Favorable outcome of patients with lung adenocarcinoma harboring POLE mutations and expressing high PD-L1

Mutations in polymerase ε (POLE) confer favorable prognosis and outcomes in various cancer types, but their role in non-small cell lung cancer (NSCLC) is unknown. Utilizing the data of 513 patients with adenocarcinoma (LUAD) and 497 patients with squamous cell carcinoma (LUSC) from The Cancer Genome Atlas (TCGA) cohort, we tested the prognostic value of POLE mutations and programmed cell death ligand 1 (PD-L1) expression in the two main subtypes of NSCLC. POLE mutation is a favorable biomarker for the improved overall survival (OS) of the LUSC patients (P = 0.033, 28 mutant vs. 469 wildtype patients), but not that of the LUAD patients (P = 0.12, 31 mutant vs. 482 wildtype patients). POLE-mutant LUAD patients with high expression of PD-L1 (Mut-High, n = 6) exhibited improved OS (P = 0.024) when compared to POLE-mutant patients with low PD-L1 expression (Mut-Low, n = 24) and other patients without POLE mutation (n = 476). This benefit was not due to the high content of the tumor infiltrating lymphocytes. Instead, the antitumor immune response was activated in Mut-High patients so that these patients were likely responding more effectively to immuno-oncology (IO) treatments; whereas genes involved with metabolic pathways were enriched in Mut-Low group, which may cause the decreased OS of these patients. Our study sheds light on the molecular basis of NSCLC and adds to our understanding of responses to chemotherapy and IO therapy.

Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancers and can be further broadly divided by histology into adenocarcinoma (LUAD), squamous cell carcinoma (LUSC), and large cell carcinoma. Therapies directed against molecular targets in NSCLC, including immuno-oncology (IO) treatments, have improved response rates and overall survival [1,2]; however, predictive markers for response and patient outcomes are still lacking.
Polymerase ε (POLE) is a DNA polymerase involved in DNA replication and repair. POLE mutation is associated with an ultra-mutated phenotype and a good prognosis in uterine corpus endometrial carcinoma (UCEC) [3] and a subgroup of colorectal tumors (CRC) [4]. In NSCLC, the mutations of POLE and DNA mismatch repair (MMR) genes result in ultra-mutation in both LUAD and LUSC [5]. Interestingly, this event was also observed in patients who had favorable responses to immunotherapy [6]. Despite these observations, little is known about POLE mutations in NSCLC.
We analyzed the relationship of POLE mutations with programmed cell death ligand 1 (PD-L1) expression in patents with LUAD or LUSC in The Cancer Genome Atlas (TCGA) cohort. PD-L1 expression was not a good prognostic predictor of patient outcomes for the two subtypes. POLE mutation alone could predict the overall survival (OS) for LUSC but not LUAD patients. Both PD-L1 expression and tumor mutation burden (TMB) have exhibited associations with better response to immunotherapies in some but not all studies. Given that POLE mutations were correlated with high mutation rates, we hypothesized that the two predictors may jointly influence response to immunotherapy and survival outcomes. We found that the combination of POLE mutations and PD-L1 expression was a favorable indicator for the improved OS of LUAD patients. Our analyses describe the molecular differences among the LUAD patients with POLE mutations and different levels of PD-L1 expression and patients without POLE mutation, which may suggest distinct responses to chemotherapy and IO treatment.

Results and discussion
POLE mutation alone is a good prognostic biomarker for patients with lung squamous cell carcinoma but not lung adenocarcinoma Analysis using genomic data across multiple types of cancers from the TCGA cohort (Additional file 1: Methods and Materials) showed that LUSC and LUAD are among the cancers with the most frequent POLE mutations (28/497 = 5.6% and 31/513 = 6.0%, respectively), which are close to the rates in UCEC (28/519 = 5. 4%) and CRC cancers (32/594 = 5.4%). However, compared to the UCEC cancer patients whose mutations mostly locate in the proofreading domain, the mutations in NSCLC patients are distributed across the POLE gene body (Additional file 2: Figure S1A).

LUAD patients with POLE mutations and PD-L1 high expression level have the best survival
We categorized POLE-mutant LUAD patients into two groups based on their PD-L1 expression levels: high (top 20%, Mut-High) and low (other 80%, Mut-Low)-PD-L1 groups (Additional file 3: Table S3). Analysis showed that all the Mut-High patients (n = 6) survived. In contrast, Mut-Low LUAD patients (n = 24) had decreased OS, which was worse than patients with wildtype POLE (n = 476, P = 0.024, Fig. 2a). Mut-High patients had a similar mutation rate as compared to Mut-Low cases (P = 0.32), and both groups were significantly higher than POLE-wild patients (P = 0.0016 and 3.3e-05, respectively, Fig. 2b). It is worth noting that PD-L1 expression could not stratify POLE-wild LUAD patients by OS (P = 0.55, 93 High (top 20%) vs. 383 Low (others), Additional file 2: Figure S2A) although high expression levels of PD-L1 was associated with higher TMB (P = 0.0051, Additional file 2: Figure S2B).
We performed the same analyses in the LUSC cancer but did not observe similar associations. The Mut-High group of LUSC patients (n = 6) had worse outcomes than the Mut-Low group (n = 22), but both were likely better than POLE-wild patients (n = 469, P = 0.094, Additional file 2: Figure S2C and Additional file 3: Table  S4). Similar to LUAD cancer, PD-L1 expression cannot stratify POLE-wild LUSC patients by OS (P = 0.76, 95 High vs. 374 Low, Additional file 2: Figure S2D).
Tumor infiltrating lymphocytes (TILs) have been identified as a good prognostic predictor in several cancer types. For LUAD patients, POLE mutations were moderately associated with higher TIL percentage (P = 0.42, Additional file 2: Figure S2E), but not favorable outcomes (Fig. 1d). We tested whether Mut-High patients had better outcomes as a result of higher numbers of TIL. The results showed that Mut-High patients indeed contained lower TIL (P = 9.0e-4, Fig. 2c) compared to Mut-Low patients, but still experienced a better outcome relative to mortality rate (0/2 = 0% vs. 10/11 = 90. 9% at 5.5-year follow-up), although survival curve comparison was not statistically significant probably due to the small sample size (P = 0.35, Additional file 2: Figure  S2F). This observation suggests that PD-L1 performs functions in POLE-mutant patients that are not due to the existence of TIL.

Genes promoting tumors are mutated in Mut-high but not Mut-low patients
We compared the mutation landscapes of Mut-High and Mut-Low LUAD patients, and identified multiple genes differentially mutated between the two groups, such as KNDC1, ENOX1 and CACNA1H (P < 0.05, Additional file 2: Figure S3). Gene Set Enrichment Analysis (GSEA) analysis showed that these genes are enriched in olfactory transduction that promote cancer cell invasiveness and metastasis emergence [9]. They also are involved in G-protein coupled receptor activity that stimulates cell proliferation in various cell types, and have a crucial role in many aggressive human cancers, including SCLC, pancreatic cancer, and prostate cancer [10]. The mutation of these genes may cause the loss of function and contribute to improved survivals upon treatment. Antitumor immune response is activated in Mut-high LUAD patients Gene expression analysis showed that signatures of immune response were upregulated in the Mut-High patients compared to the Mut-Low cases (Additional file 2: Figure S4A). Further, GSEA showed that immune related pathways, such as T cell receptor signaling, JAK-STAT signaling, and B cell receptor signaling pathways, were activated in Mut-High group ( Fig. 3a and Additional file 2: Figure S4B), indicating that the immune system was activated in this group and, therefore, benefited the patient outcomes. Metabolic pathways related to retinol metabolism, and drug and xenobiotic metabolism through cytochrome p450 were activated in the Mut-Low group ( Fig. 3b and Additional file 2: Figure  S4B). This may suggest their poor responses to IO treatment. Similar results were achieved using the gene ontology (GO) analysis (Additional file 2: Figure S4C).

Conclusions
LUSC and LUAD, as the two main subtypes of NSCLC, are distinct in disease pathology, smoking associations, metastatic trends, molecular mechanisms, treatment options, and patient outcomes. We demonstrated the distinct prognostic values of POLE mutation and PD-L1 expression in these two subtypes. Importantly, we revealed the combination of POLE mutation and PD-L1 expression as a favorable indicator for improved OS of LUAD patients and the activation of antitumor immune system. Our results identify the molecular signatures associated with POLE mutations and PD-L1 expression in LUAD and LUSC and may reveal a distinct response status to chemotherapy and immunotherapy, which needs further experiments to validate.   Figure S3. There were 96 genes that were identified to be significantly mutated in Mut-High group but not Mut-Low group with P < 0.05 (Fisher's exact test). Figure S4. Additional file 3: Table S1. Descriptive characteristics of the POLE-mutant and -wild TCGA LUSC patients. Table S2 Descriptive characteristics of the POLE-mutant and -wild TCGA LUAD patients. Table S3. Descriptive characteristics of the Mut-High and -Low TCGA LUAD patients. Table S4.
Descriptive characteristics of the Mut-High and -Low TCGA LUSC patients.