Detection of Genetic Markers of Lung Cancer

The multistage theory of carcinogenesis includes the development of multiple activating
genetic changes due to exposure to carcinogens, either primarily, or superimposed upon
pre-existing mutations in the genome. These changes result in activation of protooncogenes,
lack of expression of tumor suppressor genes, or combinations of the above, the sum of which
results in malignant transformation. Detailed analyses of chromosomal lesions in bronchogenic
lung cancer reveal several recurring abnormalities, including deletions, duplications or
polysomy of chromosomes 1, 3, 7 and 20. Aberrations in the short arm of chromosome 3, in
particular, are found in many small cell and non-small cell lung cancers, and polysomy 7 is a
frequent finding in non-small cell lung cancers. Many of these abnormalities have no
identified significance, however the application of current and evolving techniques of
molecular biology have revealed specific genomic changes leading to malignant phenotypes in
several tumors, for example, the application of polymerase chain reaction amplification
techniques has revealed a striking incidence of mutations in the h- and k-ras protooncogenes
have been discovered, associated with over-expression of growth factors or receptors, for
example epidermal growth factor receptor.

As all epithelial cells are exposed to similar environmental conditions, it seems likely that
many cells undergo mutagenesis simultaneously. Clinically, this is frequently apparent, as
10-20% of patients with lung cancer have another epithelial cancer arise, either
concurrently, or at some later time in their course. The predisposition for development of
second malignancies also affects other epithelial surfaces, for example, there is a strong
tendency for patients with cancer of the head and neck to develop a second malignancy
(bronchogenic lung cancer) in the aerodigestive tract. Despite decreases in the smoking rate
overall in the United States, projections through 2025 indicate that there will still be
100,000 deaths annually from lung cancer and other smoking-associated cancers. Therefore, it
would be of great benefit to patients at risk of developing lung cancer to identify these
changes prior to the development of invasive malignant lesions. This is particularly true of
patients who have already developed a cancer, or in patients with a strong family history who
may have occupational (eg., asbestos) or habitual (eg., cigarette smoke) exposure to
carcinogens. Identification of cancers in the pre-clinical stage has been attempted
previously, for example with screening chest x-rays or sputum cytologies, however, these
approaches have not proven to be beneficial, as current detection methods are not sensitive
enough to identify early, non-phenotypic changes. The proposal outlined herein is designed to
clarify this issue by examining bronchial tissue from patients at risk for development of a
second cancer (patients undergoing primary resection for cure of bronchogenic lung cancer)
and assessing the biopsy tissue for the presence of chromosomal abnormalities and mutations
in the h- and k-ras protooncogenes. These changes may be present for long periods of time in
airway epithelial cells prior to the development of overt pathologic changes, and methods to
recognize these changes would be useful to assess and follow patients at risk for developing
malignancy.

Importance of lymph node status in lung cancer: In patients with non-small cell lung cancer
(NSCLC), tumor stage is the strongest determinant of prognosis. Stratification of patients
into stages facilitates individual treatment decisions based on the survival statistics of a
population. Within these staged populations however, subsets of patients with apparent early
disease will still suffer cancer recurrence. This is due to the inability of current staging
methods to detect small numbers of disseminated tumor cells (micrometastases) in these
patients. Reverse transcription-PCR (RT-PCR) for cancer related messenger RNA's has been
shown to detect the presence of micrometastases in histologically negative lymph node
specimens, and these findings correlate with poor outcome. Unfortunately, routine clinical
application of this technique has been limited by "false positive" results in control tissues
and a low specificity for predicting disease recurrence. We have recently shown that
quantitative RT-PCR (QRT-PCR) can discriminate between true and false positives, and that
this results in an improved ability to predict recurrence. In this proposal we intend to
analyze lymph nodes from patients undergoing surgical resection for NSCLC using quantitative
RT-PCR. These patients will then be followed for five years to determine tumor recurrence.
The goal is to use QRT-PCR to try and identify which patients are at highest risk for disease
recurrence and who may therefore benefit from more aggressive therapies.

Specific Aims

1. To obtain and maintain in cell culture 'normal' bronchial epithelial cells (NBECs) and
tumors from patients undergoing resection for cure of bronchogenic non-small cell lung
carcinoma (NSCLC) and mesothelioma.

2. To harvest NBEC and lung tumors for evaluation of genetic abnormalities.

3. To perform molecular analysis including polymerase chain reaction (PCR) amplification,
flow cytometry, immunohistochemistry, and gene analysis of material from NBECs, tumors,
adjacent and normal lung and blood for evaluation such as mutations in the K-ras and p53
protooncogenes, as well as other candidate genes and pathways such as those involved in
epithelial-mesynchymal transition. In addition, we will look for mutations and
alterations of expression of Fas, Fas ligand, and FADD, three molecules which mediate
programmed cell death and have recently been shown to be expressed on multiple tumor
cells including lung cancer.

4. To analyze cytokines present in lavage fluid, tumors, and lung tissues.

5. To produce T cell cultures from cells present in tumor-draining lymph nodes and in tumor
tissue. To isolate, numerically expand as well as phenotypically and functionally
characterize human tumor-infiltrating lymphocytes (TILs) and tumor cells.

6. To analyze intra-pulmonary and mediastinal lymph nodes for expression of tumor related
mRNA's (such as carcinoembryonic antigen (CEA), cytokeratin-19, hepatocyte growth
factor, gastrin-releasing peptide (GRP) receptor, and the neuromedin-B (NMB) receptor)
as potential evidence of micrometastases.

7. To detect metastatic tumor in bone marrow extracted from discarded rib resection
material.

8. To analyze biomarkers in biological samples and correlate with outcomes.

Significance

Several researchers have already established that chromosomal changes occur in a non-random
pattern in non-small cell lung cancer. It appears that these changes correlate with specific
genetic changes, resulting in the malignant phenotype. Furthermore, a great deal of
experimental evidence supports the multistage theory of carcinogenesis, whereby incremental
changes in the genome accumulate, resulting in the malignant phenotype. The final product of
the accumulated changes is determined by the cell of origin and the number and severity of
changes occurring. We hope to establish that early changes (as expressed by karyotypic
changes or by particular point mutations) can be identified which would indicate the
likelihood of particular patient developing another malignancy. This information could then
be applied to clinical situations, for example, to determine the frequency of clinical
follow-up by chest x-ray, screening bronchoscopy, or sputum cytology. Furthermore, the
information gathered could help identify one or a few genetic changes necessary for
transformation, which could then be explored to further define the transformation process.

The presence of malignant cells in lymph nodes is a critical parameter in the staging of lung
cancer patients. Assessment of lymph nodes is currently done by histopathology alone. The
long-term survival of lung cancer patients who have Stage IB disease (no known lymph node
involvement with a tumor greater than 2 cm) is lower than patients who are Stage IA (no known
lymph node involvement with a tumor less than 2 cm). Likewise, the survival rates of patients
who are judged to be Stage II based on histologically positive level one lymph nodes is often
no better than that of higher stage patients who have level two lymph node involvement. These
observations suggest that micrometastases are often present in lymph nodes that are not
detectable by histological assessment. This proposal will supplement the histopathological
examination of lymph nodes with methods that detect occult metastatic cells to determine
whether assigning patients to a higher stage more accurately reflects their disease burden.
This could affect subsequent treatment and patient outcomes.

Inclusion Criteria:

- Histologic confirmation of non-small cell lung cancer, mesothelioma or a radiographic
lesion highly suspicious for malignancy

- Written informed consent.

- Must be scheduled for a biopsy or surgical resection

Exclusion Criteria:

- Subjects with any "other" prior cancer (other than lung) within 5 years of date of
surgery are NOT eligible (unless there is preoperative pathologic confirmation that
the lung mass is a second primary cancer)

- Subjects with any type of lung cancer other than non-small cell lung cancer(NSCLC)