The methodology and significance of minimal residual disease detection after allogeneic stem cell transplantation

Allogeneic stem cell transplantation (SCT) is the choice of therapy for leukemia patients who respond poorly to conventional chemotherapy. Despite high remission rates after SCT, relapse of the underlying disease remains one of the most frequent causes of treatment failure. Graftversus-host disease (GVHD), a major complication after SCT, is caused by the activation of alloreactive donor T-cells. Although being life threatening in its severe forms, GVHD has a protective effect called the graft-versus-leukemia effect (GVL). In order to use the GVL effect of donor T-cells, donor lymphocyte infusions (DLI) is now used as a treatment for relapse after SCT. Response to DLI is usually better when the tumor load is low. Therefore, sensitive methods to detect residual leukemic cells are needed in order to identify patients who are at the highest risk of relapse and to start immunotherapeutic interventions when the tumor load is still low. Minimal residual disease (MRD) refers to the presence of leukemic cells below the detection limit of standard morphological analysis. The most sensitive and widely used techniques for MRD detection are based on the PCR technology. The aim of this thesis is to evaluate the clinical significance of MRD detection in leukemia patients receiving SCT. In patients with acute myeloid leukemia (AML), we evaluated the significance of mixed chimerism (MC) analysis, the detection of recipient-derived hematopoietic cells after SCT. MC analysis was performed in the leukemia-affected cell lineage to increase the specificity and sensitivity of the method. MC was detected in 14 of 30 patients. Ten of these 14 patients relapsed as compared to 2 of 16 with donor chimerism (p<0.01). MC was detected a median time of 66 (range 23-332) days before hernatological relapse. Using immunoglobulin and T-cell receptor gene rearrangements as clonal markers, we analyzed MRD levels before and after SCT in patients with acute lymphoblastic leukemia (ALL). MRD detection before SCT was associated with increased risk of relapse. However, GVHD was shown to protect against relapse in patients with high levels of MRD. MRD detection after SCT was also associated with a high risk of relapse. Relapse occurred in 8 of 9 MRD positive patients as compared to 6 of 23 MRD negative patients (p<0.01). MRD was detected a median of 5.5 (range 0.5-30) months before relapse. In recent years, nonmyeloablative SCT (NST) has been studied as a safer approach for patients who are not eligible for the toxic conditioning regimens given before SCT. We studied the kinetics of MRD and MC in chronic myeloid leukemia (CML) patients receiving NST and compared the results to those obtained from CML patients receiving a conventional SCT (CST). A competitive PCR approach was performed for quantitative MRD analysis of BCR-ABL transcripts. In the early posttransplant period, higher incidence and levels of MC and MRD were found in NST patients as compared to CST patients. However, molecular remissions were subsequently achieved in most NST patients. Wilms tumor gene (WTI) has been reported as a "panleukemic" MRD marker in many studies. We wanted to evaluate WTI as a MRD marker by comparing the kinetics of WTI levels with that of BCR- ABL using realtime quantitative PCR. We found a background expression of WTI healthy controls. In addition, WTI analysis was not sensitive enough to predict relapse. In conclusion, MRD analysis in leukemia patients provides the possibility to identify patients at high risk of relapse after SCT. Adoptive immunotherapy based on MRD results may prevent relapse and improve outcome for patients with poor prognosis

Inhibition of Heavy Chain and β(2)-Microglobulin Synthesis as a Mechanism of Major Histocompatibility Complex Class I Downregulation during Epstein-Barr Virus Replication

The mechanisms of major histocompatibility complex (MHC) class I downregulation during Epstein-Barr virus (EBV) replication are not well characterized. Here we show that in several cell lines infected with a recombinant EBV strain encoding green fluorescent protein (GFP), the virus lytic cycle coincides with GFP expression, which thus can be used as a marker of virus replication. EBV replication resulted in downregulation of MHC class II and all classical MHC class I alleles independently of viral DNA synthesis or late gene expression. Although assembled MHC class I complexes, the total pool of heavy chains, and β(2)-microglobulin (β(2)m) were significantly downregulated, free class I heavy chains were stabilized at the surface of cells replicating EBV. Calnexin expression was increased in GFP(+) cells, and calnexin and calreticulin accumulated at the cell surface that could contribute to the stabilization of class I heavy chains. Decreased expression levels of another chaperone, ERp57, and TAP2, a transporter associated with antigen processing and presentation, correlated with delayed kinetics of MHC class I maturation. Levels of both class I heavy chain and β(2)m mRNA were reduced, and metabolic labeling experiments demonstrated a very low rate of class I heavy chain synthesis in lytically infected cells. MHC class I and MHC class II downregulation was mimicked by pharmacological inhibition of protein synthesis in latently infected cells. Our data suggest that although several mechanisms may contribute to MHC class I downregulation in the course of EBV replication, inhibition of MHC class I synthesis plays the primary role in the process

T Cell Receptor Excision Circle (TREC) Monitoring after Allogeneic Stem Cell Transplantation; a Predictive Marker for Complications and Clinical Outcome

Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment modality for a variety of malignant diseases as well as for inborn errors of the metabolism or immune system. Regardless of disease origin, good clinical effects are dependent on proper immune reconstitution. T cells are responsible for both the beneficial graft-versus-leukemia (GVL) effect against malignant cells and protection against infections. The immune recovery of T cells relies initially on peripheral expansion of mature cells from the graft and later on the differentiation and maturation from donor-derived hematopoietic stem cells. The formation of new T cells occurs in the thymus and as a byproduct, T cell receptor excision circles (TRECs) are released upon rearrangement of the T cell receptor. Detection of TRECs by PCR is a reliable method for estimating the amount of newly formed T cells in the circulation and, indirectly, for estimating thymic function. Here, we discuss the role of TREC analysis in the prediction of clinical outcome after allogeneic HSCT. Due to the pivotal role of T cell reconstitution we propose that TREC analysis should be included as a key indicator in the post-HSCT follow-up

Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report

Abstract Background A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 + 3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. Case presentation We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naïve CD4+ and CD8+ T-cells, increased number of activated effector memory CD8+ T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient’s cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. Conclusions There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing

Long-Term Stable Mixed Chimerism after Hematopoietic Stem Cell Transplantation in Patients with Non-Malignant Disease, Shall We Be Tolerant?

<div><p>Long-term stable mixed chimerism is a rare and poorly understood phenomenon post hematopoietic stem cell transplantation. This study aims to shed light on whether the two hematopoietic systems in patients with mixed chimerism remain functional. Additionally, we investigate possible immunologic differences in these individuals compared to patients with only donor derived immune cells. Patients with donor and mixed chimerism, at median 10 (5–16) years post-HSCT for non-malignant diseases, were assessed regarding clinical situation and immune system (phenotypical and functional). No difference in long-term outcome was seen in terms of general wellbeing, central phenotypic immune system features (<i>e</i>.<i>g</i>., differentiation status, CD4/CD8 ratio, B and NK-cell frequency) and antibody responses to immunizations. At a median of 10 years post transplantation, patients with mixed chimerism had significantly higher IgG3 and platelet levels. Additionally, these patients had higher NKT-cell levels (CD94+CD8+ and CD56+CD8+) than patients with donor chimerism. In depth phenotypic analysis of patients with mixed chimerism demonstrated recipient-derived fractions in most immune cell lineages (<i>e</i>.<i>g</i>., T-cell, B-cell and NK-cell subsets). Recipient cells were also capable of responding to mitogenic stimulation with production of several cytokines. In conclusion, long-term mixed chimerism did not negatively affect patient wellbeing and long-term outcome. Moreover, recipient-derived immunity may still be functional in these patients, suggesting an active state of tolerance and immunologic dependence on both hematopoietic systems.</p></div

Phenotypic comparison of cellular subsets between patients with mixed and donor chimerism.

<p>For most cellular subsets no significant differences were observed between 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients (A-C). Asterisks indicate significant <i>P</i>-values (* = <i>P</i> < .05 and ** = <i>P</i> < .01), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group. (A) The white blood cell (i), platelet (ii) and neutrophil (iii) count in the two patient groups. Platelet counts were higher in MC patients (<i>P =</i> .041). K/mL = 1 000 cells/mL. (B) Radar graphs depicting relative distribution of T, B and NK-cells (i) and T-cell subsets (ii) for DC and MC patient groups. (C) Differentiation status of total T-cells (CD3+), as defined by naïve memory (CCR7+ CD45RO-), central memory (CCR7+ CD45RO+), effector memory (CCR7- CD45RO+) and terminally differentiated memory (CCR7- CD45RO-), was found to be similar between the DC and MC patient groups. (D) Representative FACS plots of potential NKT-cells (CD56+ (i-ii) or CD94+ (iv-v)) gated on CD8+ T-cells. In the corresponding graphs (iii, vi)), individual ratios of the subsets for each group are shown (<i>P =</i> .004 and <i>P =</i> .035 respectively).</p

Comparison of soluble biomarkers between patients with mixed and donor chimerism.

<p>Concentrations of cytokines, IgG and IgG subclasses were determined in the plasma of 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients at median 10 years post-HSCT. Asterisks indicate significant <i>P</i>-values (* = <i>P</i> < .05 and ** = <i>P</i> < .01), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group. (A) A higher IgG3 concentration was seen in MC patient plasma (<i>P =</i> .027). (C-D) A lower concentration of IL-4 (B), IL-12 (p40) (C) and G-CSF (D) was observed in MC patients (<i>P =</i> .016, <i>P =</i> .003 and <i>P =</i> .022 respectively).</p