Detection of ALK fusion transcripts in plasma of non-small cell lung cancer patients using a novel RT-PCR based assay

Abstract

Background: Detection of genomic rearrangements, like anaplastic lymphoma kinase (ALK) fusions, is a pivotal requirement in non-small cell lung cancer (NSCLC) for the initiation of a targeted treatment. While tissue testing remains the gold standard, detection of these alterations using liquid biopsies is an unmet need.To enable the detection of ALK rearrangements from circulating-free RNA (cfRNA) from NSCLC patients, we have evaluated a novel reverse transcription PCR (RT-PCR) based assay.Methods: Sixty-six patients with advanced stage NSCLC were included in the study. ALK status was determined by immunohistochemistry (IHC) and/or FISH on tissue sections. For the detection of ALK rearrangements from 2ml plasma collected in EDTA or Streck BCT DNA tubes, cfRNA was extracted using a prototype cfRNA sample preparation method and tested by a novel multiplex ALK/RET RT-PCR assay (Roche).Results: Of the forty-two patients with an ALK rearrangement, 30 (71%) were included at baseline. In 10 of the baseline patients, an ALK rearrangement was detected by RT-PCR [baseline sensitivity 33.33% (95% CI: 17.29-52.81%)]. All 24 negative ALK IHC/FISH-negative patients were negative using the RT-PCR based assay (specificity =100%). Conclusions:The prototype Roche ALK/RET RT-PCR assay was able to detect ALK fusion transcripts in the plasma of NSCLC patients at baseline as well as at disease progression with limited sensitivity but high

Fusion‐positive non‐small cell lung carcinoma: Biological principles, clinical practice, and diagnostic implications

“…Besides a very low tumor mutational burden of ALK ‐positive tumors, uniquely below 3 mut/MB on average, 17 an immunosuppressive tumor microenvironment also appears to contribute to the inherent ICI resistance of these tumors 18 . The ALK status at baseline can be assessed not only on a tissue biopsy, but also using cytological specimens and blood samples, notably on free‐circulating nucleic acid 19,20 . For tissue and cytological samples, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), or RNA next‐generation sequencing (NGS) can be used 21–24 .…”

“…One of the current challenges, notably in thoracic oncology is certainly detection of gene fusions with blood samples. Thus, the sensitivity of detection with blood samples is much lower than with tissue biopsies, regardless of the gene set and the sequencing method 19,144,145 . Globally, the quantity of extracted nucleic acid is related to the kit used as well as the method of extraction.…”

“…18 The ALK status at baseline can be assessed not only on a tissue biopsy, but also using cytological specimens and blood samples, notably on free-circulating nucleic acid. 19,20 For tissue and cytological samples, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), or RNA next-generation sequencing (NGS) can be used. [21][22][23][24] Among these, FISH appears to have a lower sensitivity, and IHC a lower specificity than NGS, which represents the gold standard today.…”

“…Thus, the sensitivity of detection with blood samples is much lower than with tissue biopsies, regardless of the gene set and the sequencing method. 19,144,145 Globally, the quantity of extracted nucleic acid is related to the kit used as well as the method of extraction. In this regard, the kits that obtain DNA and RNA at the same time give a lower quantity of RNA compared to the level of RNA obtained when using a kit for RNA extraction only.…”

Liquid biopsy and non-small cell lung cancer: are we looking at the tip of the iceberg?

“…In parallel, data on PCR-based techniques for plasma detection of other important druggable gene alterations, such as ALK rearrangements, are very limited, they show very low sensitivity rates for fusion genes and prospective validation studies are not available yet [ 47 – 49 ].…”