Purification and Application of Genetically Encoded Potassium Ion Indicators for Quantification of Potassium Ion Concentrations within Biological Samples.

Vital cells maintain a steep potassium ion (K+ ) gradient across the plasma membrane. Intracellular potassium ion concentrations ([K+ ]) and especially the [K+ ] within the extracellular matrix are strictly regulated, the latter within a narrow range of ∼3.5 to 5.0 mM. Alterations of the extracellular K+ homeostasis are associated with severe pathological alterations and systemic diseases including hypo- or hypertension, heart rate alterations, heart failure, neuronal damage or abnormal skeleton muscle function. In higher eukaryotic organisms, the maintenance of the extracellular [K+ ] is mainly achieved by the kidney, responsible for K+ excretion and reabsorption. Thus, renal dysfunctions are typically associated with alterations in serum- or plasma [K+ ]. Generally, [K+ ] quantifications within bodily fluids are performed using ion selective electrodes. However, tracking such alterations in experimental models such as mice features several difficulties, mainly due to the small blood volume of these animals, hampering the repetitive collection of sample volumes required for measurements using ion selective electrodes. We have recently developed highly sensitive, genetically encoded potassium ion indicators, the GEPIIs, applicable for in vitro determinations of [K+ ]. In addition to the determination of [K+ ] within bodily fluids, GEPIIs proved suitable for the real-time visualization of cell viability over time and the exact determination of the number of dead cells. © 2019 The Authors.

Progressive Multifocal Leukoencephalopathy Following Combined Rituximab-Based Immune-Chemotherapy for Post-transplant Lymphoproliferative Disorder in a Renal Transplant Recipient: A Case Report.

BACKGROUND: Transplant recipients are at risk of developing progressive multifocal leukoencephalopathy (PML), an opportunistic infection due to reactivation of JC virus. Post-transplant lymphoproliferative disorders (PTLDs) represent a common malignancy in this population, and antiCD20-therapy has become an established component of its treatment. CASE PRESENTATION: We describe the first case of a renal allograft transplant recipient with PTLD who received rituximab-based immune-chemotherapy and developed PML shortly thereafter. Despite early suspicion and diagnosis, the disease ran a relentlessly progressive course, and the patient succumbed to his illness shortly thereafter. CONCLUSION: PML should be strongly suspected whenever unusual neurologic symptoms appear in the context of immunosuppression. Clinicians and patients should be aware of the potential for PML after rituximab therapy.

Treatment and outcome of 2904 CML patients from the EUTOS population-based registry.

The European Treatment and Outcome Study (EUTOS) population-based registry includes data of all adult patients newly diagnosed with Philadelphia chromosome-positive and/or BCR-ABL1+ chronic myeloid leukemia (CML) in 20 predefined countries and regions of Europe. Registration time ranged from 12 to 60 months between January 2008 and December 2013. Median age was 55 years and median observation time was 29 months. Eighty percent of patients were treated first line with imatinib, and 17% with a second-generation tyrosine kinase inhibitor, mostly according to European LeukemiaNet recommendations. After 12 months, complete cytogenetic remission (CCyR) and major molecular response (MMR) were achieved in 57% and 41% of patients, respectively. Patients with high EUTOS risk scores achieved CCyR and MMR significantly later than patients with low EUTOS risk. Probabilities of overall survival (OS) and progression-free survival for all patients at 12, 24 and 30 months was 97%, 94% and 92%, and 95%, 92% and 90%, respectively. The new EUTOS long-term survival score was validated: the OS of patients differed significantly between the three risk groups. The probability of dying in remission was 1% after 24 months. The current management of patients with tyrosine kinase inhibitors resulted in responses and outcomes in the range reported from clinical trials. These data from a large population-based, patient sample provide a solid benchmark for the evaluation of new treatment policies.

Clinical impact of bone marrow morphology for the diagnosis of essential thrombocythemia: comparison between the BCSH and the WHO criteria.

Essential thrombocythemia (ET) is currently diagnosed either by the British Committee of Standards in Haematology (BCSH) criteria that are predominantly based on exclusion and not necessarily on bone marrow (BM) morphology, or the World Health Organization (WHO) criteria that require BM examination as essential criterion. We studied the morphological and clinical features in patients diagnosed according either to the BCSH (n=238) or the WHO guidelines (n=232). The BCSH-defined ET cohort was re-evaluated by applying the WHO classification. At presentation, patients of the BCSH group showed significantly higher values of serum lactate dehydrogenase and had palpable splenomegaly more frequently. Following the WHO criteria, the re-evaluation of the BCSH-diagnosed ET cohort displayed a heterogeneous population with 141 (59.2%) ET, 77 (32.4%) prefibrotic primary myelofibrosis (prePMF), 16 (6.7%) polycythemia vera and 4 (1.7%) primary myelofibrosis. Contrasting WHO-confirmed ET, the BCSH cohort revealed a significant worsening of fibrosis-free survival and prognosis. As demonstrated by the clinical data and different outcomes between WHO-diagnosed ET and prePMF, these adverse features were generated by the inadvertent inclusion of prePMF to the BCSH group. Taken together, the diagnosis of ET without a scrutinized examination of BM biopsy specimens will generate a heterogeneous cohort of patients impairing an appropriate clinical management.

The EUTOS population-based registry: incidence and clinical characteristics of 2904 CML patients in 20 European Countries.

This population-based registry was designed to provide robust and updated information on the characteristics and the epidemiology of chronic myeloid leukemia (CML). All cases of newly diagnosed Philadelphia positive, BCR-ABL1+ CML that occurred in a sample of 92.5 million adults living in 20 European countries, were registered over a median period of 39 months. 94.3% of the 2904 CML patients were diagnosed in chronic phase (CP). Median age was 56 years. 55.5% of patients had comorbidities, mainly cardiovascular (41.9%). High-risk patients were 24.7% by Sokal, 10.8% by EURO, and 11.8% by EUTOS risk scores. The raw incidence increased with age from 0.39/100,000/year in people 20-29 years old to 1.52 in those >70 years old, and showed a maximum of 1.39 in Italy and a minimum of 0.69 in Poland (all countries together: 0.99). The proportion of Sokal and Euro score high-risk patients seen in many countries indicates that trial patients were not a positive selection. Thus from a clinical point of view the results of most trials can be generalized to most countries. The incidences observed among European countries did not differ substantially. The estimated number of new CML cases per year in Europe is about 6370.

Azacitidine frontline therapy for unfit acute myeloid leukemia patients: clinical use and outcome prediction.

Hypomethylating agents are able to prolong the overall survival of some patients diagnosed with acute myeloid leukemia. The aim of this study was to evaluate the clinical use of azacitidine as front-line therapy in unfit acute myeloid leukemia (AML) patients and to develop a clinical prediction model to identify which patients may benefit more from the drug. One hundred and ten untreated unfit AML patients received front-line azacitidine therapy in Spain, and response and survival were evaluated in them following European LeukemiaNet (ELN) guidelines. A clinical prediction rule was obtained from this population that was validated and refined in 261 patients treated in France, Austria and Italy. ELN response was achieved in 21.0% of the 371 patients (CI95% 17.0-25.5) and did not depend on bone marrow blast cell percentage. Median overall survival was 9.6 months (CI95% 8.5-10.8) and 40.6% of the patients were alive at 1 year (CI95% 35.5-45.7). European ALMA score (E-ALMA), based on performance status, white blood cell counts at azacitidine onset and cytogenetics, discriminated three risk groups with different survival and response rates. Azacitidine seems a reasonable therapeutic option for most unfit AML patients, i.e. those displaying a favorable or intermediate E-ALMA score.

Chemotherapy line-associated differences in quality of life in patients with advanced cancer.

PURPOSE: The aim of this study was to investigate quality of life (QOL) differences between patients receiving first, second, or third-line palliative chemotherapy (CT).Furthermore, QOL was also compared to a sex- and age-matched sample of healthy controls. METHODS: Patients with different metastatic cancers receiving palliative CT were approached to complete the EORTC QLQ-C30 questionnaire by means of touch-screen computers before the start of CT, after 3 cycles and at the end of cytostatic treatment. RESULTS: One hundred four patients were recruited for QOL assessment (56.9% of patients in first, 22.5% second and 20.6% third- or above-line palliative CT). Compared to healthy controls, they suffered from substantial QOL impairments in all EORTC QLQ-C30 sub-domains. In regard to CT lines, patients with first-line CT reached better scores in emotional and social functioning than second-line patients and less financial difficulties than third-line patients. Despite the high level of impairment in the patient sample, electronic data collection proved to be feasible and well accepted. CONCLUSIONS: The results indicate that patients receiving third- or above-line palliative CT are confronted with stronger QOL impairments than first- and second-line patients. Supported by its feasibility and acceptance of by patients, electronic QOL data capture is an attractive method to screen for symptoms and track their course within clinical routine.

Complications associated with the use of Port-a-Caths in patients with malignant or haematological disease: a single-centre prospective analysis.

Totally implantable central venous catheters are widely used in the management of patients with haematological or malignant disease. This paper investigates device-related complications and compares it with the literature. A total of 143 Port-a-Caths (PaCs) were implanted in 140 patients at a single centre during 2004 and followed until March 2005. Indication for implantation was mainly administration of chemotherapy. High standards of care were applied through intensive training of staff. Complications were registered prospectively and cross-checked with the medical records at the end of the observational period. The ports were in place for a total of 29 107 days (mean 204, range 3-443 days per port). A total of 25 complications were recorded. These included 13 infections [9.1% with 5 cutaneous (3.5%) and 8 systemic (5.6%) infections], one deep vein thrombosis (0.7%). In 6 patients (4.2%) the device had to be removed because of complications. No device-related death was observed. The use of totally implantable central venous catheters for treating haemoto-oncological patients is safe. The need for device removal due to complications was particularly low in this analysis as compared with the literature.

Tuberin, p27 and mTOR in different cells.

Mutations in the genes TSC1 or TSC2 cause the autosomal dominantly inherited tumor suppressor syndrome tuberous sclerosis, which is characterized by the development of tumors, named hamartomas, in different organs. The TSC gene products, hamartin and tuberin, form a complex, of which tuberin is assumed to be the functional component. Both, hamartin and tuberin have been implicated in the control of the cell cycle by activating the cyclin-dependent kinase inhibitor p27 and in cell size regulation by inhibiting the mammalian target of rapamycin (mTOR) a regulator of the p70 ribosomal protein S6 kinase (p70S6K) and its target the ribosomal protein S6. The tuberin/hamartin complex was shown to protect p27 from protein degradation. Within the mTOR signaling pathway tuberin harbors GTPase activating (GAP) potential toward Rheb, which is a potent regulator of mTOR. In this study, we have analyzed the protein levels of tuberin, p27, cyclin D1, mTOR and phospho mTOR Ser2448 (activated mTOR), S6 and phospho S6 Ser240/244 (activated S6) and as controls alpha-tubulin and topoisomerase IIbeta, in ten different cells, including primary normal cells, immortalized and transformed cell lines.

The severity of FIP1L1-PDGFRA-positive chronic eosinophilic leukaemia is associated with polymorphic variation at the IL5RA locus.

We have investigated the hypothesis that constitutional genetic variation in IL-5 signalling may be associated with the development or severity of FIP1L1-PDGFRA-positive chronic eosinophilic leukaemia (CEL) in humans. We genotyped six single-nucleotide polymorphisms (SNP) within or close to the IL5RA or IL5 genes in 82 patients with FIP1L1-PDGFRA-positive CEL plus, as controls, healthy individuals (n=100), patients with FIP1L1-PDGFRA-negative eosinophilia (n=100) or patients with chronic myeloid leukaemia (CML) (n=100). We found no association between SNP allele frequency between FIP1L1-PDGFRA-positive and control cases. However, for FIP1L1-PDGFRA cases, we found an association between the genotype at rs4054760, an SNP in the 5'-UTR of IL5RA and peripheral blood eosinophil count (P=0.026) as well as the presence or absence of tissue infiltration (P=0.032). Although these associations fell below the level of significance once corrected for multiple testing, no such association was seen in FIP1L1-PDGFRA-negative cases and no difference in allele frequencies for rs4054760 was seen in control populations across Europe. Furthermore, in an analysis of 112 patients with CML, IL5RA expression was strongly related to rs4054760 genotype (P<0.001). These data suggest that the variations in IL5RA expression are linked to constitutional IL5RA genotype and severity of FIP1L1-PDGFRA disease.

A randomized study comparing interferon (IFN alpha) plus low-dose cytarabine and interferon plus hydroxyurea (HU) in early chronic-phase chronic myeloid leukemia (CML).

This multicenter randomized phase III study was designed to compare the efficacy and toxicity of IFN alpha-2c (3.5 MU/d) in combination with either araC (10 mg/m(2) d1-10) or hydroxyurea (HU: 25 mg/kg per day) in newly diagnosed CML patients. A total of 114 patients were randomized. Following a median observation period of 36 (range 1-73) months the major cytogenetic response rates were 25 and 27% and the 4-year survival probabilities 62.5 and 63% for the araC and HU group, respectively. While the overall toxicity profile was comparable between both groups, patients in the HU arm exhibited a slightly higher degree of WHO grades 3 and 4 non-hematological toxicities.

Vascular endothelial growth factor in CSF: a biological marker for carcinomatous meningitis.

OBJECTIVE: To determine the value of vascular endothelial growth factor (VEGF) in CSF as a marker for carcinomatous meningitis (CM). METHODS: The concentration of VEGF was measured by ELISA in matched samples of CSF and serum collected from 162 patients. These included patients with solid tumors with CM (n = 11) or brain metastases without concomitant CM (n = 12), paraneoplastic neurologic syndromes (n = 4), viral (n = 15) and bacterial (n = 20) meningitis, and a variety of non-neoplastic and noninfectious neurologic diseases (n = 100). Using CSF/serum albumin ratios, the VEGF index was calculated to estimate the proportion of intrathecally produced VEGF. Immunohistochemical staining for VEGF was performed in a brain metastasis from a mammary carcinoma associated with CM. RESULTS: High VEGF levels (median 6,794.8 pg/mL) were found in CSF of all patients with CM, whereas VEGF levels in matched sera were comparable to other disease groups. In patients with CM, the concentration of VEGF in CSF decreased significantly following antineoplastic treatment. In CSF samples from patients with brain metastases without concomitant CM, VEGF was not detectable. Median VEGF concentration in CSF from patients with acute bacterial meningitis was 38.6 pg/mL, with only 9 of these 17 patients showing detectable VEGF levels in CSF. The VEGF indices in patients with bacterial meningitis were significantly lower than in tumor patients with CM (<22.8 versus >62.3), suggesting that the proportion of intrathecally produced VEGF is much higher in patients with CM as compared with patients with bacterial meningitis. Patients without neoplastic or infectious neurologic disorders consistently showed VEGF levels in CSF below the assay detection limit of 25 pg/mL. Immunohistochemistry revealed strong cytoplasmic staining for VEGF in a metastatic lesion from breast cancer infiltrating the meninges. CONCLUSION: In patients with carcinomatous meningitis, significant amounts of VEGF are released into CSF. This study yields preliminary evidence that VEGF in CSF may be a useful biologic marker for both the diagnosis and evaluation of treatment response in carcinomatous meningitis.