Tumor-associated autoantibodies in combination with alpha-fetoprotein for detection of early stage hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) continues to be a leading challenge in modern oncology. Early detection via blood-based screening tests has the potential to cause a stage-shift at diagnosis and improve clinical outcomes. Tumor associated autoantibodies (TA-AAbs) have previously shown the ability to distinguish HCC from patients with high-risk liver disease. This research aimed to further show the utility of TA-AAbs as biomarkers of HCC and assess their use in combination with Alpha-fetoprotein (AFP) for detection of HCC across multiple tumor stages. METHODS: Levels of circulating G class antibodies to 44 recombinant tumor associated antigens and circulating AFP were measured in the serum of patients with HCC, non-cancerous chronic liver disease (NCCLD) and healthy controls via enzyme-linked immunosorbent assay (ELISA). TA-AAb cut-offs were set at the highest Youden's J statistic at a specificity ≥95.00%. Panels of TA-AAbs were formed using net reclassification improvement. AFP was assessed at a cut-off of 200 ng/ml. RESULTS: Sensitivities ranged from 1.01% to 12.24% at specificities of 95.96% to 100.00% for single TA-AAbs. An ELISA test measuring a panel of 10 of these TA-AAbs achieved a combined sensitivity of 36.73% at a specificity of 89.89% when distinguishing HCC from NCCLD controls. At a cut-off of 200 ng/ml, AFP achieved a sensitivity of 31.63% at a specificity of 100.00% in the same cohort. Combination of the TA-AAb panel with AFP significantly increased the sensitivity for stage one (40.00%) and two (55.00%) HCC over the TA-AAb panel or AFP alone. CONCLUSIONS: A panel of TA-AAbs in combination with AFP could be clinically relevant as a replacement for measuring levels of AFP alone in surveillance and diagnosis strategies. The increased early stage sensitivity could lead to a stage shift with positive prognostic outcomes

Serum autoantibody measurement for the detection of hepatocellular carcinoma

Background: Individuals with liver disease, and especially those with Hepatitis B or C, are at an increased risk of developing hepatocellular carcinoma (HCC) which is the third most common cause of cancer-related death worldwide. Inadequate screening tests largely account for presentation of advanced tumours and high mortality rates. Early detection of HCC amongst high-risk groups is paramount in improving prognosis. This research aimed to further characterise the previously described humoral immune response raised to tumour-associated antigens (TAAs) in the serum of patients with HCC.Methods: Serum from 96 patients with confirmed HCC, 96 healthy controls matched for age and sex, 78 patients with confirmed liver cirrhosis and 91 patients with confirmed chronic liver disease were analysed for the presence of IgG autoantibodies raised to 41 recombinant TAAs/antigen fragments by ELISA.Results: Varying autoantibody specificities (97–100%) and sensitivities (0–10%) were observed to individual TAAs. A 21-antigen panel achieved a specificity of 92% and sensitivity of 45% for the detection of HCC. This same panel identified 21% of 169 high-risk controls as having elevated autoantibody levels. A reproducible panel of 10 antigens achieved a specificity of 91% and sensitivity of 41% in HCC. 15% of 152 high-risk controls gave positive results with this panel.Conclusions: This minimally invasive blood test has the potential to offer advantages over currently available tools for the identification of HCC amongst pre-disposed patients. Results are comparable to current gold standards in HCC (Ultrasonography) and to similar tests in other cancers (EarlyCDT-Lung)

Tumor-associated autoantibodies in combination with alpha-fetoprotein for detection of early stage hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) continues to be a leading challenge in modern oncology. Early detection via blood-based screening tests has the potential to cause a stage-shift at diagnosis and improve clinical outcomes. Tumor associated autoantibodies (TA-AAbs) have previously shown the ability to distinguish HCC from patients with high-risk liver disease. This research aimed to further show the utility of TA-AAbs as biomarkers of HCC and assess their use in combination with Alpha-fetoprotein (AFP) for detection of HCC across multiple tumor stages.MethodsLevels of circulating G class antibodies to 44 recombinant tumor associated antigens and circulating AFP were measured in the serum of patients with HCC, non-cancerous chronic liver disease (NCCLD) and healthy controls via enzyme-linked immunosorbent assay (ELISA). TA-AAb cut-offs were set at the highest Youden's J statistic at a specificity ≥95.00%. Panels of TA-AAbs were formed using net reclassification improvement. AFP was assessed at a cut-off of 200 ng/ml.ResultsSensitivities ranged from 1.01% to 12.24% at specificities of 95.96% to 100.00% for single TA-AAbs. An ELISA test measuring a panel of 10 of these TA-AAbs achieved a combined sensitivity of 36.73% at a specificity of 89.89% when distinguishing HCC from NCCLD controls. At a cut-off of 200 ng/ml, AFP achieved a sensitivity of 31.63% at a specificity of 100.00% in the same cohort. Combination of the TA-AAb panel with AFP significantly increased the sensitivity for stage one (40.00%) and two (55.00%) HCC over the TA-AAb panel or AFP alone.ConclusionsA panel of TA-AAbs in combination with AFP could be clinically relevant as a replacement for measuring levels of AFP alone in surveillance and diagnosis strategies. The increased early stage sensitivity could lead to a stage shift with positive prognostic outcomes

Application of a high throughput method of biomarker discovery to improvement of the EarlyCDT®-Lung Test

The National Lung Screening Trial showed that CT screening for lung cancer led to a 20% reduction in mortality. However, CT screening has a number of disadvantages including low specificity. A validated autoantibody assay is available commercially (EarlyCDT®-Lung) to aid in the early detection of lung cancer and risk stratification in patients with pulmonary nodules detected by CT. Recent advances in high throughput (HTP) cloning and expression methods have been developed into a discovery pipeline to identify biomarkers that detect autoantibodies. The aim of this study was to demonstrate the successful clinical application of this strategy to add to the EarlyCDT-Lung panel in order to improve its sensitivity and specificity (and hence positive predictive value, (PPV)). Serum from two matched independent cohorts of lung cancer patients were used (n = 100 and n = 165). Sixty nine proteins were initially screened on an abridged HTP version of the autoantibody ELISA using protein prepared on small scale by a HTP expression and purification screen. Promising leads were produced in shake flask culture and tested on the full assay. These results were analyzed in combination with those from the EarlyCDT-Lung panel in order to provide a set of re-optimized cut-offs. Five proteins that still displayed cancer/normal differentiation were tested for reproducibility and validation on a second batch of protein and a separate patient cohort. Addition of these proteins resulted in an improvement in the sensitivity and specificity of the test from 38% and 86% to 49% and 93% respectively (PPV improvement from 1 in 16 to 1 in 7). This is a practical example of the value of investing resources to develop a HTP technology. Such technology may lead to improvement in the clinical utility of the EarlyCDT­-Lung test, and so further aid the early detection of lung cancer

Summary and Comparison of Panel Performance for Cohorts 1 and 2.

<p>Data derived from application of the commercial panel cut-offs to the <i>Early</i>CDT-Lung panel are shown in bold, italic font. Cut-offs were then optimised on cohort 2 and then applied to the results obtained from cohort 1. The effect of dropping one or more antigens from the panel is shown.</p

Positivity of scaled up lung cancer leads compared with the <i>Early</i>CDT-Lung panel.

<p>Cut-offs were optimized to give the maximum number of positive cancers and negative control samples for both the leads and the <i>e</i>CDT-Lung R&D panel antigens. Overall positivity in cancers and controls is given for each lead as well as the additive positivity over the <i>e</i>CDT-Lung R&D panel. Antigens defined as confirmed leads and taken forward for validation on a separate sample cohort are shown in bold italic font.</p

Sensitivity and specificity of confirmed leads.

<p>Cut-offs that had been optimised on the cohort 2 sample set using R&D batches of protein were applied to the results of assays of the cohort 1 samples to validate sensitivity (sens) and specificity (spec) for each confirmed lead alone (bold) and in combination with the <i>e</i>CDT-Lung R&D panel (italic). Sensitivity and specificity values are also given for every combination of two or three confirmed leads in when added to the <i>e</i>CDT-Lung R&D panel.</p

K-S Score for R&D assay analysis of scaled up fusion proteins.

<p>Fusion proteins shown by HTPA to be potential leads were expressed in shake flask culture, purified and used as capture antigens for measurement of AAb by 5 point R&D assay. Calculations were based on populations of 100 lung cancers and 100 individuals with no evidence of malignant disease (cohort 2). Bars represent fusion protein plated at 160 nM (black) and 50 nM (grey).</p