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sputum test for pre-cancerous cells

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Almost three years old, but it seems not widely available

Sputum Testing for Precancerous Lung Cells Progresses Toward the Clinic

Effective sputum testing to detect pre-cancerous cells in the airways now appears likely to become an important diagnostic tool. Although obstacles to its clinical acceptance remain, technological advances and discoveries in genetics and biomarkers have enabled researchers to detect cell changes indicative of lung cancer 1 to 2 years in advance of clinical disease.

Moreover, combining sputum testing with spiral computed tomography (CT) scanning should give a two-step diagnostic battery that could save millions of lives among the 80 million US smokers and former smokers who are at high risk for developing lung cancer. "I see them as two complementary approaches for early lung cancer detection," said Melvyn S. Tockman, MD, PhD, professor of medicine and director of molecular screening at H. Lee Moffitt Cancer Center and Research Institute. "This is predicated on us changing the paradigm from detection of cancer to detection of carcinogenesis."

By definition, carcinogenesis is not simply the disease state but a process that involves both neoplastic transformation and malignant growth. "So we are looking for evidence of change toward the process of carcinogenesis rather than for a single disease entity," he said. "There is a clonal phase for preinvasive lesions. Even before these cancer cells begin to invade the basement membrane, they are detectable as preinvasive lesions. We can sample their transformation and progression because many of these cells will exfoliate before they actually begin to invade.

The concept is that these cells, all through the airway, have been subject to an insult for 20 to 30 years, and that the genetic change that occurs in these cells occurs throughout the airway. Many of these genetic changes actually preceded the change in cell structures. So we can see genetic changes that actually precede morphology.

Dr. Tockman and his colleagues have investigated a protein called hnRNP, and found that it goes through a controlled, or normal expression, and an uncontrolled expression that is associated with tumors. Both normal bronchial epithelial cells taken from lung cancer patients and grown in the laboratory and cancer lines strongly express hnRNP. Normal cells, however, significantly down-regulate hnRNP expression when they reach confluence. Cancer cells keep growing. Also, in normal cells, the hnRNP forms perinuclear granules; in cancer cells, the protein is diffused throughout the cell.

"So, just as in the culture dishes, we see signs that the tumor is showing uncontrolled growth throughout the cytoplasm," Dr. Tockman said. "Interestingly, the hnRNP protein can be seen in exfoliated cells that can be coughed up in the sputum. And even in cells that are not frankly malignant, the pattern of protein expression in the cytoplasm is the same as we see in the tumor."

hnRNP Staining Technique

Moreover, with the aid of a computer program, the presence of hnRNP can be measured in the cell cytoplasm and its morphology determined using a staining technique. In several clinical trials, researchers have detected evidence of cell transformation up to 2 years in advance of the disease. In one study, the Lung Cancer Early Detection Working Group found that 660 patients whose tumors were resected were at risk of developing a second primary cancer. Thirteen of the patients did. hnRNP staining was positive in 77% of them, and negative in 82% of the patients who did not develop a second tumor.

In a study of 6,000 Chinese miners who smoked and were exposed to radon and arsenic while working underground, 56 developed primary lung cancer. The staining technique was positive in 82% of those 56 and negative in 65% of others who did not develop a tumor. In a Liverpool, England, study, 57 individuals went on to develop lung cancer and 95% of them had a positive staining, while 84% of those who did not get the disease had a negative screening.

"We are very encouraged by the sensitivity and specificity of this technique," Dr. Tockman said. "Recently, we conducted a blinded reappraisal of these specimens and found that the result held up, with an 80% and 86% sensitivity and specificity, respectively."

Adenocarcinomas Most Commonly Detected

Both spiral CT scanning and hnRNP in sputum detect cells that are complementary in terms of both stage and cell type, Dr. Tockman said. He noted that the paper published by Claudia Henschke, MD, PhD, and her colleagues in Lancet in 1999 showed that the majority of cancers detected by spiral CT scanning were adenocarcinomas, the cell type that is associated with peripheral lesions. Eleven percent were adenosquamous, 4% were squamous, and no large- or small-cell cancers were detected. In contrast, in the Lung Cancer Early Detection Working Group study, hnRNP detected early signs of the four major cells types seen in the general population. This suggests, he said, "that many of the earlier lesions in the central airways may be better detected by mechanisms using molecular markers and sputum, than perhaps by radiography."

Sputum collection today is marked by poor specimen preservation and preparation, which results in many unsatisfactory sputum specimens, Dr. Tockman noted. Nonetheless, "industry is very aware of the problem and is developing solutions for sputum collection."

In addition to hnRNP, several other biomarkers appear to offer great potential for screening cellular RNA for signs of early cancer. And researchers are examining the usefulness of several DNA markers, including one technique that detects gene promoter hypermethylation as a sign that carcinogenesis is active in the lungs.

At the Moffitt Cancer Center, Dr. Tockman and his colleagues are currently testing the hypothesis that combining hnRNP sputum sampling and spiral CT scanning will increase the percentage of stage I lung cancers detected by at least threefold. The subject population consists of individuals at least 45 years old with a smoking history of 30 pack-years. According to Florida cancer statistics, only 20% of patients diagnosed with lung cancer have resectable (stage I) disease. The Moffitt team expects that 60% of the lung cancers they find will be stage I tumors.

"The development of computers and diagnostic platforms is making cost-effective mass screenings for lung cancer by helical CT and sputum molecular airway markers possible," Dr. Tockman said. "Compared to helical CT, molecular airway markers detect lung cancers at a complementary stage (clonal and preinvasive vs invasive), complementary location (central vs peripheral), and complementary cell types. The widespread enthusiasm for trials of helical CT screening will allow the additional assessment of several roles for sputum molecular markers at little cost."

However, he warned, physicians and the public need to temper their enthusiasm for early lung cancer testing for now, he added. "The large numbers of persons at risk for lung cancer require careful assessment of these molecular and helical CT screening techniques and management algorithms before their widespread adoption. We have got to do the science before we bring it to the public."

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