Evaluation of ALK Rearrangement in Chinese Non-Small Cell Lung Cancer Using FISH, Immunohistochemistry, and Real-Time Quantitative RT- PCR on Paraffin-Embedded Tissues

Abstract

Patients with ALK gene rearrangements often manifest dramatic responses to crizotinib, an ALK inhibitor. Accurate identification of patients with ALK-positive non-small cell lung cancer (NSCLC) is essential for the clinical application of ALK-targeted therapy. However, assessing EML4-ALK rearrangement in NSCLC remains challenging in routine pathology practice. The aim of this study was to compare the diagnostic accuracy of FISH, immunohistochemistry (IHC), and real-time quantitative RT-PCR (QPCR) methodologies for detection of EML4-ALK rearrangement in NSCLC and to appraise immunohistochemistry as a pre-screening tool. In this study, a total of 473 paraffin-embedded NSCLC samples from surgical resections and biopsies were analyzed by IHC with ALK antibody. ALK rearrangement was further confirmed by FISH and QPCR. ALK protein expression was detected in twenty patients (20/473, 4.2%). Of the 20 ALK-positive cases by IHC, 15 cases were further confirmed as ALK rearrangement by FISH, and 5 cases were not interpretable. Also, we evaluated 13 out of the 20 IHC-positive tissues by QPCR in additional to FISH, and found that 9 cases were positive and 2 cases were equivocal, whereas 2 cases were negative although they were positive by both IHC and FISH. The ALK status was concordant in 5 out of 8 cases that were interpretable by three methods. Additionally, none of the 110 IHC-negative cases with adenocarcinoma histology showed ALK rearrangements by FISH. Histologically, almost all the ALK-rearranged cases were adenocarcinoma, except that one case was sarcomatoid carcinoma. A solid signet-ring cell pattern or mucinous cribriform pattern was presented at least focally in all ALK-positive tumors. In conclusion, our findings suggested that ALK rearrangement was associated with ALK protein expression. The conventional IHC assay is a valuable tool for the pre-screening of patients with ALK rearrangement in clinical practice and a combination of FISH and QPCR is required for further confirmation.

Clinical characteristics of patients with non-small cell lung cancers harboring anaplastic lymphoma kinase rearrangements and primary lung adenocarcinoma harboring epidermal growth factor receptor mutations

“…The incidence of ALK rearrangements was found to be 9.6% (73/763) in NSCLC and 11.0% (54/493) in adenocarcinoma in our study, which was similar to the results previously published in Chinese (Shan et al, 2014;Wang et al, 2014a;Yang et al, 2014) and Asian cohorts (Kim et al, 2014). As previously reported, patients who were ALK-positive were predominantly young women and never/light smokers (Lynch et al, 2004;Rodig et al, 2009;Shaw et al, 2009;Wong et al, 2009;Jokoji et al, 2010;Takahashi et al, 2010;Zhang et al, 2013;Zhang et al, 2014). In our study, young age, female gender, and never-smokers were significantly associated with ALK rearrangement.…”

“…Recently many studies have reported the efficacy of Ventana IHC for detecting ALK rearrangements with D5F3 antibodies and have supported that Ventana IHC is a method with excellent sensitivity and specificity to identify ALK rearrangements in paraffin-embedded lung cancer specimens (Takahashi et al, 2010;Zhang et al, 2013;Shan et al, 2014;Wang et al, 2014a;Zhang et al, 2014). The sensitivity and specificity of Ventana IHC were found to be 100 and 98.2%, respectively, and the concordance rate between the fluorescence in situ hybridization (FISH) and the Ventana IHC was 98.4% (Wang et al, 2014a).…”

“…Immunohistochemistry (IHC) for ALK protein overexpression is a promising screening modality, with D5F3 antibodies showing excellent sensitivity and specificity for ALK-rearranged NSCLC (Takahashi et al, 2010;To et al, 2013;Shan et al, 2014;Zhang et al, 2013;. Furthermore, the ALK fusion protein IHC diagnostic kit developed by Roche Ventana Medical Systems (Oro Valley, AZ, USA) has been approved by the European Medicines Agency (EMEA) and the China Food and Drug Administration (CFDA) as an aid for identifying patients who are eligible for treatment with crizotinib.…”

A real‑world study of treatment patterns and survival outcome in advanced anaplastic lymphoma kinase‑positive non‑small‑cell lung cancer

“…In the current real-world study, the characteristics of patients were consistent with those reported in previous studies (16,17). More specifically, ALK rearrangement occurred in relatively young patients, >50% of the patients had no history of smoking and almost all patients were diagnosed with adenocarcinoma.…”

Anaplastic lymphoma kinase: Role in cancer and therapy perspective

“…The amplicons can be sequenced to identify the specific fusion variants. [63][64][65] The RT-PCR method is targeted to one partner gene per run; it is limited to the primer sequence located on the partner gene and ALK gene. Quantitative RT-PCR can detect mRNA amount differences, which are appropriate to test overexpression of fusion and non-fusion ALK.…”