Hematopoietic Growth Factors for the Treatment of Inherited Cytopenias

Hematopoietic Growth Factors for the Treatment of Inherited Cytopenias Cornelia Zeidler and Karl Welte The clinical availability of recombinant hematopoietic growth factors was initially thought to be breakthrough in the treatment of bone marrow failure syndromes. However, in most disorders of hematopoeisis, the clinical use was rather disappointing. Only in congenital neutropenias (CNs) has the long-term administration of granulocyte colony-stimulating factor (G-CSF) led to a maintained increase in absolute neutrophil count (ANC) and a reduction of severe bacterial infections. In other disorders of hematopoiesis, the use of lineage-specific growth factors is either not possible due to mutations in the growth factor receptor or leads to a transient benefit only. Initial clinical trials with multilineage hematopoietic growth factors, such as stem cell factor (SCF; c-kit ligand) were discontinued due to adverse events. It is well known that bone marrow failure syndromes are pre-leukemic disorders. So far, there is no evidence for induction of leukemia by hematopoietic growth factors. However, it has been shown in patients with CN and Fanconi anemia that hematopoietic growth factors might induce preferential outgrowth of already transformed cells. Thus, it is strongly recommended to monitor patients for clonal aberrations prior to and during long-term treatment with hematopoietic growth factors. Semin Hematol 44:133-137. © 2007 Published by Elsevier Inc.

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nowledge of the influence of naturally occurring proteins on the regulation of normal hematopoiesis has a history of more than 150 years, and many outstanding scientists have contributed to the biological and clinical understanding of normal and pathologic hematopoesis over this time, finally leading to the isolation and molecular cloning of a number of proteins, known as hematopoietic growth factors or cytokines. In 1906, Carnot and Deflandre1 studied the cause of blood regeneration after bleeding and described a “substance capable of activating hematopoiesis,” which they named “hemopoietine.” The subsequent investigation of hematopoiesis by in vitro cultures of bone marrow cells indicated that not only erythropoiesis is regulated by such humoral factors, and from the 1960s an increasing number of hematopoietic activities were identified (for review, see Metcalf2). Hematopoietic growth factors like erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, stem cell factor (SCF), and thrombopoietin (TPO) were purified, characterized, and cloned. The availability of re-

Department of Pediatric Hematology/Oncology, Medical School Hannover, Hannover, Germany. Address correspondence to Karl Welte, MD, PhD, Department for Pediatric Hematology/Oncology, Kinderklinik, Medizinische Hochschule Hannover, Carl-Neuberg-Str 1, 30625 Hannover, Germany. E-mail address: [email protected]

0037-1963/07/$-see front matter © 2007 Published by Elsevier Inc. doi:10.1053/j.seminhematol.2007.04.003

combinant human hematopoietic growth factors from the late 1980s and their therapeutic application has substantially benefited the lives of thousands of patients suffering from a broad spectrum of diseases, such as renal failure, malignancies, and inherited and acquired bone marrow failure syndromes. In parallel, our comprehension of the inherited cytopenias, such as Diamond–Blackfan anemia (DBA), Kostmann syndrome and other congenital neutropenias (CNs), congenital thrombocytopenia, and Fanconi anemia (FA) has greatly increased. With the availability of recombinant hematopoietic growth factors for clinical application, many patients with these rare disorders could now be treated. However, initial enthusiasm for recombinant hematopoietic growth factors as treatment for the different inherited cytopenias did not endure for all of them. In some of the diseases, the use of recombinant hematopoietic growth factors did not lead to the anticipated effect on hematopoiesis, and in others toxicities in the first trials did not allow for the continuation of their clinical use. This review will focus on the current knowledge on the use of hematopoietic growth factors with respect to their risks and benefits in inherited cytopenias.

Inherited Neutropenia Rolf Kostmann, a Swedish physician, originally described a kindred with severe CN inherited as an autosomal recessive 133

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Figure 1 Long-term course of median ANCs in severe CN patients.

trait without additional hematological changes or other congenital abnormalities.3,4 Since his observations, different genetic patterns in CN have been described despite a uniformity of phenotype, characterized by severe chronic neutropenia and a maturation arrest of myelopoiesis in the bone marrow at the promyelocyte/myelocyte stage. Molecular genetic diagnosis is now available for the majority of patients. For recessive inheritance, mutations in the HAX1 gene have been detected recently.5 In cyclic neutropenia and dominant or sporadic inheritance of CN, different mutations in the ELA2 gene occur.6 – 8 For both HAX1 and ELA2 mutations, the downstream transcription factor LEF-1 is severely downregulated.9. In addition to HAX1 and ELA2 mutations, a number of different genetic aberrations have been identified in patients suffering from other syndromes associated with severe CN.10 Acquired G-CSF receptor mutations affecting the cytoplasmic domain are found in the majority of patients with leukemic transformation independent of somatic genetic mutations.11,12 After detection of a G-CSF receptor mutation, there is no evidence for an altered clinical response to G-CSF treatment, irrespective of G-CSF dose. Bone marrow transplantation (BMT) historically was the only curative treatment option for patients with human leukocyte antigen (HLA)-compatible donors.13 The availability of recombinant human G-CSF (rHuG-CSF)14 dramatically changed both the prognosis of CN and the quality of life of affected patients.15,16

Treatment Since 1987, G-CSF has been available for treatment of CN. Phase I/II/III studies demonstrated the efficacy of G-CSF to increase the number of neutrophils, which was associated with reduction of infections.15–19 In contrast, GM-CSF treatment does not lead to an increase in blood neutrophils but only blood eosinophils.20 In long-term follow-up since 1994 by the Severe Chronic Neutropenia International Registry (SCNIR), more than 95% of patients have responded to G-CSF treatment, with an increase in absolute neutrophil counts to 1.0 x 109/L and above (Fig 1). The majority of CN patients respond to a G-CSF dose between 3 and 20 ␮g/kg/d, but approximately 25% of CN

patients require G-CSF dosages between 20 and 100 ␮g/ kg/d. G-CSF response is defined as an increase in absolute neutrophil count (ANC) to 1.0 x 109/L or above. Patients with CN due to other syndromes such as Shwachman-Diamond-syndrome, glycogen-storage-disease, type 1b, myelokathexis, WHIM syndrome (warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis), hyper-IgM syndrome, etc10 respond to G-CSF similarly as do patients with HAX1 or ELA2 mutations. In some non-responders to G-CSF, whose ANC failed to improve at G-CSF levels exceeding 120 ␮g/kg/d, and partial responders, who increase their ANC to 0.5 to 1.0 x 109/L but still have bacterial infections, a combination of G-CSF with SCF has led to a further increase in neutrophils. Because of potential allergic side effects from SCF, this treatment combination has only been used during severe infections in hospitalized patients, who must receive concomitant antihistamines.21 For those patients who do not respond to G-CSF treatment alone or in combination with SCF, hematopoietic stem cell transplantation (HSCT) is the only currently available treatment19,22; when successful, hematopoiesis normalizes and no cytokine treatment is required. Leukemic transformation has been reported in single patients with CN.23,24 The first 374 patients (1987–2000) with CN on long-term G-CSF who were enrolled in the SCNIR have been studied in detail to identify risks for leukemic transformation.25 The hazard of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) increased significantly over time, from 2.9% per year after 6 years to 8.0%/ per year after 12 years on G-CSF; after 10 years, the cumulative incidence was 21% for MDS/AML. Of importance, the hazard of MDS/AML correlated with the dose of G-CSF. Twenty-nine percent of CN patients received more than the median dose of G-CSF (⬎ 8 ␮g/kg/d) but achieved less than the median ANC response (ANC ⬍2,188 cells/␮L at 6 to 18 months); in these less responsive patients, the cumulative incidence of adverse events was highest: after 10 years, 40% developed MDS/AML compared to 11% of more responsive patients, whose ANC was above the median on doses of G-CSF below the median. Available data do not suggest that the risk of MDS/AML is lower in Shwachman-Diamond syndrome than in CN. Comparable data have been reported by the French Severe Chronic Neutropenia study group.17 It remains difficult to give a recommendation for transplantation for patients with CN who benefit from G-CSF and show no evidence of impending malignant transformation.19,22

Inherited Anemias EPO was the first cytokine made available for clinical use, for the indication of renal anemia in 1986. Initially, patients with anemia secondary to chronic renal failure, a group known to have low EPO plasma levels, were entered in clinical trials.26 For thousands of these patients, the use of recombinant human EPO (rHuEPO) has changed their lives substantially and beneficially. However, experience with EPO as a treatment for inherited anemias is very limited. Lack of EPO, as in renal failure, does not play a role in the inherited disorders with

HGFs for inherited cytopenias defective erythroid maturation or ineffective erythropoiesis, like congenital aplastic anemia, DBA, congenital dyserythropoietic anemia (CDA) types 1– 4, and others. In the inherited bone marrow failure syndromes associated with chronic anemia, endogenous serum levels of EPO are normal or high for the corresponding level of anemia.27,28 Other hematopoietic growth factors, such as IL-3, GMCSF (sargramostim), or SCF, alone or in combination, have effects on the growth of erythroid colonies in vitro, but the benefit of these hematopoietic growth factors in clinical trials was too unconvincing to lead to their recommendation for routine clinical use.

Diamond-Blackfan Anemia DBA is a rare inherited anemia associated with macrocytosis, reticulocytopenia, a selective deficiency in erythroid progenitors, and specific phenotypic abnormalities. In approximately 25% of patients, heterozygous mutations in the ribosomal proteins S19 and S24 genes have been detected.29,30 In 1991, Niemeyer et al31 reported on nine DBA patients treated with subcutaneously administered EPO, in whom no increase in reticulocyte count or hemoglobin was observed. In the early 1990s, three clinical trials of different doses of IL-3 in DBA patients were reported32–34: of 31 patients, eight (25%) experienced a significant clinical response, but IL-3 had to be discontinued in two responders because of deep venous thrombi. To date, hematopoietic growth factors have no role in the general treatment of DBA patients.

Congenital Dyserythropoietic Anemias CDAs comprise a group of rare hereditary anemias characterized by ineffective erythropoiesis causing moderate to severe macrocytic anemia, with pathognomonic morphologic abnormalities in the majority of erythroblasts in the bone marrow. In CDA type 1, Tamary et al35 reported on the clinical use of erythropoietin (rHuEpo) in eight patients. During 18 weeks of treatment with increasing doses, to 500 IU/kg three times per week, there was no substantial effect on the mean hemoglobin value. In 2006, Heimpel et al36 reported on a long-term follow-up on 21 CDA I patients; of 21 patients, five were treated with interferon-alpha-2a. All patients responded with a rise of hemoglobin concentrations between 2.5 and 3.5 g/dL within the first 4 weeks of treatment. However, two patients subsequently discontinued treatment due to toxicities that decreased their quality of life.

Inherited Thrombocytopenias The two major forms of inherited thrombocytopenias, thrombocytopenia with absent radii (TAR) and congenital amegakaryocytic thrombocytopenia (CAMT), share the clinical features of isolated thrombocytopenia, reduced or absent bone marrow megakaryocytes, impaired responsiveness to TPO, and high plasma TPO levels.37 However, their long-

135 term outcomes are strikingly different: development of pancytopenia in the CAMT patients within the first 3 years of life versus improvement of thrombocytopenia in TAR patients.37,38 In CAMT patients, different mutations in the gene for the TPO receptor c-mpl have been identified. The type of mpl mutation determines the clinical course of CAMT. Homozygous nonsense mutations lead to a total loss of the TPO receptor, homozygous missense mutations reveal residual function of the TPO receptor and a transient increase of platelets.39 The development of pancytopenia in both patient groups indicates that mpl expression is critical for the maintenance and homeostasis of hematopoietic and even for mesenchymal stem cells. In non-human primates, the expression of mpl on hemangioblasts is consistent with these broad activities.40 TPO or synthetic mpl-ligands have not been tested in patients because TPO has no effect in vitro due to a total lack of a functional c-mpl receptor. Therefore, HSCT is the treatment of choice in inherited thrombocytopenias. Gene therapy protocols using retroviral vectors containing wildtype c-mpl and expression of c-mpl in CD34⫹ cells from CAMT patients ex vivo are in preclinical testing.

Fanconi Anemia FA is an autosomal recessive disorder of hematopoiesis characterized by progressive marrow failure, hypersensitivity to DNA crosslinking agents, and a strong cancer predisposition.41 Complementation analysis has revealed mutations in up to 12 genes (FANCA, FANCB, FANCC, FANCD, and others). Early use of hematopoietic growth factors in FA did not produce sustained responses. Pilot studies of G-CSF or GMCSF in FA patients demonstrated some improvement, such as increased ANC in patients who had residual myelopoiesis but not when myeloid progenitor cells were significantly reduced.42– 44 However, long-term treatment was tested only in a GM-CSF study by Guinan and coworkers, who reported follow-up of more than 19 months in six FA patients.42 They showed a substantial increase in neutrophil counts but not in hemoglobin concentration, although the reticulocyte count increased. All other studies have used cytokines for shortterm treatment, probably because of fear of development of clonal cytogenetic aberrations. Reduction of G-CSF dose in one patient led to a marked decrease in the number of monosomy 7 cells.44 Administration of G-CSF has produced an increase in circulating CD34⫹,43 presenting a new therapeutic option for FA patients45: G-CSF–induced mobilization and collection of CD34⫹ cells prior to the onset of severe bone marrow failure for autologous stem cell transplantation might be of clinical benefit. However, the efficacy and risk of this procedure need to be investigated in controlled studies. The feasibility of collecting CD34⫹ cells after G-CSF priming might also be useful in gene therapy of FA. In one study, in vivo infusion of interferon-gamma in a preclinical Fanca-/mouse model provided evidence that this cytokine is sufficient to allow long-term engraftment of wild-type repopulating cells, suggesting the possibility of conditioning with a

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136 Table 1 Clinical Use of Hematopoietic Growth Factors in Inherited Bone Marrow Failure Syndromes Bone Marrow Failure Syndrome Congenital neutropenia (CN)

Diamond-Blackfan anemia (DBA) Thrombocytopenia with absent radii (TAR) syndrome Congenital amegakaryocytic thrombocytopenia (CAMT) Fanconi anemia (FA)

Hematopoietic Growth Factor

Clinical Response

G-CSF GM-CSF SCF plus G-CSF EPO IL-3 TPO

Long-term treatment with sustained neutrophil responses No effect on neutrophil numbers Responses in some G-CSF non-responders No effect Heterogeneous effects on erythroid responses No data

TPO

No effect expected, no data

G-CSF

Transient neutrophil response, dependent on residual progenitor cells in the bone marrow Transient hemoglobin response, dependent on residual progenitor cells in the bone marrow

EPO

nontoxic interferon-gamma regimen as myelopreparation also in FA patients.46

Summary The clinical development of recombinant hematopoietic growth factors was initially thought to be a breakthrough in the treatment of bone marrow failure syndromes. However, in most of these disorders of hematopoiesis, their effects in the clinic were rather disappointing (Table 1). Only in CNs has long-term administration of G-CSF led to a maintained increase in ANC, reduction in severe bacterial infections, and improvement of the quality of life. Long-term daily subcutaneous administration of G-CSF for up to 20 years has proven feasible, without exhaustion of myelopoiesis and without severe adverse events. In other disorders of hematopoiesis, the use of lineage-specific growth factors is either not possible, due to mutations in the growth factor receptor, as in CAMT, or is of only transient benefit only, as for DBA or FA. Clinical trials with multilineage hematopoietic growth factors, such as SCF (c-kit ligand), have been discontinued due to serious toxicities. The bone marrow failure syndromes are pre-leukemic disorders, but to date, there is no evidence for induction of leukemia by hematopoietic growth factors. However, in patients with CN and FA, hematopoietic growth factors might induce preferential outgrowth of already transformed cells. Therefore, it is strongly recommended to monitor for clonal aberrations in patients prior to and during long-term treatment with hematopoietic growth factors.

Acknowledgment The authors thank Beate Schwinzer and Gusal Pracht from the Severe Congenital Neutropenia International Registry for creating Figure 1.

References 1. Carnot P, Deflandre C: Sur l’activité hemopoietique de serum au cours de la regeneration du sang. C R Acad Sci 143:432-435, 1906 2. Metcalf D: The molecular control of cell division, differentiation commitment and maturation in haematopoietic cells. Nature 339:27-30, 1989

3. Kostmann R: Infantile genetic agranulocytosis. Acta Pediatr Scand 45: 1-78, 1956 4. Kostmann R: Infantile genetic agranulocytosis: A review with presentation of ten new cases. Acta Pediatr Scand 64:362-368, 1975 5. Klein C, Grudzien M, Appaswamy G, Germeshausen M, Sandrock I, Schaffer AA, et al: HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease). Nat Genet 39:86-92, 2007 6. Aprikyan AA, Liles WC, Boxer LA, Dale DC: Mutant elastase in pathogenesis of cyclic and severe congenital neutropenia. J Pediatr Hematol Oncol 24:784-786, 2002 7. Dale DC, Person RE, Bolyard AA, Aprikyan AG, Bos C, Bonilla MA, Boxer LA, et al: Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia. Blood 96:2317-2322, 2000 8. Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC: Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoesis. Nat Genet 23:433-436, 1999 9. Skokowa J, Cario G, Uenalan M, Schambach A, Germeshausen M, Battmer K, et al: LEF-1 is crucial for neutrophil granulocytopoiesis and its expression is severely reduced in congenital neutropenia. Nat Med 12:1191-1197, 2006 10. Welte K, Zeidler C, Dale DC: Severe congenital neutropenia. Semin Hematol.43:189-195, 2006 11. Dong F, Brynes RK, Tidow N, Welte K, Lowenberg B, Touw IP: Mutations in the gene for the granulocyte-colony stimulating factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia. N Engl J Med 333:487-493, 1995 12. Germeshausen M, Ballmaier M, Welte K: Incidence of CSF3R mutations in severe congenital neutropenia and relevance for leukemogenesis: Results of a long-term survey. Blood 109:93-99, 2007 13. Rappeport J, Parkman R, Newburger P, Camitta BM, Chusid MJ: Correction of infantile granulocytosis (Kostmann syndrome) by allogeneic bone marrow transplantation. Am J Med 68:605-609, 1980 14. Souza L, Boone T, Gabrilove J, Lai PH, Zsebo KM, Murdock DC, et al: Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 232:61-65, 1986 15. Bonilla M, Gillio A, Ruggeiro M, Kernan NA, Brochstein JA, Abboud M, et al: Effects of recombinant human granulocyte colony-stimulating factor on neutropenia in patients with congenital agranulocytosis. N Engl J Med 320:1574-1580, 1989 16. Bonilla M, Dale D, Zeidler C, Last L, Reiter A, Ruggeiro M, et al: Longterm safety of treatment with recombinant human granulocyte colonystimulating factor (r-metHuG-CSF) in patients with severe congential neutropenias. Br J Hematol 88:723-730, 1994 17. Donadieu J, Leblanc T, Meunier BB, Barkaoui M, Fenneteau O, Bertrand Y, et al: Analysis of risk factors for myelodysplasia/leukemia and infectious death among patients with congenital neutropenia: Experience of the French Severe Chronic Neutropenia Study Group. Haematologica 90:45-53, 2005

HGFs for inherited cytopenias 18. Dale D, Bonilla M, Davis M, Nakanishi AM, Hammond WP, Kurtzberg J, et al: A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (Filgrastim) for treatment of severe chronic neutropenia. Blood 81:2496-2502, 1993 19. Zeidler C, Boxer L, Dale DC, Freedman MH, Kinsey S, Welte K: Management of Kostmann syndrome in the G-CSF era. Br J Haematol 109: 490-495, 2000 20. Welte K, Zeidler C, Reiter A, Muller W, Odenwald E, Souza L, et al: Differential effects of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in children with severe congenital neutropenia. Blood 75:1056-1063, 1990 21. Zeidler C, Vogel R, Wyres M, Welte K: Beneficial effects of stem cell factor (SCF) in children with severe congenital neutropenias refractory to G-CSF. Blood 92:380a, 1998 (abstr) 22. Zeidler C, Welte K, Barak Y, Barriga F, Bolyard AA, Boxer L, et al: Stem cell transplantation in patients with severe congenital neutropenia without evidence of leukemic transformation. Blood 95:1195-1198, 2000 23. Gilman P, Jackson D, Guild H: Congenital agranulocytosis: Prolonged survival and terminal acute leukemia. Blood 36:576-585, 1970 24. Rosen R, Kang S: Congenital agranulocytosis terminating in acute myelomonocytic leukemia. J Pediatr 94:406-408, 1979 25. Rosenberg PS, Alter BP, Bolyard AA, Bonilla MA, Boxer LA, Cham B, et al: The incidence of leukemia and mortality from sepsis in patients with severe congenital neutropenia receiving long-term G-CSF therapy. Blood 107:4628-4635, 2005 26. Winearls CH, Oliver DO, Pippard MJ, Reid C, Downing MR, Cotes PM: Effect of human erythropoietin derived from recombinant DNA on the anemia of patients maintained by chronic haemodialysis. Lancet 2:1175, 1986 27. Das REG, Milne A, Rowley M, Smith ECG, Cotes PM: Serum immunoreactive erythropoietin in patients with idiopathic aplastic and Fanconi’s anemias. Br J Haematol 82:601, 1992 28. Hammond D, Keighley G: The erythrocyte-stimulating factor in serum and urine in congenital hypoplastic anemia. Am J Dis Child 100:466, 1960 29. Draptchinskaia N, Gustavsson P, Andersson B, Pettersson M, Willig TN, Dianzani I, et al: The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia. Nat Genet 21:169-175, 1999 30. Gazda HT, Grabowska A, Merida-Long LB, Latawiec E, Schneider HE, Lipton JM, et al: Ribosomal protein S24 gene is mutated in DiamondBlackfan anemia. Am J Hum Genet 79:1110-1118, 2006 31. Niemeyer CM, Baumgarten E, Holldack J, Meier I, Trenn G, Jobke A, et al: Treatment trial with recombinant human erythropoietin in children with congenital hypoplastic anemia. Contrib Nephrol 88:276-280, 1991 32. Dunbar CE, Smith DA, Kimball J, Garrison J, Nienhues AW, Young NS: Treatment of Diamond-Blasckfan anemia with hematopoiesis growth factors, granulocyte-macrophage colony-stimulating factor and inter-

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34.

35.

36.

37.

38.

39.

40.

41. 42.

43.

44.

45.

46.

leukin-3: Sustained remission following IL-3. Br J Haematol 79:316-321, 1991 Gillio AP, Faulkner LB, Alter BP, Reilly L, Klafter R, Heller G, et al: Treatment of Diamond-Blackfan anemia with recombinant human interleukin-3. Blood 82:744-751, 1993 Olivieri F, Feig SA, Valentino L, Berriman AM, Shore R, Freedman MH: Failure of recombinant human interleukin-3 therapy to induce erythropoiesis in patients with refractory Diamond-Blackfan anemia. Blood 83:2444-2450, 1994 Tamary H, Shalev H, Pinsk V, Zoldan M, Zaizov R: No response to recombinant human erythropoietin therapy in patients with congenital dyserythropoietic anemia type I. Pediatr Hematol Oncol 16:165-168, 1999 Heimpel H, Schwarz K, Ebnöther M, Goede JS, Heydrich D, Kamp T, et al: Congenital dyserythropoietic anemia type I (CDA I): Molecular genetics, clinical appearance, and prognosis based on long-term observation. Blood 107:334-340, 2006 Geddis AE: Inherited thrombocytopenia: Congenital amegakaryocytic thrombocytopenia and thrombocytopenia with absent radii. Semin Hematol 43:196-203, 2006 King S, Germeshausen M, Strauss G, Welte K, Ballmaier M: Congenital amegakaryocytic thrombocytopenia: A retrospective clinical analysis of 20 patients. Br J Haematol 131:636-644, 2005 Germeshausen M, Ballmaier M, Welte K. c-mpl mutations in 23 patients suffering from congenital amegakaryocytic thrombocytopenia: the type of mutation predicts the course of the disease. Hum Mut 27:296, 2006 Wang Z, Skokowa J, Pramono A, Ballmaier M, Welte K: Thrombopoietin regulates differentiation of Rhesus monkey embryonic stem cells to hematopoietic cells. Ann N Y Acad Sci 1044:29-40, 2005 Kutler DI, Singh B, Satagopan J: A 20-year perspective on the International Fanconi Anemia Registry (IFAR). Blood 101:1249-1256, 2003 Guinan EC, Lopez KD, Huhn RD, Felser JM, Nathan DG: Evaluation of granulocyte-macrophage colony-stimulating factor for treatment of pancytopenia in children with fanconi anemia. J Pediatr 124:144-150, 1994 Rackoff WR, Orazi A, Robinson CA, Cooper RJ, Alter BP, Freedman MH, et al: Prolonged administration of granulocyte colony-stimulating factor (filgrastim) to patients with Fanconi anemia: A pilot study. Blood 88:1588-1593, 1996 Scagni P, Saracco P, Timeus F, Farinasso L, Dall’Aglio M, Bosa EM, et al: Use of recombinant granulocyte colony-stimulating factor in Fanconi’s anemia. Haematologica 83:432-437, 1998 Croop JM, Cooper R, Fernandez C, Graves V, Kreissman S, Hanenberg H, et al: Mobilization and collection of peripheral blood CD34⫹ cells from patients with Fanconi anemia. Blood 98:2917-2921, 2001 Si Y, Ciccone S, Yang FC, Yuan J, Zeng D, Chen S, et al: Continuous in vivo infusion of interferon-gamma (IFN-gamma) enhances engraftment of syngeneic wild-type cells in Fanca-/-and Fancg-/- mice. Blood 108:4283-4287, 2006