Long term follow up of patients after allogeneic stem cell transplantation and transfusion of HSV-TK transduced T-cells.

Allogeneic stem cell transplantation (allo-HSCT) is one of the curative treatments for hematologic malignancies, but is hampered by severe complications, such as acute or chronic graft-versus-host-disease (aGvHD; cGvHD) and infections. CD34-selection of stem cells reduces the risk of aGvHD, but also leads to increased infectious complications and relapse. Thus, we studied the safety, efficacy, and feasibility of transfer of gene modified donor T-cells shortly after allo-HSCT in two clinical trials between 2002 and 2007 and here we compare the results to unmodified donor leukocyte infusion (DLI). The aim of these trials was to provide patients with the protection of T-cells after T-cell-depleted allo-HSCT in the matched or mismatched donor setting with an option to delete transduced T-cells, if severe aGvHD occurred within the trial period. Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3, expressing HSV-TK and the truncated ΔLNGFR for selection of transduced cells. Transduced cells were transfused either after day +60 (matched donors) or on day +42 (haploidentical donors). Nine patients were included in the first trial (MHH; 2002 until 2007), two were included in TK007 (2005-2009) and six serves as a control group for outcome after haploidentical transplantation without HSV-TK-transduced DLI. Three patients developed acute GvHD, two had grade I of the skin, one had aGvHD on day +131 (post-HSCT; +89 post-HSV-TK DLI) grade II, which was successfully controlled by ganciclovir (GCV). Donor chimerism was stabilized after transfusion of the transduced cells in all patients treated. Functionality of HSV-TK gene expressing T-cells was shown by loss of bcr-able gene expression as well as by control of cytomegalovirus-reactivation. To date, six patients have relapsed and died, two after a second hematopoietic stem cell transplantation without T-cell depletion or administration of unmodified T-cells. Eleven patients (seven post-HSV-TK DLI) are alive and well to date.

Kinetics of the in vivo expression of glucocorticoid receptor splice variants during prednisone treatment in childhood acute lymphoblastic leukaemia.

BACKGROUND: The in vivo glucocorticoid response in childhood acute lymphoblastic leukaemia (ALL) correlates with the response to multi-agent chemotherapy. However, it is still unclear, whether the expression levels of glucocorticoid receptor (GR) splice variants facilitate the escape from glucocorticoid-induced apoptosis and hence contribute to glucocorticoid resistance. PROCEDURE: In the present study, the initial in vivo expression of the common GR (cGR) and its splice variants GR-alpha, GR-gamma and GR-P was determined using a quantitative RT-PCR approach. Two cohorts of glucocorticoid sensitive (prednisone good responder, PGR) and resistant (prednisone poor responder, PPR) patients were compared. The kinetics of GR splice variant expression was measured during 36 hr following the first use of glucocorticoids in seven patients. RESULTS: The GR splice variant GR-gamma showed the most pronounced differential regulation comparing PPR and PGR patients in both cohorts. GR-alpha and GR-gamma were upregulated faster and to a higher level in PGR as compared to PPR in the in vivo stimulation cohort. Here as well, the most pronounced effect was observed for GR-gamma. CONCLUSIONS: Differential regulation of the cGR and its splice variants under glucocorticoid treatment rather than the expression level at diagnosis is associated with glucocorticoid response.

Expression and structural analysis of glucocorticoid receptor isoform gamma in human leukaemia cells using an isoform-specific real-time polymerase chain reaction approach.

Glucocorticoids are broadly used for chemotherapy in childhood acute lymphoblastic leukaemia (ALL). The intracellular effects of glucocorticoids are mediated through the glucocorticoid receptor. The human glucocorticoid receptor gamma isoform (hGR-gamma) differs from the main isoform (hGR-alpha) by an additional amino acid within the DNA binding domain of the receptor protein. This may decrease hGR-alpha-mediated transcriptional activation. The importance of hGR-gamma expression in childhood ALL is unknown. To evaluate hGR-gamma mRNA expression levels, a real-time polymerase chain reaction (PCR)-based approach, allowing the selective amplification of hGR-gamma, was developed and optimized. We were able to demonstrate target selectivity of hGR-gamma amplification using sequence-specific primers. Studying the structure of the 3' end of hGR-gamma, a combination of this isoform with other hGR isoforms could be demonstrated. Using analysis of hGR-gamma-specific amplification in comparison with the expression of hGR-total (all isoforms) in leukaemic blasts from patients with either a good response to prednisone (PGR) or poor-prednisone response (PPR) in vivo, relative hGR-gamma expression was observed to be lower in cells from patients with PGR compared with PPR, in particular after 10 h of dexamethasone stimulation. These data were correlated with cell survival, demonstrating a more pronounced induction of apoptosis in cells from patients with PGR as compared with PPR.