Expression of Nestin associates with BRCA1 mutations, a basal-like phenotype and aggressive breast cancer

We here examined whether Nestin, by protein and mRNA levels, could be a predictor of BRCA1 related breast cancer, a basal-like phenotype, and aggressive tumours. Immunohistochemical staining of Nestin was done in independent breast cancer hospital cohorts (Series I-V, total 1257 cases). Also, TCGA proteomic data (n = 103), mRNA microarray data from TCGA (n = 520), METABRIC (n = 1992), and 6 open access breast cancer datasets (n = 1908) were analysed. Patients with Nestin protein expression in tumour cells more often had BRCA1 germline mutations (OR 8.7, p < 0.0005, Series III), especially among younger patients (<40 years at diagnosis) (OR 16.5, p = 0.003). Nestin protein positivity, observed in 9–28% of our hospital cases (Series I-IV), was independently associated with reduced breast cancer specific survival (HR = 2.0, p = 0.035) and was consistently related to basal-like differentiation (by Cytokeratin 5, OR 8.7–13.8, p < 0.0005; P-cadherin OR 7.0–8.9, p < 0.0005; EGFR staining, OR 3.7–8.2, p ≤ 0.05). Nestin mRNA correlated significantly with Nestin protein expression (ρ = 0.6, p < 0.0005), and high levels were seen in the basal-like intrinsic subtype. Gene expression signalling pathways linked to high Nestin were explored, and revealed associations with stem-like tumour features. In summary, Nestin was strongly associated with germline BRCA1 related breast cancer, a basal-like phenotype, reduced survival, and stemness characteristics.


GSE20685
This dataset includes 327 breast cancer patients (stage I-IV). Molecular subtypes were divided into six, I-VI. Type I is similar to the basal-like subtype; II is similar to the HER2 enriched subtype; III represents a mixture of HER2 and luminal B subtypes; IV represents luminal B, and V-VI represent the luminal A subtype 12,17,18 .

GSE22358
This dataset includes 154 breast cancer patients (stage II-III) 14 , with molecular subtypes based on PAM50 classification 7 .

GSE1456
This dataset is from a series of 159 breast cancer patients (stage I-II) 15 .
Molecular subtypes were classified as described by Sørlie et al. 17,18 . The gene expression profiling of this cohort has previously been approved by the Ethics Committee at the Karolinska University Hospital together with additional amendments.
A probe presence filter had been applied to select only probes present in at least 520 of 577 assays (n=10377) 16 . Thus, signature generation was not feasible, since many of the relevant probes were not present. Only Nestin mRNA expression and its association with molecular subtypes and BRCA1 germline mutations were analysed in this dataset.

Kaplan-Meier online database
An online database, "KM-plotter" (www.kmplot.com) 19 , including EGA (European Genome-phenome Archive) and GEO datasets (Affymetrix microarrays only), was used to evaluate Nestin mRNA levels and signature score in relation to recurrence-free breast cancer survival in a merged dataset of 1660 breast cancer cases.

RNA seq analysis-TEAK
With the use of TCGA Assembler 6 , the RNA seq level 3 RSEM normalized data from 1052 tumour samples were downloaded and processed. From this cohort, 263 samples with the highest expression of Nestin mRNA (upper quartile) and 263 samples with the lowest expression (lower quartile) were retained for subpathway enrichment analysis. We filtered genes with zero counts in more than 80% of the samples. An offset of 1 was added to normalized counts and log2 transformation was applied.
The Topology Enrichment Analysis frameworK (TEAK) tool 50 was applied after downloading the updated human KEGG pathway KGML files 51 . The tool, with default parameters, was run and the "case -control" setting was implemented. "Cases" were defined as the samples with high Nestin expression and as "controls", the samples with low Nestin expression. All linear and non-linear subpathway topologies from both metabolic and nonmetabolic KEGG pathways were examined.

Triple-negative subtypes
To assess whether Nestin mRNA levels and signature score were associated with the triple-negative categories published by Lehmann et al. 52  All statistical tests were two-sided, and statistical significance was assessed at 5% level, and p-values between 5-10% were regarded as borderline significant.