title: Validation of a new algorithm for a quick and easy RT-PCR-based ALK test in a large series of lung adenocarcinomas: Comparison with FISH, immunohistochemistry and next generation sequencing assays.
abstract: Anaplastic Lymphoma Kinase (ALK) gene rearrangements have been described in 3-5% of lung adenocarcinomas (ADC) and their identification is essential to select patients for treatment with ALK tyrosine kinase inhibitors. For several years, fluorescent in situ hybridization (FISH) has been considered as the only validated diagnostic assay. Currently, alternative methods are commercially available as diagnostic tests.
  
  
  
  A series of 217 ADC comprising 196 consecutive resected tumors and 21 ALK FISH-positive cases from an independent series of 702 ADC were investigated. All specimens were screened by IHC (ALK-D5F3-CDx-Ventana), FISH (Vysis ALK Break-Apart-Abbott) and RT-PCR (ALK RGQ RT-PCR-Qiagen). Results were compared and discordant cases subjected to Next Generation Sequencing.
  
  
  
  Thirty-nine of 217 samples were positive by the ALK RGQ RT-PCR assay, using a threshold cycle (Ct) cut-off ≤35.9, as recommended. Of these positive samples, 14 were negative by IHC and 12 by FISH. ALK RGQ RT-PCR/FISH discordant cases were analyzed by the NGS assay with results concordant with FISH data. In order to obtain the maximum level of agreement between FISH and ALK RGQ RT-PCR data, we introduced a new scoring algorithm based on the ΔCt value. A ΔCt cut-off level ≤3.5 was used in a pilot series. Then the algorithm was tested on a completely independent validation series. By using the new scoring algorithm and FISH as reference standard, the sensitivity and the specificity of the ALK RGQ RT-PCR(ΔCt) assay were 100% and 100%, respectively.
  
  
  
  Our results suggest that the ALK RGQ RT-PCR test could be useful in clinical practice as a complementary assay in multi-test diagnostic algorithms or even, if our data will be confirmed in independent studies, as a standalone or screening test for the selection of patients to be treated with ALK inhibitors.
authors: Center of Predictive Molecular Medicine, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti-Pescara, Chieti, Italy.
authors: Antonio
authors: A
authors: Marchetti
authors: Center of Predictive Molecular Medicine, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti-Pescara, Chieti, Italy.
authors: Maria Vittoria
authors: MV
authors: Pace
authors: Center of Predictive Molecular Medicine, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti-Pescara, Chieti, Italy.
authors: Alessia
authors: A
authors: Di Lorito
authors: Center of Predictive Molecular Medicine, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti-Pescara, Chieti, Italy.
authors: Sara
authors: S
authors: Canarecci
authors: Oncological and Cardiovascular Molecular Medicine Unit, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti, Chieti, Italy.
authors: Lara
authors: L
authors: Felicioni
authors: Center of Predictive Molecular Medicine, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti-Pescara, Chieti, Italy.
authors: Tommaso
authors: T
authors: D'Antuono
authors: Center of Predictive Molecular Medicine, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti-Pescara, Chieti, Italy.
authors: Marcella
authors: M
authors: Liberatore
authors: Center of Predictive Molecular Medicine, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti-Pescara, Chieti, Italy.
authors: Giampaolo
authors: G
authors: Filice
authors: Department of Surgery, University of Chieti, Chieti, Italy.
authors: Luigi
authors: L
authors: Guetti
authors: Department of Surgery, University of Chieti, Chieti, Italy.
authors: Felice
authors: F
authors: Mucilli
authors: Oncological and Cardiovascular Molecular Medicine Unit, Center for Excellence on Ageing and Translational Medicine (CeSI-MeT), University of Chieti, Chieti, Italy. Electronic address: fbuttitta@unich.it.
authors: Fiamma
authors: F
authors: Buttitta
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics*; D000077192:Adenocarcinoma of Lung; D000465:Algorithms; D000077548:Anaplastic Lymphoma Kinase; D015870:Gene Expression; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics*; D012372:ROC Curve; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic
chemical_list: C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases
keywords: ALK RT-PCR; ALK expression; ALK translocation; FISH; Lung adenocarcinoma; Molecular diagnosis; NGS; NSCLC; Predictive biomarkers; Translational research