Paroxysmal Nocturnal Hemoglobinuria Clones In Patients Research Articles

HLA Class I Allele-Specific Pathology Defines Clinical Manifestations of Immune Aplastic Anemia

Leukocytes that have lost the cell surface expression of an HLA class I allele due to somatic HLA gene mutations or 6p loss of heterozygosity (LOH) are often present in patients with immune aplastic anemia (AA), consistent with T cell-mediated bone marrow cell destruction. A recent NIH study suggested that the clinical significance and underlying pathology of HLA loss may differ according to the type of HLA allele affected (Zaimoku et al. Blood 2021) because (1) the significant correlation of HLA loss with clonal evolution was primarily attributed to patients who lost HLA-B*14:02 and A*02:01; (2) all patients who lost B*40:02 and A*02:06 achieved a hematologic response to immunosuppressive therapy (IST), though HLA loss in general was not correlated with the IST response; (3) loss of B*07:02 and B*40:01 was associated with older age of onset; (4) patients who retained B*14:02, B*07:02, and B*40:01 also showed clinical features of patients who lost respective HLA alleles. We aimed to confirm the HLA class I allele-related clinical manifestations in Japanese AA patients. HLA loss was assessed by SNP array, HLA flow cytometry, digital PCR, or deep nucleotide sequencing. An HLA allele was classified as "affected by loss" in individual patients when the single allele was lost or inactivated by somatic mutations or when both HLA-A and HLA-B alleles in a haplotype were lost due to 6p LOH with no evidence of loss or inactivation of a single HLA allele. Clinical characteristics were compared according to the affected HLA alleles. In 496 Japanese patients with AA (severe, n = 368; non-severe, n = 128; median age, 59 [range, 1-89] years), there were 146 (29%) patients with HLA loss. HLA-B*40:02, A*02:06, A*31:01, and B*54:01 were more frequently affected in our cohort than in the NIH cohort, where B*14:02, A*02:01, B*08:01, and B*07:02 held the majority of lost alleles (Figure 1). The median age of patients with B*40:01 loss (75 years) was highest among the groups of patients with HLA allele loss, consistent with the NIH study. The prevalence of ≥0.003% paroxysmal nocturnal hemoglobinuria (PNH) clones was significantly reduced in patients with B*40:02 loss but not in others. Sex and severity of AA were not markedly different across the groups of patients with HLA allele loss. Outcomes after anti-thymocyte globulin (ATG)-based IST were evaluable in 263 of the 496 patients. Although HLA loss was correlated with the IST response at 6 months (as reported by our group), loss of HLA-A*02:01 did not contribute to the high response, consistent with the NIH study. Loss of frequently affected HLA alleles in Japanese patients (B*40:02, A*02:06, B*54:01, etc.) was generally correlated with the IST-response. Further, patients who carried 5 HLA-B alleles (B*40:01, B*40:02, B*44:03, B*54:01, and B*56:01) without loss still displayed higher response rates than other patients. In contrast, the favorable effect of A*02:06 loss was not seen in patients who carried A*02:06 without loss. A*02:06 loss, the presence of the 5 HLA-B alleles (irrespective of loss), and previously reported HLA class II alleles (DRB1*13 and DRB1*15) were independently correlated with the IST response. The incidence of clonal evolution to myeloid neoplasms after ATG-based IST was significantly higher in patients with HLA-A*02:01 loss than other patients (Figure 2), as observed in the NIH study; A*02:01 was affected in 4 of 7 patients showing both HLA loss and clonal evolution in our cohort; B*40:02, B*44:02, and B*52:01 were affected in other 3 patients. In summary, our study in Japanese AA patients broadly confirmed the associations of loss of specific HLA alleles with clinical manifestations observed in American patients, including older age of onset in patients with HLA-B*40:01 loss, high IST response rates in patients who lost B*40:02 and A*02:06 but not in patients with A*02:01 loss, and frequent clonal evolution in patients with A*02:01 loss. We also revealed the correlation of 5 HLA-B alleles with IST response, irrespective of loss, similar to the relationship of B*14:02 to clonal evolution in the United States, and the reduced prevalence of PNH clones in patients with B*40:02 loss. These findings strongly suggest that the heterogeneous pathology of AA can be classified by affected HLA alleles. This should be useful for studies to unravel the pathophysiology and improve the outcomes of patients with immune AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Difficult Cases of Paroxysmal Nocturnal Hemoglobinuria: Diagnosis and Therapeutic Novelties.

Paroxysmal nocturnal hemoglobinuria (PNH) is an intriguing disease that can pose many difficulties to physicians, as well as to hematologists, who are unfamiliar with it. Research regarding its pathophysiologic, diagnostic, and therapeutic aspects is still ongoing. In the last ten years, new flow cytometry techniques with high sensitivity enabled us to detect PNH clones as small as <1% of a patient’s hematopoiesis, resulting in increasing incidence but more difficult data interpretation. Particularly, the clinical significance of small PNH clones in patients with bone marrow failures, including aplastic anemia and myelodysplastic syndromes, as well as in uncommon associations, such as myeloproliferative disorders, is still largely unknown. Besides current treatment with the anti-C5 eculizumab, which reduced PNH-related morbidity and mortality, new complement inhibitors will likely fulfill unmet clinical needs in terms of patients’ quality of life and better response rates (i.e., responses in subjects with C5 polymorphisms; reduction of extravascular hemolysis and breakthrough hemolysis episodes). Still, unanswered questions remain for these agents regarding their use in mono- or combination therapy, when to treat, and which drug is the best for which patient. Lastly, long-term safety needs to be assessed in real-life studies. In this review, we describe some clinical vignettes illustrating practical aspects of PNH diagnosis and management; moreover, we discuss recent advances in PNH diagnostic and therapeutic approaches.

Aplastic Anemia Associated with PNH-Clone - a Single Centre Experience

Introduction: Pathogenetic relationship of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) was registered a long time ago. Approximately in 50-60% of patients with AA is detected PNH-clone, and during long-term observation the transformation of AA into classic PNH is likely. At the time of diagnosis PNH-clone sizes range from small proportion of PNH-cells, requiring only monitoring, up to combination of clinically manifested AA and PNH requring combined therapy. Nevertheless, the peculiar characteristics of the disease in cases of different AA-PNH-combinations still remain unclear.Aim: to evaluate the relationship between size and dynamics of PNH-clone with clinical features of AA/PNH.Materials and methods: We have evaluated blood samples of 81 patients with AA (whole group) for the presence of PNH-clone using flow cytometry according to ICCS standards. 23 patients aged 23 to 54 years (median 46 years) were under constant observation for 3 years or more, control examination was performed once every 6 months (dynamic observation group). Of them 17 had severe AA (SAA) and 6 - nonsevere AA (NAA). Twelve patients received standard immunosupressive therapy (IST) for the treatment of AA, other patients were in remission and did not require IST.Results: In the whole group of patients (n=81) 47 patients (58%) were AA/PNH-positive (PNHpos) with clone size ranging from 0.01% to 97.9%. PNH-clone less than 1% was identified in 10% of subjects; PNH-clone more than 10% was observed in 50% of subjects. In dynamic observation group 18 patients were AA/PNHpos with initial clone size from 0.1% to 95.9%, and 5 patients - AA/PNH negative (AA/PNHneg). In AA/PNHneg patients pathological clone still has not been detected in repeated studies for the observed period. Among patients with AA/PNHpos stable clone was observed in 11 subjects (61%), it increased to 10% and more in 5 subjects (28%) and decreased in 2 patients (11%) with small PNH-clone. Clinical and laboratory signs of chronic intravascular hemolysis were usually observed in patients with pathological clone over 25%. PNH-clone less than 10% was not accompanied by significant deviations in clinico-laboratory indices. Most of patients in whom PNH-clone size increased, had remission of AA. Four patients had clonal evolution to manifest classical PNH, of them in 2 patients with SAA аplasia of hematopoiesis persisted. In one patient, with complete remission of SAA lasting more than 3 years and minor PNH-clone, was observed transformation into myeloid leukemia leading to death. Ekulizumab was prescribed to 4 patients: 2 with clinically manifest hemolysis and clone size > 90% (1 - in conjunction with IST for AA), and 2 with clone size 24% and 70% and LDH > 1.5 ULN due to unplanned pregnancies (both on Cyclosporin therapy). All of them achieved the target level of LDH <1.5 ULN in the first 2 weeks of therapy. Of 2 transfusion-dependent patients, one still does not require transfusion support after the Ekulizumab initiation, and in other patient transfusion dependency decreased, despite refractory TAA. In one patient with PNH-clone 70% Ekulizumab therapy was initiated in III trimester of pregnancy, delivery was without complications, full-term baby, 8-9 points Apgar. In the 2nd pregnant patient with PNH-clone 24% Ekulizumab was prescribed because of pre-eclampsia and LDH>4 ULN at 21-22 weeks with antenatal fetal death, and it allowed for accelerating cure of pathological symptoms.Conclusion: PNH-clone was detected in 58% of patients with AA. Our data confirm the need for monitoring of PNH clone in patients with AA and for timely adequate therapy, including inhibitors of intravascular hemolysis, according to specific indications in each case. DisclosuresFominykh:Novartis Pharma: Honoraria; BMS: Honoraria.

Screening of Patients with Idiopathic Venous Thromboembolism for Paroxysmal Nocturnal Hemoglobinuria Clones

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon disorder characterized by hemolysis, thrombosis and marrow failure. Whereas venous and arterial thrombosis is a very common symptom of the disease, the frequency of PNH clones in patients with unexplained venous thromboembolism, including deep vein thrombosis and pulmonary embolism, has not been studied. We conducted a cross sectional study evaluating the presence of PNH clones in patients with prevalent venous thromboembolism using a high sensitivity flow cytometry assay for erythrocytes and neutrophils. Among the 388 patients enrolled in the study one patient had a detectable PNH clone of 0.02% in the neutrophil population (0.26%; 95% CI 0.05 to 1.45) and no detectable erythrocyte clone. We conclude that the presence of PNH clones in patients with idiopathic venous thrombosis is rare. Screening for PNH clones among VTE patients might be better reserved for patients with signs of hemolysis.

Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia.

Flow cytometry is the gold standard methodology for screening of paroxysmal nocturnal hemoglobinuria. In the last few years, proaerolysin conjugated with fluorescein (FLAER) has become an important component of antibody panel used for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study aimed to compare PNH clone detection by flow cytometry in the pre-FLAER era versus the FLAER era. This was a retrospective analysis of 4 years and included 1004 individuals screened for PNH clone, either presenting as hemolytic anemia or as aplastic anemia. In the pre-FLAER time period, the RBCs and neutrophils were screened with antibodies against CD55 and CD59. With the introduction of FLAER, neutrophils were screened with FLAER/CD24/CD15 and monocytes with FLAER/CD14/CD33 combination. A comparative analysis was done for detection of PNH clone in aplastic anemia patients versus non-aplastic anemia patients, as well as between pre-FLAER and FLAER era. Out of a total of 1004 individuals, 59 (5.8%) were detected to have PNH clone positivity. The frequency of PNH clone detected in aplastic anemia and non-aplastic anemia groups was 12.02 and 3.36%, respectively. The detection rate of PNH clone increased from 4.5% (32/711) in the pre-FLAER era to 9.2% (27/293) with the introduction of FLAER. However, this increase could be attributed to increased detection of PNH clone in the aplastic anemia group, which showed a significant increase from 8.3 to 18.2% after use of FLAER. In the non-aplastic group, PNH clone was detected with similar frequencies before and after use of FLAER (3.2 versus 3.8%, respectively). Mean PNH clone size was lower in the aplastic anemia group when compared with the non-aplastic group. RBCs always showed a lower clone size than neutrophils. PNH clone on neutrophils and monocytes was however similar. Inclusion of FLAER increases the sensitivity of the test which is especially useful in picking up small PNH clones in patients of aplastic anemia.

Management of Paroxysmal Nocturnal Hemoglobinuria in the Era of Complement Inhibitory Therapy

Despite the availability of safe, effective targeted therapy that controls intravascular hemolysis, the management of paroxysmal nocturnal hemoglobinuria (PNH) remains complicated because of disease heterogeneity and close association with BM failure syndromes. The purpose of this review is to provide a framework for individualizing treatment based on disease classification. According to the recommendations of the International PNH Interest Group, patients can be placed into one of the following 3 categories: (1) classic PNH, (2) PNH in the setting of another BM failure syndrome, or (3) subclinical PNH. The PNH clone in patients with subclinical disease is insufficiently large to produce even biochemical evidence of hemolysis, and consequently, patients who fit into this category require no PNH-specific therapy. Patients with PNH in the setting of another BM failure syndrome (usually aplastic anemia or low-risk myelodysplastic syndrome) have at least biochemical evidence of hemolysis, but typically the PNH clone is small (< 10%) so that hemolysis does not contribute significantly to the underlying anemia. In these cases, the focus of treatment is on the BM failure component of the disease. Intravascular hemolysis is the dominant feature of classic PNH, and this process is blocked by the complement inhibitor eculizumab. The thrombophilia of PNH also appears to be ameliorated by eculizumab, but the drug has no effect on the BM failure component of the disease. Low-grade extravascular hemolysis due to complement C3 opsonization develops in most patients treated with eculizumab, and in some cases is a cause for suboptimal response to treatment. Allogeneic BM transplantation can cure classic PNH, but treatment-related toxicity suggests caution for this approach to management.

Screening Patients with Myelodysplastic Syndrome and Aplastic Anemia for Paroxysmal Nocturnal Hemoglobinuria Clones: A Retrospective Study,

Abstract 3426 Introduction:Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare disorder due to a somatic mutation in the hematopoietic stem cell. The introduction of highly sensitive flow cytometric and aerolysin testing have shown the presence of PNH clones in patients with a variety of other hematological disorders such as aplastic anemia (AA) and myelodysplasic syndrome (MDS). It is hypothesized that patients with these disorders and PNH clones may share an immunologic basis for marrow failure with relative protection of the PNH clone, due to their lack of cell surface expression of immune accessory proteins. This is supported by the literature showing responsiveness in AA and MDS to immunosuppressive treatments. Preliminary results from a recent multicenter trial, EXPLORE, notes that PNH clones can be seen in 70% of AA and 55% of MDS patients, and therefore there may be utility in the general screening of all patients with bone marrow failure (BMF) syndromes. Furthermore, it has been suggested that the presence of PNH cells in MDS is a predictive biomarker that is clinically important for response to immunosuppressive therapy. Methods:Our retrospective cohort study in a tertiary care center used a high sensitivity RBC and FLAER assay to detect PNH clones as small as 0.01%. Of all patients screened with this method, those with bone marrow biopsy and aspirate proven MDS, AA, or other BMF syndromes (defined as unexplained cytopenias) were analysed. Results from PNH assays were compared to other clinical and laboratory parameters such as LDH. Results:Overall, 102 patients were initially screened over a 12 month period at our center. 30 patients were excluded as they did not have biopsy or aspirate proven MDS, AA, or other BMF syndromes. Of the remaining 72 patients, four patients were found to have PNH clones, where 2/51 had MDS (both RCMD, IPSS 0) [3.92%] and 2/4 had AA [50%]. The PNH clone sizes of these four patients were 0.01%, 0.01%, 0.02%, and 1.7%. None of the MDS patients with known recurrent karyotypic abnormalities had PNH clones present. Only one of the four patients had a markedly increased serum LDH level. Conclusions:Our retrospective study indicates much lower incidence of PNH clones in MDS patients or any patients with BMF syndromes when compared to the preliminary data from the EXPLORE trial. There is also significant disagreement in other smaller cohorts in regards to the incidence of PNH in AA and MDS. Screening for PNH clones in patients with bone marrow failure needs further study before adoption of widespread use. Disclosures:Keeney:Alexion Pharmaceuticals Canada Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wells:Alexion Pharmaceuticals Canada Inc: Honoraria. Sutherland:Alexion Pharmaceuticals Canada Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Natural History of Paroxysmal Nocturnal Hemoglobinuria Clones In Patients Presenting as Aplastic Anemia

AbstractAbstract 4428 Introduction:Acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are closely related bone marrow failure disorders. Most AA results from an autoimmune attack directed against hematopoietic stem/progenitor cells. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a PIG-A mutation. The PIG-A gene mutation leads to glycosylphosphatidylinositol-anchor protein (GPI-AP) biosynthesis deficiency and subsequent hemolysis secondary to the absence of complement regulatory proteins (CD55 and CD59). Both PNH and AA can be cured by allogeneic bone marrow transplantation (alloBMT), but only a minority of patients is offered this approach due to the potential morbidity and mortality. AA can be treated with immunosuppressive therapy (IST) and PNH can be controlled by eculizumab. It has been estimated that more than 50% of AA patients harbor small, but expandable PNH populations at diagnosis. The natural history of PNH clones in AA patients following non-transplant therapy is not well studied. The purpose of this study is to determine the fate and clinical relevance of these PNH clones in patients with AA who did not receive an alloBMT. Patients and Method:Twenty-seven patients with AA and a detectable PNH clone were monitored for a median of 5.3 years (range,1.5 to 11.5 years). The PNH granulocyte clone sizes were measured using flow cytometry and analyzed via CellQuest software. PE-conjugated anti-CD15 and fluoresceinated aerolysin variant (FLAER) staining were used to define granulocytes and GPI-AP deficient cells respectively. Serum lactate dehydrogenase (LDH) value was used as a surrogate for monitoring hemolysis and 1.5× the upper limit of normal LDH value (330mg/dL) as a cut-off point to define clinically apparent hemolysis. A PNH size change <2.5% was considered as stable. Patients were treated with IST, HiCy, or both. Result:We found a linear relationship between PNH granulocyte clone size and LDH values (Pearson correlation coefficient=0.73; P<0.0001). A PNH clone size above 23% was the threshold to identify hemolysis as measured by LDH (ROC analysis with AUC=0.88). Higher LDH values over the period of follow-up were associated with larger PNH granulocyte clone size at diagnosis (P=0.03). Patients with small (≤15%) initial PNH granulocytes had lower LDH levels at 5 years after diagnosis (mean±SD: 236.9±109.9 vs 423.1±248.8; P=0.02), and were less likely to develop hemolysis (13.3% vs 55.6%, P=0.06) comparing to those with larger (>15%) initial PNH granulocytes.Of 9 patients who initially were treated with traditional IST (ATG, CsA, and prednisone), 7 did not respond to treatment and eventually received high-dose cyclophosphamide (HiCy) salvage therapy, 2 achieved a remission and did not require further treatment though one demonstrated PNH clone size expansion to 50% after 37 months. After HiCy salvage, all 7 patients became transfusion independent and 4 of them had no further PNH clone expansion. PNH clone expansion was observed in 7 of 9 patients at a median time of 3 (range: 2 to 87) months after treatment.Of 15 patients who received HiCy as initial therapy, 14 achieved remissions. Later expansion of PNH size was observed in 7 patients, of which 5 eventually required intermittent blood transfusion but only 1 developed symptomatic hemolysis necessitating eculizumab therapy. The median time to PNH granulocyte clone expansion after HiCy was 52 (range: 18 to 106) months. In 5 patients who received HiCy and then relapsed, their PNH clone size only increased (1±0.7)% in (71±31) months observation during post treatment remission; however, their PNH clone size increase accelerated to (38±14)% in (34±21) months after AA relapse (P=0.04).Two nSAA patients with an initial PNH clone size ≤15% spontaneously recovered hematopoiesis at 84 and 56 months respectively, neither had PNH clone size expansion.In this study, 25.9% patients kept a stable PNH size, 48.1% patients increased the size, and 26% patients decreased the size. The group with small initial PNH clone sizes (≤15%) was the most stable over time. Conclusion:The risk of developing clinically significant PNH over 10 years appears to be low in AA patients with PNH clones, especially for those with small initial PNH granulocyte clones (≤15%) and for those who maintain remission following therapy. Disclosures:No relevant conflicts of interest to declare.

Changes of Clonality of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones during Clinical Courses in Patients with PNH.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder, in which almost all hematopoietic cells lack glycosylphosphatidylinositol (GPI)-anchored proteins due to phosphatidylinositol glycan-class A (PIG-A) gene abnormalities at the level of hematopoietic stem cells. It has been reported that several minor PNH clones in patients with aplastic anemia and myelodysplastic syndromes occur at random during the clinical course (Okamoto M et al, Leukemia, 2006), while one or more major PNH clones in patients with typical PNH continue to exist during their clinical courses (Nishimura J et al, Blood, 2002). To clarify how PNH clones in patients with PNH expand during their clinical courses, we examined sequence analyses of the PIG-A gene using peripheral blood granulocytes from PNH patients with high (>10%; n=5) and low (<10%; n=3) proportions of CD16− or CD59− granulocytes, which were determined by flow cytometry using CD16b or CD59 monoclonal antibody, respectively. A patient with over 1% of CD59− erythrocytes was judged to have PNH erythrocytes (Shichishima T et al, Br J Haematol, 1993). PNH clones were defined as GPI-negative hematopoietic cells with PIG-A mutations. Twenty to 40 bacterial clones of PCR or RT-PCR products of the PIG-A gene with GPI-negative or unsorted populations of peripheral blood granulocytes were analyzed by nucleotide sequencing. We examined sequence analyses of the PIG-A gene in peripheral blood granulocytes at least twice during the clinical course (the period of 1995–2007). Major PNH clones were decided by the Chi2 test or the Fisher's exact probability test. In all of 5 PNH patients with high proportions of PNH clones, major PNH clones were found at least once and were common during their clinical courses. However, the PNH clones in 3 of 5 PNH patients were transiently major during their clinical courses. In 2 of 3 PNH patients with low proportions of PNH clones, there were common PIG-A mutations during their clinical courses, but were not major clones. The PNH clone from one of the 2 PNH patients acquired a new PIG-A mutation, which was also minor. Another patient of the 3 patients with low proportions of PNH clones had a major PNH clone at the time of the first examination, but disappeared at the time of the next analysis. In conclusion, our findings suggest that the clonality of PNH clones in PNH patients with high and low proportions of PNH clones could change during their clinical courses and that especially, the clonality of PNH clones in PNH patients with low proportions of PNH clones is not necessarily definitive.PIG-A mutations in PNH patients with high and low proportions of PNH clonesCase No.Common mutationUncommon mutationPNH patients with high proportions of PNH clonesCase 1214 C to T, 5′ splicing site del G (Major)NDCase 2698 G to A (Major), 1152 del CNDCase 3548 G to A (Major)982 G to TCase 4ND491 C to TCase 5ND407 A to GPNH patients with low proportions of PNH clonesCase 5ND148 del G, 142 G to A (Major), 460 C to TCase 6ND11 G to C, 91 ins C, 916 del CCase 6ND67 del G, 72 del 13bp, 72 del 13bp/188 G to A, 358 G to A, 365 A to C, 952 del AAbbreviations: del , deletion; ND, not dectected; and ins, insertion

Decrease in Sensitivity of PIG-A-Mutant Cells to Natural Killer Cells Conferred by Missing of Stress-Inducible Membrane Proteins ULBPs.

The mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand to manifest symptoms is still unknown. PNH cells with PIG-A mutations do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of a series of GPI-linked membrane proteins. Blood cells with PNH phenotype (GPI− cells) expand in patients with bone marrow (BM) failure syndromes including PNH, aplastic anemia, and myelodysplastic syndromes. The diseases share an immune-mediated BM injury possibly by cytotoxic lymphocytes such as natural killer (NK) cells and cytotoxic T lymphocytes (CTL). It is suggested that the BM injury allows PNH clones survive selectively. Indeed, we have shown that human leukemic cells (K562) decrease their sensitivity to NK cells in vitro when K562 cells acquire PIG-A mutations (Nagakura et al, Blood 2002). In the present study, we show that the decrease in NK sensitivity of PIG-A mutant cells is ascribable to the deficiency of GPI-linked membrane proteins, ULBPs. ULBPs bind cytomegalovirus protein UL-16, consist of ULBP1-3, and emerge in cell membrane when cells are infected or transformed. They trigger off a NK activation signal, which overrides an inhibitory signal from MHC class I (Cosman et al, Immunity 2001). ULBPs also activate CTL besides NK cells. As target cells, a pair of GPI+ control and GPI− mutant cell lines was prepared from a GPI− K562 cell line bearing a PIG-A mutation by transfection with a PIG-A cDNA and a vector alone, respectively. Flow cytometry detected ULBP1-3 on the surface of GPI+ but none of GPI− K562 cells. As effector cells, we used cultured human NK cells (KHYG-1) deficient in Fc γR type III (CD16). GPI− cells were more resistant than GPI+ cells to the killing by NK cells in the 51Cr-release assay. GPI+ cells decreased their sensitivity to NK cells to the level of GPI− cells in the presence of antibodies to both ULBP1 and ULBP2, while antibodies to ULBP3 exerted no effects. None of the antibodies to ULBPs showed any effects on the killing of GPI− cells, while antibodies to NKG2D, which is a NK receptor for both ULBPs and MICA/B, inhibited the killing of both GPI+ and GPI− cells. Thus, GPI− cells that lack membrane ULBPs show a survival advantage in the setting of immune attack in vitro. Of clinical interests, ULBPs were detected on the cell surface of GPI+ but none of GPI− granulocytes of patients with PNH. GPI+ granulocytes of healthy individuals were negative for ULBPs. There appears pressure to induce membrane ULBPs in patients with PNH, leading to BM injury. These findings suggest that the failure of membrane expression of ULBPs permits selective expansion of PNH clones in patients with PNH and other BM failure syndromes.

The dual pathogenesis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease with distinct and rather peculiar characteristics that have puzzled hematologists for more than a century. PNH cells are deficient in a set of membrane proteins that have in common a glycolipid anchor. We refer to this combination of deficiencies as the PNH abnormality or the PNH phenotype. Biochemical analysis has recently made it possible to pinpoint the metabolic block in PNH cells to an early step in the biosynthesis of the glycolipid anchor. This block is due in turn to the deficiency of a protein, called PIG-A, which is encoded by an X-linked gene. Expression cloning of the PIG-A gene has been followed by the identification in patients with PNH of somatic mutations in this gene that inactivate or impair the function of the PIG-A protein. These findings explain in full the molecular basis of the PNH abnormality, but they do not explain how the PNH clone, which is biochemically defective, can expand to the extent of contributing a substantial proportion of the patient's hematopoiesis. Thus a second factor is required to explain the pathogenesis of PNH. This is most likely the coexistence of an element of bone marrow failure that produces, paradoxically, a survival or growth advantage for the PNH clone. The notion that the injury causing failure of normal stem cells spares selectively cells with the PNH phenotype is supported by a number of observations, including the finding of multiple independently arisen PNH clones in patients with PNH.