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Oncol Lett
2015 Dec 01;106:3385-3392. doi: 10.3892/ol.2015.3740.
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EML4-ALK translocation is associated with early onset of disease and other clinicopathological features in Chinese female never-smokers with non-small-cell lung cancer.
Ren W
,
Zhang BO
,
Ma J
,
Li W
,
Lan J
,
Men H
,
Zhang Q
.
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Non-small-cell lung cancer (NSCLC) with echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) translocation is resistant to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, but responds to the ALK-TKI crizotinib. Characterization of EML4-ALK translocation may provide invaluable information to facilitate disease diagnosis and improve the outcome of customized treatment. Although the occurrence of EML4-ALK translocation is likely to be affected by the smoking habits and gender of patients, the translocation has not been characterized extensively in female never-smokers with NSCLC. Therefore, 280 female never-smokers that were diagnosed with NSCLC were enrolled in the present study, and characteristics of EML4-ALK translocation, including the frequency, were determined in these NSCLC patients. EML4-ALK fusion variants were detected using Multiplex one-step reverse transcription-polymerase chain reaction and subsequently confirmed by DNA sequencing and Vysis ALK Break Apart fluorescence in situ hybridization analysis. The EML4-ALK fusion variants were detected in 21 carcinoma tissue specimens, accounting for 7.5% of the enrolled patients. Out of these patients with EML4-ALK fusion variants, EML4-ALK fusion variant 1 was identified in 12 patients, indicating that variant 1 is the most common type of EML4-ALK fusion gene in the present cohort of patients. ALK mRNA was aberrantly expressed in all the tissues with EML4-ALK translocation, but not in the carcinoma tissues without EML4-ALK translocation. In addition, the EML4-ALK translocation was more frequently found in younger patients. The median age of patients with EML4-ALK translocation was 50.95±2.29 years, which was significantly younger (P<0.01) than the median age of the patients without EML4-ALK translocation (57.15±0.56). The EML4-ALK translocation was detected exclusively in undifferentiated tumors that were graded as poorly- or moderately-differentiated carcinomas and suspected to be more malignant compared with well-differentiated tumors. In summary, the present study found that 7.5% of patients with NSCLC that are female never-smokers harbor EML4-ALK translocations, which are associated with the aberrant expression of ALK mRNA, early onset of disease and undifferentiated carcinomas.
Figure 1. Screening for EML4-ALK fusion transcripts in the tissues obtained from female never-smokers with non-small cell lung cancer. (A) Representative DNA gel images revealing the expression of the EML4-ALK fusion transcript variants V1, V3a, V3b, V4a, V5a and E17-20, and ABL controls, as determined by Multiplex one-step reverse transcription-polymerase chain reaction. (B) ALK Break Apart fluorescence in situ hybridization analysis revealed ALK inversion in carcinoma tissues. Two probes targeting the sequences prior to and following the breaking point were labeled by red and green fluorescent dye, respectively. Representative images of EML4-ALK-negative (left) and -positive (right) carcinomas are shown. The ALK inversion in EML4-ALK-positive carcinomas is indicated by two separated red and green dots (right) located at least two signal diameters apart. NC, negative case; M, DNA ladder; EML4-ALK, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase; ABL, Abelson murine leukemia viral oncogene homolog.
Figure 2. EML4-ALK fusion transcript variants in female never-smokers with non-small cell lung cancer. (A) Representative DNA sequencing chromatographs and schematic drawings revealing EML4-ALK fusion junctions. Five representative EML4-ALK fusion transcript variants were detected by Multiplex reverse transcription-polymerase chain reaction. The sequences and variant types were determined by direct DNA sequencing. The 5 detected EML4-ALK fusion variants consisted of V1, V3a/3b, V4a, V5a and E17-20. Variant 2 was not detected in the present cohort of patients. Schematic drawings are exhibited below the corresponding DNA sequences to indicate the organization of fusion junctions. (B) Pie graph showing the frequency of EML4-ALK fusion variants. A total of 5 different EML4-ALK fusion variants were detected, consisting of V1, V3a/3b, V4a, V5a, and E17-20. The number and the percent of each variant are indicated in the graph. Variant 2 was not detected in these carcinoma tissues. EML4-ALK, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase.
Figure 3. Aberrant expression of ALK mRNA and protein. (A) Scatter graph demonstrating that the expression of ALK mRNA is significantly higher (P<0.0001) in carcinoma tissues harboring EML4-ALK fusion compared with tissues without the fusion gene in female never-smokers with non-small cell lung cancer. The values are expressed as the mean ± standard error of the mean. (B) Representative images exhibiting the immunohistochemical staining for ALK in carcinoma tissues. The ALK protein was aberrantly expressed in carcinoma tissues harboring the EML4-ALK fusion gene (EML4-ALK+), but ALK protein expression was absent in tissues without the EML4-ALK fusion gene (EML4-ALK-). EML4-ALK, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase.
Figure 4. Summary of the median age of ALK+ and ALK- patients and the two combined. The median age in ALK+ was significantly lower compared with the median age in ALK- patients or in the two groups combined. The values are expressed as the mean ± standard error of the mean. *P<0.05 and **P<0.01. EML4-ALK, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase; ALK+, patients harboring EML4-ALK fusion; ALK-, patients without EML4-ALK fusion.
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