Comparison of RNA Marker Panels for Circulating Tumor Cells and Evaluation of Their Prognostic Relevance in Breast Cancer

Abstract

Liquid biopsy is a promising tool for therapy monitoring of cancer patients, but a need for further research in this field exists in order to improve sensitivity, specificity, standardization and minimize costs. In our present study, we evaluated two panels of transcripts related with the presence of circulating tumor cells (CTCs) (Panel 1: CK19, EpCAM, SCGB2A2 and Panel 2: EMP2, SLC6A8, HJURP, MAL2, PPIC and CCNE2) in two cohorts of breast cancer patients (metastatic and early). A blood cell fraction possibly containing CTCs was isolated with density gradient centrifugation, followed by RNA isolation and qPCR using TaqMan® or RT-qPCR using hybridization probes. The positivity rates of the investigated panels were similar, albeit higher in metastatic (69.4% Panel 1, 75.0% Panel 2; total 86.1%) compared to early (18.9% Panel 1, 23.3% Panel 2; total 31.1%) breast cancer patients. CK19, SCGB2A2, EMP2, HJURP, MAL2, and CCNE2 individually correlated with shorter overall survival in the metastatic patient cohort. The findings highlight the additional value of Panel 2 markers, which are in contrast to CK19 and EpCAM not solely linked to an epithelial phenotype.

Prognostic significance of circulating tumor cells and tumor related transcripts in small cell lung cancer: A step further to clinical implementation

“…However, at the individual marker level, a substantial level of concordance was only observed with the highly specific DLL3 and CK19 LC (Table S2) assays. The CK19 LC hybridization probe RT‐qPCR assay was introduced to minimize false positive samples, due to a very high specificity 25 . Compared to the CK19 TaqMan assay, the CK19 transcript was not amplified in any of the HD samples using the hybridization probe assay.…”

“…The CK19 LC hybridization probe RT-qPCR assay was introduced to minimize false positive samples, due to a very high specificity. 25 Compared to the TaqMan CK19 transcript was not amplified in any of the HD samples using the hybridization probe assay.…”

“…The targets of interest ( SYP , CHGA , EpCAM , CK19 and DLL3 ) as well as the reference gene CDKN1B were quantified as previously described 19 with or without a preceding target‐specific pre‐amplification of 10 cycles. For each marker, a sample was assigned as positive according to previously defined cut‐off thresholds 25 . With the other half of the RNA samples CK19 gene expression using hybridization probes and primers specific for the CK19 gene 26 was analyzed on the Lightcycler 480 II (LC) (Roche) according to Welsch et al 25 Due to the high specificity of the assay, any amplified transcript was considered positive.…”

“…For each marker, a sample was assigned as positive according to previously defined cut‐off thresholds 25 . With the other half of the RNA samples CK19 gene expression using hybridization probes and primers specific for the CK19 gene 26 was analyzed on the Lightcycler 480 II (LC) (Roche) according to Welsch et al 25 Due to the high specificity of the assay, any amplified transcript was considered positive.…”

Detection and Molecular Characterization of Circulating Tumour Cells: Challenges for the Clinical Setting

“…In a recent study, using RT-qPCR, two panels of transcripts related to the presence of CTCs (Panel 1: CK19 , EpCAM , SCGB2A2 and Panel 2: EMP2 , SLC6A8 , HJURP , MAL2 , PPIC and CCNE2 ), in two cohorts of breast cancer patients (metastatic and early), were evaluated. The findings indicated that Panel 2 markers that were not linked to an epithelial phenotype also provided important prognostic information [ 50 ].…”