Application of ProteinChip array profiling in serum biomarker discovery for patients suffering from severe acute respiratory syndrome.

A new strain of coronavirus has caused an outbreak of severe acute respiratory syndrome (SARS) from 2002 to 2003 resulting in 774 deaths worldwide. By protein chip array profiling technology, a number of serum biomarkers that might be useful in monitoring the clinical course of SARS patients were identified. This book chapter describes how the protein chip array profiling was carried out for this study. Briefly, SARS patients' serum samples were first fractionated in Q Ceramic HyperD ion exchange sorbent beads by buffers at different pH. Serum protein fractions thus obtained were then bound onto a copper (II) immobilized metal affinity capture (IMAC30 Cu [II]) ProteinChip Array or a weak cation-exchange (CM10) ProteinChip Array. After washing and addition of sinapinic acid, the chips were read in a Protein Biological System (PBS) IIc mass spectrometer. Ions were generated by laser shots and flied in a time of flight mode to the ion detector according to their mass over charge (m/z) ratio. The serum profiling spectra in SARS patients were acquired, baseline subtracted and analyzed in parallel with those from the control subjects by Ciphergen ProteinChip Software 3.0.2 with their peak intensities compared by a nonparametric two sample Mann-Whitney-U test. More than twelve peaks were differentially expressed in SARS patients with one at m/z of 11,695 (later identified to be serum amyloid A protein), which had increase in peak intensity correlating with the extent of SARS-coronavirus induced pneumonia as defined by a serial chest X-ray opacity score. The remaining biomarkers could also be useful in the study of other clinical parameters in SARS patients.

Deep proteome profiling of sera from never-smoked lung cancer patients.

Previous studies on the serum proteome are hampered by the huge dynamic range of concentration of different protein species. The use of Equalizer Beads coupled with a combinatorial library of ligands has been shown to allow access to many low-abundance proteins or polypeptides undetectable by classical analytical methods. This study focused on never-smoked lung cancer, which is considered to be more homogeneous and distinct from smoking-related cases both clinically and biologically. Serum samples obtained from 42 never-smoked lung cancer patients (28 patients with active untreated disease and 14 patients with tumor resected) were compared with those from 30 normal control subjects using the pioneering Equalizer Beads technology followed by subsequent analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Eighty-five biomarkers were significantly different between lung cancer and normal control. The application of classification algorithms based on significant biomarkers achieved good accuracy of 91.7%, 80% and 87.5% in class-prediction with respect to presence or absence of disease, subsequent development of metastasis and length of survival (longer or shorter than median) respectively. Support vector machine (SVM) performed best overall. We have proved the feasibility and convenience of using the Equalizer Beads technology to study the deep proteome of the sera of lung cancer patients in a rapid and high-throughput fashion, and which enables detection of low abundance polypeptides/proteins biomarkers. Coupling with classification algorithms, the technologies will be clinically useful for diagnosis and prediction of prognosis in lung cancer.

Identification of serum amyloid a protein as a potentially useful biomarker to monitor relapse of nasopharyngeal cancer by serum proteomic profiling.

PURPOSE: Nasopharyngeal cancer (NPC) is a common cancer in Hong Kong, and relapse can occur frequently. Using protein chip profiling analysis, we aimed to identify serum biomarkers that were useful in the diagnosis of relapse in NPC. EXPERIMENTAL DESIGN: Profiling analysis was performed on 704 sera collected from 42 NPC patients, 39 lung cancer patients, 30 patients with the benign metabolic disorder thyrotoxicosis (TX), and 35 normal individuals (NM). Protein profile in each NPC patient during clinical follow up was correlated with the relapse status. RESULTS: Profiling analysis identified two biomarkers with molecular masses of 11.6 and 11.8 kDa, which were significantly elevated in 22 of 31 (71%) and 21 of 31 (68%) NPC patients, respectively, at the time of relapse (RP) as compared with 11 patients in complete remission (CR; RP versus CR, P = 0.009), 30 TX (RP versus TX, P < 0.001), or 35 NM (RP versus NM, P < 0.001). The markers were also elevated in 16 of 39 (41%) lung cancer patients at initial diagnosis. By tryptic digestion, followed by tandem mass spectrometry fragmentation, the markers were identified as two isoforms of serum amyloid A (SAA) protein. Monitoring the patients longitudinally for SAA level both by protein chip and immunoassay showed a dramatic SAA increase, which correlated with relapse and a drastic fall correlated with response to salvage chemotherapy. Serum SAA findings were compared with those of serum Epstein-Barr virus DNA in three relapsed patients showing a similar correlation with relapse and chemo-response. CONCLUSIONS: SAA could be a useful biomarker to monitor relapse of NPC.

Circulating Epstein-Barr virus DNA in serum of patients with lymphoepithelioma-like carcinoma of the lung: a potential surrogate marker for monitoring disease.

PURPOSE: The purpose of this work was to study the sera of patients with lymphoepithelioma-like carcinoma (LELC) of the lung for circulating EBV DNA. EXPERIMENTAL DESIGN: Prospectively collected serum samples from five female patients with advanced, inoperable LELC of the lung were measured for free circulating EBV DNA using a quantitative PCR technique. EBV-encoded small RNA (EBER)-1 was assayed in serial serum samples of three of the five patients, either from the start or during the initial phase of chemotherapy/radiotherapy until their terminal event or last follow-up. There was only a single-point sample for analysis in the fourth and fifth patients. Six other patients with LELC of the lung were also retrospectively identified, and their sera were tested for EBER-1 at either the first visit plus the last follow-up visit (n = 2), the first visit only (n = 2), or the last follow-up visit only (n = 2). RESULTS: Prospectively collected serum samples from five patients and retrospectively collected serum samples from two patients who had clinical disease at initial serum measurement showed detectable levels of EBER-1. Retrospectively collected serum samples from four patients with no clinical disease had negative sera. There is consistent correlation between the clinical response to treatment and subsequent clinical course of LELC and serum EBER-1 levels in the three prospective patients with longitudinal serum monitoring. CONCLUSIONS: This study shows for the first time that free EBV DNA can be detected in the serum of patients with LELC of the lung and further suggests the feasibility of its use for monitoring response to therapy in advanced cases.

Prevalence and parental awareness of hearing loss in children with Down syndrome.

BACKGROUND: To establish the prevalence of hearing deficit in children with Down syndrome (DS) in Hong Kong as measured by brainstem auditory evoked potentials (BAEP). The secondary objective is to examine the agreement between BAEP and clinical questioning in detecting hearing deficit in DS. METHODS: Consecutive DS patients attending the Down's Clinic in a regional pediatric referral center were recruited into this cross-sectional study. BAEP data performed within 12 months were retrieved. The care-taker was interviewed with a structured questionnaire to detect any symptom of hearing impairment. BAEP findings and clinical questionings were compared in an agreement analysis using quadratic weighted kappa statistics. RESULTS: Fifty DS patients (35 male, 15 female, mean age 11.70 years ± 5.74 standard deviation) were recruited. Eighteen patients (36.0%) were identified having hearing deficit by BAEP. Among patients with hearing impairment, 13 patients (72.2%) had a conductive deficit, and most have mild to moderate hearing loss. Five patients (27.8%) had sensorineural deficit and most have moderate to severe degree. Eight (44.4%) had bilateral hearing deficit. Care-takers of 13 patients (26.0%) reported symptoms of hearing impairment, with 9 (69.2%) having mild symptoms, 3 (23.1%) had moderate symptoms and 1 (7.7%) had severe symptoms. The weighted kappa was 0.045 (95.0% confidence interval - 0.138-0.229), indicating very poor strength of agreement between BAEP and clinical questioning. For patients with conductive hearing impairment, only 1 patients (7.7%) recalled history of otitis media. CONCLUSIONS: The estimated point prevalence of hearing impairment in Chinese DS children in Hong Kong is 36%. Our finding of poor strength of agreement between objective testing and symptom questioning reflects significant underestimation of hearing impairment by history taking alone. In view of the high prevalence and low parental awareness, continuous surveillance of hearing is mandatory for DS patients throughout childhood and adolescence.

Prevalence and Characteristics of Chinese Patients With Duchenne and Becker Muscular Dystrophy: A Territory Wide Collaborative Study in Hong Kong.

The aim of this collaborative study on Duchenne muscular dystrophy and Becker muscular dystrophy is to determine the prevalence and to develop data on such patients as a prelude to the development of registry in Hong Kong. Information on clinical and molecular findings, and patient care, was systematically collected in 2011 and 2012 from all Pediatric Neurology Units in Hong Kong. Ninety patients with dystrophinopathy were identified, and 83% has Duchenne muscular dystrophy. The overall prevalence of dystrophinopathy in Hong Kong in 2010 is 1.03 per 10 000 males aged 0 to 24 years. Among the Duchenne group, we observed a higher percentage (40.6%) of point mutations with a lower percentage (45.3%) of exon deletions in our patients when compared with overseas studies. Although we observed similar percentage of Duchenne group received scoliosis surgery, ventilation support, and cardiac treatment when compared with other countries, the percentage (25%) of steroid use is lower.

Role of MIF/CXCL8/CXCR2 signaling in the growth of nasopharyngeal carcinoma tumor spheres.

Macrophage migration inhibitory factor (MIF) and CXCL8 (also named IL-8) are strongly expressed in the tissues of nasopharyngeal carcinoma (NPC). However, their role in the growth of NPC has not been fully examined. This study aims to evaluate the functions of MIF and CXCL8 on the growth of NPC tumor spheres. The elevated expression of CXCL8 in tumor over normal tissues was confirmed in 37 pairs of biopsies from NPC patients. In the in vitro study, all the poorly differentiated NPC cell lines, including the EBV-positive C666-1, and the EBV-negative CNE-1, CNE-2, SUNE-1, HNE-1 and HONE-1 cells, were found to express CXCL8 and MIF. Therefore, the EBV-positive C666-1 cell was selected to examine for the role of MIF and CXCL8 in the growth of the NPC tumor spheres. Functional study showed that the growth of C666-1 tumor spheres, under the nutrient poor or growth factor supplemented culture conditions, could be inhibited by the CXCL8 specific peptide inhibitor. The growth of the tumor spheres could also be reduced by the CXCR2 specific inhibitor SB225002 or the PI3K/AKT inhibitor LY294002, indicating that the endogenously produced CXCL8 plays an autocrine role in the growth of the tumor spheres. Further mechanistic studies revealed that the gene expression of CXCL8 could be reduced by the MIF specific small interfering RNA (siRNA) or NF-κB inhibitor parthenolide, and the growth of tumor spheres was also reduced after MIF siRNA transfection. Taken together, the present study highlights the role of MIF/CXCL8/CXCR2 axis in the growth of NPC tumor spheres. Chemotherapeutic interference of this signaling pathway may help to control the growth of the NPC tumor.

ProteinChip array profiling for identification of disease- and chemotherapy-associated biomarkers of nasopharyngeal carcinoma.

BACKGROUND: We previously used ProteinChip array profiling analysis to discover a serum biomarker associated with nasopharyngeal carcinoma (NPC). In this study, we used the same method to examine other biomarkers associated with NPC and response to chemotherapy (CT) in NPC patients. METHODS: We performed ProteinChip array analysis in 209 serum samples from 66 relapsed patients before and after salvage CT with gemcitabine and cisplatin or etoposide and cisplatin combinations, 11 patients in remission, and 35 healthy individuals. Intensities of the biomarker peaks were correlated with CT response of the patients and other clinical parameters. RESULTS: We discovered 13 candidate biomarkers associated with different clinical parameters. Two biomarkers (2803 and 3953 Da) were significantly increased in patients compared with controls at all stages of disease. Analysis of pre- and post-CT paired serum samples revealed 7 biomarkers correlated with impact of CT. Of these 7 biomarkers, 2 (2509 and 2756 Da) were significantly increased and 5 (7588, 7659, 7765, 7843, and 8372 Da) were significantly decreased post-CT in either 1 or both CT cohorts. Four biomarkers from pre-CT sera were correlated with CT response, with 3 (2950, 13 510, and 14 855 Da) being significantly decreased and 1 (6701 Da) significantly increased in patients who did not respond to CT. Tandem mass spectrometric sequencing and/or immunoaffinity capture assay identified the 3953 Da biomarker as a fragment of interalpha-trypsin inhibitor precursor and 7765 Da biomarker as platelet factor-4. CONCLUSIONS: Treatment-associated serum biomarkers found might serve to triage NPC patients for appropriate CT treatment.

Protein chip array profiling analysis in patients with severe acute respiratory syndrome identified serum amyloid a protein as a biomarker potentially useful in monitoring the extent of pneumonia.

BACKGROUND: A new strain of coronavirus (CoV) has caused an outbreak of severe acute respiratory syndrome (SARS), with 8098 individuals being infected and 774 deaths worldwide. We carried out protein chip array profiling analysis in an attempt to identify biomarkers that might be useful in monitoring the clinical course of SARS patients. METHODS: We performed surface-enhanced laser desorption ionization time-of-flight mass spectrometry on 89 sera collected from 28 SARS patients, 72 sera from 51 control patients with various viral or bacterial infections, and 10 sera from apparently healthy individuals. RESULTS: Nine significantly increased and three significantly decreased serum biomarkers were discovered in the SARS patients compared with the controls. Among these biomarkers, one (11,695 Da) was identified to be serum amyloid A (SAA) protein by peptide mapping and tandem mass spectrometric analysis. When we monitored the SAA concentrations longitudinally in 45 sera from four SARS patients, we found a good correlation of SAA concentration with the extent of pneumonia as assessed by a serial chest x-ray opacity score. Increased SAA occurred in three of four patients at the time of extensive pneumonia as indicated by high x-ray scores. Over the course of gradual recovery in two patients, as assessed clinically and radiologically, SAA concentrations gradually decreased. In the third patient, the concentrations were initially increased, but were further increased with superimposed multiple bacterial infections. SAA was not markedly increased in the fourth patient, who had low x-ray scores and whose clinical course was relatively mild. CONCLUSIONS: Protein chip array profiling analysis could be potentially useful in monitoring the severity of disease in SARS patients.