Background: The increase in lung cancer screening is intensifying the need for a noninvasive test to characterize the many indeterminate pulmonary nodules (IPN) discovered. Correctly identifying non-cancerous nodules is needed to reduce overdiagnosis and overtreatment. Alternatively, early identification of malignant nodules may represent a potentially curable form of lung cancer. Objective: To develop and validate a plasma-based multiplexed protein assay for classifying IPN by discriminating between those with a lung cancer diagnosis established pathologically and those found to be clinically and radiographically stable for at least one year. Methods: Using a novel technology, we developed assays for plasma proteins associated with lung cancer into a panel for characterizing the risk that an IPN found on chest imaging is malignant. The assay panel was evaluated with a cohort of 277 samples, all from current smokers with an IPN 4–30 mm. Subjects were divided into training and test sets to identify a Support Vector Machine (SVM) model for risk classification containing those proteins and clinical factors that added discriminatory information to the Veteran’s Affairs (VA) Clinical Factors Model. The algorithm was then evaluated in an independent validation cohort. Results: Among the 97 validation study subjects, 68 were grouped as having intermediate risk by the VA model of which the SVM model correctly identified 44 (65%) of these intermediate-risk samples as low (n=16) or high risk (n=28). The SVM model negative predictive value (NPV) was 94% and its sensitivity was 94%. Conclusion: The performance of the novel plasma protein biomarker assay supports its use as a noninvasive risk assessment aid for characterizing IPN. The high NPV of the SVM model suggests its application as a rule-out test to increase the confidence of providers to avoid aggressive interventions for their patients for whom the VA model result is an inconclusive, intermediate risk.

Background: In the National Lung Screening Trial, 96.4% of nodules had benign etiology. To avoid unnecessary actions and exposure to harm, individuals with benign disease must be identified. We describe herein the analytical validation of a multi-analyte immunoassay for characterizing the risk that a lung nodule found on CT is malignant. Those at lower risk may be considered for serial surveillance to avoid unnecessary and potentially harmful procedures. While those nodules characterized at higher risk may be appropriate for more aggressive actions. Objective: To validate the analytical performance of multiplexed plasma protein assays used in a novel test for lung nodule characterization. Methods: A multiplexed immunoassay panel for the measurement of plasma proteins in current smokers who present with a lung nodule on CT scan was evaluated in a clinical testing laboratory. Assay analytical sensitivity, reproducibility, precision, and recovery of Epidermal Growth Factor Receptor (EGFR), Prosurfactant protein B (ProSB), and Tissue Inhibitor of Metalloproteinases 1 (TIMP1) from human EDTA plasma samples were evaluated across multiple runs, lots, and technicians. Interfering substances and sample pre-analytical storage conditions were evaluated for their effect on analyte recovery. The lung nodule risk score reproducibility was assessed across multiple lots. Results: The assay sensitivities were 0.10 ng/mL EGFR, 0.02 ng/mL ProSB, and 0.29 ng/mL TIMP1 with over three orders of magnitude in the assay dynamic ranges. The assays and analytes are robust to pre-analytical sample handling and the plasma can be stored for up to 4 days at 4°C either when freshy collected or thawed after long-term storage at −80°C. Total imprecision after 20 days of testing remained under 9% for all three assays. Risk score variability remained within a ± 10% risk score range. Conclusions: The three protein assays comprising the multi-analyte plasma test for lung nodule characterization performed quite acceptably in a clinical laboratory.

Background: To reduce over diagnosis and overtreatment of prostate cancer, a noninvasive and easy to administer test is needed to assess the risk of clinically significant disease. Such an assay must also be able to help accurately inform whether a prostate biopsy is warranted.

Background: To reduce overdiagnosis and overtreatment of noncancerous pulmonary nodules found on CT scans, a noninvasive and easily administered test is needed to assess clinically significant disease risk. Such an assay should also accurately inform whether additional aggressive evaluation, including lung biopsy or thoracic surgery, is warranted. Objective: To determine the performance of a novel, plasma-based multiplexed protein test model when compared to the Veterans Affairs Clinical Factors Model (VA model) for discriminating between a lung cancer diagnosis established pathologically and an Indeterminate Pulmonary Nodule (IPN) found to be clinically and radiographically stable for at least one year. Method: The protein biomarker-based risk model had been trained and tested with a cohort of 277 subjects at high risk of lung cancer, aged 25-85, who were current smokers with an indeterminate lung nodule 4-30mm in diameter (121 subject training set; 59 subject test set) from eight medical centers across the US. Using retrospective plasma samples, we compared the protein biomarker model results with the malignant or benign outcomes in an independent validation cohort comprised of 97 subjects from the Vanderbilt University medical center. Result: Among the 97 validation study subjects (average age 60.1 years, range 42-83; average nodule size 16.1mm), the protein biomarker model correctly identified as benign or malignant an additional 44 of the 68 (65%) indeterminate pulmonary nodules classified as having intermediate risk by the VA model. Negative predictive value was 0.94. Only three patients with malignant disease were missed (94% sensitivity) while an additional 28 intermediate risk samples (41%) were properly classified as true positive, thus potentially avoiding aggressive interventions in those subjects with benign disease. Conclusion: This study evaluated a novel plasma protein biomarker assay model as a noninvasive risk assessment aid for characterizing indeterminate pulmonary nodules. When the model results are combined with the VA model, risk stratification for benign nodules is improved compared to current methods in clinical practice. We hypothesize patients with benign disease may benefit the most from this assay by avoiding unnecessary lung biopsy and subsequent overtreatment, while improving patient quality of care and reducing risks from these procedures. Providers and their patients in whom they suspect lung cancer may consider using this novel assay prior to proceeding with more aggressive interventions.Background: The incidence and prognosis associated with patients undergoing sub-lobar resections and having positive lymph nodes(PLN) has been rarely studied. Our investigation will retrospectively review this topic. Method: The National Cancer Database(NCDB) was queried during the years 2004-2014 to assess patients undergoing sub-lobar resection (wedge, segmentectomy, and sub-lobar-NOS, N ¼ 38,599) and specifically the patients with PLN (N ¼ 5484). Patients were excluded who had any pre-op chemo...

Background: Over diagnosis and overtreatment of indolent prostate cancer (PC) is a serious health issue. There is an unmet clinical need for noninvasive, easy to administer, diagnostic assay to help assess whether a prostate biopsy is warranted.

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BackgroundProtein biomarker profiling in biological fluids will soon become an established procedure in clinical diagnosis of neurodegenerative disease subtypes. Observed differences between technology platforms in performances can be attributed to technology principles and their development status, selection and characterization of antibody combinations, reproducibility of test results, and pre‐analytical factors and processing of samples. Combination of advantages of emerging technologies, together with availability of precision‐qualified assay formats and innovative sample processing procedures, will create additional possibilities for individual patient management using clinically validated protein biomarker panels.MethodThe Giant Magneto Resistance (GMR) technology platform was used to develop multiplex immunoassays for different biomarker proteoforms in blood (e.g., Aß, (phospho)Tau, NF‐L, and GFAP).ResultWe have developed and optimized performance of multiplex assays using a Design of Experiment (DOE) approach to generate reproducible results. The selectivity and specificity of each antibody combination was confirmed with multiplex assay designs. We have used clinical samples instead of calibrators in buffer to shorten the development time. Process optimization through DOE was done with biotinylated peptides. After optimization, reproducibility was improved by approximately 4‐fold to < 5% CV for each analyte. We have included 3D6 as a capture antibody on the printed circuit boards (PCBs), while analyzing Aß1‐42 & Aß1‐40 using 21F12 and 2G3 as capture antibodies, respectively, to identify the presence of Aß oligomers. PCB selection, printing conditions including environmental factors, buffer selections, and conjugation process were optimized in a stepwise manner while frequently checking biomarker levels in clinical samples. Sample processing is part of our roadmap to generate clinically valuable results. The diagnostic potential for each assay format to identify amyloidopathy in an early phase of the disease will be presented using blood samples from cognitively normal, MCI, and AD subjects, characterized by CSF biomarker profiles and/or amyloid PET status.ConclusionThe GMR technology allows screening of performance of antibodies in multiplex formats, shortening the assay development time. The biomarker panels and novel methods for analysis are uniquely positioned to help the healthcare community. This study was funded by Alzheimer’s Drug Discovery Foundation's Diagnostics Accelerator (DxA) Program.

Diagnosis of Gleason 6 prostate cancer can leave uncertainty about the presence of undetected aggressive disease. To reduce overdiagnosis and overtreatment of indolent disease, a noninvasive and easy-to-administer test is needed to better stratify risk in patients with clinically insignificant disease. For men on active surveillance, such an assay must also be able to help accurately inform whether a prostate biopsy is warranted, and it must be able to more closely monitor patients with non-aggressive disease.Objective: To evaluate the performance of a novel, serum-based multiplexed autoantibody assay plus age versus PSA for discriminating high-grade prostate cancer (Gleason Score 7 or greater) from Gleason 6 among patients who have positive biopsies. Methods:Using retrospective serum samples, the autoantibody assay results were compared with biopsy outcomes in 377 patients (an autoantibody-binding peptide discovery study of 146 samples, a training set of 231 total samples (90 + 141) and a validation cohort comprised of approximately 1/3-2/3 1000X protocol involving a total of 231 samples (90 + 141)) with prostate cancer (Gleason 6 or greater). Samples were sourced from two academic sites and one community clinical facility in the United States. Eligible participants included men aged 40 years or older undergoing a prostate biopsy due to suspicious digital rectal examination finding and/ or elevated PSA level.Results: Among the 377 men (median age 63.2 years; median PSA 7.4 ng/mL), the autoantibody assay plus Age showed better discrimination (Area Under the Curve (AUC) 0.83, 95% CI 0.76-0.90) than PSA alone (AUC 0.60, 95% CI 0.52-0.68) (P =0.001) for separating Gleason Score 7 or greater from Gleason Score 6 in prostate cancer positive patients. For detected Gleason Score 7 or higher test with 95% sensitivity, 24.4% of total biopsies could have been avoided, missing only 3.9% or six (6) patients with Gleason Score 7 disease. Of these six (6) patients whose cancers were missed, a sub-analysis revealed five were Gleason 7 (3+4) and only one was Gleason 7 (4+3). Conclusion:This study evaluated a novel, cancer-specific biomarker assay based on autoantibody signatures that could be used as a noninvasive risk assessment aid for men with non-aggressive disease who are not receiving treatment. When results of this assay are combined with traditional clinical risk factors, risk stratification for high-grade prostate cancer and biopsy decision making are improved compared to current methods in clinical practice. We hypothesize patients with Gleason Score 6 will exhibit the greatest benefits from employing the assay as they may likely avoid both unnecessary prostate biopsy and subsequent overtreatment. We further hypothesize the assay will thus significantly reduce costs to the healthcare system while further improving patient's quality of care. Providers and their patients diagnosed with indolent prostate cancer may consider using this novel assay prior to proceeding with prostate biopsy or other treatments.