ALK Associated NSCLC:Study And Perspective Of Immunohistochemical Screening And EML4-ALK Fusion Heterogeneity

The incidence of lung cancer had rapid increased during the past decade.A new classification for lung cancer had updated by the 2015 World Health Organization(WHO)classification of tumors of the lung,pleura,thymus and heart,which proposed a new emphasis on genetic studies,in particular,integration of molecular testing.The targeted therapy gained driver genes has been leading the new personalize treatment strategies for precision medicine,which will bring the development of detection technology for more and more potential genes with clinical significance.The result of treatment from targeted drugs for anaplastic lymphoma kinase(ALK)gene fused non-small cell lung cancer(NSCLC)was better than that of traditional chemotherapy.The accurate diagnosis is crucial to precision treatment for ALK positive NSCLC.In China,molecular detection is still in early stages,and ALK gene detection and diagnosis process still needs further optimization continuously combined with clinical situation.Objective:A variety of methods to detect ALK positive lung cancer had been tested to investigate the frequency of ALK associated NSCLC and the clinicopathological characteristics,and to explore a diagnosis process suitable for the local hospitals.EML4-ALK fusion variants were analyzed for the significance of diagnosis and treatment.Methods:(1)The test for ALK status in 566 cases of NSCLC had been performed by Ventana automated immunostainer with anti-ALK D5F3(Ventana-IHC),FISH and RT-PCR.The staining from Ventana-IHC was comparative studied.The histological features,treatment and follow-up were investigated.(2)60 NSCLCs were detected by Ventana-IHC and manual staining using 4 different antibodies of D5F3(Ventana),D5F3(Cell Signaling),1A4/1H7(OriGene),5A4(Abcam).Their expressions from 4 antibodies were compared with those by Ventana-IHC.(3)EML4-ALK fusion variants of 36 cases of ALK positive NSCLC were detected by RT-PCR and sequencing and the clinicopathological characteristics with different fusion variant were analyzed.Results:(1)38 cases(6.7%)of ALK gene fusion NSCLC were identified by Ventana-IHC.36 cases with abnormal staining from Ventana-IHC were identified to be ALK-negative NSCLC by FISH and RT-PCR.The incidence rate of ALK gene fusion NSCLC in male(7.7%)was higher than that in female patients(6.0%);Statistically,only the age difference in ? 60 years(10.6%)and>60 years(3.5%)was significantly related to ALK positive NSCLC.The number of never cigarette smokers(78.9%)was more than that of smoking patients.One case showed EGFR gene mutation coexisting with ALK gene fusion.Histologically,the ALK positive NSCLC patients were consisted mostly of adenocarcinoma(81.6%)among which 18 cases of solid predominant subtype with mucin production,9 cases of acinar predominant subtype,1 case of papillary predominant subtype,3 cases of invasive mucinous adenocarcinoma.And the non-adenocarcinoma included 3 cases of squamous cell carcinoma,3 cases of adenosquamous carcinoma and 1 case of pleomorphic carcinoma.80%patients received Crizotinib did not suffer from worsening.(2)The sensitivity of different antibodies used manual-IHC each was 93.8%,84.4%,93.8%,56.3%,and all the speciticity was 100%.The consistency with Ventana-IHC was 96.7%,91.7%,96.7%and 76.7%,respectively.The validity of immunohistochemical staining in surgical resection specimens was better than in small biopsies.(3)The detection rate of EML4-ALK fusion variant for paraffin-embedded tissue was 52.8%(19/36).The most variant was V1(52.6%),followed by V3(31.6%)and V2(15.8%).V3 included 1 case of V3a(E6;A20),1 case of 3b(E6ins33;A20)and 4 cases of V3a + V3b.The differences between various fusion variants and their clinicopathological characteristics or survival time were no statistical significance.Conclusion:(1)NSCLC with ALK gene rearrangement was an unusual lung cancer,presenting distinctive clinical and histological features.Ventana-IHC may be used as a reliable and valid technique to detect the ALK rearrangement in NSCLC.(2)Effective traditional manual IHC based on skilled and popularized technique and to be combined with high-affinity antibody clone,may be a more economic and widespread pre-screening strategy,followed by confirmatory FISH analysis in manual-IHC positive cases.(3)There was no relation with different EML4-ALK fusion variant and their clinicopathological characteristics,and our study may provide additional experimental data and reference standard for molecular heterogeneity research of EML4-ALK gene rearranged NSCLC.