Gene Mutation and Its Association with Clinicopathological Features in Young Patients with Non-Small-Cell Lung Cancer.

Background: We investigated the correlation between genetic mutations and clinical-pathological features in young patients with NSCLC.
Methods: Clinicopathologic information of 102 young NSCLC patients was collected. Direct ctDNA sequencing of a portion of these patients was performed. The correlation between EGFR mutation and ALK fusions with clinicopathologic parameters was analyzed.
Results: In young NSCLC patients, adenocarcinoma is the major histology (86.9%), and the misdiagnosis rate was as high as 45.7%. EGFR gene mutation was found in 13 patients (31.7%) and common mutations were with EGFR19del mutation (7 cases, 17.1%) and EGFR21L858R mutation (4 patients, 9.7%). EGFR mutation was constantly found in adenocarcinoma and male gender, and ever smokers (100%, P < 0.05). Furthermore, ALK fusions were found in 7 patients (31.8%), which include EML-4-ALK fusions; there was a trend that ALK fusions were associated with adenocarcinoma and female gender. However, there was no significant difference in overall survival between patients with or without gene mutations. Conclusions: EGFR mutation and ALK fusions are related to histology, gender, and smoke exposure in young NSCLC patients, and may be effective predictive factors.

1. Introduction

Lung cancer, the most major malignant tumor, has a high incidence and mortality rate worldwide. It is identified as one of the most serious diseases threatening human health and life [1]. Lung cancer occurs in adults over the age of 50, of which the age at onset is typically between 60 and 80 years [2]. The incidence of lung cancer among young people (age < 40 years) is relatively low, with an incidence rate of 1.2% to 6.2% [3, 4]. However, in recent years, epidemiological data suggest lung cancer incidence with a trend toward a younger onset worldwide [5-9], especially with a higher incidence in young females than in young males [10]. In China, the incidence rate of lung cancer in youngers is rising, and a survey from Shanghai showed that younger (<45 years old) accounted for 5.275% of all lung cancer cases [11]. The clinicopathologic features and disease progression in youngers are different from those in middle-aged and old-aged patients with lung cancer. Here, the pathogenesis of young patients (age ≤ 35 years) showing non-small-cell lung cancer (NSCLC) was researched through the clinicopathological characterization combined with analyzing the mutation profiles of serum in circulating tumor DNA (ctDNA) by next-generation sequencing. In recent years, epidermal growth factor receptor (EGFR) mutations and anaplastic large-cell lymphoma kinase (ALK) rearrangements are now routine biomarkers that have been incorporated into the practice of managing non-small-cell lung cancer (NSCLC).

2. Methods and Materials

2.1. Patients

102 lung cancer patients who underwent treatments at Fujian Cancer Hospital and Fujian Union Hospital between March 2014 and July 2018 were enrolled. The patients were diagnosed by histopathology analysis and aged less than or equal to 35 years. Clinicopathologic staging was determined according to the 8th Edition of the AJCC/UICC staging system. Patients' characteristics including demographics, smoking background, family history, and clinical data were also recorded. In addition, 10 ml of peripheral venous blood was obtained from every patient for ctDNA assay. Informed consent was gained from all patients. All procedures were approved by the Ethics Committee of Fujian Cancer Hospital and Fujian Union Hospital.

2.2. ctDNA Sequencing

Briefly, the ctDNA from blood samples was extracted by using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). DNA concentration was determined by using the NanoDrop1000 spectrophotometer (Thermo Fisher, Wilmington, USA). The library was generated to target all exons and select introns of a custom gene panel of 10, 66, 116, or 448 genes. The ctDNA was sheared, end-repaired, phosphorylated, and adaptor-ligated. Next-generation sequencing was conducted with 200 ng sheared DNA through the custom hybridization capture panel (SureSelectXT, Agilent, CA), hybrid selection, and PCR amplification. Libraries were constructed with 80 ng of DNA and amplified with 12 PCR cycles. Finally, the libraries were sequenced (sequencing depth 300×) on an Illumina NextSeq-500 platform (Illumina, San Diego, CA) for paired reads (read length 151 bp).

2.3. Gene Mutation Analysis

To identify genomic alterations and gene rearrangements, the sequencing results were trimmed by Trimmomatic for the adaptor and mapped to the human genome (hg19) with BWA aligner 0.7.10. Local alignment optimization, variant calling, and annotation were conducted by GATK 3.2, MuTect, and VarScan.

2.4. Follow-Up

The overall survival (OS) of all patients was recorded through telephone or door-to-door follow-up, and the last follow-up time was July 28, 2018. The definition of OS was the number of months from lung cancer diagnosis to the patient's death or the end of follow-up.

2.5. Statistical Analysis

Statistical analysis was conducted by IBM SPSS Statistics 21.0 software (SPSS Inc., USA). Clinical characteristics were compared using the χ2 test. Age distribution was analyzed using the unpaired t-test. OS was measured by the Kaplan–Meier method and compared using the log-rank test. P < 0.05 was considered a statistically significant difference.

3. Results

3.1. General Information

A total of 102 patients who met the inclusion criteria were enrolled. After removing small-cell lung cancer (6 patients), lung carcinoma in situ (5 patients), 4 cases of carcinoid tumor, 1 case of epithelioid hemangioendothelioma, 1 case of undifferentiated sarcoma or undifferentiated carcinoma, and 1 case of atypical adenomatous hyperplasia, 84 young patients with NSCLC were enrolled in the current assessment, including 12 stage I cases (14.3%), 4 stage II cases (4.8%), 13 stage III cases (15.5%), and 55 stage IV cases (65.5%). The NSCLC patients consisted of 37 females (44%) and 47 males (56%), with the mean age of 30.27 ± 4.195 years. Besides, no significant difference in the disease stages was found between the males and females. Patients' characteristics are shown in Table 1. Nine patients (10.7%) were ever smokers and 75 (89.3%) patients were nonsmokers. Only three patients had a family history of lung cancer. For histological classification, there were 73 cases of lung adenocarcinoma (86.9%), 8 cases of lung squamous carcinoma (9.5%), and 3 cases of other types (3.6%).

Table 1

Clinicopathological features of young patients with NSCLC in the study (N = 84 cases).

Clinicopathological features	Cases	Percentage
Gender
Male	47	56.0
Female	37	44.0
Age
14–20	2	2.4
21–25	8	9.5
26–30	29	34.5
31–35	45	53.6
Smoking
No	75	89.3
Yes	9	10.7
Histological classification
Lung adenocarcinoma	73	86.9
Lung squamous carcinoma	8	9.5
Other types	3	3.6
Family history	3	2.7
Tumor-node-metastasis staging
I	12	14.3
II	4	4.8
III	13	15.5
IV	55	65.5

3.2. Clinical Manifestations and Time of Diagnosis of Young NSCLC Patients

Among the 84 patients, 14 cases (16.7%) had no clinical symptoms and were found through physical examination; 30 cases (35.7%) had cough and expectoration as their first symptoms; 4 cases (4.7%) had neck masses; 5 cases (5.9%) had pain caused by bone metastases; 4 cases (4.7%) had chest pain. The symptoms of other patients were described as follows: hemoptysis in 5 cases (5.9%), chest oppression in 4 cases (4.7%), dizziness and headache in 4 cases (4.7%), shortness of breath in 4 cases (4.7%), numbness and weakness of the extremities in 2 cases (2.4%), dry cough in 3 cases (3.6%), fever in 1 case (1.2%), hoarseness in 1 case (1.2%), edema of the extremities in 1 case (1.2%), weight loss in 1 case (1.2%), and nausea, retching, and sudden unconsciousness in 1 case (1.2%).

Fourteen cases were found during a physical examination and were pathologically confirmed as NSCLC within 7–14 days. The time of diagnosis of the remaining 70 cases ranged from 2 weeks to 6 months, with an average of 102.5 days. Due to the relatively low incidence of lung cancer in youngers, malignant lesions were not taken into account by physicians at the first diagnosis. In this study, we found that 32 cases among the patients without physical examinations had different degrees of misdiagnosis, and the misdiagnosis rate was as high as 45.7%. The misdiagnosis included 18 cases of pneumonia, 4 cases of tuberculosis, 2 cases of lymphadenitis, 2 cases of acute bronchitis, 2 cases of upper respiratory tract infection, 1 case of cervical spondylosis, 1 case of migraine, 1 case of hyperthyroidism, and 1 case of benign lymph nodes.

3.3. Metastatic Sites

A total of 55 young NSCLC patients had distant metastasis consisting of bone metastases (15 cases, 27.3%), pleural effusion (14 cases, 25.5%), brain metastases (8 cases, 14.5%), lung metastases (8 cases, 14.5%), pericardial effusion (5 cases, 9.1%), lymph node metastases (5 cases, 9.1%), pleura metastases (3 cases, 5.5%), liver metastases (3 cases, 5.5%), adrenal gland metastases (2 cases, 3.6%), subcutaneous soft tissue metastases (1 case, 1.8%), breast metastases (1 case, 1.8%), and abdominal wall metastases (1 case, 1.8%), as shown in Table 2.

Table 2

The characteristics of the 55 young NSCLC patients with distant metastasis.

Metastatic site	Cases	Percentage
Bone	15	27.3
Pleural effusion	14	25.5
Brain	8	14.5
Lung	8	14.5
Pericardial effusion	5	9.1
Lymph node	5	9.1
Pleura	3	5.5
Liver	3	5.5
Adrenal gland	2	3.6
Subcutaneous soft tissue	1	1.8
Breast	1	1.8
Abdominal wall	1	1.8

3.4. Mutation Analysis

A total of 41 patients underwent EGFR mutation testing, and 13 patients (31.7%) suffered EGFR gene mutations consisting of 1 patient (2.4%) with EGFR18 (G719A/G719S/719C) gene mutation, 7 patients (17.1%) with EGFR19del mutation, 4 patients (9.7%) with EGFR21L858R mutation, and 1 patient (2.4%) with EGFR21L858R mutation and concurrent EGFR20 exon 768I mutation. 22 patients underwent ALK fusion gene testing, and 7 patients (31.8%) had ALK fusions including EML-4-ALK fusions, RICTOR, and TP53 gene mutations. All cases with genetic mutations were analyzed histologically and classified as adenocarcinoma.

3.5. Gene Mutation and Clinicopathologic Correlations

EGFR mutation was constantly found in adenocarcinoma and male gender of young NSCLC patients and was significantly associated with ever smokers (100%, P < 0.05) compared with never smokers (28.2%), as shown in Table 3. Moreover, 2 cases of male patients who ever smoke both showed EGFR mutation. However, there was no significant difference between EGFR mutation, disease stage, and patient age. The median survival times of young NSCLC patients with or without EGFR mutations were 16 and 28 months, respectively. But no significant change in the overall survival was found in these two groups (P=0.32) (Figure 1)(a).

Table 3

Patient characteristics and EGFR mutation.

Variables	Cases	EGFR mutation (%)	P
Gender
Female	24	5 (20.8%)	0.07
Male	17	8 (47.0%)
Smoking history
Never smoker	39	11 (28.2%)	0.03
Ever smoker	2	2 (100%)
Histology classification
Adenocarcinoma	36	13 (36.1%)	0.10
Others	5	0
Stage
I-II	8	3 (37.5%)	0.69
III-IV	33	10 (30.3%)
Age (years)
14–25	5	1 (20.0%)	0.5483
26–35	36	12 (33.3%)

P < 0.05 was considered a statistically significant difference.

Figure 1

Correlation of EGFR mutation, ALK fusions, and patient survival by the Kaplan–Meier method.

We also explored the relationship between the ALK fusions and clinicopathologic factors. ALK fusions were only found in adenocarcinoma (36.8%), We found that there was a trend that ALK fusions frequently occurred in male patients compared with female patients (P=0.29). In addition, in 6 of the 7 cases, ALK fusions were present in patients at stage IV. However, there was no significant difference between ALK fusions and smoking, ALK fusions and patient age are shown in Table 4. The median survival times of young NSCLC patients with or without ALK fusions were 11 and 10 months. And no significant difference in the overall survival was found in these two groups (P=0.73) (Figure 1)(b). There was no significant difference in the overall survival between the two groups for EGFR mutations (A, P=0.32) or ALK fusions (B, P=0.73).

Table 4

Patient characteristics and ALK fusions.

Variables	Cases	ALK fusions (%)	P
Gender
Female	13	3 (23.1%)	0.29
Male	9	4 (44.4%)
Smoking history
Never smoker	20	6 (30.0%)	0.56
Ever smoker	2	1 (50.0%)
Histology classification
Adenocarcinoma	19	7 (36.8%)	0.20
Others	3	0
Stage
I-II	2	1 (50.0%)	0.56
III-IV	20	6 (30.0%)
Age (years)
14–25	5	1 (20.0%)	0.51
26–35	17	6 (35.2%)3

P < 0.05 was considered a statistically significant difference.

4. Discussion

Lung cancer has a low incidence in youngers, with only 1.2–6.2% of cases younger than 40 years [3, 4, 12], of which 65% of cases are female, 84% of cases are adenocarcinoma, 4% of cases are squamous carcinoma, and stage IV patients accounted for 65% [13]. However, currently and for some years now, the age of onset of lung cancer is becoming younger. The clinical characteristics and genetic mutations of NSCLC patients aged less than or equal to 35 years. And there was a statistical difference in age-sex comparisons for 84 young patients with small cell lung cancer. Additionally, we found that there were slightly more young male NSCLC patients than young female patients in terms of gender. Adenocarcinoma is the most common histological type. Besides, our findings revealed that the age of NSCLC onset was lower in young females than in young male patients.

In the present study, lung adenocarcinoma was the most common histological type, accounting for 77.7% of cases, which is consistent with the previous findings [13, 14]. The reason may be that it takes a long time to develop squamous carcinoma, but adenocarcinoma or undifferentiated carcinoma derived from respiratory mucosa has a higher susceptibility to carcinogenesis. Certainly, genetic factors and environmental factors also have functional roles in young patients with lung cancer [15].

Clinical symptoms of young NSCLC patients lack in specificity, among which cough and sputum are the most common symptoms, followed by hemoptysis, chest tightness, and chest pain, which are consistent with previous reports [14, 16]. Young patients with symptoms such as cough, sputum, and chest tightness, tend to receive symptomatic treatment, but are usually misdiagnosed as pneumonia or tuberculosis. The misdiagnosis rate is high, reaching 28.6%. Due to the early onset of the patient's illness, doctors rarely carry out further clinical examinations in time, delaying the diagnosis; As a result, most patients have reached the advanced stages of NSCLC by the time they are diagnosed. There were 71 NSCLC patients (63.4%) with advanced stages, which is consistent with those previously published. Besides, Ak et al. found that the time from symptoms appearing to treatment in young patients is longer than that in elderly patients, but without a significant difference [17]. The most common distant metastasis site among advanced NSCLCs who were younger than 50 years was the bone, accounting for 26.9%. Similarly, we found that 55 of the 84 patients had distant metastasis, and the major site was the bone, followed by pleural effusion, the brain, and the lung.

Previous studies have shown that gene mutations including EGFR and ALK were related with cancer diagnosis in youngers [18]. In the present study, young NSCLC patients (histopathologic subtype was adenocarcinoma) underwent genetic testing. Among them, 13 patients (31.7%) had EGFR gene mutation; 7 cases (31.8%) had ALK gene mutation. Moreover, recent data suggest that ALK fusions are related to lung cancer development in youngers [19, 20]. Our findings suggest that young NSCLC patients with ALK fusion are mostly in advanced stages, suggesting that ALK fusion in younger patients presents NSCLC with high malignant potential, which is consistent with previous studies [21-23]. Tanaka K et al. suggested that oncogenic gene mutations were prevalent in youngers with lung adenocarcinoma, moreover, the positive rates of ALK fusions, HER-2 mutations, ROS1 fusions, and RET rearrangement were higher in younger lung cancer patients than in elderly patients, indicating that younger patients are more likely to benefit from targeted therapy [23]. However, for lung cancer patients without mutations, there may be unknown driver mutations, such as EGFR-RAD51 fusions and EGFR kinase domain duplication, that have been reported as actionable EGFR mutations [24, 25]. Recently, with the rapid development of third-generation DNA sequencing, comprehensive genetic testing is beneficial to young lung cancer patients, providing more opportunities for targeted therapy.