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Keywords:

  • paroxysmal nocturnal haemoglobinuria;
  • immunoselection;
  • clonal expansion;
  • NKG2D (KLRK1);
  • high mobility group AT-hook 2

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired stem cell disorder caused by expansion of PNH clones that harbour PIGA mutations and lack glycosylphosphatidylinositol (GPI)-linked membrane proteins, such as CD55 and CD59, leading to complement-mediated intravascular haemolysis and thrombosis (Parker & Ware, 2003). PNH also manifests immune-mediated bone marrow (BM) failure. PNH presents critical problems that need to be resolved (Luzzatto et al, 1997; Dunn et al, 2000; Inoue et al, 2003; Nakakuma & Kawaguchi, 2003; Parker & Ware, 2003): the mechanism by which PNH clones expand, the pathogenesis of BM failure, which is a major cause of death, and PNH aetiology.

Two hypotheses exist for the mechanism of clonal expansion: survival and growth advantage theories. For the first theory, PNH clones selectively survive in the setting of immune-mediated BM injury (survival advantage) (Dunn et al, 2000; Inoue et al, 2003; Nakakuma & Kawaguchi, 2003). A possible candidate for this is NKG2D (KLRK1)-mediated immunity (Hanaoka et al, 2009), which is triggered by the expression of ligands, such as major histocompatibilty complex class I chain-related peptides A and B (MICA/B) and cytomegalovirus UL-16 binding proteins (ULBPs). MICA/B and ULBPs are peptide-linked (transmembrane) and GPI-linked membrane proteins, respectively. The ligands share NKG2D as a common receptor on such lymphocytes as natural killer (NK) cells and CD8+ cytotoxic T cells. The engagement of NKG2D with its ligands that are frequently coexpressed promotes the elimination of NKG2D ligand-expressing cells by NKG2D+ lymphocytes. Then, PNH clones lacking GPI-linked ULBPs may preferentially survive by immunoselection (Hanaoka et al, 2006). The growth advantage theory is partly supported by the pathological expression of genes such as high mobility group AT-hook 2 (HMGA2), which encodes a transcription factor often found in benign tumours such as lipoma and myoma, early growth response factor 1 (EGR1), and Wilms' tumour 1 (WT1) (Inoue et al, 2003; Nakakuma & Kawaguchi, 2003; Ikeda et al, 2011; Murakami et al, 2012). Current reports suggest that the two theories are cooperative rather than mutually exclusive (Inoue et al, 2003; Nakakuma & Kawaguchi, 2003). Indeed, we here report a patient with PNH showing this cooperation.

A 47-year-old woman was diagnosed as having PNH with a coexisting congenital deficiency of C9 in 1980. She is presently 79 years old and has maintained a high quality of life for more than 32 years after PNH diagnosis. She has mild BM failure responsive to low-dose metenolone acetate (10 mg/day). She also manifests very low levels of both intra- and extravascular haemolysis, with detection of haemosiderinuria and C3d-bound erythrocytes (Hanaoka et al, 2012). Flow cytometry showed complete occupancy of her peripheral blood by PNH cells negative for both CD55 and CD59 (Fig 1A). Erythrocytes (Fig 1B) and leucocytes (Fig 1C) all had the PNH phenotype. In general, it is very rare that all lymphocytes show PNH phenotype even in patients with high population of PNH-erythrocytes and -granulocytes. Of interest, virtually all cells in the lymphocyte fraction (CD11b leucocytes) were also negative for CD55 and CD59 (Fig 1C). It is then conceivable that all haematopoietic stem cells are also affected. The blood cells were of a single PIGA-mutant clone, which has persistently maintained haematopoiesis under the treatment with low-dose metenolone acetate for more than 13 years since 1998 (data not shown). These findings prompted us to be concerned about the mechanism by which the mutant clone completely occupies blood cells in the patient.

image

Figure 1. Characterization of the PNH clone. (A-C) Lack of CD55 and CD59. (A) total peripheral blood cells (Total cells, shaded histogram), (B) erythrocytes (CD235a+ cells), (C) granulocytes (CD11b+ cells) and lymphocytes (CD11b cells, ▼). An arrow (in panel A) indicates blood cells positive for CD55 and CD59 of a healthy donor (positive control). (D) Granulocytes positive for MICA/B but negative for ULBPs. (E) Lymphocytes positive for NKG2D. (F) HMGA2 mRNA amplification in leucocytes of the present case in this report. For comparison, the data of the present case (●) were shown together with our published data of 25 patients with PNH (PNH) and 11 healthy volunteers (Normal) (Murakami et al, 2012). The value of relative expression shows the ratio of HMGA2 mRNA expression: sample/normal control. The mean relative mRNA expression (—) was 1·97 ± 1·15 (standard deviation, SD) in PNH, 2·73 in the present case of PNH, and 0·70 ± 0·26 in normal individuals. (A, D, and E) Dotted lines in the histograms indicate nonspecific background staining with isotype-matched control immunoglobulin.

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Regarding NKG2D-mediated immunity as a potential candidate that allows the immunoselection of PNH clones (Inoue et al, 2003; Nakakuma & Kawaguchi, 2003; Hanaoka et al, 2006, 2009), we confirmed the pathological expression of MICA/B, peptide-linked NKG2D ligands, on granulocytes (Fig 1D). Given that NKG2D receptor is expressed in the patient's lymphocytes (Fig 1E), NKG2D-mediated injury of the blood cells may occur in the patient (Hanaoka et al, 2006, 2009). In this setting, the PIGA-mutant clone lacking ULBPs as GPI-linked NKG2D ligands (Fig 1D) may survive and accumulate (Hanaoka et al, 2006, 2009), leading to clonal expansion. Of note, the PIGA-mutant clone also showed HMGA2 amplification about four times normal (Fig 1F), which is reported to confer the benign tumour-like growth phenotype (Ikeda et al, 2011; Murakami et al, 2012). The amplification may also support expansion of the mutant clone in the patient.

This is the first case of PNH indicating the marked expansion of a single PNH clone by combination of both survival and growth advantages.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. Author contributions
  4. Conflicts of interest
  5. References

The authors thank Tatsuya Kawaguchi of Kumamoto University for his critical discussion.

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Ministry of Labour and Welfare of Japan, and the Takeda Science Foundation.

Author contributions

  1. Top of page
  2. Acknowledgements
  3. Author contributions
  4. Conflicts of interest
  5. References

NH designed and performed research, analysed data, and wrote the paper. YM and TK performed molecular analyses. MN, KH, SN, YY, SM, and TS analysed clinical data. HN supervised the project, analysed data, and wrote the paper.

Conflicts of interest

  1. Top of page
  2. Acknowledgements
  3. Author contributions
  4. Conflicts of interest
  5. References

All authors declare no competing financial interests.

References

  1. Top of page
  2. Acknowledgements
  3. Author contributions
  4. Conflicts of interest
  5. References
  • Dunn, D.E., Liu, J.M. & Young, N.S. (2000) Paroxysmal nocturnal hemoglobinuria. In: Bone Marrow Failure Syndromes (ed. by N.S. Young), pp. 99121. Saunders, Philadelphia, PA.
  • Hanaoka, N., Kawaguchi, T., Horikawa, K., Nagakura, S., Mitsuya, H. & Nakakuma, H. (2006) Immunoselection by natural killer cells of PIGA mutant cells missing stress-inducible ULBP. Blood, 107, 11841191.
  • Hanaoka, N., Nakakuma, H., Horikawa, K., Nagakura, S., Tsuzuki, Y., Shimanuki, M., Kojima, K., Yonemura, Y. & Kawaguchi, T. (2009) NKG2D-mediated immunity underlying paroxysmal nocturnal haemoglobinuria and related bone marrow failure syndromes. British Journal of Haematology, 146, 538545.
  • Hanaoka, N., Murakami, Y., Nagata, M., Nagakura, S., Yonemura, Y., Sonoki, T., Kinoshita, T. & Nakakuma, H. (2012) Persistently high quality of life conferred by coexisting congenital deficiency of terminal complement C9 in a paroxysmal nocturnal hemoglobinuria patient. Blood, 119, 38663868.
  • Ikeda, K., Mason, P.J. & Bessler, M. (2011) 3'UTR-truncated Hmga2 cDNA causes MPN-like hematopoiesis by conferring a clonal growth advantage at the level of HSC in mice. Blood, 117, 58605869.
  • Inoue, N., Murakami, Y. & Kinoshita, T. (2003) Molecular genetics of paroxysmal nocturnal hemoglobinuria. International Journal of Hematology, 77, 107112.
  • Luzzatto, L., Bessler, M. & Rotoli, B. (1997) Somatic mutations in paroxysmal nocturnal hemoglobinuria: a blessing in disguise? Cell, 88, 14.
  • Murakami, Y., Inoue, N., Shichishima, T., Ohta, R., Noji, H., Maeda, Y., Nishimura, J., Kanakura, Y. & Kinoshita, T. (2012) Deregulated expression of HMGA2 is implicated in clonal expansion of PIGA deficient cells in paroxysmal nocturnal haemoglobinuria. British Journal of Haematology, 156, 383387.
  • Nakakuma, H. & Kawaguchi, T. (2003) Pathogenesis of selective expansion of PNH clones. International Journal of Hematology, 77, 121124.
  • Parker, C.J. & Ware, R.E. (2003) Paroxysmal nocturnal hemoglobinuria. In: Wintrobe's Clinical Hematology (ed. by J.P. Greer, F. Foerster, J.N. Leukens, G.M. Rodgers, F. Paraskevas & B. Glader), pp. 12031221. Lippincott Williams and Wilkins, Baltimore, MD.