Short summary

This is a translational study investigating the use of methylated DNA, auto-Ab, and NK cell activity in plasma as a potential screening tool for lung cancer. If the hypotheses are correct, the blood sample could be used to eliminate suspicion of lung cancer without any invasive procedures or carcinogenic radiation. It is quick and simple to perform with minimal discomfort to the patient and can be carried out in general practice settings. It could contribute to earlier diagnosis of lung cancer, which is urgently needed, since the disease is often discovered in an advanced stage. It could mean less redundant diagnostic and invasive procedures for patients, if the suspicion could be refuted by a blood sample. 

Rationale

Lung cancer is the leading cause of cancer deaths in Denmark, partly because it is often diagnosed at an advanced stage limiting curative surgery. When a lesion is detected on a CT scan the patient undergoes diagnostic procedures such as PET scan, bronchoscopy, bronchoalveolar lavage, and biopsy. 

If the results suggest benign disease and the clinical suspicion is small, further examinations are usually stopped. If the lesion is too small for biopsy the patient is enrolled in the nodule control program with CT scans. If the lesion remains unchanged during the recommended follow-up period, it is considered benign and the control program is discontinued. CT scans are done as low dose scans with minimal radiation, but there is still a small carcinogenic effect and the method has not yet been implemented as a screening tool in Denmark. A CT scan needs millions of cancer cells for detection. If a blood sample could detect a smaller amount of cancer cells, patients would be able to undergo treatment at an earlier stage, hopefully improving the prognosis. Furthermore, if the method is sensitive enough a blood sample may even exclude malignancy. 

Aberrant methylation occurs in almost all tumours, and analysis of methylated circulating tumour specific DNA has gained increasing interest. A number of studies have suggested its potential for early diagnosis and screening. The primary focus has been on methylated ctDNA as a diagnostic adjunct in manifest lung cancer. There are no studies on the possible merit in patients with an ambiguous lesion. Supplementary information may be obtained by analysis of bronchial lavage, but there are no studies on this topic.

We have developed an analysis of methylated HOXA9, which serves as an important factor in cell proliferation. Our studies have clearly indicated that methylated HOXA9 DNA is an important prognostic marker in locally advanced lung cancer. 

The immunologic status is an important factor in the biological defence against cancer. A change in the immunologic status may serve as a facilitator of an early diagnosis, and recently, detection of lung cancer specific auto-antibodies has been suggested as a biomarker. 

Natural killer (NK) cells serve as an innate immunological surveillance factor in the biological defence against cancer. Following stimulation of NK cells in whole blood with a recombinant protein, a new assay utilises the concentration of interferon gamma in plasma as a surrogate for NK cell activation. This activation seems to differ between cancer patients and the general population. Furthermore, a recent study has demonstrated that four simple proteins in plasma can predict the presence of lung cancer with a sensitivity of 0.63. Therefore, it may be applied as an additional diagnostic tool. 

Objectives

- To investigate the diagnostic sensitivity and specificity of the following markers in lung cancer individually and combined:

- - Methylated HOXA9 in plasma and BALF

- - NK cell activity in plasma and BALF

- - Auto-antibodies in plasma and BALF

- - Cancer antigen 125, carcinoembryonic antigen [CEA], cytokeratin-19 fragment, and the precursor form of surfactant protein B

- - Breath condensate measuring tumor mRNA by the Hawkeye system

- To investigate the additive diagnostic value of the same markers during follow-up of patients with clinical suspicion of lung cancer

- To investigate the lead-time between changes in plasma markers and lung cancer diagnosis in patients with an ambiguous lesion

Description of the cohort

Adults referred from general practice suspected of having lung cancer. 

Data and biological material

Blood samples, bronchial lavage samples from the patients having this procedure performed, breath condensate. 

Clinical and demographic data. 

Collaborating researchers and departments

Department of Oncology, Vejle Hospital

• Professor, MD, DMSc, Anders Jakobsen
• Associate professor, MD, PhD, Torben Frøstrup Hansen

Department of Medicine, Vejle Hospital

• Professor, MD, DMSc, Ole Hilberg

Department of Clinical Biochemistry, Vejle Hospital

• Molecular biologist, PhD, Rikke Fredslund Andersen
• Molecular biologist, PhD, Line Nederby