Brigitte Strahm

X-linked lymphoproliferative disease due to SAP/SH2D1A deficiency: a multicenter study on the manifestations, management and outcome of the disease

X-linked lymphoproliferative disease (XLP1) is a rare immunodeficiency characterized by severe immune dysregulation and caused by mutations in the SH2D1A/SAP gene. Clinical manifestations are varied and include hemophagocytic lymphohistiocytosis (HLH), lymphoma and dysgammaglobulinemia, often triggered by Epstein-Barr virus infection. Historical data published before improved treatment regimens shows very poor outcome. We describe a large cohort of 91 genetically defined XLP1 patients collected from centers worldwide and report characteristics and outcome data for 43 patients receiving hematopoietic stem cell transplant (HSCT) and 48 untransplanted patients. The advent of better treatment strategies for HLH and malignancy has greatly reduced mortality for these patients, but HLH still remains the most severe feature of XLP1. Survival after allogeneic HSCT is 81.4% with good immune reconstitution in the large majority of patients and little evidence of posttransplant lymphoproliferative disease. However, survival falls to 50% in patients with HLH as a feature of disease. Untransplanted patients have an overall survival of 62.5% with the majority on immunoglobulin replacement therapy, but the outcome for those untransplanted after HLH is extremely poor (18.8%). HSCT should be undertaken in all patients with HLH, because outcome without transplant is extremely poor. The outcome of HSCT for other manifestations of XLP1 is very good, and if HSCT is not undertaken immediately, patients must be monitored closely for evidence of disease progression.

Antiviral and Regulatory T Cell Immunity in a Patient with Stromal Interaction Molecule 1 Deficiency

Stromal interaction molecule 1 (STIM1) deficiency is a rare genetic disorder of store-operated calcium entry, associated with a complex syndrome including immunodeficiency and immune dysregulation. The link from the molecular defect to these clinical manifestations is incompletely understood. We report two patients with a homozygous R429C point mutation in STIM1 completely abolishing store-operated calcium entry in T cells. Immunological analysis of one patient revealed that despite the expected defect of T cell proliferation and cytokine production in vitro, significant antiviral T cell populations were generated in vivo. These T cells proliferated in response to viral Ags and showed normal antiviral cytotoxicity. However, antiviral immunity was insufficient to prevent chronic CMV and EBV infections with a possible contribution of impaired NK cell function and a lack of NKT cells. Furthermore, autoimmune cytopenia, eczema, and intermittent diarrhea suggested impaired immune regulation. FOXP3-positive regulatory T (Treg) cells were present but showed an abnormal phenotype. The suppressive function of STIM1-deficient Treg cells in vitro, however, was normal. Given these partial defects in cytotoxic and Treg cell function, impairment of other immune cell populations probably contributes more to the pathogenesis of immunodeficiency and autoimmunity in STIM1 deficiency than previously appreciated.

X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis.

X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.

Prevalence, clinical characteristics, and prognosis of GATA2-related myelodysplastic syndromes in children and adolescents

Germline GATA2 mutations cause cellular deficiencies with high propensity for myeloid disease. We investigated 426 children and adolescents with primary myelodysplastic syndrome (MDS) and 82 cases with secondary MDS enrolled in 2 consecutive prospective studies of the European Working Group of MDS in Childhood (EWOG-MDS) conducted in Germany over a period of 15 years. Germline GATA2 mutations accounted for 15% of advanced and 7% of all primary MDS cases, but were absent in children with MDS secondary to therapy or acquired aplastic anemia. Mutation carriers were older at diagnosis and more likely to present with monosomy 7 and advanced disease compared with wild-type cases. For stratified analysis according to karyotype, 108 additional primary MDS patients registered with EWOG-MDS were studied. Overall, we identified 57 MDS patients with germline GATA2 mutations. GATA2 mutations were highly prevalent among patients with monosomy 7 (37%, all ages) reaching its peak in adolescence (72% of adolescents with monosomy 7). Unexpectedly, monocytosis was more frequent in GATA2-mutated patients. However, when adjusted for the selection bias from monosomy 7, mutational status had no effect on the hematologic phenotype. Finally, overall survival and outcome of hematopoietic stem cell transplantation (HSCT) were not influenced by mutational status. This study identifies GATA2 mutations as the most common germline defect predisposing to pediatric MDS with a very high prevalence in adolescents with monosomy 7. GATA2 mutations do not confer poor prognosis in childhood MDS. However, the high risk for progression to advanced disease must guide decision-making toward timely HSCT.

Atypical familial hemophagocytic lymphohistiocytosis due to mutations in UNC13D and STXBP2 overlaps with primary immunodeficiency diseases

Background Familial hemophagocytic lymphohistiocytosis is a genetic disorder of lymphocyte cytotoxicity that usually presents in the first two years of life and has a poor prognosis unless treated by hematopoietic stem cell transplantation. Atypical courses with later onset and prolonged survival have been described, but no detailed analysis of immunological parameters associated with typical versus atypical forms of familial hemophagocytic lymphohistiocytosis has been performed. Design and Methods We analyzed disease manifestations, NK-cell and T-cell cytotoxicity and degranulation, markers of T-cell activation and B-cell differentiation as well as Natural Killer T cells in 8 patients with atypical familial hemophagocytic lymphohistiocytosis due to mutations in UNC13D and STXBP2. Results All but one patient with atypical familial hemophagocytic lymphohistiocytosis carried at least one splice-site mutation in UNC13D or STXBP2. In most patients episodes of hemophagocytic lymphohistiocytosis were preceded or followed by clinical features typically associated with immunodeficiency, such as chronic active Epstein Barr virus infection, increased susceptibility to bacterial infections, granulomatous lung or liver disease, encephalitis or lymphoma. Five of 8 patients had hypogammaglobulinemia and reduced memory B cells. Most patients had a predominance of activated CD8+ T cells and low numbers of Natural Killer T cells. When compared to patients with typical familial hemophagocytic lymphohistiocytosis, NK-cell cytotoxicity and NK-cell and CTL degranulation were impaired to a similar extent. However, in patients with an atypical course NK-cell degranulation could be partially reconstituted by interleukin-2 and cytotoxic T-cell cytotoxicity in vitro was normal. Conclusions Clinical and immunological features of atypical familial hemophagocytic lymphohistiocytosis show an important overlap to primary immunodeficiency diseases (particularly common variable immunodeficiency and X-linked lymphoproliferative syndrome) and must, therefore, be considered in a variety of clinical presentations. We show that degranulation assays are helpful screening tests for the identification of such patients.

Complex karyotype newly defined: the strongest prognostic factor in advanced childhood myelodysplastic syndrome

To identify cytogenetic risk factors predicting outcome in children with advanced myelodysplastic syndrome, overall survival of 192 children prospectively enrolled in European Working Group of Myelodysplastic Syndrome in Childhood studies was evaluated with regard to karyotypic complexity. Structurally complex constitutes a new definition of complex karyotype characterized by more than or equal to 3 chromosomal aberrations, including at least one structural aberration. Five-year overall survival in patients with more than or equal to 3 clonal aberrations, which were not structurally complex, did not differ from that observed in patients with normal karyotype. Cox regression analysis revealed the presence of a monosomal and structurally complex karyotype to be strongly associated with poor prognosis (hazard ratio = 4.6, P < .01). Notably, a structurally complex karyotype without a monosomy was associated with a very short 2-year overall survival probability of only 14% (hazard ratio = 14.5; P < .01). The presence of a structurally complex karyotype was the strongest independent prognostic marker predicting poor outcome in children with advanced myelodysplastic syndrome.

Preemptive immunotherapy in childhood acute myeloid leukemia for patients showing evidence of mixed chimerism after allogeneic stem cell transplantation

Previous studies have shown that children with acute myeloid leukemia (AML) who developed mixed chimerism (MC) were at high risk for relapse after allogeneic stem-cell transplantation (allo-SCT). We investigated the feasibility of intensified preemptive immunotherapy in children receiving allo-SCT for AML. Eighty-four children were registered in our trial from May 2005 to April 2009; of these, 71 fulfilled the inclusion criteria and were treated according to the study protocol. Serial and semiquantitative analyses of posttransplantation chimerism were performed. Defined immunotherapy approaches were considered in MC patients. Continuous complete chimerism (CC) was observed in 51 of 71 patients. MC was detected in 20 patients and was followed by immunotherapy in 13. Six of 13 MC patients returned to CC without toxicity and remained in long-term remission. Overall, the probability of event-free survival (pEFS) was 66% (95% confidence interval [95% CI] = 53%-76%) for all patients and 46% (95% CI = 19%-70%) in MC patients with intervention; however, this number increased to 71% (95% CI = 26%-92%) in 7 of 13 MC patients on immunotherapy who were in remission at the time of transplantation. All MC patients without intervention relapsed. These results suggest that MC is a prognostic factor for impending relapse in childhood AML, and that preemptive immunotherapy may improve the outcome in defined high-risk patients after transplantation.

Disseminated bocavirus infection after stem cell transplant.

To the Editor: Human bocavirus (HBoV) (1) is increasingly recognized as a cause of respiratory infections worldwide. Children and infants appear to be most at risk (2–7), although HBoV’s role in immunocompromised patients remains unclear. We report on a child with disseminated HBoV infection after hematopoietic stem cell transplantation (HSCT). HBoV DNA was detected at high levels in nasopharyngeal aspirates (NPAs) and in blood and stool samples. A 4.5-year-old boy with dyskeratosis congenita was brought for treatment to our hospital due to severe persistent cytopenia. Allogenic HSCT was performed in August 2006 after conditioning with total body irradiation (200 cGy, day –8 before HSCT surgery), fludarabine (days –7 to –4), antithymocyte globulin (days –4 to –1), and cyclophosphamide (days –3 to –2). He received 7.16 × 108 nucleated bone marrow cells/kg body weight from a 9/10 human leukocyte antigen–matched unrelated donor. Graft-versus-host disease (GvHD) prophylaxis consisted of a short course of methotrexate and cyclosporin A. Neutrophil and platelet engraftment occurred on days 22 and 65 after surgery, respectively. Despite pre- and post-HSCT anti-infective prophylaxis with cotrimoxazole, colistin, acyclovir, and fluconazole, Enterobacter cloacae sepsis was diagnosed on day 2. After meropenem treatment, blood cultures remained negative. On day 12, fever reoccurred, elevated C-reactive protein values (229 mg/L) and reduced general health were noted, but no bacterial pathogen was isolated. During this period, the patient received antimicrobial drug therapy with meropenem, tobramycin, vancomycin, and amphotericin B. On day 16, his body temperature peaked to 40.6°C, and a cough and dyspnea without wheezing developed. Chest radiograph results suggested pneumonia with perihilar infiltrates. Reduced oxygen saturation (pO2 86%) was recorded transcutaneously, and oxygen supplementation (maximum 4 L/min) was started by face mask (Appendix Figure). An NPA sample investigated by multiplex PCR (results provided by W. Puppe and J. Weigl; www.pid-ari.net) was negative for adenovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza viruses 1–4, influenza viruses A and B, coronavirus, reovirus, enterovirus, Clamydia pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis, B. parapertussis, and Legionella pneumophila, but positive for rhinovirus RNA. Retrospectively, the same NPA sample was reanalyzed for HBoV DNA by real-time PCR (7) and showed a viral load of 4.6 × 107 copies/mL (Appendix Figure); specificity was confirmed by sequencing. From day 19 on, the patient’s general health improved and the chest radiograph results returned to normal. After neutrophil engraftment (day 22) and addition of erythromycin to the antimicrobial drug regimen, body temperature decreased and oxygen supplementation was discontinued. However, rhinitis, cough, and low-grade fever (<38.5°C) persisted until day 50 (Appendix Figure), and HBoV DNA was detected in NPAs on days 37 and 44 at 2.4 × 1011 and 1.3 × 1014 copies/mL, respectively (Appendix Figure). The NPA sample on day 37 was still rhinovirus positive. Concurrent with the increased HBoV load in NPAs, cytomegalovirus (CMV) reactivation was first diagnosed by PCR on day 20 and peaked (58.250 copies/mL whole blood) on day 41 despite gancyclovir therapy. Switching to foscarnet led to temporary control of CMV replication (Appendix Figure). Additionally, on day 22, acute GvHD grade I with skin manifestations developed. Treatment with steroids until day 60 led to complete resolution. HBoV infection in this patient was not restricted to the respiratory tract. Diarrheic stool samples obtained on day 21 and, after resolution of respiratory symptoms, on day 75 showed substantial HBoV DNA (2.5 × 106 and 6.0 × 105 copies/mg, respectively; Appendix Figure). Tests for rotavirus and adenovirus antigens were negative, and no bacterial pathogen was isolated. Moreover, HBoV DNA was detected at lower levels (3.7 × 103 to 7.8 × 104 copies/mL) in 4 EDTA plasma samples taken days 21–47. Subsequent plasma (days 61, 68, 75, 88, 219), NPA (day 219), and stool (day 219) samples were negative for HBoV DNA. However, the ability of HBoV to cause persistent infection, as do other members of the Parvovirinae subfamily, cannot be excluded. Future investigations are needed to address this hypothesis. Here, we report on disseminated HBoV infection in an immunocompromised patient. Whether the clinical course in this case was more severe or prolonged than it would have been for HBoV infections in non-HSCT children remains unknown due to the lack of long-term observations in immunocompetent children. The dramatic increase of HBoV load in NPAs and viral dissemination most likely resulted from progressive impairment of cellular immunity as indicated by simultaneous CMV reactivation. Moreover, the increased viral load might have also been a consequence of steroid addition to immunosuppressive therapy to control GvHD. The contribution of HBoV to respiratory disease remains ambiguous because 2 NPA samples were also rhinovirus positive. Additional studies are required to investigate the pathogenic role of HBoV in double or multiple infections. Association of HBoV with the patient’s continued diarrhea is in accordance with previous studies (8–10). Prolonged fecal shedding has important implications for isolation measures in transplantation units. More studies in immunocompromised patients are required to evaluate the spectrum of pathology caused by this emerging virus.

Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome

Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of cancer, including myeloid leukemia. We hypothesized that CpG island hypermethylation also occurs in juvenile myelomonocytic leukemia (JMML) and investigated whether it is associated with clinical, hematologic, or prognostic features. Based on quantitative measurements of DNA methylation in 127 JMML cases using mass spectrometry (MassARRAY), we identified 4 gene CpG islands with frequent hypermethylation: BMP4 (36% of patients), CALCA (54%), CDKN2B (22%), and RARB (13%). Hypermethylation was significantly associated with poor prognosis: when the methylation data were transformed into prognostic scores using a LASSO Cox regression model, the 5-year overall survival was 0.41 for patients in the top tertile of scores versus 0.72 in the lowest score tertile (P = .002). Among patients given allogeneic hematopoietic stem cell transplantation, the 5-year cumulative incidence of relapse was 0.52 in the highest versus 0.10 in the lowest score tertile (P = .007). In multivariate models, DNA methylation retained prognostic value independently of other clinical risk factors. Longitudinal analyses indicated that some cases acquired a more extensively methylated phenotype at relapse. In conclusion, our data suggest that a high-methylation phenotype characterizes an aggressive biologic variant of JMML and is an important molecular predictor of outcome.

Hematopoietic stem cell transplantation for advanced myelodysplastic syndrome in children: results of the EWOG-MDS 98 study

We report on the outcome of children with advanced primary myelodysplastic syndrome (MDS) transplanted from an HLA-matched sibling (MSD) or an unrelated donor (UD) following a preparative regimen with busulfan, cyclophosphamide and melphalan. Ninety-seven patients with refractory anemia with excess blasts (RAEB, n=53), RAEB in transformation (RAEB-T, n=29) and myelodysplasia-related acute myeloid leukemia (MDR-AML, n=15) enrolled in the European Working Group of MDS in Childhood (EWOG-MDS) 98 study and given hematopoietic stem cell transplantation (HSCT) were analyzed. Median age at HSCT was 11.1 years (range 1.4–19.0). Thirty-nine children were transplanted from an MSD, whereas 58 were given the allograft from a UD (n=57) or alternative family donor (n=1). Stem cell source was bone marrow (n=69) or peripheral blood (n=28). With a median follow-up of 3.9 years (range 0.1–10.9), the 5-year probability of overall survival is 63%, while the 5-year cumulative incidence of transplantation-related mortality (TRM) and relapse is 21% each. Age at HSCT greater than 12 years, interval between diagnosis and HSCT longer than 4 months, and occurrence of acute or extensive chronic graft-versus-host disease were associated with increased TRM. The risk of relapse increased with more advanced disease. This study indicates that HSCT following a myeloablative preparative regimen offers a high probability of survival for children with advanced MDS.