Research On The Reference Material For EML4-ALK Fusion Gene Detection Based On CRISPR/CAS9 Technology

Lung cancer is one of the most common malignant tumors and one of the major causes of cancer-related death in the world.Of the patients who diagnosed with lung cancer,80%to 85%were Non-small cell lung cancer(NSCLC).Most of these cases were diagnosed in the middle or late stages,withing only 10%to 15%five-year survival rate.In the past,the traditional treatment of NSCLC including surgery,chemotherapy,and radiotherapy.While with the emerging clinical paradigm of precision medicine,molecular diagnosis and targeted drug therapy is becoming more and more significant.At present,a series of NSCLC-related targeted gene mutations and gene rearrangement have been found.Of which accurate detection of the rearrangements of echinoderm microtubule-associated protein-like 4(EML4)gene and the anaplastic lymphoma kinase(ALK)gene is important to select the subgroup patients of NSCLC patients for crizotinib therapy.Despite many attempts have been done to improve the EML4-ALK detecting in clinical practice,the inaccuracy of testing is still an important yet to be solved problem within all the methodologies,including fluorescence in situ hybridization(FISH),immunohistochemistry(IHC),real-time quantitative reverse transcription PCR(RT-qPCR)and next-generation sequencing(NGS).For the clinical laboratories,in order to improve accuracy and reliability of detection,a proper reference material for validation of laboratory-developed tests,verification of commercial detection kits,internal quality control and proficiency testing is of prime important.Therefore,to ensure the accuracy and reproducibility of detection,here we developed a kind of well-characterized candidate reference materials based on clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9(CRISPR/Cas9)editing and xenografts for various kind of EML4-ALK testing.In this study,CRISPR/Cas9 technique was used to edited three types of cell lines containing EML4-ALK rearrangements variant 1,2,and 3a/b.Single guide RNAs(sgRNAs)were designed in silico to target specific EML4 locus and ALK locus.pX330 vector expressing Cas9 and specific sgRNA was transfected into HEK293T cells.The edited individual positive clones were verified by PCR,RT-PCR,and western blotting.Followed by subcutaneous inoculation,the formalin fixed paraffin embedded(FFPE)samples based on CRISPR/Cas9 and xenograft were prepared and tested for suitability as candidate reference materials by FISH,IHC,RT-qPCR and NGS.All the results were compared with the authentic clinical specimens to assess the commutability.In addition,homogeneity and stability assessments were also performed.By editing HEK293T cells by CRISPR/Cas9 system,three kinds of cells containing EML4-ALK variant 1,2,and 3a/b have been constructed and named as 1-F8-G9,9-E11-G5,14-F9-E8,respectively.The edited cells were all verified on DNA,RNA and protein levels,and can be observed a high frequency of exact fusion events.Via subcutaneously injection,the corresponding xenograft tumors were obtained in their fourth week and embedded as FFPE blocks.All kind of FFPE samples derived from xenograft tumors were found with typical histological structures by using HE staining,such as tumor infiltrating,inflammation,and partial necrosis.In addition,by FISH,IHC,RT-qPCR,and NGS,all the materials were verified to be EML4-ALK rearrangements-positive.Among four methodologies for EML4-ALK detection,the validation test showed 100%concordance.Meanwhile,compared with clinical ALK positive-NSCLC specimens,the novel FFPE samples showed great commutability.Furthermore,the materials were also completely homogeneous and stable for at least 2 months.To sum up,we developed a kind of novel EML4-ALK rearrangements reference material in a simple,rapid and economical way.Without limitations on variant types and production,our novel FFPE samples based on CRISPR/Cas9 editing and xenograft are homogeneous,stable,and suitable for all the methodologies(especially for NGS)as candidate reference materials in the validation,verification,internal quality control and proficiency testing of EML4-ALK detection.More specifically,the method used in this study that construct artificial cell lines withing the same genomic background but different mutations or rearrangements through CRISPR/Cas9 technology can play a guiding role to develop other kind of reference materials,such as the reference materials for tumor tissue mutation detection or circulating tumor DNA mutation detection and so on.