of surgical resection, time-to-events (including EFS and OS), correlation of blood, tissue and stool biomarkers with efficacy. Exploratory endpoints included tissue-, blood-, stool-and imagingbased biomarkers. All outcomes were assessed by the study investigators using the methods and criteria detailed in the manuscript, including RECIST criteria v. 1.1, National Cancer Institute Common Terminology Criteria for Adverse Events v. 4., time-to-events (event-free survival [EFS] defined as the time from treatment initiation to any progression of primary lung cancer precluding planned surgery, any progression or recurrence (as assessed by imaging and/or histopathologically) of primary lung cancer after surgery, any progression of primary lung cancer in patients without surgery, or death from all causes, or to the time of last imaging), and overall survival [OS], defined as the time from treatment initiation to the time of death from all causes or to the time of last follow up, obituaries were cross-referenced for any unreported patient deaths), tissue scRNA-seq, tumor NanoString, tissue flow cytometry, tumor PD-L1 IHC, tissue multiplex immunofluorescence staining and 16S gut microbiome, by the study investigators.

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Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Fresh tumor tissue was disaggregated using a medimachine and subsequent filtering to generate a single cell suspension for staining. PBMCs were thawed, washed and resuspended for staining. Surface staining was performed in FACS Wash Buffer (lX DPBS with 1% Bovine Serum Albumin) for 30 min on ice using fluorochrome-conjugated monoclonal antibodies from BD Biosciences, Biolegend, and eBioscience. Cells were fixed in 1% paraformaldehyde solution for 20min at room temperature following surface staining. For panels containing transcription factors, cells were fixed and permeabilized using the BD Transcription factor kit according to the manufacturer's instructions. A complete list of the antibodies, catalog numbers, company and clones used are available. Dead cells were stained using AQUA live/dead dye (lnvitrogen)and excluded from the analysis. Gating strategy Cells were initially gated using FSC-A v SSC-A followed by singlet gates using SSC-Av SSC-H. Single cells were then gated for exclusion of dead cells. A QC metric of 100 events was required in the immediate parental gate for any subgating.

Instrument
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