Procedures for risk-stratification of lung cancer using buccal nanocytology

Abstract

Lung cancer is the leading cause of cancer deaths in the U.S. with survival dramatically depending on stage at diagnosis. We had earlier reported that nanocytology of buccal cells can accurately risk-stratify smokers for the presence of early and late-stage lung cancer. To translate the technique into clinical practice, standardization of operating procedures is necessary to consistently yield precise and repeatable results. Here, we develop and validate simple, robust, and easily implementable procedures for specimen collection, processing, etc. in addition to a commercially-viable instrument prototype. Results of this work enable translation of the technology from academic lab to physicians' office.

Keywords: (110.0180) Microscopy; (110.2960) Image analysis; (120.0120) Instrumentation, measurement, and metrology; (170.1610) Clinical applications.

Fig. 1

(a) Schematic of the Partial Wave Spectroscopic (PWS)Microscopy instrument. White light is incident from a Xenon (Xe) lamp using Kohler illumination microscope light path (PWS Optics)| through the objective (OBJ) onto the sample. Spectrally-resolved microscope image is registered on a charge coupled device (CCD)| camera using a spectral filter (SF, either a slit spectrometer or a liquid-crystal tunable filter). TL denotes atube lens. (b) Measurement work flow of the buccal partial wave spectroscopic (PWS) nanocytology.

Fig. 2

Work flow of the buccal partial wave spectroscopic (PWS) nanocytology process that will enable clinical translation of the technology.

Fig. 3

Examples of buccal squamous epithelial cells found on prepared specimen slides. (a) Representative transmission image of two overlapping cells and (d) the corresponding spatially resolved map Σ(x, y) calculated by PWS. (b) Example of a folded isolated cell and (e) the corresponding map of Σ. (c) Isolated, non-folded cell classified as “suitable” for our study and (f) the corresponding Σ(x, y).

Fig. 4

The smearing deposition is affected by an individual’s technique and a majority of the results are not reproducible as observed from intra-coordinator and inter-coordinator comparisons. (a) Clinical coordinator A’s smearing technique – the brush was smeared back-and-forth in repetitive motions producing a very poor deposition of cells, (b) Clinical coordinator B’s smearing technique – extreme pressure may have been applied on the brush head producing only a few suitable cells, (c) Clinical coordinator C’s technique was most suited in producing many suitable cells for PWS analysis

Fig. 5

Representational transmission microscope images of slides prepared following the deposition techniques that were compared for PWS System Lung test SOP optimization. (a) Aerosol deposition at 10x (left) and 40x microscope magnifications (right), (b) Smear deposition at 10x and 40x.

Fig. 6

Structured workflow representation of the final, optimized SOP for the PWS System Lung Test. Standardized parameters were developed and validated on clinical subjects for each stage in the process.

Fig. 7

3-D model of the commercial-ready HT-PWS prototype that will be implemented in the centralized lab.

Fig. 8

Results of the validation clinical study (n = 37). Bar plots display the observed statistically significant difference between mean Σ of (a) red cells and (b) blue cells obtained from the smoker control population (n = 11 subjects) and the lung cancer population (n = 26) . (c) ROC curve of the diagnostic performance of Σ based on the two parameter model (AUC = 0.85, Se = 81%, Sp = 91%).