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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.

SUBMITTER: Welsch E

PROVIDER: S-EPMC9954525 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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

Welsch Eva E Schuster Eva E Krainer Michael M Marhold Maximilian M Bartsch Rupert R Fischer Michael B MB Hermann Michael M Hastermann Gabriele G Uher Heidemarie H Sliutz Gerhard G Anker Birgit B Zeillinger Robert R Obermayr Eva E

Cancers 20230216 4

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 ...[more]

PMID: 36831613

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Project description:Circulating tumor cells (CTCs) are useful biomarkers of many solid tumors, but are infrequently detected in early stage pancreatic ductal adenocarcinomas (PDACs). The first drainage of pancreatic venous blood flow come to portal vein and pass through the liver, and they finally go out for peripheral blood. We thought that comparing CTCs from portal vein and peripheral blood could enable us to understand the clinical meaning of CTCs from each different site in PDACs. Therefore, we aimed to determine 1) whether CTCs could be reliably identified in early stages (operable) of PDACs, 2) if there are any differences in the detected number of CTC in portal vein blood and peripheral blood, and 3) whether CTCs can be sensitive biomarkers for the prognosis of resectable PDAC patients. Newly diagnosed PDAC patients who underwent operation with curative intention between 2013 and 2015 were prospectively enrolled. Blood draws from portal and peripheral vein ran through the microfabricated porous filter, and anti-epithelial cell adhesion molecule (EpCAM) and anti-Plectin-1 antibodies were used for CTC identification. Baseline clinical characteristics, tumor characteristics, treatment, and clinical outcomes were assessed. The clinical stages of the 32 enrolled patients were as follows: IA/IB 1 (3.1%); IIA 9 (28.1%); IIB 17 (53.1%); III 5 (15.6%). Twenty-seven patients (84.4%) received R0 resection, while five patients (15.6%) received R1 resection. EpCAM+ CTCs were detected in 20 portal blood (62.5%) and 22 peripheral blood (68.8%). Plectin-1+ CTCs were identified in 14 portal blood (43.8%) and 16 peripheral blood (50%). Plectin-1-expressing CTCs were picked from CTC platform (microfabricated porous filter) and we could find out all KRAS mutation. Patients with detectable EpCAM+ CTC less than one in peripheral blood showed longer overall survival (OS) compared to patients with detectable CTCs more than one (35.5 months vs. 16.0 months). EpCAM and Plectin-1 successfully identified CTCs at the early stage of PDACs. Also, the number of CTCs could be a prognostic marker for survival in resectable PDACs.

Project description:BackgroundCurrently, there is no reliable blood-based marker to track tumor recurrence in endometrial cancer (EC) patients. Liquid biopsies, specifically, circulating tumor DNA (ctDNA) analysis emerged as a way to monitor tumor metastasis. The objective of this study was to examine the feasibility of ctDNA in recurrence surveillance and prognostic evaluation of high-risk EC.MethodsTumor tissues from nine high-risk EC patients were collected during primary surgery and tumor DNA was subjected to next generation sequencing to obtain the initial mutation spectrum using a 78 cancer-associated gene panel. Baseline and serial post-operative plasma samples were collected and droplet digital PCR (ddPCR) assays for patient-specific mutations were developed to track the mutations in the ctDNA in serial plasma samples. Log-rank test was used to assess the association between detection of ctDNA before or after surgery and disease-free survival.ResultsSomatic mutations were identified in all of the cases. The most frequent mutated genes were PTEN, FAT4, ARID1A, TP53, ZFHX3, ATM, and FBXW7. For each patient, personalized ddPCR assays were designed for one-to-three high-frequent mutations. DdPCR analysis and tumor panel sequencing had a high level of agreement in the assessment of the mutant allele fractions in baseline tumor tissue DNA. CtDNA was detected in 67% (6 of 9) of baseline plasma samples, which was not predictive of disease-free survival (DFS). CtDNA was detected in serial post-operative plasma samples (ctDNA tracking) of 44% (4 of 9) of the patients, which predicted tumor relapse. The DFS was a median of 9 months (ctDNA detected) versus median DFS undefined (ctDNA not detected), with a hazard ratio of 17.43 (95% CI, 1.616-188.3). The sensitivity of post-operative ctDNA detection in estimating tumor relapse was 100% and specificity was 83.3%, which was superior to CA125 or HE4.ConclusionsPersonalized ctDNA detection was effective and stable for high-risk EC. CtDNA tracking in post-operative plasma is valuable for predicting tumor recurrence.

Project description:BackgroundThe detection of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTC) before and/or after adjuvant chemotherapy in patients with operable breast cancer is associated with poor clinical outcome. Reliable prognostic markers for late disease relapse are not available. In this study we investigated the value of CTC detection during the first five years of follow-up in predicting late disease relapse.MethodsBlood was analyzed from 312 women with operable breast cancer who had not experienced disease relapse during the first two years of follow-up. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) for CK-19 mRNA was used to detect CTC three months after the completion of adjuvant chemotherapy and every six months thereafter for a follow-up period of five years.ResultsEighty patients (25.6% of the study population) remained CTC free throughout the five-year period. A change in CTC status was observed in 133 patients (42.6%); 64 patients (20.5%) with initially CK-19 mRNA-positive CTC during the first 24 months turned CTC-negative afterwards while 69 (22.1%) who were initially CTC-negative became CTC-positive. Ninety-nine patients (31.7%) remained persistently CK-19 mRNA-positive. After a median follow-up period of 107 months (range: 38 to 161 months), the persistently CTC-positive patients with either hormonal receptor positive or negative tumors, had a higher risk of late-disease relapse compared to the persistently CTC-negative patients (36.4% versus 11.2%, P <0.001). Multivariate analysis revealed that persistently CTC-positive patients also had a shorter disease-free (P = 0.001) and overall survival (P = 0.001).ConclusionsPersistent detection of CK-19 mRNA-positive CTC during the first five years of follow-up is associated with an increased risk of late relapse and death in patients with operable breast cancer and indicates the presence of chemo-and hormonotherapy-resistant residual disease. This prognostic evaluation may be useful when deciding on subsequent adjuvant systemic therapy.

Project description:BackgroundThe isolation of cell-free DNA (cfDNA) from the bloodstream can be used to detect and analyze somatic alterations in circulating tumor DNA (ctDNA), and multiple cfDNA-targeted sequencing panels are now commercially available for Food and Drug Administration (FDA)-approved biomarker indications to guide treatment. More recently, cfDNA fragmentation patterns have emerged as a tool to infer epigenomic and transcriptomic information. However, most of these analyses used whole-genome sequencing, which is insufficient to identify FDA-approved biomarker indications in a cost-effective manner.Patients and methodsWe used machine learning models of fragmentation patterns at the first coding exon in standard targeted cancer gene cfDNA sequencing panels to distinguish between cancer and non-cancer patients, as well as the specific tumor type and subtype. We assessed this approach in two independent cohorts: a published cohort from GRAIL (breast, lung, and prostate cancers, non-cancer, n = 198) and an institutional cohort from the University of Wisconsin (UW; breast, lung, prostate, bladder cancers, n = 320). Each cohort was split 70%/30% into training and validation sets.ResultsIn the UW cohort, training cross-validated accuracy was 82.1%, and accuracy in the independent validation cohort was 86.6% despite a median ctDNA fraction of only 0.06. In the GRAIL cohort, to assess how this approach performs in very low ctDNA fractions, training and independent validation were split based on ctDNA fraction. Training cross-validated accuracy was 80.6%, and accuracy in the independent validation cohort was 76.3%. In the validation cohort where the ctDNA fractions were all <0.05 and as low as 0.0003, the cancer versus non-cancer area under the curve was 0.99.ConclusionsTo our knowledge, this is the first study to demonstrate that sequencing from targeted cfDNA panels can be utilized to analyze fragmentation patterns to classify cancer types, dramatically expanding the potential capabilities of existing clinically used panels at minimal additional cost.

Dataset Information

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

Publications

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

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