Hi-C sequencing to identify clinically actionable fusions in non–small cell lung cancer missed by other sequencing technologies.

8630 Background: For a patient with non-small lung cancer (NSCLC) to benefit from targeted therapy, an actionable driver alteration needs to be identified. DNA-based next-generation sequencing (NGS) is the standard across many clinical laboratories, however, there are known limitations in the detection of structural rearrangements. RNA-based NGS is now being increasingly utilized in cases with negative DNA NGS testing, but it is currently unknown how many driver alterations are missed with RNA NGS. Hi-C, high-throughput chromosome conformation capture, is a newer NGS method that can be performed on formalin-fixed paraffin-embedded (FFPE) tissue to detect pairwise interactions between DNA regions and may have increased sensitivity for fusion detection. Methods: We collected FFPE tissue from NSCLC cases that were previously deemed to be negative for driver mutations through standard DNA and/or RNA based NGS. We performed Hi-C sequencing on these samples with the Arima Aventa FusionPlus test. This involves digestion of DNA in situ followed by religating to nearby DNA regions and sequencing pairs of DNA tags that reveal proximal DNA sequences. RNA-Sequencing was performed via multiple commercial testing labs using either panel based or whole transcriptome sequencing. Results: In 118 NSCLC specimens that were negative on prior NGS testing, 6 samples (5%) tested positive on Hi-C sequencing for a clinically actionable alteration, including 5 samples that were negative with both DNA and RNA-based NGS. Among these 6 samples were 5 cases of NRG1 fusions and one case of an NTRK2 fusion. All cases with NRG1 fusions have breakpoints proximal to exon 2 and therefore are expected to have expression of the full EGF-like domain. RNA-Sequencing of several of these cases confirmed high expression of NRG1. As an example, one patient had prior negative sequencing with tissue-based panel DNA NGS, ctDNA NGS, an RNA fusion panel, and whole transcriptome RNA-Seq. This patient’s Hi-C sequencing identified a non-canonical NRG1 fusion with an intergenic region fused upstream of exon 2 of NRG1. After progression on chemo-immunotherapy, the patient was treated with zenocutuzumab, a recently approved bispecific antibody that blocks NRG1 from activating HER2/HER3 signaling. A CT Chest scan 6 weeks after initiation showed near complete resolution of the diffuse miliary metastases. Conclusions: Hi-C can identify clinically actionable driver alterations in NSCLC cases that were negative on other sequencing tests, highlighting the potential value of incorporating this technology into clinical practice.