Immune heterogeneity in adenocarcinoma and squamous cell carcinoma components of lung adenosquamous carcinoma.
Authors: Lin Gen , Li Chao , Li Pansong , Fang Wenzheng , Xu Hai-peng , Lv Xinze , Li Qian , Xia Xuefeng , Miao Qian , Wu Biao , Jiang Kan , Zheng Xiaobin , Huang Cheng 
Published: June 2021

e20523 Background: Lung adenosquamous carcinoma (ASC) is a relatively uncommon malignancy that comprised of both adenocarcinoma component (ACC) and squamous cell carcinoma component (SCCC). We have reported its genomic profile, evolutionary origin, and clinical management. Information of tumor immune microenvironment (TIME) of ASC, including tumor mutation burden (TMB), tumor-infiltrating lymphocytes (TILs), T cell receptors (TCR) repertoire, and PD-L1 status remains scarce. Methods: 28 Surgical ASCs were collected and identified by immunohistochemistry (IHC). ACC and SCCC were obtained separately by microdissection. Targeted sequencing was performed for the two components using a 1021-gene-panel independently. TMB of ACC and SCCC were independently calculated. TMB of ASC calculation was based on aggregation (removing duplicates) of non-synonymous mutations from both ACC and SCCC. TILs and PD-L1were examined by multiplex immunohistochemistry (mIHC). The infiltration level of immune cells in tumor, stroma, and total region (named by both tumor and stroma regions) was investigated. T cell receptor (TCR) repertoires were sequenced and clonality and Shannon index were calculated. Results: SCCC (7.2 mutations/Mb) had a higher TMB level than ACC (6.5 muts/Mb), indicating the immunogenic heterogeneity between the different pathological components. The TMB value of ACC and SCCC were modestly proportional (Spearman r = 0.56, p = 0.001), which was related to the clonal origin. SCCC TMB was lower than the archival LUSC (10.1 muts/Mb), and ACC TMB was similar to LUAD (4.3 muts/Mb). The TMB of ASC was higher than LUAD and close to LUSC. The nonidentical TIL level in ACC and SCCC showed infiltration heterogeneity. For tumor region, CD3+ total T cells, CD4+ T cells, CD8+ T cells, and PD1+ cells accounted for the most proportion of immune cells, with no differences between ACC and SCCC; FOXP3+ CD4+ Treg cells were more abundant in SCCC than ACC ( p = 0.047), as well as CD3+ LAG3+ T cell ( p = 0.029). For stroma region, ACC and SCCC had similar level of CD4+, CD8+, and PD-1 immune cell infiltration; the amount of FOXP3+ CD4 + Treg cells was also higher in SCCC than ACC, as well as CD3+ LAG3+ T cells. For the total region, FOXP3+ CD4 + Treg cells, CD3+ LAG3+ T cells, and PD-1+ T cells were enriched in SCCC compared with ACC, while CD57+ natural killer T (NKT) cells were accumulated with a marginally higher level in ACC ( p = 0.049). TCR repertoires sequencing revealed a lower Shannon’s diversity ( p = 0.029) but higher clonality ( p = 0.047) of SCCC compared with ACC. PD-L1 expression identified by mIHC in SCCC was significantly higher than ACC, in the tumor ( p < 0.001), stroma ( p < 0.001), and total regions ( p < 0.001). Conclusions: SCCC of ASC had higher levels of TMB, Treg cells, TCR clonality, and PD-L1, and similar level of CD3+, CD4+, and CD8+ TILs, comprehensively reflecting the intratumor heterogeneity of TIME.