Why Isoform-Resolved RNA?

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Full-length, Isoform-Resolved RNA Exposes the Drivers of Phenotype

A single disease-linked DNA mutation can produce 30+ protein isoforms—only isoform-resolved RNA analysis reveals what’s actually happening.

The phenotypic potential of the human genome fully arises only after complex steps of RNA processing. For each DNA gene, alternative RNA splicing and related processing create multiple gene products, often with different molecular functions.

Only after RNA processing:

Functional Diversity Emerges
Mutations take effect only after RNA processing defines which protein isoforms are produced.
Disease-Causing Isoforms Revealed
Abnormal RNA processing creates pathogenic isoforms invisible to genome sequencing.

Real-world Examples from Oncology - HER2

Oncologists have been aware of the disconnect between genes and phenotype for a long time. For example, there is a 10% to 25% discordance rate between protein-based versus gene-based tests for HER2.

Discordance between protein expression and gene status is not uncommon and has significant therapeutic implications. A HER2 IHC score of 2+, for example, can either lead to targeted therapy or not, depending on a confirmatory FISH test.
Head shot of Dr. Reis-Filho
Jorge S. Reis-Filho
Memorial Sloan Kettering Cancer Center
Breast Cancer Res, 2005
A subgroup of HER2‑overexpressing tumors also express p95HER2, an amino-terminally truncated receptor that lacks the extracellular domain but retains a highly functional HER2 kinase domain.
Head shot of Dr. Scaltriti
Maurizio Scaltriti
Vall d'Hebron University Hospital and Research Institute, Barcelona, Spain
J. Natl Cancer Inst, 2007

Real-world Examples from Oncology - Other Biomarkers

  • Biomarker
    Gene Test
    Protein Test
    Isoform / Modification Discordance
  • EGFR
    Gene Test
    Mutation panel (e.g., exon 19 del)
    Protein Test
    IHC (total protein)
    Discordance
    Some mutations may alter protein stability or epitope
  • PD-L1
    Gene Test
    mRNA expression panels
    Protein Test
    IHC (protein on membrane)
    Discordance
    PD-L1 mRNA ≠ surface protein; localization and glycosylation affect detectability
  • ER/PR
    Gene Test
    ESR1/PGR mRNA expression
    Protein Test
    IHC (nuclear receptor proteins)
    Discordance
    Receptor variants may be inactive or degraded
  • ALK, ROS1, NTRK
    Gene Test
    Fusion detected by NGS or FISH
    Protein Test
    IHC (antibody to fusion protein)
    Discordance
    Some fusions may not produce stable or detectable protein
  • TP53
    Gene Test
    Mutation (missense, nonsense)
    Protein Test
    IHC (accumulation of dysfunctional protein)
    Discordance
    Truncating mutations → no protein, despite mutation detected
  • CD20
    Gene Test
    No DNA test (expression only)
    Protein Test
    IHC / Flow
    Discordance
    Splice variants may reduce expression, affecting rituximab efficacy

Real-world Examples from Antisense Oligonucleotide (ASO) Therapies

Protein isoforms, created through alternative splicing, are key to disease pathology and therapeutic targets. ASO therapies modulate splicing to address dysfunctional isoforms, such as nusinersen (Spinraza) enhancing SMN2 exon 7 inclusion for SMA, or eteplirsen inducing exon 51 skipping for DMD, restoring functional proteins where genomic methods fall short.

Tools like JunctionSeq and BIISQ enable discovery and quantification of novel isoforms, improving cancer classification in TCGA cohorts and supporting ASO development. By early 2025, 11 ASO drugs are approved, with cases like Mila’s custom ASO for Batten disease highlighting personalized RNA interventions.



References for HER2
HER2 Isoform Issue

Scaltriti M, Rojo F, Ocana A, et al. Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer. J Natl Cancer Inst. 2007;99(8):628-638. doi:10.1093/jnci/djk134

Arribas J, Baselga J, Pedersen K, Parra-Palau JL. p95HER2 and breast cancer. Cancer Res. 2011;71(5):1515–1519. doi:10.1158/0008-5472.CAN-10-3795

Breast Cancer

Yin L, Zhang H, Liang X, et al. Clinicopathological characteristics and biological markers of primary and recurrent or metastatic breast cancer: a meta-analysis. Oncology Advances. 2024;4(1):e00027. doi:10.20801/ona.2024.00027

Press MF, Sauter G, Bernstein L, et al. Diagnostic evaluation of HER2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large prospective clinical trials. Breast Cancer Res Treat. 2005;86(1):1-13. doi:10.1007/s10549-004-4561-3

Gastric/Gastroesophageal Cancer

Park SR, Park YS, Ryu MH, et al. Extra-gastric metastases of HER2-positive gastric carcinoma: discordance of HER2 status between primary tumors and metastatic lesions. Sci Rep. 2017;7:13023. doi:10.1038/s41598-017-03304-9

Kim MA, Jung EJ, Lee HS, et al. Prognostic significance of HER2 gene amplification and protein overexpression in gastric carcinoma: a study of 587 patients. Int J Cancer. 2008;122(4):967-975. doi:10.1002/ijc.23187

Lee HE, Park KU, Yoo SB, et al. Clinical significance of intratumoral HER2 heterogeneity in gastric cancer. Eur J Cancer. 2013;49(6):1448-1457. doi:10.1016/j.ejca.2012.10.019

Other Solid Tumors / Pan-Cancer Studies

Sahin IH, Lee C, Svrcek M, et al. Discordance of HER2 expression and/or amplification between primary tumors and metastases in gastrointestinal malignancies. Mol Cancer Ther. 2023;22(8):976–985. doi:10.1158/1535-7163.MCT-22-0966

References for Other Oncology Biomarkers
EGFR

Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007;7(3):169–181.

Wei J, et al. Discordance between EGFR mutation status and EGFR protein expression in non-small cell lung cancer. Oncology Letters. 2019;17(6): 5047–5052.

PD-L1

Ilie M, Long-Mira E, Bence C, et al. Comparative study of the PD-L1 status between surgically resected specimens and matched biopsies of NSCLC patients. Ann Oncol. 2016;27(1):147–153.

Wang Y, et al. PD-L1 expression variability in lung cancer: causes and consequences. Cancer Commun. 2020;40(2):87–98.

ER/PR

van de Vijver MJ, et al. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med. 2002;347(25):1999–2009.

Shibata A, et al. Discordance in ER, PR, and HER2 between primary breast cancer and brain metastases. J Clin Oncol. 2018;36(suppl):1016.

ALK / ROS1 / NTRK

Camidge DR, Doebele RC. Treating ALK-positive lung cancer — early successes and future challenges. Nat Rev Clin Oncol. 2012;9(5):268–277.

Solomon JP, Linkov I, Rosado A, et al. NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls. Mod Pathol. 2020;33:38–46.

TP53

Soussi T, Wiman KG. TP53: an oncogene in disguise. Cell Death Differ. 2015;22(8):1239–1249.

Bártová E, et al. p53-dependent mechanisms of gene regulation and chromatin remodeling. Cell Mol Life Sci. 2019;76:2249–2261.

CD20

Tedder TF, Engel P. CD20: a regulator of cell-cycle progression of B lymphocytes. Immunol Today. 1994;15(9):450–454.

Johnson NA, Boyle M, Bashashati A, et al. Diffuse large B-cell lymphoma: reduced CD20 expression is associated with an inferior survival. Blood. 2009;113(16):3773–3780.