Gandhi, Manish J.

Abstract Abstract 467 Background: While donor-recipient disparity at HLA loci is associated with greater risk for severe acute graft vs. host disease (GVHD) and inferior survival after unrelated donor allogeneic hematopoietic cell transplantation (HCT), the impact of amino acid substitution (AAS) at peptide binding pockets of the HLA molecule is incompletely understood. Methods: Adult and pediatric patients who received myeloablative or reduced intensity/non-myeloablative first unrelated bone marrow or peripheral blood stem cell transplantation for AML, ALL, CML or MDS between 1988 and 2009 were included. Donors and patients were fully high resolution matched for HLA-A, B, C, and DRB1 (8/8) or had single mismatch (7/8) at one HLA class I locus. Among 7/8 donor-recipient pairs, cases were categorized based on the presence or absence of the AAS of interest at positions 9, 77, 99, 116, or 156 of the class I molecule. In multivariable analysis accounting for patient, disease, and transplantation variables, we studied the independent impact of AAS at these residues on risk for grade III-IV acute GVHD, chronic GVHD, treatment-related mortality, primary malignancy relapse, and overall survival. We compared 7/8 donor-recipient pairs with AAS of interest to 7/8 pairs without these AAS in the primary analyses. Additionally, we performed this analysis restricted to each HLA class I locus. Results: Donor-recipient pairs were 8/8 matched (n=5282), 7/8 with AAS of interest (n=1713), or 7/8 without AAS of interest (n=318). In multivariable analysis, AAS at position 116 was associated with increased risk for grade III-IV acute GVHD (HR 1.21, 1.04–1.42, p=0.0165). No other significant association was detected between AAS studied and clinical outcomes. In multivariable analysis restricted to each class I HLA locus, we detected the following: Among 7/8 matched pairs with mismatch at HLA-C, AAS at position 116 was associated with increased risk for severe acute GVHD (HR 1.42, 1.13–1.79, p=0.0031) and inferior OS (HR 1.2, 1.01–1.41, p=0.0343). AAS at position 99 was associated with increased TRM (HR 1.37, 1.11–1.69, p=0.0037). Of 7/8 pairs with mismatch at HLA-B, AAS at position 9 was associated with increased chronic GVHD (HR 2.19, 1.31–3.66, p=0.0029). Specific amino acid substitution pairs with frequency > 30 were tested for association with HCT outcomes. None met the significance level of 0.00125, pre-specified for multiple comparisons. Conclusions: These results support the concept that AAS at key peptide-binding residues in the HLA class I molecule are associated with increased risk for severe acute GVHD and lower survival. Disclosures: No relevant conflicts of interest to declare.

Abstract Previously in our lab, a missense mutation in the MASTL (FLJ14813) gene on human chromosome 10 was linked to an autosomal dominantly inherited thrombocytopenia in a single pedigree. The mutation results in an amino acid change from glutamic acid at position 167 to aspartic acid and correlates with the inheritance of the disorder in all the affected individuals. The patients present with mild thrombocytopenia with an average platelet count of 60,000 platelets per microliter of blood. Northern blot analysis indicates that MASTL mRNA is restricted in its expression to hematopoietic and cancer cell lines. Functional studies presented here demonstrate a direct relationship between the transient knockdown of the Microtubule Associated Serine/Threonine Like (MASTL) kinase expression and the reduction of circulating thrombocytes in zebrafish (Danio rerio) while erythrocytes development normally. This transient knockdown of MASTL in zebrafish correlates with a decrease in the expression of the thrombopoietin receptor, c-mpl, and the CD41 platelet adhesion GpIIb protein but has no effect on essential housekeeping zebrafish gene, EF1α. The transient expression of a fluorescent protein fused to MASTL and E167D MASTL kinase demonstrates that MASTL localizes to the nucleus of cells as determined by confocal microscopy and TO-PRO-3 counterstain.

Abstract Background: Platelet transfusions are important for supporting patients with hematologic disorders. Refractoriness to platelet transfusions may lead to serious complications and pose a challenge to clinical management. A variety of laboratory strategies to optimize transfusion support in this population are being used; however, data on clinical correlation and success of these interventions is limited. We report the laboratory testing, transfusion yield, and clinical correlation in a cohort of patients receiving cross-matched platelet units. Methods: All consecutive adult patients refractory to platelet transfusions (failure to increment platelet count by 10 x 10(9) times two) with platelet crossmatch performed between June 2014 and April 2016 at our institution were included. Detailed clinical information, laboratory data of the crossmatch results, and Single Antigen Bead (SAB; Luminex) for each platelet unit cross-matched was retrospectively collected. Mean fluorescence intensity (MFI) of antibodies directed towards donor platelet HLA antigens identified through SAB were grouped into zones according to previously validated laboratory data: green zone (<4,000 MFI), yellow zone (4000-10000) and red zone (>10,000). Adequate transfusion yield was defined as an absolute increase of at least 10 x 10((9)/L platelets on a post-transfusion (i.e., within 1 hour) sample. Patients with major bleeding per International Society on Thrombosis and Haemostasis (ISTH) criteria, palpable splenomegaly, fever during transfusion, DIC, active sepsis, or antibodies directed against human platelet antigens were excluded from the final analysis. Importantly, the Hematologist and Pathologist were blinded to the other's data until collection was completed. Data analysis correlating adequate transfusion yield and laboratory parameters was performed. Results: 22 patients (72% female) met our study criteria. All patients had a hematologic malignancy with prolonged pancytopenia after chemotherapy, 4 (18%) had undergone allogeneic bone marrow transplantation. A total of 422 platelet cross-matches were performed, with 17 patients ultimately receiving 46 cross-matched units. The indication for transfusion was: prophylactic 35 (76%), bleeding 4 (9%) and peri-procedural 7 (15%). Ten (22%) transfusion events were excluded: splenomegaly 3, fever 4 and active severe bleeding 2. In one case a post transfusion platelet count was not performed. The overall success rate of transfusion was 64% (23/36). All transfusions leading to an adequate yield had a negative crossmatch. Of the 13 ineffective transfusions, 2 had a positive crossmatch. Based on single antigen MFI assignment, 26 (74%) donor units were classified as green zone, 8 (23%) yellow and 1 (3%) red. The success rates according to the single MFI zone assignment are presented in Table 1. These MFI-defined assignments were predictive of clinically successful transfusions. Conclusion: Selection of appropriate donor platelet units in refractory patients is a challenge that is managed in a variety of ways. While crossmatching is sometimes necessary, a strategy of antigen-avoidance can dramatically increase the identification of potentially effective platelet units for many patients. Understanding of how to navigate HLA antibody data is essential for identifying which HLA antigens should be avoided. Based on these data, in general, our practice is to avoid transfusing a platelet when there is a donor specific antibody of 8,000 MFI or greater. Furthermore, in our dataset there were two patients with both successful and unsuccessful transfusions after receiving crossmatch-negative units with green zone assignment. This finding highlights the limitations of laboratory testing and emphasizes the importance of post-transfusion platelet counts for continuously improving the platelet support of each immune-refractory patient. Disclosures No relevant conflicts of interest to declare.

Discovery of a novel HLA‐B*45:22 allele in a Middle Eastern heart and lung transplant candidate.

A new allele, now named HLA‐C*02:138, was discovered during testing of a registry donor for possible stem cell transplantation.

Phased sequencing identified the HLA‐C*07:607 allele in an African‐American patient and sibling donor.

BACKGROUND Platelet transfusion–refractoriness is a challenging and expensive clinical scenario seen most often in patients with hematologic malignancies. Although the majority of platelet transfusion–refractory cases are due to nonimmune causes, a significant minority are caused by alloimmunization against Class I human leukocyte antigens (HLAs) or human platelet antigens (HPAs). Such platelet transfusion–refractory patients can be effectively managed with appropriate antigen‐negative products. STUDY DESIGN AND METHODS Our institution has developed a diagnostic and management algorithm for the platelet transfusion–refractory patient with an early focus on identifying those cases caused by immune‐mediated factors. Using physical platelet cross‐matches to initially classify platelet transfusion–refractory patients as immune‐mediated or not, cross‐match–compatible inventory is then provided to immune‐mediated patients, whereas subsequent HLA (with or without HPA) testing is performed. RESULTS Our blood donor program performs Class I HLA typing of all repeat platelet donors to facilitate the identification of antigen‐negative platelet units (virtual cross‐matching) as well as the recruitment of HLA‐matched donors. The platelet transfusion–refractoriness algorithm realizes an initial net cost savings once two apheresis platelets are saved from use for each newly identified, immune‐mediated platelet transfusion–refractory patient. CONCLUSION An algorithm utilizing physical platelet cross‐matches, Class I HLA and HPA antibody testing, and upfront Class I HLA typing of platelet donors leads to overall resource savings and improved clinical management for platelet transfusion–refractory patients.

SummaryBackgroundTissue transglutaminase (tTG) immunoglobulin A (IgA) testing is a sensitive adjunct to the diagnosis of coeliac disease. The threshold for positivity was developed for diagnosis, with negative results reported as below the reference value (<4 U/mL).AimTo investigate if an undetectable (tTG IgA<1.2 U/mL) is more predictive of healing compared to patients with negative but detectable serology (1.2‐3.9 U/mL).MethodsWe performed a retrospective study of 402 treated coeliac disease patients seen at the Mayo Clinic with negative tTG IgA values drawn within 1 month of duodenal biopsy between January 2009 and December 2015. The Corazza‐Villanacci score was used to assess mucosal healing. The presence of gastrointestinal symptoms was also collected. Logistic regression was used to assess the relationship of clinical variables with a normal biopsy.ResultsPatients with undetectable titres more frequently had normal duodenal histology compared to patients with detectable tTG IgA levels (117/240 vs. 53/162; OR=1.96; 1.292, 2.961). Asymptomatic patients more frequently had normal duodenum as compared to symptomatic patients (88/163 vs. 82/239; OR=2.25; CI: 1.494, 3.377). Patients with undetectable serology and on a gluten‐free diet for ≥2 years were more likely to have no villous atrophy compared to patients with detectable serology (148/192 vs. 55/88; OR=2.02; CI: 1.17, 3.49).ConclusionIn subjects recovering from coeliac disease with negative tTG IgA serology, an undetectable titre is associated with normal histology on follow‐up biopsy.

AbstractObjectiveHLA‐DPB1 matching may impact allogeneic hematopoietic stem cell transplantation (ASCT) outcomes; however, this locus is not in linkage disequilibrium with the remainder of the HLA genes. After classifying HLA‐DPB1 mismatches based on T‐cell epitope, avoiding non‐permissive mismatches may impact survival. We tested this hypothesis at a single academic institution.MethodsRetrospective HLA‐DPB1 genotyping was performed on 153 adult patients who underwent ASCT and unrelated donors matched for HLA‐A, B, C, DRB1, and DQB1 loci (10/10). Using the ImMunoGeneTics/HLA T‐cell epitope matching algorithm, mismatch status was classified as permissive or non‐permissive.ResultsOf 153 donor‐recipient pairs, 22 (14.4%) were HLA‐DPB1 matches, 64 (42.8%) permissive mismatches, and 67 (43.8%) non‐permissive mismatches. DPB1 mismatch increased risk of chronic graft‐versus‐host disease (cGVHD; RR 2.89 [1.19‐9.53], P=.016) compared with DPB1‐matched transplants, but there were no differences in overall mortality, risk of relapse, or acute GVHD (aGVHD). Combining matches and permissive mismatches and comparing to non‐permissive mismatches, there was no significant difference in overall survival or relapse; however, patients receiving non‐permissive mismatched transplants experienced greater risk of aGVHD overall and severe aGVHD (RR 1.66 [1.13‐2.44], P=.010 and RR 1.97 [1.10‐3.59], P=.024, respectively).ConclusionIn this single‐center study, HLA‐DPB1 matching influenced outcomes of patients undergoing ASCT for hematologic malignancy.

Context.— Numerous feasibility studies to type human leukocyte antigens (HLAs) by next-generation sequencing (NGS) have led to the development of vendor-supported kits for HLA typing by NGS. Some clinical laboratories have introduced HLA-NGS, and many are investigating the introduction. Standards from accrediting agencies form the regulatory framework for introducing this test into clinical laboratories. Objectives.— To provide an assessment of metrics and considerations relevant to the successful implementation of clinical HLA-NGS typing, and to provide as a reference a validated HLA-NGS protocol used clinically since December 2013 at the Children's Hospital of Philadelphia (Philadelphia, Pennsylvania). Data Sources.— The HLA-NGS has been performed on 2532 samples. The initial 1046 and all homozygous samples were also typed by an alternate method. The HLA-NGS demonstrated 99.7% concordance with the alternate method. Ambiguous results were most common at the DPB1 locus because of a lack of phasing between exons 2 and 3 or the unsequenced exon 1 (533 of 2954 alleles; 18.04%) and the DRB1 locus because of not sequencing exon 1 (75 of 3972 alleles; 1.89%). No ambiguities were detected among the other loci. Except for 2 false homozygous samples, all homozygous samples (1891) demonstrated concordance with the alternate method. The article is organized to address the critical elements in the preanalytic, analytic, and postanalytic phases of introducing this assay into the clinical laboratory. Conclusions.— The results demonstrate that HLA typing by NGS is a highly accurate, reproducible, efficient method that provides more-complete sequencing information for the length of the HLA gene and can be the single methodology for HLA typing in clinical immunogenetics laboratories.