Dimitrios Monos

Ventricular assist device-associated anti-human leukocyte antigen antibody sensitization in pediatric patients bridged to heart transplantation

BACKGROUND Ventricular assist devices (VAD) are associated with the formation of antibodies to anti-human leukocyte antigens (HLA) or sensitization. The incidence and effects of VAD-associated anti-HLA sensitization have not been well studied in the pediatric population. METHODS A retrospective review of all patients undergoing VAD implant at our institution from 1998 to 2008 was performed. Panel reactive antibody (PRA) results before VAD implant, after VAD implant, and after orthotopic heart transplantation (OHT) were recorded. Patients who became sensitized (PRA for class I and/or II immunoglobulin G antibodies >or= 10%) on VAD support were compared with non-sensitized patients with regard to demographics, diagnosis, device type, and blood product exposure on VAD support. Outcomes after OHT were also compared between groups. RESULTS VAD support was initiated in 20 patients (median age, 14.4 years), with 75% survival to OHT or recovery. PRA data before and after VAD implant were available for 17 patients. VAD-associated sensitization developed in 35% of recipients. There were no differences between those sensitized in association with VAD support and non-sensitized patients with regard to age, gender, diagnosis, device type, extracorporeal membrane oxygenation use, or blood product exposure on VAD support. Black race predicted sensitization on VAD (p = 0.02). There were no differences in survival or rejection between groups. CONCLUSIONS VAD therapy was associated with the development of anti-HLA sensitization in 35% of recipients. Black race predicted sensitization, but there were no differences in overall survival or outcomes after OHT.

Abdominal Aortic Aneurysm Is a Specific Antigen‐Driven T Cell Disease

Abstract:  To determine whether monoclonal/oligoclonal T cells are present in abdominal aortic aneurysm (AAA) lesions, we amplified β‐chain T cell receptor (TCR) transcripts from these lesions by the nonpalindromic adaptor (NPA)‐polymerase chain reaction (PCR)/V‐β‐specific PCR followed by cloning and sequencing. Sequence analysis revealed the presence of substantial proportions of identical β‐chain TCR transcripts in AAA lesions in 9 of 10 patients examined, strongly suggesting the presence of oligoclonal populations of αβ TCR+ T cells. We have also shown the presence of oligoclonal populations of γδ TCR+ T cells in AAA lesions. Sequence analysis after appropriate PCR amplification and cloning revealed the presence of substantial proportions of identical VγI and VγII TCR transcripts in 15 of 15 patients examined, and of Vδ1 and Vδ2 TCR transcripts in 12 of 12 patients. These clonal expansions were very strong. All these clonal expansions were statistically significant by the binomial distribution. In other studies, we determined that mononuclear cells infiltrating AAA lesions express early‐ (CD69), intermediate‐ (CD25, CD38), and late‐ (CD45RO, HLA class II) activation antigens. These findings suggest that active ongoing inflammation is present in the aortic wall of patients with AAA. These results demonstrate that oligoclonal αβ TCR+ and γδ TCR+T cells are present in AAA lesions. These oligoclonal T cells have been clonally expanded in vivo in response to yet unidentified antigens. Although the antigenic specificity of these T cells remains to be determined, these T cells may play a significant role in the initiation and/or the propagation of the AAA. It appears that AAA is a specific antigen‐driven T cell disease.

Persistence of anti-human leukocyte antibodies in congenital heart disease late after surgery using allografts and whole blood

BACKGROUND Allografts are used for vascular reconstruction in many forms of congenital heart disease. Although allografts induce anti-human leukocyte antibody (HLA) formation, much about this response is unknown. METHODS Three groups of patients aged 8 to 18 years old underwent analysis for class I and II anti-HLA antibodies using Luminex. Groups were defined by timing of allograft exposure and diagnosis at Norwood for hypoplastic left heart syndrome (neonatal group), at Glenn for single-ventricle lesions not requiring arch reconstruction (infant group), and cardiac defects repaired during infancy without allografts (controls). Patients had significant anti-HLA (sensitization) if mean fluorescence intensity was ≥ 1500. RESULTS The study enrolled 29 patients (median age, 10.1 years). Significant class I anti-HLA antibodies were seen in 44% (8 of 18) of the neonatal group, 25% (1 of 4) of the infant group, and 14% (1 of 7) of controls; class II anti-HLA antibodies were seen in 44% (8 of 18) of the neonatal group, 25% (1 of 4) of the infant group, and 29% (2 of 7) of controls. All patients received fresh whole blood, but the neonatal group had greater exposure (p = 0.001). There was less sensitization with increasing time from last receipt of allograft(s) or blood transfusion (p = 0.05). CONCLUSIONS Exposure to allograft at the Norwood procedure is associated with long-term sensitization to anti-HLA antibodies in 56% of patients. Sensitization also occurs in those without prior exposure to allografts, may decrease over time, and appears related to whole blood. These findings have implications for those in whom heart transplant is considered late in the clinical course.

Oligoclonal T cells are infiltrating the brains of children with AIDS: sequence analysis reveals high proportions of identical β-chain T-cell receptor transcripts

We have recently described the presence of perivascular CD3+ CD45RO+ T cells infiltrating the brains of children with AIDS. To determine whether these infiltrates contain oligoclonal populations of T cells, we amplified by PCR β‐chain T‐cell receptor (TCR) transcripts from autopsy brains of four paediatric patients with AIDS. The amplified transcripts were cloned and sequenced. Sequence analysis of the β‐chain TCR transcripts from all four patients revealed multiple identical copies of TCR β‐chain transcripts, suggesting the presence of oligoclonal populations of T‐cells. These TCR transcripts were novel. The presence of oligoclonal populations of T cells in the brains of these four paediatric patients with AIDS suggests that these T cells have undergone antigen‐driven proliferation and clonal expansion very likely in situ, in the brains of these AIDS patients, in response to viral or self‐antigens. Although the specificity of the clonally expanded β‐chain TCR transcripts remains to be elucidated, none of the β‐chain TCR transcripts identified in this study were identical to those specific for HIV‐1 antigens that are currently reported in the GENBANK/EMBL databases. Certain common CDR3 motifs were observed in brain‐infiltrating T cells within and between certain patients. Large proportions (24 of 61; 39%) of β‐chain TCR clones from one patient (NP95‐73) and 2 of 27 (7%) of another patient (NP95‐184‐O) exhibited substantial CDR3 homology to myelin basic protein (MBP)‐specific TCR derived from normal donors or TCR expressed in the brain of patients with multiple sclerosis (MS) or with viral encephalitis. These two patients (NP95‐73 and NP95‐184‐O) also shared HLA class II with the normal donors and the MS patients who expressed these homologous TCR. Pathologic examination at autopsy of the brains revealed the presence of myelin pallor only in patient NP95‐73. T‐cell clones identified in the brain of patients NP95‐73 and NP95‐184‐O may recognize MBP or another CNS self antigen and this recognition may be restricted by either DRB1*15 or DQB1*0602 specificities.

Changes in the methodology of pre‐heart transplant human leukocyte antibody assessment: an analysis of the United Network for Organ Sharing database

We sought to investigate temporal trends in the methodology of human leukocyte antibody assessment in heart transplantation.

Evaluation and optimization of variables in immunofixation electrophoresis for the detection of IgG paraproteins

We examined different antisera and varying serum dilutions for their effects on the sensitivity of immunofixation electrophoresis (IFE) for detecting and characterizing serum IgG paraproteins. Different antisera reacted unequally with a single paraprotein and conversely, a single antiserum reacted unequally with several different paraproteins. The dilution of serum was extremely important; the adoption of a single dilution for all sera could result in failure to detect low-concentration or poorly immunoreactive paraproteins. While the application of a standardized amount of paraprotein is recommended, it must be emphasized that no single antiserum can be relied on to provide unequivocal detection of all paraproteins of that particular isotype.

Characterization of a new HLA-C*04 allele, C*04:112.

HLA-C*04:112 differs from HLA-C*04:01:01:01 by one nucleotide at position 270 resulting in an amino acid change, Lysine to Asparagine, at codon 66 of exon 2.

CD3+/CD19+ depleted matched and mismatched unrelated donor hematopoietic stem cell transplant with targeted T cell addback is associated with excellent outcomes in pediatric patients with non-malignant hematologic disorders

Unrelated donor hematopoietic stem cell transplantation (HSCT) is increasingly being used to cure nonmalignant hematologic diseases (NMHD) in patients who lack HLA matched related donors. Both graft rejection and graft-versus-host disease (GVHD) remain major barriers to safe and effective transplant for these patients requiring unrelated donors. Partial T cell depletion combined with peripheral stem cell transplantation (pTCD-PSCT) has the potential advantages of providing a high stem cell dose to facilitate rapid engraftment, maintaining cells that may facilitate engraftment, and decreasing GVHD risk compared with T cell-replete HSCT. Here, we report a single-institution, retrospective experience of unrelated donor pTCD-PSCT for pediatric patients with NMHD. From 2014 to 2017, 12 pediatric patients with transfusion-dependent NMHD underwent matched unrelated donor (MUD) or mismatched unrelated donor (MMUD) pTCD HSCT in our center using disease-specific conditioning. Donor PSCs underwent CD3+ T cell and CD19+ B cell depletion using CliniMACS, followed by a targeted addback of 1 × 105 CD3+ T cells/kg to the graft before infusion. All 12 patients demonstrated rapid trilinear engraftment. At a median follow-up of 740days (range, 279 to 1466), all patients were alive with over 92% total peripheral blood donor chimerism and without transfusion dependence or recurrence of their underlying hematologic disease. Immune reconstitution was rapid and comparable with T cell-replete HSCT. No patients developed severe acute GVHD (grades III to IV) or chronic extensive GVHD, and all patients had discontinued systemic immune suppression. Viral reactivations were common, but no patient developed symptoms of life-threatening infectious disease. Our data indicate that MUD and MMUD pTCD-PSCTs are safe and effective approaches that enable rapid engraftment and immune reconstitution, prevent severe GVHD, and expand availability of HSCT to any patients with NMHD who have closely MUDs.

Value of a flow cytometry crossmatch in the setting of a negative complement-dependent cytotoxicity crossmatch in heart transplant recipients

Complement‐dependent cytotoxicity cross‐match (CDCXM) is used for evaluation of preformed HLA‐specific antibodies in patients undergoing heart transplantation. Flow cytometry cross‐match (FCXM) is a more sensitive assay and used with increasing frequency. To determine the clinical relevance of a positive FCXM in the context of negative CDCXM in heart transplantation, the United Network for Organ Sharing (UNOS) database was analyzed. Kaplan‐Meier analysis and Cox proportional hazard modeling were used to assess graft survival for three different patient cohorts defined by cross‐match results: T‐cell and B‐cell CDCXM+ (“CDCXM+” cohort), CDCXM− but T‐cell and/or B‐cell FCXM+ (“FCXM+” cohort), and T‐cell/B‐cell CDCXM− and FCXM‐ (“XM−” cohort). During the study period, 2558 patients met inclusion criteria (10.7% CDCXM+, 18.8% FCXM+, 65.5% XM−). CDCXM+ patients had significantly decreased graft survival compared to FCXM+ and XM− cohorts (P = .003 and <.001, respectively). CDCXM− and FCXM+ patients did not have decreased graft survival compared to XM− patients (P = .09). In multivariate analysis, only CDCXM+ was associated with decreased graft survival (HR 1.22, 95% CI 1.01‐1.49). In conclusion, positive FCXM in the context of negative CDCXM does not confer increased risk of graft failure. Further study is needed to understand implications of CDCXM and FCXM testing in heart transplant recipients.

Abstract 2977: Most patients with acquired aplastic anemia develop clonal hematopoiesis early in disease

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Clonal hematopoiesis is an expansion of hematopoietic stem cells, caused by somatic mutations or epigenetic changes that confer a growth advantage to the host cell. Although recently recognized as a phenomenon of aging, clonal hematopoiesis has been traditionally associated with pre-cancerous states and malignant transformation. Acquired aplastic anemia (AA), a non-neoplastic autoimmune blood disorder occurring in children and adults, has been associated with clonal hematopoietic disorders; transformation to myelodysplastic syndrome (MDS) or acute leukemia is a late complication in 10-15% of AA patients. Based on the association of AA with clonal disorders, we hypothesized that clonal hematopoiesis is a general phenomenon in AA, and can be seen in the majority of AA patients, including children. To evaluate somatic genetic changes in AA, we used a combination of single nucleotide polymorphism array (SNP-A) genotyping and comparative whole exome sequencing of paired bone marrow aspirates and skin in twenty nine patients with AA. All somatic mutations were validated by bi-directional Sanger sequencing. The median age of diagnosis was 14 years (range 1.5-65). Patients were analyzed at a median of 1.1 years from diagnosis. None of the patients had histopathological evidence of MDS at the time of analysis. Somatic mutations were identified in the majority of patients, including patients with pediatric-onset AA. Three patients (10%) had somatic loss-of-function mutations in HLA class I alleles. Although MDS-associated mutations were identified in 2 of 29 patients, the majority of mutations were not in genes associated with MDS and hematologic malignancies. Pathway analysis of mutated genes revealed an enrichment of genes in pathways of immunity and transcriptional regulation. Comparison of somatic mutations in AA to a patient with a 30-year history of AA who progressed to MDS revealed that, unlike in AA, which was characterized by diverse and frequently oligoclonal hematopoiesis, progression to MDS was associated with an expansion of a dominant clone carrying multiple classical mutations linked to malignancy: pathogenic mutations in SUZ12 (homozygous for the mutated region due to copy number-neutral loss of heterozygosity (CN-LOH) at the chromosomal region 17q11.2qter), ASXL1, RUNX1, and PHF6. In conclusion, our data show that clonal hematopoiesis emerges in the majority of patients with AA, including children and young adults, can be detected early in disease, and has a mutational spectrum largely distinct from MDS. Our results highlight that in the absence of morphologic features of myelodysplasia, the presence of clonal hematopoiesis with somatic mutations cannot be used to distinguish MDS from AA. Future longitudinal studies of clonal hematopoiesis in AA will help to explain differences in patients’ disease course, and will enable personalized treatment approaches in AA. Citation Format: Daria V. Babushok, Nieves Perdigones, Juan C. Perin, Timothy S. Olson, Wenda Ye, Jacquelyn J. Roth, Curt Lind, Carine Cattier, Yimei Li, Helge Hartung, Michele E. Paessler, Dale M. Frank, Hongbo M. Xie, Tracy M. Busse, Shanna Cross, Gregory M. Podsakoff, Dimitrios Monos, Jaclyn A. Biegel, Philip J. Mason, Monica Bessler. Most patients with acquired aplastic anemia develop clonal hematopoiesis early in disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2977. doi:10.1158/1538-7445.AM2015-2977