الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
The treatment of paediatric ALL represents one of the greatest success stories in cancer therapeutics however, the outcome in adults remains grim despite paediatric inspired protocols. The majority of adult patients do achieve CR however, this apparent clearance of the disease is often temporary and more than half the patients relapse despite intensive cycles of chemotherapy. Although several novel immunotherapeutic agents have been introduced across the past decade to try to bridge this survival gap including monoclonal antibodies, bi-specific T-cell engagers and chimeric antigen receptor T-cells, until this day, allogeneic hematopoietic stem cell transplantation represents the main potentially curative treatment for adult ALL.It is now common practice to offer allo-HSCT to all high-risk adult ALL patients (from either matched related or unrelated donors) and to standard risk adult patients if a matched sibling donor is available. The aim of the transplant is to deplete the residual leukaemic burden and patient’s haematopoiesis, followed by infusion of donor haematopoietic stem cells to repopulate the bone marrow niches recovering haematopoiesis and providing a sustained graft versus leukaemia (GvL) effect. This involves a series of complex simultaneous and consecutive biological events that can potentially proceed in a non-optimal fashion at any point of time leading to a wide array of complications. Thus, post-transplantation monitoring is mandatory to predict negative events, such as graft failure, disease relapse, graft rejection and graft-versus-host disease, in order to intervene with appropriate therapy. In this context, chimerism analysis is an important method in monitoring post HSCT outcome. A variety of methods have been described to detect the chimeric state after allo-HSCT. These included sex chromosome analysis, cytogenetic analysis, RBC phenotyping, and restriction fragment length polymorphism analysis, each of which has limitations, including low sensitivity and degree of informativeness.The application of polymerase chain reaction (PCR)–based technology has greatly enhanced the ability to detect small amounts of host or donor cells. In particular, the amplification of short number tandem repeats (STR) by PCR has been established as the standard method for monitoring chimerism status post transplantation. The advantages of STR analysis include increased sensitivity, the use of smaller quantities of DNA, easier preparation of the DNA, faster turnaround time, the elimination of restriction enzymes and radioisotopes, and overall cost reduction.The aim of this study was to assess whether the state of donor T cell chimerism after RIC allo-HSCT correlates with the outcome in adult ALL.SubjectsThis study includes 72 post RIC allo-HSCT adult ALL patients. All patients gave written, informed consent, according to the Declaration of Helsinki.All patients received standard induction phase 1 and 2 chemotherapy. Patients with Ph chromosome-positive (Ph+) disease received continuous oral Imatinib daily throughout induction. Intra-thecal prophylaxis using Methotrexate was given to all patients.All patients received the standard UKALL14 Reduced Intensity Conditioning (RIC) regimen. Conditioning was done using Fludarabine 30 mg/m2 D-6 – D-2, Melphalan 140 mg/m2 on D-2 and Alemtuzumab 30 mg on D-1 for all patients. Patients with a MUD received an extra 30 mg of Alemtuzumab on D-2.
The donor was given G-CSF 10 micrograms/kg/day s.c. from D-4 to day 0. Mononuclear cells were collected by leucapheresis on day 0 and +1 if needed. A minimum dose of 2 x106 CD34+ cells/kg was returned to the patient on Day 0 of the transplant. The target dose for returned cells was > 4 x106 CD34+ cells/kg.Chimerism analysis
Chimerism analysis was performed by an STR-PCR assay with up to 13 different STR primers with fluorescence-based detection. Peripheral blood T-cells were magnetically sorted using the AutoMACS cell separator (Miltenyi Biotec) and the CD3 human whole blood microbeads kit (Miltenyi Biotec cat number: 130- 090-874). DNA extraction was by direct Proteinase K-lysis and amplification was performed by conventional PCR. All reactions were performed in a volume of 50μL containing 5 uL MgCl2 free 10x PCR buffer (Applied Biosystems), 3-9 uL 25mM MgCl2 (Applied Biosystems), 4 uL dNTPs mix, 2.5mM each dNTP (Bioline), 1μl forward primer, 1μl reverse primer, and 5μl template DNA or RNase-free water for negative controls. Amplicons were run on the ABI3130 genetic analyser (Applied Biosystems) and interpretation was done using ‘GeneMapper’ software (Invitrogen Life Science Technologies).Classification of chimerism status Patients with no signal of host DNA during follow-up were categorized as having full donor chimerism (FC), whereas patients with a measurable host signal more than 5% were categorized as mixed chimerism (MC) and those with no sample available for that time point were designated N/A.
For sequential analysis, patients fell into one of four categories; stable FC, stable MC, increasing donor chimerism (in-DC) were donor DNA signal increased >10% or decreasing donor chimerism (de-DC) were donor DNA decreased >10%. Results The median duration of follow-up was 22 months with a range of 3.4 to 42.4 months. Engraftment was prompt in all patients and no graft failures were reported. Considering pre-transplant chimerism samples, 72 recipient/donor pairs (144 total baseline samples) were included in this study. Also, 256 post transplant chimerism samples were received from the studied 72 patients. The median number of follow-up samples per patient was 3 with a range of 1 to 8 at the scheduled time- points. Two year OS and RFS were assessed in relation to the result of T- cell chimerism analysis at Day100 post HSCT. Accordingly, the 2 year overall survival (OS) for patients with FC was 54%, for patients with MC was 86% and for those with no chimerism assessment at that time point was 58%. As for Relapse Free Survival (RFS), it was 54%, 58% and 58% for the same groups respectively.In the current study, sequential chimerism assessment was done to evaluate the impact of T-cell chimerism kinetics on outcome. The 2 year OS was 61% in patients with stable FC, 82% in those with in-DC, 85% in stable MC and 42.5% in patients with de-DC. As for RFS, the rates were 64%, 78%, 68% and zero% for the same groups respectively.