Advancements in cancer genomics have unlocked new possibilities for precision medicine. However, translating these discoveries into clinically approved tests remains challenging, especially when leveraging comprehensive tools like RNA (RNA-seq) and whole exome sequencing (WES). Our team recently published a validation study addressing this very issue — offering a clinically validated roadmap to guide cancer care and streamline drug development.
RNA-seq and WES Challenges
RNA-seq is a powerful tool that measures gene activity across the transcriptome, detects gene fusions, and reveals tumor-specific expression patterns to aid in discovering new therapeutic targets. When paired with WES — which focuses on the protein-coding portion of the genome where most actionable mutations occur — this combined approach delivers a more complete picture of a patient’s cancer biology. The integration allows clinicians to uncover both DNA- and RNA-level biomarkers from a single tumor sample.
Despite their promise, why are RNA-seq and WES still rarely used in the clinic? There are very few standardized frameworks for validating such broad and complex assays under clinical regulations, especially for achieving certifications like Clinical Laboratory Improvement Amendments (CLIA), New York State Department of Health (NYSDOH), and College of American Pathologists (CAP). In fact, as of now, only one CLIA-certified RNA-seq assay for gene expression exists in the United States.
WES faces similar challenges. Unlike small targeted panels, which focus on a few hundred known cancer-related genes, WES analyzes thousands of genes at once. But current guidelines for test validation were not designed taking this level of complexity into account. Additionally, one major roadblock is the lack of robust reference samples for somatic variant calling in cancer — in general, existing references consisting of normal DNA in various dilutions don’t adequately reflect the variability seen in tumors, particularly when it comes to detecting subclonal mutations or copy number changes.
Proposed Solution: A Clinically Validated Integrated Assay
At BostonGene, we developed the Tumor Portrait™ test — an integrated RNA-seq and WES assay designed to profile tumors alongside matched-normal tissue. We set out to validate this assay using the most comprehensive approaches to date, and it resulted in CLIA, CAP, and NYSDOH approvals for the combined approach, including RNA-seq-based components.
Key Pillars of our Validation Framework:
1. Building a Rigorous Validation Framework
We created a three-step validation process:
- Technical benchmarking with known reference materials
- Orthogonal comparison using results from other clinical methods
- Real-world validation using data from 2,230 cancer patients
2. Expanding and Sharing Genomic References
We curated a large public reference dataset with 3,042 small mutations (SNVs/INDELs) and nearly 50,000 gene amplifications/deletions across five cell lines now available on GitHub. This dataset sets a new benchmark for validating complex assays
3. Validating Gene Expression from Challenging Samples
Most real-world clinical samples come from formalin-fixed paraffin-embedded (FFPE) tissue, which often degrades RNA. We demonstrated high accuracy (correlation coefficient = 0.97) and reproducibility (<3.6% coefficient of variation at 1 TPM) in gene expression results — confirming robustness even under suboptimal conditions.
4. Delivering Real-World Impact
In our 2,230-patient cohort, 98% of tumors had at least one actionable mutation, and 89% overexpressed targets linked to antibody-drug conjugates (ADC), a fast-growing class of cancer therapies. This illustrates the clinical potential of the integrated assay not just in diagnostics, but in therapy selection.
5. RNA-seq Beyond Expression
We also proved that RNA-seq can help confirm DNA-based mutations — and even rescue ones that WES alone might miss. Up to 50% of relevant protein-coding mutations found by RNA-seq were below the WES detection threshold. This synergy between the two platforms strengthens both.
Conclusions: Looking Ahead
Our study offers a blueprint for integrating RNA-seq and WES into clinical workflows with validation rigor needed for real-world application While targeted panels remain useful, the complexity of cancer often demands broader tools — and RNA-seq and WES together provide that scope. But with complexity comes the responsibility of rigorous validation. Our study shows that it’s not only possible, but essential and can be further strengthened and customized.
This is a good starting point. As pharma companies increasingly seek to identify the right patients for the right therapies, especially in early-phase trials, robust platforms like BostonGene’s can help bridge discovery and delivery. We hope the guidelines and data we’ve shared will contribute to building stronger, more reliable assays across the field — accelerating precision oncology and ultimately bringing better treatments to patients faster.