Josh N Vo, Yi-Mi Wu, Rui Wang, Tiffany Pham, Xuhong Cao, Sophie Yeung, Mingyu Park, Yelena Kleyman-Smith, Guo Ci Teo, Alyssa J Wu, Anne Li, James C Estill, Lakshmi P Kunju, Chen Yang, Dan R Robinson, Arul M Chinnaiyan
Short-read sequencing cannot determine whether co-occurring variants within a cancer gene lie on the same allele (cis) or opposing alleles (trans), a distinction with direct therapeutic consequences: trans configurations confirm biallelic tumor suppressor inactivation, whereas cis configurations generate compound oncogenic alleles with enhanced activity. Among 768 patients with prostate, breast, or ovarian cancers, we used mutational signatures to nominate cryptic genomic instability cases lacking a causative biallelic event on short-read sequencing. Long-read nanopore sequencing resolved 32 of 46 cryptic cases (69.6%) through methylation detection, long insertion resolution, and structural variant characterization, confirming trans inactivation in every resolved tumor suppressor case. Analysis of 4,496 MiOncoSeq samples identified 17,519 multi-hit gene pairs, 78.7% of which exceeded the 500 bp short-read phasing limit, and long-read phasing revealed recurrent compound cis alleles in NOTCH1, PIK3CA, PDGFRB, and KIT. Haplotype phasing addresses an overlooked gap in cancer variant interpretation and warrants integration into precision oncology.