Shotgun Proteomics Identifies Serum Fibronectin as a Candidate Diagnostic Biomarker for Inclusion in Future Multiplex Tests for Ectopic Pregnancy

Ectopic pregnancy (EP) is difficult to diagnose early and accurately. Women often present at emergency departments in early pregnancy with a 'pregnancy of unknown location' (PUL), and diagnosis and exclusion of EP is challenging due to a lack of reliable biomarkers. The objective of this study was to identify novel diagnostic biomarkers for EP. Shotgun proteomics, incorporating combinatorial-ligand library pre-fractionation, was used to interrogate pooled sera (n = 40) from women undergoing surgery for EP, termination of viable intrauterine pregnancy and management of non-viable intrauterine pregnancy. Western blot was used to validate results in individual sera. ELISAs were developed to interrogate sera from women with PUL (n = 120). Sera were collected at time of first symptomatic presentation and categorized according to pregnancy outcome. The main outcome measures were differences between groups and area under the receiver operating curve (ROC). Proteomics identified six biomarker candidates. Western blot detected significant differences in levels of two of these candidates. ELISA of sera from second cohort revealed that these differences were only significant for one of these candidates, fibronectin. ROC analysis of ability of fibronectin to discriminate EP from other pregnancy outcomes suggested that fibronectin has diagnostic potential (ROC 0.6439; 95% CI 0.5090 to 0.7788; P>0.05), becoming significant when 'ambiguous' medically managed PUL excluded from analysis (ROC 0.6538; 95% CI 0.5158 to 0.7918; P<0.05). Fibronectin may make a useful adjunct to future multiplex EP diagnostic tests

Immediate chest X-ray for patients at risk of lung cancer presenting in primary care: randomised controlled feasibility trial

Background: Achieving earlier stage diagnosis is one option for improving lung cancer outcomes in the United Kingdom. Patients with lung cancer typically present with symptoms to general practitioners several times before referral or investigation. Methods: We undertook a mixed methods feasibility individually randomised controlled trial (the ELCID trial) to assess the feasibility and inform the design of a definitive, fully powered, UK-wide, Phase III trial of lowering the threshold for urgent investigation of suspected lung cancer. Patients over 60, with a smoking history, presenting with new chest symptoms to primary care, were eligible to be randomised to intervention (urgent chest X-ray) or usual care. Results: The trial design and materials were acceptable to GPs and patients. We randomised 255 patients from 22 practices, although the proportion of eligible patients who participated was lower than expected. Survey responses (89%), and the fidelity of the intervention (82% patients X-rayed within 3 weeks) were good. There was slightly higher anxiety and depression in the control arm in participants aged >75. Three patients (1.2%) were diagnosed with lung cancer. Conclusions: We have demonstrated the feasibility of individually randomising patients at higher risk of lung cancer, to a trial offering urgent investigation or usual care

Pooled sera from women undergoing surgical management of EP, surgical management of NVIUP and surgical termination of VIUP were affinity-purified using ProteoMiner™ spin-columns and separated by 1D SDS-PAGE, prior to trypsin digestion and LC-ESI-MS/MS.

<p>Interrogation of the SwissProt database produced a total of 118 non-redundant high confidence identities.</p

Patient recruitment: 120 patients with an initial diagnosis of a PUL were recruited to the study and grouped according to final pregnancy outcomes.

<p>Patient recruitment: 120 patients with an initial diagnosis of a PUL were recruited to the study and grouped according to final pregnancy outcomes.</p

Western blots of individual whole sera from women undergoing surgical management of EP, surgical management of NVIUP and surgical termination of VIUP were probed with antibodies specific for: HBB; ADIPO; FN1; and PSG4.

<p>Integrated density values of specifically labelled bands were normalised against a positive control (pooled VIUP serum). ROC curves were generated for ectopic vs non-ectopic patient groups.</p

Western blots of pooled whole sera and ProteoMiner™ affinity-purified serum from women undergoing surgical management of EP, surgical management of NVIUP and surgical termination of VIUP were probed with antibodies specific for proteins identified by LC-ESI-MS/MS (Table 1).

<p>Western blots of pooled whole sera and ProteoMiner™ affinity-purified serum from women undergoing surgical management of EP, surgical management of NVIUP and surgical termination of VIUP were probed with antibodies specific for proteins identified by LC-ESI-MS/MS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066974#pone-0066974-t001" target="_blank">Table 1</a>).</p

1D SDS-PAGE of ProteoMiner™ affinity-purified serum, alongside the original whole sera and flow-through, from women undergoing surgical management of EP (E), surgical management of NVIUP (N) and surgical termination of VIUP (V).

<p>Note the increased complexity and reduction in high abundance proteins in the enriched fractions. Three bands (B1, B2 and B3) appeared differentially expressed and were submitted for PMF.</p

FN1 and PSG4 ELISA of serum collected at first presentation of women with a PUL, categorized according to final pregnancy outcome: dNVIUP (n = 26); dVIUP (n = 28); dEP (n = 17); NP (n = 11); srPUL (n = 27); pEP (n = 8); and tPUL (n = 3).

<p>ROC curves were generated for ectopic vs non-ectopic patient groups. Analysis was repeated after ambiguous medically treated PUL outcomes (pEP and tPUL) were excluded (-PUL Data).</p