Cell-free total nucleic acid (cfTNA)-based liquid biopsy (LBx) offers a minimally invasive alternative to tissue-based next generation sequencing (NGS). Most NGS-based LBx assays require several days for results. This study validated a rapid NGS-based LBx assay with a two-day turnaround time (TAT). Patient plasma samples were used to validate the Oncomine Precision Assay for single nucleotide variants (SNVs), insertion deletions (indels) and fusions across 50 genes on the Genexus Sequencer. Manual extraction using the QIAamp cfTNA Kit was performed to optimize fusion detection. Analytical sensitivity in synthetic controls (N=20) using the automated workflow was 99.2% for SNVs and 95% for indels, with 100% specificity (allele frequency ≥0.5%). In clinical samples (N=107), sensitivity was 99.4% for SNVs and 100% for indels; specificity was 100% for both, compared with orthogonal assays. Manual extraction improved overall performance compared to the automated extraction and was used for the final clinical workflow. Analytical sensitivity of fusions in synthetic controls (N=22) was 98.9%, with 100% specificity (≥7 copies). In clinical samples (N=38) sensitivity and specificity for both SNVs and indels were 100% and 99.9%, respectively; sensitivity for fusions was 72.2%; specificity was 100%. Overall precision was >99% and average TAT was ≤2 days. This study reports the feasibility and validation of a two-day TAT NGS-based cfTNA assay that can potentially reduce time-to-treatment.
S. Roy-Chowdhuri, Ana Galán-Cobo, N. Agarwal et al.· Journal of Molecular Diagnos...· 0 citations
BACKGROUND
Genomic analysis has revealed that approximately 40% of bladder cancer (BLCA) tumors harbor alterations in the PI3K/AKT pathway, with PIK3CA mutations occurring in 15-25% of cases. PIK3CA, which encodes the catalytic p110α subunit of PI3K, plays a critical role in regulating cell survival, proliferation, and metabolism. However, the metabolic and functional consequences of PIK3CA mutations in BLCA remain poorly defined.
METHODS
To investigate the role of PIK3CA mutations in BLCA, we performed targeted sequencing on tumors from patients, identifying recurrent alterations. Using CRISPR/Cas9 knock-in models in SCaBER and UM-UC-3 cell lines, we introduced the PIK3CA E545K mutation to study its effects. We conducted transcriptomic profiling, targeted metabolomics, and stable isotope tracing to assess metabolic reprogramming. Functional assays measured proliferation, mitochondrial complex I activity, and glutaminolysis. Orthotopic xenografts in mice were used to evaluate in vivo tumor growth and metabolism.
RESULTS
PIK3CA mutations were present in 20% of cases, consistent with TCGA data. The E545K and E545Q hotspots accounted for 70% of these mutations. PIK3CA E545K strongly activated PI3K/AKT signaling. Transcriptomic analysis revealed enrichment of OXPHOS, fatty acid metabolism, and mTORC1 signaling. Metabolomics indicated changes in TCA cycle metabolites and enhanced reductive carboxylation of glutamine to citrate, driving fatty acid synthesis. Mutant cells showed increased expression of GLS1 and FASN, higher proliferation rates, and elevated mitochondrial complex I activity. In vivo, PIK3CA-mutant xenografts displayed significantly increased tumor growth.
CONCLUSION
PIK3CA mutations are frequent drivers of metabolic reprogramming in BLCA, leading to increased glutamine flux, elevated OXPHOS activity, and enhanced fatty acid synthesis, all of which contribute to tumor progression. These findings provide the first comprehensive evidence that PIK3CA-driven metabolic alterations are both biomarkers of aggressive disease and actionable therapeutic targets. The efficacy of PI3Kα inhibition in combination with metabolic targets may support its potential in precision medicine for PIK3CA-mutant BLCA and highlights the value of integrating metabolic biomarkers into treatment strategies for advanced BLCA.
K. R. Kami Reddy, V. Putluri, D. Piyarathna et al.· Cell communication and signa...· 0 citations
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