Thread by @TurajlicLab

📰Out NOW in @CancerCell ‼️👉cell.com/cancer-cell/fu… 🎉What underpins anti-PD-1 response in ccRCC?

Results from ADAPTeR Phase II clinical trial, bulk/single-cell analysis of longitudinal tumour samples ⤵️(1/15) @royalmarsdenNHS @TheCrick @uclcancer

Intratumour heterogeneity (ITH) is pervasive in ccRCC & longitudinal data on anti-PD1 response is scarce 🚨 Thus we performed multi-omics analysis on 115 multiregion tumour samples, taken at baseline, week-9, & PD, from 15 treatment-naïve patients treated with nivo 🔬 (2/15)

What are the determinants of anti-PD1 response? 🤔 In keeping with other studies, no nsSNV/fsINDEL/copy number event, nor TMB/wGII (measure of chromosomal complexity) correlated with response, pre or post-nivo, even accounting for ITH (3/15)

BONUS👀One case - enabled by post-mortem sampling - we saw uncharacteristic high TMB underpinned by MLH1 mutation with loss of antigen presentation (B2M) was driving differential response. Nivo-resistant metastases had these features, responsive sites did not. Interesting! (4/15)

On RNAseq, we found 1) HERVs expression signatures previously associated with IO benefit, mostly come from immune cells. Therefore, prior associations were likely indirect; 2) ccRCC-specific HERVs reflect tumour purity (5/14)

Next, pre/post-nivo RNAseq show T cell activation and upregulation of TCR signalling in responders. IHC/mIHC show responders upregulate GZMB post-nivo 🚨 These dynamic changes warranted dedicated investigation ➡️ we then performed TCRseq & single-cell sequencing, (6/15)

At baseline, high TCR clonality correlated with response. Tracking total TCR repertoires post-treatment...✔️ In responders, expanded TCRs that were maintained on nivo ❌ In non-responders, expanded TCRs were replaced. This was observable in tumours, not in PBMC (7/15)

In responders, TCR clustering analysis (of similar CDR3 sequences) show expanded TCR clones had more cluster structure than non-responders – pre & post-nivo. This supports existence of T cells likely recognising tumour antigen(s) & are maintained with nivo in responders (8/15)

BONUS 👀 2nd patient with differential response. TCRseq of nivo-responsive metastases show maintained expanded TCRs present during life & response. Resistant sites had only novel TCR clones #PEACEstudy (9/15)

Next, we isolated TILs from a responder & a non-responder who underwent nephrectomy at week 9 of nivo. This provided adequate material for flow cytometry & single-cell analysis of the exact CD8 T cells nivolumab were bound to. 🤿 A deep-dive! (10/15)

Paired scRNA/TCRseq show a ‘hyperexpanded’ population of nivolumab-bound CD8 T cells in the responder. 🔵🔵🔵These T cells had a cytotoxic phenotype and had more cluster structure. These features were not found in the nivo-bound T cells from the non-responder (11/15)

To confirm these findings, we performed a meta-analysis using external scRNA/TCRseq datasets. In >100,000 CD8 T cells, despite inherent differences in treatment regimens and sampling timepoints, we found CD8 T cells from IO responders were expanded and upregulated GZMK (12/15)

Summary - Longitudinal tumour profiling during anti-PD-1 showed ✅ Expanded TCRs pre-treatment ✅Maintenance of expanded TCRs in responders ✅ Nivo binds pre-existing expanded CD8 T cells to enhance cytotoxicity (13/15)

A big THANK YOU to @royalmarsdenNHS as study sponsor & the incredible research team. 🎉Our wonderful collaborators @uclcancer @squezadd @BennyChain and @CharlesSwanton @MariamJHanjani @kevlitchfield & George Kassiotis @TheCrick and many more 🎉(14/15)

Finally, to @lewisjwau @emine_h @MarcRdM who led on the work. Funders @royalmarsden @CR_UK @cancerBRC @UCLHresearch. But most importantly, to the patients and their families, for their generosity, and putting their trust in us in this study 🙏 Thank you (15/15)