Purpose Patients with stage III clear cell renal cell carcinoma are at high risk for recurrence after nephrectomy. To mitigate overtreatment, there is a pressing clinical need to determine which patients benefit most from perioperative immune checkpoint inhibition. We performed a multimodal digital spatial analysis of gene and protein expression in stage III primary renal cell carcinomas, a subset of which had preoperative immune checkpoint inhibition exposure.

Materials and Methods Surgically resected tumors from stage III clear cell renal cell carcinoma patients were analyzed using the Nanostring GeoMx Digital Spatial Profiler. Differential expression analysis was performed and validated using NCT02210117 trial data to identify genes associated with immune checkpoint blockade and clinical response. A gene score was then generated to predict overall survival in patients from The Cancer Genome Atlas.

Results Among 19 patients, RNA expression significantly differed based on preoperative immune checkpoint blockade and recurrence – CD8+ effector and central-memory T-cell signatures were less prevalent in the treatment-naïve with recurrence group. Three out of four patients with preoperative immune checkpoint inhibition had recurrence. External validation yielded a 4-gene set (GZMK, GZMA, ITGAL, and IL7R); higher gene expression levels predicted better overall survival in The Cancer Genome Atlas cohort (p=0.005).

Discussion Preoperative immune checkpoint blockade favorably altered the tumor microenvironment to resemble that of treatment-naïve patients without recurrence. However, this did not translate to better clinical outcomes. On external validation, the genes GZMK, GZMA, ITGAL, and IL7R were modifiable with immune checkpoint inhibition and associated with improved survival. Further investigation to assess if patients with low baseline expression of these genes may particularly benefit from perioperative immune checkpoint blockade is warranted.

The authors have declared no competing interest.

This study was supported by NIH grants R01 CA250378, R21 CA259440, P30 CA069533, and P30 CA069533 13S5 through the OHSU-Knight Cancer Institute, the Hope Foundation (SWOG), and the OHSU Department of Pathology and Laboratory faculty support (GVT). The pathology was performed at the Histopathology Shared Resource supported by the OHSU Knight Cancer Institute (NIH P30 CA069533). The University of Washington Spatial Biology Core Facility is supported in part by the Department of Laboratory Medicine and Pathology.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Ethics committee/IRB of Oregon Health & Science University gave ethical approval for this work through the protocol number OHSU-IRB #4918.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

↵* The last two authors are co-senior authors

Author emails: LL: lawrenluohsu.edu

EN: emmajunenehamicloud.com

SA: shreeramuw.edu

AEM: moranamohsu.edu

CLC: corlesscohsu.edu

LL: langmessohsu.edu

BC: cengizohsu.edu

KE: kazumiohsu.edu

JL: jenjohsu.edu

SI: isharwalohsu.edu

CLA: amlingohsu.edu

MCS: strothemohsu.edu

GVT: thomasgeohsu.edu

NHC: chakiryaohsu.edu

All data produced in the present study are available upon reasonable request to the authors.