ASPiRATION: Australian observational cohort study of comprehensive genomic profiling in metastatic lung cancer tissue.

ASPiRATION is a national prospective observational cohort study assessing the feasibility, clinical and economic value of up-front tissue-based comprehensive genomic profiling (CGP) to identify actionable genomic alterations in participants with newly diagnosed metastatic non-squamous non-small-cell lung cancer in Australia. This study will enrol 1000 participants with tumor available for CGP and standard of care molecular testing (EGFR/ALK/ROS1). Participants with actionable variants may receive novel targeted treatments through ASPiRATION-specific substudies, other trials/programs. Clinical outcome data will be collected for a minimum of 2 years. Study outcomes are descriptive, including the ability of CGP to identify additional actionable variants, leading to personalized treatment recommendations, and will describe the feasibility, efficiency, cost and utility of implementation of CGP nationally.

Lung cancer is the most common cause of cancer death in Australia and worldwide. This disease often happens due to alterations in specific genes that allow cancer cells to develop and spread. Scientists have designed targeted drugs that are better at attacking cancer cells that have specific 'actionable' gene alterations and have less effect on other cells in the body. The result is often more benefit from treatment and fewer side effects than other standard treatments (chemotherapy or immunotherapy). The targeted drugs are well established as the best initial treatments for some gene alterations, but more research is needed to know if this is true for some of the less common or recently identified gene alterations, and where the targeted drugs are very new. Comprehensive genomic profiling is a new way of testing lung cancer cells for all the gene alterations (the well-known ones as well as the rare ones) in a single test. It is expected that this test will find many more of these gene alterations, which will allow more people to have safer and more effective targeted treatments leading to potentially better outcomes, and will allow some people to join clinical trials testing newer targeted treatments. The ASPiRATION study will help work out whether comprehensive genomic profiling is better than the current way of testing for gene alterations in Australia, and if it is feasible to use in all people diagnosed with advanced lung cancer in Australia. Clinical Trial Registration: ACTRN12621000221853 (ANZCTR).

Limited clinical activity of palbociclib and ribociclib monotherapy in advanced cancers with cyclin D-CDK4/6 pathway alterations in the Dutch DRUP and Australian MoST trials.

The Dutch Drug Rediscovery Protocol (DRUP) and the Australian Cancer Molecular Screening and Therapeutic (MoST) Program are similar nonrandomized, multidrug, pan-cancer trial platforms that aim to identify signals of clinical activity of molecularly matched targeted therapies or immunotherapies outside their approved indications. Here, we report results for advanced or metastatic cancer patients with tumors harboring cyclin D-CDK4/6 pathway alterations treated with CDK4/6 inhibitors palbociclib or ribociclib. We included adult patients that had therapy-refractory solid malignancies with the following alterations: amplifications of CDK4, CDK6, CCND1, CCND2 or CCND3, or complete loss of CDKN2A or SMARCA4. Within MoST, all patients were treated with palbociclib, whereas in DRUP, palbociclib and ribociclib were assigned to different cohorts (defined by tumor type and alteration). The primary endpoint for this combined analysis was clinical benefit, defined as confirmed objective response or stable disease ≥16 weeks. We treated 139 patients with a broad variety of tumor types; 116 with palbociclib and 23 with ribociclib. In 112 evaluable patients, the objective response rate was 0% and clinical benefit rate at 16 weeks was 15%. Median progression-free survival was 4 months (95% CI: 3-5 months), and median overall survival 5 months (95% CI: 4-6 months). In conclusion, only limited clinical activity of palbociclib and ribociclib monotherapy in patients with pretreated cancers harboring cyclin D-CDK4/6 pathway alterations was observed. Our findings indicate that monotherapy use of palbociclib or ribociclib is not recommended and that merging data of two similar precision oncology trials is feasible.

A signal-seeking Phase 2 study of olaparib and durvalumab in advanced solid cancers with homologous recombination repair gene alterations.

PURPOSE: To determine the safety and efficacy of PARP plus PD-L1 inhibition (olaparib + durvalumab, O + D) in patients with advanced solid, predominantly rare cancers harbouring homologous recombination repair (HRR) defects. PATIENTS AND METHODS: In total, 48 patients were treated with O + D, 16 with BRCA1/2 alterations (group 1) and 32 with other select HRR alterations (group 2). Overall, 32 (66%) patients had rare or less common cancers. The primary objective of this single-arm Phase II trial was a progression-free survival rate at 6 months (PFS6). Post hoc exploratory analyses were conducted on archival tumour tissue and serial bloods. RESULTS: The PFS6 rate was 35% and 38% with durable objective tumour responses (OTR) in 3(19%) and 3(9%) in groups 1 and 2, respectively. Rare cancers achieving an OTR included cholangiocarcinoma, perivascular epithelioid cell (PEComa), neuroendocrine, gallbladder and endometrial cancer. O + D was safe, with five serious adverse events related to the study drug(s) in 3 (6%) patients. A higher proportion of CD38 high B cells in the blood and higher CD40 expression in tumour was prognostic of survival. CONCLUSIONS: O + D demonstrated no new toxicity concerns and yielded a clinically meaningful PFS6 rate and durable OTRs across several cancers with HRR defects, including rare cancers.

Pharmacodynamics effects of CDK4/6 inhibitor LEE011 (ribociclib) in high-risk, localised prostate cancer: a study protocol for a randomised controlled phase II trial (LEEP study: LEE011 in high-risk, localised Prostate cancer).

INTRODUCTION: Despite the development of new therapies for advanced prostate cancer, it remains the most common cause of cancer and the second leading cause of cancer death in men. It is critical to develop novel agents for the treatment of prostate cancer, particularly those that target aspects of androgen receptor (AR) signalling or prostate biology other than inhibition of androgen synthesis or AR binding. Neoadjuvant pharmacodynamic studies allow for a rational approach to the decisions regarding which targeted therapies should progress to phase II/III trials. CDK4/6 inhibitors have evidence of efficacy in breast cancer, and have been shown to have activity in preclinical models of hormone sensitive and castrate resistant prostate cancer. The LEEP trial aims to assess the pharmacodynamic effects of LEE011 (ribociclib), an orally bioavailable and highly selective CDK4/6 inhibitor, in men undergoing radical prostatectomy for high-risk, localised prostate cancer. METHODS AND ANALYSIS: The multicentre randomised, controlled 4:1 two-arm, phase II, open label pharmacodynamic study will recruit 47 men with high risk, localised prostate cancer who are planned to undergo radical prostatectomy. Participants who are randomised to receive the study treatment will be treated with LEE011 400 mg daily for 21 days for one cycle. The primary endpoint is the frequency of a 50% reduction in Ki-67 proliferation index from the pretreatment prostate biopsy compared to that present in prostate cancer tissue from radical prostatectomy. Secondary and tertiary endpoints include pharmacodynamic assessment of CDK4/6 cell cycle progression via E2F levels, apoptotic cell death by cleaved caspase-3, changes in serum and tumour levels of Prostate Specific Antigen (PSA), pathological regression, safety via incidence of adverse events and exploratory biomarker analysis. ETHICS AND DISSEMINATION: The protocol was approved by a central ethics review committee (St Vincent's Hospital HREC) for all participating sites (HREC/17/SVH/294). Results will be disseminated in peer-reviewed journals and at scientific conferences. DRUG SUPPLY: Novartis. PROTOCOL VERSION: 2.0, 30 May 2019 TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry (ACTRN12618000354280).

Correction: Associations of measures of systemic blood flow used in a randomized trial of delayed cord clamping in preterm infants.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Associations of measures of systemic blood flow used in a randomized trial of delayed cord clamping in preterm infants.

OBJECTIVE: To determine associations of low superior vena cava (SVC) flow (≤55 ml/kg/min) and low right ventricular output (RVO) (≤150 ml/kg/min) in preterm infants. DESIGN/METHODS: An observational study in infants <30 weeks gestation randomized to receive immediate (<10 s) or delayed cord clamping (DCC) (≥60 s). RESULTS: The study enrolled 265 infants with a mean (SD) gestation 28 (2) weeks. Eighty-six (33%) infants had low SVC flow and 81 (31%) infants had low RVO. In multivariate analysis, low SVC flow was associated with gestation; low RVO was associated with DCC, gender and 5-minute Apgar; whereas mean RVO was negatively associated with both FiO2 and mean airway pressure (MAP) at 9 h and 24 h. Low SVC flow was associated with ductus arteriosus (DA) treatment. Infants with low RVO had higher mortality on univariate analysis, but this was not significant after adjusting for gestation. CONCLUSIONS: SVC flow was associated with gestation, whilst RVO was associated with placental transfusion, gender, condition at birth, and early respiratory adaptation. Compared to infants with normal values, more infants with low SVC flow were treated for DA, but infants with low RVO had no significant difference in mortality or morbidity.

Cancer Molecular Screening and Therapeutics (MoST): a framework for multiple, parallel signal-seeking studies of targeted therapies for rare and neglected cancers.

BACKGROUND: Precision medicine aims to link molecular targets in tumours with corresponding therapies, particularly for patients with rare cancers. Innovative approaches are needed to translate molecular opportunities into clinical care. The Cancer Molecular Screening and Therapeutics (MoST) program employs a molecular screening platform to identify molecular changes of therapeutic relevance (actionable changes) and a master protocol for multiple, parallel signal-seeking clinical substudies, focused on therapies for patients with rare and neglected cancers. Methods and analysis: Archival pathology laboratory samples from patients with treatment-refractory advanced solid cancer of any histologic type undergo molecular tumour profiling. Following review by a Molecular Tumour Board, eligible patients are offered treatment in therapeutic substudies. This novel master protocol allows expedited addition of individual substudies; at least 12 open label, single arm, signal-seeking substudies during the initial 4 years of MoST are planned. The primary objectives are to identify signals of efficacy for developing biomarker-driven therapies and biomarkers that more accurately predict response to therapy, as well as to evaluate the MoST design. Ethics approval: The program has been approved by the St Vincent's Hospital Sydney Human Research Ethics Committee (reference, HREC/16/SVH/23). Dissemination of results: A report summarising and interpreting collected study data will be published. Our findings will be presented at national and international conferences and scientific meetings, and published in peer-reviewed journals. TRIAL REGISTRATION: Australia New Zealand Clinical Trials Registry: ACTRN12616000908437 (8 July 2016).

Interobserver agreement and image quality of functional cardiac ultrasound measures used in a randomised trial of delayed cord clamping in preterm infants.

OBJECTIVE: Functional cardiac ultrasound measures are used clinically and in trials for assessing the haemodynamic status of newborn infants. Superior vena cava (SVC) flow and right ventricular output (RVO) are established measures of systemic blood flow on the first postnatal day. The objective was to assess image quality and interobserver agreement of these measures in preterm infants enrolled in a randomised trial of immediate versus delayed cord clamping. DESIGN AND SETTING: Image quality and interobserver agreement for SVC flow, RVO and ductus arteriosus (DA) size were assessed on measurements taken at 3-6, 6-12 and 20-28 hours for the first 10 infants enrolled at each of four sites (total 40). Bland-Altman plots were constructed; mean difference (bias) and limits of agreement (LOA) were calculated. Potential sources of variation were explored. RESULTS: Quality was judged satisfactory for >97% of images. The mean difference and LOA between the observers were 5.4 mL/kg/min and -49.0 to 59.8 mL/kg/min for SVC flow, -26.6 mL/kg/min and -131.4 to 78.2 mL/kg/min for RVO, and 0 mm and -0.8 to 0.8 mm for DA diameter, respectively. The principal source of measurement error for SVC flow was diameter, and for RVO, diameter and velocity time integral. The difference between observers for both SVC and RVO was significantly associated with site. CONCLUSION: Interobserver variability for SVC flow is consistent with that previously reported, but higher for RVO. The findings should be incorporated into clinical practice, training, accreditation and trial design.

Delayed versus Immediate Cord Clamping in Preterm Infants.

BACKGROUND: The preferred timing of umbilical-cord clamping in preterm infants is unclear. METHODS: We randomly assigned fetuses from women who were expected to deliver before 30 weeks of gestation to either immediate clamping of the umbilical cord (≤10 seconds after delivery) or delayed clamping (≥60 seconds after delivery). The primary composite outcome was death or major morbidity (defined as severe brain injury on postnatal ultrasonography, severe retinopathy of prematurity, necrotizing enterocolitis, or late-onset sepsis) by 36 weeks of postmenstrual age. Analyses were performed on an intention-to-treat basis, accounting for multiple births. RESULTS: Of 1634 fetuses that underwent randomization, 1566 were born alive before 30 weeks of gestation; of these, 782 were assigned to immediate cord clamping and 784 to delayed cord clamping. The median time between delivery and cord clamping was 5 seconds and 60 seconds in the respective groups. Complete data on the primary outcome were available for 1497 infants (95.6%). There was no significant difference in the incidence of the primary outcome between infants assigned to delayed clamping (37.0%) and those assigned to immediate clamping (37.2%) (relative risk, 1.00; 95% confidence interval, 0.88 to 1.13; P=0.96). The mortality was 6.4% in the delayed-clamping group and 9.0% in the immediate-clamping group (P=0.03 in unadjusted analyses; P=0.39 after post hoc adjustment for multiple secondary outcomes). There were no significant differences between the two groups in the incidences of chronic lung disease or other major morbidities. CONCLUSIONS: Among preterm infants, delayed cord clamping did not result in a lower incidence of the combined outcome of death or major morbidity at 36 weeks of gestation than immediate cord clamping. (Funded by the Australian National Health and Medical Research Council [NHMRC] and the NHMRC Clinical Trials Centre; APTS Australian and New Zealand Clinical Trials Registry number, ACTRN12610000633088 .).

Effect of Delayed Cord Clamping on Systemic Blood Flow: A Randomized Controlled Trial.

OBJECTIVE: To determine whether delayed cord clamping improves systemic blood flow compared with immediate cord clamping in very preterm infants in the first 24 hours. STUDY DESIGN: Women delivering at <30 weeks' gestation at 5 tertiary centers were randomized to receive immediate cord clamping (<10 seconds) or delayed cord clamping (≥60 seconds). Echocardiography and cardiorespiratory data were collected at 3, 9, and 24 hours after birth. The primary outcome was mean lowest superior vena cava (SVC) flow. RESULTS: Of 266 infants enrolled, 133 were randomized to immediate cord clamping and 133 to delayed cord clamping. The 2 groups were similar at baseline, including mean gestation (immediate cord clamping 28 weeks vs delayed cord clamping 28 weeks) and birth weight (immediate cord clamping 1003 g vs delayed cord clamping 1044 g). There was no significant difference between groups in the primary outcome of mean lowest SVC flow (immediate cord clamping 71.4 mL/kg/min [SD 28.1] vs delayed cord clamping 70.2 mL/kg/min [SD 26.9]; P = .7). For secondary outcomes, hemoglobin increased by 0.9 g/dL at 6 hours in the group with delayed cord clamping (95% CI 3.9, 14.4; P = .0005, adjusted for baseline). The group with delayed cord clamping had lower right ventricular output (-21.9 mL/kg/min, 95% CI -39.0, -4.7; P = .01). Rates of treated hypotension, ductus arteriosus size and shunt direction, and treatment of the ductus arteriosus were similar. CONCLUSIONS: Delayed cord clamping had no effect on systemic blood flow measured as mean lowest SVC flow in the first 24 hours in infants <30 weeks' gestation. TRIAL REGISTRATION: Australia New Zealand Clinical Trials Registry: ACTRN12610000633088.

Precision Medicine for Advanced Pancreas Cancer: The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) Trial.

PURPOSE: Personalized medicine strategies using genomic profiling are particularly pertinent for pancreas cancer. The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) trial was initially designed to exploit results from genome sequencing of pancreatic cancer under the auspices of the International Cancer Genome Consortium (ICGC) in Australia. Sequencing revealed small subsets of patients with aberrations in their tumor genome that could be targeted with currently available therapies. EXPERIMENTAL DESIGN: The pilot stage of the IMPaCT trial assessed the feasibility of acquiring suitable tumor specimens for molecular analysis and returning high-quality actionable genomic data within a clinically acceptable timeframe. We screened for three molecular targets: HER2 amplification; KRAS wild-type; and mutations in DNA damage repair pathways (BRCA1, BRCA2, PALB2, ATM). RESULTS: Tumor biopsy and archived tumor samples were collected from 93 patients and 76 were screened. To date 22 candidate cases have been identified: 14 KRAS wild-type, 5 cases of HER2 amplification, 2 mutations in BRCA2, and 1 ATM mutation. Median time from consent to the return of validated results was 21.5 days. An inability to obtain a biopsy or insufficient tumor content in the available specimen were common reasons for patient exclusion from molecular analysis while deteriorating performance status prohibited a number of patients from proceeding in the study. CONCLUSIONS: Documenting the feasibility of acquiring and screening biospecimens for actionable molecular targets in real time will aid other groups embarking on similar trials. Key elements include the need to better prescreen patients, screen more patients, and offer more attractive clinical trial options.

Performance of the Parent Report of Children's Abilities-Revised (PARCA-R) versus the Bayley Scales of Infant Development III.

BACKGROUND: The Parent Report of Children's Abilities-Revised (PARCA-R) assesses cognitive and language development at 24 months. It was validated against the Mental Development Index of the Bayley Scales of Infant Development II (BSID II), but this has now been superseded by BSID III. OBJECTIVE: To compare the PARCA-R against the BSID III. METHODS: PARCA-R and BSID III assessments scheduled at 24 months of age (corrected for prematurity) were completed in 204 infants with suspected or proven neonatal sepsis in the International Neonatal Immunotherapy Study. Associations between the scales were measured and the predictive accuracy of the PARCA-R for moderate cognitive delay and moderate language delay was assessed using Receiver Operating Characteristic (ROC) analysis. RESULTS: Median birthweight was 911 g, median gestational age at birth was 27 weeks and 100 (49.0%) were girls. 4.4% and 8.4% met standard BSID III criteria for cognitive delay and language delay, respectively. These rates increased to 19.6% and 12.6% when an independent sample of normal term infants were used as the reference group suggesting standard BSID III reference norms may tend to underestimate delay. The Spearman correlation between PARCA-R and BSID scales were 0.43 for cognition and 0.71 for language. The PARCA-R successfully predicted cases of cognitive delay and language delay with the area under the ROC curves ranging from 0.83 to 0.97 depending on reference norms used. CONCLUSIONS: The results support the PARCA-R as a practical tool for the identification of appreciable cognitive and language delay at 24 months among critically ill premature and extremely low birthweight neonates.

Identification of infants with major cognitive delay using parental report.

AIM: The collection of data on longer-term neurodevelopmental outcomes within large neonatal randomized controlled trials by trained assessors can greatly increase costs and present many operational difficulties. The aim of this study was to develop a more practical alternative for identifying major cognitive delay in infants at the age of 24 months, based on parental reports. METHOD: A sample of 476 infants (206 female, 270 male) previously diagnosed with neonatal sepsis (mean birthweight 1329g [SD 865g], mean gestational age at birth 28.7wks [SD 4.5wks]) from the International Neonatal Immunotherapy Study were assessed using the Parent Report of Children's Abilities - Revised and the Bayley Scales of Infant Development, 2nd edition. Logistic regression was used to model the association between the risk of major cognitive delay (i.e. Bayley Scales of Infant Development Mental Development Index <55) and the Parent Report of Children's Abilities - Revised data. RESULTS: The receiver operating characteristic curves for a number of predictive models were constructed - each achieved an area under the curve of at least 90%. The sensitivity, specificity, positive predictive value, and negative predictive value of a number of points on the receiver operating characteristic curves are presented. INTERPRETATION: The Parent Report of Children's Abilities - Revised is a practical tool for identifying major cognitive delay in infants at 24 months.

Proteomic analysis of sputum from adults and children with cystic fibrosis and from control subjects.

RATIONALE: Recurrent pulmonary exacerbations are associated with progressive lung disease in cystic fibrosis (CF). Current definitions of an exacerbation, although not precisely defined, include new/worsening symptoms, declining lung function, and/or changing radiologic appearance. Early diagnosis of exacerbations by rapid noninvasive means should expedite therapeutic intervention, thereby minimizing lung damage. OBJECTIVES: To identify biomarkers of lung exacerbation for point-of-care monitoring of CF lung disease progression. METHODS: Saline-induced sputum was collected from adults with CF with an exacerbation and requiring hospitalization (FEV(1) < 60%), a subset of these adults at hospital discharge, children with stable CF and preserved lung function (FEV(1) > 70%), and control subjects (FEV(1) > 80%). Sputum was arrayed by two-dimensional electrophoresis and differentially expressed proteins were identified by proteomic analysis. MEASUREMENTS AND MAIN RESULTS: Sputum profiles from adults with CF with an exacerbation were characterized by extensive proteolytic degradation and influx of inflammation-related proteins, with some adults with CF approaching a "healthy" protein profile after hospitalization. Two children with CF showed profiles and biomarker expression resembling those of adults with an exacerbation. Levels of differentially expressed myeloperoxidase, cleaved alpha(1)-antitrypsin, IgG degradation, interleukin-8, and total protein concentration, together with their correlation to FEV(1), were statistically significant. Statistical correlation analyses indicated that changes in myeloperoxidase expression and IgG degradation were the strongest predictors of FEV(1). CONCLUSIONS: We identified extensive protein degradation and differentially expressed proteins as biomarkers of inflammation relating to pulmonary exacerbations. Prediction of exacerbation onset and more precise evaluation of the extent of resolution with treatment could be achieved by including biomarkers in standard assessment.

Optimal replication and the importance of experimental design for gel-based quantitative proteomics.

Quantitative proteomic studies, based on two-dimensional gel electrophoresis, are commonly used to find proteins that are differentially expressed between samples or groups of samples. These proteins are of interest as potential diagnostic or prognostic biomarkers, or as proteins associated with a trait. The complexity of proteomic data poses many challenges, so while experiments may reveal proteins that are differentially expressed, these are often not significant when subjected to rigorous statistical analysis. However, this can be addressed through appropriate experimental design. A good experimental design considers the impact of different sources of variation, both analytical and biological, on the statistical importance of the results. The design should address the number of samples that must be analyzed and the number of replicate gels per sample, in the context of a particular minimum difference that one is seeking to achieve. In this study, we explore the ways to improve the quality of protein expression data from 2-DE gels, and describe an approach for defining the number of samples required and the number of gels per sample. It has been developed for the simplest of situations, two groups of samples with variation at two levels: between samples and between gels. This approach will also be useful as a guide for more complex designs involving more than two groups of samples. We describe some Internet-accessible tools that can assist in the design of proteomic studies.

An immunoproteomic approach for identification of clinical biomarkers for monitoring disease: application to cystic fibrosis.

Circulating antibodies can be used to probe protein arrays of body fluids, prepared by two-dimensional gel electrophoresis, for antigenic biomarker detection. However, detected proteins, particularly low abundance antigens, often remain unidentifiable due to proteome complexity and limiting sample amounts. Using a novel enrichment approach exploiting patient antibodies for isolation of antigenic biomarkers, we demonstrate how immunoproteomic strategies can accelerate biomarker discovery. Application of this approach as a means of identifying biomarkers was demonstrated for cystic fibrosis (CF) lung disease by isolation and identification of inflammatory-associated autoantigens, including myeloperoxidase and calgranulin B from sputum of subjects with CF. The approach was also exploited for isolation of proteins expressed by the Pseudomonas aeruginosa strain PA01. Capture of PA01 antigens using circulating antibodies from CF subjects implicated in vivo expression of Pseudomonas proteins. All CF subjects screened, but not controls, were immunoreactive against immunocaptured Pseudomonas proteins, representing stress (GroES and ferric iron-binding protein HitA), immunosuppressive (thioredoxin), and alginate synthetase pathway (nucleoside-diphosphate kinase) proteins, implicating their clinical relevance as biomarkers of infection.

Mucin glycosylation changes in cystic fibrosis lung disease are not manifest in submucosal gland secretions.

SMG (submucosal gland) secretions are a major component of the airway surface liquid, are associated with innate immunity in the lung, and have been reported to be altered in lung disease. Changes in lung mucosal glycosylation have been reported in CF (cystic fibrosis), which may be responsible for differential bacterial binding to glycosylated components in the lung mucosa and hence increased pre-disposition to pulmonary infection. Glycoproteomic analysis was performed on SMG secretions collected from explanted bronchial tissue of subjects with severe lung disease, with and without CF, and controls without lung disease. Mucins MUC5B and MUC5AC were shown to be the dominant high-molecular-mass glycoprotein components, with a minor non-mucin glycoprotein component, gp-340, also present. Oligosaccharides containing blood-group determinants corresponding to subjects' blood type were abundant on MUC5B/MUC5AC, as were Lewis-type epitopes and their sialylated analogues, which are ligands for pathogens and leucocytes. No significant differences were found in the glycosylation of MUC5B/MUC5AC or gp-340 between CF and non-CF subjects with severe lung disease, implying that CF does not influence SMG secretion mucin glycosylation in end-stage lung disease. There were also no significant differences found in the glycosylation of these components in severe lung disease compared with non-diseased lungs. This suggests that previously reported changes in the glycosylation of respiratory glycoconjugates in CF, and other pulmonary conditions, are not due to the glycosylation of components in SMG secretions, but may involve other secretions, responses or extracellular factors.

Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins.

Abundant and hydrophilic nonmembrane proteins with isoelectric points below pH 8 are the predominant proteins identified in most proteomics projects. In yeast, however, low-abundance proteins make up 80% of the predicted proteome, approximately 50% have pl's above pH 8 and 30% of the yeast ORFs are predicted to encode membrane proteins with at least 1 trans-membrane span. By applying highly solubilizing reagents and isoelectric fractionation to a membrane fraction of yeast we have a purified and identified 780 protein isoforms, representing 323 gene products, including 28% low abundance proteins and 49% membrane or membrane associated proteins. More importantly, considering the frequency and importance of co- and post-translational modifications, the separation of protein isoforms is essential and two-dimensional electrophoresis remains the only technique which offers sufficient resolution to address this at a proteomic level.