Attenuated isolated 3’ signal: A highly challenging therapy relevant ALK FISH pattern in NSCLC

Abstract

Targeted therapies in the management of patients with lung cancer provide significantly better outcome compared to chemotherapy. Detection of the anaplastic lymphoma kinase (ALK) gene rearrangement has great predictive value for treatment with small molecule tyrosine kinase inhibitor (crizotinib and alectinib commonly). Fluorescent in situ hybridisation (FISH) assay is a basic diagnostic test designed for detecting ALK gene rearrangements. Although being considered as gold standard method by IASLC's guideline, it is often regarded as difficult and error prone. Our aim was to examine a unique atypical ALK FISH pattern, revealed during a systematic large-scale monitoring, which carries the great risk of misinterpretation, hence may result in loss of patients eligible for targeted therapy. Materials and Methods: Tissue and cytology samples from nearly one thousand patients with advanced stage nonsmall cell lung cancer (NSCLC, n = 996) were routinely examined by ALK FISH and immunohistochemistry (Ventana ALK-D5F3-CDx assay). Anchored Multiplex PCR based Next Generation Sequencing (AMP-NGS) was used to detect fusion gene transcripts in ambiguous cases. Results: Fifty-nine (5,9%) of the cases were positive with ALK FISH test. Three cases showed atypical pattern with a significantly reduced sized red (3′) signal and complete loss of green signals. Digital signal measurement confirmed this finding, showing consistent attenuation of 3′ signals throughout the tumours. In all three cases AMP-NGS and ALK IHC verified the presence of a fusion gene and expressed oncoprotein, respectively. Conclusion: Approximately 5% of the 59 ALK positive cases exhibited atypical attenuated isolated 3′ signal pattern. The immunohistochemistry and AMP-NGS examinations helped to clarify the presence of oncoprotein and the fusion gene, respectively. Our results emphasize the importance of extensive exploration of the genetic background of any unexpected FISH finding to avoid false diagnosis. This enables clinicians to indicate the adequate therapy with higher efficiency for patients suffering from NSCLC.

Characterization of an ETV6-NTRK3 rearrangement with unusual, but highly significant FISH signal pattern in a secretory carcinoma of the salivary gland: a case report

“…E.g., an EML4-ALK translocation with at least five different genomic break points and subsequent loss of several non-connected regions has been described [ 11 ]. Recently, three cases of functional E ML4 - ALK fusions with attenuated single orange signals in addition to fusion signals have been analyzed [ 12 ], a signal pattern which is quite similar to the pattern of our case.…”

False positivity in break apart fluorescence in-situ hybridization due to polyploidy

“…The relevance of adequate knowledge in the technical application of FISH has been described in detail by Tibiletti before (23,24) and is emphasized in a recent study on the specificity of RET FISH analysis (25) as well as in the effect of an attenuated 3' signal, which may lead to an atypical pattern and possibly reduced sensitivity (26).…”

A Novel LOC101927967 Intergenic Region ALK Fusion Identified by NGS and Validated by IHC and FISH in a Patient with Early Stage Adenocarcinoma of Lung

“…However, it is fraught with technical and financial problems and has a potential for a false-negative rate. 8 IHC is widely implemented in pathology laboratories, is relatively inexpensive, faster, and easy to use. But its interpretation remains challenging to standardize and time-consuming.…”