Fractionated Total Body Irradiation Research Articles

Incidence, Risk Factors, Characteristics, and Outcome of Chronic Graft Versus Host Disease in Children Undergoing Haploidentical Peripheral Blood Stem Cell Transplant With Post-transplant Cyclophosphamide.

Chronic graft versus host disease (cGVHD) is a major cause of morbidity postallogeneic peripheral blood stem cell transplant (PBSCT). There is paucity of literature describing incidence, risk factors, characteristics, and outcome of cGVHD in children undergoing haploidentical PBSCT with post-transplant cyclophosphamide (PTCy). Here, we describe our experience from our center regarding the same. All children who underwent haploidentical PBSCT with PTCy between January 2016 and December 2021 at our center and survived beyond day+100 post-transplant were included in this retrospective study. Conditioning regimens used were: Thiotepa-Fludarabine-Cyclophosphamide with 2Gy single fraction total body irradiation, Thiotepa-Busulfan-Fludarabine, Fludarabine-total body irradiation and Fludarabine-Melphalan. Peripheral blood was used as stem cell source in all patients. GVHD prophylaxis was PTCy 50mg/kg on day +3 and +4, Mycophenolate mofetil and Calcineurin inhibitors. Clinical and laboratory data was electronically retrieved and analyzed based on National Institute of Health Consensus Criteria-2014 at regular intervals. Impact of various patient, donor, and transplant-related factors on development of cGVHD were analyzed. Incidence of relapse, event free survival (EFS) and overall survival (OS) were calculated and compared between cGVHD and no cGVHD groups. Patients with rejection were excluded from risk factor analysis for cGVHD but were considered for survival analysis. Fifty-one children included in this study. Median age of transplant of our cohort was 7.5 years with male:female=1.6:1. Eight patients had rejection with autologous recovery. History of acute GVHD (aGVHD) was present in 15/51 (Grade III to IV in 7/51). cGVHD developed in 19/51 patients (mild-9/51, moderate-6/51, and severe-4/51). Skin was the most common organ involved (100%) followed by gastrointestinal tract (47.4%), liver (36.8%), eyes (21%), lungs (21%), mouth (15.7%), and joints (5.2%). Advanced donor age (>30y) and previous aGVHD were found to be significantly associated with increased risk of developing cGVHD. At last follow-up, complete response and partial response of cGVHD was seen in 6/19 and 4/19 patients, respectively. Overall mortality was 15/51 (cause of mortality was relapse of cancer 8/15, cGVHD-3/15, other 4/15). EFS and OS of full cohort was 55% and 70.6%, respectively. Compared with patients without cGVHD, patients with cGVHD demonstrated a lower relapse (18.2% vs. 40%, P =0.2333), higher EFS (68.4% vs. 53.1%, P =0.283), and higher OS (73.7% vs. 68.8%, P =0.708). Incidence of cGVHD was high in children undergoing haploidentical PBSCT with PTCy. Other than PBSC graft source; donor age and previous aGVHD were the risks factors for development of cGVHD. Patients with cGVHD had lower incidence of relapse translating into better survival but this difference was not statistically significant.

Incidence of Radiation Pneumonitis after a Single 550 cGy Fraction of Total Body Irradiation: Single Institutional Experience

<h3>Purpose/Objective(s)</h3> Total body irradiation (TBI) is an important component of the conditioning regimen prior to allogeneic stem cell transplant. While a variety of TBI dose and fractionation schemas have been studied, the optimal regimen is unknown. The American College of Radiology (ACR) suggests that relatively low dose rates of less than 10-20cGy per minute may provide the optimal outcome and reduce the risk of interstitial pneumonitis. However, there are no reported outcomes of patients treated with dose rates higher than 20cGy per minute to the best of our knowledge. The objective of the study is to investigate the incidence of radiation pneumonitis after a single fraction of 550 cGy TBI delivered at a high dose rate (∼30cGy per minute) at a single institution, where this regimen has been used as a component of the conditioning regimen for patients with hematological malignancies undergoing hematopoietic stem cell transplantation over the past 25 years. Radiation pneumonitis (RP) is an important potential toxicity associated with TBI and its incidence has not been previously reported for this regimen. <h3>Materials/Methods</h3> A retrospective chart review was performed for 183 consecutively treated patients (61% male, 39% female, aged 24 to 71) treated with single fraction 550 cGy TBI delivered at a high dose rate at our institution from 2010-2021 for a wide range of hematological malignancies including ALL (n=55), AML (n=49), CLL (n=7), CML (n=4), various forms of lymphoma (n=49), MDS (n=8), multiple myeloma (n=7), T-PLL (n=2), and B-PLL (n=2). Patients received one of the following conditioning regimens: 550 cGy single fraction TBI with cyclophosphamide, 550 cGy single fraction TBI with cyclophosphamide and thymoglobulin, or 550 cGy single fraction TBI with cyclophosphamide and fludarabine. Patients were treated on a stretcher with opposed lateral fields. Patients were evaluated for the development of radiation pneumonitis at 12 months based on CTCAE v5.0. <h3>Results</h3> Of the 183 patients, 96 patients survived until the 12-month time point. The cause of death includes sepsis, GVHD, and multi-organ failure, among others. The incidence of grade 2 or greater RP was 9.4% (9/96 patients) at 12 months in these patients. Only 1 patient developed grade 3 RP and 1 patient developed grade 4 RP. <h3>Conclusion</h3> The institutional regimen of 550 cGy TBI delivered in a single fraction at a high dose rate is associated with a low incidence of radiation pneumonitis at 12 months; however, the analysis was limited by competing transplant-related mortality in this patient population.

Total Body Irradiation in Haematopoietic Stem Cell Transplantation for Paediatric Acute Lymphoblastic Leukaemia: Review of the Literature and Future Directions.

Total body irradiation (TBI) has been a pivotal component of the conditioning regimen for allogeneic myeloablative haematopoietic stem cell transplantation (HSCT) in very-high-risk acute lymphoblastic leukaemia (ALL) for decades, especially in children and young adults. The myeloablative conditioning regimen has two aims: (1) to eradicate leukaemic cells, and (2) to prevent rejection of the graft through suppression of the recipient's immune system. Radiotherapy has the advantage of achieving an adequate dose effect in sanctuary sites and in areas with poor blood supply. However, radiotherapy is subject to radiobiological trade-offs between ALL cell destruction, immune and haematopoietic stem cell survival, and various adverse effects in normal tissue. To diminish toxicity, a shift from single-fraction to fractionated TBI has taken place. However, HSCT and TBI are still associated with multiple late sequelae, leaving room for improvement. This review discusses the past developments of TBI and considerations for dose, fractionation and dose-rate, as well as issues regarding TBI setup performance, limitations and possibilities for improvement. TBI is typically delivered using conventional irradiation techniques and centres have locally developed heterogeneous treatment methods and ways to achieve reduced doses in several organs. There are, however, limitations in options to shield organs at risk without compromising the anti-leukaemic and immunosuppressive effects of conventional TBI. Technological improvements in radiotherapy planning and delivery with highly conformal TBI or total marrow irradiation (TMI), and total marrow and lymphoid irradiation (TMLI) have opened the way to investigate the potential reduction of radiotherapy-related toxicities without jeopardising efficacy. The demonstration of the superiority of TBI compared with chemotherapy-only conditioning regimens for event-free and overall survival in the randomised For Omitting Radiation Under Majority age (FORUM) trial in children with high-risk ALL makes exploration of the optimal use of TBI delivery mandatory. Standardisation and comprehensive reporting of conventional TBI techniques as well as cooperation between radiotherapy centres may help to increase the ratio between treatment outcomes and toxicity, and future studies must determine potential added benefit of innovative conformal techniques to ultimately improve quality of life for paediatric ALL patients receiving TBI-conditioned HSCT.

Clinical Report on 6 Children with Hematopoietic Disease with Umbilical Cord Blood Transplantation.

To study the efficacy of hematopoietic stem cell transplant in children with umbilical cord blood 6 children received transplant in our hospital within these 3 years.Except for one patient suffered from acute severe aplastic anemia (SAA). All others had relapsed leukemia. Among them, 3 were relapsed ALL and another 2 were relapsed AML. The average age was 55 months (range, 38 months to 7 years old) and average weight was 17.8 kg (range, 13.5 ~28 kg) in this group. Only a 7 years old ALL-CR3 patient was conditioned with fraction total body irradiation, all the others were conditioned with non radiation regimen. Patients with ALL conditioned with Busulfan 16 mg + Cyclophosphomide 120 mg + VP1640 mg and patients with AML received Busulfan 16 mg + Cyclophosphomide 200 mg. Fludarabine 30 mg/m 2 × 5day +CTX 60 mg/kg × 2 day was chosen as the conditioning regimen for our SAA-I patient. 1 patient did not use ATG because the donor was matched related sibling, all the others received 6–30 mg/kg. day × 5 day ATG(Fresennius S). Only CSA was used for prophylaxis GVHD in this group.Two cord blood provided from Shanghai and Beijing cord blood bank separately and another 4 were from Guangzhou cord blood bank. The average total neuclear cell count was 10×107/kg (6.5~13×107/kg) and CD34+ cell was 0.81×106/kg (0.2~1.47×107/kg). 5 patients had hematopoitic reconstitution with the average time for absolute neutrophil over 0.1×109/L and 0.5×109/L 16 days (range, 14~19day) and 17days (range, 14~20day) respectively. The average time for transfusion independent platelet count recovered over 20×109/L, 50×109/L was 33.3 days (range, 27~39 days) and 49.3days (range, 48~51days) separately. 4 patients had grade II GVHD after transplantation. All patients infected with CMV and one died from CMV pneumonia on day +43, another one had encephalitis on day +124 and treated with anti-epilepsy drug till now. The average time for follow up was 29 months (13–41 months) in this group. One patient suffered from grade III hemorrhagic cystitis on day +60 and resolved on day +100 after being treated with hydration, alkalization and anti-virus drugs. One patient suffered from a great deal of hemorrhagic pericardial effusion on day +172, because of no response after several times of pericardial puncture, dissection of pericardium was received on day +182 and recovered since then. Another patient received transfusion with autologous back-up peripheral blood stem cells on day +40 because of graft failure confirmed by DNA chimerism study on day +34. This patient died of leukemia relapse on day +73. 4 patients have kept on disease free survival till now.These results further demonstrate umbilical cord blood transplant is a therapeutic option for children with very poor-prognosis leukemia and who lack an HLA-identical sibling or matched unrelated donor. But the decision of transplant for SAA patient with cord blood should be considered carefully.

Once-a-day fractionated total-body irradiation: A regimen tailored to local logistics in allogeneic stem cell transplantation for acute lymphoblastic leukemia.

The objective of the study was to estimate the cumulative incidence (CI) of relapse, relapse-free survival (RFS) and overall survival (OS) in ALL patients after a once-a-day fractionated TBI (F-TBI) regimen with 9.9 Gy. The secondary objectives were evaluation of short and long-term toxicity and non-relapse mortality (NRM). Total body irradiation (TBI), as a part of the conditioning regimen before allogeneic stem cell transplantation (ASCT) for acute lymphoblastic leukemia (ALL), allows disease control by eradicating residual blast cells in the transplant recipient. Retrospective study conducted in patients with ALL who received between March 2003 and December 2013 a conditioning regimen with F-TBI and chemotherapy. Irradiation was delivered with 3.3 Gy once-a-day for three consecutive days. Eighty-seven patients were included. The median age was 19 years (range: 5-49 years). The 3-year CI of relapse was 30%. The estimated 3-year RFS and OS were 54% and 58%, respectively. Cumulative incidence of acute graft-versus-host disease (aGVHD) grade II-IV and chronic GVHD (cGVHD) was 31% and 40%, respectively. Interstitial pneumonitis was observed in 2 patients. The 3-year CI of NRM was 16%. In multivariate analysis, cGVHD was associated with a lower CI of relapse (RR = 0.26, 95% CI: 0.07-0.95, p = 0.04). High-risk cytogenetics was associated with a lower RFS (RR = 2, 95 CI: 1.04-3.84, p = 0.03). Grade II-IV aGVHD was an independent predictor of higher CI of NRM (RR = 6.7, 95% CI: 1.4-31.7, p = 0.02). Once-a-day F-TBI regimen is effective, safe and practical in patients who underwent ASCT for ALL.

Myeloablative conditioning for allo-HSCT in pediatric ALL: FTBI or chemotherapy?\u2014A multicenter EBMT-PDWP study

Although most children with acute lymphoblastic leukemia (ALL) receive fractionated total body irradiation (FTBI) as myeloablative conditioning (MAC) for allogeneic hematopoietic stem cell transplantation (allo-HSCT), it is an important matter of debate if chemotherapy can effectively replace FTBI. To compare outcomes after FTBI versus chemotherapy-based conditioning (CC), we performed a retrospective EBMT registry study. Children aged 2–18 years after MAC for first allo-HSCT of bone marrow (BM) or peripheral blood stem cells (PBSC) from matched-related (MRD) or unrelated donors (UD) in first (CR1) or second remission (CR2) between 2000 and 2012 were included. Propensity score weighting was used to control pretreatment imbalances of the observed variables. 3.054 patients were analyzed. CR1 (1.498): median follow-up (FU) after FTBI (1.285) and CC (213) was 6.8 and 6.1 years. Survivals were not significantly different. CR2 (1.556): median FU after FTBI (1.345) and CC (211) was 6.2 years. Outcomes after FTBI were superior as compared with CC with regard to overall survival (OS), leukemia-free survival (LFS), relapse incidence (RI), and nonrelapse mortality (NRM). However, we must emphasize the preliminary character of the results of this retrospective “real-world-practice” study. These findings will be prospectively assessed in the ALL SCTped 2012 FORUM trial.

Long-Term Outcomes of Patients with Acute Myelogenous Leukemia Treated with Myeloablative Fractionated Total Body Irradiation TBI-Based Conditioning with a Tacrolimus- and Sirolimus-Based Graft-versus-Host Disease Prophylaxis Regimen: 6-Year Follow-Up from a Single Center.

Cyclophosphamide (Cy)/etoposide combined with fractionated total body irradiation (FTBI) or i.v. busulfan (Bu) has been the main conditioning regimens for allogeneic hematopoietic cell transplantation (alloHCT) for young patients with acute myelogenous leukemia (AML) eligible for a myeloablative conditioning (MAC) regimen. Recent data has suggested that i.v. Bu could be the preferred myeloablative regimen in patients with myeloid malignancies. However, Bu-based regimens are associated with higher rates of sinusoidal obstruction syndrome. Here we report long-term survival outcomes of patients with AML receiving FTBI combined with Cy or etoposide before undergoing alloHCT at City of Hope (COH). We obtained a retrospective review of a prospectively maintained institutional registry of clinical outcomes in 167 patients (median age, 41 years; range, 18 to 57 years) with AML in first or second complete remission who underwent alloHCT at COH between 2005 and 2015. Eligible patients received a MAC regimen with FTBI (1320 cGy) and Cy (120 mg/kg) for unrelated donor transplantation or etoposide (60 mg/kg) for related donor transplantation. Graft-versus-host disease (GVHD) prophylaxis was provided with tacrolimus and sirolimus. In this retrospective study, 6-year overall survival was 60% and nonrelapse mortality was 15%. The GRFS rate was 45% at 1 year and 39% at 2 years. We also describe late metabolic effects and report the cumulative incidence of secondary malignancies (9.5%). Overall, in this young adult patient population, our results compare favorably to chemotherapy-based (i.v. Bu) conditioning regimens without significant long-term toxicity arising from TBI-based regimens.

Single-Dose Daily Fractionation Is Not Inferior to Twice-a-Day Fractionated Total-Body Irradiation Before Allogeneic Stem Cell Transplantation for Acute Leukemia: A Useful Practice Simplification Resulting From the SARASIN Study

Total-body irradiation (TBI) is a major constituent of myeloablative conditioning regimens. The standard technique consists of 12Gy in 6 fractions over a period of 3days. The Standard-fractionation compAred to one-daily fRaction total body irrAdiation prior to tranSplant In LEUkemia patieNts (SARASIN) study aimed to compare standard fractionation with once-daily fractionation before transplant in leukemia. We retrospectively compared TBI regimens delivered in 2993 patients from the European Society for Blood and Marrow Transplantation database, who underwent transplantation between 2000 and 2014 for acute lymphoblastic leukemia (ALL, n=1729) or acute myeloid leukemia (AML, n=1264).TBI was delivered as either 12Gy in 6 fractions (group 1, considered the reference group; 1362 ALL and 857 AML patients), 9 to 12Gy in 2 fractions (group 2, 173 ALL and 256 AML patients), or 12Gy in 3 to 4 fractions (group 3, 194 ALL and 151 AML patients). The median follow-up was 60 and 84months in ALL and AML patients, respectively. At 5years, the leukemia-free survival rate, overall survival rate, relapse incidence, and nonrelapse mortality rate were 46.6%, 50.4%, 28.8%, and 24.6%, respectively, in ALL patients and 46.6%, 48.9%, 29.7%, and 23.6%, respectively, in AML patients. In multivariate analyses, the outcomes of groups 2 and 3 were not statistically different from those in group 1. The cumulative incidence of secondary malignancies (SMs) was significantly higher in group 2 (7.2%; P<10-6 for group 2 vs group 1). However, group 2 was not associated with an increase in SMs when we considered non-T-cell-depleted transplant patients. We showed that the 12-Gy fractionated TBI dose delivered either in 2 fractions or in 1 fraction per day over a period of 3 to 4days resulted in nonsignificant differences in disease control and survival. However, 1-day fractionation may be associated with a higher risk of mucositis and hemorrhagic cystitis. The absence of a significant difference in the SM incidence in the non-T-cell-depleted group should be interpreted with caution in the context of a retrospective study design. Our findings are important to consider for radiation therapy department organization. In-depth analyses of other nonlethal toxicities and late effects are required.

Alternative donor hematopoietic cell transplantation for Fanconi anemia

AbstractHistorically, alternative donor hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) patients resulted in excessive morbidity and mortality. To improve outcomes, we made sequential changes to the HCT conditioning regimen. A total of 130 FA patients (median age, 9.0 years; range, 1-48) underwent alternative donor HCT at the University of Minnesota between 1995 and 2012. All patients received cyclophosphamide (CY), single fraction total body irradiation (TBI), and antithymocyte globulin (ATG) with or without fludarabine (FLU), followed by T-cell–depleted bone marrow or unmanipulated umbilical cord blood transplantation. The addition of FLU enhanced engraftment 3-fold. The incidence of grades 2-4 acute and chronic graft-versus-host disease was 20% and 10%, respectively. Severe toxicity was highest in patients >10 years of age or those with a history of opportunistic infections or transfusions before HCT. Mortality was lowest in patients without a history of opportunistic infection or transfusions and who received conditioning with TBI 300 cGy, CY, FLU, and ATG. These patients had a probability of survival of 94% at 5 years. Alternative donor HCT is now associated with excellent survival for patients without prior opportunistic infections or transfusions and should be considered for all FA patients after the onset of marrow failure. These studies were registered at http://www.clinicaltrials.gov as NCT00005898, NCT00167206, and NCT00352976.

Palifermin for prevention of oral mucositis in allogeneic hematopoietic stem cell transplantation: a single-institution retrospective evaluation.

The purpose of this study is to assess the impact of palifermin on oral mucositis (OM) and its sequelae in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) who were conditioned with fractionated total body irradiation (FTBI) and etoposide. This retrospective chart review study compared the effect of palifermin on the development of OM in patients who received this agent during an allo-HSCT (n = 99) to those who did not (n = 30). The primary end points were severity and duration of OM. Secondary end points included requirements for opioids, total parenteral nutrition (TPN), and intensive oral care; incidence of infection; length of hospital stay; and overall survival. There was no significant difference in the incidence of all grades of OM, but incidence of severe OM was decreased in palifermin-exposed patients (34 vs 80 %, p < 0.0001). In patients who developed OM (all grades), the median duration of OM was shorter in palifermin-exposed patients (13 vs 18 days, p = 0.0001); there was no difference in the median duration of severe OM. Patients who received palifermin used less opioids and required a shorter duration of intensive oral care. There was no difference in duration of TPN, incidence of infection, length of hospital stay, and overall survival. Our findings demonstrated a significant benefit with the use of palifermin for allo-HSCT recipients who were conditioned with FTBI and etoposide. Palifermin can potentially improve quality of life for this patient population and reduce complications and resources used during the transplant process. A randomized clinical trial is required to confirm these results.

Selective Photodepletion of Recipient-Alloreactive T-Cells Enables Safe and Efficacious Haploidentical HSCT: Initial Results from a Phase 2 Trial in Patients with AML, ALL, and MDS

▪IntroductionFor patients in need of a hematopoietic stem cell transplant (HSCT) but lacking an HLA matched donor, a haploidentical family donor is a particularly appealing alternative. However, to prevent graft-versus-host disease (GVHD), haploidentical HSCT necessitates intensive in vivo or ex vivo T-cell depletion that results in frequent and often lethal infectious complications and/or high relapse rates, thus decreasing overall survival. To overcome this limitation, we have developed a strategy that photodepletes host-reactive cells from the donor T cell graft, while preserving anti-infection and anti-leukemia reactivity.Patients and MethodsIn an open-label, multi-center phase 2 clinical trial (CR-AIR-007; NCT01794299), 12 of a planned 23 patients with high-risk hematologic malignancies were treated to date with this immunotherapy approach consisting of donor lymphocytes selectively allodepleted of host-reactive T-cells using photodynamic therapy (ATIR). ATIR was infused 28-32 days after haploidentical CD34-selected HSCT. No post-transplant GVHD prophylaxis was used. These patients were compared to a control group of 28 patients treated in a previous Phase 2 study with an investigational product manufactured using a process different from the Phase 1 trial and resulting mainly in dead and inactive cells instead of ATIR (CR-AIR-004).ResultsTwelve patients, mean age of 45 (range 21-64), 6 females/6 males with AML (n=9) and ALL (n=3) were treated with ATIR so far. ATIR consisted mainly of T-cells (>90%), with residual B and NK cells (≤10%). Selective depletion of recipient-reactivity in each ATIR cell graft was assessed using a CFSE-based proliferation assay. Cell division numbers upon stimulation were analyzed using Modfit LT software (Fig 1A), which generated a proliferation index representing viable/reactive T-cells in donor cells (blue) and final ATIR product (green)(Fig 1B). Selective depletion of recipient-reactive T-cells with preservation of reactivity towards 3rd party antigens and anti-CD3/CD28 was observed in all ATIR cell grafts and used as a release criteria in the 007 study. [Display omitted] Preparative regimen consisted of A) FTBI (1200 cGy; n=5) or B) melphalan (120 mg/m2; n=7), along with thiotepa (10 mg/kg), fludarabine (30 mg/m2 x5 d) and ATG (2.5 mg/kg x4 d). Neutrophil and platelet engraftment was achieved in all patients at a median of 12 days (range: 9-35). No patient experienced graft rejection.Patients (n=28) in the 004 control group, mean age of 42 (range 18-61), 13 females/15 males had AML (n=19), ALL (n=6) or MDS (n=3). CFSE proliferation in T-cell grafts could not be assessed a posteriori due to low cell viability. These 004 patients received the same A) FTBI- (n=14), B) melphalan- (n= 10) based preparative regimen as 007 patients, except for 4 patients receiving single fraction (800 cGy) TBI. Neutrophil and platelet engraftment was achieved at a median of 16 days (range: 7-54). Three patients showed secondary graft rejection.Two patients in study CR-AIR-007 developed acute GVHD grade I (skin only) approximately 130 days post HSCT, which was of short duration, (18 and 41 days). Two patients died of infection and no patient relapsed at a mean follow-up of 8 months post HSCT (range 1-14 months). In the CR-AIR-004 control group, 2 patients developed grade I, 1 patient grade II and 3 patients grade III GvHD, none of these cases were lethal. Seventeen patients died of transplant related complications and 2 patients of relapse/disease progression. TRM is 20% in 007 group vs 63% in the 004 control group and OS is 80% in 007 group vs 35% in the 004 control group at 9 months post-transplant (Figures 2A and 2B). [Display omitted] [Display omitted] ConclusionsThese data confirm that a novel immunotherapy strategy consisting of donor lymphocytes selectively photodepleted of alloreactive cells (ATIR) can be manufactured consistently and reproducibly. Results to date show that ATIR is safe and does not cause any grade III/IV GvHD. Moreover, haploidentical HSCT patients treated with ATIR demonstrate very promising TRM and OS rates when compared to the control group. DisclosuresRoy:Kiadis Pharma: Consultancy, Research Funding. Foley:Hoffman-LaRoche: Advisory Board/Lectures Other, Honoraria; Lundbeck: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Sanofi: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Celgene: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Pfizer: Advisory Board/Lectures Other, Honoraria; Novartis: Advisory Board/Lectures Other, Honoraria; Jansen: Advisory Board/Lectures Other, Honoraria; Alexion: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Roche Canada: Honoraria, Research Funding, Unrestricted educational grant, Unrestricted educational grant Other. De Jong:Kiadis Pharma: Employment. Velthuis:Kiadis Pharma: Employment. Gerez:Kiadis Pharma: Employment. Reitsma:Kiadis Pharma: Employment. Wagena:Kiadis Pharma: Employment.

Phosphoramide Mustard As a Biomarker Of Cyclophosphamide Exposure In Adults Receiving Reduced Intensity Conditioning Hematopoietic Cell Transplantation

IntroductionThe use of RIC reduced intensity conditioning (RIC) hematopoietic cell transplant (HCT) has extended the availability of transplantation to older and less medically fit patients. Despite the increased tolerability of RIC HCT, treatment related mortality (TRM) still remains a significant problem. Biomarkers of chemotherapy exposure which are predictive of toxicity or efficacy would be clinically useful in making treatment decisions. We studied phosphoramide mustard (PM), one of the final active metabolites in the complex metabolic pathway of cyclophosphamide (CY), as a biomarker of toxicity and/or efficacy. PM is highly active and undergoes further non-enzymatic degradation to produce nornitrogen mustard which alkylates the guanine on DNA, thereby preventing DNA replication and ultimately leading to cellular death. This is the first report of PM in RIC HCT recipients. MethodsWe studied 10 patients undergoing allogeneic RIC HCT at the University of Minnesota from March 2013 to June 2013 for PM pharmacokinetics. Median age was 60 years (range 34 - 72). Four patients were male and six female. Patients were transplanted for MDS (n=3), AML (n=2), CLL, CMML, B-cell ALL, Hodgkins lymphoma and mantle cell lymphoma. Allogeneic donor sources were double umbilical cord blood (n=4), sibling peripheral blood (n=4) and matched unrelated donor peripheral blood (n=2). RIC preparative regimen consisted of CY 50 mg/kg x1 dose infused over 2 hours on day-6. CY doses were based on actual body weight except for 2 obese patients whose doses were based on an adjusted body weight and received 15 and 25% dose reductions. Preparative regimen also included fludarabine 30-40 mg/m2/day x5 on days -6 to -2 plus single fraction TBI 200 cGy. PM plasma pharmacokinetics were conducted with the single CY dose at times 0 (prior to infusion), 4, 6, 8 and 26 hours after start of CY infusion. PM was derivatised with DDTC and measured by HPLC with ultraviolet detection. Pharmacokinetic measures were calculated using noncompartmental analysis. ResultsMedian (range) total body weight was 76.7 kg (47.9-108.7) and CY dose was 3680mg (2395 - 5210mg). PM was readily measurable in all plasma samples. AUC0-inf was 108.6 hr*mcg/mL (54.0 – 133.9), Cmax was 6.3 mcg/mL (3.7-7.5), concentration at 24 hours was 0.87 mcg/mL (0.06-2.0) and half-life 7.5 hr (2.9 - 12.9). Since not all patients received the same dose, AUC0-inf was normalized to total CY dose and was 0.028 hr*mcg/mL/mg (0.019 - 0.037). There was a weak correlation between dose normalized AUC0-inf, and CrCl, SCr, BMI and BSA (all r2<0.17) but a good inverse correlation with total protein at time of transplant (r2=0.63). There was a good correlation between PM concentration at 26 hours and AUC0-inf (r² = 0.73).Seven patients are alive. Two patients died with TRM at days 37 and 55 posttransplant. Causes of death were graft failure, septic shock secondary to multiple infectious organisms (HHV6, VRE, mucor), and progressive neurological decline, HHV6 encephalopathy and recurrent PRES, respectively. A third patient died of disease progression at posttransplant day 39. The two patients with TRM had 2 of the 3 highest AUCs; 133.9 and 129.8 hr*mcg/mL, respectively. ConclusionWe have demonstrated that PM is readily measurable for at least 26 hours in the plasma after CY treatment. Preliminary analysis suggests that PM exposure may be associated with pretransplant total protein. Renal function appears to play a small role in PM exposure although the range of renal function in this cohort was limited. Additional data are needed to determine the relationship between PM exposure and clinical outcomes.Special acknowledgment for financial support from the American Society of Hematology and the Trainee Research Award program (AW). Disclosures:No relevant conflicts of interest to declare.

Ovarian function after allogeneic hematopoietic stem cell transplantation in childhood and adolescence

The aim of the study was to evaluate long-term ovarian function after allogeneic hematopoietic stem cell transplantation (HSCT) in childhood and adolescence. Predictive factors for ovarian function were evaluated among 92 adult or pubertal female survivors transplanted at Huddinge and Helsinki University Hospital during 1978-2000, at a mean age of 9±4.3 years (range 1-19). At the time of the study a mean±s.d. of 13±5.5 years (range 6-27) had elapsed since the HSCT and the mean age of the participants was 22±6.3 years (range 9-41). Spontaneous puberty based on breast development occurred in 40 and menarche in 30 of the 70 girls who were prepubertal at transplantation. Six out of 20 girls who received HSCT after initiation of pubertal development recovered their ovarian function. Younger age at HSCT, conditioning without total body irradiation (TBI), and a non-leukemia diagnosis predicted the spontaneous menarche. The incidence of menarche was higher after fractioned vs single fraction TBI (P<0.05), cyclophosphamide (Cy) vs busulfan (Bu)-based conditioning (P<0.05), and among leukemia patients transplanted at first remission vs later remissions (P<0.01) and with no cranial irradiation (cranial radiotherapy, CRT) vs given CRT (14-24 Gy) (P<0.01). The majority of recipients conditioned with only Cy vs TBI (P<0.001) or vs Bu-based regimens (P<0.01) showed preserved ovarian function and required no estrogen replacement at their latest follow-up visit at a mean age of 23±6.3 years (range 15-41). Ten women became pregnant. Patients conditioned with TBI or Bu-based regimes are at high risk of ovarian failure. Intensive anti-leukemia therapy before HSCT including CRT especially among relapsed patients may further decrease the possibility of spontaneous menarche.

Alternative Donor Hematopoietic Cell Transplantation for Patients with Fanconi Anemia.

AbstractAbstract 3081Hematopoietic cell transplantation (HCT) is the only proven curative therapy for the hematological abnormalities in patients with Fanconi anemia (FA). In the mid-1990s, survival after alternative donor (AD)-HCT was reported to be 18% with excessive rates of regimen-related toxicity, graft failure, graft-versus-host-disease (GVHD) and opportunistic infections (OI). Between 1990 and 2010, 127 FA patients underwent AD-HCT at the University of Minnesota using one of 5 consecutive, prospective phase I-II total body irradiation (TBI) containing clinical trials, representing the largest single center experience. All patients received cyclophosphamide (CY, 40 mg/kg) and single fraction TBI. Sequential changes were made to prevent GVHD, graft failure and OI and ultimately enhance survival; in 1994, all patients received T cell depleted (TCD) bone marrow (BM) or umbilical cord blood (UCB) to reduce GVHD; in 1999, fludarabine (FLU) was added to enhance engraftment; in 2003, thymic shielding (TS) was added to reduce OI risk; and in 2006, TBI dose reduction was evaluated to reduce toxicity. Over this time period, HLA-matched unrelated donor BM was the donor of choice with partially HLA matched BM and UCB only used as alternatives. Neutrophil recovery was twice as likely after FLU-containing regimens than non-FLU containing regimens and was not deleteriously affected by TS or reducing TBI dose to 300cGy. Grade II-IV acute GVHD was significantly reduced by the use of TCD (18%) compared to T replete BM or UCB (50% and 38%, respectively: p<0.01). Similarly, chronic GVHD was significantly lower with TCD BM (9%) vs T replete BM or UCB (25% and 15%, respectively; p=0.04). For the entire cohort of 127 patients, the probability of survival was 61% at 1 year and 54% at 5 years with a median follow-up of 9 years. Mortality was higher in recipients who were older (>10 years) or CMV seropositive, received any transfusions before HCT, or developed OI before HCT (Table). Mortality was lowest using CY/FLU/ATG, TBI 300 cGy with TS (Trial 5, relative risk [RR] 0.1; p<0.01). For 10 patients on Trial 5 who had no prior history of transfusions or OI, incidences of neutrophil and platelet are 100% and 100%, acute and chronic GVHD 17% and 0%, and probability of survival is 92%.Table:Factors associated with 3 year mortality: Multivariate analysisFactorsRelative Risk of Mortality (95% CI)P-valueTrial*2: CY/ATG/TBI 450-600, CSA/MP1.03: CY/FLU/ATG/TBI 450, CSA/MP0.3 (0.1–.7)<0.014: CY/FLU/ATG/TBI 450+TS, CSA/MP0.2 (0.1–0.7)0.015: CY/FLU/ATG/TBI 300+TS, CSA/MP or MMF0.1 (0.04–0.3)<0.01Donor TypeMatched URD Marrow1.0Mismatched URD Marrow0.3 (0.1–0.8)<0.01Mismatched RD Marrow0.3 (0.1–2.3)0.22Single or Double UCB2.0 (0.9–4.6)0.08Age at Transplant<10 years1.010–17 years2.4 (1.2–5.0)0.0118+ years3.1 (1.4–7.0)<0.01Prior OINone1.0Yes3.2 (1.5–6.9)<0.01Prior TransfusionsNoneYes2.4 (1.0–5.8)0.05CMV SerostatusPatient negative/donor negative1.0Patient negative/donor positive1.5 (0.7–3.4)0.34Patient positive2.2 (1.1–4.3)0.03*Trial 1 (CY/TBI 450, MTX/MP or CSA) was excluded as there were only 8 patients in this group and a number of demographic and disease factors were unknown.Substantial progress has been made in the successful application of AD-HCT for FA. These data support the urgency of transplant prior to the onset of severe pancytopenia when risk of OI and transfusions are more likely, particularly when a suitable BM donor is available. While other regimens are being explored, CY/FLU/ATG/TBI300 with TS should be considered a new standard of care for FA patients undergoing AD-HCT. Disclosures:No relevant conflicts of interest to declare.

Intestinal Microbiota as Novel Biomarkers of Prior Radiation Exposure

There is an urgent need for rapid, accurate, and sensitive diagnostic platforms to confirm exposure to radiation and estimate the dose absorbed by individuals subjected to acts of radiological terrorism, nuclear power plant accidents, or nuclear warfare. Clinical symptoms and physical dosimeters, even when available, do not provide adequate diagnostic information to triage and treat life-threatening radiation injuries. We hypothesized that intestinal microbiota act as novel biomarkers of prior radiation exposure. Adult male Wistar rats (n = 5/group) received single or multiple fraction total-body irradiation of 10.0 Gy and 18.0 Gy, respectively. Fresh fecal pellets were obtained from each rat prior to (day 0) and at days 4, 11, and 21 post-irradiation. Fecal microbiota composition was determined using microarray and quantitative PCR (polymerase chain reaction) analyses. The radiation exposure biomarkers consisted of increased 16S rRNA levels of 12 members of the Bacteroidales, Lactobacillaceae, and Streptococcaceae after radiation exposure, unchanged levels of 98 Clostridiaceae and Peptostreptococcaceae, and decreased levels of 47 separate Clostridiaceae members; these biomarkers are present in human and rat feces. As a result of the ubiquity of these biomarkers, this biomarker technique is non-invasive; microbiota provide a sustained level of reporting signals that are increased several-fold following exposure to radiation, and intestinal microbiota that are unaffected by radiation serve as internal controls. We conclude that intestinal microbiota serve as novel biomarkers of prior radiation exposure, and may be able to complement conventional chromosome aberrational analysis to significantly enhance biological dose assessments.

Results of a Phase 1 Clinical Trial of Allogeneic Tumor-Primed Natural Killer Cells in AML

Abstract 946We have previously reported that resting human NK cells can be primed to lyse NK-resistant tumor cells, including AML, by co-incubation with membrane preparations from the leukemia cell line CTV-1. Tumor-primed NK cells can be cryopreserved and remain in a primed state when thawed. Following translation of this production method to GMP-compliance we designed a phase 1 trial for AML patients. PROTOCOL: AML patients meeting the inclusion criteria were enrolled and they and their children HLA-typed and screened for medical suitability for apheresis. KIR mismatched donors in the GvL direction were preferred when available. PATIENT INCLUSION CRITERIA: >60 years in PR (blasts >5<20% in BM) after 2nd course of induction chemotherapy; >60 years in CR2 after re-induction chemotherapy; >60 years in PR or CR after 2 courses of chemotherapy with poor risk disease by conventional cytogenetic criteria; <60 years beyond CR2 but unsuitable for transplantation. CONDITIONING: Fludarabine 25mg/m2/day for 3 days plus a single fraction (2Gy) TBI on day 4. MANUFACTURE OF Tumor-primed NK: T-pNK cells were generated from single 2hr apheresis from haploidentical related donors in 14 cases; 1 donor declined and the patient was not treated. NK cells were isolated with anti-CD56 microbeads (CliniMACS, Miltenyi Biotec) with (n=2) or without (n=12) prior depletion of CD3+ cells. Purified NK cells were co-incubated with a lysate of CTV-1 cells (DSMZ repository) overnight. Lysate was removed by density gradient separation on day 2 and cells were cryopreserved in dosed aliquots and released for infusion within 15 days of manufacture. TREATMENT: Three groups of 5 patients were to receive 1×10^6/kg, 5×10^6/kg or 1×10^7/kg haploidentical related donor T-pNK as a single i.v. infusion 24 hours after completion of TBI. RESULTS: 15 patients were enrolled between July 2008 and Dec 2009 – Table 1. Mean donor T-pNK yield was 6.73×10^9 and CD56 purity 99.3%. T cell contamination was <10^4/kg. All products achieved QC release criteria. Median time from enrolment to product release was 46 days. 8/15 patients received the T-pNK therapy; 7 untreated due to relapse during product manufacture or failure to remit (6) and donor refusal (1). No infusional toxicity was observed. 7/8 patients experienced profound bone marrow suppression with median time of neutrophil recovery 55 days (19–101). Two patients who previously had an allogeneic transplant were salvaged with a CD34+ top up stem cell transfusion from their original donors. All experienced neutropenic fevers requiring antibiotic support with 2 of them requiring ICU admission. Due to the toxicities observed the dose escalation was abandoned and all treated patients received 1×10^6 cell/kg. The median duration of CR post T-pNK was 222 days (55–845) compared to previous CR median of 47 days (0–90) – Table 1. 7/8 patients experienced a longer CR post T-pNK than their previous CR; median increase in duration of CR was 242 days (range 54–815). CONCLUSIONS: T-pNK can be easily generated and administered with acceptable toxicity which was easily managed. In this non-controlled trial there was evidence of clinical efficacy but delays in donor screening and processing led to 40% attrition of enrolled patients prior to drug administration.Table 1:UPNSexAgeCytogeneticsAlloHSCTStatus at treatmentDuration of CR pre-TpNK (days)Duration CR post-TpNK01F54Trisomy 21NoCR33084503M72N/KNoPR1N/A25004M51t(9;11)YesCR33028407M71ComplexNoCR1605509F73Trisomy 13NoCR14714108M67normalNoCR26211611M61Trisomy 9YesCR39035212F67normalNoCR23098 Disclosures:Lowdell:Coronado Biosciences: Consultancy.

Hematopoietic Cell Transplantation for FA Patients with Acute Leukemia or Advanced MDS,

Abstract 4146Fanconi anemia (FA) patients with acute leukemia or advanced myelodysplastic syndrome (MDS with ≥ 5% blasts) have a poor prognosis and hematopoietic cell transplantation (HCT) experience is limited. We report on the outcomes of 21 patients with FA and acute myeloid leukemia (AML, n=16), acute lymphocytic leukemia (ALL, n=2) or advanced MDS (n=3) who underwent allogeneic HCT at the University of Minnesota from 1988–2009. Five patients had biallelic BRCA2 mutations. Only 2 of 7 patients who received chemotherapy before HCT achieved remission both of whom had biallelic BRCA-2 mutations and T-cell ALL. Median age was 15.5 (range 1.1–48.5) years. Median age for BRCA2 patients was substantially lower at 1.9 (range 1.4–6.6) years. Patients received cyclophosphamide, antithymocyte globulin and either single fraction total body irradiation (n=15) or busulfan (n=6), with (n=11) or without (n=10) fludarabine (FLU) followed by HLA-matched sibling (n=3) or alternative (n=18) donor stem cells consisting of bone marrow (BM, n=14), single (n=4) or double (n=3) umbilical cord blood (UCB). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine A and methylprednisolone. Probability of neutrophil engraftment was 75% without FLU and 100% with FLU based regimens. Probability of acute GVHD was 19%. For the entire cohort, probability of survival at 1 year was 43%. A higher rate of survival was observed in the 11 patients who received a FLU based regimens (50%; figure) and in the 5 patients with BRCA2 mutations (60%). Probability of relapse was 21% in these 11 patients. [Display omitted] These data suggest that HCT with a FLU based preparative regimen offers potential curative therapy for FA patients with acute leukemia or advanced MDS. Except perhaps for BRCA-2 patients with T cell ALL, there is no clear role for chemotherapy prior to HCT in FA patients with AML or MDS. Disclosures:No relevant conflicts of interest to declare.

Tregs combined with mature donor T cells hasten immune reconstitution without triggering GvHD in HLA haploidentical transplantation

Haploidentical transplantation, with extensive T cell depletion to prevent GvHD, is associated with a high incidence of infection-related deaths. The key challenge is to improve immune recovery with allogeneic donor T cells without triggering GvHD. As T regulatory cells (Tregs) controlled GvHD in preclinical studies, the present phase I/II clinical trial evaluated the impact of early infusion of donor CD4/CD25+ Tregs, followed by an inoculum of donor mature T cells (Tcons) and positively immunoselected CD34+ cells. Twenty-eight patients (median age 41, range 21-60) were enrolled from September 2008 onwards; 22 had AML (10 in CR1 at high risk, 10 in ≥CR2 and 2 in relapse), 5 had ALL (4 in CR1; 1 in relapse) and 1 had high grade NHL in relapse. Conditioning was: 8 Gy single fraction TBI, thiotepa (4 mg/kg × 2), fludarabine (40 mg/m2 × 5), cyclophosphamide (35 mg/kg × 2). All patients received immunoselected Tregs (CliniMACS, Miltenyi Biotec) (23/28 2 × 106/kg bw; 5/28 4 × 106/kg bw) and 4 days later positively immunoselected CD34+ cells (median 8.2 × 106/kg bw, range 5.0-19.1) together with Tcons (4/28 0.5 × 106/kg bw; 17/28 1 × 106/kg bw; 5/28 2 × 106/kg bw; 2/28 did not receive Tcons). CD4/CD25+ Tregs (purity 92.7 ± 2.1) consisted of 33.6% ± 13.1 CD25high; 58.1% ± 6.6 CD25int; 5.8% ± 2.5 CD25low; 65.7% ± 11.8 FoxP3; 17.4% ± 7.2 CD127 (mean ± SD). No GvHD prophylaxis was administered. 26/28 patients engrafted. No GvHD developed in 24/26 patients, 2 developed ≥ grade II GvHD. Ten patients died (3 VOD, 2 fungal pneumonia, 1 bacterial sepsis, 1 CNS aspergillosis, 1 systemic toxoplasmosis, 1 adenoviral infection, 1 MOF). CD4 and CD8 counts reached, respectively, 50/μL medianly on days 34 (range 19-63 days) and 24 (range 15-87); 100/μL medianly on days 47 (range 28-100 days) and 34 (range 19-95); 200/μL on days 70 (range 41-146 days) and 61 (range 21-95). A wide T-cell repertoire developed rapidly with high frequencies of specific CD4+ and CD8+ for opportunistic pathogens. Episodes of CMV reactivation were significantly fewer than after our standard haplo transplants. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved. In conclusion, in the setting of haploidentical transplantation infusion of Tregs makes administration of a high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution.

Rituximab in bronchiolitis obliterans after haematopoietic stem cell transplantation

To the Editors: Bronchiolitis obliterans (BO) following allogeneic haematopoietic stem cell transplantation (HSCT) is recognised as a pulmonary manifestation of chronic graft- versus -host disease (GVHD) [1, 2]. As lung biopsy is rarely performed in these patients, the diagnosis of BO syndrome (BOS) usually relies on pulmonary function testing (PFT) and computed tomography (CT) lung scans [1, 2]. Very little retrospective data exist regarding the treatment of BOS, which currently consists of increasing a patient’s baseline dose of systemic immunosuppressive therapy; however, this treatment strategy has been shown to have poor efficacy [2, 3]. The efficacy of new drugs for the treatment of chronic GVHD, including the anti-CD20 monoclonal antibody rituximab, must be specifically evaluated for BOS. Here, we report three cases in which patients received rituximab for BO after allogeneic HSCT. Patient 1 was a 15-yr-old male who received bone marrow from a sibling human leukocyte antigen (HLA)-matched donor for acute myeloid leukaemia after a myeloablative conditioning regimen that included 12 Gy fractionated total body irradiation (FTBI) and cyclophosphamide. 5 months after transplantation, he was diagnosed with chronic liver GVHD that led to the initiation of prednisone at a dosage of 1 mg·kg−1·day−1 as well as tacrolimus. 8 months after transplantation, as his liver GVHD was coming under control, he presented with dyspnoea, dry cough and chest pain. At this time, he was receiving tacrolimus and prednisone at a dosage of 20 mg·day−1. PFT revealed a severe obstructive pattern based on a forced expiratory volume in 1 s (FEV1) of 1.13 L (33% predicted), a forced vital capacity (FVC) of 2.79 L (65% pred) and FEV1/FVC ratio of 41%. A lung CT scan demonstrated the presence of air trapping and a …

Phase-2 trial of an intensified conditioning regimen for allogeneic hematopoietic cell transplant for poor-risk leukemia

Patients with poor-risk leukemia have a high relapse rate despite allogeneic transplant. We report on the phase II trial of an intensified allogeneic transplant regimen whose aim was tolerable toxicity and durable remission. Study patients (n=30) had unfavorable first remission cytogenetics, progression from myelodysplasia or active disease due to induction failure or relapse. Conditioning was intravenous busulfan, targeted to a first-dose plasma area under the curve (AUC) of 700–900 µM·min, VP-16 at 30 mg/kg of adjusted ideal body weight and fractionated total body irradiation (FTBI) at 1200 cGy in ten fractions. Graft-versus-host disease (GVHD) prophylaxis was cyclosporine A and mycophenolate mofetil. Regimen-related toxicities (Bearman) included grade 3 mucositis in 29 patients (97%) and grade 4 in one, grade 2–3 sinusoidal obstructive syndrome in 2 patients (7%), and grade 2–3 skin toxicity in 8 patients (27%). The 30- and 100-day transplant-related mortalities were 0% and 7% respectively. The median follow-up was 83.7 months (60.7–96.4) for surviving patients. The 5-yr overall and disease-free survival was 40% for all patients. Cumulative 5-yr relapse incidence was 23% and transplant-related mortality was 37%. We have shown promising overall survival and relapse incidence in these poor-risk patients, who typically have few curative options.