CEC Count Changes to Support GvHD Diagnosis.
Condition: Graft vs Host Disease; Complications of Organ Transplant Stem Cells; Endothelial Dysfunction
MeSH terms: Graft vs Host Disease
Study type: Observational

Summary: 
      In consideration of the fact that the vascular endothelium has been shown to be a target of
      GvHD in early stage and that the count of CEC may represent a marker of endothelial damage,
      we want to evaluate the changes in CEC counts of patients affected by hematological disorders
      undergoing allo-HSCT, as a function of endothelial damage. We will enroll 50 patients
      affected by hematologic disorders undergoing allo-HSCT. Peripheral blood will be drawn before
      (T1, baseline) and at the end of the conditioning regimen (T2, pre-transplant), upon
      confirmation of hematopoietic recovery (T3, engraftment) and thereafter at onset of GVHD
      (GVHD T4) and one week after the start of steroid therapy (T5, post-GvHD). All patients will
      also be checked for CEC at day + 28. CEC enumeration will be performed by using the
      CellSearch® System and a flowcytometry procedure.

      Through the conduct of this study, we expect to confirm our preliminary results on a larger
      series of patients, and to evaluate the predictive role of CEC on the occurrence of GvHD and
      prognostic response to treatment of GvHD. The possibility of early identification of patients
      who do not respond to traditional treatments of GvHD, and for this reason at a higher risk of
      morbidity and mortality, may allow greater individualization of the therapeutic program, for
      example with the introduction as early as possible of alternative treatments. In addition,
      the identification of patients at higher risk of non-responsiveness to steroid treatment,
      would allow, through a closer monitoring, the early introduction of additional treatment
      before the development of resistance/refractoriness to treatment of GvHD.

      The present study takes the form of a prospective study. The primary endpoint is the
      identification and enumeration of CECs in peripheral blood of patients with hematological
      disorder undergoing allo-HSCT, as a function of endothelial damage. The secondary endpoint is
      to define the prognostic and predictive value of the changes of CEC counts on the diagnosis
      of GvHD and response to treatment.
    

Detailed description: 
      The endothelial damage is a characteristic common to several complications of vascular origin
      that may occur in the course of allo-HSCT (GVHD, IPS, VOD, TMA). The diagnosis of vascular
      complications represents an interesting challenge, but unfortunately limited by the fact that
      the markers of endothelial damage are extremely scarce. In particular, the plasmatic dosage
      of von Willebrand factor, thrombomodulin and adhesion molecules generated results difficult
      to use for a potential application for routine diagnostics for the high degree of
      non-specificity and the potential influence on the assay by co-morbid conditions related to
      the recipient. Much more promising methods appear to be those that are based on the
      evaluation of microparticles derived from endothelial cells (EMP) or the direct counting of
      circulating endothelial cells (CEC). Recently, the EMP were assessed by flow cytometry
      (annexin V-FITC and anti-CD62-PE) in 19 patients undergoing allo-HSCT, showing an increase in
      patients with acute GvHD grade ≥ I, compared to patients that did not display GvHD. In
      addition, the evaluation of EMP did not appear to be affected by the radio-chemotherapy
      conditioning regimen. This method is, however, burdened with a procedural complexity and a
      certain degree of non-specificity due to the fact that the microparticles can be released by
      endothelial cells as well as also from platelets, red blood cells and leukocytes.

      Currently, the direct counting of the CEC seems to be the most reliable ways to assess the
      degree of endothelial damage. The finding of elevated numbers of CEC has been shown to
      reflect the extent of endothelial damage in numerous pathologies of autoimmune nature, but
      are still preliminary data in the course of allo-HSCT. Woywodt et al. demonstrated in
      patients with ANCA-positive vasculitis a correlation between the number of CEC and the degree
      of disease activity and response to treatment. Moreover, the number of CEC correlates, in
      patients undergoing renal transplantation, with the risk of organ rejection. In the course of
      allo-HSCT, the same authors have shown a correlation between the number of CEC and
      endothelial damage induced by radio-chemotherapy conditioning regimen. However, the lack of a
      standardized method, the use of manual procedures of immunoselection, the lack of consensus
      on the identification of CEC represent limiting factors for routine application.

      The present study takes the form of a prospective study. The primary endpoint is the
      identification and enumeration of CECs in peripheral blood of patients with hematological
      disorders undergoing allo-HSCT, as a function of endothelial damage. The secondary endpoint
      is to define the prognostic and predictive value of CEC counts changes on the diagnosis of
      GvHD and on the response to treatment.

      Peripheral blood (PB) will be drawn before (T1, baseline) and at the end of the conditioning
      regimen (T2, pre-transplant), upon confirmation of hematopoietic recovery (T3, engraftment)
      and thereafter at onset of GVHD (GVHD T4) and one week after the start of steroid therapy
      (T5, post-GvHD) for the control of GvHD. All patients will also be checked for CEC at day +
      28.

      The peripheral blood for counting CEC will be collected, respectively, in the CellSave
      Preservative Tube (Veridex, J & J, USA), containing a preservative for the stabilization of
      the cells at room temperature, for counting with the CellSearch® System and in CBC tube
      containing K2EDTA, for counting by flow cytometry.

      By the CellSearch® System an event will be classified as CEC when its morphology is
      consistent with that of a cell and simultaneously shows the following phenotype: CD146+,
      CD105+, DAPI+ and CD45-. By the flowcytometry procedure, after staining of cells with
      lyophilized antibodies of the Endo Panel tube (CD146, 7-AAD, CD34, CD309, CD45) 4x106 events
      in the lympho-monocyte gate will be immediately aquired at flowcytometry.
    

Intervention name(s): Patients undergoing allo-HSCT, who manifest GvHD.
Intervention type(s): Other
Intervention description(s): Changes in CEC counts in relation to GvHD ONSET
Observational model: Case-Only
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Patients, affected by hematologic disorders, undergoing allo-HSCT.
      
Keywords: Graft vs Host Disease; Hematopoietic Stem Cell Transplant; Biological Marker; Circulating Endothelial Cells; Endothelial Dysfunction