We previously identified a VEGFA metagene in TNBC which is associated with poor prognosis and eliminates the good prognostic effect of TILs (PMID 21978456, 28750120). But high pretreatment values of this signature predicted pCR in the neoadjuvant GeparQuinto trial (OR 2.40, P = 0.006) showing an interaction with bevacizumab treatment (P = 0.020). The main cellular sources of the transcripts that comprise the VEGFA metagene are unknown since mRNA profiling of bulk biopsies contains signals from different cell types. The individual genes have been reported to be expressed both in endothelial cells, fibroblasts, and epithelial cancer cells.
We analyzed single-cell sequencing RNA-Seq (sc-RNA-Seq) data of n = 772 single cells from TNBC and 52,698 single cells from lung cancer and non-malignant lung to identify the cellular source of the metagene expression. We then established and validated single marker IHC assays for the VEGFA metagene and applied them to tissue micro arrays (TMA).
sc-RNA-Seq from TNBC revealed co-expression of core genes of the VEGFA metagene (NDRG1, VEGFA, and DDIT4) in different cell types with highest expression in basal-like cancer cells. Strong co-expression in a subset of carcinoma cells was also seen in lung cancer sc-RNA-Seq, but only limited expression in endothelial cells. An IHC assay revealed strong para-necrotic expression in line with functions in cellular stress and hypoxia. Whole-slide-IHC showed good correlation to mRNA expression even in core biospies (Kappa=0.864, P < 0.001). However, comparison of whole-slide and TMA demonstrated a profound loss of sensitivity, while specificity remained high. Loss of sensitivity was mainly due to heterogeneity in expression and the small sized TMA cores from pretreatment biopsies. Nevertheless, still a predictive value for pCR was detected in the TMA dataset (P = 0.025).
The cellular source of the VEGFA metagene are peri-necrotic carcinoma cells. Thus, necrosis detection may help in response prediction and as stratification factor in trials. Since the signature indicates an immunosuppressive environment it should also be further studied in the context of combination therapies of anti-angiogenic and immune treatments.
University of Frankfurt.
H.W. & J. Hector-Stiftung, Mannheim, Germany.
All authors have declared no conflicts of interest.