Abstract
Ipilimumab improves clinical outcomes when combined with nivolumab in metastatic non-small cell lung cancer (NSCLC), but its efficacy and impact on the immune microenvironment in operable NSCLC remain unclear. We report the results of the phase 2 randomized NEOSTAR trial (NCT03158129) of neoadjuvant nivolumab or nivolumab + ipilimumab followed by surgery in 44 patients with operable NSCLC, using major pathologic response (MPR) as the primary endpoint. The MPR rate for each treatment arm was tested against historical controls of neoadjuvant chemotherapy. The nivolumab + ipilimumab arm met the prespecified primary endpoint threshold of 6 MPRs in 21 patients, achieving a 38% MPR rate (8/21). We observed a 22% MPR rate (5/23) in the nivolumab arm. In 37 patients resected on trial, nivolumab and nivolumab + ipilimumab produced MPR rates of 24% (5/21) and 50% (8/16), respectively. Compared with nivolumab, nivolumab + ipilimumab resulted in higher pathologic complete response rates (10% versus 38%), less viable tumor (median 50% versus 9%), and greater frequencies of effector, tissue-resident memory and effector memory T cells. Increased abundance of gut Ruminococcus and Akkermansia spp. was associated with MPR to dual therapy. Our data indicate that neoadjuvant nivolumab + ipilimumab-based therapy enhances pathologic responses, tumor immune infiltrates and immunologic memory, and merits further investigation in operable NSCLC.
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Data availability
The data supporting the findings of the present study are available within the paper and its supplementary information files. Taxonomy was assigned using the Silva database (https://www.arb-silva.de) for 16S rRNA sequences. TCR-sequencing data (supporting the findings in Fig. 5 and Extended Data Figs. 7 and 9) have been deposited and are publicly available at the immuneACCESS platform (DOI: 10.21417/TC2020NM; http://clients.adaptivebiotech.com/pub/cascone-2020-nm). The 16S fecal microbiome sequencing data (supporting the findings in Extended Data Figs. 8 and 9) have been deposited and are publicly available in the National Center for Biotechnology Information Sequence Read Archive (SRA BioProject ID PRJNA665109). All other relevant deidentified data related to the present study are available from the corresponding author (T.C.) upon reasonable academic request and will require the researcher to sign a data access agreement with the University of Texas MD Anderson Cancer Center after approval. Source data are provided with this paper.
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Acknowledgements
We thank the patients and their families for participating in this study. We thank all the members of our regulatory, clinical, data coordination and translational research teams in the Departments of Thoracic/Head and Neck Medical Oncology and Thoracic Surgery at the MD Anderson Cancer Center for their support on this trial. We thank the members of the strategic alliance teams at Bristol Myers Squibb and the MD Anderson Cancer Center (E. B. Roarty and A. Spelman) for their support. We thank the members of the Translational Molecular Pathology Immune-Profiling Laboratory (TMP-IL) B. Sanchez Espiridon, S. Wijeratne, for their assistance with sample procurement and inventory, C.-W. B. Chow, W. Lu, L. Kakarala, M. Jiang, A. Tamegnon, and J. Zhou for their technical assistance, and D. Lorenzini for pathology assistance in imaging analysis. We thank L. Little and C. Gumbs from the Department of Genomic Medicine for assistance with TCR sequencing. We thank the MD Anderson’s Program for Innovative Microbiome and Translational Research (PRIME-TR) for supporting the analysis and interpretation of the microbiome results presented herein (Drs. J. A. Wargo and N. J. Ajami are the program director and executive scientific director for PRIME-TR, respectively). We thank Mr. D. Aten, Sr. Medical Illustrator in Creative Communications at MD Anderson Cancer Center, for his assistance with figure formatting. Funding support for the clinical trial was provided by Bristol Myers Squibb. Support for the study was also partially provided by the National Institutes of Health (NIH)/National Cancer Institute (NCI) through the University of Texas Lung Specialized Program of Research Excellence (SPORE; grant no. 5P50CA070907 to T.C., L.A.B., F.S., J.M.K., J.A.R., I.I.W., D.L.G. and J.V.H.), the NIH/NCI P30 CA016672 Cancer Center Support Grant, the Conquer Cancer Foundation of the American Society of Clinical Oncology Career Development Award 2018 Project ID 12895 (to T.C.), the Connie Rasor Endowment for Cancer Research (to D.L.G.), the Bruton Endowed Chair in Tumor Biology (to J.V.H.), and the TMP-IL at the Department of Translational Molecular Pathology, the University of Texas MD Anderson Cancer Center. The study was also partially supported by the generous philanthropic contributions to the University of Texas MD Anderson Cancer Center Lung Cancer Moon Shot Program, the University of Texas MD Anderson Cancer Center Physician Scientist Program (from the T. J. Martell Foundation, to T.C). the Khalifa Bin Zayed Al Nahyan Foundation (to T.C.), the Ford Petrin Donation (to J.V.H.), the Rexanna’s Foundation for Fighting Lung Cancer (to T.C., D.L.G., J.V.H. and B.S.) and the Bob Mayberry Foundation (to T.C.).
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T.C., W.N.W.Jr, H.Y.L, J.J.L. and J.V.H. designed the study. H.Y.L. and J.J.L developed the statistical plan. C.H.L. and H.Y.L performed the statistical analyses. T.C. served as principal investigator on the study. B.S. served as co-principal investigator on the study. T.C., W.N.W.Jr, F.V.F., F.E.M, A.S.T., G.B.Jr, X.L., J.Z., F.S., J.M.K., M.A., C.L., B.S.G., L.A.B., Y.Y.E., R.J.M., D.C.R., G.L.W., W.L.H., J.A.R., M.B.A., S.G.S., D.L.G., A.A.V., J.V.H. and B.S. recruited and/or treated patients. T.C., A.W., C.H.L., H.Y.L., A.P., M.C.B.G., B.W.C., L.F., A.R., M.A.W.K., H.D., A.F.C., E.R.P., L.M.S., J.F., H.T.T., N.K., C.H., N.J.A. and B.S. collected and/or analyzed the data. T.C., W.N.W.Jr, A.W., A.P., A.R., M.A.W.K., C.H., C.B., N.J.A., R.R.J., J.A.W., D.L.G., J.V.H. and B.S. interpreted the data. H.K., A.F. and I.I.W. provided support and guidance for data processing and analysis. P.S. and J.P.A. provided intellectual contribution. All authors contributed to writing the manuscript and approved the manuscript.
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T.C. reports speaker’s fees from the Society for Immunotherapy of Cancer and Bristol Myers Squibb, consulting fees from MedImmune/AstraZeneca and Bristol Myers Squibb, advisory role fees from EMD Serono and Bristol Myers Squibb, and research funding to the MD Anderson Cancer Center from Boehringer Ingelheim, MedImmune/AstraZeneca, EMD Serono and Bristol Myers Squibb. W.N.W.Jr reports consulting or advisory role fees from Clovis Oncology and AstraZeneca, speaker’s fees from Boehringer Ingelheim, honoraria from Roche/Genentech, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Merck, Bayer, Pfizer and Eli Lilly, and research funding from OSI Pharmaceuticals, Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly and Merck. M.C.B.G. has received research funding from Siemens Healthcare. H.T.T. reports research funding from Bayer-AS, Bristol Myers Squibb, Ziopharm and Guardant Health. N.K. reports consulting or advisory role fees from Merck, Bristol Myers Squibb, Abbvie and Roche. A.S.T. reports advisory board/consultant fees from Bristol Myers Squibb, Eli Lilly, Genentech, Roche, Novartis, Ariad, EMD Serono, Merck, Seattle Genetics, AstraZeneca, Boehringer Ingelheim, Sellas Life Science, Takeda, Epizyme and Huron, and receives research grants from Eli Lilly, Millennium, Polaris, Genentech, Merck, Boehringer Ingelheim, Bristol Myers Squibb, Ariad, Epizyme, Seattle Genetics, Takeda and EMD Serono. G.B.Jr receives personal fees and research funding from Amgen, Bayer, Bristol Myers Squibb, Celgene, Daiichi Sankyo, Genentech, MedImmune, Merck, Roche and Xcovery, research funding from Adaptimmune, Exelixis, GlaxoSmithKline, Immatics, Immunocore, Incyte, Kite pharma, Macrogenics, Torque, AstraZeneca, Tmunity, Regeneron, Beigene, Novartis and Repertoire Immune Medicines, and personal fees from Abbvie, Adicet, Amgen, Araid, Clovis Oncology, AstraZeneca, Bristol Myer Squibb, Celgene, Genentech, Gilead, Merck, Novartis, Roche, Virogin Biotech, John & Johnson/Janssen and Maverick Therapeutics. X.L. receives consultant and advisory fees from Eli Lilly, AstraZeneca and EMD Serono, and research funding from Eli Lilly, Boehringer Ingelheim and Spectrum Pharmaceuticals. J.Z. reports grants from Merck, Johnson and Johnson, and consultant fees, advisory fees or honoraria from Bristol Myers Squibb, AstraZeneca, GenePlus, Innovent, OrigMed and Roche outside the submitted work. M.A. reports research funding to the MD Anderson Cancer Center from Genentech, Nektar therapeutics, Merck, GlaxoSmithKline, Novartis, Jounce therapeutics, Bristol Myers Squibb, Eli Lilly and Adaptimmune, and receives advisory fees from GlaxoSmithKline and Shattuck labs. B.G. reports research funding to MD Anderson Cancer Center from Pfizer Inc., ISA Pharmaceuticals, MedImmune/AstraZeneca and Cue Bio. L.A.B. receives advisory/consultant fees and research funding from AstraZeneca, AbbVie, GenMab, PharmaMar and Sierra Oncology, advisor/consultant fees from Genentech, Bristol Myers Squibb, Alethia, Merck and Pfizer, as well as research funding from ToleroPharmaceuticals. W.L.H. receives research funding from Johnson & Johnson. J.A.R. reports fees as consultant, scientific advisor, ownership interest, inventor on intellectual property licensed by Genprex and PI on Genprex-sponsored research. H.K. receives funding to the MD Anderson Cancer Center from Johnson and Johnson. C.H. serves as an advisory board member for Briacell. R.R.J. receives consultant role fees from Merck, Karius and Microbiome DX, advisory member role fees from Seres and Kaleido, and patent licensing fees from Seres. P.S. reports consulting, advisory roles and/or stocks/ownership for Achelois, Apricity Health, BioAlta, Codiak BioSciences, Constellation, Dragonfly Therapeutics, Forty-Seven Inc., Hummingbird, ImaginAb, Jounce Therapeutics, Lava Therapeutics, Lytix Biopharma, Marker Therapeutics, Oncolytics, Infinity Pharma, BioNTech, Adaptive Biotechnologies and Polaris, and owns a patent licensed to Jounce Therapeutics. J.P.A. reports consulting, advisory roles and/or stocks/ownership for Achelois, Apricity Health, BioAtla, Codiak BioSciences, Dragonfly Therapeutics, Forty-Seven Inc., Hummingbird, ImaginAb, Jounce Therapeutics, Lava Therapeutics, Lytix Biopharma, Marker Therapeutics, Polaris, BioNTech and Adaptive Biotechnologies, and owns a patent licensed to Jounce Therapeutics. J.A.W. is an inventor on a US patent application (PCT/US17/53.717) submitted by the University of Texas MD Anderson Cancer Center which covers methods to enhance immune checkpoint blockade responses by modulating the microbiome, reports compensation for speaker’s bureau and honoraria from Imedex, Dava Oncology, Omniprex, Illumina, Gilead, PeerView, Physician Education Resource, MedImmune, Exelixis and Bristol Myers Squibb, serves as a consultant/advisory board member for Roche/Genentech, Novartis, AstraZeneca, GlaxoSmithKline, Bristol Myers Squibb, Merck, Biothera Pharmaceuticals and Microbiome DX, and receives research support from GlaxoSmithKline, Roche/Genentech, Bristol Myers Squibb and Novartis. I.I.W. reports honoraria from Genentech/Roche, Bayer, Bristol Myers Squibb, AstraZeneca/Medimmune, Pfizer, HTG Molecular, Asuragen, Merck, GlaxoSmithKline, Guardant Health, Platform Health, Daiichi, Merck, Flame, Oncocyte and MSD, and research support from Genentech, Oncoplex, HTG Molecular, DepArray, Merck, Bristol Myers Squibb, Medimmune, Adaptive, Adaptimmune, EMD Serono, Pfizer, Takeda, Amgen, Karus, Johnson & Johnson, Bayer, Iovance, 4D, Novartis and Akoya. S.G.S. reports speaker, travel and lodging expenses—Egyptian Society of Surgical Oncology/Best of SSO Cairo; West Hawaii Cancer Symposium; review panel participant, travel and lodging expenses—Peter MacCallum Cancer Centre; unpaid advisory board participant—Ethicon. D.L.G. reports honoraria for scientific advisory boards from AstraZeneca, Sanofi, Alethia Biotherapeutics and Janssen, and research support from Janssen, Takeda, Ribon Therapeutics and AstraZeneca. J.V.H. reports fees for advisory committees from AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Catalyst, EMD Serono, Foundation Medicine, Hengrui Therapeutics, Genentech, GSK, Guardant Health, Eli Lilly, Merck, Novartis, Pfizer, Roche, Sanofi, Seattle Genetics, Spectrum and Takeda, research support from AstraZeneca, GlaxoSmithKline, Spectrum, and royalties and licensing fees from Spectrum. B.S. reports consulting fees from Bristol Myers Squibb. The remaining authors report no competing interests.
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Extended data
Extended Data Fig. 1 Trial schema.
Patients with resectable, pathologically confirmed, clinical stage I-IIIA (N2 single station) NSCLC were stratified by stage and randomized in 1:1 ratio to neoadjuvant nivolumab 3 mg/kg IV every 14 days for up to three doses (arm A; D1, D15 and D29) or ipilimumab 1 mg/kg IV every 6 weeks plus nivolumab 3 mg/kg IV every 14 days for up to three doses (arm B; ipilimumab on D1 only, nivolumab on D1, D15 and D29), followed by surgical resection (at least 3 weeks and within 6 weeks after the last dose of nivolumab). Standard of care adjuvant chemotherapy and/or postoperative radiation therapy were allowed at the discretion of the treating physician. The primary endpoint of the trial was MPR, defined as ≤10% viable tumor in resected tumor specimens. Select secondary endpoints included toxicity, perioperative morbidity and mortality, objective response rates (ORR) by RECIST v.1.1, survival outcomes, radical resection (R0) rate, pathologic complete response (pCR) rate, defined as 0% viable tumor in resected tumor specimens, and quantification of TILs in resected tumor tissues. Select exploratory endpoints included analysis of biomarkers and their modulation by treatment. Imaging studies were performed with CT and PET-CT scans pretherapy (prior to first dose) and at least 14 days after the last dose of neoadjuvant therapy before surgical resection (posttherapy). Tumor samples were collected pretherapy and at surgery together with tumor-adjacent uninvolved lung tissue. Stool samples were collected pretherapy and posttherapy (prior to surgery). Longitudinal blood samples were collected pretherapy, prior to dose 2 and 3, posttherapy (prior to surgery) and within 8 weeks after surgery. NSCLC, non–small cell lung cancer; ECOG PS, Eastern Cooperative Oncology Group performance status; MPR, major pathologic response; ORR, objective response rate; RFS, recurrence-free survival; OS, overall survival; R0, complete surgical resection; pCR, pathologic complete response; TILs: tumor-infiltrating lymphocytes. D: day of therapy. CT: computed tomography, PET-CT: positron emission tomography-computer tomography scan.
Extended Data Fig. 2 Consolidated Standards of Reporting Trials (CONSORT) flow diagram.
Flow diagram depicts the disposition of patients throughout the phases of the study, including screening, randomization to neoadjuvant treatment and surgery. Reasons for screen failures, no completion of planned neoadjuvant therapy and surgery not performed, or surgery performed off trial are shown. SAE, serious adverse event, TRAE, treatment-related adverse event; PD, progressive disease; PS, performance status.
Extended Data Fig. 3 Tumor size change from baseline after neoadjuvant ICIs by treatment arm and by MPR status.
a, b, Boxplots depict the association between percent change in tumor measurement from pretherapy (baseline) in patients treated with neoadjuvant therapy by treatment arm in ITT (a) and in resected patients by MPR status (b). In one patient, the solid lesion was <1 cm following three doses of nivolumab monotherapy and did not change compared to baseline; response was considered SD. One patient developed TRAE (SAE) after one dose of nivolumab plus ipilimumab and RECIST response and percent change in tumor size from baseline were not evaluable. ITT patients: Nivo, n = 22; Nivo plus Ipi, n = 20. Resected patients: MPR, n = 13; No MPR, n = 23. Data are presented as median with minima, lower and upper quartiles, and maxima. The ends of the box are the upper and lower quartiles (75th and 25th percentiles), the median is the horizontal line inside the box. The whiskers are the two lines outside the box that extend to the maxima and minima. Two-sided P value is from Wilcoxon rank-sum test. c,d, Examples of radiographic (CT scan) and pathologic (H&E) images of NSCLC pre- and post-nivolumab (c) and pre- and post-nivolumab plus ipilimumab (d). CR, complete response; PR, partial response; MPR, major pathologic response; pCR, pathologic complete response; VT, viable tumor; CT, computed tomography; H&E, hematoxylin and eosin.
Extended Data Fig. 4 Impact of histology, stage, smoking status, responses and postoperative treatment on lung cancer-related RFS after neoadjuvant nivolumab and nivolumab plus ipilimumab.
a, Kaplan-Meier curves of probability of lung cancer-related RFS after neoadjuvant nivolumab and nivolumab plus ipilimumab by tumor histology. Among 26 patients with adenocarcinoma (26/44, 59%), four patients (4/26, 15%) progressed, and among 18 patients with SCC/ASC (18/44, 41%), three patients (3/18, 17%) progressed/died. b, Kaplan-Meier curves of probability of lung cancer-related RFS after neoadjuvant nivolumab and nivolumab plus ipilimumab by stage. Among 23 patients with stage I disease (23/44, 52%), one patient (1/23, 4%) progressed, among 12 patients with stage II disease (12/44, 27%), two patients (2/12, 17%) progressed/died, and among nine patients with stage IIIA disease (9/44, 20%), four patients (4/9, 44%) progressed. c, Kaplan-Meier curves of probability of lung cancer-related RFS after neoadjuvant nivolumab and nivolumab plus ipilimumab by smoking status. Among 36 former/current smokers (36/44, 82%), three patients (3/36, 8%) progressed, and among eight never smokers (8/44, 18%), four patients (4/8, 50%) progressed/died. d, Kaplan-Meier curves from landmark analysis performed to explore the effects of radiographic (RECIST) responses to neoadjuvant nivolumab and nivolumab plus ipilimumab on lung cancer-related RFS. Among nine patients with CR/PR (9/44, 20%), one patient (1/9, 11%) died following steroid-treated pneumonitis complicated with BPF and empyema and respiratory failure, and among 34 patients with SD/PD (34/44, 77%), six patients (6/34, 18%) experienced disease recurrence, and, among those, one later died from the disease. One patient was not evaluable due to development of grade 3 TRAE after one dose of nivolumab plus ipilimumab. e, Kaplan-Meier curves from landmark analysis performed to explore the effects of pathologic response (MPR vs. No MPR) to neoadjuvant nivolumab and nivolumab plus ipilimumab on lung cancer-related RFS. Among 13 resected patients with MPR (13/37, 35%), one patient (1/13, 8%) died 2.2 months after surgery, and among 24 resected patients with no MPR (24/37, 65%), three (3/24, 13%) patients progressed 15.0, 16.4, and 17.9 months after surgery. f, Kaplan-Meier curves from landmark analysis performed to explore the effects of PORT on lung cancer-related RFS after neoadjuvant nivolumab and nivolumab plus ipilimumab. Among four resected patients who received PORT (4/37, 11%), two patients (2/4, 50%) progressed, and among 33 resected patients who did not receive PORT (33/37, 89%), two patients (2/33, 6%) progressed/died. g, Kaplan-Meier curves from landmark analysis performed to explore the effects of adjuvant chemotherapy on lung cancer-related RFS after neoadjuvant nivolumab and nivolumab plus ipilimumab. Among 17 resected patients who received adjuvant chemotherapy (17/37, 46%), two patients (2/17, 12%) progressed, and among 20 resected patients who did not receive adjuvant chemotherapy (20/37, 54%), two patients (2/20, 10%) progressed/died. SCC, squamous cell carcinoma; ASC, adenosquamous carcinoma; Never, never smokers; Former/Current, Former smokers/Current smokers; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. MPR, major pathologic response; N/E, not evaluable. PORT, postoperative radiation therapy. Two-sided P value is from logrank test.
Extended Data Fig. 5 Association between tumor PD-L1 expression in malignant cells and response to neoadjuvant nivolumab and nivolumab plus ipilimumab.
a,b, Percent viable tumor in tumor specimens resected after nivolumab and nivolumab plus ipilimumab according to pretherapy (a) and posttherapy (b) tumor PD-L1 IHC expression (< 1% vs. ≥ 1%) in malignant cells. Pretherapy tumor PD-L1: < 1%, n = 16; ≥ 1%, n = 8. Posttherapy tumor PD-L1: < 1%, n = 13; ≥ 1%, n = 10. Data are presented as median with minima, lower and upper quartiles, and maxima. All violin plots show single data points, dashed line shows the median value, dotted lines show lower quartile and upper quartile values of the range; top and bottom of the violin plots indicate the minima and maxima. Experiments and scorings related to the presented results were conducted once. Two-sided P value is from Wilcoxon rank-sum test.
Extended Data Fig. 6 Multiplex immunofluorescence (mIF) VECTRA staining of immune infiltrates in pre- and posttherapy tumors.
a–f, Staining of cell populations identified with co-expression markers in mIF VECTRA panel 1 as (a) PD-L1+ malignant cells (%), (b) CD3+CD8+ T cells (n/mm2), (c) CD3+PD-1+ T cells (n/mm2), (d) CD3+CD8+PD-1+ T cells (n/mm2), (e) CD68+ cells (n/mm2), (f) CD68+PD-L1+ cells (%) in resected (surgery) vs. pretherapy tumors treated with nivolumab (n = 8) or nivolumab plus ipilimumab (n = 7). g–i, Staining of cell populations identified with co-expression markers in mIF VECTRA panel 2 as (g) CD3+ T cells (n/mm2), (h) CD3+CD8+GZB+ T cells (n/mm2), (i) CD3+CD8-FOXP3+ T cells (n/mm2) in resected (surgery) vs. pretherapy tumors treated with nivolumab (n = 8) or nivolumab plus ipilimumab (n = 7). Experiments and scorings related to the presented results were conducted once. Two-sided P values are from Wilcoxon signed-rank test.
Extended Data Fig. 7 Changes in T cell clones after neoadjuvant treatment and correlation with tumor pathologic regression.
a,b, Changes in TCR repertoire richness (a) and clonality (b) in matched blood samples from pre- to posttherapy (prior to surgery) after neoadjuvant nivolumab (n = 4, blue) or nivolumab plus ipilimumab (n = 3, red). c–f, Correlation between percent viable tumor at surgery and T cell clonality in tumor (c,d) or blood (e,f) pretherapy (c,e) and posttherapy (d,f) with neoadjuvant nivolumab (blue) and nivolumab plus ipilimumab (red). Two-sided P value is from Spearman rank-order correlation. g,h, Number of significantly (two-sided P < 0.01 with Benjamini-Hochberg adjustment for false-discovery rate) expanded (g) and contracted (h) T cell clones in matched resected (surgery tumor) vs. pretherapy tumors (n = 7), matched resected tumors (surgery tumor) vs. tumor-adjacent uninvolved lungs (surgery uninvolved) (n = 12), matched posttherapy (prior to surgery) vs. pretherapy blood samples (n = 7), matched resected tumors (surgery tumor) vs. pretherapy blood samples (n = 7) and tumor-adjacent uninvolved lungs (surgery uninvolved) vs. pretherapy blood samples (n = 7) after neoadjuvant nivolumab (blue) and nivolumab plus ipilimumab (red). Data are presented as median with minima, lower and upper quartiles, and maxima. All violin plots show single data points, dashed line shows the median value, dotted lines show lower quartile and upper quartile values of the range; top and bottom of the violin plots indicate the minima and maxima. Closed dots: MPR; Open dots: No MPR.
Extended Data Fig. 8 Association between fecal microbiome diversity and tumor pathologic responses and TRAEs.
a, Ordination plots from principal coordinate analysis (PCoA) demonstrating clustering patterns of fecal microbiomes of patients at pre- (n = 30) and posttherapy (n = 28) using Weighted UniFrac distance. Two-sided P value is from analyses of similarities (ANOSIM) test performed with 999 permutations to calculate whether taxonomic composition between these two categories were significantly different. b, Box-and-whisker plots of pairwise distances between pre- and posttherapy samples within response and toxicity groups of patients having microbiome data (n = 25, MPR=9, No MPR=16; treatment-related adverse events (TRAEs) >2 = 12, TRAEs ≤2 = 13). The box portion of the plot is drawn from the first quartile to the third quartile with inside line indicating the median value. The whiskers extend from the ends of the box to the minimum and maximum data values. Two-sided P value is from Mann-Whitney U rank-sum test. c, Box-and-whisker plots of the relative distributions of the top ten most abundant bacteria at family level observed in MPR (n = 3) and No MPR (n = 15) in nivolumab-treated patients (top panel). The box portion of the plot is drawn from the first quartile to the third quartile with inside line indicating the median value. The whiskers extend from the ends of the box to the minimum and maximum data values. d, Inverse Simpson index estimating fecal bacterial diversity between MPR (n = 3) and No MPR (n = 15) in nivolumab-treated patients (bottom panel). The box portion of the plot is generated from the first quartile to the third quartile with inside line indicating the median value. The whiskers extend from the ends of the box to the minimum and maximum data values. Two-sided P value is from Mann-Whitney U rank-sum test. e, Box-and-whisker plots of the relative distributions of the top ten most abundant bacteria at family level observed in MPR (n = 7) and No MPR (n = 8) in nivolumab plus ipilimumab-treated patients. The box portion of the plot is generated from the first quartile to the third quartile with inside line indicating the median value. The whiskers extend from the ends of the box to the minimum and maximum data values. f, Inverse Simpson index estimating fecal bacterial diversities between MPR (n = 7) and No MPR (n = 8) in nivolumab plus ipilimumab-treated patients. The box portion of the plot is generated from the first quartile to the third quartile with inside line indicating the median value. The whiskers extend from the ends of the box to the minimum and maximum data values. Two-sided P value is from Mann-Whitney U rank-sum test. g, Linear Discriminant Analysis (LDA) Effective Size (LEfSe) used to estimate discriminative features in fecal microbiomes at genus level between MPR (n = 3) and No MPR (n = 15) in nivolumab (top) and nivolumab plus ipilimumab-treated patients (bottom; MPR, n = 7 and No MPR, n = 8) pretherapy. The length of the bar indicates the effect size associated with a genus. Alpha value of 0.05 for the factorial Kruskal-Wallis test and logarithmic LDA score of 2 were used to calculate the discriminative features. h, LDA Effect Size (LEfSe) plot of pairwise comparisons of bacterial taxa at genus level dichotomized by TRAE categories in nivolumab-treated patients (top panel) (TRAEs ≤2 (n = 10) and TRAEs >2 (n = 10)) and in nivolumab plus ipilimumab-treated patients (bottom panel) (TRAEs ≤2 (n = 9) and TRAEs >2 (n = 10)). Alpha value of 0.05 for the factorial Kruskal-Wallis test and logarithmic LDA score of 2 were used to calculate the discriminative features.
Extended Data Fig. 9 Association between fecal microbiome diversity and tumor TCR clonality and richness.
a,c, Heatmaps showing pretherapy taxonomic abundances at various levels in nivolumab (n = 10) and nivolumab plus ipilimumab (n = 9) arms and posttherapy tumor TCR clonality (a) and richness (c). b,d, The relationships between the microbiome and TCR clonality (b) and richness (d) were conducted using the linear regression model. Spearman correlation test (two-sided) was used to calculate the rho and P values. Here, the unadjusted P value cutoff of 0.05 was used.
Extended Data Fig. 10 Flow cytometry gating strategy for CD103 T cell and T cell memory panels.
The gating strategy is shown including initial QC gates (SSC singlets, FSC singlets, live cells) followed by immune cell subsets included in the panel. The frequencies referenced for each subgated cell population shown are from the parental gate. a, Subgating is performed on CD4+ Tregs, CD4+ non-Tregs and CD8+ T cell subsets as shown. Subgating of checkpoint receptors was also assessed on the tissue-resident memory (TRM) (CD103+) and non-TRM (CD103-) T cell subsets. Arrows indicate the transition through the individual gates. b, Fluorescence minus one (FMO) gating for CTLA-4, FoxP3, CD25, TIM3, PD-1 and CD103 controls are shown. c, Subgating for T cell memory panel is performed on CD4+ and CD8+ T cell subsets as shown. Arrows indicate the transition through the individual gates. d, Fluorescence minus one (FMO) gating for CD45RA, CCR7, CD28 and CD27 controls are shown. Experiments and gating related to presented results were conducted once. Subgating was only performed when more than 100 events were present in parental gate.
Supplementary information
Supplementary Table 1
Radiographic responses by RECIST in 44 patients treated on trial. ┼In one patient the solid lesion was <1 cm and did not change in size after three doses of nivolumab compared with baseline; radiographic response was considered to be the SD. #In one patient RECIST response was not evaluable on the study due to TRAE after one dose of combined therapy.
Supplementary Table 2
Radiographic responses by RECIST in 37 patients resected on trial. The two-sided P value is from Fisher’s exact test.
Supplementary Table 3
Association between RECIST and pathologic responses in patients resected on trial. Proportion of resected patients with CR/PR by RECIST who achieved MPR compared with that of patients with SD/PD who achieved MPR. One patient was not evaluable for RECIST due to TRAE after one dose of nivolumab + ipilimumab. The two-sided P value is from Fisher’s exact test.
Supplementary Table 4
TRAEs by grade in patients treated with neoadjuvant nivolumab and nivolumab + ipilimumab.
Supplementary Table 5
SAEs in patients treated with neoadjuvant nivolumab and nivolumab + ipilimumab.
Supplementary Table 6
Pathologic responses to neoadjuvant nivolumab by tumor histology, staging and smoking status. The two-sided P value is from Fisher’s exact test.
Supplementary Table 7
Radiographic responses to neoadjuvant nivolumab by tumor histology, staging and smoking status. The two-sided P value is from Fisher’s exact test.
Supplementary Table 8
Pathologic responses to neoadjuvant nivolumab + ipilimumab by tumor histology, staging and smoking status. The two-sided P value is from Fisher’s exact test.
Supplementary Table 9
Radiographic responses to neoadjuvant nivolumab + ipilimumab by tumor histology, staging and smoking status. The two-sided P value is from Fisher’s exact test.
Supplementary Table 10
Immune cell densities in tumors treated with neoadjuvant nivolumab and nivolumab + ipilimumab. Differences in densities of tumor-infiltrating immune cells between post- and pretherapy values at mIF staining. For malignant cells and immune cells expressing (membranous expression), the values are shown as percentages. The two-sided P value is from Wilcoxon’s rank-sum test.
Source data
Source Data Fig. 1
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Fig. 2
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Fig. 3
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Fig. 4
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Fig. 5
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Extended Data Fig. 3
Source data with relevant annotations used to generate a and b and to perform statistical analyses.
Source Data Extended Data Fig. 4
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Extended Data Fig. 5
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Extended Data Fig. 6
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Extended Data Fig. 7
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Extended Data Fig. 8
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
Source Data Extended Data Fig. 9
Source data with relevant annotations used to generate each panel and to perform statistical analyses.
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Cascone, T., William, W.N., Weissferdt, A. et al. Neoadjuvant nivolumab or nivolumab plus ipilimumab in operable non-small cell lung cancer: the phase 2 randomized NEOSTAR trial. Nat Med 27, 504–514 (2021). https://doi.org/10.1038/s41591-020-01224-2
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DOI: https://doi.org/10.1038/s41591-020-01224-2
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