-------------------------------------------------------------------------------- TITLE: GENETICS IN OTOLARYNGOLOGY SOURCE: Dept. of Otolaryngology, UTMB, Grand Rounds DATE: November 10, 1993 RESIDENT PHYSICIAN: Chris H. Tompson, M.D. FACULTY: Ronald W. Deskin, M.D. DATABASE ADMINISTRATOR: Melinda McCracken, M.S. -------------------------------------------------------------------------------- "This material was prepared by resident physicians in partial fulfillment of educational requirements established for the Postgraduate Training Program of the UTMB Department of Otolaryngology/Head and Neck Surgery and was not intended for clinical use in its present form. It was prepared for the purpose of stimulating group discussion in a conference setting. No warranties, either express or implied, are made with respect to its accuracy, completeness, or timeliness. The material does not necessarily reflect the current or past opinions of members of the UTMB faculty and should not be used for purposes of diagnosis or treatment without consulting appropriate literature sources and informed professional opinion." GENETICS IN OTOLARYNGOLOGY I. Introduction In my limited experience in otolaryngology it seems that our only contact with genetics comes around inservice time when we memorize all of the genetic syndromes that most of us will never see. And although I've included these syndromes in my handout, I'm going to devote more time to the genetics of cancer which is going to have a huge impact on our field. I'm also going to briefly go through the basics of genetics and terminology and then spend some time reviewing the techniques which are used in the study of molecular genetics. II. History A. August Weissman (1834-1914) proposed the Germ Plasm theory B. Gregor Mendel 1866: Origin of Mendelian Genetics describing the basic rules of inheritance, those of independent assortment and random segregation C. Hugo deVries, Carl Correns, and Erich von Tschermak rediscover Mendel's works in the early 1900's D. Walter S. Sutton and Theodor Boveri in 1902 propose the chromosome theory of heredity E. Thomas Hunt Morgan and Calvin Bridges in 1916 explain that specific genes on specific chromosomes obey classical Mendelian rules F. Wendell M. Stanley 1953 discovered that viruses are nucleic acid and proteins G. James D Watson in 1953 proposes model form the structure of DNA H. Tigo and Levan in 1956 develop techniques of chromosome study and find the human karyotype to contain forty-six chromosomes I. Philadelphia chromosome discovered in 1960 J. Saiki develops the polymerase chain reaction in 1985 III. Common Genetic Disorders A. Autosomal Dominant 1. Pierre Robin (cleft palate, micrognathia, glossoptosis) a. Systemic manifestations: cyanosis at birth, respiratory distress in the supine position b. Facies: small and receded mandible, low set ears and ocular anomalies common c. Skeletal system: congenital amputations, syndactyly, hip dislocation, and talipes equinovarus d. Nervous system: 20% exhibit mental retardation e. Oral manifestations: failure of mandibular development, tongue drops into post-pharyngeal space producing airway obstruction 2. Treacher Collins Syndrome (mandibulofacial dysostosis or first branchial arch syndrome) a. Facies: down sloping palpebral fissures, depressed cheek bones, deformed pinnas, large fish-like mouth, malar bones under-developed, small paranasal sinuses b. Eyes: coloboma present 75% of time, normal vision, eyelashes absent medial to coloboma c. Ears: pinna deformed, EAC absent 33%, ossicular chain anomalies, cochlear and vestibular deformities, skin tags and blind fistulas anywhere from tragus to angle of mouth d. Nose: wide bridge e. Oral: hypoplastic mandible, cleft palate in 30% of cases, macrostomia in 15% 3. Waardenberg Syndrome a. Facies: broad nasal root, heterochromic irides, prognathism, and white forelock b. Eyes: widened interpupillary distance, confluence of brows c. Hair and skin: poliosis 40%, vitiligo 15% d. Ears: SNHL 20%, normal high frequency hearing 4. Alport's Syndrome a. Ears: SNHL at age ten and progessive, stria vascularis and organ of corti degeneration, all males lose hearing, some females with hearing loss, vestibular hypofunction b. Renal: hematuria, proteinuria, and death from uremia by age thirty in males 5. Apert's Syndrome (acrocephalosyndactyly) a. Facies: congenital craniosynostosis leading to turribrachycephaly, hypertelorism, parrot nose, low set ears b. Oral: small maxilla, high arching palate, 33% cleft palate c. Skeletal: syndactyly and synostosis of hands and feet d. Ears: flat CHL, stapes footplate fixation, patent cochlear aqueduct 6. Crouzon's Syndrome (craniofacial dysostosis) a. Facies: synostotic malformation, bilateral exophthalmous, external strabismus, parrot-beak nose b. Ears: Mixed hearing loss, atretic auditory canal 7. Van der Hoeve's Syndrome (osteogenesis imperfecta) a. Eyes: blue sclera b. Ears CHL secondary to stapes fixation c. Skeletal: fragile bones and loose ligaments B. Autosomal Recessive 1. Goldenhar syndrome (occuloauriculovertebral dysplasia) a. Facies: hemifacial microsomia b. Nervous system: ten percent mental retardation c. Ear: dysplastic auricle, CHL d. Eye: epibulbar dermoids at the limbus on the lower outer quadrant, unilateral coloboma e. Skeletal: vertebral anomalies in 50-60%, anomalous ribs and vertebrae f. Heart: 45-55% with VAD, PDA, and right-sided aortic arch g. Oral: absent glenoid fossa, narrowed maxilla, hypoplastic tongue 2. Usher's syndrome a. Eyes: retinitis pigmentosa with progressive blindness b. Ears: congenital deafness, ten percent of all hereditary deafness, frequent vestibular involvement IV. Basic Genetics A. Karyotyping The human karyotype, which is pictured, gives us an overview of the genetic information contained in a cell. The study of karyotypes, cytogenetics, is diagnostic of many genetic diseases and is making great advances in cancer research. Basically, to produce a karyotype a mitotic inhibitor is added to the cell culture stopping the dividing cells in metaphase. The cell walls are disrupted and a Giemsa stain is added. One example of each of the twenty-three different chromosomal pairs is photographed and put together in the karyotype. The banding pattern for each chromosome pair is similar among individuals so that each of the twenty-three pairs is easily identified and any structural abnormalities are detected. B. Nomenclature The chromosome pairs are numbered from one to twenty-two in order of decreasing size, the sex chromosomes designated X or Y. This gives us a total of forty-six, which is referred to as the diploid number of chromosomes. Haploid then refers to half, or 23, and tetraploid refers to four times the normal number. Each chromosome pair has one maternal and one paternal representative and the two are joined together by a centromere. The short arms of each pair are denoted 'p', the long arms 'q'. A standard numbering system is applied to the bands. C. Chromosome structure As we all know, a chromosome is two long strands of DNA each of which is a series of repeating nucleotides. Each nucleotide has a five carbon sugar ring, a phosphate group, and one of four bases. The sugar rings of each nucleotide are joined to form the strand and the bases join the two strands together. D. Replication The key to biologic function is the base pairing mechanism. The four bases are: the purines adenine and guanine; and the pyrimidines cytosine and thymine. Adenine always binds to thymine, guanine with cytosine. For replication to occur, the two strands become disassociated, and loose nucleotides in the vicinity will be joined to the complimentary nucleotide on the strand by replicating enzymes present. When complete, each of the two original DNA strands have a new complementary strand. E. Transcription The coding function of DNA operates in a similar fashion, allowing the messenger RNA to transcribe the sequence of bases coded on the DNA into proteins in the cytoplasm. For this to occur a DNA-binding enzyme identifies the appropriate location on the DNA strand and causes the two strands to unwind for the length of the coding sequence. Again, free nucleotides in the vicinity are joined to their corresponding bases on the coding sequence. However, these nucleotides differ from those of the replicating process in that their five carbon sugar ring lacks an oxygen molecule. Once completed, the ribonucleic acid sequence then separates from the DNA and travels out of the nucleus into the cytoplasm. Here the ribosomal RNA translates the mRNA into a protein by joining amino acids together in a sequence complimentary to the sequence of bases in the mRNA. In this process, every three mRNA bases codes for a specific amino acid. These coding triplets are called 'codons'. The joined amino acids produce a polypeptide and ultimately a mature protein once the entire sequence is converted. F. Genes With this background, the definition of a gene is easily explained as a segment of the DNA sequence that codes for a particular protein. To give a perspective on the relationship between the genes and chromosomes, sixty to one hundred genes are found on each of the chromosome bands. The structure of a gene is shown here. The only portion of the gene that is actually transcribed is the exon. The flanking regions regulate transcription activity by carrying the start and stop sequences for transcription. They also contain the enhancer regions which control transcription frequency. The intron regions are nonfunctional and may represent extinct genetic material, remnants from retroviral gene insertion, or segments known as repeats. Repeats are stretches of tens to thousands of copies of some short sequence. The shorter repeat sequences have proved to be very unique from individual to individual and are the target of the process known as DNA fingerprinting. G. Oncogenes Oncogenes have become a household term and are often used synonymously with cancer. These are genes normally found in the human genome, and whose product is involved in cell proliferation. Expression out of normal context, due to a mutation, can contribute to transformation. These genes were discovered in viral genomes, and later found to exist normally in the human genome. The term oncogene now refers to those genes found in the viral genome, and proto-oncogene describes the endogenous versions of these genes. H. Mutations 1. Macro-mutations Mutations are classified into macro and micro. Macro- mutations describe chromosomal breaks which do not rejoin normally. Their product may be a deletion, duplication, translocation, an inversion or an insertion. An example of a deletion is cri du chat syndrome, characterized by the loss of the short arm of chromosome five. Insertion mutations are unlikely to alter the phenotype. Translocations are divided into two types, reciprocal and Robertsonian. Reciprocal translocation is the exchange of blocks of chromatin between two non-homologous chromosomes. The process requires breakages of both chromosomes with repair in an abnormal arrangement. The Philadelphia chromosome of chronic myelogenous leukemia and the eight-fourteen translocation in Burkitt's lymphoma are examples. The Robertsonian translocation describes the fusion of two acrocentric chromosomes. These are chromosomes that have the centromere close to one end. The short arms are lost and the two long arms join to form a new chromosome. Macro-mutations are easily detected on karyotype analysis, and a lot of recent work looking at karyotypes in solid tumor cell lines has been done. Deletion, duplication, and translocation mutations are commonly seen in these tumor karyotypes''. 2. Micro-mutations Micro-mutations then, involve changes in the nucleotide sequence and can be subdivided into point and frameshift. The most common type, the point mutation, occurs once every one to ten million meioses and involves the replacement of one nucleotide base with another. Such a substitution would likely have little or no effect on the protein product, but in the appropriate location, this alteration will seriously impact the phenotype. An example would be the sickle cell hemoglobin point mutation. The other micro-mutation, the frameshift, involves an insertion or deletion of a nucleotide. All of the downstream codons are disrupted and the transcribed protein will have no activity. Although I have described mutations as occurring spontaneously, their frequency has been shown to increase dramatically in the presence of ionizing or ultraviolet radiation and chemical mutagens. The association between tobacco and alcohol and squamous cell carcinoma of the upper aero-digestive tract has long since been established. But recent work by Wong and Biswas demonstrates the over-expression and amplification of specific genes in response to a chemical mutagen in hamster cheek pouch carcinomas. Another proto-oncogene product the P53 protein, which I will describe later, has been found to be elevated in patients with squamous cell carcinomas of the head and neck and a history of heavy smoking. In addition to confirming the chemical mutagenesis theory, these studies bring out the important point that mutations associated with cancer involve the regulatory sequences of the gene. Instead of a qualitative alteration in the protein product, the result is a quantitative change in output of the normal protein product. With the recent advances in the field of molecular genetics our ability to examine mutations in individual genes is quickly growing. The discovery of over and under-expression of such genes is steadily uncovering the molecular mechanisms of carcinogenesis. V. Molecular Genetics A. Introduction Karyotype analysis, as described previously, is the level at which the field of cytogenetics deals. Molecular genetics covers the structure and function of the genes themselves. Most of what we know in this field has come about through the application of recombinant DNA technology, so it's important to have an overview of the recombinant techniques for intelligent discussion. B. Recombinant DNA technology The inception of molecular genetics coincided with the discovery of bacterial proteins called restriction nucleases. When a bacterium is invaded by an organism containing DNA, like a virus, it can defend itself by producing one or more proteins known as restriction endonucleases. These enzymes break DNA wherever, and only wherever, a specific, short string of nucleotides are found. The cut pieces of DNA are called restriction fragments. Each restriction enzyme is specific for a particular recognition sequence called the restriction site or cutting site. For instance, the HpaI restriction enzyme cuts chromosomes everywhere the sequence GTTAAC exists. Because the bacterium would quickly digest its own DNA with these enzymes, it has developed a method of protection. This mechanism requires another series of enzymes which methylate all restriction sites on its own DNA, making the sequence unrecognizable to the restriction enzymes. When the DNA is purified from a collection of human cells, and combined with a certain restriction enzyme, it is cut into thousands of pieces whose number and length depend on the number of, and distance between, the restriction sites. Because individuals differ at thousands of nucleotides across the genome, each person will produce different sets of restriction fragments. The resulting differences among individuals in a given gene region are known as restriction fragment length polymorphisms, or RFLPs. In order to identify these fragments they must be separated from one another and they must be stained. To separate, the fragments are placed in a gel. Because the fragments have an associated electrical charge, the application of an electric field to either end of the gel will cause migration of the fragments. The larger fragments will have more drag as they move through the gel, and will therefore move a shorter distance. Additionally, fragments of similar size with different charges will experience different degrees of attraction and will also separate. Now that they are separated it is necessary to apply a staining method such that only the fragments of interest become visible. Looking at the entire set of restriction fragments, we would find a continuous line of staining along the gel. Instead, probes have been developed which bind gene regions known to have great variability among individuals. In this way we can look at those fragments which will be unique to an individual and his family. Staining methods differ in the substance used to make up the probe. The most common method, Southern blotting, uses DNA probes to visualize the fragments of interest. One of the problems working with DNA is the small quantities involved. The polymerase chain reaction, developed in 1985, permits targeted amplification of specific nucleic acid sequences. A prerequisite in this technique is that the nucleotide sequence of both ends be known exactly. Oligonucleotide "primers"(short strands of nucleotides complementary to the known end-sequences) must then be synthesized to begin the reaction. With the appropriate primers and proper buffer and temperature conditions, synthesis of a complementary strand can proceed automatically by joining free nucleotides together, using a polymerizing enzyme (DNA polymerase). For this elongation step, the exact sequence of the template in question does not need to be known; the reaction will proceed with great fidelity, regardless. After sufficient elongation time to generate the entire complementary strand, the reaction mixture can be heated to denature the synthesized strand from the template, completing the first cycle. Each succeeding cycle will then double the amount of DNA, and millions of copies can be made in a few hours. This technique has made it possible to study DNA from before birth to millions of years after death. DNA from smears, hair roots, blood spots, paraffin sections, and even single cells can be analyzed to find gene defects, viruses, and activated oncogenes. VI. Cytogenetics of Cancer A. Cytogenetic application With the powerful techniques available now in molecular genetics, cytogenetic analysis might seem outdated. In fact the level of resolution with molecular methods is at the base pair level, whereas with cytogenetics, the minimum change detectable is about 1.5 million base pairs. However, cytogenetics remains invaluable to localize sites of chromosomal abnormalities, so that molecular techniques can be applied to specific areas, and not the entire genome. B. Leukemias Initial cytogenetic applications to cancer resulted in the discovery of chromosomal translocations responsible for Burkitt's lymphoma, and chronic myelogenous leukemia (CML). Molecular techniques later demonstrated that in Burkitt's lymphoma, the c-myc gene was disrupted. This translocated gene, responsible for control of the cellular replicating process, continues to produce a normal product; but it acquires new regulatory sequences which promote over-production. Similar studies in CML demonstrated that an abnormal protein product was formed from the fusion of two different genes, and this protein somehow initiated carcinogenesis. Experimentation with cells from retinoblastoma and Wilm's tumor identified deletional abnormalities which were later determined to represent losses of tumor suppressor genes. This phenomenon was predicted by Knudson's (Sharon) "two hit hypothesis" which says that Mr. retinoblastoma is more likely to occur in individuals who inherit only one copy of this tumor suppressor gene because of a deletion in the parental genome. These individuals cannot tolerate another 'hit', as it would render them without either copy of the gene. C. Solid tumors Work with solid tumors was not as popular because karyotype analysis identified more than just one chromosomal change. So it was not until 1988 that a solid tumor model was proposed. Vogelstein et al, using colorectal tumors, analyzed DNA from early lesions, late metastatic tumors, and stages in between and found that early lesions had few genetic changes, whereas late lesions had at least four genetic changes. Most of the advanced tumors demonstrated similar changes while the early lesions had different combinations of those same changes. This flow chart illustrates their observations of the steps of genetic alteration present in colorectal carcinoma. They then proposed that the precise sequence of events is less important than the net accumulation of genetic change. From this evidence, it seems that neoplasia is the result of genetic changes, which may include (1) overproduction of a gene product; (2) production of an abnormal gene product; and (3) loss of a gene product through inactivation by mutation, translocation, or deletion of the gene. Solid tumors appear to result from accumulation of more genetic changes than leukemias, and the changes are a mixture of the three events described. D. Head and neck tumors The work done in the field of head and neck cancer has moved slowly because of the complexity of the tumor cell karyotypes. Approximately ten chromosomes have been suggested as the site of the head and neck cancer gene including 1,3,4,7,8,9,10,11,13, and 18''''. Additionally, one study demonstrated the presence of two breakpoint hot spots on chromosomes 1 and 11. Hot spots are regions on a chromosome which are found to be mutated much more frequently than could be attributed to by chance. To give you an idea of the complexity, a recent study by Van Dyke illustrates the karyotype summary from twenty-nine squamous cell carcinomas of the head and neck. Each mark indicates one breakpoint found on that region of the chromosome and a total of 697 were found. In a similar study by Cowan using cell lines from sixteen head and neck squamous cell carcinomas, deletions of chromosomes 18, 13, 10, 8, and 22 were found in over seventy-five percent of tumors. Duplication of chromosome 7p occurred in eighty-seven percent. The results of their breakpoint analysis was as complex as those of Van Dyke, but they postulated that since many of the breakpoints localized to non gene-containing regions, the field could be significantly reduced. However, sites on ten chromosomes still remained as potential proto-oncogenes. This complexity indicates that many of the mutations have happened by chance and do not provide any growth advantage or transforming potential; but among them are the mutations that are necessary for transformation as well as those that do provide growth advantages. These studies provide three sets of data. The first set points to chromosome regions frequently deleted, possibly indicating sites of tumor suppressor genes. The second are locations frequently duplicated, representing possible proto-oncogenes. The third is identification of chromosomal bands frequently involved in rearrangement, and may be the site of proto-oncogene activation. These types of studies, using large numbers of tumors, are infrequent in the current literature, and much needs to be done in order to identify the important mutations in these complex karyotypes. VII. Molecular studies in head and neck cancer A. C-myc gene Without the assistance of cytogenetic analysis, many of the early molecular genetic studies were aimed at identifying proto-oncogenes known to have importance in other tumors. The c-myc gene involved in cell growth and differentiation in normal cells has been implicated in a number of different cancers; however in most studies, the gene has not been shown to be an important feature of head and neck cancer in the western world. One recent study by Haughey did demonstrate increased c-myc in two of eight tumors, both of which had positive neck nodes with increased c-myc expression. This may indicate a relationship between metastatic potential and c-myc expression. B. Ras oncogene The ras oncogene is mutated in a wide variety of primary human tumors and was an early step in the colorectal model of carcinogenesis proposed by Vogelstein. As a result, this gene has received attention in the study of head and neck carcinomas. However, recent studies in the western world have demonstrated the ras mutation in less than five percent, and a study by Irish several months ago showed no evidence of ras mutations in seventeen tumor specimens. An interesting geographic split, however, has been illustrated by studies in India that demonstrate ras gene mutations in thirty-five percent of oral squamous cell carcinomas. Theories on the differences include the prevalent use of betal nut chewing and reverse smoking in the area. C. Chromosome 11q13 breakpoints With the evidence from the cytogenetic studies, several labs have investigated the region of chromosome eleven which, frequently houses a breakpoint site. Somers has identified a cluster of proto-oncogenes localized on chromosome eleven band q13 which is amplified in approximately thirty percent of squamous cell carcinomas of the head and neck. Two of the oncogenes, int-2 and hst-1, are members of the fibroblast growth factor gene family. Overexpression of these genes could provide tumors with autonomous growth factors and angiogenic signals. The other two on the cluster are the prad-1 and the bcl-1 genes. These have been identified as the cyclin gene which produces proteins that give positive regulation to the cell cycle, an obvious advantage in tumor transformation. D. P53 gene The p53 gene, which has undoubtedly received the most attention in the literature, was discovered fifteen years ago as a host cell protein bound to T antigen, the dominant transforming oncogene of the SV40 virus. It is a tumor suppressor gene localized to chromosome 17p, and has been found to be mutated or deleted in about one-half of all adult cancers. Much of the attention is the result of a study by Field which linked cigarette smoking with elevated p53 expression in head and neck tumors. Although it seems ironic that a tumor suppressor gene product would be elevated in smokers, previous studies had shown that the half-life of the mutated p53 protein was roughly sixteen times longer than that of the normal protein. Additional evidence linking p53 mutation with smoking came when Somers and Schechter demonstrated that the most common p53 mutation was a G to T transversion. This has previously been shown to be induced by benzo(a)pyrene, an element of tobacco smoke. Many studies have looked at p53 overexpression in head and neck squamous cell cancers and the results vary from thirty-three to one-hundred percent of tumors exhibiting this overexpression. This variation could be explained by deletional mutations in tumors not overexpressing the p53 protein, so that instead of making an abnormal product, no product at all is made. Enough work has been done on the gene so that clustering of mutations has been localized to the codon level. The mutations generally come in the form of single substitutions, multiple deletions, and occasionally additions. As to the chronology of this mutation in the evolution of a tumor, the debate continues. Many researchers propose a role in the later stages, while others are convinced that the mutation plays a part in early transformation. E. HPV and the p53 gene Another area of p53 investigation concerns its interaction with the human papilloma virus (HPV). In vitro studies have demonstrated the E6 protein of HPV-16 to promote degradation of the p53 protein. This is felt to be the mechanism by which the HPV promotes the development of cervical carcinoma. In head and neck carcinoma, the reports of papilloma viruses in both benign and malignant lesions may indicate a similar pathogenesis. However, the area remains controversial. Another area of recent study concerning HPV is its association with tonsillar carcinoma. Snijders detected HPV DNA in all of the ten cases of tonsillar carcinomas they investigated, and found no evidence of HPV infection in seven cases of tonsillitis. They isolated HPV 16 and HPV 33 in equal numbers. Another significant finding was that the HPVs were producing the p53 degrading protein, E6, in all of the tumors studied. This adds further support to the association between the viral infection and tonsillar cancer. E. E-cadherin gene The cadherin gene family is responsible for epithelial cell-to-cell adhesion. This adhesion mechanism seems to operate incorrectly during malignant transformation and down regulation of the E-cadherin gene has been associated with both invasion and metastasis. Recent work by Schipper has demonstrated that E-cadherin is expressed in well and moderately well differentiated squamous cell carcinomas of the head and neck, but no detectable E-cadherin was found in the poorly differentiated tumors. Additionally, seven out of eight lymph nodes were found to have down-regulated E-cadherin regardless of the differentiation grade of the primary tumor. This gene has been located to chromosome 16 and has recently been found to be the site of translocation in a karyotype analysis. VIII. Ploidy analysis Ploidy refers to the number of chromosomes in the nucleus; twenty-three pairs are found in the normal cell, and such a cell is said to be diploid. Cells with more or less than normal are known as aneuploid, and polyploid refers to those cells with more than twenty-three pairs. Ploidy study began in 1979 with a paper by Atkin and Kay looking at uterine cancers, and was accelerated by the discovery of flow cytometry. low cytometry is a process by which cells passing in a fluid stream through a glass tube are struck by a laser. Because the cells are stained prior to the process, the chromosomes fluoresce in the presence of the laser. The fluorescence, which is directly proportional to the amount of DNA, is measured and the DNA content is calculated. Many studies have been done in this field, and a variety of conclusions have been drawn. Stell in 1991 published a meta-analysis of twenty-six series looking at ploidy in squamous carcinoma of the head and neck. A total of 1984 patients were included and a variety of variables were analyzed. Two-thirds of the tumors were non-diploid and there was no relation between host factors and ploidy. Tumors with a poorer degree of differentiation were more likely to be non-diploid but there was no relation with stage group, although many authors had stated that there was. The correlation with nodal metastases found in the analysis is likely due to the fact that tumors with less differentiation are more likely to metastasize and also more likely to be non-diploid. Survival was found to be significantly better for diploid tumors, but subgroup analysis demonstrated that this was due to mouth cancers, whereas ploidy did not effect the outcome in laryngeal cancer. Ploidy was not found to influence the response to radiotherapy, but a radiotherapy recurrence was more likely to be diploid. Chemotherapy response was found to be improved in patients with end-stage cancers that were non-diploid. The final word on ploidy analysis is still out. Although it does provide us with unique information, it has not yet proven to yield any pertinent clinical direction. However, newer studies into cell cycle analysis, s-phase fractions, and proliferating cell nuclear antigens may shed new light on the subject. In the future, ploidy analysis may be a valuable prognostic tool. IX. Future Direction The field of cancer genetics is in its infancy, but studies thus far in epidemiology, cytogenetics, and molecular genetics support the concept that human cancer is a multistep process resulting in the accumulation of successive genetic alterations. Vogelstein's model of colon cancer will eventually be duplicated and applied to head and neck cancer, and the chronology of the mutations will be determined. The potential will then exist to target early genetic changes with gene therapy and interrupt the transformation process. Such work has already been done with regard to the prophylaxis of genetic mutations, as it has been shown that vitamin E demonstrates a dose-dependant protection against mutation in lymphocytes of head and neck cancer patients. In the field of molecular genetics, processes have already been developed to provide 'gene' therapy. Using viruses as vectors to carry a therapeutic gene, it is possible to integrate such a gene into the human genome. The virus, which has been freed of its detrimental properties, is allowed to infect human cells, and in doing so, splices its genetic information into the DNA of the human cell. Not only will the infected cell express the new genetic material, but the following generations of offspring will also carry the gene. Another method of introducing genes into human cells is through the use of plasmids. These are cyclic DNA molecules usually found in bacteria. Specific genes can be spliced into the plasmids, which can then be introduced into human cells. These cells will then express the gene product, but unlike the virally infected cell, the information will not be passed on to the offspring. These technologies obviously have great potential in the therapy of cancer. With the identification of mutations in specific tumor suppressor genes, such as p53, it may become possible to exogenously replace the gene and interrupt the carcinogenesis. Alternatively, by introducing genetic material which down-regulates neighboring genes, proto-oncogenes would be silenced. A great deal of work remains before such therapies exist, specifically in the identification of the important genetic mutations that occur early in the transformation process. Although we are probably many years from genetic therapy for cancer, genetic studies will likely have clinical significance in the near future. As more mutations are identified, studies will begin to document the prognostic significance of their existence. Certain mutations may be identified that endow radio, or chemo-sensitivity to a tumor, while others may provide the tumor with a greater metastatic potential. With a better understanding of the chronology of the mutations we may be able to determine an early cancer versus a late one by identifying which mutations it contains. Obviously, an entire staging system with very specific prognosticating information could be built on such technology. ------------------------------------END------------------------------------