Limits...
Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression.

Zhang X, Zhang S, Yang X, Yang J, Zhou Q, Yin L, An S, Lin J, Chen S, Xie Z, Zhu M, Zhang X, Wu YL - Mol. Cancer (2010)

Bottom Line: Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer.However, expression of EML4 did not differ between the groups.RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK-positive patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Medical Research Center of Guangdong General Hospital, Guangdong Lung Cancer Institute, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.

ABSTRACT

Background: The anaplastic lymphoma kinase (ALK) gene is frequently involved in translocations that lead to gene fusions in a variety of human malignancies, including lymphoma and lung cancer. Fusion partners of ALK include NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC. Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer.

Results: RACE-coupled PCR sequencing was used to assess ALK fusions in a cohort of 103 non-small cell lung carcinoma (NSCLC) patients. Within this cohort, the EML4-ALK fusion gene was identified in 12 tumors (11.6%). Further analysis revealed that EML4-ALK was present at a frequency of 16.13% (10/62) in patients with adenocarcinomas, 19.23% (10/52) in never-smokers, and 42.80% (9/21) in patients with adenocarcinomas lacking EGFR and KRAS mutations. The EML4-ALK fusion was associated with non-smokers (P = 0.03), younger age of onset (P = 0.03), and adenocarcinomas without EGFR/KRAS mutations (P = 0.04). A trend towards improved survival was observed for patients with the EML4-ALK fusion, although it was not statistically significant (P = 0.20). Concurrent deletion in EGFR exon 19 and fusion of EML4-ALK was identified for the first time in a Chinese female patient with an adenocarcinoma. Analysis of ALK expression revealed that ALK mRNA levels were higher in tumors positive for the EML-ALK fusion than in negative tumors (normalized intensity of 21.99 vs. 0.45, respectively; P = 0.0018). However, expression of EML4 did not differ between the groups.

Conclusions: The EML4-ALK fusion gene was present at a high frequency in Chinese NSCLC patients, particularly in those with adenocarcinomas lacking EGFR/KRAS mutations. The EML4-ALK fusion appears to be tightly associated with ALK mRNA expression levels. RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK-positive patients.

Show MeSH

Related in: MedlinePlus

Assessment of ALK fusion status by RACE-coupled PCR sequencing. (A) Representative image showing gel electrophoresis results for first-round PCR products. Lane M, marker (200 bp ladder); lanes P, positive controls (2 lanes); lane NTC, no template control; other lanes, samples 1 through 45. (B) Representative image showing gel electrophoresis results for second-round PCR products. Lane organization is identical to (A). (C) Representative sequencing chromatograph showing ALK fusion with EML4. (D) RT-PCR validation for samples positive for ALK fusion expression. RT-PCR was used to confirm EML4-ALK fusion transcript expression in 12 samples. Sample 68 is the V5 variant (E2;A20, 408 bp). Samples 268, 171, and 320 are V9 variants (E18;A20, 2256 bp). Samples 446, 433, 138, and 347 are V1 variants (E13;A20, 1689 bp). Sample 534 is the V2 variant (E20;A20, 2445 bp). Samples 131, 421, and 431 are V3a/b variants (E6;A20 or E6 ins33;A20, 867 and 900 bp, respectively). Lane M, marker (100 bp ladder).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2908583&req=5

Figure 1: Assessment of ALK fusion status by RACE-coupled PCR sequencing. (A) Representative image showing gel electrophoresis results for first-round PCR products. Lane M, marker (200 bp ladder); lanes P, positive controls (2 lanes); lane NTC, no template control; other lanes, samples 1 through 45. (B) Representative image showing gel electrophoresis results for second-round PCR products. Lane organization is identical to (A). (C) Representative sequencing chromatograph showing ALK fusion with EML4. (D) RT-PCR validation for samples positive for ALK fusion expression. RT-PCR was used to confirm EML4-ALK fusion transcript expression in 12 samples. Sample 68 is the V5 variant (E2;A20, 408 bp). Samples 268, 171, and 320 are V9 variants (E18;A20, 2256 bp). Samples 446, 433, 138, and 347 are V1 variants (E13;A20, 1689 bp). Sample 534 is the V2 variant (E20;A20, 2445 bp). Samples 131, 421, and 431 are V3a/b variants (E6;A20 or E6 ins33;A20, 867 and 900 bp, respectively). Lane M, marker (100 bp ladder).

Mentions: RNA samples from a total of 103 NSCLC cases were reverse-transcribed to make cDNA, followed by oligo-dC tailing. Two successive rounds of PCR were used to identify potential fusion fragments (Fig. 1A, B). BigDye3.1-labeled products were then sequenced to identify fusions between ALK and potential partners (Fig. 1C). Based on a sequence alignment with the ALK reference sequence (NCBI accession number: NM_004304.3), in total, 12 samples were identified as ALK fusion-positive (Table 1).


Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression.

Zhang X, Zhang S, Yang X, Yang J, Zhou Q, Yin L, An S, Lin J, Chen S, Xie Z, Zhu M, Zhang X, Wu YL - Mol. Cancer (2010)

Assessment of ALK fusion status by RACE-coupled PCR sequencing. (A) Representative image showing gel electrophoresis results for first-round PCR products. Lane M, marker (200 bp ladder); lanes P, positive controls (2 lanes); lane NTC, no template control; other lanes, samples 1 through 45. (B) Representative image showing gel electrophoresis results for second-round PCR products. Lane organization is identical to (A). (C) Representative sequencing chromatograph showing ALK fusion with EML4. (D) RT-PCR validation for samples positive for ALK fusion expression. RT-PCR was used to confirm EML4-ALK fusion transcript expression in 12 samples. Sample 68 is the V5 variant (E2;A20, 408 bp). Samples 268, 171, and 320 are V9 variants (E18;A20, 2256 bp). Samples 446, 433, 138, and 347 are V1 variants (E13;A20, 1689 bp). Sample 534 is the V2 variant (E20;A20, 2445 bp). Samples 131, 421, and 431 are V3a/b variants (E6;A20 or E6 ins33;A20, 867 and 900 bp, respectively). Lane M, marker (100 bp ladder).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908583&req=5

Figure 1: Assessment of ALK fusion status by RACE-coupled PCR sequencing. (A) Representative image showing gel electrophoresis results for first-round PCR products. Lane M, marker (200 bp ladder); lanes P, positive controls (2 lanes); lane NTC, no template control; other lanes, samples 1 through 45. (B) Representative image showing gel electrophoresis results for second-round PCR products. Lane organization is identical to (A). (C) Representative sequencing chromatograph showing ALK fusion with EML4. (D) RT-PCR validation for samples positive for ALK fusion expression. RT-PCR was used to confirm EML4-ALK fusion transcript expression in 12 samples. Sample 68 is the V5 variant (E2;A20, 408 bp). Samples 268, 171, and 320 are V9 variants (E18;A20, 2256 bp). Samples 446, 433, 138, and 347 are V1 variants (E13;A20, 1689 bp). Sample 534 is the V2 variant (E20;A20, 2445 bp). Samples 131, 421, and 431 are V3a/b variants (E6;A20 or E6 ins33;A20, 867 and 900 bp, respectively). Lane M, marker (100 bp ladder).
Mentions: RNA samples from a total of 103 NSCLC cases were reverse-transcribed to make cDNA, followed by oligo-dC tailing. Two successive rounds of PCR were used to identify potential fusion fragments (Fig. 1A, B). BigDye3.1-labeled products were then sequenced to identify fusions between ALK and potential partners (Fig. 1C). Based on a sequence alignment with the ALK reference sequence (NCBI accession number: NM_004304.3), in total, 12 samples were identified as ALK fusion-positive (Table 1).

Bottom Line: Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer.However, expression of EML4 did not differ between the groups.RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK-positive patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Medical Research Center of Guangdong General Hospital, Guangdong Lung Cancer Institute, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.

ABSTRACT

Background: The anaplastic lymphoma kinase (ALK) gene is frequently involved in translocations that lead to gene fusions in a variety of human malignancies, including lymphoma and lung cancer. Fusion partners of ALK include NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC. Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer.

Results: RACE-coupled PCR sequencing was used to assess ALK fusions in a cohort of 103 non-small cell lung carcinoma (NSCLC) patients. Within this cohort, the EML4-ALK fusion gene was identified in 12 tumors (11.6%). Further analysis revealed that EML4-ALK was present at a frequency of 16.13% (10/62) in patients with adenocarcinomas, 19.23% (10/52) in never-smokers, and 42.80% (9/21) in patients with adenocarcinomas lacking EGFR and KRAS mutations. The EML4-ALK fusion was associated with non-smokers (P = 0.03), younger age of onset (P = 0.03), and adenocarcinomas without EGFR/KRAS mutations (P = 0.04). A trend towards improved survival was observed for patients with the EML4-ALK fusion, although it was not statistically significant (P = 0.20). Concurrent deletion in EGFR exon 19 and fusion of EML4-ALK was identified for the first time in a Chinese female patient with an adenocarcinoma. Analysis of ALK expression revealed that ALK mRNA levels were higher in tumors positive for the EML-ALK fusion than in negative tumors (normalized intensity of 21.99 vs. 0.45, respectively; P = 0.0018). However, expression of EML4 did not differ between the groups.

Conclusions: The EML4-ALK fusion gene was present at a high frequency in Chinese NSCLC patients, particularly in those with adenocarcinomas lacking EGFR/KRAS mutations. The EML4-ALK fusion appears to be tightly associated with ALK mRNA expression levels. RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK-positive patients.

Show MeSH
Related in: MedlinePlus