Expansion Of Paroxysmal Nocturnal Hemoglobinuria Clone Research Articles

Insights from Trajectories of PIGA-Mutated Clones in Paroxysmal Nocturnal Hemoglobinuria

Background: Patients with paroxysmal nocturnal hemoglobinuria (PNH) exhibit clonal expansion of hematopoietic cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Mutations in PIGA gene, involved in the biosynthesis of GPI-APs, result in the loss of GPI-APs from hematopoietic cells. However, PIGA mutations alone are insufficient to drive this clonal expansion. The pathogenesis of clonal expansion in PNH remains elusive, while several hypotheses or factors have been proposed, including immune attacks with concomitant aplastic anemia (AA) or additional mutations offering intrinsic growth advantage. Recently high-sensitivity flow cytometry and next-generation sequencing have allowed us to evaluate the clonal dynamics and architecture more precisely. The method has the potential to clarify the trajectories of PIGA mutated clones before the significant clonal expansion or the diagnosis, thereby to gain insight into the pathogenesis of the clonal expansion in PNH. Methods: To estimate the trajectories of PIGA-mutated clones, we constructed phylogenetic trees using whole genome sequencing (WGS) data of single cell-derived colonies of bone marrow samples from two PNH patients (53 colonies for Case 1 and 75 for Case 2). We used the targeted-sequencing and exome sequencing to detect driver mutations after isolating GPI-deficient and GPI-positive cells using flow cytometry. Results: Case 1 was diagnosed with AA at the age of 28. He developed hemoglobinuria at the age of 40 and was diagnosed with PNH at the age of 45. Bone marrow and peripheral blood samples were collected when he was 47 years old. The PNH clone sizes in granulocytes and red blood cells were 88.7% and 46.1%, respectively. Targeted-sequencing revealed six PIGA mutations in GPI-deficient cells and three BCOR mutations in GPI-positive cells. Case 2 was diagnosed with AA at the age of 33 and with PNH at the age of 34. Bone marrow and peripheral blood samples were collected when she was 59 years old. The PNH clone sizes in granulocytes and red blood cells were 98.6% and 96.3%, respectively. We previously identified chromosomal abnormalities in chr12, with the breakpoint located in an HMGA2 gene (Inoue et al. Blood. 2006). Targeted-sequencing detected no other driver mutations than single PIGA mutation and the HMGA2 mutation.In Case 1, Phylogenetic tree analysis constructed with WGS of single cell-derived colonies revealed multiple PIGA-mutated and BCOR-mutated clones that expanded independently of each other. All BCOR-mutated clones were identified in the GPI-positive population. In Case 2, all PIGA-mutated clones harbored the HMGA2 mutation, while no GPI-positive populations acquired the HMGA2 mutation.In both cases, the numbers of passenger single nucleotide variants per year were higher in PIGA-mutated clones than in PIGA-unmutated clones (16 vs. 11 mutations/year for Case 1 and 14 vs. 12 for Case 2). Assuming that variant acquisition rates per cell division are similar in both clones, the result suggests that PIGA-mutated clones had growth advantage against PIGA-unmutated clones.The acquisition of PIGA mutations was estimated to have occurred more than ten years prior to the PNH diagnosis. In Case 1, following the AA diagnosis, both PIGA-mutated clones and BCOR-mutated clones started expansion simultaneously, which suggests an association with immune attacks. Interestingly, even wild type clones expanded after AA diagnosis, suggesting bottle-neck effects of post-AA hematopoietic recovery. In Case 2, PIGA-mutated clones began expanding more than ten years before the AA diagnosis. Either a subclinical immune attack that might have existed before the diagnosis of clinical AA or the concomitant HMGA2 mutation conferred survival advantage for PIGA-mutated clones in Case 2. Conclusions: This estimation of the trajectories of the PIGA-mutated clone indicates that the pathogenesis of clonal expansion exhibits heterogeneity between two PNH patients.

Insights Into the Emergence of Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease as simple as it is complex. PNH patients develop somatic loss-of-function mutations in phosphatidylinositol N-acetylglucosaminyltransferase subunit A gene (PIGA), required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. Ubiquitous in eukaryotes, GPI anchors are a group of conserved glycolipid molecules responsible for attaching nearly 150 distinct proteins to the surface of cell membranes. The loss of two GPI-anchored surface proteins, CD55 and CD59, from red blood cells causes unregulated complement activation and hemolysis in classical PNH disease. In PNH patients, PIGA-mutant, GPI (-) hematopoietic cells clonally expand to make up a large portion of patients’ blood production, yet mechanisms leading to clonal expansion of GPI (-) cells remain enigmatic. Historical models of PNH in mice and the more recent PNH model in rhesus macaques showed that GPI (-) cells reconstitute near-normal hematopoiesis but have no intrinsic growth advantage and do not clonally expand over time. Landmark studies identified several potential mechanisms which can promote PNH clonal expansion. However, to what extent these contribute to PNH cell selection in patients continues to be a matter of active debate. Recent advancements in disease models and immunologic technologies, together with the growing understanding of autoimmune marrow failure, offer new opportunities to evaluate the mechanisms of clonal expansion in PNH. Here, we critically review published data on PNH cell biology and clonal expansion and highlight limitations and opportunities to further our understanding of the emergence of PNH clones.

Leveraging Whole Genome Sequencing to Define the Mutational Landscape in Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a prototypic disorder of hematopoietic stem cells leading to recurrent complement-mediated intravascular hemolysis. The development of PNH has been linked to somatic mutations in the PIGA gene, coding for an enzyme critically involved in GPI anchor synthesis. While mutations in PIGA are pathognomonic for PNH, other mutations of GPI biosynthetic pathways have been described in isolated cases. Moreover, additional genetic determinants of clonal advantage and other genetic modifiers of the clinical course have not been sufficiently clarified. Aim: This study was performed to investigate the molecular pathogenesis of PNH in a large patient cohort. Methods: Using a typical myeloid targeted panel, mutations in PIGA gene can be identified 60-70% of cases and thus we performed whole genome sequencing (WGS, mean. coverage 100x). To further clarify the molecular pathogenesis of PIGA WT cases. A cohort of 94 patients (irrespective of PIGA mutations) with a confirmed hemolytic PNH, primary or evolved from antecedent AA was studied. The average clone size at diagnosis was 73.5%, measured by granulocyte flow cytometry. Before extracting all exomic variants, including small variants and large-scale insertions/deletions, primary analysis was performed for initial variant calling and filtering of putative sequencing artifacts (1A). Results: As expected the majority of these patients (73.4%) harbored genetic lesions or mutations in PIGA with a mean variant allele frequency (VAF) of 29% and clone size of 69% by flow. Of these, 45% carried frameshift mutations, while 19% had splice variants and 14.5% carried missense mutations. Lastly, 14.5% of these mutations were based on gained stop codons or in-frame deletions while in 7% large scale genomic deletions in the PIGA locus were found (1B). The majority of these patients (67%) harbored a single genetic lesion, but >1 mutation was found in 23% of patients. Thus flow cytometric clone size may be a result of multiple genetically diverse PIGA mutant clones as indicated by the correlation between PNH clone size and number of PIGA mutants (P=4.14x10-7) (1C). In patients with PNH in whom PIGA mutations were not found, the average clone size was smaller (average 15.0%). We thus subjected them to targeted deep sequencing (coverage mean 1000x) to ascertain that inability to detect mutations was not due to technical sensitivity. Indeed, 8/25 cases turned out to have a PIGA mutation. In addition we studied expression of PIGA in PIGAmut versus WT cases, but no significant decrease in PIGA RNA was found. To further clarify the pathogenesis, we analyzed a gene panel of 33 proteins currently known to be involved in GPI anchor biosynthesis. We found a PIGO nonsense mutation (c.2593C>T) and a PIGV missense variant (c.467G>A) in two WT patients. These two variants were unique in PNH compared to a large cohort of myeloid neoplasias (N=2630) and appeared to be functionally consequential according to stringent in silico predictor and quality filter pipelines. Since the progression of PNH into frank myeloid neoplasia can occur and the dynamics of the expansion of PNH clones is unpredictable and thus may be influenced by additional hits, we queried if additional lesions could explain these two phenomena. We also investigated the concomitant presence of myeloid mutation in these cases. We found additional somatic mutations (panel of 77 genes) in 47.8% of all PNH patients including ASXL1 (N=10), BCOR (N=8), KMT2D (N=6), and RUNX1 (N=4) (1D). Analyzing the VAF revealed that the majority of these events were subclonal in relation to PIGA mutations. Since we studied the patients with already highly expanded clones and due to assertion bias no difference in clone size and additional mutations was found but an ongoing study in early PNH will determine whether subclonal hits predict progression to full blown PNH (1E). Conclusion: Our study shows that WGS can faithfully detect PIGA defects in a majority of PNH patients but this detection may be low in patients with smaller clones. Second, subclonal mutations linked to myeloid neoplasia are frequent events in PNH. Lastly we found two novel mutations in GPI synthetic pathway. Since none of these genes have been implicated in the pathogenesis of PNH, these findings extend our understanding of the pathogenesis of PNH and make the case for a targeted PIG pathway-wide sequencing of affected patients. Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Paroxysmal Nocturnal Hemoglobinuria Clones Are Infrequent in Hepatitis-Associated Aplastic Anemia at the Time of Diagnosis

Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and < 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (< 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

Extent and Clinical Implications of Subclonal Diversity in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal hemoglobinuria (PNH) has been traditionally considered a monogenic disease due to somatic mutations in PIGA gene. While selective immune pressure was implicated in PNH evolution via expansion of a PIGA mutant clone in a privileged environment, history of aplastic anemia (AA) is not always present in a manifest hemolytic PNH. Similarly, expansion of PNH clone after AA therapy does not seem to correlate with the strength of immunosuppression and the quality of response. Our initial studies (J.Clin.Invest.2014; 124 :4529-4538) indicated that additional mutational events may be present in PNH and act as intrinsic factors modifying clinical features and leading to a differential expansion. We hypothesize that subclonal hits may be present in some patients with PNH to explain their expansion dynamics and clinical features. To that end we have collected 319 patients with bone marrow failure of whom 202 had PNH clone including 70 hemolytic PNH cases. For a subgroup of these patients (n=197), deep multi-targeted NGS has been performed to identify mutations in 78 myeloid genes of which 38 genes were found to be mutated (≥1) in this PNH spectrum disease. 116 serial samples for 33 patients were also sequenced. As a control group, we have included AA stably negative for PNH clone (n=113). For analytic purposes, on clinical grounds we have sub grouped patients with PNH clone into AA/PNH (31%) defined as patients with presence of bone marrow failure but no clinical or laboratory evidence of hemolysis and WBC PNH clone <20%, and PNH group (69%). The latter (69%) consisted of patients with primary hemolytic PNH (pPNH; 60%) or secondary PNH (sPNH;40%). sPNH is defined as a group showing hemolysis with an antecedent history of AA and clone size >20%.Our analysis focused on driver mutations. In cross-sectional analysis, in addition to PIGA we have identified somatic hits in 10/26 (38%) of AA/PNH pts. (On average 2.1 hits/per positive patient) compared to 27/58(47%) of PNH patients (on average 1.22 hits per positive patients. Subdividing PNH patients, 20/35(57%) had a mutation in primary PNH compared to 7/23(30%) in the sPNH group. In contrast, 42/113(37%) patients had a mutation in the control AA group with a (2.1 mutations per positive patient). The mean VAF for AA is 29% compared to 23% in AA/PNH vs. 34% in PNH vs. 31%in sPNH.In all patients with PNH clone, 44% had a mutation vs. 37% in the AA group. AA/PNH subcohort contained hits in cohesins genes (RAD21-SMC3-STAG2 (8 %), ZRSR2 (8%), BCORs (4%) and TET2 (4%). PNH group had mutations in BCOR/BCORL1 (BCORs) (11%), cohesions, ZRSR2 and TET2 (6% each). In sPNH, a similar trend was observed with the most common hits in EZH2-SUZ12 (9%), ZRSR2 (9%), and BCORs, TET2, LUC7L2 (4% each). In contrast, AA without PNH, the 5 most frequently mutated genes were TET2, ASXL1, EZH2-SUZ12 AND BCORs (5% each) and RUNX 1 (4%). BCORs mutations were more frequent in PNH group compared to AA, (11% vs. 5%). In summary, BCOR/L and ZRSR genes showed more mutations in PNH clone cohort while AA cohort did not show any dominant hits.To determine whether somatic hits observed cross-sectionally led to establishment of stable subclones or were merely transient, we performed a longitudinal analysis. In PNH, 3/4 clones were transient which included EZH2, PHF6 and STAG2. In AA/PNH patients, 9/14 mutational hits appeared in the course of their disease (TET2, CEBPA, CUX1, STAG2, BCOR, SMC3) but 10/14 faded away in subsequent course and thus only 4 were stable clones. In AA cohort, 32 additional mutational hits were noted in 77 serial samples and 34 mutations vanished in subsequent course.In sum, our results show that in PNH, similar to AA, additional somatic hits are common and involve some of the commonly mutated myeloid genes seen in MDS. While likely, many of these subclonal events are transient, seemingly lacking the proper ancestral context, some of the hits resulted in significant expansions (sweeping mutations) and thus may modify the clinical course including speed of the PNH clone evolution. DisclosuresNo relevant conflicts of interest to declare.

Ineffective Erythropoiesis With Increased Neutrophils In Old Mice Overexpressing Hmga2 cDNA

HMGA2 is frequently overexpressed in hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and primary myelofibrosis (PMF), and occasionally in myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). We recently reported that transgenic mice overexpressing truncated Hmga2 cDNA (ΔHmga2 mice) showed increased numbers in all lineages of peripheral blood (PB) cells, hypercellular bone marrow (BM), and splenomegaly in young adults of 3 months old (Ikeda et al, Blood, 2011). ΔHmga2 mice also showed growth advantage of hematopoietic stem cells (HSCs) in serial BM transplants and of committed progenitors in colony-replating assays. Thus, overexpression of HMGA2 may contribute to clonal expansion in PNH or myeloproliferation in PMF and MDS/MPN. Although these disorders show disease progression and development of leukemia in the long term, consequences of long-term overexpression of HMGA2 in hematopoiesis is unknown. Therefore, in this study, we investigated hematopoiesis in old ΔHmga2 mice at 15 months old. We found an increase in PB neutrophils in 16 old ΔHmga2 mice compared with 8 old wild-type (WT) mice (mean ± SD; 4.3 ± 1.7 vs. 2.4 ± 0.6 x109/L, p<0.01) as well as young mice. Strikingly, in contrast to young mice, we found a decrease in PB red blood cells of older ΔHmga2 mice compared with WT mice (8.5 ± 0.5 vs. 9.3 ± 0.4 x1012/L, p<0.01), which was associated with an increase in the mean corpuscular volume (MCV, 48.5 ± 3.7 vs. 42.1 ± 1.8 fL, p<0.01). PB total leukocyte and platelet counts were similar between old ΔHmga2 mice and old WT mice. Total BM cell counts were higher in 11 old ΔHmga2 mice compared with 12 WT mice (2.1 ± 0.4 vs. 1.4 ± 0.5 x107/femur, p<0.01). Spleens were 2-fold heavier in old ΔHmga2 mice (n=11) compared with old WT mice (n=12, p<0.01). Histology showed hypercellular BM with erythroid dysplasia, but neither fibrosis nor leukemia, in 5 old ΔHmga2 mice. Absolute numbers of BM lineage-Sca-1+c-kit+ HSCs were higher in 5 old ΔHmga2 mice compared with 3 old WT mice (73 ± 31 vs. 23 ± 10 x103/femur, p=0.036). In addition, proportions of CD71+Ter119+ erythroblasts were higher in 3 old ΔHmga2 mice compared with 3 WT mice (11.8 ± 1.2 vs. 5.1 ± 1.1%, p<0.01), although absolute number of megakaryocyte-erythrocyte progenitors did not show a statistical difference (137 ± 36 vs. 96 ± 18 x103/femur, p=0.1). Progenitor assay also showed increased numbers of BFU-E colonies in 3 old ΔHmga2 mice compared with 3 WT mice (25 ± 2 vs. 13 ± 2, p<0.01), but new BFU-E colonies did not grow from replating of these colonies of old ΔHmga2 mice, in contrast to young ΔHmga2 mice. Annexin V staining showed increased spontaneous apoptosis after 24-hour incubation in Ter119+CD71+ cells of 3 old ΔHmga2 mice compared with 3 old WT mice (26.6 ± 2.7 vs. 14.3 ± 7.0%, p=0.033). In MACS-sorted Ter119+ erythroid cells, expression of anti-apoptotic Bcl-xl mRNA in old ΔHmga2 mice was lower compared with old WT mice (3.3 ± 1.2 vs. 5.3 ± 0.3, p=0.016), while that in young ΔHmga2 mice were higher than young WT mice (6.9 ± 0.4 vs. 3.5 ± 0.3, p<0.01), suggesting the possibility that some changes, including expression of Bcl-xl affected sensitivity of erythroid cells to apoptosis during aging, resulted in ineffective erythropoiesis. In conclusion, long-term expression of HMGA2 may lead to neutrophilia with ineffective erythropoiesis, which may mimic MDS/MPN rather than myelofibrosis, possibly through impairment of Bcl-xl in erythroid cells during a long course. Ineffective erythropoiesis may also in part explain BM failure in PNH. Disclosures:No relevant conflicts of interest to declare.

Mechanism of paroxysmal nocturnal hemoglobinuria clonal dominance: possible roles of different apoptosis and CD8 + lymphocytes in the selection of paroxysmal nocturnal hemoglobinuria clones

Paroxysmal nocturnal hemoglobinuria (PNH), a clonal hematopoietic stem cell disorder, manifests when the PNH clone populates in the hematopoietic compartment. We explored the roles of different apoptosis of GPI+ and GPI- (glycosylphosphatidylinositol) cells and CD8+ lymphocytes in a selection of PNH clones. Granulocytes from PNH patients and normal controls were subjected to an apoptosis assay using annexin V. Hematopoietic cell in semisolid media were cultured with or without CD8+ lymphocytes. In PNH, CD59+ granulocytes exhibited more apoptosis than their CD59- counterparts, after 0 or 4 hours in liquid growth culture system (mean [standard error of mean]: 2.1 (0.5) vs 1.2 (0.2), P=.01 at 0 hour and 3.4 [0.7] vs 1.8 [0.3], P=.03 at 4 hour, respectively). The presence of mononuclear cells (MNCs) rendered a greater difference in apoptosis. The percentages of apoptotic CD59+ granulocytes measured at 4 hours with or without MNC fraction were correlated with the sizes of PNH clones (r=0.633, P=.011; and r=0.648, P=.009; respectively). The autologous CD8+ lymphocytes inhibited CFU-GM and BFU-E colony formation in PNH patients when compared with normal controls (mean [SEM] of percentages of inhibition: 61.7 (10.4) vs 11.9 (2.0), P=.008 for CFU-GM and 26.1 (6.9) vs 4.9 (1.0), P=.037 for BFU-E). Increased apoptosis of GPI+ blood cells is likely to be responsible in selection and expansion of PNH clones. MNCs or possibly CD8+ lymphocytes may play a role in this phenomenon.

3′UTR-truncated Hmga2 cDNA causes MPN-like hematopoiesis by conferring a clonal growth advantage at the level of HSC in mice

AbstractOverexpression of high mobility group AT-hook 2 (HMGA2) is found in a number of benign and malignant tumors, including the clonal PIGA− cells in 2 cases of paroxysmal nocturnal hemoglobinuria (PNH) and some myeloproliferative neoplasms (MPNs), and recently in hematopoietic cell clones resulting from gene therapy procedures. In nearly all these cases overexpression is because of deletions or translocations that remove the 3′ untranslated region (UTR) which contains binding sites for the regulatory micro RNA let-7. We were therefore interested in the effect of HMGA2 overexpression in hematopoietic tissues in transgenic mice (ΔHmga2 mice) carrying a 3′UTR-truncated Hmga2 cDNA. ΔHmga2 mice expressed increased levels of HMGA2 protein in various tissues including hematopoietic cells and showed proliferative hematopoiesis with increased numbers in all lineages of peripheral blood cells, hypercellular bone marrow (BM), splenomegaly with extramedullary erythropoiesis and erythropoietin-independent erythroid colony formation. ΔHmga2-derived BM cells had a growth advantage over wild-type cells in competitive repopulation and serial transplantation experiments. Thus overexpression of HMGA2 leads to proliferative hematopoiesis with clonal expansion at the stem cell and progenitor levels and may account for the clonal expansion in PNH and MPNs and in gene therapy patients after vector insertion disrupts the HMGA2 locus.

T cells expressing the activating NK-cell receptors KIR2DS4, NKG2C and NKG2D are elevated in paroxysmal nocturnal hemoglobinuria and cytotoxic toward hematopoietic progenitor cell lines

To investigate the presence of T cells with natural killer cell receptors (NKR) in paroxysmal nocturnal hemoglobinuria (PNH), and their potential involvement in clonal expansion of glycosylphosphatidylinositol (GPI)-deficient hematopoietic stem cells by selective immune attack to normal and not GPI-deficient hematopoietic stem cells. By flow cytometry, the frequency and number of T cells expressing NKR was evaluated in 39 PNH patients and compared to healthy controls. Elevated T cell subsets in PNH were assessed for differential cytotoxic lysis of GPI(+) and GPI(-) CD34(+) hematopoietic progenitor cell lines. In PNH patients, the frequency (p < 0.001) and absolute number of T cells expressing the NKR CD56 (p = 0.01) were significantly increased. Furthermore, a higher percentage of T cells expressed the activating NKR NKG2D (p < 0.01), NKG2C (p < 0.01), and KIR2DS4 (p = 0.01). Further characterization showed that these populations predominantly consist of CD8(+) effector memory CD45RA(+) T cells (T(EMRA)). NKR(+) cytotoxic T-lymphocyte lines isolated from PNH patient peripheral blood and bone marrow displayed high cytotoxicity towards CD34(+) hematopoietic progenitor cell lines and K562 cells, suggesting major histocompatibility complex class I-independent cytotoxicity. These cytotoxic T lymphocyte (CTL) lines are capable of differential lysis of GPI(+) and GPI(-) hematopoietic cell lines, however, not in all cases. This suggests that multiple factors, such as the highly activated status of in vitro cultured CTLs, influence whether GPI-dependent lysis occurs. The increased frequency of CD8(+) effector-memory T cells with activating NKR and cytotoxicity toward hematopoietic cell lines suggests involvement in bone marrow failure and clonal expansion in PNH.

Features of Myeloproliferative Neoplasms and Clonal Advantage of Hematopoietic Stem Cells In Mice Expressing An Hmga2 cDNA Lacking the 3′UTR.

AbstractAbstract 1161Overexpression of high mobility group AT-hook 2 (HMGA2) has been identified in patients with paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndromes, and myeloproliferative neoplasms (MPN). Overexpression is often associated with truncation or deletions of the 3′ untranslated region (UTR) of HMGA2 and is regulated by the binding of let-7-family micro RNAs to the 3′UTR of HMGA2 mRNA. PNH is an acquired hemolytic anemia caused by clonal expansion of a hematopoietic cell (PNH cell) with a somatic mutation in the X-linked PIGA gene occurring in the background of bone marrow (BM) failure. The PIGA gene is essential in the synthesis of GPI-floats, and therefore progeny cells of this clone lack all GPI-linked proteins. However, the nature of the clonal growth advantage of a PNH cell is unclear. Here, to clarify the role of expression of 3′UTR-truncated HMGA2 in hematopoiesis and clonal expansion of hematopoietic cells, we generated transgenic mice (ΔHmga2 mice) expressing an Hmga2 cDNA with a truncation which removes six of the seven let-7-complementary sequences in the 3′UTR, which is similar to the truncations of HMGA2 found in two patients with PNH (Inoue et al., Blood, 2006). Expression of Hmga2 mRNA and protein were both significantly higher in BM, spleen, and thymus of ΔHmga2 mice compared to wild-type (WT) mice, with overexpression being more prominent in protein rather than mRNA. Unexpectedly, ΔHmga2 mice showed proliferative hematopoiesis that mimicked an MPN-like phenotype with increased numbers of peripheral white blood cells, red blood cells, and platelets, and low serum erythropoietin (EPO) values, hypercellular BM, and splenomegaly with extramedullary erythropoiesis compared to WT mice. Furthermore, colony assays of BM cells from ΔHmga2 mice showed significantly higher numbers of burst forming-unit erythroid, colony forming-unit erythroid (CFU-E), and CFU granulocyte-macrophage compared to those from WT mice. Formation of EPO-independent CFU-E was also observed. In competitive repopulation assays after BM transplant (BMT), BM cells from ΔHmga2 mice had a growth advantage over those from WT mice in all lineages. Surprisingly, cells derived from ΔHmga2 BM took over most of hematopoiesis during serial BMT with a ratio of 1:9 donor:recipient cells at the initial BMT, suggesting that overexpression of Hmga2 confers a clonal advantage to hematopoietic cells at the level of the hematopoietic stem cell (HSC). We found significantly higher expression of phosphorylated Stat3 in BM HSC-enriched KLS (c-kit+lineage-Sca-1+) cells and phosphorylated Akt in total BM cells from ΔHmga2 mice compared to WT mice without stimulation by cytokines, suggesting that intrinsic activation of Stat3 and Akt may contribute to proliferative hematopoiesis in ΔHmga2 mice. Microarray analysis of KLS cells and megakaryocyte-erythrocyte progenitor (MEP) cells showed a wide range of differences, in part mimicking MPN. When we bred our mice with circulating PNH cells (Piga- mice) with ΔHmga2 mice, the ΔHmga2+Piga+ cells and ΔHmga2+Piga- cells showed similar growth kinetics, indicating that the lack of GPI-linked proteins did not add an additional clonal advantage to the ΔHmga2+ cells. In summary, our results showed that the overexpression of a 3′UTR-truncated Hmga2 leads to proliferative hematopoiesis with clonal advantage at the level of HSC, which may also explain the effect of HMGA2 expression on clonal expansion in PNH or MPN. Disclosures:Bessler:Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Taligen: Consultancy.

T Cells Expressing the Activating NK Cell Receptors KIR2DS4, NKG2C and NKG2D Are Elevated In Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and Cytotoxic towards Hematopoietic Progenitor Cell Lines

AbstractAbstract 2235 Background:Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease characterized by hemolysis due to an acquired mutation in the X-linked PIG-A gene in the hematopoietic stem cell (HSC). This leads to a clone of hematopoietic cells with deficient expression of glycosyl phosphatidyl inositol (GPI) anchored proteins at the cell membrane. The clinical evolution of PNH arises through clonal expansion of PIG-A mutated HSC which is insufficiently explained by the PIG-A mutation alone. Hypothetically, clonal expansion could result from autoreactive T cells selectively attacking normal HSC, whereas GPI deficient HSC are unharmed. Methods and Results:we investigated the presence of potentially autoreactive T cells in peripheral blood of patients with PNH (n = 39) by flow cytometry. We compared T cell subset frequencies and absolute numbers with healthy controls (n = 25) using Mann-Whitney U test. In PNH patients, T cells expressing the NK cell marker CD56 were significantly elevated, both in percentage (p < 0.001) and in absolute numbers (p < 0.01). Furthermore, the frequency of T cells expressing the activating NK cell receptors (NKRs) NKG2D (p < 0.01), NKG2C (p < 0.01), and KIR2DS4 (p = 0.01) was significantly increased. KIR2DS4+, NKG2C+ and NKG2D+ T cells mainly consist of highly differentiated effector memory CD45RA+ T cells (TEMRA) (KIR2DS4: median 90%, range 70–96%, NKG2C: median 83%, range 49–99%, NKG2D: median 40%, range 38–66%). A highly variable proportion of these T cell populations consists of γδ T cells (KIR2DS4: median 28%, range 6–72%, NKG2C: median 36%, range 3–75%, NKG2D: median 11%, range 7–40%). By 10 color flow cytometry, we examined NKR coexpression patterns. KIR2DS4+ and NKG2C+ T cells mainly coexpress either only NKG2D (KIR2DS4+ T cells: median 24%, range 2 – 74%, NKG2C+ T cells: median 21%, range 3–71%), or a combination of NKG2D, NKG2C and CD158b1/b2,j, but not inhibitory NKG2A (KIR2DS4+ T cells: median 16%, range 1 – 55%, NKG2C+ T cells: median 20%, range 1–60%). In contrast, NKG2D+ T cells generally do not express any other NKRs tested (median 77%, range 53–84%). NKG2D+ KIR2DS4+ cytotoxic T lymphocyte (CTL) lines isolated from PNH patient peripheral blood and bone marrow display high cytolytic activity towards CD34+ hematopoietic progenitor cell lines and MHC class I deficient K562 cells, suggesting T cell receptor independent cytolytic activity. These NKR+ CTL lines are capable of differentially lysing GPI+ and GPI- hematopoietic cell lines, however not in all cell line models and CTL lines. This suggests that multiple factors, as for example the highly activated status of in vitro cultured CTLs, influence whether or not GPI dependent lysis occurs. Conclusion:The increased frequency of T cells expressing activating NK cell receptors KIR2DS4, NKG2C, and NKG2D, with a CD8+ effector-memory phenotype and differences in cytotoxicity towards GPI+ and GPI- hematopoietic cell lines suggests that these T cell populations may be involved in bone marrow failure and expansion of PNH clones. Disclosures:Muus: Alexion: member of advisory board.

GPI-Anchored Protein Deficiency Confers Resistance to Apoptosis in PNH.

OBJECTIVE: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).INTRODUCTION: Whether PNH cells are inherently less susceptible to apoptosis remains controversial. Studies using PNH mouse models and lymphoid cell lines have failed to find a difference in apoptosis, but reports using K562 cells have found a relative resistance. Studies using primary CD34+ cells from PNH patients have also found a relative resistance of PNH cells, but not in comparison to CD34+ cells from normal controls. We designed experiments 1) to control for genetic heterogeneity among PNH and control cell lines, 2) to address the potential methodological issues associated with studying lymphocytes, which are rarely affected in PNH, and 3) address inconsistency comparing normal and PNH progenitors that have experienced different in vivo environments.METHODS: GPI-anchored protein (GPI-AP) positive and negative primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune selection in vivo, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Using a doxycycline-inducible wild type PIG-A tet-SUPER transgene expression system, in which GPI-AP− and GPI-AP+ cells are isogenic, we assessed apoptosis resistance and clonal expansion in response to various pro-apoptotic stimuli. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 effectors, TNF-α, and γ-irradiation. Apoptosis was measured by annexin V/PI staining (effector experiments) or caspase 3/7 activity (TNF-α and γ-irradiation experiments). Blocking experiments of NK92-mediated killing utilized mAb to ULBP1 and ULBP2, as per Hanaoka et al. Clonal competition experiments tracked wild type and PIG-A mutant TF-1 cells using flow cytometry after exposure to TNF-α as a surrogate for immune attack.RESULTS: In PNH patients, GPI-AP− CD34+ hematopoietic progenitors were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% versus 49%, p<0.05) and allogeneic (28% versus 58%, p<0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP− TF-1 cells exhibited less apoptosis than GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing (35% less), NK92-mediated killing (24% less), TNF-α (14% less), and γ-irradiation (24% less). All differences were statistically significant by the paired t test with p<0.05. For allogeneic effector killing experiments, GPI-AP− TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP− cells, arguing against a GPI-AP being the target of immune attack in PNH. In the NK92-mediated killing model, 24% of the apoptosis resistance was lost with ULBP1 and ULBP2 blockade (p<0.05). No change in ULBP1 and ULBP2 expression was observed in TNF-α and γ-irradiation experiments. Clonal competition experiments demonstrate that the mutant clone expands by 72% over two days under pro-apoptotic conditions with TNF-α (p<0.05).CONCLUSIONS: PIG-A mutations contribute to the clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under pro-apoptotic stresses. Lack of GPI-anchored ULBP1 and ULBP2 expression contributes to the apoptosis resistance in a cell-mediated killing model but is not solely responsible for the apoptosis resistance. The global apoptosis resistance translated into expansion of the PNH clone in an in vitro model of immune attack.

Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH

Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH). Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity. In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha. PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.

Pathogenesis of selective expansion of PNH clones.

Hemolysis, a characteristic of paroxysmal nocturnal hemoglobinuria (PNH), is caused by the expansion of an affected stem cell with a mutation of the PIG-A gene. Increasing evidence has shown that the presence of the PIG-A mutation alone does not induce the expansion. Two theories have been proposed. One, the growth advantage hypothesis, is supported by current data indicating the presence of several intrinsic alterations that might confer a proliferative advantage to PNH clones over normal cells. Alternatively, the PIG-A mutation might confer a relative survival advantage to PNH clones. This theory is supported by clinical observation indicating that PIG-A mutant cells survive immune-mediated bone marrow injury in patients with aplastic anemia, PNH, and myelodysplastic syndromes. The latter theory is also supported by current experimental data indicating that PIG-A mutant cells are relatively resistant to cytotoxic attack by natural killer cells and cytotoxic T-lymphocytes. The 2 theories appear complementary rather than mutually exclusive. Rapid progress in this field can be expected in the near future.

Paroxysmal nocturnal hemoglobinuria: Differential gene expression of EGR-1 and TAXREB107.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect of hematopoietic stem cells characterized by deficiency in GPI-anchored surface proteins. It is not yet known how GPI-deficient stem cells are able to expand within the bone marrow and contribute considerably to the hematopoiesis. In PNH, as well as in AA and MDS, genetic instability and increased mutation frequency have been detected. Therefore, a second event is very likely, such as additional mutations, leading to clonal expansion of GPI-deficient bone marrow stem cell in PNH. In order to elucidate the molecular basis of clonal expansion in PNH, we identified several genes differentially expressed in normal and GPI-deficient cells of PNH patients by combination of RNA fingerprinting and cDNA array hybridization. Expression of two of these genes, EGR-1 and TAXREB107, has been further investigated. EGR-1 is upregulated in granulocytes of all PNH patients analyzed so far. In contrast, significant upregulation of TAXREB107 is present only in some of our PNH patients. Further analysis confirmed their overexpression in PNH and excluded a possible secondary event character of observed overexpression. Moreover, similar levels of expression in cases of other clonal diseases, such as MPS and MDS, has been identified. Our data suggest that additional genetic alterations apart from PIG-A mutations could be present in PNH granulocytes. In addition, these genetic changes might contribute to clonal expansion of GPI-deficient cells in PNH.

New Insights into the Pathophysiology of Acquired Cytopenias

This review addresses three related bone marrow failure diseases, the study of which has generated important insights in hematopoiesis, red cell biology, and immune-mediated blood cell injury. In Section I, Dr. Young summarizes the current knowledge of acquired aplastic anemia. In most patients, an autoimmune mechanism has been inferred from positive responses to nontransplant therapies and laboratory data. Cytotoxic T cell attack, with production of type I cytokines, leads to hematopoietic stem cell destruction and ultimately pancytopenia; this underlying mechanism is similar to other human disorders of lymphocyte-mediated, tissue-specific organ destruction (diabetes, multiple sclerosis, uveitis, colitis, etc.). The antigen that incites disease is unknown in aplastic anemia as in other autoimmune diseases; post-hepatitis aplasia is an obvious target for virus discovery. Aplastic anemia can be effectively treated by either stem cell transplantation or immunosuppression. Results of recent trials with antilymphocyte globulins and high dose cyclophosphamide are reviewed. Dr. Abkowitz discusses the diagnosis and clinical approach to patients with acquired pure red cell aplasia, both secondary and idiopathic, in Section II. The pathophysiology of various PRCA syndromes including immunologic inhibition of red cell differentiation, viral infection (especially human parvovirus B19), and myelodysplasia are discussed. An animal model of PRCA (secondary to infection with feline leukemia virus [FeLV], subgroup C) is presented. Understanding the mechanisms by which erythropoiesis is impaired provides for insights into the process of normal red cell differentiation, as well as a rational strategy for patient management. Among the acquired cytopenias paroxysmal nocturnal hemoglobinuria (PNH) is relatively rare; however, it can pose formidable management problems. Since its first recognition as a disease, PNH has been correctly classified as a hemolytic anemia; however, the frequent co-existence of other cytopenias has hinted strongly at a more complex pathogenesis. In Section III, Dr. Luzzatto examines recent progress in this area, with special emphasis on the somatic mutations in the PIG-A gene and resulting phenotypes. Animal models of PNH and the association of PNH with bone marrow failure are also reviewed. Expansion of PNH clones must reflect somatic cell selection, probably as part of an autoimmune process. Outstanding issues in treatment are illustrated through clinical cases of PNH. Biologic inferences from PNH may be relevant to our understanding of more common marrow failure syndromes like myelodysplasia.

New Insights into the Pathophysiology of Acquired Cytopenias

AbstractThis review addresses three related bone marrow failure diseases, the study of which has generated important insights in hematopoiesis, red cell biology, and immune-mediated blood cell injury. In Section I, Dr. Young summarizes the current knowledge of acquired aplastic anemia. In most patients, an autoimmune mechanism has been inferred from positive responses to nontransplant therapies and laboratory data. Cytotoxic T cell attack, with production of type I cytokines, leads to hematopoietic stem cell destruction and ultimately pancytopenia; this underlying mechanism is similar to other human disorders of lymphocyte-mediated, tissue-specific organ destruction (diabetes, multiple sclerosis, uveitis, colitis, etc.). The antigen that incites disease is unknown in aplastic anemia as in other autoimmune diseases; post-hepatitis aplasia is an obvious target for virus discovery. Aplastic anemia can be effectively treated by either stem cell transplantation or immunosuppression. Results of recent trials with antilymphocyte globulins and high dose cyclophosphamide are reviewed.Dr. Abkowitz discusses the diagnosis and clinical approach to patients with acquired pure red cell aplasia, both secondary and idiopathic, in Section II. The pathophysiology of various PRCA syndromes including immunologic inhibition of red cell differentiation, viral infection (especially human parvovirus B19), and myelodysplasia are discussed. An animal model of PRCA (secondary to infection with feline leukemia virus [FeLV], subgroup C) is presented. Understanding the mechanisms by which erythropoiesis is impaired provides for insights into the process of normal red cell differentiation, as well as a rational strategy for patient management.Among the acquired cytopenias paroxysmal nocturnal hemoglobinuria (PNH) is relatively rare; however, it can pose formidable management problems. Since its first recognition as a disease, PNH has been correctly classified as a hemolytic anemia; however, the frequent co-existence of other cytopenias has hinted strongly at a more complex pathogenesis. In Section III, Dr. Luzzatto examines recent progress in this area, with special emphasis on the somatic mutations in the PIG-A gene and resulting phenotypes. Animal models of PNH and the association of PNH with bone marrow failure are also reviewed. Expansion of PNH clones must reflect somatic cell selection, probably as part of an autoimmune process. Outstanding issues in treatment are illustrated through clinical cases of PNH. Biologic inferences from PNH may be relevant to our understanding of more common marrow failure syndromes like myelodysplasia.

New Insights into the Pathophysiology of Acquired Cytopenias

This review addresses three related bone marrow failure diseases, the study of which has generated important insights in hematopoiesis, red cell biology, and immune-mediated blood cell injury. In Section I, Dr. Young summarizes the current knowledge of acquired aplastic anemia. In most patients, an autoimmune mechanism has been inferred from positive responses to nontransplant therapies and laboratory data. Cytotoxic T cell attack, with production of type I cytokines, leads to hematopoietic stem cell destruction and ultimately pancytopenia; this underlying mechanism is similar to other human disorders of lymphocyte-mediated, tissue-specific organ destruction (diabetes, multiple sclerosis, uveitis, colitis, etc.). The antigen that incites disease is unknown in aplastic anemia as in other autoimmune diseases; post-hepatitis aplasia is an obvious target for virus discovery. Aplastic anemia can be effectively treated by either stem cell transplantation or immunosuppression. Results of recent trials with antilymphocyte globulins and high dose cyclophosphamide are reviewed.Dr. Abkowitz discusses the diagnosis and clinical approach to patients with acquired pure red cell aplasia, both secondary and idiopathic, in Section II. The pathophysiology of various PRCA syndromes including immunologic inhibition of red cell differentiation, viral infection (especially human parvovirus B19), and myelodysplasia are discussed. An animal model of PRCA (secondary to infection with feline leukemia virus [FeLV], subgroup C) is presented. Understanding the mechanisms by which erythropoiesis is impaired provides for insights into the process of normal red cell differentiation, as well as a rational strategy for patient management.Among the acquired cytopenias paroxysmal nocturnal hemoglobinuria (PNH) is relatively rare; however, it can pose formidable management problems. Since its first recognition as a disease, PNH has been correctly classified as a hemolytic anemia; however, the frequent co-existence of other cytopenias has hinted strongly at a more complex pathogenesis. In Section III, Dr. Luzzatto examines recent progress in this area, with special emphasis on the somatic mutations in the PIG-A gene and resulting phenotypes. Animal models of PNH and the association of PNH with bone marrow failure are also reviewed. Expansion of PNH clones must reflect somatic cell selection, probably as part of an autoimmune process. Outstanding issues in treatment are illustrated through clinical cases of PNH. Biologic inferences from PNH may be relevant to our understanding of more common marrow failure syndromes like myelodysplasia.