Development of Resistant GvHD in a Patient Treated with Nivolumab for Hodgkins Lymphoma Relapse after Allogeneic Unrelated Transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation is one of the therapeutic options for patients with relapsed or refractory classic Hodgkins lymphoma (cHL). In the case of dis-ease relapse after transplant, other treatment options are still limited (for example donor lymphocyte infusion, and chemother-apy with brentuximab, bendamustine, or other agents) with uncertain outcomes in terms of patient tolerance and long-term dis-ease remission. One way to achieve remission is administration of the PD-1 inhibitor nivolumab, a PD-1 checkpoint inhibitor. Nivolumab is also indicated for the treatment of cHL relapses after autologous hematopoietic stem cell transplantation. Since September 2018, nivolumab has been approved by the State Institute for Drug Control in the Czech Republic for treatment of cHL autologous hematopoietic stem cell transplantation relapse; however, treatment with nivolumab is accompanied by an increased risk of develop-ing fatal, acute graft-versus-host dis-ease. CASE: The article describes the development of resistant acute graft-versus-host disease in a patient who had received allogeneic-unrelated transplantation and nivolumab treatment for Hodgkins lymphoma relapse. CONCLUSION: Our case study, as well as the literature review, demonstrates the excellent efficacy of PD-1 inhibitors, but also cautions against the administration of these agents in patients follow-ing allogeneic hematopoietic stem cell transplantation. Administration of nivolumab to these patients should be done on a strictly individual basis in the context of known risks, and consideration should be given to other treatment options. Key words Hodgkins lymphoma -  PD-1 inhibitor -  nivolumab -  GvHD -  transplantation.

Comparison of autologous hematopoietic cell transplantation performed in tandem and in disease relapse in multiple myeloma patients.

Multiple myeloma is a malignant hemato-oncological malignancy that affects up to 600 people in the Czech Republic every year. Treatment options are under constant improvement and the autologous hematopoietic cell transplantation (Tx) remains a part of treatment protocols. Despite modern drug administration, the autologous Tx keeps its irreplaceable position and when ensuring two autologous Tx, the studies confirm a survival time more than twice as long as in non-transplant patients. However, there are no standardized procedures specifying the period in between the transplantations in more detail. Within our group, we compared the total of 66 patients who were administered a double transplant. One group underwent both planned tandem autologous Tx within a median of six months and mostly achieved just partial remission (PR) and less after the first transplant and out of disease progression. The other group only underwent the second Tx within a median of up to 14 months during a progression period or disease relapse. Both groups were comparable as far as basic parameters are concerned (age, type of induction therapy and cytogenetic risk). A significantly better treatment free survival (TFX) and overall survival (OS) were observed in the group where tandem Tx was administered. TFS was 18 months and median OS was not reached for the group of patients who received tandem Tx, while TFS was 10 months (p=0.04) and median OS was 57 months (p=0.005) for those who received delayed second Tx. In the group of patients who received second Tx during relapse, we observed that TFS and OS were shorter in those with a higher paraprotein level, thus suggesting the potential role of paraprotein level as a prognostic marker. The TFS in the subgroup with a high initial level was 4 months vs. 11 months (p=0.0016) and OS 44 months vs. 65 months (p=0.03).

Biochemical responses induced in galls of three Cynipidae species in oak trees.

Gall-making Cynipidae manipulate the leaves of host plant to form galls where offspring find shelter and food. The relationship between oak gallwasp and biochemical mechanisms of galls still requires a better understanding. So, in this research, protein and phenolic compound contents, as well as the activity of antioxidative enzymes and pathogenesis-related (PR) proteins were determined. Galls caused by asexual generation of Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and N. quercusbaccarum L., as a model were used. All cynipid species modified the protein levels of gall tissues, but they cannot be treated as protein sinks. Significantly higher levels of phenols were observed in the galled leaves and galls of all cynipid species when compared with the control tissues. Peroxidase and polyphenol oxidase activity was usually low or showed no activity in galled tissues of all species. PR proteins, such as chitinase and ß-1,3-glucanase, had a similar activity profile. Their activity significantly increased in the leaves with galls of all cynipid species, especially those infested with C. quercusfolii. Data generated in this study clearly indicate that galling Cynipidae manipulate the biochemical machinery of the galls for their own needs. However, the pattern of the biochemical features of leaves with galls and galled tissues depends on gall-making species.

Risk factors for treatment failure after allogeneic transplantation of patients with CLL: a report from the European Society for Blood and Marrow Transplantation.

For young patients with high-risk CLL, BTK-/PI3K-inhibitors or allogeneic stem cell transplantation (alloHCT) are considered. Patients with a low risk of non-relapse mortality (NRM) but a high risk of failure of targeted therapy may benefit most from alloHCT. We performed Cox regression analyses to identify risk factors for 2-year NRM and 5-year event-free survival (using EFS as a surrogate for long-term disease control) in a large, updated EBMT registry cohort (n= 694). For the whole cohort, 2-year NRM was 28% and 5-year EFS 37%. Higher age, lower performance status, unrelated donor type and unfavorable sex-mismatch had a significant adverse impact on 2-year NRM. Two-year NRM was calculated for good- and poor-risk reference patients. Predicted 2-year-NRM was 11 and 12% for male and female good-risk patients compared with 42 and 33% for male and female poor-risk patients. For 5-year EFS, age, performance status, prior autologous HCT, remission status and sex-mismatch had a significant impact, whereas del(17p) did not. The model-based prediction of 5-year EFS was 55% and 64%, respectively, for male and female good-risk patients. Good-risk transplant candidates with high-risk CLL and limited prognosis either on or after failure of targeted therapy should still be considered for alloHCT.

Long-term survival of patients with CLL after allogeneic transplantation: a report from the European Society for Blood and Marrow Transplantation.

Even with the availability of targeted drugs, allogeneic hematopoietic cell transplantation (allo-HCT) is the only therapy with curative potential for patients with CLL. Cure can be assessed by comparing long-term survival of patients to the matched general population. Using data from 2589 patients who received allo-HCT between 2000 and 2010, we used landmark analyses and methods from relative survival analysis to calculate excess mortality compared with an age-, sex- and calendar year-matched general population. Estimated event-free survival, overall survival and non-relapse mortality (NRM) 10 years after allo-HCT were 28% (95% confidence interval (CI), 25-31), 35% (95% CI, 32-38) and 40% (95% CI, 37-42), respectively. Patients who passed the 5-year landmark event-free survival (N=394) had a 79% probability (95% CI, 73-85) of surviving the subsequent 5 years without an event. Relapse and NRM contributed equally to treatment failure. Five-year mortality for 45- and 65-year-old reference patients who were event-free at the 5-year landmark was 8% and 47% compared with 3% and 14% in the matched general population, respectively. The prospect of long-term disease-free survival remains an argument to consider allo-HCT for young patients with high-risk CLL, and programs to understand and prevent late causes of failure for long-term survivors are warranted, especially for older patients.

Prognostic factors to predict outcome of reduced intensity allogeneic haematopoietic cell transplantation for chronic lymphocytic leukemia.

Despite advances in immunochemotherapy CLL remains an incurable disease.. Allogeneic haematopoietic cell transplantation (HCT) has proven curative potential with ability to overcome adverse prognostic factors, however due to its toxicity it is generally perceived as the last option. We performed retrospective study to explore the outcomes and possible determinants of survival in the unselected consecutive cohort of 68 CLL patients (median age 59 years) receiving reduced intensity HCT as a part of salvage therapy in 2 Czech centers. The median interval from diagnosis to HCT was 69 months with median 3 of prior regimens, all patients were refractory to purine analogues. 49% of patients were transplanted with advanced (i.e. refractory or progressive disease or CR/PR>3), 38% had high risk cytogenetics. With median follow-up of 35 months the 3-year Kaplan-Meier survival probability for OS and PFS were 39% and 26%, respectively. Altogether 18 patients (26%) have relapsed or progressed. During the follow-up 41 patients died, 32 (78%) of transplant related factors (NRM), the others of relapse or disease progression.Univariate analysis failed to identify any clinical and pre- or post-transplant variables having clear prognostic significance for OS or PFS. The marginal OS advantage favoring HCT performed recently was detected (3-year OS: 31% for HCT until 2006 and 47% thereafter, p=0.0923). In multivariable hazards model only the female donors were associated with shorter OS (HR 2.278, p=0.016) whereas transplanted T-cell> 2.75x108/kg predicted inferior PFS(HR 1.957, p=0.035). No prognostic impact of donor type, age of donor and recipient, HLA mismatch, disease status pre-HCT, number of previous therapy lines, interval from dg. to HCT and number of transplanted hematopoietic cells was found. Our findings support the conclusion that alloHCT is able to overcome well known negative cytogenetic prognostic factors and that preferring male to female donors could be beneficial.

Allogeneic stem cell transplantation can improve outcome of AML patients without complete cytogenetic response after induction and consolidation treatment.

UNLABELLED: Our retrospective analysis was performed on 376 consecutive patients diagnosed with AML. A total of 256 (68%) were treated with standard "7+3" induction and high-dose cytarabine and mitoxantrone containing "4+3" consolidation/intensification regimens. Our study focused on patients with presumably very poor prognosis - patients, who did not achieve complete cytogenetic remission (CRc). Twenty-five AML patients without CRc were further analysed for clinical and laboratory parameters. Firstly, the subgroups with or without morphologic CR were compared. Similar cytogenetic abnormalities were observed in both with myelodysplasia related changes being the most common. Complex karyotype with deletion of 5q constituted approximately a third of all karyotypes in both subgroups. There were 1 patient with intermediate risk cytogenetics in the subgroup without morphologic CR and 5 patients in the subgroup with morphologic CR. Interestingly, in 4/25 patients subclones were diminished by the chemotherapy treatment, however cytogenetically less advanced clones proliferated. Secondly, transplanted or nontransplanted patients were analysed. Allogeneic stem cell transplantation (allo-SCT) was found to be the only curative treatment for patients without CRc after 7+3 and 4+3 regimens. In our cohort, 40% of the patients, who underwent allo-SCT, are alive. Importantly, 67% of the patients, who died after allo-SCT, died of causes unrelated to progression of AML. Nonrelapse mortality is therefore one of the fields where survival could be further improved. KEYWORDS: acute myeloid leukaemia, complete cytogenetic remission, cytogenetic abnormalities, stem cell transplantation, nonrelapse mortality.

[Spontaneous remission of acute myeloid leukemia - a single center case reports]. / Spontánní remise akutní myeloidní leukemie - klinické prípady jednoho centra.

BACKGROUND: Acute myeloid leukemia is a malignant disease characterized by clonal expansion of immature hematopoietic cells - myeloblasts - in the bone marrow. Intensive chemotherapy treatment in elderly patients (over 60) has disappointing results. In these patients, conservative treatment, including compensation of deficiency of red blood cells and platelets by transfusions and treatment of infectious complications is recommended. Also, relatively new treatment with hypometyl agents (azacytidine, decitabine) could be used. DESIGN: The idea of this article is to present a spontaneous remission phenomenon, which has not been published in Czech literature yet. In this article, we present 2 case studies of our patients who were diagnosed with acute myeloid leukemia, were not treated with chemotherapy and spontaneously reached remission of acute myeloid leukemia. CONCLUSION: The mechanisms of the spontaneous remission remain unclear, but we assume positive effect of a severe systemic infection or previous applications of blood transfusions. Antibodies in blood transfusions and a strong immune response to sepsis may have contributed to spontaneous remission.

Proteomic analysis of the presynaptic active zone.

Synaptic vesicles are key organelles in chemical signaling, allowing neurons to communicate with each other and with neighboring cells. Vesicle integral or membrane-associated proteins mediate the various tasks the organelle fulfills during its life cycle. These include organelle transport, interaction with the nerve terminal cytoskeleton, uptake and storage of low molecular weight constituents, and the regulated interaction with the presynaptic plasma membrane, the active zone, during exo- and endocytosis. Converging work from several laboratories within the last 30 years resulted in the molecular and functional characterization of the protein inventory of the synaptic vesicle compartment. Nowadays advances in membrane protein separation and mass spectrometry have dramatically promoted this field resulting in a detailed description of the synaptic vesicle proteome and making synaptic vesicles the best characterized organelles. Recently, the proteome of the active zone was identified using the docked synaptic vesicles as target for immunoisolation. Combining gel-based protein separation techniques, mass spectrometry, and immunodetection, a considerable variety of proteins has been detected in the active zone. This includes synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular signal transduction, a large variety of adhesion molecules and proteins potentially involved in regulating the functional and structural dynamics of the presynapse. Here, we discuss recent information concerning the proteome of the presynaptic active zone, focusing on proteins that are potentially involved in the short- and long-term structural modulation of the mature presynaptic compartment. In addition, we discuss the functional relevance of amyloid precursor protein in these membrane fractions and the putative interplay with direct or indirect interaction partners in the active zone.

The leukemogenic AF4-MLL fusion protein causes P-TEFb kinase activation and altered epigenetic signatures.

Expression of the AF4-MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4-MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4-MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4(me3) and H3K79(me3) histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.

Characteristics and risk factors of oral mucositis after allogeneic stem cell transplantation with FLU/MEL conditioning regimen in context with BU/CY2.

The fludarabine (FLU)/melphalan (MEL) conditioning regimen containing FLU and high-dose MEL was analyzed in comparison with the BU/CY2 regimen to characterize oral mucositis (OM) and risk factors. OM incidence significantly varied between BU/CY2 and FLU/MEL (100 vs 78%, P=0.004), but the incidence of severe OM grades 3-4 WHO and kinetics of OM were fully comparable. Patients with OM persisting on day +21 had more acute GVHD (68 vs 32%, P=0.005), which tended to occur earlier than among those without such prolonged OM. Multivariate analysis showed significant dependency of acute GVHD on severity and prolonged duration of OM and significant correlation between OM severity and its prolonged duration. Body surface area-based dosing in the FLU/MEL regimen led to a wide range of MEL doses administered per kilogram body weight (2.5-5.2 mg/kg, median 3.5). In multivariate analysis, MEL dose per kilogram of body weight was found to be a significant predictor of OM incidence and severity. Female gender and lower body mass index were less important variables than the fact that the actual dose of MEL administered per kilogram of body weight was relatively high when the dosage was calculated on the basis of body surface area.

Differential activity profiles of translation inhibitors in whole-cell and cell-free approaches.

AIMS: Evaluation of the activity profiles of standard prokaryotic translation inhibitors with different physicochemical properties under whole-cell and cell-free conditions. METHODS AND RESULTS: The minimal inhibitory concentration values (cell-free/whole-cell microg ml(-1)) for three aminoglycosides (neomycin, 0.01/6.92; paromomycin, 0.7/1.96; streptomycin 1.45/1.57), three macrolides (erythromycin, 1.53/56.9; josamycin, 1.61/87.7; oleandomycin, 5.12/565.9), chloramphenicol (11.9/3.04), and two tetracyclines (tetracycline hydrochloride, not determined/0.63; minocycline hydrochloride, 2.53/1.09), towards Escherichia coli A19 cells were determined with a microtitre plate-based broth dilution method and compared with values determined in a coupled transcription/translation system based on a S30 extract of the same E. coli strain (cell-free) for the production of the green fluorescent protein. CONCLUSIONS: The analysed prokaryotic translation inhibitors showed substance-specific activity profiles under cell-free vs whole-cell conditions that are explainable by the physicochemical properties of the molecules. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the advantages and limits of cell- free transcription/translation (CFTT) experiments for the discovery of novel antimicrobials. The main advantage is the direct access of the target structures (ribosomes) for the inhibitors, and our results provide an estimation of the concentration necessary to detect new agents. The main limitations are that the inhibitory properties of different agents in CFTT experiments do not necessarily reflect their growth inhibition activity in cell cultures.

[Treatment of invasive candidiasis--recommendations of professional]. / Lécba invazivní kandidózy--doporucení odborných spolecností.

National working group representing clinicians (hematologists, oncologists, infection diseases and ICU specialists), microbiologists, and different special medical societies and working groups prepared evidence-based guidelines for the treatment established fungal infection--invasive candidiasis in the adult hematology and ICU patients. These guidelines updated those published in the Czech Republic in 2003-2004. Evidence criteria of the Infectious Diseases Society of America (IDSA) were used for assessing the quality of clinical trials, and EORTC/MSG Consensus Group for definitions of invasive fungal disease.

[Treatment for invasiveness aspergillosis--recommendations of professionals]. / Lécba invazivní aspergilózy--doporucení odborných spolecností.

An increasing incidence of invasive aspergillosis is observed in most immunocompromised patients, and especially patients with acute leukemia and after hematopoietic stem cell transplantation. In order to decrease the mortality due to this infection, the clinicians need to optimise their treatment choice. The objective of these guidelines is to summarize the current evidence for treatment of invasive aspergillosis. The recommendations have been developed by an expert panel following an evidence-based search of literature with regard to current recommendation of European Conference in Infections in Leukemia and Infectious Diseases Society of America.

Influence of the matrix on analyte fragmentation in atmospheric pressure MALDI.

In this paper, we report the measurement of the degree of analyte fragmentation in AP-MALDI as a function of the matrix and of the laser fluence. The analytes include p-OCH3-benzylpyridinium, three peptides containing the sequence EEPP (which cleave very efficiently at the E-P site), and three deoxynucleosides (dA, dG, and dC), which lose the neutral sugar to give the protonated base. We found that the matrix hardness/softness was consistent when comparing the analytes, with a consensus ranking from hardest to softest: CHCA >> DHB > SA approximately THAP > ATT > HPA. However, the exact ranking can be fluence-dependent, for example between ATT and HPA. Our goal here was to provide the scientific community with a detailed dataset that can be used to compare with theoretical predictions. We tried to correlate the consensus ranking with different matrix properties: sublimation or decomposition temperature (determined using thermogravimetry), analyte initial velocity, and matrix proton affinity. The best correlation was found with the matrix proton affinity.

[Frequency view on genome changes testing]. / Frekvencní pohled na vysetrení odchylek genomu.

Laboratories dealing with human genome, both inherited and acquired changes, dispose with similar methods and technology. The spectrum of genetic tests is relatively broad and the number of mutations or variants tested differs substantially. Also the number of examinations carried out in individual laboratories varies. Data presented in the tables come from the year 2004 and indicate the number of examinations requested and number of positive results. Many laboratories mentioned in the registry CZDDNAL (http://www.uhkt.cz/lab_a_vysetreni/nr lab_dna_diag/dna_lab_db) perform the same tests but there is also a great number of tests carried out by only one laboratory. Reasons of the request, cost-effectiveness and clinical utility of genetic testing is being discussed.

Identification of inducible protein complexes in the phenol degrader Pseudomonas sp. strain phDV1 by blue native gel electrophoresis and mass spectrometry.

Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.

Quality control of tablets by Near Infrared (NIR)-Spectroscopy.

Today, NIR-spectroscopy is an established analytical technique not only in the identification of raw materials but also in the quantification of active ingredients in tablets. In this work calibration models were set up with tablets of the same active ingredient but of miscellaneous origin and manufacturess. Consequently the tablets had different excipients and appearance. The pharmaceutical preparations used included atenolol 100 mg tablets, enalapril 20 mg tablets and acetylsalicylic acid (ASS) tablets of different dosage units. In order to proof if the calibration models set up are generally feasible the assay declared by the manufacturer was used to calculate the partial least square (PLS) calibration. With respect to enalapril tablets simultaneous analysis by HPLC, according to USP 26 was carried out. It was investigated if such methods allow a determination of active ingredients in tablets within limits of +/- 10% of declaration. It was shown that it is possible to set up calibration models to quantify active ingredients in tablets independent of adjuvants or optical appearance. Additionally it could be shown that NIR-spectroscopy is also applicable to determine the concentration of active ingredients in blister-packed tablets.

Nanospray 'taxation' and how to avoid it.

In mass spectrometric analysis with nanospray ionization, some analytes were found to appear in spectra with a delay of tens of minutes, while a few others could not be detected at all. The effect was found to be related to cation-exchange chromatography with negative charge on the glass surface, and with the most affected peptide or protein ions having strong localization of positive charge in blocks of two or more adjacent basic amino acid residues (e.g. melittin). The 'affinity' to the glass surface was studied with a peptide mixture and bovine serum albumin (BSA) tryptic digest solutions at sub-micromolar concentration. About 20% fewer tryptic peptides could be identified from spectra recorded with a glass nanospray capillary compared to those acquired with either conventional 1 microL/min electrospray or a quartz nanospray capillary. Protein identification studies are not likely to be seriously affected by this loss, but other protein applications, such as investigations of mutations or post-translational modifications, may suffer due to reduced sequence coverage. Ways to avoid losses of useful ions are discussed.