Neoadjuvant nivolumab or nivolumab plus ipilimumab in operable non-small cell lung cancer: the phase 2 randomized NEOSTAR trial

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.

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 (75^th and 25^th 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 (T_RM) (CD103⁺) and non-T_RM (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.

Fig. 1.. Pathologic and radiographic responses to neoadjuvant nivolumab and nivolumab + ipilimumab in ITT and resected patients on trial.

a, Pathologic responses in ITT population (resected and not resected patients, nivolumab, n = 23, nivolumab + ipilimumab, n = 21). Primary endpoint: MPR (≤ 10% viable tumor cells) consists of pCR, that is, 0% viable tumor, + 1-10% viable tumor. The MPR and pCR rates are estimated with exact 95% CIs from the binomial distribution. The two-sided P value is from Chi-square test for MPR and Fisher’s exact test for pCR. ^╬Seven patients did not undergo surgery after neoadjuvant therapy on trial. Of these, five patients (one nivolumab, four nivolumab + ipilimumab) had no surgery; two patients had surgery off trial after additional systemic therapies (one nivolumab; one nivolumab + ipilimumab). ^a0% viable tumor (pCR); ^b1–10% viable tumor. b,c, Waterfall plots of radiographic percentage change in overall tumor size from baseline at least 14 d after the last dose of neoadjuvant therapy in nivolumab (b) and nivolumab + ipilimumab (c) groups (nivolumab, n = 23, 22% ORR; nivolumab + ipilimumab, n = 21, 19% ORR; ^#one patient was not evaluable on the study). Dashed black line at 20% point depicts cutoff for PD. Dashed black line at −30% point depicts cutoff for PR. *Indicates overall response of PD due to presence of enlarging and/or new lesions; ^┼Indicates that the solid lesion was <1 cm, considered to be SD. The white arrows indicate the patients who did not undergo surgery; the black arrow indicates one patient who underwent surgery off trial after chemoimmunotherapy. ^#One patient was not radiographically evaluable on the study due to development of grade 3 diarrhea/colitis after one dose of nivolumab + ipilimumab (restaging scans were performed during and after neoadjuvant platinum doublet chemotherapy followed by surgery, both administered off trial). d, Proportion of MPR/no MPR (≤10% viable tumor/>10% viable tumor) and pCR/no pCR (0% viable tumor/>0% viable tumor) in resected patients on trial after neoadjuvant nivolumab (n = 21, bright blue/light blue) and nivolumab + ipilimumab (n = 16, bright red/light red). The two-sided P value is from Chi-square test for MPR and Fisher’s exact test for pCR. e, Percentage viable tumor in resected tumor specimens after nivolumab (n = 21, blue) and nivolumab + ipilimumab (red, n = 16). Median percentage viable tumor: nivolumab 50% (range 0-97.5%, n = 21), nivolumab + ipilimumab 9% (range 0-74.5%, n = 16). Data are presented as the median with minima, lower and upper quartiles, and maxima. The dashed line indicates the median; the dotted lines indicate the lower quartile and upper quartile values; the top and bottom of the violin plots indicate the minima and maxima. The two-sided P value is from Wilcoxon’s rank-sum test. f,g, Waterfall plots of pathologic tumor regression (% viable tumor – 100%) in resected patients after neoadjuvant nivolumab (f; n = 21) and nivolumab + ipilimumab (g; n = 16). Dashed black line represents MPR.

Fig. 2.. Survival outcomes in patients treated with neoadjuvant nivolumab and nivolumab + ipilimumab.

a, Kaplan-Meier curve of probability of OS in patients treated with neoadjuvant nivolumab (n = 23) from randomization to death. Median OS was not reached. One patient treated with neoadjuvant nivolumab and eventually diagnosed with pneumonitis requiring steroids, died within 90 d of surgery and 4.1 months after randomization due to BPF and empyema resulting in respiratory failure. b, Kaplan-Meier curve of probability of OS in patients treated with neoadjuvant nivolumab + ipilimumab (n = 21) from randomization to death. Median OS was not reached. One patient treated with combination therapy had PD 2.6 months after randomization (post-neoadjuvant therapy) and did not undergo surgery. This patient died from lung cancer 17.1 months after randomization following additional therapies. c, Kaplan-Meier curve of probability of lung cancer-related RFS in patients treated with neoadjuvant nivolumab (n = 23) from randomization to recurrence or death. Median lung cancer-related RFS was not reached. One patient died as reported above. Two patients experienced lung cancer-related recurrence due to local and/or distant metastatic disease after 20.0 months (n = 1) and 12.6 months (no surgery on trial, n = 1) after randomization. d, Kaplan-Meier curve of probability of lung cancer-related RFS in patients treated with neoadjuvant nivolumab + ipilimumab (n = 21) from randomization to recurrence or death. Median lung cancer-related RFS was not reached. Four patients experienced disease recurrence at 2.6 months (no surgery, n = 1, died 17.1 months after randomization), 18.5 months (n = 1), 20.1 months (no surgery, n = 1), and 17.1 months after randomization (n = 1).

Fig. 3.. Association of tumor PD-L1 expression in malignant cells and response to neoadjuvant nivolumab and nivolumab + ipilimumab.

a,b, Pretherapy tumor PD-L1 IHC membranous expression (%) in malignant cells from responders and nonresponders treated with nivolumab and nivolumab + ipilimumab by RECIST (a; CR/PR versus SD/PD; n = 4 versus n = 23) and MPR status (b; n = 8 versus n = 19). Data are presented as the median with minima, lower and upper quartiles, and maxima. Individual data points are shown, dashed line shows the median value, and dotted lines show lower quartile and upper quartile values; the top and bottom of the violin plots indicate the minima and maxima. The two-sided P value is from Wilcoxon’s rank-sum test. c,d, Post-therapy (resected) tumor PD-L1 IHC expression in malignant cells from responders and nonresponders treated with nivolumab and nivolumab + ipilimumab by RECIST (c; n = 3 versus n = 20) and MPR status (d; n = 4 versus n = 19). Data are presented as the median with minima, lower and upper quartiles, and maxima. Individual data points are shown; the dashed line shows the median value, and dotted lines show the lower quartile and upper quartile values of the range; the top and bottom of the violin plots indicate the minima and maxima. The two-sided P value is from Wilcoxon’s rank-sum test. e,f, Examples of hematoxylin and eosin (H&E) micrographs (top panels) of pathologic response (MPR or pCR) in patients treated with nivolumab (e) or nivolumab + ipilimumab (f) with elevated pretherapy tumor PD-L1 expression in malignant cells (bottom panels). Experiments and scorings related to the presented micrographs were conducted once. g,h, Examples of H&E micrographs (top panels) of lack of pathologic response (no MPR) in patients treated with nivolumab (g) or nivolumab + ipilimumab (h) with lack of pretherapy tumor PD-L1 expression in malignant cells (bottom panels). Experiments and scorings related to the presented micrographs were conducted once. N/E, not evaluable PD-L1 due to lack of viable tumor cells in the analyzed tissue section.

Fig. 4.. Immune correlates of response to neoadjuvant nivolumab and nivolumab + ipilimumab.

a-d, Frequencies (top panels) and representative gates (bottom panels) of CD3⁺ T cells (of CD45⁺) (a), CD103⁺ T_RM cells (of CD3⁺ (from CD45⁺) CD4⁺) (b), CD103⁺ effector T_RM cells (of CD3⁺ (from CD45⁺) CD8⁺) (c), and CD27⁻CD28⁺ effector memory T cells (of CD3⁺ (from CD45⁺) CD4⁺) (d) by flow cytometry in tumors resected after neoadjuvant nivolumab (blue; n = 13 (a); n = 12 (b); n = 13 (c); n = 12 (d)) and nivolumab + ipilimumab (red; n = 10 (a); n = 10 (b); n = 10 (c); and n = 9 (d)). Data are presented as the median with minima, lower and upper quartiles, and maxima. All violin plots show single data points; the dashed line shows the median value, and dotted lines show the lower quartile and upper quartile values of the range; the top and bottom of the violin plots indicate the minima and maxima. The two-sided P value is from Wilcoxon’s rank-sum test. Experiments and gating related to presented results were conducted once. Subgating was only performed when more than 100 events were present in parental gate. e, Examples of micrographs of mIF staining of immune markers (panels 1 and 2, respectively) in pretherapy and resected tumors after neoadjuvant nivolumab and nivolumab + ipilimumab. Experiments and scorings related to the presented micrographs were conducted once. f-h, Quantification of CD3⁺ T-cell (panel 1) (f), CD3⁺CD8⁺ T-cell (panel 2) (g), and CD3⁺CD8⁺CD45RO⁺ T-cell (h) densities (no. per mm²) by mIF staining in paired pretherapy and resected tumors after nivolumab (in blue; n = 8) and nivolumab + ipilimumab (in red; n = 7). The two-sided P value is from Wilcoxon’s signed-rank test.

Fig. 5.. TCR changes in blood and tumors treated with neoadjuvant nivolumab and nivolumab + ipilimumab.

a, Correlation of TCR repertoire richness between the pretherapy (baseline) peripheral blood and resected (surgery) tumors after neoadjuvant nivolumab (n = 4, blue) and nivolumab + ipilimumab (n = 3, red). The two-sided P value is from Spearman’s rank-order correlation. b,c, TCR repertoire richness (b) and clonality (c) between resected tumor-adjacent, uninvolved lungs (n = 12) and resected tumors (n = 20) after neoadjuvant nivolumab (blue) and nivolumab + ipilimumab (red). Data are presented as the median with minima, lower and upper quartiles, and maxima. All violin plots show single data points; the dashed line shows the median value and dotted lines show lower quartile and upper quartile values of the range; the top and bottom of the violin plots indicate the minima and maxima. The two-sided P value is from a two-sample Student’s t-test. d,e, Proportion of patients with increased and decreased TCR repertoire richness (d) and clonality (e) in resected tumors compared with their matched resected, tumor-adjacent, uninvolved lungs (n = 12, purple), after nivolumab (N, n = 5, blue) or nivolumab + ipilimumab (NI, n = 7, red). f,g, Changes in TCR repertoire richness (f) and clonality (g) between matched resected tumors and tumor-adjacent, uninvolved lungs after neoadjuvant nivolumab (n = 5, blue) or nivolumab + ipilimumab (n = 7, red). h,i, Changes in TCR repertoire richness (h) and clonality (i) in matched pretherapy and resected (surgery) tumors after neoadjuvant nivolumab (n = 4, blue) or nivolumab + ipilimumab (n = 3, red). Closed dots: MPR; Open dots: No MPR.