Assay for ALK Rearrangement in Primary Lung Adenocarcinoma
Assay for ALK Rearrangement in Primary Lung Adenocarcinoma
Background: To evaluate the diagnostic value of a novel fully automated immunohistochemistry (IHC) assay for detection of anaplastic lymphoma kinase (ALK) fusion in a large number of ALK-positive lung adenocarcinoma (ADC) patients.
Patients and methods: We tested 196 lung ADCs for ALK rearrangement by two IHC assays (Ventana pre-diluted ALK D5F3 antibody with the Optiview DAB IHC detection kit and Optiview Amplification kit, D5F3 by Cell Signaling Technology (CST) with Ultraview DAB detection kit by Ventana), fluorescence in situ hybridization (FISH) and real-time reverse transcription–PCR (RT–PCR). CST ALK IHC was scored using the scoring scheme of 0, no staining; 1+, faint; 2+, moderate; and 3+, strong cytoplasmic reactivity in ≥10% of tumor cells. As for Ventana IHC, a binary scoring system (positive or negative for ALK status) was adopted for evaluating the staining results.
Results: Among 196 cases tested, 63 (32%), 65 (33%), 70 (36%), and 69 (35%) cases were ALK positive by FISH, Ventana IHC, CST IHC, and RT–PCR, respectively. The sensitivity and specificity of Ventana IHC were 100% and 98%, respectively. Two Ventana IHC-positive cases, which were also CST IHC score of 3+, showed FISH negative, but their ALK rearrangement was confirmed by RT–PCR and direct sequencing. The sensitivity and specificity of CST IHC with staining intensity score of 1+ or more were 100% and 95%, respectively. Five (25%, of 20) patients with CST IHC score of 1+ were both FISH and RT–PCR negative. The sensitivity and specificity of RT–PCR for detection of ALK fusion were 98% and 95%, respectively. The total accordance rate between ALK RT–PCR and Ventana IHC was 97%.
Conclusions: The novel fully automated IHC assay is a reliable screening tool in routine pathologic laboratories for identification of patients with ALK rearrangement for targeted therapy in lung ADC.
Lung cancer is the most frequent cause of cancer-related death worldwide, accounting for about 1.4 million deaths per year. signaling pathways within cancer cells has led to the development of new targeted therapies in a subset of patients with NSCLC. For example drugs that target epidermal growth factor receptor (EGFR) have been proven more effective than cytotoxic chemotherapy in advanced NSCLC patients with sensitizing EGFR mutations. More recently, a similarly marked response to an anaplastic lymphoma kinase (ALK) inhibitor was demonstrated in patients with ALK fusion-bearing NSCLC.
The echinoderm microtubule-associated protein-like 4 –ALK (EML4-ALK) fusion in NSCLC was first described in 2007. This fusion results from a small inversion within chromosome 2p, resulting in the expression of a chimeric tyrosine kinase in which the N-terminal half of the EML4 is fused to the intracellular ALK kinase domain. Although EML4 is the predominant fusion partner, other fusion partners have also been reported in lung cancer, including TFG,KIF5B, and KLC1. The incidence of ALK gene rearrangement appears to range from 2% to 7%, and they are rarely coincident with EGFR, KRAS mutations. Recently, results from clinical trials evaluating an ALK inhibitor, Crizotinib, in patients with ALK positive locally advanced or metastatic NSCLC demonstrated promising results. However, lung cancer with the ALK fusion constitutes only a small fraction of lung cancers, therefore, efficient determination of ALK status in NSCLC patients is critical for directing patient care.
Currently, fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and real-time reverse transcription–PCR (RT–PCR) are commonly used to detect the ALK fusion. Each of these methods has their own advantages and disadvantages. Although FISH is currently the gold standard method used in clinical trials to detect fusion gene, and it was the first FDA-approved method for detecting the ALK fusion, IHC analysis is technically easy because it is integrated into routine pathological diagnosis. Recent studies indicate that IHC is sensitive and specific for determination of ALK status, and is a viable alternative to ALK FISH. However, studies comparing IHC and with FISH have used different clones with different detection systems and scoring methods, and only small number of ALK-positive cases were collected in these studies, due to its low incidence. In this study, we introduced a fully automated IHC assay to determine ALK status, together with another reported IHC assay, FISH, and RT–PCR in a large collection of ALK-positive cases, and compared the specificity and sensitivity of this novel IHC assay with other methods for the detection of ALK fusion in patients with primary lung ADC.
Abstract and Introduction
Abstract
Background: To evaluate the diagnostic value of a novel fully automated immunohistochemistry (IHC) assay for detection of anaplastic lymphoma kinase (ALK) fusion in a large number of ALK-positive lung adenocarcinoma (ADC) patients.
Patients and methods: We tested 196 lung ADCs for ALK rearrangement by two IHC assays (Ventana pre-diluted ALK D5F3 antibody with the Optiview DAB IHC detection kit and Optiview Amplification kit, D5F3 by Cell Signaling Technology (CST) with Ultraview DAB detection kit by Ventana), fluorescence in situ hybridization (FISH) and real-time reverse transcription–PCR (RT–PCR). CST ALK IHC was scored using the scoring scheme of 0, no staining; 1+, faint; 2+, moderate; and 3+, strong cytoplasmic reactivity in ≥10% of tumor cells. As for Ventana IHC, a binary scoring system (positive or negative for ALK status) was adopted for evaluating the staining results.
Results: Among 196 cases tested, 63 (32%), 65 (33%), 70 (36%), and 69 (35%) cases were ALK positive by FISH, Ventana IHC, CST IHC, and RT–PCR, respectively. The sensitivity and specificity of Ventana IHC were 100% and 98%, respectively. Two Ventana IHC-positive cases, which were also CST IHC score of 3+, showed FISH negative, but their ALK rearrangement was confirmed by RT–PCR and direct sequencing. The sensitivity and specificity of CST IHC with staining intensity score of 1+ or more were 100% and 95%, respectively. Five (25%, of 20) patients with CST IHC score of 1+ were both FISH and RT–PCR negative. The sensitivity and specificity of RT–PCR for detection of ALK fusion were 98% and 95%, respectively. The total accordance rate between ALK RT–PCR and Ventana IHC was 97%.
Conclusions: The novel fully automated IHC assay is a reliable screening tool in routine pathologic laboratories for identification of patients with ALK rearrangement for targeted therapy in lung ADC.
Introduction
Lung cancer is the most frequent cause of cancer-related death worldwide, accounting for about 1.4 million deaths per year. signaling pathways within cancer cells has led to the development of new targeted therapies in a subset of patients with NSCLC. For example drugs that target epidermal growth factor receptor (EGFR) have been proven more effective than cytotoxic chemotherapy in advanced NSCLC patients with sensitizing EGFR mutations. More recently, a similarly marked response to an anaplastic lymphoma kinase (ALK) inhibitor was demonstrated in patients with ALK fusion-bearing NSCLC.
The echinoderm microtubule-associated protein-like 4 –ALK (EML4-ALK) fusion in NSCLC was first described in 2007. This fusion results from a small inversion within chromosome 2p, resulting in the expression of a chimeric tyrosine kinase in which the N-terminal half of the EML4 is fused to the intracellular ALK kinase domain. Although EML4 is the predominant fusion partner, other fusion partners have also been reported in lung cancer, including TFG,KIF5B, and KLC1. The incidence of ALK gene rearrangement appears to range from 2% to 7%, and they are rarely coincident with EGFR, KRAS mutations. Recently, results from clinical trials evaluating an ALK inhibitor, Crizotinib, in patients with ALK positive locally advanced or metastatic NSCLC demonstrated promising results. However, lung cancer with the ALK fusion constitutes only a small fraction of lung cancers, therefore, efficient determination of ALK status in NSCLC patients is critical for directing patient care.
Currently, fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and real-time reverse transcription–PCR (RT–PCR) are commonly used to detect the ALK fusion. Each of these methods has their own advantages and disadvantages. Although FISH is currently the gold standard method used in clinical trials to detect fusion gene, and it was the first FDA-approved method for detecting the ALK fusion, IHC analysis is technically easy because it is integrated into routine pathological diagnosis. Recent studies indicate that IHC is sensitive and specific for determination of ALK status, and is a viable alternative to ALK FISH. However, studies comparing IHC and with FISH have used different clones with different detection systems and scoring methods, and only small number of ALK-positive cases were collected in these studies, due to its low incidence. In this study, we introduced a fully automated IHC assay to determine ALK status, together with another reported IHC assay, FISH, and RT–PCR in a large collection of ALK-positive cases, and compared the specificity and sensitivity of this novel IHC assay with other methods for the detection of ALK fusion in patients with primary lung ADC.
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