Novel human-derived EML4-ALK fusion cell lines identify ribonucleotide reductase RRM2 as a target of activated ALK in NSCLC.

INTRODUCTION: Echinoderm microtubule-associated protein-like 4 (EML4)-Anaplastic Lymphoma Kinase (ALK) rearrangements occur in 3% to 7% of lung adenocarcinomas and are targets for treatment with tyrosine kinase inhibitors (TKIs). Here we have developed three novel EML4-ALK-positive patient-derived Non-Small-Cell-Lung-Cancer (NSCLC) cancer cell lines, CUTO8 (variant 1), CUTO9 (variant 1) and CUTO29 (variant 3) and included a fourth ALK-positive cell line YU1077 (variant 3) to study ALK-positive signaling and responses. Variants 1 and 3 are the most common EML4-ALK variants expressed in ALK-positive NSCLC, and currently cell lines representing these EML4-ALK variants are limited. MATERIALS AND METHODS: Resazurin assay was performed to evaluate cell viability. Protein levels were determined using western blotting. RNA sequencing was performed in all four cell lines to identify differentially expressed genes. Whole-genome sequencing was performed to determine the presence of EML4-ALK fusion and ALK tyrosine kinase inhibitor resistance mutations. RESULTS: In this study, we have confirmed expression of the corresponding ALK fusion protein and assessed their sensitivity to a range of ALK tyrosine kinase inhibitors. These patient derived cell lines exhibit differential sensitivity to lorlatinib, brigatinib and alectinib, with EML4-ALK variant 3 containing cell lines exhibiting increased sensitivity to lorlatinib and brigatinib as compared to alectinib. These cell lines were further characterized by whole genome sequencing and RNA-seq analysis that identified the ribonucleotide reductase regulatory subunit 2 (RRM2) as a downstream and potential therapeutic target in ALK-positive NSCLC. CONCLUSION: We provide a characterization of four novel EML4-ALK-positive NSCLC cell lines, highlighting genomic heterogeneity and differential responses to ALK TKI treatment. The RNA-Seq characterization of ALK-positive NSCLC CUTO8, CUTO9, CUTO29 and YU1077 cell lines reported here, has been compiled in an interactive ShinyApp resource for public data exploration (https://ccgg.ugent.be/shiny/nsclc_rrm2_2022/).

Community-acquired pneumonia in primary care: clinical assessment and the usability of chest radiography.

OBJECTIVES: To investigate the diagnostic value of different clinical and laboratory findings in pneumonia and to explore the association between the doctor's degree of suspicion and chest X-ray (CXR) result and to evaluate whether or not CXR should be used routinely in primary care, when available. DESIGN: A three-year prospective study was conducted between September 2011 and December 2014. SETTING: Two primary care settings in Linköping, Sweden. SUBJECTS: A total of 103 adult patients with suspected pneumonia in primary care. MAIN OUTCOME MEASURES: The physicians recorded results of a standardized medical physical examination, including laboratory results, and rated their suspicion into three degrees. The outcome of the diagnostic variables and the degree of suspicion was compared with the result of CXR. RESULTS: Radiographic pneumonia was reported in 45% of patients. When the physicians were sure of the diagnosis radiographic pneumonia was found in 88% of cases (p < 0.001), when quite sure the frequency of positive CXR was 45%, and when not sure 28%. Elevated levels of C-reactive protein (CRP) ≥ 50mg/L were associated with the presence of radiographic pneumonia when the diagnosis was suspected (p < 0.001). CONCLUSION: This study indicates that CXR can be useful if the physician is not sure of the diagnosis, but when sure one can rely on one's judgement without ordering CXR. KEY POINTS: There are different guidelines but no consensus on how to manage community-acquired pneumonia in primary care. When the physician is sure of the diagnosis the judgement is reliable without chest X-ray and antibiotics can be safely prescribed. Chest X-ray can be useful in the assessment of pneumonia in primary care, when the physician is not sure of the diagnosis.

p37δ is a new isoform of PI3K p110δ that increases cell proliferation and is overexpressed in tumors.

The phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, proliferation and survival, and are frequently affected in human cancer. PI3K is composed of a catalytic subunit, p110, and a regulatory subunit, p85. The PI3K catalytic subunit p110δ is encoded by PIK3CD and contains p85- and RAS-binding domains, and a kinase domain. Here we present an alternatively spliced PIK3CD transcript encoding a previously unknown protein, p37δ, and demonstrate that this protein is expressed in human ovarian and colorectal tumors. p37δ retains the p85-binding domain and a fraction of the RAS-binding domain, lacks the catalytic domain, and has a unique carboxyl-terminal region. In contrast to p110δ, which stabilizes p85, p37δ promoted p85 sequestering. Despite the truncated RAS-binding domain, p37δ bound to RAS and we found a strong positive correlation between the protein levels of p37δ and RAS. Overexpressing p37δ, but not p110δ, increased the proliferation and invasive properties of HEK-293 cells and mouse embryonic fibroblasts. Cells overexpressing p37δ showed a quicker phosphorylation response of AKT and ERK1/2 following serum stimulation. Ubiquitous expression of human p37δ in the fruit fly increased body size, DNA content and phosphorylated ERK1/2 levels. Thus, p37δ appears to be a new tumor-specific isoform of p110δ with growth-promoting properties.

Similar inhibition of platelet adhesion, P-selectin expression and plasma coagulation by ioversol, iodixanol and ioxaglate.

Contrast media (CM) are reported to possess both prothrombotic and anticoagulant properties. The mechanisms are not clearly understood, and early reports are contradictory. To study the effects of CM on haemostasis, we analysed the ex vivo effects of ioversol and iodixanol on platelet adhesion and P-selectin expression, and the in vitro effects of ioversol, iodixanol and ioxaglate on platelet adhesion, P-selectin expression and plasma coagulation. A novel enzymatic assay was used to measure platelet adhesion to protein surfaces, and an enzyme-linked immunosorbent assay was used to measure platelet P-selectin surface expression. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were used to measure plasma coagulation. The ex vivo study consisted of blood from 27 outpatients administered ioversol and 9 patients administered iodixanol intravenously. Samples were collected before and 5 min after CM administration. Healthy donors were used for the in vitro studies on the effects of CM. The ex vivo study showed significantly (p<0.05) decreased platelet adhesion and P-selectin expression after administration of ioversol and iodixanol. Adhesion was more affected than P-selectin expression. The in vitro study showed that ioversol, iodixanol and ioxaglate significantly (p<0.05) and dose-dependently (beginning at 3 mg ml(-1)) decreased platelet adhesion and P-selectin expression. APTT and PT were significantly (p<0.01) prolonged at concentrations of 10 mg ml(-1) and 30 mg ml(-1), respectively. In conclusion, ioversol, iodixanol and ioxaglate inhibit platelet adhesion and P-selectin expression, as well as plasma coagulation. Platelets are more sensitive in relation to the inhibiting effect on plasma coagulation.

Genetic and epigenetic changes in the common 1p36 deletion in neuroblastoma tumours.

Chromosome 1p is frequently deleted in neuroblastoma (NB) tumours. The commonly deleted region has been narrowed down by loss of heterozygosity studies undertaken by different groups. Based on earlier mapping data, we have focused on a region on 1p36 (chr1: 7 765 595-11 019 814) and performed an analysis of 30 genes by exploring features such as epigenetic regulation, that is DNA methylation and histone deacetylation, mutations at the DNA level and mRNA expression. Treatment of NB cell lines with the histone deacetylase inhibitor trichostatin A led to increased gene transcription of four of the 30 genes, ERRFI1 (MIG-6), PIK3CD, RBP7 (CRBPIV) and CASZ1, indicating that these genes could be affected by epigenetic downregulation in NBs. Two patients with nonsynonymous mutations in the PIK3CD gene were detected. One patient harboured three variations in the same exon, and p.R188W. The other patient had the variation p.M655I. In addition, synonymous variations and one variation in an intronic sequence were also found. The mRNA expression of this gene is downregulated in unfavourable, compared to favourable, NBs. One nonsynonymous mutation was also identified in the ERRFI1 gene, p.N343S, and one synonymous. None of the variations above were found in healthy control individuals. In conclusion, of the 30 genes analysed, the PIK3CD gene stands out as one of the most interesting for further studies of NB development and progression.

Inverse relationships between coronary blood flow obstruction and platelet reactivity in stable angina pectoris.

This study investigates relationships between platelet reactivity and coronary blood flow obstruction in stable angina pectoris. Consented were 36 patients with single-vessel disease. The subjects were divided into two groups. One group (n=14) had less severe (<=80%) and the second group (n=22) had severe coronary flow impairment (90%). Before elective coronary angiography platelet in vitro reactivity in venous whole blood was determined using a flow cytometry technique. A thrombin-receptor activating peptide (TRAP-6) (0.77 and 0.06 g/l) and ADP (8.5 and 1.7 micromol/l) were used to activate platelets. The number of fibrinogen positive cells (%) i.e., activated platelets after stimulation was employed as experimental parameter. Less severe flow obstruction was associated with more reactive platelets. When stimulating with 0.77 g/l TRAP-6 the number of activated platelets was 64+/-15 (SD)%. The corresponding value for the group with severe flow obstruction was 40+/-17(SD)%. The difference is significant (P<0.001). 0.06 g/l TRAP-6 yielded similar results (P<0.01). Also when using 8.5 micromol/l ADP to challenge platelets less severe flow obstruction was associated with enhanced reactivity (P<0.01). 1.7 micromol/l ADP generated comparable results (P<0.05). Thus, in stable angina pectoris coronary flow obstruction is inversely related to platelet reactivity.

Individual variations of platelet inhibition after loading doses of clopidogrel.

OBJECTIVE: To investigate individual variations of platelet inhibition after clopidogrel-loading doses. SETTING: Department of Cardiology, Linköping University Hospital, Linköping, Sweden. SUBJECTS: Individuals with stable angina pectoris (n = 18) subject to percutaneous coronary interventions (PCI) and subsequent stenting were investigated. METHODS AND EXPERIMENTAL PROTOCOL: A 300-mg clopidogrel loading dose was administrated immediately after stenting (day 1) followed by an additional 75 mg clopidogrel after 24 h (day 2). The ADP-evoked platelet fibrinogen binding was analysed to estimate platelet reactivity immediately before angiography and on day 2. A flow cytometry technique was used with two ADP solutions (final concentrations 0.6 and 1.7 micromol L-1) employed as platelet activating agents. Soluble P-selectin was used as a marker of platelet activity. RESULTS: When using 1.7 micromol L-1 ADP to activate platelets four individuals had a strong inhibition (i.e. platelet reactivity <10% of the day 1-value day 2). In contrast, five patients demonstrated a weak inhibition (i.e. platelet reactivity >60% of the day 1-value day 2). Similar results were obtained when using 0.6 micromol L-1 ADP as a platelet-activating agent. Clopidogrel, however, fails to suppress platelet activity as estimated from soluble P-selectin. CONCLUSIONS: Clopidogrel evoked platelet inhibition exhibits a considerable individual heterogeneity. Some individuals only had weak responses whereas others displayed strong platelet inhibition. The present flow cytometry technique appears suitable for identifying patients with abnormal reactions after clopidogrel exposure.

Management of life-threatening haemoptysis.

Massive haemoptysis represents a major medical emergency that is associated with a high mortality. Here we present two cases of life-threatening haemoptysis, the first caused by rupture of an aortic aneurysm into the lung in a 37-yr-old woman with polyarteritis nodosa and the second caused by massive bleeding from an angiectatic vascular malformation in the right main bronchus in a 21-yr-old woman. Fibreoptic bronchoscopy played an essential role in the diagnostic process and management of the respiratory tract. Diagnosis in the first case was obtained by CT scan and the aneurysm was treated surgically. In the second case, bronchial arteriography contributed to both definitive diagnosis and treatment. Initial cardiorespiratory management, diagnostic procedures and definitive therapy are described and reviewed. Adequate early management of the cardiorespiratory system is essential to the outcome. Aggressive measures to elucidate the cause of haemoptysis and prompt therapy are warranted because of the high risk of recurrence.

Heart rescue in acute left main coronary artery occlusion with CABG using initial retrograde perfusion.

Acute occlusion of the left main coronary artery (LMCA) is a rare and almost invariably fatal condition. Here, we report on heart salvage in two such cases with CABG aided by emergent retrograde reperfusion as the initial operative step. Both cases were extremely unusual. The first patient had twice survived LMCA occlusion; the second also had right coronary artery occlusion. We will also review the literature on acute LMCA occlusion and coronary venous retroperfusion.

Inverse relationship between platelet density and reactivity alterations at coronary angiography.

This work investigates relationships between platelet density and reactivity. 21 individuals subject to coronary angiography were studied. Peak platelet density was analyzed using a newly developed electronic device. The apparatus measures light transmission through test tubes containing density-separated platelets, thus allowing an estimation of the platelet distribution in the gradient. A flow cytometry technique was used for determining platelet reactivity after stimulating with ADP. Platelet counts, mean platelet volumes, peak platelet density and platelet reactivity were determined immediately before (day 1) and 24 h after cardiac catheterization (day 2). For all parameters changes during the day of angiography were compared with platelet density alterations. The subjects were divided into two groups according to density changes at angiography. Group 1 individuals showed density alterations (i.e. day 2 - day 1 value) > or = -8 x 10(-5) kg/l. In contrast, group 2 subjects either displayed density changes < -8 x 10(-5) kg/l or grossly disturbed platelet density patterns on day 2. Before angiography both groups had similar platelet counts and volumes. Then platelet reactivity when stimulating with ADP did not differ significantly between the two groups. After angiography, the number of fibrinogen-positive cells when stimulating with ADP rose by 6 +/- 8% for group 2 patients. The corresponding figure for group 1 was -1 +/- 6%. The difference was significant (p = 0.01). No such relationships were found when comparing density alterations and changes of platelet counts and volumes. We conclude that in this study platelet density alterations at coronary angiography are inversely related to variations of platelet reactivity.

Doppler myocardial imaging in the assessment of regional myocardial function in longitudinal direction pre- and post-PTCA.

AIMS: Doppler myocardial imaging is potentially a sensitive tool to assess regional myocardial velocities pre- and post-percutaneous transluminal coronary angioplasty (PTCA) as a marker of contractility to evaluate short- to medium-term information on functional myocardial recovery following the release of ischaemia. METHODS: Thirty patients with single vessel disease were studied to assess regional myocardial peak systolic velocity, systolic velocity time integral and mitral valve plane excursion in longitudinal direction one day pre-, one day post- and 3 months post-PTCA. The patients were assigned to group A with coronary stenoses >70% and group B with stenoses < 70%. RESULTS: In group A pre-PTCA the ischaemic segments showed a significantly lower peak systolic velocity and velocity time integral compared with the values one day after PTCA (5.8 +/- 1.4 vs 7.7 +/- 1.4cm.s(-1); 1.06 +/- 0.22 vs 1.23 +/-0.28cm;P< 0.03). In contrast, mitral valve plane excursion in this group remained unchanged after PTCA for both the ischaemic and non-ischaemic left ventricular wall. In group B no changes of these parameters and no differences in mitral valve plane excursion of the ischaemic and the non-ischaemic left ventricular wall could be seen. CONCLUSION: With Doppler myocardial imaging it was possible to quantify a number of indices which changed due to the successful release of ischaemia.

A comparison of analogue and digital techniques in upper gastrointestinal examinations: absorbed dose and diagnostic quality of the images.

This study was performed to investigate whether patient exposure and diagnostic quality of the image is significantly influenced by the introduction of digital image acquisition techniques. Evaluation was performed for three different techniques (analogue, analogue fluoro + digital radiography, digital) in examination of the upper gastrointestinal tract. The evaluation was done from data acquired in three different departments. Patient exposure was recorded as KERMA-area product (KAP) and the individual patient readings were normalised to a standard size patient. Image quality was assessed using visual grading with a reference image. The recorded KAP values were significantly higher (22.3 Gycm2) for the fully digital technique compared to the others (analogue 6.8 Gycm2, analogue + digital 3.6 Gycm2). This was due mostly to an increased number of exposures. The diagnostic quality of the image was, however, also regarded to be slightly lower for the technique giving the lowest patient dose with the smallest number of exposures (analogue + digital). The digital examination technique, as used in this study, thus resulted in significantly higher patient dose without any significant gain in diagnostic quality of the image.

Patient radiation exposure during coronary angiography and intervention.

PURPOSE: To prospectively register fluoroscopic and cine times in a random fashion, and to measure patient radiation exposure from routine coronary angiography and coronary balloon angioplasty. We also evaluated an optional dose reduction system used during interventions. MATERIAL AND METHODS: The incident radiation to the patient was measured as kerma area product (KAP) in Gycm(2), obtained from an ionisation chamber mounted on the undercouch tube during 65 coronary angiography procedures and another 53 percutaneous transluminal coronary angioplasties (including 29 stent procedures), mostly directly following complete coronary angiography. RESULTS AND CONCLUSION: The values from coronary angiography were comparable to other reports with a mean fluoroscopic time of 4.4 min and a mean KAP value of 62.6 Gycm(2). The corresponding figures from coronary balloon angioplasty without stenting were lower than otherwise reported, with 8.2 min and 47.9 Gycm(2), respectively. The use of coronary stents did prolong the mean fluoroscopic time (10.5 min) but did not significantly enhance the patient mean radiation dose (51.4 Gycm(2)). The dose reduction technique resulted in a significant KAP value reduction of 57%. In conclusion, with regard to radiation exposure, coronary angiography and balloon angioplasty are considered safe procedures.

Femoral artery haemostasis with a pneumatic compression device versus a clamp after coronary angiography.

To evaluate the safety and efficacy of a new pneumatic compression device for achieving haemostasis after femoral artery catheterization, 1,017 patients undergoing selective coronary angiography by a 5F unilateral femoral route were prospectively randomised to pneumatic or the routinely used clamp compression technique. All initial bleedings could be controlled in the pneumatic group, whereas in 38 patients (8%) of the clamp group the initial positioning of the clamp was unsuccessful or was not tolerated by the patient (p < 0.05). Ultrasound Doppler study of the puncture site because of suspected postcatheterization vascular complication revealed two haematomas which needed no further measure and two pseudoaneurysms which were successfully treated with ultrasound-guided compression or surgical repair. The rate of complications requiring treatment (pseudoaneurysms) was 0.2% overall, 0.5% in the clamp group and nil in the pneumatic compression group (NS). We conclude that the pneumatic compression device is effective, convenient and at least as safe as the clamp and, by shortening the time in the catheterization laboratory, offers time for further angiograms.