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.

Therapeutic Modification of Hypoxia.

Regions of reduced oxygenation (hypoxia) are a characteristic feature of virtually all animal and human solid tumours. Numerous preclinical studies, both in vitro and in vivo, have shown that decreasing oxygen concentration induces resistance to radiation. Importantly, hypoxia in human tumours is a negative indicator of radiotherapy outcome. Hypoxia also contributes to resistance to other cancer therapeutics, including immunotherapy, and increases malignant progression as well as cancer cell dissemination. Consequently, substantial effort has been made to detect hypoxia in human tumours and identify realistic approaches to overcome hypoxia and improve cancer therapy outcomes. Hypoxia-targeting strategies include improving oxygen availability, sensitising hypoxic cells to radiation, preferentially killing these cells, locating the hypoxic regions in tumours and increasing the radiation dose to those areas, or applying high energy transfer radiation, which is less affected by hypoxia. Despite numerous clinical studies with each of these hypoxia-modifying approaches, many of which improved both local tumour control and overall survival, hypoxic modification has not been established in routine clinical practice. Here we review the background and significance of hypoxia, how it can be imaged clinically and focus on the various hypoxia-modifying techniques that have undergone, or are currently in, clinical evaluation.

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.

IMRT - Biomarkers for dose escalation, dose de-escalation and personalized medicine in radiotherapy for head and neck cancer.

Radiotherapy (RT) is an integral component in the management of head and neck cancer. Despite progress in several respects, a noteworthy proportion of the treated patients do not achieve complete response after RT. Regardless of novel dose delivery technologies, RT for head and neck cancer is still associated with acute as well as late toxicity. These challenges could potentially be addressed by means of personalized treatment. In this paper, we discuss the possibilities for dose escalation, dose de-escalation and allocation to systemic concomitant treatment based on prognostic and predictive markers for tumor control as well as predictive markers for normal tissue radiosensitivity.

Methodological development and biological observations of cell free DNA with a simple direct fluorescent assay in colorectal cancer.

BACKGROUND: Cell free DNA (cfDNA) has shown promising utility as prognostic biomarker for patients with colorectal cancer (CRC), with an ongoing need to optimize and validate the laboratory methodology. Here, we report our optimization and validation of a direct fluorescent assay and display the potential utility in patients with colorectal cancer. METHODS: Plasma cfDNA was analyzed by a direct fluorescent assay (DFA) and compared to quantification by droplet digital PCR (ddPCR). For clinical validation, baseline blood samples were available for a total of 273 patients from six different Nordic trials, covering patients with locally advanced rectal cancer (n = 176, cohorts A + B), liver limited metastatic CRC (n = 75C + D) and wide spread metastatic CRC (n = 22 E + F). RESULTS: Validating the DFA analysis with ddPCR revealed a strong correlation with an R2 of 0.81. For the clinical cohorts, the levels of cfDNA were: 0.8 ng/uL (95%CI 0.75-0.83) (A + B), 0.93 ng/uL (95%CI 0.86-1.02) (C + D) and 1.2 ng/uL (95%CI 0.85-1.47) (E + F), respectively (p < 0.01). All cohorts of colorectal cancer had higher levels of cell free DNA than healthy individuals (n = 94) (p < 0.01). CONCLUSION: Analysis of cell free DNA by a direct fluorescent assay could be an attractive laboratory option for a rapid inexpensive quantification of cell free DNA.

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.

MET amplification and epithelial-to-mesenchymal transition exist as parallel resistance mechanisms in erlotinib-resistant, EGFR-mutated, NSCLC HCC827 cells.

Although many epidermal growth factor receptor (EGFR)-mutated lung cancer patients initially benefit from the EGFR-inhibitor erlotinib, all acquire resistance. So far, several mechanisms implicated in resistance have been identified, but the existence of multiple resistance mechanisms in parallel have only been sparsely investigated. In this study, we investigated parallel resistance mechanisms acquired by HCC827, an EGFR-mutated adenocarcinoma cell line dependent on EGFR activity and sensitive to erlotinib. The cell line was treated with erlotinib by stepwise escalation of the drug-concentration and erlotinib-resistant (HCC827ER) cells created. HCC827ER cells depicted a mixed epithelial and mesenchymal phenotype. To clarify potential parallel resistance mechanisms, 14 resistant subclones were established by limited dilution. Interestingly, all HCC827ER subclones harbored either a MET-amplification (6/14) or underwent EMT (8/14), mechanisms both found in previous studies, but not in co-occurrence. Both subclone-types were resistant to erlotinib, but only MET-subclones responded to the MET-inhibitors crizotinib and capmatinib. EMT-subclones on the other hand had markedly increased FGFR1 expression and responded to the FGFR-inhibitor AZD4547, whereas MET-subclones did not. Monitoring gene expression through the development of HCC827ER revealed upregulation of FGFR1 expression as an early response to erlotinib. In addition, FGFR1 expression increased upon short-term erlotinib treatment (48 h) identifying a physiological role immediately after erlotinib exposure. The high FGFR1 expression seen in EMT-subclones was stable even after five passages without erlotinib. Here we show, that parallel resistance mechanisms appear during erlotinib-resistance development in EGFR-mutated NSCLC cells and highlight a role for FGFR1 expression changes as an early response to erlotinib as well as a bypass-signaling mechanism.

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.

Exosomal proteins as prognostic biomarkers in non-small cell lung cancer.

BACKGROUND: Use of exosomes as biomarkers in non-small cell lung cancer (NSCLC) is an intriguing approach in the liquid-biopsy era. Exosomes are nano-sized vesicles with membrane-bound proteins that reflect their originating cell. Prognostic biomarkers are needed to improve patient selection for optimal treatment. We here evaluate exosomes by protein phenotyping as a prognostic biomarker in NSCLC. METHODS: Exosomes from plasma of 276 NSCLC patients were phenotyped using the Extracellular Vesicle Array; 49 antibodies captured the proteins on the exosomes, and a cocktail of biotin-conjugated antibodies binding the general exosome markers CD9, CD81 and CD63 was used to visualise the captured exosomes. For each individual membrane-bound protein, results were analysed based on presence, in a concentration-dependent manner, and correlated to overall survival (OS). RESULTS: The 49 proteins attached to the exosomal membrane were evaluated. NY-ESO-1, EGFR, PLAP, EpCam and Alix had a significant concentration-dependent impact on inferior OS. Due to multiple testing, NY-ESO-1 was the only marker that maintained a significant impact on inferior survival (hazard rate (HR) 1.78 95% (1.78-2.44); p = 0.0001) after Bonferroni correction. Results were adjusted for clinico-pathological characteristics, stage, histology, age, sex and performance status. CONCLUSION: We illustrate the promising aspects associated with the use of exosomal membrane-bound proteins as a biomarker and demonstrate that they are a strong prognostic biomarker in NSCLC.

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.

Related hematopoietic cell donor care: is there a role for unrelated donor registries?

In almost half of allogeneic hematopoietic progenitor cell (HPC) transplants, a related donor (RD) is used, yet a lack of standardized guidelines means that their care is heterogeneous. Changes to regulatory standards aim to improve uniformity, but adherence to these regulations can prove logistically difficult for the transplant centers (TCs) managing RDs. Discussion has ensued around possible alternative models of related donor care and a session at the European Society for Blood and Marrow Transplantation (EBMT) annual meeting in 2013 debated the question of whether a role exists for unrelated donor registries in the management of 'related' donors. In this overview, we discuss the issues raised at this debate and the pros and cons of donor registry involvement in various aspects of RD management. By examining existing models of related donor care that have been adopted by members of the World Marrow Donor Association (WMDA), we look for ways to enhance and homogenize RD care, while also enabling transplant centers to meet standards required for mandatory accreditation.

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.

PET imaging of patients with non-small cell lung cancer employing an EGF receptor targeting drug as tracer.

BACKGROUND: We have previously developed (11)C-erlotinib as a new positron emission tomography (PET) tracer and shown that it accumulates in epidermal growth factor receptor (EGFR)-positive lung cancer xenografts in mice. Here, we present a study in patients with non-small cell lung cancer (NSCLC) investigating the feasibility of (11)C-erlotinib PET as a potential method for the identification of lung tumours accumulating erlotinib. METHODS: Thirteen patients with NSCLC destined for erlotinib treatment were examined by contrast-enhanced computed tomography (CT), (11)C-erlotinib PET/low-dose CT and (18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) PET/low-dose CT before start of the erlotinib treatment. After 12 weeks treatment, they were examined by (18)F-FDG PET/contrast-enhanced CT for the assessment of clinical response. RESULTS: Of the 13 patients included, 4 accumulated (11)C-erlotinib in one or more of their lung tumours or lymph-node metastases. Moreover, (11)C-erlotinib PET/CT identified lesions that were not visible on (18)F-FDG PET/CT. Of the four patients with accumulation of (11)C-erlotinib, one died before follow-up, whereas the other three showed a positive response to erlotinib treatment. Three of the nine patients with no accumulation died before follow-up, four showed progressive disease while two had stable disease after 12 weeks of treatment. CONCLUSION: Our data show a potential for (11)C-erlotinib PET/CT for visualizing NSCLC lung tumours, including lymph nodes not identified by (18)F-FDG PET/CT. Large clinical studies are now needed to explore to which extent pre-treatment (11)C-erlotinib PET/CT can predict erlotinib treatment response.

Complications related to blood donation: a population-based study.

BACKGROUND: Population-based data on the rate and outcome of complications related to blood donation are sparse. STUDY DESIGN AND METHODS: Data from a survey conducted in 2003 in Aarhus County, Denmark, were used to assess the overall rate of donor complications. Additional nationwide data on moderate and severe donor complications were obtained from the Danish Register of Complications Related to Blood Donation, with records of all moderate and severe donor complications in Denmark occurring during the period 1997-2003. RESULTS: In the regional survey, we identified 340 complications of any type among 41 274 donations, corresponding to a rate of 824/100,000 donations [95% confidence interval (CI): 741-916]. All complications were either needle injuries or vasovagal reactions. In the nationwide register, a total of 752 moderate and severe complications were recorded among 2,575,264 donations, corresponding to a rate of 29/100,000 donations (95% CI: 27-31). The rates of complications leading to long-term morbidity or disablement (> 5% loss of working capacity) were 5/100,000 donations (95% CI: 4.2-5.9) and 2.3/100,000 donations (95% CI: 1.8-2.9), respectively. CONCLUSION: The risk of complications related to blood donation is low. However, attention towards donor complications is warranted, given the non-negligible rate of complications resulting in long-term morbidity and disablement.

The temporal development in a hybridizing population of wild and cultivated chicory (Cichorium intybus L.).

Hybridization and its possible impacts is a subject of increased attention in connection with the risk of unintended gene flow from cultivated (including genetically modified) plants to wild relatives. Whether such gene flow by hybridization is likely to take place depends among other things on the persistence of the hybrids in a natural environment over time. To evaluate this, we studied an experimental hybridizing population of wild and cultivated chicories (Cichorium intybus) relative to a previous study on the same population 2 years earlier. We compared the genetic composition, morphology and fitness traits of plants from 2004 to the plants in the same plot in 2002. The majority of the plants in 2004 was more morphologically and genetically intermediate than in 2002. This indicates that no selection towards being wild-like or cultivar-like was present over the period of 2 years. Furthermore, no distinct fitness differences existed between the plants of 2004, probably due to most of the plants being intermediate. No hybridization barriers appeared to be present between wild and cultivated chicories beyond the F1 generation, since F2 hybrids and backcrosses were in abundance; in fact, hybrids of probably fourth or fifth generation were present. In conclusion, all results indicate that no barriers exist to the temporal persistence of chicory hybrids in a natural environment.

The relation between survival and expression of HER1 and HER2 depends on the expression of HER3 and HER4: a study in bladder cancer patients.

Increased expression of the epidermal growth factor (EGF) receptors, HER1 and HER2 are related to poor prognosis in most cancers studied. Recently, a high expression of the two remaining receptors of the EGF system, HER3 and HER4 has been related to a favourable prognosis. However, prognostic significance of HER1 and HER2 receptors in bladder cancer is controversial and the effect of the expression of different combinations of these receptors on patient survival is not well understood. Therefore, we examined the mRNA expression of all four EGF receptors with real-time polymerase chain reaction in biopsies from 88 patients with bladder cancer, where the survival was followed for a median of 38.5 months (range 1-117 months). Expression of HER1 and HER2 alone showed no correlation with survival. However, a high expression of HER1 together with high expression of HER3 and HER4 correlated to a better prognosis compared to the high expression of HER1 together with low expression of HER3 and HER4 (P=0.0006). Also, a significantly longer survival was observed in patients expressing high HER2 when coexpressed with high HER3 and HER4, as compared to the survival in patients with tumours expressing high HER2 but low HER3 and HER4 (P=0.0005). Our results suggest that the final outcome of patients with high HER1- and HER2-expressing tumours depends on the expression of HER3 and HER4.

Expression of the epidermal growth factor system in human endometrium during the menstrual cycle.

The epidermal growth factor (EGF) system is ubiquitous in humans and plays fundamental roles in embryogenesis, development, proliferation and differentiation. As the endometrium of fertile women is characterized by proliferation and differentiation, we hypothesize a role for the EGF system. Fourteen premenopausal women had endometrial samples removed on day 6 +/- 1 and day 6 +/- 1 and 12 +/- 1 after ovulation during one menstrual cycle. RNA was extracted and analysed by real-time PCR, and immunohistochemistry was performed to localize the components of the EGF system. Human EGF Receptor 1 (HER1) showed highest expression during the proliferative phase, HER2 and HER4 during the early and HER3 during the late secretory phase. Amphiregulin (AR) and transforming growth factor alpha (TGFalpha) expression is highest in proliferative phase. Heparin binding (HB)-EGF and betacellulin (BCL) show no variation. Epiregulin (EP) is detectable in some samples. EGF is undetectable. HER1, HER2, HER3 and HER4 were localized to the epithelium and glands HER3 and HER4 solely in the secretory phase. Amphiregulin was seen in leucocytes and stromal cells, TGFalpha and betacellulin in the epithelial lining, epiregulin in stromal cells whereas HB-EGF and EGF are undetectable. In conclusions, we observed cyclical expression of the four EGF receptors and two ligands and localized all four receptors and four ligands in endometrial biopsies. This suggests a role for the EGF system in growth of the endometrium.