Abstract
Background
The echinoderm microtubule-associated protein-like-4-anaplastic lymphoma kinase (EML4-ALK) fusion gene defines a novel molecular subset of non-small-cell lung cancer (NSCLC). However, the clinicopathological features of patients with the EML4-ALK fusion gene have not been defined completely.
Methods
Clinicopathological data of 200 Chinese patients with advanced NSCLC were analyzed retrospectively to explore their possible correlations with EML4-ALK fusions.
Results
The EML4-ALK fusion gene was detected in 56 (28.0%) of the 200 NSCLC patients, and undetected in 22 (11.0%) patients because of an insufficient amount of pathological tissue. The median age of the patients with positive and negative EML4-ALK was 48 and 55 years, respectively. Patients with the EML4-ALK fusion gene were significantly younger (P< 0.001). The detection rate of the EML4-ALK fusion gene in patients who received primary tumor or metastatic lymph node resection was significantly higher than in patients who received fine-needle biopsy (P= 0.003). The detection rate of the EML4-ALK fusion gene in patients with a time lag from obtainment of the pathological tissue to EML4-ALK fusion gene detection ≤48 months was significantly higher than in patients >48 months (P= 0.020). The occurrence of the EML4-ALK fusion gene in patients with wild-type epidermal growth factor receptor (EGFR) was significantly higher than in patients with mutant-type EGFR (42.5% [37/87] vs. 6.3% [1/16], P= 0.005).
Conclusions
Younger age and wild-type EGFR were identified as clinicopathological characteristics of patients with advanced NSCLC who harbored the EML4-ALK fusion gene. The optimal time lag from the obtainment of the pathological tissue to the time of EML4-ALK fusion gene detection is ≤48 months.
Keywords: ALK, EGFR, non-small-cell lung cancer (NSCLC)
Introduction
Lung cancer is one of the most common malignant tumors and the leading cause of cancer death worldwide.1Although tremendous progress has been made in the comprehensive treatment of malignant tumors in recent years, advanced non-small cell lung cancer (NSCLC) remains almost incurable. Although pathological subtypes remain the important factors in selecting chemotherapy agents, spectacular progress has been made in NSCLC molecular targeted therapy owing to the deeper understanding and research of the molecular biology of tumor cells in the last two decades. We have been gradually aware of the key function of tumor cell gene mutation or chromosomal rearrangement in predicting the efficacy of molecular targeted agents. For instance, NSCLC patients who harbored epidermal growth factorreceptor (EGFR) mutations could apparently benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy. More importantly, these results suggest that we should make efforts to seek effective target points of molecular targeted therapy by identifying and confirming molecular subtypes of lung cancer patients.
The echinoderm microtubule associated protein-like-4-anaplastic lymphoma kinase (EML4-ALK) fusion gene is the second target for NSCLC treatment at present. Previous studies using tumor specimens from different races of NSCLC patients showed that the occurrence of EML4-ALK gene rearrangement was only 2.6–6.7%,2–6and 2.4–5.6% in lung adenocarcinoma patients.7–10Subsequent studies found that a positive EML4-ALK fusion gene was more frequently seen in young lung adenocarcinoma patients with a never or light-smoking history.5,9–12However, other studies have reported that the EML4-ALK fusion gene was not associated with smoking state and did not co-exist with EGFR and KRAS mutations.8Whether there is a gender difference remains disputable. The corresponding target inhibitor Xalkori (crizotinib) has been approved for ALK-positive NSCLC patients.
In the present study, we analyzed the clinicopathological features in 200 Chinese patients with advanced NSCLC in an attempt to define clinical features affecting ALK gene rearrangement in Chinese NSCLC patients and factors affecting the outcome of ALK gene rearrangement detection, in order to provide references for further research on ALK gene rearrangement.
Patients and methods
Inclusion criteria and test methods
Two hundred NSCLC patients who received treatment in the CancerInstitute and Hospital of the Chinese Academy of Medical Sciencesbetween January 2011 and December 2012 were included in this retrospective study. The diagnosis of NSCLC was confirmed by cytology or pathology in all patients with stage IIIB or IV, according to the International Association for the Study of Lung Cancer (IASLC)-proposed 7thedition of the Tumor Node Metastasis (TNM) Classification for NSCLC. All included patients had complete clinical data, and received fluorescence in situ hybridization (FISH) for the EML4-ALK fusion gene. Some of the patients also received DNA sequencing for EGFR and KRAS mutations.
Statistical analysis
Based on the EML4-ALK fusion gene assay findings, an analysis of variance was performed with respect to gender, age, smoking history, pathological type, methods of obtaining the pathological tissue, the site of pathological tissue, and the time lag from obtainment of the pathological tissue to ALK detection. Differences between the EML4-ALK fusion gene positive and negative groups were compared by χ2 test, ttest, and Mann Whitney U test. Bilateral P-values of <0.05 were considered statistically significant. All data were treated using SPSS 16.0 statistical software.
Results
Clinical features and echinoderm microtubule-associated protein-like-4-anaplastic lymphoma kinase (EML4-ALK) gene rearrangement
Clinical data of the 200 patients with advanced NSCLC who met the inclusion criteria were analyzed retrospectively (Table 1). All patients received FISH detection for the EML4-ALK fusion gene, but no result was obtained in 22 of the 200 patients because of the small proportion of tumor cells in the sections. The 178 patients in whom the EML4-ALK fusion gene was detectable were separated into two groups: the positive group (n= 56), and the negative group (n= 122). The median age of the two groups was 48 (range 25–74) and 55 (range 27–76) years, respectively, the difference being statistically significant (P< 0.001). The EML4-ALK fusion gene was detectable in 78 of the 90 male patients, with a positive rate of 25.6% (20/78), and detectable in 100 of the 110 female patients, with a positive rate of 36.0% (36/100). The positive rate in female patients was insignificantly higher than in male patients (P= 0.140). The positive rate of the EML4-ALK fusion gene in the 143 smokers and 35 non-smokers was 34.3% (49/143) and 20.0% (7/35), respectively (P= 0.103).
Table 1.
Correlations between the clinicopathological features and the anaplastic lymphoma kinase fusion gene in 200 patients with advanced non-small cell lung cancer
| Features | Total | EML4-ALK | P-value† | ||
|---|---|---|---|---|---|
| Positive | Negative | UN | |||
| n(%) | 200 (100) | 56 (28.0) | 122 (61.0) | 22 (11.0) | |
| Age (year) | |||||
| Median | 53 | 48 | 55 | 53 | <0.001 |
| Range | 25–76 | 25–74 | 27–76 | 26–60 | |
| Gender n(%) | |||||
| Male | 90 (45.0) | 20 (35.7) | 58 (47.5) | 12 (54.5) | 0.140 |
| Female | 110 (55.0) | 36 (64.3) | 64 (52.5) | 10 (45.5) | |
| Smoking status n(%) | |||||
| Never/light-smoking | 162 (81.0) | 49 (87.5) | 94 (77.0) | 19 (86.4) | 0.103 |
| Smoking | 38 (19.0) | 7 (12.5) | 28 (23.0) | 3 (13.6) | |
| Pathology n(%) | |||||
| Adenocarcinoma | 183 (91.5) | 52 (92.8) | 109 (89.3) | 22 (100) | 0.438‡ |
| Adenosquamous carcinoma | 9 (4.5) | 3 (5.4) | 6 (4.9) | 0 (0) | |
| Squamous carcinoma | 3 (1.5) | 0 (0) | 3 (2.5) | 0 (0) | |
| LCC/NOS | 5 (2.5) | 1 (1.8) | 4 (3.3) | 0 (0) | |
| Methods of obtaining pathological tissue n(%) | |||||
| Lung tumor resection | 100 (50.0) | 24 (42.9) | 65 (53.3) | 11 (50.0) | 0.003§ |
| LN resection | 71 (35.5) | 25 (44.6) | 44 (36.0) | 2 (9.1) | |
| Puncture | 19 (9.5) | 4 (7.1) | 9 (7.4) | 6 (27.3) | |
| Others | 10 (5.0) | 3 (5.4) | 4 (3.3) | 3 (13.6) | |
| Site of pathological tissue n= (%) | |||||
| Lung tumor | 116 (58.0) | 27 (48.2) | 72 (59.0) | 17 (77.3) | 0.217¶ |
| Metastatic LN | 74 (37.0) | 26 (46.4) | 46 (37.7) | 2 (9.1) | |
| Pleura | 4 (2.0) | 1 (1.8) | 1 (0.8) | 2 (9.1) | |
| Other metastases | 6 (3.0) | 2 (3.6) | 3 (2.5) | 1 (4.5) | |
| Pathology-ALK detection interval (month) | |||||
| Median | 7.9 | 7.0 | 7.7 | 18.0 | 0.727 |
| Range | 0.1–118.4 | 0.1–63.3 | 0.1–80.9 | 0.2–118.4 | 0.065†† |
| Pathology-ALK detection interval n= (%) | |||||
| ≤24 | 155 (77.5) | 49 (87.5) | 92 (75.4) | 14 (63.7) | 0.140 |
| >24 ≤48 | 29 (14.5) | 6 (10.7) | 20 (16.4) | 3 (13.6) | 0.020‡‡ |
| >48 | 16 (8.0) | 1 (1.8) | 10 (8.2) | 5 (22.7) | |
Comparison between the positive and negative groups.
Comparison between adenocarcinoma and NAC.
Comparison between lung tumor/LN resection and puncture/others.
Comparison between lung tumor and metastatic LN.
Comparison in time between UN and detectable specimens.
Comparison of the time of ALK detection ≤48 and>48 months between UN, detectable specimens. ALK, anaplastic lymphoma kinase; EML4, echinoderm microtubule-associated protein-like-4; LCC, large cell carcinoma; LN, lymph node; NAC, non-adenocarcinoma; NOS, not otherwise specified; UN, unknown (either the result was uninformative or the section was undetectable).
Pathological features and EML4-ALK gene rearrangement
The pathological types of the included patients were adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, large cell carcinoma (LCC) or not otherwise specified (NOS). After statistical treatment, the latter three types were finally entered into the non-adenocarcinoma (NAC) group. The EML4-ALK fusion gene was detectable in 161 adenocarcinoma patients and the 17 NAC patients, with positive rates of 32.3% (52/161) and 23.5% (4/17), respectively (P= 0.438). The pathological tissues were obtained by surgical resection of the primary tumors in 100 cases, resection of the metastatic lymph nodes in 71 cases, bronchoscopic or CT-guided percutaneous fine-needle biopsy of the lung tumors or metastatic lymph nodes in 19 cases, and other methods in the remaining 10 cases, including metastatic brain tumor resection in two cases, metastatic bone tumor fine-needle biopsy in three cases, percutaneous soft tissue fine-needle biopsy in one case, and ultrasound-guided pleural metastasis fine-needle biopsy in four cases. In the 171 patients who received lung tumor and metastatic lymph node resection, the detection rate of the EML4-ALK fusion gene was significantly higher than in the 29 patients who received biopsy and other methods (92.4% [158/171] vs. 24.1% [7/29]), P= 0.003]. There was no statistical difference in the detection rate of the EML4-ALK fusion gene between the primary lung tumor group and the metastatic lymph node group. The median time lag from obtainment of the pathological tissue to the time of ALK detection in the 178 detectable patients and the 22 undetectable patients was 7.4 months and 18.0 months, respectively (P= 0.065). When the time lag from obtainment of the pathological tissues to ALK detection was classified into two groups (≤48-month group and >48-month group), there was a significant difference in the detection rate of the EML4-ALK fusion gene between the two (90.8% [167/184] vs. 68.8% [11/16], P= 0.020).
Epidermal growth factor receptor (EGFR) and KRAS mutations or clinical benefit time from EGFR-tyrosine kinase inhibitor treatment and EML4-ALK gene rearrangement
EGFR and KRAS mutations in 103 of the 200 patients were detected using the DNA sequencing technique (Table 2). The existence of the EML4-ALK fusion gene in patients with wild-type EGFR was significantly higher than in patients with EGFR mutation (42.5% [37/87] vs. 6.3% [1/16], P= 0.005). No EML4-ALK fusion gene was detectable in the four patients with KRAS mutation, while the occurrence of the EML4-ALK fusion gene was 38.4% (38/99) in patients with wild-type KRAS. EGFR and KRAS mutations were not detected in 97 patients; 34 of these patients received EGFR-TKI treatment, including gefitinib or erlotinib. When the patients were classified into two groups (≤6- and >6-month) according to the clinical benefit time for EGFR-TKI treatment, the occurrence of the EML4-ALK fusion gene was 33.3% (9/27) in the ≤6-group, while no EML4-ALK fusion gene was detected in the seven patients of the >6-month group.
Table 2.
Correlations between epidermal growth factor receptor (EGFR) and KRAS mutations or EGFR-tyrosine kinase inhibitor therapeutic efficacy and the anaplastic lymphoma kinase fusion gene
| Feature | Total | Positive | Negative | P-value† |
|---|---|---|---|---|
| EGFR n(%) 103 (100) | ||||
| Mutation | 16 (15.5) | 1 (2.6) | 15 (23.1) | 0.005 |
| Wild | 87 (84.5) | 37 (97.4) | 50 (76.9) | |
| KRAS n(%) 103 (100) | ||||
| Mutation | 4 (3.9) | 0 (0) | 4 (6.2) | 0.294 |
| Wild | 99 (96.1) | 38 (100) | 61 (93.8) | |
| Time of benefiting from EGFR-TKI treatment n(%) 34 (100) | ||||
| ≤6 months | 27 (79.4) | 9 (100) | 18 (72.0) | 0.151 |
| >6 months | 7 (20.6) | 0 (0) | 7 (28.0) |
Comparison between positive and negative groups. EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor.
Selective populations and EML4-ALK rearrangement
In the 129 patients with a never- or light-smoking history, the occurrence of EML4-ALK rearrangement was 34.9% (45/129). When these patients were classified according to gender, the occurrence of EML4-ALK rearrangement was 30.0% (12/40) in male patients and 37.1% (33/89) in female patients, with no significant difference between the two groups (P= 0.435). When the result of EGFR detection was added, there were 71 patients with a never- or light-smoking history and the wild-type EGFR, in whom the occurrence of EML4-ALK rearrangement was 39.4% (28/71). When they were further classified into the male and female groups, the occurrence of EML4-ALK rearrangement was 20.0% (6/30) in the male group and 53.7% (22/41) in the female group, and there was notably a significant difference between the two groups (P= 0.004).
Discussion
ALK gene rearrangement, activation, mutation or amplification mostly occurs in human tumors, including NSCLC, lymphoma, and other malignant tumors.13As early as 2007, Japanese researchers detected the EML4-ALK fusion gene in NSCLC patients.3,14Previous studies reported that the occurrence of the EML4-ALK fusion gene was relatively low in NSCLC. But as the overall incidence of lung cancer is relatively high, EML4-ALK gene rearrangement has become a new lung cancer subtype that has unique clinicopathological features.
Studies have reported that more than 50% of advanced NSCLC patients were older than 65 years of age at the time of diagnosis.15Most studies found that the median age of patients with the EML4-ALK fusion gene was younger than that of patients with negative ALK, which is consistent with our finding that patients with the EML4-ALK fusion gene were relatively younger.5,10–12As is the case with EGFR mutation, the EML4-ALK fusion gene was more frequently seen in never- or light-smoking individuals.2,5,9–12,16However, other studies have argued that the EML4-ALK fusion gene is not correlated with smoking status.8In our study, the occurrence of the EML4-ALK fusion gene in never-smokers was slightly higher than in light-smokers and smokers (34.3% vs. 20.0%, P= 0.103). Some studies have also maintained that the occurrence of the EML4-ALK fusion gene is not correlated with gender.9,10,12However, in a study mainly including patients of Asian origin, Shaw et al. found that the EML4-ALK fusion gene seemed more likely to occur in male patients.11There are still controversies over whether there are gender or racial differences in the occurrence of the EML4-ALK fusion gene.
In clinical practice, it is difficult to obtain a sufficient amount of pathological tissue for EGFR and EML4-ALK gene detection in inoperable patients with advanced NSCLC. When selecting patients, clinicians are also concerned whether the results of EML4-ALK detection in these patients will affect medical treatment. Shaw et al. found that the therapeutic outcome of EGFR-TKI treatment was not sufficient in 10 patients with EML4-ALK fusion gene: stable disease (SD) was achieved in only four patients, and progressive disease (PD) was observed in the remaining six.11In our study, the EML4-ALK fusion gene was detected in about one-third of our patients who clinically benefited from EGFR-TKI treatment of ≤6 months, while the EML4-ALK fusion gene was not detected in patients who clinically benefited from EGFR-TKI treatment of >6 months.
Although the occurrence of the EML4-ALK fusion gene was generally low in NSCLC, the detection rate may be increased in selected patients. In selected patients who had at least two of the four features (female, Asian origin, a never- or light-smoking history, and adenocarcinoma), the occurrence of the EML4-ALK gene rearrangement was 13%.11Another study reported that in adenocarcinoma patients who smoked fewer than 10 packs of cigarettes per year and who had no EGFR or KRAS mutation, the occurrence of the EML4-ALK gene rearrangement was as high as 44.8%.16In our study, occurrence of the EML4-ALK gene rearrangement was 39.4% in such patients, which is similar to the finding of the previous study. It is noteworthy that when these patients were re-grouped according to gender, occurrence of the EML4-ALK gene rearrangement in female patients was significantly higher than that in male patients (P= 0.004).
We found that the detection rate of the EML4-ALK fusion gene in patients who received lung tumor resection or metastatic lymph node resection was significantly higher than in patients who received fine-needle biopsy and other methods (92.4% vs. 24.1%, P= 0.003), indicating that a sufficient amount of pathological tissue is a key factor affecting the detection rate of the EML4-ALK fusion gene. More recent studies have demonstrated that immunohistochemistry (IHC) also has high sensitivity and specificity in detecting ALK.17–19As IHC is a quick and inexpensive technique and needs a relatively small amount of pathological tissue, it may prove to be an important method for improving the detection rate of the EML4-ALK fusion gene. The European Medicines Agency (EMA) has approved the use of Ventana IHC for the diagnosis of ALK-positive NSCLC patients. In addition, we also found that there was no significant difference in the detection rate of the EML4-ALK fusion gene between the pathological specimens from primary lung tumors and those from metastatic lymph nodes, indicating that either primary pathological lesions or metastatic lymph nodes could be used for EML4-ALK fusion gene detection. There have been no previous studies detailing whether the time lag from obtainment of the pathological tissue to the time of EML4-ALK fusion gene detection affects the outcome of detection. In this study, the detection rate in cases when the time lag from the obtainment of the pathological tissue and EML4-ALK fusion gene detection was ≤48 months was significantly higher than that in cases >48 months (P= 0.020), suggesting that clinicians should advise patients of the possible benefits of undergoing EML4-ALK fusion gene detection as early as possible, especially in young adenocarcinoma patients with a never- or light-smoking history, as well as in dominant groups with wild-type EGFR ALK gene potential.
Conclusion
In summary, the EML4-ALK fusion gene is more likely to be detected in young NSCLC patients, especially in dominant groups with adenocarcinoma who smoke fewer than 10 packs of cigarettes per year and with EGFR or KRAS mutations. Either primary or metastatic lesions could be used for EML4-ALK fusion gene detection. The optimal time lag from the obtainment of the pathological tissue to the time of EML4-ALK fusion gene detection is ≤48 months.
Acknowledgments
This study was financially supported by the Chinese National Major Project for New Drug Innovation (2008ZX09312, 2012ZX09303012), the Chinese Central Health Authority Special Fund (B2009B124), the Chinese National High Technology Research and Development Program (863 Program) (2011AA02A110), the Beijing Municipal Science and Technology Commission Major Project for New Drug Innovation (Z111102071011001), and the Special Foundations of Wu Jieping Medical Foundation of China (320.6799.1109).
Disclosure
No authors report any conflict of interest.
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