Background
Lung cancer is one of the most common cancers and is by far the leading cause of cancer-related deaths worldwide [
1]. Nearly 80% of lung cancer patients are diagnosed as non-small cell lung cancer (NSCLC) at advanced stages of the disease [
2]. The average survival time is usually short after diagnosis, largely due to unsatisfactory outcomes from conventional chemotherapeutic regimens, especially in patients with metastatic cancer [
3]. However, the treatment outcome has been improved significantly in the past several years owing to increased understanding on molecular mechanisms of tumorigenesis. It is highlighted by the finding that the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) including gefitinib and erlotinib have shown superior improvement on survival time and life quality in a subset of patients harboring EGFR activating mutations [
4,
5]. Thus, current strategies to improve treatment outcomes have been focused on target-specific and customized treatment according to the patient’s molecular profile [
6].
The fusion of the anaplastic lymphoma kinase (
ALK) with the echinoderm microtubule-associated protein-like 4 (
EML4) on chromosome 2p was first identified as oncogenic driver mutations in 2007 in Japanese NSCLC patients [
7]. Out of 75 examined Japanese patients, 5 patients (6.7%) were found carrying the
EML4–ALK fusion transcript, which resulted from a small inversion within chromosome 2p [
7]. Multiple studies have been carried out to determine the frequency of
EML4–ALK translocation occurrences in patients with NSCLC, ranging from 1.6% to 11.7% in individual studies [
7‐
18] with an averaged frequency at about 5%, estimated from published results [
6]. The huge variation among these studies is likely due to the differences in patient selection criteria such as disease status, race, country, gender, and/or smoking habit. Other
ALK-fusion genes including
KIF5B-ALK have also been identified in patients with NSCLC [
8,
19‐
21]. It has been suggested that patients with
ALK rearrangement are resistant to EGFR TKIs [
22]. However, crizotinib (XALKORI®, Pfizer Inc.), an ALK tyrosine kinase activity inhibitor, has been approved by the FDA in the United States for treating patients with ALK + advanced NSCLC [
23] as well as in other countries, including China.
Although
EML4–ALK translocation was first identified from a lung adenocarcinoma specimen surgically resected from a 62-years-old man with a history of smoking [
7], increased evidence suggests that it is much more common in never-smokers based on the studies performed in different countries [
10,
15,
16,
22]. As estimated, the incidence of
EML4-ALK fusion in never-smokers is 9.4% vs. 2.9% in smokers [
6]. In addition to smoking habit, studies also suggest that the frequency of the incidence is different between male and female patients [
17,
18]. However, based on the available data from these publications, it is not clear what the frequency is in either male or female never smokers who were diagnosed as NSCLC. A recent study has reported that the incidence could be as high as 15.2% (5/33) in a small cohort of Chinese female adenocarcinoma patients who are never-smokers [
18]. However, it is not clear whether the incidence is also high in male never-smokers with NSCLC. To address this question, we assembled 95 Chinese male patients who are never smokers and diagnosed with NSCLC. We used one-step reverse transcription polymerase chain reaction (RT-PCR) to screen
ELM4-ALK fusion genes in these patients. We have identified 8 (8.42%) cases with
ALK rearrangement, which is significantly higher than estimated 2.9% in the smokers with NSCLC worldwide [
6]. Interestingly, our study suggests that
EML4-ALK rearrangements in Chinese male never-smokers with NSCLC are more frequently detected in younger patients and in less-differentiated carcinomas.
Discussion
In this study, we have determined the frequency of EML4-ALK translocation and its associated clinical features in Chinese male never smokers with NSCLC. A previous study has shown that the frequency of EML4-ALK translocation in Chinese female patients with NSCLC is higher in never-smokers. Similar to that, we have found that the incidence in Chinese male NSCLC patients is also significantly higher in never-smokers by comparing to published data. Interestingly, we have also found that these Chinese male never-smoker patients in our study are strongly associated with younger-onset and less-differentiated carcinomas, which are likely caused by aberrant expression of ALK mRNA and protein.
Since
EML4-ALK fusion genes were first identified as potential driver mutations in NSCLC [
7], multiple studies have been carried out to determine the frequency of
EML4-ALK translocation. The frequencies of
EML4-ALK may vary substantially among different publications, ranging from 1.6% in a cohort of Japanese patients [
15] to 11.7% in a cohort of Chinese patients [
16]. The large variation is likely contributed by multiple factors, including race, gender, smoking habit, etc. Several groups have estimated the frequency of
EML4-ALK translocation worldwide by combining published data and found the value at 5% [
6], 3.7% [
17], or 4.8% [
18], respectively. The differences among these values are caused by differences in the selection of data sources. To exclude the influence caused by differences in races (genetic factors) and countries (geographic, cultural, environmental, diet, etc.), and also to better determine the influence of gender and smoking habit in the occurrence of
EML4-ALK translocation, we performed an analysis using the data extracted from published studies that were done exclusively in Chinese patients (Table
5) [
8,
14,
16‐
18]. The average frequency of
EML4-ALK in non-selected Chinese patients is 5.50% (Table
5), which is similar to the estimated number using published studies in the patients from multiple countries. This result suggests that race and country have the least effect in the occurrence of
EML4-ALK translocation in NSCLC. Interestingly, our analysis has shown that both gender and smoking habit are important factors affecting the frequency of
EML4-ALK translocation in Chinese patients.
EML4-ALK translocation is more frequent in Chinese never-smokers than in Chinese smokers, which is consistent with the result in patients worldwide [
6]. Our analysis has also clearly shown that the difference between Chinese male and Chinese female patients is highly significant (
p < 0.002, Table
5).
Interestingly, a previous study has found that the frequency of
EML4-ALK translocation is as high as 15.2% (5/33) in Chinese female non-smokers with NSCLC [
18]. Although double the amount, it is not significantly different (by Fisher’s exact) when compared to the frequency in Chinese male never-smokers from our study (8.42% or 8/95). Therefore, more patients are needed to draw a solid conclusion on whether there is a significant difference in the frequency of
EML4-ALK translocation between male and female never-smokers. In addition, authors in this study detected zero patients with
EML4-ALK translocation in 147 Chinese male patients, including 45 never-smokers (calculated from available data in the paper) [
18]. The failure to detect any positive cases in male patients is likely due to the small sample size and low frequency (3.44%, Table
5) in Chinese male patients with NSCLC. As determined in our current study, the frequency in Chinese male never-smokers with NSCLC (8.42%) is significantly higher than that in all Chinese male patients with NSCLC (2.25%, Table
5). It should be noted that although we enrolled consecutive patients who met our criteria, our samples are predominantly collected from adenocarcinomas (88.4%), which may bias our results and interpretations.
We have detected 5 different types of
EML4-ALK variants (V1, V2, V3a, V4a, and V5a). It was the first time that V4 was detected in Chinese NSCLC patients based on previously published results [
8,
14,
16‐
18]. Both ALK mRNAs and proteins are highly expressed in these
EML4-ALK positive carcinomas (Figures
3 and
4), which is consistent with the notion that
EML4-ALK rearrangements cause aberrant expression of
EML4-ALK fusion oncogene and overactivation of ALK tyrosine kinase, which in turn leads to the inhibition of apoptosis and promotion of tumor cell proliferation [
25,
29,
30]. Crizotinib, a selective inhibitor of
ALK and mesenchymal epithelial growth factor tyrosine kinases, has shown significant improvement in response rates and response duration in
ALK-positive patients in clinical trials [
25]. Meanwhile, it has been shown that
ALK-positive patients are resistant to EGFR TKIs [
22]. It is then possible that nearly one-tenth of male never-smokers with NSCLC (8.42%) would likely respond to crizotinib, but not to EGFR TKIs (gefitinib and erlotinib) or drugs targeting other tyrosine kinases. Therefore, at least among Chinese patients, identification of
EML4-ALK translocation is crucial for predicting the resistance to EGFR TKIs and the responsiveness to ALK TKIs. Ultimately, it will facilitate the planning of an effective treatment and improve the outcome of such treatment in NSCLC.
In this study, we have identified clinical features that are associated with
EML4-ALK translocation in Chinese male never-smokers with NSCLC. We have found that the median age in these patients (50.63 yrs) is significantly younger than in all studied patients (58.76 yrs). Thus,
EML4-ALK translocation in Chinese male never-smokers is more likely to occur in younger patients. In other words, the disease onset is much earlier in
EML4-ALK-positive patients in a subset of Chinese NSCLC patients who are male never-smokers. A previous study has reported that the
EML4-ALK-positive lung adenocarcinomas are characterized as less-differentiated [
31]. In that report, the differentiation levels were determined in 11
EML4-ALK positive carcinomas, which were identified by the same group previously [
32]. While only one patient carried the carcinomas that were well-differentiated, 10 other patients carried tumors that were poorly or moderately differentiated. In line with this result, our study has shown that
EML4-ALK rearrangements are predominantly found in carcinomas with less (poorly or moderately) differentiated cells, with the exception of one with well differentiated cells (Table
4). However, there is a difference between the two studies. While all patients in our study are never-smokers, the 11
EML4-ALK positive patients in other study include 6 never-smokers and 5 smokers. Thus, it is possible that the differentiation of carcinomas, regardless of smoking history, is associated with the expression of
EML4-ALK mutant genes. In other words, the aberrant activity of ALK tyrosine kinase induced by
EML4-ALK translocation results in less-differentiated carcinomas, more dangerous and aggressive types of cancers.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YG, JM, and WB contributed to the experimental design, specimen collection, data acquisition. BW, JZ, NZ, and SF participated in data analyses, interpretation of results. LD participated in the design of the study and carried out data interpretation. JZ contributed to conception, experimental design, data acquisition, analyses, and interpretation, and manuscript preparation. All authors read and approved the final manuscript.