Quality control of allergen products with mass spectrometry part I: Positioning within the EU regulatory framework.

Mass spectrometry (MS) has advanced greatly and many of its applications are ready for utilization within regulatory procedures and could significantly contribute to overcome challenges in standardization of allergen products. It seems sensible to discuss MS within the regulatory framework, before addressing technical questions. While the application to purified proteins is well established from product development to manufacturer's release analytics, its application to complex products such as allergen products is still under development. It needs to be determined where it can complement or replace established methods or where MS offers limited improvement. Despite its technical appeal and versatility, currently MS is mentioned in regulatory guidelines only as one possible measurement method. For example, no specific MS method is given in the European Pharmacopoeia. We discuss applications of MS within the EU regulatory framework. This includes their advantages and disadvantages and their positioning between research, characterization, manufacturer's release analytics and official batch testing. We discuss the qualitative detection of single and multiple allergens as proof of identity, qualitative to semi-quantitative protein profiles for batch to batch consistency testing, and quantification of allergens to state mass units of allergens. MS may also facilitate standardization of allergen products, reference products and reference standards.

[Recombinant allergens, peptides, and virus-like particles for allergy immunotherapy]. / Rekombinante Allergene, Peptide und Virus-like Particles in der Immuntherapie von Allergien.

Currently, extract-based therapeutic allergens from natural allergen sources (e.g., house dust mites, tree and grass pollen) are used for allergen-specific immunotherapy (AIT), the only causative therapy that can exhibit positive disease-modifying effects by tolerance induction and prevention of disease progression. Due to variations in the natural composition of the starting materials and different manufacturing processes, there are variations in protein content, allergen composition, and allergenic activity of similar products, which poses specific challenges for their standardization. The identification of the nucleotide sequences of allergenic proteins led to the development of molecular AIT approaches. This allows for the application of exclusively relevant structures as chemically synthesized peptides, recombinant single allergens, or molecules with hypoallergenic properties that potentially allow for an up-dosing with higher allergen-doses without allergic side effects leading more quickly to effective cumulative doses. Further modifications of AIT preparations to improve allergenic and immunogenic properties may be achieved, e.g., by including the use of virus-like particles (VLPs). To date, the herein described therapeutic approaches have been tested in clinical trials only. This article provides an overview of published molecular approaches for allergy treatment used in clinical AIT studies. Their added value and challenges compared to established therapeutic allergens are discussed. The aim of these approaches is to develop highly effective and well-tolerated AIT preparations with improved patient acceptance and adherence.

Report from the GHPP-BloodTrain inspection workshop in Harare the 20th to the 24th of May 2019.

During the training workshop on Inspection of Blood Establishments, which was hosted by the PEI GHPP BloodTrain in Harare from the 20th to the 24th of May 2019, participants from the National Regulatory Authorities from seven Sub-Sahara African countries presented their current experiences related to regulation and inspection of blood establishments in their respective countries. While in all seven countries regulation and inspection of conventional medicinal products manufacturer is performed, the regulatory situation of blood and blood components as well as inspection of blood establishments is still heterogeneous.

Does rehabilitation pose a risk to patients suffering from haemato-oncological diseases? Results of a monocentric, retrospective analysis in Germany.

OBJECTIVE: Patients suffering from haemato-oncological diseases tend to have a weakened immune system after the end of their therapy. To avoid infections, patients are advised to limit contact with other people. This poses the question whether a stay at a rehabilitation facility can be recommended. METHODS: We report about 134 rehabilitation stays of patients. Premature discontinuation of the rehabilitation stay was selected as the criterion for a serious complication during the rehabilitation, and the underlying reasons were analysed. RESULTS: Compared to the discontinuation rates of patients suffering from solid tumours (2.4%), the percentage of haemato-oncological patients ending prematurely their rehabilitation stay (8.2%) is significantly increased. This rises to 17.1% for patients who have undergone an allogeneic stem cell transplantation. The analysis of the discontinuation reasons revealed that they were not directly connected to the rehabilitation. Apart from the already known risk factors for premature termination of the rehabilitation stay, we have identified the period (days) between the last therapy and the beginning of the rehabilitation stay as a risk factor. CONCLUSIONS: We show for the first time that a rehabilitation stay does not pose additional risks for patients suffering from haemato-oncological diseases.

Circulating t(14;18)-positive cells in healthy individuals: association with age and sex but not with smoking.

Abstract t(14;18)-positive cells can be detected not only in patients with follicular lymphoma (FL) but also in healthy individuals (HIs). We used epidemiological data and blood samples of the population-based Study of Health in Pomerania (SHIP) to analyze associations of FL risk factors and t(14;18)-positive cells in HIs. Buffy coat samples from 4152 study participants were tested by real-time polymerase chain reaction (PCR) for t(14;18)-positive cells. Of 3966 evaluable subjects, 1526 were t(14;18)-PCR positive [38.5%, median 3.9 t(14;18)-positive per million nucleated cells, range 0.6-9299]. In multivariable analyses, age and sex but not parameters of smoking exposure were significantly associated with t(14;18) prevalence (logistic regression, p < 0.001). Multivariable analyses of t(14;18)-frequency showed a positive association with age but not with sex or smoking. These age and sex associations in HIs require careful control in future studies of t(14;18) as a potential biomarker of lymphoma risk.

Statistical analysis of results obtained by real-time PCR for improvement of absolute quantification of target sequences.

BACKGROUND: Real-time quantitative PCR is increasingly used in clinical laboratories. Genomic DNA or plasmids containing cloned target sequences are necessary to generate data for standard curves. These data must be analysed to obtain the relative or absolute quantity of the target concentration in a sample. The method chosen for data analysis can strongly influence results of the quantification. Absolute quantification is important especially in clinical settings. For different reasons estimating the copy number of the gene of interest based on DNA concentration measurements is vague and tends toward overestimation, especially if cell lines are used. METHODS: Data gained by limiting dilution and multiple-tube approach were analyzed using our new Poisson distribution based software and were compared with results from DNA concentration measurement. Data from different cell sources (peripheral blood mononuclear cells and two cell lines) were compared: RESULTS: Limiting dilution and multiple-tube approach analyzed by a Poisson distribution simplifies and improves the generation of standard curves for real time PCR if cell lines are used. The absolute target copy number in a sample, the standard deviation, and a 95% confidence interval are calculated by the software. CONCLUSIONS: With this easy to use program a target copy number can be reliably quantified. The program is available free of charge from: http://www.medizin.uni-greifswald.de/InnereC/index.php?id=18 (link will be activated after acceptance of the paper).

Delayed processing of bone marrow samples reveals a prognostic pattern of NME mRNA expression in cytogenetically normal acute myeloid leukemia.

Improvements in the therapy of cytogenetically normal acute myeloid leukemia (CN-AML) will depend largely on the characterization of functional subtypes identified by prognostic markers. Exposing leukemic cells to stress ex vivo may reveal relevant phenotypic markers not apparent in freshly explanted cells. Here, we assess the prognostic relevance of expression of the nucleoside diphosphate kinase genes NME1 and NME2 in a cohort of 78 patients with CN-AML aged < 60 years using archived mononuclear cell samples originally prepared from bone marrow either directly (n = 25) or following 2-3 days of transport (n = 53). The stress conditions arising during transport resulted in the development of a prognostic pattern of NME mRNA with maintenance of high NME2 mRNA being a strong indicator of increased event-free survival independent of FLT3-internal tandem duplication. Prospective analysis of CN-AML bone marrow (n = 7) confirmed that NME1 mRNA is always decreased during storage, while NME2 mRNA is either decreased or maintained. We conclude that ex vivo stress can reveal novel prognostic markers.

Upfront immunization with autologous recombinant idiotype Fab fragment without prior cytoreduction in indolent B-cell lymphoma.

Idiotype vaccination for follicular lymphoma is primarily being developed as remission consolidation after chemotherapy. We investigated idiotype vaccination as primary intervention for treatment-naive indolent B-cell lymphoma and in a separate cohort as remission consolidation after chemotherapy to assess immunization-induced immune responses in relation to progression-free survival (German Clinical Trials Register, DRKS00000227). Twenty-one patients in each cohort received 6 intradermal injections of adjuvanted recombinant idiotype Fab fragment (Fab(Id)); 76% of patients in both groups developed anti-idiotype antibodies and/or cellular immunity as measured by enzyme-linked immunosorbent assay and interferon-γ ELISpot. In treatment-naive patients, only cellular responses correlated with superior progression-free survival (P < .002) and durable objective remissions (P = .04). Immunization-induced T cells recognized hypermutated or complementarity-determining region 3 epitopes. After remission consolidation immunization, induction of anti-idiotype antibodies correlated with progression-free survival. Low B-cell counts after rituximab therapy predicted for failure to develop anti-idiotype antibodies. These results are similar to published trials showing an association of humoral immunity with control of residual lymphoma. In contrast, effective immunity against untreated lymphoma appears to be dependent on idiotype-specific T cells. Sustained remissions in patients with vaccination-induced cellular immunity suggest clinical benefit and warrant a randomized comparison of this vaccine with expectant management for asymptomatic follicular lymphoma.

Prevalence and frequency of circulating t(14;18)-MBR translocation carrying cells in healthy individuals.

The t(14;18) translocation is a common genetic aberration that can be seen as an early step in pathogenesis of follicular lymphoma (FL). The significance of low level circulating t(14;18)-positive cells in healthy individuals as clonal lymphoma precursors or indicators of risk is still unclear. We determined the age dependent prevalence and frequency of BCL2/IgH rearrangements in 715 healthy individuals ranging from newborns to octo- and nonagenarians. These results were compared with number of circulating t(14;18)-positive cells in 108 FL patients at initial presentation. The overall prevalence of BCL2/IgH junctions in this large sample was 46% (327/715). However, there was a striking dependence upon age. Specifically, among individuals up to 10 years old, none had detectable circulating t(14;18)-positive cells. In the age groups representing 10-50 years old, we found a steady elevation in the prevalence of BCL2/IgH junctions up to a prevalence of 66%. Further increases of the prevalence in individuals older than 50 years were not seen. The mean frequency of BCL2/IgH junctions in healthy individuals > or =40 years (18-26 x 10(-6)) was significantly higher than in younger subjects (7-9 x 10(-6)). Four percent (31/715) of individuals carried more than one t(14;18)-positive cell per 25,000 peripheral blood mononuclear cells (PBMNCs). In comparison, 108 stage III/IV FL patients had a median number of circulating t(14;18)-positive malignant FL cells of about 9200/1 million PBMNCs (range 7-1,000,000). These findings will further improve the understanding of the relevance of t(14;18)-positive cells in healthy individuals as a risk marker toward the development into lymphoma precursors.

Mobilization of hemopoietic stem cells with high-dose methotrexate plus granulocyte-colony-stimulating factor in patients with primary central nervous system lymphoma.

BACKGROUND: High-dose therapy with autologous stem cell support after standard dose induction is a promising approach for therapy of primary central nervous system lymphoma (PCNSL). High-dose methotrexate (HD-MTX) is a standard drug for induction of PCNSL; however, data about the capacity of HD-MTX plus granulocyte-colony-stimulating factor (G-CSF) to mobilize hemopoietic progenitors are lacking. STUDY DESIGN AND METHODS: This investigation describes the data from stem cell mobilization and apheresis procedures after one or two cycles of HD-MTX for induction of PCNSL within the East German Study Group for Haematology and Oncology 053 trial. Eligible patients proceeded to high-dose busulfan/thiotepa after induction therapy and mobilization. RESULTS: Data were available from nine patients with a median age of 58 years. The maximal CD34+ cell count per microL of blood after the first course of HD-MTX was 13.89 (median). Determination was repeated in six patients after the second course with a significantly higher median CD34+ cell count of 33.69 per microL. Five patients required two apheresis procedures and in four patients a single procedure was sufficient. The total yield of CD34+ cells per kg of body weight harvested by one or two leukapheresis procedures was 6.60 x 10(6) (median; range, 2.68 x 10(6)-15.80 x 10(6)). The yield of CD34+ cells exceeded the commonly accepted lower threshold of 3 x 10(6) cells per kg of body weight in eight of nine cases. Even in the ninth, hemopoietic recovery after stem cell reinfusion was rapid and safe. CONCLUSION: HD-MTX plus G-CSF is a powerful combination for stem cell mobilization in patients with PCNSL and permits safe conduction of time-condensed and dose-intense protocols with high-dose therapy followed by stem cell reinfusion after HD-MTX induction.

Age-dependent prevalence and frequency of circulating t(14;18)-positive cells in the peripheral blood of healthy individuals.

Circulating t(14;18)-positive cells were detected by quantitative real-time polymerase chain reaction on DNA isolated from peripheral blood mononuclear cells (PBMNCs) from 644 healthy individuals between <1 and 91 years of age. In all, 45% of all samples (287/644) were positive, and 40% of the positive samples (114/287) contained more than one positive clone. The prevalence of t(14;18)-positive cells showed a strong correlation with age. A total of 36 cord blood samples and 48 PBMNCs from children <10 years were negative. The prevalence of circulating positive cells increased from the second to fifth decade of life from 20% to 66% and remained stable thereafter. Also the median frequency of circulating t(14;18)-positive cells as well as the prevalence of multiple clones showed an increase with age. In all, 4% (24/644) of all blood samples contained >1 positive cell in 25,000 cells, a finding restricted to healthy individuals >40 years. These results are discussed in relation to the low incidence of follicular lymphoma.

Rapid and sustained clearance of circulating lymphoma cells after chemotherapy plus rituximab: clinical significance of quantitative t(14;18) PCR monitoring in advanced stage follicular lymphoma patients.

This study of first-line treatment in advanced-stage follicular lymphoma patients analysed the effects of MCP (mitoxantrone, chlorambucil and prednisolone) chemotherapy alone or in combination with rituximab (R-MCP) on circulating lymphoma cells (CLC) and assessed the prognostic value of a quantitative monitoring of CLC. CLC numbers were determined by quantitative polymerase chain reaction (PCR) for the t(14;18)-translocation or by allele-specific PCR for rearranged immunoglobulin heavy chain genes. We analysed blood samples from 43 patients treated in a randomized trial comparing eight cycles of MCP versus R-MCP. Clearance of CLC at the end of therapy was achieved in 21/25 patients (84%) treated with R-MCP compared with 0/18 after MCP alone (P < 0.0001). A > or = 2 log CLC reduction was associated with a favourable clinical response (P = 0.0004) and prolonged event-free survival (P = 0.02). In R-MCP patients, stable CLC numbers or consistently PCR-negative blood samples were associated with a continuing clinical remission whereas in two patients a relapse was preceded by a > or = 2 log CLC increase. This study demonstrated that R-MCP led to a rapid and sustained eradication of CLC and a > or = 2 log CLC reduction was associated with a superior quality and duration of the clinical response.

A human Burkitt's lymphoma cell line carrying t(8;22) and t(14;18) translocations.

A combination of chromosomal translocations associated with bcl-2 re-arrangement (t(14;18)) and c-myc re-arrangement (t(8;14), t(8;22), or t(2;8)) is a rare event. We describe the first cell line exhibiting t(14;18) and t(8;22), which will enable us to study the interactions of bcl-2 and c-myc systematically. Cell culture was started with circulating lymphoma cells from the peripheral blood of an adult male Caucasian patient with Burkitt's lymphoma after the second relapse. The cells grew spontaneously without cytokines, fulfilled all criteria of a cell line and were analysed. An Epstein-Barr virus (EBV) genome-negative cell line (DoGKiT) has been established. RC-banding analysis of the chromosomes showed a complex karyotype with a modal number of 48, XY, dup(1)(q31;q44), t(8;22)(q24;q11), der(10), t(14;18)(q32;q21), add(16)(pter), dup(17)(q12q24), +der(18), +20. The combination of t(8;22)(q24;q11), a variant translocation of Burkitt's lymphoma and t(14;18)(q32;q21), typical for follicular lymphoma (FL), was confirmed by FISH and SKY-analysis. Surface marker studies of the cell line showed that the cells were positive for CALLA (CD10), CD19, cyCD22, cyCD79a and HLA-DR and negative for TdT, IgM, CD5 and CD23. To our knowledge, this is the first established cell line carrying these two translocations. In contrast to already established cell lines carrying the more common combination of t(8;14)(q24;q32) and t(14;18)(q32;q21) with both IgH alleles being involved in translocations, the cell line DoGKiT carries only one translocated IgH allele. This cell line may serve as an important tool in the study of the combination of the chromosomal translocations t(14;18) and t(8;22) and in molecular genetic studies of transformed FL.

Complete remission and early relapse of refractory plasma cell leukemia after bortezomib induction and consolidation by HLA-mismatched unrelated allogeneic stem cell transplantation.

BACKGROUND: Chromosomal deletion of q13 (del q13) and plasma cell leukemia predict both a worse prognosis in myeloma. Experiences with bortezomib in plasma cell leukemia prior to allogeneic stem cell transplantation (SCT) have not yet been reported. CASE REPORT: A 66- year-old male patient was admitted for IgA myeloma(IIIA) with del q13. The myeloma progressed to plasma cell leukemia. Bortezomib was given, free light chain (FLC) excretion decreased, and myeloma cells disappeared from blood and decreased in marrow. An unrelated, mismatched allogeneic SCT was performed. The patient was discharged after engraftment with full chimerism without signs of GvHD. FLC excretion increased, and immunosuppression was discontinued. From day +84 on, bortezomib was infused again and FLC excretion decreased rapidly. Relapse was confirmed in marrow, and bortezomib was continued. Donor lymphocyte infusions (DLI) had no effect, and a further cycle of bortezomib and thalidomide had only minor effects. On day +209, the patient died from myeloma. CONCLUSION: This case gives evidence for an excellent initial response of plasma cell leukemia to bortezomib, however, early relapse after SCT clearly indicates limitations of both bortezomib therapy and allogeneic SCT in high-risk myeloma. Future developments are mandatory and could include combination of bortezomib with other cytostatics and early DLI in the absence of GvHD. In addition, there is need to clarify if graft-versus-myeloma effect is diminished by bortezomib therapy after allogeneic SCT.

No evidence for simian virus 40 DNA sequences in malignant non-Hodgkin lymphomas.

DNA sequences coding for simian virus 40 (SV40) large T antigen have been detected at different frequencies in human non-Hodgkin's lymphomas (NHL) by PCR techniques as well as immunohistochemistry. A highly sensitive quantitative real-time PCR specific for a sequence of SV40 large T antigen was established to test whether SV40 DNA is present in malignant lymphomas of German patients. Thirty-three lymph node samples obtained from 27 patients with NHL and 6 patients with Hodgkin's disease (HD) were tested in addition to 48 samples of peripheral blood mononuclear cells (PBMNC) from patients with NHL containing between 0.1% and >90% circulating lymphoma cells determined by PCR. Fourteen lymph nodes obtained from patients with other diseases than malignant lymphomas and 47 PBMNC samples from healthy volunteers served as controls. All samples from patients with malignant lymphomas and all controls were negative for SV40 DNA by quantitative real-time. In contrast, EBV-DNA could be detected in 29 of 46 DNA preparations isolated from lymph nodes (63%) and in 20 of 47 DNA preparations from PBMNC. EBV-positive samples contained between 5 and 80,000 EBV copies per 100,000 cells. Our results do not support the hypothesis that SV40 plays a major role in the etiology of malignant lymphomas and, in addition, they exclude a clonal SV 40 infection of malignant lymphoma cells in all samples investigated.

Multiple osteolytic lesions and testicular involvement at first relapse of follicular lymphoma grade 1 in transformation.

A 62-year-old man was initially diagnosed with stage IA follicular lymphoma grade 1 of the left tonsil. Shortly after radiotherapy he rapidly developed multiple painful acroosteolytic lesions and testicular involvement. The histological examination revealed a transformed lymphoma in the testis (DLCL) and follicular lymphoma in the acroosteolytic lesions. The clonal identity of lymphoma cells within the primary biopsy as well as in the two sites at relapse was shown by PCR and nucleotide sequence analysis of the lymphoma clone specific B-cell receptor rearrangement. Chemotherapy with six cycles of CHOP followed by high dose chemotherapy and autologous blood stem cell transplantation led to a complete clinical remission with disappearance of all osteolytic lesions.

Detection and monitoring of minimal residual disease by quantitative real-time PCR.

BACKGROUND: The detection of malignant cells by quantitative real-time PCR has become state of the art for diagnosis, monitoring response to treatment and detection of minimal residual disease (MRD) in patients with leukemia or lymphoma. In order to be used in high-throughput analyses technical details have to be standardized to improve reproducibility and comparability of quantitative results obtained in different laboratories. METHODS: Molecular monitoring of disease activity during and after treatment based on the detection of malignant cells in circulation or bone marrow by quantitative real-time PCR will be helpful to develop individualized treatment strategies for every patient. CONCLUSIONS: The effectiveness of any kind of innovative treatment with specific antibodies, cellular immunotherapy or molecules designed for specific targets of tumor cells can be controlled at a very high level of sensitivity and accuracy. Based on quantitative results indicative for success or treatment failure, therapeutic changes upon the detection of progressive disease at the molecular level can be made even before symptoms or signs of clinical relapse occur. Hopefully, this will lead to higher cure rates and improved long-term survival.

Combined immunoradiotherapy induces long-term remission of CNS relapse of peripheral, diffuse, large-cell lymphoma after allogeneic stem cell transplantation: case study.

Relapse of peripheral non-Hodgkin's lymphoma (NHL) in the central nervous system commonly has a poor prognosis. Graft-versus-leukemia effects (GvL) contribute substantially to eradication of hematological malignancies after allogeneic stem cell transplantation. Few data are available describing GvL activity within the brain. We report the case of a man allografted for peripheral NHL. On day +83 after transplantation a CNS relapse of the lymphoma occurred. The brain was irradiated with 44 Gy, anti-CD20 antibodies were given, and the immunosuppression was withdrawn. Subsequently, limited-stage, chronic graft-versus-host disease occurred. The lymphoma regressed completely, and the patient has been in continuous complete remission for 30 months. The favorable course suggests substantial contribution of immunomodulation to excellent outcome.