Originally published as JCO Early Release 10.1200/JCO.2003.07.976 on September 24 2003

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Early Detection of Lung Cancer Using Serum RNA or DNA Markers: Ready for Prime Time or for Validation?

University of Colorado Cancer Center, Denver, CO

THERE ARE no established methods to screen smokers or other high-risk subjects for lung cancer. Previous studies of sputum cytolgy or annual chest x-rays showed no benefits in lung cancer mortality reduction, despite improved survival for detected cases.¹ Previous studies of serum protein markers, such as carcinoembryonic antigen and other tumor antigens, have failed to yield sufficient sensitivity or specificity for routine screening use.² More recent studies have reported the presence of DNA markers in the serum of patients with a variety of cancers, including lung cancers.

In this issue of the Journal of Clinical Oncology, Sozzi et al³ present the results of the largest of such studies. Using real-time quantitative polymerase chain reaction (PCR) and human telomerase catalytic component (hTERT) primers, they report elevated (> 15 ng/mL) levels in 78% of lung cancer patients, compared with zero controls. Those reports are an important step forward, but much remains to be done before such tests become standard. A PubMed search for serum DNA markers and lung cancer revealed the studies summarized in Table 1.^3–16 These studies can be divided into studies measuring: (1) total DNA^3–5; (2) gene expression levels using quantitative PCR techniques^5–7; (3) methylation of the promoter of various tumor suppressor genes, alone or in combination^8–11; (4) microsatellite alterations using several markers, alone or in combination^4,10,12–16; and (5) mutations in specific oncogenes such as K-ras.^9,10 These studies have several common features. First, the case numbers are small, varying from 16 to 100. Second, the number of control cases is even smaller (n = 0 to 100), and the controls were not matched for obvious clinical features such as age, sex, smoking history, and pulmonary function. Third, abnormalities were found in 25% to 78% of the cases, but in only 0% to 12% of the controls. Fourth, standardized methods and validation of the methods have not been performed.

Methylation-specific PCR techniques have been used to quantify the methylation of the promoter regions of a number of oncogenes.^8–11 Four studies^8–11 used this technology to assess the frequency of abnormalties in the plasma of lung cancer cases and controls. Usadel et al⁸ detected methylation of adenomatous polyposis coli (APC) in 95 (96%) of 99 cancers, primarily lung cancers, of which 47% had detectable amounts of methylated APC promoter DNA in plasma or serum. In contrast, no methylated APC promoter DNA was detected in serum samples from 50 healthy controls. However, other authors have reported far lower frequencies of methylated APC promoter DNA in primary lung cancers. Ramirez et al⁹ used similar techniques to assess promoter methylation of the TMS-1, RASSF1A, and DAPK genes and reported abnormalties in 34% to 40% of 50 lung cancer cases (Table 1). Bearzatto et al¹⁰ used methylation-specific PCR to look for methylation of the p16 promoter in 35 patients. Methylation was found in 34%. Esteller et al¹¹ combined methylation studies of four oncogenes and found that one or more were detectable in the plasma of 11 (50%) of 22 patients but in none of 11 controls.

Loss of heterozygosity and the presence of allele shifts indicating genomic instability have been studied in at least seven reports.^4,10,12–16 All series used two to five markers to increase the percentage of abnormal findings. Sozzi et al conducted allele shift analysis of D21S1245 with LOH of the FHIT locus, and found microsatellite alteration in 35 (40%) of 87 plasma samples.¹² Bruhn et al,¹³ Cuda et al,¹⁴ and Chen et al¹⁵ each used three markers and reported microsatellite alterations in 33% of 43 cases, 61% of 28 cases, and 71% of 21 cases, respectively. These microsatellite alterations were not found in 41 controls. Gonzalez et al¹⁶ used four markers and found that a higher percent were abnormal (71% of 35 patients). However, Bearzatto et al¹⁰ and Sozzi et al⁵ used five markers and found alterations in only 32% and 24% of cases, respectively. There was no uniformity of the selection of the markers used in these studies.

Two studies looked for K-ras mutations in serum or plasma. Ramirez et al⁹ reported finding mutations in 12 of 50 cases, while Bearzatto et al¹⁰ found no mutations among 35 cases.

Given the diversity of findings in these studies, where do we go from here? The results from the study of Sozzi et al in this issue of the Journal of Clinical Oncology are certainly provocative, with a sensitivity of 78% and a specificity of 95%, at a cutoff of 15 ng/mL; however, several steps are necessary before such tests are ready for "prime time." Within the studies presented in Table 1, there are many inconsistencies. For example, assays for hTERT varied in positivity from 25% to 69%, methylations of APC in the literature varies from 0% to more than 90%, K-ras mutations varied from 0% to 24%, and microsatellite alterations also varied considerably. These differences likely reflect variations in the manner of which the blood specimens were collected and handled, and variations in the methods by which the DNA/RNA assay were conducted. Thus, validation of collection and assay methods in different laboratories is critical. The cases and controls in these studies were not well matched. Pulmonary disease and changes in all epithelia induced by tobacco carcinogens should produce serum alterations. Thus, larger, better-matched control series are needed. A large, nested case-control series would be of considerable value.

Spiral computed tomography scanning is being assessed as another potential screening method for lung cancer in the National Lung Screening Trials (NLST) in the United States and in several trials in Europe. Serial blood samples are being collected in several, but not all of these trials (an unfortunate cost-cutting measure by the National Cancer Institute). When one of these DNA or RNA tests becomes validated by consistent results in several labs and confirmed in a large, matched control series, it will be important to study the samples being collected in the NLST and other spiral computed tomography trials to determine whether these serum analyses can help identify subjects at risk for lung cancer, compared with those with benign lesions. Serial analyses will be important in this regard. Ultimately, a prospective mortality reduction study like the National Cancer Institute’s Prostate, Lung, Colorectal, and Ovarian screening trial will be necessary to validate the use of these markers to reduce lung cancer mortality.

Finally, there is the issue of using these assays to observe patients, to predict their clinical outcomes. In the study of Sozzi et al,³ 35 cancer patients had a second analysis 3 to 15 months after surgery. Median DNA concentrations fell from 24.5 ng/mL to 8.4 ng/mL (P < .001) after surgery. However, DNA concentrations fell in four of the five subjects who relapsed, and rose in three of the 28 subjects without relapse. Follow-up, however, is short, and additional time and subjects will be necessary to further evaluate changing levels as a prognostic tool.

In summary, tests for DNA or RNA alterations in plasma have great potential for early detection and follow-up. This study by Sozzi et al is a step forward in developing such a test. However, for lung cancer, much needs to be done in validation, and much larger series must be completed before these tests are ready for prime time.

AUTHOR’S DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The author indicated no potential conflicts of interest.

REFERENCES

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