Targeted next generation sequencing panel for HPV genotyping in cervical cancer.

Cervical cancer are generally caused by a persistent infection with the oncogenic virus, HPV. Patients with HPV integration are more prone to develop cervical cancer than patients without integration. In this proof-of-concept study, we aimed to develop a sensitive method based on targeted amplicon based NGS for early and precise detection of high-risk HPV-genotypes that are highly associated with the development of cervical cancer. Furthermore, we aimed to investigate if amplicon based NGS allowed for HPV genotyping in cervical lesions and whether it could detect HPV integration. The cohort included a group of CIN3+ biopsies (n = 64), CIN2 samples that progressed (n = 5), CIN2 samples that regressed (n = 3), healthy controls (n = 10), and plasma samples (n = 10) from cervical cancer patients. Sequencing was performed using a custom targeted NGS panel designed to detect all 25 high-risk and probably high-risk and two low-risk HPV genotypes. The method was validated by the SPF10 PCR-DEIA-LiPA25 assay. In the cohort, the following HPV genotypes were identified: HPV-16, 18, 31, 33, 35, 45, 51, 52, 56, 58, and 59. When comparing the results from the SPF10 PCR-DEIA-LiPA25 analyses with the NGS analyses, there was close to a perfect agreement (K = 0.92) among the genotyped HPV types, while in the two cases with complete disagreement, a third assay was applied, and here the results of the NGS analyses were confirmed. Whereas multiple HPV types were detected by the SPF10 PCR-DEIA-LiPA25 assay, the NGS analysis clearly suggest that there is one predomentant HPV type. The NGS assay was capable of detecting HPV-16 in a previous false-negative sample classified by the INNO-LiPA assay, emphasizing the importance of including multiple regions of the HPV genome when genotyping. For the 10 plasma samples, our NGS analyses showed full agreement with the digital droplet PCR (ddPCR) analyses of HPV positive as well as negative plasma samples. Lastly, the custom panel was capable of detecting the integration of HPV-16 in the SiHa cell line. The HPV panel provides a highly cost-effective method for HPV detection and genotyping, as exemplified by a list price of around 75 € per sample. In conclusion, the current study demonstrates that targeted NGS is capable of detecting and genotyping HPV in both FFPE biopsies and plasma samples. This method provides for early diagnosis and prognosis of cervical cancer disease progression, thereby optimizing the potential of recovery and survival for these patients.

Circulating cell-free DNA as predictor of treatment failure after neoadjuvant chemo-radiotherapy before surgery in patients with locally advanced rectal cancer.

Background: Treatment of patients with locally advanced rectal cancer (LARC) is based on a combination of chemo-radiotherapy (CRT) and surgery. The rate of distant recurrences remains over 25%. Circulating cell-free DNA (cfDNA) in plasma is a mixture of normal and cancer-specific DNA segments and is a promising biomarker in patients with colorectal cancer. The aim of our study was to investigate plasma cfDNA as a prognostic marker for outcome in patients with LARC treated with neoadjuvant CRT and surgery. Patients and methods: In total, 123 patients with LARC were included in 2 biomarker studies. Patients were treated with neoadjuvant CRT before TME surgery. Fifty-two (42%) of the patients received induction chemotherapy with capecitabine + oxaliplatin. Total cfDNA was measured by direct fluorescent assay in EDTA plasma samples obtained at baseline, after induction chemotherapy, and after CRT. Serial samples 5 years after surgery were collected in 51 patients (41%). Results: Median follow-up was 55 months. Distant or local recurrence was seen in 30.9% of the patients. Patients with baseline cfDNA levels above the 75th quartile had a higher risk of local or distant recurrence and shorter time to recurrence compared with patients with plasma cfDNA below the 75th percentile (HR = 2.48, 95% CI: 1.3-4.8, P = 0.007). The same applied to disease-free survival (DFS) (HR = 2.43, 95% CI: 1.27-4.7, P = 0.015). In multivariate analysis, a high cfDNA level was significantly associated with time to progression and DFS. During follow-up, the association remained significant regardless of time point for sample analysis. Conclusion: We have demonstrated an association between a high baseline plasma level of cfDNA and increased risk of recurrence, shorter time to recurrence, and shorter DFS in patients with LARC. Consequently, cfDNA could potentially improve pre- and post-treatment risk assessment and facilitate individualized therapy for patients with LARC.

Concizumab, an anti-tissue factor pathway inhibitor antibody, induces increased thrombin generation in plasma from haemophilia patients and healthy subjects measured by the thrombin generation assay.

AIMS: Concizumab, a humanized monoclonal antibody against tissue factor pathway inhibitor (TFPI), is being developed as a subcutaneously (s.c.) administered treatment for haemophilia. It demonstrated a concentration-dependent procoagulant effect in functional TFPI assays; however, global haemostatic assays, such as the thrombin generation assay (TGA), offer a more complete picture of coagulation. We investigated how concizumab affects thrombin generation following ex vivo spiking in plasma from haemophilia patients using the TGA, and if the assay can detect the effect of multiple s.c. concizumab doses in healthy subjects. METHODS: For the ex vivo spiking study, platelet-poor plasma (PPP) from 18 patients with severe haemophilia was spiked with 0.001-500 nm concizumab. For the multiple-dosing study, four healthy males received concizumab 250 µg kg-1 s.c. every other day for eight doses; blood was collected before and after dosing and processed into PPP. In both studies, thrombin generation was measured using a Calibrated Automated Thrombogram® system with 1 pm tissue factor. RESULTS: In spiked samples from haemophilia patients, peak thrombin and endogenous thrombin potential (ETP) increased concentration dependently, reaching near-normal levels at concizumab concentrations >10 nm. Repeated s.c. doses of concizumab in healthy subjects increased both peak thrombin and ETP; these effects were sustained throughout the dosing interval. CONCLUSIONS: Thrombin generation assay demonstrated increased thrombin generation with concizumab after ex vivo spiking of haemophilia plasma and multiple s.c. doses in healthy subjects, supporting both the utility of the TGA in evaluating concizumab treatment and the potential of s.c. concizumab as a novel haemophilia therapy.

Serological biomarkers detect active joint destruction and inflammation in patients with haemophilic arthropathy.

INTRODUCTION: Progressive arthropathy caused by recurrent joint bleeds is a severe complication in haemophilia. AIM: We investigated whether biomarkers of cartilage and bone degradation, and inflammation were altered in haemophilia patients and whether these biomarkers could identify haemophilia patients with arthropathy. METHODS: Serum from 35 haemophilia patients with varying degrees of arthropathy and 43 age- and gender-matched control subjects were analysed. Biomarkers of cartilage degradation (C2M, COMP, CTX-II, ADAMTS5), cartilage formation (PRO-C2), bone formation (PINP), bone resorption (CTX-I) and inflammation (hsCRP, CRPM) were measured by ELISA. Arthropathy was assessed by radiological evaluation (Pettersson score) and physical examination (Gilbert score). RESULTS: In patients with haemophilia, cartilage degradation, measured by C2M, CTX-II and COMP, was increased by 25% (P < 0.05) compared with control subjects. Levels of the cartilage degradation enzyme, ADAMTS5, were 10% lower in haemophilia patients (P < 0.05). Bone formation (PINP) was reduced by 25% (P < 0.05) in haemophilia patients, whereas bone resorption (CTX-I) was increased by 30% (P < 0.001). Acute inflammation (hsCRP) was increased by 50% (P < 0.01), whereas chronic inflammation (CRPM) was decreased by 25% (P < 0.0001). The hsCRP/CRPM ratio was 60% higher (P < 0.001) in haemophilia patients relative to control subjects. A biomarker panel combining C2M, CRPM, and ADAMTS5 could distinguish haemophilia patients from control subjects with 85.3% accuracy (P < 0.0001). We found no strong correlation between biomarkers and radiological and physical examination of the joint. CONCLUSION: Biomarkers detect increased cartilage and bone degradation, and altered inflammatory activity in haemophilia patients with arthropathy. These biomarkers could potentially be used to identify patients with progressing joint disease.

Post-cardiac arrest level of free-plasma DNA and DNA-histone complexes.

BACKGROUND: Plasma DNA-histone complexes and total free-plasma DNA have the potential to quantify the ischaemia-reperfusion damages occurring after cardiac arrest. Furthermore, DNA-histone complexes may have the potential of being a target for future treatment. The aim was to examine if plasma DNA-histone complexes and the levels of total free-plasma DNA were elevated in post-cardiac arrest patients compared with healthy individuals, and to examine if these biomarkers were capable of predicting mortality. METHODS: We included 42 comatose out-of-hospital cardiac arrest patients and collected blood samples after 22, 46 and 70 h. Samples for DNA-histone complexes were quantified by Cell Death Detection ELISAplus . The total free-plasma DNA analyses were quantified with qPCR by analysing the Beta-2 microglobulin gene. The control group comprised 40 healthy individuals. RESULTS: We found no difference in the level of DNA-histone complexes between the 22-h sample and healthy individuals (P = 0.10). In the 46-h sample, there was an increased level of DNA-histone complexes in non-survivors compared with survivors 30 days after the cardiac arrest (P < 0.01) and the area under the ROC curve was 0.78 (95% confidence interval: 0.59;0.96). The level of total free-plasma DNA was increased in the 22-h sample compared with healthy individuals (P < 0.001) but no significant difference was found between non-survivors and survivors 30 days after the cardiac arrest (all P ≥ 0.06). CONCLUSION: An increased level of DNA-histone complexes was associated with increased mortality and that the level of total free-plasma DNA was elevated post-cardiac arrest.

Monitoring bypassing agent therapy - a prospective crossover study comparing thromboelastometry and thrombin generation assay.

The aim of this study was to evaluate the capability of thromboelastometry (ROTEM) and thrombin generation assay (TGA) to monitor the treatment response of bypassing agent (BPA) therapy and to study whether one method is superior to another. In a prospective crossover study haemophilia A patients with high titre inhibitors were included to receive a dose of 75 U kg(-1) activated prothrombin complex concentrates (aPCC) intravenously. Blood sampling was performed at baseline, 15, 30 min, 1, 2, 3 and 4 h post-infusion for TGA and ROTEM analysis. After a washout period of 14 days the subjects received recombinant FVIIa (rFVIIa) at a dose of 90 µg kg(-1) and similar blood sampling was performed. Healthy subjects were used as controls. Six haemophilia A patients with inhibitors were included. We found that TGA parameters endogenous thrombin potential (ETP) and peak thrombin increased 2-3 folds from baseline 15-30 min after infusion. ROTEM parameters MaxVel and maximum clot firmness increased to a level comparable to that of healthy controls. An individual difference in response was observed for different parameters among participants. ETP and peak thrombin were almost two-fold greater following aPCC infusion compared to rFVIIa, whereas ROTEM parameters showed no difference in response between the two products. The study showed that ROTEM and TGA have a great potential to evaluate the effect of BPA in haemophilia patients with inhibitors. TGA seemed to be more sensitive than ROTEM in reflecting the difference in treatment response between aPCC and rFVIIa. Additional prospective clinical studies are needed to clarify which assay and what parameters are clinically predictive.

Pain following blood donation: a questionnaire study of long-term morbidity (LTM) in blood donors.

BACKGROUND: Nerve injury is a main cause of long-term morbidity following blood donation, but little is known about symptoms, impact, prognosis and underlying cause. MATERIALS AND METHODS: A questionnaire, designed to characterize pain and estimate the prevalence of neuropathic pain, was sent to all blood donors registered with a complication related to 3 297 674 blood donations in Denmark from 2000-2009, with a local complication mainly characterized by pain, with severity grade 'long-term morbidity' and imputability grade 'definite' or 'probably'. RESULTS: The questionnaire was sent to 152 donors (4·6 per 100 000 donations). Response rate was 88/152 (57·9%). At the time of the questionnaire, which was between 12 months and 10 years after the blood donation, 69/88, who responded (78·4%) still experienced symptoms. Of the 69 donors with persistent symptom, pain occurred in 51 donors (74%) was moderate to severe in 24/69 donors (35%) and had an impact on daily activity in 17/69 (25%). Neuropathic pain was estimated to be the underlying cause of symptom in 30-52% of the 69 donors with persistent symptoms, using three different systems for estimation, corresponding to 0·6-1·1/100 000 donations. DISCUSSION: Although a rare complication, nerve injury after blood donation may lead to long-term morbidity and may become chronic in a small proportion of donors. The most common symptoms are pain, and we estimate that neuropathic pain can be the underlying cause.

Damage tolerance and structural monitoring for wind turbine blades.

The paper proposes a methodology for reliable design and maintenance of wind turbine rotor blades using a condition monitoring approach and a damage tolerance index coupling the material and structure. By improving the understanding of material properties that control damage propagation it will be possible to combine damage tolerant structural design, monitoring systems, inspection techniques and modelling to manage the life cycle of the structures. This will allow an efficient operation of the wind turbine in terms of load alleviation, limited maintenance and repair leading to a more effective exploitation of offshore wind.

Long-standing prophylactic therapy vs. episodic treatment in young people with severe haemophilia: a comparison of age-matched Danish and Russian patients.

Two distinctly different substitution principles are commonly used in haemophilia: treatment at bleeding episodes only referred to as on-demand treatment, and prophylactic factor administration. The aim of the cross-sectional study which was undertaken in young patients suffering severe haemophilia A was to challenge our hypothesis that on-demand treatment is inferior to prophylactic substitution in prevention of chronic joint disease at young age. The method involved an investigation of 40 patients from Russia (n = 27) and Denmark (n = 13) born between 1975 and 1990 with no history of inhibitors; Russian patients had exclusively received factor VIII on demand, while Danish patients were managed with prophylactic treatment during a mean period of 16 years since median age of 5 years. The study endpoints were clinical joint scores, Quality of Life scores and functional independence scores. Matched by identical age (±1 year) 13 Danish and 13 Russian patients were compared, while 14 age similar Russian patients served as controls. Demographic data among all groups were quite comparable. The results are that Russian patients presented with clinical joint scores at 27 ± 8.5 (mean ± SD) while matched Danish counterparts scored 3.8 ± 5.3 (mean ± SD), differences being highly significant. The number of joint bleeds in recent 5 years were 199.5 ± 135 (mean ± SD) vs. 8.1 ± 8.7 (mean ± SD). Likewise, Quality of Life and functional independence scores were significantly higher in patients on prophylaxis as compared to on-demand treatment. In conclusion, the study outcomes confirmed our hypothesis. Longer term prophylactic factor administration during childhood and adolescence prevents joint destruction.

Tranexamic acid as adjunct therapy to bypassing agents in haemophilia A patients with inhibitors.

Haemophilia patients with inhibitors require bypassing agents (BPA) like activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rFVIIa) to control bleeds. Adjunct tranexamic acid (TXA) may improve haemostasis. The objective of this study was to investigate safety and haemostatic effect of TXA given in combination with BPA. Healthy volunteers (N = 5) and haemophilia inhibitor patients (N = 6) were enrolled in a prospective case crossover design. Controls were treated with TXA 20 mg kg(-1) orally (O.R.) Patients were treated with aPCC 75 IU kg(-1) intravenous (I.V.) on day 1 followed by TXA 20 mg kg(-1) O.R. combined with aPCC 75 IU kg(-1) I.V. on day 2. A 14-day washout occurred before crossover to rFVIIa 90 µg kg(-1) I.V. ±TXA. Safety evaluation and blood sampling processes were performed at baseline, 15, 30, 60, 120, 180 and 240 min post treatment. Primary outcome was maximum clot firmness (MCF) evaluated by whole blood thromboelastometry using a TF + tissue plasminogen activator-based assay. Healthy controls showed a 20-fold increase in MCF following TXA. Adjunct TXA to aPCC or rFVIIa induced a significant increase in MCF (P < 0.0001) reaching levels indistinguishable from healthy controls treated with TXA (P > 0.05). Infusion of aPCC or rFVIIa alone induced only 3-10 fold increase in MCF from baseline, with a decline in MCF starting after 60-120 min. TXA did not increase the endogenous thrombin potential. No clinical or laboratory signs of thromboembolic events, disseminated intravascular coagulation, or hypercoagulability were observed. Combination of aPCC or rFVIIa with TXA normalizes clot stability in haemophilia patients with inhibitor as compared to healthy controls. No clinical or laboratory adverse events were observed.

Transfusion-associated anaphylaxis during anaesthesia and surgery--a retrospective study.

BACKGROUND AND OBJECTIVES: Transfusion-associated anaphylaxis (TAA) is a severe adverse reaction reported to occur in 1:9000-90,000 transfusions. According to the Danish Registration of Transfusion Risks (DART), the frequency is 1:300,000 transfusions, which suggests insufficient reporting of TAA in Denmark. Our aims were to identify possible cases of TAA, to characterize their symptoms and tryptase levels and to investigate the reporting of TAA to the haemovigilance systems. MATERIAL AND METHODS: We reviewed 245 patients with suspected allergic reactions during anaesthesia and surgery, investigated at the Danish Anaesthesia Allergy Centre (DAAC). Based on the outcome of this investigation, the patients were classified as DAAC positive (confirmed hypersensitivity to identified agent, n = 112), or DAAC negative (no confirmed hypersensitivity, n = 133). Data on case history, details of blood transfusion and results of laboratory and clinical investigations were collected. TAA cases were identified according to the recommendations of the International Society of Blood Transfusion (ISBT). RESULTS: Ten possible TAA cases (30% of all transfused patients) were identified, all DAAC negative. The frequency of elevated serum tryptase, hypotension and male sex was significantly higher among these cases compared with the remaining DAAC negative (P < 0·05), but not different from the DAAC-positive patients. One case had been reported to the Blood Bank haemovigilance system and none to DART. CONCLUSION: We identified unreported cases of possible TAA, which resembled the DAAC-positive patients with respect to elevated tryptase and symptoms. By applying the ISBT criteria of adverse transfusion reactions, we conclude that TAA during anaesthesia and surgery is likely to be underreported in Denmark.

A five-gene qPCR signature can classify type 2 asthma comparably to microscopy of induced sputum from severe asthma patients.

Asthma is a heterogenous disease characterized by airway inflammation and variable expiratory airflow limitation resulting in variable respiratory symptoms. Characterization of airway inflammation is important to choose the optimal treatment for severe asthma patients eligible for biological treatment. However, counting cells in induced sputum samples are a time-consuming process, highly dependent on personal skills. Replacing eosinophil and neutrophil cell counting with qPCR for transcripts of selected mast cell, and basophil genes may provide more reproducible results. Aims: The objective of this study was to compare qPCR with microscopy in asthma endotyping. Methods: A qPCR method measuring five mast cell/basophil genes was applied on induced sputum samples from 30 severe asthma patients and compared with microscopy. Target gene Ct-values (CPA3, GATA2, HDC, MS4A2, TPSAB1/TPSB2) were referenced to household ß-actin Ct values as a measure of relative mRNA abundance of the target in each sample. Target/ß-actin-ratios in eosinophilic and non-eosinophilic groups determined by microscopy with an eosinophil threshold of 3% in 400 cells were compared using Mann-Whitney U Test. Spearman´s correlations were used to test for correlation between targets vs. FENO and targets vs. blood eosinophil counts. Results: The study demonstrated a statistical difference in relative mRNA abundance for four mast cell/basophil specific genes. CPA3, GATA2, HDC and MS4A2 were elevated in eosinophilic asthma versus non-eosinophilic asthma patients. The study found that GATA2, CPA3, MS4A2 and TPSAB1/TPSB2 transcripts are positively correlated with FENO. Neither the five mast cell genes nor the five-gene signature correlated with blood eosinophils. The five-gene signature with a target/ß-actin-ratio cut-off ≥2 generated sensitivity = 87%, specificity = 94%, NPV = 88% and PPV = 92% compared to microscopy. Conclusion: This study confirms the contribution of mast cells in the pathogenesis of EA and suggests that mast cell mRNA markers could be one of the biomarkers used to identify EA.

Directing HER4 mRNA expression towards the CYT2 isoform by antisense oligonucleotide decreases growth of breast cancer cells in vitro and in vivo.

BACKGROUND: The tyrosine kinase receptor HER4 is a member of the epidermal growth factor receptor (EGFR) family. It plays diverse roles in cancer development and cancer progression and can both exert oncogenic and tumour-suppressive activities. Alternatively spliced isoforms of HER4 are critical to the different signalling possibilities of HER4. METHODS: We use a splice-switching oligonucleotide (SSO) to direct the alternative splicing of HER4 from the CYT1 to the CYT2 isoform in HER4-expressing breast cancer cells. RESULTS: Treatment with a target-specific SSO was accompanied by a decreased growth of the cells (P<0.0001). In addition, the SSO treatment induced a decreased activity of Akt. We confirmed the SSO-dependent switching of the HER4 isoform CYT1 to CYT2 expression in a xenografted mouse tumour model driven by subcutaneously injected MCF7 cells. We hence demonstrated the feasibility of SSO-directed splice-switching activity in vivo. Furthermore, the SSO treatment efficiently decreased the growth of the xenografted tumour (P=0.0014). CONCLUSION: An SSO directing the splicing of HER4 towards the CYT2 isoform has an inhibitory effect of cancer cell growth in vitro and in vivo. These results may pave the way for the development of new anticancer drugs in HER4-deregulated cancers in humans.

Forward error correction supported 150 Gbit/s error-free wavelength conversion based on cross phase modulation in silicon.

We build a forward error correction (FEC) module and implement it in an optical signal processing experiment. The experiment consists of two cascaded nonlinear optical signal processes, 160 Gbit/s all optical wavelength conversion based on the cross phase modulation (XPM) in a silicon nanowire and subsequent 160 Gbit/s-to-10 Gbit/s demultiplexing in a highly nonlinear fiber (HNLF). The XPM based all optical wavelength conversion in silicon is achieved by off-center filtering the red shifted sideband on the CW probe. We thoroughly demonstrate and verify that the FEC code operates correctly after the optical signal processing, yielding truly error-free 150 Gbit/s (excl. overhead) optically signal processed data after the two cascaded nonlinear processes.

Platelet activation and aggregation: the importance of thrombin activity--a laboratory model.

This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated by tissue factor evaluated by means of impedance aggregometry. Citrated whole blood from healthy volunteers and haemophilia A patients with the addition of inhibitors of the contact pathway and fibrin polymerization was evaluated. In healthy persons, a second wave of platelet aggregation was found to coincide with the thrombin burst and to be abolished by thrombin inhibitors. In this system, platelet aggregation in severe haemophilia A (n = 10) was found to be significantly decreased as compared with healthy individuals (912 ± 294 vs. 1917 ± 793 AU × min, P = 0.003), most probably due to the weak level of thrombin generation. For the first time, analysis of platelet aggregation as induced by endogenously generated thrombin was demonstrated. The new method makes it possible to explore the influence of the coagulation system on platelet function. In contrast to the general understanding, the data suggest that the impaired thrombin generation in haemophilia may affect platelet activation. Future studies will address whether our results may contribute to understanding differences in bleeding phenotypes and response to haemostatic substitution observed among patients.

Novel coagulation factor concentrates: issues relating to their clinical implementation and pharmacokinetic assessment for optimal prophylaxis in haemophilia patients.

Prophylaxis is considered the optimal treatment regimen for patients with severe haemophilia, and may be especially important in the prevention of joint disease. Novel coagulation factor concentrates with prolonged half-lives promise to improve patient treatment by enabling prophylaxis with less frequent dosing. With the call to individualize therapy in haemophilia, there is growing awareness of the need to use pharmacokinetic (PK) assessments to tailor prophylaxis. However, for new factor concentrates, it is not yet known which PK values will be most informative for optimizing prophylaxis. This topic was explored at the Eighth Zurich Haemophilia Forum. On the basis of our clinical experience and a discussion of the literature, we report key issues relating to the PK assessment of new coagulation factors and include suggestions on the implementation of PK data to optimize therapy. As both inter- and intra-individual variability in factor half-life have been reported, we suggest that frequent PK assessments should be conducted. However, to diminish the burden of more frequent sampling, sparser sampling strategies and the use of population modelling should be considered. Guidelines on how to assay new factor concentrates, and which PK parameters should be measured, are needed. Concerns were raised regarding the possibility of breakthrough bleeding, and current thinking on how to prevent breakthrough bleeding may no longer be appropriate. Finally, as treatment adherence may be more important to ensure that a therapeutic level of a new coagulation factor concentrate is maintained, behavioural techniques could be implemented to help to improve treatment adherence.

Natural killer cell activity in metastatic castration resistant prostate cancer patients treated with enzalutamide.

Metastatic castration resistant prostate cancer (mCRPC) is still the lethal stage for the whole spectrum of prostate cancer disease. Even though different treatment options have been introduced in the last decade with a significant survival improvement for this population, a lack of more reliable prognostic and predictive markers is still one of the main clinical challenges in management of mCRPC. The aim of this study was to investigate the correlation between Natural Killer cell activity (NKA) and both treatment effect and outcomes in patients with mCRPC treated with enzalutamide. A total of 87 patients with mCRPC treated with enzalutamide as the first line treatment were enrolled. NKA was estimated at baseline and prior to each treatment cycle. Endpoints included both treatment effect with biochemical response (BR), biochemical progression (BP) and radiological progression (RP), as well as outcome data with overall survival (OS), radiologic progression free survival (rPFS), and time to next treatment (TTT). At the time of BR, interferon-gamma (IFNγ) decreased significantly compared to levels detected at baseline (z-score = 2.33, p = 0.019). Regarding outcome data, the whole cohort was divided into four groups according to the change of IFNγ level during the first 3 cycles of enzalutamide treatment. In group 1 (n = 42) the IFNγ level remained within a normal range (≥ 250 pg/mL),while in group 2 (n = 7) it increased from an abnormal (< 250 pg/mL) to a normal level. In group 3 (n = 13) it dropped to an abnormal level, and it remained at an abnormal level during treatment in group 4 (n = 17). Patients in group 2 showed the worst prognosis with shorter both rPFS and TTT (HR 4.30, p = 0.037; and HR 6.82, p = 0.011, respectively). In this study inverse correlations between NKA and both treatment response and outcomes was observed in mCRPC patients receiving enzalutamide, suggesting an unfavourable role of NK cells in the late stage of PCa.

Emerging treatment strategies for trauma-induced coagulopathy.

BACKGROUND: Trauma-induced coagulopathy has a multifactorial aetiology. Coagulopathy is related to blood loss including consumption of clotting factors and platelets and haemodilution. Additionally hyperfibrinolysis, hypothermia, acidosis and metabolic changes affect the coagulation system. METHODS: This is a review of pathophysiology and new treatment strategies for trauma-induced coagulopathy. RESULTS: Paradigms are actively changing and there is still a shortage of data. The aim of any haemostatic therapy is to control bleeding and minimize blood loss and transfusion requirements. Transfusion of allogeneic blood products as well as trauma-induced coagulopathy cause increased morbidity and mortality. Current opinion is based on present studies and results from small case series, combined with findings from experimental studies in animals, in vitro studies and expert opinions, as opposed to large, randomized, placebo-controlled studies. A summary of new and emerging strategies, including medical infusion and blood products, to beneficially manipulate the coagulation system in the critically injured patient is suggested. CONCLUSION: Future treatment of trauma-induced coagulopathy may be based on systemic antifibrinolytics, local haemostatics and individualized point-of-care-guided rational use of coagulation factor concentrates such as fibrinogen, prothrombin complex concentrate, recombinant factor VIIa and factor XIII. The authors speculate that timely and rational use of coagulation factor concentrates will be more efficacious and safer than ratio-driven use of transfusion packages of allogeneic blood products. Copyright © 2011 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Global haemostasis and point of care testing.

The evaluation of the coagulation profile has used so far either clotting-based or chromogenic assays with different endpoints. Clotting-based techniques are the most used worldwide, and they certainly are useful for diagnosis of clotting factor deficiencies. However, the information provided is relatively limited, and therefore the individual profile of coagulation is poorly assessed. This is reflected by the weak correlation between the results of these assays and the clinical phenotype. Among the assays that benefited from technological advances, thrombin generation and thromboelastography are probably the most actively investigated, but they require specific instruments and are not fully automated. Their standardisation level is rapidly progressing, and they are progressively entering the clinical scene, with the attempt to provide additional information on the coagulation process and a meaningful clinical correlation. These inherited bleeding disorders frequently require replacement therapy using clotting factor concentrates that increase the plasma level of the missing clotting factor. The classical adjustment of the therapy is mainly based on the measurement of the plasma clotting activity of the protein administered. If one considers that a certain level of thrombin generated would predict clinical efficacy, monitoring of thrombin formation might offer new possibilities to individually predict the bleeding phenotype, select the most adapted therapeutic product and tailor the dose. The same holds true for thromboelastography/thromboelastometry which evaluate fibrin formation as well as clot resistance to fibrinolytic challenge, one step further down in the coagulation process. In this regard, these 2 assays could be seen as complementary in terms of information provided on the coagulation profile at the individual level.

On-demand treatment of bleeds in haemophilia patients with inhibitors: strategies for securing and maintaining predictable efficacy with recombinant activated factor VII.

On-demand therapy with recombinant activated factor VII (rFVIIa) can provide effective haemostasis for spontaneous bleeds in haemophilia patients with inhibitors. However, treatment approaches vary amongst physicians, positively or negatively affecting outcomes. A panel of physicians proposed recommendations for securing and maintaining predictable efficacy with rFVIIa, comparing these with 'real-life' patient management, using a questionnaire circulated to other expert physicians from haemophilia care centres in Europe and the United States. For rFVIIa treatment of spontaneous bleeds in inhibitor patients, early intervention with the highest appropriate dose is recommended. Home-based therapy can facilitate early intervention. If additional rFVIIa therapy is required after the initial dose, rFVIIa 90 µg kg(-1) may be administered at 2-3 h intervals. Treatment should be tailored to bleed site/severity, recognizing the advantages of appropriate adjunct therapy. Questionnaire results suggested that many respondents adopted strategies in line with the recommendations. Most (36/46) recommended initial therapy within 1 h of bleed onset. rFVIIa 270 µg kg(-1) was the most frequently prescribed/recommended initial dose for paediatric (aged ≤ 15 years; 22/44 respondents) and adult (aged > 15 years; 23/44 respondents) patients. However, there may be opportunity for improved bleed management on occasion, with regard, for instance, to dosing and dose interval. To secure and maintain predictable efficacy with rFVIIa, judicious dose selection and treatment timing are important, together with adjunct therapy where necessary. As inhibitor patients present with different bleeding scenarios, a tailored treatment approach should be adopted.