Multiplex RT-PCR-Based Sequencing Assay to Detect Kidney Cancer-Specific Splice Variants in Tumor Tissues and Plasma Cell-Free RNAs.

Authors

Kapil K Avasthi, Kendrick H Yim, Youngchul Kim, Paola M Ramos Echevarria, Liz M Darst, Nicholas Abreu, Kelly P Zea, Mitchell T Hayes, Aram Vosoughi, Brandon J Manley, Liang Wang

Published in

Molecular diagnosis & therapy. Jul 06, 2026. Epub Jul 06, 2026.

Abstract

Clear cell renal cell carcinoma (ccRCC) lacks sensitive molecular biomarkers for early detection of disease recurrence and monitoring. Recurrent splice variants (SVs) offer promising diagnostic potential for capturing tumor-specific signals in circulating blood. In this study, we report a multiplex reverse transcription-polymerase chain reaction sequencing assay to quantify ccRCC-specific SVs in tumor tissue and plasma samples.
A 56-target reverse transcription-polymerase chain reaction panel was designed to amplify 27 previously identified ccRCC-associated SVs, 24 matched wild-type transcripts, and five housekeeping genes. Endogenous SVs were validated in ccRCC cell lines using nanopore long‑read sequencing. Analytical sensitivity was assessed using synthetic oligos, and translational potential of these SVs was evaluated in 47 prospectively collected matched tumor-plasma pairs from patients undergoing curative surgery. Normalized SV counts were analyzed for tumor-plasma detectability, agreement, bootstrap stability, and depth robustness.
Long-read sequencing confirmed multiple SVs and demonstrated cell line-specific isoform patterns. The serial dilution of synthetic oligos demonstrated strong analytical performance, including linear quantification over 4 orders of magnitude (R² > 0.99) and a limit of detection of approximately 100 molecules. Principal component analysis demonstrated distinct global expression patterns between tumor tissue RNA and plasma cell‑free RNA, consistent with differences in sample origin. Several SVs exhibited high tumor-to-plasma concordance, including TTCB-SV, MVK-beta-SV, MVK-alpha-SV, and MCCC-SV, with positive percent agreement that ranged from 0.87 to 0.98. Downsampling analyses showed strong depth stability, with leading SVs retaining most detectability at 0.25× plasma sequencing depth (TTCB-SV = 0.99, MVK-beta-SV = 1.00, MVK-alpha-SV = 0.87, MCCC-SV = 0.93). A composite ranking of agreement, stability, prevalence, and robustness consistently identified a core set of highly reliable circulating SVs suitable for future clinical development.
This multiplex reverse transcription-polymerase chain reaction sequencing assay enables sensitive detection of ccRCC-associated SVs from both tumor RNA and plasma cell-free RNA. Its high analytical fidelity, strong tumor-plasma concordance, and depth robustness support advancement of a focused SV panel for non-invasive molecular profiling, with potential applicability to longitudinal monitoring in ccRCC.

PMID:
42410176
Bibliographic data and abstract were imported from PubMed on 07 Jul 2026.

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