Myeloid Nuclear Differentiation Antigen Expression Research Articles

MNDA expression and its value in differential diagnosis of B-cell non-Hodgkin lymphomas: a comprehensive analysis of a large series of 1293 cases.

MNDA (myeloid nuclear differentiation antigen) has been considered as a potential diagnostic marker for marginal zone lymphoma (MZL), but its utility in distinguishing MZL from other B-cell non-Hodgkin lymphomas (B-NHLs) and its clinicopathologic relevance in diffuse large B-cell lymphoma (DLBCL) are ambiguous. We comprehensively investigated MNDA expression in a large series of B-NHLs and evaluated its diagnostic value. MNDA expression in a cohort of 1293 cases of B-NHLs and 338 cases of reactive lymphoid hyperplasia (RLH) was determined using immunohistochemistry and compared among different types of B-NHL. The clinicopathologic relevance of MNDA in DLBCL was investigated. MNDA was highly expressed in MZLs (437/663, 65.9%), compared with the confined staining in marginal zone B-cells in RLH; whereas neoplastic cells with plasmacytic differentiation lost MNDA expression. MNDA expression was significantly higher in mantle cell lymphoma (MCL, 79.6%, p = 0.006), whereas lower in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, 44.8%, p = 0.001) and lymphoplasmacytic lymphoma (LPL, 25%, p = 0.016), and dramatically lower in follicular lymphoma (FL, 5.2%, p < 0.001), compared with MZL. 29.6% (63/213) of DLBCLs were positive for MNDA. The cases in non-GCB group exhibited a higher rate of MNDA positivity (39.8%) compared to those in GCB group (16.3%) (p < 0.001), and MNDA staining was more frequently observed in DLBCLs with BCL2/MYC double-expression (50%) than those without BCL2/MYC double-expression (24.8%) (p = 0.001). Furthermore, there was a significant correlation between MNDA and CD5 expression in DLBCL (p = 0.036). MNDA was highly expressed in MZL with a potential utility in differential diagnosis between MZL and RLH as well as FL, whereas its value in distinguishing MZL from MCL, CLL/SLL is limited. In addition, MNDA expression in DLBCL was more frequently seen in the non-GCB group and the BCL2/MYC double-expression group, and demonstrated a correlation with CD5, which deserves further investigation. The clinical relevance of MNDA and its correlation with the prognosis of these lymphomas also warrant to be fully elucidated.

MNDA, a PYHIN factor involved in transcriptional regulation and apoptosis control in leukocytes.

Inflammation control is critical during the innate immune response. Such response is triggered by the detection of molecules originating from pathogens or damaged host cells by pattern-recognition receptors (PRRs). PRRs subsequently initiate intra-cellular signalling through different pathways, resulting in i) the production of inflammatory cytokines, including type I interferon (IFN), and ii) the initiation of a cascade of events that promote both immediate host responses as well as adaptive immune responses. All human PYRIN and HIN-200 domains (PYHIN) protein family members were initially proposed to be PRRs, although this view has been challenged by reports that revealed their impact on other cellular mechanisms. Of relevance here, the human PYHIN factor myeloid nuclear differentiation antigen (MNDA) has recently been shown to directly control the transcription of genes encoding factors that regulate programmed cell death and inflammation. While MNDA is mainly found in the nucleus of leukocytes of both myeloid (neutrophils and monocytes) and lymphoid (B-cell) origin, its subcellular localization has been shown to be modulated in response to genotoxic agents that induce apoptosis and by bacterial constituents, mediators of inflammation. Prior studies have noted the importance of MNDA as a marker for certain forms of lymphoma, and as a clinical prognostic factor for hematopoietic diseases characterized by defective regulation of apoptosis. Abnormal expression of MNDA has also been associated with altered levels of cytokines and other inflammatory mediators. Refining our comprehension of the regulatory mechanisms governing the expression of MNDA and other PYHIN proteins, as well as enhancing our definition of their molecular functions, could significantly influence the management and treatment strategies of numerous human diseases. Here, we review the current state of knowledge regarding PYHIN proteins and their role in innate and adaptive immune responses. Emphasis will be placed on the regulation, function, and relevance of MNDA expression in the control of gene transcription and RNA stability during cell death and inflammation.

Myeloid nuclear differentiation antigen: an aid in differentiating lymphoplasmacytic lymphoma and splenic marginal zone lymphoma in bone marrow biopsies at presentation

The differential diagnosis between lymphoplasmacytic lymphoma (LPL) and marginal zone B-cell lymphoma, particularly splenic type (SMZL), can be challenging on onset of bone marrow biopsy (BMB) since morphology and phenotype are not specific and clinical features can overlap or be mildly developed at diagnosis. The LPL-specific L265P mutation in the MYD88 gene is not available in all laboratories, and genetic aberrancies identified in SMZL (del7q, mutations of NOTCH2 and KLF2) are seldom searched in routine practice. The study aim is to investigate the potential role of myeloid nuclear differentiation antigen (MNDA) expression in this specific differential diagnosis. We report MNDA reactivity in 559 patients with small B-cell lymphoma including bone marrow biopsies from 90 LPL and 91 SMZL cases. MYD88 p.Leu265Pro mutation status was assessed and confirmed as positive in 24 of 90 LPL cases, which served as the test set. MNDA staining was negative in 23 of 24 LPL cases in the test set (96%). In the 157 remaining cases (66 LPL, 91 SMZL), which served as the validation set, the MYD88 p.Leu265Pro mutation was unavailable and MNDA was more frequently expressed in SMZL (p<0.00001). In addition, immunohistochemical features more consistent with SMZL (i.e., presence of CD23+ follicular dendritic cell meshworks, polytypic plasma cells, DBA44 reactivity) were more often present in MNDA-positive cases (statistically significant for 2 such parameters). On the widest case series so far published focusing on LPL and SMZL immunohistochemical diagnosis at onset of BMB, we demonstrated that MNDA expression significantly support the diagnosis of SMZL. This observation may be of particular help in cases where the MYD88 p.Leu265Pro mutational status and/or SMZL-related genetic aberrations are unavailable.

MNDA controls the expression of MCL-1 and BCL-2 in chronic lymphocytic leukemia cells

The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course.

IRTA1 and MNDA Expression in Marginal Zone Lymphoma: Utility in Differential Diagnosis and Implications for Classification.

To evaluate the clinical utility of immune receptor translocation-associated protein 1 (IRTA1) and myeloid nuclear differentiation antigen (MNDA) expression in the diagnosis and classification of marginal zone lymphomas (MZLs). IRTA1 was examined using a novel RNA in situ hybridization assay and MNDA expression determined by immunohistochemistry in 127 small B-cell neoplasms, including 80 cases of MZL. IRTA1 expression was detected in 31 (42%) of 74 MZLs vs one (2%) of 43 other small B-cell neoplasms (P < .001). MNDA staining was positive in 51 (64%) of 79 MZLs vs 21 (45%) of 46 non-MZLs (P = .06). MNDA expression was particularly uncommon in follicular lymphoma (3/14, 21%; P = .003 vs MZL). There was no association between MNDA and IRTA1 expression and the presence of monocytoid cytology. IRTA1 expression was less frequent in cases with a diffuse growth pattern. IRTA1 and MNDA are useful markers in the differential diagnosis of MZLs.

Value of Quantitative assessment of Myeloid Nuclear Differentiation Antigen expression and other flow cytometric parameters in the diagnosis of Myelodysplastic syndrome.

The diagnosis of myelodysplastic syndrome (MDS) based on morphology is particularly difficult in low-grade MDS. Thus, the role of myeloid nuclear differentiation antigen (MNDA) and other flow cytometric (FCM) parameters in MDS was evaluated. Bone marrow aspirates (BMA) collected from 52 patients with unexplained persistent cytopenias were divided into three groups: (i) proven MDS (n = 12) based on morphology and/or cytogenetics; (ii) suspected MDS (n = 6), noncontributory morphology, and cytogenetics; and (iii) non-MDS (n = 34). Sixteen control BMA were studied. Cases were analyzed for MNDA expression (on granulocytes, blasts, monocytes, and lymphocytes) and for seven quantitative parameters: CD34(+) myeloblasts % in nucleated cells, CD34(+) B-cell progenitor% in CD34(+) cells, lymphocyte/myeloblast CD45 MFI ratio, granulocyte/lymphocyte SSC peak channel ratio and the proportion of CD34(+) myeloblasts expressing CD15, CD11b, and CD56. A score of 1 was given to each parameter beyond the cutoff, and score ≥3 was considered FCM positive. MNDA expression on granulocytes and blasts was significantly lower in proven MDS and suspected MDS vs. non-MDS. Quantitative FCM parameters successfully distinguished MDS and suspected MDS from non-MDS. MNDA expression is an independent marker for the evaluation of dyspoiesis and may be added to the standard panel for quantitative assessment by FCM.

Myeloid Nuclear Differentiation Antigen (MNDA) Expression: A Useful Marker in the Diagnosis of Myelodysplastic Syndromes By Flow Cytometry

Background: Diagnostic workup for suspected myelodysplastic syndromes (MDS) is increasingly done with the aid of multiparamter flow cytometry (MFC) detecting aberrant antigen expression. Myeloid nuclear differentiation antigen (MNDA) is a hematopoietic protein expressed strongly in mature myeloid cells, but only weakly in myeloid progenitor cells. Recently, reduced MNDA expression was detected by MFC in monocytes (M), granulocytes (G) and myeloid progenitor cells (MP) in patients (pts) with MDS and MNDA as additional marker for MFC was proposed to improve diagnostic capabilities of MFC.Aim: To verify differences in MNDA expression in M, G and MP of pts with MDS vs. those without MDS and to test its value as additional MFC parameter in a cohort with suspected MDS.Patients and methods: We analysed bone marrow from 131 pts (median age 74, range 17-93 years) with suspected MDS by cytomorphology (CM), standard 10-color-MFC and cytogenetics (CG) in parallel. For detection of MNDA expression we applied a readily available five color intracellular staining assay using monoclonal antibodies against MNDA, CD45, CD64, CD15 and myeloperoxidase (MDS-Quant, Trillium Diagnostics, Bangor, ME). Different gating strategies to best define M [SSC/CD45 plot (A) vs CD16/CD15 plot (B)], G (A vs B) and MP (A) were used. MNDA expression in pts diagnosed with no MDS (n=35), possible MDS (n=40; based on morphologic dysplastic changes insufficient for diagnosis of MDS) and MDS (n=56) by CM was compared. We also correlated results to findings in standard MDS-MFC (n=128) and CG (n=120).Results: Based on CM diagnosis, pts with MDS showed higher percentages of G and M with weak expression of MNDA (%dimG and %dimM) than no-MDS pts irrespective of the gating strategy [mean±SD, %dimG: 16±17 vs 6±7, p<0.001 (A) and 3±3 vs 1±2, p=0.011 (B); %dimM: 22±21 vs 15±12, p=0.054 (A) and 14±25 vs 4±4, p=0.004 (B)]. Diagnostically challenging cases with "possible MDS" by CM also displayed significantly higher %dimG and %dimM than no-MDS cases [%dimG 13±14 vs 6±7, p=0.006 (A), %dimM: 23±20 vs 15±12, p=0.027 (A) and 17±24 vs 4±4, p=0.002 (B)]. Differences between MDS and possible MDS existed only in higher %dimGra in MDS gated in plot B (2.7±3.3 vs 1.6±1.9, p=0.036). Conversely, MDS pts and pts with possible MDS had higher percentages of MP with high MNDA expression (%hiMP) than no-MDS pts (17±16 vs 9±8, p=0.005 and 14±13 vs 9±8, p=0.041, respectively). MNDA expression levels measured by mean fluorescence intensity (MFI) were diminished in M of pts with MDS vs no-MDS [26±12 vs 33±9, p=0.006 (A) and 30±14 vs 39±10, p=0.002 (B), respectively] and in pts with possible MDS vs no-MDS pts [26±12 vs 33±9, p=0.004 (A) and 30±2 vs 39±10, p=0.005, respectively]. Looking at the low level MNDA MP (dimMP), higher MFIs were found in MDS compared to no-MDS (1±0.8 vs 0.7±0.4, p=0.002) and in possible MDS vs no-MDS (1.0±0.7 vs 0.7±0.4, p=0.011). Comparing results of MNDA MFC and standard MFC we also found higher MFI in dimMP (1.0±0.8 vs 0.7±0.3, p=0.039) in pts diagnosed MDS by MFC (n=63) vs those without signs of MDS (n=14). In the inexplicit cases diagnosed MDS possible by MFC (aberrant antigen expressions not sufficient for MDS diagnosis, n=51) higher percentages of dimG [16±18 vs 9±10, p=0.015 (A) and 3±3 vs 1±2, p=0.005 (B)] and dimM [16±25 vs 7±13, p=0.02 (B)] were seen as compared to no-MDS, in line with CM results. Moreover, a lower MFI in M [26±12 vs 31±11, p=0.024 (A) and 29±14 vs 36±13, p=0.02 (B)] and a higher MFI in dimMP (1.0±0.8 vs 0.9±0.6, p=0.021) could be detected. Considering CG, cases with an aberrant karyotype (n=41) had higher values for %dimM (27±23 vs 18±16, p=0.035) as compared to those with a normal karyotype (n=79). Including the most significant markers [%dimG>12 (A), %dimM>13 (B), %hiMP>15 (A), 1 point each], we created a new MNDA score with a score of ≥2 indicating MDS. Of 14 pts with a score of ≥2, 13 pts were concordantly diagnosed MDS using CM for validation; 1 pt had no MDS by CM but was classified MDS by standard MDS-MFC.Conclusions: Reduced MNDA levels and higher percentages of M and G with low MNDA expression as well as a higher percentage of MP with high MNDA expression could be confirmed in pts with MDS. Applying our newly defined MNDA score for MFC, identification of a subset of pts with MDS was possible at high specificity. Further analyses will have to evaluate this MNDA score incorporated into standard MDS-MFC panels to improve MFC-based diagnostic approaches for MDS. DisclosuresBellos:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Davis:Trillium Diagnostics: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Evaluation of flow cytometric assessment of myeloid nuclear differentiation antigen expression as a diagnostic marker for myelodysplastic syndromes in a series of 269 patients

Myeloid nuclear differentiation antigen (MNDA) is expressed in myelomonocytic cells with highest levels in mature granulocytes and monocytes. It is suggested to be expressed more weakly in patients with myelodysplastic syndromes (MDS). The analysis of MNDA therefore may improve diagnostic capabilities of multiparameter flow cytometry (MFC) in MDS. We used MFC for detection of MNDA expression in 269 patients with suspected or known MDS, acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMML), cytopenia of unknown cause or without malignancy (negative controls). Results were compared with the diagnoses revealed by cytomorphology (CM) and cytogenetics (CG). Percentages of granulocytes and monocytes with diminished MNDA expression (dimG and dimM) were higher in patients with MDS (mean ± SD, 20% ± 20%, P < 0.001 and 31% ± 24%, P < 0.001) and AML (27% ± 27%, P = 0.007 and 45% ± 31%, P = 0.001) diagnosed by CM, vs. patients without MDS (8% ± 10% and 16% ± 11%), respectively. Significant differences were also found for mean fluorescence intensity (MFI) of MNDA in monocytes which was lower in MDS (mean ± SD, 71 ± 36, P = 0.004) and AML (55 ± 39, P < 0.001) vs. no MDS samples (85 ± 28), respectively. Within patients with MDS, cases with cytogenetic aberrations showed a trend to higher %dimG (24% ± 18%, P = 0.083) compared with those without (16% ± 21%). Cut-off values for %dimG (12%) and %dimM (22%) as well as for MFI in monocytes (72) were defined capable of discriminating between MDS and non-MDS. MNDA expression in bone marrow cells can be assessed reliably by MFC and may facilitate evaluation of dyspoiesis when added to a standard MDS MFC panel.

Flow Cytometric Assessment of Myeloid Nuclear Differentiation Antigen (MNDA) Expression in Myelodysplastic Syndromes As a Diagnostic Marker,

Abstract 3805 Background:Detection of aberrant antigen expression by multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of myelodysplastic syndromes (MDS). Myeloid nuclear differentiation antigen (MNDA) has been described to be strongly expressed in mature myeloid cells while myeloid progenitor cells show a weak expression only. MNDA has been suggested to be aberrantly expressed in MDS and therefore may improve the diagnostic capabilities of MFC. Aim: To analyze differences in the expression of MNDA between non-MDS cytopenias, MDS, and AML. Patients and methods: Bone marrow samples from a total of 269 patients with cytopenias and suspected MDS were analyzed by cytomorphology and MFC in parallel for diagnostic purposes. MNDA expression was determined by MFC in granulocytes, monocytes, and myeloid progenitor cells using the antibody clone 3C1 (provided by Trillium Diagnostics, Bangor, ME). MNDA expression was compared between patients grouped according to cytomorphology as: no MDS (n=103), MDS (n=85), CMML (n=9), AML (n=19), and possible MDS (n=53, based on cytomorphologic features of dysplasia but not sufficient to diagnose MDS). In addition, MNDA expression was compared between patients with aberrant and normal karyotype (cytogenetics was available in 238/269 patients, an aberrant karyotype was present in 59 patients). Results: A higher percentage of granulocytes (G) and monocytes (M) with weak expression of MNDA (%dimG and %dimM) was consistently found in MDS/AML samples as compared to samples without MDS. In detail, %dimG (mean±SD, 20±20% vs. 8±10%, p<0.001) and %dimM (31±24% vs. 16±11%, p<0.001) were significantly higher in cases with MDS as compared to those without. Furthermore, in patients with MDS, as compared to those without, the mean fluorescence intensity (MFI) of MNDA expression was found to be lower in monocytes (mean MFI, 71±36 vs. 85±27, p=0.004) as well as in myeloid progenitor cells with bright (67±24 vs. 86±66, p=0.007) or dim (2.5±0.9 vs. 2.8±1.2, p=0.037) expression of MNDA. In AML cases, as compared to cases with no MDS, similar figures were observed with higher values for %dimG (27±27% vs. 8±10%, p=0.007) and for %dimM (45±31% vs. 16±11%, p=0.001). In addition, in AML cases MFI of MNDA expression was lower in monocytes (55±39 vs. 85±27, p<0.001). Considering the diagnostically challenging cases with dysplastic features by cytomorphology but not sufficient to diagnose MDS similar although smaller differences were seen as compared to cases with no MDS with higher values for both %dimG (15±22% vs. 8±10%, p=0.026) and %dimM (25±19% vs. 16±11%, p=0.002) as well as a lower MFI of MNDA expression in monocytes (73±26 vs. 85±27, p=0.010). Interestingly, in CMML cases no reduced expression of MNDA could be found in any of the cell lines analyzed which might argue in favour of regarding CMML as myeloproliferative rather than myelodysplastic disease. From the cytogenetic point of view cases with an aberrant karyotype had higher values for both %dimG (22±20% vs. 12±18%, p=0.001) and %dimM (33±24% vs. 23±21%, p=0.004) as compared to those with a normal karyotype. Conclusions: These data suggests that MDS, as compared to non-MDS bone marrow, is associated with a reduced expression of MNDA in different cell lines which in part also applies to AML. Further analyses should define the potential of MNDA assessment to improve the MFC-based approach to diagnose MDS. Disclosures:Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bellos:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Quantitative Assessment of Myeloid Nuclear Differentiation Antigen Distinguishes Myelodysplastic Syndrome From Normal Bone Marrow

By using flow cytometry, we analyzed myeloid nuclear differentiation antigen (MNDA) expression in myeloid precursors in bone marrow from patients with myelodysplastic syndrome (MDS) and control samples from patients undergoing orthopedic procedures. The median percentage of MNDA-dim myeloid precursors in MDS cases was 67.4% (range, 0.7%-97.5%; interquartile range, 44.9%-82.7%) of myeloid cells, with bimodal MNDA expression in most MDS samples. Control samples demonstrated a median MNDA-dim percentage in myeloid precursors of 1.2% (range, 0.2%-13.7%; interquartile range, 0.6%-2.7%), with no bimodal pattern in most samples. The area under the receiver operating characteristic curve for MNDA-dim percentage in myeloid precursors was 0.96 (P = 9 × 10(-7)). Correlation of MNDA-dim levels with World Health Organization 2008 morphologic diagnoses was not significant (P = .21), but correlation with patient International Prognostic Scoring System scores suggested a trend (P = .07). Flow cytometric assessment of MNDA in myeloid precursors in bone marrow may be useful for the diagnosis of MDS.

Quantitative Assessment of Myeloid Nuclear Differentiation Antigen Distinguishes Myelodysplastic Syndrome From Normal Bone Marrow.

Abstract 1754▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-780 IntroductionDefinitive diagnosis and classification of MDS are often difficult because of variable presence of diagnostic criteria and imprecision and ambiguities of interpretation of both morphologic and ancillary data. An objective criterion that reliably distinguishes MDS from normal marrow would greatly facilitate diagnosis, and might contribute to subclassification of MDS. Gene expression profiling has identified marked (7 fold) down-regulation of myeloid nuclear differentiation antigen (MNDA) message in cases of MDS (Hofmann WK, et al, Blood 2002;100:3553-60), and we have previously shown down-regulation of MNDA protein expression in random MDS cases using immunohistochemistry and flow cytometry (Briggs RC, et al. Cancer Research 2006;66:4645-51). To continue our previous analyses, we evaluated MNDA expression in myeloid progenitors using quantitative flow cytometric analysis in MDS and normal control marrow samples. Patients and MethodsThe study included 20 MDS patients receiving only supportive care undergoing bone marrow sampling for clinical purposes, and 19 frequency age-matched normal controls undergoing orthopedic surgery with no antecedent primary hematologic abnormalities. MDS diagnosis was based on 2008 WHO criteria. Quantitative flow cytometric analysis was performed with an FC500 flow cytometer (Beckman Coulter, Fullerton, CA). Cells were treated with CD45-PE and CD34-ECD (Beckman Coulter), washed in PBS with 2% FCS, and permeabilized with PermiFlow. MNDA-Alexa-488 was added at a granulocyte-monocyte specific concentration, with analysis using Winlist 5.0 software (Verity Software, Topsham, ME) with DDE links to ModFitLT 3.0 using modifications of published methods. Differentiating myeloid progenitors were identified as high side scatter/intermediate CD45/CD34-negative cells, with lymphocytes (low side scatter/high CD45/CD34-negative cells) serving as an internal dim MNDA control in each sample. ResultsMDS cases consisted of 12 patients with refractory cytopenia with multilineage dysplasia (RCMD), 3 with refractory anemia with excess blasts-1(RAEB-1), 4 with RAEB-2, and 1 with therapy-related MDS. In myeloid progenitors in MDS patients, the median percent of MNDA-dim cells was 67.4% (range 0.7-97.5%, interquartile range 44.9-82.7%). The analogous median percent of MNDA-dim cells in control patients was 1.2% (range 0.2-13.7%, interquartile range 0.6-2.7%). The area under the ROC curve was 0.96 (p = 9×10-7), indicating almost complete discrimination between cases and controls. 19 of 20 MDS patients demonstrated bimodal distribution of MNDA expression comprising a distinct MNDA-dim population and a separate MNDA-normal population, suggesting an admixture of MDS and normal cells. 18 of 19 control patients demonstrated a single population of MNDA-normal cells without evidence of a bimodal distribution. The single MDS patient with normal MNDA expression had prior clinical and laboratory features suggestive of refractory anemia with ringed sideroblasts. The single pediatric MDS patient had reduced MNDA expression similar to other high grade MDS samples. Patients showed trends for MDS subtype (p = 0.21) and IPSS score (p = 0.07) versus percent MNDA-dim myeloid progenitors. These data demonstrate remarkable sensitivity and specificity for use of MNDA expression to detect MDS. ConclusionsQuantitative flow cytometric analysis of MNDA expression in marrow myeloid progenitors is a promising objective test for diagnosis of MDS, demonstrating a striking difference of MNDA expression in myeloid progenitors in MDS versus control patients. Our results require elaboration with analysis of low grade cases. A single possible low grade MDS case in this series demonstrated MNDA expression identical to normal control samples. Our results also require elaboration in cases constituting the differential diagnosis of MDS to evaluate the clinical utility of MNDA evaluation. The biological significance of down-regulation of MNDA in MDS is uncertain, although MNDA has been implicated in regulation of programmed cell death. Our results suggest a mix of normal and abnormal myelopoiesis in MDS patients, as predicted by cytogenetics in many cases of MDS. In summary, testing of MNDA expression in myeloid progenitors shows great promise as an objective test for diagnosis of MDS in marrow samples. DisclosuresNo relevant conflicts of interest to declare.

Dysregulated Human Myeloid Nuclear Differentiation Antigen Expression in Myelodysplastic Syndromes: Evidence for a Role in Apoptosis

Reduced levels of human myeloid nuclear differentiation antigen (MNDA) gene transcripts have been detected in both familial and sporadic cases of myelodysplastic syndromes (MDS). Numerous reports implicate elevated apoptosis/programmed cell death and death ligands and their receptors in the pathogenesis of MDS. MNDA and related proteins contain the pyrin domain that functions in signaling associated with programmed cell death and inflammation. We tested the hypothesis that MNDA is involved in the regulation of programmed cell death in human myeloid hematopoietic cells. Clones of K562 cells (MNDA-null) that expressed ectopic MNDA protein were established using retroviral transduction. MNDA-expressing K562 clones were resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, but were not protected from programmed cell death induced with genotoxic agents or H(2)O(2). MNDA protein expression assessed in control and intermediate and high-grade MDS marrows showed several patterns of aberrant reduced MNDA. These variable patterns of dysregulated MNDA expression may relate to the variable pathophysiology of MDS. We propose that MNDA has a role regulating programmed cell death in myeloid progenitor cells, and that its down-regulation in MDS is related to granulocyte-macrophage progenitor cell sensitivity to TRAIL-induced programmed cell death.

Dysregulated Expression of Myeloid Nuclear Differentiation Antigen (MNDA) in MDS: Possible Role in TRAIL-Induced Apoptosis.

A reduced level of MNDA expression has been reported with gene expression profiling in both sporadic and familial MDS. We undertook studies to evaluate MNDA protein expression in MDS, and its possible functional significance. Patients (n=7) clinical histories, lab data, cytogenetics, and morphology were reviewed blinded to study results to confirm diagnoses. Patients had Refractory Cytopenia with Multilineage Dysplasia (n=6) or Refractory Anemia with Excess Blasts-1 (n=1)(WHO). Patients lacking primary marrow disease (n=5) were used as controls. Samples were assessed with multiparametric flow cytometry (MPFC) for CD45 antigen density, log side scatter, CD34, CD71, and MNDA (using a specific rat monoclonal antibody) with an FC500 (Beckman-Coulter) flow cytometer, and analyzed with Winlist 5.0 and ModFitLT 3.0 (Verity Software) using published methods and ECFI-generated algorithms. Analyses were blinded to clinical results. Immunohistochemistry (IHC) for MNDA was performed using the same antibody and published methods. Both MPFC and IHC demonstrated MNDA expression in MDS predominantly in granulocyte-macrophage (G-M) progenitors, similar to controls. G-M progenitors in MDS demonstrated reduced MNDA protein expression compared to controls, with 3 patterns: uniform reduced MNDA protein, a mix of normal and absent MNDA protein, or a mix of reduced and absent MNDA protein. We interpret the normal expression in some cases as consistent with presence of a mixture of normal and MDS cells, while the mix of reduced and absent expression may represent clonal evolution of MDS. To evaluate possible functional significance of MNDA expression we used stable K562 clones that express ectopic MNDA protein via retroviral transduction. Parental K562 cells lack MNDA expression. Compared to parental K562, ectopic expression of MNDA failed to protect cells from intrinsic path apoptosis induced by genotoxic agents (mechlorethamine, etoposide, cisplatin) or H2O2. Apoptosis was evaluated by morphology, TUNEL assay, trypan blue exclusion, and phosphorylation of Histone H2B at serine 14 (a specific marker of apoptosis). In sharp contrast, ectopic MNDA expression provided marked protection of cells from extrinsic path apoptosis induced by TRAIL (TNF-related apoptosis inducing ligand) exposure. Disordered apoptosis is hypothesized to be a factor in MDS pathogenesis. Our results indicate MNDA promotes viability in myeloid progenitors, and its down-regulation in MDS might relate to the G-M progenitor sensitivity to TRAIL-induced apoptosis that has been reported in MDS.

Variable expression of human myeloid specific nuclear antigen MNDA in monocyte lineage cells in atherosclerosis

MNDA (human myeloid nuclear differentiation antigen) is expressed in specific lineages of hematopoietic cells and most notably at high levels in macrophages at sites of inflammation. MNDA and related proteins appear to modulate the activity of transcription factors and in some cases have a role in mediating cell death. The expression of MNDA was characterized in normal and diseased human aorta. MNDA positive cells double labeled for CD68 in all tissue examined. Twenty percent of normal aortas were negative or contained rare MNDA positive cells while other normal aorta contained more frequent positive cells. In atherosclerotic aorta, the number of MNDA positive cells increased with progression of disease. In normal and early lesions, MNDA positive cells adjacent to the endothelium generally displayed a strong MNDA reactivity associated with small amount of CD68 reactive cytoplasm. In the same sections, MNDA positive cells at increasing distances from the endothelium displayed lower MNDA reactivity and were associated with larger amounts of CD68 reactive cytoplasm. Foam cells in fatty streaks exhibited MNDA reactivity that ranged from strong to weak or negative. In advanced lesions, cells in the shoulder and those in fibrous tissue surrounding an atheroma were highly reactive for MNDA. However, only a fraction of the CD68 positive foam cells near the lipid core under the cap and shoulder contained MNDA reactivity. The variation in MNDA expression appeared to change with phenotypic specialization of monocytes in atherosclerosis consistent with its association with inflammation and suspected roles in regulating gene expression or in mediating cell death.

Myeloid Nuclear Differentiation Antigen (MNDA) Protein Expression in Myelodysplastic Syndrome (MDS) Is Reduced in Maturing Myeloid Cells.

Expression of the human MNDA protein is restricted to hematopoietic cells, and occurs at uniformly high level only in the normal granulocyte/monocyte lineage. Myeloblasts and all later stages of the lineage express MNDA, while stem cells do not (Hum Pathol 1999; 30:1040). Gene expression analyses of MDS cases have shown that MNDA mRNA is significantly down regulated in familial MDS and in high-risk sporadic MDS cases (Blood 2000; 100:3553 and Leukemia 2004; 18:449). We evaluated MNDA protein expression in patients with MDS.Methods: We directly conjugated Alexa 488 (Molecular Probes) to a rat monoclonal antibody specific for MNDA for use in multiparameter cytometric analysis with CD45/CD34 and DRAQ5 (DNA content). Patients' (n=17) clinical histories, laboratory data, cytogenetics, and morphology were reviewed for confirmation of diagnoses and blinded to study results. Patients lacking primary marrow disease (n=9) were used as controls. Analysis was performed using Winlist 5.0 software (Verity Software) with DDE links to ModFitLT 3.0 using modifications of published methods along with Esoterix Center for Innovation generated algorithms. Analyses were blinded to clinical results.Results: The normal dataset revealed MNDA expression in expected cell populations, with only a small subset of CD34+ cells exhibiting MNDA (7.5% on average). Expression of MNDA protein in maturing myeloid forms in normals was consistently greater than in MDS samples (MFI=802 vs. 569 in MDS; MFI=mean fluorescence intensity defined by the linear mean of each population). Six of 17 MDS cases displayed greater than 10% maturing myeloid forms lacking MNDA protein expression (never observed in normals). The % MNDA+/CD34+ cells was identical in the two datasets (7.5% in normal vs. 7.6% in MDS).Discussion: Our results suggest that MNDA protein expression in MDS patients follows a pattern similar to that in gene expression analyses. We additionally demonstrate that MNDA protein expression at the cellular level is variable in MDS, versus uniform expression in normals; parallel data for MNDA message expression at the cellular level is not available. The finding of MNDA-negative myeloid forms in MDS may have diagnostic utility, and may provide insight into both impaired myeloid differentiation in MDS and the biologic role of MNDA.

Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: evidence for the role of Sp1 and not of c-Myb or PU.1 in myelomonocytic lineage-specific expression.

The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelomonocytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in bone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to -16) that did not include the cluster of c-Myb sites. A 4-bp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22) including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4-bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super-shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene.