Hematopoietic Stem Cell Transplantation in Omenn Syndrome: a Single-Center Experience

Hematopoietic stem cell transplantation in Omenn syndrome: a single-center experience

E Mazzolari1, D Moshous1, C Forino1, D De Martiis1, C Offer1, A Lanfranchi1, S Giliani1, L Imberti2, S Pasic3, AG Ugazio1,4, F Porta1 and LD Notarangelo1

1Department of Pediatrics, Children’s Hospital, University of Brescia, Italy; 2Laboratorio di Biotecnologie, Spedali Civili, Brescia, Italy; and 3Mother and Child’s Institute, Beograd, Yugoslavia

Summary: pathy.1 Other common symptoms include diarrhea, alope- cia, and failure to thrive.2 Occasionally, a similar clinical We retrospectively analyzed the outcome of hematopoietic phenotype may be noticed in other immunodeficiency stem cell transplantations (HSCT) performed at our syndromes,3–6 and particularly in SCID with maternal Center between 1991 and 2002 in 11 unselected patients T-cell engraftment. with Omenn syndrome, a variant of severe combined Laboratory findings of OS include hypogammaglobulin- immunodeficiency. The patients’ mean age at the time of emia with elevated serum IgE, defective antibody produc- the first HSCT was 8.4 months. Two patients received two, tion, marked eosinophilia, and a variable number of and one patient three, HSCT procedures. The resulting 15 autologous, activated, and oligoclonal T lymphocytes, that HSCT derived in seven cases from HLA-haploidentical tend to infiltrate target organs and are generally poorly parents, in four patients from matched unrelated donors, in responsive to mitogens.7–9 Villa et al10 showed that OS is three cases from an HLA phenotypically identical related usually caused by hypomorphic mutations in the recombi- donor, and in one case from an HLA genotypically nation activating gene (RAG)1 or RAG2 that result in identical family donor. Nine out of 11 patients are alive residual protein expression and function. More recently, we and immunoreconstituted 30–146 months after transplan- found that patients with OS have defective thymic expres- tation. At the time of the most recent evaluation, all of the sion of Aire, a transcription factor that controls expression nine survivors had normal T-cell function, and eight of of tissue-specific peptides by thymic medullary epithelial them had developed normal antibody production. This cells.11 This defect is likely to result in defective negative study demonstrates an overall mortality of 18.2%, which is selection of autoreactive T cells in OS, that can therefore substantially lower than previously reported. Early recog- expand in the periphery in response to autoantigens. nition of OS, rapid initiation of adequate supportive In the absence of hematopoietic stem cell transplantation treatment and HSCT lead to improved outcome for this (HSCT), OS remains a fatal disorder due to increased otherwise fatal disease, regardless of the origin and susceptibility to infections with a high lethality early in life matching of hematopoietic stem cells. despite intensive supportive care. In contrast, transplant- Bone Marrow Transplantation (2005) 36, 107–114. ation of allogeneic hematopoetic stem cells can provide doi:10.1038/sj.bmt.1705017 permanent cure.12 In the past, satisfactory results have been Published online 23May 2005 obtained mostly with HSCT from HLA-matched family Keywords: Omenn syndrome; Severe Combined Immuno- donors, whereas significant mortality and long-term deficiency; stem cell transplantation morbidity have been reported in the literature for HSCT from mismatched family donors or from matched unrelated donors (MUD).2,12,13 In particular, experience at a single center using related donors has resulted in 66.7% overall survival;12 a lower success rate (16.7% survival) has been reported in another Omenn syndrome (OS) is a rare autosomal recessive study focusing on mismatched related or matched unrelated disorder characterized by severe combined immunodefi- bone marrow transplantation.13 A recent review analyzed ciency (SCID) typically associated with the triad of the outcome of 68 OS patients reported in the literature erythrodermia, hepatosplenomegaly, and lymphadeno- between 1965–1999.2 The overall mortality of 41 evaluable patients was 59%; in particular, 69% of the untransplanted patients died within the first year of life, whereas mortality Correspondence: Professor LD Notarangelo, Department of Pediatrics of the 28 transplanted OS patients was shown to be 46%. and ‘Angelo Nocivelli’ Institute for Molecular Medicine, University of We report here our single-center experience of allogeneic Brescia, 25123Brescia, Italy; E-mail: [email protected] 4Current address: Ospedale Pediatrico ‘Bambino Gesu` ’, Rome, Italy HSCT in 11 unselected patients with OS who were Received 14 February 2005; accepted 31 March 2005 transplanted in the period between October 1991 and July Published online 23May 2005 2002. Stem cell transplantation in Omenn syndrome E Mazzolari et al 108 Patients and methods 1-M; for three patients (OS4, OS5, and OS6), positively selected CD34 þ BMSC and/or PBSC were obtained using Patients the Isolex 300 system (Baxter). Patient OS8 received three attempts of haploidentical HSCT (one from her mother We studied retrospectively 11 unselected patients with the and two from her father), using positively selected CD34 þ clinical diagnosis of OS referred to the Department of BMSC and PBSC, purified with the Clinimacs System Pediatrics, Children’s University Hospital, Brescia, Italy, (Miltenyi), as described previously.14 who received allogeneic HSCT between October 1991 and Three patients (OS2, OS3, and OS11) received a total of July 2002. Inclusion criteria were based on typical clinical four attempts of HSCT from MUD; in all of these cases, and laboratory features of OS (Table 1), associated with unmanipulated bone marrow stem cells (BMSC) were used. absence of maternal T-cell engraftment which may other- Two patients (OS9, OS10) received three HSCT with wise mimic OS and result in an ‘Omenn-like’ phenotype. unmanipulated BMSC from an HLA phenotypically The procedures for genetic analysis, HSCT, immunological identical parent. Finally, one patient (OS7) received follow-up, and data monitoring were approved by the local unmanipulated BMSC from an HLA-identical sibling. A Ethical Committee, and informed consent was obtained median of 6 Â 108 nucleated bone marrow cells per from the patients’ parents or their legal guardians. kilogram of body weight were infused (range: 3–11.3 Â 108/kg). In the six transplants in which PBSC were also 6 Hematopoietic stem cell transplantation infused, the median dose of PBSC was 4.1 Â 10 CD34 þ cells/kg (range: 1–15 Â 106/kg). HLA typing to ascertain recipient–donor compatibility was performed according to standard serologic methods in five Conditioning regimen, supportive care, and patients and high-resolution molecular typing methods in graft-versus-host disease (GVHD) prophylaxis six cases. The median age of recipients at the time of the first HSCT was 8.4 months (range: 4–18 months). Details As illustrated in Table 2, myeloablative conditioning of the HSCT procedures are reported in Table 2. In all regimens were mainly based on busulfan and cyclophos- cases, the bone marrow was used as the source of phamide; the dose of the former was adjusted according to hematopoietic stem cells; furthermore, in three cases plasma levels after the first administration. Thio-Tepa (5– donors agreed to donate also peripheral blood stem cells 20 mg/kg) was used in six patients, most often in associa- (PBSC). Mobilization of PBSC was induced by adminis- tion with busulfan and cyclophosphamide. Seven patients tration of granulocyte colony-stimulating factor (G-CSF) received rabbit antithymocyte globulin (ATG, 10 mg/kg) (10 mg/kg daily for 5 days); the PBSC were then harvested daily for 3days. A conditioning regimen based on by leukapheresis. fludarabine (200 mg/m2) and melphalan (100 mg/m2) was Seven haploidentical transplants were performed in five used in two patients. All patients had a central venous line infants, one of whom received three transplants. In one of and were nursed in protective isolation (laminar airflow) these patients (OS1), HSCT was performed with bone before and throughout the HSCT period. Appropriate marrow cells that had been T-cell depleted using Campath antibiotic, antiviral, antifungal, and supportive prophy-

Table 1 Clinical features at presentation in 11 patients with OS

Patient Age at Erythrodermia Liver and/or Lymphadenopathy Diarrhea Other infections Failure to Associated features onset spleen thrive (days) enlargement

OS 1 70 + À + + Candidiasis, + interstitial pneumonia (CMV) OS 2 3+ + + + + OS 340 + + À + + Arthritis, elevated liver enzymes OS 4 50 + À + + + Hypotonia, acute renal failure, thrombocytopenia OS 5 20 + + À + Oral candidiasis, + Elevated liver enzymes, arthritis otitis, interstitial pneumonia (RSV) OS 6 30 + À + + Otitis (S. + pneumoniae) OS 7 45 + + + + + OS 8 15 + ÀÀ+ Candidiasis, + Seizures mucositis OS 9 14 + À + + Interstitial + Thrombocytopenia, anemia pneumonia (CMV), BCGitis OS 10 1 + + + À Candidiasis À Elevated liver enzymes OS 11 60 + + + + Salmonella +

CMV ¼ cytomegalovirus; RSV ¼ respiratory syncytial virus; BCG ¼ Bacillus Calmette-Guerin.

Bone Marrow Transplantation Stem cell transplantation in Omenn syndrome E Mazzolari et al 109 laxis/therapies were administered, along with adequate parenteral nutrition when necessary. Surveillance for

busulfan; Cytomegalovirus (CMV), Herpes Simplex Virus (HSV), ¼ Human Herpes Virus 6 (HHV 6), Adenovirus, and Epstein–Barr virus (EBV), as well as for Aspergillus antigen, was performed regularly. Immunoglobulins were None None None liver (4) administrated intravenously weekly at 200 mg/kg in the early post-transplant period, and then at 400 mg/kg/21 days until evidence for endogenous immunoglobulin production was achieved. Single-donor platelets were administered to ; ATG None 2 maintain platelets counts 420 Â 109/l, and red blood cell

2 transfusions were used to keep hemoglobin levels 48.0 g/ matched unrelated donor; Bu

¼ dl. Prophylaxis of graft-versus-host (GvH) disease was used

2 in all patients, and was based on cyclosporin A per os, adjusting the dose based on blood levels. Survival was estimated by the Kaplan–Meier method,

2 taking into consideration the event time for survival from ; Melphalan 100 mg/m 2 date of transplantation to the date of death or date of last contact. mononuclear cells; MUD ¼ Engraftment and chimerism analysis Regular blood cell counts were performed to survey ; Melphalan 100 mg/m 2 engraftment. Myeloid engraftment was considered to have taken place on the ﬁrst of three consecutive days during which the absolute neutrophil count (ANC) exceeded 0.5 Â 109/l. Platelet recovery was deﬁned to be achieved

Conditioning regimen Acute GvHD(grade) 9 peripheral blood; MNC the day when the platelet count exceeded 20 Â 10 /l without ¼ transfusion within the preceding 3days. Patients dying within 28 days after HSCT were considered not evaluable for engraftment. Graft failure was deﬁned as the absence of /kg) or 8 engraftment within 30 days post HSCT. 10 þ Â Chimerism was assessed on positively selected CD3 T þ bone marrow; PB lymphocytes and CD19 B lymphocytes, as well as on /kg BM MNC Bu 16 mg/kg; Cy 200 mg/kg; VP-16 120 mg/m ¼ 8 /kg CD34+ cells/kg CD34+ cells Bu/kg 16 BM mg/kg; MNC Cy 200 mg/kg; ATG;Thio-Tepa 10 mg/kg Bu 16 mg/kg; Cy 200 mg/kg; Thio-Tepa 10 mg/kg Bu 8 mg/Kg; Cy 200 mg/kg None Skin (++) Skin (++); gut (1) /kg CD34+ cells/kg CD34+ cells Bu 16 mg/kg; Cy 200 mg/kg; Thio-Tepa 5 mg/kg; Flu 200 mg/m /kg BM MNC Thio-Tepa 20 mg/kg; Bu 20 mg/kg; Cy 100 mg/kg Skin (++++) Skin (++++); gut, 6 6 8 granulocytes, by microsatellite analyses using probes for 6 6 8 CD34+/kg) /kg BM MNC/kg BM MNC/kg CD34+ Bu cells 20 mg/kg; Cy 200 m g/kg; ATG; Bu VP-16 16 120 mg/kg mg/m ;Cy Bu 200 mg/kg; 16/kg mg/kg; ATG CD34+ Cy cells 200 mg/kg; Thio-Tepa 10 mg/kg/kg BM MNC/kg BM OKT3; MNC Cy/kg 120 BM mg/kg MNC/kg BM MNC Flu 200 mg/m Bu 16 mg/kg; Cy Bu 100 mg/kg; 16 mg/kg; ATG Skin Cy (+) Thio-Tepa 200 mg/kg; 10 mg/kg, ATG ATG, Cy 120 mg/kg Skin (++); liver (1) None None None Skin (+); liver (1) 10 6 8 8 6 6 8 8 8 8 10 10 10 10 10 10

Â DQ alpha, D1S80 and ApoB. For chimerism analysis, 10 10 10 10 10 10 10 10 10 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â positive selection of T and B lymphocytes was accom- ( plished using monoclonal antibody-coated magnetic beads with the Minimacs System (Miltenyi).

Immunological reconstitution

graft-versus-host disease; BM Immunological reconstitution in the T-, B-, and natural ¼ killer (NK)-cell compartments was monitored by ﬂow cytometry using a FACScalibur instrument, and monoclonal antibodies to CD3, CD4, CD8, CD19, and CD16. Naı¨ ve and memory T cells were distinguished by their expression of CD45RA and CD45RO, respectively. The function and diversity of T lymphocytes were assessed by Mother 6/6 (full marrow) 3 Donor (source) BM cell dose ( analyzing PHA-induced lymphocyte proliferation and the antithymocyte globulin. T-cell receptor repertoire, as described previously.10 Thy- ¼ mic output was evaluated by measuring T-cell receptor 15 5 Father 3/6 (BM CD34+ cells) 6

10 Father 3/6 (BM and PB CD34+ cells)15 20 MUD 6/6 (full marrow)excision 10 circles (TRECs), as described. HSCT (months) Mutation analysis HSCT and GvHD in 11 patients with OS Prior to HSCT, blood samples were obtained upon hematopoietic stem cell transplantation; GvHD

¼ informed consent from all patients and, when available, cyclophosphamide; ATG

¼ from their parents and their siblings. Following DNA Cy OS 9 M 6 Mother 6/6 (full marrow) 3 OS 2OS 3F M 8 8 MUD 6/6 (full marrow) MUD 6/6 (full marrow) 5 11.3 Table 2 Patient Sex Age at HSCT OS 5OS 7OS F 8 F F 18 9 Mother 3/6 (BM 4 CD34+ cells) Sister 6/6 (full marrow) Mother 3/6 (BM and PB CD34+ cells) 4 12 4.5 OS 1OS F 4OS 6 M 5 M 6 Mother 3/6 (T-cell-depleted BM) 12OS 10 Father 3/6OS (BM 11 and PB CD34+ Mother cells) 6 3/6 M (BM CD34+ cells) F 8.4 4 12 4.2 Father 6/6extraction, (full MUD marrow) 6/6 (full marrow) PCR 5 ampliﬁcation 8 and sequencing of the RAG1

Bone Marrow Transplantation Stem cell transplantation in Omenn syndrome E Mazzolari et al 110 and RAG2 gene coding sequences were performed as described.7

Results

Clinical and laboratory features at presentation 1 49 Low 1 83Absent 1 70 Absent 11 n.d. 43Normal Low 1 25 Low o o o o o All patients included in this study had typical clinical o features of OS (Table 1). The median age at onset of symptoms was 30 days (range: 1–70 days). Generalized

erythrodermia was present in all patients. Other common (%) (%) (%) to PHA signs and symptoms included diarrhea and failure to thrive (90.9% of the patients), a history of recurrent infections 13 157 o (81.8%), lymphadenopathy (72.7%), and liver and/or o spleen enlargement (54.5%). Interstitial pneumonia was present in three patients, and was due to CMV in two, and to respiratory syncytial virus in one. Patient OS9 had been vaccinated with Bacillus Calmette-Guerin (BCG) at birth, and showed signs of BCGosis. RO (%) RA (%) All of the patients were of Italian origin, with the

exception of patients OS9 and OS11 who were Slavians; (%) parental consanguinity was not reported. Laboratory features at presentation are shown in Table 3. 1 16 2 12 33 15 24 Low 18180 11 91 15.3ND 20.7 ND n.d. ND n.d. 21.4 n.d. 22 10 12 40 Absent 1107 9 o o o o o White blood cell count (WBC) ranged from 3.1 to 45.1 Â 10 /l, o with a mean value of 16.6 Â 109/l. With the exception of patient OS9, eosinophilia was present in all patients. The absolute lymphocyte count was variable, as typically

observed in OS. Six of the patients (OS2, OS3, OS5, OS8, RO (%) RA (%) OS9, OS11) were lymphopenic. Furthermore, significant abnormalities in the distribution of lymphocyte subsets were documented, including a skewed distribution of CD4 þ and CD8 þ T cells, an excess of activated/memory (CD45R0 þ ) vs naı¨ ve (CD45RA þ ) T cells, and a low/ undetectable number of circulating B lymphocytes. In vitro proliferative response to PHA was abrogated in five patients, and reduced in other five patients. Only patient OS10 had a normal in vitro response to PHA. Three l (%) (%)

patients (OS1, OS5, and OS7) had a normal to increased m proportion of circulating B lymphocytes, and two of them also had normal to increased serum IgM and IgA levels (Table 3). Low IgG serum levels were present in patient l

OS1, and had been documented at other referring centers in m patients OS3and OS8, who were receiving substitution therapy with intravenous immunoglobulins. Five additional patients had low IgA and/or IgM serum levels. Increased 3,000 20,295 6,314 83 43 42 1 29 26 3 7 3,000 1,015 784 20 17 15 IgE serum levels were documented in three patients. 5,000 7,507 1,916 88.1 67.8 65.1 4 4 4 55 163663 86 64.2 42.2 40 6.7 ND 4,000 720 48 26 ND ND 8 ND n.d. 8 37 n.d. Low 6.7 ND 5,494 14,391 93 9 9 Mutation analysis at the RAG1 and RAG2 loci 6.7 50 604 1,367 16 11 9 23.9 13.8 584 1,176 65 64 64 0 5 ND n.d. 15 1 34 Absent o o o o o Analysis of the coding sequence demonstrated mutations of o the RAG1 gene in seven patients, and of the RAG2 gene in

one patient (mutation data are available as Supplementary 5 o information). No mutations of the RAG genes were found phytohemagglutinin. in three patients (OS1, OS7, and OS8). ¼ Hematological and immunological features at presentation in 11 patients with OS HSCT: engraftment, hematopoietic recovery, and GvHD (mg/dl) (mg/dl) (mg/dl) (IU/ml) proliferative response to PHA was considered low if lower than 30% of normal control, and absent if lower than 3% of normal control.

As shown in Table 2, HSCT was performed at a mean age not done; PHA

of 8.4 months (range: 4–18 months). The mean age at ¼ OS 1OS 2OS 3OS 4 100 248 523* 776 21 5 20 74 42 4 54 37 73 760 660 1,960 1,480 35 84 30 31 n.d. 30 n.d. 1 25 51 n.d. 44 n.d. 41 4 10 2 n.d. Absent OS 5 848 102 78 In vitro Table 3 Patient IgG IgM IgAOS 6 IgE 310 Lymph./ Eosinophils/ CD3 8 *Indicates IgG levels whileND on intravenous immunoglobulin substitution CD4 treatment. CD4CD45 CD8 CD8CD45 CD16 CD19 DR Response OS 7 257 102 58 11 2,692 4,420 6358 57 HSCT in the three patients undergoing MUD HSCT was OS 8OS 9OS 10OS 11 523* 508 445 350 32.6 5 10

Bone Marrow Transplantation Stem cell transplantation in Omenn syndrome E Mazzolari et al 111 9.3months. Three patients required multiple attempts of a 10000 HSCT. In particular, patient OS8 showed rejection of L) maternal stem cells shortly after developing acute GvHD; she received a second transplant with paternal CD34 þ cells and because of slow engraftment received a third 1000 transplantation with paternal CD34 þ cells. Graft failure was documented in patient OS9 after the ﬁrst attempt with maternal bone marrow cells; for this reason, this patient received a second HSCT. Finally, patient OS11 also 100 lymphocytes (cells/ received two attempts of HSCT because of slow engraft- + ment, followed by rejection, after the ﬁrst procedure. No CD3 toxicity related to the conditioning regimen was recorded in 10 any of the patients. 3 6 12 24 36 48 72 Taking into account only the successful procedures, the Time (months) median time needed to achieve neutrophil recovery was 16.6 (range: 11–27) days. Withdrawal of red blood cell b 1000 transfusions was possible after a median time of 17.1 (range: 8–31) days. Platelet engraftment was achieved at a median of 17.7 (range: 11–30) days. )

Of the 11 patients, 10 showed evidence for engraftment 3 of donor-derived cells. Patient OS1 died at day þ 63due to − 100 graft failure and interstitial pneumonia. Of the 10 patients who were successfully engrafted, three patients (OS2, OS4, and OS10) showed no signs of acute GvHD (aGvHD), whereas seven patients developed aGvHD that was mostly 10 conﬁned to the skin. As shown in Table 2, aGvHD greater to PHA (cpm x 10 than grade 2 was observed in two patients (OS8 and OS11), Proliferative response and was the cause of death in one of them (OS11), who had severe skin, gut, and liver involvement. No cases of chronic GvHD were documented among the 1 nine patients who survived beyond 100 days (Tables 2 and 3). 36 12 24 36 48 72 Time (months)

Immune reconstitution c 10000 Nine of the 11 patients survived more than 2 years after HSCT, and were therefore analyzed for the quality of L) immune reconstitution. The number of circulating CD3 þ lymphocytes increased rapidly after HSCT, and normalized 1000 in most patients within 1 year (Figure 1a). The kinetics of T-cell reconstitution were slower in most of the infants who were treated by haploidentical HSCT, since they presented a low T-cell count at 3months after HSCT. Distribution of CD4 þ and CD8 þ T lymphocytes, that was highly skewed 100 lymphocytes (cells/ at diagnosis, was also already normal at 3months after + HSCT (data not shown). Furthermore, with the exception of patient OS6, all long-term survivors were able to mount CD19 a vigorous T-lymphocyte proliferation in vitro in response 10 to PHA already at 3months of age (Figure 1b). In all cases, the quality of T-cell reconstitution was sustained with time. 36 12 24 36 48 72 Evaluation of thymic output, as measured by TRECs Time (months) analysis at the last examination (mean interval from HSCT: Figure 1 Immunological reconstitution following HSCT for OS. Number 69.1 months; median: 72.5, range: 9–119 months), was of circulating CD3 þ lymphocytes (panel a), in vitro proliferative response normal or nearly normal in all nine long-term survivors to phytohemagglutinin (PHA) (panel b), and number of circulating CD19 þ lymphocytes (panel c) following HSCT for OS. In each of these (see: Supplementary data). These data were confirmed by panels, filled squares, filled triangles, empty triangles, and empty circles þ þ the predominance of CD45RA cells within both CD4 identify patients treated by HSCT from HLA-identical family donors, and CD8 þ lymphocytes (data not shown). Moreover, MUD, phenotypically-identical related donors, and haploidentical family analysis of T-cell diversity showed a fully polyclonal T-cell donors, respectively. repertoire in four patients; four additional patients had a polyclonal repertoire with few dominant clones, and only While seven of the nine survivors had low or undetect- one patient (OS3) displayed a restricted T-cell repertoire able circulating B lymphocytes at diagnosis, all of them (data not shown). showed reconstitution of the B-lymphocyte compartment

Bone Marrow Transplantation Stem cell transplantation in Omenn syndrome E Mazzolari et al 112 after HSCT (Figure 1c). Withdrawal of intravenous manifestations (hemolytic anemia, hypothyroidism, and immunoglobulin substitution was possible in all patients, myasthenia gravis) at 7, 15, and 24 months after at a time interval from HSCT that ranged from 14 days to 4 haploidentical HSCT. Interestingly, this patient belongs years. Antibody response to vaccine antigens (tetanus to a family with predisposition to autoimmunity, as the toxoid and hepatitis B antigen) was tested after suspension patient’s mother has exophthalmos, moderately raised TSH of IVIG substitution therapy at least 1 year after HSCT, levels, and positive antithyroid antibodies. Furthermore, and was shown to be positive in all nine survivors. Seven her sister displays auto-immune thyroiditis and insulin- patients also received conjugated pneumococcal vaccine, dependent diabetes mellitus, while a ﬁrst cousin has insulin- and all of them produced speciﬁc antibodies. dependent diabetes mellitus. Analysis of lymphoid chimerism in the nine survivors Of the nine survivors, seven are in excellent clinical showed full donor chimerism in six and mixed chimerism in conditions at home. Two patients have complications that three patients (OS4, OS7, and OS10), with a proportion of are unrelated to HSCT. In particular, patient OS4 suffers donor-derived T cells ranging from 50% (in patient OS7) to from familial diplegia. Patient OS10 has reduced vision due 490% (in patients OS4 and OS10), and a proportion of to bilateral optical neuritis, an adverse effect of ethambutol donor B lymphocytes 480% in all three. This degree of that was required to treat disseminated BCG infection. mixed chimerism was stable over time.

Outcome of HSCT Discussion Out of the 11 patients treated with HSCT, nine are alive at Since the first reports on successful allogeneic bone marrow a median time of 94 months since HSCT (range: 30–146 transplantation for otherwise lethal immunodeficiencies in months) (Figure 2). Seven of these nine patients are more 1968,16,17, this procedure has been applied successfully for than 6 years after HSCT. more than 30 years. While overall results of HSCT in SCID Acute GvHD developed in seven out of 10 patients in have shown significant improvement in recent years, less whom engraftment of donor-derived lymphocytes was satisfactory data have been reported on long-term survival demonstrated. Patient OS1 died at day þ 63due to graft after HSCT in OS. A recent review analyzed the outcome of failure and interstitial pneumonia without evidence of 68 OS patients reported in the literature between 1965 and engraftment. Among the seven patients who developed 1999:2 the overall mortality of the 28 transplanted OS aGvHD, this was mostly confined to the skin, and usually patients was shown to be 46%. In particular, both pre- and mild (Table 2). However, severe aGvHD with gut and liver post-transplant complications appear to be significantly involvement was the cause of death in patient OS11. None more common and severe in patients with OS as compared of the nine patients who survived for at least 100 days after to other forms of SCID, contributing significantly to post- HSCT developed chronic GvHD. transplant mortality.13,18 In this regard, recently nonmyelo- Three patients developed autoimmune manifestations ablative stem cell transplantation has been proposed in following HSCT, but all of them are currently well, without order to reduce mortality.19,20 Furthermore, inadequate any evidence of autoimmunity, and do not require any restoration of humoral immunity has been often reported, treatment. In particular, patient OS4 at 5 months after particularly after haploidentical HSCT. HSCT developed idiopathic thrombocytopenic purpura At our center, during the last 13years, we have been able that lasted for 13months, and was followed by sponta- to obtain sustained engraftment and long-term immune neous remission. Patient OS11 developed autoimmune reconstitution in nine out of 11 consecutive infants treated hepatitis at 8 months after HSCT, and hemolytic anemia by HSCT for OS. Importantly, donor–recipient HLA 1 year after transplant; in both cases, steroids were mismatch did not significantly affect the outcome. Several effective. Finally, patient OS6 developed autoimmune factors may account for the improved outcome of HSCT for OS in recent years. 100 Development of ex vivo procedures for isolation of CD34 þ stem cells has extended the potential benefit of 80 HSCT from partially-matched related donors. Further- more, co-infusion of donor PBSC (obtained after mobiliza- 60 tion with granulocyte colony-stimulating factor, 10 mg/kg for 5 days) and bone marrow stem cells has been recently 40 proposed, with the aim of increasing the cell dose and thus achieve faster engraftment after intensive conditioning Survival (%) 20 regimen.14 We have used this procedure in three of our transplants, but did not observe significant advantages. 0 Severe infections, hypovolemic shock, and multiple 0 20 40 60 80 100 120 140 160 organ failure are important causes of death early in life in Time (months) patients with OS. Immune dysregulation may also lead to significant clinical problems in OS, as in patient OS4 (who Figure 2 Event-free survival in 11 children treated by HSCT for OS. For patients who received multiple transplants, survival is calculated from the presented with nephritic–nephrotic syndrome and acute day of the last transplantation. Bars identify duration of follow-up in the renal failure without infection), OS3and OS5 (both of nine survivors. which showed signs of polyarthritis), and in patient OS8

Bone Marrow Transplantation Stem cell transplantation in Omenn syndrome E Mazzolari et al 113 (who developed nonfebrile seizures, that were associated (RBNE0189JJ_003), European Union project EURO-POL- with signs of systemic inflammation without obvious ICY-PID (grant 006411), and MIUR-COFIN 2004 (to LDN). infection). Adequate supportive therapy and protective environ- ment are essential to improve the clinical status of OS References patients prior to HSCT. Isolation in laminar flow units 1 Omenn G. Familial reticuloendotheliosis with eosinophilia. since referral to our center, immunoglobulin substitution N Engl J Med 1965; 273: 427–432. therapy, vigorous treatment with antibiotics, parenteral 2 Aleman K, Noordzij JG, de Groot R et al. Reviewing Omenn nutrition and albumin infusion (when needed), and topical syndrome. Eur J Pediatr 2001; 160: 718–725. or systemic immune suppression with steroids and/or 3Arbiser JL. Genetic immunodeficiencies: cutaneous manifesta- cyclosporin A (to control immune dysreactivity) have been tions and recent progress. J Am Acad Dermatol 1995; 33: consistently used in all of our patients. This has allowed 82–89. achievement of better and stable clinical conditions, and 4 Glover MT, Atherton DJ, Levinsky RJ. Syndrome of has often allowed search of MUD panels. The importance erythroderma, failure to thrive, and diarrhea in infancy: a of aggressive therapeutic and supportive measures in manifestation of immunodeficiency. Pediatrics 1988; 81: 66–72. et al. infants with OS is also highlighted by the observation that 5 O’Driscoll M, Cerosaletti KM, Girard PM DNA ligase IV mutations identified in patients exhibiting developmental during the same period (1991–2002) only one other infant delay and immunodeficiency. Mol Cells 2001; 8: 1175–1185. with OS was referred to our center at 3months of age in 6 Markert ML, Alexieff MJ, Li J et al. Complete DiGeorge very severe clinical conditions, and died 2 weeks after syndrome: development of rash, lymphadenopathy, and admission with respiratory distress and myocarditis due to oligoclonal T cells in 5 cases. J Allergy Clin Immunol 2004; adenovirus. 113: 734–741. Finally, age at HSCT is another important factor that 7 Villa A, Sobacchi C, Notarangelo LD et al. V(D)J recombina- may affect outcome. Myers et al.21 have recently shown tion defects in lymphocytes due to RAG mutations: severe that early HSCT in the neonatal period leads to superior immunodeficiency with a spectrum of clinical presentations. thymic output and thus improved survival in infants with Blood 2001; 97: 81–88. et al. SCID. While early recognition is essential to maximize the 8 Rieux-Laucat F, Bahadoran P, Brousse N Highly restricted human T cell repertoire in peripheral blood and chances of successful outcome, we have been able to obtain tissue-infiltrating lymphocytes in Omenn’s syndrome. J Clin prolonged survival and sustained immune reconstitution Invest 1998; 102: 312–321. even in patients transplanted as late as at 18 months of life. 9 Brugnoni D, Airo P, Facchetti F et al. In vitro cell death of This observation indicates that clinical status at the time of activated lymphocytes in Omenn’s syndrome. Eur J Immunol HSCT is more important than age among factors that may 1997; 27: 2765–2773. affect outcome of HSCT for OS. 10 Villa A, Santagata S, Bozzi F et al. Partial V(D)J recombina- Prior to this study, other reports of HSCT in series of tion activity leads to Omenn syndrome. Cell 1998; 93: 885–896. patients with OS had not evaluated the possible role of the 11 Cavadini P, Vermi W, Facchetti F et al. AIRE deficiency in genetic defect. Although in most cases OS is due to thymus of 2 patients with Omenn syndrome. J Clin Invest 2005; 115: 728–732. mutations of either RAG1 or RAG2 genes,7,10,22 it is now 12 Gomez L, Le Deist F, Blanche S et al. 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