Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting.

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 5.2 × 10-3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10-4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

Correction: Minimal residual disease quantification by flow cytometry provides reliable risk stratification in T-cell acute lymphoblastic leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

T-cell acute lymphoblastic leukemia in patients 1-45 years treated with the pediatric NOPHO ALL2008 protocol.

The NOPHO ALL2008 is a population-based study using an unmodified pediatric protocol in patients 1-45 years of age with acute lymphoblastic leukemia. Patients with T-ALL were given a traditional pediatric scheme if fast responding (minimal residual disease (MRD) < 0.1% day 29), or intensive block-based chemotherapy if slow responding (MRD > 0.1% day 29). Both treatment arms included pediatric doses of high-dose methotrexate and asparaginase. If MRD ≥ 5% on day 29 or ≥0.1% after consolidation, patients were assigned to allogeneic hematopoietic stem cell transplantation. The 5-year overall survival of the 278 T-ALL patients was 0.75 (95% CI 0.69-0.81), being 0.82 (0.74-0.88) for patients 1.0-9.9 years, 0.76 (0.66-0.86) for those 10.0-17.9 years, and 0.65 (0.55-0.75) for the older patients. The risk of death in first remission was significantly higher in adults (12%) compared with the 1-9 years group (4%). The MRD responses in the three age groups were similar, and only a nonsignificant increase in relapse risk was found in adults. In conclusion, an unmodified pediatric protocol in patients 1-45 years is effective in all age groups. The traditional pediatric treatment schedule was safe for all patients, but the intensive block therapy led to a high toxic death rate in adults.

Minimal residual disease quantification by flow cytometry provides reliable risk stratification in T-cell acute lymphoblastic leukemia.

Minimal residual disease (MRD) measured by PCR of clonal IgH/TCR rearrangements predicts relapse in T-cell acute lymphoblastic leukemia (T-ALL) and serves as risk stratification tool. Since 10% of patients have no suitable PCR-marker, we evaluated flowcytometry (FCM)-based MRD for risk stratification. We included 274 T-ALL patients treated in the NOPHO-ALL2008 protocol. MRD was measured by six-color FCM and real-time quantitative PCR. Day 29 PCR-MRD (cut-off 10-3) was used for risk stratification. At diagnosis, 93% had an FCM-marker for MRD monitoring, 84% a PCR-marker, and 99.3% (272/274) had a marker when combining the two. Adjusted for age and WBC, the hazard ratio for relapse was 3.55 (95% CI 1.4-9.0, p = 0.008) for day 29 FCM-MRD ≥ 10-3 and 5.6 (95% CI 2.0-16, p = 0.001) for PCR-MRD ≥ 10-3 compared with MRD < 10-3. Patients stratified to intermediate-risk therapy on day 29 with MRD 10-4-<10-3 had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10-4 or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10-4 had a cumulative incidence of relapse of 2.3% (95% CI 0-6.8, n = 59). Thus, FCM-MRD allows early identification of patients eligible for reduced intensity therapy, but this needs further studies. In conclusion, FCM-MRD provides reliable risk prediction for T-ALL and can be used for stratification when no PCR-marker is available.

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.

Case Report: Renal Transplantation in Patients with Pre-existing Hypogammaglobulinemia.

Hypogammaglobulinemia (HGG) is well-characterized as a common phenomenon after kidney transplantation. However, no reports of pre-existing HGG from kidney transplantation seem to be available. We have reviewed three patients who developed HGG prior to kidney transplantation, and all three were treated successfully with immunoglobulin replacement therapy before and after kidney transplantation. The kidney grafts were functioning at follow-up 1.5-8 years (mean: 3.6 years) after transplantation, and there were no diagnosed episodes of clinical rejections and no severe infection complications post-transplantation.

Genomic profiling of thousands of candidate polymorphisms predicts risk of relapse in 778 Danish and German childhood acute lymphoblastic leukemia patients.

Childhood acute lymphoblastic leukemia survival approaches 90%. New strategies are needed to identify the 10-15% who evade cure. We applied targeted, sequencing-based genotyping of 25 000 to 34 000 preselected potentially clinically relevant single-nucleotide polymorphisms (SNPs) to identify host genome profiles associated with relapse risk in 352 patients from the Nordic ALL92/2000 protocols and 426 patients from the German Berlin-Frankfurt-Munster (BFM) ALL2000 protocol. Patients were enrolled between 1992 and 2008 (median follow-up: 7.6 years). Eleven cross-validated SNPs were significantly associated with risk of relapse across protocols. SNP and biologic pathway level analyses associated relapse risk with leukemia aggressiveness, glucocorticosteroid pharmacology/response and drug transport/metabolism pathways. Classification and regression tree analysis identified three distinct risk groups defined by end of induction residual leukemia, white blood cell count and variants in myeloperoxidase (MPO), estrogen receptor 1 (ESR1), lamin B1 (LMNB1) and matrix metalloproteinase-7 (MMP7) genes, ATP-binding cassette transporters and glucocorticosteroid transcription regulation pathways. Relapse rates ranged from 4% (95% confidence interval (CI): 1.6-6.3%) for the best group (72% of patients) to 76% (95% CI: 41-90%) for the worst group (5% of patients, P<0.001). Validation of these findings and similar approaches to identify SNPs associated with toxicities may allow future individualized relapse and toxicity risk-based treatments adaptation.

Long-term follow-up on affinity maturation and memory B-cell generation in patients with common variable immunodeficiency.

PURPOSE: Common variable immunodeficiency (CVID) comprises a heterogeneous group of primary immunodeficiency disorders. Immunophenotyping of memory B cells at the time of diagnosis is increasingly used for the classification of patients into subgroups with different clinical prognoses. The EUROclass classification is a widely used method. Levels of somatic hypermutation (SHM) have proven useful as a prognostic marker for recurrent respiratory tract infections. As time of presentation and diagnosis is highly variable in CVID patients, and diagnostic delay is a common problem, it is important to know whether classification parameters are stable over time. The purpose of the study was to address this question in a cohort of 33 CVID patients followed from 3 to 19 years after diagnosis (average follow-up 8.8 years). METHODS: Levels of class-switched memory B cells were analyzed using flow cytometric immunophenotyping, and patients were classified according to the EUROclass criteria. Affinity maturation of B cells was measured using Igκ-REHMA, which assesses somatic hypermutation in kappa light chain transcripts. Clinical manifestations in terms of splenomegaly, autoimmune disease and granulomatous disease were also determined. RESULTS: Switched memory B cells and levels of SHM were not consistently stable markers in a long-term follow-up setting. At a given time during follow-up, 60% of the patients were assigned to the EUROclass group SmB- (less than 2% switched memory B cells), but only 23% were consistently assigned to this group. Associations between clinical manifestations and levels of switched memory B cells or SHM were not observed in our study. CONCLUSION: Based on our findings, we suggest that immunologic characteristics in CVID patients should be evaluated several times after diagnosis using internationally standardized methods.

Combined IL-12 receptor and IgA deficiency in an adult man intestinally infested by an unknown, non-cultivable mycobacterium.

Interleukin-12 receptor deficiency is a well-described cause of human susceptibility to infection with low-virulent mycobacteria and Salmonella species. We identified a male patient presenting in his late forties with severe gastroenteropathy because of outbred infestation by a previously unknown mycobacterium. In addition to selective IgA deficiency, the patient was found to carry a not previously described R283X homozygous mutation in his IL12RΒ1 gene. Two of his sisters, a brother, and his four children were healthy, heterozygous carriers of the mutation. In this patient, the combination of two deficiencies could promote illness. Even though the IgA deficiency in itself does not predispose to mycobacterial disease, the lack of secreted IgA may have disturbed the intestinal homoeostasis and increased the susceptibility to the low-virulent mycobacterium that the patient was not able to clear because of his IL12R deficiency. Antimycobacterial chemotherapy and interferon-γ treatment for 2 years significantly improved his condition. This is the first description of IL12RΒ1 deficiency combined with another immunodeficiency, and we suggest that combinatory defects may circumvent the otherwise low penetrance of IL12RB1 deficiency.

Complement factor H deficiency and endocapillary glomerulonephritis due to paternal isodisomy and a novel factor H mutation.

Complement factor H (CFH) is a regulator of the alternative complement activation pathway. Mutations in the CFH gene are associated with atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II and C3 glomerulonephritis. Here, we report a 6-month-old CFH-deficient child presenting with endocapillary glomerulonephritis rather than membranoproliferative glomerulonephritis (MPGN) or C3 glomerulonephritis. Sequence analyses showed homozygosity for a novel CFH missense mutation (Pro139Ser) associated with severely decreased CFH plasma concentration (<6%) but normal mRNA splicing and expression. The father was heterozygous carrier of the mutation, but the mother was a non-carrier. Thus, a large deletion in the maternal CFH locus or uniparental isodisomy was suspected. Polymorphic markers across chromosome 1 showed homozygosity for the paternal allele in all markers and a lack of the maternal allele in six informative markers. This combined with a comparative genomic hybridization assay demonstrated paternal isodisomy. Uniparental isodisomy increases the risk of homozygous variations in other genes on the affected chromosome. Therefore, we analyzed other susceptibility genes on chromosome 1 and found no sequence variation in membrane cofactor protein, but homozygosity for the common deletion of CFH-related proteins 1 and 3, which may contribute to the early onset of disease.

Use of read-across and tiered exposure assessment in risk assessment under REACH--a case study on a phase-in substance.

REACH requests the exploration of alternative strategies for hazard identification before resorting to (in vivo) testing. Here, we combined read-across as non-testing strategy with a tiered exposure assessment for the risk characterisation of 1-methoxypropan-2-ol (PGME) as a representative for phase-in substances to be registered under REACH. Read-across from the selected source substances provided data which were comparable with experimental data available for target substance PGME, resulting in a realistic starting point for both qualitative and quantitative risk assessment. Greater variability was observed in the exposure estimates from a first Tier model (ECETOC TRA) or less conservative further Tier models (Stoffenmanager; RISKOFDERM), when these results were compared with results from a data-rich approach using measured data. When safe use of chemicals cannot be demonstrated with these approaches, refinement can be introduced in the estimation of hazard and exposure, or both. In view of the variability associated with exposure modeling, it may often add more value to invest in realistic exposure data than in toxicity studies, apart from animal welfare considerations.

Proposal for an approach with default values for the protection offered by PPE, under European new or existing substance regulations.

Introduction of personal protective equipment (PPE) in the process of quantitative exposure and risk assessment should be addressed carefully. PPE which have been designed and manufactured according to CE-criteria and have proved to pass relevant test criteria, can be classified as "proper functioning". However, test criteria for PPE are not equal to levels of protection which can be achieved in the workplace, because actual workplace exposure scenarios, fit, maintenance and storage may differ substantially from the test conditions. The proper use of PPE is related to issues which form a part of a PPE-programme. Such a programme should be implemented in a company to ensure selection of proper PPE and information, training and instruction of employees how to wear PPE properly. Assigned protection factors (APFs) for different designs of respiratory protective devices (RPD) have been introduced to quantify effectiveness of RPD in the workplace. Similar APFs are proposed for dermal protection (gloves and clothing). In general biological monitoring studies show lower reduction of internal exposure than estimated by reduction of external exposure. Therefore, conservative estimates of protection by PPE, i.e. the lowest APFs, are proposed for risk assessment purposes if "proper use of proper functioning" PPE as part of a PPE-programme cannot be demonstrated.

Recording of data of individual measurements of occupational exposure: guideline of the Dutch Society of Occupational Hygiene (October 1999).

Following the recommendations of the European Working Group on Exposure Databases, a Working Group (on Storage of Data of Measurements of Occupational Exposure) of the Dutch Occupational Hygiene Society has developed a Guideline which was presented at the International Symposium on Occupational Exposure Databases and Their Application for the Next Millennium, November 1-3, 1999, London. To establish the present situation, a small-scale telephone survey of monitoring practices and storage of data was done within the Society. The results of the telephone survey and the draft guidance document were discussed with the occupational hygienists and other stakeholders (e.g., authorities, industry, labor unions, and occupational physicians) in a society meeting. This meeting was used to gather ideas on the need and support for a guidance document and to get input for improving the draft guidance document and for implementation of the Guideline. After this meeting, the Guideline was further developed and published by the Dutch Occupational Hygiene Society. The Guideline concentrates on the data elements required when storing exposure data. The data elements presented are the minimum and should be stored minimally to ensure proper interpretation of results at present and in the future and definitions of the items used are given. The Guideline does not prescribe how the data should be stored, or which procedures need to be used to guarantee the quality of the recorded data elements.

Assessing reasonable worst-case full-shift exposure levels from data of variable quality.

Exposure assessors involved in regulatory risk assessments often need to estimate a reasonable worst-case full-shift exposure level from very limited exposure information. Full-shift exposure data of very high quality are rare. A full-shift value can also be calculated from (short term) task-based values, either derived from measured data or from models. The most simple option is to use the task based exposure levels as the full-shift value. A second option is to calculate a time-weighted average (TWA), using (reasonable worst case) estimates of the duration and the exposure level of the relevant tasks. The third option is to use a Monte Carlo analysis with estimated input distributions for exposure level and duration of exposure. If an estimated distribution of respiratory volume is also included, this leads to a distribution of inhaled amounts. The 90th percentile of such a distribution is generally substantially lower than the fixed point estimates calculated using high end values for each parameter. This technique can thus prevent unnecessary conservative estimates in risk assessment. The output distribution can also be used as valuable input to the risk management process, because it provides information on probabilities of exposure levels, that can influence the cost-benefit analysis of the risk management process. Finally, the sensitivity analysis of Monte Carlo simulation can give guidance for further studies to increase the accuracy of the exposure assessment.

Harmonization of future needs for dermal exposure assessment and modeling: a workshop report.

Dermal exposure assessment and modeling is still in early phases of development. This article presents the results of a workshop organized to harmonize the future needs in this field. Methods for dermal exposure assessment either assess the mass of contaminant that is transferred to the skin, or the transfer of contaminant through the skin. Models for dermal exposure are either knowledge-based or deterministic. Any method or model should be transparent, validated, and open to further development. Some (partly) validated and standardized methods are available for measuring or modeling permeation of the skin or of personal protective equipment (PPE). Further validation and standardization is necessary. More research is needed on permeation of dusts and aerosols and more realistic tests should be developed and used for PPE. Several methods have been developed to measure contamination of surfaces or skin, but they are not validated or standardized. A number of non-validated models exist to assess dermal exposure. A clear need exists for more studies of dermal exposure, regarding measurement methods, models and actual exposure levels. A running four-year European study will greatly expand the knowledge in this field. Simple tools to assess and control the risks of dermal exposure in small and medium sized enterprises are also needed. Increasing the general knowledge of practitioners (e.g., safety professionals, occupational hygienists and physicians) in the field of dermal exposure is a first requirement. Available data, for example, on the permeation of PPE, should be made more readily available, using modern information technology. When information on dermal exposure is gathered and stored, the core information needs are partly the same as those for inhalation exposure. Some elements of process and activity, substance and product or worker, specific for dermal exposure, have been suggested by the workshop.

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.

Two edged role of mannose binding lectin in rheumatoid arthritis: a cross sectional study.

OBJECTIVE: We investigated whether polymorphisms in the gene of mannose binding lectin (MBL) may be associated with onset of rheumatoid arthritis (RA), and whether MBL in conjunction with aggregated agalactosyl IgG (IgG-G0) may be associated with clinical and paraclinical variables. METHODS: MBL genotypes and serum concentrations were measured by polymerase chain reaction and ELISA in 189 patients with established RA. Binding of purified MBL to IgG-G0 in serum was assessed and clinical and paraclinical variables were recorded. RESULTS: The median age at onset of RA in the 3 genotypes (normal: A/A, hetero: A/0, and homozygous: 0/0 for variant alleles) was 54.1 (n = 108), 47.0 (n = 68), and 38.4 years (n = 13), respectively (p = 0.01). The frequency of variant alleles in patients with onset below the median age (50.8 yrs) was 0.32, but was 0.17 in patients with onset above 50.8 years (p = 0.003) and 0.20 in 250 controls (p = 0.001). Stratification according to erosion score (no, small, large) revealed an increasing tendency among the different groups in binding of MBL to IgG-G0, increased Health Assessment Questionnaire score, and acute phase reactants in A/A individuals, while no difference was seen among carriers of variant alleles. This effect was most pronounced in those with late onset RA. CONCLUSION: Presence of MBL variant alleles was associated with early onset of RA. MBL deficiency may, therefore, accelerate the disease. However, in patients with late onset and advanced disease our results indicate that the A/A type may be associated with additional inflammation different from that seen in carriers of variant alleles.