Cytotoxic drug sensitivity of Epstein-Barr virus transformed lymphoblastoid B-cells

BACKGROUND: Epstein-Barr virus (EBV) is the causative agent of immunosuppression associated lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), AIDS related immunoblastic lymphomas (ARL) and immunoblastic lymphomas in X-linked lymphoproliferative syndrome (XLP). The reported overall mortality for PTLD often exceeds 50%. Reducing the immunosuppression in recipients of solid organ transplants (SOT) or using highly active antiretroviral therapy in AIDS patients leads to complete remission in 23–50% of the PTLD/ARL cases but will not suffice for recipients of bone marrow grafts. An additional therapeutic alternative is the treatment with anti-CD20 antibodies (Rituximab) or EBV-specific cytotoxic T-cells. Chemotherapy is used for the non-responding cases only as the second or third line of treatment. The most frequently used chemotherapy regimens originate from the non-Hodgkin lymphoma protocols and there are no cytotoxic drugs that have been specifically selected against EBV induced lymphoproliferative disorders. METHODS: As lymphoblastoid cell lines (LCLs) are well established in vitro models for PTLD, we have assessed 17 LCLs for cytotoxic drug sensitivity. After three days of incubation, live and dead cells were differentially stained using fluorescent dyes. The precise numbers of live and dead cells were determined using a custom designed automated laser confocal fluorescent microscope. RESULTS: Independently of their origin, LCLs showed very similar drug sensitivity patterns against 29 frequently used cytostatic drugs. LCLs were highly sensitive for vincristine, methotrexate, epirubicin and paclitaxel. CONCLUSION: Our data shows that the inclusion of epirubicin and paclitaxel into chemotherapy protocols against PTLD may be justified

Diagnostic accuracy of point-of-care testing for acute coronary syndromes, heart failure and thromboembolic events in primary care: a cluster-randomised controlled trial

Background: Evidence of the clinical benefit of 3-in-1 point-of-care testing (POCT) for cardiac troponin T (cTnT), N-terminal pro-brain natriuretic peptide (NT-proBNP) and D-dimer in cardiovascular risk stratification at primary care level for diagnosing acute coronary syndromes (ACS), heart failure (HF) and thromboembolic events (TE) is very limited. The aim of this study is to analyse the diagnostic accuracy of POCT in primary care. Methods: Prospective multicentre controlled trial cluster-randomised to POCT-assisted diagnosis and conventional diagnosis (controls). Men and women presenting in 68 primary care practices in Zurich County (Switzerland) with chest pain or symptoms of dyspnoea or TE were consecutively included after baseline consultation and working diagnosis. A follow-up visit including confirmed diagnosis was performed to determine the accuracy of the working diagnosis, and comparison of working diagnosis accuracy between the two groups. Results: The 218 POCT patients and 151 conventional diagnosis controls were mostly similar in characteristics, symptoms and pre-existing diagnoses, but differed in working diagnosis frequencies. However, the follow-up visit showed no statistical intergroup difference in confirmed diagnosis frequencies. Working diagnoses overall were significantly more correct in the POCT group (75.7% vs 59.6%, p = 0.002), as were the working diagnoses of ACS/HF/TE (69.8% vs 45.2%, p = 0.002). All three biomarker tests showed good sensitivity and specificity. Conclusion: POCT confers substantial benefit in primary care by correctly diagnosing significantly more patients

Allogeneic stem cell transplantation in children : Identification and prevention of complications : adoptive transfer of EBV-immunity

Allogeneic stem cell transplantation (SCT) can cure leukaemia, aplastic anaemia, and some inborn errors of metabolism and immunodeficiencies in children. As only 113 of patients who need SCT have an HLA-identical sibling, the use of alternative donors has increased, to over 50% of all paediatric SCTs at our centre. The immunological interactions and the clinical course are much more complex in such transplants. The aim of this study is to investigate and find means to manage some of the problems in paediatric SCT, particularly those inherent in matched unrelated donor (MUD) or partially mismatched donor transplants. In a single centre study, we compared the outcome of all 59 children, who underwent MUD SCT, with case controls receiving sibling donor grafts. The 5-year probability of survival was 52 % for MUD vs. 77% for sibling recipients (p= 0.014). In ALL, the survival of the MUD (77%) and sibling group was equal. In SAA, survival was 43% vs. 86% (p=0.09) and in metabolic disorders 63% vs. 89% (p=0.025). The incidence of acute graft-versushost-disease (GVHD and the transplant related mortality (TRM) were higher in the MUD group, while eight relapses occurred in each group. The relapse rate was lower in children with chronic GVHD. These results support the use of MUDs when a sibling donor is not available. They also prompted us to examine further whether GVHD is associated with a graft- versus-leukaemia (GVL) effect, also in childhood acute leukaemia. In all 169 children, who had SCT for ALL and AML at our centre, median time to relapse was 24 months in patients with chronic GVHD and 6 months in those without. The 5-year probabilities of relapse were 30 and 45% (p=0.01). Patients with chronic GVHD had a better survival, 77 vs. 51% (p=0.01). In a Cox regression model, chronic, but not acute GVHD decreased the risk of relapse (RR 0.44) and was predictive of an increased relapsefree survival (RFS) (RR 1.7), most apparent in late-stage disease and in ALL. This is in support of a GVL-effect in childhood leukaemia related to chronic GVHD. To investigate further which factors impact on survival, relapse, TRM and GVHD we included all 181 children transplanted due to leukaemia at our unit. At the end of follow up 54% of the patients were alive, 27% had died in relapse, while 19% had died of other causes. The 5-year probabilities of acute GVHD grade II-IV, TRM, and RFS were 21%, 18% and 49%. Survival was similar in recipients of related (55%) and unrelated grafts (48%). In multivariate analysis, stage of disease (>CR 2) was an independent predictor for relapse and death. The risk of relapse increased significantly after 1992. A donor positive for 3-4 herpes viruses, increased the risk of acute GVHD, TRM and death, while a female to male transplant increased the risk of TRM, particularly in combination with a mismatch. HLA- matching independently improved survival, RFS and TRM. In children with leukaemia, an unrelated donor was not a risk factor for any of the five endpoints analysed. Instead, other donor characteristics such as HLA- matching, herpes virus serology, immunisation and sex were more important for outcome. In mismatched donor SCT, the graft is T-cell depleted (TCD) to prevent life-threatening GVHD. TCD, however, increases the risk of Epstein-Barr virus (EBV)-associated lymphoma (PTLD), rejection and relapse. For six recipients at risk to develop PTLD, EBV-specific cytotoxic donor T lymphocyte (CTL) lines were generated by stimulation with EBV-transformed lymphoblasts, several weeks before SCT. Monitoring of the EBV-DNA load with semiquantitative PCR demonstrated that 4 of 5 recipients of TCD grafts and one Wiskott-Aldrich patient had a 4 to 5-log increase of EBV-DNA within 1-3 months after SCT, predicting a high risk of PTLD. In the other three recipients of unmanipulated grafts, the increases were moderate. Two to 4 infusions Of 1x107of the EBVCTLs / m2 resulted in a 2- to 3-log decrease of EBV-genomes in four patients and in stabilisation of the virus load on a moderate level in one case. One child, who received a T-cell culture lacking in EBV-specificity, progressed to fatal PTLD. The results suggest that a rapid increase of the EBV-load occurs in the absence of EBV-specific T-cell precursors, after TCD or in the presence of immunodeficiency. Infusion of EBV-CTLs early after SCT appears to prevent EBV-PTLD. To investigate the increased EBV-DNA load and the state of viral latency in these patients, we analysed EBVDNA in serum, applied limiting dilution analysis of EBV-DNA in mononuclear cells and RT-PCRs for viral gene expression . The increased virus load is suggested to be due to an expansion of a latently infected B-cell compartment that contains less than 10 EBV genome copies per cell and expresses EBERs, LMP-2A and occasionally LMP 1 and EBNA 1. This is compatible with latency forms I-II in non-proliferating B-cells, but not latency Ill. Most likely this is due to proliferation of EBV+ B cells somewhere in the lymphatic compartment, outside peripheral blood, as we did not find evidence of active replication of virus

Predictive Value of the Spinal Instability Neoplastic Score for Survival and Ambulatory Function After Surgery for Metastatic Spinal Cord Compression in 110 Patients with Prostate Cancer

STUDY DESIGN: We retrospectively analyzed Spinal Instability Neoplastic Score (SINS) in 110 patients with prostate cancer operated for metastatic spinal cord compression (MSCC). OBJECTIVE: We aimed to investigate the association between SINS and clinical outcomes after surgery for MSCC in patients with prostate cancer. SUMMARY OF BACKGROUND DATA: The SINS is a useful tool for assessing tumor-related spinal instability, but its prognostic value regarding survival and neurological outcome is still controversial. METHODS: We analyzed 110 consecutive patients with prostate cancer who underwent surgery for MSCC. The patients were categorized according to their SINS. Patients with castration-resistant prostate cancer (CRPC, n = 84) and those with hormone-naïve disease (n = 26) were analyzed separately. RESULTS: In total, 106 of 110 patients met the SINS criteria for potential instability or instability (scores 7-18). The median SINS was 10 (range 6-15) for patients with CRPC and 9 (7-16) for hormone-naïve patients. In the CRPC group, the SINS was classified as stable (score 0-6) in 4 patients, as potentially unstable (score 7-12) in 70 patients, and as unstable (score 13-18) in 10 patients. In the hormone-naïve group, 22 patients met the SINS criteria for potential instability and 4 patients for instability. There was no statistically significant difference in the overall risk for death between the SINS potentially unstable and unstable categories (adjusted hazard ratio 1.3, P = 0.4), or in the risk of loss of ambulation 1 month after surgery (adjusted odds ratio 1.4, P = 0.6). CONCLUSION: The SINS is helpful in assessing spinal instability when selecting patients for surgery, but it does not predict survival or neurological outcomes. Patients with a potential spinal instability benefit equally from surgery for MSCC as do patients with spinal instability.Level of Evidence: 3

Design and rationale of the Danish trial of beta-blocker treatment after myocardial infarction without reduced ejection fraction: study protocol for a randomized controlled trial.

Treatment with beta-blockers is currently recommended after myocardial infarction (MI). The evidence relies on trials conducted decades ago before implementation of revascularization and contemporary medical therapy or in trials enrolling patients with heart failure or reduced left ventricular ejection fraction (LVEF ≤ 40%). Accordingly, the impact of beta-blockers on mortality and morbidity following acute MI in patients without reduced LVEF or heart failure is unclear. The Danish trial of beta-blocker treatment after myocardial infarction without reduced ejection fraction (DANBLOCK) is a prospective, randomized, controlled, open-label, non-blinded endpoint clinical trial designed to evaluate the efficacy of beta-blocker treatment in post-MI patients in the absence of reduced LVEF or heart failure. We will randomize 3570 patients will be randomized within 14 days of index MI to beta-blocker or control for a minimum of 2 years. The primary endpoint is a composite of all-cause mortality, recurrent MI, acute decompensated heart failure, unstable angina pectoris, or stroke. The primary composite endpoint will be assessed through locally reported and adjudicated endpoints supplemented by linkage to the Danish national registers. A number of secondary endpoints will be investigated including patient reported outcomes and cardiovascular mortality. Data from similar ongoing trials in Norway and Sweden will be pooled to perform an individual patient data meta-analysis. DANBLOCK is a randomized clinical trial investigating the effect of long-term beta-blocker therapy after myocardial infarction in patients without heart failure and reduced LVEF. Results from the trial will add important scientific evidence to inform future clinical guidelines. Clinicaltrials.gov, NCT03778554. Registered on 19 December 2018. European Clinical Trials Database, 2018-002699-42, registered on 28 September 2018.The trial has received grants from the Novo Nordisk Foundation and the Danish Heart Foundation. The Novo Nordisk Foundation and the Danish Heart Foundation are not otherwise involved in the trial.S

Both high and low levels of cellular Epstein-Barr virus DNA in blood identify failure after hematologic stem cell transplantation in conjunction with acute GVHD and type of conditioning

The level of Epstein-Barr virus DNA in blood has proven to be a biomarker with some predictive value in allogeneic hematopoietic stem cell transplantation patients (HSCT). We evaluated the impact of EBV load on survival of 51 patients (32M/19F, median age: 32 years, from < 1 to 68 years old), who had received HSCT for different types of malignancies (49 cases) or non-malignancies (2 cases). The overall survival [1]was compared between patients with extreme and moderate cell bound EBV DNA levels. Different sources of stem-cells (peripheral blood stem, n = 39; bone marrow, n = 9; or umbilical cord blood, n = 3) were used. Twenty patients received reduced-intensity conditioning regimen while the other 31 received myeloablative conditioning. Patients with high or very low level of cell bound EBV-DNA levels had a shorter OS than those with moderate EBV load: OS at 5 years was 67% vs 90% (p < 0.03). There was a conspicuous relationship between EBV load and the reconstitution dynamics of total and EBV-specific T cells, CD4+ and CD4- CD8- (double negative) T cells in the few patients where it was analyzed. This was not statistically significant. Two other factors were associated to early mortality in addition to high or low EBV load: acute GVHD II-IV (p < 0.02) and pre-transplant conditioning with total body irradiation (TBI) ≥6 Gy, (p < 0.03). All the patients meeting all three criteria died within two years after transplantation. This points to a subgroup of HSCT patients which deserve special attention with improvement of future, personalized treatment