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Chimerism Analysis and Graft Rejection after Allogeneic Hematopoietic Cell Transplantation
with Nonmyeloablative Conditioning.


Tania Masmas  


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Accepted by: Faculty of Health Sciences University of Copenhagen
Defended on: February 26, 2009
Official opponents: Anders Elm Pedersen , Jonas Mattsson , Mehmet Uzunel
Tutors: Arne Svejgaard , Lars Vindeløv

Published in the PhD Database: March 4, 2009


English abstract
This PhD thesis, consisting of two studies, is based on work performed in The Allogeneic Hematopoietic Cell Transplantation Laboratory, Department of Hematology, and The Tissue Typing Laboratory, Department of Immunology, Rigshospitalet.

Hematopoietic cell transplantation (HCT) is a potential curative treatment modality for several hematological disorders where allogeneic hematopoietic cells are infused into a recipient prepared with a conditioning regimen. This thesis will focus on the novel treatment modality, nonmyeloablative-conditioning HCT (NMC-HCT), where reduced conditioning consisting of low-dose radio-chemotherapy results in less toxicity but an increased risk of graft rejection in comparison to conventional myeloablative conditioning HCT. The curative principle of NMC-HCT is the graft-versus-tumor effect. Measurement of coexisting recipient and donor cells by chimerism analysis is an important tool in the follow up of these patients.

The aims of the thesis were to implement and evaluate a high-resolution real time quantitative polymerase chain reaction (RQ-PCR) method for chimerism analysis, and to analyze graft rejection episodes after NMC-HCT in a cohort of Danish patients treated for hematological malignancies.

Implementation of RQ- PCR based chimerism analysis with an improved detection limit of 0,1% cells was successful and results were comparable with results by standardized chimerism analysis based on short tandem repeats. Application of chimerism analysis of CD34+ cells as a measure of minimal residual disease had to be abandoned because of scarcity of CD34+ cell in the samples. Low donor T cell chimerism was confirmed as a risk factor for graft rejection and storage of donor cells at room temperature was identified as a new risk factor. Storage of donor cells is now being performed at 5°C. Retransplantation with NMC-HCT with increased total body irradiation was well-tolerated but with decreased overall- and progression-free survival as compared to patients with successful primary engraftment. There was no effect of pentostatin and donor lymphocyte infusion in patients with imminent graft rejection. Retransplantation as soon as possible seems to be the treatment of choice of graft rejection after NMC-HCT.



Danish abstract
Denne Ph.d. afhandling består af to studier og er baseret på arbejde udført i Laboratoriet for Allogen Hæmatopoietisk Celle Transplantation, Hæmatologisk Klinik og Vævstypelaboratoriet, Klinisk Immunologisk Afdeling på Rigshospitalet.

Hæmatopoietisk celle transplantation (HCT), hvor allogene hæmatopoietiske celler infunderes efter forbehandling med konditionering, er potentiel kurativ ved flere hæmatologiske sygdomme. Denne afhandling vil fokusere på en ny behandlings-modalitet, mini-transplantation, hvor reduceret konditionering bestående af lav-dosis radio-kemoterapi medfører mindre toksicitet, men afstødningsraten af donorcellerne til gengæld er forøget i forhold til HCT med myeloablative konditionering. Det kurative princip ved mini-transplantation er graft-versus-tumor effekten. Bestemmelse af forholdet mellem recipient og donor celler ved kimærisme analyse er et vigtigt redskab i opfølgningen af disse patienter.

Formålet med dette projekt har været at implementere og evaluere en høj-opløselig real-time kvantitativ polymerase chain reaction (RQ-PCR) metode til kimærisme analyse, samt at analysere afstødningsepisoder efter mini-transplantation i en kohorte af danske patienter med hæmatologiske lidelser.

Implementering af RQ-PCR baseret kimærisme analyse med en forbedret detektionsgrænse på 0,1% celler var vellykket og resultaterne var sammenlignelige med resultater fra standardiseret kimærisme analyse baseret på ¿short tandem repeats¿. Anvendelse af kimærisme-analyse af CD34+ celler som mål for minimal residual sygdom måtte opgives pga. for få CD34+ celler i prøverne.
Lav donor T celle kimærisme blev konfirmeret som risikofaktor for afstødning af transplantatet og opbevaring af donorceller ved stuetemperatur blev identificeret som ny risikofaktor. Opbevaring af donorceller foregår nu ved 5°C. Retransplantation efter afstødning med ny mini-transplantation med forøget bestrålings-intensitet var veltolereret, men med mindsket total og progressionsfri overlevelse i forhold til blandt patienter uden afstødning. Vi fandt ingen effekt af pentostatin og donor lymfocyt infusion ved truende afstødning. Retransplantation hurtigst mulig ved afstødning ser ud til at være det optimale behandlingsvalg.