Lunx is a superior molecular marker for detection of non-small cell lung cancer in peripheral blood [corrected]

Abstract

The clinical management of non-small cell lung cancer (NSCLC) would benefit greatly by a test that was able to detect small amounts of NSCLC in the peripheral blood. In this report, we used a novel strategy to enrich tumor cells from the peripheral blood of 24 stage I to IV NSCLC patients and determined expression levels for six cancer-associated genes (lunx, muc1, KS1/4, CEA, CK19, and PSE). Using thresholds established at three standard deviations above the mean observed in 15 normal controls, we observed that lunx (10 of 24, 42%), muc1 (5 of 24, 21%), and CK19 (5 of 24, 21%) were overexpressed in 14 of 24 (58%) peripheral blood samples obtained from NSCLC patients. Patients who overexpressed either KS1/4 (n = 2) or PSE (n = 1) also overexpressed either lunx or muc1. Of patients with presumed curable and resectable stage I to II disease (n = 7), at least one marker was overexpressed in three (43%) patients. In advanced stage III to IV patients (n = 17), at least one marker was overexpressed in 11 patients (65%). These results provide evidence that circulating tumor cells can be detected in NSCLC patients by a high throughput molecular technique. Further studies are needed to determine the clinical relevance of gene overexpression.

Figure 1.

Reliable detection of 20 gene copies in a single round of PCR. Real-time RT-PCR reactions were performed in triplicate as described in Materials and Methods using the lunx primer pair and the lunx synthetic sequence listed in Materials and Methods. Gene copy number was determined by UV absorbance measurements at 260 nm. The line through the data points was obtained by linear regression analysis using Microsoft Excel software.

Figure 2.

Multimarker real-time RT-PCR analysis of NSCLC in peripheral blood. Real-time PCR analyses of peripheral blood specimens from 15 healthy volunteers (open triangles), and 24 NSCLC patients (open diamonds) were performed as described in the text using primer pairs for the indicated genes. Threshold levels of marker positivity for each gene were calculated as described in the text and are depicted by the horizontal line on the left side of each data set. Expression levels of each gene were calculated with Q-gene software and are expressed as the ratio of the target gene relative to β₂-microglobulin.