We have previously shown that expression of the IL8/VEGFA-metagene eliminates the good prognostic effect of TILs in TNBC (PMID 21978456, 28750120). We also showed that the VEGFA metagene predicted response to neoadjuvant bevacizumab in the GeparQuinto trial (Karn 2017 SABCS #851166). 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.
Individual genes that comprise the VEGFA metagene were measured in bulk tissue- and single cell-RNA-Seq from breast cancer subtypes and normal cells on different platforms (Affymetrix n=4915, Agilent n=597, Illumina n=2433, RNA-Seq n=1215, Exome Capture RNA-Seq n=226, HTG-Seq n=243, sc-RNA-Seq n=24710). For blinded, orthogonal validation we performed immunohistochemistry. Effect of neoadjuvant chemotherapy with or without bevacizumab was studied by RNA-Seq and IHC on samples from GeparQuinto trial. SWOG S0800 (GSE114403), PROMIX (GSE87455), and GeparSixto trials were used for validation. TCGA was mined for mutations and somatic CNA. RNA-Seq from GeparNuevo was used for correlation with checkpoint inhibitor treatment.
We identified a stable core of six genes (VEGFA, ANGPTL4, ADM, NDRG1, DDIT4, CSTB) in different cohorts. Strong expression of this signature was mainly restricted to TNBC subtype and associated with poor prognosis within this subgroup. Single cell RNA-Seq of breast epithelial cells from 4 reduction mammoplasties and 4 TNBC revealed that these genes are coexpressed in individual epithelial cells and not associated with endothelial cells. In line with their presumed functions in cellular stress and hypoxia, immunohistochemistry revealed strong para-necrotic expression in TNBC. Moreover, high gene expression in TNBC was associated with mutations in DNA damage control pathways, somatic copy number alterations, and lower TILs. While chemotherapy led to downregulation, bevacizumab increased expression. In multivariate analysis, high pretreatment values predict pCR to both bevacizumab and chemotherapy (OR 2.40, P=0.006), which may be explained by sensitivity of tumors which are already under cellular stress. On the other hand, expression of the VEGFA metagene seems to create an immunosuppressive environment that counteracts the positive prognostic effect of TILs. In pre-treatment biopsies from the GeparNuevo checkpoint inhibitor trial we found a negative correlation of VEGFA metagene expression with the amount of the recently identified tissue-resident memory T cell subset (CD8TRM, PMID 29942092; P=0.002), while the subsequent increase of CD8TRM during treatment was larger in tumors with high VEGFA (P=0.019).
Perinecrotic carcinoma cells under stress from hypoxia and or chromosomal instability are the source of the VEGFA metagene signature. Its predictive value in TNBC suggests estimating and reporting the amount of necrosis in the pathology report may be helpful in predicting response to preoperative chemotherapy, and could be used as stratification factor in clinical trials. The signature indicates an immunosuppressive environment and should be further studied in the context of immune therapies in combinations with anti-angiogenic treatment.
Citation Format: Karn T, Denkert C, Sinn BV, Weber K, Nekljudova V, Rody A, Meissner T, Hatzis C, El-Balat A, Becker S, Solbach C, Untch M, Schneeweiss A, von Minckwitz G, Loibl S, Pusztai L, Holtrich U. Single-cell profiling identifies hypoxic carcinoma cells as source of an immunosuppressive VEGFA metagene [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-01.