Donor Choice Research Articles

Influence of donor and harvesting protocol on 30 day mortality after heart transplantation

Abstract Purpose Heart transplantation continues to be the gold standard for the treatment of advanced HF, as it promotes increased survival, exercise capacity and quality of life. The choice of the potential donor, as well as the safety of the harvesting process, graft preservation and transportation are fundamental to the success of the procedure. With the territorial extension of Brazil and a large number of donations in other states and regions, evaluating donors remotely, as well as structuring an adequate fundraising plan are fundamental. Therefore, the study of these variables and their influence on the outcome at 30 days was the objective of this study. Methods A retrospective cohort was conducted involving patients aged 18 or over, who underwent heart transplantation from July 2012 to July 2022 at our center. Patients who underwent re-transplantation and those who not used the bicaval technique were excluded. Statistical analysis was performed using STATA Software version 18. The Kaplan-Meier method was used to obtain the overall survival curve and, to identify risk factors for death, Cox proportional hazards. The Log test -rank was used for comparisons between groups. Results 255 cases of heart transplantation were studied, with a 30-day survival rate of 86.6%. The average age of donors was 29 years old and the main cause of death was traumatic brain injury.Regarding the factors that increase the risk of mortality in 30 days, the longest CPB time (HR 1.01; 95% CI 1.002-1.014; p = 0.011) and the donor's age (HR 1.08; 95% CI 1.023-1.139; p = 0.005) were statistically significant. In the univariate analysis, the donor's nutritional status (overweight or obese) was also significant (p = 0.03).The donor's length of hospital stay, use of vasoactive drugs or antimicrobials, serum sodium level or history of previous PCR did not influence the recipient's 30-day survival. Distance organ retrival surgery did not influence the outcome as long as the ischemia time of less than 240 minutes was respected. Conclusion The distance harvesting protocol used in our institution proved to be safe and effective. Adequate evaluation of the donor through echocardiography as well as management of vasoactive drugs and electrolytes may have influenced the non-relevance of classic factors that affect the outcomes of patients after heart transplantation.

Transplant Outcomes in Myelofibrosis: Impact of Donor Type (Cord Blood Grafts Supported by CD34+ selected Cells [Haplo-Cord] Versus Matched Donors)

BackgroundDespite the established potentially curative role of allogeneic hematopoietic cell transplantation (alloHCT) in managing MF, the choice of alternative donors for patients lacking matched donors remains a challenge, and the optimal graft source in this disease entity continues to be an ongoing debate. ObjectivesWe aimed to evaluate the impact of donor type: umbilical cord blood transplant supported CD34+ haploidentical donor (haplo-cord) vs adult matched related donor (MRD) and matched unrelated donor (MUD) in 40 adult patients with primary or secondary MF, including those progressing to accelerated phase (AP) or blast phase (BP), who underwent their first allo-HCT. The primary objective of this study was to analyze the impact of stem cell source on primary endpoints of overall survival (OS), graft versus host disease (GVHD) and non-relapse mortality (NRM). ResultsMedian follow-up for all alive patients was 53 months (range 0.3-63 months). Nine patients (22.5%) underwent an MRD allo-HCT, 15 patients (37.5%) underwent a MUD allo-HCT, and 16 patients (40%) underwent a haplo-cord allo-HCT. Four patients died without neutrophil engraftment: 3 (19%) in haplo-cord group and one (4%) in MRD/MUD group. The cumulative incidence of ANC engraftment by day 60 was 80% (95%CI 45-94) in the haplo-cord group and 92% (95%CI 65-98) in the MRD/MUD group (p=0.09). The cumulative incidence of platelet engraftment by day 60 was 59% (95%CI 27-81) in haplo-cord group and 75% (95%CI 51-88) in MRD/MUD group (p=0.4). OS was 62% at 1 year (95%CI 49-79) and 34% at 3 years (95%CI 21-55). The 3-yr OS was similar between the haplo-cord group and the MRD/MUD (37% vs 32%, p=0.9). The 1-yr OS for accelerated/blast phase AP/BP patients was 50% (95%CI 27-93) in the haplo-cord group, compared to 40% (95%CI 19-86) in the MRD/MUD. The 1-yr OS for chronic phase CP patients was 83% (95%CI 58-100) in the haplo-cord group, compared to 79% (95%CI 60-100) in the MRD/MUD group. The cumulative incidence of relapse at 3 years in the haplo-cord group was 13% (95%CI 1.8-34), and in the MRD/MUD group was 28% (95%CI 10-49) (p=0.36). 1-yr non-relapse mortality (NRM) was 38% (95%CI 15-61) in the haplo-cord group and 33% (95%CI 15-52) in the MRD/MUD group. 3-yr NRM was 48% (95%CI 19-72) in the haplo-cord group and 54% (95%CI 29-73) in MRD/MUD group(p=0.95). ConclusionWe showed no significant difference in OS, relapse, and NRM outcomes after haplo-cord transplant compared to adult matched donors’ grafts (MRD or MUD) in MF patients. However, there were more graft failures in patients transplanted with a haplo-cord transplants and delayed engraftments with inadequate haplo myeloid bridges. Despite the small sample size in our study, considering our findings and the availability of other alternative donors, using haplo-cord platforms may no longer be justified for myelofibrosis unless the UCB engraftment dynamics can be optimized.

Allogeneic hematopoietic cell transplantation from alternative donors in acute myeloid leukemia.

Allogeneic hematopoietic cell transplantation (HCT) potentially provides a cure for patients with acute myeloid leukemia (AML) who are unlikely to be cured with chemotherapy alone. Previously, human leukocyte antigen (HLA)-matched related donors were used exclusively, which made the procedure available for a limited proportion of patients. The introduction of high-resolution HLA-typing technology, innovations in immunosuppressive therapy, and improved supportive care measures have significantly changed the situation. Now, patients without a matched related donor have an ample opportunity to receive allogeneic HCT with the use of matched or mismatched unrelated donors, umbilical cord blood grafts, or haploidentical related donors. The outcomes of alternative donor transplantations have improved over the past decades, and the growth of unrelated donor registries as well as the donor diversification have enhanced the chance of finding a suitable donor. With multiple alternative donor choices available for most patients, the donor selection is becoming increasingly important. To discuss the optimal donor choice in case of unavailability of an HLA-matched related donor, this article reviews the existing literature of retrospective and prospective comparisons of different alternative donor transplantations in AML and discusses the current state-of-art modalities in allogeneic HCT using alternative donors.

Total Body Irradiation and Fludarabine with Post-Transplantation Cyclophosphamide for Mismatched Related or Unrelated Donor Hematopoietic Cell Transplantation

Allogeneic hematopoietic cell transplantation (HCT) remains the sole curative treatment for most patients with hematologic malignancies. A well-matched donor (related or unrelated) remains the preferred donor for patients undergoing allogeneic HCT; however, a large number of patients rely on alternative donor choices of mismatched related (haploidentical) or unrelated donors to access HCT. In this retrospective study, we investigated outcomes of patients who underwent mismatched donor (related or unrelated) HCT with a radiation-based myeloablative conditioning MAC regimen in combination with fludarabine, and post-transplantation cyclophosphamide (PTCy) as higher-intensity graft-versus-host disease (GVHD) prophylaxis. We retrospectively assessed HCT outcomes in 155 patients who underwent mismatched donor HCT (related/haploidentical versus unrelated [MMUD]) with fractionated-total body irradiation (fTBI) plus fludarabine and PTCy as GVHD prophylaxis at City of Hope from 2015 to 2021. Diagnoses included acute lymphoblastic leukemia (46.5%), acute myelogenous leukemia (36.1%), and myelodysplastic syndrome (6.5%). The median age at HCT was 38 years, and 126 patients (81.3%) were an ethnic minority. The Hematopoietic Stem Cell Transplantation Comorbidity Index was ≥3 in 36.1% of the patients, and 29% had a Disease Risk Index (DRI) of high/very high. The donor type was haploidentical in 67.1% of cases and MMUD in 32.9%. At 2 years post-HCT, disease-free survival (DFS) was 75.4% and overall survival (OS) was 80.6% for all subjects. Donor type did not impact OS (hazard ratio [HR], .72; 95% confidence interval [CI], .35 to 1.49; P = .37) and DFS (HR, .78; 95% CI, .41 to 1.48; P = .44), but younger donors was associated with less grade III-IV acute GVHD (HR, 6.60; 95% CI, 1.80 to 24.19; P = .004) and less moderate or severe chronic GVHD (HR, 3.53; 95% CI, 1.70 to 7.34; P < .001), with a trend toward better survival (P = .099). The use of an MMUD was associated with significantly faster neutrophil recovery (median, 15 days versus 16 days; P = .014) and platelet recovery (median, 18 days versus 24 days; P = .029); however, there was no difference in GVHD outcomes between the haploidentical donor and MMUD groups. Nonrelapse mortality (HR, .86; 95% CI, .34 to 2.20; P = .76) and relapse risk (HR, .78; 95% CI, .33 to 1.85; P = .57) were comparable in the 2 groups. Patient age <40 years and low-intermediate DRI showed a DFS benefit (P = .004 and .029, respectively). High or very high DRI was the only predictor of increased relapse (HR, 2.89; 95% CI, 1.32 to 6.34; P = .008). In conclusion, fludarabine/fTBI with PTCy was well-tolerated in mismatched donor HCT, regardless of donor relationship to the patient, provided promising results, and increased access to HCT for patients without a matched donor, especially patients from ethnic minorities and patients of mixed race.

Donor selection in allogeneic stem cell transplantation.

Recent progress in human leukocyte antigen (HLA) characterization, increased accrual of unrelated donors and cord blood units, and a new platform for haploidentical transplantation have resulted in the widespread availability of donors for allogeneic hematopoietic stem cell transplantation. Advances in HLA typing have identified an increasing number of loci and alleles that are crucial for successful transplantation. Newer HLA A, B, C, DRB1, and DQB1 alleles, DPB1 mismatches, and HLA B leader sequence matching are incorporated into donor selection algorithms. Donor selection is highly relevant because of recently published conflicting studies using different donor types. These studies are largely retrospective and compare patients with different diseases and stages, conditioning regimens, graft versus host disease (GVHD) prophylaxis, and time periods. A broad consensus indicates that the best donor is an available matched sibling, followed by a matched unrelated donor, and then alternative donors such as haploidentical, mismatched unrelated, and cord blood units. This consensus is being challenged by other factors, such as donor age, patient condition, urgency of transplantation, and costs involved. In this review, we will analyze the unique characteristics of each donor type, the HLA and non HLA factors that affect donor choices, and the outstanding comparative outcome studies of different donor usage in hematologic malignancies.

Haploidentical stem cell donor choice for patients with acute myeloid leukemia: a study from the ALWP of the EBMT

AbstractThere is a paucity of information to guide the selection of the most suitable donor in haploidentical (Haplo) hematopoietic stem cell transplantation (HSCT). For this reason, from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation, we conducted a retrospective analysis to evaluate the impact of Haplo donor characteristics on outcomes in patients with acute myeloid leukemia (AML) who received graft-versus-host disease prophylaxis with posttransplant cyclophosphamide (PTCy). The primary end point was graft-versus-host disease (GVHD)-free and relapse-free survival (GRFS). Overall, 2200 patients were included. The median age of donors was 37 years (range, 8-71); 820 (37%) were females, including 458 (21%) who were used for male recipients. In addition, 1631 donors (74%) donated peripheral blood (PB). Multivariable analysis identified certain donor-related risk factors with a detrimental impact on transplant outcomes. The use of PB, older donors’ ages (>37 years), and female donors to male recipients negatively affected GRFS. Donor’s age and female donor-to-male recipient combination also affected nonrelapse mortality, leukemia-free survival, and overall survival. In conclusion, donor-related variables significantly influence outcomes in patients with AML after Haplo-HSCT with PTCy. When possible, younger donors and male donors for male recipients should be prioritized. The use of bone marrow can additionally prevent GVHD.

Recent advances in prevention and treatment of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Chronic graft-versus-host disease (cGVHD) negatively impacts long-term survival and quality of life (QOL) after allogeneic hematopoietic cell transplantation. Corticosteroids are the first-line treatment for cGVHD, but approximately 30% to 70% of patients develop steroid-refractory cGVHD (SR-cGVHD), which has an extremely poor prognosis. The pathophysiology of cGVHD is more complicated than acute GVHD, but recent advances using murine models in conjunction with human studies indicate three major phases: 1) acute inflammation, 2) chronic inflammation with loss of immune tolerance, and 3) disrupted target organ homeostasis and fibrosis. Strategies that help prevent cGVHD include optimal donor selection and choice of conditioning regimen as well as pharmacologic and graft manipulation strategies. The key cellular mediators of SR-cGVHD are T cells, B cells, antigen-presenting cells, and fibroblasts. T cells and B cells are now targetable with the inhibitors ibrutinib and ruxolitinib, respectively. Recently, promising results have been obtained by modulating pathologic T cell responses with Rock2 inhibitors and targeting fibrosis with CSF-1R inhibitors. To optimize the use of these medications, a better understanding of the biological and target organ-specific mechanisms of cGVHD is needed. Here we review recent advances in cGVHD pathogenesis and discuss how best to implement recently approved biology-driven treatments for cGVHD.

Strategy for Remediation of Polychlorinated Biphenyls-Contaminated Soil Through Redox Management Based on Electronegativity of the Contaminants.

Literature review reveals that Persistent Organic Pollutants (POPs), such as polychlorinated biphenyls (PCBs), are electron deficient compounds due to the presence of highly electronegative groups. Hence, they are more amenable to anaerobic biodegradation rather than oxidative metabolism. However, the studies on PCBs bioremediation are more inclined towards aerobic treatment. Besides, the past studies are mainly centered on screening and application of PCB-degrading microorganisms. In our opinion the degradative capacity is already present in the native microflora, and choice of electron donor is of paramount importance for faster reductive metabolism of PCBs. In this study, the use of methanol as electron donor with cow dung as the general microbial inoculum resulted in high specific rate of degradation (0.0542-0.0637 /day) for high-chlorinated biphenyls. The % removal of PCBs ranged between 67.7 and 71.7%. It may be the first study on the application of methanol as a cheap electron donor for PCBs biodegradation without bioaugmentation with specifically selected microorganisms.

Machine Learning Accelerated Exploration of Ternary Organic Heterojunction Photocatalysts for Sacrificial Hydrogen Evolution.

Donor-acceptor heterojunctions in organic photocatalysts can provide enhanced exciton dissociation and charge separation, thereby improving the photocatalytic activity. However, the wide choice of possible donors and acceptors poses a challenge for the rational design of organic heterojunction photocatalysts, particularly for large ternary phase spaces. We accelerated the exploration of ternary organic heterojunction photocatalysts (TOHP) by using a combination of machine learning and high-throughput experimental screening. This involved 736 experiments in all, out of possible 4320 ternary combinations. The top ten most active TOHPs discovered using this strategy showed outstanding sacrificial hydrogen production rates of more than 500 mmol g-1 h-1, with the most active ternary material reaching a rate of 749.8 mmol g-1 h-1 under 1 sun illumination. These rates of photocatalytic hydrogen generation are among the highest reported for organic photocatalysts in the literature.

Haploidentical and Matched Sibling Transplantation for Acute Myeloid Leukemia: A Hospital-Based Study.

Allogeneic peripheral blood stem cell transplantation (PBSCT) has been increasing for the last years in Latin America. The objective of this study was to describe clinical outcomes in acute myeloid leukemia (AML) receiving allogeneic PBSCT between 2013 and 2019 in a single center of Cali, Colombia. A retrospective cohort study was conducted in Fundacion Valle del Lili. Patients diagnosed with AML who received an allogeneic PBSCT between 2013 and 2019 using human leukocyte antigen (HLA)-matched sibling donors (MSDs) or haploidentical related donors (HRDs) with myeloablative conditioning regimen were included. Cases with diagnosis of promyelocytic leukemia, myelodysplastic syndrome-related AML and therapy-related AML were excluded. Data were obtained directly from the hospital PBSCT database and clinical records. A total of 50 patients were included (HRD, n = 32; MSD, n = 18). Sixty-two percent was in the first complete remission (CR1) at the time of the transplant, of which 26% were MSD and 74% were HRD. The European Group for Blood and Marrow Transplantation (EBMT) risk score was: 44% vs. 50% low, 28% vs. 28% intermediate and 28% vs. 22% high for MSD vs. HRD, respectively. Overall survival at 5 years for MSD was 62% (95% confidence interval (CI): 31-83%) and 43% (95% CI: 25-60%) for HRD. Event-free survival was 56% (95% CI: 26-78%) and 35.6% (95% CI: 18-53%), respectively. Non-relapse mortality at day-100 was 6% (95% CI: 0.8-35%) and 20% (95% CI: 9-39%). Relapse at5 years was 18% (95% CI: 4-58%) and 25% (95% CI: 10-52%). Overall mortality rate was 46%. The grade II-IV, III-IV acute graft-versus-host disease and severe chronic graft-versus-host disease was 44%, 11% and 12% for MSD, and 43%, 9% and 0% for HRD. These results underline that MSD remains the first donor choice for AML patients in CR1 when available. HRDs are still our next option among alternative donors. It is necessary to find strategies that have a positive impact on those outcomes that markedly affect the quality of allogeneic PBSCT and the prognosis of patients. Comparative, randomized, prospective studies with longer follow-up of haploidentical allogeneic PBSCT with other donor types are required to definitely establish its role among alternative donors.

Graft-Versus-Host Disease (GvHD) Prophylaxis after Haploidentical Peripheral Blood Stem Cell Transplantation (haplo-PBSCT) Using Abatacept in Combination with Post-Transplant Cyclophosphamide (PTCy)

Background: Multiple studies demonstrated the efficacy of PTCy in achieving successful multiple-HLA-antigen mismatched (haploidentical) donor transplantation. However, acute GvHD (aGvHD) remains a major cause of morbidity and mortality after transplantation. Furthermore, many patients (pts) experience severe cytokine release syndrome (CRS) immediately after haplo-donor transplantation, which may be a greater risk after haplo-PBSCT. We hypothesized that adding co-stimulation blockade with abatacept (Abat) starting before transplantation to a standard GvHD prophylaxis regimen of PTCy, tacrolimus, and mycophenolate mofetil in haplo-PBSCT would be safe and effective in aGvHD prevention and decrease the incidence of cytokine release syndrome (CRS) associated with use of this cell source. Methods: This retrospective study included 50 recipients of haplo-PBSCT between 1/1/20, and 3/31/23, who received standard PTCy GvHD prophylaxis with (PTCY/Abat, n=22) or without (PTCy, n=28) the addition of Abat at a planned dose of 10 mg/kg/dose IV on days -1, +5, +14 (n=10) and +28 (n=12). Choice of donor, conditioning regimen, and use of Abat were per physician discretion. The PTCy GvHD regimen and pre- and post-transplant care of all pts were as previously described (Baker et al. Biol Blood Marrow Transplant. 2016;22:2047-55). Survival development of aGvHD or chronic GvHD (cGvHD), and engraftment success and kinetics were evaluated. CRS (Lee, et al. Blood 2014;124:188-95), aGvHD (Magic criteria, Harris et al. Biol Blood Marrow Transplant. 2016;22:4-10) and cGvHD (NIH consensus criteria, Jagasia et al. Biol Blood Marrow Transplant 2015;21:389-401) were scored per citations. Results: In total, 22 pts received PTCy/Abat. Ten pts did not receive day +28 Abat for insurance reasons. Twenty-eight pts received PTCy without abatacept. Pt characteristics and CRS grading are described in the Table. Although distributions of diagnosis, sex, and pt or donor ages were similar between cohorts, there was some imbalance, particularly in higher use of TBI myeloablative conditioning regimen for PTCy cohort (Table). The median follow-up of survivors is 10 months. In this analysis we found a non-significant trend to lower incidence (65% vs 88% p=0.079) and severity of CRS for PTCy/Abat recipients vs PTCy recipients (Table). Only 1 of 20 evaluable PTCy/Abat recipients developed grade 2 CRS vs 6 of 26 PTCy pts. PTCy/Abat recipients achieved faster ANC engraftment (median: 15 days, 95% CI: 14-16 vs 17 days, 95% CI: 16-18; p&amp;lt;0.0001 (Figure 1A)) and platelet recovery (median: 15 days, 95% CI 13-22 vs 24 days 95% CI 19-29; p=0.002). No pt in either group developed 1 o or 2 o graft failure. Median peripheral blood CD3 chimerism at day +28 was 100% (range, 57-100%) for PTCy/Abat pts and 100% (range, 85-100%) for PTCy pts. The cumulative incidence of any aGvHD was 71% for PTCy/Abat pts vs 73% for PTCy pts, (p=0.9). Grade II or III aGvHD occurred in 13/21 and 1/21 evaluable PTCy/Abat pts, and 12/26 and 3/26 evaluable PTCy pts, respectively. No grade IV or liver aGvHD occurred in either group. The median time of onset of aGvHD was 54 days for PTCy/Abat pts and 36 days for PTCy pts (p= 0.08). The cumulative incidence of any grade cGvHD trended higher in the PTCy/Abat group (81% vs 50% p=0.052). Also, the PTCy/Abat group had a higher incidence of severe grade cGvHD (6/13 vs 2/10 evaluable pts). The 200-day cumulative incidence of relapse was 35% for PTCy/Abat and 11% for PTCy (p=0.2). No statistically significant differences in overall survival, or relapse-free survival (Figure 1B) were detected for these two populations. Conclusions:The addition of Abat starting before haplo-PBSCT did not interfere with the aGvHD and engraftment protective effects of PTCy in this cohort. Abat initiated before PBSC infusion may reduce the risk and severity of CRS and was associated with earlier neutrophil and platelet recoveries. Although the incidence of aGvHD did not differ between the two groups, time of onset trended later among PTCy/Abat recipients. Larger pt populations will be needed to determine more accurately the effects of adding Abat to PTCy on relapse risk and cGvHD incidence and severity.

Score to Guide Donor Choice in Haploidentical Stem Cell Transplant Using Post-Transplant Cyclophosphamide for Patients with Acute Myeloid Leukemia: A Study from the Acute Leukemia Working Party of the EBMT

Introduction: There is paucity of information to guide selection of the most appropriate stem cell donor for haploidentical (Haplo) hematopoietic stem cell transplant (HSCT). Patients and Methods: We retrospectively analyzed the characteristics of haploidentical family donors that may affect transplant outcomes in patients with acute myeloid leukemia (AML) who received graft-versus-host disease (GVHD) prophylaxis with postransplant cyclophosphamide (PTCy) registered in the EBMT database. The primary endpoint was GVHD and relapse-free survival (GRFS). Results: Overall, 2200 patients were included with a median age of 56 years (range, 18-75); 1742 (79%) were in complete remission and 1246 (56.6) received reduced intensity conditioning. Regarding donors, the median age was 37 years (range, 8-71), 820 (37%) were females of which 458 (21%) were used for male recipients, 1252 (57%) had positive CMV serostatus, 1631 (74%) donated peripheral blood (PB) and 1638 (75%) had ≥ 4/8 HLA mismatch with the recipient. 100-day acute GVHD grade II-IV and III-IV and 2-year chronic and chronic extensive GVHD were 28% (95% CI 26-30), 11% (95% CI 10-12), 33% (95% CI 31-35) and 14% (95% CI 12-15), respectively. After median follow-up of 24 months, the cumulative incidence of relapse and non-relapse mortality (NRM) and the probability of leukemia-free survival (LFS), overall survival (OS) and GRFS were 26% (95% CI 24-28), 22% (95% CI 20-24), 52% (95% CI 50-55), 57% (95% CI 55-60) and 41% (95% CI 39-43), respectively. In multivariable analysis, donor-related risk factors with a negative impact on GRFS were older age (HR 1.1; 95% CI 1.04-1.15), use of PB (HR 1.19; 95% CI 1.04-1.37), and female donors to male recipients (HR 1.25; 95% CI 1.09-1.43). We further developed a score that distinguished 4 groups with 0 (241), 1 (913), 2 (863) or 3 (183) donor-related risk factors. GRFS was 57% (95% CI 50-63), 43% (95% CI 39-46), 37% (95% CI 33-40), and 30% (95% CI 23-38), respectively (P &amp;lt; 0.001) (Figure 1). The score was also able to stratify risk groups for acute and chronic GVHD, NRM, LFS and OS. Conclusion: Donor variables have an important impact on AML patient's outcome after Haplo-HSCT using PTCy. With three simple donor characteristics, age, gender and stem cell source, we were able to generate a score that may help to select the most appropriate donor in clinical practice.

TCRαβ +/CD19 + - Depleted HLA-Haploidentical Hematopoietic Stem Cell Traansplantation in Pediatric Patient with Fanconi Anemia

Background Allogeneic hematopoietic stem cell transplantation (HSCT) currently represents the only proven treatment able to rescue bone marrow failure and prevent the occurrence of clonal evolution in patients with Fanconi Anemia (FA). If HSCT from HLA-identical related donor (MRD) is reported to be associated with better outcomes, this is not available for most patients. On the other hand, the identification of a fully matched unrelated donor (MUD) may be unsuccessful, or require times incompatible with a prompt treatment of the haematological condition. For patients who lack a MRD or MUD, the preferred donor choice is debated. HLA partially matched haploidentical donor (haplo) is virtually immediately available for all patients and haplo-HSCT has been reported over the last few years as a promising alternative for those patients. Following the favorable results already reported from our group of a cohort of pediatric and young adult FA patients underwent haplo-HSCT, based on the selective depletion of TCRαβ + and CD19 + cells graft manipulation, here we present the outcome of an enlarged cohort of patients who received HSCT in 3 different centers. Methods Patients diagnosed with FA and eligible for an allogeneic transplantation, but lacking a MRD or MUD, and with a suitable HLA-haploidentical donor were eligible to this study. The conditioning regimen included intravenous fludarabine, cyclophosphamide and single-dose total body irradiation for most patients. Pretransplant anti-T-lymphocyte globulin was administered in all patient to control in vivo bidirectional donor-recipient alloreactivity. Apheresis and graft manipulation were performed as previously reported (Li Pira, Biol Blood Marrow Transplant 2016). Results Thirty-five patients (19 males and 16 females) underwent haplo-HSCT between September 2011 and April 2023 in 3 different centers (Bambino Gesù Children's Hospital, Rome, Italy; Policlinico San Matteo, Pavia, Italy; Tokyo; National Center for Child Health and Development, Children's Cancer Center, Tokyo, Japan). Twenty-four patients have been previously reported (Strocchio, Blood Adv 2021). Details on patients and donor characteristics, graft composition and conditioning regimen are listed in table 1. One patient with clonal evolution and 1 with AML received fludarabine and AraC 3 weeks before the start of the conditioning. Primary engraftment was achieved in 33 patients (94.3%), with median time for neutrophil and platelet recovery of 12 days (range, 9-20 days) and 10 days (range, 7-14 days), respectively. Primary graft failure occurred in 2 patients and both were successfully transplanted from the other haploidentical parent. The monitoring of chimerism analysis confirmed a stable engraftment of donor hemopoiesis in all engrafted patients. Within 33 evaluable patients, 9 (27.3%) experienced grade 1-2 aGvHD (7 with stage I-II skin-only involvement, 1 with stage I lower gastrointestinal involvement) at a median time of 35.5 days (range, 16-67 days) after HSCT. One patient developed late-onset skin only grade 2 aGvHD at day +211 after HSCT. One patient (3%) experienced grade IV aGvHD (liver and gastrointestinal involvement) at day + 100 from HSCT, 4 weeks after the infusion of adenoviral-specific donor T cells; he is currently receiving ruxolitinib and the complete resolution of GvHD was obtained. Only one patient (3%), developed cGvHD with mild skin involvement. Thirteen patients (39.4%) experienced one or more viral infections or reactivations (6 cytomegalovirus, 4 herpesvirus-6, 2 adenovirus, 1 parainfluenzae); the specific antiviral treatment was administered with a complete resolution of infection in all cases. With a median follow up of 5.5 years (range, 0.6-11.4 years) all the patients are alive and with resolution of hematological complications. Except for the already reported vulvar intraepithelial neoplasia, no additional posttransplant malignancy was observed Conclusions Our data obtained from enlarged cohort of patients previously reported, confirm excellent results in terms of survival and a good rate of engraftment also in a multicenter setting. The low incidence of acute and chronic GvHD, a particularly detrimental complication in patients with DNA repair disorder, makes this approach attractive in this patient population. In FA patients lacking a standard donor, TCRab +/CD19 + haplo-HSCT is confirmed to be a promising strategy.

Comparison of Stem Cell Transplantation Using Unrelated, Haploidentical, and Sibling Donors for Patients with Acquired Severe Aplastic Anemia: A Single-Center Retrospective Cohort Study

The preferred donor (haploidentical vs. matched unrelated donor) choice in patients with acquired severe aplastic anemia (SAA) who lack a human leukocyte antigen (HLA)-matched sibling donors (MSDs) and fail upfront immunosuppressive treatment (IST) therapy is unknown. We retrospectively investigated SAA patients (n=58) who underwent allogeneic stem cell transplantation (allo-SCT) between January 2012 and October 2022. The 5-year overall survival (OS) (87.5% ± 11.7% vs. 98.0% ± 6.5% vs. 83.3% ± 7.6%, respectively, p= 0.926) and 5-year failure-free survival (FFS) (60.0% ± 18.2% vs. 87.0% ± 7.0% vs. 78.3% ± 8.6%, respectively, p= 0.222) were comparable among the unrelated donors (URD; n=8), haploidentical donors (HID; n=25), and MSD cohorts (n=25). Multivariate analysis revealed that primary engraftment failure independently predicted OS, and secondary graft failure predicted FFS among SAA patients who underwent allo-SCT, although donor type and age did not predict the outcomes. An urgent second SCT for patients with engraftment failure may be an effective salvage treatment. Summarily, our findings showed that an alternative donor SCT is indicated for eligible SAA patients without MSDs even if these patients are aged ⩾ 40 years.

Evaluating the Performance of Large Language Models in Hematopoietic Stem Cell Transplantation Decision Making

Introduction: Large Language Models (LLMs) are a form of Artificial Intelligence (AI), by identifying patterns and connections within data, they can predict the most likely words or phrases in specific contexts. Previous studies have indicated that GPT (Generative Pre-trained Transformer; OpenAI) performs well in answering single-choice clinical questions. However, its performance seems to be less satisfactory when dealing with multiple-choice questions and more intricate clinical cases (Cosima et al. 2023 EAO; Cascella et al. 2023 J Med Syst). Notably, no study has evaluated LLMs responses in the context of Transplantation Decision Making, a complex process heavily reliant on physician expertise. Additionally, most studies focused solely on GPT's performance, without considering other competitive LLMs like Llama-2 or VertexAI. Our study aims to assess the performance of LLMs in the domain of hematopoietic stem cell transplantation. Methods: We modified and anonymized the clinical histories of six hematological patients. An experienced hematologist reviewed and validated these modified clinical histories, which included demographic data, past medical history, hematology disease features (genetic data and MRD when available), treatment responses, adverse events from previous therapies, and potential donor information (related/unrelated, HLA, CMV status). We presented these clinical cases to six experienced bone marrow transplant physicians from two major JACIE accredited hospitals and 11 hematology residents from the University Milano-Bicocca. LLMs employed for the analysis were: GPT-4, VertexAI Palm 2, Llama-2 13b and 70b. LLMs were configured with different temperature settings to control token selection randomness, always maintaining low levels for more deterministic responses. A triple-blinded survey was conducted using Typeform, where both senior hematologists and residents provided anonymized responses with personal tokens. The senior hematologists, residents, and LLMs testers were unaware of the responses provided by the other groups. We calculated Fleiss K (K) and overall percentage of agreement (OA) between residents and LLMs, considering the consensus answer (CoA) among experts as the most frequent response. Subsequently, OA and K values for both residents and LLMs were compared using T- or Mann-Whitney tests with Graphpad v 10.0.1. Results: The results showed perfect agreement among experts in patient transplant eligibility assessment (K=1.0) and substantial agreement in the choice of donors and conditioning regimens (K=0.62 for both questions). Fair agreement was observed in Transplant Related Mortality (TRM) estimation (K=0.22). The median OA and K value between residents and the CoA of experts were 76.5% (range 52.9-88.2%) and 0.61 (range 0.4-0.8), respectively. The median OA and K value between LLMs answers and experts were 58.8% (range 47-71%) and 0.45 (range 0.3-0.61), respectively. The mean OA and K value of residents were significantly higher compared to LLMs (p=0.02). Specifically, residents showed higher median OA and K values in patient eligibility assessment (median OA 100 vs. 83% and K 1 vs. 0.78; p=0.01). However, there was no significant difference in median K for donor choice (0.56 vs. 0.56), conditioning regimen (0.67 vs. 0.33), and TRM evaluation (0.33 vs. 0) (Table 1). The median K values of GPT-4, Palm-2, Llama2-13b, and Llama2-70b were 0.49, 0.53, 0.33, and 0.53 respectively (Figure 1). Conclusion: Our study sheds light on the potential and limitations of LLMs in complex hematopoietic stem cell transplantation decision-making. While LLMs showed promising results with a median OA of 59%, residents demonstrated superior performance. LLMS displayed good performances in patients' eligibility and donor choice but showed shortcomings in conditioning regimens and TRM evaluation. Not using a rating scale from experts when evaluating LLMs responses aimed to avoid potential bias. However, it is important to note that the consensus answer, even though it was the most frequent, does not necessarily imply that other responses provided by the experts were incorrect. Therefore, the lower consensus among the experts in TRM evaluation, possibly due to the challenge of precisely calculating TRM in a survey-based evaluation, should also lead to a cautious approach when evaluating residents and LLMs answers in this setting.

Role of Polyomavirus BK and Adenovirus in the Development of Urinary Tract Complications in Pediatric Hematopoietic Stem Cell Transplantation

One of the major challenges in pediatric hematopoietic stem cell transplantation (HSCT) is the susceptibility to viral infections and reactivations that can lead to potentially life-threatening complications and increase the transplant-related mortality (TRM). Polyomavirus BK (BKPyV) and Adenovirus (AdV) represent the predominant causes of urinary tract complications, in particular hemorrhagic cystitis (HC), in HSCT recipients. Treatment options for BKPyV and AdV infections are limited and there is no accepted standard of care for the prevention and treatment of these HSCT complications. The aim of our retrospective study was to analyze the incidence of BKPyV and AdV infection in urinary and plasma samples of pediatric patients undergoing HSCT for both malignant and non-malignant disease at a single pediatric Hematology-Oncology unit. Endpoints were the cumulative incidence of infection, measured on blood and urine samples, in the first 100 days after HSCT, prevalence of asymptomatic and symptomatic infection, and correlation between BKPyV and AdV DNAemia and DNAuria and the development of HC. A total of 79 patients were analyzed. Thirty-seven (47%) patients were positive for BKPyV on urine samples and 20 (30%) on blood samples within the first 30 days after transplantation. As for AdV, 11 (9%) patients were positive on urine samples and 18 (15%) on blood samples. In our pediatric cohort, BKPyV DNAuria was a frequent event after HSCT, while BKPyV DNAemia was observed less frequently, and was accompanied by higher viruria values (p-value &amp;lt;0,001, with a median viral load in the urine of 3.8 x 10 9 copies/mL). Conversely, AdV positivity in urine is rare, while AdV DNAemia was more frequent, due to the modality of BKPyV and AdV distribution (urothelium is the main reservoir for BKPyV, while intestinal lymphocytes represents the main reservoir for AdV). Of the 37 patients with BKPyV DNA on urine samples, 11 (30%) developed a symptomatic infection with grade II (5 patients, 40%) and grade III (6 patients, 60%) HC. We did not observe any correlation between BKPyV DNA urinary levels and the development of HC, as higher viral loads have not been shown to be good predictors of occurrence or severity of HC. Cumulative incidence (CI) of BKPyV infection was higher in haploidentical HSCT (51%) and matched unrelated donors (MUD) (48%) than in HSCT from matched familiar donors (31%). Similarly, CI of AdV reactivation was higher in haploidentical HSCT (16%) than MUD (6%) and matched familiar donors (9%). Choice of donor, the type of immunosuppression used and the intensity of conditioning regimen represented predictors of both blood and urine DNA positivity for both viruses. In our cohort, positivity for BKPyV on blood and/or urine did not increase the risk of transplant-related mortality and did not affect the overall survival after HSCT. On the basis of our data, we now perform screening of BKPyV viruria only in symptomatic patients or in transplant recipients with specific risk factors (type of donor, severity of immunosuppression). Similarly, search for BKPyV DNAemia is reserved to subjects with a high viral load on urine. Likewise, search for AdV on urine samples is performed on symptomatic patients with high-load AdV DNAemia.

Deciphering the Differential Diagnosis of Inherited Versus Acquired Bone Marrow Failure Syndromes: A Large Cohort Study

The wide overlaps in clinical and hematologic features of inherited (iBMF) and acquired (aBMF) bone marrow failures are often the cause of diagnostic difficulties and delay in proper therapeutic approaches. Screening tests for iBMF are complex and expensive and have limited availability. Data-driven machine learning algorithms may reveal useful tools in clinical practice to guide differential diagnosis. We collected data about clinical, laboratory and natural history of 597 patients with BMF (55 iBMF; 542 aBMF). iBMFs were typically younger (30 y, 0.1-71 vs 44 y, 2-85, p &amp;lt;0.001) and presented higher baseline blood counts (reticulocytes 62x10^9/L, 31-205 vs 35x10^9/L, 0-216; neutrophiles 1.4x10^9/L, 0-4.3 vs 0.6x10^9/L, 0-4.6; platelets 93x10^9/L, 7-356 vs 23x10^9/L, 1-355, p &amp;lt;0.001), and more cytogenetic aberrations (32% vs 13%, p 0.002). Conversely, PNH clones were detected and confirmed overtime mainly in aBMF (51% vs 9% p &amp;lt;0.001), with median clone size of 0.6% (range 0.02-3.8) in iBMF. By NGS analysis, no differences in acquired myeloid gene mutations incidence were detected between the groups (25% vs 13%, p 0.1). Evolution to MDS occurred in 20% of iBMF vs 11% of aBMF (p 0.04), and to AML in 3.6% vs 1.5% (p 0.2). Time between diagnosis and evolution was shorter in iBMF (2 y, 0-14 vs 5.4 y, 0.2-32 p 0.01). Survival was similar between the 2 groups with median values of 7 y (95%CI, 6.1-7.6), reaching 8 y (95%CI, 6.9-9.2) in transplanted patients. Factors significantly associated with a better survival were female gender (HR: 0.73, 95%CI: 0.59-0.90, p 0.004), BMT (HR: 0.62, 95%CI: 0.47-0.83, p 0.001), number of therapy lines (HR: 0.67, 95%CI: 0.51-0.87, p 0.003), response to 1 st line (HR: 0.53, 95%CI: 0.41-0.69, p &amp;lt; 0.001), and evolution to PNH (HR: 0.61, 95%CI: 0.40-0.96, p 0.03). Conversely, a lower survival was associated with older age: patients aged 31-60 had 2-fold risk of death compared to those under 30 (HR 1.8, 95%CI 1.15-2.75, p &amp;lt;0.001); patients over 60 had a 3.2 times higher risk compared to under 30 (HR 3.2, 95%CI 2.35-4.45, p &amp;lt;0.001). Presence of trilineage cytopenia also predicted poorer survival (HR: 2.92, 95%CI: 2.25-3.79, p &amp;lt;0.001 versus single- or bi-lineage cytopenias). No clear association with survival was found regarding karyotype and presence of mutations at myeloid NGS panel (Table 1). Finally, via a 10-folds cross-validation, we built a multivariate logistic model to predict the probability of having an iBMF. A dataset composed of 139 patients (120 aBMF and 19 iBMF) was used to train and validate the model, while subjects with missing information were excluded from the analysis. The obtained classifier was tested on an independent test set, resulting in 96% (81-100) accuracy, with 26/27 patients correctly assigned to their true class, 100% sensitivity and 96% specificity and having area under the ROC curve (AUC) equal to 0.97. Factors showing an impact on the predictive model were as follows: higher Hb levels (OR 2.35, range 0.98-5.60, p 0.05), reticulocytes (OR 1.01, range 0.99-1.04, p 0.29), platelets (OR 1.05, range 1.01-1.09, p 0.005), and erythropoietin (OR 1.0, range 1.00-1.01, p 0.009) were associated with IBMF; whilst older age (OR 0.91, range 0.84-0.99, p 0.03), normal NGS myeloid panel (OR 0.09, range 0.01-1.52, p 0.09), and the presence of a PNH clone (OR 0.46, range 0.04-4.83, p 0.52) showed a negative association (Figure 1). In conclusion, iBMF and aBMF differ in clinical presentation and risk of clonal evolution, and deserve specific treatments (IST in aBMF vs BMT and androgens in iBMF) and special considerations (i.e. choice of BMT donor and conditioning in iBMF). The proposed model includes few and routinely available variables and may be a practical tool to be implemented in the clinic, though it will require validation on an independent cohort to confirm its feasibility and prediction accuracy.

Allogeneic Hematopoietic Cell Transplantation for Vexas Syndrome: Results of a Multicenter Study from the Chronic Malignancies and Autoimmune Disease Working Parties of the EBMT

VEXAS is a new entity encompassing a variety of clinical manifestations spanning from autoimmune/auto-inflammatory to hematologic conditions including MDS, plasma cell dyscrasias, and recurrent thrombosis. Somatic mutations of the X-linked UBA1 gene in hematopoietic stem and progenitor cells constitute the molecular underpinnings of VEXAS, typically occurring in men during the 6 th decade of life. Over the last 2 years since its first description, VEXAS has elicited wide medical interest resulting in the identification of pleomorphic clinical phenotypes, which make this syndrome a “big masquerader” and a clinical diagnostic challenge. Treatment strategies aim at suppression of the UBA1-mutant clone (e.g., with azacitidine or allo-HCT) or blockade of the downstream pleiotropic effects of hypercytokinemia. However, no treatment guidelines exist to date, and patients may manifest life-threatening inflammatory symptoms frequently refractory to many therapies. As few literature reports have described the successful use of transplant, we here report the outcomes of patients with VEXAS in a multicenter EBMT registry-based study. Leveraging a multidisciplinary liaison of EBMT and key rheumatology centers across Europe, we disseminated a survey to gather further information on patients with VEXAS undergoing allo-HCT to explore overall survival (OS), transplant-specific outcomes, and reversal of the VEXAS clinical phenotype. We evaluated a total of 13 patients, all males and with a median age of 58 years (IQR, 52-63) at the time of transplant. The majority had concomitant MDS (n=10) while 2 presented with only autoinflammatory manifestations, and 1 had MPN. In line with the genomics of the disease, UBA1 mutations consisted of missense substitutions at the Met41 hotspot in 10 cases (n=6 Thr/3 Val/1 Leu), splice site (n=2 c.118-2A&amp;gt;C, c.118-1G&amp;gt;C), and the rare p.Ser56Phe at a median VAF of 73% (IQR, 45-84). All patients presented with anemia and had concomitant rheumatologic manifestations ( Fig1A) requiring specific treatment. VEXAS symptoms preceded the diagnosis of MDS/MPN in all but one case. Cytogenetics was normal in all VEXAS/MDS cases, which also displayed lower-risk IPSS-R scores with a median bone marrow blast of 2% (IQR, 1-4). Allo-HCT was performed at a median of 46 months (IQR, 22-59) from VEXAS onset. Before transplant, patients received a variety of both rheumatological (steroids/anti-interleukins) and hematological (azacitidine/ruxolitinib) treatments, with a high refractoriness (median number of lines 5, range 1-13). In all cases, stability of the clinical phenotype with no flares/uncontrolled manifestations was achieved at the time of the procedure. Conversely, in 3 MDS cases, clonal progression with acquisition of additional chromosomal alterations [+8, +21, t(12;16)] was evident. Overall, 62% of patients had a Karnofsky score &amp;gt;80% and low/intermediate (&amp;lt; 3) HCT-CI scores. Donor choice consisted of matched related in 23%, matched unrelated in 54%, mismatched related in 15%, and mismatched unrelated in 8% of the cohort. Peripheral blood was the preferred stem cell source in all but one case and conditioning regimen consisted of a variety of fludarabine-based strategies (n=4 MAC, n=9 RIC). GvHD prophylaxis was based on post-transplant cyclophosphamide (n=4), or serotherapy (n=8 ATG, n=1 alemtuzumab), in combination with cyclosporine and short-course methotrexate (n=5) or mycophenolate (n=4). All patients engrafted at a median time of 16 and 14 days for neutrophils and platelets, respectively. Overall, 92% of patients achieved full donor chimerism at last-follow-up. Grade II-IV acute GvHD occurred in 23% of cases, whereas only 1 patient presented with chronic extensive GvHD, which resolved 1-year after transplant. With a median time of 19 months (IQR, 8-42) from allo-HCT, 2-year OS was 82% ( Fig1B). Of note, 1 patient died because of infection and 1 because of CNS toxicity, whereas all cases with prolonged follow-up (&amp;gt;10 months) displayed resolution of the VEXAS clinical phenotypes and were able to discontinue immunosuppression between 2-8 months post-transplant. Allo-HCT remains the only curative option for MDS and is used to treat rheumatologic conditions refractory to conventional treatments. We present the largest series of allografted VEXAS cases. Given the hemato-inflammatory nature of the disease, allo-HCT may be a viable option in selected patients.

A-150 Abo Subgroup Incompatibility Leading to Red Cell Engraftment Failure and Fatal Outcome: A Rare But Preventable Complication in Related Allogeneic HSCT

Abstract Background Pre-allogeneic hematopoietic stem cell transplant (HCST) testing includes, among others, ABO and HLA typing and testing for anti-HLA antibodies. ABO subgroup typing is not routinely performed, as A2 subgroup representes 1% of the total group A population and only 0.4% of A2 individuals have anti-A1 in their serum. ABO antigens are also found on the platelet surface. We describe a case of alloHSCT performed between siblings, the donor A1 and the recipient A2, with a fatal outcome caused by red cell and platelet engraftment failure due to anti-A1 antibodies, and emphasize the need to routinely investigate subgroup incompatibility in the pre-alloHSCT workup. Methods Patient data was collected from hospital and laboratory electronic records. Blood typing and immunohematological investigation was performed at the hospital blood bank using gel cards for ABO typing, DAT and antibody screening panels (Grifols, Barcelona). Tube method was used for A subgroup typing (Fresenius, Germany). Bone marrow analysis and other blood tests were performed at local hospital laboratory. HLA typing and antibody screening (Luminex, USA) were sent to a specialized laboratory. Results A 51 year-old male with Non-Hodgkin lymphoma underwent a full-match ABO compatible allogeneic HSCT, both brother/donor and recipient group A. Pre-transplant anti-HLA or ABO antibodies were negative. WBC recovery was achieved on D + 22. The patient had a catheter infection on D + 15 (Permcath removed), CMV infection on D + 24 (treated with gancyclovir) and received several blood transfusions. On D + 22, he had a hemolytic transfusion reaction with fever (38.5°C), chills and a decline in hemoglobin (7.3 g/dL to 6.6 g/dL in the following day), low haptoglobin and increased indirect bilirrubin levels. Immunohematological investigation revealed a positive DAT with an anti-A antibody in the eluate. Subgroup incompatibility was suspected and the patient was found to be subgroup A2, whereas the donor typed A1. Three days later the DAT became negative and anti-A1 was no longer detected. Other RBC antibodies were also excluded. Only group O RBCs and A / AB platelets were transfused thereafter to prevent further hemolysis. On D + 35 the patient was diagnosed with GI-GVHD, which responded to corticosteroids. He never achieved transfusion independence and received RBC and platelet transfusions on a regular basis. Failure of RBC and platelet engraftment was confirmed by a bone marrow aspirate on D + 45, showing only 20% erythroid lineage and absence of megakaryocytes. Marrow chimerism revealed 83% of donor origin. Sepsis by S. aureus ensued on D + 57 and three days later he had fever (38.2°C) and dyspnea after a platelet transfusion. Despite entubation and mechanical ventilation, he evolved to RCP and died on D + 60. Conclusion ABO subgroup typing is a simple and low-cost technique routinely available in the blood bank. Detection of A subgroup incompatibility impacts on key decisions, such as donor choice (if more than one candidate) and the need for erythrocyte depletion of donor stem cells and recipient plasmapheresis if the anti-A1 titer is high. Since graft failure due to ABO subgroup incompatibility can be hazardous, we recommend routinely ABO subgroup typing in the pre-alloHSCT workup to increase donor safety.

The Influence of Brand Equity Dimensions on Donor’s Decisions in Paying Alms

The purpose of this research is to analyze the influence of brand equity dimensions simultaneously and also partially on donors' decisions in giving alms at UPZIS NU CARE-LAZISNU MWCNU Wonocolo. The method of analysis of this research is quantitative with the technical analysis tool used is multiple linear regression, to determine the direction and how much influence the independent variables have on the dependent variable. This research is using primary data obtained from the results of filling out the questionnaire. Based on the findings of data analysis using SPSS version 26, it shows that brand awareness and brand loyalty factors have a significant influence on the choice of donors partially. In addition, the brand equity aspect also has a simultaneous impact on donors' decisions to make donations at UPZIS NU CARE-LAZISNU MWCNU Wonocolo. This research, is expected that UPZIS NU CARE-LAZISNU MWCNU Wonocolo will continue to improve and maintain the brand that has been built.