Expression of tumor markers in oropharyngeal cancer



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Expression of tumor markers

in oropharyngeal cancer


David Lindquist



BACKGROUND
There are approximately 644,000 new cases of head and neck cancer diagnosed in the world each year excluding oesophageal cancer (1). In Sweden, head and neck cancer comprises 3-4% of all cancer cases, and the most common sub-group is tonsillar cancer (2). Known risk factors for head and neck cancer are mainly alcohol and smoking (3), but there is also substantial evidence that high risk human papilloma virus (HPV) is involved in the development of oropharyngeal cancer. This evidence consists of several retrospective studies (4-7), summarized in one meta-analysis (8), one large serological study (9), one case-control study (10), presence of HPV in precancerous lesions (11), localization of HPV to the nucleus (12, 13) and presence of HPV in nodal metastasis (12-14). It has recently been reported that tonsillar cancer is increasing in Sweden and other countries (2, 15, 16), despite a decrease in smoking prevalence (2). HPV is likely to be a risk factor for this increase. It has been shown that HPV is also commonly found in base of tongue cancer, but less often in tongue cancer localized to the oral cavity (17), thus tonsillar cancer and base of tongue cancer is now usually studied together as oropharyngeal cancer.
In Dalarna, 68 patients were diagnosed with oropharyngeal squamous cell carcinoma during 1970-2007 (18) and there is a tendency for an increase over this period of time (fig1). It has been shown that there is a high prevalence of Chlamydia (another sexually transmitted infection) in Dalarna (19), but little is known about the prevalence of HPV.

Fig 1. The number of cases diagnosed with

oropharyngeal squamous cell carcinoma is increasing in Dalarna (18).


There is strong evidence for presence of HPV as a positive prognostic factor in oropharyngeal cancer. However, the biological reason for this remains to be elucidated. High expression of the tumor suppressor gene p16 has been shown to correlate to presence of HPV and a better disease specific survival in oropharyngeal cancer (20-23). Other tumor markers which has been studied in cervical caner, also known to harbour high risk HPV, are c-myc, COX-2, Ki-67, CD4+, EGFR, VEGF, E-cadherin, CD44 and p27 (24). These tumor markers represent different functions in carcinogenesis (table 1). The tumor markers COX-2, EGFR and VEGF are of particular interest since targeted treatment now is available. Little is known about the expression of these tumor markers in oropharyngeal cancer, especially with regard to comparing HPV positive and negative tumors.
Table 1. Functions of common tumor markers.


Tumor marker

Major functions

c-myc

Cell-cycle progression, malignant transformation

COX-2

Prostaglandin synthesis, angiogenesis, decreased apoptosis

Ki-67

Proliferation

CD4+

Immune response

EGFR

Proliferation

VEGF

Angiogenesis

E-cadherin

Cell-cell adhesion

CD44

Cell-cell adhesion

p27

Cell cycle arrest


AIM
The aim of this project is:

  • to examine the presence of HPV in oropharyngeal cancer in Dalarna

  • to investigate if there are differences in expression of biological tumor markers in HPV positive and HPV negative oropharyngeal tumors

  • to correlate the presence of HPV and expression of tumor markers to clinical outcome



MATERIAL, METHODS AND COMMENT ON PREVIOUS EXPERIENCE OF THESE METHODS
The diagnostic biopsy and clinical data for all patients diagnosed with oropharyngeal squamous cell carcinoma in Dalarna during 1970-2007 will be obtained after approval from the local ethical committee. From the paraffin blocks material will be taken for analysis with either polymerase chain reaction for detection of HPV or immunohistochemical analysis for the tumor markers presented in table 1.

Detection of HPV by PCR

A total of 30 um from the paraffin embedded biopsies will be cut and then DNA will be extracted using High Pure RNA kit (Roche Diagnostics, Stockholm, Sweden) excluding DNAse treatment. A negative control between each sample will be included to avoid cross contamination. High risk HPV DNA will be detected using the PCR method with general prmiers Gp5+/6+ and CpI/IIG, targeting the L1 and E1 region of the genome respectively. The HPV type will then be determined using type specific primers or direct sequencing of the genome, all this as previously described (5, 25, 26).


The HPV detection will be performed in cooperation with Professor Tina Dalianis, Karolinska Insititutet. The Dalianis group has long experience of analyzing presence of HPV (5, 7, 27-30), and this analysis has also been performed by the applicant. The security in the lab is ensured by a standardized education of all new staff working in the lab.

Immunohistochemical analysis

Three micrometer sections from the paraffin blocks will be reviewed by a senior pathologist and a representative area of the tumor will be marked for tissue micro array (TMA). Punch biopsies will be taken from the blocks and be joined together into TMA paraffin block, including controls. Immunohistochemsitry will then be performed with the Dako Autostainer as previously described (31).


The immunohistochemistry will be performed in cooperation with Associate Professor Tibor Tot, Falu Lasarett. This analysis will be performed in a clinical pathology laboratory with long experience of this type of analysis. The analysis is performed with same safety equipment and by the same staff working in the clinical laboratory, the safety of this work is considered high.

Statistical analysis

The results from the HPV detection and the expression of the different tumor markers will then be correlated to clinical data retrieved from the patient files. The correlation between HPV status and different tumor markers will be investigated by either Fisher´s exact 2-tailed test, a Chi square test or by logistic regression. The survival analysis will be performed with a multivariate Cox proportional hazard regression model controlling for known confounders. The data will be presented in a Kaplan-Meier graph where differences will be analyzed by the Log-rank test. All significance testing will be performed at the 0,05 level and two sided p-values will be used where applicable.



CLINICAL SIGNIFICANCE
It has been suggested that HPV positive oropharyngeal cancer have different tumor biology with a genetic profile providing better conditions for adequate response to the cellular stress induced by treatment with radiotherapy and chemotherapy, and that this would explain the better survival (32). Therefore, it is important to study if there are other tumor markers that differ in expression when comparing HPV positive and HPV negative oropharyngeal cancer. If differences in tumor markers can be detected they can be used for diagnosing HPV positive oropharyngeal cancer, which is not as expensive as the methods used today, and could therefore be valuable for clinical practice.
The prognostic value of different tumor markers should also be evaluated and would provide more prognostic information to the patient if used in clinical practice. Further studies of the differences in biology of HPV positive and HPV negative oropharyngeal cancer could also lead to individualized treatment as discussed above.
Finally, before including the HPV vaccines in the general vaccination program, it is important to further investigate the HPV status in oropharyngeal cancer which has been done in the Stockholm area but not in Dalarna. A decrease in the incidence of oropharyngeal cancer after the vaccine has been introduced would provide more evidence of HPV as an etiological agent in oropharyngeal cancer.


ETHICAL CONSIDERATIONS
This project requires no participation from the patients except for their approval for using the material from their biopsies and collecting data from the patient files, thus the patients are not exposed to any risks.
The result of this project will not be of value for the participants with regard to knowledge about treatment or prognosis, only hopefully for patients diagnosed with oropharyngeal cancer in the future.

REFERENCES


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