Immune mechanisms are involved in the pathophysiology of aplastic anemia (AA) and myelodysplastic syndrome (MDS) .

In myelodysplastic syndromes (MDS) increased chromosomal breaks point toward defects in DNA repair machinery including base excision repair (BER) pathway involved in handling of oxidative DNA damage. We investigated whether defects in this pathway can be found in MDS. Elevated levels of 8-oxoguanine (8-OG) were found in a significant proportion of MDS patients, indicating increased oxidative DNA damage or defective handling of oxidative load. In a distinct subgroup of patients, increased 8-OG content was associated with increased hOGG1 mRNA expression and activity. In some patients, increased numbers of abasic sites (AP sites) correlated with low levels of POLb. To further investigate the nature of this defect, we examined genetic lesions potentially explaining accumulation of 8-OG and AP sites. We genotyped a large cohort of MDS patients and found a correlation between increased oxidative damage and the presence of the hOGG1-Cys326 allele suggesting inadequate compensatory feedback. Overall, this hOGG1 variant was more frequent in MDS, particularly in advanced forms, as compared to controls. In summary, we demonstrated that BER dysfunction in some MDS patients may be responsible for the increased 8-OG incorporation and explains one aspect of the propensity to chromosomal breaks in MDS but other mechanisms may also be involved.

T cell large granular lymphocyte leukemia (T-LGL) is a chronic clonal lymphoproliferation of CTL. In many ways, T-LGL clones resemble terminal effector CTL, including down-modulation of CD28 and overexpression of perforin, granzymes, and CD57. We studied the transcriptome of T-LGL clones and compared it with healthy CD8+CD57+ effector cells as well as CD8+CD57− populations. T-LGL clones were sorted based on their TCR variable β-chain restriction, and controls were obtained by pooling cell populations from 14 donors. Here, we focus our analysis on immunological networks, as immune mechanisms play a prominent role in the etiology of bone marrow failure in T-LGL. Informative genes identified by expression arrays were studied further in an independent cohort of patients using Taqman PCR, ELISA assays, and FACS analysis. Despite a strikingly similar gene expression profile between T-LGL clones and their healthy counterparts, important phenotypic differences were identified, including up-modulation of TNFRS9, myeloid cell leukemia sequence 1, IFN-γ, and IFN-γ-related genes, and several integrins/adhesion molecules. In addition, T-LGL clones were characterized by an overexpression of chemokines and chemokine receptors that are typically associated with viral infections (CXCL2, Hepatitis A virus cellular receptor 1, IL-18, CCR2). Our studies suggest that immunodominant LGL clones, although phenotypically similar to effector CTL, show significantly altered expression of a number of genes, including those associated with an ongoing viral infection or chronic, antigen-driven immune response.

Summary T‐cell large granular lymphocyte leukaemia (T‐LGL) is a chronic clonal proliferation of cytotoxic T lymphocytes (CTL). T‐LGL presents with cytopenias, often accompanied by autoimmune diseases, suggesting clonal transformation arising from an initially polyclonal immune response. Various immunogenetic predisposition factors, previously described for both immune‐mediated bone marrow failure and autoimmune conditions, may promote T‐LGL evolution and/or development of cytopenias. The association of T‐LGL was analysed with a number of immunogenetic factors in 66 patients, including human leucocyte antigen (HLA) and killer‐cell immunoglobulin‐like receptor (KIR) genotype, KIR/KIR‐L mismatch, CTLA‐4 (+49 A/G),CD16−158V/F, CD45 polymorphisms, cytokine single nucleotide polymorphisms including: TNF‐α (−308G/A), TGF‐β1 (codons 10 C/T, 25 G/C), IL‐10 (−1082 G/A), IL‐6 (−174 C/G), and IFN‐γ(+874 T/A). A statistically significant increase in A/A genotype for TNF‐α−308, IL‐10–1082, andCTLA‐4 +49 was observed in T‐LGL patients compared with control, suggesting that the G allele serves a protective role in each case. No association was found between specific KIR/HLA profile and disease. KIR/KIR‐L analysis revealed significant mismatches between KIR3DL2 and KIR2DS1 and their ligands HLA‐A3/11 and HLA‐C group 2 (P = 0·03 and 0·01 respectively); the biological relevance of this finding is questionable. The significance of additional genetic polymorphisms and their clinical correlation to evolution of T‐LGL requires future analysis.

We observed increased ferritin levels in newly diagnosed MDS-RARS patients without transfusional ironoverload. Hence, we hypothesized RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this myelodysplastic syndromes (MDS) subset. We studied a cohort of 140 MDS patients: 42 with RARS, 10 with increased ringed sideroblasts, and 96 with other forms of MDS (43 RA, 27 RAEB, 17 RAEB-T, 8 MDS/MPD, 1 CMML). Patients were genotyped using restriction fragment length polymorphism, designed to detect C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygosity for C282Y mutation in RARS patients compared with a large control population of matched race individuals (21 vs. 9.8% in controls, P 5 0.03); H63D genotype was not significantly increased. Frequency of HFE variation in other MDS subtypes failed to differ significantly from controls. Within this group, we included patients with a rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt). 10/14 RARSt patients were carriers of either C282Y or H63D allele significantly increased compared with the combined prevalence in a healthy population (71 vs. 33%, P < 0.01). We found expected distribution of mutant HFE alleles in patients with other forms of MDS (9.1 vs. 9.8%, P 5 0.82). Increased prevalence of HFE gene mutations is not a generalized feature of MDS, but some subgroups of MDS, especially those characterized by excessive accumulation of ringed sideroblasts, exhibit C282Y mutations at a higher frequency than in other forms of MDS and healthy controls. Am. J. Hematol. 82:1076Hematol. 82: -1079Hematol. 82: , 2007

Objective-T-cell-mediated autoimmunity may be involved in some cases of idiopathic neutropenia. We hypothesized that a precise T-cell receptor repertoire analysis may uncover cytotoxic T-cell (CTL) expansions that are less pronounced than those seen in T large granular lymphocyte leukemia (T-LGL), but are pathophysiologically analogous and thus can serve as markers of a T-cell-mediated process. Materials and Methods-Using rational algorithms for T-cell receptor analysis and in vivotracking of CTL responses previously established in our laboratory, we studied patients with unexplained chronic neutropenia (n = 20), T-LGL (n = 15), and healthy controls (n = 12). We further investigated the involvement of soluble inhibitory factors by coculture assays. To determine the level of immune activation, we studied interferon-g expression in CD8 + cells using Taqman polymerase chain reaction.Results-Fifteen expanded (immunodominant) CTL clones were detected in 12 of 20 patients. In comparison to LGL leukemia, these clones were less immunodominant, but clearly discernible from subclinical lymphoproliferations in controls. As a surrogate of cytotoxic activity, we found markedly increased production of interferon-γ in most of the neutropenia patients, irrespective of the presence of immunodominant CTL clones.Conclusions-These results suggest that, while immunodominant CTL clones are detectable in a proportion of patients only, CTL-mediated pathophysiology may be a general mechanism operating in idiopathic neutropenia. Oligogoclonal CTL expansions in chronic neutropenia may indicate an ongoing autoimmune process, while highly polarized monoclonalities in a subset of neutropenic LGL patients may represent the "extreme" end of the clonal continuum.Drugs, hereditary factors, infections, and intrinsic bone marrow diseases explain the etiology of most neutropenias. The inability to detect an immune-mediated process or other obvious cause often leads to diagnosis of chronic idiopathic neutropenia (CIN) [1][2][3][4][5][6]. Similarly, secondary autoimmune neutropenia (AIN), usually associated with collagen vascular diseases, lymphoproliferative disorders and viruses, is typically diagnosed by exclusion and thus relies on the quality of clinical and laboratory workups [6][7][8][9]. Lineage-restricted cytopenias, including neutropenia, have been associated with T-cell large granular lymphocyte leukemia It is likely that patients with AIN/CIN may belong to a heterogeneous group of diseases combining various autoimmune etiologies. Appearance of the bone marrow may reveal some clues as to the cellular targets of the autoimmune process; left shift could indicate that more mature cells are targets, while pure white cell aplasia suggests that very early myeloid precursors are affected. The similar target spectrum in AIN/CIN and T-LGL suggests that cytotoxic T-cell (CTL)-mediated autoimmunity can also operate in AIN/CIN. Consequently, detection of highly polarized CTL expansions in some cases of neutropenia patients may be consistent wi...

Myelodysplastic syndromes (MDS) are characterized by the presence of clonal chromosomal abnormalities detectable by traditional cytogenetics in around 50% of patients. We demonstrated that a higher percentage of unbalanced clonal chromosomal defects and uniparental disomy (UPD) can be identified in MDS using high-density SNP arrays (50 or 250K SNP-A; Gondek et al, 2005). The higher detection rate may have important clinical consequences. However, such findings must be considered in the context of normal karyotypic variation. Before the clinical relevance of new lesions identified by SNP-A can be presumed, several issues must be addressed. The increased precision of karyotypic analysis may lead to the detection of lesions in normal bone marrow. This is especially relevant to elderly patients with MDS for whom adequate age-matched comparisons should be performed. The normal distribution of chromosomal changes across the genome must be defined as it may overlap with that in disease. The minimal clonal size detectable by SNP-A analysis is of importance, as hematopoiesis may be oligo- rather than monoclonal in many conditions. We stipulated that the clinical applicability of SNP-A-based karyotyping will depend upon the findings in healthy controls and have studied 36 normal bone marrows using SNP-A karyotype analysis. We utilized the Affymetrix 250K SNP chip and the CNAG software for copy number and LOH analysis. Using a stringent set of criteria, suspicious lesions were identified in 83% of samples. Loci altered in 2 or more samples, most likely reflecting copy number polymorphisms (CNP), were identified in 70% of individuals. With these CNP excluded, 69% of controls harbored putative lesions. 11 marrows contained 1 chromosomal change, while 2-4 were found in 14 marrows. Both loss and gain of sequences were detected. The size of the largest deletion and duplication was 1.8 Mb and 1.64 Mb, respectively. Karyotypic abnormalities identified in control samples appeared to be randomly distributed across the genome. No lesions were identified on chromosome 8 or chromosome 5q, although one deletion on chromosome 5p was found. A small deletion of 7q was detected (1.2 Mb); overall, the regions frequently affected in MDS were rarely altered in controls. Finally, we identified LOH due to UPD spanning 13q21.3 to 13q32.1 (4.95 Mb) in one healthy control. To assess the minimal detectable clonal size, we next examined the sensitivity of SNP-A analysis to the admixture of normal cells. When samples identified as abnormal (deletion of 7q, and trisomy 8) by traditional cytogenetics were serially diluted with normal genomic DNA, the previously identified lesions could still be detected when the sample contained 25% normal genomic DNA, but at 50% the copy number analysis appeared normal. Our result underscores that, only significantly expanded clones can be systematically detected by SNP-A analysis. Pathogeneic defects have to exclude regions of CNP and must be larger than changes in healthy controls. Additionally, they would preferentially occur in regions not frequently affected in normal bone marrow. Our studies reveal the physiologic level of chromosomal abnormalities present in healthy controls and allow us to design criteria for defining abnormal karyotypes as measured by SNP-A.

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