Evaluation of ALK in Non-Small Cell Lung Cancer Using FISH and RT-PCR
Kenneth J Bloom, John Glassco, Paul Choppa. Clarient, A GE Healthcare Company, Aliso Viejo, CA
Background: Translocations involving the kinase domain of the anaplastic lymphoma kinase (ALK) gene have been identified in a number of different malignancies including non-small cell lung cancer (NSCLC). Although ALK has been demonstrated to fuse with a number of different genes, the echinoderm microtubule-associated protein-like 4 (EML4) gene is commonly associated with ALK in NSCLC. Crizotinib was recently approved for the treatment of ALK positive NSCLC patients as determined by the presence of an ALK gene rearrangement based on a companion diagnostic FISH assay. The frequency of ALK related NSCLC reported in the literature varies widely ranging from approximately 2-12%. Part of this variability is likely due to the testing method. We sought to compare FISH with an RT-PCR reaction capable of detecting the 10 most common EML4-ALK variants.
Design: 133 formalin fixed paraffin embedded lung samples were assessed using the Vysis LSI ALK break apart rearrangement FISH probe and a variant specific RT-PCR which is capable of detecting the 10 most common EML4-ALK variants. For FISH, 50 tumor cells were enumerated for the presence of a break apart signal which was considered as present when at least one set of orange and green signals were 2 or more signal diameters apart or when a single orange signal without a corresponding green signal was observed in more than 15% of tumor cells. The RT-PCR reaction uses one-step chemistry and is capable of detecting each of the 10 fusion transcripts at a sensitivity of approximately 1%.
Results: Of the 133 cases, 7 (5.3%) samples had a detectable break apart by the FISH assay. Four of these samples were confirmed as a form of EML4-ALK by the RT-PCR while the other 3 did not generate a signal from any of the 10 EML4-ALK variant specific reactions. The four samples that were confirmed by RT-PCR consisted of 2 variant ones, 1 variant two and 1 variant three. The samples that were not confirmed by the RT-PCR suggest the possibility of an ALK translocation partner other than EML4. In addition to alternative ALK rearrangements, the FISH test identified 3 (2.3%) samples as having ALK gene amplification.
Conclusions: FISH detected a break apart signal in 5.3% of samples, consistent with the reported literature. Attempting to detect the specific EML4-ALK variant by RT-PCR failed to identify 3 (42.8%) of the 7 ALK positive tumors identified by FISH suggesting that this method should not be used as the sole means of identifying patients who may benefit from Crizotinib therapy.
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 303, Tuesday Morning