Nonmyeloablative allogeneic hematopoietic stem cell transplantation ...

Bone Marrow Transplantation (2003) 31, 407–410 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00

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Case report Nonmyeloablative allogeneic hematopoietic stem cell transplantation for treatment of Dyskeratosis congenita T Gu¨ngo¨r1, S Corbacioglu1, R Storb2 and RA Seger1 1 Division of Immunology, Hematology, Oncology and Bone marrow transplantation, University Children’s Hospital, Zu¨rich, Switzerland; and 2Clinical Research Division, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, USA

Summary: We describe the treatment of a 10-year-old girl with autosomal recessive Dyskeratosis congenita (DC), neutropenia, thrombocytopenia and combined immunodeficiency by nonmyeloablative hematopoietic stem cell transplantation. The conditioning regimen consisted of fludarabine 30 mg/m2/day (days 5, 4, 3) and 2 Gy TBI (0.07 Gy/min; day 0). For graft-versus-host disease (GVHD) prophylaxis a course of intravenous MMF and CSA was administered. At 2 years after transplantation of granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells from a healthy 11-year-old HLA-identical brother, peripheral blood counts and Tand B-cell functions have completely normalized and donor chimerism was 100% in all cell lineages. No GVHD occurred. Neurological examination and lung function remained normal. The current transplantation regimen appears suitable, safe and efficacious in patients with DC. Bone Marrow Transplantation (2003) 31, 407–410. doi:10.1038/sj.bmt.1703844 Keywords: non-myeloablative stem cell transplantation; Dyskeratosis congenita; Hoyeraal–Hreidarsson syndrome; combined immunodeficiency; hematopoietic stem cell transplantation (HSCT)

Dyskeratosis congenita (DC) is an inherited disease characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. Bone marrow failure with an aplastic anemia is the principal cause of early mortality with an additional predisposition to malignancy and fatal pulmonary fibrosis.1 Combined immunodeficiency with opportunistic infections may contribute to early death from the disease.2 The X-linked form of the disease is because of mutations in the gene DKC1 in the long arm of the X-chromosome (Xq28).3 It has been shown that Hoyeraal–Hreidarsson (HH) syndrome, a multisystem disorder characterized by aplastic anemia, immunodeficiency, microcephaly, cerebellar hypoplasia

Correspondence: Dr T Gu¨ngo¨r, Division of Immunology, Hematology, Oncology and Bone marrow transplantation; University Children’s Hospital, Zu¨rich, Switzerland Received 26 June 2002; accepted 1 October 2002

and growth retardation, is also caused by mis-sense DKC I mutations.4 The occurrence of DC and HH in males and females of the same family suggests autosomal recessive and dominant inheritance.1,5,6 The affected protein in Xlinked DC is dyskerin, which is a nucleolar protein associated with human telomerase RNA (hTR). Until now, the only treatment option for combined immunodeficiency and/or aplastic anemia in DC patients has been allogeneic bone marrow transplantation (BMT). Unfortunately, the results have been very poor. Unusual early and late toxicities with interstitial pneumonitis, lung fibrosis, veno-occlusive disease (VOD) and secondary tumors have been described, especially after myeloablative conditioning regimens.7–9 Here, we report on the use of a nonmyeloablative hematopoietic transplant regimen for the treatment of DC.

Case report The 10-year-old girl is the fourth child of a Swiss consanguineous family. Their first child, a boy, died because of progressive bone marrow failure and disseminated Aspergillus infection. He had symptoms of cerebellar atrophy that were consistent with HH syndrome as previously reported.10 Their daughter born 1 year after his death developed normally for the first 6 years of her life. Thereafter, she suffered from recurrent bacterial pneumonias and disseminated therapy-refractory molluscum contagiosum skin infection. At the age of eight (Table 1; day 365), leukopenia, moderate neutropenia and thrombocytopenia were found. Fluorescence-activated cell sorting (FACS) analyses revealed a B-cell lymphopenia and low normal CD4 cells counts with an inverted CD4/CD8 count ratio. Immunoglobulin titers were low, with IgG and IgM below the third percentiles. Antibody production after immunization with tetanus toxoid and pneumococcal polysaccharides was abnormally low. Mitogen-induced in vitro lymphocyte stimulation assays were normal for phytohemagglutinin (PHA), but abnormal for anti-CD3. In vitro lymphocyte stimulation assays showed nonresponsiveness to tetanus toxoid and candidin. Results of natural killer cell cytotoxicity assays using K562 target cells were normal. Bone marrow smears and biopsies were negative for malignancy or myelodysplasia with cytogenetic abnormalities. Cytogenetic and chromosomal breakage stu-

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dies after clastogenic agents (Mitomycin C, diepoxybutan and 1.5 Gy X-radiation) excluded other chromosomal breakage syndromes. Neuropsychological and neurological evaluation were normal. She was attending the second class of a primary school with satisfactory school results. A CT scan of the brain showed no abnormalities, such as cerebellar hypoplasia. At age 8.5 years she developed a Pneumocystis carinii pneumonia, suggesting profound functional T-cell deficiency. She was successfully treated with high doses of cotrimoxazole. Subsequently, leukocyte, neutrophil and platelet counts further decreased and B-cell counts disappeared completely (Table 1; day 90). We hypothetized, but could not prove, that this girl and her deceased brother with HH/DC had the same disease with presumed autosomal recessive inheritance. Mutation analyses excluded autosomal dominant and X-linked DC. Given the severity of the immunodeficiency and the absence of other treatment options, we performed hematopoietic stem cell transplantation (HSCT) after parental consent. A healthy HLA-identical 11-year-old brother with no clinical, hematological, cytogenetic or immunological abnormalities was identified as a potential hematopoietic stem cell donor. We chose to use peripheral stem cells in order to obtain the largest possible number of CD34-positive cells. After parental consent, her brother was given 5 days of granulocyte colony-stimulating factor (G-CSF) stimulation (10 mg/kg bw/day) with a yield of 10 106/kg CD34positive stem cells and 96 106/kg CD3-positive T cells. For stem cell collection, a Baxter CS 3000 apheresis

machine was used. Central venous canulation was not necessary because of satisfactory peripheral venous access. Non-myeloablative conditioning was given following the Seattle protocol, in order to reduce toxicity and tissue damage. Fludarabine, 30 mg/m2/day, was administered i.v. on days 4, 3, and 2. Total body irradiation (TBI), 2 Gy, was delivered from a linear accelerator at 0.07 Gy/ min on day 0. Donor peripheral blood stem cells (PBSC) were infused within hours of TBI. Postgraft immunosuppression consisted of Mycophenolate Mofetil (MMF), 15 mg/kg/b.i.d. i.v., given from the afternoon of day 0 until day +27. Cyclosporine A (CSA) was initially administered (day 3 to +27) at 2–3 mg/kg/day as a continuous infusion and continued at 5–6/mg/kg/b.i.d. orally until day +100. This conditioning regimen had already been shown to be efficacious in elderly patients with various diseases with minimal toxicity.11,12 No early toxicities (mucositis, hair loss, VOD) occurred during or after conditioning. VOD prophylaxis consisted of 100 U/kg/day i.v. Heparin and antithrombin (AT) IIIconcentrate (Kybernins; Aventis Behring) were given when ATIII activity fell below 70% (VOD-prophylaxis until day +30). Neutrophil (4500/ml) and platelet (450 000/ml) engraftment was observed on days +16 and +22, respectively. At day +16, fluorescence in situ hybridization (FISH analysis of the donor Y-chromosome) and variable number tandem repeat (VNTR) analyses revealed 100% donor chimerism in neutrophils and mononuclear cells. These chimerism studies were repeated every month and

PBSCT 5

300 platelets (G/l) 250

Hematological reconstitution

Neutrophils (G/l) 4

Neutro

200

Platelets

3 100 %

100 %

100 %

100 %

100 %

100 %

150

2 Donor chimerism % 1

100 50 0

0 -2

-0.5

0

0.5

1

2

3

4.5

2500

6

7

8

9

10

11.5 Mo

10

11.5 Mo

Immunological reconstitution CD3 CD4 CD8 CD20

2

3

100 %

1

100 %

1000 Donor chimerism % 500

100 %

Cand. + Tet. + 100 %

1500

100 %

2000

100 %

Lymphocytes/µl

0 -2

-0.5

0

0.5

4.5

6

7

SZT Figure 1

Hematological/immunological reconstitution and chimerism after stem cell transplantation.

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8

9

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Figure 2 (a, b) Reversible lingual leukoplakia at day +75 (a) and +365 (b) after peripheral stem cell transplantation.

Table 1

Immunological and hematological parameters prior and after PBSCT (day 0) Day 365

Hemoglobin (g/l) Leukocyte count (g/l) Neutrophil count (g/l) Platelet count (g/l) Lymphocyte count (/ml) Lymphocyte subpopulation (/ml) CD4+ CD8+ CD45+CD4RA+ CD45+CD4RO+ CD19+ Lymphocyte proliferation Phytohemagglutinin (dpm) Anti-CD3 (dpm) Candida albicans (dpm) Tetanus toxoid (dpm) Immunoglobulins (g/l) IgG IgM IgA Specific antibody titres Anti-IgG-tetanus (U/l) Anti-pneumococcal-IgG (mg/ml)

90

0

+10

+30

+365

+500

111 2.7 0.7 156 1350

112 1.7 0.4 127 1100

102 0.5 0.3 82 200

81 0.4 0.02 32 300

89 2.5 1.23 151 640

125 7.2 4.7 220 2300

130 8.0 4.0 210 3000

340 907 246 91 30

353 640 221 121 0

nd nd nd nd nd

nd nd nd nd nd

289 307 nd nd 9

937 1119 484 393 484

1200 880 710 490 510

52056 2074 272 1068

88848 1790 352 733

nd nd nd nd

nd nd nd nd

nd nd nd nd

73181 49 424 12 942 16 229

5.71 (oP10)a 0.35 (oP10) 0.96

2.5 (oP10)a 0.27 (oP10) 0.29 (oP10)

nd nd nd

nd nd nd

nd nd nd

8.6 0.45 0.52

o100 o1

o100 o1

n* n*

n* n*

n* n*

N* N*

nd nd nd nd

7.5 0.8 0.9

2500 >10

a

IgG1 and IgG2 were below the normal range. Normal values Pathologic values are typed in bold; n*=values under IVIG treatment; nd=not done. Mitogen (PHA, anti-CD3) in vitro lymphocyte proliferation test: >25 000 dpm. Antigen-stimulation (Candida albicans, Tetanus toxoid) in vitro lymphocyte proliferation test: >10 000/dpm. Anti-IgGtetanus toxoid: >100 U/l. Anti-pneumococcal-IgG (subtypes 4, 9V, 19, 23F): >1 mg/ml.

continued to show sustained 100% donor engraftment in all myeloid and lymphatic blood cells (Figure 1). After 57 days the patient was discharged from the hospital in good clinical condition. At 75 days after peripheral stem cell transplantation, lingual leukoplakia and dystrophy of the nails occurred, confirming the diagnosis of DC (Figure 2a). A tongue biopsy excluded malignancy. Lingual leukoplakia and nail dystrophy showed marked regression after cessation of CSA (Figure 2b). Immunological function was restored (Table 1) based on normal results in mitogen- and antigen-stimulation

assays (day +365). B and T-cell numbers had normalized and naive CD45 RA-positive CD4 cells were present in considerable numbers indicating thymic T-cell neogenesis. Antibody response after diphtheria and tetanus toxoid as well as pneumococcal polysaccharide vaccination was normal. At 2 years after transplantation, she is in an excellent clinical condition with no neuropsychological or neurological abnormalities. Her pulmonary function remained completely normal. Currently, she is attending the fourth class of a primary school with good results. Bone Marrow Transplantation

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Discussion The conditioning regimen including fludarabine 3 30 mg/ m2 and 2 Gy TBI was chosen to minimize toxicity, taking into account the importance of dyskerin and hTR for chromosomal stability, telomere length and tissue repair. Other nonmyeloablative conditioning regimens13 were considered too toxic for this patient. The finding of 100% donor chimerism as early as day +16 emphasizes the previously reported selection advantage of normal donor cells vs DC cells.1,3,5 We believe that the high number of CD3-positive T cells in our graft contributed to the rapid replacement of recipient lympho- and myelopoiesis. High CD3-T cell numbers might also contribute to a higher incidence of graft-versus-host disease (GHVD). In order to minimize GVHD because of variable gastrointestinal MMF absorption, in a nonmalignant disease, we favored intravenous administration.14 It is possible that even low-toxicity conditioning regimens like the current one may accelerate the natural course of the disease. Previous reports have described progression of the clinical signs of DC after transplantation which was indistinguishable from the natural course of disease.1,9 Other reports have described bleomycin-induced hypersensitivity of DC fibroblasts, lymphocytes, transformed lymphoblasts and enhanced G2 chromatid radiosensitivity of DC fibroblasts.15 Thus, it cannot be excluded that even low doses of TBI or chemotherapy may be harmful to DC patients. However, cell cycle studies after 1.5 Gy radiation (detecting a G2 cell cycle arrest) and other clastogenic agents had been normal in our patient. As her immunodeficiency had progressed rapidly just prior to transplantation, the occurrence of leukoplakia and nail dystrophies just after transplantation may have been coincidental or induced by other factors (such as immunosuppressive drugs interfering with telomerase activity) since the leukoplakia reversed itself after cessation of CSA. In summary, while bone marrow hypoplasia and combined immunodeficiency were successfully treated with HSCT, it is as yet not known whether the risks of lung fibrosis or cancer remain because of persisting telomerase dysfunction in other tissues. We suggest that the current transplantation regimen is suitable, safe and efficacious in patients with DC. Larger studies are required to reveal the short- and long-term sequelae of this regimen. Further investigation of its efficacy and safety in other breakage syndromes and DNA-repair disorders than DC (eg Fanconi anemia) is required.

Acknowledgements We thank Dr Inderjeet Dokal (Department of Haematology, Imperial College School of Medicine, London, UK) for performing DC mutation analyses; Dr Detlev Schindler (Labor fu¨r Chromosomenbruchanalyse, Biozentrum Am Hubland; D097074 Wu¨rzburg/Germany) for performing cell cycle studies;

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Dr David Betts for performing FISH analyses and Dr JP Hossle for performing VNTR analyses. Rainer Storb was supported by Grant HL 36444 from the National Institutes of Health, Bethesda, Maryland. Tayfun Gu¨ngo¨r’s grant support by Aventis Behring, Zu¨rich, Switzerland.

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