Autoantibody Signature Enhances the Positive Predictive Power of Computed Tomography and Nodule-Based Risk Models for Detection of Lung Cancer.

INTRODUCTION: The incidence of pulmonary nodules is increasing with the movement toward screening for lung cancer by low-dose computed tomography. Given the large number of benign nodules detected by computed tomography, an adjunctive test capable of distinguishing malignant from benign nodules would benefit practitioners. The ability of the EarlyCDT-Lung blood test (Oncimmune Ltd., Nottingham, United Kingdom) to make this distinction by measuring autoantibodies to seven tumor-associated antigens was evaluated in a prospective registry. METHODS: Of the members of a cohort of 1987 individuals with Health Insurance Portability and Accountability Act authorization, those with pulmonary nodules detected, imaging, and pathology reports were reviewed. All patients for whom a nodule was identified within 6 months of testing by EarlyCDT-Lung were included. The additivity of the test to nodule size and nodule-based risk models was explored. RESULTS: A total of 451 patients (32%) had at least one nodule, leading to 296 eligible patients after exclusions, with a lung cancer prevalence of 25%. In 4- to 20-mm nodules, a positive test result represented a greater than twofold increased relative risk for development of lung cancer as compared with a negative test result. Also, when the "both-positive rule" for combining binary tests was used, adding EarlyCDT-Lung to risk models improved diagnostic performance with high specificity (>92%) and positive predictive value (>70%). CONCLUSIONS: A positive autoantibody test result reflects a significant increased risk for malignancy in lung nodules 4 to 20 mm in largest diameter. These data confirm that EarlyCDT-Lung may add value to the armamentarium of the practitioner in assessing the risk for malignancy in indeterminate pulmonary nodules.

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).

Signal stratification of autoantibody levels in serum samples and its application to the early detection of lung cancer.

BACKGROUND: Further signal stratification for the EarlyCDT®-Lung test should facilitate interpretation of the test, leading to more precise interventions for particular patients. METHODS: Samples were measured for the presence of autoantibodies to seven tumor-associated antigens (TAAs) (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, MAGE A4, and HuD). In addition to the current test cut-offs (determined using a previously reported Validation case-control sample set, set A; n=501), new high and low cut-offs were set in order to maximize the test's positive and negative predictive values (PPV and NPV, respectively). All three sets of cut-offs were applied to two confirmatory datasets: (I) the case-control set B (n=751), and (II) Population-derived set C (n=883), and all three datasets combined (n=2,135). RESULTS: For the Validation dataset, cancer/non-cancer positivity for current cut-offs was 41%/9% (PPV =0.109, 1 in 9). The high positive stratum improved this to 25%/2% (PPV =0.274, 1 in 4). The low negative stratum improved this to 8%/23% (NPV =0.990, 1 in 105). This provides a 25-fold difference in lung cancer probability between the highest and lowest groups. The test performs equally well in subjects who fulfilled the entry risk criteria for the National Lung Screening Trial (NLST) and subjects who did not meet the NLST criteria. CONCLUSIONS: The EarlyCDT®-Lung test has been converted to a four-stratum test by the addition of high and low sets of cut-offs: patients are thus stratified into four risk categories. This stratification will enable personalization of subsequent screening and treatment programs for high risk individuals or patients with lung nodules.

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

BACKGROUND: 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)). METHODS AND FINDINGS: 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). CONCLUSION: 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.

Development and validation of a high throughput system for discovery of antigens for autoantibody detection.

An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor-associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment.

EarlyCDT®-Lung test: improved clinical utility through additional autoantibody assays.

Tumor-associated autoantibodies (AAbs) have been described in patients with lung cancer, and the EarlyCDT®-Lung test that measures such AAbs is available as an aid for the early detection of lung cancer in high-risk populations. Improvements in specificity would improve its cost-effectiveness, as well as reduce anxiety associated with false positive tests. Samples from 235 patients with newly diagnosed lung cancer and matched controls were measured for the presence of AAbs to a panel of six (p53, NY-ESO-1, CAGE, GBU4-5, Annexin I, and SOX2) or seven (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, HuD, and MAGE A4) antigens. Data were assessed in relation to cancer type and stage. The sensitivity and specificity of these two panels were also compared in two prospective consecutive series of 776 and 836 individuals at an increased risk of developing lung cancer. The six-AAb panel gave a sensitivity of 39% with a specificity of 89 %, while the seven-AAb panel gave a sensitivity of 41 % with a specificity of 91 % which, once adjusted for occult cancers in the population, resulted in a specificity of 93 %. Analysis of these AAb assays in the at-risk population confirmed that the seven-AAb panel resulted in a significant increase in the specificity of the test from 82 to 90 %, with no significant change in sensitivity. The change from a six- to a seven-AAb assay can improve the specificity of the test and would result in a PPV of 1 in 8 and an overall accuracy of 92 %.

EarlyCDT-Lung: an immunobiomarker test as an aid to early detection of lung cancer.

Recent publications have reported the technical and clinical validation of EarlyCDT-Lung, an autoantibody test which detected elevated autoantibodies in 40% of lung cancers at diagnosis. This manuscript reports the results of EarlyCDT-Lung run on four new (postvalidation) data sets. The following four cohorts of patients (n = 574) with newly diagnosed lung cancer were identified: group 1 (n = 122), 100% small cell lung cancer (SCLC); group 2 (n = 249), 97% non-small cell lung cancer (NSCLC); group 3 (n = 122), 100% NSCLC; group 4 (n = 81), 62% NSCLC. Serum samples were obtained after diagnosis, prior to any anticancer treatment. Autoantibody levels were measured against a panel of six tumor-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1, and SOX2) in the EarlyCDT-Lung panel and previously established cutoffs applied. In groups 2, 3, and 4, patients were individually matched by gender, age, and smoking history to a control individual with no history of malignant disease. Assay sensitivity was tested in relation to cancer type and stage, and in the matched normals to demographic variables. The autoantibody panel showed sensitivity/specificity of 57%/n.d (not done) for SCLC in group 1, 34%/87% for NSCLC in group 2, 31% and 84% for NSCLC in group 3, and 35%/89% for NSCLC and 43%/89% for SCLC in group 4. There was no significant difference in positivity of EarlyCDT-Lung and different lung cancer stages. These studies confirm the value of an autoantibody assay, EarlyCDT-Lung, as an aid to detecting lung cancer in patients at high risk of the disease.