Research Papers:

Sensitive and affordable diagnostic assay for the quantitative detection of anaplastic lymphoma kinase (ALK) alterations in patients with non-small cell lung cancer

Elisa Dama, Micol Tillhon, Giovanni Bertalot, Francesca de Santis, Flavia Troglio, Simona Pessina, Antonio Passaro, Salvatore Pece, Filippo de Marinis, Patrizia Dell’Orto, Giuseppe Viale, Lorenzo Spaggiari, Pier Paolo Di Fiore, Fabrizio Bianchi, Massimo Barberis and Manuela Vecchi _

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Oncotarget. 2016; 7:37160-37176. https://doi.org/10.18632/oncotarget.9471

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Elisa Dama1,2, Micol Tillhon1, Giovanni Bertalot1, Francesca de Santis1,8, Flavia Troglio1,9, Simona Pessina3, Antonio Passaro4, Salvatore Pece1,5, Filippo de Marinis4, Patrizia Dell’Orto3, Giuseppe Viale3,5, Lorenzo Spaggiari5,6, Pier Paolo Di Fiore1,5,7, Fabrizio Bianchi1,10, Massimo Barberis3, Manuela Vecchi1,7

1Molecular Medicine Program, European Institute of Oncology, Milan, Italy

2Division of Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy

3Department of Pathology, European Institute of Oncology, Milan, Italy

4Division of Thoracic Oncology, European Institute of Oncology, Milan, Italy

5DIPO, Department of Hemato-Oncology and Oncology, University of Milan, Milan, Italy

6Division of Thoracic Surgery, European Institute of Oncology, Milan, Italy

7IFOM, the FIRC Institute of Molecular Oncology, Milan, Italy

8Present address: Advanced Cell Diagnostics, Segrate, Milan, Italy

9Present address: Division of Immunology, Transplantantion and Infectious Disease, Leukocyte Biology Unit, San Raffaele Scientific Institute, Milan, Italy

10Present address: Institute for Stem-cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), IRCCS Casa Sollievo della Sofferenza, Foggia, Italy

Correspondence to:

Manuela Vecchi, email: [email protected]

Keywords: ALK, RT-qPCR, inverse prediction, FFPE NSCLC, cytology specimens

Received: January 04, 2016     Accepted: April 25, 2016     Published: May 19, 2016


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.

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