Hybridization-Induced Aggregation Technology for Practical Clinical Testing

Abstract

KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in <10 minutes after PCR. Here, we evaluate the clinical utility of the HIA method for mutation detection (HIAMD). In the analysis of 20 lung and colon tumor pathology specimens, we observed a 100% correlation between the KRAS mutation statuses determined by HIAMD and sequencing. In addition, we were able to detect KRAS mutations in a background of 75% wild-type DNA-a finding consistent with that reported for sequencing. With this, we show that HIAMD allows for the rapid and cost-effective detection of KRAS mutations, without compromising analytical performance. These results indicate the validity of HIAMD as a mutation-testing technology suitable for practical clinical testing. Further expansion of this platform may involve the detection of mutations in other key oncogenic pathways.

An enzymatic on/off switch‐mediated assay for KRAS hotspot point mutation detection of circulating tumor DNA

“…Screening the KRAS mutations by HRM had a sensitivity of 5%‐6% 10,28 . Hillary et al were able to detect KRAS mutations by allele‐specific hybridization‐induced aggregation (HIA) of oligonucleotide probe‐conjugated microbeads with a sensitivity of 25% 29 . Our multiplex real‐time PCR method can detect KRAS mutation as few as 100 copies in at least 10 5 wild‐type counterparts.…”

Desarrollo de métodos integrados para la determinación de biomarcadores genéticos

“…Recientemente el grupo de Lapitan detectó mutaciones del gen KRAS mediante un ensayo combinado de MNPS y ligación (Lapitan et al, 2019), mientras que el grupo de Hui también detectó esta mutación a partir de producto de PCR mediante ensayo de LFA (Hui et al, 2016). Mutaciones de este mismo gen también han sido detectadas con este tipo de plataformas mediante procesos de agregación, distinguiendo entre partículas conjugadas con sondas nativas y con sondas mutantes (Sloane et al, 2015), (Sloane et al, 2016). También, el grupo de Chen aplicó la misma estrategia para la detección de esta mutación mediante ligación (Chen et al, 2012).…”