Tapering Of Immunosuppression Research Articles

Donor Lymphocyte Infusion (DLI) post allogeneic stem cell transplant (allo-SCT) in Acute Myeloid Leukemia (AML) and High-Grade Myelodysplastic Syndrome (MDS). A longitudinal retrospective study using peripheral blood (PB) CD34+ and CD3+ donor chimerism (DC) monitoring

IntroductionThis longitudinal study was based on the outcomes of Donor Lymphocyte Infusion (DLI) for falling peripheral blood (PB) CD34+ and CD3+ donor chimerism (DC). MethodsFrom 2012 to 2018, data was collected from the BMT database and electronic medical records (EMR). The primary objective was to compare the indication for DLI based on falling PB CD34+ or CD3+ DC in patients post allo-SCT for AML and MDS and their overall survival (OS). Results18/70 patients met the inclusion criteria. Indications for DLI were i) falling PB CD34+ DC ≤ 80 % with morphological relapse, ii) falling PB CD34+ DC ≤ 80 % without morphological relapse and iii) falling PB CD3+ DC ≤ 80 % without falling PB CD34+ DC. Log rank analysis showed falling PB CD34+ DC and morphological relapse had significantly lower OS. Linear regression demonstrated better OS post DLI if there was PB CD34+ and CD3+ chimerism response at 30 days (p = 0.029), GVHD (p = 0.032) and tapering immunosuppression at the time of falling DC (p = 0.042). ConclusionDLI for PB CD34+ DC values ≤ 80 % and morphological relapse had the lowest OS. In this study, full DC was achieved after DLI even with a PB CD3+DC value as low as 13 %, provided the PB CD34+ DC remained > 80 %. Further research is vital in CD34+ DC as a biomarker for disease relapse and loss of engraftment.

Progressive demyelinating polyneuropathy after hematopoietic cell transplantation in metachromatic leukodystrophy: a case series

Metachromatic leukodystrophy (MLD) is a neuro-metabolic disorder due to arylsulfatase A deficiency, causing demyelination of the central and peripheral nervous system. Hematopoietic cell transplantation (HCT) can provide a symptomatic and survival benefit for pre-symptomatic and early symptomatic patients by stabilizing CNS disease. This case series, however, illustrates the occurrence of severely progressive polyneuropathy shortly after HCT in two patients with late-infantile, one with late-juvenile, and one with adult MLD, leading to the inability to walk or sit without support. The patients had demyelinating polyneuropathy before HCT, performed at the ages of 2 years in the first two patients and at 14 and 23 years in the other two patients. The myeloablative conditioning regimen consisted of busulfan, fludarabine and, in one case, rituximab, with anti-thymocyte globulin, cyclosporine, steroids, and/or mycophenolate mofetil for GvHD prophylaxis. Polyneuropathy after HCT progressed parallel with tapering immunosuppression and paralleled bouts of infection and graft-versus-host disease (GvHD). Differential diagnoses included MLD progression, neurological GvHD or another (auto)inflammatory cause. Laboratory, electroneurography and pathology investigations were inconclusive. In two patients, treatment with immunomodulatory drugs led to temporary improvement, but not sustained stabilization of polyneuropathy. One patient showed recovery to pre-HCT functioning, except for a Holmes-like tremor, for which a peripheral origin cannot be excluded. One patient showed marginal response to immunosuppressive treatment and died ten months after HCT due to respiratory failure. The extensive diagnostic and therapeutic attempts highlight the challenge of characterizing and treating progressive polyneuropathy in patients with MLD shortly after HCT. We advise to consider repeat electro-neurography and possibly peripheral nerve biopsy in such patients. Nerve conduction blocks, evidence of the presence of T lymphocytes and macrophages in the neuronal and surrounding nerve tissue, and beneficial effects of immunomodulatory drugs may indicate a partially (auto)immune-mediated pathology. Polyneuropathy may cause major residual disease burden after HCT. MLD patients with progressive polyneuropathy could potentially benefit from a more intensified immunomodulatory drug regime following HCT, especially at times of immune activation.

Integrative Single-Cell Tracking of Clonal Evolution, Leukemia Phenotypes and Donor Engraftment Following Allogeneic Stem Cell Transplant Using Mitochondrial DNA Mutations

Mitochondrial DNA (mtDNA) mutations can distinguish cells from unrelated individuals and their spectrum per patient sample can evolve following therapeutic bottlenecks. As such, these natural barcodes can potentially enable integrated single cell tracking of chimeric cellular populations and clonal evolution following unrelated allogeneic hematopoietic stem cell transplantation (HSCT). However, the feasibility of mtDNA-based donor-recipient deconvolution or identification of leukemia-specific phenotypes and the extent of co-evolution with somatic mutations remains unknown. If confirmed, post-transplant monitoring using mtDNA mutations could enable detection of early post-HSCT relapse and provide qualitative information on leukemia phenotypes, clonal evolution, or residual donor engraftment via a combined single cell assay. To investigate the extent of co-evolution of somatic nuclear mutations with mtDNA mutations, we generated 33,796 single cell DNA sequencing profiles (Tapestri) from 3 sample pairs of circulating chronic lymphocytic leukemia (CLL) collected before and after HSCT. The samples were characterized using a primer panel (n=67 nuclear and 21 mtDNA) targeting single nucleotide variants previously identified by whole-exome sequencing. We detected 8 mtDNA mutations that defined CLL subclones together with a median of 14 (8-19) somatic mutations from these cases. Clonal shifts defined by somatic mutations discernable pre- or post-HSCT tracked with analogous changes in mtDNA mutations and included clonal replacement, selection of a resistant subclone or skewing of CLL subclones. We thus provide clear evidence of co-segregation between somatic nuclear and mtDNA mutations. To assess the sensitivity of mtDNA-based donor-recipient deconvolution, we performed an in-silico titration and spiked 1-1,000 single cell mtDNA profiles (mtscATAC-seq) from one CLL case ('CLL4') into 7,579 profiles from another ('CLL5'). Overall, we recovered 98.3-100% of CLL4, identifying up to 1 in 7,579 cells (0.13%). Only 123 cells (1%) failed deconvolution, due to insufficient mtDNA coverage. In an in-vitro titration of expanded T cells (donor1) with peripheral blood mononuclear cells (donor2) at ratios of 1:3, 1:30 and 1:300, we recovered 1,449 of 5,345 (27%), 150 of 4,262 (3.5%) and 14 of 4,381 (0.3%) T cells of donor1. In sum, with current sequencing throughput, mtDNA-based donor-recipient deconvolution was assessed to have a sensitivity of ~0.1%. To determine if mtDNA-based deconvolution can track early AML relapse and identify leukemia phenotypes, we analyzed peripheral blood and bone marrow of 4 patients with disease recurrence 87-185 days post-HSCT and subsequent reinstatement of remission 105-184 days after tapering of immunosuppression (IST). We obtained 19,143 ATAC with select antigen profiling by sequencing (ASAP-seq) profiles at incipient relapse (pre-IST) and following response to IST (post-IST). Donor-recipient deconvolution detected recipient-derived cells in progenitor, erythroid and monocytic populations pre-IST but no recipient-derived cells post-IST, consistent with elimination of AML. Overall, chromatin accessibility of donor- and recipient-derived cells was very similar, reflecting that AML recapitulates hematopoietic differentiation states. Nevertheless, we observed differences in accessibility of genes such as IL1B, CD36, chemokine or homeobox protein genes in recipient- compared to donor-derived cells. Together, mtDNA-based donor-recipient deconvolution reflected clinical responses and identified leukemia-specific chromatin accessibility profiles. Finally, we tracked clonal evolution and donor-derived hematopoiesis in two non-responder cases of overt AML relapse following 3-10 cycles of decitabine and ipilimumab (NCT02890329). We defined 12 and 7 mtDNA-based AML subclones and observed contraction and expansion of subclones during treatment. Donor-derived non-T cells were exceedingly rare (12 of 10,708 [0.1%] and 34 of 5,863 [0.6%]), mainly of erythroid origin (12 and 23 cells) and disappeared during treatment. Thus, mtDNA mutations can monitor donor- and recipient-derived hematopoiesis and resolve short-term changes in AML subpopulations during therapy. As sequencing throughput improves, we envision single cell-based post-transplant monitoring as a powerful approach for guiding clinical decision making.

GO-8: Stable Expression of Factor VIII over 5 Years Following Adeno-Associated Gene Transfer in Subjects with Hemophilia a Using a Novel Human Factor VIII Variant

Background: GO-8 (ClinicalTrials.gov: NCT03001830) is a study of liver-directed adeno-associated virus (AAV) gene therapy for haemophilia A (HA) that uses a factor VIII (FVIII) variant containing a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain (AAV-HLP-hFVIII-V3). In preclinical studies, AAV-HLP-hFVIII-V3 mediated a 3-fold higher FVIII expression when compared to an identical AAV construct encoding the hFVIII-SQ variant used in most HA gene therapy trials. Methods: In a multi-centre, open-label, non-randomised, phase I/II clinical trial, we assessed the safety and efficacy of escalating doses of AAV-HLP-hFVIII-V3 pseudotyped with an AAV8 capsid in adults with severe haemophilia A (FVIII activity ≤1%). All participants received prophylactic glucocorticoids, with or without tacrolimus, with the aim of reducing the risk of vector-related transaminase elevation. The primary endpoints were safety and efficacy. Efficacy was assessed by measuring FVIII activity (FVIII: C) using both chromogenic and one-stage clotting assays and factor consumption pre and post-gene therapy. Results: As of May 31 2023, 12 participants were enrolled sequentially into one of four vector doses: 6×1011 vector genomes (vg)/kg body weight (n=1), 2×1012 vg/kg (n=3), 4×1012 vg/kg (n=3), or 6×1012 vg/kg (n=5). All participants were on FVIII prophylaxis prior to gene therapy. The most common vector-related adverse event was an elevation in liver aminotransferase levels, which occurred in 10 of 12 participants. In 7 of the 8 participants treated at doses ≥4×1012 vg/kg, recurrent elevation in aminotransferase levels was observed during the first 12 months, often associated with tapering of immunosuppression. This resulted in a reduction in transgene expression from peak levels in all participants, with a complete loss of transgenic protein in one participant. Vector-related elevation in aminotransferase was not observed after the 12-month time point in long-term follow-up. Mean chromogenic FVIII: C levels at 12 months after gene therapy were 3 IU/dL in the 6×1011 vg/kg cohort, 13±9IU/dL (range: 2-19 IU/dl) in the 2×1012 vg/kg cohort, 8±1IU/dl in the 4×1012 vg/kg cohort (range: 7-9 IU/dl) and 22±34 IU/dl in the 6×1012 vg/kg cohort (range 1-82 IU/dl). Transgene expression was then stably maintained over a median follow-up of 3 years (range: 0.2-5 years) from the level achieved 1-year post-infusion, best illustrated by the data from the 2×1012 and 4×1012 vg/kg cohorts shown in Figure 1. FVIII: C was, on average 2-fold higher when measured using a one-stage clotting assay compared to the chromogenic method. Nine of the 12 participants remained off prophylaxis after gene therapy for the duration of the follow-up period. Baseline mean and median annualised factor VIII use was 4097 and 4657 IU/kg per year before gene therapy. Following gene therapy, the mean and median annualised factor VIII concentrate use reduced across all participants to 1186 and 61 IU/kg (One sample t-test p=0.0009), respectively. No FVIII inhibitors or thrombotic events were reported for the duration of the study. Conclusion: A single infusion of AAV-HLP-hFVIII-V3 resulted in stable FVIII expression over a follow-up period of up to 5 years in participants with severe haemophilia A. A high rate of liver aminotransferase elevation following gene transfer impacted transgene expression. However, 9 of the 12 participants were able to discontinue FVIII prophylaxis over the duration of the study, resulting in a significant reduction in FVIII concentrate usage.

Full CD34 Donor Chimerism Following CD34 Chimerism Directed Donor Lymphocyte Infusion Results in Reduced Relapse and Superior Overall Survival Compared with Mixed Chimerism Status in Allogeneic Haemopoietic Stem Cell Transplant Recipients

Introduction Peripheral blood CD34 donor chimerism (DC) is a clinically valuable method for detecting relapse of myeloid malignancies following allogeneic hematopoietic stem cell transplantation (alloHSCT). Our institution has recently shown that CD34 DC ≤80% was superior to CD3 DC for predicting relapse (1). While pre-emptive DLI has demonstrated efficacy in preventing relapse in the setting of falling whole unfractionated and CD3 DC, little has been reported about its utility in falling CD34 DC. Methods We conducted a retrospective analysis of consecutive patients who received DLI at the Alfred Hospital in Melbourne, Australia between January 2012 and January 2023. Peripheral blood CD34 DC was monitored in alloHSCT patients transplanted for myeloid malignancies or acute lymphoblastic leukaemia (ALL). CD34+ cells were enriched using magnetic beads and DC measured using an STR-PCR method, which had a sensitivity of 1%. DLI was administered to patients with CD34 ≤80% that did not respond to immunosuppression taper. DLI doses were based on donor type and recipient weight, with subsequent doses administered incrementally according to a predefined protocol. Starting doses were 3x10 6/kg for sibling donors, 1x10 6/kg for unrelated donors and 0.5x10 6/kg for haploidentical donors. DLI was discontinued upon achieving full DC (FDC) defined as CD34 DC > 95%, clinically significant graft-versus-host disease (GVHD), disease progression, or lack of available donor cells. Results One hundred and six patients received DLI between 2012 and 2023. Twenty-five of these patients received DLI for CD34 DC ≤80% without morphologic relapse. Eleven of 25 (44%) patients received DLI alone, while 14 (56%) patients received it in combination with other therapies, primarily a hypomethylating agent. At the time of DLI, 5 (20%) of the 25 patients had concurrent decreased CD3 DC ≤ 80%. The median follow-up period was 1.84 years (range 0.58 to 8.43 years). Sixteen of 25 (66.7%) patients achieved CD34 FDC while 8 (33.3%) had persistent CD34 mixed DC (CD34 MDC). One patient could not be assessed due to death from infection. Patient characteristics and outcomes according to response to DLI are presented in Table 1. The cumulative incidence of relapse was significantly lower in the CD34 FDC group (12.5% vs 75% at 2 years, p<0.001). Patients achieving CD34 FDC had improved progression-free survival (PFS) and overall survival (OS) compared to the CD34 MDC group (PFS median years not reached [95% CI 1.94-ND] vs 0.29 years [95% CI 0.09-ND], p<0.001; OS median years not reached vs 0.68 years [95% CI 0.58-ND], p=0.003; Figure 1). Rates of grade II-IV acute graft-versus-host-disease (aGVHD) post-DLI were 12.5% in those achieving FDC and MDC (Table 1). A significant proportion of patients (37.5%) achieving FDC developed moderate-severe cGVHD while the MDC group did not survive long enough to develop cGVHD (Table 1). The non-relapse mortality observed in the CD34 FDC group was 12.5% at 3 years post-DLI. Conclusion Our centre experience demonstrated that CD34-directed DLI reduced relapse risk and improved overall survival when FDC is restored in alloHSCT patients, with an acceptable toxicity profile. Only 20% of patients with CD34 MDC had concurrent CD3 MDC, highlighting the additional value of CD34 monitoring in guiding salvage DLI administration. In patients with CD34 MDC ≤80%, the target of DLI should be to achieve FDC (>95%) given the clear association with improved outcomes in this group. References 1/ Das TP, North D, Fleming SA, Tan JLC, Ivey A, Cummings NJ, Spencer A, Patil SS, Widjaja JML, Swain MI, Bourke C, O'Brien ME, Kliman DS, Curtis DJ. Peripheral Blood CD34 Donor Chimerism has Greater Clinical Utility Than CD3 for Detecting Relapse after Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia or Myelodysplastic Syndrome. Transplant Cell Ther. 2023 Jul;29(7):454.e1-454.e8. doi: 10.1016/j.jtct.2023.03.025.

A Novel, Closed-Line Mini Extracorporeal Photopheresis (ECP) Technique Using the Therakos TM CellEx TM Photopheresis System, Developed for Patients with Contraindications Against Conventional ECP Therapy

Introduction: Conventional ECP has proven efficacy for treating several diseases, most significantly steroid-refractory chronic GVHD. Unfortunately, body weight and sufficient venous access limit the availability of this treatment modality. Children, in particular, usually have limited access to ECP. A simplified mini buffy coat ECP technique that allows treating small children and patients with apheresis contraindications has been tested in selected cases during the last 20 years. However, this method relies on an open-line system that requires a longer time and a cell processing laboratory, making this approach challenging. A new -closed-line- method, using the Therakos TM CellEx TM Photopheresis System, has been developed and evaluated in our Center. This method requires no processing laboratory or other special equipment and takes only 60-120 minutes, making the procedure potentially usable in routine practice. Design and methods: A 7-year-old girl had been treated at our Pediatric Bone Marrow Transplantation Unit with grade IV chronic steroid-refractory GVHD (skin, and less severe liver involvement). As all other options were exhausted, ECP became a desirable form of treatment. However, the patient's low body weight and serious venous access problems hindered conventional ECP treatment. We developed a solution that requires 200-250 ml whole blood collection from the patient, takes less than 2 hours, does not need special cell processing equipment, and allows the ECP treatment to commence in the form of a regular autotransfusion. Consent forms were obtained from the patient's caregivers as well as from the local Ethical Committe. The patient's age at the start of treatment was seven years, and her body weight was 28 kg. A total of 17 mini-ECPs were performed weekly up until the submission of this abstract, and the number of treatments per patient was 33. The duration of mini-ECP treatment has been five months. 5 to 8 mL/kg of whole blood was drawn from the patient. Separation of the white blood cell (WBC) rich buffy coat of the patient's blood - with donor blood product supplementation as needed - was performed through an individualized use of the CellEx TM photopheresis kit and blood prime program. The buffy coat was ultraviolet A (UVA)-irradiated after adding 8-methoxypsoralen (8-MOP) within the Therakos TM CellEx TM Photopheresis System and divided into two fractions under a laminar air flow cabinet. The autologous residual blood with the first fraction of the UVA irradiated buffy coat was transfused immediately. The second buffy coat fraction was stored at 4 °C and transfused the following morning through a peripheral vein. Results : The median volume of the collected whole blood was 250 mL (203-250 mL). The median volume was 72 ml (48-88 mL) for the first fraction of the irradiated buffy coat and 71 ml for the second fraction (48- 88 mL). Apoptosis and cell death were analyzed by annexin V staining; apoptosis and lymphocyte proliferation was measured by flow cytometry. UVA irradiation of the 8-MOP infused buffy coat resulted in significant induction of WBC apoptosis at 24 hours. No clinical side effects were observed during mini ECP treatment; all microbiological control tests were negative. Partial response was observed within three months (skin), and the tapering of systemic immunosuppression (methylprednisolone and ruxolitinib) began at five months. Conclusion: Mini whole blood ECP induces apoptosis and lymphocyte proliferation inhibition, both occurring after standard ECP. The closed-line method requires no special equipment and is not demanding for the patient, providing a viable solution to the technical difficulties children with the need for ECP treatment face.

Flares of Acute Graft-Versus-Host Disease: Mount Sinai Acute Gvhd International Consortium (MAGIC) Study

[Background] The current standard first-line treatment for acute graft-versus-host disease (GVHD), systemic high-dose steroids, induces clinical responses in a majority of patients. Flares of GVHD after initial improvement often occur during tapering or discontinuation of immunosuppression. There is no consensus regarding the definition of GVHD flare and little has been published regarding its natural history. [Methods] We retrospectively evaluated clinical data and blood samples from 968 allogeneic hematopoietic cell transplantation (HCT) recipients from 23 Mount Sinai Acute GVHD International Consortium (MAGIC) transplant centers who achieved complete response (CR) or very good partial response (VGPR) within 4 weeks of acute GVHD treatment between 2014 and 2021. VGPR was defined as the complete resolution of acute GVHD manifestations except residual stage 1 skin disease. Flares were defined as a recurrence of acute GVHD after achievement of CR/VGPR if (1) patients had an increased symptom severity of at least 1 organ stage; (2) received intensified treatment (increase in steroid dose ≥ 0.25 mg/kg or initiation of additional systemic immunosuppression); and (3) had no prior history of primary disease relapse or donor lymphocyte infusion preceding the flare. Serum samples obtained at the time of CR/VGPR and at the time of flares were analyzed for ST2 and REG3α concentrations that generated MAGIC algorithm probabilities (MAPs) resulting in previously validated Ann Arbor (AA) scores (1, 2, and 3). [Results] The median recipient age at HCT was 55 years (range: 0 to 79); 18.6% of patients had grades III-IV acute GVHD before CR/VGPR. The median maximum daily dose of corticosteroids before CR/VGPR was 1 mg/kg methylprednisolone equivalent (range, 0.1 to 3.2 mg/kg). Flares developed within 6 months following CR/VGPR in 210/968 (21.7%) patients with a median onset of 28 days (range: 2 to 448). Symptom severity at the onset of flare symptoms varied widely (grade I: 22%; grade II: 38%; grade III-IV: 41%) and the development of a flare was associated with a five-fold increase in NRM (hazard ratio [HR], 4.84 [95% CI, 3.19-7.36], P < 0.001) when considered as a time-dependent covariate in a multivariate regression model. The large majority of non-relapse deaths after flares (54/71, 76%) were due to acute GVHD or complications from its treatment. The AA scores at the time of first CR/VGPR successfully stratified patients for risk of six-month NRM (AA1: 5%, AA2: 11%, AA3: 34%, P < 0.001) (Figure A). Increasing AA score at CR/VGPR was also associated with significantly increased risk of GVHD flares (Figure B). Not only did the risk of flare increase with each rising AA score, but the number of patients with severe (grade III/IV) symptoms at flare onset also increased (AA1: 34%, AA2: 43%, AA3: 54%). We next generated a multivariate regression model of risk factors for the development of flares. High AA scores at the time of CR/VGPR (AA2: HR, 1.81 [95% CI, 1.32-2.48], P = 0.001; AA3: HR, 3.14 [95% CI, 1.98-4.98], P < 0.001), HCT from HLA mismatched unrelated donor (HR, 1.74 [95% CI, 1.00-3.02], P = 0.049), and interestingly, rapid achievement of CR/VGPR from the time of initial treatment (≤14 days) (HR, 1.84 [95% CI, 1.21-2.80], P = 0.004) were all identified as significant risk factors. Meanwhile, no other transplant, GVHD, or treatment characteristics associated with the development of flares including maximum GVHD severity, maximum steroid dose, and use of additional immunosuppressive agents other than steroids before CR/VGPR. AA scores measured at the onset of flare symptoms in 98 patients also predicted six-month NRM (AA1, 6%; AA2, 19%; AA3 42%, P = 0.01). [Conclusion] Despite advances in treatment, flares of GVHD symptoms following excellent (CR/VGPR) responses still occur in over one fifth of patients in current practice and are associated with a five-fold increase in six-month NRM. AA scores from serum biomarkers predict outcomes despite the absence of symptoms, suggesting that MAGIC biomarkers detect subclinical damage to GI crypts that can drive later recrudescence of clinical GVHD. Measurement of the MAP at the time of response to GVHD treatment may thus help to risk-stratify tapering strategies of immunosuppressive therapy.

Blast Clearance after High Dose-Melphalan Conditioning for Allogeneic Stem Cell Transplantation in Relapsed and Refractory Acute Myeloid Leukemia

Introduction: For patients (pts) with relapsed or refractory acute myeloid leukemia (r/r AML) allogeneic stem cell transplantation (ASCT) has become an established highly effective and potential curative treatment option. Even for pts with active disease, direct ASCT using sequential conditioning regimens has shown promising outcomes. In this retrospective study, we examined prognostic factors for long-term survival in r/r AML pts with active disease with special focus to the prognostic value of blast clearance during condition therapy. Patients and Methods: 186 r/r AML pts treated with sequential conditioning regimens at our center between 2014-2023 were included in our analysis. Patients underwent high-dose melphalan (100 - 140 mg/m 2, day -11 prior ASCT) followed by either fractionated total body irradiation with 8 Gy (TBI, n = 71) or non-TBI regimens (busulfan, n = 101, or treosulfan, n = 14) in combination with fludarabin (120 - 150 mg/m 2) from day -6 before ASCT. TBI-based conditioning was applied almost exclusively in patients aged < 60 years. Graft versus host-disease (GvHD) prophylaxis consisted of calcineurin inhibitors, mycophenolate and anti-T-lymphocyte globulin. In 180 out of 186 patients, blast clearance was assessed by cytologic and flow cytometric blast count via bone marrow (BM) aspirate in median 5 days after Melphalan and prior to continuation of the sequential conditioning therapy. Blast clearance was defined as cytologic BM blast count < 5% and < 0.1% BM cells with leukemia-associated immunophenotype in flow cytometry. Results: Median pts age was 67 years (range 45 - 76 years) in the non-TBI group and 51 years (range 19 - 71 years) in the TBI group, respectively. Median follow up of surviving patients was 46 months. As expected, comorbidities and disease-specific factors varied between these groups. HCTCI scores > 3 points were observed in 14% of TBI pts compared to 32% in non-TBI pts ( p .01) and 45% versus 54% harbored adverse risk genetics according to European Leukemia Net (ELN) 2017 classification ( p .41), respectively. In the non-TBI group, 38% of the pts had a secondary or therapy-related AML compared to 20% of pts in the TBI group ( p .03). HLA-mismatched (<10/10 matched) donor grafts were transplanted in 21% (TBI) vs. 20% (non-TBI) of the pts. 185 of 188 pts received GCS-F mobilized stem cell grafts; 3 pts received a bone marrow graft. Kaplan-Meier estimates for overall survival (OS) with and without blast clearance at 2 years were 69.6% (95% CI 60.1-80.6%) vs. 58% (95% CI 48.8-69.1%, p .33), for event free survival (EFS, event defined as death or overt relapse) 58.9% (95% CI 49-70.8%) vs. 46.1% (95% CI 36.7-58.1%, p .2), respectively. We included the re-occurrence / persistence of defined molecular and cytogenetic markers or decreasing CD34 + bone marrow chimerism (< 95%) after transplant as measures for impending relapse as a modified EFS. The EFS including impending relapse was 49.8% (95% CI 40-62%) vs. 36.4% (27.7-47.8%) after 2 years. A non-significant trend with reduced relapse risk for those patients who achieved blast clearance after Melphalan ( p .1, HR 0.73) was observed. Most patients with impending relapse showed similar survival as patients without evidence of relapse after ASCT. In multivariate cox regression, age, HCTCI scores > 3 points, adverse risk according to ELN 2017 definitions and donor mismatch < 10/10 resulted in adverse prognosis. Especially, patients with mismatched donors displayed highly significant inferior outcomes. Both leukemic burden prior to ASCT as well as blast clearance after melphalan showed no significant correlation with clinical outcome parameters. Conclusion: Sequential conditioning regimens followed by ASCT offer long-term survival for more than 50% of pts with active relapsed or refractory AML. Patients who did not achieve complete blast clearance during conditioning therapy showed a trend towards higher relapse risk after ASCT which did not result in inferior survival. This underscores the importance of close monitoring and early interventions such as accelerated immunosuppression tapering, preemptive donor lymphocyte infusions and targeted therapies in case of impending relapse after ASCT.

Bath psoralen plus ultraviolet-A photochemotherapy for chronic graft-versus-host disease: a retrospective cohort study.

Chronic graft-versus-host disease is a severe complication of allogeneic stem cell and bone marrow transplantation. First-line immunosuppressive agents, such as steroids, are used to prevent this disease; however, they have multiple side effects. Therefore, bath psoralen plus ultraviolet-A (PUVA) is an alternative second-line treatment. This study aimed to evaluate the clinical efficacy of bath PUVA for managing chronic graft-versus-host disease. This retrospective, case-control study included 14 patients with extensive cutaneous chronic graft-versus-host disease, resistant to systemic corticosteroid, treated with bath PUVA. Major and partial responses were defined as clinical improvements of >70% and 50-70%, respectively. We analyzed the graft-versus-host disease clinical presentation and timing after allogeneic stem cell and bone marrow transplantation, bath PUVA doses, background diseases, additional treatments, and adverse effects. We observed eight major (three lichenoid and five sclerodermatoid) and six partial (three lichenoid and three sclerodermatoid) responses after a mean of 28 treatment sessions. After 6 to 25 months, four of the eight patients with sclerodermatoid lesions and all those with lichenoid lesions experienced relapse but responded to additional treatment cycles. Bath PUVA is well-tolerated and effective for extensive cutaneous chronic graft-versus-host disease. It allows rapid tapering of adjuvant immunosuppressants; however, most patients require prolonged maintenance phototherapy.

The Role of Allograft Nephrectomy in the Failing Kidney Transplant.

Patients with failed renal allografts have associated increased morbidity and mortality. The individualization of immunosuppression taper is the key element in managing these patients to avoid graft intolerance and sensitization while balancing the risk of continued immunosuppression. Most patients with uncomplicated chronic allograft failure do not require allograft nephrectomy (AN), and there is no clear evidence that it improves outcomes. The AN procedure is associated with variable morbidity and mortality. It is reserved mainly for early technical graft failure or in symptomatic cases associated with allograft infection, malignancy, or graft intolerance syndrome. It may also be considered in those who cannot tolerate immunosuppression and are at high risk for graft intolerance. AN has been associated with an increased risk of sensitization due to inflammatory response from surgery, immunosuppression withdrawal with allograft failure, and retained endovascular tissue. Although it is presumed that for-cause AN after transplant failure is associated with sensitization, it remains unclear whether elective AN in patients who remain on immunotherapy may prevent sensitization. The current practice of immunosuppression taper has not been shown to prevent sensitization or increase infection risk, but current literature is limited by selection bias and the absence of medication adherence data. We discuss the management of failed allografts based on retransplant candidacy, wait times, risk of graft intolerance syndrome, and immunosuppression side effects. Many unanswered questions remain, and future prospective randomized trials are needed to help guide evidence-based management.

Diagnosis and treatment of 26 cases of adenovirus infection after allogeneic hematopoietic stem cell transplantation

Objective: To analyze the clinical characteristics and prognosis of adenovirus infection after allogeneic hematopoietic stem cell transplantation. Methods: A total of 26 patients with adenovirus infection admitted to the posttransplant ward of the First Affiliated Hospital of Soochow University from 2018 to 2022 were enrolled. Their data on baseline and clinical characteristics, treatment, and follow-up were analyzed. Results: The median patient age was 30 (22, 44) years. Twenty-two patients received related haploid stem cell transplantation, three received unrelated stem cell transplantation, and one received umbilical cord stem cell transplantation. Antithymocyte globulin was included in the conditioning regimen in 25 patients. The median time of adenovirus infection was +95 (+44, +152) days. The median peripheral blood lymphocyte count was 0.30 (0.11, 0.69) × 10(9)/L. Twelve patients had acute graft-versus-host disease. Twenty-four patients received antirejection therapies at diagnosis. Sixteen cases had combined infection with other pathogens with adenovirus infection. Eight cases were diagnosed as asymptomatic infection, and 18 were diagnosed as adenovirus disease, including pneumonia (38.89% ) , gastrointestinal disease (38.89% ) , encephalitis (33.33% ) , hepatitis (5.56% ) , and urinary tract inflammation (5.56% ) . The age of >30 years was a risk factor for adenovirus disease (P=0.03) . Eighteen patients received tapering of immunosuppression, and all 26 patients received at least one antiviral drug. Other treatments included high-dose gamma globulin and donor lymphocyte infusion. Adenovirus infection improved in 10 cases and progressed in 16 cases. The median follow-up time was 30 (7, 237) days. Twenty-two patients died. The all-cause mortality rate was (88.5±7.1) % , and the attributable mortality rate was 45.5% . There was no significant difference in the 100 d survival rate between asymptomatic infected patients and patients diagnosed with adenovirus disease (37.5% vs 22.2% , HR=1.83, 95% CI 0.66-5.04, P=0.24) . Conclusion: The age of >30 years was a risk factor for adenovirus disease. Mortality was high in patients with adenovirus infection after allogeneic hematopoietic stem cell transplantation.

An 8-month-old boy with infantile nephrotic syndrome caused by semaphorin 3B-associated membranous nephropathy.

We present a case of nephrotic syndrome caused by semaphorin 3B-associated membranous nephropathy. The patient was an 8-month-old male infant who presented with severe proteinuria and hypertension. He was treated with prednisolone (PSL) for nephrotic syndrome; however, remission was not achieved within 4weeks. He was diagnosed with steroid-resistant nephrotic syndrome and underwent kidney biopsy. Pathological examination revealed membranous nephropathy with IgG deposits on both the glomerular basement membrane (GBM) and the tubular basement membrane (TBM). He was treated with cyclosporine (CsA) in addition to PSL and achieved complete remission. However, frequent relapses occurred after the discontinuation or tapering of immunosuppressants. Two years after treatment initiation, a second biopsy was performed and showed worsening of the disease, which required treatment with several immunosuppressants to achieve complete remission. After that, we performed additional immunostaining for semaphorin 3B, which confirmed the diagnosis of semaphorin 3B-associated membranous nephropathy. Although extremely rare in infantile cases, semaphorin 3B-associated membranous nephropathy should be considered in the differential diagnosis, as strong treatment with immunosuppressants might be needed. In addition, mycophenolate mofetil showed an effective clinical response in this case, indicating that it can be considered for future treatment strategies.

Immunosuppression Boost With Mycophenolate Mofetil for Mixed Chimerism in Thalassemia Transplants

Declining mixed chimerism (MC) portending impending graft failure is an undesirable outcome. However, for hemoglobinopathies in a stable state of MC, residual host cells persist without rejection in 30% to 40% of patients after hematopoietic stem cell transplantation (HSCT). Early detection and level of MC have been attributed to be significant in predicting the outcome of MC. Common clinical approach on MC is removal of immunosuppression. We retrospectively evaluated MC in transfusion dependent thalassemia patients who underwent HSCT in our institution between September 2013 and January 2022 to determine the outcome of MC on the basis of our approach of immunosuppression boost in comparison to conventional approach of immunosuppression tapering. Among 90 patients, 22 (24.4 %) had MC at some time point after transplantation with a median follow-up of 496 (67-1492) days. Immunosuppression withdrawal was done in 12 (54.5%) patients, whereas immunosuppression boost was given in 8 (36.3%) patients. In the immunosuppression withdrawal group, 2 (16.6%) patients evolved to complete chimerism, 5(41.6%) patients had persistent MC (PMC), whereas 5 (41.6%) patients had secondary rejection. All these rejections were at median of 186 (89-251) days after transplantation. In the immunosuppression boost group, all patients (n=8) had PMC with no secondary rejection until median follow-up of 255(97-812) days after transplantation. We acknowledge that we need more experience with our unconventional approach of immunosuppression boost to obtain statistical significance in comparison to the conventional approach of tapering of immunosuppression.

The use of transient elastography in identifying sub-clinical chronic ductopenic rejection in adult liver transplant recipients: A case series

BackgroundTransient elastography has become a standard tool for the accurate non-invasive assessment of liver stiffness and fat content. Liver transplant recipients can develop allograft fibrosis during long-term follow-up despite normal or mildly abnormal liver chemistries. Tapering of immunosuppression in long-term liver transplant survivors is performed relying solely on liver chemistries. It is important to know if underlying liver histology would be abnormal or if rejection was present as this would alter the desire to decrease stable maintenance doses of immunosuppression. MethodsWe present our experience of five liver transplant recipients who had transient elastography performed prior to consideration of weaning of their immunosuppression. ResultsAll five patients showed signs of elevated liver stiffness on transient elastography in the setting of normal to slightly abnormal liver tests with very stable immunosuppressant doses. This prompted the performance of liver biopsies which demonstrated immune-mediated liver injury and thus negated the immunosuppression withdrawal. ConclusionTransient elastography has utility as a non-invasive method to evaluate allograft health in long-term liver transplant survivors and can be useful in the decision-making process for immunosuppression weaning.

Critical Illness Immunosuppression: Ultra-Rapid Taper to Monotherapy

<h3>Purpose</h3> Immunosuppression (IS) is a critical component leading to the success of heart transplantation (HT). However, clinicians need to balance the risks of infection with the need for adequate rejection prophylaxis. Most programs maintain the highest levels of IS in the first 2-3 months following HT. With recent allocation changes which prioritize critically ill candidates, the acuity of such patients (pts) is rising. <h3>Methods</h3> At our institution, standard IS consists of tacrolimus (TAC), steroids and mycophenolate mofetil (MMF). Induction therapy is given for selected pts, particularly with severe hemodynamic instability or multiorgan dysfunction. For pts where the risk of rejection is outweighed by the risk of infection, we have established a critical illness IS protocol where MMF and steroids are stopped within 2-3 weeks of txp. Rejection was defined as ISHLT grade 2R or higher. Outcomes are described. <h3>Results</h3> From May '19 to July '21 there were 64 adult HT performed. Of these, 6 pts were given critical illness IS. The median recipient age was 63.5 years old (IQR 50.1-66.6), 2 were male, most dilated cardiomyopathy. Half were UNOS Status 1 pre-HT, one was status 3 and two were status 4 (stable ventricular assist device). 4/6 had severe primary graft dysfunction (PGD) following HT requiring ECMO and the others had severe coagulopathy and massive bleeding. 4 of 6 received at least one dose of induction antibody. Pts were weaned to TAC monotherapy over a mean of 18.2 +/- 4.4 days post-HT (range 13-25). The pts underwent 11.7 +/- 3.7 biopsies. One pt died of overwhelming sepsis at day 42 but the others are all alive with median follow-up of 282 days (IQR 77.2-568.2). No pt required resumption of corticosteroids or MMF. <h3>Conclusion</h3> Transplant IS is a delicate balance between the risk of severe rejection and the risk of infection. We describe ultra-fast tapering of IS in a cohort of mostly severe PGD recipients. It is feasible to treat highly selected pts with single-drug IS when the risk/benefit equation favors this approach.

Pulmonary Alveolar Proteinosis After Lung Transplantation

<h3>Introduction</h3> Secondary pulmonary alveolar proteinosis (PAP) is a minor subtype of the disease entity with poorer prognosis than common autoimmune PAP. It has been reported to complicate hematologic disorders, immunodeficiency, and infections. However, PAP associated with lung transplantation is not well recognized. Here, we report two cases of PAP after lung transplantation. <h3>Case Report</h3> Case 1: A four-year-old boy underwent a double-lung transplantation. He was maintained standard immunosuppressive regimen. The immediate post-operative course was uneventful. On post-operative day (POD) 23, chest computer tomography (CT) scan showed ground-glass opacities (GGOs) with interlobular septal thickening, along with worsening oxygenation. An acute rejection was clinically suspected, and the patient underwent steroid pulse therapy. It improved temporarily, but worsened again within a week. Despite the repeated steroid pulse, administration of anti-thymocyte globulin and inhibitors of the mammalian target of rapamycin, the situation gradually deteriorated and eventually complicated by infectious diseases. The patient died from respiratory failure on POD 248. At autopsy, diffuse intra-alveolar eosinophilic materials stained by surfactant apoprotein A immunohistochemistry were found and led to a diagnosis of PAP. Case 2: A 52-year-old man underwent double-lung transplantation. He was maintained standard immunosuppressive regimen. The post-operative course was uneventful. On POD 99, routine chest CT scan showed bilateral centrilobular ground-glass opacities mimicking rejection. A diagnostic trial of steroid increase was ineffective. Bronchoalveolar lavage fluid analysis and transbronchial biopsy demonstrated intra-alveolar eosinophilic materials which stained by periodic acid-Schiff. He was diagnosed with PAP and followed up with tapering immunosuppression in the standard manner, resulting in a clinical and radiological improvement. <h3>Summary</h3> PAP is a rare cause of graft dysfunction after lung transplantation. In case 1, the patient was misdiagnosed as acute rejection, and immunosuppression was intensified, which led to fatal consequences. PAP after lung transplantation is potentially a transient or slowly progressive pathology as seen in case 2. Transplant physicians should be aware of this rare complication.

Pharmacologic Strategies for Post-Transplant Maintenance in Acute Myeloid Leukemia: It Is Time to Consider!

Simple SummaryAllogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapeutic procedure for patients with high risk acute myeloid leukemia. Its anti-leukemic activity is mainly derived from the intensity of the conditioning regimen used and the graft-versus-leukemia effect exerted by the donor T lymphocytes. Despite major progress in the allo-HCT procedure in recent years with the achievement of lower non-relapse mortality and long-term disease control. However, up to 45% of patients still experience relapse. Therapeutic strategies post-transplant aiming at preventing disease relapse are discussed in this review.Patients with high-risk acute myeloid leukemia are offered allogeneic hematopoietic cell transplantation (allo-HCT) in first remission to reduce risk of relapse. However, disease recurrence remains the major reason of allo-HCT failure, occurring in around 35–45% of patients, and leading to dismal outcomes. Strategies to reduce the risk of relapse are greatly needed, especially in the early post-transplant phase where the graft-versus-leukemia (GVL) effect is not yet activated. Some practices include the use of myeloablative conditioning regimens, close monitoring of measurable residual disease and donor chimerism, rapid tapering of immunosuppression, and implementation of pre-emptive strategies as the use of donor lymphocyte infusion. However, it’s time to consider prophylactic pharmacologic interventions post allo-HCT that aim at maintaining leukemic clones under control by both direct cytotoxic activity and by enhancing the GVL effect. In this current review, available data on drugs targeting epigenetic pathways like azacitidine, or actionable mutations like FLT3 and IDH1/2 inhibitors used as maintenance post allo-HCT, will be discussed.

Hepatitic Variant of Graft-vs-Host Disease.

Graft-vs-host disease (GVHD) of the liver is a complication of allogeneic hematopoietic stem cell transplantation with hepatitic and classic variants. We determined the percentage of hepatitic variant cases, compared clinicopathologic features of the two groups, and assessed prognostic factors. Fifty liver biopsy specimens from 40 patients with GVHD were studied. Fifteen (30%) cases had moderate to marked lobular inflammation and were classified as a hepatitic variant. Bile duct damage was present in all cases. Ductular reaction, apoptosis. and endotheliitis were more commonly seen in the hepatitic variant. Hepatocyte ballooning was an independent poor prognostic factor. The median aspartate aminotransferase and alanine aminotransferase were higher in the hepatitic variant while alkaline phosphatase and bilirubin were higher in the classic group. Forty (80%) GVHD cases were more than 100 days after transplant, correlating to immunosuppression taper. There was response to treatment with increased immunosuppression in both groups, but time to normalization of liver function tests was higher in the hepatitic variant. Bile duct damage was the most consistent pathologic finding in our cohort and was present in all cases of GVHD. Moderate to marked lobular inflammation can be seen in GVHD in up to 30% of cases without any other coexisting cause. Hepatocyte ballooning is an independent poor prognostic factor.

Acquired Hemophilia A associated with bullous pemphigoid: A rare combination

Acquired Hemophilia A (AHA) is a rare autoimmune disorder, caused by the development of circulating autoantibodies against coagulation factor VIII (FVIII). AHA is associated with bullous pemphigoid in 2% of patients. We report a case of a 74-year-old man admitted with anemia and a tense subcutaneous and muscular hematoma in the right thigh. Blood analysis confirmed AHA. The patient had a recent diagnosis of bullous pemphigoid. Response to bypass agents and corticosteroids was good with bleeding control and normalization of FVIII and negative autoantibodies, respectively. In a 3-month follow-up period after tapering and stopping prednisolone, a relapse occurred, and immunosuppression was reinitiated. An early diagnosis and effective treatment in AHA are essential to reduce morbimortality. A careful tapering of immunosuppression is important to minimize FVIII inhibitor recurrence, as observed in this case.

Post-Transplant MRD Negativity on Day +100 Predicts Outcomes for Pre-Transplant Relapsed/Refractory AML Patients

IntroductionPatients with relapsed/refractory (r/r) AML have in general a dismal prognosis. Allogeneic stem cell transplantation (allo-SCT) provides a curative approach for these patients, however the overall survival (OS) remains still low achieving 20-40%. Though post-transplant minimal residual disease (MRD) monitoring has been shown to be predictive for development of post-transplant relapses and lower survival, data focusing on pre-transplant relapsed/refractory AML patients is scarce. In this study, we investigated the impact of achieving MRD negativity on day +100 for relapses and survival for this high risk patients.Patients and MethodsWe analyzed post-transplant outcomes for pre-transplant r/r AML patients depending on their post-transplant MRD status at day +100. The day +100 was chosen concerning the possibility of early post-transplant interventions (e.g. tapering of immunosuppression or administration of donor lymphocytes).Fifty six consecutive adult patients (≥18 years old) with r/r AML (median age 58, range 20-76; male, n=34, 61%) who underwent allo-SCT (first, n=44, 79%; second, n=12, 21%) between 2015-2020 at the Department for Stem Cell Transplantation at the University Medical Center Hamburg (Germany) were included. The MRD was assessed on day +100 using multiparameter flow cytometry according to “different from normal” strategy.The patients experienced rather primary refractory disease (64%), secondary/therapy-related AML (55%) and abnormal cytogenetics (59%) at diagnosis. The median pre-transplant blast count was 25% (6-91%). A number of 29 patients (52%) showed blasts in peripheral blood. Myeloablative conditioning was used in 31 (55%) patients, whereas 25 (45%) patients received reduced intensity regimens. A number of 29 patients (52%) received a FLAMSA-based conditioning. Post-transplant donor lymphocyte infusions as well as other treatment were given to 13 (23%) and 17 (30%) patients, respectively.ResultsThe median follow up was 20 months (range 4-66). Forty patients (71%) achieved MRD negativity on day +100 and 16 (29%) remained MRD positive. The 2-year OS, LFS, relapses and NRM at 2 years for day +100 MRD negative patients were: 76% (95% CI: 60-87%), 59% (95% CI: 41-75%), 31% (95% CI: 17-50%) and 8% (95% CI: 3-19%), respectively. The 2-year OS, LFS, relapses and NRM at 2 years for day +100 MRD positive patients were: 35% (95% CI: 17-59%, p=p=0.001), 23% (95% CI: 9-46%, p<0.0001), 70% (95% CI: 45-87%, p=0.0002) and 6% (95% CI: 1-28%, p=0.88), respectively.Several factors were evaluated for possible association with day +100 MRD negativity (Table 1). There were no significant associations. Further, the incidence of acute (grade II-IV) GvHD at 100 days was not significantly different between the day 100 MRD positive und negative patients.Following factors had impact on post-transplant outcomes in multivariate analysis: presence (no vs yes) of peripheral blasts prior to allograft (OS: HR 0.3, 95% CI: 0.1-0.9, p=0.03; LFS: HR 0.4, 95% CI: 0.1-0.9, p=0.03; relapses: HR 0.4, 95% CI: 0.1-0.99, p=0.048; NRM: HR 0.5, 95% CI: 0.2-1.3, p=0.17), FLAMSA vs other preparative regimens (OS: HR 0.4, 95% CI: 0.1-0.93, p=0.03; LFS: HR 0.4, 95% CI: 0.2-0.98, p=0.04; relapses: HR 0.4, 95% CI: 0.2-1.0, p=0.05; NRM: HR 0.4, 95% CI: 0.2-1.0, p=0.06), and day +100 MRD (negative vs positive) (OS: HR 0.3, 95% CI: 0.1-0.7, p=0.009; LFS: HR 0.2, 95% CI: 0.1-0.6, p=0.001; relapses: HR 0.2, 95% CI: 0.1-0.4, p=0.0001; NRM: HR 0.2, 95% CI: 0.1-0.5, p=0.0006).ConclusionsPost-transplant MRD detection plays predictive role in pre-transplant r/r AML patients and may help to define possible candidates for early post-transplant interventions. DisclosuresNo relevant conflicts of interest to declare.