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Article

Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients

1
Biomarker Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Via Giovanni Antonio Amadeo 42, 20133 Milano, Italy
2
Division Personalized Tumor Therapy, Fraunhofer-Institute for Toxicology and Experimental Medicine, Biopark 1|Am Biopark 9, 93053 Regensburg, Germany
3
Experimental Medicine and Therapy Research, University Regensburg, Franz-Josef-Strauss Allee 11, 93040 Regensburg, Germany
4
Unit of Biostatistics, Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25125 Brescia, Italy
5
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, 171 77 Stockholm, Sweden
6
Breast Unit, Fondazione IRCCS Istituto Nazionale Dei Tumori di Milano, Via Venezian 1, 20133 Milano, Italy
7
Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Via Giacomo Venezian 1, 20133 Milan, Italy
8
Oncology and Hemato-Oncology Department, University of Milan, Via Festa del Perdono 7, 20122 Milano, Italy
*
Author to whom correspondence should be addressed.
Equal contribution M.S. and C.R. are co-first authors.
Equal contribution B.P. and V.C. are co-last authors.
Academic Editors: Fabio Puglisi and Lorenzo Gerratana
Cancers 2021, 13(6), 1409; https://doi.org/10.3390/cancers13061409
Received: 28 January 2021 / Revised: 26 February 2021 / Accepted: 15 March 2021 / Published: 19 March 2021
(This article belongs to the Special Issue Liquid Biopsy in Cancer)
Distant metastases derive from the shedding and dissemination of single cancer cells (CTCs) or circulating tumor emboli (CTMs) into circulation. Previous studies on CTMs were mainly run in patients with metastatic disease; however, we observed that CTMs are more frequently detected in patients with early-stage breast cancer. Here, we collected single CTMs and their relative primary tumor tissue samples in early-stage patients. By studying genomic aberrations, present in tumors cells and absent in normal cells, we predicted the tumor fraction thanks to a statistical model developed from a calibration curve with breast cancer cell lines. The tumor fraction ranged from 8% to 48% and CTMs contained specific and shared alterations with respect to tissue. Thus, CTMs may derive from different regions of the primary tumor or from occult micrometastases. Moreover, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be further explored in follow-up and mechanistic studies.
Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0–100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31–71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30–63%), whereas primary tumor-private alterations were rare (4–12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies. View Full-Text
Keywords: breast cancer; circulating tumor microemboli; metastatic dissemination; tumor fraction; copy number alteration; low-pass whole genome sequencing breast cancer; circulating tumor microemboli; metastatic dissemination; tumor fraction; copy number alteration; low-pass whole genome sequencing
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MDPI and ACS Style

Silvestri, M.; Reduzzi, C.; Feliciello, G.; Vismara, M.; Schamberger, T.; Köstler, C.; Motta, R.; Calza, S.; Ferraris, C.; Vingiani, A.; Pruneri, G.; Daidone, M.G.; Klein, C.A.; Polzer, B.; Cappelletti, V. Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients. Cancers 2021, 13, 1409. https://doi.org/10.3390/cancers13061409

AMA Style

Silvestri M, Reduzzi C, Feliciello G, Vismara M, Schamberger T, Köstler C, Motta R, Calza S, Ferraris C, Vingiani A, Pruneri G, Daidone MG, Klein CA, Polzer B, Cappelletti V. Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients. Cancers. 2021; 13(6):1409. https://doi.org/10.3390/cancers13061409

Chicago/Turabian Style

Silvestri, Marco, Carolina Reduzzi, Giancarlo Feliciello, Marta Vismara, Thomas Schamberger, Cäcilia Köstler, Rosita Motta, Stefano Calza, Cristina Ferraris, Andrea Vingiani, Giancarlo Pruneri, Maria Grazia Daidone, Christoph A. Klein, Bernhard Polzer, and Vera Cappelletti. 2021. "Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients" Cancers 13, no. 6: 1409. https://doi.org/10.3390/cancers13061409

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