TERT Mutation was Associated with the Risk of Recurrence in Lung Adenocarcinoma with A Micropapillary Pattern

Background: Lung adenocarcinoma with a micropapillary pattern (MPPAC) is the histological subtype of lung cancer. It has attracted increasing attention, especially regarding its association with poor prognosis, including the predisposition towards recurrence and metastasis. Although MPPAC has been described in early-stage cases, only a few studies have reported the correlation between disease-specic prognosis and gene mutation of MPPAC. This study aimed to clarify the common genetic mutations and the prognostic characteristics in MPPAC patients. Methods: A total of 17 patients whose surgical pathology was dened as MPPAC were followed up, the molecular characteristics were elucidated by next-generation sequencing, and the prognostic characteristics were analyzed. Results: Epidermal growth factor receptor (EGFR) mutations were identied in 11/17 (65%) of patients. TP53 alterations were identied in 10/17 (59%). Other common mutations include ATM (18%), KRAS (18%), SDHA (18%), and TERT (18%). MPPAC patients harboring EGFR and TERT mutations were at a high risk of tumor recurrence, while TP53 might be associated with a low risk of recurrence. Conclusions: TERT mutation was more frequently harbored in MPPAC patients than in the other histological type of lung cancer, and such patients were at a high risk of recurrence. So TERT mutation might be associated with adverse prognosis in MPPAC patients.

Several studies have shown that MPP is associated with lung cancer prognosis, and even a minimal proportion of the tumor can lead to poor prognosis [7,11,12,14,[35][36][37].

Sample collection
Formalin-xed para n-embedded (FFPE) blocks from 17 MPPAC patients between 2012 and 2017 were collected retrospectively from Zhejiang Cancer Hospital in China. The pathological diagnosis was based on the standard criteria de ned by the International Association for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society (IASLC/ATS/ERS) [2]. The classi cation of stages was de ned by the eighth edition of the TNM (tumor, node, metastasis) classi cation for lung cancer [38].
The clinical characteristics, such as gender, age, and clinical stage, are listed in Table 1 Next-generation sequencing (NGS) The genomic alterations (GAs) in formalin-xed, FFPE tissue samples obtained from 17 MPPAC patients were subjected to comprehensive pro ling using NGS-based cancer gene panel, as described previously [39]. TP53 mutation is one of the most common genetic abnormalities found in all types of human tumors and has been reported in approximately half of the lung cancer [46]. In various malignancies, TP53 mutations have been associated with tumor progression, metastasis, resistance to chemotherapy and radiation, and reduced overall survival (OS) [47,48]. Quinlan et al. [49] rst reported TP53 oncoprotein expression as a poor prognostic factor in non-small cell lung carcinoma (NSCLC). The percentage of TP53 mutation varies by tumor type and ranges from 10-80% (http://p53.free.fr). In previous studies, TP53 alterations, mainly missense mutations, are found in 35-55% of NSCLC and are more prevalent in squamous cell carcinoma than AC [50,51]. In this study, the mutation frequency of TP53 was similar to that observed previously. Herein, we detected TP53 mutations in tumor samples from 10/17 (59%) MPPAC patients.
Also, we identi ed that TERT frequently mutated in MPPAC patients. 3/17 (18%) MPPAC harbored TERT mutation. TERT activities are frequently upregulated in many human cancers and might contribute to human tumorigenesis [52]. The prevalence of TERT promoter mutations (pTERTm) was rst identi ed in melanoma and subsequently detected in bladder cancer, glioblastoma, thyroid cancer, and other cancers [53][54][55][56][57]. pTERTm is a moderately prevalent genetic event in NSCLC. Also, the prevalence and association of pTERTms in NSCLC patients have been studied. Yuan et al. [58] identi ed a low frequency of pTERTm (5.8%) in NSCLCs, and found that patients who carry pTERTm are older than noncarriers, male patients were more likely to carry pTERTm and that pTERTm had a signi cant association with distant metastasis.
Hence, it could be a poor prognostic factor for cancer patients. Ma et al. [59] demonstrated that 2.67% of NSCLC patients in their cohort had pTERTm, and those with TERT promoter mutation were signi cantly associated with older age. We also found that the mutation frequency of TERT was higher than that reported previously. However, due to the small sample size, we could not link TERT promoter mutations to the age and gender of patients.
The tumor mutation burden (TMB) is 0.7-24.7 (median 5.7) in these patients (Fig. 1b). According to the progression-free survival (PFS) results of Checkmate 026 [60] and 227 [61], TMB was considered as a potentially new and independent biomarker, the high TMB patients could choose nivolumab plus ipilimumab combination treatment, irrespective of PD-L1 expression level. TMB-positive advanced NSCLC patients who have no oncogenic drivers choose the immune checkpoint inhibitors as rst-line treatment, while platinum-based chemotherapy may represent only the rst-line option for patients with no PD-L1 expression and TMB-negative [62]. Nevertheless, the predictive value of TMB should be further investigated in future randomized trials. In the current study, the TMB was not signi cantly associated with disease-free survival (DFS). DFS was de ned as the interval from the surgery to the point of any de nite clinical or pathological evidence of local or distant disease recurrence or last evaluation.
Genomic alterations associated with the risk of recurrence In this study, all patients were subjected to surgically and pathologically determined stages I, II, and III, which accounted for 4 (23%), 3 (18%), and 10 (59%) of the cohort. Thirteen patients received adjuvant chemotherapy, local radiotherapy, and EGFR TKIs after surgery. These patients presented different genomic alterations, and the DFS in patients ranged from 13 to > 72 months (alive at the time of testing) after diagnosis (Fig. 1c), suggesting that their genetic background might be related to the differences in their prognosis.
Some studies reported a higher than usual incidence of EGFR mutations in papillary and micropapillary AC of the lung [63,64]. In this study, 11/17 (65%) MPPAC harbored EGFR mutations. Three cases with EGFR mutation were at stage I disease, 1 case was at stage II disease, and 7 cases were at stage III. The 2stage I patients received no other treatment after surgery, 1 stage II patient received chemotherapy, and 1 stage III patient received no other treatment after surgery, 1 received TKI treatment, and 5 received chemotherapy. The DFS of the patients received TKI treatment for > 46 months (alive at the time of the return visit), while the DFS in patients who received chemotherapy ranged from 13 to > 42 months. MPPAC patients harboring EGFR mutation might be good candidates for the treatment with EGFR TKIs. According to the recurrence status, 17 patients were divided into two groups: recurrence and non-recurrence. One patient was lost to follow-up, and disease recurrence was found in 10 patients; 8/10 carried the EGFR mutation. Thus, MPPAC patients harboring the EGFR mutation might face a high risk of tumor recurrence. However, due to the small sample size, we did not observe a signi cant association between the risk of recurrence and EGFR mutation (P = 0.299451, Table 2). Kishi et al. [65] demonstrated that the MPPAC patients harboring L858R were at a high risk of recurrence in the pN0M0 lung AC. In previous studies, the correlation between EGFR mutations and the risk of recurrence of the MPPAC patients was controversial. Some investigators showed a positive correlation [63,64,66,67], whereas one proposed that there was no correlation [12]. Sumiyoshi et al. [17] established a positive correlation between EGFR mutations and micropapillary component and speculated that MPPAC was biologically aggressive but could be controlled with EGFR-TKIs. The study recommended that to avoid the progression of the disease, the MPPAC patients harboring EGFR mutation should be actively treated with EGFR-TKIs. The current results showed that the EGFR mutation might be associated with the risk of recurrence in MPPAC patients. The risk of disease recurrence might be predicted by EGFR mutations, and the MPPAC patients harboring the EGFR mutation would be subjected to EGFR TKIs treatment. In this retrospective study, we reviewed and analyzed the risk of recurrence in 17 MPPAC patients with documented tumor p53 mutational status (mutant-type [mtp53] vs. wild-type [wtp53]). Ten patients had mtp53, 1 was lost to follow-up, and 7 did not carry the TP53 mutation. 6/10 harbored the TP53 mutation showed a DFS of > 33 months, while 1/7 with wpt53 had a DFS > 33 months. Thus, TP53 could be speculated as a positive prognostic factor for enhanced DFS (P = 0.06014, Fisher's test), indicating that it is associated with a low risk of recurrence in MPPAC patients. However, due to the limited sample size, we did not observe a signi cant association between the risk of recurrence and TP53 mutation. In contrast to our data, a previous study showed that TP53 positive or overexpression is found to be signi cantly associated with short OS in NSCLC [68][69][70], and differences were observed between the various subtypes of NSCLC. Intriguingly, Nishio et al. [71] found an association between TP53 abnormalities and poor prognosis of patients with ACs but not with the squamous cell carcinomas. TP53 mutations are widespread in NSCLC, but to date, there are no approved agents that speci cally target TP53 in NSCLC. The Wee-1 inhibitor AZD1775 is currently under investigation, but mainly with respect to small cell lung cancer [72].
In addition, 3/17 (18%) MPPAC patients harbored TERT mutation. BRAF and NRAS mutations activate the expression of TERT via MAPK pathway [54]. 68.6% of the lung ACs in the Chinese population carry EGFR or KRAS mutations [73], which were the known oncogenes that drive lung cancer by the MAPK/AKT pathway. In the present study, all 3 patients with TERT mutations had a concurrent EGFR mutation and also showed a disease recurrence. This phenomenon could be attributed to the TERT mutation in MPPAC patients, which elevated the risk of tumor recurrence in this group. However, due to the limited sample size, we did not observe a signi cant association between the risk of recurrence and TERT mutation (P = 0.25, Table 2). Another study showed that there was no signi cant difference in the OS or relapse-free survival (RFS) between TERT with and without mutation [54]. Therefore, additional studies and large sample sizes are needed to determine the correlation between TERT mutation and the risk of tumor recurrence. Previous ndings of the associations of TERT expression and telomere length with the survival length of NSCLC patients also remained controversial. Several molecular epidemiology studies have associated TERT overexpression with poor prognosis in NSCLC patients [74][75][76][77][78][79]. Also, a signi cant association between high TERT levels and short periods of DFS and OS and high TERT levels in breast cancer were found to be signi cantly associated with short RFS [80]. A correlation between real-time quantitative measurement of TERT and the clinicopathological parameters of poor prognoses, such as histologic grade and muscle invasion, has been observed in bladder urothelial cell carcinomas [81], albeit with different conclusions [82][83][84][85]. The telomerase activity is one of the critical prognostic factors in NSCLC patients, as assessed by multivariate analysis. Additionally, TERT was not associated with the prognosis, as depicted in another study.

Discussion
Previous studies have demonstrated close correlations between the presence of MPP and lymph node metastasis, lymphatic invasion, venous invasion, differentiation grade, and TNM stage, suggesting that MPP display highly aggressive biological behavior. Thus, MPP in lung AC is considered as a distinct histopathological variant with biological and prognostic signi cance. The presence of an MPP could serve as an accurate indicator of t prognosis in lung AC patients.
In this study, we analyzed the genomic pro les of 17 MPPAC patients by targeted sequencing of pancancer genes in archived primary FFPE tissue samples. Thus, we obtained an overall pro le of the genomic alterations for this distinctive subtype of invasive lung ACs and analyzed the prognostic characteristics of these patients. The mutational pro les of MPPAC patients were compared to that from a previous study on lung ACs. It was found that these distinctive micropapillary subtypes had similarities but also differed in genomic alterations as compared to those with lung ACs. EGFR was the most commonly mutated gene. TP53 alterations were more prevalent in MPPAC than lung ACs. The mutation rate of KRAS gene was similar in MPPAC and lung ACs, while the mutation frequency of TERT was higher than that reported previously.
Herein, we investigated the effect of EGFR, TP53, and TERT mutations on DFS in MPPAC patients. In the placing them at a high risk of tumor recurrence. Also, the MPPAC patients harboring TERT mutation were at similar risk, while TP53 mutation served as a positive prognostic factor for improved DFS. However, large sample size is required to con rm the above conclusion.

Conclusions
MPPAC is determined as a new histological subtype and a highly malignant potential type. TERT mutation was more frequently harbored in MPPAC patients than the other histological type of lung cancer, and those harboring TERT mutation were at a high risk of recurrence in patients. Thus, TERT mutation might be associated with the adverse prognostic in MPPAC patients. In the current study, EGFR, TP53, and TERT were the common mutations in MPPAC patients, and recognition of the common mutations would guide the treatment and predict the outcomes in MPPAC patients. According to targeted therapy, MPPAC patients harboring EGFR mutation were promising candidates for the treatment with EGFR TKIs. However, the high prevalence of TP53 and TERT mutations in MPPAC poses a challenge because of the absence of effective therapy for TP53 and TERT mutations.

Disclosure
The authors report no con icts of interest in this study. Figure 1