English abstract
Background
Busulfan (Bu) an alkylating drug, belongs to the Alkyl methanesulfonates group, works directly by alkylating DNA, and results in cross-linking of DNA strands leading to apoptosis. Bu is a vital drug in most of the conditioning regimens prior to Hematopoietic Stem Cell Transplant (HSCT). Bu metabolism occurs in the liver, where it initially gets conjugated to glutathione catalyzed by mainly Glutathione S-transferase isoenzyme Al-1 (GSTAI). There are other isoenzymes (GSTM1, GȘTT1 and GSTP1) involved but in a lower extent. As soon as Busulfan is conjugated, it loses its alkylating property. The hematopoietic stem cells have very minimal to none isoenzymes in the cell leading to an increased Bu effect in them. The Glutathione family has a high chance of polymorphism, hence affecting some pharmacokinetic profiles of Bu, and clinical outcomes of the HSCT.
The study aim is to assess the role of GST genetic variants in affecting the clearance of Bu and clinical outcomes of patients undergoing HSCT. This will allow us to observe the frequency of GST polymorphism and would better explain the busulfan clearance and hematopoietic stem cell transplant outcomes.
Methods
A single center retrospective cohort study with genotyping of previously collected samples (as standard of care) for the known polymorphism of GST related to Bu metabolism. Inclusion criteria were 135 adult and pediatric patients who received IV Bu prior to HSCT at SQUH from January 2003 - October 2016. Patient's Bu clearance was calculated using Kinetica software. Genotyping for polymorphism was done using Capillary Electrophoresis for GSTM1 and GSTT1 (insertion or deletion), and DNA sequencing for GSTA1 (G-52A, C-69G, A-513G, T-567G, G -631T and G-1142C) and GSTP1 (A313G and C341T). Each GST polymorphism was analyzed and checked if it was a factor causing a difference in busulfan clearance and HSCT outcomes such as aGvHD, VOD, graft loss, relapse, and mortality. Standard descriptive and analytical statistics and graphs were created using STATA.
Results
The total number of patients that were included is 135, of which 63 males and 72 females. The mean Bu clearance in our population was found to be 3.7 +0.98 ml/min/kg. Thirty patients developed a GvHD, 9 developed hepatic VOD, 4 developed
graft loss, and 5 died. While from the acute leukemia and MDS patients, 5 relapsed. The
frequency of GSTMI deletion in our population is 41%, GSTTI deletion is.16% and double insertion of GSTMI and GSTT) combined is 50%. The wild type, heterozygous and homozygous genotypes of each location of GSTAI are as following, G-52A, 35%, 48% and 17%, C-69G; 38%, 48% and 14%, A-513G; 0%, 4% and 96%, T-567G; 18%, 44% and 38%, G -631T;60%, 33%, and 7% and G-11420, 42%, 41% and 17%. The wild type, heterozygous and homozygous genotype of each location of GSTPI is as follows, A313G; 47%, 40% and 13%, and C341T; 84%, 16% and 0%. Patients with GSTAI A-513G heterozygous (AG) were found to have higher incidence of graft loss (p= 0.006), and with double GSTMI and GSTT1 double deletion, there was a higher incidence of aGvHD (p=0.04). Double insertion of GSTMI and GSTTI significantly increased the risk of mortality p=0.034, and GSTM1 insertion is also associated with increased mortality p=0.021.
Conclusions From these results we propose that GST genotyping prior to HSCT influences clinical outcomes, and might improve clinical outcomes for known genetic background.
