Cancer gene panel analysis of cultured circulating tumor cells and primary tumor tissue from patients with breast cancer

223 Background: Recently, we identified a six gene panel (CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8) for the RT-qPCR based detection of circulating tumor cells (CTC) in breast cancer patients. The aim of the present study was to evaluate the gene panel in further blood samples. Methods: Blood samples were taken from breast cancer patients with metastatic disease (MBC, N=10) or with no evidence of disease (NED, N=30). Putative CTC were enriched by Oncoquick density gradient centrifugation. Total RNA was isolated with RNeasy Micro Kit (QIAgen). Template cDNA was generated with M-MLV Reverse Transcriptase, RNase H Minus (Promega) and random nonamers as primers. RT-qPCR was performed in duplicate reactions using TaqMan Assays (Applied Biosystems) with default thermal cycling parameters. Raw data were analyzed with the AB7900 Sequence Detection Software version 2.2.2 using automatic baseline correction and manual cycle threshold setting. Gene expression was normalized to GAPDH expression. A threshold value TX for each gene X was set at two standard deviations above the mean dCtX value in the healthy control group. A patient was defined as CTC-positive, if at least one gene marker was over-expressed compared to the defined threshold. Results: The gene panel consisting of CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8 identified 4/11 MBC but only 5/27 NED patients as CTC positive (p=0.163). By adding known CTC markers (SCGB2A2, TFF1, FXYD3, AGR2, S100A18, and EPCAM) to the panel, 7/11 MBC but only 6/27 NED patients were CTC positive (p=0.018). The presence of CTC in NED patients correlated with pN staging (p=0.026). Only one out of the six CTC positive NED patients relapsed within the observation period (median 35 months, range 25-39 months from blood sampling). We observed no correlation of CTC positivity and recurrence in NED patients. Conclusions: The sensitivity of the RT-qPCR based CTC detection in breast cancer patients may be enhanced by adding known CTC markers (SCGB2A2, TFF1, FXYD3, AGR2, S100A18, and EPCAM) to the six gene panel (CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8). Longer follow-up times are needed to evaluate the predictive value of the gene markers on survival.

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Comparison of the PI3KCA pathway in circulating tumor cells and corresponding tumor tissue of patients with metastatic breast cancer

Molecular Medicine Reports ◽

10.3892/mmr.2017.6415 ◽

2017 ◽

Vol 15 (5) ◽

pp. 2957-2968 ◽

Cited By ~ 7

Author(s):

Maren Bredemeier ◽

Sabine Kasimir-Bauer ◽

Hans-Christian Kolberg ◽

Thomas Herold ◽

Sarah Synoracki ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Tumor Tissue ◽

Metastatic Breast

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Features of tumor heterogeneity in regional metastasis of breast cancer

Kazan medical journal ◽

10.17816/kmj2021-716 ◽

2021 ◽

Vol 102 (5) ◽

pp. 716-725

Author(s):

K K Konyshev ◽

S V Sazonov

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Primary Tumor ◽

Tumor Heterogeneity ◽

Tumor Tissue ◽

Clonal Evolution ◽

Clinical Manifestations ◽

Progesterone Receptors ◽

Primary Tumors ◽

Regional Metastases

The review looked at the issues of tumor heterogeneity in breast cancer. Tumor heterogeneity is classified according to the main feature demonstrating regional differences within a tumor (for example, heterogeneity of clinical manifestations, histological heterogeneity, heterogeneity of protein expression, etc.) and by tumor regions (differences between primary tumors and metastases, differences between cell clones within a single tumor node, etc.). Temporal heterogeneity is also distinguished, which manifests itself in the clonal evolution of tumor cells. The review covers the heterogeneity in the expression of four biomarkers from the gold standard for immunohistochemical staining of breast cancer: estrogen receptors, progesterone receptors, Her2/neu and Ki67 in primary tumor tissue and regional metastases. According to various studies, discordance in estrogen receptor status of primary tumor cells and metastases was observed with a frequency of 4 to 62%, progesterone receptors from 12 to 54%, Her2/neu from 0 to 24%, Ki67 from 4 to 39%. The results of studies of changes in the expression levels of individual markers in breast cancer metastases, as well as the heterogeneity of surrogate subtypes of tumor tissue in metastasis, are briefly described. Possible reasons for heterogeneity in the expression of key prognostic and predictive markers by primary tumor and metastatic cells, such as artificial factors at the preanalytic and analytic stages of the study, polyclonality of the primary tumor before metastasis, clonal evolution of tumor cells during metastasis, selection of tumor clones under the therapy are highlighted.

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Whole Genome Sequencing in single CTC improves clinical outcome in Her-2 negative breast cancer patients

10.21203/rs.3.rs-15473/v1 ◽

2020 ◽

Author(s):

Bo Yu ◽

Yongping Li ◽

Hao Yuan ◽

Bin Zhang ◽

Xiaofei Jiang ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Genome Sequencing ◽

Primary Tumor ◽

Tumor Tissue ◽

Whole Genome ◽

Breast Cancer Patients ◽

Her2 Gene ◽

Her 2

Abstract Background Tumor tissues are usually highly heterogeneous and difficult to characterize which could mislead treatment strategy. Circulating tumor cells (CTCs) represent the most active and invasive tumor cells. This study explored the feasibility of individualized treatment of breast cancer patients based on genome sequencing of single cell CTC. Methods Twenty-four CTCs were identified in three patients with breast cancer. For each patient, one polyploid CTC was captured and on which the whole genome sequencing (WGS) was performed. Based on the histopathological Her-2 status in tumor tissue and the HER2 gene status in WGS results of CTC, we adjusted treatment strategies, and monitored disease progression. Results Patient ID1 and ID2 are Her-2 positive in both primary tumor and HER2 abnormal in the DNA of CTC. In patient ID3, histological examination of primary tumor and liver metastases revealed Her-2 negative, but the WGS analysis of CTC showed that the HER2 gene was amplified and mutated. After adjusting treatment according to the results of CTC sequencing, the liver metastases and pleural effusion were significantly reduced, CTC number and ctDNA burden were decreased. In addition, some potential therapeutic targets and mutations in drug-resistant genes were found. Conclusion The results of CTC sequencing effectively guided treatment of a patient with HER2 gene amplification/mutation in CTC but with Her-2 negative on tumor tissue. CTC sequencing is useful in resolving the heterogeneity of tumors and providing precision medicine for patients.

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The influence of removal of primary tumor on incidence and phenotype of circulating tumor cells in primary breast cancer

Geburtshilfe und Frauenheilkunde ◽

10.1055/s-0031-1286423 ◽

2011 ◽

Vol 71 (08) ◽

Author(s):

FA Taran ◽

A Hartkopf ◽

M Banys ◽

N Krawczyk ◽

S Becker ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Primary Tumor ◽

Primary Breast Cancer

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Clinical Relevance of Immune Checkpoints on Circulating Tumor Cells in Breast Cancer

Cancers ◽

10.3390/cancers12020376 ◽

2020 ◽

Vol 12 (2) ◽

pp. 376 ◽

Cited By ~ 6

Author(s):

Maria A. Papadaki ◽

Anastasios V. Koutsopoulos ◽

Panormitis G. Tsoulfas ◽

Eleni Lagoudaki ◽

Despoina Aggouraki ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Tumor Tissue ◽

Mononuclear Cells ◽

Immune Checkpoints ◽

Peripheral Blood Mononuclear ◽

Increased Risk

The role of CD47 and PD-L1 expression on circulating tumor cells (CTCs) remains unclear, and it is currently unknown whether their distribution varies between the blood and tumor tissue in breast cancer (BC). In this study, CD47 and PD-L1 expression was investigated a) on peripheral blood mononuclear cell (PBMC) cytospins from early (n = 100) and metastatic (n = 98) BC patients, by triple immunofluorescence for CD47/PD-L1/Cytokeratins, and b) on matched primary and/or metastatic tumor tissue from CTC-positive patients using immunohistochemistry. CD47+and/orPD-L1+ CTCs were detected in 11%, 16.9%, and 29.6% of early, recurrent, and de novo metastatic patients (p = 0.016). In metastatic disease, CD47highand/orPD-L1high CTCs were associated with disease progression (p = 0.005) and shorter progression-free survival (PFS) (p = 0.010), and independently predicted for an increased risk of relapse (HR: 2.719; p = 0.008) and death (HR: 2.398; p = 0.034). PD-L1 expression rates differed between CTCs and tissue tumor cells and between peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) (positive concordance of 3.8% and 4%, respectively). CD47 expression also differed between CTCs and tumor cells (positive concordance of 11.5%). In conclusion, CTCs expressing CD47 and PD-L1 have independent poor prognostic implications in metastatic BC, indicating a potential role of innate and adaptive immune evasion mechanisms in their metastatic potential. The clinical value of the parallel assessment of the peripheral and local immune response merits further evaluation in BC.

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