Trial Information

Detection of Genetic Markers of Lung Cancer Initiation and Progression

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).

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

3. To perform polymerase chain reaction (PCR) amplification of DNA harvested from NBECs,
tumors, adjacent and normal lung and white blood cells as a control for evaluation for
mutations in the K-ras and p53 protooncogenes, as well as other candidate lung cancer
genes. 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.

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.

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 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.