A Decade of Molecular Preimplantation Genetic Diagnosis of 350 Blastomeres for Beta-Thalassemia Combined with HLA Typing, Aneuploidy Screening and Sex Selection in Iran

Background: Preimplantation genetic diagnosis (PGD) has been developed to detect genetic disorders before pregnancy which is usually done on blastomeres biopsied from 8-cell stage embryos obtained from in vitro fertilization method (IVF).Here we report molecular PGD results for diagnosing of beta thalassemia (beta-thal) which are usually accompanied with evaluating chromosomal aneuploidies, HLA typing and sex selection.Methods: In this study, haplotype analysis was performed using short tandem repeats (STRs) in a multiplex nested PCR and the causative mutation was detected by Sanger sequencing.Results: We have performed PGDs on 350 blastomeres from 55 carrier couples; 142 blastomeres for beta-thal only, 75 for beta-thal and HLA typing, 76 for beta-thal in combination with sex selection, and 57 for beta-thal and aneuploidy screening. 150 blastomeres were transferable, 15 pregnancies were happened, and 11 babies born.We used 6 markers for beta-thal, 36 for aneuploidy screening, 32 for sex selection, and 35 for HLA typing. To our knowledge combining all these markers together and the number of STR markers are much more than any other studies which have ever done.Conclusions: PGD is a powerful diagnostic tool for carrier couples who desire to have a healthy child and wish to avoid medical abortion.

Improve in-depth immunological risk assessment to optimize genetic-compatibility and clinical outcomes in child and adolescent recipients of parental donor kidney transplants: protocol for the INCEPTION study

Background Parental donor kidney transplantation is the most common treatment option for children and adolescents with kidney failure. Emerging data from observational studies have reported improved short- and medium-term allograft outcomes in recipients of paternal compared to maternal donors. The INCEPTION study aims to identify potential differences in immunological compatibility between maternal and paternal donor kidneys and ascertain how this affects kidney allograft outcomes in children and adolescents with kidney failure. Methods This longitudinal observational study will recruit kidney transplant recipients aged ≤18 years who have received a parental donor kidney transplant across 4 countries (Australia, New Zealand, United Kingdom and the Netherlands) between 1990 and 2020. High resolution human leukocyte antigen (HLA) typing of both recipients and corresponding parental donors will be undertaken, to provide an in-depth assessment of immunological compatibility. The primary outcome is a composite of de novo donor-specific anti-HLA antibody (DSA), biopsy-proven acute rejection or allograft loss up to 60-months post-transplantation. Secondary outcomes are de novo DSA, biopsy-proven acute rejection, acute or chronic antibody mediated rejection or Chronic Allograft Damage Index (CADI) score of > 1 on allograft biopsy post-transplant, allograft function, proteinuria and allograft loss. Using principal component analysis and Cox proportional hazards regression modelling, we will determine the associations between defined sets of immunological and clinical parameters that may identify risk stratification for the primary and secondary outcome measures among young people accepting a parental donor kidney for transplantation. This study design will allow us to specifically investigate the relative importance of accepting a maternal compared to paternal donor, for families deciding on the best option for donation. Discussion The INCEPTION study findings will explore potentially differential immunological risks of maternal and paternal donor kidneys for transplantation among children and adolescents. Our study will provide the evidence base underpinning the selection of parental donor in order to achieve the best projected long-term kidney transplant and overall health outcomes for children and adolescents, a recognized vulnerable population. Trial registration The INCEPTION study has been registered with the Australian New Zealand Clinical Trials Registry, with the trial registration number of ACTRN12620000911998 (14th September 2020).

Myelodysplastic syndromes (MDS)

MDS in children is a rare group of hematopoietic stem cell clonal disorder. Allogeneic HSCT is the only curative treatment. HLA typing and the search for a compatible donor must be carried out upon diagnosis, for all patients. However, patients with refractory cytopenia of childhood without an unfavorable karyotype can keep the disease stable for a long time. Thus, in the absence of transfusion dependence or severe neutropenia, a careful observation strategy without treatment is recommended. The treatment of children diagnosed with MDS with excess blasts remains a major challenge. Allogeneic HSCT is the only curative treatment, although the data published in the literature generally include a small number of patients, heterogeneously transplanted. For children with MDS secondary to therapy, despite HSCT, the evolution is generally unfavorable.

Rigorous Benchmarking of HLA Callers for RNA Sequencing Data

Although precise identification of the human leukocyte antigen (HLA) allele is crucial for various clinical and research applications, HLA typing remains challenging due to high polymorphism of the HLA loci. However, with Next-Generation Sequencing (NGS) data becoming widely accessible, many computational tools have been developed to predict HLA types from RNA sequencing (RNA-seq) data. However, there is a lack of comprehensive and systematic benchmarking of RNA-seq HLA callers using large-scale and realist gold standards. In order to address this limitation, we rigorously compared the performance of 12 HLA callers over 50,000 HLA tasks including searching 30 pairwise combinations of HLA callers and reference in over 1,500 samples. In each case, we produced evaluation metrics of accuracy that is the percentage of correctly predicted alleles (two and four-digit resolution) based on six gold standard datasets spanning 650 RNA-seq samples. To determine the influence of the relationship of the read length over the HLA region on prediction quality using each tool, we explored the read length effect by considering read length in the range 37-126 bp, which was available in our gold standard datasets. Moreover, using the Genotype-Tissue Expression (GTEx) v8 data, we carried out evaluation metrics by calculating the concordance of the same HLA type across different tissues from the same individual to evaluate how well the HLA callers can maintain consistent results across various tissues of the same individual. This study offers crucial information for researchers regarding appropriate choices of methods for an HLA analysis.

Severe delayed hypersensitivity reactions to IL-1 and IL-6 inhibitors link to common HLA-DRB1*15 alleles

ObjectivesDrug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, delayed hypersensitivity reaction (DHR). We observed DRESS to inhibitors of interleukin 1 (IL-1) or IL-6 in a small group of patients with Still’s disease with atypical lung disease. We sought to characterise features of patients with Still’s disease with DRESS compared with drug-tolerant Still’s controls. We analysed human leucocyte antigen (HLA) alleles for association to inhibitor-related DHR, including in a small Kawasaki disease (KD) cohort.MethodsIn a case/control study, we collected a multicentre series of patients with Still’s disease with features of inhibitor-related DRESS (n=66) and drug-tolerant Still’s controls (n=65). We retrospectively analysed clinical data from all Still’s subjects and typed 94/131 for HLA. European Still’s-DRESS cases were ancestry matched to International Childhood Arthritis Genetics Consortium paediatric Still’s cases (n=550) and compared for HLA allele frequencies. HLA association also was analysed using Still’s-DRESS cases (n=64) compared with drug-tolerant Still’s controls (n=30). KD subjects (n=19) were similarly studied.ResultsStill’s-DRESS features included eosinophilia (89%), AST-ALT elevation (75%) and non-evanescent rash (95%; 88% involving face). Macrophage activation syndrome during treatment was frequent in Still’s-DRESS (64%) versus drug-tolerant Still’s (3%; p=1.2×10−14). We found striking enrichment for HLA-DRB1*15 haplotypes in Still’s-DRESS cases versus INCHARGE Still’s controls (p=7.5×10-13) and versus self-identified, ancestry-matched Still’s controls (p=6.3×10−10). In the KD cohort, DRB1*15:01 was present only in those with suspected anakinra reactions.ConclusionsDRESS-type reactions occur among patients treated with IL-1/IL-6 inhibitors and strongly associate with common HLA-DRB1*15 haplotypes. Consideration of preprescription HLA typing and vigilance for serious reactions to these drugs are warranted.

Epstein–Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report

EBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.

Single nucleotide polymorphisms within HLA region are associated with the outcomes of unrelated cord blood transplantation

Cord blood transplantation (CBT) provides a treatment scheme for hematologic diseases and leukemia in both children and adults. However, adverse reactions and transplantation-related death may still occur in patients receiving CBT even when donor and recipient have fully matched HLA in high-resolution HLA typing analysis. Single nucleotide polymorphisms (SNPs) of HLA-related and unrelated genes are known to associate with disease status of patients with unrelated stem cell transplantation. In this study, the genomic regions ranging from 500 base pairs upstream to 500 base pairs downstream of the eight SNPs that were reported as transplantation determinants by Petersdorf et al. were analyzed to evaluate whether genetic variants were associated with the survival status of patients, and the risk for severe (grades 3–4) graft-versus-host disease (GVHD) or cytomegalovirus (CMV) infection/reactivation. The analyses were performed in the mode of recipient genotype, donor genotype, and recipient-donor mismatching, respectively. By analysis of sixty-five patients and their HLA-matched unrelated donors, we found that five SNPs were associated with patient survival which included the recipient genotype with SNPs of rs107822 in the RING1 gene, and rs2070120, rs17220087 and rs17213693 in the HLA-DOB gene; and the recipient-donor mismatching with SNPs of rs9282369 in HLA-DOA gene, and rs2070120, rs17220087 and rs17213693 in the HLA-DOB gene. Five SNPs were associated with the risk for severe GVHD which included the donor genotype with SNPs of rs213210 and rs2523675; the recipient genotype with SNPs of rs9281491 in the HCP5 gene; and the recipient-donor mismatching with SNPs of rs209130 in the TRIM27 gene, and rs986522 in the COL11A2 gene. Six SNPs were related to the risk for CMV infection/reactivation which included the donor genotype with SNPs of rs435766, rs380924, and rs2523957; and the recipient-donor mismatching with SNPs of rs2070120, rs17220087, and rs17213693 in the HLA-DOB gene; and rs435766 and rs380924 in the MICD gene. This study provides the basis for larger analyses and if the results are confirmed, a way of selecting better unrelated CBT candidate donors.

Celiac Disease Prevalence Among Children with Dermatologic Pathology: Cross Sectional Study with Clinical Case Series

Background. Celiac disease (gluten enteropathy) is relatively rare disease. However, such patients have higher risk of skin pathology than general the population, and their therapy efficacy is limited by the use of gluten-free diet. Therefore, screening of dermatologic patients on celiac disease may be relevant. Objective. Our aim was to study the prevalence of celiac disease among children with skin pathology. Methods. The study included children hospitalized in dermatology department. Screening for celiac disease included detection in blood serum of antibodies (IgA, IgG, IgM) to tissue transglutaminase via rapid tests. In case of positive result of rapid test, we have repeated the estimation of antibodies (IgA, IgG) to tissue transglutaminase via immunochemiluminescent method with ImmunoCAP technology or via enzyme immunoassay. In case of positive serological test, we have performed HLA typing to determine haplotypes of DQ2 and DQ8, as well as esophagogastroduodenojejunoscopy (EGDJS) with biopsy of the duodenal and jejunal mucosa for further histological verification of the diagnosis. Results. We examined 1,000 children with various dermatologic pathologies. Rapid tests showed positive result in 21 patients (2.1%; 95% C11.3-3.2%). The presence of antibodies to tissue transglutaminase was confirmed via additional serological examination in all cases. HLA-haplotypes DQ2/8 were revealed in all patients with positive rapid test results. Typical form of gluten enteropathy was confirmed in 18/21 patients (86%) according to a histological study, thus, estimated prevalence of celiac disease is 1.8% (95% C11.1-2.8%). Conclusion. The prevalence of celiac disease remains underestimated among children with skin diseases. More studies are needed on the diagnostic features of rapid tests on tissue transglutaminase, as well as the benefits of screening for celiac disease to achieve patient-relevant clinical outcomes of skin pathology with wider gluten-free diet.

Combination of Haploidentical Hematopoietic with Low-Dose TBI and Cord Blood Transplantation for Hematologic Malignancies

Objective: To evaluate the efficacy and safety of the combination of haploidentical hematopoietic with low-doseTotal body irradiation（TBI）and cord blood transplantation for hematologic malignancies. Methods: This study was conducted as a retrospective review of medical records of 5 patients with hematologic malignancies who received a combination of haploidentical hematopoietic with low-dose TBI and cord blood transplantation at Affiliated HangZhou first people's hospital of Zhejiang University school of medicine, from March to June 2021. Results: 5 patients were included for the analysis. There were 1 acute myeloid leukemia (AML),2 acute lymphoblastic leukemia (ALL) and 2 Non-Hodgkin's Lymphoma(NHL). The minimal residual disease (MRD) of the two patients with ALL was positive before allo-HCT.Median age of patients at the time of allo-HCT was 30 years (range 21-63 years). All patients received low-dose TBI(4-6GY) +antithymocyte globulin（ATG）based conditioning.Cord blood units were selected based on the results of HLA typing and cell doses evaluated before freezing. Units with at least 4/6 matched HLA loci became the candidates. The median values of absolute total nucleated cell counts were 139.0 (80.8-240.0) × 10 7 / kg in The haploidentical grafts and 2.25 (1.32-3.10)× 10 7 / kg in the cord blood units,respectively. The median doses of CD34+ cells infused were 28.6 (22.0-51.1) × 10 5 / kg in the haploidentical grafts and 1.5 (1.0-3.5)×10 5/kg in the cord blood units, respectively.All patients attained complete engraftment,of which 3 were haploidentical engraftment and 2 were mixed hematopoietic chimerism that included haploidentical and cord blood engraftment.The median time to neutrophil engraftment was 12 (10-22) days and 13 (11-22) days for platelets. All patients were in complete remission with MRD-negetive during a median follow-up of 83 (34-136) days.No patients developed grade II-IV acute graft versus host desease. Conclusion:The results suggested that the combination of haploidentical hematopoietic with low-dose TBI and cord blood transplantation may potentially improve the outcome of HSCT. It offers a transplant alternative for patients with hematologic malignancies who lack matching donors. Disclosures No relevant conflicts of interest to declare.

Barriers to and Results of Allogeneic Hematopoietic Transplantation for Adult Patients with Sickle Cell Disease: An Analysis of Referrals and Transplants Performed at a Single Center in the Era of Haploidentical Donor Use

Allogeneic hematopoietic cell transplantation (alloHCT) is the only proven curative treatment for homozygous sickle cell disease (SCD) with debilitating symptoms. alloHCT may ideally be performed in children with severe manifestations of SCD before they develop irreversible organ dysfunction. However, many adult patients may also be appropriate for alloHCT. The historic lack of HLA-matched donors for this population has recently been ameliorated by the use of HLA-haploidentical donors with a T-replete graft and post-transplant cyclophosphamide (HAPLO). However, patients with SCD belong to ethnically and socio-economically underserved populations and may face several additional barriers to accessing complex and expensive curative strategies such as alloHCT. We analyzed SCD patients referred between January 2012 and April 2021 for alloHCT to our center- an adult only center with special expertise in HAPLO transplantation, located in an area with a large population of black patients. Success and barriers to progression from referral to alloHCT were assessed using our institutional database and coordinator records where they had been prospectively entered. Numbers of patients advancing from referral through stages of pre-transplant workup to alloHCT are shown in Table 1. Fifty-nine patients referred to our program for consideration of alloHCT for SCD during the period. The number of annual referrals increased during the studied period from 7 in 2012-2014 to 35 in 2018-2021. Of referred patients, 78% had an initial consult, 41% underwent high resolution HLA typing, 34% also had HLA typing of family members, 31% had an acceptable donor (HLA-identical, 8/8 matched MUD or HAPLO without donor-specific antibodies) and 14% underwent alloHCT. No significant differences were seen among the above categories with respect to age, gender, self-assigned race, year of referral and insurance status. Referrals came from a limited subset of community hematologists- with one physician each referring 9, 6, and 4 patients. Three physicians referred 3 patients, six referred 2 patients and nineteen referred 1 each. Reasons for referred patients not proceeding to alloHCT were: psychosocial issues including lack of caregiver support and history of non-compliance with treatments (n=15), insufficient disease severity by institutional criteria (n=14), failure of referred patient to attend initial consult (n=13), medical comorbidities precluding transplant (n=9), refusal of alloHCT by clinically and psycho-socially suitable patient (n=7), lack of acceptable donor despite suitable patient and search (n=4), refusal of relatives to undergo HLA-typing for an otherwise suitable patient (n=2). Patients (n=8) who underwent alloHCT a median of 253d post referral and 232d post initial consult. Transplant characteristics were: donor- HAPLO (6), MRD (1), MUD (1); graft source BM (8); intensity - NMA (4), RIC (4); median CD34 dose- 3.6x 10e6/kg (1.5-4.9); median CD3 dose 3.3x 10e7/kg (2.4-4.5); HCT-CI - <3 (2), >4(6); CMV pos patient with neg donor (3); ABO major incompatible (3). Engraftment was assessed using T-cell (CD3) and myeloid (CD33) donor chimerism. Median CD33 chimerisms were 100% on each assessment (d30, 60, 100, 180 and 360). Median CD3 chimerisms were 52%, 100%, 100%, 100%, 93% respectively. All evaluable patients achieved > 95% CD33 chimerism by d 30. Two patients (one MRD, one HAPLO) transplanted on an earlier protocol without ATG and thiotepa subsequently rejected their graft. All five HAPLO patients transplanted using the ATG/thiotepa based regimen used in BMTCTN 1507 had sustained high level engraftment (>95% CD33 chimerism) with freedom from SCD symptoms post-transplant. With a median follow-up of 39 months, three-year cumulative incidences of grade 2-4 acute GVHD, and moderate to severe NIH grade chronic GVHD were 12.5% and 0% respectively. Estimated 3 year overall survival was 85%. These data show that while most referred patients lack HLA matched donors, allo-HCT performed using HAPLO donors and RIC conditioning incorporating ATG and thiotepa with a BM graft is highly effective at eradicating SCD. However, many barriers are faced by referred patients resulting in only a small percentage of referred patients proceeding to transplant. Some of these barriers may be overcome by education of referring physicians, patients and their families Figure 1 Figure 1. Disclosures Solh: Jazz Pharmaceuticals: Consultancy; Partner Therapeutics: Research Funding; BMS: Consultancy; ADCT Therapeutics: Consultancy, Research Funding. OffLabel Disclosure: Preparative regimens for allogeneic transplantation typically involve off-label use of chemotherapy drugs