Efficacy Evaluation Of ALK Fusion Non-small Cell Lung Cancer And Mechanism Of Action Of Rare Fusion GALNT7-ALK

Background and ObjectiveA large number of clinical studies have shown that the efficacy of targeted therapy in patients with Anaplastic Lymphoma Kinase(ALK)positive in non-small cell lung cancer is significantly superior to chemotherapy,but there is still a lack of domestic studies on the efficacy and prognosis of ALK fusion combined therapy(combination of targeted therapy and chemotherapy).The purpose of this study was to explore the efficacy and prognosis of different treatment modalities,the differences between ALK fusion gene profiles and related clinical information,in order to find the most appropriate treatment modalities for ALK positive patients with advanced non-small cell lung cancer(NSCLC)and molecular markers related to efficacy prediction at the molecular level of targeted therapy.In addition,previous studies have shown that different ALK gene fusion ligand will lead to different sensitivity to ALK tyrosine kinase inhibitors,so the fusion type is closely related to clinical efficacy.To explore the influence of rare fusion on ALK transcription and expression,as well as the sensitivity to ALK Tyrosine Kinase inhibitors(ALK-TKIs),so as to verify the novel ALK fusion genes at the DNA,RNA and ALK protein levels,and provide an important basis for the selection of treatment methods for patients with this type of ALK fusion in the future.Methods1.Exploration of the clinical features,treatment methods,efficacy and prognosis for advanced NSCLC with positive ALK gene fusionCollect and screen the data of patients with advanced non-small cell lung cancer with ALK protein-positive immunohistochemical results from January 2016 to December 2019 in the Department of Pathology,Henan Cancer Hospital,and enroll the patients according to the screening conditions.According to the treatment method,they are divided into targeted therapy group(treatment based on crizotinib),chemotherapy group(treatment based on double platinum chemotherapy),and combination therapy group(treatment based on targeted combined chemotherapy).Samples of enrolled patients were collected for next-generation sequencing(NGS),and the identification analysis of gene fusion was carried out on the sequencing results.The clinical characteristics,treatment methods,mutation types and survival prognosis of these patients were analyzed statistically.2.Identification the architecture of the novel ALK fusions.Analyze mutation sites,fusion methods and mutation abundance through NGS sequencing results.The integrated genomics viewer(Integrative Genomics Viewer,IGV)visualizes the fusion base sequence.3.Exploreation of ALK expression modification influenced by novel ALK fusion.Probe was designed in the ALK kinase domain and non-kinase domain,and the expression levels of GALNT7-ALK domain and non-kinase domain were compared by RT-PCR method.Immunohistochemistry was used to determine whether the fusion promoted ALK expression at the protein level.4.Investigatation of the clinical response and prognosis of the patient treated with ALK-TKIsClinical medical records and CT,MRI and other information of this patients in different periods were collected in detail,and patients were followed up strictly to evaluate the therapeutic response of this fusion to ALK-TKIS.Results1.A total of 193 patients with positive ALK protein were screened out in this study,among which 88 patients met the inclusion requirements.The results of NGS sequencing were positive for ALK fusion.2.The relationship between various clinical data and the patient's progression-free survival(PFS):The results of Kaplan-Meier survival curve analysis show that age,smoking status,and Eastern Cooperative Oncology Group(Eastern Cooperative Oncology Group,ECOG)score,clinical stage,EML4-ALK variant subtype were not statistically different with PFS(P>0.05).However,we found that the PFS of adenocarcinoma and non-adenocarcinoma patients was statistically different,and the PFS of adenocarcinoma was significantly prolonged(P=0.023,median PFS was 262 days vs 179 days,respectively).3.Three different treatment methods were compared for PFS.Using Kaplan-Meier method analysis and multivariate cox regression analysis and correction,the PFS of the three different treatments were significantly different(Log-rank P<0.001).There were differences in the PFS comparison between the targeted therapy group and the chemotherapy group(Log-rank P=0.006)and the comparison between the chemotherapy group and the combination therapy group(Log-rank P<0.001).Although the median PFS of the combination therapy group was higher than that of the targeted group(439 days vs 346 days),there was no significant difference in PFS between the combination therapy group and the targeted therapy group(Log-rank P=0.170).4.The Disease Control Rate(DCR)of chemotherapy group,targeted therapy group and combination therapy group were 80.0%,84.0%and 87.5%,respectively(P=0.802).The Objective Response rates(ORR)of the three groups were 46.7%,68.0%and 81.3%(P=0.064),respectively.5.Among the 88 ALK-positive specimens tested by NGS,EML4-ALK accounted for 89.8%(79/88)of the total(73 cases were a single EML4-ALK fusion,6 cases were a double fusion containing EML4-ALK).EML4-ALK is dominated by V1,V2 and V3,which account for 38.0%(30/79),16.5%(13/79)and 31.6%(25/79)respectively.The three types account for 86.1%of EML4-ALK.6.Chi-square test was used to compare the clinical data of patients in the single EML4-ALK type group and the group carrying other variation types(including rare fusion and double fusion).It was found that the age,gender,smoking status,pathological classification,clinical stage and Progression location of patients were not correlated with the two groups of genotypes.However,patients with EML4-ALK had better clinical status compared to those with other types of variation,which was correlated with ECOG score(P=0.049).7.There were no significant differences in PFS between patients with single EML4-ALK type group and those with other variant types(P=0.064,median PFS was 278 days vs 210 days,respectively).8.There was no significant difference in progression-free survival among different variants of EML4-ALK type(V1,V3 and Others)(P=0.229).9.The sequencing results by the DNA-based NGS(Burning Rock,Guangzhou,China)revealed a novel ALK rearrangement by a fusion of the GALNT7 intergenic region on 4q34.1 to the intron 19 of ALK on 2p23(GALNT7-ALK)and the breakpoint of GALNT7-ALK fusion was G2:A20 and the abundance rate was 15.33%10.RT-PCR results showed mRNA levels Expression of GALNT7-ALK at the C-terminal region(backward from the breakpoint at exon 20 in ALK rearrangement)was 13.2-fold(223.89:16.91)than expression at region across 18 and 19 exons,which showed a great expression in ALK kinase domain.11.The verification result of FFPE samples via Ventana-IHC Immunohisto-chemical staining(ALK D5F3 antibody)showed a strong expression of ALK protein.It proved that this ligand can activate ALK,make ALK expression,and cause the production of carcinogenic fusion protein.12.The mass of this patient was significantly reduced after first-line crizotinib targeted therapy,and the patient achieved a partial response(PR)to crizotinib and usutained response for 7 months.After brain metastatic were observed,the patient was treated with Alectinib 600mg twice daily,and the disease was significantly controlled,which has been maintained for 7 months.The efficacy was evaluated as PR.This patient showed no clinical symptom of progression and good tolerance till January 2021,the last follow-up date.Conclusions1.Comparing the clinical characteristics and prognosis of the three treatment methods,the targeted therapy group and the combination therapy group have longer PFS than chemotherapy patients.However,there was no significant difference in median PFS between the combination therapy group and the targeted therapy group,and combination therapy was not superior to targeted therapy alone.2.A novel fusion gene,GALNT7-ALK,was identified.Bioinformatics analysis showed that exon 2 of GALNT7 fuses with exon 20 of ALK,which specifically activates transcription and expression of the ALK kinase domain.Moreover,the fusion gene achieved beneficial effects with ALK-TKIs and patients have significant benefits.