Background Emerging evidence from studies in sarcoma and melanoma immune checkpoint blockade(ICB) trials demonstrated enhanced efficacy in tumors harbouring tertiary lymphoid structures(TLS)^1–3 - lymph node like aggregates in the tumor microenvironment postulated to recruit lymphocyte infiltration and coordinate tumor antigen presentation and lymphocyte priming. Previously, our group described a 12-chemokine metagene signature that reflects strong intratumoral chemotactic signalling and accurately predicts the presence of TLS in colorectal carcinoma.⁴ Grounded in these works, we attempted to correlate high 12CK signature with TLS formation in the context of muscle invasive bladder cancer(MIBC), and then sought to define its prognostic implications and its ability to predict response to ICB.

Methods A total of 130 MIBC samples were arrayed on Affymetrix microarrays and 12CK scores were assessed. Scores were normalized using 12CK>1 to denote 12CK-High(n=24). We then investigated the the presence of TLS with the associated immune cellular infiltration evaluated by immunohistochemistry and gene signature deconvolution method (xCell).⁵ 12CK scores were also correlated with survival in our institutional cohort and validated using data from TCGA. Finally, 12CK scores were extracted from the IMVIGOR210 study to examine its ability to predict response to ICB.⁶

Results Type III TLS, consisting of germinal center-like structures and discrete T-cell zones were found in 7/22 12CK-High vs. 0/21 12CK-Low tumors (p=0.009) (figure 1a). Additionally, a more robust immuno-environment was seen in 12CK-High tumors, consisting of increased infiltration of CD4+ T lymphocytes (p=0.1), CD8+ T lymphocytes (p=0.02), activated dendritic cells (p=0.047), and B lymphocytes (p=0.006) on immunohistochemistry (figure 1b). Furthermore, on xCell deconvolution, M1 macrophage, NK cells, CD8+ Tem, CD4+ Tem, and memory B cells were enriched in 12CK-High tumors, suggesting both a heightened innate and adaptive immune response (figure 1c, d). Kaplan-Meier survival analyses of our internal cohort revealed improved PFS (HR 0.25, p=0.003 ), CSS (HR 0.25, p=0.003), and OS (HR 0.55, p=0.03) amongst 12CK-High patients (figure 2a-c). From the TCGA, similar improvements were found in PFS (HR 0.55, p=0.007), CSS (HR 0.40, p=0.002), and OS (HR 0.59, p=0.01) in 12CK-High patients (figure 2d-f). From the IMVIGOR 210 study, complete responders exhibited significantly higher 12-CK scores than all other groups (figure 2g). Strikingly, the 12CK-High signature conferred a median overall survival benefit of almost 1 year in the atezolizumab-treated patients (figure 2h).

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Abstract 68 Figure 1

High 12-chemokine score (12-CK) is associated with the presence of tertiary lymphoid structures (TLS) in the tumor microenvironment (TME) and a more robust peritumoral inflammatory response. (A) H&E and immunohistochemistry (IHC) stains were performed against CD4, CD8, CD20, and LAMP3 to define populations of CD4+ T lymphocytes, CD8+ T lymphocytes, CD20+ B lymphocytes, and activated dendritic cells in the TME, respectively. Type III tertiary lymphoid structures, consisting of prominent B-cell follicles with germinal center-like structures and discrete T-cell zones, were found within tumors as well as at the tumor invasive front. (B) Higher densities of CD4+ T lymphocytes (p=0.1), CD8+ T lymphocytes (p=0.02), B lymphocytes (p=0.008), and activated dendritic cells (p=0.047) were found in the 12CK-High TME. (C) Findings using the deconvolution tool xCell demonstrated heightened signatures related to immune cells found within the (C) adaptive and (D) innate immune responses. 12-CK – 12-Chemokine score; TLS – Tertiary lymphoid structure; GC – germinal center; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

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Abstract 68 Figure 2

The prognostic and predictive implications of 12-CK score. Kaplan-Meier survival analyses revealed improved overall survival (OS, HR 0.55, p=0.03) (A), disease-specific survival (DSS, HR 0.25, p=0.003) (B), and progression-free survival (PFS, HR 0.25, p=0.003) (C) in 12CK-High muscle invasive bladder cancer patients treated with radical cystectomy at Moffitt Cancer Center. The favorable prognosis in OS (HR 0.59, p=0.010) (D), DSS (HR 0.40, p=0.002) (E), and PFS (HR 0.55, p=0.007) (F) were confirmed in patients from TCGA. (G) From the IMVIGOR-210 study testing the efficacy of atezolizumab in chemotherapy refractory locally advanced or metastatic bladder cancer patients, complete responders were found to have significantly higher 12CK scores than the other cohorts. (H) Stratified by the 12-CK score, 12CK-High patients were found to have a 11.2mo overall survival benefit after treatment using atezolizumab

Conclusions In muslce invasive bladder cancer, 12CK-High scores corresponded with formation of TLS in the TME; favourable prognosis in surgically treated MIBC patients; and CR in atezolizumab-treated patients.

References

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