The incidence of larynx cancer in the United States is around 4.5 cases per 100,000 per year (1). The 5-year relative survival percentage for localized disease has been stable at around 70-80% for the last 20 years (1). In early laryngeal cancer, radiotherapy is an effective treatment modality, with local control rates between 80-90% for T1 tumors (2). Partial laryngectomy or CO2 laser resection are alternative treatments with comparable survival rates, although when used as salvage after a failed radiotherapy course they have a higher complication rate (3). Treatment choice is mainly based on the estimated functional outcome and the preferences of the clinician. It would therefore be useful to predict beforehand which patients will benefit from radiotherapy. Prediction of resistance is also likely to be increasingly useful in the development of biological modifiers which increase the effects of radiation, providing an alternative treatment for resistant tumors.
Important clinical factors associated with local recurrence after radiotherapy are tumor stage, tumor size, radiotherapy fraction size and year of treatment (4). Treatment choice is now mainly based on T-stage (5), although this is still a relatively poor indicator of survival (6). Since clinical factors cannot provide an accurate prediction, it is likely that recurrence of a tumor can partly be explained by tumor biology. Three biological processes known to influence response to radiotherapy are intrinsic radiosensitivity (7), hypoxia (8) and repopulation (9). For each of these processes, individual markers (mainly immunohistochemical) have been investigated and found to be of predictive value (10–12), although none have been sufficiently validated or are in routine use. Since many genes are involved in each process, in addition to single markers representing these processes, sets of markers (gene sets) for hypoxia (13, 14), intrinsic radiosensitivity (15–17) and repopulation (18) have also been defined. Another factor more recently hypothesized to play a role in response to therapy is the number of stem cells, ultimately determining repopulation of the tumor (19, 20) and so eradication of this subpopulation is of prime importance.
To date, no studies have investigated all these processes simultaneously. Microarrays have been used to measure gene expression (mRNA) on a genome wide scale, and can in principle monitor all the above-mentioned processes concurrently. However, only one microarray study with 14 patients has been carried out for patients treated with radiotherapy alone (21). Several expression profiling studies have been carried out on patients treated with radiotherapy in combination with surgery or chemotherapy (22–26). However, these have often included heterogeneous groups of patients and cannot address the question of factors affecting the response of laryngeal cancer to radiation alone.
Our objective was to find a gene expression profile that will accurately predict local recurrence after radiotherapy in a homogeneous group of patients with early laryngeal carcinoma. We chose to study early stage tumors, since these are likely to be more homogeneous than advanced tumors and also technically easier to treat, minimizing the chance of geographical misses. Treatment failure is then highly likely to be due to biological rather than technical factors. In addition to giving more insight into the molecular processes underlying treatment failure, accurate prediction would enable treatment to be individualized, leading to increased survival and less unnecessary morbidity. We studied two series of early stage larynx cancer patients treated with radiotherapy alone. The first was a test series of frozen tumor specimens used to study global gene expression to discover predictive markers for local control, which were then validated on a second series by immunohistochemistry.