Jean-Marie Bruey

Exon scanning by reverse transcriptase–polymerase chain reaction for detection of known and novel EML4–ALK fusion variants in non–small cell lung cancer

Chromosomal inversions within chromosome 2p, resulting in fusions between the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes, are a recent focus of treatment options for non-small cell lung cancer. Thirteen EML4-ALK fusion variants have been identified, affecting eight EML4 exons. We have developed an exon scanning approach using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify known and potential variants involving the first 22 EML4 exons. A total of 55 formalin-fixed, paraffin-embedded lung cancer tumors were screened, of which 5 (9%) were positive for EML4-ALK fusions. Four positive cases harbored known fusion variants: variant 3a, 3b, or both in three cases and variant 1 in one case. The fifth positive specimen harbored two novel variants, designated 8a and 8b, involving exon 17 of EML4. Fluorescence in situ hybridization confirmed the presence of EML4-ALK fusions in three of the four RT-PCR-positive specimens with sufficient tissue for examination, and also confirmed absence of fusions in all 19 RT-PCR-negative specimens tested. Immunohistochemistry analysis confirmed ALK protein expression in the sample containing the novel 8a and 8b variants. This RT-PCR-based exon scanning approach avoids the limitations of screening only for previously identified EML4-ALK fusions and provides a simple molecular assay for fusion detection in a clinical diagnostics setting.

Plasma tyrosine kinase activity as a potential biomarker in BCR-ABL1-targeted therapy

In targeted therapy using tyrosine kinase inhibitors (TKIs), measurement of TK activities could be beneficial for diagnosis, identification of potential responders, and monitoring treatment efficacy. Here we evaluated the utility of measuring circulating TK (cTK) activity directly from plasma in leukemia patients positive for the BCR-ABL1. Plasma cTK activity was measured from 46 patients with newly diagnosed chronic myelogenous leukemia (CML), 24 with multidrug-resistant CML, 24 with BCR-ABL1-positive acute lymphocytic leukemia (ALL), and 38 healthy donors. Circulating TK activity was significantly higher in CML (median 801.93 U/mL, range 18.10-3932.30 U/mL) and BCR-ABL1-positive ALL patients (median 659.55 U/mL, range 0-1626.90 U/mL) than in healthy donors (median 82.85 U/mL, range 0.63-852.80 U/mL) (P < 0.001). Plasma cTK activity was closely correlated with cellular BCR-ABL1 kinase activation as indicated by phosphorylation of the downstream signaling proteins CRKL (P < 0.001) and STAT-5 (P= 0.003). However, cTK activity was not associated with BCR-ABL1 transcript level and was independent of BCR-ABL1 mutation type. Ex vivo inhibition of imatinib and dasatinib on plasma cTK activity was severely diminished in patients harboring T315I mutation. Ex vivo testing measuring the effect of TKIs on plasma cTK activity thus hold promise as drug sensitivity tests for predicting and monitoring response to specific TKIs.