Sandeep Nagra

Tolerability and Clinical Activity of Post-Transplantation Azacitidine in Patients Allografted for Acute Myeloid Leukemia Treated on the RICAZA Trial

Disease relapse is the major causes of treatment failure after allogeneic stem cell transplantation (SCT) in patients with acute myeloid leukemia (AML). As well as demonstrating significant clinical activity in AML, azacitidine (AZA) upregulates putative tumor antigens, inducing a CD8+ T cell response with the potential to augment a graft-versus-leukemia effect. We, therefore, studied the feasibility and clinical sequelae of the administration of AZA during the first year after transplantation in 51 patients with AML undergoing allogeneic SCT. Fourteen patients did not commence AZA either because of transplantation complications or withdrawal of consent. Thirty-seven patients commenced AZA at a median of 54 days (range, 40 to 194 days) after transplantation, which was well tolerated in the majority of patients. Thirty-one patients completed 3 or more cycles of AZA. Sixteen patients relapsed at a median time of 8 months after transplantation. No patient developed extensive chronic graft-versus-host disease. The induction of a post-transplantation CD8+ T cell response to 1 or more tumor-specific peptides was studied in 28 patients. Induction of a CD8+ T cell response was associated with a reduced risk of disease relapse (hazard ratio [HR], .30; 95% confidence interval [CI], .10 to .85; P = .02) and improved relapse-free survival (HR, .29; 95% CI, .10 to .83; P = .02) taking into account death as a competing risk. In conclusion, AZA is well tolerated after transplantation and appears to have the capacity to reduce the relapse risk in patients who demonstrate a CD8+ T cell response to tumor antigens. These observations require confirmation in a prospective clinical trial.

Factors predicting long-term survival after T-cell depleted reduced intensity allogeneic stem cell transplantation for acute myeloid leukemia

Background Reduced intensity conditioning regimens permit the delivery of a potentially curative graft-versus-leukemia effect in older patients with acute myeloid leukemia. Although T-cell depletion is increasingly used to reduce the risk of graft-versus-host disease its impact on the graft-versus-leukemia effect and long-term outcome post-transplant is unknown. Design and Methods We have characterized pre- and post-transplant factors determining overall survival in 168 patients with acute myeloid leukemia transplanted using an alemtuzumab based reduced intensity conditioning regimen with a median duration of follow-up of 37 months. Results The 3-year overall survival for patients transplanted in CR1 or CR2/CR3 was 50% (95% CI, 38% to 62%) and 44% (95% CI, 31% to 56%), respectively compared to 15% (95% CI, 2% to 36%) for patients with relapsed/refractory disease. Multivariate analysis demonstrated that both survival and disease relapse were influenced by status at transplant (P=0.008) and presentation cytogenetics (P=0.01). Increased exposure to cyclosporine A (CsA) in the first 21 days post-transplant was associated with an increased relapse risk (P<0.0001) and decreased overall survival (P<0.0001). Conclusions Disease stage, presentation karyotype and post-transplant CsA exposure are important predictors of outcome in patients undergoing a T-cell depleted reduced intensity conditioning allograft for acute myeloid leukemia. These data confirm the presence of a potent graft-versus-leukemia effect after a T-cell depleted reduced intensity conditioning allograft in acute myeloid leukemia and identify CsA exposure as a manipulable determinant of outcome in this setting.

Chemokine-mediated tissue recruitment of CXCR3+ CD4+ T cells plays a major role in the pathogenesis of chronic GVHD.

Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8(+) T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4(+) cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4(+) cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4(+) T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3(+) T cells in cGVHD.

Role of HCT-comorbidity index, age and disease status at transplantation in predicting survival and non-relapse mortality in patients with myelodysplasia and leukemia undergoing reduced-intensity-conditioning hemopoeitic progenitor cell transplantation

Reduced-intensity-conditioning (RIC) regimens have allowed older patients to have allogeneic hemopoietic progenitor cell transplantation (HCT). This retrospective study was done to assess the impact of the HCT-comorbidity index (HCT-CI) in addition to other pre-transplant factors on the outcome of RIC transplants. In all 121 such patients were transplanted between 2002 and 2008 at two centers using fludarabine, melphalan and alumtuzumab conditioning. The OS and non-relapse mortality (NRM) were 56% and 30% at 2 years, respectively. The NRM of patients with HCT-CI ⩾3 was not significantly different from the NRM of those with HCT-CI 0–2 (P value 0.24). Age and disease status at transplantation were significant factors affecting OS (P value 0.07 and 0.008, respectively), with no impact on NRM (P value 0.14 and 0.24, respectively). Although HCT-CI on its own did not independently predict NRM or survival, taken together with age and disease status at transplantation, it can be utilized to further delineate RIC allograft recipients into groups with different outcomes. Patients with none or one of these three adverse factors (age⩾60 years, leukemia in second CR or PR/high-risk myelodysplasia (MDS) and HCT-CI⩾3) had a 2-year NRM and survival of 18% and 80%, respectively, which was significantly better than those of patients with two or more of these adverse factors with 2-year NRM and survival of 46% (P value 0.03) and 40% (P value 0.02), respectively. None of the patients with all three adverse factors (age⩾60 years, leukemia in second CR or PR/high-risk MDS and HCT-CI⩾3) had survived for 2 years (median survival 12 months). This information can be used to guide patient selection for RIC transplants and to appropriately counsel patients of the risks and benefits of this treatment.

Post-transplant T cell chimerism predicts graft versus host disease but not disease relapse in patients undergoing an alemtuzumab based reduced intensity conditioned allogeneic transplant

In this multicentre retrospective study we have studied the impact of T cell chimerism on the outcome of 133 patients undergoing an alemtuzumab based reduced intensity conditioning allograft (RIC). The median age of the patients was 50 years (range 42-55 years). 77 patients were transplanted using an HLA identical sibling donor while 56 patients received a fully matched volunteer unrelated donor graft. 64 patients had a lymphoid malignancy and 69 were transplanted for a myeloid malignancy. 38 patients (29%) relapsed with no significant difference in risk of relapse between patients developing full donor and mixed donor chimerism in the T-cell compartment on D+90 and D+180 post transplant. Day 90 full donor T cell chimerism correlated with an increased incidence of acute GVHD according to NIH criteria (p=0.0004) and the subsequent development of chronic GVHD. Consistent with previous observations, our results confirmed a correlation between the establishment of T cell full donor chimerism and acute GVHD in T deplete RIC allografts. However our study failed to identify any correlation between T cell chimerism and relapse risk and challenge the use of pre-emptive donor lymphocyte infusions (DLI) in patients with mixed T cell chimerism transplanted using an alemtuzumab based RIC regimen.

Memory B-cell reconstitution following allogeneic hematopoietic stem cell transplantation is an EBV-associated transformation event.

Allogeneic stem cell transplantation (allo-HSCT) provides a unique opportunity to track Epstein-Barr virus (EBV) infection in the context of the reconstituting B-cell system. Although many allo-HSCT recipients maintain low or undetectable levels of EBV DNA posttransplant, a significant proportion exhibit elevated and rapidly increasing EBV loads which, if left untreated, may lead to potentially fatal EBV-associated posttransplant lymphoproliferative disease. Intriguingly, this high-level EBV reactivation typically arises in the first 3 months posttransplant, at a time when the peripheral blood contains low numbers of CD27+ memory cells which are the site of EBV persistence in healthy immunocompetent donors. To investigate this apparent paradox, we prospectively monitored EBV levels and B-cell reconstitution in a cohort of allo-HSCT patients for up to 12 months posttransplant. In patients with low or undetectable levels of EBV, the circulating B-cell pool consisted predominantly of transitional and naive cells, with a marked deficiency of CD27+ memory cells which lasted >12 months. However, among patients with high EBV loads, there was a significant increase in both the proportion and number of CD27+ memory B cells. Analysis of sorted CD27+ memory B cells from these patients revealed that this population was preferentially infected with EBV, expressed EBV latent transcripts associated with B-cell growth transformation, had a plasmablastic phenotype, and frequently expressed the proliferation marker Ki-67. These findings suggest that high-level EBV reactivation following allo-HSCT may drive the expansion of latently infected CD27+ B lymphoblasts in the peripheral blood.

Prognostic value of monitoring a candidate immunophenotypic leukaemic stem/progenitor cell population in patients allografted for acute myeloid leukaemia

It is postulated that disease relapse in patients with acute myeloid leukemia (AML) is consequent upon chemoresistance within leukemic stem/progenitor cell (LSC) populations from which bulk blasts arise1. In adults with high risk AML, allogeneic hematopoietic cell transplantation (HCT) has become a central component of the treatment algorithm to overcome this chemoresistance as it delivers maximal anti-leukemic activity through both dose intensification and by the genesis of a potent graft-versus-leukemia (GVL) effect2-4. However relapse still occurs in a significant proportion of allografted patients and now represents the major cause of treatment failure particularly with reduced intensity conditioning (RIC) regimens5. Whilst minimal residual disease (MRD) from the bulk leukemic population is known to be prognostic, more accurate predictors of relapse risk might be developed from detection of putative LSC populations pre- or post-transplant. However, to date an association between LSC and transplant outcome remains uncertain.

Prediction of intravenous cyclosporine area under the concentration-time curve after allogeneic stem cell transplantation

Currently, routine monitoring of cyclosporine in patients undergoing allogeneic stem cell transplantation is based on analysis of trough, or C0, predose concentrations. However, recent studies in solid organ transplant recipients have demonstrated that monitoring cyclosporine exposure by analyzing 2-hour postdose concentrations (C2) or area under the concentration-time curve (AUC) may improve clinical outcome. This study investigated the ability of single samples to predict exposure to intravenous cyclosporine in eight patients undergoing allogeneic stem cell transplantation. Patients received cyclosporine at a starting dose of 2.5 mg/kg 12-hourly by intravenous infusion over 4 hours. Blood samples were taken at 0, 1, 2, 3, 4, 4.17, 4.33, 4.67, 5, 6, 8, and 12 hours after the start of the infusion. Linear regression was undertaken to investigate the relationship between AUC and concentrations measured at individual time points; bias and precision were also examined. Cyclosporine doses ranged from 250 mg to 430 mg/day, AUC from 3.85 to 8.39 mg·h/L, clearance from 19.1 to 48.1 L/h, and elimination half-life from 3.7 to 15.5 hours. Although Cmax and the concentration measured at 3 hours (C3) provided the best prediction of AUC (r2 = 0.90 and r2 = 0.87, respectively), the infusion protocol made the time of Cmax difficult to predict. Concentrations measured at the end of the infusion (Cend) and 12 hours postdose (C12) gave similar results (r2 = 0.87 and 0.77, respectively). These data suggest that C12 concentrations provide an acceptable marker of total exposure to intravenous cyclosporine in patients undergoing allogeneic stem cell transplantation.

Outcome of Azacitidine Therapy in Acute Myeloid Leukemia Is not Improved by Concurrent Vorinostat Therapy but Is Predicted by a Diagnostic Molecular Signature.

Purpose: Azacitidine (AZA) is a novel therapeutic option in older patients with acute myeloid leukemia (AML), but its rational utilization is compromised by the fact that neither the determinants of clinical response nor its mechanism of action are defined. Co-administration of histone deacetylase inhibitors, such as vorinostat (VOR), is reported to improve the clinical activity of AZA, but this has not been prospectively studied in patients with AML. Experimental Design: We compared outcomes in 259 adults with AML (n = 217) and MDS (n = 42) randomized to receive either AZA monotherapy (75 mg/m2 × 7 days every 28 days) or AZA combined with VOR 300 mg twice a day on days 3 to 9 orally. Next-generation sequencing was performed in 250 patients on 41 genes commonly mutated in AML. Serial immunophenotyping of progenitor cells was performed in 47 patients. Results: Co-administration of VOR did not increase the overall response rate (P = 0.84) or overall survival (OS; P = 0.32). Specifically, no benefit was identified in either de novo or relapsed AML. Mutations in the genes CDKN2A (P = 0.0001), IDH1 (P = 0.004), and TP53 (P = 0.003) were associated with reduced OS. Lymphoid multipotential progenitor populations were greatly expanded at diagnosis and although reduced in size in responding patients remained detectable throughout treatment. Conclusions: This study demonstrates no benefit of concurrent administration of VOR with AZA but identifies a mutational signature predictive of outcome after AZA-based therapy. The correlation between heterozygous loss of function CDKN2A mutations and decreased OS implicates induction of cell-cycle arrest as a mechanism by which AZA exerts its clinical activity. Clin Cancer Res; 23(21); 6430–40. ©2017 AACR.

Greatly reduced risk of EBV reactivation in rituximab-experienced recipients of alemtuzumab-conditioned allogeneic HSCT.

EBV-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). We retrospectively analysed the incidence and risk factors for EBV reactivation in 186 adult patients undergoing consecutive allo-HSCT with alemtuzumab T-cell depletion at a single centre. The cumulative incidence of EBV reactivation was 48% (confidence interval (CI) 41–55%) by 1 year, with an incidence of high-level EBV reactivation of 18% (CI 13–24%); 8 patients were concurrently diagnosed with PTLD. Amongst patients with high-level reactivation 31/38 (82%) developed this within only 2 weeks of first EBV qPCR positivity. In univariate analysis age⩾50 years was associated with significantly increased risk of EBV reactivation (hazard ratio (HR) 1.54, CI 1.02–2.31; P=0.039). Furthermore, a diagnosis of non-Hodgkin lymphoma (NHL) was associated with greatly reduced risk of reactivation (HR 0.10, CI 0.03–0.33; P=0.0001) and this was confirmed in multivariate testing. Importantly, rituximab therapy within 6 months prior to allo-HSCT was also highly predictive for lack of EBV reactivation (HR 0.18, CI 0.07–0.48; P=0.001) although confounding with NHL was apparent. Our data emphasise the risk of PTLD associated with alemtuzumab. Furthermore, we report the clinically important observation that rituximab, administered in the peri-transplant period, may provide effective prophylaxis for PTLD.