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Multi-State Modeling Identifies Determinants of Successful Immune Suppression Discontinuation: Secondary Analysis of BMT CTN 0201 and 0402 Trials
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Abstracts / Biol Blood Marrow Transplant 23 (2017) S18–S391
recipients. KIR-B haplotype donors were associated with alloreactivity in the form of aGVHD, but the specific use of KIR 2DS2 donors may be beneficial in myeloid malignancies to decrease NRM and increase OS. Younger donors should be favored over their older counterparts. We will continue to study the impact of these characteristics to help inform donor selection as our population grows.
LATE EFFECTS/QUALITY OF LIFE/PSYCHOSOCIAL ISSUES
81 Multi-State Modeling Identifies Determinants of Successful Immune Suppression Discontinuation: Secondary Analysis of BMT CTN 0201 and 0402 Trials Joseph A. Pidala 1, Michael Martens 2, Jeanette Carreras 3, Claudio Anasetti 1, Corey S. Cutler 4, Stephanie J. Lee 5, Joseph H. Antin 6, Mary M. Horowitz 7, Brent R. Logan 2. 1 Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL; 2 Division of Biostatistics, Medical College of Wisconsin, Milwaukee, WI; 3 IBMTR, Medical College of Wisconsin, Milwaukee, WI; 4 Dana-Farber Cancer Institute, Boston, MA; 5 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 6 Hematologic Malignancies, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; 7 Department of Medicine, CIBMTR and Medical College of Wisconsin, Milwaukee, WI The likelihood of immune suppression discontinuation (ISD) over time after allogeneic hematopoietic cell transplantation (HCT) and factors that predict successful ISD vs. the development of graft vs. host disease (GVHD) after ISD are poorly understood. Clinical practice of ISD is variable, allowing us to examine these questions in a multi-center study population (n = 827) comprised of BMT-CTN 0201 and 0402 trials, supplemented with long-term follow up data from the CIBMTR. ISD after HCT was represented using a multistate model with 6 states: immune suppression (IS)/no GVHD, acute GVHD, chronic GVHD, off IS without GVHD, resumed IS for GVHD, and death/ relapse/second HCT. Likelihood of each state over time was estimated using differences in Kaplan-Meier estimators for transient states and cumulative incidence for absorbing states. Likelihood of being off IS without GVHD was modeled using pseudo-value regression with results summarized using odds ratios (OR). Hazard ratios for the risk of resuming IS for GVHD were estimated using Cox regression, resetting the clock at ISD. Adjusted cumulative incidences for resuming IS were summarized for the effect of prior GVHD history and timing of ISD due to non-proportional hazards.
Figure 1. State probabilities over time.
Abstracts / Biol Blood Marrow Transplant 23 (2017) S18–S391
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Figure 2. (A) Pseudo-value model for off IS, no GVHD. (B) Cox model for Resuming IS for GVHD.
Median (range) follow up time for surviving patients was 79 months (11-124) for CTN 0201, and 61 months (24-97) for CTN 0402. Patients were median age 44 years (range <1-67). CIBMTR disease risk was early (58%), intermediate (21%), or advanced (21%). Graft was peripheral blood (PBSC) stem cells (68%) vs. bone marrow (BM) (32%). Donors were sibling (MRD) (36%), matched unrelated (49%), or mismatched unrelated (14%). GVHD prophylaxis was tacrolimus or cyclosporine with methotrexate (CNI/MTX) (79%), or tacrolimus/sirolimus (18%), or others (3%). ATG was used in 17%. Over the total follow up period, 342 (41%) reached ISD at least once. The proportion off IS over time is presented in Figure 1. Median days from HCT to ISD varied according to prior
GVHD history: No prior GVHD 190, prior acute GVHD 276, prior chronic GVHD 871. Baseline variables associated with successful ISD were younger age, MRD, ATG use, CNI/MTX, BM graft type, and early disease risk (Figure 2A). Of the 342 that stopped IS, 127 (37%) subsequently needed to restart IS for GVHD. Resumption of IS after attempted ISD was associated with increased age, no ATG use, parous female donor, and PBSC (Figure 2B), and varied according to prior GVHD history and time of ISD post-HCT (Figure 3), with shorter duration of initial IS predicting higher risk for resuming IS. Our analysis provides the first comprehensive multi-state modeling-based analysis of ISD and subsequent GVHD, and identifies several variables predictive of these important HCT outcomes. Major applications of this work include
Figure 3. Adjusted incidence of resuming IS for GVHD by state history and timing of discontinuation of IS.
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Abstracts / Biol Blood Marrow Transplant 23 (2017) S18–S391
patient education on ISD rates, evidence to support riskadapted IS management for clinicians, and robust ISD estimates for use in HCT protocol design.
GVH/GVL
82 Deletion of AMP-Activated Protein Kinase (AMPK) in Donor T Cells Protects Against Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia Responses Kevin Beezhold 1, Nathan Moore 2, Pailin Chiaranunt 1, Rebecca Brown 1, Craig Alan Byersdorfer 1. 1 Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA; 2 University of Pittsburgh School of Medicine, Pittsburgh, PA
Figure 1. AMPK−/− T cells cause less severe GVHD. (A) wt or AMPK−/− T cells (blue) were transferred to C3H. SW recipients and monitored for GVHD up to 10 weeks post-BMT. (B) splenocytes were harvested from Allo or syngeneic (Syn) recipients on day 7 post-BMT and the total number of donor T cells quantitated, *** P < .001.
Figure 2. AMPK−/− cells maintain anti-leukemia properties. wt or AMPK−/− T cells (blue) were transferred to B6xDBA2 F1 recipients (red = no T cells) and leukemia cell percentages assessed in PBMCs on day 13 post-transplant (A). Similar results seen in liver, spleen, and bone marrow. Alternatively, recipients were followed for survival out to day 56 post-transplant (B).
Allogeneic transplantation remains a curative treatment for high-risk leukemia, but its broader use is limited by acute graft-versus-host disease (GVHD). We have previously shown that during GVHD, alloreactive T cells increase their dependence on the oxidation of fat. To explore this mechanistically, we studied the role of AMPK, an intracellular energy sensor, in GHVD-causing T cells. Alloreactive T cells increased phosphorylation of AMPK as early as day 3 post-transplant, with levels up to 8-fold higher than naive cells (P = .0003). We then confirmed AMPK’s necessity during GVHD by depleting AMPK in donor T cells. AMPK-/- cells caused significantly less disease in two models of GVHD (Figure 1A), and decreased the number of donor T cells recovered on day 7 post-transplant (3.15 ± .49 × 106 vs.1.87 ± .53 × 106, P = .0006, wildtype (wt) vs. AMPK-/- respectively). Importantly, syngeneic T cell expansion was unaffected by AMPK deficiency (Figure 1B). In contrast to their GVHD potential, AMPK -/- T demonstrated equivalent cytotoxicity against MHC-mismatched targets, both in vitro and in vivo, equal clearance of p815 leukemia cells in a model with high tumor burden (Figure 2A), and similar extension of survival compared to wt T cells in this same model (Figure 2B). To elucidate possible mechanisms explaining these findings, we evaluated metabolic pathways adopted by T cells post-transplant. To our surprise, rates of fatty acid oxidation, T cell autophagy, and mammalian target of rapamycin signaling were identical between wt and AMPK-/- cells. We next quantitated levels of regulatory T cells (Treg). In contrast to expectation, both the percentage and total number of Treg increased in mice receiving AMPK-/- cells (.85 ± .32 × 104 vs. 1.69 ± .34 × 104, wt vs. AMPK-/-, P = .004). To note, Treg levels were equivalent in wt vs. AMPK-/- donors. AMPK deficiency facilitated expansion of donor T reg , because elimination of FoxP3 + cells prior to transplantation abrogated the AMPK-/-benefits. Finally, we assessed the ability of AMPK-/T cells to infiltrate GVHD target organs. As shown in Figure 3, peri-portal infiltration of AMPK-/- cells was reduced compared to wt T cells, and liver infiltrates in mice receiving AMPK-/- cells contained many fewer CD3+ T cells per highpowered field. Decreased hepatic infiltration also correlated with lower levels of the integrin pair α4β7 (55.2 ± 1.4% vs. 47.2 ± 2.8% α4β7Hicells, P = .0017, wt vs. AMPK-/-). In conclusion, AMPK deletion in donor T cells decreases GVHD severity but spares anti-leukemia responses and preserves homeostatic immune reconstitution. Mechanistically, this occurs through an increase in Treg numbers and percentages and decreased infiltration of donor T cells into target organs. From these findings, we conclude that AMPK represents a clinically relevant target in donor T cells to effectively separate GVHD and GVL effects.
Figure 3. Decreased hepatic infiltration of AMPK−/− T cells. (A) wt or AMPK−/− T cells were transplanted into B6xDBA2 F1 recipients and frozen sections of liver tissues obtained on day 7, followed by immunofluorescence for DAPI, CD3, and CD68. (B) The total number of CD3+ (left panel), or percentage of CD3+ cells from total cells (right panel), was quantitated per high-powered field (hpf). Numbers are quantitated from 24 slides, with 3 sections reviewed from each of 8 independent recipients of wt or AMPK−/− donor cells. ***P < .001.
Abstracts / Biol Blood Marrow Transplant 23 (2017) S18–S391
recipients. KIR-B haplotype donors were associated with alloreactivity in the form of aGVHD, but the specific use of KIR 2DS2 donors may be beneficial in myeloid malignancies to decrease NRM and increase OS. Younger donors should be favored over their older counterparts. We will continue to study the impact of these characteristics to help inform donor selection as our population grows.
LATE EFFECTS/QUALITY OF LIFE/PSYCHOSOCIAL ISSUES
81 Multi-State Modeling Identifies Determinants of Successful Immune Suppression Discontinuation: Secondary Analysis of BMT CTN 0201 and 0402 Trials Joseph A. Pidala 1, Michael Martens 2, Jeanette Carreras 3, Claudio Anasetti 1, Corey S. Cutler 4, Stephanie J. Lee 5, Joseph H. Antin 6, Mary M. Horowitz 7, Brent R. Logan 2. 1 Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL; 2 Division of Biostatistics, Medical College of Wisconsin, Milwaukee, WI; 3 IBMTR, Medical College of Wisconsin, Milwaukee, WI; 4 Dana-Farber Cancer Institute, Boston, MA; 5 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 6 Hematologic Malignancies, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; 7 Department of Medicine, CIBMTR and Medical College of Wisconsin, Milwaukee, WI The likelihood of immune suppression discontinuation (ISD) over time after allogeneic hematopoietic cell transplantation (HCT) and factors that predict successful ISD vs. the development of graft vs. host disease (GVHD) after ISD are poorly understood. Clinical practice of ISD is variable, allowing us to examine these questions in a multi-center study population (n = 827) comprised of BMT-CTN 0201 and 0402 trials, supplemented with long-term follow up data from the CIBMTR. ISD after HCT was represented using a multistate model with 6 states: immune suppression (IS)/no GVHD, acute GVHD, chronic GVHD, off IS without GVHD, resumed IS for GVHD, and death/ relapse/second HCT. Likelihood of each state over time was estimated using differences in Kaplan-Meier estimators for transient states and cumulative incidence for absorbing states. Likelihood of being off IS without GVHD was modeled using pseudo-value regression with results summarized using odds ratios (OR). Hazard ratios for the risk of resuming IS for GVHD were estimated using Cox regression, resetting the clock at ISD. Adjusted cumulative incidences for resuming IS were summarized for the effect of prior GVHD history and timing of ISD due to non-proportional hazards.
Figure 1. State probabilities over time.
Abstracts / Biol Blood Marrow Transplant 23 (2017) S18–S391
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Figure 2. (A) Pseudo-value model for off IS, no GVHD. (B) Cox model for Resuming IS for GVHD.
Median (range) follow up time for surviving patients was 79 months (11-124) for CTN 0201, and 61 months (24-97) for CTN 0402. Patients were median age 44 years (range <1-67). CIBMTR disease risk was early (58%), intermediate (21%), or advanced (21%). Graft was peripheral blood (PBSC) stem cells (68%) vs. bone marrow (BM) (32%). Donors were sibling (MRD) (36%), matched unrelated (49%), or mismatched unrelated (14%). GVHD prophylaxis was tacrolimus or cyclosporine with methotrexate (CNI/MTX) (79%), or tacrolimus/sirolimus (18%), or others (3%). ATG was used in 17%. Over the total follow up period, 342 (41%) reached ISD at least once. The proportion off IS over time is presented in Figure 1. Median days from HCT to ISD varied according to prior
GVHD history: No prior GVHD 190, prior acute GVHD 276, prior chronic GVHD 871. Baseline variables associated with successful ISD were younger age, MRD, ATG use, CNI/MTX, BM graft type, and early disease risk (Figure 2A). Of the 342 that stopped IS, 127 (37%) subsequently needed to restart IS for GVHD. Resumption of IS after attempted ISD was associated with increased age, no ATG use, parous female donor, and PBSC (Figure 2B), and varied according to prior GVHD history and time of ISD post-HCT (Figure 3), with shorter duration of initial IS predicting higher risk for resuming IS. Our analysis provides the first comprehensive multi-state modeling-based analysis of ISD and subsequent GVHD, and identifies several variables predictive of these important HCT outcomes. Major applications of this work include
Figure 3. Adjusted incidence of resuming IS for GVHD by state history and timing of discontinuation of IS.
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Abstracts / Biol Blood Marrow Transplant 23 (2017) S18–S391
patient education on ISD rates, evidence to support riskadapted IS management for clinicians, and robust ISD estimates for use in HCT protocol design.
GVH/GVL
82 Deletion of AMP-Activated Protein Kinase (AMPK) in Donor T Cells Protects Against Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia Responses Kevin Beezhold 1, Nathan Moore 2, Pailin Chiaranunt 1, Rebecca Brown 1, Craig Alan Byersdorfer 1. 1 Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA; 2 University of Pittsburgh School of Medicine, Pittsburgh, PA
Figure 1. AMPK−/− T cells cause less severe GVHD. (A) wt or AMPK−/− T cells (blue) were transferred to C3H. SW recipients and monitored for GVHD up to 10 weeks post-BMT. (B) splenocytes were harvested from Allo or syngeneic (Syn) recipients on day 7 post-BMT and the total number of donor T cells quantitated, *** P < .001.
Figure 2. AMPK−/− cells maintain anti-leukemia properties. wt or AMPK−/− T cells (blue) were transferred to B6xDBA2 F1 recipients (red = no T cells) and leukemia cell percentages assessed in PBMCs on day 13 post-transplant (A). Similar results seen in liver, spleen, and bone marrow. Alternatively, recipients were followed for survival out to day 56 post-transplant (B).
Allogeneic transplantation remains a curative treatment for high-risk leukemia, but its broader use is limited by acute graft-versus-host disease (GVHD). We have previously shown that during GVHD, alloreactive T cells increase their dependence on the oxidation of fat. To explore this mechanistically, we studied the role of AMPK, an intracellular energy sensor, in GHVD-causing T cells. Alloreactive T cells increased phosphorylation of AMPK as early as day 3 post-transplant, with levels up to 8-fold higher than naive cells (P = .0003). We then confirmed AMPK’s necessity during GVHD by depleting AMPK in donor T cells. AMPK-/- cells caused significantly less disease in two models of GVHD (Figure 1A), and decreased the number of donor T cells recovered on day 7 post-transplant (3.15 ± .49 × 106 vs.1.87 ± .53 × 106, P = .0006, wildtype (wt) vs. AMPK-/- respectively). Importantly, syngeneic T cell expansion was unaffected by AMPK deficiency (Figure 1B). In contrast to their GVHD potential, AMPK -/- T demonstrated equivalent cytotoxicity against MHC-mismatched targets, both in vitro and in vivo, equal clearance of p815 leukemia cells in a model with high tumor burden (Figure 2A), and similar extension of survival compared to wt T cells in this same model (Figure 2B). To elucidate possible mechanisms explaining these findings, we evaluated metabolic pathways adopted by T cells post-transplant. To our surprise, rates of fatty acid oxidation, T cell autophagy, and mammalian target of rapamycin signaling were identical between wt and AMPK-/- cells. We next quantitated levels of regulatory T cells (Treg). In contrast to expectation, both the percentage and total number of Treg increased in mice receiving AMPK-/- cells (.85 ± .32 × 104 vs. 1.69 ± .34 × 104, wt vs. AMPK-/-, P = .004). To note, Treg levels were equivalent in wt vs. AMPK-/- donors. AMPK deficiency facilitated expansion of donor T reg , because elimination of FoxP3 + cells prior to transplantation abrogated the AMPK-/-benefits. Finally, we assessed the ability of AMPK-/T cells to infiltrate GVHD target organs. As shown in Figure 3, peri-portal infiltration of AMPK-/- cells was reduced compared to wt T cells, and liver infiltrates in mice receiving AMPK-/- cells contained many fewer CD3+ T cells per highpowered field. Decreased hepatic infiltration also correlated with lower levels of the integrin pair α4β7 (55.2 ± 1.4% vs. 47.2 ± 2.8% α4β7Hicells, P = .0017, wt vs. AMPK-/-). In conclusion, AMPK deletion in donor T cells decreases GVHD severity but spares anti-leukemia responses and preserves homeostatic immune reconstitution. Mechanistically, this occurs through an increase in Treg numbers and percentages and decreased infiltration of donor T cells into target organs. From these findings, we conclude that AMPK represents a clinically relevant target in donor T cells to effectively separate GVHD and GVL effects.
Figure 3. Decreased hepatic infiltration of AMPK−/− T cells. (A) wt or AMPK−/− T cells were transplanted into B6xDBA2 F1 recipients and frozen sections of liver tissues obtained on day 7, followed by immunofluorescence for DAPI, CD3, and CD68. (B) The total number of CD3+ (left panel), or percentage of CD3+ cells from total cells (right panel), was quantitated per high-powered field (hpf). Numbers are quantitated from 24 slides, with 3 sections reviewed from each of 8 independent recipients of wt or AMPK−/− donor cells. ***P < .001.