Multi-omic analysis of the prognostic and predictive value of LAG3 expression in urothelial carcinoma.
Authors: Ayanambakkam Adanma , Lam Anh B. , Sweeney Kieran , Xu Chao , Elliott Andrew , Tomek Seven , Nabhan Chadi , Gupta Sumati , Chahoud Jad , Sundi Debasish , Skelton William Paul , Graham Laura , Berim Lyudmyla Derby , Salhia Bodour , Kern Sean , Edge Stephen B. , Zakharia Yousef , Tripathi Abhishek , McKay Rana R. , Naqash Abdul Rafeh 
Published: June 2024

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Background: Lymphocyte-activation gene 3( LAG3) is an immune checkpoint protein expressed on immune cells that inhibits T-cell function. Despite its established prognostic significance in other malignancies, the role of LAG3 as a prognostic or predictive biomarker in urothelial carcinoma (UC) remains inadequately studied. Methods: DNA (592-gene or whole exome) and RNA (whole transcriptome) sequencing were performed for patient tumors submitted to Caris Life Sciences. PD-L1+ status (22c3, combined positive score≥10) was determined by IHC. TMB-High (TMB-H) was defined as ≥10 mutations/Mb. LAG3 high ( LAG3-H) and low ( LAG3-L) groups were defined by the top and bottom quartiles of LAG3 RNA transcripts per million, respectively. Tumor microenvironment (TME) cell fractions were estimated by RNA deconvolution using quanTIseq. Significance was tested using Mann-Whitney U and 𝜒2 tests as appropriate. Real-world median overall survival (mOS) was obtained from insurance claims data and calculated from treatment start to last contact, while time-on-treatment (ToT) was calculated from treatment start to end. Hazard ratio (HR) was calculated using the Cox proportional hazards model, and p-values were calculated using the log-rank test. Clinical and RNA-seq data from patients enrolled in the Oncology Research Information Exchange Network (ORIEN) were used to validate the analysis. Results: Among 3343 UC cases, LAG3-H was associated with increased TP53 (70.6% vs 49.4%, q<10-4) and RB1 mutations (31.0% vs 18.0%, q<10-4), decreased FGFR3 mutations (6.0% vs 18.1%, q<10-4), and increased TMB-H (50.1% vs 36.7%, q<10-4) and PD-L1+ status (71.0% vs 16.2% 22c3, q<10-4). LAG3-H tumors had increased infiltration of CD8+ (1.5% vs 0.0%, q<10-4) and NK (2.3% vs 1.6%, q<10-4) cells, but also higher levels of inhibitory Tregs (3.3% vs 1.4%, q<10-4). No significant difference was found in mOS of patients with LAG3-H, when comparing low vs high levels of FGL1 expression (HR=0.934, p=0.51). Among patients who received an immune checkpoint inhibitor (ICI; N=675), multivariate analysis using Cox proportional hazard regression revealed that LAG3-H had improved mOS vs LAG3-L (HR=0.720, p=0.002) after accounting for potential confounders (sex, TMB, TP53, and FGFR3). LAG3-H also had significantly improved mOS vs LAG3-L in PD-L1+ patients given ICI (N=291, HR=0.605, p=0.009). LAG3 expression was also higher in patients with above median ToT on avelumab (1.4-fold, p=0.049) and pembrolizumab (1.2-fold, p=0.022). Improved mOS for LAG3-H vs LAG3-L patients (HR=0.65, p=0.036) were validated using the ORIEN database. Conclusions: Increased LAG3 expression in UC correlates with a distinct mutational landscape, an inflamed TME characterized by augmented immune cell infiltration, prolonged ICI ToT, and significant improvements in mOS. These findings corroborate the potential for dual LAG3 PD1 blockade in UC.