Germline mutation analyses of malignant ground glass opacity nodules in non-smoking lung adenocarcinoma patients

Introduction

Though therapeutic advances have been made using targeted therapy and immunotherapy, lung cancer continues to be the most common cause of cancer-related deaths worldwide (Siegel, Miller & Jemal, 2015). The majority of lung cancers are caused by somatic mutations that accumulate with age and germline mutations could explain a predisposition to cancer development.

Lung cancer is a complex disease that is mainly attributed to smoking (Hung et al., 2008). However, over 10% of lung cancer patients are non-smokers (Subramanian & Govindan, 2007). The development of lung cancer in never-smokers is associated with several potential risk factors, including environmental pollution and genetic predisposition (Malhotra et al., 2016). Germline mutations in lung cancer have been studied to some extent (Ikeda et al., 2014; Liu et al., 2016; Shukuya et al., 2018; Zhang et al., 2017), including some in familial settings (Kanwal et al., 2018; Tomoshige et al., 2015). There are also studies on non-smoking lung cancer cohorts (Donner et al., 2018; Renieri et al., 2014). Nonetheless, studies on germline mutations in lung cancer patients fall far short when compared to those on somatic mutations. There is a need to study germline mutations in lung cancer since they are related to the pharmacodynamics, prognosis, and interactions with somatic mutations (Bartsch et al., 2007; Erdem et al., 2012; Wang et al., 2018; Winther-Larsen et al., 2015).

GGOs observed on computed tomography are described as hazy areas but preserved broncho-vascular markings (Austin et al., 1996; Lee et al., 2014). Advances in high resolution computed tomography and its application in lung cancer screening have led to an increased detection rate of GGOs, with an estimated prevalence of 0.2–0.5% (Henschke et al., 2006). While many GGOs are benign and disappear with time, some are persistent and turn malignant. These tumours are frequently found in non-smokers and women lung cancer patients (Blons et al., 2006; Raz et al., 2006).

Here, we recruited a total of 13 non-smoking patients with malignant GGO nodules to study their germline mutations using whole exome sequencing (WES). The results provide a better understanding of molecular mechanisms underlying the development of GGOs and their predisposition to turn cancerous.

Materials & Methods

Results

Clinical information of patients was summarised in Table 1. The mean age at onset of non-small cell lung cancer (NSCLC) in the cases was 61.5 years (range 48–79 years). All cases were non-smokers and 84.6% were females.

Two computed tomographic images are shown as representative of GGO nodules in the study cohort (Fig. 1). Of the 13 cases, 12 were diagnosed as lung adenocarcinomas while one was diagnosed as an atypical adenomatous hyperplasia. Five cases were adenocarcinoma in situ, four were invasive, and three were minimally invasive. Eight of the GGO nodules were located at the right upper lobe, two were at the right lower lobe, and three were at the left upper lobe. Detailed histologic information is presented in Table 2.

We used a stepwise filtering strategy to screen for potential candidate variants (Fig. 2). Of 83,302 single-nucleotide variants (SNVs) located in exons of the whole exome, our filtering strategy identified 17 potential candidate variants (Table 3). Of the 17 candidate variants in 17 genes, NM_000700:exon6:c.A418T in AXAN1, NM_001184790:exon8:c.C1070T in PARD3, NM_001170721:exon4:c.C392T in BCAR1, NM_001127221:exon46:c.G6587A in CACNA1A, NM_001170634:exon5:c.G383A in FUS, and NM_002451:exon6:c.C538T in MTAP were present in three cases each. In addition, rs756875895 frameshift in MAX, NM_001199292:exon7:c.C482G in HSD17B4, NM_005732:exon13:c.2165_2166insT in RAD50, NM_001350128:exon11:c.T1172C in PPOX, NM_001098816:exon28:c.A4751G in TENM4, NM_004004:exon2:c.235delC in GJB2, and NM_001142316:exon2:c.G203C in LMO2 were present in two cases each. The remaining variants were present in one case each. Of the 17 variants, 12 were nonsynonymous mutations, four were frameshift deletions, and one was a stopgain. The distribution of mutations in each patient was shown in Fig. 3.

We identified four potential deleterious frameshift deletions: rs80338943 in GJB2, rs587781454 in RAD50, rs756875895 in MAX, each occurring in two cases, and a frameshift in LDLRAP1, occurring in one case. The first frameshift, rs80338943 in GJB2, causing a p.L79fs fusion, was annotated as uncertain significance by InterVar. The second frameshift, rs587781454 in RAD50 caused a p.K722fs fusion and was annotated as pathogenic from InterVar. The third frameshift, rs756875895 in MAX, was annotated as likely pathogenic in InterVar, causing a fusion of p.L52fs. The frameshift in LDLRAP1 caused a fusion of p.W22fs in one case and was interpreted as pathogenic from InterVar, it might be deleterious in the development of lung cancer. Another potential deleterious variant was a stopgain, rs7755898 in CYP21A2, causing a protein change of p.Q289X which was likely pathogenic according to InterVar.

The other interesting candidates were four likely pathogenic SNVs annotated from InterVar: NOTCH 3:p.T357M (present in one case), HSD17B4 p.A161G (present in two cases), PPOX p.L391P (present in two cases), and TENM4 p.Q1584R (present in two cases).

There were five SNVs annotated as uncertain significance by InterVar that were present in three patients: ANXA1:p.I140 F, BCAR1:p.P131L, CACNA1A:p.R2196Q, FUS:p.S128N, and MTAP:p.R180W.

There were two additional candidate variants, LMO2 p.G68A and TTN p.R18629C, that were present in two cases and one case, respectively (Table 3). Their annotations by InterVar were of uncertain significance.

KEGG analysis did not indicate pathways that were related to AXAN1, TENM4 and GJB2. Pathways of BCAR1, CYP21A2, LPLRAP1, HSD17B4, MTAP, PPOX and TTN were not associated with cancer. Pathways derived from NOTCH3, PARD3, CACNA1A, MAX, RAD50, FUS and LMO2 were cancer-related. The details were shown in a (Table S). Mutations in these genes were considered unlikely to cause cancer, therefore they would not be discussed here.

Discussion

Although there are studies available on genetic mutations of lung cancer, the heritability of lung cancer, especially for GGO nodules, remains understudied compared to sporadic lung cancer. Using WES, our study reports germline mutations in GGO nodules of non-smoker lung cancer patients, largely females.

The discovery of germline mutations is very significant for both basic research and clinical treatment of lung cancer. First, germline mutations may play a role in tumorigenesis. Wang et al. (2018) reported that germline mutations interacted with somatic mutations, indicating their role in lung tumorigenesis. Tomoshige et al. (2015) also reported that germline mutations could cause familial lung cancer. Second, germline mutations are valuable for prognosis (Erdem et al., 2012). For example, a study by Winther-Larsen et al. (2015) found that genetic polymorphism in the epidermal growth factor receptor could predict the outcome in advanced NSCLC patients treated with erlotinib. Third, germline mutations are closely associated with a genetic predisposition to cancer, and screening for germline mutations is beneficial to the susceptible population (Chen et al., 2015) and for their health management.

In this study, we used a highly selective population, lung adenocarcinoma patients with GGOs, to investigate germline mutations and their possible role in the predisposition to lung cancer. In our cohort, 11 of 13 were females and all were non-smokers. The ethnicity of all patients was Han Chinese. The aforementioned facts were consolidated with the notion that malignant GGO nodules occur frequently in non-smokers and women (Blons et al., 2006; Raz et al., 2006).

Strong evidence for two deleterious germline mutations (rs587781454 in RAD50 and rs756875895 in MAX) has been shown in lung cancer patients. rs587781454 in RAD50 was reported as a hereditary predisposition and labelled as pathogenic in ClinVar (Nykamp et al., 2017). rs756875895 in MAX was labelled as likely pathogenic by InterVar. Both variants occurred simultaneously in two females (WL-5 and WL-6). Both had minimally invasive GGO nodules. How these mutations in the same patient affected lung tumorigenesis is worth examining.

There was one likely pathogenic variant in NOTCH 3 (WL-13). The expression of NOTCH 3 was inversely associated with the sensitivity to platinum-based chemotherapy in patients with NSCLC. The NOTCH 3 protein, rather than the gene polymorphism, is associated with the chemotherapy response and prognosis of advanced NSCLC patients (Shi et al., 2014).

Though annotated as uncertain significance by InterVar, three patients carried variants in BCAR1 (WL-7, WL-10 and WL-13) and CACNA1A (WL-5, WL-6 and WL-9). Increased expression of BCAR1 was associated with poor prognosis and carcinogenesis in NSCLC (Deng et al., 2013; Huang et al., 2012). Overexpression of CACNA1A predicted a poor prognosis in NSCLC (Zhou et al., 2017). There were one additional candidate variants, LMO2 p.G68A in WL-1 and WL-8. Collectively, these findings suggest that germline mutations may function by regulating gene expression and thereby affect cancer development and/or prognosis.

Our study has limitations. First, the sample size is small. In our study, only non-smoker patients with malignant GGOs were enrolled. Second, gene expression was not investigated. Finally, the identified germline mutations have not been validated. These limitations restrict conclusions about their causative effects on tumorigenesis and their roles as biomarkers for prognosis or for treatment response.

Conclusions

In summary, our results demonstrate potentially deleterious germline mutations in GGO nodules in non-smoking lung adenocarcinoma patients. These findings significantly expand the spectrum of genetic variants that may affect the response to therapies and patient survival and possibly increase the risk of being germline mutation carriers. However, due to the small patient samples, our observations encourage further studies. In future, prospective studies, expanding enrolled patients and functional studies should be performed to better understand their causative roles in tumorigenesis and prognosis, and to better manage patients’ health.

Supplemental Information