Development of a multiplexed tumor-associated autoantibody-based blood test for the detection of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies worldwide, and early diagnosis is vital to improving prognoses. We explored the diagnostic potential of a multiplex autoantibody panel as a biomarker for the detection of CRC by ELISA. METHODS: In total, 192 serum samples (92 CRC and 100 matched controls) were tested against a panel of 12 tumor-associated antigens (TAAs): RPH3AL, RPL36, SLP2, p53, survivin, ANAXA4, SEC61B, CCCAP, NYCO16, NMDAR, PLSCR1, and HDAC5. Individual and combined autoantibody signatures were examined. RESULTS: Compared to individual autoantibody markers, the combinations of TAAs provided better discrimination between tumorous and normal sera. The overall sensitivity of a selected panel of four antibodies (anti-SLP2, -p53, -SEC61B, and -PLSCR1) was 64.1%, with a specificity of 80% that increased to 83.7% when carcinoembryonic antigen (CEA) measurement was added. Furthermore, the sensitivity of the panel of four antibodies for early and advanced stages of CRC was 66.7% and 62%, increasing to 88.3% and 84%, respectively, when CEA was added. CONCLUSIONS: We identified a panel of four antibodies as a promising diagnostic biomarker for the detection of CRC.

Rapid identification of HPV 16 and 18 by multiplex nested PCR-immunochromatographic test.

Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application.

Comparison of KRAS mutation analysis of primary tumors and matched circulating cell-free DNA in plasmas of patients with colorectal cancer.

Colorectal cancer (CRC) patients with KRAS mutations do not benefit from epidermal growth factor receptor (EGFR) targeted therapy. In clinical practice, identifying patients with KRAS mutations is critical prior to EGFR targeting therapy, and gene testing is generally performed using the DNA extracted from tumor tissue. The aim of this study was to compare the presence of KRAS mutations in circulating cell-free DNA (cfDNA) and primary tumor tissue using a peptide nucleic acid mediated polymerase chain reaction. We extracted and analyzed the DNA from plasmas and corresponding primary tumor samples from 52 patients with CRC. The results demonstrated that the detection rate of KRAS sequence variations was 50% (26 of 52) in plasma samples and 28.8% (15 of 52) in resected primary tumor tissue samples. The majority of KRAS mutations detected in tumors were also found in matched plasma specimens with an agreement rate of 78.8%. Eleven plasma cfDNA were found positive for KRAS mutation but not in their corresponding tissue. In conclusion, our results suggest that circulating cfDNA provides a better representation of the malignant disease as a whole and could be a reliable source of diagnostic DNA to replace the tumor tissue in a diagnostic setting.

Antibody against N-terminal domain of phospholipid scramblase 1 induces apoptosis in colorectal cancer cells through the intrinsic apoptotic pathway.

Phospholipid scramblase 1 involve in biological processes including phospholipid movement, proliferation, and apoptosis. Treatment with an antiphospholipid scramblase 1 antibody (NP1) has been demonstrated to inhibit cell proliferation in colorectal cancer. This study aimed to explore the role of NP1 treatment in the apoptosis of colorectal cancer cells. Results showed that NP1 treatment significantly increases the apoptosis of colorectal cancer cells via the activation of caspase 8, caspase 9, and caspase 3. Moreover, pretreatment with a caspase 8 inhibitor did not fully prevent the apoptotic effects of NP1. Taken together, these data indicate NP1 induces cell apoptosis primary through the intrinsic apoptotic pathway. NP1 may serve as a potential therapeutic agent.

Association and prognostic value of serum inflammation markers in patients with leukoplakia and oral cavity cancer.

BACKGROUND: Oral cavity cancer ranks as the fourth leading cancer in men in Taiwan. The development of a serum biomarker panel for early detection and disease monitoring is, therefore, warranted. METHODS: Nine inflammation-associated markers were investigated in 46 patients with leukoplakia, 151 patients with untreated oral cavity squamous cell carcinoma (OSCC), and 111 age- and gender-matched healthy controls using enzyme-linked immunosorbent assay. During a subsequent 28-month surveillance of OSCC patients, serum samples were prospectively collected at predetermined intervals following the completion of therapy. RESULTS: Logistic regression analysis showed matrix metalloproteases (MMP)-2, MMP-9, C-reactive protein (CRP), transforming growth factor-ß1 (TGF-ß1), and E-selectin having the best discrimination power between groups and significant elevation trends of those five markers were noted from control to OSCC. By combining those five markers, a 0.888 and 0.938 area under curve by ROC curve analysis with 67.4% and 80% overall sensitivity and fixed 90% specificity for leukoplakia and OSCC groups were demonstrated. In the follow-up period, 25 OSCC patients developed recurring or secondary tumors. All examined markers had decreased in relapse-free patients following treatment. However, in patients with relapse, interleukin-6, CRP, and serum amyloid A remained at elevated levels. Statistical analysis showed that patients with CRP ≧2 mg/L and E-selectin ≧85 ng/mL at baseline had highest probability of relapse (odds ratio=3.029, p<0.05). CONCLUSIONS: The results indicate that inflammation plays a crucial role in the pathogenesis process of OSCC. By examining the inflammation markers, physicians could potentially identify patients at risk of cancer transformation or relapse.

Blockade of phospholipid scramblase 1 with its N-terminal domain antibody reduces tumorigenesis of colorectal carcinomas in vitro and in vivo.

BACKGROUND: Membrane-bound phospholipid scramblase 1 (PLSCR1) is involved in both lipid trafficking and cell signaling. Previously, we showed that PLSCR1 is overexpressed in many colorectal carcinomas (CRCs). In the present study, we investigated the tumorigenic role of PLSCR1 in CRC and suggest that it is a potential therapeutic target. METHODS: To identify PLSCR1 as a therapeutic target, we studied the tumorigenic properties of CRC cell lines treated with a monoclonal antibody (NP1) against the N-terminus of PLSCR1 in vitro and in vivo. We also investigated cell cycle status and epidermal growth factor receptor-related pathways and downstream effectors of PLSCR1 after blocking its function with NP1. RESULTS: Treating CRC cells with NP1 in vitro and in vivo decreased cell proliferation, anchorage-independent growth, migration, and invasion. Adding NP1 to the CRC cell line HT29 caused arrest at G1/S. Treating HT29 cells with NP1 significantly decreased the expression of cyclin D1 and phosphorylation levels of Src, the adaptor protein Shc, and Erks. The reduced level of cyclin D1 led to an increase in the activated form of the tumor suppressor retinoblastoma protein via dephosphorylation. These actions led to attenuation of tumorigenesis. CONCLUSIONS: Therefore, PLSCR1 may serve as a potential therapeutic target for CRC.

New enzyme-linked immunosorbent assay for detection of antibodies against hepatitis delta virus using a hepatitis delta antigen derived from a Taiwanese clone and comparison to the Abbott radioimmunoassay.

An anti-hepatitis delta (HD) enzyme-linked immunosorbent assay (ELISA) using a specific recombinant hepatitis delta antigen derived from a local dominant hepatitis delta virus (hepatitis D virus; HDV) strain in Taiwan has been established. The detection efficiency of this assay was comparable to that of the commercially available Abbott anti-HD radioimmunoassay (RIA) and could be useful in routine laboratory diagnoses of HDV infection.

Detection of autoantibodies against Rabphilin-3A-like protein as a potential biomarker in patient's sera of colorectal cancer.

BACKGROUND: Rabphilin-3A-like (RPH3AL) protein functions in the regulation of hormone exocytosis, and mutations in the RPHA3L gene have been associated with tumorigenesis in colorectal cancer (CRC). We evaluated the potential use of anti-RPH3AL autoantibodies as a marker for CRC detection. METHODS: Sera from 84 patients with CRC and 63 healthy controls were analysed for the presence of RPH3AL autoantibodies with a Western blotting assay. RESULTS: The frequencies of RPH3AL autoantibodies in the early stage, advanced stage and all CRC patients were 64.7%, 78.0% and 72.6%, respectively. These values are significantly higher than the frequency of RPH3AL autoantibodies in healthy controls (15.9%, P<0.001). Although the presence of RPH3AL autoantibodies did not correlate with clinical parameters, RPH3AL autoantibodies were found in 69.4% (34/49) of CRC patients who were negative for carcinoembryonic antigen. The value of the area under the receiver operating characteristic curve of RPH3AL autoantibody was 0.84, which suggests that screening for these autoantibodies could potentially be used for CRC diagnosis. CONCLUSION: Circulating RPH3AL autoantibodies are prevalent in patients with CRC, and detection of these autoantibodies might provide a novel non-invasive approach for CRC diagnosis.

Performance of rapid-test kits for the detection of the pandemic influenza A/H1N1 virus.

The early detection of pandemic influenza strains is a key factor for clinicians in treatment decisions and infection control practices. The aims of this study were to determine the analytical sensitivity and clinical performance of the commercially available influenza rapid tests in Taiwan. Four rapid tests for influenza virus (BinaxNow test, QuickVue test, TRU test, and Formosa Rapid test) were evaluated for their detection limit against four influenza viruses (the 2009 pandemic influenza A virus H1N1, seasonal influenza virus H1N1, H3N2, and influenza B virus) circulating in Taiwan. The viral load of these isolates were quantified by rtRT-PCR and then diluted 2-fold serially for the comparison. The lowest detectable viral load of the pandemic influenza A virus H1N1 by the Formosa Rapid test, QuickVue test, TRU test, and Binax Now test was 5.3×10(4), 1.0×10(5), 1.0×10(5), and 4.2×10(5)copies/µL, respectively. Of these four tests, the two most sensitive tests (the QuickVue test and the Formosa Rapid test) were chosen to evaluate 62 nasopharyngeal specimens from patients who were suspected of infection with pandemic influenza A virus H1N1. The positive rate for the Formosa Rapid test and the QuickVue test were 53.2% (33/62) and 45.2% (28/62) (McNemar's test, P=0.125), respectively. In conclusion, the Formosa Rapid test was the most sensitive test in the present study for the detection of influenza antigens and its clinical performance was similar to that of the QuickVue test (Kappa=0.776). This suggests that the Formosa Rapid test could be used to aid clinical decision making in primary health care settings during outbreaks of influenza.

An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer.

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48-70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.

Identification of SEC61ß and its autoantibody as biomarkers for colorectal cancer.

BACKGROUND: To identify novel serological biomarkers for human colorectal cancer (CRC), we analyzed CRC tissues using gel-assisted digestion and isobaric tags with related and absolute quantitation (iTRAQ) labeling mass spectrometry (MS). By comparing pairs of tumor tissues and matched normal tissues, we discovered the SEC61ß with expression changes 3.3-fold and a marginal statistical significance (p=0.052) previously. METHODS: SEC61ß expression in CRC tissues was further analyzed by western blotting and immunohistochemistry. We next assessed the putative diagnostic value of the SEC61ß autoantibody as a serum marker. RESULTS: Using western blotting analysis, SEC61ß expression was increased 1.9-fold in tumor tissues. Immunohistochemical analysis of 64 CRC specimens showed that SEC61ß was positively detected in 64% of the tumors, but weakly or not detected in >80% of the adjacent nontumor epithelial cells. Western blot analysis with plasma samples showed that the sensitivity and specificity of the SEC61ß autoantibody from patients with CRC were 79% and 75%, respectively. Importantly, the results of the SEC61ß autoantibody for early detection of colorectal cancer revealed a higher sensitivity of 77% than the carcinoembryonic antigen (CEA) assay. CONCLUSIONS: Measurement of SEC61ß autoantibody levels may provide an alternative detection indicator for CRC, particularly among early-stage patients.

Identification of phospholipid scramblase 1 as a biomarker and determination of its prognostic value for colorectal cancer.

The purpose of this study was to examine the expression of phospholipid scramblase 1 (PLSCR1) in tumor tissues and plasma specimens of patients with colorectal cancer (CRC), as well as analyze its association with clinical parameters. The expression levels of PLSCR1 protein in 104 matched CRC and adjacent normal tissue sections and 50 pairs of CRC tissue blocks were determined by use of immunohistochemical and Western blot analyses, respectively. To evaluate the diagnostic potential of PLSCR1, the plasma levels of PLSCR1 were investigated in 111 additional subjects (59 CRC patients and 52 healthy controls) by Western blot. PLSCR1 was overexpressed in malignant adenocarcinoma tissues compared with normal colorectal mucosa (P < 0.001). In addition, the plasma level of PLSCR1 was not only significantly elevated in CRC patients compared with healthy individuals (P < 0.001), but it was also substantially increased in early stage CRC (P < 0.001). Importantly, the overall sensitivity and specificity of PLSCR1 for CRC detection were 80% and 59.6%, respectively. The area under the ROC curve of PLSCR1 for CRC diagnosis is 0.75, which increases to 0.8 if combined with the measurement of carcinoembryonic antigen. Univariate analysis with the Cox regression model revealed that elevated PLSCR1 expression indicated a poor prognosis for CRC. This study showed that PLSCR1 protein levels were significantly elevated in both the cancer tissue and plasma of CRC patients. Moreover, the plasma levels of PLSCR1 were significantly elevated in patients with early stage CRC compared with healthy individuals, suggesting that PLSCR1 might be used as a noninvasive serological diagnostic and prognostic biomarker for CRC.

Prevalence of human papillomavirus genotypes in northern Taiwanese women.

The prevalence of Human Papillomavirus (HPV) in the general population of northern Taiwan is described. A total of 343 consecutive cervical swabs from women visiting the medical center for routine gynecologic care were included. Cervical cell cytology was examined by the Papanicolaou (Pap) test, and a PCR-based hybridization gene chip analysis was used to identify HPV genotypes. The HPV prevalence in the overall population was 32.4%. When divided into two groups according to cytology, 20.9% of women with normal cytology were HPV positive while 75.3% of women with abnormal cytology were HPV positive. Among positive samples, 68.5% were single type infections while 31.5% harbored multiple HPV types. A total of 32 types of HPV were identified; the leading five were HPV16 (5.8%), HPV58 (5.3%), HPV53 (4.1%), HPV52 (3.8%), and HPV18 (2.3%). Our results constitute baseline data and may provide important implications for future prophylactic programs. The relatively high prevalence of HPV 58, 53, and 52 among northern Taiwanese women has important implications for vaccine development.

Identification and clinical association of anti-cytokeratin 18 autoantibody in COPD.

The etiology of chronic obstructive pulmonary disease (COPD) remains unclear. A mechanism involving the autoimmune reaction in the pathogenesis of COPD has been proposed but not confirmed. The aim of this study was to investigate whether serum autoantibodies against pulmonary cellular proteins are present in COPD patients and to identify their autoantigens if possible. Samples from 50 COPD patients and 42 control subjects were studied. Circulating autoantibodies were detected by Western blot. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were used to identify the autoantigens. Autoantibodies against pulmonary cellular antigens were found in the sera of COPD patients. Specifically, an autoantibody against the 45-kDa human cytokeratin 18 protein was found in 76.0% of COPD patients and 23.8% of control subjects (p<0.001). Furthermore, the cytokeratin 18 autoantibody level was positively correlated with the FEV(1) (L) (p=0.013) and FEV(1) (%pred.) (p=0.043) values observed in COPD patients. This study identified the pulmonary epithelial cytokeratin 18 protein as a COPD-associated autoantigen and found that anti-cytokeratin 18 autoantibodies were prevalent in COPD patients. Our results support the hypothesis that humoral autoimmunity may be involved in the pathogenesis of COPD.

Multiple serological biomarkers for colorectal cancer detection.

The aim of this study was to initiate a survey of human autoantibody responses to a panel of select colorectal tumor-associated antigens identified by previous serological analysis of a cDNA expression library and to subsequently identify multiple serological biomarkers for the detection of colorectal cancer. For screening of autoantibodies against colorectal tumor-associated antigens, sera from 94 colorectal cancer patients and 54 normal controls were analyzed by enzyme-linked immunosorbent assay using recombinant rCCCAP, rHDAC5, rP53, rNMDAR and rNY-CO-16 proteins as coating antigens. Seropositivity among colorectal cancer patients to the 5 individual coating antigens varied from 18.1% to 35.1%. Seropositivity to any of the 5 coating antigens was 58.5% and combining this analysis with evaluation of serum carcinoembryonic antigen (> or =5 ng/ml) significantly increased the seropositivity to 77.6%. Seropositivity of early-stage (Dukes' Stages A and B) colorectal cancer patients to CEA was 21.9%, and seropositivity to any of the 5 colorectal cancer-associated antigens was 53.7%, and the combination of these 2 measurements resulted in a higher diagnostic capacity (65.9%) than either marker alone. In conclusion, these results collectively indicated that combined detection of serum autoantibody profiles against our panel of colorectal tumor-associated antigens and the analysis of carcinoembryonic antigen provides a promising diagnostic biomarker for colorectal cancer, particularly among early-stage patients.

Quantitative detection of survivin in malignant pleural effusion for the diagnosis and prognosis of lung cancer.

In the present study, pleural effusions are the first time to be used as the specimens for detection of survivin expression in lung cancer patients. We demonstrated that by quantifying survivin expression with enzyme-linked immunosorbent assay (ELISA) in the 80 effusion samples exhibited a diagnostic power of 85% and 75% in sensitivity and specificity, respectively. A multivariate analysis with the Cox regression model revealed that both high survivin expression and cancer cells of stage IV were the indicators for poor prognosis of lung cancer. In conclusion, quantitative assay of survivin in pleural effusion could be useful both in diagnosis and prognosis for lung cancer.