Encode
DNA defines potential
The genome provides the sequence, but not the final molecular product.
The missing biological layer
Genes set the possibility.
Isoforms shape the phenotype.
A single disease-linked DNA mutation can produce 30+ protein isoforms. Only full-length, isoform-resolved RNA analysis shows which molecules biology actually produced.
Alternative splicing, promoter usage, UTR selection, and other processing steps transform each gene into multiple RNA products—often with different molecular functions and clinical consequences.
From sequence to function
The phenotype emerges after the gene is read.
Gene-level counts compress this biology. Isoform resolution preserves the molecular decisions that occur between DNA and function.
Process
RNA creates diversity
Splicing and transcript processing determine which full-length molecules exist.
Resolve
Isoforms expose function
Complete transcripts reveal the disease-driving and therapeutically relevant forms.
Functional diversity becomes visible
Mutations take effect only after RNA processing defines which protein isoforms are produced.
Disease-causing isoforms are resolved
Pathogenic RNA products can remain invisible to genome sequencing and gene-level summaries.
Localization-associated signals remain visible
Isoform-specific UTR features can support hypotheses about RNA localization and trafficking.
Clinical evidence / HER2
One gene. Different molecular realities.
Oncologists have long observed a 10% to 25% discordance rate between protein-based and gene-based tests for HER2. The gap is not noise—it can reflect different isoforms, including p95HER2, a truncated receptor that retains an active kinase domain.
10–25%reported discordance between HER2 gene- and protein-based tests
“Discordance between protein expression and gene status is not uncommon and has significant therapeutic implications.”
Jorge S. Reis-FilhoMemorial Sloan Kettering Cancer CenterBreast Cancer Res, 2005
“A subgroup of HER2-overexpressing tumors also express p95HER2, an amino-terminally truncated receptor.”
Maurizio ScaltritiVall d'Hebron University HospitalJ. Natl Cancer Inst, 2007
Beyond HER2
The same gap appears across oncology.
Across major biomarkers, DNA, RNA, and protein tests answer different questions. Isoform resolution explains part of the distance between them.
Biomarker
Gene test
Protein test
What can diverge
EGFR
Gene testMutation panel (e.g., exon 19 del)
Protein testIHC (total protein)
What can divergeSome mutations may alter protein stability or epitope
PD-L1
Gene testmRNA expression panels
Protein testIHC (protein on membrane)
What can divergePD-L1 mRNA ≠ surface protein; localization and glycosylation affect detectability
ER/PR
Gene testESR1/PGR mRNA expression
Protein testIHC (nuclear receptor proteins)
What can divergeReceptor variants may be inactive or degraded
ALK, ROS1, NTRK
Gene testFusion detected by NGS or FISH
Protein testIHC (antibody to fusion protein)
What can divergeSome fusions may not produce stable or detectable protein
TP53
Gene testMutation (missense, nonsense)
Protein testIHC (accumulation of dysfunctional protein)
What can divergeTruncating mutations → no protein, despite mutation detected
CD20
Gene testNo DNA test (expression only)
Protein testIHC / Flow
What can divergeSplice variants may reduce expression, affecting rituximab efficacy
Therapeutic relevance
Isoforms are not just biomarkers. They are therapeutic targets.
Antisense oligonucleotide therapies modulate RNA splicing to restore or suppress specific molecular forms—intervening at a layer gene-level methods cannot resolve.
Tools such as JunctionSeq and BIISQ have helped discover and quantify novel isoforms. By early 2025, 11 ASO drugs had been approved, while individualized cases such as Mila’s custom ASO demonstrated the potential of personalized RNA intervention.
Spinal muscular atrophy
Nusinersen
Promotes SMN2 exon 7 inclusion to increase functional protein.
Duchenne muscular dystrophy
Eteplirsen
Induces exon 51 skipping to restore a functional reading frame.
Source material
References
The studies supporting the biological, clinical, and biomarker examples above.
HER2 and breast cancer
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.
Press MF, Sauter G, Bernstein L, et al. Diagnostic evaluation of HER2 as a molecular target. Breast Cancer Res Treat. 2005;86(1):1-13.
Gastric and pan-cancer HER2
Park SR, Park YS, Ryu MH, et al. Extra-gastric metastases of HER2-positive gastric carcinoma. Sci Rep. 2017;7:13023.
Kim MA, Jung EJ, Lee HS, et al. Prognostic significance of HER2 gene amplification and protein overexpression in gastric carcinoma. Int J Cancer. 2008;122(4):967-975.
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.
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.
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. 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.
UTRs and RNA localization
Salehi S, Zare A, Prezza G, et al. Cytosolic Ptbp2 modulates axon growth in motoneurons through axonal localization and translation of Hnrnpr. Nat Commun. 2023;14:4158. doi:10.1038/s41467-023-39787-6.
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