Unsupervised Clustering Analysis of Regimen and HLA Characteristics in Pediatric Umbilical Cord Blood Transplantation.

BACKGROUND: Human leukocyte antigen (HLA) matching is a critical factor in allogeneic unrelated hematopoietic cell transplantation (HCT) due to its impact on post-transplant survival and quality of life. Umbilical cord blood transplantation (UCBT) offers unique advantages but the optimal approach to graft selection and immunosuppression remains challenging. Unsupervised clustering, a machine learning technique has potential in analyzing transplant outcomes but its application in investigating leukemia outcomes has been limited. OBJECTIVE: To identify optimal combinations of HLA/KIR donor-patient pairing, conditioning, and immunosuppressive regimens in pediatric patients with acute lymphoblastic (ALL) or acute myeloblastic (AML) leukemia undergoing umbilical cord blood transplantation (UCBT). STUDY DESIGN: Outcome data for single, unmanipulated UCBT in pediatric AML (n=708) and ALL (n=1034) patients from the Eurocord/EBMT registry were analyzed using unsupervised clustering. Resulting clusters were used to inform post-hoc competing risks and Kaplan-Meier analyses. RESULTS: In AML, single HLA-C mismatches with other loci fully matched (7/8) associated with poorer relapse-free survival (RFS) (p=0.039), but a second mismatch at any other locus counteracted this effect. In ALL, total body irradiation (TBI) effectively prevented relapse mortality (p=0.007). KIR/HLA-C match status affected RFS in AML (p=0.039) but not ALL (p=0.8). Anti-thymocyte globulin (ATG) administration substantially increased relapse, with no relapses occurring in the 85 patients not receiving ATG. CONCLUSIONS: Our unsupervised clustering analyses generate several key statistical and mechanistic hypotheses regarding the relationships between HLA matching, conditioning regimens, immunosuppressive therapies, and transplantation outcomes in pediatric AML and ALL patients. HLA-C and killer immunoglobulin receptor (KIR) combinations significantly impact RFS in pediatric AML, but not ALL. ATG use in fully matched pediatric patients is associated with late-stage relapse. TBI regimens appear beneficial in ALL, with efficacy largely independent of histocompatibility variables. These findings reflect the distinct genetic and biological profiles of AML and ALL.

Secondary Oral Squamous Cell Carcinoma following Haematopoietic Stem Cell Transplantation.

We report 10 cases of oral squamous cell carcinoma (SCC) post-haematopoietic stem cell transplant (HSCT).. Median latency from HSCT to oral SCC diagnosis was 10 years (range: 4-17 years) with 90% reporting a history of chronic graft-versus-host disease (cGVHD) and 40% exhibited active severe manifestations of oral GVHD. Clinical findings at diagnosis included induration, ulceration, tenderness, bleeding, hyperkeratosis, speckling and lymphadenopathy. The tongue and buccal mucosa were the commonest sites affected. Disease stage at presentation ranged from T1N0M0 to T4N2M0. Management included surgical resection in 90% of cases ± chemo/radiotherapy. Median follow-up for the cohort was l years with 50% mortality rate. SCC-specific mortality was 30%. Our data highlight the importance of regular, active oral and cutaneous surveillance of post-HSCT patients in specialised dermatology clinics, irrespective of GVHD severity and length of iatrogenic immunosuppression.

Characterisation of RAET1E/ULBP4 exon 4 and 3' untranslated region genetic architecture reveals further diversity and allelic polymorphism.

NKG2D is a natural killer cell activating receptor recognising ligands on infected or tumorigenic cells, leading to their cytolysis. There are eight known genes encoding NKG2D ligands: MICA, MICB and ULBP1-6. MICA and MICB are highly polymorphic and well characterised, whilst ULBP ligands are less polymorphic and the functional implication of their diversity is not well understood. Using International HLA and Immunogenetics Workshop (IHIW) cell line DNA, we previously characterised alleles of the RAET1E gene (encoding ULBP4 proteins), including the 5' UTR promoter region and exons 1-3. We found 11 promoter haplotypes associating with alleles based on exons 1-3, revealing 19 alleles overall. The current study extends this analysis using 87 individual DNA samples from IHIW cell lines or cord blood to include RAET1E exon 4 and the 3' UTR, as polymorphism in these regions have not been previously investigated. We found two novel exon 4 polymorphisms encoding amino acid substitutions altering the transmembrane domain. An amino acid substitution at residue 233 was unique to the RAET1E*008 allele whereas the substitution at residue 237 was shared between groups of alleles. Additionally, four haplotypes were found based on 3' UTR sequences, which were unique to certain alleles or shared with allele groups based on exons 1-4 polymorphisms. Furthermore, putative microRNAs were identified that may interact with these polymorphic sites, repressing transcription and potentially affecting expression levels.

Impact of donor NKG2D and MICA gene polymorphism on clinical outcomes of adult and paediatric allogeneic cord blood transplantation for malignant diseases.

OBJECTIVES: NKG2D is an activating receptor expressed by natural killer (NK) and CD8+ T cells and activation intensity varies by NKG2D expression level or nature of its ligand. An NKG2D gene polymorphism determines high (HNK1) or low (LNK1) expression. MICA is the most polymorphic NKG2D ligand and stronger effector cell activation associates with methionine rather than valine at residue 129. We investigated correlation between cord blood (CB) NKG2D and MICA genotypes and haematopoietic stem cell (HSC) transplant outcome. METHODS: We retrospectively studied 267 CB HSC recipients (178 adult and 87 paediatric) who underwent transplant for malignant disease between 2007 and 2018, analysing CB graft DNA for NKG2D and MICA polymorphisms using Sanger sequencing. Multivariate analysis was used to correlate these results with transplant outcomes. RESULTS: In adult patients, LNK1 homozygous CB significantly improved 60-day neutrophil engraftment (hazard ratio (HR) 0.6; 95% confidence interval (CI) 0.4-0.9; p = .003). In paediatrics, HNK1 homozygous CB improved 60-day engraftment (HR 0.4; 95% CI 0.2-0.7; p = .003), as did MICA-129 methionine+ CB grafts (HR 1.7 95% CI 1.1-2.6; p = .02). CONCLUSION: CB NKG2D and MICA genotypes potentially improve CB HSC engraftment. However, results contrast between adult and paediatric recipients and may reflect transplant procedure disparities between cohorts.

Impaired humoral and cellular response to primary COVID-19 vaccination in patients less than 2 years after allogeneic bone marrow transplant.

Allogeneic haematopoietic stem cell transplant (HSCT) recipients remain at high risk of adverse outcomes from coronavirus disease 2019 (COVID-19) and emerging variants. The optimal prophylactic vaccine strategy for this cohort is not defined. T cell-mediated immunity is a critical component of graft-versus-tumour effect and in determining vaccine immunogenicity. Using validated anti-spike (S) immunoglobulin G (IgG) and S-specific interferon-gamma enzyme-linked immunospot (IFNγ-ELIspot) assays we analysed response to a two-dose vaccination schedule (either BNT162b2 or ChAdOx1) in 33 HSCT recipients at ≤2 years from transplant, alongside vaccine-matched healthy controls (HCs). After two vaccines, infection-naïve HSCT recipients had a significantly lower rate of seroconversion compared to infection-naïve HCs (25/32 HSCT vs. 39/39 HCs no responders) and had lower S-specific T-cell responses. The HSCT recipients who received BNT162b2 had a higher rate of seroconversion compared to ChAdOx1 (89% vs. 74%) and significantly higher anti-S IgG titres (p = 0.022). S-specific T-cell responses were seen after one vaccine in HCs and HSCT recipients. However, two vaccines enhanced S-specific T-cell responses in HCs but not in the majority of HSCT recipients. These data demonstrate limited immunogenicity of two-dose vaccination strategies in HSCT recipients, bolstering evidence of the need for additional boosters and/or alternative prophylactic measures in this group.

Pruritic rash following haploidentical stem cell transplantation.

A woman who had undergone haematopoietic stem cell transplantation presented with cutaneous features suggestive of graft-versus-host disease. Histopathological examination revealed a diffuse dermal infiltration of atypical monomorphic cells with a high proliferative index. Immunohistochemistry revealed positivity for monocytic markers, but negativity for T-cell markers.

"Is this the GVHD?" A qualitative exploration of quality of life issues in individuals with graft-versus-host disease following allogeneic stem cell transplant and their experiences of a specialist multidisciplinary bone marrow transplant service.

BACKGROUND: Graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following allogeneic stem cell transplantation. These patients face unique challenges due to the complexity of GVHD which can affect multiple organ systems, and the toxicity of treatments. Despite the known impact on quality of life (QOL), qualitative data within the bone marrow transplantation (BMT) literature is rare, and there has been no qualitative work exploring patient experience of specialist healthcare provision for GVHD in the United Kingdom. METHODS: We conducted a primary explorative qualitative study of the experience of QOL issues and multidisciplinary care in patients with chronic GVHD following allogeneic stem cell transplantation. Eight patients were identified using convenience sampling from specialist BMT outpatient clinics. Following consent, patients were interviewed individually via telephone. Transcripts of interviews were analyzed using an inductive thematic approach. RESULTS: Mean participant age was 61-years-old (range 45-68), with a mean time post-transplant of 3 years at time of interview (range 3 months-15 years). Five key QOL themes were identified: (1) 'Restricted as to what I can do'; (2) Troubling symptoms-'you can sort of get GVHD anywhere'; (3) Confusion/uncertainty over GVHD symptoms-'Is this the GVHD?'; (4) Unpredictable course and uncertainty about the future; and (5) Adapting to the sick role. In addition, four themes related to experience of service provision were identified: (1) personal care and close relationship with BMT nurses; (2) efficiency versus long waits-'On the case straight away'; (3) information provision-'went into it with a bit of a rosy view'; and (4) the role of support groups. CONCLUSIONS: These qualitative data reflect the heterogeneity of experiences of the GVHD patient population, reflecting the need for a flexible and nuanced approach to patient care with emphasis on comprehensive information provision. We have identified the key role that BMT specialist nurses within the multidisciplinary team play in supporting patients. We advocate future research should focus on ways to meet the complex needs of this patient group and ensure that the personal care and close relationships are not lost in service redesigns embracing remote consultations.

A multicenter comparative acute myeloid leukemia study: can we explain the differences in the outcomes in resource-constrained settings?

Outcomes in acute myeloid leukemia (AML) are dependent on patient- and disease-characteristics, treatment, and socioeconomic factors. AML outcomes between resource-constrained and developed countries have not been compared directly. We analyzed two cohorts: from São Paulo state, Brazil (USP, n = 312) and Oxford, United Kingdom (OUH, n = 158). USP cohort had inferior 5-year overall survival compared with OUH (29% vs. 49%, adjusted-p=.027). USP patients have higher early-mortality (23% vs. 6% p<.001) primarily due to multi-resistant Gram-negative bacterial and fungal infections. USP had higher 5-year cumulative incidence of relapse (60% vs. 50%, p=.0022), were less likely to undergo hematopoietic stem cell transplant (HSCT) (28% vs. 75%, p<.001) and waited longer for HSCT (median, 23.8 vs. 7.2 months, p<.001). Three-year survival in relapsed patients was worse in USP than OUH (10% vs. 39%, p<.001). Our study indicates that efforts to improve AML outcomes in Brazil should focus on infection prevention and control, and access to HSCT.

Enumerating regulatory T cells in cryopreserved umbilical cord blood samples using FOXP3 methylation specific quantitative PCR.

BACKGROUND: Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders. However, it carries significant risk of morbidity and mortality. To improve patient outcomes, better graft selection strategies are needed, incorporating HLA matching with clinically important graft characteristics. Studies have shown that the cellular content of HCT grafts, specifically higher ratios of T regulatory (Tregs)/T cells, are important factors influencing outcomes when using adult peripheral blood mobilised grafts. So far, no equivalent study exists in umbilical cord blood (CB) transplantation due to the limitations of cryopreserved CB samples. STUDY DESIGN AND METHODS: To establish the most robust and efficient way to measure the Treg content of previously cryopreserved CB units, we compared the enumeration of Treg and CD3+ cells using flow cytometry and an epigenetic, DNA-based methodology. The two methods were assessed for their agreement, consistency and susceptibility to error when enumerating Treg and CD3+ cell numbers in both fresh and cryopreserved CB samples. RESULTS: Epigenetic enumeration gave consistent and comparable results in both fresh and frozen CB samples. By contrast, assessment of Tregs and CD3+ cells by flow cytometry was only possible in fresh samples due to significant cell death following cryopreservation and thawing. CONCLUSION: Epigenetic assessment offers significant advantages over flow cytometry for analysing cryopreserved CB; similar cell numbers were observed both in fresh and frozen samples. Furthermore, multiple epigenetic assessments can be performed from DNA extracted from small cryopreserved CB segments; often the only CB sample available for clinical studies.

The Migratory Properties and Numbers of T Regulatory Cell Subsets in Circulation Are Differentially Influenced by Season and Are Associated With Vitamin D Status.

The control of peripheral immune responses by FOXP3+ T regulatory (Treg) cells is essential for immune tolerance. However, at any given time, Treg frequencies in whole blood can vary more than fivefold between individuals. An understanding of factors that influence Treg numbers and migration within and between individuals would be a powerful tool for cellular therapies that utilize the immunomodulatory properties of Tregs to control pathology associated with inflammation. We sought to understand how season could influence Treg numbers and phenotype by monitoring the proportion of natural thymus-derived Tregs (nTregs) defined as (CD3+CD4+CD25+FOXP3+CD127-/low ) cells as a proportion of CD4+ T cells and compared these to all FOXP3+ Tregs (allTregs, CD3+CD25+FOXP3+CD127-/low ). We were able to determine changes within individuals during 1 year suggesting an influence of season on nTreg frequencies. We found that, between individuals at any given time, nTreg/CD4+ T cells ranged from 1.8% in February to 8.8% in the summer where median nTreg/CD4 in January and February was 2.4% (range 3.75-1.76) and in July and August was 4.5% (range 8.81-3.17) p = 0.025. Importantly we were able to monitor individual nTreg frequencies throughout the year in donors that started the year with high or low nTregs. Some nTreg variation could be attributed to vitamin D status where normal linear regression estimated that an absolute increase in nTreg/CD4+ by 0.11% could be expected with 10 nmol increase in serum 25 (OH) vitamin D3 (p = 0.005, 95% CI: 0.03-0.19). We assessed migration markers on Tregs for the skin and/or gut. Here cutaneous lymphocyte associated antigen (CLA+) expression on CD25+FOXP3+CD4+/CD4+ was compared with the same population expressing the gut associated integrin, ß7. Gut tropic CD25+FOXP3+ß7+Tregs/CD4+ had similar dynamics to nTreg/CD4+. Conversely, CD25+FOXP3+CLA+Tregs/CD4+ showed no association with vitamin D status. Important for cellular therapies requiring isolation of Tregs, the absolute number of ß7+CD4+CD25+FOXP3+Tregs was positively associated with 25(OH)vitamin D3 (R2 = 0.0208, r = 0.184, p = 0.021) whereas the absolute numbers of CLA+CD4+CD25+FOXP3+Tregs in the periphery were not influenced by vitamin D status. These baseline observations provide new opportunities to utilize seasonal variables that influence Treg numbers and their migratory potential in patients or donors.

Corrigendum: Functional Characterisation and Analysis of the Soluble NKG2D Ligand Repertoire Detected in Umbilical Cord Blood Plasma.

[This corrects the article DOI: 10.3389/fimmu.2018.01282.].

Nanopore metagenomic sequencing to investigate nosocomial transmission of human metapneumovirus from a unique genetic group among haematology patients in the United Kingdom.

BACKGROUND: Human metapneumovirus (HMPV) infection causes a spectrum of respiratory tract disease, and may be a significant pathogen in the context of immunocompromise. Here, we report direct-from-sample metagenomic sequencing of HMPV using Oxford Nanopore Technology. METHODS: We applied this sequencing approach to 25 respiratory samples that had been submitted to a clinical diagnostic laboratory in a UK teaching hospital. These samples represented 13 patients under the care of a haematology unit over a 20-day period in Spring 2019 (two sampled twice), and ten other patients elsewhere in the hospital between 2017-2019. RESULTS: We generated HMPV reads from 20/25 samples (sensitivity 80% compared to routine diagnostic testing) and retrieved complete HMPV genomes from 15/20 of these. Consensus sequences from Nanopore data were identical to those generated by Illumina, and represented HMPV genomes from two distinct sublineages, A2b and B2. Sequences from ten haematology patients formed a unique genetic group in the A2b sublineage, not previously reported in the UK. Among these, eight HMPV genomes formed a cluster (differing by ≤3 SNPs), likely to reflect nosocomial transmission, while two others were more distantly related and may represent independent introductions to the haematology unit. CONCLUSION: Nanopore metagenomic sequencing can be used to diagnose HMPV infection, although more work is required to optimise sensitivity. Improvements in the use of metagenomic sequencing, particularly for respiratory viruses, could contribute to antimicrobial stewardship. Generation of full genome sequences can be used to support or rule out nosocomial transmission, and contribute to improving infection prevention and control practices.

Molecular MRD status and outcome after transplantation in NPM1-mutated AML.

Relapse remains the most common cause of treatment failure for patients with acute myeloid leukemia (AML) who undergo allogeneic stem cell transplantation (alloSCT), and carries a grave prognosis. Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry before alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays, which have far greater sensitivity. We analyzed pretransplant blood and bone marrow samples by reverse-transcription polymerase chain reaction in 107 patients with NPM1-mutant AML enrolled in the UK National Cancer Research Institute AML17 study. After a median follow-up of 4.9 years, patients with negative, low (<200 copies per 105ABL in the peripheral blood and <1000 copies in the bone marrow aspirate), and high levels of MRD had an estimated 2-year overall survival (2y-OS) of 83%, 63%, and 13%, respectively (P < .0001). Focusing on patients with low-level MRD before alloSCT, those with FLT3 internal tandem duplications(ITDs) had significantly poorer outcome (hazard ratio [HR], 6.14; P = .01). Combining these variables was highly prognostic, dividing patients into 2 groups with 2y-OS of 17% and 82% (HR, 13.2; P < .0001). T-depletion was associated with significantly reduced survival both in the entire cohort (2y-OS, 56% vs 96%; HR, 3.24; P = .0005) and in MRD-positive patients (2y-OS, 34% vs 100%; HR, 3.78; P = .003), but there was no significant effect of either conditioning regimen or donor source on outcome. Registered at ISRCTN (http://www.isrctn.com/ISRCTN55675535).

Immune reconstitution following umbilical cord blood transplantation: IRES, a study of UK paediatric patients.

To obtain a qualitative as well as quantitative view immune reconstitution following umbilical cord blood (UCB) transplantation of paediatric patients, we utilised a broad panel of flow cytometry markers to monitor the phenotypes of lymphoid and myeloid cells at 1-12 months post-transplant. Samples were received from 46 patients with a median age of 3.3 years and survival was 76% at 1 year. Monocytes were at similar or higher median levels than in adult controls at all times tested, with a high CD16+ proportion in the first 3 months. NK cells were also within adult ranges, with a CD56++ high proportion in the first 6 months. B cell recovery was seen from 2 months in most patients and T cells from 3 months, both were delayed with anti-thymocyte globulin (ATG) treatment. CD4:CD8 ratios were high in the first 6 months, and the proportion of T cells with recent thymic emigrant and naïve phenotypes rose from 3 months. NK and plasmacytoid dendritic cell numbers remained at reduced levels in patients not surviving to 1 year. Our results can serve as a useful reference for detailed monitoring of immune reconstitution in paediatric recipients of UCB.

The fight against cancer: is harnessing the immune system the ultimate strategy?

Malignancy is a micro-evolutionary phenomenon shaped by selection pressures. Chief among these is the adaptive immune system, which recognizes malignant cells as a threat and attempts to eradicate them. The task is not easily achieved - if it were, cancer would not be a part of our human experience. The field of immunotherapy has rapidly expanded over the last two decades. It has produced some of the most exciting results of 21st century medicine, and has deepened clinicians' understanding of the relationship between malignancy and the immune response. This review discusses this relationship and analyses key tools in the immunotherapy arsenal.