Abstract
Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency and variable degrees of neutropenia. SDS patients are at risk of developing myelodysplasia, aplastic anemia, and leukemic transformation. The role and timing of allogeneic hematopoietic stem cell transplantation (HSCT) in SDS remain controversial. We report three SDS patients with severe aplasia transplanted using unrelated umbilical cord blood (UCB). Patients received melphalan (180 mg/m 2 ), etoposide (1200 mg/m 2 ), antithymocyte globulin (90 mg/kg), and total lymphoid irradiation (500 cGy); graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and prednisone. Myeloid engraftment occurred promptly with absolute neutrophil count 4500 cells/mm 3 on day 1575 and all patients displayed 100% donor chimerism by 2 months post transplant. The major complication of transplant was GVHD, with all patients developing grade II or III acute GVHD, one progressing to chronic extensive GVHD. Patients are alive 309, 623, and 2029 days post transplant. Factors important in HSCT outcome for SDS may include transplantation at a young age, avoidance of cyclophosphamide, and adequate GVHD prophylaxis. Importantly, these cases also suggest that unrelated UCB, in the absence of a matched family member, is an excellent alternative stem cell source for SDS patients undergoing HSCT. Bone Marrow Transplantation (2005) 36, 855-861.

poietic stem cell transplant; umbilical cord blood; bone marrow failure Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive bone marrow failure disorder characterized by pancreatic insufficiency and neutropenia. 1 Patients with SDS also exhibit short stature, skeletal abnormalities and defects in neutrophil chemotaxis. 2, 3 Importantly, up to 20% of patients with SDS ultimately develop bone marrow aplasia, myelodysplasia, or leukemic transformation. 4 Boocock et al 5 initially described the association of mutations in the previously unidentified ShwachmanBodian-Diamond syndrome (SBDS) gene in patients with SDS and their families. SBDS is a member of a highly conserved protein family hypothesized to play a role in RNA metabolism. More recently crystal structure of SBDS orthologues support the hypothesis that SBDS is involved in RNA metabolism. 6, 7 However, the pathobiological basis of SDS remains unclear. There are no obvious genotypephenotype correlations. 8, 9 While granulocyte cell motility is abnormal in SDS patients 3 other defects are less well defined. It is unclear whether apoptosis or aberrant stromal cell function plays a role in marrow failure in SDS. Thus, while progress in understanding the biological function of the SBDS gene has recently been made, the implications for therapy are uncertain.

Long-term survival of patients with SDS is variable and often related to the level of bone marrow dysfunction. In patients who develop aplastic anemia, survival is significantly shortened averaging 14 years. 4 Supportive therapies including transfusions, cytokine growth factors, and antibiotics are all aimed at maintaining quality of life. However, these do not alter the risk of developing severe hematological complications. 4, 10 Patients who develop malignancy or leukemic transformation respond poorly to chemotherapeutic agents and generally have a poor prognosis. 11 Allogeneic bone marrow transplantation has been reported as a curative modality for hematological disturbances in patients with SDS. 12, 13 Unfortunately allogeneic bone marrow transplantation in SDS has been poorly tolerated due to excessive organ toxicities (ie cardiac toxicity). 13 Owing to the rarity of this syndrome and the lack of consensus regarding transplant indications, there are no standardized transplant regimens. More recently, hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) as a source of donor cells has been used successfully to treat children with malignant diseases, marrow failure syndromes, immunodeficiencies as well as inborn errors of metabolism.

14,15 UCB transplants are now being performed in adults with a variety of hematologic conditions, making cord blood a viable alternative source of hematopoietic stem cells for many conditions. 16, 17 Here, we report our experience using UCB as a source of donor cells for HSCT in children with SDS, with all patients receiving a uniform myeloablative conditioning regimen. The major complications of transplant were infections and graft versus host disease (GVHD), and all patients remain alive with excellent performance scales.
Methods
All patients were enrolled on an IRB-approved research protocol at the University of Iowa Hospitals & Clinics and a legally authorized representative gave written informed consent.
Methods
Case reports UPN #1. Patient 1 presented at 3 months of age with fever, diarrhea, failure to thrive, and severe neutropenia. Diagnostic testing revealed undetectable serum pancreatic amylase and lipase suggesting pancreatic insufficiency. Owing to persistent neutropenia, a bone marrow aspiration was performed, revealing a normocellular marrow with decreased granulopoiesis, decreased megakaryocytes, and normal cytogenetics. The patient's diarrhea resolved following pancreatic enzyme replacement therapy. Despite therapy with high-dose granulocyte colony-stimulating factor (G-CSF, 5-50 mg/kg) the patient continued to have significant neutropenia (absolute neutrophil counts 100-500/mm 3 ) with recurrent life-threatening infections, including intestinal candidiasis, staphylococcal soft tissue abscesses, and necrotizing enterocolitis. The patient was formally diagnosed with SDS at 7 months of age based upon the clinical and laboratory findings of pancreatic insufficiency, short stature, bony abnormalities, persistent neutropenia, and abnormal neutrophil chemotaxis (Table 1) . Owing to the ongoing neutropenia and recurrent life threatening infections the patient was referred for an allogeneic HSCT. At the time of transplant the patient was 15 months of age with the following complete blood count (CBC): hemoglobin 9.8 g/dl, MCV 89, platelets 109 000/mm 3 , WBC 10 600/mm 3 , ANC 212/mm 3 . The patient lacked an HLA identical family member and bone marrow registries failed to reveal any 6/6 HLA matched unrelated donors. Umbilical cord blood from an unrelated partially matched donor was chosen as the stem cell source. As shown in Table 2 , the cord blood unit had an excellent CD34 þ stem cell dose and was matched at 5/6 HLA antigens with a single mismatch at the A loci. UPN #2. Patient 2 had a protracted history of pancreatic insufficiency as evidenced by failure to thrive, chronic diarrhea, and near absent serum amylase and lipase levels. She also had intermittent neutropenia (ANC o200/mm 3 ) during the first year of life. Based upon these clinical findings she was given the diagnosis of SDS at 12 months of age, which was later confirmed following identification of a mutation in the SBDS gene. The neutropenia persisted with ANCs ranging from 50 to 500/mm 3 . She had multiple infections throughout the first 7 years of life. Annual bone marrow aspirate examinations showed progressive hypoplasia (15-30% cellularity), without evidence of myelodysplasia or cytogenetic abnormalities. The patient also received immunosuppressive therapy (ATG) at 7 years of age without improvement. She was ultimately referred for allogeneic HSCT at 8 years of age due to worsening thrombocytopenia and transfusion dependency. There was no organ dysfunction or cardiac abnormalities noted on the pre transplant workup. There was no HLA matched sibling or 6/6 matched unrelated donor available. However, a 5/6 HLA-matched (A mismatch) umbilical cord blood unit was identified and used as the donor source for HSCT ( Table 2) . Table 1 Demographic data on SDS patients ) and anemia (hemoglobin 8 g/dl) necessitating several transfusions (Table 1) . Owing to pancytopenia, a bone marrow examination was performed, demonstrating hypocellularity with paucity of megakaryocytes and normal cytogenetics. The patient's neutropenia responded transiently to G-CSF and the diarrhea resolved following pancreatic enzyme replacement therapy. The diagnosis of SDS was suspected based upon clinical findings and confirmed by molecular testing, revealing two common point mutations in both SBDS alleles. Although an HLA matched sibling was available, this sibling shared many of the phenotypic features of SDS and was excluded from marrow donation. A 6/6 HLA-matched cord unit was identified within the National Marrow Donor Program registry, and this was used as the donor source for HSCT (Table 2) .
Preparative regimen
A cardiac sparing myeloablative conditioning regimen was used for transplantation, as previously described. 13 Briefly, all patients received melphalan (60 mg/m 2 /dose daily for 3 days), etoposide (400 mg/m 2 /dose daily for 3 days), and total lymphoid irradiation (500 cGy in two fractions). Patients also received anti-thymocyte globulin (30 mg/kg/ day) for 3 days beginning at day À3 as shown in Figure 1 .
GVHD prophylaxis
In addition to the anti-thymocyte globulin, acute GVHD prophylaxis consisted of cyclosporine. Cyclosporine was given i.v. beginning on day À1, and then converted to the oral route upon patient tolerance. Levels were checked every 3-7 days and serum levels were maintained between 200 and 300 mg/ml. Patients received prednisone for acute GVHD flares and the steroid dose was adjusted for clinical outcomes. Acute and chronic GVHD were graded per the Seattle criteria. 18 
Supportive care
All patients were admitted and hospitalized within the University of Iowa Pediatric Blood and Marrow Transplant Unit. Patients were housed in single rooms ventilated with high efficiency air filtration systems. Granulocyte colony stimulating factor (G-CSF) was given to all patients starting at day þ 5 until neutrophil recovery. Intravenous immunoglobulin (250 mg/kg) was administered to each patient weekly for 1 month then changed to 500 mg/kg every 3 weeks until patients were weaned of immunosuppressive medications. All patients were started on broad-spectrum prophylactic antibiotics once their ANC was o500/mm 3 . Patients also received voriconazole or fluconazole for antifungal prophylaxis and trimethoprim/ sulfamethoxazole for Pneumocystis carinii prophylaxis. Additionally, patients that were sero-positive for herpes simplex virus or cytomegalovirus received prophylactic acyclovir.
Hematological reconstitution
Neutrophil engraftment was defined as the first of three consecutive days where the ANC was 4500/mm 3 . Platelet recovery was defined as the first day to reach a platelet count 420 000/mm 3 in the absence of platelet transfusions for 7 days. All patients underwent a bone marrow aspirate 3-4 weeks post HSCT for analysis of engraftment and chimerism.
Results

UPN #1
Myeloid engraftment occurred on day þ 18 with platelet reconstitution achieved on day þ 46 ( Table 3 ). The patient developed grade III mucositis secondary to the preparative regimen and was on i.v. narcotics for 14 days post HSCT for pain control. The mucositis and diarrhea resolved upon myeloid engraftment. Transient tachypnea and tachycardia developed on day þ 5 and was presumed to be an early engraftment syndrome. An echocardiogram and the chest X-ray performed at that time were normal, and the tachycardia and tachypnea resolved without further (Table 3 ). The patient was discharged 33 days post transplant and did well as an outpatient without any subsequent hospitalizations, and was weaned off prednisone and cyclosporin by 6 months post HSCT. She is now 42000 days post transplant and is doing well without evidence of chronic GVHD. The patient has required no further transfusions and CBCs are entirely normal. Immune reconstitution was demonstrated by 1 year post HSCT, and she has protective titers to all immunizations. Cardiac evaluation reveals a normal echocardiogram without any evidence of cardiac dysfunction. She remains quite short (o5th percentile) and is now weaning off pancreatic enzyme supplementation. Her Lansky play scale is 100.
UPN #2
Myeloid reconstitution occurred on day þ 17 and platelet recovery on day þ 58. Bone marrow aspirate on day þ 22 revealed trilineage engraftment with 20% cellularity, and fluorescence in situ hybridization (FISH) revealed 100% donor chimerism (male donor, female recipient). The patient developed grade IV mucositis with significant oral pain, necessitating IV narcotics for several weeks post transplant (Table 3) . Of note, she developed a transient increase in her total bilirubin, which reached 6.3 mg/dl on day þ 13. She also had mild abdominal pain, ascites, and hepatomegaly consistent with VOD. She developed significant anasarca, leaky capillary syndrome, and abdominal distention that ultimately resulted in her being transferred to the Pediatric Intensive Care Unit where she was mechanically ventilated for 10 days and stayed a total of 12 days. During that time she developed significant renal insufficiency with a BUN of 141 mg/dl and a serum creatinine of 1.4 mg/dl on day þ 16, although never became anuric and did not require dialysis. She subsequently underwent paracentesis, and the VOD symptoms slowly resolved with diuretics and sodium restriction therapy. The total bilirubin again rose abruptly on day þ 46 (14.8 mg/dl) and acute cholecystitis was diagnosed, with normalization of the bilirubin by 70 days post transplant. From an infection standpoint, she was treated with several prolonged courses of antibiotics for Pseudomonas aeruginosa tracheitis and bacteremia, Staphylococcus epidermidis bacteremia and Clostridium difficile enterocolitis. The patient also had significant symptoms of acute GVHD, consisting of voluminous stools and abdominal cramping. On day þ 22 she underwent a rectal biopsy that was diagnostic of acute GVHD. Despite therapy with cyclosporine and IV methylprednisolone her symptoms persisted, and on day þ 38 she was treated with infliximab (Centocor, USA) 5 mg/kg. She had a gradual response, and upon discharge 65 days post HSCT was on oral cyclosporine, prednisone, and mycophenolate mofetil. She ultimately developed signs of chronic GVHD 5 months post transplant, involving both the gut and skin, and was subsequently treated with tacrolimus, prednisone, and hydroxychloroquine. She has had significant improvement in her GI symptoms although continues to receive antacids and gastric feeds for optimal caloric intake. She remains on pancreatic enzyme supplementation, and is now transfusion independent with a Lansky play scale of 90.
UPN #3
Myeloid reconstitution occurred on day þ 14 post transplant and platelet independence on day þ 69 ( Table 3 ). The patient developed grade III mucositis requiring IV narcotics for 2 weeks post transplant. She also developed mild anorexia and was supplemented with central venous nutrition for approximately 1 month. She had several fevers requiring broad-spectrum antibiotics, however, blood cultures were never positive, and the only infectious complication was C. difficile enterocolitis that promptly resolved with oral antibiotics. She was discharged from the hospital 24 days post HSCT. A bone marrow examination 29 days post transplant revealed trilineage engraftment and FISH analysis revealed 100% donor chimerism. The patient was readmitted to the hospital 1 week later with symptoms of diarrhea and a skin rash consistent with stage II acute GVHD. A colon biopsy demonstrated apoptotic cellular changes of acute GVHD, with overall grade II acute GVHD. She responded well to IV methylprednisolone, and was slowly tapered off immunosuppression by 300 days post HSCT. She currently is transfusion independent; taking full oral feeds, and has a Lansky play scale of 100.
Discussion
SDS was originally described in 1964 as a syndrome involving bone marrow dysfunction, multiple skeletal abnormalities, as well as pancreatic insufficiency. 19 Since the original description it has become clear that a wide variety of hematological abnormalities occur in SDS. 4 Some of these abnormalities include pancytopenia, bone marrow aplasia, and myelodysplasia, and there is an appreciable risk of leukemic transformation, often to acute myelogenous leukemia (AML). 4, 20 In the most comprehensive study to date, Ginzberg et al 2 found neutropenia in 98% of patients with SDS. Anemia and thrombocytopenia occur in 42% and 34% of patients, respectively. Importantly, pancytopenia occurred in approximately 20% of the 88 patients reported in this study. Several of these patients also developed clonal cytogenetic abnormalities, primarily involving chromosome 7. These clonal cytogenetic abnormalities are sometimes associated with a myelodysplastic phase. 21 Smith et al 4 noted that 24% of their SDS patients developed leukemia following myelodysplasia. Also, in a recent review of congenital neutropenia patients, the cumulative risk of developing AML/MDS in SDS was 36% at 30 years. 22 Nonetheless there are several reports in the literature that suggest that monosomy 7 does not always progress to myelodysplasia or AML in SDS. 23, 24 However, those that ultimately develop
Discussion
Cord blood transplant in SDS R Vibhakar et al AML with abnormalities of chromosome 7 are generally poorly responsive to chemotherapy. 21 To date, allogeneic bone marrow transplantation has been the only definitive treatment for severe bone marrow dysfunction and clonal disorders in SDS patients. 12, 13 However, the overall experience has been limited with between 20 and 30 cases being described in the literature (Table 4) . 12, 13, 25, 26 As reviewed by Fleitz et al 13 and shown in Table 4 , indications for transplantation in some of these patients included marrow aplasia, myelodysplastic syndrome, and AML. With the exception of one patient, all received bone marrow as a source of hematopoietic stem cells, and most were from unrelated donors. 13 Importantly, long-term survival data have not been available for the majority of these cases, and at the time of reporting less than 50% of patients were noted to be alive. Many patients died in the early post transplant phase, and causes of death included congestive heart failure, infection, multiorgan failure, and pulmonary hemorrhage (Table 4) . 13 In our series, all three patients were transplanted using umbilical cord blood as the hematopoietic stem cell source. The preparative regimen was standardized and designed to decrease cardiac toxicity and of note, no patient developed signs of myocardial dysfunction. All patients engrafted promptly and displayed 100% donor chimerism. Peritransplant complications consisted primarily of acute GVHD and bacterial infections, and one patient developed significant VOD. All patients are now alive and well, although one patient continues to exhibit chronic GVHD symptoms requiring ongoing immunosuppression.
Discussion
Based upon the available literature and our own data, it is quite evident that SDS patients with hematopoietic abnormalities can benefit from allogeneic HSCT. Nonetheless, due to a still poorly understood predisposition to myeloablative-related morbidity, several factors must be considered in the decision to transplant patients with SDS. Regarding HSCT indications, the existence of leukemia would appear to mandate transplant assuming the patient has good organ function. The development of severe aplastic anemia and transfusion dependency (or imminent transfusion dependency) is also a transplant indication, as there is no role for immunotherapy (ie anti-thymocyte globulin) in this disease. However, intermittent neutropenia and mild to moderate anemia/thrombocytopenia may not require transplant, and it is not possible to accurately predict when and if a patient will progress to an aplastic state. In addition, myelodysplasia or marrow hypocellularity are not absolute transplant indications, as these findings may persist for several years. Certain cytogenetic abnormalities, specifically isochromosome 7q, are commonly seen in SDS and rarely progress to malignant transformation. A recent report of nine SDS patients with isochromosome 7q found only one of three survivors in those undergoing HSCT, while six nontransplanted patients were alive without progressive aplasia or leukemia. 27 With regards to the optimal myeloablative preparative regimen in SDS, consideration should be given to avoid potentially cardio-toxic agents (ie cyclophosphamide) due to several reports of SDS patients developing cardiac insufficiency following HSCT. 13, 28 However as summarized in Table 4 several patients have been successfully transplanted with cyclophosphamide containing regimens. 29, 30 Although our series was limited to three patients, several other SDS patients have also been transplanted with the preparative regimen of Melphalan, VP16, TLI and ATG (personal communications) and no cardiac complications have been reported. Careful pre-transplant evaluation is important in planning the preparative regimen as some SDS patients could have pre-existing organ dysfunction (hepatic, cardiac, renal). Furthermore some SDS patients have transaminitis of unknown etiology that may be important for risk of VOD post transplant.
Discussion
The issue of age with regard to timing of transplant remains controversial. Faber et al 29 suggested that transplantation at an earlier age might result in favorable outcomes prior to the development of leukemic transformation or organ dysfunction that may impact tolerance of the myeloablative preparative regimen.
Discussion
Finally, the source of hematopoietic stem cells is another important factor in transplant. While the use of a matched related nonSDS affected donor is optimal, they often do not exist, making unrelated transplants a necessity. There will remain some debate as to whether there is an advantage to using cord blood versus marrow for the source of hematopoietic stem cells in this condition. However, there has been a trend towards fewer transplant related morbidity with cord blood transplants, although GVHD does not appear to be markedly different between these two groups. 31 In fact, GVHD was the major complication in our patients, and further strategies need to be implemented to decrease the incidence and severity of this condition.
Discussion
In conclusion, allogeneic HSCT can be successfully employed in SDS patients that develop aplastic anemia or have severe cytopenia-mediated complications. In the absence of a matched sibling donor, strong consideration should be given to the use of umbilical cord blood as the stem cell source. Ultimately, controlled prospective studies are needed to validate the optimal conditioning regimen and timing of HSCT in SDS.

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