ALK Testing in Non-Small Cell Lung Carcinoma: What Now?

Abstract

®he rapid evolution in personalized medicine for patients with advanced non— smallcell lung cancer (NSCLC) has brought considerable benefits for those whose tumors are addicted to a particular ongogenic driver, but posed new challenges for pathologists in identifying those cases. Epidermal growth factor receptor (EGFR) mutation testing, to underpin the prescription of EGFR tyrosine kinase inhibitors (TKIs), is now a standard of care, where a range of methods may be used to detect and identify the mutation, subject to appropriate validation. 1 The anaplastic lymphoma kinase (ALK) TKIs are the second class of biomarker-selected “molecularly tar geted” treatments to reach maturity in NSCLC therapy, for patients whose tumors harbor an ALK gene rearrangement. 2–4 The laboratory identification of ALK gene rearrangements has, however, been a rather more contentious issue. These rearrangements are relatively heterogeneous; at least 27 variants have now been described, the vast majority in patients with adenocarcinoma. 5 Most involve an inversion in chromosome 2p, placing a variable length of the echinoderm microtubule-associated protein-like 4 (EML4) gene and its promoter upstream of the ALK tyrosine kinase domain. Other fusion partners from other chromosomes have the same effect—overexpression of a constitutively activated ALK tyrosine kinase with pro-oncogenic effects. Crizotinib was the “first in class” ALK TKI approved by the U.S. Federal Drug Administration (FDA), in conjunction with a specific companion diagnostic, the Vysis LSI ALK Dual Colour, Break-Apart Rearrangement Probe (Abbott Molecular, Abbott Park, IL). This was the methodology used in the trials that led to the approval of crizotinib; thus, the ALK fluorescence in situ hybridization (FISH) test became the “standard procedure” for patient identification. FISH testing, and ALK FISH testing in NSCLC in particular, is generally acknowledged as being relatively costly, time-consuming, requires specialized equipment, and requires considerable expertise. 6 Inversions in 2p in particular lead to relatively small physical separation of the two ALK fragments, sometimes making the “break apart” difficult to identify. In addition, the requirement for 50 assessable cells to be read, of which at least 15% should show rearrangement for a “positive test,” means that as many as 20% of routine diagnostic lung cancer samples requiring testing fall short of this requirement. 6 Although not “FDA approved,” alternative methods for detecting ALK fusions are attractive. Reverse-transcriptase polymerase chain reaction (RT-PCR) was the method originally used to identify the EML4-ALK gene rearrangement in NSCLC. 7 It is a highly sensitive and specific technique, but it has drawbacks. The ever-increasing number of fusion partners for the ALK gene mandates a multiplex RT-PCR approach with multiple specific primers, a relatively “specialist” technique. A greater problem, however, is the availability of adequate quality messenger RNA from tumor samples. Most published studies used fresh-frozen tumor samples. Formalin-fixed, paraffin-embedded samples generally are a poor, unreliable source of messenger RNA. Unfixed cytology type samples have been used in some specialized centers but questions arise over the presence of tumor cells in unexamined test samples, and this approach also poses logistical issues. RT-PCR is not recommended in the College of American Pathologists/International Association for the Study of