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Arch Pathol Lab Med. 2014 Jun;138(6):794-802. doi: 10.5858/arpa.2012-0762-OA.

Immunohistochemistry, fluorescence in situ hybridization, and reverse transcription-polymerase chain reaction for the detection of anaplastic lymphoma kinase gene rearrangements in patients with non-small cell lung cancer: potential advantages and methodologic pitfalls.

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From the Laboratory of Molecular Diagnostics (Drs Demidova and Gagarin and Mr Barinov), the Departments of Pathology (Drs Savelov and Grinevitch), Chemotherapy (Dr Stroiakovaski), and Thoracic Surgery (Dr Popov), Moscow Oncological Hospital 62, Moscow, Russia; the Departments of Thoracic Oncology (Dr Laktionov) and Chemotherapy (Dr Gutorov), N. N. Blokhin Russian Cancer Research Center of Russian Academy of Medical Sciences, Moscow, Russia; the Department of Radiology, N. N. Burdenko Central Military Hospital, Moscow, Russia (Dr Smolin); the Laboratory of Cytogenetics and Molecular Genetics, Russian Federal Center for Pediatric Hematology, Oncology and Immunology, Moscow, Russia (Ms Olshanskaya); and the Laboratory of Cytogenetics, Russian Scientific Center for Hematology, Moscow, Russia (Ms Obukhova).



Echinoderm microtubule-associated protein-like 4 gene (EML4) and anaplastic lymphoma kinase gene (ALK) fusion was shown to be the driver of tumorigenesis in approximately 3% to 5% of patients with non-small cell lung cancer (NSCLC) and is associated with response to inhibition with crizotinib. However, no complete agreement regarding the best diagnostic test for identification of ALK rearrangements has been achieved yet.


To investigate the concordance, sensitivity, and specificity of immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and reverse transcription-polymerase chain reaction (RT-PCR) for detection of ALK rearrangements.


Thirty-six prospectively tested patients with NSCLC who had adenocarcinoma and 10 ALK-positive samples were included in the study. All samples were tested by IHC (ALK1 clone, 5A4 clone, D5F3 clone), FISH (LSI ALK Break Apart and ALK FISH Probe), and multiplexed RT-PCR.


Immunohistochemistry staining was successful in all samples.. Clone D5F3 showed the best sensitivity and specificity of 100%; clones ALK1 and 5A4 showed sensitivities of 91% with specificity of 100%. Both FISH probes showed concordance with sensitivity and specificity of 100%. Hybridization and RT-PCR were successful in 98% and 93.4% of samples, respectively, with sensitivity of 88% and specificity of 100%. Frequent artifacts leading to misinterpretation were observed with all 3 methodologies.


All 3 methodologies showed good sensitivity, specificity, and concordance, when artifacts were characterized and excluded. However, all ambiguous cases have to be confirmed as ALK rearranged by at least 2 of the 3 methods.

[Indexed for MEDLINE]

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