title: Sensitive and affordable diagnostic assay for the quantitative detection of anaplastic lymphoma kinase (ALK) alterations in patients with non-small cell lung cancer.
abstract: Accurate detection of altered anaplastic lymphoma kinase (ALK) expression is critical for the selection of lung cancer patients eligible for ALK-targeted therapies. To overcome intrinsic limitations and discrepancies of currently available companion diagnostics for ALK, we developed a simple, affordable and objective PCR-based predictive model for the quantitative measurement of any ALK fusion as well as wild-type ALK upregulation. This method, optimized for low-quantity/-quality RNA from FFPE samples, combines cDNA pre-amplification with ad hoc generated calibration curves. All the models we derived yielded concordant predictions when applied to a cohort of 51 lung tumors, and correctly identified all 17 ALK FISH-positive and 33 of the 34 ALK FISH-negative samples. The one discrepant case was confirmed as positive by IHC, thus raising the accuracy of our test to 100%. Importantly, our method was accurate when using low amounts of input RNA (10 ng), also in FFPE samples with limited tumor cellularity (5-10%) and in FFPE cytology specimens. Thus, our test is an easily implementable diagnostic tool for the rapid, efficacious and cost-effective screening of ALK status in patients with lung cancer.
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.|Division of Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Dama
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.
authors.forename: Micol
authors.initials: M
authors.lastname: Tillhon
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bertalot
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.|Present address: Advanced Cell Diagnostics, Segrate, Milan, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: de Santis
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.|Present address: Division of Immunology, Transplantantion and Infectious Disease, Leukocyte Biology Unit, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Flavia
authors.initials: F
authors.lastname: Troglio
authors.affiliation: Department of Pathology, European Institute of Oncology, Milan, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Pessina
authors.affiliation: Division of Thoracic Oncology, European Institute of Oncology, Milan, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Passaro
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.|DIPO, Department of Hemato-Oncology and Oncology, University of Milan, Milan, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Pece
authors.affiliation: Division of Thoracic Oncology, European Institute of Oncology, Milan, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: de Marinis
authors.affiliation: Department of Pathology, European Institute of Oncology, Milan, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Dell'Orto
authors.affiliation: Department of Pathology, European Institute of Oncology, Milan, Italy.|DIPO, Department of Hemato-Oncology and Oncology, University of Milan, Milan, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Viale
authors.affiliation: DIPO, Department of Hemato-Oncology and Oncology, University of Milan, Milan, Italy.|Division of Thoracic Surgery, European Institute of Oncology, Milan, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Spaggiari
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.|DIPO, Department of Hemato-Oncology and Oncology, University of Milan, Milan, Italy.|IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy.
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: Di Fiore
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.|Present address: Institute for Stem-cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), IRCCS Casa Sollievo della Sofferenza, Foggia, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Bianchi
authors.affiliation: Department of Pathology, European Institute of Oncology, Milan, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Barberis
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy.|IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Vecchi
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000077548:Anaplastic Lymphoma Kinase; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002889:Chromosomes, Human, Pair 2 / Q000235:genetics; D015331:Cohort Studies; D005260:Female; D015321:Gene Rearrangement; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic; D015854:Up-Regulation
chemical_list: D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases
keywords: ALK; FFPE NSCLC; RT-qPCR; cytology specimens; inverse prediction