The role of ERBB4 and TP53 mutation in the prognosis of NSCLC patients analyzed by cBioPortal
Mutation frequencies of TP53 and ERBB4 was 54% and 8% in NSCLC, respectively (Fig. 1A). In the both cohorts, mutual exclusive analysis in cBioPortal indicated that ERBB4 does show co-occurencing mutations with TP53 (p < 0.01). Moreover, TP53 mutation was found in all ERBB4-mutated patients of the both cohorts. Then, relationship between the survival of NSCLC patients and ERBB4 mutation statues was further analyzed. According to the ERBB4 gene mutation status, advanced NSCLC patients treated with ICIs were divided into ERBB4-MT and ERBB4-WT groups, and then KM analysis was performed. We found that the PFS and OS time of the ERBB4-MT NSCLC patients treated with ICIs was longer than that of the ERBB4-WT patients (the median PFS: 9.2 VS 3.17 months, p = 0.0360; the median OS: 21 VS 11 months, p = 0.0378, Fig. 1B and C). In addition, both TCGA-LUAD and -LUSC somatic mutation and survival data was downloaded from the Genomic Data Commons. We found that ERBB4 gene mutation only predict prognosis in the TCGA-LUSC patients who did not receive ICI treatment (LUSC: p = 0.014; LUAD: p = 0.785, Fig. 1D and E). Therefore, ERBB4-mutated patients exhibited a better prognosis than those with ERBB4 wild type treated with ICIs.
The role of ERBB4 and TP53 mutation in outcome for ICIs treatment analyzed by cBioPortal
Firstly, due to TP53 mutation found in all ERBB4-mutated patients of the both cohorts, we divided patients harboring TP53 mutation into ERBB4-MT group (ERBB4 and TP53 comutation),TP53-MT group (TP53 mutation with ERBB4-wildtype) and WT group ( without TP53 or ERBB4 mutation). Analysis of the datasets from the cBioPortal showed that ERBB4 and TP53 comutation was associated with an improved sensitivity to immunotherapy. Although the difference of PFS between ERBB4-MT and TP53-MT was not significant (p = 0.135, Fig. 2A), the median PFS in ERBB4-MT group was 9.2 months compared to 4.33 months in TP53-MT group. In addition, patients harboring ERBB4 mutation benefited more from ICIs treatment and achieved a longer OS time than those in the TP53-MT group (21 months VS 8 months, p = 0.021, Fig. 2B). As depicted in Fig. 2C, the percentage of DCB were high in the ERBB4-MT group than that in the TP53-MT group and WT group (60% VS 31.9% VS 24%, ERBB4-MT VS TP53-MT group: p = 0.035; ERBB4-MT VS WT group: p = 0.021).
The role of ERBB4 and TP53 mutation in the TMB level and PD-L1 expression analyzed by cBioPortal and TCGA
In addition, the mutation status of ERBB4 and TP53 is closely associated with the TMB level and PD-L1 expression (Fig. 2D, E, F, G and H). ERBB4 and TP53 comutation is related to a higher PD-L1 score (ERBB4-WT VS MT group: p = 0.022, Fig. 2D) in advanced NSCLC patients. However, there is no significant difference of PD-L1 expression between ERBB4-WT group and TP53-WT group (p = 0.609). In the both cohorts, a higher TMB value were confirmed in the ERBB4-MT group compared to TP53-MT and WT group (Rizvi’s research: TP53-MT group: p = 0.002, WT group: p < 0.001; Samstein’s research: TP53-MT group: p < 0.001, WT group: p < 0.001, Fig. 2E and 2F). Moreover, ERBB4 and TP53 mutation were associated with a higher TMB value in both TCGA-LUAD and -LUSC cohorts, respectively (p < 0.001, Fig. 2G and 2H).
Construction of nomogram for the prognosis of immunotherapy
Nomogram to predict the PFS of eighty-three NSCLC patients of the selected cohort was based on integrated information of clinicopathologic features, targeted sequencing and PD-L1 expression. Firstly, we identified variables for nomogram construction by univariate analyses. Multiple variables were proved to be significantly linked to the PFS of NSCLC patients with ICI treatment, including ERBB4 mutation (p = 0.0079), EGFR mutation (p = 0.0152), smoking (p = 0.0040), treatment lines (p = 0.0097), TMB (p = 0.0059) and PD-L1 expression (p = 0.0113 ) (Fig. 3A–F ). Moreover, the univariate analyses indicated that the advanced NSCLC patients with ERBB4 mutation, ever smoking, first-line ICIs administration, elevated expression of PD-L1 (≥ 50% percentage), or a high TMB score (≥ 75th percentage) could derive survival benefits from immunotherapy. However, EGFR mutation might predict the poor prognosis of NSCLC treated by ICIs. A systematic nomogram was formulated based on these variables (Fig. 3G ) This novel nomogram could help clinical physicians to easily obtain a point of each variable, and then the total point was evaluated as the sum of all variable points. Therefore, we could assess the efficacy of ICIs therapy for advanced NSCLC patients in advance. The nomogram demonstrated good accuracy in estimating the PFS of advanced NSCLC patients with ICIs therapy, with a bootstrap-corrected C index of 0.75 (95% CI, 0.72to 0.78). In addition, calibration plots graphically showed great predictive performance in Fig. 3H (the dashed lines in the calibration plots correspond to a 10% margin of error). Therefore,ERBB4 mutation could be regarded as a novel prognostic biomarker for ICIs treatment, and could have a connection with factors that closely associated with efficacy of immunotherapy.
The role of ERBB4 mutation in Tumor-infiltrating immune cell modulation and enrichment pathway analysis of ERBB4 mutation
The relationship between ERBB4 mutation and tumor-infiltrating immune cells was analyzed using CIBERSORT algorithm. As shown in Fig. 4A, we discovered that CD8 T cells, activated memory CD4 T cells, follicular helper T cells and M1 macrophages were more enriched in ERBB4 mutant LUAD group, nevertheless, memory resting CD4 T cells, dendritic cells and Mast cells were enriched in wild-type LUAD group. As for LUSC group, activated memory CD4 T cells, follicular helper T cells and M1 macrophages were more enriched in ERBB4 mutant group(Fig. 4B). Therefore,ERBB4 deficiency might activate the antigen presentation process and cellular immunity and led to the change in the sensitivity to immunotherapy in advanced NSCLC patients.
GSEA analysis performed with TCGA-LUAD revealed that Cell Cycle, Oocyte Meiosis, Spliceosome, RNA Degradation, Nucleotide Excisio, Repair DNA Replication, Notch Signaling Pathway Progesterone Mediated Oocyte Maturation, Pyrimidine Metabolism, Ubiquitin Mediated Proteolysis pathway significantly enriched in samples with ERBB4 mutation (Fig. 5A). As for TCGA-LUSC samples, Porphyrin and Chlorophyll Metabolism, Galactose Metabolism, Sphingolipid Metabolism Pentose and Glucuronate Interconversions, Glutathione Metabolism Pathway significantly enriched in samples with ERBB4 mutation (Fig. 5B). However, Glioma pathway were enriched in samples with wild type.
Prognostic value of ERBB4 in pan-cancer
Prognostic value of ERBB4 was analyzed as an independent external validation in the cohort [TMB and Immunotherapy (MSKCC, Nat Genet 2019)]  in cBioPortal for Cancer Genomics. The mutation of ERBB4 led to a higher TMB level in various cancers (p < 0.0001) (Fig. 5C). The Kaplan–Meier survival analysis indicated that the mutation status of ERBB4 was associated to the prognosis of cancer patients (p = 0.0130) (Fig. 5D).