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Updated: Feb 20, 2026
Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material
Published on: August 1, 2018
Reverse phase protein array identification of triple-negative breast cancer subtypes and comparison with mRNA
Hiroko Masuda1,2,3, Yuan Qi4, Shuying Liu1
1Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Background:
Reverse phase protein array (RPPA) analysis, allows investigation of potential targets at the functional protein level,. We identified TNBC subtypes at the protein level using RPPA and compared them with mRNA molecular subtypes (TNBCtype, TNBCtype-4, and PAM50) that is unique in its availability of both RPPA and mRNA analyses.
Methods:
We classified the samples from 80 TNBC patients using both k-means and hierarchical consensus clustering analysis and performed Ingenuity Pathway Analysis. We also investigated whether we could reproduce the mRNA molecular subtypes using the RPPA dataset.
Results:
Both clustering methods divided all samples into 2 clusters that were biologically the same. The top canonical pathways included inflammation, hormonal receptors, and MAPK signaling pathways for the first cluster ["inflammation and hormonal-related (I/H) subtype"] and the GADD45, DNA damage, and p53 signaling pathways for the second cluster ["DNA damage (DD)-related subtype"]. Further k-means cluster analysis identified 5 TNBC clusters. Comparison between sample classification using the 5 RPPA-based k-means cluster subtypes and 6 gene-expression-based TNBCtype molecular subtypes showed significant association between the 2 classifications (p = 0.017).
Conclusions:
The I/H and DD subtypes identified by RPPA advance our understanding of TNBC's heterogeneity from the functional proteomic perspective.
Insights
Reverse phase protein array (RPPA) analysis identified two main subtypes of triple-negative breast cancer (TNBC): inflammation and hormonal-related (I/H) and DNA damage (DD)-related. These RPPA-based subtypes correlate with gene-expression subtypes, enhancing understanding of TNBC heterogeneity.
Area of Science:
- Biomedical research
- Cancer biology
- Proteomics
Background:
- Triple-negative breast cancer (TNBC) exhibits significant heterogeneity.
- Reverse phase protein array (RPPA) enables functional protein-level target investigation.
- This study uniquely integrates RPPA and mRNA analyses for TNBC subtyping.
Purpose of the Study:
- To classify TNBC subtypes at the protein level using RPPA.
- To compare RPPA-derived subtypes with established mRNA molecular subtypes.
- To explore the functional proteomic basis of TNBC heterogeneity.
Main Methods:
- RPPA analysis of 80 TNBC patient samples.
- K-means and hierarchical consensus clustering for sample classification.
- Ingenuity Pathway Analysis to identify key signaling pathways.
- Comparison of RPPA-based clusters with TNBCtype, TNBCtype-4, and PAM50 mRNA subtypes.
Main Results:
- Two biologically consistent clusters emerged: inflammation and hormonal-related (I/H) and DNA damage (DD)-related subtypes.
- Key pathways identified include inflammation, hormonal receptors, MAPK signaling (I/H), and GADD45, DNA damage, p53 signaling (DD).
- Five TNBC clusters were identified using k-means RPPA analysis, showing significant association with gene-expression-based TNBCtype molecular subtypes (p = 0.017).
Conclusions:
- RPPA analysis successfully identified distinct I/H and DD subtypes in TNBC.
- These findings provide a functional proteomic perspective on TNBC heterogeneity.
- The identified subtypes offer potential for refined TNBC classification and targeted therapies.
