ADVANCES IN MOLECULAR BIOLOGY

Feb 7, 2009

The discovery of the DNA double-helix structure by Watson and Crick in 1953 was the most important step toward understanding genetics, protein regulation, and normal cell function. As technology advanced beyond the gross and histologic levels, the cell and its underlying genetic machinery became targets for exploration. It is well established that disease essentially begins at the gene level, and resulting aberrations in gene product expression are what are presented to clinicians. Molecular biology techniques developed for the detection and manipulation of proteins, RNA, and DNA are now commonly used in research directed at the diagnosis and understanding of an array of diseases. The field of hereditary deafness, for example, has benefited from the development of advanced genetic techniques. Further discoveries in the molecular pathogenesis of diseases will allow physicians to improve prevention, diagnosis, prognosis, and treatment. This chapter provides a brief description of recent advances in molecular biology within otolaryngology.
HEAD AND NECK CANCER
Cancer has long been suspected to be a disease ultimately caused by the loss of genetic control. In the last several years, many studies have identified genetic markers that can be used in the prognosis of cancers in the breast, lung, and other sites. The search for gene markers in head and neck cancer lagged. Recent studies have identified markers that may serve as prognostic factors in the management of head and neck cancer.
Normal cell life is regulated by specific genes that code for a variety of proteins that affect homeostasis. Alterations in genes that regulate cellular proliferation, differentiation, and apoptosis (programmed cell death) differentiate neoplastic cells from normal cells. Gene alterations can occur in several ways, such as genome damage by means of mutation or deletion, imprinting, chromosomal rearrangement, or mitotic recombination. Gene activity also can be affected by interaction with viral oncoproteins or carcinogens. In general, any such changes can cause aberrant gene expression and tumorigenesis.
The regulatory genes can be divided into two main categories. Protooncogenes encode proteins that stimulate cellular proliferation. In most cases, they code for growth factors, receptors, and other molecules involved in signal transduction pathways or for transcription factors that regulate gene expression. Oncogenes are protooncogenes that have a mutation that causes malignant transformation when they are inappropriately expressed. Tumor suppressor genes normally hinder the growth of uncontrolled cell proliferation driven by oncogenes. The double-hit theory of cancer development holds that both alleles of a tumor suppressor gene must be inactivated for the cell to proliferate. These abnormal cells thus are allowed to reproduce and expand unchecked.
Several gene regulators have been identified, particularly in regard to squamous cell carcinoma. The function of these genes typically is evaluated indirectly by means of detection of the associated gene product. Continued identification of specific genes, gene products, and their roles in tumor regulation may lead to new preventive techniques, improved diagnostic capabilities, reliable prognostic markers, and specific treatment strategies.
B-cell Lymphoma/Leukemia-2 Gene
The B-cell lymphoma/leukemia-2 gene (bcl-2) is a tumor-suppressor gene and primary regulator of apoptosis. Normal bcl-2 expression inhibits apoptosis and counteracts the effects of p53. The bcl-2 proteins are present predominantly in the mitochondrial membrane and have been found in a variety of tissues, including lymphoid tissue, bronchial epithelium, skin, intestine, breast, prostate, thyroid, and nasopharynx (1). Under healthy conditions, bcl-2 proteins are present only in the basal or proliferating cells of these tissues.
The expression of bcl-2 protein has been studied in tumors of the breast, lung, and prostate as well as in tumors of the head and neck. Although bcl-2 is a relative newcomer to the group of recognized gene markers, it is emerging as a marker of clinical significance. Numerous studies have been performed to identify other gene markers, such as p53, as prognostic indicators. None of the investigators found consistent, statistically relevant predictors of outcome.
In early studies, Friedman et al. (2) identified bcl-2 as a highly sensitive marker for predicting prognosis in early squamous cell carcinoma of the larynx. This was especially important because the group of patients who participated in the study included those treated with either radiation or surgery alone. In a retrospective study of early-stage squamous cell carcinoma of the head and neck (T1N0 or T2N0 glottic larynx; T1N0 oral cavity, pharynx, supraglottic larynx), overexpression of bcl-2 correlated with a significantly reduced cure rate, 50% versus 90%, which is expected in the management of these localized lesions. There was no significant difference in recurrence rate with regard to treatment modality (surgery or radiation therapy).
Other studies have shown similar results among patients treated with radiation only. Gallo et al. (3) conducted a study with a group of patients who had tumors of the head and neck at all sites; 70% of the tumors were located in the larynx. The investigators showed that overexpression of bcl-2 correlated with a shortened disease-free interval and decreased overall survival rate. These results are greatly encouraging but not conclusive. In a study involving 70 patients with squamous cell carcinoma of the larynx and several tumor markers identified, bcl-2 was not a prognostic discriminator (4). Additional studies by Friedman et al. (5) showed that bcl-2 is not a prognostic indicator in advanced laryngeal carcinoma.
Two mechanisms have been suggested by which disordered bcl-2 expression can cause resistance to treatment and shorter survival times. Overexpression of bcl-2 may prevent spontaneous apoptosis and lead to more rapid accumulation of tumor cells for a given proliferation rate. Spontaneous apoptosis is known to be an important factor in tumor-volume doubling time. Another possibility is that bcl-2 confers resistance to therapy by blocking treatment-related apoptosis. Radiation therapy and chemotherapy are directed at inducing apoptosis.
The prognostic significance of bcl-2 overexpression alone may not apply to all tumors. In one study, patients with squamous cell carcinoma of the lung and overexpression of bcl-2 had a better survival rate than those not expressing bcl-2 (6). A similar observation was reported for breast cancer (7). These contradictory findings may be the result of different methods of assessing bcl-2 expression, but the more likely explanation may involve the influence of other closely related gene products, such as bax, bcl-sl, bcl-xs, and bad (8). For example, bcl-2 is thought to function largely by means of antagonizing the cell death–inducing effect of bax. If bax expression is somehow impaired, cancer cells may be resistant to apoptosis even in the presence of very low levels of bcl-2. Abnormalities of bax expression have been documented for breast cancer (8). Assessing the family of related genes instead of bcl-2 alone in squamous cell carcinoma of the head and neck may make it easier to ascertain who will have a poor response to standard treatment.
p53
The p53 tumor suppressor gene is responsible for arrest in the cell cycle after genetic injury. It allows the cell to repair the DNA defect before the next cell division. The gene also induces apoptosis. Alteration in the p53 gene locus is the most commonly identified genetic mutation in squamous cell carcinoma of the head and neck and in all types of cancer among humans.
Mutations of p53 and overexpression of p53 protein have been found in approximately 40% of invasive squamous cell carcinomas of the head and neck and in more than 50% of malignant neoplasms of the mouth (9). Overexpression of p53 also has been observed in dysplasia and carcinoma in situ of the larynx (10). Cigarette smoking is known to cause p53 mutations (11). Squamous cell carcinomas with p53 mutations are more common among persons who smoke and among those who drink heavily. The mutations among these persons have been found over a broad area of the chromosome, rather than at limited sites, as is characteristic of spontaneous mutations in abstainers. Mutations of the p53 gene are less frequent among patients with squamous cell carcinoma who are older than 75 years than among those 40 to 70 years of age (12). This finding implies that squamous cell carcinoma of the upper aerodigestive tract among elderly patients may be a result of a longer period of exposure to genetic injuries from spontaneous mutation or environmental factors combined with an aging repair process. Koch et al. (12) found a loss of heterozygosity of markers on a number of chromosomal arms in specimens of squamous cell carcinoma. This finding indicated the possible involvement of several suppressor genes.
Overexpression of p53 has had mixed results as a prognostic factor. In a review of T1 squamous cell carcinoma of the floor of the mouth and ventral tongue, no statistically significant correlation was found between the level of p53 expression and tumor aggressiveness (13). In contrast, a review of oropharyngeal squamous cell carcinoma specimens showed that p53 protein was predictive of increased risk of death independently of tumor grade, stage, and lymph node status (14). Expression of p53 seems to correlate with a poor prognosis, particularly in advanced squamous cell carcinoma of the head and neck. The full negative effect of overexpression p53 may occur only very late in disease, thus correlating with survival in end-stage disease. The function of p53 protein can be interrupted with viral oncoprotein binding, such as the E6 protein of human papillomavirus types 16 and 18, and thus potentiate carcinogenesis (15).
Human Papillomavirus
Human papillomavirus (HPV) has been linked to development of papilloma in the nose and respiratory tract and to carcinogenesis in the genitourinary tract. Known oncogenic types 16, 18, and 31 have been found in squamous cell carcinoma of the tongue, tonsil, larynx, and pharynx. Human papillomavirus DNA was detected in 46% of archival tissue specimens of laryngeal and hypopharyngeal carcinoma, and the presence of this DNA appeared to correlate with a poorer prognosis than among cases in which there was no detectable HPV (16). Portugal et al. (17) detected HPV (11%) and p53 mutation (66%) within the same specimens of squamous cell carcinoma of the oral cavity and tonsil, which showed that neither p53 gene mutation nor HPV infection serves as a prognosticator of tumor behavior, although survival rates were higher among persons with HPV-infected cancer of the tonsil (17). Among patients with a history of low alcohol and tobacco use, HPV infection was an independent risk factor for squamous cell carcinoma of the oral cavity and tonsil.
The exact role of HPV in carcinogenesis in the upper aerodigestive tract is unknown. Binding of E6 HPV proteins to the p53 tumor suppressor gene may lead to gene product degradation and unchecked cell proliferation. The E7 HPV protein is known to form complexes with the retinoblastoma tumor suppressor gene product pRB, and this process leads to tumorigenesis (18). No role for the retinoblastoma gene has been found in squamous cell carcinoma of the head and neck. An association of HPV with mutated H-ras oncogene has been suggested in squamous cell carcinoma of the mouth (19). However, the ras oncogene group is infrequently involved in head and neck cancer (20).
Thyroid Cancer
Quantification of nuclear DNA in papillary carcinoma reveals a close correlation between DNA ploidy and the aggressiveness of thyroid lesions (21). Two groups of patients have been compared, one with noninvasive disease and another with invasion of the thyroid. Forty percent of invasive lesions were aneuploid, whereas all tumors without such extent were diploid. The thyroid has been considered an advantageous target for somatic gene therapy because of its great capacity for protein synthesis, high blood flow, and sensitivity to hormonal regulation. O’Malley et al. (22) developed a method for transferring genes into cultured human thyroid follicular cells with the use of retroviral vectors. Cells were transfected with either the LNL6 vector, carrying the gene for neomycin resistance, or the zen-B-gal vector, carrying the b-galactosidase marker. In this experimental model, transfection rates ranged from 0.1% to 3%.
Salivary Gland Neoplasms
The factors of prognostic significance for salivary gland neoplasms are well known and are based mainly on histologic features, including status of surgical margins, perineural invasion, and lymph node metastasis as well as histologic type and grade. In the future, prognostic indicators found at the gene level may provide the most accurate information. DNA ploidy was examined as a prognostic indicator for adenoid cystic carcinoma in a small series of 20 patients (23). All tumors with DNA aneuploidy recurred; only two of the 14 DNA diploid lesions recurred. These results may have an effect on treatment planning, particularly because DNA aneuploid tumors are more radiosensitive than are DNA diploid tumors.
Overexpression of c-erb-b2 oncoprotein, a receptor of growth factors, in adenocarcinoma of the major salivary glands may be an indicator of aggressiveness. In 59 cases of malignant tumors of the major salivary gland that also included squamous cell, adenoid cystic, and mucoepidermoid carcinoma, only adenocarcinoma produced a positive staining result (24). The tumors with c-erb-b2 overexpression were more difficult to resect, were associated with more frequent nodal metastasis, and resulted in a markedly lower disease-free survival than did adenocarcinomas that did not show overexpression.
OTOLOGIC DISEASE
Acoustic Neuroma
Acoustic neuroma accounts for about 8% of all intracranial tumors. These tumors are bilateral and familial in 4% of cases, a condition associated with neurofibromatosis type 2. The neurofibromatosis type 2 gene has been localized to chromosome 22. Genetic analysis of both familial and sporadic acoustic neuroma has revealed frequent loss of alleles at chromosome 22 in both types (25,26). Greater extent of chromosome 22 deletions was associated with larger tumor size, but this finding was not statistically significant. Further work is being directed at identifying a prognostic indicator for tumor aggressiveness.
Otitis Media
Clinical research suggests that susceptibility to microorganisms that cause acute otitis media may be hereditary. Kalm et al. (27) tested for several human lymphocyte antibody (HLA) antigens in patients with recurrent acute otitis media. HLA-A2 antigen was present more frequently in the recurrent otitis media group than in controls, indicating the existence of a relation between recurrent acute otitis and the HLA locus.
Hearing Loss
Nowhere has the development of molecular techniques had more consequence than with hearing loss, particularly hereditary deafness. About 50% of congenital diseases of the inner ear are genetically acquired. Genetic mapping relied mainly on kindred and linkage analyses until the recent increase in the number of available genetic markers (see Chapter 2). Research in otolaryngology directed at the molecular level continues at an accelerated pace. Future advances are certain to advance the understanding and management of maladies such as cancer. Somatic gene therapy, with targeted gene inactivation and insertional mutagenesis, ultimately may provide the means to alter disease.

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