Download Expression of tumor markers in orophyaryngeal cancer

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 34% 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, Ki67, 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
c-myc
COX-2
Ki-67
CD4+
EGFR
VEGF
E-cadherin
CD44
p27
Major functions
Cell-cycle progression, malignant transformation
Prostaglandin synthesis, angiogenesis, decreased apoptosis
Proliferation
Immune response
Proliferation
Angiogenesis
Cell-cell adhesion
Cell-cell adhesion
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, 2730), 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 pvalues 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.
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