Tracking of allogeneic haematopoietic stem cells transplantation success rate at the level of genomic DNA with real-time PCR

Presaditev alogenskih krvotvornih matičnih celic je način zdravljenja določenih malignih in nemalignih bolezni. Stanje po alogenski presaditvi, pri katerem so v prejemnikovem telesu sočasno prisotne celice prejemnika in darovalca krvotvornih matičnih celic, imenujemo himerizem. Z določitvijo himerizma lahko zgodaj zaznamo zaplete kot so ponovitev bolezni, bolezen presadka proti gostitelju in zavrnitev presadka. Sledenje himerizma je zato pomembno za oceno in napoved uspešnosti presaditve krvotvornih matičnih celic in omogoča hitrejše ukrepanje ter poveča možnost ozdravitve bolnika. V nalogi smo za sledenje himerizma po presaditvi krvotvornih matičnih celic uporabili metodo PCR v realnem času in ocenili primernost kompleta setov AlleleSEQR (Celera, ZDA) ter programske opreme AlleleSEQR Suite v1.1 Chimerism Analysis Software (Celera, ZDA) in KMRengine Chimerism Analysis Software (GenDx, Nizozemska). Za testiranje smo uporabili vzorce genomske DNA, izolirane iz periferne krvi 23 bolnikov in njihovih 23 darovalcev. Pri vseh parih smo med 34 bialelnimi polimorfizmi insercij/delecij določili informativne označevalce, ki omogočajo razlikovanje med prejemnikovo in darovalčevo genomsko DNA. Nato smo s testom za kvantitativno določanje himerizma v več zaporednih vzorcih prejemnika, odvzetih v različnih časovnih intervalih po presaditvi krvotvornih matičnih celic, določili delež prejemnikove genomske DNA. Rezultate določitve himerizma smo primerjali s klinično sliko bolnika. Informativnost nabora označevalcev je bila 100 % za vse pare prejemnikov in darovalcev ter je bila po pričakovanju za nesorodne pare večja kot za sorodne. Pri 19 od 23 bolnikov se je klinična slika popolnoma ujemala z rezultati določitve himerizma. Pri štirih bolnikih se klinična slika ni vedno ujemala z rezultatom določitve himerizma. Pri petih bolnikih bi bilo za popolno interpretacijo rezultatov določitve himerizma potrebnih več zaporednih vzorcev. Bolezen presadka proti gostitelju je bila prisotna pri večjem deležu bolnikov s popolnim himerizmom kot pri bolnikih z mešanim himerizmom. Občutljivost metode je bila visoka, saj smo zaznali že 0,02 % himerizem, kar daje zgodnjo informacijo o dinamiki deleža prejemnikove genomske DNA. S testnim sistemom smo uspešno opravili tudi zunanjo kontrolo kakovosti. Zaključimo lahko, da je testni sistem primeren za sledenje himerizma po presaditvi alogenskih krvotvornih matičnih celic pri slovenski populaciji prejemnikov krvotvornih matičnih celic.Allogeneic hematopoietic stem cell transplantation is used in treating certain malignant and non-malignant diseases. Chimerism after allogeneic haematopoietic stem cell transplantation is a condition, in which recipient and donor cells are present in recipient’s body. Chimerism analysis enables early detection of complications, such as disease relapse, graft-versus-host disease and graft rejection. Chimerism monitoring is therefore important for assessment and prediction of successfulness of hematopoietic stem cell transplantation and enables earlier intervention and thus the possibility of a patient being cured is higher. In our study we determined the usefulness of the AlleleSEQR (Celera, USA) kit and software AlleleSEQR Suite v1.1 Chimerism Analysis Software (Celera, USA) and KMRengine Chimerism Analysis Software (GenDx, Netherlands) for chimerism monitoring after hematopoietic stem cell transplantation with the use of quantitative PCR. Genomic DNA samples isolated from peripheral blood of 23 patients and their 23 donors were used for testing. Among 34 biallelic insertion/deletion polymorphisms informative markers were determined, which enables differentiation between donor and recipient genomic DNA. The proportion of recipient genomic DNA in several successive recipient samples, collected in different time intervals after transplantation, was then determined using the test for quantitative chimerism monitoring. The chimerism results were compared with patient’s clinical picture. The informativity of the assay was 100 % for all included donor/recipient pairs and was higher for unrelated pairs than for related. The results of the determination of proportion of genomic DNA matched with the patient’s clinical picture in 19 patients out of 23. The chimerism results did not always match with the clinical picture in four patients. In five patients we would need more successive samples to completely interpret the chimerism results. A smaller proportion of patients with mixed chimerism had graft-versus-host disease compared to the patients with complete chimerism. The sensitivity of the assay is high, as we detected 0,02 % chimerism, which enables earlier information about the change in proportion of recipient genomic DNA. The external quality control was successfully passed with the tested system. We can conclude that the test system is suitable for chimerism monitoring after allogeneic haematopoietic stem cell transplantation for Slovenian population of haematopoietic stem cell recipients