Preoperative ipilimumab plus nivolumab in locoregionally advanced urothelial cancer: the NABUCCO trial

Abstract

Preoperative immunotherapy with anti-PD1 plus anti-CTLA4 antibodies has shown remarkable pathological responses in melanoma1 and colorectal cancer2. In NABUCCO (ClinicalTrials.gov: NCT03387761), a single-arm feasibility trial, 24 patients with stage III urothelial cancer (UC) received two doses of ipilimumab and two doses of nivolumab, followed by resection. The primary endpoint was feasibility to resect within 12 weeks from treatment start. All patients were evaluable for the study endpoints and underwent resection, 23 (96%) within 12 weeks. Grade 3–4 immune-related adverse events occurred in 55% of patients and in 41% of patients when excluding clinically insignificant laboratory abnormalities. Eleven patients (46%) had a pathological complete response (pCR), meeting the secondary efficacy endpoint. Fourteen patients (58%) had no remaining invasive disease (pCR or pTisN0/pTaN0). In contrast to studies with anti-PD1/PD-L1 monotherapy, complete response to ipilimumab plus nivolumab was independent of baseline CD8+ presence or T-effector signatures. Induction of tertiary lymphoid structures upon treatment was observed in responding patients. Our data indicate that combined CTLA-4 plus PD-1 blockade might provide an effective preoperative treatment strategy in locoregionally advanced UC, irrespective of pre-existing CD8+ T cell activity.

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Fig. 1: NABUCCO study design and baseline study population characteristics.
Fig. 2: Pathological tumor regression and outcome to preoperative ipilimumab plus nivolumab.
Fig. 3: pCR to ipilimumab plus nivolumab is independent of baseline CD8+ T cells and inflammatory signatures.
Fig. 4: B cell analysis and assessment of TLS dynamics upon preoperative ipilimumab plus nivolumab.

Data availability

DNA and RNA sequencing data have been deposited in the European Genome-phenome Archive under the accession code EGAS00001004521 and will be made available upon reasonable request for academic use and within the limitations of the provided informed consent by the corresponding author upon acceptance. Every request will be reviewed by the institutional review board of the Netherlands Cancer Institute; the researcher will need to sign a data access agreement with the Netherlands Cancer Institute after approval. Sequencing data correspond with Figs. 3 and 4. Multiplex immunofluorecence raw quantification data corresponding to Figs. 3d and 4 will be made available upon reasonable academic request within the limitations of informed consent by the corresponding author upon acceptance.

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Acknowledgements

We acknowledge the clinical trial data managers (C. Hagenaars and J. Kant), clinical trial nurses (A. Lechner, E. van der Laan and S. van der Kolk), the Genomics Core Facility and the Core Facility Molecular Pathology and Biobanking (I. Hofland, S. Cornelissen, L. Braaf and J. Sanders) for support, all at the Netherlands Cancer Institute. We thank B. Stegenga at Bristol-Myers Squibb. M.v.d.B. was financially supported by the Swiss National Science Foundation (CRSII5_177208 and 310030_175565), the University of Zurich Research Priority Program ‘Translational Cancer Research’, the Cancer Research Center Zurich and Worldwide Cancer Research (18−0629).

Author information

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Authors

Contributions

The trial protocol was designed and written by the authors (N.v.D., K. Sikorska, C.U.B., B.W.v.R. and M.S.v.d.H.). N.v.D. coordinated the trial initiation and study procedures. K. Sikorska analyzed the clinical data. N.v.D., C.U.B., B.W.v.R. and M.S.v.d.H. interpreted clinical data. N.v.D., A.G.-J., K. Silina, M.v.d.B. and M.S.v.d.H. analyzed translational data. A.G.-J., D.J.V., Y.L., L.F.A.W. and K. Silina performed and interpreted bioinformatics analyses. D.P. and C.v.R. performed immunohistochemical staining, multiplex immunofluoresence staining and HALO image analysis. Work related to tertiary lymphoid structures was conducted and supervised by K. Silina and M.v.d.B. RNA and DNA sequencing preparations and multiplex/immunohistochemical assessments were supervised by E.H. and A. Broeks. L.A.S. and M.L.v.M. performed histopathological assessment and assessed tissue availability and immunopathological scoring. K. Sikorska performed statistical analyses. N.v.D., J.M.d.F. and M.S.v.d.H. informed patients and were responsible for patient care. B.W.v.R., K.H. and H.G.v.d.P. performed surgery. T.N.B. and A. Bruining assessed CT and MRI scans and analyzed tumor volumes. P.K. and T.N.S. provided scientific input during protocol writing and design of the study. The manuscript was written by N.v.D., A.G.-J. and M.S.v.d.H., together with all co-authors, who vouch for the accuracy of the data reported and adherence to the protocol. All authors edited and approved the manuscript.

Corresponding author

Correspondence to Michiel S. van der Heijden.

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Competing interests

T.N.S. is a consultant for Adaptive Biotechnologies, AIMM Therapeutics, Allogene Therapeutics, Amgen, Merus, Neon Therapeutics and Scenic Biotech. T.N.S. has received grant and research support from Merck, Bristol-Myers Squibb and Merck KGaA. T.N.S. is a stockholder in AIMM Therapeutics, Allogene Therapeutics and Neon Therapeutics. L.F.A.W. reports research funding from Genmab. C.U.B. reports personal fees for advisory roles for Merck Sharp & Dohme, Bristol-Myers Squibb, Roche, GlaxoSmithKline, Novartis, Pfizer, Genmab and Eli Lilly and grants from Bristol-Myers Squibb, NanoString and Novartis. M.S.v.d.H. has received research support from Bristol-Myers Squibb, AstraZeneca and Roche and consultancy fees from Bristol-Myers Squibb, Merck, Merck Sharp & Dohme, Roche, AstraZeneca, Seattle Genetics and Janssen (all paid to the Netherlands Cancer Institute). No other authors have disclosures relevant to this work.

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Extended data

Extended Data Fig. 1 Assessment of response discrepancies between the responding primary bladder tumor and unresponsive local lymph node micrometastases upon ipilimumab plus nivolumab.

a1-2. Imaging shows a cT4aN1 bladder tumor (enlarged lymph node not shown). Pathological assessment revealed a complete response in the bladder, as shown by multiplex immunofluorescent analysis (a-3) and a non-responding lymph node micrometastasis, annotated by a red arrow (a-4). Experiments and scorings related to the presented micrographs were conducted once. b, Whole-exome sequencing was employed to assess whether lymph node micrometastases are genetically distinct from the primary tumor and could explain unresponsiveness (n=2 patients). The figure shows overlap between somatic variants identified in the baseline tumor (purple) and the post-treatment lymph node metastasis (green). c, Genomic alterations in genes related to interferon gamma signaling, JAK/STAT signaling or antigen presentation machinery for baseline tumor samples (purple, n=2) and matching lymph nodes metastases (green, n=2). Sample labels and genetic alteration type are displayed in figure legends. No specific genetic cause for resistance could be identified in the discrepant mutations.

Extended Data Fig. 2 Analysis of baseline genomic alterations by whole-exome sequencing.

Whole-exome sequencing of pretreatment tumor tissue and germline DNA was employed to identify somatic mutations in baseline tumors (n=24). An oncoprint figure of genetic alterations in significantly mutated bladder cancer genes (by TCGA, Robertson et al, Cell 2018) is shown. Alterations are clustered by response categories, including 14 CR and 10 non-CR tumors. Sample labels and genetic alteration type are displayed in the figure. Abbreviations: CR: complete response, non-CR: non complete response, CNA: copy number alterations.

Extended Data Fig. 3 Assessment of interdependence between baseline B cell presence and CD8 T cell infiltration.

a, Average expression of B-cell-related differentially expressed genes at baseline stratified by intratumoral and stromal CD8+ higher and lower than median groups on multiplex immunofluorescence (mIF). Gene expression levels are represented as transcripts per million (TPM), mean-centered and scaled (Z-scores). b, Intratumoral CD8 density per mm2 by multiplex immunofluorescence, stratified by stromal CD20 higher and lower than median groups on multiplex immunofluorescence (mIF). c, Average expression of B-cell-associated genes at baseline stratified by average TGE8 signature groups (higher or lower than median) showing similar expression of B-cell-related genes in the TGE8 signature groups. Gene expression levels are represented as transcripts per million (TPM), mean-centered and scaled (Z-scores). A Wilcoxon signed tank test was used for all comparisons. The P-value is presented in-between boxplots. All statistical tests were two-sided. Analyses include CR (blue; n=11) and non-CR (orange; n=7) tumors. All boxplots display the median and 25th and 75th percentiles. The whiskers expand from the hinge to largest value not exceeding 1.5× IQR from the hinge. No adjustments were made for multiple comparisons. Abbreviations: CR: complete response, non-CR: non complete response.

Extended Data Fig. 4 Dynamics of tertiary lymphoid structure spectrum upon immunotherapy for response and steroid groups.

Upon multiplex immunofluorescent staining and segregation of tertiary lymphoid structure (TLS) areas, a, Early-TLS, b, Primary follicle-like TLS and c, Secondary follicle-like TLS were quantified as normalized TLS area (square microns per tissue square centimeter). For each TLS maturation stage, four different analysis were performed; 1) Comparison of normalized TLS areas in baseline and post-therapy samples between response groups by Mann Whitney test, 2) Normalized TLS area assessed as fold change (post/pre) upon treatment between response groups, 3) Normalized TLS area comparison in post-therapy samples between patients receiving steroids and no steroids, 4) Normalized TLS area assessed as fold change (post/pre) upon treatment in patients that received steroids and no steroids. Unless otherwise noted, all boxplots display the median and 25th and 75th percentiles. The whiskers expand from the hinge to largest value not exceeding 1.5× IQR from the hinge. A Mann Whitney test was used for comparisons between response and steroid groups. The P-value is presented in-between boxplots. All statistical tests were two-sided. Analyses have 14 CR and 10 non-CR, or 9 steroids (>20mg a day prior to surgery) and 15 no steroid patients. No adjustments were made for multiple comparisons. Abbreviations: TLS: tertiary lymphoid structures, CR: complete response, non-CR: no complete response.

Extended Data Fig. 5 Exploratory assessment of TLS markers and a TLS signature upon immunotherapy for response groups.

a, Average gene expression for TLS-related genes (CCL19, CCL21, CXCL13, CCR7, SELL, LAMP3, CXCR4, CD86, BCL6), as published by Cabrita et al. 19. Pre- and post-treatment samples were compared for CR (n=11 pre-treatment, n=8 post-treatment) and non-CR (n=7 pre-treatment, n=10 post-treatment) tumors using a two-sided t-test. Baseline expression was not significantly different between CR and non-CR (p=0.28). b, TLS gene signature (Cabrita et al., Nature 2020) derived from genes specifically upregulated in CD8+CD20+ metastasized melanoma tumors (CD79B, CD1D, SKAP1, CETP, EIF1AY, RBP5, PTGDS). LAT and CCR6 genes were lowly expressed and thus removed from the analysis. Gene signatures were compared between baseline and post- treatment samples for CR and non-CR tumors. Baseline expression was not significantly different between CR and non-CR (p=0.05). Boxplots in all panels represent the median and 25th and 75th percentiles. The whiskers expand from the hinge to largest value not exceeding 1.5× IQR from the hinge. CR samples are marked in blue, while non-CR samples are displayed in orange. A t-test test was used for comparisons between CR and non-CR. The p-value is presented in-between boxplots. All statistical tests were two-sided. All analyses involved 11 CR and 7 non-CR tumors in pre-treatment samples, and 10 CR and 8 non-CR tumors in post-treatment samples. Abbreviations: TLS: tertiary lymphoid structures, CR: complete response, Non-CR: non complete response.

Extended Data Fig. 6 Assessment of CD27+ B-cells in TLS upon immunotherapy for response groups.

a, Example images of CD20 (yellow) and CD27 (purple) IHC co-staining, revealing CD27+ B-cells (CD20+CD27+) in red, as indicated by the black arrow. Experiments and scorings related to the presented micrographs were conducted once. b/c. Baseline and post-treatment comparison of the mean percentage of CD20+CD27+ cells in germinal center (GC) negative (b) and GC+ (c) TLS per patient between CR (n=9 GC- pre-treatment, n=6 GC+ pre-treatment, n=11 GC- post-treatment, n=8 GC+ post-treatment) and non-CR (n=6 GC- pre-treatment, n=3 GC+ pre-treatment, n=8 GC- post-treatment, n=5 GC+ post-treatment) tumors. The percentage CD20+CD27+ cells in the CD20+ population in TLS was estimated by a pathologist (L.S.). Boxplots represent the median and 25th and 75th percentiles. The whiskers expand from the hinge to largest value not exceeding 1.5× IQR from the hinge. Complete responders are marked in blue, while non-responders are displayed in orange. A Wilcoxon signed tank test was used to compare the percentage of CD20+CD27+ between CR and non-CR tumors. The P-value is presented in-between boxplots. All statistical tests were two-sided. No adjustments were made for multiple comparisons. Abbreviations: CR: complete response, NR: no response, GC: germinal center.

Extended Data Fig. 7 Assessment of CD4+ T-cells and CD4+BCL6+ follicular T helper cells in tumor and TLS regions upon immunotherapy.

a, Example images of CD4 (yellow) and BCL6 (purple) IHC co-stainings, showing CD4+BCL6+ follicular T helper cells pre- and post-treatment, characterized by a purple nucleus and deep orange cytoplasmatic staining. b, Mean absolute CD4+BCL6+ cell counts in co-stains for mature and immature TLS and CD4BCL6+ cell counts for mature TLS only; for CR tumors (n=7 GC- pre-treatment, n=5 GC+ pre-treatment, n=10 GC- post-treatment, n=8 GC+ post-treatment) and non-CR tumors (n=4 GC- pre-treatment, n=3 GC+ pre-treatment, n=8 GC- post-treatment, n=5 GC+ post-treatment). Co-stainings were assessed and scored (number of cells per TLS) by a pathologist. c, Representative example images of CXCL13 (brown) in TLS for CR and non-CR tumors by immunohistochemistry in pre- and post-treatment specimens. CXCL13 positivity is clearly present in TLS, emphasizing that TLS are characterized by CXCL13 expression. No post-treatment differences were observed between response groups (Quantified data not shown). Unless otherwise noted, all boxplots display the median and 25th and 75th percentiles. The whiskers expand from the hinge to largest value not exceeding 1.5× IQR from the hinge. Complete responders are marked in blue, while non-responders are displayed in orange. A Wilcoxon signed tank test was used to compare the CD4+BCL6+ counts between CR and non-CR tumors. The P-value is presented in-between boxplots. All statistical tests were two-sided. No adjustments were made for multiple comparisons. Experiments and scorings related to the presented micrographs in A and C were conducted once. Abbreviations: CR: complete response, non-CR: non complete response, GC: germinal center.

Extended Data Fig. 8 Exploratory assessment of cellular distribution within TLS regions using a bioinformatic algorithm to analyse multiplex immunofluorescence slides.

a, Data map displaying immune cell subsets by digital multiplex immunofluorescent analysis in baseline tissue in a spatial context. A bioinformatic algorithm (methods) was developed to identify and segment TLS-like structures, as annotated in red in the example map. Tumor is depicted in gray (panCK) b, Distribution of CD3+CD8 and CD20+ cells over the analysed area, between algorithm assigned tumor bed (orange) and TLS (grey) pre- and post-therapy, in CR (n=7 pre, n=5 post) and non-CR (n=7 pre, n=4 post) patients. c, The ratio of FOXP3+/FOXP3- cells was calculated within the TLS CD3+CD8- compartment upon algorithmic TLS segmentation and quantitation of immune cell subsets in multiplex immunofluorescence images (Methods). Pre-treatment and post-therapy samples were compared in the complete cohort (C; n=14 pre, 9 post) p=0.0086) or between CR (n=7 pre, 5 post; p=0.073) and non-CR (n=7 pre, 4 post; p=0.11) d, by Mann Whitney test. Unless otherwise noted, all boxplots display the median and 25th and 75th percentiles. The whiskers expand from the hinge to largest value not exceeding 1.5× IQR from the hinge. Complete responders are marked in blue, while non-responders are displayed in orange. A Wilcoxon signed tank test was used to compare the CD4+BCL6+ counts between CR and non-CR tumors. The P-value is presented in-between boxplots. All statistical tests were two-sided. No adjustments were made for multiple comparisons. Abbreviations: CR: complete response, non-CR: non complete response, GC: germinal center.

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Supplementary Table 1, CONSORT diagram, CONSORT checklist and NABUCCO study protocol

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van Dijk, N., Gil-Jimenez, A., Silina, K. et al. Preoperative ipilimumab plus nivolumab in locoregionally advanced urothelial cancer: the NABUCCO trial. Nat Med (2020). https://doi.org/10.1038/s41591-020-1085-z

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