PD-L1 targeting and subclonal immune escape mediated by PD-L1 mutations in metastatic colorectal cancer

Abstract

Background: In patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC), immune checkpoint blockade is ineffective, and combinatorial approaches enhancing immunogenicity need exploration.

Methods: We treated 43 patients with predominantly microsatellite stable RAS/BRAF wild-type mCRC on a phase II trial combining chemotherapy with the epidermal growth factor receptor antibody cetuximab and the programmed cell death ligand 1 (PD-L1) antibody avelumab. We performed next-generation gene panel sequencing for mutational typing of tumors and liquid biopsy monitoring as well as digital droplet PCR to confirm individual mutations. Translational analyses included tissue immunohistochemistry, multispectral imaging and repertoire sequencing of tumor-infiltrating T cells. Detected PD-L1 mutations were mechanistically validated in CRISPR/Cas9-generated cell models using qRT-PCR, immunoblotting, flow cytometry, complement-dependent cytotoxicity assay, antibody-dependent cytotoxicity by natural killer cell degranulation assay and LDH release assay as well as live cell imaging of T cell mediated tumor cell killing.

Results: Circulating tumor DNA showed rapid clearance in the majority of patients mirroring a high rate of early tumor shrinkage. In 3 of 13 patients expressing the high-affinity Fcγ receptor 3a (FcγR3a), tumor subclones with PD-L1 mutations were selected that led to loss of tumor PD-L1 by nonsense-mediated RNA decay in PD-L1 K162fs and protein degradation in PD-L1 L88S. As a consequence, avelumab binding and antibody-dependent cytotoxicity were impaired, while T cell killing of these variant clones was increased. Interestingly, PD-L1 mutant subclones showed slow selection dynamics reversing on avelumab withdrawal and patients with such subclones had above-average treatment benefit. This suggested that the PD-L1 mutations mediated resistance to direct antitumor effects of avelumab, while at the same time loss of PD-L1 reduced biological fitness by enhanced T cell killing limiting subclonal expansion.

Conclusion: The addition of avelumab to standard treatment appeared feasible and safe. PD-L1 mutations mediate subclonal immune escape to avelumab in some patients with mCRC expressing high-affinity FcγR3a, which may be a subset experiencing most selective pressure. Future trials evaluating the addition of avelumab to standard treatment in MSS mCRC are warranted especially in this patient subpopulation.

Trial registration number: NCT03174405.

Keywords: biomarkers; clinical trials; gastrointestinal neoplasms; immunotherapy; phase II as topic; translational medical research; tumor.

Conflict of interest statement

Competing interests: AS received institutional research grants from Merck, BMS, Roche, Sanofi, Servier and honoraria for lectures and advisory board meetings by Merck, Roche, Amgen, Lilly, Sanofi-Aventis, Servier, Bayer, BMS, MSD and Sirtex. S-E A-B has an advisory role with Merck, Roche, Celgene, Lilly, Nordic Pharma, Bristol-Myers Squibb, Astellas and MSD Sharp & Dohme; is a speaker for Roche, Celgene, Lilly, Nordic Pharma, AIO gGmbH, MCI, promedicis, Forum für Medizinische Fortbildung and Taiho pharma; he is CEO/founder of IKF Klinische Krebsforschung GmbH at Northwest Hospital; and has received research grants from Sanofi, Merck, Roche, Celgene, Vifor, Medac, Hospira, Lilly, Eurozyto, German Cancer Aid (Krebshilfe), German Research Foundation and the Federal Ministry of Education and Research. UP received institutional research grants from Celgene, BMS, Amgen, Lilly, Roche, Sanofi and Servier and honoraria for lectures and advisory board meetings by Roche, Celgene, Amgen, Lilly, Sanofi-Aventis, Servier, Bayer and BMS. AH received honoraria for lectures from Roche. CB received institutional research grants and honoraria for lectures and advisory board meetings from Merck, BMS, Roche, Sanofi, Servier, Bayer, BMS, Astrazeneca, Lilly, Mundipharma, Hexal, MSD and GSO. MB received institutional research grants from Merck, BMS, Hexal, German Cancer Aid (Krebshilfe), German Research Foundation and the Federal Ministry of Education and Research as well as honoraria for lectures and advisory board meetings by Celgene, Janssen, Gilead, Merck, Roche, Amgen, Sanofi-Aventis and BMS.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Efficacy of AVETUX protocol. (A) Consort diagram. (B) Kaplan-Meier estimates of progression-free survival. (C) Waterfall plot of best responses in target lesions (PD in responding patient was new lesion). (D) Spider plot depicting tumor measurements over time. Patients with MSI are marked with asterisk. (E) Kaplan-Meier estimates of overall survival. PD, progressive disease.

Figure 2

Mutational profiling and liquid biopsy disease monitoring. (A) Distribution of mutation spectra in FFPE tumor tissue and liquid biopsy at baseline evaluation. (B) Venn diagram of patients of which tumor driver mutations were detected by gene panel sequencing in FFPE tissue and/or liquid biopsy, respectively. (C) Circulating tumor (CT) DNA clearance from baseline to week 4 after treatment initiation. (D) Serial liquid biopsy testing in patients with disease progression during observational period. Gray box: increase or reappearance of ctDNA prior to clinical PD in weeks. Line indicates median. (E) KRAS and NRAS circulating tumor DNA monitoring during AVETUX therapy regimen. Respective patient number in bold. Patients with MSI are marked with asterisk. CR, complete response; FFPE, formalin-fixed paraffin-embedded; PR, partial response; PD, progressive disease; SD, stable disease; VAF, variant allele frequency; SFU, safety follow-up.

Figure 3

Selection of resistance variants on AVETUX protocol and clearance after avelumab withdrawal. (A) Overview of B2M, JAK1 and PD-L1 mutations in baseline and on-treatment tumor and liquid biopsy samples. Tumor samples collected under treatment or at EOT originate from: patient 12: liver metastasis, patient 22: tumor DNA 1: rectum and tumor DNA 2: bladder (same metastatic site as used for baseline testing),. (B) Localization of B2M, JAK1 and PD-L1 mutations. (C) Distribution of emerging resistance mutations to avelumab and cetuximab as well as FcγR3a genotype (rs396991) over the cohort. Asterisk on KRAS indicates that mutation disappeared in follow-up liquid biopsy samples. In total five on-treatment tumor samples were available. (D) ddPCR validation of immune checkpoint blockade resistance variants in patient 22. (E) PFS of patients with F/F versus F/V or V/V FcγR3a genotype (rs396991). Dotted line indicates median PFS of entire patient cohort. Statistical test: one-sided, unpaired t-test. (F) Longitudinal ctDNA and biopsy (hemicolectomy) mutational and CEA profile of patient 21 with a treatment-induced PD-L1 K162fs mutation and patient 30 with a treatment-induced PD-L1 L88S mutation. Treatment is indicated above each plot. Highlighted area indicates time during AVETUX regimen. Patients with MSI are marked with asterisk. APC, adenomatous polyposis coli; B2M, β2-microglobulin; CEA, carcinoembryonic antigen; JAK1, Janus kinase 1; PD-L1, programmed cell death ligand 1; PFS, progression-free survival; PD, progressive disease; PR, partial response; TP53, tumor protein p53;

Figure 4

IHC staining of PD-L1 in pre-treatment and on-treatment tumor tissue of patient 22 with genetic evidence of a treatment-induced PD-L1 L88S mutation. Representative micrographs of H&E and PD-L1 stainings of tumor tissue at 100× and 400× magnification, respectively. Moreover, multiplex IHC is shown using antibodies against PanCK (tumor cells, green), CD3 (T cells, turquoise) and PD-L1 (magenta). Nuclei are stained with DAPI. IC, immune cell score; IHC, immunohistochemistry; PD-L1, programmed cell death protein ligand 1; TPS, Tumor proportional score; Tx, treatment.

Figure 5

Selected PD-L1 mutations reduce protein abundancy, surface expression, ADCC and T cell suppression. (A) Schematic representation of gRNAs targeting the PD-L1 coding sequence (CDS). (B) CRISPR/Cas9-mediated depletion of PD-L1 as detected in crude cell extracts via immunoblotting. (C) PD-L1 protein levels after lentiviral redelivery of PD-L1 variants into PD-L1KO2 human cell lines and the murine Ba/F3 cell line as detected by immunoblotting. (D) Enrichment of PD-L1 in the membranous fraction (M) of HT-29 cell variants from panel C. (C) Cytosolic, non-membranous fraction. (E) Flow cytometric detection of PD-L1 surface expression displayed as mean relative fluorescence intensity (RI) after staining with avelumab. n(DLD-1)=7, n(HT-29)=8, n(UT-SCC-14)=9, n(UT-SCC-29)=8, n(Ba/F3)=4. (F) NK cell degranulation induced by coculturing primary NK cells and cell lines expressing indicated PD-L1 variants in the presence of avelumab (n=4 for DLD-1, HT-29, UT-SCC-29; n=5 for UT-SCC-14). Percent degranulated NK cells normalized to spontaneous NK degranulation is shown for all cocultures. (G) Representative images and time course of T cell mediated tumor cell killing. CD8+ T cells were cocultured with HT-29, DLD-1 and UT-SCC-14 cells expressing WT PD-L1 or the L88S and K162fs variants (red fluorescence). Caspase 3/7 activity (relative intensity of green fluorescence) was monitored every 90 min for 24 hours with an Incuyte S3. Scale bar, 200 µM. Asterisks indicate p value range (*p<0.05; **p<0.01; ***p<0.001; ***p<0.0001). Statistics for time course: two-tailed paired t test. All other statistics: two-tailed unpaired t test. NK, natural killer; PD-L1, programmed cell death protein ligand 1.

Figure 6

PD-L1 L88S exhibits enhanced phosphorylation-dependent proteasomal degradation. (A) immunoblot analysis of PD-L1 abundancy in HT-29 (n=6) and UT-SCC-14 (n=4) cells overexpressing PD-L1 variants after blocking protein synthesis with 20 µM CHX for 3, 6 and 9 hours (h). (B) Quantification of (A) and four replicates using ImageJ. (C) Relative stability of PD-L1 protein (=signal intensity relative to control as quantified using ImageJ) in PD-L1 overexpressing cells after abrogation of N-glycosylation for 18 hours using tunicamycin (Tm) as determined by immunoblotting (n=5). (D) Quantification of PD-L1 protein abundancy in PD-L1 overexpressing cells after 2-hour and 4-hour blocking of AMPK with 10 µM compound C (Comp C) as determined by immunoblotting (n=6). (E) Enrichment of PD-L1 variants after inhibition of the proteasome using 20 µM MG132 for 4 hours. Quantification of glycosylated (·) and non-glycosylated (··) PD-L1 from four replicates of PD-L1 transduced HT-29 and UT-SCC-14 cells. Statistics: two-tailed unpaired t-test. Asterisks indicate p value range (*p<0.05; **p<0.01; ***p<0.001; ***p<0.0001; ns>0.05). AMPK, AMP-activated protein kinase; PD-L1, programmed cell death protein ligand 1.