Hanne Vibeke Marquart

Risk group assignment differs for children and adults 1–45 yr with acute lymphoblastic leukemia treated by the NOPHO ALL‐2008 protocol

The prognosis of acute lymphoblastic leukemia is poorer in adults than in children. Studies have indicated that young adults benefit from pediatric treatment, although no upper age limit has been defined.

Genetic, molecular and functional analyses of complement factor I deficiency

Complete deficiency of complement inhibitor factor I (FI) results in secondary complement deficiency due to uncontrolled spontaneous alternative pathway activation leading to susceptibility to infections. Current genetic examination of two patients with near complete FI deficiency and three patients with no detectable serum FI and also close family members revealed homozygous or compound heterozygous mutations in several domains of FI. These mutations were introduced into recombinant FI and the resulting proteins were purified for functional studies, while transient transfection was used to analyze expression and secretion. The G170V mutation resulted in a protein that was not expressed, whereas the mutations Q232K, C237Y, S250L, I339M and H400L affected secretion. Furthermore, the C237Y and the S250L mutants did not degrade C4b and C3b as efficiently as the WT. The truncated Q336x mutant could be expressed, in vitro, but was not functional because it lacks the serine protease domain. Furthermore, this truncated FI was not detected in serum of the patient. Structural investigations using molecular modeling were performed to predict the potential impact the mutations have on FI structure. This is the first study that investigates, at the functional level, the consequences of molecular defects identified in patients with full FI deficiency.

Properdin deficiency associated with recurrent otitis media and pneumonia, and identification of male carrier with Klinefelter syndrome

Properdin is an initiator and stabilizer of the alternative complement activation pathway (AP). Deficiency of properdin is a rare X-linked condition characterized by increased susceptibility to infection with Neisseria meningitidis associated with a high mortality rate. We report properdin deficiency in a large Pakistani family. The index cases were found by screening for immunodeficiency due to a history of recurrent infections. This revealed absent AP activity, but normal classical and lectin pathway activity. Sequencing of the properdin gene (PFC) revealed a novel frameshift mutation. When all available relatives (n=24) were screened for the mutation, four affected males, four female carriers and a male heterozygous carrier were identified. He was subsequently diagnosed with Klinefelter syndrome. A questionnaire revealed a striking association between properdin deficiency and recurrent otitis media (P=0.0012), as well as recurrent pneumonia (P=0.0017). This study is the first to show a significant association between properdin deficiency and recurrent infections.

Common variable immune deficiency and lung transplantation

We report on a male patient with bronchiectasis secondary to common variable immune deficiency (CVID) receiving lung transplantation. The patient had been diagnosed with CVID many y prior to right-sided single lung transplantation and was receiving appropriate immunoglobulin substitution therapy. He received antithymocyte globulin induction and maintenance triple therapy with cyclosporine, azathioprine and prednisolone. The early post-operative course was complicated by the development of severe acute cellular rejection and organizing pneumonia. Despite immunoglobulin replacement and antifungal prophylaxis and treatment, Aspergillus fumigatus was repeatedly cultured from bronchoalveolar lavage fluid, 18 months after transplantation. The patient died following a protracted period of repeated hospital admissions, 46 months after transplantation. A review of the literature suggests that many CVID patients appear to have had a complicated post-operative course after lung- and other solid-organ transplantation, and highlights the need for the establishment of international registries for transplanted patients with uncommon conditions.

Quality control of flow cytometry data analysis for evaluation of minimal residual disease in bone marrow from acute leukemia patients during treatment.

Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered to be a powerful indicator of treatment response in acute lymphatic leukemia (ALL). A Nordic quality assurance program, aimed on standardization of the flow cytometry MRD analysis, has been established before implementation of MRD at cutoff level 10−3 as one of stratifying parameters in next Nordic Society of Pediatric Hematology and Oncology (NOPHO) treatment program for ALL. In 4 quality control (QC) rounds 15 laboratories determined the MRD levels in 48 follow-up samples from 12 ALL patients treated according to NOPHO 2000. Analysis procedures were standardized. For each QC round a compact disc containing data in list-mode files was sent out and results were submitted to a central laboratory. At cutoff level 10−3, which will be applied for clinical decisions, laboratories obtained a high concordance (91.6%). If cutoff level 10−4 was applied, the concordance would be lower (85.3%). The continuing standardization resulted in better concordance in QC3 and QC4 compared with QC1 and QC2. The concordance was higher in precursor B as compared with T-cell ALL. We conclude that after standardization, flow cytometry MRD detection can be reliably applied in international, multicenter treatment protocols.

Deficiency of Somatic Hypermutation of Immunoglobulin G Transcripts Is a Better Predictor of Severe Respiratory Tract Infections than Lack of Memory B Cells in Common Variable Immunodeficiency

Defects of memory B cells and of somatic hypermutation (SHM) are involved in the pathogenesis of common variable immunodeficiency (CVID). Here we report for the first time a systematic study of the relationship between memory B cell deficiency and SHM abnormalities in CVID, and relate these variables to prediagnostic infections. Isotype switched Vh3-23 transcripts were undetectable or low in 30% (IgG) and 63% (IgA) of the patients, but never in controls (P < 0.001). When measurable, the SHM fraction of transcripts was significantly lower in patients (IgM: median 32% vs. 56% (P = 0.0002); IgG: 72% vs. 87% (P = 0.0002); IgA: 81% vs. 88% (P = 0.04)). The concentration of switched (CD19+/CD27+/IgG+) and unswitched (CD19+/CD27+/IgM+/IgD+) memory cells was reduced in 75% and 58% of the patients, respectively. Patients with reduced concentrations of switched memory B cells had normal or low SHM, and only the IgG SHM fraction correlated with prediagnostic incidence of severe respiratory tract infections (P = 0.004).

The role of complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) in promoting C3 fragment deposition and membrane attack complex formation on normal peripheral human B cells

Normal human B lymphocytes are known to activate the alternative pathway (AP) of complement, leading to C3‐fragment deposition and membrane attack complex (MAC) formation. The process is mediated via complement receptor type 2 (CR2, CD21), with complement receptor type 1 (CR1, CD35) playing a subsidiary role. In this study, we examine the relative contributions of CR1 and CR2 to the deposition of C3 fragments and MAC on B lymphocytes under circumstances where all complement pathways are operational. C3‐fragment deposition and MAC formation were assessed on human peripheral B lymphocytes in the presence of 30% autologous serum. Blocking the CR2 ligand‐binding site with monoclonal antibody (mAb) FE8 resulted in significant reduction (37.9±11.9%) in C3‐fragment deposition, whereas MAC formation was only marginally affected (12.1±22.2% reduction). Blocking the CR1 binding‐site resulted in significant reduction of both C3‐fragment deposition (22.0±14.5%) and MAC formation (47.4±13.8%). Both the lack of CR2 influence on MAC formation and the promotion of C3‐fragment deposition by CR1 are in striking contrast to the situation where only the AP is operational. The presence of erythrocytes (E) bearing CR1, however, markedly reduced both C3‐fragment deposition and MAC formation. Our data suggest that C3‐fragment deposition and MAC formation on B lymphocytes in vivo may involve both AP and classical pathway activation, with CR1 contributing significantly to the latter. On the other hand, the presence of extrinsic CR1, on E, may serve to limit spontaneous MAC formation and thereby ensure cell survival in the circulation.

Residual disease detected by flow cytometry is an independent predictor of survival in childhood acute myeloid leukaemia; results of the NOPHO-AML 2004 study.

Early response after induction is a prognostic factor for disease outcome in childhood acute myeloid leukaemia (AML). Residual disease (RD) detection by multiparameter flow cytometry (MFC) was performed at day 15 and before consolidation therapy in 101 patients enrolled in the Nordic Society of Paediatric Haemato‐Oncology AML 2004 study. A multicentre laboratory approach to RD analysis was used. Event‐free survival (EFS) and overall survival (OS) was significantly different in patients with and without RD at both time points, using a 0·1% RD cut‐off level. RD‐negative and ‐positive patients after first induction showed a 5‐year EFS of 65 ± 7% and 22 ± 7%, respectively (P < 0·001) and an OS of 77 ± 6% (P = 0·025) and 51 ± 8%. RD‐negative and ‐positive patients at start of consolidation therapy had a 5‐year EFS of 57 ± 7% and 11 ± 7%, respectively (P < 0·001) and an OS of 78 ± 6% and 28 ± 11%) (P < 0·001). In multivariate analysis only RD was significantly correlated with survival. RD before consolidation therapy was the strongest independent prognostic factor for EFS [hazard ratio (HR):5·0; 95% confidence interval (CI):1·9–13·3] and OS (HR:7·0; 95%CI:2·0–24·5). In conclusion, RD before consolidation therapy identifies patients at high risk of relapse in need of intensified treatment. In addition, RD detection can be performed in a multicentre setting and can be implemented in future trials.

CR2‐mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane‐bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4·4 mm MgCl2/20 mm EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand‐binding site with the monoclonal antibody FE8 resulted in 56 ± 13% and 71 ± 9% inhibition of the C3‐fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 ± 5%) and C9 (95 ± 3%). The kinetics of C3‐fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation.

Results of NOPHO ALL2008 treatment for patients aged 1–45 years with acute lymphoblastic leukemia

Adults with acute lymphoblastic leukemia (ALL) do worse than children. From 7/2008 to 12/2014, Nordic and Baltic centers treated 1509 consecutive patients aged 1–45 years with Philadelphia chromosome-negative ALL according to the NOPHO ALL2008 without cranial irradiation. Overall, 1022 patients were of age 1–9 years (A), 266 were 10–17 years (B) and 221 were 18–45 years (C). Sixteen patients (three adults) died during induction. All others achieved remission after induction or 1–3 intensive blocks. Subsequently, 45 patients (12 adults) died, 122 patients relapsed (32 adults) with a median time to relapse of 1.6 years and 13 (no adult) developed a second malignancy. Median follow-up time was 4.6 years. Among the three age groups, older patients more often had higher risk ALL due to T-ALL (32%/25%/9%, P<0.001), KMT2A rearrangements (6%/5%/3%, P<0.001) and higher day 29 residual leukemia for B-lineage (P<0.001), but not T-ALL (P=0.53). Event-free survival rates (pEFS5y) were 89±1% (A), 80±3% (B) and 74±4% (C) with significant differences only for non-high risk groups. Except for thrombosis, pancreatitis and osteonecrosis, the risk of 19 specified toxicities was not enhanced by age above 10 years. In conclusion, a pediatric-based protocol is tolerable and effective for young adults, despite their increased frequency of higher risk features.