Chimerism Status Research Articles

Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation

Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R2=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.

Densitometry of STR-PAGE for donor chimerism in acute leukemia's: A simple method for routine use.

To evaluate a PCR based method of polyacrylamide gel electrophoresis of short tandem repeats and its quantification for detecting donor chimerism after haematopoietic stem cell transplantation in acute leukaemias. The descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Feb 2018 - Nov 2020. A total of twenty patients with acute leukaemias having undergone HSCT were selected and assessed for the analysis of chimerism status. DNA extraction from the whole blood was done by chelex method and short tandem repeats were amplified by using conventional STR- PCR assay. Electrophoresis was carried out and 6% polyacrylamide gels were used for the resultant amplified DNA products and then followed by their densitometry. These patients had undergone HSCT from Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre. The peaks in the PAGE densitometry represented the donor chimerism in all post transplant samples of the patients. Our study showed that densitometry of STR PCR PAGE is a useful and cheaper method for demonstration of donor chimerism in acute leukaemia patients having undergone HSCT. Hence this method can be a valuable option in the monitoring of chimerism status in these patients and therefore helps in preventing graft failure by fast and early treatment strategies for these patients.

Risk factors for graft-versus-host-disease after donor lymphocyte infusion following T-cell depleted allogeneic stem cell transplantation.

Unmodified donor lymphocyte infusions (DLI) after allogeneic stem cell transplantation (alloSCT) can boost the beneficial Graft-versus-Leukemia (GvL) effect but may also induce severe Graft-versus-Host-Disease (GvHD). To improve the balance between GvL and GvHD, it is crucial to identify factors that influence the alloreactivity of DLI. We investigated the effects of the presence of patient-derived antigen-presenting cells at time of DLI as estimated by the bone marrow (BM) chimerism status, lymphopenia as measured by the absolute lymphocyte count (ALC) at time of DLI, and the presence of a viral infection (de novo or reactivation) close to DLI on the risk of GvHD after DLI. The cohort consisted of patients with acute leukemia or myelodysplastic syndrome who prophylactically or pre-emptively received DLI as standard care after alemtuzumab-based alloSCT. In patients at high risk for relapse, DLI was administered at 3 months after alloSCT (n=88) with a dose of 0.3x106 or 0.15x106 T cells/kg in case of a related or unrelated donor, respectively. All other patients (n=76) received 3x106 or 1.5x106 T cells/kg, respectively, at 6 months after alloSCT. For both DLIs, patients with reduced-intensity conditioning and an unrelated donor had the highest risk of GvHD. For DLI given at three months, viral infection within 1 week before and 2 weeks after DLI was an additional significant risk factor (hazard ratio (HR) 3.66 compared to no viral infection) for GvHD. At six months after alloSCT, viral infections were rare and not associated with GvHD. In contrast, mixed BM chimerism (HR 3.63 for ≥5% mixed chimerism compared to full donor) was an important risk factor for GvHD after DLI given at six months after alloSCT. ALC of <1000x106/l showed a trend for association with GvHD after this DLI (HR 2.05 compared to ≥1000x106/l, 95% confidence interval 0.94-4.45). Furthermore, the data suggested that the presence of a viral infection close to the DLI at three months or ≥5% mixed chimerism at time of the DLI at six months correlated with the severity of GvHD, thereby increasing their negative impact on the current GvHD-relapse-free survival. These data demonstrate that the risk factors for GvHD after DLI depend on the setting of the DLI.

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 &amp;gt; 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&amp;lt;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&amp;lt;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 (&amp;gt;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.

HLA Class I Allele Loss and Bone Marrow Transplantation Outcomes in Immune Aplastic Anemia

In patients with immune-mediated acquired aplastic anemia (AA), HLA class I alleles often disappear from the surface of hematopoietic progenitor cells, potentially enabling evasion from cytotoxic T lymphocyte-mediated pathogenesis. Although HLA class I allele loss has been studied in AA patients treated with immunosuppressive therapy (IST), its impact on allogeneic bone marrow transplantation (BMT) has not been thoroughly investigated. The purpose of this study was to evaluate the clinical implications of HLA class I allele loss in patients with acquired AA undergoing allogeneic BMT. The study enrolled acquired AA patients who underwent initial BMT from unrelated donors through the Japan Marrow Donor Program between 1993 and 2011. The presence of HLA class I allele loss due to loss of heterozygosity (HLA-LOH) was assessed using pretransplantation blood DNA and correlated with clinical data obtained from the Japanese Transplant Registry Unified Management Program. A total of 432 patients with acquired AA were included in the study, and HLA-LOH was detected in 20 of the 178 patients (11%) available for analysis. Patients with HLA-LOH typically presented with more severe AA at diagnosis (P=.017) and underwent BMT earlier (P < .0001) compared to those without HLA-LOH. They also showed a slight but significant recovery in platelet count from the time of diagnosis to BMT (P=.00085). However, HLA-LOH status had no significant effect on survival, engraftment, graft failure, chimerism status, graft-versus-host disease, or other complications following BMT, even when the 20 HLA-LOH+ patients were compared with the 40 propensity score-matched HLA-LOH- patients. Nevertheless, patients lacking HLA-A*02:06 or HLA-B*40:02, the alleles most frequently lost and associated with a better IST response, showed higher survival rates compared to those lacking other alleles, with estimated 5-year overall survival (OS) rates of 100% and 44%, respectively (P=.0042). In addition, in a specific subset of HLA-LOH- patients showing clinical features similar to HLA-LOH+ patients, the HLA-A*02:06 and HLA-B*40:02 allele genotypes correlated with better survival rates compared with other allele genotypes, with estimated 5-year OS rates of 100% and 43%, respectively (P=.0096). However, this genotype correlation did not extend to all patients, suggesting that immunopathogenic mechanisms linked to the loss of certain HLA alleles, rather than the HLA genotypes themselves, influence survival outcomes. The survival benefit associated with the loss of these two alleles was confirmed in a multivariable Cox regression model. The observed correlations between HLA loss and the pretransplantation clinical manifestations and between loss of specific HLA class I alleles and survival outcomes in AA patients may improve patient selection for unrelated BMT and facilitate further investigations into the immune pathophysiology of the disease.

Treosulfan Exposure Predicts Thalassemia-Free Survival in Patients with Beta Thalassemia Major Undergoing Allogeneic Hematopoietic Cell Transplantation.

A toxicity-reduced conditioning regimen with treosulfan, fludarabine, and thiotepa in patients with high-risk β-thalassemia major has significantly improved hematopoietic stem cell transplantation (HCT) outcomes. However, complications resulting from regimen-related toxicities (RRTs), mixed chimerism, and graft rejection remain a challenge. We evaluated the dose-exposure-response relationship of treosulfan and its active metabolite S, S-EBDM, in a uniform cohort of patients with β-thalassemia major to identify whether therapeutic drug monitoring (TDM) and dose adjustment of treosulfan is feasible. Plasma treosulfan/S, S-EBDM levels were measured in 77 patients using a validated liquid chromatography with tandem mass spectrometry method, and the pharmacokinetic parameters were estimated using nlmixr2. The influence of treosulfan and S, S-EBDM exposure, and GSTA1/NQO1 polymorphisms on graft rejection, RRTs, chimerism status, and 1-year overall survival (OS), and thalassemia-free survival (TFS) were assessed. We observed that treosulfan exposure was lower in patients with graft rejection than those without (1,655 vs. 2,037 mg•h/L, P = 0.07). Pharmacodynamic modeling analysis to identify therapeutic cutoff revealed that treosulfan exposure ≥1,660 mg•hour/L was significantly associated with better 1-year TFS (97% vs. 81%, P = 0.02) and a trend to better 1-year OS (90% vs. 69%, P = 0.07). Further, multivariate analysis adjusting for known pre-HCT risk factors also revealed treosulfan exposure <1,660 mg•h/L (hazard ratio (HR) = 3.23; 95% confidence interval (CI) = 1.12-9.34; P = 0.03) and GSTA1*B variant genotype (HR = 3.75; 95% CI = 1.04-13.47; P = 0.04) to be independent predictors for inferior 1-year TFS. We conclude that lower treosulfan exposure increases the risk of graft rejection and early transplant-related mortality affecting TFS. As no RRTs were observed with increasing treosulfan exposure, TDM-based dose adjustment could be feasible and beneficial.

Analysis of the Impact of Whole Blood and T-Cell Donor Chimerism after Allogeneic Stem Cell Transplant

Background: Allogeneic stem cell transplant (alloSCT) is one of the few currently available therapies to cure acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Positive outcomes are dependent on engraftment of donor cells to provide hematopoiesis as well as a graft-versus-tumor effect. Measurement of whole blood (WB) and T-cell (TC) chimerism at routine intervals post alloSCT allows for monitoring of engraftment. Development of mixed donor chimerism (MC) may indicate impending graft failure or disease relapse. The ideal frequency for chimerism measurements as well as the role of WB versus TC chimerism remains unclear. In this study, we analyzed the impact of WB versus TC mixed chimerism on survival outcomes in patients with AML and MDS. Furthermore, we studied baseline factors associated with the development of MC. Methods: In this retrospective analysis, adults with AML or MDS who received an alloSCT between January 1, 2016 and February 1, 2022 that had at least 1 donor chimerism measured on Day 30 (D30), Day 60 (D60), Day 90 (D90) at Juravinski Hospital and Cancer Centre (Hamilton, ON, Canada). Baseline demographic and transplant-related characteristics were collected. AML and MDS diagnoses were based on WHO 2016 classification of myeloid neoplasm and acute leukemia. Conditioning intensities were classified according to CIBMTR definitions. A total of 271 transplants were completed during the study period for AML or MDS. 128 were excluded from the analysis (chimerism testing not performed (n=121), second transplants (n=7)). We analyzed a total of 143 patients with median age of 63 years (19 - 76) who were transplanted for AML (n=105) and MDS (n=38). Donors were HLA-matched siblings (MRD, n=26), unrelated donors (MUD (8/8 or 7/8), n=85) or haploidentical related donor (MMRD, n=32). All patients except one received peripheral blood stem cell grafts. For alloSCT, 20 % of patients received myeloablative conditioning (MAC) and 80% received reduced-intensity conditioning (RIC). Complete chimerism (CC) was defined as ≥95%, whereas MC was defined as ≤95%. All chimerism measurements were measured using a PowerPlex® 16 System (Promega) (Hamilton Regional Laboratory Medicine Program, Hamilton, ON). Results: In our cohort, OS at 2 years was 57% and RFS 49%. Of the baseline factors, only disease-risk based on ELN 2022 for RFS with a hazard ratio (HR) of 1.43, 95% CI [0.83, 2.49] for adverse risk and HR 0.53 95% CI [0.23, 1.03] (p=0.03) for favourable risk. WB MC was detected in 22%, 22%, 23% of patients on D30, D60, and D90, respectively. TC MC was detected in 48%, 46%, and 43% of patients on D30, D60, and D90, respectively. WB MC had a strong negative impact on RFS, with a HR of 2.81, 95% CI [1.69, 4.66; p&amp;lt;0.001] on D30, 3.07, 95% CI [1.67, 5.66; p&amp;lt;0.001] on D60 and 4.77, 95% CI [2.47, 9.22; p&amp;lt;0.001] on D90. The influence of TC MC on RFS was also significant at all time points. Of 101 patients who were alive and did not have a relapse prior to D90 and had D30 chimerism and D90 chimerism evaluated, the patients who had a mixed WB and TC chimerism on D90 had a HR of 6.13, 95% CI [2.23, 16.85] and HR 2.73, 95% CI [1.24, 5.98] for RFS, which was statistically significant even after adjusting for known information of D30 WB/TC chimerism status (Table 1). WB MC had a strong negative impact on OS with a HR of 2.67, 95% CI (1.54, 4.64; p=0.001) on D30, 2.81, 95% CI (1.47, 5.38; p=0.002) on D60 and 4.78, 95% CI (2.47, 9.24; p&amp;lt;0.001) on D90. Again, the later time points remained statistically significant, when adjusting for known D30 chimerism status. The influence of TC MC on OS was also significant at all time points, however only a non-significant trend remained at later time points when adjusting for D30 TC chimerism (Table 2). We analyzed baseline factors predictive for D30 chimerism. For WB chimerism, only donor source remained statistically significant (p=0.038) with MMRD having a lower HR for MC 0.39, 95% CI [0.11, 1.41] and MRD having a higher HR for MC 2.33, 95% CI [0.9 - 5.99]. Conclusions: Our data confirm that a mixed chimerism as early as D30 post-alloSCT can have a detrimental impact on outcomes (RFS and OS) in alloSCT. Patients receiving haploidentical alloSCT had a lower risk to develop mixed chimerism in our cohort. These dismal outcomes of patients with mixed chimerism in our cohort provides a strong rationale to study interventions such as novel maintenance strategies or donor-lymphocyte infusions in this patient group.

Assessing cell chimerism in an acute graft-versus-host disease model established in TLR4 knockout mice.

Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation. To elucidate the role of Toll-like receptor 4 (TLR4), the major receptor for bacterial lipopolysaccharide, in the development of GVHD, we constructed a GVHD model in TLR4 knockout (TLR4-/-) mice and monitored the cell chimerism. In this study, we used polymerase chain reaction to identify whether TLR4 knockout (TLR4-/-) mice were established. Before transplantation, we pretreated mice with irradiation so as to obtain an appropriate irradiation dose. Flow cytometry was applied to measure the chimerism status, the distributions of antigen-presenting cells (APCs), and T-cells in TLR4+/+ and TLR4-/- recipient mice. The general condition of TLR4-/- recipients was better than that of TLR4+/+ recipients, and the TLR4-/- recipient mice showed less severe GVHD manifestations than the TLR4+/+ recipient mice. Most of the APCs and T-cells in the host mouse spleen were derived from donor cells, and CD4+ T-cells, including memory T-cells, were in the majority in host mice. In general, our data show that TLR4 deletion attenuated GVHD development, which suggests that TLR4 could be used as a novel target and therapeutic paradigm in GVHD therapies.

Is Mixed Chimerism Post-allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Acute Lymphoid Leukemia a Prognostic Factor for Relapse?

Hematopoietic stem cell transplantation (HSCT) has been considered curative for children with high-risk acute leukemia (ALL), offering better survival. Short tandem repeat has been used as a marker of chimerism status after HSCT. The appearance of recipient cells >1% post-allogeneic stem cell transplant is defined as mixed chimerism (MC). Chimeric studies post-HSCT are dynamic. This study aimed to investigate the significance of recipient cells in post-HSCT pediatric ALL patients as a predictor of relapse of their primary disease. The rate of MC was 51.4% (19 out of 37 recipients). It was 48.6% (n = 18) during Day+100 and 12.9% (4 out of 31 recipients) during post-Day+100 follow-up until two years. No significant association was noted between MC and all grade overall acute graft-versus-host disease. A mortality rate of 35.1% (n = 13) and a median follow-up of 56.9 months (95% CI: 39.7-74.2) were observed for all but four (16.7%) of the survivors in remission. Regarding causes of death, transplant-related mortality was recorded in only 2 of 13 expired patients (15.4%); both succumbed to sepsis. No significant association was found between MC and primary causes of death. The cumulative probability of five-year overall survival and event-free survival was not found to be statistically significantly different for MC (≤1.0% vs. > 1.0%). In conclusion, our data did not show MC testing alone as an effective prognostic marker for detecting relapse; molecular and flow cytometric analyses should be considered in children with ALL post-HSCT for monitoring relapse.

Posttransplant MRD and T-cell chimerism status predict outcomes in patients who received allografts for AML/MDS.

Allogeneic stem-cell transplant allows for the delivery of curative graft-versus-leukemia (GVL) in patients with acute myeloid leukemia/myelodysplasia (AML/MDS). Surveillance of T-cell chimerism, measurable residual disease (MRD) and blast HLA-DR expression may inform whether GVL effectiveness is reduced. We report here the prognostic impact of these biomarkers in patients allografted for AML/MDS. One hundred eighty-seven patients from FIGARO, a randomized trial of reduced-intensity conditioning regimens in AML/MDS, were alive and relapse-free at the first MRD time-point and provided monitoring samples for flow cytometric MRD and T-cell chimerism, requested to month+12. Twenty-nine (15.5%) patients had at least 1 MRD-positive result posttransplant. MRD-positivity was associated with reduced overall survival (OS) (hazard ratio [HR], 2.18; P= .0028) as a time-varying Cox variable and remained significant irrespective of pretransplant MRD status in multivariate analyses (P< .001). Ninety-four patients had sequential MRD with T-cell chimerism results at months+3/+6. Patients with full donor T-cell chimerism (FDTC) had an improved OS as compared with patients with mixed donor T-cell chimerism (MDTC) (adjusted HR=0.4; P=.0019). In patients with MDTC (month+3 or +6), MRD-positivity was associated with a decreased 2-year OS (34.3%) vs MRD-negativity (71.4%) (P= .001). In contrast, in the group with FDTC, MRD was infrequent and did not affect the outcome. Among patients with posttransplant MRD-positivity, decreased HLA-DR expression on blasts significantly reduced OS, supporting this as a mechanism for GVL escape. In conclusion, posttransplant MRD is an important predictor of the outcome in patients allografted for AML/MDS and is most informative when combined with T-cell chimerism results, underlining the importance of a GVL effect in AML/MDS.

A redescription of Palaeogekko risgoviensis (Squamata, Gekkota) from the Middle Miocene of Germany, with new data on its morphology.

After its original description, the Middle Miocene gekkotan Palaeogekko risgoviensis remained an enigma for palaeontologists due to a rather poor knowledge of its osteology and relationships. Coming from a single locality in southern Germany, this gecko lived in central Europe during a period when a single gekkotan lineage (i.e., euleptine sphaerodactylids) is confidently reported to have inhabited the continent. However, it is unclear whether P. risgoviensis may represent a member of this same lineage or a second clade of Gekkota. In order to shed light on this issue, the type material of P. risgoviensis is here redescribed, refigured and extensively compared with extinct and extant geckos from Europe. A phylogenetic analysis is also conducted in order to investigate its relationships. The new observations confirm the validity of the German species as a distinct taxon, and exclude the previously-suggested chimeric status of the type material of this gecko (with the exception of a single dentary included in the type series, which clearly belong to a different lizard). Phylogenetic relationships of Palaeogekko are still unclear, though, with different positions within the gekkotan tree recovered for the taxon. Nevertheless, it is confidently supported as a non-eublepharid gekkonoid, in agreement with hypothesys presented by other scholars.

Two decades of stem cell transplantation in patients with Fanconi anemia: Analysis of factors affecting transplant outcomes.

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for the hematological complications of patients with Fanconi anemia (FA). Over the last two decades, HSCT outcomes have improved dramatically following the development of regimens tailored for FA patients. In this study, we analyzed genetic, clinical, and transplant data of 41 patients with FA who underwent HSCT at Hadassah Medical Center between November 1996 and September 2020. Overall survival (OS) was 82.9% with a median follow-up time of 2.11-years (95% CI, .48-16.56). Thirteen patients (31.7%) developed acute graft-versus-host disease (GVHD), three of them with grades 3-4. Nine patients developed chronic GVHD, five had extensive disease. Twelve patients (29.3%) developed stable mixed-chimerism with complete resolution of bone marrow failure (BMF); none of them had acute nor chronic GVHD. Significantly higher GVHD rates were observed in transplants from peripheral blood stem cell grafts as compared to other stem cell sources (p=.002 for acute and p=.004 for chronic GVHD). Outcome parameters were comparable between HSCT from matched-sibling (n=20) to other donors (n=21), including survival rates (p=.1), time to engraftment (p=.69 and p=.14 for neutrophil and platelet engraftment time, respectively), chimerism status (p=.36 and p=.83 for full-donor and mixed chimerism, respectively), and GVHD prevalence (p=1). Our results demonstrate the vast improvements in HSCT outcomes of patients with FA, narrowing the gap between matched-sibling versus alternative donor transplantations. Our data identifies factors that may significantly affect transplant outcomes such as graft source and chimerism status.

A Retrospective Cohort Study of Donor Cell Leukemia

Rationale: Donor cell leukemia (DCL) is formally defined as the new onset of hematological malignancy following an allogeneic hematopoietic stem cell transplantation (alloHSCT) that is derived from the donor. DCL is a rare but increasingly recognized complication with more than 100 cases being reported in the literature and an incidence between 0.12 to 5% (1-6). The identification of DCL is crucial in order to determine the most appropriate therapy for the patient. The underlying mechanisms that contribute to DCL remain largely unknown with no formal guidelines pertaining to prevention. Objective: To determine the incidence of DCL in a patient cohort and better characterize this patient population in terms of natural history, response to therapy, clinical course, and outcomes. Methods: A retrospective chart review was conducted to identify DCL cases that were diagnosed between 2012 to 2022 at the Juravinski Cancer Center (JCC) in Hamilton, Ontario. All patients were 18 years of age or older and diagnosed with a hematological malignancy that was treated with a sex mismatched alloHSCT. DCL was defined as a new presentation of hematological malignancy as per the 2022 WHO classification (7), with the source being from the donor, as confirmed by chimerism status testing with short tandem repeat polymerase chain reaction (STR-PCR) and karyotyping. Results: We identified four patients with confirmed DCL (Table 1). These patients previously received a sex mismatched alloHSCT between 2005 to 2017. A total of 205 patients at JCC received a sex mismatched alloHSCT during the same time span of 2005 to 2017. The incidence of DCL in this cohort was 1.95%. Three patients developed AML and one patient had B-ALL. Three patients were female, and one was male, with a median age of 64.5 years. Three cases were from sibling-matched donors. All patients had normal clonal cell next generation sequencing using a NN-gene myeloid panel (OGT), whereas two patients had an abnormal donor clonal karyotype (inversion 16, deletion 20q, trisomy 21). The latency period between initial malignancy and DCL was between 8 months to 13 years post-transplant. Three patients were alive at time of chart review, with a 25% mortality rate. Conclusions: The time span of the DCL latency period is broad, ranging from months to upwards of a decade. In patients with leukemia diagnosed following sex mismatched alloHSCT, regardless of when it occurs following the initial malignancy, both relapse of leukemia and DCL should be considered as potential differentials. DCL may be the product of somatic and/or germline mutations that warrants additional genetic testing and counselling prior to transplantation. The concept of more comprehensive molecular and cytogenetic testing to identify somatic and germline mutations in related donors prior to alloHSCT to assist with prevention of DCL needs to be explored further.

Dynamics and Predictive Value of T-Cell Chimerism after Haploidentical Hematopoietic Cell Transplantation Using Post-Transplant Cyclophosphamide

Background: T-cell chimerism after allogeneic hematopoietic cell transplantation (alloHCT) is an important tool to investigate T-cell reconstitution. Haploidentical HCT (haplo-HCT) using post-transplantation cyclophosphamide (PTCy) has been increasingly applied due to readily available donors and favorable outcomes. PTCy induces immune tolerance by depleting expansion of alloreactive T-cells. However, limited clinical data exist regarding the impact of PTCy on the dynamics of T-cell chimerism. Methods: Adult patients receiving haplo-HCT with PTCy and reduced-intensity conditioning regimens were retrospectively reviewed. Patients with primary graft failure or receiving donor-lymphocyte infusion prior to +90 days were excluded. T-cell chimerism was measured from sorted CD3+ cells collected from peripheral blood or bone marrow at 1, 2, 3, 6, and 12 months. Complete chimerism was defined as < 5% donor T-cells. Patients were divided into full and partial T-cell chimerism groups based on their T-cell chimerism status at Month 1 and 3. Results: From 1/2017 to 6/2021, 34 patients underwent haplo-HCT and met inclusion criteria (Table 1). One month post HCT, 29 (85.3%) patients achieved complete T-cell chimerism; 3 months post HCT, 31 (93.9%) patients achieved complete T-cell chimerism (Figure A). In univariate logistic regression analysis, peripheral blood rather than bone marrow as the donor source, and the occurrence of cytokine-release syndrome (CRS), were significantly associated with higher incidences of full T-cell chimerism at Month 1 (Table 2). In multivariable analysis, only the occurrence of CRS was significantly associated with full T-cell chimerism at Month 1 (odds ratio 18.7, 95% confidence interval [CI] 1.30 to 641, p=0.04, Table 2). Next, we investigated whether early achievement of full T-cell chimerism would affect clinical outcomes. After a median follow up of 363 and 530 days for patients with full and partial T-cell chimerism at Month 1, respectively, there was no significant difference of progression-free survival (PFS) (median PFS not reached v.s. 367 days, hazard ratio [HR] 0.54, 95%CI 0.14 to 2.07, Figure B) or overall survival (OS) (median OS not reached vs 530 days, HR 0.41, 95%CI 0.10 to 1.69, Figure C). In contrast, patients who achieved full T-cell chimerism at Month 3 had significantly longer OS than those with partial T-cell chimerism (median OS not reached v.s. 492 days, HR 0.18, 95%CI 0.03 to 0.90; p=0.04, log-rank test; Figure E). Of note, for the two deaths in the partial T-chimerism group, 1 died from relapse and the other from sepsis in the setting of Grade 4 acute graft-versus-host disease. Moreover, patients who achieved full T-chimerism at Month 3 had a trend of improved PFS over those with partial T-chimerism, although the difference was not statistically significant (HR 0.23, 95%CI 0.05 to 1.11; p=0.07; Figure D). Lastly, achieving partial T-cell chimerism at Month 1 or Month 3 was not associated with an increased risk of bacteremia (p=0.83 for Month 1; p=0.99 for Month 3; Chi-square test) or pneumonia (p=0.31 for Month 1; p=0.38 for Month 3), compared with those with full T-cell chimerism. Conclusion: For patients receiving haplo-HCT with PTCy, the majority achieved full T-cell chimerism within the first month. The occurrence of CRS was associated with a significantly higher chance of achieving full T-cell chimerism at Month 1. Achieving a full T-cell chimerism at Month 3 was associated with significantly improved OS compared with those with partial T-cell chimerism. These data should be confirmed in a larger patient cohort. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Post-Transplantation Day +100 Minimal Residual Disease Detection Rather Than Mixed Chimerism Predicts Relapses after Allogeneic Stem Cell Transplantation for Intermediate-Risk Acute Myelogenous Leukemia Patients Undergoing Transplantation in Complete Remission

Chimerism and minimal residual disease (MRD) are suggested to be prognostic for post-transplantation relapse in patients with acute myelogenous leukemia (AML). Nevertheless, the predictive value of both approaches in homogeneous populations remains underinvestigated. Here we suggest that MRD may have greater predictive value for relapse than mixed chimerism (MC) in intermediate-risk AML patients. Seventy-nine patients with intermediate-risk AML (40 males; median age, 57 years [range, 19 to 77 years]) were included. MRD detection on day +100 was performed in bone marrow via multiparameter flow cytometry (MFC) and quantitative real-time PCR (qPCR) for patients with an NPM1 mutation. Chimerism analysis was performed in peripheral blood. MC was defined as the persistence of <99.9% of donor alleles. The area under the receiver operating characteristic curve was highest for qPCR-MRD (.93), followed by MFC-MRD (.80) and MC (.65). The highest rate of relapse at 3 years was observed in day +100 qPCR-MRD-positive patients (100%), followed by MFC-MRD-positive patients (55%; P < .001). No patients with MC and without detectable MRD experienced relapse. The median 3-year overall survival (OS) and leukemia-free survival (LFS) for patients with MC without detectable MRD were both 86% (range, 61% to 96%), significantly higher than the values in day +100 MFC-MRD-positive patients (OS, 61% [range, 36% to 84%]; LFS: 30% [range, 11% to 59%]) and with day +100 qPCR-MRD-positive patients (OS: 17% [range, 3% to 56%], P = .001; LFS: 0%, P < .001). In patients with intermediate-risk AML, the qPCR-MRD on day +100 was highly predictive for relapse and long-term survival after allogeneic stem cell transplantation, followed closely by MFC-MRD. In contrast, chimerism status had limited predictive potential. Thus, molecular and flow cytometry MRD monitoring rather than MC in the first several months post-transplantation can identify patients at increased risk of relapse who may benefit from early post-transplantation preemptive intervention.

Association of early donor chimerism status with survival outcomes in post allogeneic haematopoietic stem cell transplant patients of nonmalignant diseases.

To highlight the association of early donor chimerism status at 2nd month with various survival outcomes. The retrospective study was conducted at the Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan, and comprised patient data from January 2011 to July 2016. Data related to participants who underwent human leukocyte antigen-matched transplants for bone marrow failure syndrome and beta thalassemia major. Short tandem repeat-based polymerase chain reaction was used to assess donor chimerism status. Overall survival, disease-free survival, relapse-free survival, and graft versus host disease-free survival rates were noted. Data was analysed using SPSS 23. Of the 106, 64(60.4%) had bone marrow failure syndrome and 42(39.6%) had beta thalassemia major. The overall median follow-up was 13.53 months (range: 1.81-62.73 months). Early donor chimerism status was associated with overall survival (p=0.02) and disease-free survival (p=0.01). Mixed donor chimerism was less hazardous in terms of overall survival (p=0.04) and disease-free survival (p=0.02). Early mixed donor chimerism contributed to optimal survival in nonmalignant disease.

D1S111 and 33.4 Polymorphism as a Tool of Genetic Identity among Egyptians

Abstract Background The variable number tandem repeats (VNTR) are highly polymorphic markers. Due to their high polymorphic content, VNTR constitute useful tools in population genetic studies in understanding population and ethnic migrations throughout history. They also constitute preferred systems for DNA “fingerprinting”, criminal and forensic examinations and paternity testing, evaluating hematopoietic chimerism and in determining the origin of leukemic cells in patients with recurrent disease after BMT. Aim of the Work To standardize PCR protocols of 2 VNTR loci (D1S111/33.6 and 33.4) in order to detect allelic polymorphism among Egyptians as a tool in following up the chimeric status after allogenic stem cell transplantation. Patients and Methods The current study was conducted at Clinical Pathology Department of Ain Shams University Hospital between January 2018 and December 2019, a total of 130 subjects [65 pairs (allogenic stem cell transplantation donors and their recipients)] were included. D1S111/33.6 and 33.4 VNTR loci were evaluated in every subject by PCR technique to study their relative distribution and number of informative alleles detected in order to be used in chimerism follow up and population studies. Results Five alleles were found at D1S111/33.6 in 130 subjects with molecular weight ranging from 364 to 993 bp, frequency of homozygous alleles were 30.8% and it was 69.2% for heterozygous, 90 out of the total 130 subjects were heterozygous for 33.6, that means that the heterozygosity index for D1S111/33.6 is 69.2%, from the 65 pairs tested 31 pair were informative or discriminative with discriminative power 47.69%. For 33.4 we tried many PCR programs but no bands were detected at all. Conclusion D1S111/33.6 VNTR polymorphism is a dependable cost effective method in following up of allogenic stem cell transplantation recipients.

Transfusion Dependence at Day +100 Predicts Poorer Outcomes in Patients Undergoing Allogeneic Stem Cell Transplantation for Myelofibrosis

Introduction - Various pre-transplantation factors help predict outcomes in patients (pts) with myelofibrosis (MF) undergoing allogeneic stem cell transplantation (HCT). However, early post-HCT characteristics have not been correlated with outcomes. Our study aims at identifying day +100 characteristics that are indicative of poorer outcomes.Methods - Pts undergoing HCT for MF at the 3 sites of Mayo Clinic in Rochester, Arizona and Florida, between January 2005 and August 2016, were retrospectively reviewed for collecting baseline, disease, transplantation and outcome data. Day +100 data was collected as available between day +80 through day +120. Day +100 characteristics included molecular status, WHO grade of bone marrow fibrosis, spleen size, red blood cell (RBC) transfusion dependence and platelet transfusion dependence. Transfusion dependence was defined as RBC or platelet transfusions requirements of 2 units in a 4 week period. Since there was limited data on donor chimerism and molecular marker status over the years, we used a variable called “molecular status” to reflect on the molecular response after HCT. Molecular status was defined as favorable if either 100% donor chimerism in CD33 fraction from blood or molecular marker negativity was known and as unfavorable if donor chimerism <100% or molecular marker positivity was known at day +100. Survival analysis was conducted using Kaplan-Meier (K-M) survival curve and a series of univariate and multivariate Cox proportional hazard regression models. Patient characteristics measured at day +100 along with age at HCT, gender, Dynamic International Prognostic Scoring System (DIPSS)-Plus score, conditioning regimen (myeloablative vs reduced intensity) and donor source (related vs unrelated) were used to examine group differences in K-M curves and to predict overall survival in the regression models.Results -Baseline/ Transplantation characteristics : A total of 87 pts underwent HCT for MF, of which, 79 were included in our study. Eight pts who died within 80 days from HCT were not included in the analysis. Median age at HCT was 58 years (range, 19-73 years). Forty seven (60%) were men and MF was primary in 54 (68%) pts. DIPSS-Plus score was high in 34 (43%), intermediate-2 in 40 (51%) and intermediate-1 in 5 (6%) pts. Conditioning regimen was myeloablative in 14 (18%) and reduced intensity in 65 (82%). Anti-thymocyte globulin was used in 35 (44%) pts. Donor source was related in 39 (49%) and unrelated in 40 (51%) pts. Of the related donors, 2 were haploidentical donor HCTs. JAK2 mutation was positive in 41 (54%) out of 76, CALR was positive in 8 (38%) out of 21 and MPL was positive in 4 (13%) out of 31 pts, in whom the status was available. Forty three (57%) pts were ABO compatible while 19 (25%) had minor ABO incompatibility, 12 (16%) had minor incompatibility and 1 (1%) had bidirectional ABO incompatibility. Ten (13%) pts had undergone splenectomy prior to HCT.Survival analysis : Twenty-six (33%) pts had died at the time of this study. Cause of death was HCT-related in 19 (73%) and disease relapse/ progression in 7 (27%) pts. RBC transfusion dependence, platelet transfusion dependence and unfavorable molecular status at day +100 were associated with increased risk of death in the univariate model (Table 1). The significant and nearly significant day +100 predictors from the univariate models were included in the multivariate model (table 2) along with pre-transplant characteristics. RBC and platelet transfusion dependence were significant predictors associated with poorer overall survival in the multivariate analysis (hazard ratios 30.18 and 8.43, respectively). Bone marrow fibrosis of grade ≥ 2 showed a trend towards poorer OS in the univariate analysis, but was not statistically significant in the multivariate model (hazard ratio 5.44, p= 0.08).Conclusions - In this exploratory analysis of day +100 variables for predicting long-term outcomes in pts undergoing HCT for MF, we report a strong relationship between RBC/ platelet transfusion dependence at day +100 and increased risk of death (figure 1). The mechanism of this relationship is not clear. These pts could, perhaps, benefit from close monitoring or early intervention. Given the scope of the current study, larger studies are warranted to further investigate these findings and identify the cause of this association. [Display omitted] DisclosuresKhera:Novartis: Consultancy. Mesa:Novartis Pharmaceuticals Corporation: Consultancy; Gilead Sciences, Inc.: Research Funding; Incyte Corporation: Research Funding; Ariad: Consultancy; Promedico: Research Funding; Celgene Corporation: Research Funding; CTI BioPharma Corp.: Research Funding; Galena Biopharma, Inc.: Consultancy.

Non-Myeloablative Transplant in Severe Sickle Cell Disease Is Safe & Effective. Single Center Experience from Saudi Arabia

Background:Sickle cell disease (SCD) is a severe monogenic disorder in the world with >300,000 newly born afflicted children per year. Prevalence of this disorder is high in sub-Saharan Africa, Mediterranean basin, Middle East and India. Patients with severe disease phenotype manifested with stroke or other complications may benefit from allogeneic hematopoietic stem cell transplant (SCT). Recent reports in pediatric patients using myeloablative regimen showed excellent outcomes but similar progress in adults has been hampered by higher rates of transplant related mortality (TRM) and infertility.Methods:We conducted a study of adult patients at our institution with SCD whom underwent a matched related SCT during the period of 2014-2017. Medical records of eligible patients were retrospectively reviewed and extracted. All patients received hydroxyurea (HU) at maximally tolerated doses with hyper-transfusion 2-3 months prior to SCT. Iron chelation was given for patients with ferritin > 1000 ug/L. The preperative regimen was non-myeloablative (NMA) consisting of alemtuzumab 1 mg/kg divided over 5 days (day -7 to -3) followed by Total Body Irradiation (TBI) 300 cGy on day -1. Stem cells were collected from peripheral blood via apheresis after 5 days of GCSF with target CD34 cells of 10×106/kg. Graft vs. host disease (GVHD) prophylaxis consisted of sirolimus starting from day -2 and continued for one year post SCT with dose modification based on chimerism. Event free survival (EFS) was defined from time to SCT to graft failure, development of GVHD or death from any cause. EFS and overall survival (OS) via time to end point analysis was computed using the Kaplan-Meir test.Results:A total of 18 adults patients were eligible and further analysed. Median age was 23.5 (14 - 39) years with 14 males and 4 females. All patients were on HU prior to SCT. Median ECOG performance status was 1 (0-1). Indications for transplant were recurrent vaso-occlusive crisis (VOC) refractory to standard therapy in 61%, stroke or other central nervous system (CNS) complication in 38%, recurrent avascular necrosis of joints in 38%, recurrent acute chest syndrome in 22% and severe disease requiring recurrent intensive care unit admission (ICU) in 17%. All donors were related siblings fully matched at 10/10 human leucocyte antigen (HLA) loci with median age of 23.6 years. A total of 11 (61%) of donors were sickle cell carriers with average Hgb S 33%. Stem cell mobilization was with 5 ug/kg GCSF in all donors and the average stem cell collected 11.6 x 106/kg. No significant complications resulted from GCSF use in donors with sickle cell trait.17 patients were conditioned with alemtuzumab / TBI regimen and GVHD prophylaxis with sirolmus. One patient who failed prior transplant received fludarabine, busulfan and thymoglobulin with GVHD prophylaxis as cyclosporine and methotrexate. Median duration of neutrophil engraftment was 20 days and platelet engraftment 2.6 days with half of patient's platelet nadir > 20. Average hemoglobin (Hb) pre- and post-SCT was 83 g/L and 142 g/L respectively. Average hemoglobin (Hb) S pre- and post-SCT was 77% and 13% respectively. Peri-SCT adverse events were 9 (50%) patients experienced neutropenic fever, 12 (67%) mucositis, 6 (33%) diarrhea. None of the patients developed acute or chronic GVHD. No SCD crises developed post SCT. One patient developed aplastic anemia while retaining 71% donor cell chimerism and underwent a second successful SCT from the same donor with fludarabine, cyclophosphamide and thymoglobulin. All patients have stable mixed chimerism resulting in full donor myeloid engraftment and normalization of blood count. With a median follow up of 9.1 months (2.3-32), all patients are alive and free of sickle cell crises. At last follow up, OS and EFS were 100% and 88.9%, respectively as shown in Figure 1.Conclusion:Herein, we report that SCT in adults with severe SCD using NMA regimen is safe, feasible and results in amelioration of disease severity. These excellent results are encouraging given the high prevalence and debilitating nature of SCD in our region. Follow up is on-going to assess the feasibility of sirolimus tapering and sustainability of donor chimerism status over time. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Establishment of A Mixed Chimeric Mouse Model of Allogeneic Bone Marrow Transplantation and Its Influencing Factors

To establish a mouse mixed chimerism (MC) model of nonmyeloablative allogeneic bone marrow transplantation(allo-BMT) and explore its affecting factors. The MC model was established by nonmyeloablative allo-BMT followed by high-dose post-transplant cyclophosphamide (PTCY). 123 mice in the experiments was retrospectively analyzed, and the factors related with the chimerism were explored with the univariate and multivariate logistic regression analysis. A multivariate linear regression was performed by R project to obtain a mathematical model for predicting the chimeric level with relevant affecting factors. The model presented mixed chimerism on day 14 after transplantation, and was characterized by a donor lymphocyte infusion (DLI) which significantly promoted donor engraftment on day 15, but transfplantation of PBS in control group was failed. Among 123 mice, 47 (38.21%) mice were MC, while 76 (61.79%) mice were non-MC in 123 mice, respectively; univariate analysis showed that the baseline body weight of mice (P=0.001, 17.84±1.19 g vs 18.50±0.94 g), total body irradiation(TBI，P=0.048) and the using of cyclophosphamide (P=0.16) were affected the chimeric state of mice, while the number of infusing cells and the time of detection showed no significant effects. Multivariate regression analysis showed that under certain conditions, the body weight of mice on day 0 was an independent factor affecting chimeric levels (OR=0.493, 95% CI 0.307-0.791, P=0.003). Through R project multiple linear regression, the math model was achieved, which was chimerism=6.09-12×weight(g)+80.03×TBI(Gy)-4.4×cell-counts (× 107) +0.38×CTX (mg/kg), R2=0.5841, P<0.001. The experiment presents a method for establishing a mixed chimeric mice model after non-myeloablative bone marrow transplantation and constructs a mathematical model with relevant factors affected chimerism status.