Post-transplant Time Points Research Articles

Impact of induction agents and maintenance immunosuppression on torque teno virus loads and year-one complications after kidney transplantation

BackgroundTorque teno virus load (TTVL) is gaining importance as a surrogate parameter to assess immunocompetence in kidney transplant recipients. Although the dynamics of TTVL have been investigated before, the impact of different induction agents and variations in immunosuppressive maintenance therapies on TTVL remain unknown.MethodsIn this retrospective study, TTVL was quantified in 537 plasma or serum samples from 134 patients transplanted between 2018 and 2021. TTVL was examined pre-transplantation and 30-, 90-, 180-, and 360-days post-transplant. To assess the influence of induction therapy on TTVL, 67 patients receiving anti-thymocyte globulin (ATG) induction were matched with 67 patients receiving an interleukin-2 receptor antagonist (IL2-RA) induction in terms of age, sex, and donor modality.ResultsFollowing transplantation, there was a steep increase in TTVL post-transplant for all patients with peak viral loads at 90 days post-transplant (median TTVL [IQR] 7.97×106, [4.50×105–1.12×108]) followed by subsequently declining viral loads. Compared to patients receiving IL2-RA as induction therapy, patients receiving ATG had significantly higher peak viral loads 3 months post-transplant (median TTVL [IQR] 2.82×107 [3.93×106–1.30×108] vs. median TTVL [IQR] 2.40×106 [5.73×104–2.60×107]; P<0.001). Throughout all post-transplant time points, patients receiving additional rituximab for induction along with higher tacrolimus target levels exhibited the highest TTVL.Patients whose TTVL 3-months post-transplant exceeded the currently proposed cutoff to predict infections within the first year post-transplant [6.2 log10] showed a trend towards a higher risk of being hospitalized with an infection in the following 9 months, albeit without being statistically significant (HR=1.642, P=0.07).ConclusionsHigher TTVL reflects the greater immunosuppressive burden in immunological high-risk patients receiving intensive immunosuppression. The choice of induction agent and intensified immunosuppressive maintenance therapy notably affects TTVL at 3 months post-transplant and beyond, necessitating careful consideration when interpreting and applying TTVL cutoffs to monitor immunocompetence post-transplant.

Performance of risk prediction models for post-liver transplant patient and graft survival over time.

Given liver transplantation organ scarcity, selection of recipients and donors to maximize post-transplant benefit is paramount. Several scores predict post-transplant outcomes by isolating elements of donor and recipient risk, including the donor risk index, Balance of Risk, pre-allocation score to predict survival outcomes following liver transplantation/survival outcomes following liver transplantation (SOFT), improved donor-to-recipient allocation score for deceased donors only/improved donor-to-recipient allocation score for both deceased and living donors (ID2EAL-D/-DR), and survival benefit (SB) models. No studies have examined the performance of these models over time, which is critical in an ever-evolving transplant landscape. This was a retrospective cohort study of liver transplantation events in the UNOS database from 2002 to 2021. We used Cox regression to evaluate model discrimination (Harrell's C) and calibration (testing of calibration curves) for post-transplant patient and graft survival at specified post-transplant timepoints. Sub-analyses were performed in the modern transplant era (post-2014) and for key donor-recipient characteristics. A total of 112,357 transplants were included. The SB and SOFT scores had the highest discrimination for short-term patient and graft survival, including in the modern transplant era, where only the SB model had good discrimination (C ≥ 0.60) for all patient and graft outcome timepoints. However, these models had evidence of poor calibration at 3- and 5-year patient survival timepoints. The ID2EAL-DR score had lower discrimination but adequate calibration at all patient survival timepoints. In stratified analyses, SB and SOFT scores performed better in younger (< 40 y) and higher Model for End-Stage Liver Disease (≥ 25) patients. All prediction scores had declining discrimination over time, and scores relying on donor factors alone had poor performance. Although the SB and SOFT scores had the best overall performance, all models demonstrated declining performance over time. This underscores the importance of periodically updating and/or developing new prediction models to reflect the evolving transplant field. Scores relying on donor factors alone do not meaningfully inform post-transplant risk.

Patient-Reported Outcomes of BMT CTN 1703: A Randomized Phase III Study for Gvhd Prophylaxis - a Quality of Life Evaluation

Introduction A randomized phase III trial of post-transplantation cyclophosphamide (PTCy), tacrolimus, (Tac), and mycophenolate mofetil (MMF) versus tacrolimus/methotrexate (Tac/MTX) for graft-versus-host disease (GVHD) prophylaxis showed superior graft-versus-host disease-free, relapse-free survival (GRFS) in the PTCy-containing arm (NEJM, 2023). Here, we report the effect of GVHD prophylaxis on patient-reported outcomes and impact on quality of life (QOL). Methods The QOL study component of BMT CTN 1703 employed four primary endpoints: Lee Chronic GVHD Symptom Score and the PROMIS subscales of physical function, gastrointestinal symptoms, and satisfaction with participation in social roles. Secondary endpoints included hemorrhagic cystitis symptoms and Lee symptom subscale differences, with questionnaires to English and Spanish-speaking participants at enrollment and on Days 100, 180, and 365 post-transplant. Given the risk of missing assessments, an inverse probability weighted-independent estimating equations (IPW-IEE) model was applied for score comparison between arms, adjusting for baseline score, assessment time, age, donor type, disease risk index (DRI), planned conditioning regimen, and planned use of post-transplant maintenance therapy. Significance was declared at a 1.25% level for score comparisons between arms after Bonferroni correction for multiple testing. The analysis approach classified patients according to the intention-to-treat principle. Results QOL survey completion rates for patients at each timepoint were similar, exceeding 70% in each study arm. With the Lee Chronic GVHD Symptom Scale, higher scores indicate more severe symptoms. The IPW-IEE models indicated that the Lee Chronic GVHD Symptom Scale total scores were significantly lower in the PTCy arm compared to Tac/MTX at day 100 (mean difference [MD] -2.22, 99% confidence interval [CI] -5.05 to 0.61) and day 365 (MD -1.84, 99% CI -5.13 to 1.45, p=0.01 for overall treatment effect across all timepoints, Figure 1). Nutrition subscores were significantly better with PTCy at day 100 (MD -2.41, 99% CI -5.06 to 0.24, p=0.0014). Mouth scores were significantly better in the PTCy arm at day 365 (MD -7.09, 99% CI -13.21 to -0.98, p=0.0035). Patients over 65 years of age had better psychological subscores across the study population (MD -4.80, 99% CI -8.91 to -0.69, p=0.0026); no interaction was found between age and study treatment. Patient-reported hemorrhagic cystitis symptoms showed no significant difference between the study arms at any post-transplant time point. There was no significant treatment effect observed on the 3 PROMIS subscales. Across the entire study population, both relapse and acute GVHD were associated with worse scores on the Lee Symptom Scale, and PROMIS physical function, gastrointestinal symptoms, and social functions subscales. Chronic GVHD was associated with worse scores on the Lee Symptom Scale only, not the PROMIS subscales. No associations were found between patient-reported QOL at baseline and 1-year GRFS, disease-free survival, or overall survival. Conclusions The PTCy arm of BMT CTN 1703 showed superior GRFS, and this study confirms better patient-reported outcomes with PTCy. Patients who received PTCy showed better nutrition and mouth subscores on the Lee Chronic GVHD Symptom Scale compared to those who received Tac/MTX, possibly due to lower observed rates of severe acute and chronic GVHD in the PTCy arm. No association was found between patient-reported baseline quality of life and survival endpoints, suggesting the necessity of further research to elucidate the interplay of treatment strategies, patient factors, and their impact on long-term outcomes.

Using Mass Spectrometry to Assess Disease Status in Treated Multiple Myeloma Patients

Introduction As multiple myeloma (MM) is a bone-marrow based disease, the majority of MRD studies have focused on sensitive bone-marrow based assays. However, to effectively monitor patients this would require sequential invasive procedures at semi-frequent intervals. Sensitive, non-invasive, blood-based assays which can detect the presence of MRD are needed to optimize the management of MM patients. Mass spectrometry (MS) is currently being investigated for its clinical utility at different points in the monoclonal gammopathy pathway, from screening to MRD detection. This study aimed to compare the sensitivity and prognostic utility of MS with other blood-based M-protein detection methods and bone marrow flow cytometry at active disease, best response and MRD timepoints. Methods MS analysis took place at The Binding Site laboratory (The Binding Site, UK) using a pre-commercial quantitative immunoprecipitation (QIP-MS) system. Mass spectra was acquired over an m/z range of 10,000 to 30,000 and analysis was performed by two independent readers. Both transplant eligible and non-transplant chemotherapy only patients were recruited at varying stages of disease and treatments. Median follow up was 18 months. A total of 190 serum samples from 87 individual patients were tested for the presence of M-protein by conventional methods (serum electrophoresis (SPEP), immunofixation (IFE), free light chain (FLC)) and MS. A bone marrow flow cytometry (NGF) assay was performed up to a sensitivity of 10 -6 at pre- and post-transplant for a subset of patients. Disease response was determined according to the IMWG criteria. Results and Discussion When compared to conventional techniques MS showed increased sensitivity over SPEP, IFE and FLC (Table 1). The 1 M-protein detected by SPEP and IFE but not by MS was a post-transplant sample which showed multiple oligoclonal peaks by MS where the original M-protein could not be determined due to not having a diagnostic MS result available. When evaluating the 37 samples from 19 individual patients which were MS positive and IFE negative it was found that 7/19 (37%) patients in this group relapsed during the course of the study. As SPEP, IFE and FLC analyses are often performed together, the utility of using MS alone was compared to an algorithm of SPEP/IFE/FLC tests, where if any of the conventional tests showed abnormality this was considered positive. Using this scenario, MS outperforms the conventional tests in the 190 samples evaluated - with 7 samples being MS positive only and 1 sample (discussed above) being negative by MS but positive by all SPEP/IFE/FLC. As expected, upon analysing the percentage of samples within each IMWG clinical response group which had a positive or negative MS results the percentage of samples with a negative MS result increased with greater depth of response whilst those in partial response or worse had no negative MS samples. However, even in the deepest response group, complete response, 77% of samples were MS positive, showing how MS outperforms the IMWG criteria. Progression free survival (PFS) analysis at the best response time point showed a significant survival advantage of having a MS negative result (p=0.007). When comparing MS against NGF results at bone marrow testing time points agreement at pre-transplant was 88%, with all samples being NGF positive but 22/25 being MS positive and 1 of these 3 discordant patients relapsing during the study. At post-transplant agreement was only 56%. However, evaluating PFS at the post-transplant time point showed that MS status had a significant effect on survival (p=0.02) (Figure 1) where IFE (p=0.44) did not and NGF only approached significance (p=0.051). Conclusion This study aimed to evaluate the viability of using MS to identify M-proteins in treated MM patients. In this study the MS method had a higher number of positive results than any of the conventional methods, and outcompeted the algorithm of SPEP/IFE/FLC in detecting the presence of M-protein. This study has shown a survival benefit of being MS negative at both post-ASCT and best response time points across a range of treatments. There were a high number of patients who were MS positive throughout monitoring and it will be important to determine how such a sensitive method can be used to provide clinically useful information to clinicians to give the best balance between treatment and quality of life for patients.

Non-Invasive MRD Monitoring in Multiple Myeloma Patients By Heavy/Light Chain Analysis

Introduction The majority of multiple myeloma (MM) patients are now achieving very deep responses. The most sensitive disease detection techniques are currently bone marrow assays. However, as bone marrow sampling is invasive and unpleasant for patients it is important to investigate whether blood-based assays could be equally or more informative of disease status. The heavy/light chain assay (Hevylite TM, The Binding Site, UK) evaluates serum heavy/light chains (HLC) for IgG, IgA and IgM M-proteins. This study assessed whether the HLC assay could act as an effective marker for disease in the bone marrow of treated MM patients and evaluated the prognostic utility of HLC measurements at best response and relapse time points. Methods 104 IgG and IgA MM patients were recruited either prior to transplant or during chemotherapy. Median follow up was 18 months. Routine M-protein tests were performed alongside HLC testing with parameters including involved HLC (iHLC), uninvolved HLC (uHLC) and kappa/lambda HLC ratio (HLCr). A bone marrow flow cytometry (NGF) assay was performed up to a sensitivity of 10 -6 and was considered to be the gold standard assay. Disease response was determined according to the IMWG criteria. Results and Discussion At pre- and post-transplant time points there was significant agreement between HLC measurements and NGF, with uHLC being the most sensitive measurement (86%) and HLCr the most specific (100%). Both uHLC and HLCr show substantial agreement with the bone marrow NGF method whilst iHLC cannot be used to indicate bone marrow status. Survival analysis showed significantly inferior progression free survival (PFS) in patients with an abnormal uHLC (p=0.01) (Figure A) or HLCr (p=0.02) result post-transplant but no survival difference based on iHLC status (p=0.78) (Figure B). Interestingly, performing survival analysis on these same samples using serum immunofixation (IFE) status (positive or negative) did not produce a significant PFS difference between participants (p=0.21). During long term follow up, HLC values showed a correlation with depth of response, with uHLC being abnormal in 60% of complete response (CR) patients. This suggests that those participants currently grouped as CR may be able to be further subdivided according to uHLC level and this could have a prognostic value in the monitoring and treatment of these patients. At best response uHLC was the only HLC or free light chain (FLC) measurement which gave a significant difference in PFS for all patients in partial response (PR) or better (p=0.005), remaining true when analysing patients in ≥CR only (p=0.002). The addition of free light chain measurements into the HLC analysis did not improve the significance of these survival differences. In relapsed patients, paired comparisons of M-protein and disease activity markers at best response vs. prior to clinical relapse were performed. uHLC was the only marker which was significantly different between the two time points in IgG and IgA patients. The significance of this was confirmed by showing that those patients in stable disease did not experience a significant change in uHLC levels. For other M-protein variables such as total immunoglobulin, IFE status and FLC values there was an increase in levels or abnormality between best response and pre-relapse but these were not significant. The paired comparison suggests that monitoring an individual using HLC could help to detect relapse earlier than conventional methods. In all analyses performed the HLC assay showed increased sensitivity and utility over the IFE technique. Conclusion This study suggests that the HLC assay can be used prior to bone marrow analysis to help influence decisions and improve the patient and laboratory experience. These results can be put into practice during patient follow up post initial treatment in the following ways: At deepest response an abnormal uHLC or HLCr suggests a positive MRD status, meaning a possible reduction or delay in the requirement for bone marrow assessment.All patients in ≥VGPR should have HLC performed alongside conventional assays during follow up.If uHLC values become abnormal during follow up then it should be assumed that the patient is about to undergo relapse. The measurement of HLC values have been shown to be a significant marker in this study, for sensitively detecting the M-protein, giving further clarification of response status and predicting early relapse.

Tagraxofusp in Combination with Azacitidine and Venetoclax in Newly Diagnosed CD123+ Acute Myeloid Leukemia, Expansion Cohort of a Phase 1b Multicenter Trial

Background: CD123 is a subunit of the interleukin 3 (IL3) receptor expressed on the surface of blasts in most acute myeloid leukemias (AMLs). Tagraxofusp (TAG), a CD123-directed therapy, is a recombinant protein drug consisting of IL3 fused to a truncated diphtheria toxin payload approved for treatment of blastic plasmacytoid dendritic cell neoplasm. We found TAG resistance in AML cells was mediated by DNA methylation and silencing of diphthamide genes (e.g., DPH1), which causes resistance to diphtheria toxin (Togami, JCI 2019). The hypomethylating agent azacitidine (AZA) reversed TAG resistance, and TAG plus AZA prolonged survival vs. either alone in xenograft models. TAG-exposed AML cells were also sensitized to the BCL2 inhibitor venetoclax (VEN). We performed a phase 1b trial combining these agents in myeloid malignancies and reported acceptable safety in dose escalation of TAG with AZA-VEN (ASH 2021). Here, we report an expansion cohort in newly diagnosed AML treated with TAG-AZA-VEN. Methods: Patients (pts) were adults with AML ineligible or declining intensive induction chemotherapy. CD123+ blasts were required (tested locally by flow or IHC, any level). Eligibility included albumin &amp;gt;3.2 g/dL and normal cardiac ejection fraction. Pts were treated on 28-day cycles with AZA 75 mg/m2 d1-7, VEN 400 mg d1-21 with ramp up in cycle 1, and TAG 12 µg/kg d4-6 ( Fig A), with cycle 1 hospitalization to mitigate risks of capillary leak syndrome (CLS). Also included are 3 pts who received TAG 7 µg/kg (n=2) or 9 µg/kg (n=1) in escalation. TAG was given on d4 to avoid VEN ramp up overlap and because preclinical data showed AZA pretreatment can sensitize AML cells to TAG. Results: 26 pts with AML were treated; median age 71 (range 60-81). All were adverse risk per ELN 2022 criteria, including 13 (50%) with a mutation in TP53 and others with 1 or more of ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, or U2AF1. Eight (31%) had complex karyotype, 8 (31%) secondary AML, and 5 (19%) therapy-related AML. Adverse events (AEs) were like those seen with TAG or AZA/VEN, without evidence of exacerbation by combination. Grade 3+ AEs occurring in &amp;gt;15% of pts regardless of attribution included thrombocytopenia (54%), leukopenia (54%), febrile neutropenia (35%), and anemia (31%). CLS, a known TAG side effect, occurred in 19% (n=5; three grade 2, one gr 3, one gr 4; all in cycle 1) and was manageable by albumin supplementation and diuresis. Mortality at 30 days was 11.5%, with causes of death of sepsis (n=2), multiorgan system failure (n=1). 18 of 26 pts (69%) achieved a best response of CR (10; 39%), CRi (5; 19%), or MLFS (3; 12%). Of the 13 with TP53 mutation, 7 (54%) achieved CR/CRi/MLFS (CR=4, CRi=2, or MLFS=1). Bone marrow blasts decreased in all pts who had a biopsy after starting treatment ( Fig A). For the 18 achieving CR/CRi/MLFS, the median duration of response was 12.4 months (mo) (95% CI, 6.1-NA). 13 of 26 (50%) proceeded to allogeneic stem cell transplantation, which included 6/13 (46%) with TP53 mutation. The median number of cycles was 3 for both pts who did (range 1-15) and did not (range 2-4) proceed to transplant. The median follow-up was 10.7 mo, including post-transplant timepoints. Median overall survival (OS) was 14 mo (95% CI, 9.5-NA) and progression-free survival (PFS) was 8.5 mo (95% CI, 5.1-NA). In pts with TP53 mutation, median OS was 9.5 mo (95% CI, 1.8-NA); and not reached (NR) without ( Fig B). PFS for pts with TP53 mutation was 5.1 mo (95% CI, 1.8-NA), and 13.3 mo (95% CI, 8.6-NA) without. Measurable residual disease (MRD) was assessed by flow cytometry in 17 pts who achieved CR/CRi/MLFS; 12/17 (71%) were MRD negative (&amp;lt;0.1%). 4/7 (57%) with TP53 mutation achieving CR/CRi/MLFS were MRD negative. Median OS for pts who became MRD negative was NR. Median OS for the 13 pts who received allogeneic transplant was 18.2 mo (95% CI, 14.0-NA). Median PFS for the transplanted pts was 13.3 mo (95% CI, 8.2-NA). Conclusions: Treatment of AML with triplet TAG-AZA-VEN is feasible and shows encouraging activity as up-front therapy in adverse risk AML, including in pts with TP53 mutation. These data (OS 14 mo; TP53 mutant OS 9.5 mo) compare favorably to HMA/VEN in ELN adverse risk (OS 12 mo; Lachowiez, BloodAdv 2023) or TP53 mutant AML with poor risk cytogenetics (OS 5.2 mo; Pollyea, CCR 2022), albeit with some differences between populations. No new safety signals were observed. Further development of this combination is warranted, including directly compared to AZA-VEN.

Time of Origin and Environmental Drivers of Leukemia Immune Escape after Transplantation

CONCLUSIONS One of the key questions regarding cancer therapies is whether treatment-resistant subclones pre-exist therapy or originate at a later time-point from residual cancer cells that were kept under control until that time. Here, we addressed this issue in HLA loss leukemia post-transplantation relapses, representing one of the best characterized mechanisms of selective immune escape from the adoptive immune therapy of allogeneic hematopoietic cell transplantation (allo-HCT) ( Vago, NEJM, 2009). Serial purified acute myeloid leukemia (AML) samples from 11 different patients, collected before allo-HCT and at the time of the post-transplant HLA loss relapse, were subjected to high-depth (average 100x) whole genome sequencing (WGS). Patient and donor germline controls were obtained through in vitro expansion of T cells collected from patients before and after allo-HCT, respectively, and sequenced at an average 30x depth. Starting from the notion that mutations can be employed as molecular clocks to date the age of cells ( Alexandrov, Nature Genetics, 2015) and that mutational signatures recapitulate their exposure to mutagens ( Alexandrov, Nature, 2020), we combined WGS with Poisson-based modelling to perform subclonal deconvolution ( Caravagna, Bioinformatics, 2020), and derive the timing of the HLA loss rearrangement, intersecting results with the patient clinical history. The AML cell line OCI-AML3 was employed for validation experiments, assessing in vitro the ability of selected compounds to induce DNA damage (quantifying ϒH2Ax through immunofluorescence and flow cytometry) and specific mutational signatures (by the WGS of single cell clones, performed at an average depth of sequencing of 30x). By WGS data of longitudinal AML samples we successfully reconstructed the clonal dynamics leading to 12 HLA loss relapses, and in each subclone deconvolved mutational signatures. By applying Poisson-based timing models to homozygous (i.e. occurred before the copy neutral loss of hetherozygosity - cn-LOH - rearrangement) and heterozygous (i.e. occurred after the event) mutations in the rearranged region, in 10/12 HLA loss relapses we confidently dated the event to a post-transplant time-point (Fig. 1A). In all HLA loss subclones we could detect a C&amp;gt;A mutational signature that was absent in pre-transplant samples (Fig. 1B): combining literature data ( Kanter, Cell Stem Cell, 2021) and in in vitro experiments on the OCI-AML3 cell line, we reconducted this signature to the antiviral drug ganciclovir (GCV). By in vitro experiments, we showed that GCV induces DNA damage and homologous recombination repair in AML, an effect that could be possibly potentiated by the concomitant exposure to the immunosuppressant mycophenolate ( Fang, Genome Medicine, 2022). Concordantly, by retrospective analysis of 126 AML relapses after a family haploidentical transplantation occurred at our Institute between August 2002 and December 2020, we observed that the incidence of HLA loss was 41/95 (43.1%) in patients exposed to GCV and 5/31 (16.1%) in unexposed ones (OR: 3.94, 95%CI 1.39 to 11.16, p=0.009). Our data indicate that in the majority of patients HLA loss variants originate after transplant, and that the genotoxicity of ganciclovir is a potent facilitator of the phenomenon. Defining the time of origin of therapy resistance has fundamental clinical consequences, implying that immune escape clones can only be targeted by post-transplant interventions, and warranting the development of new tools to anticipate the rapid detection of relapse.

Time-dependent variations in BK polyomavirus genome from kidney transplant recipients with persistent viremia

BK polyomavirus (BKPyV) is a human DNA virus that resides latent in the host’s renal tissue. Reactivation occurs occasionally and in case of kidney transplantation, it can lead to polyomavirus-associated nephropathy (PVAN). Due to the lack of specific antivirals for BKPyV and despite the risk of allograft rejection, reduction of immunosuppression remains the main approach for treating PVAN. Current data suggests that mutations can accumulate over time in the major capsid protein VP1 and can lead to neutralization escape in kidney transplant recipients. Herein, we show that mutations occur throughout the entire BKPyV genome, including in VP1. Changes were identified by per-patient comparison of viral genome sequences obtained in samples from 32 kidney recipients with persistent viremia collected at different post-transplant time-points. Amino acid changes were observed in both earlier and later post-transplant samples, although some of them were only found in later samples. Changes in VP1 mainly consisted in the introduction of a new amino acid. A switch back to the conservative amino acid was also observed. This should be considered in future approaches for treating BKPyV infection in kidney transplant recipients.

Rapid Immune Reconstitution and Elevated Regulatory T Cell Frequencies in Patients Treated with Orca-T

BACKGROUND Immune reconstitution following myeloablative allogeneic hematopoietic stem cell transplant (MA-alloHSCT) is significantly delayed for T cell depleted allografts when compared to T-cell replete allografts, a feature that has been implicated in higher rates and grades of infection, less GVT, and worse overall survival. Orca-T is a high precision cell therapy currently being investigated for the treatment of certain hematological malignancies otherwise treated with MA-alloHSCT. The cellular drug products of Orca-T (HSPCs, Tregs, and Tcons) are administered at high purity, in controlled doses, and on an established schedule with the intent to reconstitute the blood and immune system while controlling GVHD. Here, we present data on the immune reconstitution in 100 adult patients who received Orca-T. METHODS In the context of an ongoing multicenter Phase Ib clinical trial of Orca-T in recipients with hematologic malignancies (NCT04013685), we performed longitudinal measurements (days -28, 28, 56, 100, 180, and 365 post-transplant) of immune reconstitution in the first 100 consecutive patients. With fresh whole blood, clinical 5-part leukocyte differentials were performed at clinical sites, and lymphocyte subset frequencies were measured by flow cytometry in a central lab. Principal component analysis (PCA) was performed to investigate potential associations with recipient sex (male vs. female) and donor relation (related vs. unrelated). RESULTS Longitudinal peripheral blood counts of platelets, WBCs, neutrophils, lymphocytes, monocytes, T cells, B cells, and NK cells are presented in Table 1. T cell and B cell counts were readily observed at days 28 and 56 respectively, and increased with each subsequent time point. Median NK cell levels were observed to be in the normal range at all post-transplant time points. CD4+ T cell and Treg cell counts exhibited similar post-transplant patterns with both being appreciably present at d28 and increasing with each subsequent time point. Strikingly, relative to the level measured in 75 corresponding PBSC donors, the Treg frequency among CD4+ T cells was significantly elevated at all time points post-transplant (Figure 1). Median CD8+ T cell counts increased for the first 6 months post-transplant and then plateaued in the normal range. Upon PCA, very few significant differences were observed in 2-group comparisons of recipient sex and donor relation. CONCLUSIONS Orca-T patients exhibit early immune reconstitution of each of the major leukocyte and lymphocyte subsets hypothesized to control relapse and infection. Concomitantly, elevated Treg frequencies were also observed. This feature of immune reconstitution profiles of Orca-T recipients may be correlated to the reduced occurrence and severity of acute and chronic GVHD in these patients (Meyer et al., EHA 2022, #S237). Similar immune reconstitution profiles were observed in patients of disparate sex and regardless of donor relation. Prospective comparisons of immune reconstitution between Orca-T and standard-of-care patients will be performed in our ongoing phase 3 clinical trial (NCT05316701). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Visual evoked potentials as a method for the prospective assessment of tacrolimus neurotoxicity in patients after kidney transplantation

IntroductionNeurotoxicity, including optic nerve injury, is one of the most common adverse effects of tacrolimus, the principal calcineurin inhibitor used after kidney transplantation (KTx). The electrophysiologic measurements of both pattern visual evoked potentials (PVEP) and flash visual evoked potentials (FVEP) are valuable when drug-induced optic neuropathy is suspected.ObjectivesTo determine whether VEP measurement is a sensitive and repeatable method for monitoring tacrolimus neurotoxicity.Material and methodsThis prospective study focused on 35 patients (20 M, 15F, 69 eyes, mean age 43 ± 11 years) who were at a median of 3.0 (IQR, 2.2–3.7) months after KTx at the time of the initial VEP evaluation and were treated with tacrolimus since KTx. The follow-up VEP examination was done after a median of 24 (22–27) months (both VEP measurements followed the ISCEV standards). The P100 wave latency and amplitude for the 1° and 15’ PVEP simulations, and the P2 wave latency and amplitude for the FVEP were analyzed.ResultsFor the 1° checks, the P100 wave latency and amplitude values were significantly worse in the follow-up examination compared to the early post-transplant time-point. Independent associations between FVEP parameters and the tacrolimus blood trough level were observed in the follow-up examination but not at the early post-transplant period. The P2 wave latency correlated with the tacrolimus trough level only in patients treated with the twice-daily, but not the once-daily, tacrolimus formulation. The brain derived neurotrophic factor (BDNF) level correlated with the P100 (15’) latency (R = 0.499; p = 0.005) and the P2 latency (R = 0.409; p = 0.025) only in patients treated with the once-daily, but not the twice-daily, tacrolimus formulation.ConclusionThe observations in this study may support the rationale for the use of VEP measurements as non-invasive monitoring of subclinical tacrolimus neurotoxicity.

Prognostic role of serum cytokines and soluble HLA-G levels in children with leukemia who undergo allogeneic stem cell transplantation

This is the first study to have investigated the prognostic role of cytokines and soluble human leukocyte antigen-G (sHLA-G) levels in pediatric leukemia patients who have undergone allogeneic stem cell transplantation (allo-SCT). Forty-one patients with acute leukemia (n = 28, acute lymphoblastic leukemia (ALL) and n = 13, acute myeloblastic leukemia) were recruited. Patients were examined at diagnosis (n = 26), in the pre-transplantation period (PreTx) (n = 26), on the day of transplantation (Tx0) (n = 41), and on post-transplantation Days 14 (PostTx14) (n = 41) and 28 (PostTx28) (n = 41). Serum levels of pro-inflammatory cytokines (interleukin [IL]-1, IL-2, IL-6, Tumor necrosis factor [TNF]-α), anti-inflammatory cytokines (IL-4, IL-10), and sHLA-G were measured by Enzyme-Linked ImmunoSorbent Assay. Median levels of all cytokines tested and sHLA-G were significantly higher at diagnosis and at the post-transplant time points than at PreTx (all p < 0.05). At the time of diagnosis (specifically ALL) and at PostTx14, elevated IL-4, IL-10, and/or sHLA-G were associated with higher post-transplant relapse rates (all p < 0.05). Elevated IL-2 and TNF-α at Tx0 were associated with lower survival rates (both p < 0.05). Levels of serum cytokines and sHLA-G may be useful predictors of survival and relapse in pediatric leukemia patients who undergo allo-SCT.

Adiponectin is unrelated to kidney function or injury markers in renal transplant recipients: A one-year follow-up study

BackgroundAdiponectin (ADPN) is a biologically active cytokine produced by adipose tissue. This protein exhibits anti-inflammatory, antioxidant, antifibrotic, and insulin-sensitizing properties. As ADPN is primarily eliminated by the kidneys, it is a potential biomarker of chronic kidney disease progression. This study aimed to analyze the fluctuations in ADPN levels after kidney transplantation during a one-year follow-up and to compare them to significant renal (eGFR, NGAL) and metabolic (insulin, glucose, lipids, HOMA-IR) markers. MethodsInsulin, ADPN, NGAL, and basic biochemical parameters were evaluated in 51 healthy controls and 39 patients right before kidney transplantation and at five time points following transplantation (5–7 days, one month, three months, six months, and twelve months). ResultsMean ADPN levels dropped significantly right after transplantation (from 35.449 to 30.920 μg/mL, p = 0.001) and decreased gradually over a year. From the third month after the transplantation, ADPN levels were comparable to healthy individuals. At the pre-transplant time point, ADPN correlated only with insulin (r = −0.60, p < 0.001) and HOMA-IR (r = −0.55, p < 0.001). At the timepoints after transplantation, ADPN correlated only with NGAL at three months (r = −0.70, p = 0.048). The correlation of ADPN with HOMA-IR found at pre-transplant was not significant at any post-transplant time point, but at one and three months after transplant, the correlations reached a borderline significance (p = 0.07 and p = 0.08, respectively). ConclusionsSuccessful kidney transplantation is followed by a gradual and significant ADPN decrease. In pre- and post-transplant patients, ADPN is unrelated to kidney function defined by GFR, but to glucose metabolism. Most of the analyzed metabolic and kidney parameters, apart from NGAL, stabilize within three months after transplantation.

The Positive Impact of Donor Bone Marrow Cells Transplantation into Immunoprivileged Compartments on the Survival of Vascularized Skin Allografts

Using the vascularized skin allograft (VSA) model, we compared the tolerogenic effects of different allogeneic bone marrow transplantation (BMT) delivery routes into immunoprivileged compartments under a 7-day protocol immunosuppressive therapy. Twenty-eight fully MHC mismatched VSA transplants were performed between ACI (RT1a) donors and Lewis (RT11) recipients in four groups of seven animals each, under a 7-day protocol of alfa/beta TCRmAb/CsA (alpha/beta-TCR monoclonal antibodies/Cyclosporine A therapy). Donor bone marrow cells (BMC) (100 × 106 cells) were injected into three different immunoprivileged compartments: Group 1: Control, without cellular supportive therapy, Group 2: Intracapsular BMT, Group 3: Intragonadal BMT, Group 4: Intrathecal BMT. In Group 2, BMC were transplanted under the kidney capsule. In Group 3, BMC were transplanted into the right testis between tunica albuginea and seminiferous tubules, and in Group 4, cells were injected intrathecally. The assessment included: skin evaluation for signs and grade of rejection and immunohistochemistry for donor cells engraftment into host lymphoid compartments. Donor-specific chimerism for MHC class I (RT1a) antigens and the presence of CD4+/CD25+ T cells were assessed in the peripheral blood of recipients. The most extended allograft survival, 50–78 days, was observed in Group 4 after intrathecal BMT. The T cells CD4+/CD25+ in the peripheral blood were higher after intrathecal BMC injection than other experimental groups at each post-transplant time point. Transplantation of BMC into immunoprivileged compartments delayed rejection of fully mismatched VSA and induction of robust, donor-specific chimerism.

Impact of antibody induction on the outcomes of new onset diabetes after kidney transplantation: a registry analysis.

We conducted this observational study to examine the impact of antibody inductions administered at kidney transplant (KT) on outcomes of 5year exposure to post-transplant diabetes (PTDM) in adult deceased-donor kidney transplant recipients (DDKTRs). We also studied the risk of PTDM associated with antibody inductions. Using 2000-2016 Organ Procurement Transplantation Network data, we employed multivariable Cox models to determine the adjusted hazard ratios (HR) of death, and overall and death-censored graft loss (OAGL, DCGL; respectively) at the 5year landmark period in antibody induction cohorts with and without PTDM at the 1year post-transplant index time point. We used multivariable logistic regression in determining the risk factors for PTDM. All multivariable analyses were adjusted for the potential confounding effects of maintenance immunosuppression, steroid regimens, and other relevant covariates. 48,031 adult DDKTRs were classified into cohorts based on antibody induction at transplant: (anti-thymocyte globulin) ATG (n = 26, 788); (alemtuzumab) ALM (n = 5916); and interleukin-2 receptor antagonist (IL-2RA) (n = 15,327). PTDM was a risk factor for 5year OAGL and death, not DCGL [(HR = 1.25, CI = 1.16-1.36), (HR = 1.13, CI = 1.06-1.21), and (HR = 1.05, CI = 0.96-1.16); respectively]. Induction regimens were not risk factors for 5year outcomes in DDKTRs with and without PTDM. Risk factors for PTDM included DDKTR obesity, age > / = 50years, acute rejection, and ATG induction, among others. In adult DDKTRs, after controlling the confounding effects of clinically relevant variables including maintenance and steroid regimens, PTDM at 1year post-transplant is associated with death and OAGL, not DCGL in the following 5 years: induction received at KT did not modify these associations.

Accelerated T Cell Senescence after Allogeneic Hematopoeitic Stem Cell Transplantation

Long-term survivors of allogeneic hematopoietic stem cell transplantation (HSCT) remain susceptible to opportunistic infections, relapse of primary malignancy and development of secondary malignancy, features that are consistent with continued immune deficiency. Reconstitution of T cell populations following HSCT and the preceding conditioning regimens occurs via two main routes: a thymic-dependent generation of naive T cells from progenitors via thymopoeisis, and a thymic-independent peripheral expansion of adoptively transferred, mature T cells in the graft. We hypothesized that dependence on peripheral expansion for reconstitution in individuals over forty years old leads to T cell populations that are prematurely senescent. In this retrospective study of patients who underwent matched, related allogeneic transplantation, we compared PBMCS from a cohort of long-term (4-5 years) survivors of transplant and their original donor infusion products (n=13), evaluating the expession of markers of immunosenescence and aging (CD28, KLRG1, CD57 and PD-1) at each time point. Using multi-parameter flow cytometry, we determined that CD8 cells expressed significantly higher levels of markers of T cell terminal differentiation and senescence post-transplant. Both the frequency and absolute number of Effector Memory and TEMRA, as well as the incidence of cells expressing senescence markers increased. Although there was no significant change in frequency of CD8+ T-cells actively proliferating (% Ki-67+), TREC analysis revealed that the frequency of recent thymic emigrants in the peripheral T-cell pool was reduced long-term. Additionally, we assessed the telomere lengths of CD8+ T-cells using Flow-FISH and, in line with our hypothesis that reliance on peripheral expansion results in replicative senescence, found that they were significantly shorter post-transplant. In conclusion, our study suggests that a contributory factor underlying the disposition for opportunistic infections in long-term survivors of transplant may be premature T cell dysfunction resulting from immune reconstitution-related senescence.Figure Captions:Figure I. Immune reconstitution post-transplant(A) Absolute T, B, and NK cell counts were measured at the donor infusion and post-transplant time points in 13 donor-recipient pairs. Lines indicate median values for each time point. (B) Individual CD4+ and CD8+ T cell changes post-transplant relative to the donor infusion time point. (C) Inversion of the normal CD4:CD8 ratio at the post-transplant time point. (D) Median values for absolute CD4 and CD8 cell counts per uL for the patient cohort throughout the post-transplant time period.Figure II. Alterations in T cell subsets post-transplant(A) Frequencies of Naive (CCR7+ CD45RA+), Central Memory (CCR7+ CD45RA-), Effector Memory (CCR7- CD45RA-), and Effector Memory cells that re-express CD45RA (TEMRA; CCR7- CD45RA+) subsets for both CD4 and CD8 cells at the donor infusion and post-transplant time points. Boxes indicate the 25th quartile, median, and 75th quartile values; whiskers indicate the minimum and maximum values. (B) Absolute cell numbers per uL for each CD4 and CD8 T cell subset. (C) Ratio of absolute cell number for Naïve/Central Memory + Effector Memory populations at the donor infusion and post-transplant time points. (D) TREC analysis of the CD4 and CD8 populations assessing thymic generation of naive T cells at the donor infusion and post-transplant time points (n=6). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Circulating Donor Lung-specific Exosome Profiles Enable Noninvasive Monitoring of Acute Rejection in a Rodent Orthotopic Lung Transplantation Model.

There is a critical need for development of biomarkers to noninvasively monitor for lung transplant rejection. We investigated the potential of circulating donor lung-specific exosome profiles for time-sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. Left lungs from Wistar transgenic rats expressing human CD63-GFP, an exosome marker, were transplanted into fully MHC-mismatched Lewis recipients or syngeneic controls. Recipient blood was collected between 4 h and 10 d after transplantation, and plasma was processed for exosome isolation by size exclusion column chromatography and ultracentrifugation. Circulating donor exosomes were profiled using antihuman CD63 antibody quantum dot on the nanoparticle detector and via GFP trigger on the nanoparticle flow cytometer. In syngeneic controls, steady-state levels of circulating donor exosomes were detected at all posttransplant time points. Allogeneic grafts lost perfusion by day 8, consistent with acute rejection. Levels of circulating donor exosomes peaked on day 1, decreased significantly by day 2, and then reached baseline levels by day 3. Notably, decrease in peripheral donor exosome levels occurred before grafts had histological evidence of acute rejection. Circulating donor lung-specific exosome profiles enable an early detection of acute rejection before histologic manifestation of injury to the pulmonary allograft. As acute rejection episodes are a major risk factor for the development of chronic lung allograft dysfunction, this biomarker may provide a novel noninvasive diagnostic platform that can translate into earlier therapeutic intervention for lung transplant patients.

Physical Performance Following Hematopoietic Stem Cell Transplantation: A Prospective Observational Study

Background and Purpose/Objective: Patients undergoing hematopoietic stem cell transplantation (HSCT) are at risk for strength impairments and activity limitations. Research describing how HSCT affects physical performance is limited. The purposes of this study were to report the short-term (≤30 days) effects of HSCT on physical performance and to compare changes among patients during the engraftment and recovery periods following HSCT. Methods: Design: Prospective observational study. Setting: Academic medical center. Patients: Twenty-five adults 21 years and older undergoing HSCT. Intervention: None. Measurements: Measures were collected at 3 time points: (1) day of transplant; (2) 8 to 14 days posttransplant; and (3) 15 to 30 days posttransplant. Beside demographic data, the authors collected Short Physical Performance Battery (SPPB) scores, Functional Comorbidity Index scores, resting heart rate, and hospitalization status. Longitudinal changes in SPPB scores were analyzed with a generalized linear model (α = 0.05). Results: SPPB scores were negatively associated with patients' body mass index values and resting heart rate measurements. Additionally, the hospitalization × time interaction was statistically significant. SPPB scores among nonhospitalized patients remained consistent across the testing periods but among hospitalized patients decreased from the day of transplant to both the 8 to 14 days (mean change = 4.0 points) and 15 to 30 days (mean change = 4.1 points) posttransplant time points. Conclusion: Physical performance scores decreased in the first 30 days of recovery among patients who were hospitalized following HSCT, but not among patients who received transplant on an outpatient basis. Future studies to examine causes of and interventions for the reduction in physical performance among hospitalized patients post-HSCT are warranted.

Total Eyelid Transplantation in a Face Transplant: Analysis of Postoperative Periorbital Function

BackgroundProlonged impairment of protective ocular functions can compromise vision and lead to blindness if uncorrected. Several facial transplants have incorporated periorbital structures with variable eyelid preservation, but objective assessment of post-transplant periorbital function has been limited. Materials and methodsKinematic data were collected from a full-face recipient that included the fist total eyelid transplantation at 5 separate pre-transplant (PRE) and post-transplant time points (T1-T4). Using optical facial tracking, eyelid movements were tracked during involuntary blinking and compared with controls. ResultsThere was significant improvement in right eye aperture from PRE to T1 (β = 5.54, P < 0.001), with no change between T1 and T4. Aperture fluctuated in the left eye, with a temporary decrease between T2 and T3 corresponding with revision brow lift (β = −4.57, P < 0.001). Although improved from the pre-transplantation, right and left eye apertures remained significantly smaller than controls at T1 and T4 (P < 0.001). Similarly, spatial coupling increased from PRE to T1 (β = 0.63, P < 0.001) and remained high at T4, albeit significantly less than controls (P < 0.001). Temporal coupling improved from PRE to T2 (β = 2.29, P < 0.02) and was sustained at subsequent time points, with no difference relative to controls at T4. Considerable improvement was observed on clinical examination, with full functional status. ConclusionsApplication of a novel method for assessing functional eyelid recovery using facial tracking technology to the first total eyelid transplantation in the setting of a full facial transplant shows clear functional improvement after transplantation and suggests revisions can be performed safely to optimize aesthetic outcomes without permanent negative functional impact.

Impact of autologous stem cell transplant in myeloma patients on renal function and overall survival.

e19535 Background: Renal impairment has been negatively associated with progression free survival and overall survival in patients diagnosed with multiple myeloma (MM). Autologous stem cell transplant (ASCT) has become a standard of care for treatment in MM. Most previous studies have assessed the safety of ASCT and the improvement in renal function in patients with MM. However, a critical question about renal function over time, and its impact on survival after ASCT has rarely been addressed. In our study we sought to understand how renal function was impacted over time, the predictors of renal function, and overall survival after ASCT. Methods: We performed a retrospective review of all MM patients who underwent ASCT at MDACC from January1, 2008 through December 31, 2013. A total of 885 none dialysis patients who received Melphalan alone as the conditioning regimen were identified. We collected demographic information, ISS stage, and disease status at time of transplant and at last follow up. Creatinine, GFR (calculated using CKD Epi equation), calcium, and LDH were also collected at day 0, 100, 180, &amp; 365. Given the longitudinal nature of the data, linear mixed effect models were used to study the change of GFR over time. A joint model approach for longitudinal and survival data was used to assess association between overall survival and GFR with patient’s age at transplant, ISS, disease status, disease risk category and response to treatment prior to transplant included in the linear mixed effect models and hazard models. As sensitivity analyses, landmark analyses were conducted with day 0, 100, 180, and 365 days post-transplant as landmark time points. Results: Patients’ GFR at post-transplant time points were significantly higher (p ≤ 0.025) compared to the day of ASCT. A higher ISS stage at diagnosis was significantly associated with a lower GFR (p &lt; .0001) at all stages of chronic kidney disease. Specifically, compared to the patients with ISS stage I, those with ISS stage II or higher had lower GFR across all time points (p ≤ .0003). GFR value was not significantly associated with OS in any of the analyses described above. In contrast, disease status, ISS stage, response to induction prior to SCT were all associated with shorter OS. Conclusions: The study demonstrates in a large cohort and in a longitudinal manner that MM patients who underwent ASCT did not have further decline in GFR over time. In addition, GFR was strongly associated with ISS stage. As far as OS, MM-related factors significantly impacted the survival while GFR did not.

StemPro™ HSC expansion medium (Prototype) supports superior expansion of human hematopoietic stem-progenitor cells

Background & Aim Since numbers of harvested hematopoietic stem cells (HSCs) continues to limit not only laboratory research but also clinical HSC transplantation and gene therapies, development of ex vivo culture systems to expand harvested human hematopoietic stem-progenitor cells (HSPCs) remains a critical translational research quest. To address the barrier that HSCs generally die or differentiate in current ex vivo culture media, we have developed an xeno-free, serum-free medium - StemPro™ HSC Expansion Medium (Prototype) by extensive evaluation of iterative modification of medium constituents. Methods, Results & Conclusion Cultures of primary human CD34+ HSPCs immunopurified from cord blood, mobilized peripheral blood and bone marrow harvests generated greatly increased numbers of immunphenotyped-defined HSPC in StemPro™ HSC Expansion Medium (Prototype) supplemented with FLT3L, KITL (also known as SCF), TPO, IL-3, and IL-6 (FKT36), as compared to either uncultured day 0 cells or cells cultured in industry-standard culture media containing FKT36. For example, culture of primary human CD34+ cells from mobilized peripheral blood for 7 days in FKT36-containing StemPro™ HSC Expansion Medium (Prototype) resulted in ∼100-fold increased numbers of CD34+CD45+Lin− cells and ∼2000-fold increased numbers of CD34+Lin−CD90+CD45RA− cells (an early HSPC immunophenotype), as compared to uncultured day 0 cells. The ex vivo-cultured cells contained high frequencies of aldehyde dehydrogenase-containing cells and formed erythroid and non-erythroid hematopoietic colonies in vitro. In an ongoing in vivo hematopoietic chimera experiment, ex vivo-cultured mPB CD34+ HSPCs harbored robust in vivo-engrafting capacity at the 8-week post-transplant short-term HSC time point evaluated to date. Thus, it appears that StemPro™ HSC Expansion Medium (Prototype) supports superior HSPC expansion that includes self-renewal of short-term-HSCs. Evaluation of long-term-HSC capacity is in progress.