Circulating endothelial cells (CECs) in allo-transplantation – Dr. Camillo Almici, Head of Laboratory for Manipulation and Cryopreservation of Stem Cells, Immunohaematology and Transfusion Medicine Service, AO Spedali Civili Brescia, Italy

Our first interview of the new year features Dr. Camillo Almici. Read on to learn more about the research Dr. Almici’s team is carrying out to understand the role of circulating endothelial cells (CECs) in allo-transplantation.

Almici C. et al. Counting circulating endothelial cells in allo-HSCT: an ad hoc designed polychromatic flowcytometry-based panel versus the CellSearch™ System. Scientific Reports 2019. doi:10.1038/s41598-018-36442-9.

The primary task of our laboratory is to support the BMT program at the ASST Spedali Civili in Brescia. We are in charge of the manipulation and cryopreservation of hematopoietic progenitor cell units from the patients, both autologous and from HLA-matched familiar or unrelated donors in the allogeneic setting. Besides our routine clinical duties, our research activity concerns mesenchymal stromal cells expansion from different sources, and more recently, our efforts have focused on the role of circulating endothelial cells (CEC) in allo-transplantation. By using different CEC identification and counting procedures, we have been involved in studying the possible correlations of CEC count changes with the endothelial complications of allogeneic hematopoietic stem cells transplantation (allo-HSCT).


What is the incidence of GVHD in allo-HSCT?

Although allo-HSCT is considered a potential curative therapy for patients affected by different hematologic disorders and has improved the survival expectations of many patients, it is still not without risks.  Graft-versus-host disease (GVHD) is the most life-threatening complication that can arise from allo-HSCT. In fact, the overall incidence of GVHD ranges between 30% and 60%, accounting for a 50% mortality rate, particularly in its more severe presentation. Therefore, clinical researchers are engaged in seeking specific and objective biomarkers in peripheral blood for improving GVHD diagnostic definition.


Which GVHD biomarkers are available in routine clinical practice?

The need of GVHD biomarkers in allo-HSCT is crucial because the identification of patients at higher risk to develop GVHD could envisage more stringent monitoring and/or potentially preventive care. To date, the more promising data on GVHD biomarkers identification has come from the large-scale proteomics approach. However, despite the first identification of a four-protein (sIL-2Ra, TNFR1, HGF, IL-8) biomarker panel1 and the tremendous expansion in the last decade of the identification and validation of GVHD proteomics biomarkers (i.e., ST2, IL-6, TIM3, REG3a, Elafin), none of these blood tests have yet entered routine clinical practice. To date, no laboratory test can predict, on a routine basis, the risk of developing GVHD or the responsiveness to treatment. Therefore, GVHD diagnosis still almost completely relies on clinical symptoms, clinician expertise and should be confirmed by histology of the affected tissues.


What is the role of the endothelium in GVHD pathogenesis?

The current GVHD pathogenesis model represents a multiphase process in which the result of the conditioning regimen, the release of cytokines and the antigenic differences between recipient and donor, lead donor lymphocytes to attack epithelial cells and mucous membranes in target organs (i.e., skin, gut, liver). The release of cytokines and the immune attack result in ongoing tissue damage with further cytokine production and a continuous inflammatory positive feedback loop. However, T-lymphocytes require a tight contact with cells of the vascular endothelium to reach the epithelial target organs. Therefore, the vascular endothelium is currently considered an early phase target of donor T-lymphocytes. During allo-HSCT, endothelial cells are affected by a series of events that result in endothelial damage, which may contribute to the development of immune reactions related to life-threatening complications, including GVHD. Current findings indicate that inflammation and neovascularization are functionally related and so inflammatory neovascularization represents a central event during GVHD occurrence. As neovascularization and endothelial damage are early steps in GVHD presentation, it is evident that markers of endothelial activity, such as CEC count changes, could be considered a valuable support to confirm GVHD diagnosis. Moreover, the physio-pathologic interrelationships between endothelium and GVHD have led to define the entity “endothelial GVHD” as an essential early phase prior to the clinical presentation of acute GVHD.


What are CECs?

CECs are a rare cellular subpopulation in blood, which come about due to detachment from vessel walls as a consequence of vascular damage or their physiological turnover. Together with endothelial progenitor cells (EPCs), CECs provide a measure of vascular health balance. CECs are markers of ongoing endothelial damage, whereas EPCs provide information about endothelial repair suitability. In several clinical conditions, CEC counts have been exploited as valuable biomarkers to monitor vascular damage and treatment response. However, due to their rareness and complex immunophenotype, no consensus has yet been fully reached on their identification and way of count. Moreover, the translation of CEC counts into clinical practice has also been weakened by the inconsistent results available till now. Therefore, the fine tuning of a standardized approach for CEC identification and count is crucial in order to move their monitoring into clinical practice. We have recently correlated CEC count changes to GVHD onset and response to treatment in 90 allo-HSCT patients, by using the commercially available rare cell isolation platform CellSearch™. 2 We confirmed that CEC change is a suitable biomarker to monitor endothelial damage in patients undergoing allo-HSCT and holds the potential to become a useful tool to support GVHD diagnosis. More recently, we joined the S.C.EN.I.C. network that has developed a highly optimized polychromatic flowcytometry (PFC) protocol for accurate CEC identification and counting3 by defining CEC as live and nucleated CD34bright/CD45neg/CD146pos events. Moreover, a physiological baseline range for healthy subjects has been established, suitable as starting point for CEC monitoring in endothelial dysfunctions.


What could be the impact of CEC monitoring in the allo-HSCT setting?

We believe that CEC count monitoring, as function of endothelial damage, may facilitate diagnosis of acute GVHD and might help identify non-responders before the overt development of refractory disease. Although CEC changes in allo-HSCT represent a dynamic phenomenon that can be influenced by many variables (among others, conditionings, immunosuppressive treatments, engraftment syndrome, infections), we previously showed that CEC peaks were constantly seen prior to the onset of acute GVHD and invariably returned to pre-transplant values after treatment response. Consequently, through the monitoring of CEC counts, it could become possible to guide the timely introduction, in non-responders, of additional immunosuppressive treatments whereas actions might be shifted to less aggressive approaches in responder patients.


Which are the aims of your present work?

We designed a comparative study of CEC counts in a cohort of 50 patients undergoing allo-HSCT with the aims to: (i) test an ad hoc designed PFC-based panel for CEC counting; (ii) evaluate the agreement of the PFC-based panel with the validated CellSearch™ system; (iii) confirm that CEC changes in allo-HSCT patients represents a suitable tool to support clinicians in the GVHD diagnosis.


Are the two CEC counting procedures comparable?

By comparing CEC values (PFC Lyotube™ versus CellSearch™) in allo-HSCT patients at five different timepoints, we have highlighted a significant comparability at all timepoints, except for the pre-transplant one (T2 timepoint). This lack was mainly related to the dual platform calculation procedure for PFC counts, as CEC determinations are heavily affected at T2 by the unreliability of lymphocyte absolute numbers obtained by standard cell counter in a very deep leukopenia phase. However, when lymphocyte absolute numbers at T2 is determined by PFC using a dedicated TruCount™ tube containing CD45 and absolute counting beads (CD45pos events in lympho-monocyte gate) a satisfactory correlation between the two counting methods is obtained.

Despite the significant correlation and the satisfactory degree of agreement between PFC Lyotube™ and CellSearch™, the two CEC counting procedures maintain pros and cons that make them not mutually exclusive, but rather complementary. The weaknesses of one CEC counting procedure are strengths of the other one and vice versa.


Which are the major pros/cons of each CEC counting procedure?

The CellSearch™ system has the great advantage, by using dedicated and specific tubes, of allowing CEC counting within 96 hours of blood drawing. This makes it possible to easily pull together samples from different days or to send them to a centralized lab-facility. However, the CellSearch™ system, besides APC and PE fluorescence channels dedicated to anti-CD45 and anti-CD105, has only one more channel available dedicated to FITC fluorescence detection. Therefore, availability of fluorescence combinations represents a fairly cumbersome limitation, particularly when dealing with phenotypically complex cellular events to identify.

PFC Lyotube™ has the more valuable advantage of the possibility to study additional antigens within the assay format (i.e. VEGFR2, for EPC identification and count) or to easily implement from the standard 6-color configuration up to combinations of 8-10 fluorescences. However, a disadvantage is the need to perform counting quickly (within 6 hours) after blood drawing, to avoid unpredictable decrease in CEC values over time. Furthermore, cryopreservation has detrimental effects, making CEC counting unreliable on thawed samples.


Are one procedure’s results superior to those of the other?

Our results strengthen the linear correlation between the two methods, but also point out the intrinsic limitations of each procedure.


What is the take home message of your work?

The present study represents the only comparison trial, performed to date, that confirms the potential role of CEC count changes as a suitable tool to support clinicians in GVHD diagnosis. We show that CEC counts, generated with either the CellSearch™ system or the PFC-Lyotube™, have a superimposable kinetic in allo-HSCT patients and that both counting procedures hold the potential to enter clinical routine as suitable tools to assist clinicians in GVHD diagnosis.


1 Paczesny S. et al. Blood 2009

2 Almici C et al. Bone Marrow Transplantation 2017

3 Lanuti P. et al. Scientific Reports 2018

Learn more about Dr. Almici here


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