Noninvasive Detection Of EML4-ALK Fusion Gene

Anaplastic lymphoma kinase(ALK),a receptor tyrosine kinase(RTK),was first found in anaplastic large cell lymphoma.The ALK gene can fuse with the echinoderm microtubule-associated protein-like 4(EML4)gene to form the EML4-ALK fusion gene by chromosome inversion.ALK kinase is activated by EML4 promoter,leading to carcinogenesis and promoting the development and progression of cancer.Recent studies have shown that EML4-ALK fusion gene exists in a variety of solid tumors including lung cancer,which has the highest detection rate in non-small cell lung cancer(NSCLC).In addition,the patients with EML4-ALK fusion gene have distinctive clinical features,suggesting that the target of EML4-ALK fusion gene is a characteristic molecular biomarker of lung adenocarcinoma.At present,at least 14 kinds of EML4-ALK variant isoforms have been found.All of these fusion genes have biological functions and are able to express a chimeric tyrosine kinase which can activate the membrane catalytic region of the ALK gene and the signal pathway,then promote cell proliferation,survival or migration,and finally achieve the effect of cell carcinogenesis.Therefore,rapid,accurate and early detection of EML4-ALK fusion gene is essential for early diagnosis,molecular typing and targeted therapy of lung cancer.Currently,all of the EML4-ALK fusion gene detection methods are designed for the tumor tissue obtained from surgery or biopsy.Therefore,early detection and real-time monitoring cannot be achieved,and the detection results may be false negative because of the tumor heterogeneity.In recent years,liquid biopsy provides a new idea for the detection of genes,which can effectively avoid the limitations of biopsy.Exosomes are one of the main detection objects in liquid biopsy.The exosomes are formed and secreted out of the cells through several processes.First of all,the early endosomes are formed by endocytosis and become multivesicular endosomes later.Since the combination of multivesicular endosomes and cell membrane,the vesicles in the multivesicular endosomes are delivered to the outside of cells,and finally form the exosomes.Therefore,exosomes contain many signal molecules reflecting the physiological state of the secretory cells,like proteins,DNA,microRNA,et al.As a result,the exosomes contain the molecular information related to cell pathology and provide a rich source of potential biomarkers.Because different types of cells in different diseases can secrete exosomes containing specific components.It enables the isolation and purification of exosomes from the body fluid,analyzing their composition and genetic information,which is expected to be used in the diagnosis and monitoring of the diseases,especially in the detection of tumor markers.Therefore,exosomes are very likely to be used as a non-invasive method for the diagnosis of the diseases,which can replace the tissue biopsy under certain conditions to alleviate the pain of patients and improve the early diagnosis rate of the diseases.At present,the protein and microRNA derived from tumor cells have been found in exosomes,but whether exosomes contain mRNA of fusion gene has not been reported yet.There are three cell lines with this fusion gene,H3122,H2228 and DFCI032 cells now.H3122 cells and H2228 cells are selected as the experimental objects,and A549 cells without the EML4-ALK fusion gene are selected as negative control,preliminarily verifying the feasibility of detection of fusion gene mRNA in exosomes.First,the EML4 and ALK gene sequences were searched on NCBI,and the primers were designed according to the different fusion forms of these genes.The total RNAs extracted from H3122 and A549 cells were processed by reverse transcription PCR(RT-PCR)to produce cDNA.The sequencing results confirmed that the thirteenth exon of EML4 in H3122 cells was fused with the twentieth exon of ALK.This fusion form was also known as the V1 fusion form.Meanwhile,the proteins extracted from these two cells were tested by Western Blot to detect the expression of ALK fusion protein.The results of Western Blot showed that H3122 cells had a specific band at 120 kD,which was consistent with the size of EML4-ALK V1 fusion protein.Then,after 24-48 hours of starvation treatment,H3122 and A549 cell culture supernatants were collected.And exosomes were extracted from these supernatants.Through the morphological observation under electron microscope and the analysis of the specific surface membrane protein of exosomes,it was confirmed that exosomes were successfully extracted.Then,RNAs extracted from exosomes were processed by RT-PCR to produce c DNAs.And the EML4-ALK fusion gene was successfully amplified by cDNA from exosomes isolated from H3122 cell culture supernatant.The sequencing results showed that the fusion form in the exosomes was V1,which was consistent with that in H3122 cells.Using the same experimental method for the detection of EML4-ALK fusion gene in H2228 cells,the sequencing results confirmed that the fusion form of H2228 was V3 a and V3 b.And the EML4-ALK fusion gene could also be detected in exosomes isolated from the cell supernatant of H2228 cells,and its fusion form was consistent with that in H2228 cells.In addition to detect the lung cancer cells H3122 and H2228,the chronic leukemia cells K562 with BCR-ABL fusion gene were also detected,in the exosomes of which the BCR-ABL fusion gene was also successfully detected.The above studies confirmed that it was a common phenomenon that mRNA of fusion gene was secreted by exosomes into the extracellular matrix,which laid the foundation for the clinical detection of exosomes.By RT-PCR,IHC and Western Blot,EML4-ALK fusion gene was detected in tumor tissues of 89 patients with NSCLC.Finally,5 patients with V1 fusion form,1 patient with V2 fusion form,8 patients with V3 fusion form and 1 patient with V5 fusion form were screened out.Then,the EML4-ALK fusion gene was detected in the serum exosomes of these 15 patients positive in histological examination.The EML4-ALK fusion gene was successfully detected in serum exosomes of 14 patients.And the fusion form of EML4-ALK fusion gene in the serum exosomes was consistent with that in the tumor tissues.Among the 74 patients negative in histological examination,4 positive patients were screened out by the method of detecting serum exosomes.To further expand the scope of histological examination of these 4 patients,they were confirmed to be positive of EML4-ALK.The experimental results showed that the detection method of serum exosomes not only was noninvasive,but also could overcome the disadvantages of tissue detection caused by tumor heterogeneity,and improve the accuracy of EML4-ALK fusion gene detection.Because these 89 patients were in the late stage of lung cancer,the experimental study about the early diagnosis cannot be carried out by using these serum samples.Therefore,we established a tumor bearing nude mouse model of lung cancer cells.H3122 cells,H2228 cells and A549 cells(1×106 cells per mouse)were vaccinated in subcutaneous back of 4-6 weeks old female nude mice.After vaccination,the tumor volume was measured regularly.At set intervals,4 mice were taken from H3122 experimental groups and H2228 experimental groups respectively,and 1 mouse was taken from the A549 control group.Blood was extracted from the eyeballs of these mice.Then the serum exosomes were isolated from blood and used to detect EML4-ALK fusion gene.The experimental results showed that the tumors were able to be measured on back of the nude mice inoculated with H3122 cells on the fourth day after vaccination.However,the EML4-ALK fusion gene could be detected in serum exosomes on the second day after vaccination.In the early stage of tumor formation,the EML4-ALK fusion gene could also be detected in the serum exosomes of the nude mice inoculated with H2228 cells.The tumor bearing test in nude mice provides the experimental basis for the study of the early detection of EML4-ALK fusion gene with serum exosomes as target.In conclusion,the research of this topic first confirmed that the exosomes derived from tumor cells contained mRNA of the fusion gene.This topic also provides a certain laboratory reference for the establishment of a new detection method for detecting fusion genes using serum exosomes and the use of targeted drugs.