ALBERT

Description: About fifteen percent of patients with severe aplastic anemia (sAA) who undergo immunosuppression therapy (IST) develop clonal disease in the decade following treatment. Of 122 patients treated with horse ATG/CsA at NIH, 13 developed cytogenetic abnormalities (monosomy 7 in 9 patients, 7p deletion in one, and trisomy 8 in 2) (Rosenfeld et al: JAMA289:1130; 2003). Monosomy 7 usually occurs in patients with sAA who are unresponsive to IST. Factors responsible for clonal progression in bone marrow failure are still unclear. We and others have reported that monosomy 7 may be detected by fluorescent in situ hybridization (FISH) months before conventional cytogenetics and that high levels of GCSF foster preferential expansion of the monosomy 7 clone in vitro (Sloand E et al; Proc. Natl. Acad. Sci2006;103:14483). We hypothesized that a clone of monsomy 7 cells might indicate an underlying stem cell disorder unlikely to respond to immunosuppression or reflect more severe disease marked by higher endogenous GCSF levels. FISH was undertaken on 40 bone marrow samples obtained from aplastic anemia patients at presentation and before immunosuppressive treatment. Bone marrow mononuclear cells were hybridized with centromeric probes specific for chromosome 7 and chromosome 8 (to control for hybridization efficiency); samples were assessed on duplicate slides by three investigators who were unaware of the diagnoses and outcomes. Twenty-five healthy controls were tested concurrently. The upper limit of normal was set at 6% based on control data. Of twenty-one patients with 〉 6% monosomy 7 cells, response to IST was observed in 12 (57%), while 6 of 19 (84%) with normal FISH responded to IST (p=0.08). Thirteen patients had monosomy 7 frequencies of 〉10%. Of these 5 (38%) responded to IST, compared with 23/27 (85%) with ≤10% monosomy 7. The proportion of monosomy 7 cells in the bone marrow correlated with age (R2 =0.6, p10% monosomy 7 by FISH at presentation, but normal cytogenetics. The two responding patients tested with 〉6% monosomy 7 prior to IST demonstrated

Description: Telomeres that cap the ends of chromosomes, protect them from recombination, end-to-end fusion, and recognition as damaged DNA. Telomere shorteningmay result in cell cycle arrest and senescence or in genomic instability that precedes malignancy. Maintenance of the integrity of telomeres requires the telomerase ribonucleoprotein complex, mainly the telomerase reverse transcriptase (TERT) and its integral RNA template (TERC). Mutations in TERC and TERT cause progressive telomere shortening; short telomeres are found in constitutional marrow failure syndromes and also in apparently acquired aplastic anemia (Fogarty PF et al, Lancet362:1628, 2003;. Yamaguchi H et al, NEJM352:1413, 2005). Myelodysplasia and acute leukemia are often present in these kindreds and associated with pathogenic mutations. The mechanism resulting in genomic instability in TERT and TERC-deficient individuals is unclear. End-to-end chromosome fusion in TERT-”knock-out” embryonic mouse stem cells are seen after multiple passages in culture and are associated with profound telomere shortening and the development of aneuploidy (Lee HW et al. Nature 9:569–74, 1998). End-to-end fusion of sister chromatids presumably results in disruption of chromosome segregation during metaphase. In the present study, we examined blood and marrow cells from 7 patients with TERT, and TERC gene mutations; 10 healthy young controls; and 2 individuals older than age 60 years of age, as well as 6 umbilical cord bloods. Leukocytes with TERT or TERC mutations had short telomeres as previously measured by flow-fluorescent in situ hybridization (FISH) and confirmed by Southern hybridization (mean telomere shortening in comparison to age-matched controls, 3.4 ± 1.7 kb) and low telomerase enzymatic activity (144±13 vs. 270±56 respectively; P

Description: Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

Description: Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated immunologically-mediated pathophysiology in some patients. Indeed, among 129 patients treated with immunosuppression at NIH, trisomy 8 was associated with hematologic response to immunosuppression. We have demonstrated that trisomy 8 patients have oligoclonal CD8 T cell expansion, as determined by increased proportions of one or more T cell receptor (TCR) V beta subfamilies (Sloand et al. Blood106:841; 2005) which had cytotoxic activity toward trisomy 8 cell progenitors. Here we examine the response of cytotoxic T cells to cyclin D1 and Wilms tumor protein (WT-1); these candidate autoantigens had earlier been observed to be over-expressed in trisomy 8 CD34 cell in our microarray analysis (Chen et al. Blood 104:4210, 2004 ). Bone marrow mononuclear cells from 19 trisomy 8 patients (defined by cytogenetics and FISH) had significantly increased levels of WT-1 mRNA and protein expression compared to normal CD34 cells by real time PCR and immunoblot, respectively (p

Description: Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated autoimmune process in some patients. Among 133 patients treated at NIH, predominantly with antithymocyte globulin, trisomy 8 as the sole karyotypic abnormality was specifically and significantly associated with favorable hematologic response to immunosuppresssion, as compared to patients with other cytogenetic abnormalities or with a normal karyotype. Trisomy 8 MDS patients show stable increases in the proportion of aneuploid bone marrow cells following immunosupppression (IST). We have reported that all of thirty patients with trisomy 8 had significant CD8 T cell expansion of one or more T cell receptor (TCR) Vβ subfamilies as measured by flow cytometry, and expanded subfamilies showed complimentary determining region 3 (CDR3) skewing by spectratyping. Sorted T cells of the expanded Vβ subfamilies, but none of the remaining subfamilies, specifically inhibit hematopoietic colony formation by trisomy 8 cell progenitors. Colony formation of cytogenetically normal cells from the same individuals was less or not inhibited. Here, we examined protein expression levels and measured the response of cytotoxic T cells to two antigens which we had found to be overexpressed in trisomy 8 CD34 cells by microarray analysis (Chen G et al, Blood In press): cyclin D1 (CD1) and Wilms tumor-1 antigen (WT1). Peripheral blood and bone marrow granulocytes of six trisomy 8 patients with refractory anemia all demonstrated WT1 expression levels 100–1000 x normal (N=38) by real time PCR; the three patients with normal cytogenetics had levels comparable to normal. Immunoblotting confirmed massively increased WT1 peptide in all six trisomy 8 patients tested, with five MDS patients of normal karyotype showing normal levels. Patient and control CD8 cells were cultured for 6 hours with WT1 and CD1-loaded HLA-A2-restricted antigen-presenting cells using three different peptides for WT1 and four for CD1. Cytotoxic CD8 T cells responses were identified by flow cytometric analysis of intracellular interferon-γ. Eight of 17 trisomy 8 patients showed significant responses (〉8% CD8 cells activated compared to unstimulated samples; mean =5%) to WT1 but not to CD1 or non-peptide loaded antigen presenting cells. CD8 cell responses to WT1 measured using MHC class I A2-restricted tetramers were concordant with the results by intracellular staining in all 15 patients tested. In contrast, no responses to WT1 were seen in normal controls and five MDS patients lacking cytogenetic abnormalities. While PB of one of the three monosomy 7 patients showed upregulation of WT1, there was no cytotoxic lymphocyte response against the peptide; unlike trisomy 8 CD34 cells those from monosomy 7 patients did not express co-stimulatory molecules, HLA class I and B7.1. These data suggest an autoimmune pathophysiology for the cytopenia of trisomy 8 myelodysplasia with the following scenerio: WT1 is overexpressed by the trisomy 8 clone, resulting in a specific cytotoxic CD8 T cell immune response to WT1. Apoptotis of marrow cells results, either by cross reactivity or as a bystander effect. Improvement in hematopoiesis is seen following IST.

Description: Clinically, bone marrow failure syndromes show significant overlap. Newer analytic approaches may lead to improved diagnosis and better understanding of underlying pathophysiology. In contrast to traditional biomarker diagnostic principles, proteomic technologies such as surface-enhanced laser desorption/ionization (SELDI) method enable screening of protein patterns without knowledge of the identity of targets. The recent refinement of SELDI mass spectrometry has allowed for the early diagnosis of occult solid tumors. We explored the potential application of SELDI proteomics to the study of bone marrow failure states. An extensive sample library included 28 Aplastic anemia, 129 Myelodysplastic syndrome (MDS), 28 Paroxysmal Nocturnal Hemoglobinuria (PNH) patients and 36 control specimens. Within the MDS cohort, subgroups with RA (N=77), RARS (N=15) and RAEB/t (N=37) were studied. Analyses were also performed for patients with specific cytogenetic abnormalities, including those with 5q-syndrome (N=21), trisomy-8 (N=15), aberrations of chromosome 7 (N=15) and compared to MDS patients with normal cytogenetics (N=57). In preliminary experiments, H4 chips were selected for further study; spectra with 〉80 peaks were considered as informative. Initially, our analysis included learning sets in which 20 randomly selected samples from individual categories were compared to each other in order to identify specific peptide peaks. Subsequently, the results obtained in the learning set were confirmed using separate validation sample sets. A scoring system was applied for analysis in which signal/noise ratio was a sole criterion for peak recognition and assignment of non-parametric scores. Reproducibility was assured by repeating measurements of standard plasma sample and multiple determinations. When AA spectra were compared to controls, AA could be distinguished with 87% sensitivity and 85% specificity based on the presence of a single peak at M/Z value of 3.095kDa. When compared to RA, AA was discriminated with 95% specificity and 80% sensitivity (1.02kDa). Globally, we devised a decision tree (using 3 different peaks in 6 steps of separation) that allows for the plasma-based discrimination of MDS and controls. Within MDS group, RA could be further differentiated from a more advanced MDS with 95% specificity and 90% sensitivity. A single peak at M/Z value of 3.88kDa distinguished RA from RAEB/t. Similar learning data sets performed for RARS patient can be obtained. Not overlapping sample sets are used for validation of obtained results. Finally, samples with atypical spectra can be investigated for features that may prompt diagnostic reassignment; e.g., in patients with hypocellular MDS to AA, and PNH to AA/PNH syndrome. Most intriguing is the possibility that specific cytogenetic defects may result in proteomic patterns that may provide important clinical clues. A large sample collection allows for subanalyses based on karyotype: for example, data and result sets exist for patients with 5q-, trisomy-8 and other more common chromosomal changes. Our ongoing study demonstrates the feasibility of SELDI technology in detecting diagnostic protein signatures for individual bone marrow failure states. In the future, proteomic biomarker pattern-based analysis may allow for more precise definition and supplement the traditional diagnostic approaches.

Description: The WT1 gene contributes to leukemogenesis and all adult ALL, AML and CML express WT1 RNA by quantitative real-time reverse transcription polymerase chain reaction (qPCR) techniques. WT1 may therefore be a useful antigenic target for immunotherapy. Four HLA-A*0201-restricted WT1 T cell epitopes have been identified: Db126 (RMFPNAPYL), WH187 (SLGEQQYSV), WT37-45 (VLDFAPPGA) and WT235 (CMTWNQMNL), but only Db126 has been extensively studied in myeloid leukemias. Here, we sought CD8+ T cells directed against these epitopes in 12 healthy SCT donors, 6 patients with AML, 8 with, CML and 6 with ALL prior to SCT. All patients tested with myeloid or lymphoid leukemias expressed MHC class I, B7.1 and WT1. To detect very low frequencies of WT1-specific CD8+ T cells, we used qPCR for interferon-g (IFN-g) mRNA. After isolation, 106 CD8+ T cells were stimulated with C1R-A2 cells (MHC class I-defective LCL cells transfected with HLA-A*0201) loaded with test peptides at concentrations of 0.1, 1 and 10 mM to determine their functional avidity. CD8+ T cells were also stimulated with CMV pp65 (positive control) and gp100 (209-2M) (negative control) peptides. After 3 hr coculture, cells were harvested for RNA extraction and cDNA synthesis. qPCR was performed for IFN-g mRNA and normalized to copies of CD8 mRNA from the same sample. Parallel assays using tetramers demonstrated that the IFN-g copy number was linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. A positive response was defined as a threshold of 100 or more IFN-g mRNA copies/104 CD8 copies and a stimulation index (SI) of 2 or more, where SI = IFN-g mRNA copies/104 CD8 copies in peptide pulsed/unpulsed cultures. Responses to at least one WT1 peptide were detected in 5/8 CML patients, 4/6 patients with AML and 7/12 healthy donors. Notably, a response was not elicited to WT1 in any of the 6 patients with ALL, despite evidence of immune competence as shown by a normal CMV response. Five of five CML responders and 3/4 AML responders recognized 2 or more WT1 epitopes, while the 7 healthy donors recognized only one WT1 epitope. The range of IFN-g mRNA copies/104 CD8 copies was 289–13584, 418–45891 and 160–2683 for CML, AML and healthy donors respectively. WT1-specific tetramer-positive CD8+ T cells displayed both central memory (CD45RO+CD27+CD57−) and terminally differentiated effector memory phenotypes (CD45RO-CD27−CD57+). As multiple WT1-derived epitopes can be targeted simultaneously, it is likely that T cell response to WT1 is polyclonal. These results are important because the presence of memory WT1 responses in patients with myeloid leukemias and healthy individuals should favor vaccination as a means to expand immune responses to leukemia in the autologous and allogeneic transplant setting. Furthermore, the presence of CD8+ T cell responses to multiple WT1 epitopes should favor robust polyclonal immune responses to leukemia. However, failure to detect CD8+ T cell responses to WT1 in ALL patients suggests that WT1 may not be a useful antigen to target for immunotherapeutic purposes in this patient group.

Description: Clinical observations and laboratory evidence link bone marrow failure in myelodysplastic syndrome (MDS) to a T cell–mediated immune process that is responsive to immunosuppressive treatment (IST) in some patients. Previously, we showed that trisomy 8 MDS patients had clonally expanded CD8+ T-cell populations that recognized aneuploid hematopoietic progenitor cells (HPC). Furthermore, microarray analyses showed that Wilms tumor 1 (WT1) gene was overexpressed by trisomy 8 hematopoietic progenitor (CD34+) cells compared with CD34+ cells from healthy donors. Here, we show that WT1 mRNA expression is up-regulated in the bone marrow mononuclear cells of MDS patients with trisomy 8 relative to healthy controls and non–trisomy 8 MDS; WT1 protein levels were also significantly elevated. In addition, using a combination of physical and functional assays to detect the presence and reactivity of specific T cells, respectively, we demonstrate that IST-responsive MDS patients exhibit significant CD4+ and CD8+ T-cell responses directed against WT1. Finally, WT1-specific CD8+ T cells were present within expanded T-cell receptor Vβ subfamilies and inhibited hematopoiesis when added to autologous patient bone marrow cells in culture. Thus, our results suggest that WT1 is one of the antigens that triggers T cell–mediated myelosuppression in MDS.

Description: Administration of granulocyte colony stimulating factor (GCSF) has been linked to development of monosomy 7 in some patients with aplastic anemia (AA) and congenital neutropenia. We assessed the effect of GCSF on monosomy 7 to determine if this chromosomal abnormality developed de novo or if GCSF simply favored expansion of a pre-existing clone. Bone marrow mononuclear cells (BMMNC) were co-cultured for 14 days with pharmacological doses of GCSF and examined by fluorescent in situ hybridization (FISH). No karyotypically healthy control, AA, or myelodysplastic syndrome (MDS) bone marrow showed development of monosomy 7. In BMMNC cultures from thirteen patients with MDS and monosomy 7, all developed substantial increases in numbers of monosomy 7 cells after prolonged exposure to GCSF. Experiments on stored AA samples obtained six months prior to the development of abnormal karyotype showed monosomy 7 on FISH ,with expansion of that clone on co-culture with GCSF. GCSFR mRNA was increased in BM from monosomy 7 patients as measured by real time PCR, and monosomy 7 cells expressed more GCSFR on the cell surface than did normal cells. As a truncated GCSFR isoform is associated with a hyperproliferative response to G-CSF and prolonged activation of signal transducer and activator of transcription (STAT) complexes, we sequenced the GCSFR DNA of monosomy 7 cells and measured the expression of the truncated GCSFR mRNA relative to full length GCSFR in patients with monosomy 7. While genomic GCSFR DNA showed no abnormalities, mRNA demonstrated increased proportions of the truncated isoform IV in all six patients tested. We examined GCSF-mediated GCSFR signal transduction of the Jak/Stat system in monosomy 7 CD34 cells. STAT-1 was increased and the STAT 5: STAT 3 rato was increased by ten-fold in all patients with monosomy 7 compared to normals and to MDS with normal cytogenetics. In conclusion, pharmacologic doses of GCSF appear to increase the proportion of monosomy 7 cells; this heightened sensitivity to GCSFmay be related to altered amounts of the truncated form of the receptor, with changes in GCSF signal transduction resulting in expansion of the monosomy 7 clone.