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

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.